Nano Bio Sensing
Nano Bio Sensing
Nano Bio Sensing
BIOMEDICAL ENGINEERING
BIOLOGICAL AND MEDICAL PHYSICS,
BIOMEDICAL ENGINEERING
The fields of biological and medical physics and biomedical engineering are broad, multidisciplinary and
dynamic. They lie at the crossroads of frontier research in physics, biology, chemistry, and medicine. The
Biological and Medical Physics, Biomedical Engineering Series is intended to be comprehensive, cover-
ing a broad range of topics important to the study of the physical, chemical and biological sciences. Its
goal is to provide scientists and engineers with textbooks, monographs, and reference works to address
the growing need for information.
Books in the series emphasize established and emergent areas of science including molecular, membrane,
and mathematical biophysics; photosynthetic energy harvesting and conversion; information processing;
physical principles of genetics; sensory communications; automata networks, neural networks, and cel-
lular automata. Equally important will be coverage of applied aspects of biological and medical physics
and biomedical engineering such as molecular electronic components and devices, biosensors, medicine,
imaging, physical principles of renewable energy production, advanced prostheses, and environmental
control and engineering.
Editor-in-Chief:
Elias Greenbaum, Oak Ridge National Laboratory, Oak Ridge, Tennessee, USA
NanoBiosensing
Principles, Development and Application
Huangxian Ju Xueji Zhang
Nanjing University World Precision Instruments, Inc.
Nanjing, P.R. China Sarasota, FL, USA
[email protected] and
University of Science & Technology
Joseph Wang Beijing, P.R. China
University of California [email protected]
San Diego, CA, USA
[email protected]
ISSN 1618-7210
ISBN 978-1-4419-9621-3 e-ISBN 978-1-4419-9622-0
DOI 10.1007/978-1-4419-9622-0
Springer New York Dordrecht Heidelberg London
Library of Congress Control Number: 2011932683
The first decade of the 21st century has been labeled the sensing decade. Biosensing,
based on nanomaterials, is one of the hottest topics in nanotechnology and nanosci-
ence. The unique properties of nanomaterials offer excellent platforms as electronic
and optical signal transduction to design a new generation of biosensing devices.
Thus, nanobiosensing opens up the novel concepts for basic research and new tools
for ultrasensitive biosensing in clinical, environmental, and industrial applications.
With the achievements of nanotechnology and nanoscience, a wide variety of
nanoscale materials with different sizes (1100 nm), shapes, and compositions
have been introduced to biosensing. The small sizes of nanoparticles break through
the limitation of structure miniaturization, leading to lower detection limits, even
reaching zeptomolar concentrations. Furthermore, the biofunctional nanoparticles
can produce a synergic effect among catalytic activity, conductivity, and biocom-
patibility to accelerate the signal transduction. Most importantly, nanoscale materials
are in direct contact with the environment, which permits them to act as chemical
and biological sensors in the single molecular detection of biomolecules. As of this
writing, nanobiosensing is routinely being applied in biological systems. Future
efforts on nanomaterial-based biosensing will involve invivo detection with less
cytotoxicity, high sensitivity, and long-term stability for early screening of disease
biomarkers and reliable point-of-care diagnostics.
This book introduces novel principles and detection strategies in the area of bio-
sensing based on nanomaterials. Each chapter provides a theoretical overview of a
different topic and the interesting bioanalytical application of nanobiosensing
devices. The most exciting and unique aspect of the book is that the utilization of
nanomaterials not only enhances the biosensing capabilities, but also brings out
newer approaches such as biomimetic, reagent-less biosensing, and single molecular
detection.
The material is presented in 18 chapters, covering the most successful nanoma-
terials used so far in biosensing. The first four chapters of the book, contributed by
Huangxian Ju (Chap. 1), Songqin Liu and Huangxian Ju (Chap. 2), Zhihui Dai and
v
vi Preface
Huangxian Ju (Chap. 3), and Jianping Lei and Huangxian Ju (Chap. 4), describe the
biofunctionalization of nanomaterials, signal amplification strategies for nanobio-
sensing, and nanostructured mimicking enzymes. The next six chapters focus on
ultrasensitive platforms using various nanomaterials, such as carbon nanofiber,
nanoporous silica, carbon nanotubes, quantum dots, molecularly imprinted nanopar-
ticles, and solgel nanoparticles; they are contributed by Xueji Zhang and Lei Su
(Chap. 5), Shuo Wu and Huangxian Ju (Chap. 6), Jianping Lei and Huangxian Ju
(Chap. 7), Guizheng Zou and Huangxian Ju (Chap. 8), Ruizhuo Ouyang and
Huangxian Ju (Chap. 9), and Jiuhong Yu and Huangxian Ju (Chap. 10). The last eight
chapters illustrate the successful applications of nanobiosensing in environmental
screening and clinical diagnosis and have been contributed by Xueji Zhang and
Chunyan Wang (Chap. 11), Sichun Zhang and Huangxian Ju (Chap. 12), Dan Du and
Huangxian Ju (Chap. 13), Zong Dai, Joseph Wang, and Huangxian Ju (Chap. 14),
Zhifeng Fu and Huangxian Ju (Chap. 15), Yongkang Ye, Joseph Wang, and Huangxian
Ju (Chap. 16), Lin Ding and Huangxian Ju (Chap. 17), and Jie Wu, Joseph Wang, and
Huangxian Ju (Chap. 18). We are very grateful to all the authors who contributed
their high-quality work. We also thank Jie Wu for her help in editing the whole book.
This book involves a broad audience, such as those involved in the research,
teaching, learning, and practice of biosensing, based on various nanomaterials, in
biomedical, military, industrial, and clinical applications. We are fortunate to have
had the opportunity to undertake this enormous project. We warmly acknowledge
the gracious support of our families. Finally, we also thank Springers editors for
doing a remarkable job to publish this book.
1 Biofunctionalization of Nanomaterials.................................................. 1
1.1 Introduction....................................................................................... 1
1.2 Biofunctionalization Method of Nanomaterials................................ 2
1.2.1 Biofunctionalization by Noncovalent Assembly................... 2
1.2.2 Covalent Route for the Biofunctionalization
of Nanomaterials................................................................... 5
1.3 Biofunctional Nanomaterials............................................................ 10
1.3.1 Carbon-Based Nanomaterials............................................... 10
1.3.2 Metal Nanoparticles.............................................................. 12
1.3.3 Semiconductor Nanoparticles............................................... 14
1.3.4 Magnetic Nanoparticles........................................................ 15
1.3.5 Other Biofunctional Nanomaterials...................................... 17
1.4 Characterization of Biofunctional Nanomaterials............................. 19
1.5 Applications of Biofunctional Nanomaterials................................... 21
1.5.1 Optical Sensing..................................................................... 21
1.5.2 Electrochemical Sensing....................................................... 27
1.6 Conclusions....................................................................................... 32
References.................................................................................................. 32
2 Signal Amplification for Nanobiosensing............................................... 39
2.1 Introduction....................................................................................... 39
2.2 Nanoparticle-Amplified Optical Assay............................................. 40
2.2.1 Colloidal Gold Nanoparticle-Based Amplification............... 40
2.2.2 Semiconductor Nanoparticle-Based Amplification.............. 48
2.2.3 Nanoparticle-Amplified Chemiluminescence
and Electrogenerated Chemiluminescence Assay................. 49
2.3 Nanoparticle-Amplified Electrochemical Detection......................... 54
2.3.1 Enhanced Conductivity with Nanoparticles.......................... 54
2.3.2 Detection of Nanoparticle Label with
Stripping Voltammetry.......................................................... 57
2.3.3 Nanoparticle-Enhanced Impedance Signal........................... 58
2.3.4 Nanoparticle-Enhanced Voltammetric Signal....................... 62
vii
viii Contents
Index.................................................................................................................. 569
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Chapter 1
Biofunctionalization of Nanomaterials
1.1Introduction
1.2.1.1Physical Adsorption
The simple adsorption of biomolecules on NPs has frequently been performed for
the biofunctionalization of NPs with biomolecules, which range from small organic
substances to large protein/enzyme molecules [2]. In the case of NPs that are stabi-
lized by anionic ligands, such as carboxylic acid derivatives, citrate, tartrate, and
lipoic acid, the functional NPs allow effective binding to the positively charged
amino acid side chains of the protein by the negatively charged anionic groups on
their surface (Fig.1.1). For example, gold (Au) NPs produced by citrate reduction
can be functionalized with carcinoembryonic antibody molecules at pH values that
lie slightly above the isoelectric point of the citrate ligand [16]. Another example of
1.2 Biofunctionalization Method of Nanomaterials 3
Affinity interaction is very effective for the bioconjugation of targeting ligands to NPs
due to specific and strong complementary recognition interactions such as
antigenantibody, nucleic acidDNA, lectinglycan, streptavidinbiotin, aptamer
protein, aptamersmall biomolecule, and hormonereceptor interactions. Moreover,
1.2 Biofunctionalization Method of Nanomaterials 5
various biomolecules contain several binding sites; for example, antibodies exhibit two
Fab (antigen-binding fragment) sites, whereas streptavidin or concanavalin A each dis-
plays four binding domains. This allows the multidirectional growth of NP structures.
As shown in Fig.1.1, the surfaces of NPs can be modified with streptavidin, which
specifically binds biotinylated molecules. The linkage formed is highly stable and the
strongest of all noncovalent linkages. Unlike hydrophobic and electrostatic interac-
tions, affinity binding is not sensitive to environmental conditions such as changes in
pH, salinity, or hydrophilicity. For example, avidin-functionalized Ce/Tb-doped LaPO4
NPs have been used for the superassembly of biotinylated proteins and oligonucle-
otides [27]. Since biotinylated molecules are easily obtained, the applicability of these
bioconjugates in biosensing and biolabeling has proved to be useful.
Aptamers, the short, single-stranded nucleic acid sequences originated from
in vitro selection (SELEX), have attracted much interest as new recognition ele-
ments of proteins and other small biomolecules. The competitive advantages of
aptamers over antibodies, such as high specificity and affinity, chemical stability,
ready availability, and high flexibility, make them promising tools for protein detec-
tion. Many explorations have been achieved in the development of aptamer-based
technologies for highly sensitive protein detection by combining them with poly-
merase chain reaction (PCR), electrochemistry, capillary electrophoresis, mass
spectrometry, and quartz crystal microbalance. The aptamer-functionalized NPs
will be a promising platform for constructing new biosensing and bioanalytical sys-
tems by the specific recognition of the aptamer toward target biomolecules.
Recently, based on specific recognition between lectins and sugar epitopes, a
concanavalin Afunctionalized Au NP as nanoprobe was developed for the highly
sensitive and selective in situ evaluation of carbohydrates on living cells by electro-
chemical tracing of the enzyme coimmobilized on the NPs [28]. Coupled with the
efficient capture of cells on electrodes modified with arginine-glycine-aspartic acid-
serine tetrapeptidefunctionalized SWNTs by the specific affinity interaction
between the tetrapeptides and the integrins on the cell surface, the dynamic changes
in four kinds of cell surface carbohydrates during drug treatment can be monitored
by four horseradish peroxidase (HRP)-labeled lectins [29].
Fig. 1.3 Preparation of Ab2-MWNT-HRP bioconjugates via amide bond formation. Reprinted
with permission from Yu etal. [34]. 2006, American Chemical Society
Fig. 1.4 Bioconjugation strategies for chemical modification on a nanoparticle (NP) surface.
Reprinted with permission from Liu etal. [6]. 2010, Hindawi Publishing Corporation
When the NP surfaces are primarily functionalized with some groups, such as the
sulfhydryl, amine, carboxylic, and hydroxyl groups, the further biofunctionalization
of these NPs sometimes needs a step to activate these groups, which enables the
groups to react with ligands of choice. The method selected for the activation of a
functional NP surface must be compatible with both the ligand and the NPs. The
general methods can be summarized as follows.
The amine reactive chemistries are the first methods introduced to activate the NP
surfaces. Here cyanogen bromide (CNBr) can be used for the activation of hydroxyl
groups. It can react with the OH group at a high pH to produce a very reactive
cyanate ester, which then reacts directly with amine groups. This method can be used
almost universally to activate NPs containing hydroxyl groups. CNBr-activated NPs
can be used to couple to small ligands as well as to high-molecular-weight biopolymers
containing primary amine groups. The procedure is relatively simple to carry out and
is very reproducible for coupling sensitive biomolecules such as enzymes and anti-
bodies to NPs. Another method of amine reactive chemistries is the use of N-hydroxy
succinimide (NHS) esters as active groups to form amide bonds with primary amines.
NHS can activate both OH and COOH groups. The produced NHS ester will react
quite efficiently with primary amine-containing ligands to form a stable amide bond.
For the activation of a COOH group, N,N-carbonyl diimidazole (CDI) and EDC are
highly reactive carbonylating reagents. In the presence of a primary amine-containing
compound, the CDI-introduced imidazole is displaced to form a stable amide bond.
Sometimes, CDI can also be used to activate hydroxyl-containing NPs. The EDC-
mediated linkages can be done in two ways: Amine-containing NPs can be used to
couple a carboxyl-containing ligand, or NPs with the carboxyl group available can be
used to couple an amine-containing ligand.
Aldehydes and ketones can react with the primary and secondary amino groups to
form reversible Schiff bases, which can then be reduced and stabilized as covalent
linkages by using a reducing agent such as sodium borohydride. Thus, the primary or
secondary amino group-containing biomolecules can be introduced to NPs by these
reactions. The key step is the generation of aldehyde groups on the NP surface, which
can be created by the mild oxidation of adjacent diols of agarose with NaIO4. Thus,
the first step is to functionalize the NPs with agarose-based gels or glycidol. The
latter can react with OH to introduce adjacent hydroxyls to the NP surface.
Hydrazide reactive chemistry permits the coupling of aldehyde- or ketone-
containing ligands through the formation of stable hydrazone linkages. Thus, glyco-
proteins in particular may be introduced to the NP surface using this procedure after
being oxidized with NaIO4 to produce formyl groups on their carbohydrate chains.
This method is powerful for functionalizing hydrazide-activated NPs with proteins
while leaving critical active sites free.
Sulfhydryl reactive chemistry is another way to introduce biofunctional molecules
to the NP surface. It can be carried out by the iodoacetyl and bromoacetyl activation
method. The iodoacetyl- and bromoacetyl-activated NPs containing the NH2 group
can be coupled to sulfhydryl groupcontaining ligands by extremely stable thioether
10 1 Biofunctionalization of Nanomaterials
1.3Biofunctional Nanomaterials
1.3.1Carbon-Based Nanomaterials
Fig.1.6 1-Pyrenebutanoic acid, succinimidyl ester irreversibly adsorbing onto the sidewall of an
SWNT via pp stacking. Reprinted with permission from Chen et al. [20]. 2001, American
Chemical Society
sp2 structure and conjugation. Most notably, small aromatic molecules and conjugated
polymers have been used to decorate the nanotube surface via electrostatic interac-
tion, pp stacking, or van der Waals force, and to improve the solubility and elec-
tronic properties of CNTs. The first demonstration of SWNT functionalization with
proteins via pp stacking was reported by Dais group at Stanford University [20]. A
bifunctional molecule, 1-pyrenebutanoic acid, succinimidyl ester, was first irrevers-
ibly adsorbed onto an SWNT via pp stacking between the pyrenyl group and the
sidewall of the SWNT (Fig.1.6). Proteins were then immobilized through a nucleo-
philic substitution of NHS by the amino group of the proteins to form an amide bond.
Alternatively, a direct assembly of water-insoluble porphyrin on SWNT was designed
based on the pp noncovalent interaction between porphyrin and CNTs, resulting in
a novel biosensor for trichloroacetic acid [43].
12 1 Biofunctionalization of Nanomaterials
Fig.1.7 Schematic illustration of the dehydration and carbonization process of fructose and the
following enzyme-antibody-functionalized CNSs. Reprinted with permission from Du etal. [46].
2010, American Chemical Society
1.3.2Metal Nanoparticles
Metal NPs involving Au, Ag, Pd, and Pt NPs can provide three-dimensional architec-
tures, which have attracted widespread interest since their nanosized physical
1.3 Biofunctional Nanomaterials 13
Fig.1.8 Schematic illustration of the different stability of Au NPs with folded and unfolded ade-
nosine binding DNA aptamer. Reprinted with permission from Zhao etal. [32]. 2008, American
Chemical Society
p roperties are quite different from those of the bulk materials. Metal NPs have mainly
been modified with thiols, disulfides, amines, nitriles, carboxylic acids, and phos-
phines [8]. First, multipoint metalS bond formation is a straight-forward way to fix
functional molecules in the desired geometry. Second, some of the thiolate ligands in
alkanethiolate-stabilized Au NPs can be substituted by reaction with other thiols at
rates depending on the chain length and steric bulk of the leaving thiolate and incom-
ing thiols and on the charge of the Au NPs. Thiolthiol exchange mostly requires a
considerable excess of incoming ligand to render the exchange complete, but a delib-
erate partial exchange may also be useful. Third, carboxylic acidamine coupling
can be used to link any protein bearing a primary amine to Au NPs [7].
Conjugates of Au NPDNA are of great current interest because of the potential
use of the programmability of DNA base pairing to organize nanocrystals (NCs) in
space and the multiple ways of providing a signature for the detection of precise
DNA sequences. Adenosine aptamers have been chemically coupled onto Au NPs
using AuS chemistry. Before the addition of adenosine, the ssDNA aptamer on Au
NPs adopts a loose random coil structure, since there is no strong intramolecular
base pairing. By contrast, the aptamer folds into a well-characterized tertiary struc-
ture in the presence of adenosine (1 mM) in a buffer containing 4 mM MgCl2,
100mM NaCl, and 20mM Tris-HCl (Fig.1.8). On the basis of this unique phenom-
enon, colorimetric biosensors have been developed for the detection of adenosine,
K+, adenosine deaminase, and its inhibitors [32]. Further, coupled with PCR and
rolling-circle amplification, the three-dimensional positioning of Au NPs has been
realized for the development of negative-index materials [51] and unique scaffolds
in nanotechnology and biodiagnostics [52].
Metal NPs are often combined with other materials, such as solgel matrices,
polymers, and other nanomaterials, which can provide a network structure or a basal
matrix that immobilizes metal NPs onto the electrode surface. For example, a novel
Au NPbacteria cellulose nanofiber has been synthesized using bacterial cellulose
nanofibers as robust biotemplates via a one-step method for the detection of H2O2
14 1 Biofunctionalization of Nanomaterials
Fig.1.9 Modification of semiconductor QDs with functional encapsulating layers for water solu-
bilization and preservation of luminescence properties and/or secondary covalent modification of
the surface with biomolecules. Reprinted with permission from Gill etal. [5]. 2008, Wiley
with a detection limit lower than 1mM [53]. Pd NPs have also been electrodeposited
on SWNTs from a PdCl2 solution, leading to 1,000-fold increases in resistance to H2
gas [54].
1.3.3Semiconductor Nanoparticles
such as amines or thiols, may interact directly with the QDs surface as ligands.
When biomolecules do not contain the groups for direct QD binding, they may be
modified to introduce this functionality. For example, nucleic acids and peptides
have been modified to introduce thiol groups for binding to QDs. Since the QDs are
negatively charged in neutral or basic buffers, the positively charged molecules can
be used for the electrostatic functionalization of QDs, especially for macromole-
cules such as enzymes and antigenantibody. Another method for the surface modi-
fication of semiconductor NPs has also become modular through high-affinity
streptavidinbiotin binding. QDstreptavidin conjugates are convenient for indirect
binding to a broad range of biotinylated biomolecules. The coating of the QDs with
protective silicon oxide films or polymer films has also been an alternative method
to keep the biocompatibility of QDs [5].
In addition, amine-modified QD705 (emission maximum at 705nm) has been
conjugated to a heterobifunctional cross-linker, 4-maleimidobutyric acid-NHS ester,
yielding a maleimide-NC surface for integrin-targeted near-infrared optical imaging,
which will be useful in cancer detection [55].
The main compounds used for modifying metal oxide NPs are phosphonates,
carboxylate, and silanes. Carboxylate ligands, especially fatty acids, are often used
for the functionalization of metal oxide NPs. The binding of carboxylate ligands can
be formed on the surface of titania NPs via physical adsorption, monodentate coor-
dination, bridging chemisorption, and chelating chemisorption [8]. A new proce-
dure based on photodeposition of nano-Ag on a TiO2-coated piezoelectric quartz
crystal (PQC) electrode has been developed to fabricate a highly sensitive PQC/
DNA biosensor [56].
1.3.4Magnetic Nanoparticles
Fig.1.10 Schematic illustration of the functionalized magnetic particle fabrication process. (a)
Fe3O4 NPs; (b) PBFe3O4 NPs; (c) the Au NP seeds coating PBFe3O4 NPs; and (d) AuPBFe3O4
NPs. Reprinted with permission from Zhuo etal. [59]. 2009, Elsevier
drop by drop with the help of a slight excess of H2O2. Subsequently, the surface of
PBFe3O4 NPs is chemically modified with bovine serum albumin (BSA) to obtain
the amido and disulfide groupmodified PBFe3O4 NP. Finally, the PBFe3O4 par-
ticles covalently attract 13-nm Au NP seeds, which act in a next step as nucleation
sites for the formation of a continuous gold outer layer by citrate reduction of Au3+
to Au0. The multilabeled AuPBFe3O4 NPs exhibit satisfying redox electrochemi-
cal activity and high enzymatic activity for an ultrasensitive and reproducible elec-
trochemical immunosensor [59]. Based on magnetic beads (MBs) and HRP-labeled
anti-AFP antibody-modified Au NPs, a novel and sensitive chemiluminescence
(CL) immunoassay has been developed by employing a new CL enhancer, bro-
mophenol blue, for the determination of AFP, a tumor marker, with a linear range
from 0.1 to 5.0ngmL1 and a detection limit of 0.01ngmL1 [60]. With a multiple-
enzyme-labeled antibody-MB bioconjugate (7,500 HRP on 1mm MBs), an ultra-
sensitive electrochemical immunosensor has been constructed to detect cancer
biomarkers in serum [61].
The nucleic acid- or antigen-functionalized magnetic particles, together with the
naphthoquinone 4-modified magnetic particles, can yield enhanced electrogenerated
CL in the presence of HRP-bioconjugates and luminol. Using higher rotation speeds,
the sensitivity of the antibody detection can be further improved, with a detection
limit of 50100pgmL1 at 2,000rpm [62]. A signal amplification strategy based on
bio-barcodefunctionalized magnetic NPs as labels holds promise to improve the
sensitivity and detection limit of the detection of DNA hybridization and single-
nucleotide polymorphisms by flow-injection CL assays [63]. A sandwich-type
detection strategy is employed as shown in Fig.1.11. Biotinylated capture probes
are loaded on avidin-modified magnetic microparticles (MMPs), while thiolated
detection probes are assembled on Au NPs via AuS bonds. In the presence of
targetDNA, the capture probe recognizes the target DNA, along with the detection
probe, to the proximity of MMPs, and this complex is then magnetically separated
1.3 Biofunctional Nanomaterials 17
for subsequent optical detection. This novel method can conveniently detect as few
as 100pM target DNA with the naked eye, and this sensitivity can be significantly
improved by instrument-based assays [64].
Fig.1.12 Schematic of the immobilization of gold nanoparticles (Au NPs) by DNA hybridization
on silicon dioxide surfaces. Reprinted with permission from Koplin et al. [82]. 2006, Royal
Society of Chemistry
Fig.1.14 (a) TEM and (c) SEM images of colloidal CNSs, and (b) TEM and (d) SEM images of
the Au NP/CNS hybrids. Reprinted with permission from Cui etal. [84]. 2008, Wiley
1.5 Applications of Biofunctional Nanomaterials 21
Fig.1.15 X-ray
photoelectron spectra
of the surfaces of the
(a) antibody-deposited
CNP-PEI/SPGE and the
(b) CNP-PEI/SPGE.
Reprinted with permission
from Ho etal. [85].
2009, American Chemical
Society
1.5.1Optical Sensing
Fig.1.17 Schematic illustration showing a cascade procedure used to amplify fluorescence signal
depending on the proteolytic activity of MMP-2. Reprinted with permission from Kim and Chung
[95]. 2010, Wiley
1.5.1.2Fluorescence Detection
A new strategy for highly sensitive and rapid protease assay is developed by
mediating the proteolytic formation of oligonucleotide duplexes and using the
duplexes for signal amplification (Fig.1.17). In the presence of matrix metallopro-
tease-2 (MMP-2), fragmentation of the intact DNApeptide on Au NPs by hydro-
lytic cleavage of a peptide bond within the substrate allows diffusion of the DNA
away from the Au NPs and the formation of a DNA/RNA heteroduplex, leading to
digestion of RNA by RNase H. Because of the high quenching efficacy of Au NPs
to the fluorophore in RNA and multiple digestions of the RNA, the fluorescence
signal recovery is amplified. This method permits the assessment of the activity of
MMP-2 at concentrations as low as 10pM within 4h. Compared with the reported
protease nanosensors using QDs, Au NPs, and magnetic NPs with the same peptide
sequence, the assay time of this method is 6 times faster and the limit of detection
is 100 times more sensitive [95]. A near-infrared fluorescence imaging probe based
on Au NPs functionalized with self-assembled heterogeneous monolayers of dye-
labeled peptides and poly(ethylene glycol) has been developed to visualize prote-
olytic activity invivo [96]. And a metal-enhanced fluorescence platform has been
designed to develop a sensing surface by water-soluble polyelectrolytes with under-
lying Ag NP arrays for optically amplified DNA detection [97].
A much simpler and milder strategy to amplify fluorescence signals by using ionic
NCs with no special optical properties has been suggested. The cation-exchange reac-
tion with ionic NCs can release thousands of divalent cations, which can in turn trigger
the fluorescence from thousands of nonfluorescent metal-sensitive dyes to obtain large
fluorescence amplification. The NCdye set of CdSe and fluo-4 used in the present
study led to a 60-fold enhancement of the fluorescence signal and a limit in protein
detection 100 times lower than that of the organic fluorophore Alexa 488 [98]. Lately,
the cation exchangebased fluorescence amplification method has been designed for
24 1 Biofunctionalization of Nanomaterials
Fig. 1.18 Schematic presentation of the small RNA detection assay using CXFluoAmp. MP
magnetic particles; CP capture probe; DP detection probe. Reprinted with permission from Li
etal. [99]. 2009, American Chemical Society
the detection of low abundance and short strand length of small RNA molecules
(Fig.1.18). Nonfluorescent, ionic NCs of CdSe are conjugated to detection probes and
immobilized onto the array surface via ligation with the target small RNA, miR21,
which is bound to the capture probe complementarily. Each binding event induced by
one target miR21 molecule is then amplified by the release of thousands of Cd2+ from
one NC. The free Cd2+ immediately turns on the fluorescence of thousands of fluoro-
genic Rhod-5N molecules. With such a powerful signal-amplification strategy, the
assay achieves a limit of detection of 35fM and the signals are detectible with analyte
concentrations spanning over seven orders of magnitude [99].
FRET-based probes incorporated with single-molecule fluorescence detection
technologies have allowed the detection of DNA with low abundance without unam-
plification. As shown in Fig.1.19, the QD acts as both a FRET energy donor and a
1.5 Applications of Biofunctional Nanomaterials 25
1.5.2Electrochemical Sensing
Electrochemical transducers are very attractive for such bioassays, due to their high
sensitivity, inherent simplicity and miniaturization, and low cost and power require-
ments. The biofunctionalized nanomaterials provide an excellent platform to con-
struct ultrasensitive electrochemical sensors for the detection of biomolecules such
as DNA, proteins, cells, and other signal biomolecules.
1.5.2.1DNA
The development of highly sensitive and selective DNA sensors to pico- and femto-
molar levels is a field of ever-increasing interest for various applications, including
the diagnosis and treatment of genetic diseases, drug discovery, and warning against
bio-warfare agents. CNTs make them extremely attractive for electrochemical DNA
sensors due to the unique electronic, chemical, and mechanical properties. Most
CNT-sensing work has focused on the ability of surface-confined CNTs to promote
electron-transfer reactions involved in biocatalytic devices. Wangs group [13] has
demonstrated that CNTs play a dual-amplification role in both the recognition and
transduction events, namely, as carriers for numerous enzyme tags and for accumu-
lating the product of the enzymatic reaction. The 104-fold improvement in the sen-
sitivity is in good agreement with the estimated ALP loading per CNT. The favorable
response of the 5fgmL1 DNA target indicates a remarkably low detection limit of
around 1fgmL1 (54aM), i.e., 820 copies or 1.3zM in the 25-mL sample [13].
Asensitive electrochemical DNA biosensor has been successfully realized on poly-
aniline nanofiber (PANI)-coated MWNTs in chitosan filmmodified carbon paste
electrode (CPE). The immobilization of the probe DNA on the surface of the elec-
trode is largely improved due to the unique synergistic effect of PANI and MWNTs.
Under the optimal conditions, the dynamic detection range of this DNA electro-
chemical biosensor is from 1.01013 to 1.0107molL1, with a detection limit of
2.71014molL1, for the detection of DNA-specific sequences of the phosphino-
thricin acetyltransferase gene [111].
Figure1.21 represents a schematic view of a sandwich-type DNA sensor employ-
ing Pd NPs as electrocatalytic labels. To achieve low levels of nonspecific binding
of DNA-conjugated Pd NPs, ITO electrodes are modified with a silane copolymer
28 1 Biofunctionalization of Nanomaterials
Fig.1.21 Schematic view of DNA detection using the catalytic and electrocatalytic oxidation of
NaBH4 on Pd NPs and the rapid enhancement of the electrocatalytic activity of DNA-conjugated
Pd NPs. Reprinted with permission from Das etal. [112]. 2009, Royal Society of Chemistry
containing poly(ethylene glycol) and carboxylic acid. In this solution, the fast
catalytic hydrolysis of NaBH4 on Pd NPs generates many atomic hydrogens, which
are rapidly sorbed into Pd NPs (Fig.1.21). The fast hydrogen sorption induces the
rapid enhancement of the electrocatalytic activity of Pd NPs. Finally, the electro-
catalytic oxidation current of NaBH4 by Pd NPs is measured. Because NaBH4
undergoes multielectron (maximum 8e) oxidation on Pd NPs, higher current signals
can be obtained than those in the electrochemical reactions involving one- or two-
electron oxidation [112]. A DNA biosensor has been fabricated by immobilizing
capture-probe DNA on the nanoporous gold (NPG) electrode and hybridization
with target DNA, which further hybridizes with the reporter DNA loaded on the Au
NPs. Electrochemical signals of [Ru(NH3)6]3+ bound to the reporter DNA via elec-
trostatic interactions are measured by chronocoulometry. Taking advantage of the
dual-amplification effects of the NPG electrode and multifunctional encoded Au
NPs, this DNA biosensor can detect the DNA target quantitatively, in the range of
8.010171.61012 M, with a limit of detection as low as 28 aM, and exhibits
excellent selectivity even for single-mismatched DNA detection [30].
A new metal sulfide NPbased electrochemical detection method has been pro-
vided with the detection capability down to 100aM of target DNA. The setup is
constructed to give a signal-off response with a built-in control signal. The control
signal eliminates the disadvantages commonly associated with signal-off sensors
[113]. Multianalyte aptamer-based devices, with lower detection limits, are highly
desired for measuring a large panel of disease markers present at ultralow levels
during early stages of the disease progress. Four encoding NPs (cadmium sulfide,
zinc sulfide, copper sulfide, and lead sulfide) have been used to differentiate the
signals of four DNA targets in connection to stripping voltammetric measurements
of the corresponding metals [14].
The remarkable synergistic effects of the ZnO NPs and MWNTs have been devel-
oped for the ssDNA probe immobilization and fabrication of the electrochemical
1.5 Applications of Biofunctional Nanomaterials 29
DNA biosensor. Under optimal conditions, the dynamic detection range of the sensor
to PAT gene complementary target sequence is from 1.01011 to 1.0106molL1,
with a detection limit of 2.81012molL1 [114].
Of the diverse DNA detection techniques, ECL-based biosensing has received
considerable attention due to its versatility, simplified optical setup, and good tem-
poral and spatial control [115]. The quenching of ECL from a CdS:Mn NC film by
proximal Au NPs is observed as a result of Frster energy transfer, while an enhance-
ment of ECL takes place after hybridization with target DNA due to the energy
transfer of ECL-excited surface plasmon resonances in Au NPs to the CdS:Mn NCs
at large separation, based on which an ultrasensitive and specific DNA biosensor
has been constructed. The relationship between the increase in ECL peak height
before and after hybridization and target DNA concentration shows a linear range
from 50aM to 5.0fM. The favorable response of 50-aM target DNA indicates a
remarkably low detection limit (S/N=3), i.e., 2,100 copies in 70 mL of sample
[116]. A novel PCR-free ECL-based bio-barcode assay is also reported for the
quantitative detection of genetically modified organisms from raw materials without
additional purification [117].
A novel and sensitive flow-injection CL assay is reported for sequence-specific
DNA detection. The hybridization events are monitored by the CL intensity of lumi-
nolH2O2Cu2+ after the cupric ions are dissolved from the hybrids. The CL inten-
sity increases with the increase in the concentration of target DNA in the range of
2.010142.01012M, with a detection limit of 4.81015M [118].
1.5.2.2AntibodyAntigen
1.5.2.3Cells
CNTs and a redox mediator toluidine blue O (TBO) have been coimmobilized in a
matrix of the biopolymer chitosan and used for the oxidation of the enzyme cofactor
b-nicotinamide adenine dinucleotide (NADH) [126]. The integration of CNTs and
redox mediators can provide a remarkable synergistic augmentation of the current
because of the oxidation of redox-active species. In particular, it amplifies the NADH
current approximately 60 times while reducing the response time from approximately
50s for CHIT-TBO to approximately 5s for CHIT-TBO/CNT films.
A highly sensitive electrochemical sensor has been developed for the detection
of Hg2+ ions in aqueous solution by using a thymine (T)-rich, mercury-specific oli-
gonucleotide probe and Au NP-based signal amplification via the Hg2+-mediated
coordination of THg2+T base pairs [127]. This Au NP-based sensing strategy
brings about an amplification factor of more than three orders of magnitude, leading
to a limit of detection of 0.5nM [128].
32 1 Biofunctionalization of Nanomaterials
1.6Conclusions
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Chapter 2
Signal Amplification for Nanobiosensing
2.1Introduction
One of the major goals in developing novel biological assay methods for the
detection of biomolecules and DNA hybridization is achieving high sensitivity. The
need for ultrasensitive bioassays is of major importance in view of the growing
trend toward miniaturized assays. Highly sensitive methods, which are urgently
required for measuring disease diagnosis markers present at ultralow levels during
early stages of disease progression, can facilitate the treatment of diseases. For
example, polymerase chain reaction (PCR) amplification has revolutionized genetic
testing. However, it is somewhat restricted because of its complexity, potential con-
tamination, and cost. On the other hand, the ultrasensitive monitoring of proteins is
particularly challenging due to the absence of PCR-like amplification protocols.
Conventional (optical and electronic) sandwich bioaffinity assays have the disad-
vantage of capturing a small number of labels per binding event. Recently, signal
amplification has attracted considerable attention for developing ultrasensitive
detection methods for biothreats and infectious agents. Such kinds of highly sensi-
tive bioagent detection schemes provide an early warning of their release and pre-
vent outbreaks of foodborne illnesses, hence minimizing human casualties.
The achievement of ultrahigh sensitivity requires innovative approaches that
couple with different amplification platforms and amplification processes.
Nanotechnology offers unique opportunities for creating highly sensitive innovative
biosensing devices and ultrasensitive bioassays. The unique optical [14],
photophysical [5], electronic [6], and catalytic [79] properties of metal and semi-
conductor nanoparticles (NPs) turn them into ideal labels for biorecognition and
biosensing processes. For example, the unique plasmon-absorbance features of gold
(Au) NPs and specifically the interparticle-coupled plasmon absorbance of conju-
gated particles have been widely used for DNA [10] and antibodyantigen [1113]
analyses. Similarly, the tunable fluorescence properties of semiconductor NPs have
been used for the photonic detection of biorecognition processes [14]. This chapter
focuses on signal amplification based on nanobiotechnologies for highly sensitive
nanobiosensing.
Among all detection methods used in biosensors, the optical-based technique is the
most popular one because of its high sensitivity and the ability to remotely interro-
gate the information on the biosensor using light or laser. Metal NPs, such as Au and
silver NPs, exhibit plasmon absorbance bands in the visible spectral region that are
controlled by the size of the respective particles. Numerous studies on the labeling of
biomaterials and the staining of biological tissues by metal particles as a means to
image and visualize biological processes have been reported [15, 16]. The spectral
shifts originating from adjacent or aggregated metal NPs, such as Au NPs [17], have
led to increasing interest in the development of optical biosensors based on
biomaterial-NP hybrid systems. Similarly, semiconductor NPs exhibit size-dependent
tunable absorbance and fluorescence. Due to the high-fluorescence quantum yields,
photostability, and tunable fluorescence bands, semiconductor NPs are attracting
substantial research interest as fluorescence labels for biorecognition processes.
Fig.2.1 Diagram of the three types of fluorescence probes used for the LSPCF biosensor. Reprinted
with permission from Hsieh etal. [27]. 2007, American Chemical Society
Fig. 2.2 Schematic illustration of the RCA reaction and Au NP assembly-based assay: (1)
hybridization between padlock probe and target and ligation; (2) RCA and digestion; (3) colori-
metric detection via the assembly of Au NP-tagged DNA probes. Reprinted with permission from
Li etal. [28]. 2010, American Chemical Society
detection of mutant targets even when the ratio of the wild type to the mutant is
10,000:1. The developed RCA-based colorimetric detection scheme has been dem-
onstrated for SNP typing of the thalassemia gene at position 28 in genomic DNA.
A sandwich-type assay for the optical detection of DNA using multicomponent
cross-linked Au NP aggregates was reported by Fan (Fig.2.3) [29]. In their work, a
DNA-bridged multi-functional Au NP aggregate, which integrated DNA recogni-
tion (detection probe [DP]), signal amplification (enzyme, horseradish peroxidase,
HRP), and nonspecific blocking (bovine serum albumin, BSA) section, was
employed as the DP. In a typical sensing process, the Au aggregates DP was brought
to the proximity of magnetic particles through the DNA hybridization. Once the
magnetic field was added, these sandwich complexes were magnetically separated.
As a result, HRP that was confined at the surface of Au aggregates could catalyze
the enzyme substrate and generate an optical signal. This assay was employed for
the detection of breast cancerassociated BRCA-1 gene. The detection limit was
about 1fM, which was significantly improved compared with the results obtained
from individual Au NP-labeled assays.
Figure2.4 shows a sensitive detection method for adenosine (AD) in human urine
by using enhanced-resonance light scattering (RLS) [30]. It is based on the specific
recognition and signal amplification of AD aptamer (Apt) coupled with Au NPs via
G-quartetinduced NP assembly, which is fabricated by triggering a structureswitch-
ing of the 30 terminus G-rich sequence and Apt duplex. The RLS signal linearly
2.2 Nanoparticle-Amplified Optical Assay 43
Fig.2.3 Schematic for the amplified sandwich-type detection assay of the BRCA-1 gene using
multifunctional cross-linked Au aggregates and magnetic particles. Note: The drawing is not to
scale. Reprinted with permission from Li etal. [29]. 2009, Elsevier
Fig.2.4 Schematic diagram for analytical principle. (a) DNA duplex; (b) procedure of Au NPs
aggregate. (a) G-quartets stacked perpendicularly to the column axis; (b) four guanines fold into a
G-quadruplex structure by Hoogsteen hydrogen bonds. Reprinted with permission from Zhang
etal. [30]. 2010, Elsevier
correlates with the concentration of AD over the range of 6115nM. It has been
applied to detect AD in real human urine, and the obtained results are in good agree-
ment with those obtained by the HPLC method. This study illustrates that the com-
bination of the excellent selectivity of Apt with the high sensitivity of the RLS
technique provides promising potential for Apt-based small-molecule detection, and
may be beneficial in extending the applications of RLS.
Based on Au NP probes, a one-step, washing-free, and amplification-free assay for
protein analysis via dynamic light scattering (DLS) has been developed (Fig.2.5) [31].
The concentration of the target protein is determined by analyzing the level of Au NP
44 2 Signal Amplification for Nanobiosensing
Fig.2.5 Illustration of a one-step homogeneous biomolecular assay using gold nanoparticle (NP)
probes as light-scattering enhancers coupled with dynamic light-scattering detection. Reprinted
with permission from Liu and Huo [31]. 2009, Elsevier
Fig. 2.6 Scanometric immunoassay. Reprinted with permission from Kim et al. [32]. 2009,
American Chemical Society
Fig. 2.7 Nicking endonuclease-assisted NP amplification for target DNA detection. Reprinted
with permission from Xu etal. [34]. 2009, Wiley
Fig.2.8 Photograph showing colorimetric responses of a NEANA detection system. The labeled
concentrations (20nM, 2nM, 200pM, 20pM, and 10pM) are the calculated final target concen-
trations in solutions. The NEase recognition site of the target is highlighted in red. Reprinted with
permission from Xu etal. [34]. 2009, Wiley
Fig. 2.9 (a) Chemical structure of OFP; (b) HR-TEM image of OFP; (c) normalized UVvis
absorption spectra of the arm 4, OFP, and EB (dashed lines), and PL spectra of 4 and OFP (solid
lines) in water. Reprinted with permission from Pu etal. [54]. 2010, Wiley
Using hybrid nanomaterials as the signal amplifiers, Pu etal. provided a new way
to improve the performance of fluorescence technologies for biological imaging
through a fluorescence resonance energy transfer (FRET) approach [54]. From the
materials viewpoint, the emission wavelength, charge nature, and diameter of poly-
hedral oligomeric silsesquioxane (POSS)-based fluorescent NPs can be easily
adjusted through chemical modification of fluorescent arms so as to fulfill the dif-
ferent requirements of specific applications. In terms of materials applications, the
high quantum yields and good signal amplification capability of POSS-based mol-
ecules allow high-quality biological imaging even with a small amount of indicator
dyes, consequently avoiding the side effect of elevated dye concentrations. In view
of their aggregation-inhabited nanostructures and environment-resistant fluores-
cence, POSS-based nanomaterials are also appropriate for signal amplification in
various biological assays, such as DNA and protein microarrays (Fig.2.9).
48 2 Signal Amplification for Nanobiosensing
Fig.2.10 (a) SEM photomicrograph of beads in anisotropically etched silicon chip. (b) Chip (iv)
is fitted between double-sided adhesive layer (ii) and cover slip (i) with laminate layers (iii, v, vi)
included to direct fluid flow through the PMMA base (viii) and inlet and outlet ports (vii). (c)
Sealed LOC assembly. (d) Fluorescent image of beads after immunoassay, including negative
controls as imaged with 1s of CCD camera integration (exposure) time. Reprinted with permission
from Jokerst etal. [55]. 2009, Elsevier
Fig.2.11 Schematic of CL SNP quantitative assay based on Au and CuS NP probe and one-step
DNA hybridization reaction. Reprinted with permission from Ding etal. [56]. 2010, Elsevier
2.2.3Nanoparticle-Amplified Chemiluminescence
and Electrogenerated Chemiluminescence Assay
The functionalized NPs have been used to enhance the chemiluminescence (CL) or
electrogenerated chemiluminescence (ECL) intensity. An ultrasensitive CL method
based on the Au NP amplification for the quantitative detection of SNPs in genomic
DNA has been accomplished by the DNA polymerase I (Klenow fragment)-induced
coupling of the nucleotide-modified NP probe to the mutant sites of duplex DNA
under the WatsonCrick base-pairing rule [56]. As shown in Fig. 2.11, Au NPs
50 2 Signal Amplification for Nanobiosensing
Fig. 2.12 (a) Schematic representation of the CL detection of DNA hybridization based on
bio-barcode-functionalized magnetic nanoparticle labels (bbcMNPs) (upper part: carboxyl-coated
MNPs (carboxyl-MNPs) are functionalized with amino-modified probe DNA (amino-pDNA) and
amino-modified bio-barcode DNA (amino-bbcDNA), fabricating bbc-p-DNA-MNPs). (b)
Schematic diagram of the FI-CL detection system for the determination of Fe3+. Reprinted with
permission from Bi etal. [58]. 2009, Royal Society of Chemistry
are first electrodeposited on the surface of the Au electrode for DNA probe
immobilization, followed by the hybridization between the DNA probe and the mix-
ture of the single-base-mismatched tDNA and complementary tDNA. Au NP probes
modified with CuS NPs and a base (guanine, G) that is complementary to the muta-
tion site (cytosine, C) are coupled to the formed duplex DNA in the presence of DNA
polymerase. CuS NPs and Au NPs are linked by an amidization reaction between
mercaptoacetic acid on the surface of Au NPs and aminoethanethiol on the surface
of CuS NPs. The base G and the Au NP are linked by DNA with a sequence of
5-SH-(CH2)6-ATG TCC CTC AGA CCC TTT-(CH2)6-NH2-3. The amount of the
SNPs is monitored by the CL intensity of luminol-CN-Cu2+ after the cupric ions are
dissolved from the hybrid (Fig.2.11). A preconcentration processing of cupric ions
is performed by anodic stripping voltammetric (ASV) technology to improve the
sensitivity of the method. The mechanism of the luminolCNCu2+ CL system to
produce CL signal is based on coupling the complex-formation reaction of cupric
ions and cyanide with the CL reaction of luminal and Cu(CN)42, which has a high
oxidation potential. As a single Au NP can be loaded with 77 CuS NPs, the incorpo-
ration of Au NPs significantly enhances the sensitivity. Moreover, the preconcentra-
tion processingof cupric ions can further increase the sensitivity about tenfold. As a
result of these two combined effects, this method could detect as low as 19aM SNPs,
and the linear range for SNPs was from 8.01017 to 1.01014M. Based on this
2.2 Nanoparticle-Amplified Optical Assay 51
Fig. 2.13 CL immunoassay of IgG using CdTe QDs as label. Reprinted with permission from
Wang etal. [59]. 2009, Elsevier
Fig.2.14 CL spectra of luminolKMnO4 in the presence of CdTe QDs (CL1) and luminolKMnO4 in
the absence of CdTe QDs (CL2). Conditions: luminol, 1105 M (in 0.01 M NaOH); KMnO4,
1105M; CdTe QDs, 1103M. Reprinted with permission from Wang etal. [59]. 2009, Elsevier
Zhus group [60] indicated that the ECL of CdSe QDs could be greatly enhanced
by combining carbon nanotubes (CNTs) and poly (diallyldimethylammonium chlo-
ride) (PDDA) in CdSe QD film. Based on this phenomenon, they developed a sensi-
tive ECL immunosensor for the detection of human IgG (Ag). The fabrication
procedures for CdSe QDCNT conjugates and the ECL immunosensor were shown
in Fig.2.15. Where PDDA as a binding linker was conjugated to the CdSe QDCNT
composite film on the electrode, the ECL signal was significantly enhanced.
Subsequently, Au NPs assembled onto the CdSe QDCNT/PDDAmodified elec-
trode amplified the ECL signal once again. After antibody (Ab) was immobilized
onto the electrode through Au NPs, the ECL immunosensor was fabricated. The
principle of ECL detection for target Ag was based on the increment of steric hin-
drance after immunoreaction, which resulted in a decrease in the ECL intensity
(Fig. 2.16). The Ag concentration was determined in the linear range of 0.002
500ng/L, with a detection limit of 0.6pg/mL.
When nanoporous gold leaf (NPGL) electrodes are used, the sensitivity of the
ECL assay can be remarkably increased due to ultrathin nanopores. Based on
this phenomenon, Hu et al. [61] developed a sensitive ECL DNA assay
(Fig. 2.17). In this assay, tDNA was hybridized with capture DNA (cDNA)
bound on the NPGL electrode, which was fabricated by conjugating amino-
modified cDNA to TGA modified at the activated NPGL electrode. Following
that, amino-modified probe DNA was hybridized with the tDNA, yielding
2.2 Nanoparticle-Amplified Optical Assay 53
Fig. 2.16 ECLpotential curves of (a) CdSe QDsCNTs; (b) (a)+PDDA; (c) (b)+GNPs;
(d) (c)+Ab; (e) (d)+BSA; and (f) (e)+Ag-modified Au electrodes in 0.1 M PBS (pH 7.4)
containing 0.1M KCl and 0.1M K2S2O8. Scan rate: 100mV/s. Reprinted with permission from Jie
etal. [60]. 2009, Elsevier
Fig.2.17 Schematic representation of the process of DNA determination. Reprinted with permis-
sion from Hu etal. [61]. 2010, Elsevier
Fig. 2.18 (a) IECLE curves of CdTe QD-labeled DNA hybrids immobilized on an NPGL
electrode in PB (pH 7.4) containing 0.1mol/L K2S2O8 and 0.1mol/L KNO3 for different t-DNA
concentrations (1015mol/L): (1) 0, (2) 5.0, (3) 10, (4) 30, (5) 100, (6) 500, (7) 1,000, (8) 2,000,
(9) 4,000, (10) 6,000, (11) 8,000, and (12) 10,000. (b) Magnification of the IECLE curves
indicated in (15). Inset: Relationship between IECL and t-DNA concentration. Reprinted with per-
mission from Hu etal. [61]. 2010, Elsevier
favorably with colorimetric ELISA assays. This study also indicated that labeling the
antibody with gold NPs had no apparent effect on their interaction with their antigen.
Further enhancements to sensitivity could be achieved by the cyclic accumulation of
gold NPs [76] or the catalytic deposition of metals on core gold NP tags [77, 78].
Recent activities have demonstrated that inorganic nanocrystals offer an elec-
trodiverse population of electrical tags for multiplexed bioanalysis. For example,
Wangs group used encoding NPs (cadmium sulfide, zinc sulfide, copper sulfide,
and lead sulfide) to the multiplexed detection of DNA targets [79], SNPs [80], and
antigens [81]. The multitarget electrical detection capability was coupled to the
amplification feature of electrochemical stripping transduction (to yield fM detec-
tion limits) and with an efficient magnetic separation (to minimize nonspecific
adsorption effects). Each biorecognition event yielded a distinct voltammetric peak,
whose position and size reflected the identity and level of the corresponding target.
Recently, Wangs group designed a QD/aptamer-based ultrasensitive electrochemi-
cal biosensor to detect multiple protein targets [82]. As shown in Fig. 2.19, the
protocol was based on a simple single-step displacement assay involving the coim-
mobilization of several thiolated aptamers, along with binding of the corresponding
QD-tagged proteins on a gold surface (a), addition of the protein sample (b), and
monitoring of the displacement through electrochemical detection of the remaining
nanocrystals (c). Such an electronic transduction of aptamerprotein interactions
was extremely attractive for meeting the low-power, size, and cost requirements of
decentralized diagnostic systems. A detection limit of 20ng/L (0.5pM) was obtained
by this biosensor, which was 34 orders of magnitude lower than those (16.4nM)
obtained with other advanced aptamer biosensors.
With the use of the RCA technique, a cascade signal amplification strategy was
proposed for detection of the protein target at an ultralow concentration [83]. In this
assay, the ultrasensitive detection was achieved by combining the RCA technique
with oligonucleotide-functionalized QDs, multiplex binding of the biotin
streptavidin system, and ASV measurement. As shown in Fig.2.20, the RCA prod-
uct containing tandem-repeat sequences could serve as an excellent template for the
periodic assembly of QDs, which present per protein recognition event to numerous
QD tags for electrochemical readout. Both the RCA and the multiplex binding sys-
tem showed remarkable amplification efficiency, with very little nonspecific adsorp-
tion and low background signal (Fig.2.21). With human vascular endothelial growth
factor (VEGF) as a model protein, the designed strategy could quantitatively detect
protein down to 16 molecules in a 100-mL sample with a linear calibration range
from 1aM to 1pM and was amenable to quantification of the protein target in com-
plex biological matrices. The proposed cascade signal-amplification strategy seems
to be a powerful tool for proteomics research and clinical diagnostics.
Due to the electrochemical properties of Au NPs, they have been used as signal
amplifiers in many electrochemical DNA biosensors. Wang etal. [84] demonstrated
2.3 Nanoparticle-Amplified Electrochemical Detection 59
that Au NPs could amplify the electrochemical impedance and capacitance signals
for the model fluorescein/antifluorescein system. Following the immobilization of
fluorescein onto Au through the formation of a self-assembled monolayer, goat anti-
fluorescein conjugated with 10-nm Au NPs was introduced into the system. This
resulted in an increase of 400nF/cm2 in the capacitance, whereas no change could
be observed for goat antifluorescein without the Au NP conjugate. This allowed the
construction of high-sensitivity electrochemical impedance biosensors at a single
low frequency, where the signal was sensitive to the interfacial Rct.
The impedance detection of CEA, a glycoprotein involved in cell adhesion
produced only during fetal development, was recently reported [85]. The CEA
antibodywas first bound through its surface amino groups to glutathione-modified Au
NPsof151.5-nmdiameterbyamide-bondformationusing N-(3-dimethylaminopropyl)-
N-ethylcarbodiimide hydrochloride (EDC) and N-hydroxysulfylsuccinimide sodium
60 2 Signal Amplification for Nanobiosensing
Fig. 2.20 Schematic representation of the cascade signal-amplification strategy for protein
detection. Reprinted with permission from Cheng etal. [83]. 2010, American Chemical Society
Fig.2.21 Anodic stripping voltammograms of cadmic cation responding to 1fM of VEGF (a)
with and (b) without RCA. Reprinted with permission from Cheng etal. [83]. 2010, American
Chemical Society
2.3 Nanoparticle-Amplified Electrochemical Detection 61
Fig.2.23 (a) Schematic conformation of four individually addressable MEA platforms (left) with
graphite particles coated with gold layers by electrodeposition (right); (b) a typical SEM image of
the MEA platform after gold modification; (c) CV characterization of the above MEA platform
measured in the presence of 10mM [Fe(CN)6]3/4 with a scan rate of 0.1V/s. Reprinted with per-
mission from Zhang etal. [87]. 2009, Elsevier
particles coated with gold layers as microelectrodes [87]. As shown in Fig.2.23, the
graphite particles available on the common pencil are utilized to direct the
electrodeposition of gold layers with uniform microstructures, which displays a
well-defined sigmoidal voltammetric response. In the concept-of-proof experi-
ments, the resulting MEA platform is modified with a functionalized monolayer, on
which antihuman IgG antibodies can be stably immobilized in a site-selective way
through binding chemistry to selectively capture human IgG antigens from the sample
media. The subsequent introduction of antihuman IgG antibodies (conjugated with
15-nm electroactive gold NPs), which recognize the captured IgG proteins, results
in a significant decrease in the interfacial electron-transfer resistance. As shown in
Fig.2.24, a highly sensitive electrochemical quantification can be obtained through
gold NPamplified impedance responses. Thus, the MEA sensor can detect human
IgG with a wider linear range (0.05100ng/mL) and a sensitivity over 103 larger
than that of the conventional, bulk gold electrode.
Fig.2.24 (a) Schematic representation of successive fabrication, human IgG detection (5ng/mL),
and regeneration of an MEA platform-based immunosensor. (b) The corresponding Nyquist plots
(Zim vs. Zre) for Faradaic impedance spectra in the presence of 10mM [Fe(CN)6]3/4 at the MEA
platform after (a) electrodeposition of the gold layer, (b) assembly of the 11-MUA monolayer, (c)
covalent immobilization of antihuman IgG antibody, (d) binding of human IgG antigen, and (e)
regeneration treatment. Reprinted with permission from Zhang etal. [87]. 2009, Elsevier
Fig.2.25 Cyclic
voltammograms obtained at
(a) an ITO electrode modified
with detection probe
(DP)-conjugated Au NPs,
(b) an Au NP-modified ITO
electrode, and (c) an ITO
electrode sequentially
modified with Au NPs and
DPs, in a 0.1M phosphate
buffer solution (pH 8)
containing 2mM hydrazine
(at a scan rate of 50mV/s)
before and after NaBH4
treatment for 15min in Tris
buffer (pH 9) containing
10mM NaBH4. Reprinted
with permission from Das
and Yang [92]. 2009,
American Chemical Society
Fig.2.26 Sensing assembly: (a) top three bilayers of PAH/GOD; (b) three bilayers of PAH/PSS;
and (c) 12 bilayers of PAH/CdTe QDs. Reprinted with permission from Li etal. [93]. 2009,
American Chemical Society
NaBH4 treatment produces a high signal current, and the low intrinsic electrocata-
lytic activity of ITO electrodes results in a low background current. This high sig-
nal-to-background ratio enables a detection limit of 1 fM DNA without target
amplification or enzymatic signal amplification.
Li and coworkers [93] developed a blood glucose sensor based on the multilayer
films of CdTe QDs and glucose oxidase (GOD) by using a layer-by-layer assembly
technique (Fig.2.26). When the composite films were contacted with glucose solu-
tion, the photoluminescence of QDs in the films was quickly quenched because the
enzyme-catalyzed reaction product (H2O2) of GOD and glucose gave rise to the for-
mation of surface defects on QDs. The quenching rate was a function of the concen-
tration of glucose. The linear range and sensitivity for glucose determination could be
adjusted by controlling the layers of QDs and GOD (Fig.2.27). The biosensor could
determine the concentration of blood glucose in real serum samples without sample
pretreatment and exhibited satisfactory reproducibility and accuracy.
Au NPs can also be used as carriers of the signaling molecules for amplification
detection of DNA [94] and protein targets [9597]. For example, Au NPs have
been used as carriers of the signaling antibody anti-CA 15-3-HRP in order to
achieve an amplification analysis of CA 15-3 antigen (Fig.2.28) [97]. In the range
Fig.2.27 UV-vis spectra of
growing PAH/CdTe QD
multilayers (112). Inset
shows a plot of l=567nm
vs. the number of bilayers.
Reprinted with permission
from Li etal. [93]. 2009,
American Chemical Society
Fig.2.28 Schematic (not to scale) of (a) the preparation of the Auanti-CA153-HRP complex
and (b) the sandwich-type ELISA procedure without (IIIa) and with (IIIb) the application of
AuNPs as the signal enhancer. Reprinted with permission from Ambrosi et al. [97]. 2010,
American Chemical Society
2.4 Nanoparticles as Carrier for Signal Amplification 67
Fig.2.29 Schematic diagrams of the preparation of the (a) enzyme-labeled Au NP probes and (b)
MMP probes. (c) Schematic illustration of the enzyme-labeled Au NP probe-based immunoassay
processes. Reprinted with permission from Liu etal. [98]. 2010, Royal Society of Chemistry
Fig.2.30 Schematic illustration of the stepwise process of the modified electrode. Reprinted with
permission from Ding etal. [99]. 2010, Elsevier
2.4 Nanoparticles as Carrier for Signal Amplification 69
Fig.2.31 The schematic illustration of the determination of AFP based on the sandwich-type chemi-
luminescence immunoassay. Reprinted with permission from Bi etal. [100]. 2009, Wiley
Fig.2.32 The schematic diagram of the fabrication of the ECL aptasensor. Reprinted with permis-
sion from Fang etal. [101]. 2008, Elsevier
Fig. 2.33 (a) Directly immobilized MSO probe and (b) Au NP-mediated immobilized MSO
probe. Reprinted with permission from Zhu etal. [102]. 2009, American Chemical Society
with the MSO probe and a linking probe that is complementary to a cDNA probe
immobilized on gold electrodes (Fig.2.33b).
Using Au NPs to load many CdS NP-labeled linker DNA, a significant amplifi-
cation for the detection of thrombin has been obtained [104]. As shown in Fig.2.34,
aptamers are immobilized on the Au NP-modified electrode to construct the sand-
wich-type detection strategy. The concentration of thrombin is monitored based
upon the concentration of dissolved Cd2+ formed in the dissolution of CdS by acid
treatment and quantified by differential pulse voltammetry. Thrombin can be
detected in the linear range of 1.010151.01011 M, with a detection limit of
5.51016 M. A similar strategy has been conducted by Zhangs group based on
bio-barcode techniques [105].
2.4 Nanoparticles as Carrier for Signal Amplification 71
Fig. 2.34 The scheme of the electrochemical determination of thrombin based on aptamer.
Reprinted with permission from Ding etal. [105]. 2010, Elsevier
Taking the advantage of the catalytic reaction of DNAzyme upon its binding to
Pb2+ and the use of DNAAu bio-barcodes to achieve signal enhancement, an electro-
chemical DNAzyme sensor for the sensitive and selective detection of Pb2+ has been
developed [106]. As shown in Fig.2.35, a specific DNAzyme for Pb2+ is immobilized
onto the Au electrode surface via a thiolAu interaction. The DNAzyme hybridizes to
a specially designed complementary substrate strand that has an overhang, which in
turn hybridizes to the DNAAu bio-barcode. A redox mediator, Ru(NH3)63+, which
can bind to the anionic phosphate of DNA through electrostatic interactions, serves as
the electrochemical signal transducer. Upon binding of Pb2+ to the DNAzyme, the
DNAzyme catalyzes the hydrolytic cleavage of the substrate, resulting in the removal
of the substrate strand along with the DNAAu bio-barcode and the bound Ru(NH3)63+
from the Au electrodes surface. The release of Ru(NH3)63+ results in a lower electro-
chemical signal of Ru(NH3)63+ confined on the electrodes surface. The differential
pulse voltammetric signals of Ru(NH3)63+ provide quantitative measurements of the
Pb2+ concentrations, with a linear calibration range from 5nM to 0.1mM. Because
each NP carries a large number of DNA strands that bind to the signal transducer
molecule Ru(NH3)63+, the use of DNAAu bio-barcodes enhances the detection sensi-
tivity, enabling the detection of Pb2+ at a very low level (1nM).
A densely packed gold NP platform combined with a multiple-enzyme-labeled
detection antibodyMB bioconjugate has been used as the basis for preparing an
ultrasensitive electrochemical immunosensor to detect cancer biomarkers in serum
[107]. As shown in Fig.2.36, the sensor is fabricated by alternate layer-by-layer
electrostatic adsorption of a dense glutathione-decorated Au NP and an underlying
layer of cationic poly(diallyldimethyl ammonium chloride) on a pyrolytic graphite
72 2 Signal Amplification for Nanobiosensing
Fig.2.35 The principle of the electrochemical DNAzyme sensor for Pb2+. Reprinted with permission
from Shen etal. [106]. 2008, American Chemical Society
Fig.2.36 Au NP immunosensor with Ab1 attached that has captured an antigen from a sample
after treating with Ab2magnetic bead (MB)HRP providing multiple enzyme labels for each
PSA. The detection step involves immersing the immunosensor into buffer containing mediator,
applying voltage, and injecting H2O2. Reprinted with permission from Mani etal. [107]. 2009,
American Chemical Society
2.4 Nanoparticles as Carrier for Signal Amplification 73
CNTs are a good carrier for biomolecules and signal molecules [108, 109]. As shown
in Fig.2.37, the immunosensor array is constructed by coating layer by layer colloi-
dal Prussian blue (PB), gold NPs, and capture antibodies on screen-printed carbon
electrodes. The preparation of GOD-functionalized nanocomposites and the labeling
of antibody are performed by the one-pot assembly of GOD and antibody on gold
NP-attached CNTs (Fig.2.37b). The PB immobilized on the immunosensors sur-
face acts as a mediator to catalyze the reduction of H2O2 produced in the enzymatic
cycle. Both the high-content GOD and CNTs in the tracer amplify the detectable
signal for the sandwich-type immunoassay. The simultaneous multiplexed immuno-
assay method can offer linear ranges of three orders of magnitude, with the detection
limits down to 1.4 and 2.2pg/mL for CEA and a-fetoprotein, respectively.
Using CNT-based labels, Lee etal. developed an amplified nucleic acid detection
[110]. As shown in Fig.2.38, the CNT-based labels were synthesized based on diim-
ideactivated amidation. The DPs were prelabeled with HRP enzymes cross-linked by
glutaraldehyde. The carboxylated SWNTs were covalently functionalized with prela-
beled DPs by reacting with the amine group at the 5-end of DPs with carboxylic acid
74 2 Signal Amplification for Nanobiosensing
Fig.2.39 Schematic representation of the signal amplification for sandwich hybridization assay
performed on MBs. The signal-to-single hybridization event ratio of (a) the conventional HRP
label is amplified by (b) multiple HRP and DP-conjugated CNT-based labels. Reprinted with per-
mission from Lee etal. [110]. 2007, IOP Publishing Ltd.
Due to the small size, high surface-to-volume ratio, and good biocompatibility,
silica NPs have become another normally used carrier for biomolecule
immobilization. For example, based on silica NPs, Wang and Liu [111] developed a
fluoroimmunoassay for antigen detection and quantification. As shown in Fig.2.40,
after immobilization of the prime antibody on the silica NP surface, the NPs were
used to capture antigen and Cy3-labeled secondary antibody in a sandwich assay
format. The presence of target antigen in solution brought the fluorescent Cy3 mol-
ecules to the NPs surface. The addition of a cationic conjugated polymer (CCP)
further amplified the fluorescence signal of the dye and improved the assay sensitiv-
ity. Due to the pink color of the Cy3 molecules, the assay allowed a detection limit
of 50ng/mL of IgG by naked-eye detection.
Mesoporous silica nanoparticles (MSN) have also been used as molecule carrier
to improve detection sensitivity. For example, Yang etal. [112] designed an MSN-
based label by loading MSN with mediator thionine (TH), enzyme HRP, and
secondary antihuman IgG antibody for IgG detection (Fig.2.41). The sensitivity of
the sandwich-type immunosensor could greatly improved, leading to a detection
range of 0.0110ng/mL of human IgG.
The electrochemiluminescence of doped silica nanoparticles (DSNPs), prepared
by a reverse microemulsion method that leads to the covalent incorporation of the
Ru(bpy)32+, has been investigated in acetonitrile and aqueous buffers [113].
76 2 Signal Amplification for Nanobiosensing
Fig. 2.41 Schematic representation of the preparation of the (a) MSNTHHRPAb2 and (b)
immunosensor. Reprinted with permission from Yang etal. [112]. 2010, Elsevier
Fig. 2.42 Preparation process of Si/QD/Ab2. Inset: TEM image of the resultant Si/QD/Ab2.
Reprinted with permission from Chen etal. [114]. 2009, Royal Society of Chemistry
Fig. 2.43 Sandwiched immunoassay process using Si/QD/Ab2 as labels. Inset: TEM image of
MB/Ab1AgSi/QD/Ab2. Reprinted with permission from Chen etal. [114]. 2009, Royal Society
of Chemistry
Fig.2.44 Schematic diagram of the assembly process for the preparation of streptavidin/CdTe-
tagged polybeads. Reprinted with permission from Dong etal. [115]. 2010, Wiley
Fig.2.45 Construction of the immunosensing probe and recognition element and measurement
protocol of the NP-based electrochemical immunoassay with a sandwich-type format. Reprinted
with permission from Tang etal. [116]. 2010, American Chemical Society
2.5Conclusions
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Chapter 3
Nanostructured Mimic Enzymes
for Biocatalysis and Biosensing
3.1Introduction
Accurate, rapid, inexpensive, and selective analysis is required today for use in
clinical diagnostics and the food industry. The majority of known electrochemical
biosensors are based on immobilized specific biomolecules, such as proteins,
enzymes, nuclear acids, antibodies, and antigens on the modified electrodes [18].
These resulting biomolecule-based devices usually show high sensitivity and spec-
ificity due to the high loading of enzymes on the nanoparticles [912]. However,
their stability is limited due to easy denaturation [13] and leakage of biomolecules
during their storage and immobilization procedure. Furthermore, the preparation
and purification of biomolecules are usually time-consuming and expensive [14].
Therefore, the syntheses of artificial biomolecules with highly catalytic properties
and a wide range of practical applications are becoming a significant field for dif-
ferent purposes.
Artificial enzyme mimetics have attracted considerable interest because they can
overcome the disadvantages of the natural enzyme [1518]. Up to now, some coor-
dination compounds have been prepared as artificial enzyme mimetics [1923].
Through invitro selection, nonnatural ribozymes, deoxyribozymes (catalytic DNA
molecules) [2427], and many other enzyme mimetics [2836] have been devel-
oped, such as cytochrome P450 mimetics [3739], serine proteases mimetics [40],
dioxygenase mimetics [41, 42], phosphodiesterase mimetics [43], ligase mimetics
[44], nuclease mimetics [45], and methanogenesis mimetics [46]. Among these, a
lot of research has focused on peroxidase mimetics, including hemin [47, 48],
hematin [49], hemoglobin [50], cyclodextrin [51], and porphyrin [52, 53], which
have been used for hydrogen peroxide (H2O2) and ascorbic acid detection [54].
Due to their unique optical, electric, chemical, and mechanical properties,
nanostructured materials have been widely applied in biocatalysis and biosensing
Peroxidase mimetics, including hematin [49], porphyrin [52], and cyclodextrin [42],
were already applied in different fields. Peroxidases are responsible in nature for the
reduction of H2O2. Some iron-based compounds, such as Prussian blue (PB)
[7380], have been denoted as electrocatalysts in H2O2 reduction due to the expected
conversion of Fe3+/Fe2+, which occurs in the reaction centers of a number of peroxi-
dase enzymes [8183]. PB has good stability and highly catalytic properties. Hence,
it has a wide range of practical applications.
Although Fe3O4 nanoparticles had been used for the cathodic determination of
H2O2 [84], their peroxidase activity was always ignored until the release of a recent
report in which the intrinsic peroxidase-like activity of Fe3O4 nanoparticles was
observed to catalyze the breakdown of H2O2 [85]. Afterward, Fe3O4 nanoparticles
were presented as a mimic peroxidase for the detection of H2O2 and glucose [86].
Because FeS shows typical MichelisMenten kinetics and a good affinity to
both H2O2 and 3,3,5,5-tetramethyl benzidine (TMB), it is also used as a mimic
enzyme for the development of biocatalysts and amperometric biosensors. The
H2O2 sensor based on FeS shows better stability than that based on horseradish
peroxidase when they are exposed to solutions with different pHs and tempera-
tures. These excellent characters make the nanostructured FeS powerful for a
wide range of potential applications as an artificial peroxidase in biosensors and
biotechnology [87].
The new nanoenzyme model with a glutathione peroxidase-like active site has
been constructed on the polystyrene nanoparticle via microemulsion polymeriza-
tion. The polystyrene nanoparticle exhibits an obvious enhancement in catalytic
activity and can be developed as an excellent model for combining most of the
catalytic factors of the enzyme into one scaffold [88].
Natural enzymes are macromolecules, while many enzyme models and mimics
are small molecules. Breslow etal. have done extensive studies on reactions related
to those catalyzed by enzymes that used pyridoxal phosphate and pyridoxamine
phosphate, the central coenzymes for amino acid metabolism [89106]. As a typi-
cal example, they catalyzed the transamination between an amino acid and a keto
acid, which is the most important form of nitrogen transfer in diverse biological
systems.
3.2 Nanostructure Used in Artificial Mimic Enzymes 87
3.2.1K3Fe(CN)6
H 2 O 2 + 2e 2OH
k
cat
(3.3)
88 3 Nanostructured Mimic Enzymes for Biocatalysis and Biosensing
The electrochemical rate constants for H2O2 reduction have been found to be
dependent on the amount of PB deposited on the electrode, confirming that the H2O2
penetrates the films, and inner layers of the polycrystal take part in catalysis. For
46 nmol/cm2 of PB, the electrochemical rate constant exceeds 0.01 cm/s [115],
which is higher than that for all the other known H2O2 transducers. For comparison,
the electrochemical rate constant for H2O2 oxidation on platinum in neutral media is
less than 7106cm/s [116]. The activity of Pt in H2O2 reduction is even lower.
Due to both high activity and high selectivity, PB was denoted as an artificial
peroxidase [77]. Using PB as a transducer for H2O2, it was possible to achieve a
sensitivity of 0.6/AM/cm2 in flow-injection mode [75, 77], which takes into account
the dispersion coefficient [117] corresponding to the sensitivity of 1/AM/cm2, either
in batch regime or under continuous flow.
This is pertinent to the chemical synthesis of a PB-based H2O2 transducer, which
can be used in both screen-printed and carbon paste electrodes. Both the commer-
cially available and commonly precipitated PB showed a minor electrocatalytic
activity in H2O2 reduction [118]. A successful chemical synthesis of the electrocata-
lyst has recently been carried out by open-circuit deposition onto graphite powder
from a ferricyanide solution [119]. The resulting carbon pastePB transducer for
H2O2 exhibited high operational stability in neutral and weakly basic media up to
pH 9. The sensitivity of the PB-based carbon paste sensors for H2O2 was rather
satisfactory, but 20200 times lower than the sensitivity of the PB-modified glassy
carbon electrodes.
There were several attempts to use other metal hexacyanoferrates as H2O2 trans-
ducers. Cupric hexacyanoferrate was integrated in a biosensor, but the resulting
sensitivity was three orders of magnitude lower than that of a similar biosensor
based on PB [120]. Moreover, cupric hexacyanoferrate has to be poised to high
cathodic overvoltages (>0.7 V vs. its redox potential) to achieve a considerable
rate of H2O2 reduction. These observations indicated that cupric hexacyanoferrate
was a poorer electrocatalyst than PB.
Another transition metal-based transducer for H2O2 was made by cycling a tita-
nium dioxide electrode in ferricyanide solution [121]. Although the authors did not
claim the synthesis of a new transition metal hexacyanoferrate, the cyclic voltam-
mograms were similar to PB. Unfortunately, the titanium dioxide electrode modi-
fied with hexacyanoferrate did not show high activity to H2O2 reduction: The
sensitivity calculated from the data in the paper was 0.8 mA/M/cm2. Two metal
hexacyanoferrates with extremely high sensitivity to H2O2 (>1/AM/cm2) were
recently reported: Co-hexacyanoferrate [112] and Cr-hexacyanoferrate [29].
There was also doubt concerning the high catalytic activity of cohexacyanofer-
rate. Lin and Jan reported two redox potentials for the electroactivity of cobalt
hexacyanoferrate: at 449 and 595mV [112]. However, after the addition of H2O2,
electrocatalysis was observed at a shoulder, with a potential of about 0.2V [112],
which has never been reported for Co-hexacyanoferrate, but was similar to one
attributed to PB with the precision of the reference electrode used. It should be
noted that the deposition of Co-hexacyanoferrate [112] included 6h of cycling in
solution containing ferricyanide, and, thus, the resulting film obviously contained
3.2 Nanostructure Used in Artificial Mimic Enzymes 89
some amount of PB being deposited even from a single ferricyanide solution [122].
There was no experimental evidence showing that the high catalytic activity in H2O2
reduction was peculiar to Co-hexacyanoferrate rather than to PB. This study con-
firmed the unique catalytic properties of PB. As a conclusion from this section, PB
has to be considered the most advantageous H2O2 transducer over all other existing
systems.
Overall, a PB-based electrocatalyst is a truly good choice for the development of
oxidase-based biosensors. PB-modified electrodes are (1) selective electrocatalysts
for electrochemical reduction of H2O2 in the presence of oxygen, which is not a
particular property of platinum; (2) more stable and active than peroxidase-modified
electrodes; and (3) less expensive than both platinum and peroxidase electrodes.
3.2.2Fe3O4
Fig. 3.1 Fe3O4-catalyzed oxidation of various peroxidase substrates in the presence of H2O2 to
produce different color reactions. Reprinted with permission from Gao etal. [85]. 2007, Nature
A detection limit of 7.6mM was also obtained (S/N=3). The precision value from
RSD was 2.2% by 20 successive measurements of 0.5mM H2O2. A typical response
time between 10 and 90% of the steady-state response was 5.2s at the injection of
0.5mM H2O2.
Fe3O4 possesses an intrinsic enzyme mimetic activity similar to that found in
natural peroxidases. Peroxidase activity has a wide range of practical applications.
For example, the ability to catalyze the oxidation of organic substrates to reduce
their toxicity and/or to produce a color change was frequently used in wastewater
treatment or as a detection tool. Fe3+/Fe2+ ions in solution (Fentons reagent) are
known to catalyze the breakdown of H2O2. A number of peroxidase enzymes
(including the heme-containing enzyme HRP) and enzyme mimetics contain Fe2+ or
Fe3+ in their reaction centers. However, the fact that Fe3O4 nanoparticles have been
conjugated to HRP to introduce peroxidase activity in a number of applications,
including commercially available magnetic enzyme-linked immunosorbent assay
(ELISA) kits, demonstrates that the presence of this activity has so far been ignored.
Taking HRP as a comparison, the peroxidase-like activity of Fe3O4 was character-
ized by Gao etal. [85].
Fe3O4 catalyzed the reaction of TMB in the presence of H2O2 to produce a blue-
colored reaction (Fig.3.1 left), with a maximum absorbance at 652nm. Like enzy-
matic peroxidase activity, such as that observed for the commonly used enzyme
HRP, this color reaction was quenched by adding H2SO4. To further characterize the
peroxidase-like activity of Fe3O4, other peroxidase substrates, including di-azo-
aminobenzene (DAB) and o-phenylenediamine (OPD), were used in place of TMB.
The middle photo in Fig.3.1 shows that the Fe3O4 not only catalyzed the oxidation
of TMB, producing a blue color, but also catalyzed DAB to give a brown color and
OPD to give an orange color. These results indicated that the Fe3O4 had peroxidase-
like activity toward typical peroxidase substrates (Fig.3.1, far right).
To investigate the mechanism of the peroxidase activity of Fe3O4, the apparent
steady-state kinetic parameters for the reaction were determined. Within a suitable
3.2 Nanostructure Used in Artificial Mimic Enzymes 91
Fig. 3.2 Immunoassays based on the peroxidase activity of Fe3O4 magnetic nanoparticles. (a)
immunosensor preparation, and (b) immunoassay process. Reprinted with permission from
Gao etal. [85]. 2007, Nature
nonspecific binding was removed. The Fe3O4 with immobilized protein A and the
substrate TMB were then added, so that protein A bound to the primary anti-pre
antibody and Fe3O4 catalyzed a color reaction in the presence of H2O2. The reaction
was measured using an ELISA reader at 652nm. The results demonstrated that the
intrinsic peroxidase-like activity of the Fe3O4 can still be detected after surface modi-
fication. On the other hand, the magnetic properties of the Fe3O4 could potentially be
used for recovery or recycling of the Fe3O4. As shown in Fig.3.2b, the two intrinsic
properties of Fe3O4, namely, magnetism and peroxidase activity, were combined in a
novel capturedetection immunoassay format. First, an antibody to cardiac troponin
I (TnI), a well-known biomarker for myocardial infarction, was immobilized on the
Fe3O4. The antibody-labeled Fe3O4 was then mixed with serum, allowing capture of
the target TnI in the sample. The TnI captured by Fe3O4 was easily separated from
the sample using a magnet. After washing off contaminants, the Fe3O4 with target
bound was transferred onto a plate coated with another anti-TnI antibody. After
washing off nonbound Fe3O4, the substrate TMB was added in the presence of H2O2
and the bound Fe3O4 catalyzed a color reaction. These assays demonstrated the versa-
tility and power of Fe3O4 as both a capture agent and a detection tool, due to its intrinsic
dual functionality. This can be compared with traditional magnetic ELISA, in which
Fe3O4 captures targets and an additional step is required to introduce a secondary
antibody carrying, for example, HRP to allow detection. This method was easier,
faster, and more economical and provided greater sensitivity. Furthermore, the intrin-
sic peroxidase-like activity of Fe3O4 should be taken into account when it is used in
standard magnetic ELISA: The conjugation of the secondary antibody to HRP for
detection is likely to lead to high background. In fact, it was this observation that
discovered the peroxidase-like activity of the Fe3O4.
More importantly, a sensitive and selective method for glucose detection was
developed using glucose oxidase (GOx) and Fe3O4 [86]. Such detection platforms
for H2O2 and glucose not only confirmed the intrinsic peroxidase-like activity of
Fe3O4, but also showed great potential applications in varieties of simple, robust,
and easy-to-make analytical approaches in the future. With a combination of the
catalytic reaction of glucose with GOx and the Fe3O4 catalytic reaction ((3.4) and
(3.5)), the developed method exhibited a sensitive and selective response toward
glucose detection.
Fe3 O4
H 2 O 2 + ABTS 2H 2 O + oxidized ABTS (3.4)
O 2 + glucose
GOx
H 2 O 2 + gluconic acid (3.5)
The Fe3O4 was prepared via a coprecipitation method. The as-prepared Fe3O4
was then used to catalyze the oxidation of a peroxidase substrate 2,2-azino-bis
(3-ethylbenzo-thiazoline-6-sulfonic acid) diammonium salt (ABTS) by H2O2. The
oxidized colored product (3.4) provided a colorimetric detection of H2O2. A linear
range from 5106 to 1104mol/L, with a detection limit of 3106mol/L H2O2,
could be detected.
3.2 Nanostructure Used in Artificial Mimic Enzymes 93
When the catalytic reaction shown in (3.4) is coupled with the glucose catalytic
reaction by GOx (3.5), the observation of a colorimetric glucose detection occurred
as a result. A typical absorption profile for glucose detection using the colorimetric
method is shown in Fig.3.3. Because GOx could be denatured in pH 4.0 buffer solu-
tion, the glucose detection was performed in two separate steps. When the reaction
of (3.5) was finished in a pH 7.0 buffer solution, the H2O2 produced by the glucose
oxidation with GOx was detected using the as-prepared Fe3O4 (3.4). A typical glu-
cose concentration response curve as low as 3105mol/L glucose could be detected,
with a linear range from 5105 to 1103mol/L. In order to test if the detection of
glucose is specific, control experiments were taken using fructose, lactose, and
maltose. As high as 5mM control samples were investigated, and no detectable sig-
nals were obtained, showing high selectivity toward glucose detection.
3.2.3FeS
Because of its high activity and selectivity toward the reduction of H2O2, FeS can
also be considered an artificial enzyme peroxidase and has been used in the con-
struction of electrochemical biosensors [87]. FeS possesses specific electron-
transfer ability [12] and good adsorption and, more importantly, has a lower band gap
than FeO [13], which is in favor of facilitating the electron transfer. A nanostructure
of sheet-like FeS that was prepared by a simple micelles-assisted synthetic method
was designed as a novel mimic peroxidase. This nanostructured FeS could provide
the enzymatic active center of Fe2+/Fe3+ for electron transfer. Such a nanostructure
had a large specific surface area and high peroxidase-like activity, allowing it to be
used as a mimic enzyme for the development of biocatalysts and amperometric bio-
sensors. The synthesized sheet-like FeS nanostructure showed attractive performance
of intrinsic peroxidase-like activity, which was confirmed by evaluating the ability to
catalyze the oxidation of organic substrates to produce a color change and developing
94 3 Nanostructured Mimic Enzymes for Biocatalysis and Biosensing
Fig.3.4 The electrocatalytic activity of sheet-like FeS nanostructure (red line) and HRP (black
line) to H2O2 at pH 7.0 and 40C after exposure to different pHs (a) and temperatures (b) for 2h.
Reprinted with permission from Dai etal. [87]. 2009, Wiley
a fast, sensitive, and low-cost electrochemical sensor for H2O2. It is well known that
the peroxidase can catalyze the oxidation of a peroxidase substrate to produce a color
change and the color reaction can generally be quenched by H2SO4. The sheet-like
FeS nanostructure showed typical MichelisMenten kinetics and a good affinity to
both H2O2 and TMB. The Kmapp for the sheet-like FeS with TMB was 0.13mM, while
the value of Kmapp for HRP with TMB was 0.4mM. The latter coincided with that
reported previously, and the former was slightly larger than that of Fe3O4 nanoparti-
cles. The Kmapp value of the sheet-like FeS nanostructure suggested that it had a higher
affinity to TMB than to HRP. This may be due to the fact that an HRP molecule has
only one iron ion, in contrast to the surface of a sheet-like FeS nanostructure. On the
other hand, the Kmapp value of the FeS with H2O2 was 7.2mM, slightly higher than
that of 3.7mM for HRP in solution. But the Kmapp value was much lower than that of
154mM for Fe3O4 nanoparticles, indicating a better affinity of the sheet-like FeS
nanostructure to H2O2 than Fe3O4 nanoparticles.
At pH 7.0, the constructed amperometric sensor showed a linear range for the
detection of H2O2 from 0.5 to 150 mM, with a correlation coefficient of 0.9998,
without the aid of any electron-transfer mediator. Also, this H2O2 sensor had a more
sensitive response than those based on spherical FeS nanoparticles.
The FeS nanostructure is an inorganic nanomaterial and is expected to be more
stable than natural peroxidases. To examine its stability, both HRP and the sheet-
like FeS nanostructuremodified electrodes were exposed to different temperatures,
ranging from 10 to 70C, and solutions, with pH ranging from 2.0 to 10.0 for 2h,
and their relative activities toward the electrocatalytic reduction of H2O2 were then
measured at pH 7.0 and 40C. The sheet-like FeS nanostructure showed much better
stability than HRP in the measured temperature and pH ranges (Fig. 3.4). After
exposure to pH 2.0 and 10.0 solutions for 2h, the nanostructure could maintain 40
and 56% of the peroxidase activity, respectively, while the HRP denatured
completely at pH lower than 3.0 and maintained only 30% of its activity at pH 10.0.
3.2 Nanostructure Used in Artificial Mimic Enzymes 95
3.2.4Polystyrene
A new nanoenzyme model with a glutathione peroxidase-like active site was con-
structed on a polystyrene nanoparticle (PN1) via microemulsion polymerization
[88]. In this model system, two functional monomers were designed: One was a
tellurium-containing compound that was introduced on the surface of the nanopar-
ticle and acted as a catalytic center; the other was an arginine-containing compound
designed as a binding site for the complexation of the carboxyl group of substrate
3-carboxy-4-nitrobenzenethiol (ArSH, 1). As a new glutathione peroxidase (GPx)
mimic, it demonstrated excellent catalytic activity and substrate specificity. In the
ArSH assay system, it was at least 316,000-fold more efficient than diphenyl disele-
nide (PhSeSePh) for the reduction of cumene hydroperoxide (CUOOH) by ArSH.
To further promote the catalytic efficiency, a substrate ArSH surface-imprinted
nanoenzyme model (I-PN) was developed. The polymerization process is depicted
in Fig.3.5. By correctly incorporating and positioning the catalytic center tellurium
and functional binding factor guanidinium, a 596,000-fold continuative activity
enhancement for the reduction of CUOOH was observed by catalyst I-PN rather
than PhSeSePh. The results clearly show that a polymeric nanoparticle can be devel-
oped as an excellent model for combining most of the catalytic factors of an enzyme
into one scaffold.
96 3 Nanostructured Mimic Enzymes for Biocatalysis and Biosensing
Fig. 3.5 Polymerization process of the surface-imprinted nanoenzyme model. Reprinted with
permission from Huang etal. [88]. 2008, American Chemical Society
3.2.5Breslows Mimics
studied some polymeric enzyme models. They reported a great increase in the
transamination rate for the pyridoxamine-keto acid system when they attached pyri-
doxamine to polyethylenimine (PEI) carrying some attached lauryl groups. They
also reported that hydrophobic effects exert profound effects on rates and substrate
selectivities in the PEIpyridoxamine transaminase mimics.
A variety of mono- and unsymmetrical bifunctional b-CD have been developed
as efficient mimics of aldolases, some of which have shown a large rate of accelera-
tion and substrate selectivity [91]. A novel catalyst has been synthesized in which a
manganeseporphyrin unit was linked to four hydrophobic cyclophane-binding
groups [92]. The Cu(II) complex of a cyclodextrin dimer linked by a bipyridyl unit
catalyzed the hydrolysis of an unactivated doubly bound benzyl ester [93]. A cyclo-
dextrin dimer with a linking bipyridyl group was synthesized as a catalyst precursor,
a holoenzyme mimic. It bound both ends of potential substrates into the two differ-
ent cyclodextrin cavities, holding the substrate ester carbonyl group directly above
a metal ion bound to the bipyridyl unit. The result was very effective ester hydroly-
sis with good turnover catalysis. For example, a Cu(II) complex accelerated the rate
of hydrolysis of several nitrophenyl esters, with at least 50 turnovers and no sign of
product inhibition. In the best case, with an added nucleophile that also bound to the
metal ion, a rate acceleration of 1.45107 over the background reaction rate was
observed. Hydrolysis by a catalyst with only one cyclodextrin-binding group was
significantly slower than in the bidentate-binding cases. As expected, the binding of
a transition state analog to these catalysts with the presence of the metal ion was
stronger than those without the metal ion. This and kinetic evidence point to a
mechanism in which the metal ion played a bifunctional acid-base role, enforced by
the binding geometry that held the substrate functionality right on top of the cata-
lytic metal ion [98]. Zn(II) complexes of monomers and dimers derived from
1,4,7-triazacyclododecane and 1,5,9-triazacyclotetradecane were examined as cata-
lysts for the hydrolyses of p-nitrophenyl phosphate and for the cyclizations of
p-nitrophenyl 2-hydroxypropyl phosphate and 3,5-uridyluridine (UpU). The dim-
ers with 1,3-phenyl linkers were more effective than monomers or a longer dimer
with a 4,4-biphenyl linker in the hydrolysis of p-nitrophenyl phosphate, suggesting
that two Zn(II) ions coordinated to the phosphate group, as in the enzyme alkaline
phosphatase. However, for the hydrolysis or cyclization of the phosphate diesters,
the longer biphenyl linker was preferred. In this case, one Zn(II) coordinated to the
phosphate group, while the other delivered a nucleophilic oxide anion. Bell-shaped
pH vs. rate profiles were seen in both cases [101].
Synthetic organic chemistry normally achieves selectivity by manipulating the
intrinsic reactivity of the substrate, but enzyme use is quite a different principle. The
geometry of the enzymesubstrate complex determines enzymatic selectivity, com-
pletely overwhelming any normal selective reactivities. Biomimetic chemistry aims
to imitate the enzymatic style. Some early approaches used attached reagents or
templates to direct photochemical and free radical processes, with a combination of
geometric and reactivity control. Recent work used a mimic of the enzyme class
cytochrome P-450 to achieve the selective hydroxylations of steroids with complete
domination by the geometry of the catalystsubstrate complex [103]. The ability of
98 3 Nanostructured Mimic Enzymes for Biocatalysis and Biosensing
3.3.1H2O2 Sensors
An alternative way for a low-potential and selective detection of H2O2 was recently
demonstrated by Karyakin [7477, 111, 115, 125]. PB polycrystals deposited in a
defined way onto the electrode surface were shown to be active and selective electro-
catalysts for H2O2 reduction. Due to their catalytic ability, which is reminiscent of
biological catalysts, the specially deposited PB was denoted artificial peroxidase.
Figure3.6 shows the experimental curves obtained for the amperometric detec-
tion of H2O2 performed with electrodes of different amounts of PAH/PB nanoparti-
cle bilayers. A good linear range up to 0.4 mmol/L of H2O2 was obtained on all
electrodes. On the other hand, the analytical response depended strongly on the
number of bilayers. The sensitivities obtained by the electrode containing 5, 10, and
15 bilayers were 34.5, 76.8, and 103.5 mA mmol/L/cm2, respectively, showing a
linear increase in sensitivities with the amount of immobilized PB. These results
clearly indicated electrical connections among nanoparticles in different bilayers in
the sense of obtaining an amplification of the analytical response with the amount
of catalyst controlled at the nanoscale level.
3.3.2Glutamate Sensors
Fig.3.6 Linear-range
analytical curves obtained for
H2O2 detection for LBL films
of 5, 10, and 15 PAH/PB
nanoparticle bilayers.
Reprinted with permission
from Fiorito etal. [78].
2005, Royal Society of
Chemistry
3.3.3Glucose Sensors
MWCNTs and PB. A fast amperometric response to H2O2 was observed, with a
detection sensitivity of 1.3mA/mM/cm2 and a detection limit of 25nM. These results
were much better than those reported for PB-based amperometric sensors. In addi-
tion, a glucose biosensor fabricated by casting an additional GOx-contained Nafion
film above the MWCNT/PVP/PB composite film showed promise for the sensitive
and fast detection of glucose. The high stability, high sensitivity, and high reproduc-
ibility of the MWCNT/PVP/PB composite films are promising for the reliable and
durable detection of H2O2 and glucose.
The ability of metal hexacyanoferrates to oxidize some organic and inorganic com-
pounds was used in the 1990s for analytical applications. Despite some of the compounds
being tested in real objects, the cross-selectivity of such sensors must be low.
3.3 Mimic Enzymes for Sensors 101
The PB films grown through interdigitated arrays were used as a humidity sensor
[145] as well as a sensor for vapors of methanol and dichloroethane [146]. A non-
conventional optical pH sensor based on PB was also reported [147, 148].
Oxidase
Analyte Oxidized
Analyte
O2 H2O2
biosensors as an example: Due to the low potential of the indicator electrode, the
influence of interferents on the PB-based biosensor [75] was similarly low as that
in the case of a biosensor based on peroxidase wired in osmium hydrogel [160].
However, PB-based electrodes offered a detection limit of 1107M glutamate in
the flow-injection mode, which was one order of magnitude lower than that for
known biosensors.
3.4Conclusions
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wwwwwwwwwwwww
Chapter 4
Porphyrin-Based Nanocomposites
for Biosensing
4.1Introduction
Fig.4.1 (a) Crystal structure of the active site of cytochrome c oxidase (CcO) from the bovine
heart; (b) biomimetic analogs of the heme/Cu site of CcO. Reprinted with permission from
Collman etal. [3]. 2007, Science
donor moiety have been expected to lead the construction of a novel electron
donoracceptor composite system for the development of photoelectrochemical and
electrochemical biosensing.
4.2.1Carbon Nanotubes
The unique structural, mechanical, and electronic properties of CNTs have made
these promising materials for device fabrication. To effectively utilize CNTs as
building blocks for nanotechnology, nanotubes have been covalently and noncova-
lently functionalized in a number of ways to render them soluble in aqueous or
organic solutions and to gain precise control over nanotube orientation and location.
CNTs are usually divided into single-walled carbon nanotubes (SWCNTs) and mul-
tiwalled carbon nanotubes (MWCNTs) according to the number of layers of curved
graphene sheets. The functionalization of SWCNTs with porphyrins has been inves-
tigated with increasing frequency because these flat, planar aromatic structures are
ideal for p-stacking interactions with the sidewalls of SWCNTs. Generally, this
procedure can be performed by covalent or noncovalent routes.
Noncovalent methods, including electrostatic interactions, pp interactions, and
axial coordination, are more appealing because they do not significantly perturb the
electronic structure of the nanotubes. In 2003, the first hybrid nanomaterials of
SWCNTsporphyrin were reported in Nakashimas group. As shown in Fig.4.2a, b,
the solid purified SWCNTs solely are insoluble in DMF, and the DMF solution of
zinc protoporphyrin IX (ZnPP) shows a red color. After the sonication of SWCNTs
in ZnPP DMF solution, a reddish-black-colored transparent solution is observed
(Fig. 4.2c), strongly suggesting that ZnPP can disperse/dissolve p-SWCNTs. No
precipitation is noticed in the SWCNTZnPP DMF solution even after a 2-month
storage at 5C [11]. Density functional theory calculation proves that the chemical
reactivity of semiconducting SWCNTs toward metalloporphyrin is stronger than
that of metallic SWCNTs [12]. Therefore, semiconducting SWCNTs can be sepa-
rated from metallic SWCNTs through the adsorption of metalloporphyrin due to the
difference in charge transfer and hybridization between metalloporphyrin molecules
and SWCNTs [13].
Protonated porphyrin provides another convenient way to construct ordered
molecular assemblies. The ordered assembly of SWCNTs-protonated porphyrin
can be obtained by adding 1 mg of purified SWCNTs in tetrahydrofuran (THF)
containing 1.0% H2SO4 (v/v) and 0.2mM porphyrin. The pp interaction between
porphyrins and SWCNTs plays an important role in achieving the ordered assembly
of protonated porphyrin in the form of J- and H-type aggregates on the SWCNTs
surface (Fig. 4.3). This unusual molecular aggregation phenomenon driven by
SWCNTs further assembles in the form of linear bundles [14]. This simple method
of designing supramolecular assembly can pave the way for developing light-
harvesting assemblies and optoelectronic devices.
114 4 Porphyrin-Based Nanocomposites for Biosensing
Fig.4.2 Photos of (a) DMF dispersion of SWCNTs, (b) DMF solution of ZnPP, and (c) a transparent
DMF dispersion of SWCNTsZnPP. Reprinted with permission from Murakami etal. [11]. 2003,
Elsevier
CNTs to form electron donor for the self-assembly of porphyrin molecules. Similarly,
nitrogen-doped MWCNTs (CNx-MWCNTs) containing the nitrogen atom in a CNT
structure provide the electron donor to form axial coordination for the preparation of
a functional nanocomposite of picket-fence porphyrin, bromo-[iron(III)-5,10,15,20-
tetrakis(a,a,a,a-2-pivalamidopheny) porphyrin] (FeTpivPP). The functional nano-
composite exhibits a promising tool to assemble the CNTs via Fe-N axial coordination
(Fig.4.4). This approach provides a facile avenue for the direct axial assembly of
porphyrin and the design of novel biofunctional materials [18].
Compared to noncovalent methodologies, the formation of SWCNT conjugates
employing covalent methods bears a number of advantages. First, the spacer that is
used to link SWCNT and the photoactive molecule is stable and well defined.
Second, the number of functional groups is controlled by fine-tuning the function-
alization processes. The covalently connected porphyrins on CNTs can enhance the
efficiency of photoinduced electron transfer and energy transfer [19]. Typically,
SWCNTs are purified using stepwise wet-air oxidation and shortened using a
sulfuric acid/nitric acid (3:1) treatment to introduce carboxylic acid groups on the
surface of CNTs. Then, CNTs treated with thionyl chloride are reacted with excess
5-phydroxyphenyl-10,15,20-tritolylporphyrin (por-OH) in toluene in the presence
of triethylamine at 100C for 24h under a pure nitrogen atmosphere. To remove the
unreacted por-OH, the tubes are washed thoroughly with plenty of methanol, fol-
lowed by a small amount of acetic acid and triethylamine, and finally with THF. The
final products are then dried at 40C for 5h under vacuum (Fig.4.5). Steady-state
fluorescence reveals that covalently connected porphyrins act as energy-absorbing
and electron-transferring antennae, and the CNTs act as electron acceptors [20].
116 4 Porphyrin-Based Nanocomposites for Biosensing
Fig.4.5 Porphyrin-grafted CNTs and the photoinduced electron transfer. Reprinted with permission
from Baskaran etal. [20]. 2005, American Chemical Society
4.2.2.2H2O2
H2O2 is a product of the enzymatic reactions between most oxidases and their sub-
strates; thus, its detection is very interesting for the development of biosensors for
oxidase substrates. Iron(III) protoporphyrin IX [Fe(III)PP], adsorbed either on
SWCNTs or on hydroxyl-functionalized SWCNTs (SWCNTs-OH), has been
incorporated within a Nafion matriximmobilized graphite electrode. Both the
SWCNTFe(III)PP- and SWCNTOHFe(III)PP-modified graphite electrodes exhibit
electrocatalytic activity toward H2O2 reduction. The sensitivities of the modified elec-
trodes for H2O2 are found to vary in the following sequences: SWCNTOHFe(III)
PP=2.45 mA/MSWCNTFe(III)PP=2.95 mA/M>free Fe(III)PP=1.34 mA/M
[30]. In addition, an electrochemiluminescent (ECL) biosensor of H2O2 has been con-
structed based on cobalt(II) meso-tetraphenylporphrine/MWCNT (CoTPP/MWCNT)
modified GCE. Under the optimum conditions, the enhanced ECL intensity shows a
linear relationship with the concentration of H2O2 in the range of 1.0107
8.0108mol/L, with a detection limit of 5.0109mol/L [31].
4.2.2.3DNA
4.2.2.4Sudan I
rapid, and convenient method for the determination of Sudan dyes is of great
importanceand interest. In pH 7.0 TrisHCl buffers, Sudan I shows a sensitive cata-
lytic reduction peak at 0.08V on the iron-porphyrin (5,10,15,20-tetraphenyl-21H,
23H-porphine iron(III) chloride)SWCNTDMFmodified GCE. Using square-
wave voltammetry, the linear relationship of Sudan I is 5.031082.01106mol/L,
with a detection limit of 1108mol/L [33]. This biosensor has been successfully
applied in the determination of Sudan I in hot chili powder, hot chili juice, and
ketchup samples.
4.2.2.5l-Glutathione
4.2.3Carbon Nanohorns
4.2.4Graphene Sheets
Semiconductors are an attractive material for a broad range of electronic, optical, and
piezoelectric applications due to their direct band gap and excellent thermal, chemical,
and structural properties. There are various ways to anchor porphyrins onto semicon-
ductors host surfaces: (1) covalent attachment by anchoring groups; (2) electrostatic
interactions, via ion exchange, ion pairing, or donoracceptor interactions; (3) hydro-
phobic interactions; (4) hydrogen bonding; (5) van der Waals interactions; and
(6) physical entrapment inside the pores or cavities of hosts. Semiconductors are
usually divided into two parts: metal oxide and quantum dots (QDs).
4.3.1TiO2Porphyrin Nanocomposite
TiO2 is a most useful semiconductor, but its wide band gap (3.2eV) limits its use as
a visible-light photocatalyst. The main drawback is low quantum yield, and the lack
4.3 Assembly of Porphyrins on Semiconductor Nanoparticles 123
Fig.4.10 Structures of the porphyrins and the anticipated binding geometries of the COOH and
COOEt3NH derivatives on metal oxide surfaces. Reprinted with permission from Rochford etal.
[41]. 2007, American Chemical Society
with an isophthalic acid anchoring unit, have been prepared as model dyes for the
study of sensitization processes on metal oxide semiconductor nanoparticle (such as
TiO2, ZnO, and insulating ZrO2) surfaces for photoelectrochemical cells [42].
In an alternative approach, short aromatic amines have been tethered to TiO2
nanoparticles and used to scavenge porphyrin (Ru(CO)OEP, Ru(CO)TPP, and
ZnTPP) from solution and anchor the porphyrin, via axial ligation, in close proxim-
ity to the electrode surface. The attachment of porphyrins via axial coordination
provides a number of advantages, including the ability to control chromophore ori-
entation relative to the surface, and simplifies syntheses using commercially avail-
able materials for the modular assembly of porphyrin sensitization, reduction of
surface aggregation, and a controllable mechanism for stepwise construction of
multiporphyrin arrays. The simple method is highly adaptable for use in dye-
sensitized solar cells [43]. In addition, TiO2 nanoparticles have been found to
enhance the formation of J-aggregates of water-soluble porphyrin [44].
The main disadvantage of these anchoring groups is instability against water,
acids, and bases in their applications. To overcome this limitation, catechol, ethers,
acetylacetonate, and salicylates have been explored. For example, the introduction
of catechol-anchoring groups (3,4-dihydroxybenzo compounds) for grafting por-
phyrins onto metal oxide surfaces is relatively stable and soluble [45]. Using cate-
chol groupterminated Zn(II)-porphyrin to functionalize the single-crystalline TiO2
nanoleaves along the face results in a facet-selective, self-assembled, 2D stacking
structure [46]. Tyrosine methyl ester can be used as a bridge between TiO2 nano-
clusters and tetratolylporphyrin in neutral ethanol solution to enhance the photoin-
duced electron transfer in a heterogeneous system [47].
Fig. 4.11 TEM image of (a) H2P-COO-TiO2 tube, and (b) H2P-COO-TiO2Pa. (c, d) are SEM
images of OTE/SnO2/(H2P-COOTiO2 tube+C60)n and OTE/SnO2/(H2P-COO-TiO2Pa+C60)n.
Reprinted with permission from Hasobe etal. [48]. 2007, Wiley
architecture. The efficiency of light energy conversion of these solar cells has been
explained on the basis of the geometrical orientation of the porphyrins with respect
to the TiO2 surface and the supramolecular complex formed with C60.
XPS has been used to distinguish the surface change of pure TiO2 and N-doped
TiO2 (N-TiO2) nanoparticles after they adsorbed Zn porphyrin. Figure4.12 shows O
1s XPS spectra of pure TiO2 and N-TiO2 before and after they adsorb ZnTPP and
ZnTCPP, respectively. The O 1s peaks of N-TiO2-ZnTCPP are 0.50.7eV higher
than those of pure TiO2 and N-TiO2, while no shift of O 1s peaks for the N-TiO2-
ZnTPP is observed. This result suggests that the ZnTCPP molecules bind more
slightly on the surface of TiO2 samples than ZnTPP, and their structure may change
after their adsorption on the surface of TiO2. The characterization shows that
the ZnTCPP is chemisorbed on the surface of TiO2 through an O=COTi bond,
while the ZnTPP is physically adsorbed. N-TiO2 sensitized by Zn porphyrin exhib-
its higher absorption in the visible-light region and higher photocatalytic degrada-
tion efficiency of methylene blue under visible-light irradiation than N-TiO2 and
TiO2 sensitized by ZnTCPP [49].
Figure4.13 displays the XRD patterns of the bare TiO2 and the Nb-, Ge-, and
Zr-added TiO2. All the peaks in each sample can be assigned to anatase. No XRD
pattern arising from rutile is observed. It should be noted here that all the samples
126 4 Porphyrin-Based Nanocomposites for Biosensing
Fig.4.13 XRD patterns of (a) TiO2, (b) Nb-added TiO2, (c) Ge-added TiO2, and (d) Zr-added
TiO2. Reprinted with permission from Imahori etal. [50]. 2006, American Chemical Society
4.3 Assembly of Porphyrins on Semiconductor Nanoparticles 127
exhibit a similar XRD pattern. This implies that the TiO2 anatase nanocrystalline
structure is retained after doping a small amount (5mol%) of Nb, Ge, or Zr in the
TiO2 structure [50]. It is the first systematic comparison of the electrode structures
and photovoltaic properties of porphyrin-sensitized solar cells with TiO2 composite
electrodes in which the Ti atom is partially substituted with other metals (i.e., Nb,
Ge, and Zr).
The electron transfer at the semiconductordye interface has been successfully uti-
lized in the development of solar cells, electronic devices, heterogeneous photoca-
talysis, and biosensing. Manganese oxides, having economic and environmental
advantages, have been used for a long time in air electrodes as electrocatalysts for
the reduction of O2. Combined with porphyrin, a binary catalyst composed of elec-
trodeposited manganese oxide nanoparticles (nano-MnOx) and cobalt porphyrin
has been proposed for the efficient four-electron reduction of molecular oxygen to
water in acidic media [51]. The modification of GCE with cobalt porphyrin alone
results in a significant positive shift of the oxygen reduction reaction (ORR) com-
pared to the unmodified GCE, which maintained a two-electron reduction. A posi-
tive shift of the onset potential of the ORR of ca. 450mV is achieved at the former
electrode. The modification of the GCE with nano-MnOx alone does not affect the
ORR peak potential, but causes a remarkable increase in the reduction peak current
due to the catalytic disproportionation of the electrogenerated hydrogen peroxide
into water and oxygen. The modification of a GCE with both cobalt porphyrin and
nano-MnOx results in the occurrence of the ORR at a significantly positive potential
with almost double the peak current compared to the unmodified GCE (Fig.4.14),
suggesting a promising procedure for developing electrocatalysts to oxygen reduc-
tion in replacement of costly Pt.
An improved photocurrent generator can be prepared easily by spin coating a
Nafion/porphyrin/TiO2 mixture onto an ITO substrate. The generated photocurrent
density is about 10 times higher than that in the absence of TiO2 (Fig.4.15). The
photocurrent density increases linearly concomitantly with high surface concentra-
tions of porphyrin and high membrane thickness. It becomes evident that TiO2 con-
tributes to more efficient photocurrent generation by intramembrane electron
mediation [52].
SiO2/TiO2/phosphate can be obtained by the solgel processing method, and then
H2TMPyP is immobilized on the matrix surface by an ion-exchange reaction and
metallated in situ with Co(II), resulting in an SiO2/TiO2/phosphate/CoTMPyP mate-
rial. The amount of CoTMPyP incorporated into the matrix is 35.0 mmol/g. The
immobilized complex catalyzes O2 reduction to H2O at 0.22 V in 1 mol/L KCl
solution at pH 6.8 (Fig.4.16). The cathodic current intensities plotted against O2
concentrations between 1 and 11ppm show a linear correlation [53]. Hematoporphyrin
IX and protoporphyrin IX are efficiently immobilized on a cellulose/titanium (IV)
oxide composite fiber surface by the reaction of the porphyrin COOH groups with
128 4 Porphyrin-Based Nanocomposites for Biosensing
Fig. 4.14 Cyclic voltammetries obtained at (a) bare GC and modified with (b) nano-MnOx,
(c) cobalt porphyrin, and (d) cobalt porphyrin and nano-MnOx GCE in O2-saturated 0.1M H2SO4.
Scan rate: 100mV/s. Reprinted with permission from EI-Deab etal. [51]. 2008, Springer
Fig. 4.15 Photoelectrochemical responses of the Nafion-TiO2-porphyrin (solid line) and the
Nafion-porphyrin (dashed line) membranes on ITO electrodes. Reprinted with permission from
Ikeda etal. [52]. 2005, American Chemical Society
TiO2, presumably by forming the COO-Ti chemical bond. The resulting sensors
show a linear range, from 0.5 to 13mg/L, to the O2 reduction [54].
The quantification of phenolic derivatives is of great importance since many of
these compounds, even in small proportions, easily penetrate through the skin and
4.3 Assembly of Porphyrins on Semiconductor Nanoparticles 129
Fig.4.16 Cyclic
voltammetric curves obtained
for SiO2/TiO2/phosphate/
CoTMPyP in various
dioxygen concentrations: (a)
desaerated; (b) 3.6, (c) 7.1,
and (d) 10.0ppm. Reprinted
with permission from
Castellani and Gushikem
[53]. 2000, Academic
Press
4.3.2Quantum Dots
IIVI semiconductor nanoparticles, due to the wide band gap and controlling emis-
sion, are particularly interesting for the development of novel optoelectronic devices
like light-emitting diodes, lasers, and transistors. Hetero-nanoassemblies in toluene
solution have been formed via anchoring pyridyl substituted free-base porphyrin mol-
ecules (Pyr)nH2P on the colloidal core-shell semiconductor nanocrystals CdSe/ZnS
QDs. Only one molecule is estimated to anchor on one nanocrystal even at high molar
ratios [56]. Then, the above-outlined self-assembly principle is organized where
molecular arrays anchor on semiconductor QD surfaces in a systematic way. The
quenching of the fluorescence is partly related to fluorescence resonance energy trans-
fer from the QD to H2P and can be explained according to the Frster model [57].
A new type of self-assembled film has been prepared by alternating the deposi-
tion of oppositely charged meso-tetra-(4-trimethylaminophenyl) porphyrin
nickel iodide(NiTAPPI) and citrate-stabilized CdSe nanoparticles [58]. The SEM
images show the formation of densely packed 2D arrays and the conversion
from disorder to order of CdSe nanoparticles on the quartz substrate modified by
poly(diallyldimethylammonium) chloride (PDDA) when depositing positively
130 4 Porphyrin-Based Nanocomposites for Biosensing
Fig. 4.17 Mode of interaction between porphyrins and thioglycolic acid-capped CdTe QDs.
Reprinted with permission from Jhonsi and Renganathan [59]. 2010, Academic Press
charged NiTAPPI. Placed in ambient air, the self-assembled film exhibits a significant
enhancement in fluorescence intensity.
The photoinduced interaction of thioglycolic acid-capped CdTe QDs with
porphyrins provides a promising way to assemble porphyrin on QDs (Fig.4.17). The
QDs surface is negatively charged since the thiol capping agent contains a carboxylic
group. Positively charged TMPyP interacts with QDs through charge-transfer mech-
anism, negatively charged porphyrins (TCPP and TSPP) interact through an energy-
transfer mechanism, and the neutral TPP does not have any interaction [59].
4.4 Assembly of Porphyrins on Metal Nanoparticles 131
4.3.3Fe3O4 Nanoparticles
Porphyrin derivatives and iron oxides are complementary in both properties and
functions. The conjugation of porphyrin and iron oxide nanoparticles may lead to a
bimodal anticancer agent that can be used in the combinational treatment of photo-
dynamic therapy and hyperthermia therapy. Figure 4.18 illustrates the synthetic
pathway for making the conjugation of Fe3O4porphyrin nanoparticles. After an
N-hydroxysuccinimide-activated derivative of dopamine reacts with the diaminopo-
rphyrin, a simple deprotection is used to remove the benzyl groups and affords com-
pound 4 in good yield (65%). Reacting compound 4 (5 mg, in 2 mL of MeOH/
CHCl3 1:1) with magnetite nanoparticles (30mg, in 5mL of hexane) in an ultrasonic
bath for 60min gives a reddish-brown mixture, which is centrifuged and redissolved
in methanol. After the methanol solution is washed 3 times using chloroform, high-
speed centrifugation yields the final product for bimodal anticancer therapy [60].
The SAMs of porphyrins on flat metal substrates or equivalents have been exten-
sively studied, with the aim of developing artificial photosynthetic materials.
However, the light-harvesting efficiency in the 2D systems has so far been limited
due to the porphyrin monolayer, which can absorb little light. Metal nanoparticles
involving Au, Ag, and Pt nanoparticles can provide three-dimensional (3D) archi-
tectures, which have attracted widespread interest, since their nanosized physical
properties are quite different from those of the bulk materials. The potential applica-
tions of metal nanoparticles have been extensively applied in biochemical sensors,
nanostructure fabrication, and optoelectronic devices.
4.4.1Au Nanoparticles
Fig.4.19 The structure of the composite and TEM images with size distribution. Reprinted with
permission from Ohyama etal. [61]. 2008, Royal Society of Chemistry
porphyrin receptors in solution, and have been shown to recognize anions for
chloride and dihydrogen phosphate [62]. To assess the quantitative significance of
multiple interactions, monolayer-protected gold nanoclusters (MPCs) with a
mixed monolayer containing different loadings of N-methylimidazole as ligand
have been exploited for the recognition of discrete porphyrin arrays, which
increases the binding strength by up to three orders of magnitude with respect to
a monovalent system [63].
In addition, alkanethiolate MPCs are stable in air, and soluble in both nonpolar
and polar organic solvents; therefore, they are capable of facile modification with
other functional thiols through exchange reactions or by couplings and nucleophilic
substitutions. Thus, constructing the 3D architectures of porphyrin MPCs, which
have a large surface area, would improve the light-harvesting efficiency as com-
pared to the 2D porphyrin SAMs [64]. The first successful synthesis and the photo-
physical properties of porphyrin MPC were reported by Fukuzumis group. The
gold nanoparticles, unlike their bulk counterparts, do not quench the fluorescence of
porphyrin MPCs intensively [65]. Furthermore, under IR light irradiation, a mixed
toluene solution of ammonium saltstabilized gold nanoparticles with (3.80.8)-
nm core diameter and a porphyrin thioacetate derivative afford a thin photoactive
film of the clusterporphyrin network [66].
Gold nanoparticles functionalized with imidazolylporphyrinatozinc(II) have
been bridged by successive imidazole-to-zinc coordinations of bidirectional
porphyrinatozinc(II) units. AFM images of samples prepared from two different
concentrations in (CHCl2)2 (9103 and 3104 M) are shown in Fig. 4.20. The
higher-concentration sample formed long and overlain wires in AFM images,
whereas much shorter and nonoverlain wires are mostly observed in the other AFM
image. The average length of the supramolecular wires in the right-hand AFM
image is 7635nm, but wires with lengths greater than 200nm are also observed
[67]. The supermolecular electronics is ready to use in next-generation electronic
devices.
4.4 Assembly of Porphyrins on Metal Nanoparticles 133
Fig.4.20 AFM images of the composite on mica; (left) 9103M and (right) 3104M (CHCl2)2
solutions were deposited. Reprinted with permission from Satake etal. [67]. 2009, Royal Society
of Chemistry
The first example of the cocatalyst effects of gold clusters in the enhanced activity
of Mn-porphyrin catalyst was reported by Konishi in 2007. The Au cluster led to
appreciable acceleration of the catalytic reaction of Mn(TPP)Cl (TPP) tetraphe-
nylporphinato toward styrene oxidation (Fig.4.21). The major role of the Au cluster
was to regenerate the active catalytic path involving Mn(III) and Mn(V) from the
deactivated Mn(IV) species [68].
Through electrostatic layer-by-layer (LBL) assembly, AuCl4 anions and CoTMPyP
cations have been alternately deposited on ITO substrates and 4-aminobenzoic acid
modified GCE. The electrochemical reduction of AuCl4 anions sandwiched between
CoTMPyP layers leads to the in situ formation of Au nanoparticles in the multilayer
films. The resulting composite films containing Au nanoparticles with high stability
exhibit high electrocatalytic activity with the two-electron reduction of O2 to H2O2 in
O2-saturated 0.1M H2SO4 solution [69]. A simple, efficient, and sensitive sensor for
dissolved oxygen has been proposed by combining a self-assembly monolayer of
mono-(6-deoxy-6-mercapto)-b-cyclodextrin, FeTMPyP, and cyclodextrin-functional-
ized gold nanoparticles. The supramolecular-modified electrode shows excellent cata-
lytic activity for oxygen reduction, with a 200-mV positive shift of the reduction
potential compared with bare gold electrode. The ORR probably involves four elec-
trons with a rate constant of 7104 mol/L/s. Alinear response range from 0.2 to
6.5mg/L, with a sensitivity of 5.5mAL/mg and a detection limit of 0.02mg/L, is
obtained. The repeatability of the sensor, evaluated in terms of RSD, is 3.0% for ten
measurements of a solution of 6.5mg/L of oxygen [70].
134 4 Porphyrin-Based Nanocomposites for Biosensing
4.4.2Ag Nanoparticles
Fig.4.22 SERRS spectra of TPP measured from Ag surfaces; soaking concentrations from bot-
tom to top: 1107M, 4107M, 8107M, 2106M, 3106M, and 6106M. Reprinted with
permission from Hajdukov etal. [73]. 2008, Elsevier
4.4.3Pt Nanoparticles
Pt nanoparticles (Pt NPs) are usually used as catalysts for hydrogen evolution.
A tetraphenylporphyrin bearing four naphthalene donor moieties 5,10,15,20-
tetrakis(4-(naphthalen-1-ylmethoxy)phenyl)porphyrin has been synthesized to form
functionalized platinum nanocomposite. The photoreceptive dye forms a shell con-
taining a nanosized Pt core (~2.8nm). The photocatalytic activity of the Pt nano-
composite toward water reduction to hydrogen is twice stronger than that of a
Ptnaphthalene-free porphyrin system [75].
136 4 Porphyrin-Based Nanocomposites for Biosensing
Fig. 4.23 Amperometric current response vs. concentration for five organohalides using a Pt
NPZn porphyrin nanocomposite in 0.1M TBAP/ACN. Reprinted with permission from Wiyaratn
etal. [78]. 2005, American Chemical Society
4.5Other Nanomaterials
4.5.1Polymer Nanoparticles
Polymers permit sufficient flexibility to assist the transport of the substrates, which
may be essential for artificial enzymes. For example, a new anionic water-soluble
polythiophene and a cationic porphyrin have been synthesized through electrostatic
interactions. Based on the enhanced energy transfer offered by light-harvesting con-
jugated polymers, the singlet oxygen is produced and effectively kills the bacteria,
with about a 70% reduction of bacterial viability in only 5min of irradiation under
white light (400800nm) [79]. The polymer nanocomposite of the polyelectrolytes
poly(allylamine hydrochloride) and poly(styrene sulfonate) and TPPS are fabricated
as photoactive microcapsules via LBL self-assembly [80]. In addition, a formylpor-
phyrin has been covalently bound to poly(allylamine hydrochloride) via a Schiff base
intermediate for the electrocatalytical oxidation of the sulfite and nitrite [81].
Oxygen is a critical component for many physiological and pathological processes in
living cells. A novel nanoparticle architecture, consisting of p-conjugated polymer mol-
ecules doped with an oxygen-sensitive phosphorescent dye, has been described for oxy-
gen sensing. The conjugated polymers employed as the doping host are the polyfluorene
derivatives poly(9,9-dihexylfluorene) (PDHF) and poly(9,9-dioctylfluorene) (PFO).
Platinum(II) octaethylporphine (PtOEP) serves as the oxygen-sensitive dye (Fig.4.24).
The rapid addition of a solution of polyfluorene and PtOEP in THF to water results in
nanoparticle formation and the simultaneous entrapment of the hydrophobic PtOEP
molecules inside the nanoparticles via the collapse of polymer chains. AFM results for
PDHF-based particles indicate that the resulting particles are approximately spherical in
shape, with particle heights (diameters) of 255nm. Upon the light excitation, the poly-
mer efficiently transfers the energy to phosphorescent dye, resulting in a bright phospho-
rescence that is highly sensitive to the concentration of dissolved oxygen [82]. A
polystyrene-based oxygen nanosensor has been constructed using platinum(II)meso-
tetra(pentafluorophenyl)porphine via spin-coating the polymer on glass plates. The sen-
sor response is assessed in aqueous solution as well as in yeast culture [83].
A porphyrin-containing copolymer having dual sensing in response to metal ions
and temperature has been used to fabricate a novel nanosensor [84]. First, a specific
triblock copolymer is designed and synthesized by sequential reversible-addition-
fragmentation chain-transfer polymerization. Below 32C, the addition of different
metal ions to the solution of copolymer leads to an unprecedented full spectral color
range (Fig. 4.25). Most surprisingly, upon heating, the multicolored nanosensors
display discrete thermochromic characteristics in the temperature range of 3561C,
with the phase-transition point dependent on the metal ion. The thermochromism
speed was typically less than ~30s, and the temperature range of the color transition
is smaller than 0.4C. This sensor displays an isothermal thermochromic point as
an ultrasensitive thermometer.
Of recent interest are the magnetic polymer nanospheres, where the core is com-
posed of numerous Fe3O4 particles and the shell is composed of a copolymer of
138 4 Porphyrin-Based Nanocomposites for Biosensing
Fig. 4.24 (a) Schematic illustration of the formation of conjugated polymer dots for oxygen
sensing. (b) AFM image of PtOEP-doped PDHF dots dispersed on a mica substrate. (c) Histogram
of particle-height data obtained from AFM image in (b). Reprinted with permission from Wu etal.
[82]. 2009, Wiley
4.5.2Silica Nanomaterials
Amorphous silica is one of the popular traditional materials used as a matrix for
incorporating functional molecules. The most attractive features of silica include
nontoxicity, high porosity, and effective transparency. In general, the 3D space in
silica is sufficiently adjustable to hold various functional molecules. Porphyrins
embedded in silica are used as biomimetic catalysts in various sensor devices for
4.5 Other Nanomaterials 139
Fig.4.25 Simplex-stimuli-sensing thermochromic sensor in the absence of metal ions (left), and
dual-sensing optical sensors (full-spectrum colorimeter and ultrasensitive thermometer) upon
introduction of different metal ions (right). Reprinted with permission from Yan et al. [84].
2010, Royal Society of Chemistry
Fig.4.26 (a) Chemical structure of the zinc(II) porphyrin derivative; (b) schematic drawing of the
SiNW-FET device from silicon-on-insulator. Reprinted with permission from Winkelmann etal.
[90]. 2007, American Chemical Society
4.6Conclusions
Porphyrins are the mimics of many important enzymes and can be modified with
other functional moiety on the porphyrin ring to enhance the selectivity toward
biomolecular catalysis. Furthermore, depending on the nanomaterials properties,
the hybrid nanocomposite of porphyrin nanomaterials can be designed and achieved
via either noncovalent or covalent interactions. The former can keep the electronic
structure of the nanomaterials, and the latter can more efficiently obtain the defined
3D superstructure of the hybrid nanocomposite. Combined with the unique struc-
ture of nanomaterials, the resulting nanocomposite demonstrated excellent optical
and electrochemical biosensing toward life-relating molecules and provided a prom-
ising application potential in bioanalysis.
In order to improve the performance of the nanocomposite in biosensing, two
problems should be addressed: The first is improving the binding between porphy-
rins and nanomaterials since many anchoring groups for assembly are instable
against water, acids, and bases in their applications. The emerging field of click
chemistry has the potential to provide an elegant protocol to prepare porphyrin-
based functional nanomaterials since the reaction is versatile and clean, and can be
operated in very mild conditions without significantly disturbing the conjugated
p-system [92]. Second, it is highly desirable to seek a novel material with a suffi-
cient binding site for functionalization. The doping heteroatom nanostructure, such
as nitrogen-doping CNTs, may be the best candidate for functionalization. The
ordered assembly of porphyrins on the nanomaterials provides a powerful platform
to construct the biosensor with high selectivity and sensitivity and shows promising
applications in biosensing.
142 4 Porphyrin-Based Nanocomposites for Biosensing
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Chapter 5
Carbon Nanofiber-Based Nanocomposites
for Biosensing
5.1Introduction
The history of carbon nanofiber (CNF) can go back more than a century. It was
reported in a patent published in 1889 that carbon filaments are grown from carbon-
containing gases using a metallic crucible as the probably unintentional
catalyst [1]. In 1950, a Russian group performed the first electron microscopy
observations of CNFs. For the first 80 years of the twentieth century, however, the
occurrence of CNFs then often referred to as carbon filaments or filamentous
carbon was considered a nuisance. For example, in FischerTropsch or steam-
methane reforming reactions, the fibers often occurred in metallic catalysts used for
the conversion of carbon-containing gases. In 1991, carbon nanotubes (CNTs) were
first discovered as a new member of the carbon allotrope family. This discovery and
other nanostructures triggered an outburst of interest in CNTs and nanofibers [2].
Generally, the nanotubes can be divided into two categories: single-walled car-
bon nanotubes (SWNTs) and multiwalled carbon nanotubes (MWNTs). MWNTs
are composed of coaxial, multilayer graphene tubes with an interlayer space of
0.34nm, and the diameter for MWNTs varies from 1.4 to 100nm. CNFs are similar
to a large-diameter MWNT; however, CNFs are not continuous like the tubes, and
their surfaces show steps at the termination of each tube wall, forming cylindrical
nanostructures. CNFs can be divided into platelet CNFs, tubular CNFs, and her-
ringbone CNFs, according to the different arrangement of graphene layers. As
shown in Fig.5.1, the graphene layers of platelet CNFs are vertical in relation to the
fiber axis, and the exposed surfaces are mainly occupied by edge atoms. The gra-
phene layers of tubular CNFs are parallel to the fiber axis, and many basal atoms are
exposed; the graphene layers of herringbone CNF incline toward the fiber axis, and
the ratio of edge atoms to basal atoms can be adjusted by controlling the angle of
graphene layers to the fiber axis [3].
CNFs can be produced by various methods, such as arc-discharge [4], laser
ablation [5], chemical vapor deposition (CVD) methods [6, 7], and others [8, 9].
Fig.5.1 Schematic representation of different types of CNTs and CNFs. Reprinted with permission
from Serp etal. [3]. 2003, Elsevier
The arc-discharge and laser ablation methods lead to mixtures of carbon materials
and thus to a cumbersome purification to obtain nanofibers or nanotubes. From an
application point of view, the catalytic growth of nanofibers should be most promis-
ing [1, 6]. CNFs also can be grown in such a way that the nanofibers are all
vertically aligned to form vertically aligned carbon nanofibers (VACNFs), which
are emerging as a useful material for applications such as chemical/biochemical
sensing [1012]. VACNFs are aligned with each fiber approximately perpendicular
to the underlying growth substrate, providing each nanofiber with a direct electrical
connection to an underlying electrode. Although the more commonly studied
SWNTs and MWNTs expose primarily basal-plane graphite, VACNFs consist of
nested cones of graphene that expose large amounts of edge-plane graphite along
their sidewalls. Electron transfer (ET) rates at edge-plane graphite are ~105 times
faster than those at the basal plane [13]; this implies that VACNFs may have out-
standing properties as supports, for example, for electrocatalytic reactions.
CNFs are very promising materials for the development of biosensor systems
since they possess several striking properties that have proven to be very suitable for
the construction of biosensor systems, such as excellent electrical conductivity,
unique structural and catalytic properties, high loading of biocatalysts, good stabil-
ity, and so on. These striking properties of CNFs have triggered intensive research
efforts to utilize them to develop various kinds of advanced biosensors with higher
sensitivity, faster response, biocompatibility, and cost-effectiveness [1417]. This
chapter surveys the current status of CNF-based nanocomposites for biosensor
5.2 Synthesis of Carbon Nanofiber 149
In CVD methods, the most important metals to catalyze the growth of graphitic
CNFs are (alloys of) iron, cobalt, and nickel; chromium, vanadium, and molybde-
num have also been studied [1, 6, 18]. The metals have been used both as bulk
particles (size typically 100nm) and as supported particles (1050nm). All of these
metals can dissolve carbon and/or form metal carbonides. Typically, methane,
carbon monoxide, synthesis gas (H2/CO), ethyne, and ethene in the temperature
range of 7001,200K are employed to provide carbon atoms [1].
A model for the nucleation and growth of CNFs is shown in Fig.5.2 [1]. Methane
decomposes into carbon and hydrogen atoms at the nickel surface (see step 2 in
Fig.5.2). H2 molecules desorb and carbon dissolves and forms (substoichiometric)
nickel carbide (see step 3 in Fig.5.2). This nickel carbide is metastable with respect
to nickel metal and graphite. After, say, 10min, the carbide phase decomposes into
metallic nickel and graphite, which encapsulates the nickel particle in question (see
step 4 in Fig. 5.2). According to this model, the metal particle is squeezed out
because of pressure buildup due to the formation of graphite layers at the internal
surface of the graphite envelope (see step 5 in Fig.5.2). As soon as the metal is
pushed out, the fresh surface is exposed to the methane and growth continues.
Finally, a steady-state process occurs with either pulsed growth (see step 6a in
Fig.5.2) or smooth growth of a straight fiber (see step 6b in Fig.5.2). This model
also explains why, more often than not, metal particles are found at the tip of the
carbon fiber: The graphite fiber pushes the metal particle from the support and con-
tinues to grow at the back of the particle.
It is known that the diameter of the nanofibers is governed by that of the catalyst
particle responsible for their growth, and the structure of CNFs can vary depending
on the type of catalyst and precursor used during synthesis. It has been reported that
the CNF morphology consists of revealed graphite platelets stacked perpendicularly
150 5 Carbon Nanofiber-Based Nanocomposites for Biosensing
Fig.5.2 Mechanism for the nucleation and growth of a carbon nanofiber (CNF) from methane
catalyzed by a supported metal particle. Reprinted with permission from De Jong and Geus [1].
2000, Marcel Dekker
to the fiber axis when produced from iron catalyst and carbon monoxide/hydrogen
precursor (4:1) at 600C. The graphite platelets were parallel to the fiber axis with
silica-supported iron catalyst, even though the same carbon precursor and synthesis
conditions were used [19, 20]. Currently, most of the catalyst methods for growing
CNTs and CNFs use transition metal particles (Fe, Co, Ni) in the presence of hydro-
carbons at high substrate temperatures ranging between 450 and 1,250C. Boskovic
etal. reported a large-area synthesis of CNFs at room temperature for the first time
[6]. They used hydrocarbon plasmas to provide the energy dynamics necessary for
the dissociation of carbon and the subsequent catalytic growth of CNF on transition
metal particles at room temperature. CNFs were synthesized from radio-frequency
plasma-enhanced CVD at room temperature with Ni powder catalyst placed on
graphite, silicon and plastic substrates at the earthed electrode, and methane or
methane/hydrogen as carbon sources. CNFs synthesized by this method are consi
dered to be potential candidates for many possible applications, such as large-area
flat-panel displays, electrochemical cells, and nanoelectronics.
Very recently, the effects of catalyst support and carbon source on the yield and
structure of carbon were reported [18, 21]. Yamada etal. quantitatively examined
CNF formation on an iron group metal loaded on spherical silica. They found that,
at optimal conditions, the amount of CNF increased in the following order:
FeCo<Ni, irrespective of carbon sources. They also found that on Ni/SiO2, poorly
crystallized CNFs were produced by the tip-growth mechanism. However, Co/SiO2
gave tip- and bottom-growth CNFs, and on Fe/SiO2 small amounts of bottom-growth
5.4 Carbon Nanofiber-Based Electrochemical Biosensors and Bioassays 151
CNFs were produced with cylindrical graphene sheets. Yamada concluded that the
growth mechanism of CNF was strongly affected by the interaction between iron
group metals and SiO2 [18].
5.4.1Glucose Sensors
Fig. 5.3 (a) SEM image, (b) TEM image, and (c) EDX spectra of the NiCF nanocomposite.
Reprinted with permission from Liu etal. [35]. 2009, Elsevier
simplicity, reproducibility, cost reduction, and the fact that it is free of oxygen
limitations [27]. For an amperometric glucose sensor, however, there are two key
issues that should be addressed properly: enhancing the sensitivity to glucose, and
the interference by electroactive species. Generally, the direct oxidation of most
carbohydrates, including glucose, requires a large overpotential at conventional
electrodes. Such an overpotential limits the selectivity of the determination of glu-
cose, causing tough interference problems [27]. Recently, combining the unique
properties of CNF with the catalytic activity of electrocatalysts has been expected to
oxidize glucose effectively. These electrocatalyst candidates consist of precious
metals such as Pt, Au, and alloy [28] and relatively low-cost catalysts such as metal
oxide and complex catalysts, i.e., CuO [29], NiO [30], MnO2 [31], Co(II) phthalo-
cyanine tetrasulfonate (CoPcTS) [32], Cu nanocluster/MWNTs [33, 34], and MWNTs
[34]. Liu et al. demonstrated such expectations by fabricating Ni nanoparticle-
loaded CNF paste electrode for the nonenzymatic oxidation of glucose [35].
They used SEM, TEM, and EDX to investigate the morphology and microstructure
of the as-prepared NiCF nanocomposite, and each element existed in the nano-
composite (see Fig.5.3). They found that Ni nanoparticles with a diameter of about
5.4 Carbon Nanofiber-Based Electrochemical Biosensors and Bioassays 153
Fig.5.4 Schematic picture of the immobilization of the model enzyme GOx on CNFs and single-
walled carbon nanotubes. Reprinted with permission from Vamvakaki etal. [22]. 2006, American
Chemical Society
50nm are embedded in the CNF matrix, and although the concentration of Ni-based
catalyst on the Ni-CNF paste electrode is much less than that on the Ni bulk elec-
trode, the sensitivity of the Ni-CNF paste electrode is about 1.5 times higher than
the bulk electrode. This phenomenon was attributed to the high electrocatalytic
activity of the Ni nanoparticles embedded in the Ni-CNF paste electrode with a
large electroactive surface area.
Though the electrochemical determination of glucose concentration without
using an enzyme is one of the dreams that many researchers have been trying to
realize, at present, the most common strategy for glucose determination is based on
glucose oxidation-catalyzing enzymes [26]. Two families of enzymes, glucose oxi-
dases (GOx) and PQQ-glucose dehydrogenases (PQQ-GDH), are most widely used
in the electrooxidation of glucose. Because the immobilization procedure of
enzymes affects the bioactivity of enzymes, several research groups have examined
the immobilization and stabilization efficiency of these enzymes on the CNF. For
example, in 2006, Vamvakaki etal. used GOx as the model enzyme to compare the
potentiality of CNF, CNT and graphite powder as the matrix for the development of
biosensors. They demonstrated that CNFs have a much larger functionalized surface
area compared to that of single-walled CNTs and are expected to immobilize and
better stabilize the enzyme GOx, as illustrated in Fig.5.4. Their experimental results
led to an interesting conclusion that CNF is the best matrix so far for the develop-
ment of biosensors, far superior to CNT or graphite powder [22].
154 5 Carbon Nanofiber-Based Nanocomposites for Biosensing
5.4.2Ethanol Sensors
The measurement of trace ethanol plays an important role in different industries and
biotechnological processes such as the production of alcoholic beverages, food-
stuffs, pharmaceutical products, and clinical and forensic analysis [36]. Two
enzymes have been extensively used in the determination of alcohols, namely,
alcohol oxidase (AOX) and alcohol dehydrogenase (ADH). Almost all AOX-based
ethanol sensors developed so far have been based on monitoring O2 consumption or
H2O2 formation. This has been mostly achieved using amperometric electrodes set
at appropriate potentials, namely, 600mV for O2 monitoring or +600mV for H2O2
monitoring. When a potential of 600 mV relative to Ag/AgCl is applied to the
platinum electrode, O2 is reduced according to the equation O2 + 4H + + 4e - 2H 2 O
and a current proportional to O2 concentration is produced [36]. However, the
cathodic current observed at 600mV often expresses the combined reduction of
both dissolved oxygen and other redox species, such as hydrogen peroxide pro-
duced by the enzyme cycle. Therefore, a decreased overpotential for the dissolved
oxygen-reduction reaction is required to eliminate the interference. Recently, Wu
etal. demonstrated a new kind of CNF-based ethanol amperometric biosensor. CNFs
were functionalized noncovalently with the thionine molecule. The noncovalent
functionalization of CNFs with thionine improved the dispersion of CNFs due to the
fact that thionine can be electrochemically polymerized, which is similar to some
kinds of heterocyclic aromatic dyes such as methylene blue (MB) and methylene
green (MG). AOx could be effectively incorporated to form a stable poly(thionine)
CNF/AOx biocomposite film on an electrode surface. Though poly(thionine) did
not show any catalytic activity toward the reduction of oxygen, the CNFs could
effectively catalyze the oxygen reduction reaction at a relatively low overpotential,
such as 0.3V vs. Ag/AgCl in pH 7.0 phosphate buffer solution [37]. Furthermore,
Wus group demonstrated the noncovalent functionalization of CNFs with water-
soluble porphyrin, i.e., iron(III) mesotetrakis(N-methylpyridinum-4-yl) porphyrin
(FeTMPyP), to construct another kind of CNF-based ethanol biosensor. The formed
CNFFeTMPyP nanocomposite (see Fig. 5.5) showed better catalytic activity
toward the reduction of dissolved oxygen than the bare CNFs, so that the constructed
ethanol biosensor could be operated at a lower overpotential (0.2V vs. Ag/AgCl
in the same conditions) [38]. The detection limit was found to be lower than that
obtained at an ADH/CNF-modified electrode by monitoring the electrocatalytic
oxidation current of NADH [39].
5.4 Carbon Nanofiber-Based Electrochemical Biosensors and Bioassays 155
Fig.5.5 Steady-state
absorption spectra of
(a) 5 mM FeTMPyP and
(b) 0.5mgmL/L CNF-
FeTMPyP in aqueous
solution. Inset: Chemical
structure of FeTMPyP.
Reprinted with permission
from Wu etal. [38]. 2008,
Elsevier
Ethanol biosensors based on ADH have been reported to be more stable and
specific than those based on AOX [36]. Recently, the biocompatibility of CNFs
toward ADH has been studied with the FT-IR spectrum, of which the amide I and
amide II infrared absorption bands of ADH can provide detailed information on the
secondary structure of the polypeptide chain. It was found that the absorption bands
of ADH in the CNF film were nearly the same as those at 1650.8 and 1541.4/cm
obtained for the protein itself, indicating that CNF film showed good biocompatibility
and retained the native structure of the ADH [39]. Such biocompatibility of CNF
makes it very attractive in the development of an ADH-based ethanol biosensor.
5.4.3Acetylthiocholine Sensors
Fig.5.6 Preparation of Try/PANIILCNFbased biosensor. (a) GCE pretreatment; (b) the for-
mation of PANI-IL-CNF composite film on GCE surface; (c) the addition of tyrosinase solution
onto the composite film; (d) the as-prepared Tyr/PANI-IL-CNF-based biosensor. Reprinted with
permission from Zhang etal. [43]. 2009, Elsevier
5.4.4Phenol Sensors
Phenolic compounds, which are highly toxic, are widely used in wood preserva-
tives, textiles, herbicides, and pesticides and are released into the ground and sur-
face water. The determination of these compounds is of great importance in
environment monitoring. Such a determination is commonly based on monitoring
the reduction signal of their enzymatic oxidation products, o-quinones, by molecu-
lar oxygen in the presence of tyrosinase (Tyr). Thus, for the construction of a phenol
biosensor with CNFs, it is essential to examine the biocompatibility of the CNFs
toward Tyr and to immobilize Tyr on CNF effectively. Recently, Zhang etal. dem-
onstrated a novel PANI-nanocomposite, polyanilineionic liquidcarbon nanofiber
(PANIILCNF) composite, by the in situ one-step electropolymerization of aniline
in the presence of IL and CNFs (see Fig.5.6). The newly designed PANIILCNF
composite showed a fibrillar morphology, which was beneficial to the loading of
enzyme, and thus improved the capacity for immobilization of Tyr. The prepared
Tyr/PANIILCNF-modified electrode exhibited highly sensitive amperometric
responses to the analogs of phenolic compounds such as catechol, p-cresol, phenol,
and m-cresol. With 0.3mM catechol as a model, the amperometric response of the
Tyr/PANIILCNF-modified electrode showed good resistance against 3mM ascor-
bic acid, 30mM uric acid, and 30mM caffeine, which was attributed to the use of a
relatively low operating potential (0.05V vs. Ag/AgCl, pH 7.0) [43].
The electrochemical determination of phenol without using enzyme is also
attractive. For example, Jamal et al. prepared a series of conductive polymer-
modified CNF electrodes and examined the ability of these electrodes to detect
para-aminophenol (p-AP). After poly[N-vinylcarbazole-co-vinylbenzene sulfonic
acid], poly[carbazole-co-methylthiophene], and polycarbazole were coated electro-
chemically on CNF microelectrodes, they found that these modified CNF electrodes
were effective systems for the determination of p-AP and thin-film-coated
5.4 Carbon Nanofiber-Based Electrochemical Biosensors and Bioassays 157
Fig.5.7 Cyclic
voltammograms obtained at
(a) p[NVCzVBSA]-coated
carbon fibers and (b)
untreated carbon fibers in
buffer solution containing
1mM p-aminophenol at scan
rates of 20, 40, 60, 100, 120,
140, and 160mV/s.
Reprinted with permission
from Jamal etal. [44].
2004, Elsevier
p[NVCzVBSA] was the most suitable modified electrode for the detection of p-AP.
It can be seen in Fig.5.7 that quasi-reversible behavior was obtained (anodic to a
cathodic peak current ratio of 1.0) and plots of peak current vs. v1/2 were linear,
indicating that the process was controlled under a diffusion step [44].
H2O2 is a product of the enzymatic reactions between most oxidases and their sub-
strates; its detection is very interesting for the development of biosensors for oxi-
dase substrates and monitoring the activity of oxidases. Generally, high overpotentials
are required for the reduction or oxidation of H2O2 on many electrode materials.
Such a high overpotential causes a problem of electrochemical interference due to
the presence of reducing compounds present in real sample matrices (e.g., ascorbic
acid, uric acid, and acetaminophen), which are also oxidized at that potential.
Furthermore, slower responses are observed.
It is well known that the electrocatalytic reactions are very common on carbon
materials, and the negative surface charge resulting from some surface oxides on a
158 5 Carbon Nanofiber-Based Nanocomposites for Biosensing
carbon surface can have significant electrochemical effects on ET rates. Due to the
existence of a large number of edge sites and the oxygen-containing groups at the
CNF surface, CNF is strongly expected to show catalytic ability for the reduction or
oxidation of hydrogen peroxide. Recently, Wu etal. demonstrated the electrocata-
lytic ability of the CNF for the reduction of hydrogen peroxide in phosphate buffer
solution (pH 7.0). They found that upon adding H2O2, the reduction current at the
CNF-modified glassy carbon electrode dramatically increased and the oxidation
peak current of the oxygen-containing groups on the CNF surface decreased, while
the naked glassy carbon electrode did not show any response to H2O2 (see Fig.5.8).
They attributed such changes in both the reduction and oxidation peak currents to
the synergetic effect of the electrocatalytic action of the oxygen-containing groups
to the reduction of H2O2 and the facilitation of ET kinetics of the electroactive H2O2
by the edge sites on the outer wall of CNFs. This is similar to the observation at
other CNF-modified electrodes. The prepared CNF-modified glassy carbon elec-
trode showed a high sensitivity and good selectivity for the determination of H2O2,
with two common physiological compounds such as uric acid and ascorbic acid as
interferences. The demonstrated electrocatalytic activity of CNF for the reduction
of H2O2 gives CNF a promising application for the development of a nonenzymatic
H2O2 sensor [45].
Additionally, Li etal. studied the effect of CNF microstructure on the catalysis
of CNF for H2O2. CNFs with three microstructures, including platelet-carbon nano-
fibers (PCNFs), herringbone-carbon nanofibers (HCNFs), and tube-carbon nanofi-
bers (TCNFs), were synthesized, characterized, and evaluated for electrochemical
sensing of hydrogen peroxide. Sensors based on PCNFs/GC, HCNFs/GC, and
TCNFs/GC were used in the amperometric detection of H2O2 in solution by applying
a potential of +0.65V vs. Ag/AgCl at the working electrode. The highest electrocatalytic
performance was observed for PCNFs/GC among the three types of hydrogen
peroxide sensors, which was attributed to the highest ratio of edge atoms to basal
atoms of PCNFs [46].
5.4 Carbon Nanofiber-Based Electrochemical Biosensors and Bioassays 159
Fig.5.9 CVs of CNF-modified electrode in (a) 0.2M pH 7.0 PBS and (b) (a) +2.0mM NADH.
Inset: CVs of bare (lower) and untreated CNF-modified (upper) electrodes in (a) 0.2M pH 7.0
PBS and (b) (a) +2.0mM NADH. Scan rate, 10mV/s. Reprinted with permission from Wu etal.
[39]. 2007, American Chemical Society
5.4.6NADH Sensors
showed two oxidation peaks, at +0.062 and +0.361V, which could be ascribed to
the oxidation of NADH at the edge plane sites on the CNF and those of the underly-
ing GCE. The catalytic activity was generally evident from the defined peak at
+0.062V. Compared to peptide nanotube-based and CNT-based NADH sensors, the
oxidation overpotential and applied potential are further decreased by more than
340 and 240 mV, respectively. They attributed the accelerated ET kinetics to the
formation of a high amount of oxygen-rich groups on the CNF, which resulted from
the oxidation treatment of CNF with nitric acid [39].
Furthermore, Perez et al. compared CNTs, CNFs, and carbon microparticles
(CMPs) as an electrocatalysts to modify the electrode substrate for the oxidation of
NADH using CVs technology. They found that though higher currents for the
NADH oxidation peak have been observed for these electrodes, the CNF film pro-
motes better electron transfer (ET) of NADH, minimizing the oxidation potential at
+0.352V. Figure5.10 shows the possible sites of attachment of NADH molecules
that can be introduced onto the surface of the carbon materials [24].
Most of the electrochemical biosensors at present can be categorized into the so-
called first- or second-generation biosensors, in which enzymes or proteins utilized
as the biocatalyst do not directly communicate electron transfer with the electrode
substrate. They are based on the consumption of the natural cosubstrate, such as O2,
or the formation of the product of enzymatic catalytic cycles, or the introduction of
artificial redox mediators. The third-generation electrochemical biosensors are
based on the concept of the direct electron transfer between the enzymes and the
electrode substrate, and a few examples of the third-generation electrochemical
biosensors have demonstrated their advantages over the former two kinds of biosensors
[47, 48]. These include horseradish peroxidase [49], cellobiose dehydrogenases
[48, 50], superoxide dismutase [51], laccase [52], and others [47]. However, the
redox centers of most enzymes are located sufficiently far from the outermost sur-
face to be electrically inaccessible, and consequently these enzymes cannot
efficiently communicate electron transfer with conventional electrode materials
[53, 54] and be applied to construct the third-generation electrochemical biosensors.
CNTs have been demonstrated to exhibit the promotion for the direct ET of enzymes
such as hemoglobin [55], cytochrome c [55], horseradish peroxidase [56], laccase
[52], and so on, and such promotion has been attributed both to CNTs serving as the
nanoscaled electrical wire and to the interaction between enzymes and CNTs
strongly hydrophobic sidewall. CNF can also act as nanoscaled electrical wire and
has the hydrophobic sidewall, just like CNTs; thus, CNF is expected to prompt the
direct ET of enzymes. Recently, Wu etal. observed the direct ET of AOx confined
onto CNF, which has never been directly observed, probably due to steric hindrances
and large reaction barriers [37]. Such an observation indicated CNF could provide
5.4 Carbon Nanofiber-Based Electrochemical Biosensors and Bioassays 161
Fig.5.10 Schematic representation (not to scale) of the possible interactions of the NADH mol-
ecules with graphene layers for the (a) CMPs and (b) CNFs. Reprinted with permission from Perez
etal. [24]. 2007, Elsevier
5.4.8Immunosensors
Fig.5.11 Preparation and detection procedures of the CA125 immunosensor. Reprinted with per-
mission from Wu etal. [61]. 2007, Elsevier
be important to detect molecules of a wide range of sizes. The ends and sidewalls of
the nanofibers provide a very high surface area and, consequently, a very high
number of biological binding sites. This high density of binding sites may increase
sensitivity in the same manner as in porous materials, such as porous carbon and
silicon [11, 12, 62]. The sidewalls of nanofibers can also be insulated, leaving
exposed electrodes of extremely small size that have been used for highly sensitive
detection of DNA and glucose via measurements of the electrochemically active
molecules in solution. These striking properties make VACNF very promising for
the development of electrochemical biosensors [63].
Most applications of CNFs have been based on bare fibers with little or no
chemical modification of the surface. In many applications, however, it is important
to functionalize the electrodes with molecules of interest in order to provide them
with specific chemical and/or electrical properties [63]. Therefore, we will focus on
the functionalization of CNF for the development of CNF-based biosensors.
Baker etal. studied covalent functionalization of VACNFs for biomolecular rec-
ognition and compared two different strategies for covalently modifying CNFs with
biological molecules, such as DNA. One method begins with a photochemical reac-
tion between the nanofibers and molecules bearing both a terminal olefin group and
a protected amine group, followed by deprotection to yield the free primary amine.
The second method uses a chemical reaction of an aryldiazonium salt with the nano-
fibers, followed by electrochemical reduction to the primary amine. Both methods
then link the primary amines to thio-terminated DNA oligonucleotides. Their mea-
surements show that both methods yield DNA-modified CNFs exhibiting excellent
specificity and reversibility in binding to DNA probe molecules in solution having
complementary vs. noncomplementary sequences [64].
Landis and Hamers used ferrocene as a model system to understand the ET prop-
erties of redox-active molecules covalently linked to the surface of VACNFs.
Ultraviolet-initiated grafting of organic alkenes was used to prepare carboxylic
acid-terminated layers, and ferrocene was then linked to these layers via amide
groups (see Fig.5.13). Their results showed that molecular layers grafted to CNFs
were sparse and disordered compared with those commonly studied on planar sur-
faces [65].
Previous studies have grafted molecular layers to VACNFs using the photo-
chemical grafting of alkenes or via reaction with diazonium compounds. The
photochemical method uses ultraviolet light to link terminal alkenes to the surface,
but is limited to alkenes that are stable under ultraviolet light and may not be effective
on thick nanofiber arrays because of the strong optical absorption of the nanofibers.
The diazonium method frequently forms multilayers due to the radical intermediates
involved. Although several other techniques, including oxidative methods and acyla-
tion in concentrated nitric acid, have been used to functionalize VACNF surfaces,
these methods require harsh reaction conditions and are difficult to control [66].
Landis and Hamers proposed a gentle method for covalently functionalizing
VACNFs through a copper-catalyzed azidealkyne cycloaddition (CuAAC) reac-
tion in which an azide group is bound to the surface and then linked with an alkyne
[66]. The CuAAC has come into wide use since its introduction in 2002 [67].
5.4 Carbon Nanofiber-Based Electrochemical Biosensors and Bioassays 165
VACNF arrays (Fig.5.15). They demonstrated that flexible and convenient photoresist
techniques provided site-specific physical, chemical, and electrochemical function-
alization of unprotected, exposed regions of the nanofiber both along its length as
well as at discrete locations across an array of nanofibers [68].
5.5Conclusions
This chapter introduced the synthesis of CNF and VACNF and their applications in
the development of electrochemical biosensors. CNF has been demonstrated to be
very promising as a material for the development of biosensor systems since it pos-
sesses several striking properties that have been proven to be very suitable for the
construction of biosensor systems, such as excellent electrical conductivity, unique
structural and catalytic properties, high loading of biocatalysts, good stability, and
so on. These demonstrations propose that CNF should be better than CNTs for the
development of electrochemical biosensors. VACNFs consist of nested cones of
References 167
graphene that expose large amounts of edge-plane graphite along their sidewalls.
VACNFs may also have outstanding properties for electrocatalytic reactions.
Therefore, the functionalization of VACNFs will endow VACNFs with more
striking properties suitable for the development of electrochemical biosensors.
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Chapter 6
Biosensors Based on Nanoporous Materials
6.1Introduction
the introduction of functional groups into the porous walls possible. And the
modified nanoporous materials could be rendered with many new functions
[2023]. Third, the great porosity and uniform structure would facilitate the fast
transport of the target analytes to the active sites in the nanopores [14, 15]. Fourth,
the inorganic matrices of most nanoporous materials are stable due to their highly
cross-linked structure and therefore can resist biodegradation under extreme condi-
tions. In addition, the structure, pore size, hydrophilic/hydrophobic character, water
insolubility, charge distribution, pH environment, conductivity, and catalytic ability
can be tailored to satisfy the practical applications by using different kinds of tem-
plates or by changing the composition of the material.
In this chapter, we intend to review some of the major advances and milestones
in nanoporous material-based electro-biosensors, classify their functions, and sum-
marize the influence of their components, structure, pore size, and particle size on
the final biosensor performance. In addition, the applications of mesoporous mate-
rial in particular are focused on in a separate section. Recent advances in other
nanoporous metals or metal hybridization are also mentioned.
6.3.1.1Pore Size
Balkus found that the protein loading amount into mesoporous silica MCM-41 in a
limited contact time decreased with increasing protein molecular weight [50].
Wright and coworkers [46] studied the adsorption and leaching of trypsin on MCM-
41, MCM-48, and SBA-15 and found that about 72% of the trypsin was leached
from MCM-48 due to the smaller pore size dimension (2.4nm) than that of trypsin
(3.8 nm). However, due to the similar dimension of trypsin and MCM-41 pores
(3.5nm), only 46% of the trypsin was leached from MCM-41, which was much
lower than those of MCM-48. All these results proved that the pore size should be
in good match with the biomolecules size. By varying the template with different
lengths or by introducing some swelling agent with strong hydrophobicity, the pore
size of the mesoporous material can be tailored [41, 51]. In general, the pore size of
MCM series mesoporous materials is relatively small due to the use of short-chain
templates. Normally, they are used to immobilize small proteins with dimensions
less than 4nm. While the pore size dimension of the mesoporous materials synthe-
sized by polymers involving SBA-15, SBA-16, and FDU-5 is in the range of
525nm, they could be applied for the entrapment of relatively large proteins. Once
the protein dimension is larger than 10nm, the mesocellular foam with a window
size greater than 10nm is preferred. For convenience, the normally used protein
dimensions are summarized in Table6.1 [52].
Except for the adsorbed amount and stability, there are close relationships
between the pore size and the enzyme activity [53]. The majority of enzymes
undergo inactivation upon isolation from their native biological environment. Recent
theoretical models suggest that the stabilization of proteins against unfolding can be
achieved by physical confinement inside relatively small cages [54], therefore main-
taining the activity of enzymes. The calculation has recently been proven by an
experimental study in which a stabilization effect was indeed observed based on the
6.3 Biosensors Based on Mesoporous Materials 175
6.3.1.2Surface Characteristics
with transition metal elements [68], such as Ti, V, Pt, Pd, and so on, thus making the
enzyme stably adsorbed on the mesowalls containing transition elements. For
example, Jus group [14] prepared a conductive nanocage comprised of palladium
nanoparticles (PdNPs) homogeneously distributed on the mesowall and used it for
the entrapment and biosensing of glucose oxidase (GOD). They found the introduc-
tion of PdNPs on the mesowalls could provide sufficient catalytic sites for lowering
the detection overpotential of glucose. In addition, the interaction between palla-
dium and COOH or NH2 led to stable immobilization of enzymes even on meso-
cellular foam with relatively large mesopore size.
Besides that, organic groups can be introduced into the mesoporous surfaces via
postgrafting, therefore changing the surface characteristics. Lei etal. [62] reported
that a suitable organically functionalized mesoporous host could provide a higher
affinity for protein molecules and a more favored microenvironment, resulting in
exceptional immobilizing efficiency. Wright and coworkers [48] investigated the
adsorption and desorption behavior of protein on SBA-15 functionalized by thiol,
chloride, amine, and carboxyl groups and found that the interactions of the enzyme-
support depended strongly on the nature of the functional groups attached to the
surface.
6.3.1.3Morphology
The morphology of the mesoporous material affects the final protein adsorption
amount. Usually, mesoporous material with a relatively small particle size can pro-
vide more entrances to entrap enzymes and will result in an exceptionally high
immobilization capacity and very rapid adsorption rate. Zhao and coworkers stud-
ied the adsorption of lysozyme in mesoporous silica with controlled morphologies
(including conventional SBA-15, 20 mm in length; rod like SBA-15, 12 mm in
length; mesoporous monolith; and the macroporous-mesoporous membranes) [69].
They found that the lysozyme entrance amount increased with the decreasing size
of mesoporous nanoparticles. The rod-like SBA-15 showed a faster adsorption rate
and larger immobilization amount than those of conventional SBA-15. Although no
systemic study on the application of nanoscale mesoporous materials in protein
adsorption has been reported [70], from the advantages the rod-like SBA-15 showed,
it could be concluded that the nanoparticle with a shorter nanoscale mesochannel
will be an advantage for a high protein loading amount and fast substrate diffusion.
6.3.1.4Mesostructure
Fig. 6.1 Structure models of 2D hexagonal p6mm (MCM-41), 3D cubic Ia3d (MCM-48), and
layer mesophase (MCM-50)
silica (HMS), and SBA-15, are mostly used. Their channels are straight and parallel
with each other, which are unfavorable for protein adsorption and substrate diffu-
sion. Since their channel length is always in the micrometer range, when proteins
are loaded onto the mesopores, they block the channel and make further protein
adsorption difficult, resulting in the lowly effective occupation of the mesopores. In
comparison, three-dimensional mesostructures can facilitate protein immobilization
and substrate diffusion due to their interconnected mesopores [4] and therefore are
more suitable for protein immobilization and biosensing applications.
Four methods are usually used for the preparation of mesoporous-based electro-
chemical biosensors. In the first method, mesoporous molecular sieves are added to
protein solutions, followed by stirring or shaking in a shaking bath and centrifuging
the mixture. Then the sediment is washed with deionized water and vacuum-dried.
Thus, the protein- or enzyme-adsorbed mesoporous molecular sieves are obtained,
which are cast on an electrode surface via an adhesive polymer.
The second method mixes mesoporous molecular sieves in carbon paste (CP) to
prepare mesoporous molecular sievemodified CP electrodes. Mesoporous molecu-
lar sieve suspension is thoroughly mixed with graphite carbon powder. Then paraf-
fin oil is added to the mixture, followed by evaporation of water for 3h in air. The
immobilization of protein on the surface of the mesoporous molecular sieve
modified CP electrode can be achieved by dropping the protein solution onto the
pretreated mesoporous molecular sievemodified CP electrode or dipping this mod-
ified CP electrode into the protein solution [71].
The third method is to prepare mesoporous molecular sieves colloid in polyvi-
nyl alcohol (PVA) for immobilization of protein. First, mesoporous molecular
sieves are dispersed into water to obtain a suspension. The suspension is then
mixed with a PVA solution of ethanol and water to produce mesoporous molecular
sieve solution. Then mesoporous molecular sieve solution and the protein solution
are dropped and mixed on a pretreated glassy carbon electrode surface and allowed to
178 6 Biosensors Based on Nanoporous Materials
Fig. 6.2 (a) The immobilization scheme based on magnetic materials on an Au electrode.
Reprinted with permission from Lee etal. [45]. 2005, Wiley. (b) The immobilization scheme
based on magnetic materials on a magnetic electrode. (a) Water drop with CNT/Fe3O4 composite
dispersed inside; (b) graphite-epoxy composite electrode; (c) water drop; and (d) CNT/Fe3O4 com-
posite loaded on the electrode. Reprinted with permission from Qu etal. [74]. 2007, Elsevier
dry under ambient conditions. Finally, the modified electrode should be rinsed with
doubly distilled water two or three times to get rid of the nonfirmly adsorbed pro-
teins [19, 72, 73].
The fourth method is based on the special mesoporous material with favorable
magnetism, as shown in Fig. 6.2. Usually, a homemade magnetic electrode first
needs to be prepared with the help of a magnet, and then the magnetic mesoporous
material is loaded onto the electrodes surface by the magnetic attraction. Compared
with the methods mentioned above, the immobilization method with the help of an
external field has the advantages of easy regeneration and reposition and can be well
controlled, which holds promise in the near future for the fabrication of magneti-
cally controlled biosystems.
Due to the good hydrophilicity and biocompatibility, mesoporous silica has exten-
sively been used in protein immobilization and biosensing. The use of mesoporous
silica as an enzyme-immobilized matrix was pioneered by Jus group in 2004 [19].
6.3 Biosensors Based on Mesoporous Materials 179
Fig.6.4 CV curves of Hb/HMS-modified GCE in 0.1M pH 7.0 PBS at 20, 30, 50, 80, 100, 120,
150, 180, and 200mVs1. Inset: plot of peak current vs. scan rate. Reprinted with permission from
Dai etal. [19]. 2004, Elsevier
e lectrodes. These proteins were encapsulated into the mesopores through physical
adsorption and hydrophobic/hydrophilic or electrostatic interaction without the con-
ventional cross-linking interaction, which are favorable for enzymes to maintain
their bioactivities.
Additionally, Lis group [83] also reported the ET of Hb in bimodal mesoporous
silica (BMS) and chitosan inorganicorganic hybrid film. The BMS possesses a
three-dimensional disordered pore structure with bimodal pore sizes, i.e., smaller
pores in the 23-nm range and larger pores between 10 and 40nm. They demon-
strated that the larger pore size compared with that of conventional protein-facilitated
BMS to effectively immobilize a large amount of proteins.
Significantly, the direct ET between immobilized redox protein and the electrode
surface without the need for any electron-transfer mediator or promoter offers an
opportunity to build mediator-free sensors for hydrogen peroxide (H2O2), nitrite, and
other biocomponents, which is promising for the construction of the third-generation
electrochemical biosensors. As shown in Fig.6.5, typical cyclic voltammetric (CV)
curves for the electrocatalysis response of Mb/HMS/GCE to H2O2 are observed [73].
Upon the addition of H2O2 to the solution, the shape of the CV for Mb changes
dramatically, with an increase in reduction current and a decrease in oxidation
current, while no electrocatalytic current is observable at a bare GCE or the HMS/
GCE (inset), displaying an obvious electrocatalytic behavior of the Mb intercalated
in the mesopores of HMS to the reduction of H2O2. Under optimal conditions
6.3 Biosensors Based on Mesoporous Materials 181
Fig.6.5 (a) CVs of Mb/HMS/GCE in 0.1M pH 7.0 PBS containing 0, 0.01, 0.02, 0.03, 0.04, 0.05,
0.07, and 0.09mM H2O2 at 20mVs1. Inset: cyclic voltammograms of HMS/GCE in 0.1M 7.0
PBS containing 0, 0.02, 0.04, 0.07, and 0.09mM H2O2 at 20mVs1, and (b) in 0.1M pH 7.0 PBS
containing 0 and 0.1mM NaNO2 at 20mVs1. Reprinted with permission from Dai etal. [73].
2004, Elsevier
(400mV, pH 7.0 PBS), the linear response range of the sensor to H2O2 concentration
is from 4.0 to 124mM, with a correlation coefficient of 0.9999. The enzyme elec-
trodes achieve 95% of the steady-state current in less than 10s, indicating HMS is an
effective material for the construction of sensors. The detection limit is 6.2108M
at a signal-to-noise ratio of 3. When the H2O2 concentration is higher than 124mM,
a response platform is observed, showing a characteristic of the MichaelisMenten
kinetics mechanism. The apparent MichaelisMenten constant for the electrocata-
lytic activity of Mb/HMS/GCE to H2O2 is determined to be 0.0650.005mM. After
a month, the Hb/HMS/GCE can retain 96% of its initial current response to H2O2.
The fabrication reproducibility of six Mb/HMS/GCE electrodes shows a RSD of
4.1% for the current determined at 4.0mM H2O2. Similar results are obtained in the
Hb/HMS/GCE and HRP/HMS/GCE electrodes, indicating proteins entrapped in the
HMS matrix are suitable for constructing H2O2 sensors [73].
Xings group found that the mesoporous silica could also facilitate the ET of
GOD [84]. They immobilized GOD on the SBA-15 and Nafion matrices; the immo-
bilized GOD had undergone a direct and nearly reversible electrochemical reaction,
containing a two-electron and two-proton exchange, with good stability. GOD
immobilized on the SBA-15 and Nafion matrices had a wide linear response to glu-
cose in the positive potential range.
The large surface areas of mesoporous molecular sieves make the simultaneous
immobilization of multiple kinds of proteins possible. Jus group prepared two
highly sensitive electrochemical sensors based on the coimmobilization of two
enzymes for the detection of phenol and glucose, respectively. Compared with
182 6 Biosensors Based on Nanoporous Materials
the detection based on a single enzyme, the two-enzyme synergy always exhibits
some exciting performance such as an extremely extended linear range and high
sensitivity. For example, a phenol electrochemical biosensor [85] without the addi-
tion of mediator was fabricated by the coimmobilization of tyrosinase and HRP in
the mesoporous silica. In air-saturated PBS solution, the electrochemical response
of phenol on the enzyme electrode was based on the following equations:
The immobilization of HRP results in an increase in the sensitivity, since the catechol
formed from the electrochemical reduction of o-quinone could be oxidized by
theH2O2 produced from the reduction of dissolved O2 [86], which was catalyzed
bythe coimmobilized HRP. And the regeneration of o-quinone would result in an
increase in reduction current. As shown in Fig. 6.6, the response of tyrosinase/
MCM-41/GCE to 0.2mM phenol was 5.4 times larger than that of tyrosinase/GCE
(Fig. 6.6a, b), while being about two times smaller than that of tyrosinase-HRP/
MCM-41/GCE (Fig.6.6b, c), indicating that the MCM-41 played an important role
in enhancing the enzymatic activity of tyrosinase and the two-enzyme system
possessed better electrocatalytic efficiency.
Recently, another electrochemical biosensing system based on the coimmobiliza-
tion of GOD and HRP was reported by Jus group for the detection of glucose [87].
The HRP immobilized in the mesopores of SBA-15 showed the direct ET. In pres-
ence of glucose, the enzymatic reaction of the GOD-glucose-dissolved oxygen system
could generate hydrogen peroxide, which was immediately reduced at 0.40V by an
electrocatalytic reaction with the HRP entrapped in the same mesopore, leading to a
highly sensitive and selective glucose biosensor without the addition of any mediator.
The mechanism could be shown by (6.1)(6.5):
Fig. 6.6 Cyclic voltammograms of (a) tyrosinase/GCE, (b) tyrosinase/MCM-41/GCE, and (c)
tyrosinase-HRP/MCM-41/GCE in the absence (solid line) and presence (dotted line) of 0.2mM
phenol in air-saturated 0.1M pH 7.0 PBS at 100mVs1. Scan from negative to positive direction.
Reprinted with permission from Dai etal. [85]. 2005, Wiley
The detection limit of the biosensor was down to 2.7107M. Compared with the
glucose biosensor based on a single GOD, the bienzyme system showed an extremely
wide linear range from 3.0106 to 3.4102M, proving the advantage of SBA-15
as enzyme a host material for the fabrication of electrochemical biosensors.
Antigenantibody and DNA could also be incorporated into the mesoporous matrix
with a suitable pore size and applied to biosensing. He and coworkers [10] prepared
a mesoporous material-modified CP electrode for the determination of cardiac tro-
ponin I (cTnI) by anodic stripping voltammetry. The cTnI in the serum of patients
has been considered as the gold standard for the diagnosis of myocardial injury
[88]. Detection of cTnI in the serum is helpful for the diagnosis of acute myocardial
infarction (AMI), the identification of myocardial injury, and the stratification of
184 6 Biosensors Based on Nanoporous Materials
Fig.6.7 Protocol format of cTnI analytical procedure. Reprinted with permission from Guo etal.
[10]. 2005, Elsevier
cardiac risk. The influence of different kinds of mesoporous materials with different
pore sizes was studied on the response of the immunosensor, and SBA-15 was found
to be more suitable for the immobilization of IgG1 than MCM-41 and Y-type zeolite
due to the larger mesopores as well as the larger dimension of cTnI (10145nm)
[10]. The pore size was the crucial factor for the current response, since the larger
the pore size, the easier the immobilization of the IgG1 and the higher IgG1 immo-
bilization efficiency. Figure6.7 shows the protocol of the cTnI analytical procedure;
the response of the cTnI is based on the anodic stripping peak of the adsorbed silver
on the cAu-IgG2. The nonspecific adsorption of the multipore materials is controlled
by selecting suitable concentration of cAu-IgG2 and adding gelatin as blocking
reagent. Under optimal conditions, a linear relationship between the anodic strip-
ping peak current of silver and the concentration of cTnI from 0.5 to 5.0ng/mL is
acquired. In addition, the established method is tested by determining cTnI in AMI
samples using enzyme-linked immunoadsorbent assay for comparison analysis,
with good results.
Recently, based on the decrease in conductivity resulting from pore blocking,
mesoporous silica film-modified electrodes have been applied to labeling-free
biomolecular recognition [89]. The working principle is as follows: First, the
electrodes modified with amine functional groups of hybrid mesoporous silica film
(0.1mm thick) are attached with biotin groups or oligonucleotides, and then they
react with streptavidin or complementary DNA molecules, respectively, followed
by measurement in an electrolyte solution containing indicator molecules. Before
specific binding, the indicator molecules can freely access the conductive surface
and produce a typical redox response. After specific binding, the indicator molecules
can no longer freely access the conductive surface, resulting in a reduction in,
6.3 Biosensors Based on Mesoporous Materials 185
ordisappearance of, the redox current. From the decrease or disappearance of the
indicator current response before and after the specific binding, the target biomole-
cules from nanomole to femtomole levels can be recognized. In addition, the
response due to the complementary DNA is much lower than that of the noncomple-
mentary DNA, indicating a good selectivity.
peak is present at either the bare or chitosan-modified electrode from 0.3 to 0.9V.
With the introduction of Hb and the CMK-3 layer onto the electrode surface
(Fig.6.9), a pair of well-defined redox peaks is observed at about 377 and 296mV
in pH 7.0 PBS under the optimized modification conditions, which is attributed to
the ET of Hb. In addition, the {Hb/CMK-3}n film electrode shows strong catalytic
reduction toward both H2O2 and O2. The better CV performance and the good cata-
lytic behavior of the electrode suggest that CMK-3 has an obvious effect to improve
the reversibility of the electrode reaction for Hb.
Liu etal. [91, 92] confirmed the advantage of OMC in promoting the ET of pro-
teins. Her group dispersed bicontinuous gyroidal mesoporous carbon (BGMC) or
mesoporous carbon nanocomposite (CMM) in the Nafion solution and used them for
the immobilization of Mb and GOD, respectively. The cyclic voltammograms showed
those OMCs could facilitate the ET of Mb and GOD. The Mb surface coverage on
Mb/BGMC(2.8nm)/GCE was calculated to be 2.01010molcm2. The value was 19
times larger than the theoretical value of a full-packed monolayer (1.081011molcm2).
From the value, they assumed that the long axis of the Mb molecule was parallel to the
electrode surface. In addition, they also observed the influence of pore size on the
electron-transfer behavior of Mb, as shown in Table6.2. They found that the surface
6.3 Biosensors Based on Mesoporous Materials 187
Table6.2 (A) The characteristic results of BGMCs and (B) the loading amounts and electron-transfer
properties of Mb immobilized on BGMCs (at 10mVs1)
Pore size (nm) 2.8 3.4 4.6 7.1
(A) BET surface area (m2g1) 1,472 1,410 1,241 1,116
Mesopore volume (cm3g1) 1.22 2.1 1.59 1.87
Electroactive surface area (cm2)a 2.720.04 3.350.06 1.690.12 1.440.08
(B) GMb (1010molcm2) 2.00.4 1.80.3 1.70.3 1.80.4
0
EMb (mV vs. SCE) 3291 3371 3383 3425
DEpMb (mV) 80 160 331 481
ket0 Mb (s1) 10.40.3 8.70.2 7.00.2 4.70.1
Reprinted with permission from You etal. [91]. 2009, Elsevier
a
Electroactive surface area and apparent geometric surface area of bare GCE are 1.10.01,
0.07cm2, respectively
coverage of Mb immobilized onto BGMC matrices with various pore sizes was
similar, indicating that the immobilization capacity for proteins was mainly influ-
enced by the close surface areas of BGMCs, and not very related to the pore sizes. The
immobilized Mb exhibited the smallest peak potential separation (DEp) and the largest
apparent heterogeneous electron-transfer rate constant ket0 in the BGMC matrix with
a pore size of 2.8nm, suggesting that such pore size made a good match to the size
of the Mb molecule (2.13.54.4 nm); thus, the shortest distance between the
redox center of Mb and the electrode surface could be achieved, giving the fastest
ET. The hypothesis was confirmed by the fact that the larger GOD molecule achieved
the smallest DEp and the largest ket0 in the BGMC matrix of a corresponding pore
size of 4.6nm.
Dongs group [93] recently presented an advanced electrochemical biosensing
platform for the detection of ethanol and glucose based on OMCs with a 2D
hexagonal p6mm mesostructure. They found that except for the advantage of con-
ductivity, OMCs also showed excited electrochemical responses to NADH and H2O2
at low oxidation overpotential, which exhibited more favorable electron-transfer
kinetics compared with carbon nanotube (CNT). In detail, Fig.6.10ac show the
cyclic voltammograms for NADH oxidation at glassy carbon electrode, CNT/GCE,
and OMC/GCE, respectively. The oxidation of NADH occurs at +0.65, +0.45, and
+0.2V, respectively. In addition, the peak current for NADH at the OMC/GCE is
2.35 times larger than that of the CNT/GCE, while the peak current at CNT/GCE
increases 2.44-fold compared with the bare electrode. Figure 6.10df show the
cyclic voltammograms of (d) GCE, (e) CNT/GCE, and (f) OMC/GCE in the pres-
ence of H2O2. With the addition of H2O2 to the solution, the anodic current begins
increasing from +0.30, +0.50, and +0.70V at OMC/GCE, CNT/GCE, and GCE,
respectively. These results confirmed that OMC/GCE had higher electrocatalytic
activity for NADH and H2O2 oxidation compared with CNT/GCE and GCE.
Obviously, the presence of OMCs can accelerate the ET.
Several factors may contribute to the good electrochemical performance of
OMCs. The first factor is the nanostructured channel of OMCs. The large surface
188 6 Biosensors Based on Nanoporous Materials
Fig.6.10 CVs for 2mmolL1 NADH at (a) GE, (b) CNTs/GE, and (c) OMCs/GE. CVs for
4.2mmolL1 H2O2 at (d) GE, (e) CNTs/GE, and (f) OMCs/GE. Dotted lines represent the background
response. Scan rate: 50mVs1. Reprinted with permission from Zhou etal. [93]. 2008, Elsevier
area may increase the apparent electroactive surface area of OMCs/GCE, which
offers a favorable microenvironment for transferring species in solution through the
pores of OMCs, and also is beneficial for accelerating ET between the electrode and
species in solution. The second factor is the uniform-pore-size ordered arrangement
of carbon nanorods and pieces of OMCs homogeneously covered on GCE, which
are very attractive for various electrochemical applications, especially for the devel-
opment of electrochemical sensors and biosensors. Additionally, the good electro-
catalytic ability is also important, which can be increased by increasing the number
of edge-plane-like defective sites, proved by FT-IR, XPS, and Raman spectra. As
shown in Fig.6.11, FT-IR spectra (a) indicated that the bands around 3,435, 1,688,
1,550, 1,515, and 1,106cm1 were attributed to the oxygen-containing functional
groups (OFGs) on CNT and OMC. XPS spectra (b) showed the O/C ratio from XPS
for CNTs and OMCs was 0.098 and 0.032, respectively, which means CNTs contain
more OFGs than OMCs. The Raman spectra of CNTs (e) and OMC exhibited the
presence of two scattering peaks centered at 1,348 and 1,595 cm1, which were
attributed to the sp3-hybridized (D band) and E2g zone center mode of crystalline
graphite (G band) [96, 97], respectively. The relative intensity ratio of the D and G
lines (ID/IG ratio) was proportional to the number of defective sites in carbon materi-
als, which was responsible for the electrochemical activity of the materials. The
ID/IG ratio of OMCs (f) and CNTs (e) was 1.82 and 0.58, respectively. This means
the OMCs contained more edge-plane-like defective sites than CNTs without puri-
fication or end-opening processing, and hence had better catalytic activity. Based on
6.3 Biosensors Based on Mesoporous Materials 189
the greatly enhanced electrochemical reactivity of NADH and H2O2, two biosensors
were fabricated by the entrapment of GOD and alcohol dehydrogenase (ADH),
respectively, in the mesopores of OMCs for the detection of glucose and alcohol,
respectively. Both of them showed a good analytical performance, indicating OMCs
are good matrices for protein immobilization and biosensing.
Fig.6.12 XRD patterns of GMC-13, GMC-6, and CNTs. Reprinted with permission from Lu etal.
[94]. 2009, Royal Society of Chemistry
Fig.6.13 CVs of
(a) HbNafionGMC6/GC,
(b) HbNafionGMC13/
GC, (c) HbNafionCNT/
GC, and (d) HbNafion/GC
in pH 7.0 PBS with scan rate
of 0.2Vs1. Reprinted with
permission from Lu etal.
[94]. 2009, Royal Society
of Chemistry
the larger effective conductive surface areas of the GMC. GMC-6 offers significant
advantages over GMC-13 and CNTs in facilitating the ET of entrapped Hb and
improving the performance of the fabricated biosensors, indicating that suitable
pore size is important for Hb immobilization. The biosensor based on GMC-6 dis-
plays excellent analytical performance over a wide linear range (1180mM, n=19,
R=0.9894) along with good stability and selectivity for the detection of H2O2, and
suggesting graphitized-ordered mesoporous carbons with good pore size matching
for enzymes will be a promising electrochemical biosensing platform.
Besides the large surface area and narrow pore size distribution, mesoporous metal
oxides may have some specific characteristics. For example, it has been reported that
TiO2 can coordinate with COOH and NH2 groups, thus facilitating the immobiliza-
tion of proteins without leaching from the pores [76]. MnO2 can catalyze the electro-
chemical oxidation of H2O2 at pH values close to neutral [101, 102]. Once those
metal oxides own the mesostructure, those specific characteristics can be amplified
due to the large surface area, therefore indicating that mesoporous metal oxides are
promising enzyme-immobilized hosts for electrochemical biosensing applications.
In 2008, mesoporous MnO2 [103] was synthesized by Zengs group through a sol
gel process using nonionic surfactant polyxyethylene fatty alcohol as template.
Although the obtained MnO2 material presented disordered porous structure, the
pore size was appropriate and suitable for the immobilization of GOD. An ampero-
metric glucose biosensor based on GOD entrapped in MnO2 was fabricated, in which
MnO2 also acted as catalyst for the electrochemical oxidation of H2O2 produced by
enzymatic reactions. The biosensor showed a fast and sensitive current response to
glucose in the linear range of 0.00092.73mM with a sensitivity of 24.2mAcm2mM1.
192 6 Biosensors Based on Nanoporous Materials
Fig.6.14 (a) TEM, (b) EDX spectrum, (c) nitrogen adsorption/desorption isotherms, and (d) pore
size distributions of the MSCF. Reprinted with permission from Wu etal. [4]. 2007, Wiley
fact that the reduction of FAD groups or the oxidation of FADH2 groups inside the
peptides of GOD is easier or more difficult than free FAD or FADH2 due to the pres-
ence of positively charged amino acid residues of these peptides at pH 7.0, the redox
couple of the MSCF/GOD/Nafion-modified electrode is not free FAD. The ket0 is
calculated to be 16.4s1, and it is the largest value reported, confirming the good
microenvironment and conductivity MSCF provided for GOD loading and biosens-
ing. Based on a decrease in the electrocatalytic response of the reduced form of
GOD to dissolved oxygen, the proposed biosensor shows a linear response to glu-
cose concentration ranging from 50mM to 5.0mM, with a detection limit of 34mM
at an applied potential of 0.4V. It has good stability and selectivity, and can exclude
the interferences from ascorbic acid and uric acid that always coexist with glucose
in real samples. All these results confirm that the nanocomposite foam is a good
matrix for protein immobilization and biosensor preparation.
By introducing transition metal elements into the mesostructure, the catalytic abil-
ity of mesoporous hybrid material can be improved. For example, Jus group designed
a conductive nanocage composed of PdNPsdoped MSCF [14] by a designed wetness
chemical process followed by electrochemical reduction. The TEM image (Fig.6.16a)
demonstrated the formation of PdNPs (black dots) with sizes of 13nm on the meso-
walls. The corresponding EDX (Fig. 6.16b) showed the typical peak of palladium
with the content of 6.1% at 2.85keV, which confirmed the existence of palladium. The
introduction of PdNPs significantly improved the electrochemical activity of the
nanocage. At the Pd-MSCF-modified electrode, the anodic oxidation of H2O2 occurred
at +0.25V, while it occurred at potentials more positive than +0.51V at the MSCF-
modified electrode (Fig.6.17a). Obviously, the presence of PdNPs made the oxidation
overpotential decrease for 260mV. In addition, the amperometric responses of the
Pd-MSCF-modified electrodes to 1.0mM H2O2 in pH 7.0 PBS at +0.3V was 24.3mA,
more than 120 times larger than 0.201mA obtained at the MSCF-modified electrode
(Fig.6.17b). After GOD entrapment and under the optimized conditions, the glucose
6.3 Biosensors Based on Mesoporous Materials 195
Fig. 6.16 Morphology and composition of Pd-MSCF. (a) TEM of Pd-MSCF; (b) EDXS of
Pd-MSCF. Reprinted with permission from Wu etal. [14]. 2008, American Chemical Society\
Fig.6.17 (a) CVs of Pd-MSCF- and MSCF- (inset) modified electrodes in 0.2M pH 7.0 PBS in
the absence (solid) and presence (dash) of 3.0 mM H2O2. (b) Amperometric responses of the
MSCF- (dash) and Pd-MSCF- (solid) modified electrodes to 1.0mM H2O2 in 0.2M PBS at +0.3V.
Reprinted with permission from Wu etal. [14]. 2008, American Chemical Society
biosensor showed a surprisingly fast response, which achieved 95% of the steady-state
current in less than 3s, a linear range from 5.0mM to 4.0mM, a limit of detection of
1.0 mM, and a high sensitivity of 2.12 mA mM1. All those results confirmed the
advantages of the hybrid nanocomposites.
Another example of hybrid nanoporous materials with improved catalytic ability
was reported by Dai etal. [108], who prepared Ti-containing MCM-41 and found
the material could significantly catalyze the electrochemical oxidation of NADH
with an overpotential decrease of about 400mV. The author ascribed the decrease
in the potential of NADH oxidation to the higher hydrophilicity induced by
Ti-MCM-41 at the modified electrode, which offered the electrode surface a better
contact with the solution containing NADH and consequently could participate in
increasing the electron-transfer rate.
196 6 Biosensors Based on Nanoporous Materials
NPG is a good conductor and has a suitable pore size distribution and large surface.
It can greatly enhance the electrochemical response toward the enzymatic substrates
NADH and H2O2 based on their low-coordinated Au atoms. All these advantages
make it attractive for the construction of dehydrogenase- and oxidase-based biosen-
sors [111, 112], which may have improved sensitivity and anti-interference ability.
The great application of NPG was reported by Huang etal. [5], who developed two
NPG-based electrochemical biosensors. The NPG was prepared simply by dealloy-
ing Ag from Au/Ag alloy. The selective dissolution of silver from Ag/Au alloy
resulted in an open bicontinuous nanoporous microstructure comprised almost
entirely of gold, as shown in the inset of Fig.6.18. The dealloying mechanism was in
accord with an atomistic model proposed by Erlebacher etal. [109]. The nanoporous
structure made NPG more active than gold sheet based on the fact that both the oxida-
tion and reduction peaks of the NPG/GCE were negatively shifted as compared with
the gold sheet electrode. The NPG-modified glassy carbon electrode (NPG/GCE)
exhibited high electrocatalytic activity toward the oxidation of NADH and H2O2. The
anodic peaks of NADH and H2O2 occurred at 0.52 and 0.4 V at the NPG/GCE-
modified electrode, which were more negative than the value of 0.72 and 0.8V at the
gold sheet-modified electrodes. In addition, the peak currents of NADH and H2O2 at
the NPG/GCE were much larger than those at the gold sheet electrode (Fig.6.19).
The high density of edge-plane-like defective sites and large specific surface area of
NPG should be responsible for the electrocatalytic behavior. Such electrocatalytic
6.4 Biosensors Based on Nanoporous Gold 197
Fig.6.18 CVs of (a) NPG/GCE and (b) gold sheet electrode in 0.1M PBS. Scan rate: 50mVs1.
The inset is the SEM image of the NPG with a pore size of 40nm. Reprinted with permission from
Qiu etal. [5]. 2009, Elsevier
Fig.6.19 CVs of (a) NADH (1mM) and (b) H2O2 (4mM) at (a) NPG/GCE and (b) gold sheet
electrode in 0.1M PBS (pH 7.2). Scan rate: 50mVs1. Reprinted with permission from Qiu etal.
[5]. 2009, Elsevier
In recent years, the development of highly sensitive and selective DNA sensors to
bring down the limit of detection to pico- and femtomolar levels has been a field of
ever-increasing interest since the genoassays are suitable for various applications,
including clinical diagnosis, environmental control, and forensic analysis [113
115]. For the DNA biosensor preparation, the immobilization of biomolecular
probes on a desired substrate is a very important process since the sensitivity, detec-
tion resolution, and reproducibility are all significantly affected by these steps. Gold
substrates have attracted special attention as an electrode material for the construc-
tion of DNA electrochemical biosensors due to their strong interaction with thio-
lated DNA via Authiol binding. Thiolated DNA can be monolayered on gold in a
self-assembly manner, which provides stable and structurally well-defined electro-
chemical interfaces [116]. Recently, Zhangs group [115] developed a sensitive
electrochemical DNA sensor based on an NPG electrode prepared by dealloying Ag
from Au/Ag alloy and multifunctional encoded Au nanoparticles (Au NPs)
(Fig.6.20). The active surface area of the NPG electrode was 9.2 times higher than
that of a bare flat electrode as characterized by CVs. A DNA biosensor was fabricated
6.4 Biosensors Based on Nanoporous Gold 199
Fig.6.21 SEM images of (a) Au nanoporous film and (b) PGNF. Reprinted with permission from
Yang etal. [9]. 2009, Elsevier
by immobilizing capture probe DNA on the NPG electrode and hybridization with
target DNA, which further hybridized with the reporter DNA loaded on the Au NPs.
The Au NP contained two kinds of bio-barcode DNA; one was complementary to
the target DNA, while the other was reducing the cross-reaction between the targets
and reporter DNA on the same Au NP. Electrochemical signals of [Ru(NH3)6]3+
bound to the reporter DNA via electrostatic interactions were measured by
chronocoulometry.
Taking advantage of the dual-amplification effects of the NPG electrode and
multifunctional encoded Au NP, this DNA biosensor could detect the DNA target
quantitatively, in the range of 8.010171.61012M, with a limit of detection as
low as 28aM, and exhibited excellent selectivity even for single-mismatched DNA
detection.
which was the electroactive substance produced when b-galactosidase catalyzed the
hydrolysis of p-aminophenyl-b-d-galactopyranoside (PAPG) in the culture medium
of E. coli, and the quantity of PAP was proportional to the concentration of E. coli.
The linear range for PAP was from 10 nM to 40 mM, with a detection limit of
1108M. Based on a sensitive detection of PAP, this PGNF electrode could suc-
ceed in detecting E. coli rapidly and sensitively. The linear range for E. coli was
from 2101 to 1106CFUmL1, with a detection limit of 10CFUmL1 (S/N=3).
The analytical performances as well as the repeatability of its E. coli response dem-
onstrated that this PGNF electrode would be suitable for the fabrication of portable
amperometric E. coli sensor.
6.5Conclusions
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wwwwwwwwwwwww
Chapter 7
Electrochemical Biosensing Based
on Carbon Nanotubes
7.1Introduction
7.1.1Structure of CNTs
Since their discovery, carbon nanotubes (CNTs) have been extensively investigated
as essential platforms in constructing electrochemical biosensors. CNTs can be
classified into two basic varieties: single-wall carbon nanotubes (SWCNTs), which
are a single layer of graphene sheet rolled into cylindrical tubes, and multiwall car-
bon nanotubes (MWCNTs) comprised of multiple layers of concentric cylinders
with a spacing of about 0.34nm between the adjacent cyclinders (Fig.7.1). The
lengths of the nanotubes can range from several hundred nanometers to several
micrometers, and the diameters from 0.22nm for SWCNTs and from 2 to 100nm
for MWCNTs [1]. CNT synthesis techniques can be classified into three major
categories: laser ablation, catalytic arc discharge, and chemical vapor deposition [2].
Due to the diameters being similar to or smaller than those of individual biomole-
cules, CNTs are expected to serve as high-performance electrical conduits for
interfacing with biological systems. Therefore, there is enormous interest in utiliz-
ing CNTs for biosensor applications [3, 4].
The insolubility of the SWCNTs in most solvents and the difficulties of handling
these highly intractable carbon nanostructures, however, are restricting their real-
life applications. In order to improve the properties of CNTs, two approaches for the
functionalization of CNTs have been involved: noncovalent interaction, including
physical adsorption and entrapment of biomolecules around the nanotubes [5], and
covalent interaction to the functional groups produced via chemical reactions on the
p-conjugated skeleton of a CNTs sidewall [6]. The noncovalent functionalization
of the SWCNTs can preserve the desired electronic properties of the CNTs while
remarkably improving their solubility. Coupled with immobilized biomolecules as
the recognition element, the functionalized CNTs as a sensing platform exhibit high
sensitivity and selectivity in electrochemical biosensing.
Fig.7.1 The structures of SWCNTs (left) and MWCNTs (right). Reprinted with permission from
Kim etal. [1], 2007, Wiley
Typically, there are three advantages for CNT-based electrochemical sensors: (1)
The high length-to-diameter ratios provide high surface-to-volume ratios for the
immobilization of functional molecules. In fact, the high surface area of SWCNTs
is estimated to be as high as 1,600m2/g; (2) CNTs have an outstanding ability to
mediate fast electron-transfer kinetics for a wide range of electroactive species,
leading to an enhanced electrochemical signal; (3) the small size of CNTs is suitable
to miniaturize the device for in situ detection.
Due to these unique properties, CNTs have attracted much attention on their
potential application in biosensing. In this chapter, two functionalization categories,
noncovalent interaction and covalent binding, are introduced to enhance the CNTs
hydrophobility for bioaffinity and realize efficient electrical communication, leading
to the amplification of signal transduction. Further, CNT-based biosensing method-
ologies are highlighted and discussed for the biosensing of DNA, antigenantibody,
cells, and other biological molecules. Finally, field-effect transistors and CNT
forests are also summarized for ultrasensitive detection. Electrochemical biosensing
based on CNTs provides a powerful way to miniaturize the device and achieve the
real-time detection of biomolecules.
Chemical functionalization offers an efficient route not only to modify the chemical
and physical properties of SWCNTs but also to further expand their potential
application areas as recognition elements [7]. The main approaches for the
functionalization of SWCNTs can be divided into two broad categories, namely,
covalent and noncovalent approaches.
7.2 Functionalization Strategy of CNTs 209
Fig.7.2 Schematic
representation of
N-succinimidyl-1-
pyrenebutanoate-decorated
SWCNTs. Reprinted with
permission from Zhao and
Stoddart [10], 2009,
American Chemical Society
7.2.1Noncovalent Interaction
Aromatic molecules, such as pyrene, porphyrin, and their derivatives, can interact
with the sidewalls of SWCNTs by means of pp stacking interactions, thus opening
up the way for the noncovalent functionalization of SWNTs. Dai and coworkers
reported a general and attractive approach to the noncovalent functionalization of
SWCNTs sidewalls and the subsequent immobilization of biological molecules onto
SWCNTs with a high degree of control and specificity [9]. The biofunctional mole-
cule N-succinimidyl-1-pyrenebutanoate was irreversibly adsorbed (Fig.7.2) onto the
hydrophobic surfaces of SWCNTs in either N,N-dimethylformamide (DMF) or
MeOH, which led to the further functionalization of SWCNTs with succinimidyl
ester groups that were reactive to nucleophilic substitution by primary and secondary
amines of some proteins, such as ferritin, streptavidin, and biotiny l-3,6-dioxaoctane-
diamine [10]. This technique has enabled the immobilization of a wide range of
biomolecules on the SWCNTs sidewalls, with high specificities and efficiencies,
and applications that may be useful for the development of biosensors.
Recently, Guldis group designed several new hybrid materials of CNTs and
electron donors via two steps. They first prepared SWCNT/pyrene+ hybrids via pp
stacking, and then immobilized negatively charged electron donors onto SWCNT/pyrene+
via electrostatic interaction to yield the electron donoracceptor nanohybrids [11].
210 7 Electrochemical Biosensing Based on Carbon Nanotubes
Fig.7.3 Photographs of vials containing 0.5mg/mL of SWCNTs in different solutions: (a) phos-
phate buffer (0.05M, pH 7.4); (b) 98% ethanol; (c) 10% ethanol in phosphate buffer; (d) 0.1%
Nafion in phosphate buffer; (e) 0.5% Nafion in phosphate buffer; and (f) 5% Nafion in ethanol.
Reprinted with permission from Wang etal. [14], 2003, American Chemical Society
7.2.1.2Entrapment
One simple and versatile method to assemble dispersed CNTs into thin films is
using layer-by-layer (LBL) assembly, which consists of the repeated, sequential
immersion of a substrate into aqueous solutions of complementarily functionalized
materials. This technique can produce conformal ultrathin films and highly tunable
surfaces using various nanomaterials on geometric surfaces. For example, positively
charged PtCNTCHIT solution and negatively charged poly(sodium-p-styrenesul-
fonate) salt (PSS) have been employed to fabricate stable ultrathin multilayer films
on gold electrode and quartz glass slides in an LBL fashion. With the immobiliza-
tion of cholesterol oxidase onto the electrode surface using GDI, a biosensor that
responds sensitively to cholesterol has been constructed due to the synergistic action
of Pt nanoparticles and CNTs. In pH 6.98 phosphate buffer, the almost interference-
free determination of cholesterol has been realized at 0.1V versus SCE with a linear
range from 0.01 to 3mM and response time <30s [40].
More recently, negatively and positively charged MWCNTs have been function-
alized on their exterior walls with carboxylic acid groups yielding MWCNTs-
COOH, and with amine groups yielding MWCNTs-NH2. LBL MWCNT films,
which consist of well-dispersed MWCNTs, are produced by LBL using stable dis-
persions of negatively and positively charged MWCNTs (Fig. 7.4). Unlike other
multilayer assemblies that contain CNTs as one of two or more components, these
systems incorporate MWCNTs without the incorporation of additional organic
materials. This difference enables the development of 100% CNT thin films with
properties that can be manipulated using assembly conditions. In particular, the
irregular shape of the CNTs enables the direct formation of porous, all-carbon
nanostructures with a high surface area. Sheet resistance and cyclic voltammetric
7.2 Functionalization Strategy of CNTs 213
Fig. 7.4 Layer-by-layer assembled MWCNT thin film with positively and negatively charged
MWCNTs. Reprinted with permission from Lee etal. [41], 2009, American Chemical Society
measurements show that these MWCNT thin films are promising electrode materials
for high-power and high-energy electrochemical devices [41].
A glucose biosensor was constructed based on an electrostatic LBL technique.
Gold electrodes were initially functionalized with negatively charged 11-mercap-
toundecanoic acid followed by alternate immersion in solutions of a positively
charged redox polymer, poly[(vinylpyridine)Os(bipyridyl)2Cl2+/3+], and a negatively
charged enzyme, GOD, or a GOD solution containing SWCNTs. The incorporation
of SWCNTs led to a dramatic (617-fold) increase in the current response, which
depended on the number of multilayers [42].
7.2.2Covalent Interaction
Fig.7.5 Synthetic route of the polyether-grafted and MIM-tethered MWCNTs. Reprinted with
permission from Xiang etal. [48], 2008, American Chemical Society
Fig.7.6 (a) SEM and (b) TEM images of aligned carbon nanotubes. Reprinted with permission
from Alonso-Lomillo etal. [52], 2007, American Chemical Society
When MWCNTS are grown on gold electrodes by microtechnology, the SEM image
can display their length of approximately 50mm (Fig.7.6a). The TEM study verifies
the presence of MWCNTs with average outer and inner diameters of 1525 and
512nm, respectively (Fig.7.6b). The vertically aligned CNTs with a high density
can be employed as a platform for the oriented and stable immobilization of an
Ni-Fe hydrogenase. A sigmoidal shape of the cyclic voltammogram is observed, in
which the current density above 550mV almost reaches a plateau, which is related
to the H2 concentration in the range of 1.3310.74mM [52].
assigned to the carbonyl stretch mode of -COOH and -COO. After covalent conju-
gation of the RGDS tetrapeptide to the SWCNT, the vibration of amide I and amide
II of the RGDS tetrapeptide can be observed at 1,645 and 1,533cm1 (Fig.7.7b),
whereas the peaks assigned to the carbonyl stretch mode decreased greatly, indicat-
ing the effective reaction of the carboxylic acid groups of SWNTs with the amino
groups of the RGDS. The conjugated RGDS shows a predominant ability to capture
cells on the electrode surface by the specific combination of RGD domains with
integrin receptors on the cell surface [53].
7.3.4Raman Spectrum
Fig.7.9 Raman spectra of (a) SWCNTsPLL assembly and (b) shortened SWCNTs. Reprinted
with permission from Zhang etal. [55], 2004, American Chemical Society
7.5.1Deoxyribonucleic Acid
Electrochemical DNA sensors are regarded as a particularly suitable tool for direct
and fast biosensing since they can convert the hybridization event into an electro-
chemical signal. DNA-sensing approaches include the intrinsic electroactivity of
DNA, electrochemistry of DNA-specific redox indicators, electrochemistry of
enzymes, and conducting polymers and ionic liquid (RTIL).
The direct electrochemical oxidation of guanine or adenine residues of ssDNA
leads to an indicator-free DNA biosensor. A composite screen-printed carbon elec-
trode modified with MWCNTs has been used for the detection of calf thymus
ssDNA ranging from 17.0 to 345mg/mL, with a detection limit of 2.0mg/mL at 3s,
and yeast tRNA ranging from 8.2mg/mL4.1mg/mL [63].
Many cationic metal complexes and intercalating organic compounds can be
used as redox indicators for the development of highly sensitive DNA biosensors.
7.5 Electrochemical Biosensing Based on Functional CNTs 221
Fig.7.12 (af) Square-wave voltammograms for 0 (control), 1, 5, 10, 20, and 40fg/mL, DNA
target. Also shown (insets) are the resulting calibration plot and the response for a 0.2-fg/mL target
solution, and the protocol. Reprinted with permission from Munge etal. [66], 2005, American
Chemical Society
For example, a novel approach was used to fabricate DNA biosensors, in which self-
assembled MWCNTs directly grew vertically to the Au substrates, followed by
DNA adsorption. Hybridization between the probe and target DNA oligonucleotides
was confirmed by the changes in the voltammetric peak of the indicator of methyl-
ene blue. The DNA biosensors based on self-assembled MWCNTs achieved a
higher hybridization efficiency than those based on random MWCNTs [64].
Signal amplification using enzyme multilayers on CNT templates has been
shown to yield a remarkably sensitive electrochemical detection of nucleic acids.
Hellers group demonstrated that a highly sensitive amperometric monitoring of
DNA hybridization (down to 5zmol) could be achieved in connection with an HRP-
labeled target [65]. Figure7.12 displays the potential of the ALP-LBL-CNT ampli-
fication route for the ultrasensitive electrochemical detection of DNA hybridization
in connection with sandwich assays, with a second probe conjugated to the CNT-
(PDDA/ALP)4PDDA/streptavidin label, along with enzymatic reaction of the
R-naphthyl phosphate substrate. It shows the voltammetric hybridization response
for extremely low DNA target concentrations, ranging from 1 to 40 fg/mL
(542,160aM). These aM concentration changes result in well-defined square-wave
voltammetric signals for the R-naphthol product. The favorable response for the
0.2-fg/mL target solution indicates a detection limit of approximately three
copies/mL (0.1fg/mL, 5.4aM) based on the signal-to-noise characteristics (S/N=3).
222 7 Electrochemical Biosensing Based on Carbon Nanotubes
Given the enormous amplification afforded by the new CNT-LBL biolabel, such a
route offers great promise for the ultrasensitive detection of infectious agents and
disease markers [66].
A sensitive electrochemical DNA biosensor was successfully realized on poly-
aniline nanofibers (PANI), MWCNTs, and CHIT-modified carbon paste electrode
(CPE) based on the synergistic effect between PANI and MWCNTs in CHIT film.
PANI and MWCNT nanocomposites resulted in highly enhanced electron conduc-
tive and biocompatible nanostructured film. The immobilization of the probe DNA
on the surface of the electrode was largely improved due to the unique synergistic
effect of PANI and MWCNTs. Under optimal conditions, the dynamic detection
range of this DNA electrochemical biosensor for the phosphinothricin acetyltrans-
ferase gene was from 1.010131.0107 mol/L, with a detection limit of
2.71014mol/L [67].
Hydrophobic room-temperature ionic liquid of BMIMPF 6, combined with
nanosized shuttle-shaped cerium oxide (CeO2) and SWCNTs, has been applied
in electrochemical sensing of the immobilization and hybridization of DNA.
The remarkable difference between the Ret value at the probe DNA-immobilized
electrode and that at the hybridized electrode can be used for the label-free
electrochemical impedance spectroscopy detection of the target DNA. Under
optimal conditions, the dynamic range for detecting the sequence-specific
DNA was from 1.010121.0107 mol/L, and the detection limit was
2.31013mol/L [68].
7.5.2AntigenAntibody
Fig.7.13 Schematic
illustration of an
electrochemical
immunosensor for detecting
mouse IgG. Reprinted with
permission from Aziz etal.
[57], 2007, Royal Society
of Chemistry
In addition, a sensitive method for the detection of cholera toxin (CT) using an
electrochemical immunosensor with liposomic magnification has been proposed
[70]. The sensing interface consisted of monoclonal antibody against the B subunit
of CT that was linked to poly(3,4-ethylenedioxythiophene) coated on Nafion-
supported MWCNT caste film on a GCE. The sandwich assay provided the ampli-
fication route for the detection of CT ranging from 1014107g/mL, with a detection
limit of 1015g/mL [70]. A disposable electrochemical immunosensor for carcino-
embryonic antigen using ferrocene liposome and MWCNT-modified screen-printed
carbon electrode was also developed recently [71].
The label-free immunosensor shows a convenient fabricating and detection pro-
cedure. For example, several label-free peptide-coated CNT-based immunosensors
have been proposed for the direct assay of human serum sample using square-wave
stripping voltammetry [72], quartz crystal microbalance measurements [73], and
differential pulse voltammetry [74]. Based on carbon nanotube field-effect transis-
tor (CNT-FET), a label-free protein biosensor functionalized with a solution con-
taining various linker-to-spacer ratios was prepared for the detection of a prostate
cancer marker [75].
Recently, protein arrays that measure multiple protein cancer biomarkers in clin-
ical samples have been seen to hold great promise for reliable early cancer detec-
tion. A prototype four-unit electrochemical immunoarray was reported based on
SWCNT forests for the simultaneous detection of multiple protein biomarkers for
prostate cancer. After the final washing, the immunoarray was placed into an elec-
trochemical cell containing the mediator hydroquinone in buffer, and hydrogen per-
oxide was injected to develop the amperometric response (Fig.7.14). The steady-state
current increased linearly in clinically relevant ranges for PSA (140 ng/mL),
prostate-specific membrane antigen (PSMA) (10250ng/mL), and platelet factor-4
(PF-4) (140ng/mL). The calibration plot for interleukin-6 (IL-6) (50500pg/mL)
was biphasic, with a better sensitivity below 350pg/mL, but the overall sensitivity
was appropriate for accurate measurements in the clinical range [76].
224 7 Electrochemical Biosensing Based on Carbon Nanotubes
Fig. 7.14 Amperometry at 0.3 V and 2,500 rpm for SWCNTs immunoarrays: (a) PSA;
(b) PSMA; (c) PF-4; and (d) IL-6. Reprinted with permission from Chikkaveeraiah etal. [76],
2009, American Chemical Society
7.5.3Cells
Fig.7.15 Schematic representation of the electrochemical cytosensor array for cell-surface gly-
can analysis, and close-up illustrations of (a) cells captured on RGDS-SWCNTs/SPCE and (b)
HRP-lectin binding with cell-surface glycans. Reprinted with permission from Cheng etal. [79],
2009, Wiley
7.5.4Other Biomolecules
7.5.4.1Nitric Oxide
Nitric oxide (NO), a simple gas molecule and a free radical, was biologically
identified as an endothelium-derived relaxing factor in 1987. Some pathological
processes, such as acute hypertension, diabetes, ischemia, and atherosclerosis, are
found to be associated with abnormalities of NO release. However, it is hard to
accurately measure the NO level due to its fast reaction with oxygen and short
lifetime in biological systems. The noncovalent nanoassembly of porphyrin on
SWCNTs manifested the efficiently electrocatalytic reduction of nitric oxide [82].
In that case, NO sensors were made by modifying the surface of GCE. However,
the big size of GCE is not suitable for the accurate quantitative determination of
NO on the single-cell level. A novel NO electrochemical microsensor was fabri-
cated by modifying the surface of a carbon fiber microdisk electrode (diameter:
57mm) with SWCNTs and a Nafion membrane. The modification of SWCNTs
dramatically improved the sensitivity, with a detection limit of 4.3nM for NO,
which was nearly 10 times lower than that from the bare one and lower than most
NO electrochemical sensors previously reported [83]. The Nafion membrane
offered a good barrier to some interferents, such as nitrite and ascorbic acid, with-
out losing response speed to NO. The sensor has been successfully applied to the
measurement of NO release from single isolated human umbilical vein endothe-
lial cells.
7.5.4.2Glucose
applications. Due to the synergy between the CdTe QDs and CNTs, this novel
biosensing platform based on a QD/CNT electrodes responded even more sensi-
tively to glucose than that based on a GC electrode modified by CdTe QDs or
CNTs alone [90].
A bienzymatic glucose biosensor was proposed for the selective and sensitive
detection of glucose [91, 92]. This mediatorless biosensor was made by the simul-
taneous immobilization of GOD and HRP in an electropolymerized pyrrole film on
SWCNT-coated electrode. The linear response range of the bienzymatic sensor was
from 0.03 to 2.43mM, with a correlation coefficient of 0.998, while the monoenzy-
matic sensor was only from 0.93 to 4.93mM, with a correlation coefficient of 0.996.
The detection limits, based on a signal-to-noise ratio of 3, were estimated to be
around 0.030.009 and 0.90.07 mM for the bienzymatic and monoenzymatic
sensors, respectively [92].
7.5.4.3Hydrogen Peroxide
It is very interesting to detect H2O2 since H2O2 is a product of the enzymatic reac-
tions between most oxidases and their substrates. An H2O2 biosensor has been fab-
ricated based on the direct electrochemistry and electrocatalysis of myoglobin (Mb)
immobilized on silver nanoparticle-doped CNT film with hybrid solgel techniques.
A pair of redox peaks with a peak separation of 160mV and a formal potential of
0.295V were observed at this composite film, corresponding to the direct electro-
chemistry of Mb. The heterogeneous rate constant was estimated to be 0.41 s1.
Under optimum conditions, the amperometric determination of H2O2 was performed
with a linear range of 2.01061.2103 mol/L and a detection limit of
3.6107 mol/L (S/N=3). The MichealisMenten constant was estimated to be
1.62mmol/L [93].
The polyanilinePB/MWCNT hybrid amplified the sensitivity greatly due to the
synergy between the polyanilinePB and MWCNTs [94]. Recently, a new composite
electrode has been fabricated using MWCNTs and the ionic liquid n-octylpyridinum
hexafluorophosphate (OPFP). Compared to other electrodes modified with CNTs
and other ionic liquids, one major advantage of this electrode is its extremely low
capacitance and background currents. The MWCNT/OPFP electrode responded
very rapidly to changes in the level of H2O2, producing steady-state signals within
46s [95].
The utilization of iron-based species (mainly metallic iron, hematite, and
magnetite) encapsulated into MWCNTs as reactants provides a new platform to
monitor H2O2. PB is electrosynthesized in a heterogeneous reaction between
ferricyanide ions in aqueous solution and the iron species encapsulated into
CNTs, resulting in an intimate contact between the PB and the CNTs. The elec-
trode exhibited a low detection limit (1.94108mol/L) and a high sensitivity
(15.3A/cm2M) [96].
228 7 Electrochemical Biosensing Based on Carbon Nanotubes
7.5.4.4Organohalide Pollutant
7.5.4.5Epinephrine
Fig.7.18 Electrical conductance of CNTFET after the introduction of PBS buffer solution, non-
target proteins (bovine serum albumin, fibrinogen, streptavidin), and target protein onto the IgG
Fab-modified CNTFET. Reprinted with permission from Kim etal. [106], 2008, Elsevier
7.7 SWCNT Forest in Electrochemical Biosensing 231
The detection limit for IgE was determined as 250 pM [107]. The label-free
SWCNT-FETs with molecular-scale sensitivity are attractive for point-of-care
applications in immunoassay.
The label-free CNT-based filed-effect sensor offers a new approach for a next gen-
eration of DNA biosensing [108, 109]. For example, a simple and generic protocol
for the label-free detection of DNA hybridization was demonstrated with a random
sequence of 15 and 30 mer oligonucleotides by detecting the charge transfer
inherent to the hybridization reaction [110]. A network CNT-based field-effect
transistor (NTNFETs) was also reported by Star et al. as selective detectors of
DNA immobilization and hybridization [111]. NTNFETs with immobilized syn-
thetic oligonucleotides have been shown to specifically recognize target DNA
sequences for label-free DNA detection at picomolar to micromolar concentra-
tions. The sensing mechanism attributed the strong effect of DNA counterions on
the electronic response, thus suggesting a charge-based mechanism of DNA detec-
tion using NTNFET devices [111].
On the other hand, aptamers are artificial oligonucleotides that can bind to a wide
variety of entities with high selectivity, specificity, and affinity, equal to or often
superior to those of antibodies. The first SWCNT-FET-based biosensor comprising
aptamer was proposed by Lees group [112]. Adding thrombin to the thrombin
aptamer-functionalized SWCNT-FET surface caused a sharp decrease in conduc-
tance, thereby demonstrating the selectivity of the immobilized thrombin aptamers
[112]. The aptamer-modified SWCNT-FETs are promising candidates for the devel-
opment of label-free protein biosensors.
Au- and Cr-contacted FET based on SWCNT networks was designed for the
detection of DNA hybridization. The contribution of electrode-SWCNT junctions
to DNA sensing was up to 6 times more pronounced than that of the SWCNT
channel [113].
For electrochemical biosensors, the ordered CNT forest will be necessary to obtain
the best sensitivities and detection limits. The highly ordered CNT array was fabri-
cated by chemical vapor deposition within a hexagonally ordered, anodized, alumi-
num oxide nanopore template. These vertically aligned MWCNT arrays showed
dense packing on the order of 1010/cm2, and a tight, even plane of nanotube tips,
which are highly suitable for interfacing with biomolecules (Fig.7.19). The site-
selective, covalent docking of the enzyme GOD on the CNT tips had a marked
232 7 Electrochemical Biosensing Based on Carbon Nanotubes
Fig.7.19 SEM image of highly ordered CNT array. Reprinted with permission from Withey etal.
[114], 2006, Elsevier
Fig. 7.20 Tapping-mode AFM of the surface of SWCNT array electrode: left, height images;
right, 3D images. Reprinted with permission from Zhang etal. [117], 2009, American Chemical
Society
the GCE surface. Under optimum conditions, the response is proportional to the
concentration of target DNA in the range of 40110nM, with a detection limit of
20nM. Ready renewal is the outstanding merit of this label-free biosensor, which
can be reused more than 3,000 times [117].
7.8Conclusions
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wwwwwwwwwwwww
Chapter 8
Biosensing with Nanoparticles as
Electrogenerated Chemiluminsecence Emitters
8.1Introduction
Fig.8.1 Time line of ECL: 19641965, first experiments; 1965, theory; 1966, transients; 1969,
magnetic field effects; 1972, Ru(bpy)32+; 1977, oxalate; 1981, aqueous; 1982, Ru(bpy)32+ polymer
and persulfate; 1984, Ru(bpy)32+ label; 1987, tri-n-propylamine (TPrA); 1989, bioassay; 1993,
ultramicroelectrodes; 1998, laser action; 2002, semiconductive nanocrystals. Reprinted with per-
mission from Miao [8], 2008, American Chemical Society
Although the ECL of NPs was first reported for the elemental semiconductor Si NPs
[12], the following research soon proved that many compound semiconductors (e.g.,
CdS [18], CdSe [19, 20], CdTe [21], ZnS [22], PbS [23, 24], CuSe [25], carbon NPs
[26, 27], CdO [28], core/shell CdSe/ZnS [29], nanotube CdSe [30], and thiol-capped
CdSe [31]) as well as the elemental semiconductor NPs (e.g., Si [12] and Ge [32])
can also produce ECL emission. Similarly to other common ECL emitters [7, 8], the
NP ECL is usually generated in two routines as reported by Bards group [12].
8.2 Principle of ECL from Nanoparticles 243
the electrode surface, which then react in solution to give the excited state R*.
Scheme8.1
solution, ECL will only require hole injection and can be easily obtained by simply
oxidizing the NPs. In this case, the oxidation of oxalate produces a strong reducing
agent, CO2, which can inject an electron into the LUMO of an oxidized Si NP to
produce an excited state that then emits light. The corresponding mechanism is
shown in Scheme8.2.
Scheme8.2 (5)
(6)
(7)
Similar to the coreactant for common ECL emitters [8], the coreactant is useful
especially when either R+ or R is not stable enough for NP ECL reaction or when
the ECL solvent has a narrow potential window so that R+ or R cannot be formed.
However, a number of criteria also need to be met for a good coreactant of NP ECL
[8], which includes solubility, stability, electrochemical properties, kinetics, quenching
effect, ECL background, etc. For example, the coreactant should be easily oxidized
or reduced at or near the electrode and undergo a rapid chemical reaction to form an
intermediate that has sufficient reducing or oxidizing energy to react with the oxi-
dized or reduced NPs to form the excited state. As many NP-based ECL sensors
have been developed with the coreactant routine [8, 1416], the coreactant routine
is of great importance for the biosensing application of NP ECL.
The electron and hole injections for NP ECL favorably occur on the NC surface due
to the large surface area of NPs and many possible dangling bonds on the surfaces.
ECL mainly results from electron and hole functions at the particle surface. As the
electrochemical potentials found for reduction and oxidation can provide data for
the band gap of the NPs, the stepwise addition (or removal) of charge from NPs by
the electrochemical method can yield information for the energy required for elec-
tron transfer and ECL emission. With different energies required for the ECL emis-
sions, there are two different generation types for the ECL reaction of NPs. Also,
the different spectra features of NP ECL also provide unambiguous evidence that
NP ECL occurs in the two different models.
One type is the ECL originated from surface states of NPs. The spectra of this type
of NP ECL show red-shifted ECL maxima with respect to their PL spectra. This
surface-state model for ECL is demonstrated nicely in Fig.8.2. Photoluminescence
(PL) mainly occurs through excitation and emission within the NP core, while the
electron and hole wave functions can interact strongly with the NC surface. Different
energies are required to achieve the emitting states of PL and ECL processes
(Fig.8.3). Because less energy is required for the electron (or hole) injections, red-
shifted ECL maxima with respect to their PL spectra are featured for this type of
8.2 Principle of ECL from Nanoparticles 245
Fig.8.2 Schematic
representation of PL and ECL
in semiconductor NPs.
Reprinted with permission
from Miao [8], 2008,
American Chemical Society
ECL emission. The red-shifted ECL maxima with respect to their PL obtained from
Si [12], CdSe [19], and Ge [32] NPs have also proved that the emitting states of ECL
from NP surface states are different from those of photoluminescence (PL) from
NPs. As PL occurs mainly from the interior of the NPs, PL spectroscopy can be used
to probe the interior of the particle and provide information about the electronic tran-
sition (band gap) of material. Because charge injection into the NPs generally occurs
via surface states, electrochemistry and ECL studies are mainly used to probe the
particle surface. This also means that ECL that originated from surface states gener-
ally is not sensitive to the NP size and capping agent used, but depends more sensi-
tively on the surface chemistry and the presence of surface states [13].
246 8 Biosensing with Nanoparticles as Electrogenerated Chemiluminsecence Emitters
This surface-state model for ECL has been further verified via the ECL study of
CdSe/ZnSe core-/shell-type NPs [29]. Unlike the spectra from the ECL of Si or CdSe
NPs, where the emission occurs in a single peak that is significantly red-shifted from
the PL peak, the CdSe NPs are well passivated with a shell of ZnSe, and thus show a
large ECL peak at the wavelength of band-edge PL plus a red shift by ~200nm from
the PL peak (Fig.8.3). The red shift is deduced from the unpassivated CdSe surface
because the same red-shift extent can be found in the ECL spectra from CdSe [19] and
Si [12] NPs. The large ECL peak at the wavelength of band-edge PL suggests that the
surfaces of CdSe/ZnSe core-/shell-type NPs have been largely passivated; ECL emis-
sion cannot be generated from the surface states on the NPs. Thus, ECL emission
from CdSe NPs whose surfaces have been largely passivated is dependent on the bulk
of NPs, which is another ECL generation type, known as band-gap ECL.
As another type of NP ECL, band-gap ECL mainly corresponds to the bulk of NPs:
Its ECL spectrum matches the PL spectrum, and therefore is size-dependent and tun-
able [21, 29, 31, 39, 40]. To achieve band-gap ECL, a common method is to passivate
the surface state of NPs [29], because the charge injection in an NP is generally assumed
to occur via its surface states, which results in a surface-state model as the main genera-
tion model for NP ECL. Bard and coworkers found that surface states of CdTe NPs
could be easily passivated and then result in band-gap ECL [21]. Recently, Ju and
coworkers also observed the band-gap ECL from CdSe and CdTe QDs in aqueous
solution by capping the QDs with thioglycolic acid (TGA) [31] and mercaptopropionic
acid (MPA), respectively [39, 40]. As shown in Fig.8.4, the ECL maximum of TGA-
capped CdSe QDs is located at the peak position of the PL, indicating that the excited
state in the ECL emission was the same as that in the PL, in which the CdSe/TGA*
could be considered the emitter to produce 1Se1Sh transition emission [31].
The two generation types of NP ECL have many different applications. The
surface-state model ECL is sensitive and can be used to probe the surface states of
NPs, while the band-gap model ECL cannot. However, the ECL spectrum of the
surface-state model will be red-shifted and not size-dependent, which may limit its
8.2 Principle of ECL from Nanoparticles 247
applications for multiplexing. The band-gap model ECL has potential in corresponding
applications. Recently, Han demonstrated that the surface-state model ECL is as effi-
cient and sensitive as the band-gap model ECL for general biosensing schemes, such
as H2O2 sensing [41]. As to the great importance of biosensing in modern times, the
applied system for NP ECL will be discussed in the following section.
There are a wide variety of NPs that exhibit ECL, suggesting NP ECL systems can
be classified by the different features of emitters. For example, NP ECL systems can
be classified as the ECL of bare QDs [12, 19], spherical QDs [42], hollow spherical
QDs [43, 44], QD film [28, 31, 4547], shell/core structured QDs [29, 48, 49],
nanotubes [30, 50, 51], nanorods [52], and nanoflakes [53] by the NP structure. NP
ECL systems can also be classified as the ECL of elemental NPs (e.g., Si [12] and
Ge [32]), of compound NPs (e.g., CdSe [19]), and even of aromatic hydrocarbon
NPs [52] and of polymer NPs [54].
A more scientific way to define different NP ECL is based on the ECL mecha-
nism; that is, NP ECL systems are reasonably classified as annihilation NP ECL
systems and coreactant NP ECL systems. Due to the merits discussed above, the
overwhelming majority of publications concern coreactant ECL and its analytical
applications. This section mainly concerns the typical coreactant NP ECL systems
and their mechanisms.
Oxalate (C2O42) system. Oxalate is the first account of a coreactant ECL system
reported by Bards group in 1977 [55]. It is often referred to as an oxidative or
oxidative-reductive coreactant due to its ability to form a strong reducing agent
(CO2) upon electrochemical oxidation. As a classical coreactant ECL system, the
electrode oxidizes both the oxalate and the ECL reactant D; the reducant, CO2, is then
generated upon bond cleavage of oxalate. This strategy has been widely used in ana-
lytical and biotechnological fields, with the reactant D being Ru(bpy)32+ [7, 8]. By
adding excess C2O42 to the NP solution, Bard and his coworkers reported that this
typical coreactant can result in coreactant anodic ECL for Si NPs by the electron trans-
fer between CO2 and oxidized Si NPs [12]. We detailed the general mechanism for an
oxalate system in Scheme8.2. Recent work further proves that oxalate can also work
as an efficient ECL coreactant for 9,10-diphenylanthracene (DPA) nanorods [45]. The
classical coreactant, oxalate, works well as the coreactant for NP ECL emissions.
Tri-n-propylamine (TPrA) system. TPrA is another important popular oxidative
reductive coreactant for ECL systems, especially the Ru(bpy)32+. The majority of
ECL applications reported so far involve Ru(bpy)32+ or its derivatives as an emitter
(or label) and TPrA as a coreactant because the Ru(bpy)32+/TPrA system exhibits the
highest ECL efficiency. This system has become the basis of commercial systems for
ECL immunoassay and DNA analysis [8]. Electrochemical studies of various ali-
phatic amines indicate a possible reaction pathway for oxidizing TPrA to produce a
248 8 Biosensing with Nanoparticles as Electrogenerated Chemiluminsecence Emitters
strong reducing agent [56]. Upon oxidation, the short-lived TPrA radical cation
(TPrA+) is believed to lose a proton from an a-carbon to form the strongly reducing
intermediate TPrA (Fig. 8.5). ECL emission resulted from the electrooxidized
rubrene NPs (R+), and TPrA suggests that TPrA can also work as a coreactant for
NP ECL emissions [52].
Recently, Wang and coworkers demonstrated the anodic ECL emission of 3-mer-
captopropionic acid (MPA)-capped CdTe/CdS QDs with TPrA as the coreactant in
aqueous solution [49], as shown in Fig.8.6.
The whole procedure for the TPrA-involved NP ECL is deduced as follows:
The cation QDs+ and TPrA+ are anodically produced by the electrochemical oxi-
dation of CdTe/CdS QDs and TPrA, respectively; TPrA+ loses a proton from an
a-carbon to form TPrA, and then reduces QD+ to QDs*; finally, the ECL is gener-
ated from QDs*. The general mechanism for the TPrA system can be depicted as
Scheme8.3.
8.2 Principle of ECL from Nanoparticles 249
Scheme8.3
Scheme8.4
Scheme8.5
250 8 Biosensing with Nanoparticles as Electrogenerated Chemiluminsecence Emitters
Fig.8.7 Cyclic voltammograms and ECL curves of (a) bare and (b) CdSe nanocrystal thin-film-
modified PIGE in air-saturated 0.1M pH 9.3 PBS containing 0.1 M KNO3, (c) solution (b) bubbled
with N2 for 25min, and (d) solution (b) +100mM H2O2. Scan rate: 20mV/s. Reprinted with per-
mission from Zou and Ju [45], 2004, American Chemical Society
Scheme8.6
8.3 Biosensing Strategy and Corresponding Application 251
Scheme8.7
Although ECL analytical techniques coupled with QDs have been rapidly developed
and extensively studied in both organic and aqueous media, it is clear that a normal
coreactant for common molecules as ECL emitters still works for the study of NP
ECL. Since the ECL of NPs has only recently been explored, it is hard to overstate
the importance of coreactants to the growth and development of NP ECL.
ECL is mainly realized with the coreactant ECL routine. According to the different
ECL mechanisms of NPs [8, 1517], their biosensing is mainly realized by the fol-
lowing strategy.
This strategy is mainly based on either the enhancing or inhibiting effect of the
analyte on the NP ECL. According to this strategy, a series of ECL sensors have
been developed with the interaction between analytes and the species in the ECL
process [31, 39, 7984].
For exampleit is believed that the intermediate OH radical in the TGA-capped
CdSe QD film/peroxide ECL system is the key species for producing hole-injected
QDs. ECL sensors for biologically important scavengers of hydroxyl radicals, such
as g-l-glutamyl-l-cysteine-glycine (GSH), are thus developed based on inhibition
effect [31]. As l-cysteine is involved in many biological processes, such as
Parkinsons and Alzheimers diseases, the high sensitivity obtained suggests their
sensing potential for both scavengers and generators of hydroxyl radicals in clinical
samples.
A stable anodic ECL emission can be detected from MPA-capped CdTe QD/
sulfite system. Using tyrosine as a model compound, whose electrooxidized product
can quench the excited QDs and thus the ECL emission, an MPA-capped CdTe
QD-based ECL sensor for tyrosine with a wide concentration range has been devel-
oped [39]. Based on the ECL of CdTe QDs using carbon nanotube-modified glassy
carbon electrode, a highly sensitive method for the determination of methimazole
has also been developed. The ECL intensity decreases linearly in the concentration
range of 1.01094.0107M for methimazole, with a relative coefficient of 0.995,
which shows a finer sensitivity than that at a bare electrode [73].
As room-temperature ionic liquid (RTIL) film on the glassy carbon electrode can
greatly enhance the ECL intensity of CdTe QDs, the highly sensitive sensing of gos-
sypol content using CdTe QD ECL with RTIL-modified glassy carbon electrode has
been achieved. The sensor shows a good linear relationship in the gossypol concen-
tration range of 5.01075.0109M, with a detection limit of 5.0109M. The
sensor has been used to detect gossypol in cottonseed oil with satisfactory results.
The RTIL-modified electrode may extend the analytical applications of QD ECL
systems [82].
An anodic ECL-sensing strategy based on a TGA-capped CdSe QD/sulfite
system in a neutral medium has been developed in a similar way for detecting ECL
8.3 Biosensing Strategy and Corresponding Application 255
As the ECL emitter for NP ECL is the excited NPs, the possible energy transfer
from excited NPs can also provide a strategy for biosensing [40, 47]. This detection
strategy mainly works in two ways:
1. ECL energy is directly transferred from the excited CdTe QDs to analyte, the
quencher. For example, intensive anodic ECL emission from MPA-capped CdTe
QDs can be obtained with a peak value at +1.17V (vs. Ag/AgCl) in pH 9.3 PBS
at an indium tin oxide (ITO) electrode, and the ECL energy transfer from the
excited CdTe QDs to catechol derivatives, such as dopamine or l-adrenalin, can
produce decreased ECL emission for sensing [40]. In the case of dopamine and
l-adrenalin, this ECL method shows wide linear ranges, from 50nM5mM and
80nM30mM, respectively. Both ascorbic acid and uric acid, which are common
interferences, do not interfere with the detection of catechol derivatives in practical
biological samples.
2. ECL energy is transferred to a quencher (not to analyte). The amount of analytes
plays an important role for the energy transfer. For example, ECL energy transfer
from CdS:Mn NP film to proximal Au NPs quenched the ECL emission. Ahybrid-
ization with target DNA (t-DNA) results in an enlarged distance between Au NPs
and CdS:Mn NPs, and hence enhanced the ECL emission of the CdS:Mn NP
film, which can be used for sensing the t-DNA [47] (Fig.8.9).
Fig.8.9 ECL sensing strategy with energy transfer between CdS:Mn NPs and Au NPs. Reprinted
with permission from Shan etal. [47], 2009, Royal Soc Chemistry
Fig.8.10 Procedure of the highly sensitive detection of tyrosine based on the enhanced anodic
ECL and catalytic oxidation of tyrosinase in air-saturated PBS. Reprinted with permission from
Liu and Ju [39], 2008, American Chemical Society
8.3.6.1DNA Assay
A common way for ECL DNA sensing is using ECL emitter as label. This technique
can work for the QD-based ECL assays.
For example, Hu etal. [87] developed a DNA assay by using TGA-capped CdTe
QDs as DNA labels. t-DNA was hybridized with capture-DNA (c-DNA) bound on
the nanoporous gold leaf (NPGL) electrode, which was fabricated by conjugating
amino-modified c-DNA to TGA-modified NPGL electrode. Following that, amino-
modified probe DNA was hybridized with t-DNA, yielding sandwich hybrids on the
NPGL electrode. Then, MPA-capped CdTe QDs were labeled to the amino group
end of the sandwich hybrids. ECL emission of the QD-labeled DNA hybrids on the
NPGL electrode was measured for DNA sensing (see Fig. 2.17).
Huang developed a novel biosensor for thrombin by the QD ECL technique. The
thiol-terminated aptamer with 15 nucleotides (probe I) was first immobilized on an
Au electrode, and then thrombin was imported to form the aptamerthrombin bioaf-
finity complexes. Another 5-biotin-modified aptamer (29 nucleotides, probe II) was
next hybridized with the combined thrombin to form a sandwich-type structure.
Streptavidin-modified QDs (avidin-QDs) were bound to probe II via the biotinavidin
system. The QD ECL signal was responsive to the amount of probe II, which was
indirectly proportional to the amount of combined thrombin. In addition, the biosensor
exhibited excellent selectivity responses and good stability toward the target analyte
[88]. A facile strategy for the fabrication of aptamer-based adenosine 5-triphosphate
(ATP) biosensor was developed by a QD ECL technique in a similar way [88].
A QD ECL biosensor for the detection of lysozyme has been developed by forming
the aptamerlysozyme bioaffinity complexes at the Au electrode. The free probes
are hybridized with the 5-biotin-modified cDNA oligonucleotides to form double-
stranded DNA (ds-DNA) oligonucleotides. Avidin QDs are bound to these hybridized
cDNA through the biotinavidin system. The ECL signal of the biosensor is
responsive to the amount of QDs bonded to the cDNA oligonucleotides, which
isindirectly inversely proportional to the combined target protein [89].
258 8 Biosensing with Nanoparticles as Electrogenerated Chemiluminsecence Emitters
Fig.8.11 Scheme for the label-free NP ECL immunoassay strategy. Reprinted with permission
from Gie etal. [76], 2008, American Chemical Society
8.3 Biosensing Strategy and Corresponding Application 259
The ECL of CdSe QDs could be greatly enhanced by the combination of CNTs
and PDDA in the CdSe QD film. A sensitive ECL immunosensor for the detection
of human IgG (Ag) was proposed with CdSe QDCNT composites [75]. After
PDDA as a binding linker was conjugated to the CdSe QDCNT composite film on
the electrode, the ECL signal was significantly enhanced. Subsequently, gold nano-
particles AuNPs assembled onto the CdSe QDCNT/PDDA-modified electrode
could further amplify the ECL signal. After antibody (Ab) was immobilized onto
the electrode through AuNPs, the ECL immunosensor was successfully fabricated.
The principle of ECL detection for target Ag was based on the increment of steric
hindrance after immunoreaction, which resulted in a decrease in ECL intensity. The
Ag concentration was determined in the linear range of 0.002500 ng/L, with a
detection limit of 0.6pg/mL. This work opened up new avenues for applying QD
ECL in highly sensitive bioassays [75].
More recently, based on the ECL emission of bidentate chelate CdTe QDs at
relatively low cathodic potential [90], a quantum dot-based electrochemiluminescent
immunosensor was developed by coupling enzymatic amplification with self-
produced coreactant from oxygen reduction [91]. The bidentate chelate CdTe QDs
exhibit surface traps to produce a narrower band gap than the core. Using the elec-
trochemically produced H2O2 as coreactant, a strong ECL emission is observed,
which can be quenched by introducing hydroquinone and horseradish peroxidase in
the solution. The quenching effect has been applied for the ECL detection of hydro-
quinone [90]. After the QDs and human IgG (HIgG) are coimmobilized on an elec-
trode surface, upon the immunorecognition of the immobilized HIgG to its antibody
labeled with horseradish peroxidase, the enzyme is introduced to the electrode surface,
as shown in Fig.8.12. In the presence of hydroquinone, the enzymatic cycle thus
consumes the self-produced coreactant H2O2, leading to a wide calibration range of
Fig.8.12 (a) Construction and (b) incubation of the immunosensor, and ECL detection (c) with-
out and (d, e) with the enzymatic amplification by the consumption of H2O2 as the coreactant.
Reprinted with permission from Liu etal. [91], 2010, American Chemical Society
260 8 Biosensing with Nanoparticles as Electrogenerated Chemiluminsecence Emitters
8.4Conclusions
The fundamental and biosensing applications with NPs as ECL emitters have been
reviewed in this chapter. As a new kind of ECL emitter, NPs have provided an
entirely different sensing paradigm. There are still many disadvantages to over-
come; for instance, a high negative potential is required before ECL is produced,
and the intensity is not at the level of Ru(bpy)32+ or luminol ECL. With further
understanding of NP ECL mechanisms, new highly efficient and tunable NP ECL
systems (emitters and coreactants) and combined analytical techniques should be
developed to further improve the analytical performance of NP-based ECL
biosensors.
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Chapter 9
Biosensing Applications of Molecularly
Imprinted Nanomaterials
9.1Introduction
Chemical sensors and biosensors have attracted considerable attention within the
field of modern analytical chemistry, as seen both from the number of publica-
tions and from the diversity of approaches and techniques. This is essentially due
to new demands and opportunities that are appearing particularly in clinical settings,
(e.g., routine blood testing), environmental monitoring, warfare protection, home-
land security, food analysis, illicit drug detection, and genotoxicity. Sensor devel-
opment has been driven, in large part, by the need for new devices that are less
expensive and simpler to construct and operate, that provide adequate detection
limits and selectivity, and that are accurate and reliable.
The central part of a biosensor is the recognition element (e.g., an antibody,
aptamer, cell, DNA oligonucleotide, enzyme, lectin, protein), which is in close contact
with an interrogative transducer and serves to recognize specifically the target analyte
(Fig.9.1a). The binding or conversion (if the analyte is a substrate) event is used to
produce an optical, mass, thermal, or electrochemical signal that is related to the
analyte concentration in the sample [1, 2]. The biosensor concept has been developed
in parallel to the development of elegant biosensor arrays [36] for simultaneous
multianalyte detection. However, despite the progress that has been made on bio-
sensors and microarrays based on biological recognition elements, there are well-
documented limitations associated with many types of biorecognition elements
[79]. Aptamers can address several of these issues [10], but they are not yet available
for a wide range of analytes and can be expensive to produce for most analyses.
Although biologically based recognition elements are clearly important and attractive
for the design of biosensors for continuous process or environmental monitoring,
the poor chemical and physical stability of biomolecules sometimes generally pre-
vents their use in harsh environments, which ultimately leads to a compelling case
for developing inexpensive, robust, and reusable alternatives for these expensive/
labile biorecognition elements. Molecular imprinted polymers (MIPs), as purely
Fig.9.1 (a) Schematic representations of antibody-based chemosensor and (b) MIP-based biomi-
metic sensor. Inset in (b) shows the concept of molecular imprinting. Reprinted with permission
from Guan etal. [2]. 2008, Molecular Diversity Preservation Int
synthetic materials, can, in principle, be designed from scratch to act not only as
simple affinity adsorbents, but also as smart reporters responsive both to targeted
molecules [11] and to different environmental stimuli (Fig.9.1b) [1215]. Table9.1
shows a basic comparison of MIPs with antibodies and aptamers [16].
Molecular imprinting has been widely recognized as the most promising meth-
odology for the preparation of a tailor-made artificial receptor that can selectively
bind predetermined target molecules [9]. Molecular imprinting is a technique
involving the formation of binding sites in a synthetic polymer matrix that are of a
complementary functional and structural character to its substrate molecule. The
principle of molecular imprinting is illustrated in Fig.9.2, which refers to arranging
polymerizable functional monomers around a template, followed by polymerization
and template removal. The arrangement is typically achieved by noncovalent inter-
actions (e.g., H-bonds, ion pairing) or reversible covalent interactions. The binding
sites that are generated during the imprinting process often have affinities and selec-
tivities approaching those of antibodyantigen systems, and the properly designed
MIPs have therefore been dubbed antibody mimics [17], which can then bind the
template or structurally similar analytes. When the combination of MIP materials
with a transducer is in a suitable format, the sensors with MIP recognition can iden-
tify and quantify a target species by converting the analyteMIP binding event into
a physically readable signal, as shown in Fig.9.1b. Except for possessing antibody-
like molecular selectivity, the major advantages of using MIPs over their biological
counterparts include physical robustness, high strength, resistance to elevated
temperatures and pressures, and inertness to acids, bases, metal ions, and organic
solvents, as well as low production cost and easy preparation [18].
Table9.1 Comparison of MIPs with antibodies and aptamers
9.1 Introduction
Fig. 9.2 Schematic representation of the two most common MIP fabrication strategies.
(a)Noncovalent imprinting; (b) covalent imprinting. Reprinted with permission from Holthoff and
Bright [44]. 2007, American Chemical Society
These mimics display some clear advantages over real antibodies for sensor
technology [19]: (1) binding affinities comparable to a biological recognition ele-
ment; (2) robustness and stability under a wide range of chemical and physical
conditions; and (3) easy design of recognition sites for analytes that lack suitable
biorecognition elements [9]. Even though the great potential of this technology has
only been recognized recently, in particular after the introduction of synthetic
organic polymers as imprinting matrices, there is now a strong development toward
the use of MIPs as recognition elements in biosensing techniques [20, 21].
Fig.9.3 (a) Examples of organic polymerizable functional monomers that can be used alone or in
combination in noncovalent molecular imprinting. (b) Examples of cross-linking monomers that
can be used for the synthesis of molecularly imprinted polymers. (c) Examples of organic polym-
erizable functional monomers that can be used in covalent molecular imprinting. Reprinted with
permission from Holthoff and Bright [19]. 2007, Elsevier
Two distinct approaches have been followed for obtaining MIPs, as depicted in
Fig.9.2a, b. The selection depends upon interactions between the template and the
functional monomers involved in the imprinting and rebinding steps. These
approaches include (1) self-assembly or noncovalent approaches a prepolymer-
ization complex between the imprint molecule and functional monomers can be
formed via noncovalent interactions; and (2) covalent approach monomers can be
covalently coupled to the imprint molecule, that is, a polymerizable derivative of
the imprint molecule is synthesized.
9.2.1Noncovalent Approach
The noncovalent approach (Fig.9.2a) was first reported by Mosbach [17]; various
polymerizable functional monomers commonly used for noncovalent molecular
imprinting are illustrated in Fig.9.3a [9]. A cross-linking monomer is generally
used to form a three-dimensional rigid structure around the template molecule and
produce stable binding cavities. Figure9.3b illustrates several of the more common
cross-linker monomers that have been used [9]. Noncovalent forces, such as
hydrogen bonds, van der Waals forces, ionic interactions, and hydrophobic effects,
270 9 Biosensing Applications of Molecularly Imprinted Nanomaterials
are utilized. This process is more similar to natural processes, in which most
biomolecular interactions are noncovalent [17, 23]. Following the polymerization
and template removal, the functional groups within the templated polymeric
matrix can subsequently recognize and bind the target analyte using the same
noncovalent interactions. Arrangement using noncovalent interactions requires
the template and target analyte to form a sufficient number of noncovalent inter-
molecular interactions to produce the binding pocket during polymerization.
Because the relative interactions involved are weak, an excess of functional
monomers is often added to stabilize the templatefunctional monomer complex
during polymerization, which can result in heterogeneous binding sites sometimes
requiring subsequent purification. However, the commercial availability of a large
number of functional monomers and the ease of preparation have attracted the
widespread use of this approach. The combinatorial synthesis [24, 25] and silica
screening methodology [26] developed recently have also accelerated the process
of obtaining optimal noncovalently imprinted polymers. The rational design of
more potent functional monomers, such as via metal coordination interactions to
bind specific amino acid sequences, leads to the binding sites becoming more
homogeneous [27]. New potent functional monomers based on polymerizable
amidines and urea have been developed, which allow stoichiometric amounts of
functional groups to be incorporated into imprinted polymers to reduce nonspe-
cific adsorption [28, 29].
9.2.2Covalent Approach
In the covalent approach, pioneered by Wulff etal. [30], reversible chemical bonds
are maintained between the template and the functional monomers during imprinting
polymerization, which can be used to overcome the limitations of noncovalent
imprinting (Fig.9.2b). The same driving force is used for the MIPs to subsequently
bind the template. This approach creates strong interactions due to the restoration of
the covalent bond between the matrix and the target; however, it is limited by the
rather small number of useful reversible covalent interactions that can be used [31].
Binding site monomers having a boronic acid, diol, aldehyde, or amine functional
group have been successfully used for covalent molecular imprinting (Fig.9.3c) [9].
Cross-linking monomers (Fig.9.3b) are also utilized in this approach to help stabi-
lize and define the molecularly templated sites within the final material. This
approach can lead to homogeneous binding sites, since the template-functional
monomer complex can be kept intact during the polymerization reaction. However,
removal of the chemically bonded template from the highly cross-linked polymer
matrix is difficult, and the rebinding process is normally very slow because of the
necessary formation of covalent bonds between the target compound and the MIP.
Furthermore, prior modification of the template is needed, which can require strin-
gent synthetic conditions.
9.3 Types of MIP Materials 271
9.2.3Other Approaches
9.3.1Organic Materials
9.3.2Inorganic Materials
The solgel technique offers a wide range of processing approaches that can pro-
duce three-dimensional matrices in different configurations such as thin films,
porous materials, and bulk structures [43], and therefore is attractive for the prepa-
ration of MIPs [4451]. The application of imprinted solgel materials to the pro-
duction of sensors is in its infancy. Most of the accounts describing these materials
end with a statement that suggests the potential application for chemical sensing. So far,
several research groups have dealt with the application of imprinted solgel films as
recognition layers applied on various transduction systems. For example, in 1949,
Dickey [52] first imprinted silica to create materials that were 420 times more effec-
tive than nonimprinted silica controls on binding the target molecules. Much has trans-
pired since Dickeys seminal work, and solgel processing has allowed researchers
to create a wide variety of molecularly imprinted solgel-based sensors. Lulka etal.
[53] described the properties of surface-imprinted silicas using NATA or fluorescein
as templates. Silica gel was mixed with bis(2-hydroxyethyl) aminopropyl triethox-
ysilane as functional monomer and TEOS as the template. The slurry was allowed
to polymerize. After extraction of the template and nonspecifically bound material,
9.4 Development of MIP Nanomaterials 273
the polymers were glued to cards and their properties were examined in a right-angle
fluorescence-detection configuration. Quenching experiments using KI and acryl-
amide suggested that MIP-bound substrates were somewhat protected from their
solvent environments. The results illustrated an alternative method for the determi-
nation of inherently fluorescent molecules.
As mentioned above, molecular imprinting can produce MIPs with a built-in func-
tionality for the recognition of a particular chemical substance (template molecule)
with its complementary cavity. The synthesis of MIPs typically consists of the copo-
lymerization of functional and cross-linking monomers in the presence of a template
molecule [9]. However, the effectiveness of this technique is greatly dependent on
three major factors: the interaction nature between the template and monomer [5456];
the formation of imprinted materials [5760]; and the rigidity of the polymeric matrix
[6167]. Because of the nature of the highly cross-linked polymer network, it is dif-
ficult to thoroughly remove the template taken up in the preparation stage. Therefore,
crushed materials are needed in the application, during which the MIPs suffer from
the gradual elution of the template. This incomplete extraction of the template from
the network can easily result in significant problems in the assessment of the rebind-
ing assay, separation efficiency, etc. [68]. Therefore, the imprinted materials that are
ideally suitable for molecular recognition elements have yet to be explored.
The most common methodology for molecular imprinting is the monolithic
approach, where MIPs are prepared in bulk and subsequently ground and sieved to
the desired size [6972]. Despite being a simple and convenient approach, the wide
applications of MIPs as artificial receptors in analytical chemistry are still limited
by several critical factors. First, because of the highly cross-linked structure with its
irregular shape, most imprinted bulky polymers usually need a grinding process to
make the removal of the template easier and more efficient. However, the extraction
of original templates located at the interior area of bulky MIPs is still quite difficult.
Sometimes the imprinted cavities in the cross-linking network can be damaged by
the grinding process [6163]. The issue of poor accessibility of the imprinted cavities,
which are often created within the polymer bulk, is a major problem of this method.
It could result in slow kinetics of the target analyte binding [64, 65]. This is especially
important in the imprinting of macromolecules, such as oligosaccharides and pro-
teins. Additionally, the rigidity of the polymeric matrix greatly reduces the confor-
mational freedom of molecular recognition by excluding any further chain mobility
[66, 67]. Second, the uncontrollable random polymerization always gives rise to the
heterogeneity of the imprinted sites in the formed polymer matrix [73, 74], which
can be ascribed to the different polymerizing abilities of vinyl functional monomer
and divinyl cross-linking agent, and to the much larger amount of the cross-linking
274 9 Biosensing Applications of Molecularly Imprinted Nanomaterials
agent than that of the functional monomer. The final problem is the lack of signal
output of the analyte binding at MIPs due to the poor assembly ability at the surface
of the transducer, which has a negative effect on their use in chemical detections or
bioassays [19]. Therefore, the design and development of MIP materials at a molecular
level so as to provide a better antibody/enzyme mimic are of necessity and a great
challenge.
Many efforts have been made to prepare MIPs in an optimizing form that controls
templates to be situated at the surface or in the proximity of the materials surface
[7587] so as to solve the problems of limited mass transfer and template removal
often associated with the traditional technique of molecular imprinting. Surface
imprinting has been widely studied as an effective approach. For example, Mosbach
and coworkers [75] first reported the surface molecular imprinting strategy by the
covalent immobilization of template molecules at the surface of a solid substrate.
After the imprinting polymerization and the removal of substrate, all the templates
were located on the surface of imprinted materials, providing a possibility for the
complete removal of templates, excellent accessibility to target species, and confor-
mational flexibility of recognition. This improvement is especially valuable when
imprinting macromolecules, such as proteins [7678], cells [79, 80], and viruses
[81]. As templates, these molecules with large sizes are more difficult to remove
from traditional highly cross-linked MIPs. The common approach of surface
imprinting is the creation of thin MIP films on suitable substrates. An advantage of
this approach is the thin film could be formed directly on different sensor substrates,
such as gold and glassy carbon electrodes [88, 89], quartz crystal microbalance
(QCM) [90, 91], and surface plasmon resonance (SPR) [92]. Various methodologies
have been used to prepare such MIP films, including spin-coating, surface-initiated
atom-transfer radical polymerization (ATRP), and electropolymerization.
In this approach, the control of MIP film thickness is the major issue, as it should
be sufficiently thin for rapid mass transfer. This is especially important in sensor
application, where a high sensitivity and a short response time are desired [87].
Accordingly, the controlled deposition of MIP nanostructures for sensor applications
has also been investigated. For example, Huang etal. constructed an on-chip sensor
device by photoirradiation of a cross-linkable polymer [93]. Such a cross-linkable
polymer in solution can be deposited more easily than a simple monomer mixture
on a flat surface by spin-coating and other printing techniques. By combing solgel
chemistry with spin-coating deposition, Marx et al. [94] coated a thin MIP film
(70nm) on ITO electrodes to fabricate electrochemical sensors. The designed sen-
sors displayed very high sensitivity and selectivity for the model analytes. By using
ATRP, Li etal. [95] synthesized a series of linear copolymers of 2-methacryloyl-
ethyl methacrylate and methacrylic acid. The linear copolymers were subsequently
allowed to form stable complexes with the templates before the formation of a
9.4 Development of MIP Nanomaterials 275
Fig.9.5 Schematic illustration of the distribution of effective binding sites in the imprinted bulky
materials and the nanosized, imprinted particles after the removal of templates. Reprinted with
permission from Gao etal. [59]. 2007, American Chemical Society
binding of target species. In the case of nanosized particles, most of the imprinted
sites are located at the surface or in the proximity of surface. Therefore, the forms
of imprinted materials are expected to greatly improve the binding capacity, kinetics,
and site accessibility of imprinted materials. In comparison with bulky MIP
materials, the imprinted nanomaterials have a higher affinity and sensitivity to target
analyte, and a more homogeneous distribution of recognition sites [2].
For the low-dimensional MIPs with nanostructures, it is easy to obtain regular
shapes and sizes, and the tunable flexibility of shapes and sizes. The nanosized MIP
materials also have better dispersibility in analyte solutions and thus greatly improve
the mass transfer, exhibiting fast binding kinetics [57, 58, 82, 83, 101]. In particular,
novel nanostructure assembly technologies have achieved wide success in building
various nanodevices [102, 103]. The imprinted nanomaterials with well-defined
morphologies can be feasibly installed onto the surface of devices in a required
form for many applications in nanosensors and molecular detection.
9.5MIP-Based Biosensors
Table9.2 Different approaches to the transduction of the binding signal in MIP sensors
Signal is generated
Directly through the
binding event By the analyte By the polymer
What is measured Change in general Specific property Change in the signal
physiochemical of the analyte emitted by reporter
properties of the groups incorporated
system into the polymer
Examples Mass change (QCM), Fluorescence, Fluorescence, scintillation,
capacitance change electrochemical spectral shift, proton
activity, IR release (pH)
spectrum
Reprinted with permission from Ye etal. [106]. 2004, Springer
p reparation. Three issues are critically important for the successful design of MIP
sensors: (1) the development of a highly sensitive transducer, capable of monitoring
the binding process and transforming it into a processable signal; (2) the develop-
ment of MIPs capable of interacting with the template-analyte under the required
conditions with the required affinity and specificity; (3) the integration of the MIP
with the transducer. Therefore, a major concern for the development of MIP-based
sensors is how to measure the analyte binding at MIP materials. Typically, MIP-
based sensors are fabricated by assembling MIP materials onto the surface of trans-
ducer; thus, the analyte binding is converted into a measurable signal (Fig.9.1b).
Table9.2 depicts the three different possibilities for the transduction of the binding
events in MIP sensors [106]. In the simplest case, a change in one or more physico-
chemical parameters of the system upon analyte binding (such as mass accumula-
tion) is used for detection. This principle is widely applicable and more or less
independent of the nature of the analyte. In general, the efficiency of sensors depends
not only on the selectivity and sensitivity of MIPs to target species, but also on the
approaches of signal output. The optimal transduction approach to a readable signal
output can be expected to maximize the selectivity and sensitivity of sensors.
In principle, many physical measurements, such as electrochemical voltammetry,
fluorescence, piezoelectricity, and SPR, can be used for the signal detection in MIP-
based sensors [12, 20, 44, 105]. The choice of the transducer is based on the proper-
ties of target analytes and the forms of MIPs. For example, mass-sensitive acoustic
transducers, such as QCM [107112], have become increasingly popular for the
design of MIP sensors based on mass-accumulation binding. An alternative way of
detecting mass accumulation at a surface is by optical means, such as ellipsometry
or SPR [113, 114]. If the target analyte exhibits a special property such as fluores-
cence or electrochemical activity, this can be exploited for the design of MIP-based
optical or electrochemical sensors. For example, a fluorescence sensor for dansyl-
phenylalanine, a fluorescent analyte, has been constructed [115]. The fluorescence
of the MIP after analyte binding is measured using fiber optics, and the signal is
found to be a function of the analyte concentration. The sensor shows a certain
degree of stereoselectivity for the l-form of the analyte. Electrochemical voltammetry
278 9 Biosensing Applications of Molecularly Imprinted Nanomaterials
9.5.3Electrochemical Sensors
and MIP nanomaterials, greatly extending the range of detected targets and improving
the sensitivity, selectivity, and simplicity of electrochemical sensors [20, 118124,
162165]. For example, a successful potentiometric sensor was reported for the
detection of glucose [166]. The sensor operation was based on measuring the con-
centration of protons released during the interaction of metal-complexing imprinted
polymer with glucose. The proposed sensor was capable of measuring the glucose
concentration in a clinically relevant range (025mM) in plasma. Kitade etal. [162]
prepared an interesting potentiometric artificial immunosensor based on an MIP as
the detecting element in micro-total analysis systems with the intent of providing
easy clinical analysis. In this study, the sensing element of serotonin-imprinted
polymer was absorbed and immobilized in a plasma-polymer layer by a method of
swelling and polymerization. The obtained sensor was highly responsive to sero-
tonin in water but not to tryptamine, acetaminophen, or procainamide, with a detec-
tion limit of 100pmol/L. The electrochemical sensors are most commonly fabricated
by installing MIP nanomaterials, as recognition elements, onto the surface of the
electrode [88, 119]. The changes of current and peak voltage at cyclic voltammetry
upon the analyte binding can sensitively respond to the concentration and kind of
analytes, respectively, due to the oxidation or reduction of analytes at the MIP-
modified electrode. Kan etal. [119] constructed a novel electrochemical sensor to
monitor the neurotransmitter dopamine by modifying the glassy carbon electrode
with a composite of multiwalled carbon nanotubes (MWNTs) and dopamine-imprinted
polymers. The MWNT-MIP-modified electrode not only possessed a rapid dynamic
binding with an equilibrium period of 30min, but also exhibited a high selectivity
and sensitivity toward dopamine, with a linear range of 5.01072.0104 M.
Zhang and coauthors [88] reported a surface molecular self-assembly strategy for
molecular imprinting in electropolymerized polyaminothiophenol (PATP) mem-
branes at the surface of gold nanoparticle (Au NP)modified glassy carbon elec-
trode for the electrochemical detection of the pesticide chlorpyrifos (CPF), as shown
in Fig.9.6a, b. The SEM image (Fig.9.6c) confirmed the formation of PATP/Au NP
membranes on the Au NPGC electrode surface, where the rough surface provided
a large surface area for the adsorption of target species at the modified electrode.
The cyclic voltammetric response of the imprinted PATPAuNP-based sensor to
CPF was about 3.2-fold more than that of the imprinted PAPTAu sensor (Fig.9.6d),
and the detection limit for CPF was about two orders of magnitude lower than by
the imprinted PAPTAu sensor.
However, not all analytes can be electroactive and hence detected using amperom-
etry. A method for the displacement of nonspecific electroactive markers from an
MIP has been developed for the detection and quantification of ligand-polymer bind-
ing events, which can be used for the development of multisensors [167]. Additionally,
the direct detection of an inert template required MIPs to possess the ability to
change conformation or surface potential upon binding with template. Sensors spe-
cific for l-phenylalanine and cholesterol [168] and sugars show high selectivity and
sensitivity at the micromolarnanomolar range. Another approach in MIP sensor
design for inert analyte detection is based on capacitance measurements [169, 170].
9.5 MIP-Based Biosensors 281
Fig.9.6 (a) Preparation procedures of the imprinted PATPAu NPGC electrode; (b) schematic
illustrations for the adsorption of the ATP molecule at the Au NP surface and the further self-
assembly of CPF at ATP-modified Au NP electrode; (c) typical SEM image of the imprinted PATP
membrane; and (d) cyclic voltammograms of (a) imprinted PATPAu NPGC electrode, (b) non-
imprinted PATPAu NPGC electrode, and (c) the imprinted PATPAu electrode in the presence
of CPF. Reprinted with permission from Xie etal. [88]. 2010, American Chemical Society
Mosbach et al. reported the first capacitive sensor by using MIP membranes as a
sensing layer in a field-effect device consisting of silicon wafers with an SiO2 coating
[116]. Upon specific binding of the original print molecule to the MIP membrane, a
reduction in the capacitance was observed. Based on this detection technique, Jus
group also designed an MIP capacitive sensor for enantioselective recognition of Glu
[169]. In this study, o-phenylenediamine and dopamine were chosen as monomers
and electrochemically copolymerized on gold electrode surface in the presence of
nonelectroactive template l- or d-Glu (Fig. 9.7a). The designed MIP capacitance
sensor displayed high enantioselectivity and sensitivity to the stereoselective rebind-
ing of l- or d- to their corresponding receptor-mimicking MIP films due to the exact
definition of the imprint cavity (Fig.9.7b, c).
Although many successful studies on the development of MIP-based electro-
chemical sensors have been reported, the current level of commercial activity related
to their development and marketing still cannot meet the requirements. Several critical
problems associated with MIP development need to be addressed before successful
commercialization can start [20]. They include (1) the development and validation
of a general protocol for MIP design; (2) the need for a substantial increase in poly-
mer affinity and improvement of the ratio between specific and nonspecific binding;
(3) the development of effective immobilization protocols. To realize these goals, a
better understanding needs to be established between polymer chemists working on
MIP development and engineers developing sensors.
282 9 Biosensing Applications of Molecularly Imprinted Nanomaterials
Fig.9.7 (a) Fabrication process of MIP film and the dependences of relative capacitance change
on the concentrations of (b) l-Glu at (a) l-Glu-imprinted and (b) nonimprinted sensors, and (c)
d-Glu at l-Glu-imprinted sensor as well as (c) (a) d- and (b) l-Glu at the d-Glu-imprinted sensors.
Reprinted with permission from Ouyang etal. [169]. 2007, Wiley
9.5.4Optical Sensors
Fig.9.8 Schematic illustration for the molecular imprinting (a) process and (b) mechanism of
LC-imprinted silica nanospheres embedded CdSe quantum dots, and (c) TEM images of (a) origi-
nal CdSe QDs, (b) CdSe-SiO2-MIP, inset: TEM image of a single CdSe-SiO2-MIP particle.
(c) SEM image of CdSe-SiO2-MIP, inset: SEM image of single CdSe-SiO2-MIP particle. Reprinted
with permission from Li etal. [137]. 2010, American Chemical Society
Fig. 9.9 The recognition of dansyl-proline with (a) proline MIP and (b) dansyl-proline MIP.
Reprinted with permission from Lin and Yamada [130]. 2000, American Chemical Society
showed good recognition behavior toward the analyte. Similarly, Li et al. [189]
developed an SPR-MIP senor by using the nanosized MIP films for the detection of
l-phenylalanine ethyl ester. The sensor showed good enantioselectivity during
rebinding events. However, in these experiments, the sensitivity of the SPR-MIP
sensors was not enough for the analysis of real samples. So an improved method
was developed by Kugimiya and Takeuchi [190]. They used a covalent approach
to graft a sialic acid MIP directly onto an allyl derivatized gold-coated SPR sur-
face to prepare an SPR-MIP sensor for ganglioside GM1, a sialic acidterminating
glycolipid. The resulting sialic acidimprinted SPR sensor responded linearly to
GM1, while it could not be used to detect concentrations lower than 0.1mg/mL.
Using a similar approach, Nishimura etal. [191] prepared an MIP-SPR sensor for
tetracaine. They grafted a methacrylic acidco-ethyleneglycol dimethacrylate
cinchonidineimprinted film onto an SPR transducer by using a covalent approach.
The sensor showed a linear response to tetracanine in the concentration range of
0.010.04mol/L.
In 2007, Raitman etal. [192] reported a very interesting SPR-MIP sensor for the
measurement of NADH. They combined the NADHimprinted polyacrylamide
polyacrylamidophenylboronic acid copolymer with SPR transducer to measure
NADH unambiguously at the 10-mM level. The catalytic properties of the MIP film
have been studied by detecting the oxidation of lactate to pyruvate by NAD+ in the
presence of lactate dehydrogenase. A number of recent studies have further
demonstrated the efficacy and adaptability of MIP-SPR sensor [193, 194]. For
example, Choi etal. [193] investigated the feasibility of employing the imprinting
technique for the detection of the mycotoxin zearalenone using an SPR transducer.
The molecularly imprinted polypyrrole (MIPPy) film was prepared by electropoly-
merization of pyrrole onto the bare Au chip in the presence of a template zearale-
none molecule (Fig.9.10). The MIPPy SPR sensor exhibited a linear response for
zearalenone in the range of 0.33,000ng/mL, with a detection limit of 0.3ng/mL.
The selectivity efficiencies of zearalenone and other structurally related analogs
were 1.0 and 0.150.27, respectively. These results suggested that a combination
of SPR sensing with MIPPy film was a promising alternative method for the detection
of zearalenone.
Willner etal. [194] first functionalized Au nanoparticles with thioaniline elec-
tropolymerizable groups and (mercaptophenyl)boronic acid, and then elctropoly-
merized the functionalized Au NPs in the presence of antibiotic substrate neomycin
(NE), kanamycin (KA), or streptomycin (ST) to yield bisaniline-cross-linked Au
NP composites. After the removal of the ligated antibiotics, the molecularly
imprinted matrixes were prepared and the antibiotics were sensed by SPR spectros-
copy. The resulting SPR-MIP sensor revealed high sensitivities and a linear response
toward the sensing of the antibiotic analytes, with detection limits of 2.00.21pM
for NE, 1.00.1pM for KA, and 20030fM for ST, respectively. The imprinted
Au NP composites were successfully used to analyze the antibiotics in milk samples.
Despite the inherent limitations in sensitivity arising when targeting low-molecular-
weight molecules, the combination of the two technologies still provided a promising
potential for its wide applications in biosensors.
288 9 Biosensing Applications of Molecularly Imprinted Nanomaterials
Fig.9.10 Schematic diagrams of the setup for electropolymerization and SPR detecting zearale-
none. (a) Preparation of molecularly imprinted polypyrrole on bare Au using a three-electrode
electrochemical system; (b) the shift in resonance angle of the SPR sensor resulting from the
rebinding of zearalenone to the MIPPy film. Reprinted with permission from Choi etal. [193].
2009, American Chemical Society
9.5.5Mass-Sensitive Devices
Fig.9.11 (a) Tapping-mode AFM picture showing the ongoing imprinting process on a coated
QCM electrode and (b) solgel layer from titaniumIV ethylate imprinted with S. cetrvisiae.
Reprinted with permission from Dickert and Hayden [196]. 2002, American Chemical Society
solution between a gold-coated QCM chip and the surface of a UV lamp. The
resultantsensor showed good enantioselectivity for the S enantiomer when com-
pared to the R enantiomer. Dickert and Hayden [196] modified a QCM surface
with a surfaced-imprinted solgel using whole yeast cells. In this study, the MIP
layer showed honeycomb-like structures (Fig.9.11). Figure9.11a shows the atomic
force microscopy (AFM) image of the ongoing imprinting process on a coated
QCM electrode. The polymerizing material incorporates half of single S. cerevisiae
cells and forms packed and highly ordered biomimetic receptors. Figure 9.11b
shows the solgel layer from titaniumIV ethylate imprinted with S. cerevisiae. The
coating is extremely robust and scratch-resistant. The reinclusion of cells allows a
selective online monitoring of these microorganism concentrations in water over
five orders of magnitude. The sensitivity to cells held up in growth media up to
21g/L. Even cell fragments could be detected in flowing conditions. Krozer etal.
[138] reported the QCM sensor with dissipation (QCM-D) by coating the sensor
surface with premade molecularly imprinted nanoparticles. The nanoparticles were
physically entrapped into a thin poly(ethylene terephthalate) (PET) layer spin-
coated on the transducer surface. By controlling the deposition conditions, a high
nanoparticle loading can be gained in the stable PET layer, allowing the recognition
sites in nanoparticles to be easily accessed by the test analytes. The highest uptake
of the nanoparticle film to propranolol corresponded to approximately 2nmol/cm2,
or about 11015molecules/cm2. The detection limit of the QCM-MIP sensor was
about 10mM, and chiral recognition and discrimination between R- and S-propranolol
can also be achieved. Similarly, Chan etal. [111] described the use of the thin per-
meable films of MIPs as biomimetic recognition materials on QCM surface. The
sensor can provide a high enantioselectivity and sensitivity for the discrimination of
l- and d-tryptophan enantiomers, with a detection limit of 8.8mM.
290 9 Biosensing Applications of Molecularly Imprinted Nanomaterials
Based on this approach, this group recently fabricated another QCM-MIP sensor
in the presence of epitope peptides for the detection of the protective antigen of
anthrax [200]. Those five peptides, such as (N-Acr-l-Cys-NHBn)2, are linear or
conformational epitopes of the anthrax-protective antigen PA83, which made the
resulting epitope-cavity show high affinity for the corresponding template, as shown
in Tables9.3 and 9.4. The affinities of the peptide to their corresponding MIPs were
more closely related to the molecular weight of the analyte than to the number of
residues. All epitope-cavities differentiated their epitope region on the protective
antigen PA83 as well as the corresponding furin-cleavage fragments PA63 and PA20.
The differential response to the protective antigen fragment could be observed in
the picomolar range.
292 9 Biosensing Applications of Molecularly Imprinted Nanomaterials
In order to prevent the spread of infection in animal and plant populations, the
rapid and inexpensive screening of viral and bacterial infections is urgently needed
[201204]. However, the detection of viruses and bacteria is generally time-
consuming and expensive because the biological detection needs a complex process,
including incubation, separation, dyeing, and microscopy. The bioimprinted QCM
sensors may provide a fast and selective biodetection. Hayden and coworkers
recently developed surface-imprinting techniques on polymer-coated QCM to detect
tobacco mosaic viruses (TMV) [109, 205] and living yeasts [196] in aqueous
media. The imprinting cavities or trenches on the polymer surface mimicking the
shape and surface functionality of the viruses and bacteria served as recognition
sites for their rebinding. The sensors were applicable to TMV detection ranging
from 100ng/mL to 1mg/mL [109], and allowed a selective online monitoring of the
yeast cell concentrations in water over five orders of magnitude [196]. Moreover,
they expended the bioimprinting concept to the recognition of mammalian cells,
which was a greater challenge because of their lower mechanical stability compared
to microorganisms [54, 130, 196, 206]. Erythrocyte-specific interactions with rec-
ognition sites on surface-imprinted polyurethane were applicable for blood-group
typing of the main ABO antigens [54]. The interesting finding in QCM-MIP sensors
was highly relevant for clinical applications in serology.
Recently, Pietrzyk etal. [112] devised a histamine piezoelectric sensor using an
electrodeposited MIP film as the recognition element. The preparation of the
sensing film involved two consecutive electrochemical polymerizations. First, a
poly(bithiophene) barrier film was deposited by electropolymerization on the Pt/
quartz resonator to prevent histamine electrooxidation and avoid possible
9.5 MIP-Based Biosensors 293
Fig.9.13 (a) Schematic of pollen stamp preparation: The pollen grains adhere to PDMS due to
adhesive forces. Excess pollen is blown off with pressurized air, resulting in a monolayer stamp;
(b) contact-mode AFM image of a polyurethane imprinted with pollen. Reprinted with permission
from Jenik etal. [206]. 2009, Springer
9.6Conclusions
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wwwwwwwwwwwww
Chapter 10
Biosensors Based on SolGel
NanoparticleMatrices
10.1Introduction
inert, porous, transparent, and mechanically stable nanostructures [19, 21, 22],
which are very suitable for biosensor fabrication [2325]. This chapter highlights
the advantages, recent developments, and future perspectives of biosensing applica-
tions based on solgel nanoparticle matrices.
10.2SolGel Chemistry
10.2.1What Is SolGel?
Sols are dispersions of colloidal particles in a liquid. Colloids are solid particles
with diameters of 1100nm. A gel is an interconnected, rigid network with pores of
submicrometer dimensions and polymeric chains whose average length is greater
than a micrometer. Three approaches are normally used to make solgel monoliths
[26]: (1) gelation of a solution of colloidal powders; (2) hydrolysis and polyconden-
sation of alkoxide or nitrate precursors followed by hypercritical drying of gels; (3)
hydrolysis and polycondensation of alkoxide precursors followed by aging and drying
under ambient atmospheres.
Fig.10.1 Structural relationship among a gel, a xerogel, and an aerogel (reprinted with permission
from Cushing etal. [33]. 2004, American Chemical Society)
i nterparticle necks and decreases the porosity. The strength of the gel thereby
increases with aging. An aged gel must develop sufficient strength to resist
cracking during drying.
Step 5: Drying. During drying, the liquid is removed from the interconnected pore
network. When a gel is evaporated to dryness, either by thermal evaporation or by
supercritical solvent extraction, the gel structure changes substantially in some
cases. During thermal drying or room-temperature evaporation, capillary forces
induce stresses on the gel that increase the coordination numbers of the particles and
induce collapse of the network. The increase in particle coordination numbers
results in the formation of additional linkages that strengthen the structure against
further collapse and eventually lead to the formation of a rigid pore structure.
Thestructure can therefore be considered as a collapsed and highly distorted form
of the original gel network, which results in xerogel.
The supercritical extraction of solvent from a gel does not induce capillary
stresses due to the lack of solventvapor interfaces. As a result, the compressive
forces exerted on the gel network are significantly diminished relative to those
created during formation of a xerogel, and lead to aerogels [33] (Fig.10.1).
Step 6: Dehydration or chemical stabilization. Through impregnated with optically
active polymers such as fluors, wavelength shifters, dyes, or nonlinear polymer,
surface silanol (SiOH) bonds can be removed from the pore network. This results
in a chemically stable ultraporous solid.
Step 7: Densification. Heating the porous gel at high temperatures causes densifica-
tion. The pores are eliminated, and the density ultimately becomes equivalent to
fused quartz or fused silica. The densification temperature depends considerably on
the dimensions of the pore network, the connectivity of the pores, and the surface
area [35]. The purity and homogeneity of dense gel-silica made by method (3) are
superior to other silica glass-processing methods. The ability to produce optics with
nearly theoretical limits of optical transmission, lower coefficients of thermal expan-
sion, and greater homogeneity, along with net shape casting, represents major
advances from solgel processing of monolith.
10.3 Biosensors Based on SolGel Nanoparticle Matrices 309
Fig.10.2 Schematic diagram showing the solgel process and its various products (reprinted with
permission from Gupta and Kumar [36]. 2008, IOP)
The solgel technique is the most widely used method to prepare various kinds of
nanoparticles (Fig.10.2) [36, 37]. First, sols themselves are dispersions of colloi-
dal particles with diameters of 1100nm in liquids, which can be used directly or
coated on a substrate though spinning, dipping, and spraying to get a nanoparticle
film [3840]. In addition, colloidal particles can be isolated from sols by drying,
centrifugation, and coprecipitation [4143] and so on. Second, by covering a com-
plete solgel process, through hydrolysis, gelation, aging, drying, stabilization,
and densification at high temperature, we can obtain nanoparticles from xerogel or
aerogel [26, 33].
Silica nanoparticles prepared by a solgel process have a high specific surface area
as well as good biocompatibility, which is favorable to biomolecule loading for
biosensors [13, 5559]. Biomolecules can attach to silica nanoparticles through an
electrostatic force. He et al. [55] assembled heme protein hemoglobin (Hb) or
myoglobin (Mb) and silica nanoparticles in a variety of charge states layer by layer
into films on solid surfaces to investigate the driving forces for film assembly. They
found that when the proteins and silicas were both negatively charged, stable layer-
by-layer films were successfully fabricated, although the amounts of proteins were
smaller than that in the films assembled by opposite charged nanoparticles and pro-
teins. This demonstrated the importance of localized Coulombic attractions between
the negatively charged nanoparticle surface and the positively charged amino acid
residues on the Mb or Hb surfaces in the assembly and for the stability of films.
Based on this finding, many kinds of biosensing methods have been developed.
Qhobosheane etal. [13] used freshly prepared pure silica nanoparticles to immo-
bilize two different enzymes, glutamate dehydrogenase (GDH) and lactate dehy-
drogenase (LDH), for biosensors, in which enzymes were involved in a similar
enzymatic reaction with their cosubstrates, glutamate and lactate, respectively.
10.3 Biosensors Based on SolGel Nanoparticle Matrices 311
Luckarift etal. [56] reported a simple and rapid method for the deposition of silica
nanoparticles coated with lysozyme onto a gold surface. The method was based
on the ability of lysozyme to mediate the formation of silica nanoparticles.
A monolayer of lysozyme was deposited via nonspecific binding to gold. The
lysozyme then mediated the self-assembled formation of the silica monolayer.
The silica layer significantly increased the surface area compared to the gold sub-
strate and was directly compatible with a detection system. Organophosphate
hydrolase was successfully encapsulated within the silica particles, and a detec-
tion limit for the substrate, paraoxon, using the surface-encapsulated enzyme was
found to be 20mM.
Grant etal. [57] utilized silica nanoparticles as an optical platform for the devel-
opment of a protease biosensor based on the chemical transduction method and
fluorescence resonance energy transfer (FRET). Tang et al. [58] formed glucose
biosensors with glucose oxidase (GOD) immobilized in a composite matrix, which
was composed of hydrophobic silica nanoparticles and polyvinyl butyral (PVB) by
a solgel method. The experiments showed that nanoparticles can significantly
enhance the catalytic activity of the immobilized enzyme. The current response can
be increased from tens of nanoamperometers to thousands of nanoamperometers
with the same glucose concentration, and the electrodes responded very quickly, to
about 1min. Tsagkogeorgas etal. [59] presented the encapsulation of anti-diclofenac
antibodies in silica nanoparticles by a combination of reverse-micelle and solgel
techniques. The antibody source was a purified IgG fraction originating from a
polyclonal rabbit antiserum. Tetramethyl orthosilicate was used as the precursor.
Rather uniform, monodispersed, and spherical silica particles of about 70-nm-diam-
eter size were fabricated. The biological activity of the encapsulated antibodies was
evaluated, which demonstrated an obvious advantage of this approach for a mild
immobilization of different antibody species.
Some small organic molecules, with optical and electrochemical activities, can
be doped into the mesopores of silica nanoparticles during the solgel process
[60, 61]. The doping procedures are shown in Fig.10.3. The dopant can enhance
the silica nanoparticle stability and generate or amplify useful biosensing signals
[13, 54, 62, 63].
Tapec etal. [62] utilized the combination of two silica precursors, tetraethy-
lorthosilicate and phenyltriethoxysilane, to synthesize organic dye-doped silica
nanoparticles. The nanoparticles could be synthesized in the nanometer range
with high photostability and minimal dye leakage. The silica matrix of the nano-
particles allowed different surface biomolecular modifications for biosensor and
bioanalysis applications. Biotin interaction of avidin-coated nanoparticles can
be used for the determination of biotinylated bovine serum albumin, and the
immobilization of GDH on the nanoparticle surfaces enabled the determination
of glutamate.
312 10 Biosensors Based on SolGel Nanoparticle Matrices
Fig.10.3 Procedure for the surface modification of dye-doped silica nanoparticles using a water-
in-oil microemulsion (reprinted with permission from Bagwe et al. [61]. 2006, American
Chemical Society)
Fig.10.4 Coimmobilization of HRP and anti-AFP on monodisperse SiO2 spheres (reprinted with
permission from Wu etal. [41]. 2009, American Chemical Society)
Due to some functional groups, for example, hydroxyl residues on the surface, silica
nanoparticle is easily derived through cross-linking [61, 67, 68]. This makes the
biofunctionalization process more convenient and diversified by using some chemi-
cal linkages.
Qian et al. [69] synthesized and characterized silica nanoparticles doped with
Nile red and further conjugated with biomolecules (such as apo-transferrin and folic
acid). In vitro experiments revealed that these functionalized nanoparticles can
serve as effective optical probes for specific targeting of cancer cells such as HeLa
cells. These doped silica nanoparticles may serve as a robust tool for early diagnosis
and therapy of cancer and other diseases. Lius group [41] reported a novel strategy
for the sensitive detection of biomarkers using horseradish peroxidase (HRP)-
functionalized silica nanoparticles as the label, which were fabricated by the
coimmobilization of HRP and r-fetoprotein antibody onto the surface of SiO2 nano-
particles using g-glycidoxypropyltrimethoxysilane (GPMS) as the linkage. The
electrochemical and chemiluminescence measurements showed 29.5- and 61-fold
increases in detection signals, respectively, in comparison with the traditional sand-
wich immunoassay (Fig.10.4).
Endo etal. [70] developed a localized surface plasmon resonance (LSPR)-based
label-free optical DNA biosensor based on gold-capped silica nanoparticles using a
silane-coupling reagent. The detection of PNA-DNA hybridization with target
oligonucleotides and PCR-amplified real samples were performed with a limit of
detection value of 0.677pM target DNA. Selective discrimination against a single-
base mismatch was also achieved. The LSPR-based biosensor was applicable to
monitor the interaction of biomolecules, such as proteins, whole cells, or receptors,
with a massively parallel detection capability in a highly miniaturized package.
In addition, the surface-functionalized mesoporous silica nanoparticle materials
can be readily internalized by animal and plant cells without posing any cytotoxicity
314 10 Biosensors Based on SolGel Nanoparticle Matrices
Fig. 10.5 Transmission electron microscopy images of human cervical cancer (HeLa) cells in
contact with MSN. (a) an MSN entering through the cell membrane. (b) an MSN trapped in vesicles
inside the cell (reprinted with permission from Slowing etal. [71]. 2007, Wiley)
Fig.10.6 TEM images of nanoparticle with 13nm Fe3O4 core, and (a) 5 and (b) 20nm silica shell,
(c) TEM image at high magnification of (b) (reprinted with permission from Won etal. [72].
2010, American Chemical Society)
Using some metal alkoxide, such as Ti, Al, and Zr, as precursors, through a solgel
process, metal oxide nanoparticles can be obtained. These oxide nanoparticles can
easily adsorb biomolecules for biosensing constructions. This has been discussed in
some review articles [36, 45, 78].
Topoglidis etal. [79, 80] prepared solgel-derived porous TiO2 nanoparticles for
protein immobilization. As optically transparent, semiconductor TiO2 could be used
to carry out direct spectroelectrochemistry of Hb (Fig. 10.7) and cytochrome c,
showing that the immobilized Hb retained its chemical functions of oxidation/
reduction (achieved using chemical oxidants and reductants) and ligand binding
(O2, CO, NO). Such solgel nanoparticle-immobilized Hb films could be used to
determine quantitatively the concentration of these dissolved gases.
Zhang etal. [81] used solgel-derived TiO2 nanoparticle film to immobilize HRP
on a pyrolytic graphite (PG) electrode. The enzyme incorporated in TiO2 nanopar-
ticle film retained its bioactivity. The direct electron transfer between HRP and PG
electrodes was greatly enhanced, indicating that the TiO2 nanoparticle could provide
10.3 Biosensors Based on SolGel Nanoparticle Matrices 317
Fig.10.7 Absorption spectra are shown for both (a) oxidized met Fe(III) and (b) electrochemi-
cally reduced deoxy Fe(II) -immobilized Hb/TiO films (reprinted with permission from Topoglidis
etal. [79]. 2000, Royal Society of Chemistry)
Fig.10.8 Cyclic voltammetric response of TiO2 nanoparticle/HRP biosensor to (A) H2O2 and
(B) dissolved O2 at different concentrations (reprinted with permission from Zhang etal. [81].
2004, Elsevier)
a favorable microenvironment for HRP to exchange electrons with the electrode and
facilitate the rate of electron transportation. Based on the direct electrochemistry of
HRP, a third-generation biosensor for H2O2 and dissolved O2 was presented
(Fig. 10.8). Obviously, in the above case, the biomolecules were immobilized
through direct physical adsorption.
Yu etal. [82] synthesized facilely mesoporous MnO2 through a solgel process
using nonionic surfactant polyxyethylene fatty alcohol as a template. The mesopo-
rous MnO2 material presented a disordered porous structure and an appropriate pore
size suitable for the immobilization of GOD. An amperometric glucose biosensor
based on GOD entrapped in mesoporous MnO2 was fabricated, in which mesopo-
rous MnO2 also acted as a catalyst for the electrochemical oxidation of H2O2
318 10 Biosensors Based on SolGel Nanoparticle Matrices
Fig.10.9 (A) Amperometric response of (a) gelatin/GCE and (b) mesoMnO2gelatin/GCE to the
addition of 0.98mM H2O2; (B) amperometric response of (c) GODgelatin/GCE, (d) mesoMnO2
gelatin/GCE, and (e) GODmesoMnO2gelatin/GCE (reprinted with permission from Yu et al.
[82]. 2008, Elsevier)
p roduced by enzyme reaction. The biosensor showed a fast and sensitive current
response to glucose in the linear range of 0.00092.73mM. The response time (t95%)
was less than 7 s (Fig. 10.9). The sensitivity and detection limit were 24.2 mA/
cm2mM and 1.8107M (S/N=3), respectively, indicating that mesoporous MnO2
has promising application in enzyme immobilization and biosensor construction.
Liu etal. [83] investigated a biosensor based on the use of ZrO2 solgel matrix
for enzyme immobilization in the mild condition. This bioceramic zirconia alcogel
was prepared by the novel alcohothermal route with a cheap inorganic salt
Zr(NO3)45H2O with several desirable features, including a large surface area (about
460m2/g) as well as pore volume and a well-developed textural mesoporosity, and
HRP was selected as a model enzyme. The results showed that the as-prepared zir-
conia matrix has an advantageous microenvironment and a large surface area avail-
able for high enzyme loading. The resulting biosensor exhibited a high sensitivity
of 111 mA/mM for hydrogen peroxide over a wide range of concentration from
2.5107 to 1.5104mol/L, a quick response of less than 10s, and good stability.
Kim etal. [84] developed an amperometric glucose biosensor by using the nano-
porous composite film of solgel-derived zirconia and perfluorosulfonated ionomer,
Nafion, for the encapsulation of GOD on a platinized glassy carbon electrode.
Zirconium isopropoxide (ZrOPr) was used as a solgel precursor for the preparation
of zirconia/Nafion composite film. The glucose biosensor based on the zirconia/
Nafion composite film could reach 95% of steady-state current in less than 5s. In
addition, the biosensor responded to glucose linearly in the range of 0.0315.08mM,
with a sensitivity of 3.40mA/mM and the detection limit of 0.037mM (S/N=3).
Moreover, the biosensor exhibited good sensor-to-sensor reproducibility (similar to 5%)
and long-term stability (90% of its original activity retained after 4 weeks) when
stored in 50mM phosphate buffer at pH 7 at 4C.
10.3 Biosensors Based on SolGel Nanoparticle Matrices 319
Fig.10.10 GNPs embedded in a 3D solgel network (reprinted with permission from Li etal.
[87]. 2010, World Gold Council)
As solgel matrix has a porous structure, nanoparticles can be incorporated inside the
pores in a solgel network to form nanocomposites or hybrids [8688], e.g., as shown
in Fig.10.10. Using organically modified solgel, termed Ormosil, to incorporate
nanoparticles, one can realize the surface assembly of solgel, which supplies an
alternative method for nanoparticle binding to solgel matrix [89, 90]. Moreover,
solgel matrices are highly biocompatible, which do not harm the bioactivities of
biomolecules. Nanoparticles in a solgel matrix can not only realize a better biomol-
ecule immobilization, but also play their own important roles, such as accelerating
electron transfer. In recent years, most reported solgel nanoparticle biosensors have
been built upon nanoparticle incorporation in solgel matrices [9196].
Among the metal nanoparticles used in biosensors, Au NPs have received greatest
interest because they have intriguing properties that can be applied to many fields,
320 10 Biosensors Based on SolGel Nanoparticle Matrices
Fig.10.11 Detection of ethanol vapors by a Pd/SnOx (2%) chemiresistor. Top: typical response
curve; Bottom: sequence of analyte injections in the measuring chamber (reprinted with permis-
sion from Cioffi etal. [126]. 2006, Elsevier)
titania nanoparticles (nano-TiO2) on the direct electron transfer between LDH and
the silica solgel-modified gold electrode by adding nano-TiO2 (50nm) in the modi-
fication process. This nano-TiO2-LDH electrode showed a pair of quasi-reversible
cyclic voltammetry peaks with the formal potential of 70mV (vs. SCE). Compared
to the pure LDH solgel-modified electrode, on this nano-TiO2-LDH solgel elec-
trode, results demonstrated that the direct electrochemistry of LDH was enhanced
by nano-TiO2. This electrode can be used as a biosensor for the determination of
lactic acid. The calibration range of lactic acid was 1.020mmol/L, and the detec-
tion limit was 0.4mmol/L. Meanwhile, the small Kmapp value (2.2mmol/L) suggested
that LDH possessed high enzymatic activity and good affinity to lactic acid due to
the promotion effect of nano-TiO2.
Carbon nanotubes are widely used for biosensor constructions [104, 128, 129]. The
presence of carbon nanotubes in solgel can facilitate electron transfer [92]. On the
other hand, the carbon/sol composite, called carbon ink, can be easily filled into the
tubes of different shapes or screened onto surfaces [130] (Fig.10.12). As the gela-
tion process is going, carbon nanomaterials, biofunctionalized or not, can be incor-
porated in the solgel matrix, to supply a versatile platform for biosensor construction.
Moreover, the composite surface can be renewed through polish, which provides a
chance to fabricate disposable biosensors, which are convenient for everyday life.
Many researchers use carbon nanotube solgel composite to construct biosen-
sors, especially electrochemical biosensors [131134]. For example, based on
CNTs self-catalytic activity, Abbaspour and Ghaffarinejad [131] used microwave
10.3 Biosensors Based on SolGel Nanoparticle Matrices 323
Fig.10.12 (a) Eight biogel-based strip electrodes produced by a screen-printing process; (b) SEM
image of the strip electrodes in (a) (reprinted with permission from Wang etal. [130]. 1996,
American Chemical Society)
Table10.1 Summary of the cyclic voltammetric data for several redox systems at
NCE and CCE
NCE CCE
Ep (mV) Ip (mA) Ep (mV) Ip (mA)
CySH 0.432 9.951 0.632 3.991
AA 0.161 21.22 0.242 11.94
UA 0.335 9.032 0.370 6.807
DA 0.176/0.123 13.99/2.723 0.193/0.130 8.654/0.6477
EP 0.430/0.09 63.94/40.22 0.460/0.070 43.02/22.61
AP 0.385/0.249 32.34/16.22 0.434/0.098 18.78/9.882
Reprinted with permission from Zhu etal. [132]. 2007, Elsevier
Fig.10.13 (A) Typical currenttime response curve and (B) calibration curve for successive addi-
tion of 0.5 mM cholesterol using (A) GC/PB/CS-SiO2-COX and (B) GC/PB/CS-SiO2-COX-
MWCNT electrode (reprinted with permission from Tan etal. [93]. 2005, Elsevier)
through direct electron transfer at the carbon nanotube electrode surface. In this
nanocomposite approach, the silica matrix was designed to be sufficiently porous
for substrate molecules to have access to the enzyme and yet provided a protective
cage for immobilization without affecting biological activity. The incorporation of
carbon nanotubes enhanced the electrical connectivity and increased the active elec-
trode surface area. The carbon nanotube sidewalls are primarily responsible for
direct electron-transfer processes.
In some cases, very complex solgel composites, which contain two or more kinds
of nanoparticles, are used for biosensor fabrication. Liu and Hu [139] fabricated an
H2O2 biosensor based on the direct electrochemistry and electrocatalysis of Mb
immobilized on silver nanoparticle-doped carbon nanotube film with hybrid solgel
techniques. A pair of redox peaks with peak separation of 160mV and formal poten-
tial of 0.295V was observed at this composite film, corresponding to the direct
electrochemistry of Mb. Under optimum conditions, the amperometric determination
of H2O2 was performed, with a linear range of 2.0 1061.2103 mol/L and a
detection limit of 3.6107mol/L (S/N=3). The MichaelisMenten constant was
also estimated to be 1.62mmol/L.
Kang et al. [24] presented a sensitivity-enhanced glucose biosensor based on
multiwalled carbon nanotubes, Pt nanoparticles (Pt NPs), and a solgel of chitosan
(CS)/silica organicinorganic hybrid composite. The CS/silica hybrid solgel was
produced by mixing methyltrimethoxysilane (MTOS) with the CNTPt NPCS
solution. With the immobilization of GOD into the solgel, the glucose biosensor of
GODCNTPt NPCSMTOSGCE was fabricated. The properties of the result-
ing glucose biosensor were measured by electrochemical impedance spectroscopy
and cyclic voltammetry. In phosphate buffer solutions (PBS, pH 6.8), the nearly
interference-free determination of glucose was realized at a low applied potential of
0.1V, with a wide linear range of 1.21066.0103M, a low detection limit of
3.0107M, a high sensitivity of 2.08mA/mM, and a fast response time (within
5s). The biosensor provided a highly synergistic electrocatalytic action, and exhib-
ited good reproducibility and long-term stability.
Guo etal. [140] employed an approach combining sonication and solgel chem-
istry to synthesize silica-coated CNT coaxial nanocables. It was found that a homo-
geneous silica layer can be coated on the surface of the CNTs. Au NPs-supported
coaxial nanocables were facilely obtained using amino-functionalized silica as the
interlinker. Furthermore, to reduce the cost of Pt in fuel cells, designing a Pt shell on
the surface of a noble metal such as gold or silver is necessary. High-density gold/
platinum hybrid nanoparticles were located on the surface of 1D coaxial nanocables
with high surface-to-volume ratios. It was found that this hybrid nanomaterial
exhibited a high electrocatalytic activity for enhancing oxygen reduction (a low
overpotential was associated with the oxygen reduction reaction and an almost four-
electron electroreduction of dioxygen to water).
326 10 Biosensors Based on SolGel Nanoparticle Matrices
10.4Conclusions
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Chapter 11
Nanostructure for Nitric Oxide Electrochemical
Sensing
11.1 Introduction
Fig.11.1 Schematic drawing of an integrated NO nanoelectrode showing the surface coated with
Nafion (dashed line) and WPI selective membrane (solid line). Reprinted with permission from
Zhang etal. [6], 2002, Elsevier
Fig.11.2 Typical amperograms at +860mV for additions of 50mM (AA), 50mM nitrite, 100mM
l-arginine (l-arg), 10mM DA, 100nM NO, and 200nM and 400nM NO, respectively, in a stirred,
saturated CuCl solution with an NO nanosensor coated with Nafion/WPI membranes. Reprinted
with permission from Zhang etal. [6], 2002, Elsevier
Besides the ultrasmall size of the electrodes that need to be developed, the sensitivity
and selectivity should be improved by modifying the electrodes. Malinski and Taha
developed a sensitive NO sensor by modifying the carbon fiber electrode with
Ni-porphyrin and Nafion and found that Ni-porphyrin had a catalytic effect on the
electrooxidation of NO [19]. Since then, many materials have been found to catalyze
11.3 Nanomaterials for Modification of NO Electrochemical Sensors 337
Fig.11.4 Formation of the NO sensor and measurement of NO in rat liver cells. Reprinted with
permission from Zheng etal. [28], 2008, Elsevier
NO in the range of 0.22120mM. The sensitivity and the determination limit were
0.25mA/(mmol/L) and 28nM, respectively. The sensor also showed good selectiv-
ity, stability (95% of its initial response remained after being kept for one week),
and reproducibility (parallel detection 10 times, with a relative standard deviation of
2.1%). This sensor was further used for measuring NO released from rat liver cells,
and results showed that the sensor could have practical applications in an NO moni-
toring system (Fig.11.4).
Not only carbon nanotubes, but also the organic nanotubes, such as Poly-
CuTAPc nanotube, can catalyze the electrooxidation of NO. Phthalocyanines are
weakly semiconducting organic dye materials and can be substituted with different
metals. CuPc is one of the most interesting MPc materials studied in the literature.
The structure of CuTAPc is demonstrated in Fig.11.5. Poly-CuTAPc has been used
for the determination of oxidizing gases due to the good p-electron donating prop-
erties of the Pc rings [29, 30]. Poly-CuTAPc is electropolymerized on the surface
of the electrode for NO sensor application [31]. Poly-MTAPc nanotubes are pre-
pared through Pt-coated anodic aluminum oxide (AAO) template-assisted elec-
tropolymerization. The SEM pictures of the synthesized Poly-MTAPc nanotubes
after the Pt and AAO have been washed out are shown in Fig.11.6. The oxidation
peak of NO on the Nafion/poly-CuTAPc nanotube-modified electrode is at +0.72V,
which is much lower than the values for bare Pt or carbon fiber electrodes
(0.90.95V). The negative shift in oxidation potential implied that poly-CuTAPc
11.3 Nanomaterials for Modification of NO Electrochemical Sensors 339
Fig.11.6 FE-SEM images of poly-CuTAPc nanotubes [A, B top views]. Reprinted with permis-
sion from Gu etal. [31], 2009, IOP
NO(aq) NO(Au)
NO(Au) NO + (Au) + e -1
NO + (Au) NO + (aq)
NO + (aq) + H 2 O NO 2 - (aq) + 2H + (aq)
4NO + (Au) + 2OH - (Au) 4NO(Au) + O 2 (aq) + 2H + (aq)
11.3 Nanomaterials for Modification of NO Electrochemical Sensors 341
Fig.11.7 (a) (a, c, e) Schematic diagram illustrating the process of modifying the carbon fiber
microdisk electrode (CFMDE). (b, d, f) The signal display provided by an oscilloscope-like win-
dow in PLUSE corresponding with three processes in (a), (c), and (e), respectively. (b) SEM
images of (a) the surface of a CFMDE and (b) the surface of an SWNT/Nafion modified CFMDE.
Reprinted with permission from Du etal. [14], 2008, Elsevier
Fig.11.8 (a) View of the calibration system to calibrate the CFMDE modified by SWNTs and
Nafion with NO solution of different concentrations. The microcapillary and the microelectrode
were both placed in a chamber filled with 1mL of PBS. The PBS was aerated to be in accordance
with physiological conditions in cell experiments. (b) View of the detection system in single-cell
experiments. A CFMDE modified by SWNTs and Nafion was placed at a fixed distance (generally
1 mm) away from the surface of a cell (HUVEC, human umbilical vein endothelial cells). The
outlet of the microcapillary rightly faced the cell to inject a stimulus toward it. Reprinted with
permission from Du etal. [14], 2008, Elsevier
Fig.11.9 SEM images of (a) the general view of carbon fiber ultramicroelectrode (CFUE), (b) the
midsection of bare CFUE, (c) the midsection of CFUE/MWNTs, and (d) the tip and cross-section
of CFUE/MWNTs. Reprinted with permission from Wang etal. [27], 2005, Elsevier
11.4 Conclusions
Nitric oxide acts as a very important role in organisms, such as vasodilatory mes-
senger, endothelium-derived relaxing factor, neurotransmitter, among other roles.
In order to elucidate the function of NO in the biology, different electrochemical
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wwwwwwwwwwwww
Chapter 12
Assembly of Nanostructures for Taste Sensing
12.1Introduction
Taste and smell are the two human senses that are chemical in nature. However, they
have not been as successfully replicated with sensors, probably because of the
complexity of the human system [1]. Although the science behind taste is still not fully
elucidated, it is known that it relies on a series of taste cells densely packed in taste buds
mainly located on the tongue and palate and in the pharynx. These taste buds are
connected to nerve fibers that carry the signals of the chemical environment to the brain
stem, where central processing of the information occurs [2]. It is commonly accepted
that humans distinguish at least five basic taste qualities: saltiness, sourness, bitterness,
sweetness, and umami, although some consider this model too simplified [3].
The electronic counterpart of tongues is an array of chemical sensors through which
a raw signal output is transferred to a computer system and the data are processed. As
an analogy to nature, it is generally assumed that taste sensors have to rely on sensors
with a rather broad or relatively low chemical selectivity [4]. This philosophy is also
supported by IUPAC in a technical report on electronic tongues [5]. The artificial
tongues are dedicated to the automatic analysis of complicated composition samples,
to the recognition of their characteristic properties, and to fast qualitative analysis.
Several approaches have been discussed for the transduction mechanism and
chemical selectivity. A wide variety of chemical sensors can be employed in the
design of electronic tongues: electrochemical (voltammetric, potentiometric), opti-
cal, or enzymatic sensors (biosensors). However, the majority of taste-sensing
systems for food and beverage analyses rely on arrays of electrochemical sensors.
In 1985, the first system for liquid analysis based on the multisensor array was
presented by Otto and Thomas [6]. Since then, a few devices of electronic tongues
have been presented. Most of these systems are based on potentiometric sensors,
especially ion-selective electrodes. The potential of the ion-selective electrodes is a
function of the activity of ionic species in a sample solution and is formed in the
ion-sensitive membrane, where selective complexation (ion recognition) of the
The detection and quantification of cells, proteins, and other biosystems in complex
matrices are important for disease detection. A wealth of methods is available to
attain this goal, including antibodies used in ELISA-type tests, proteomics and
related approaches coupled with mass spectrometry, as well as widely employed
techniques such as gel electrophoresis to detect serum imbalances in patients with
liver failure or other gross metabolic problems. The chemical nose/tongue
approach provides an alternative for the sensing protocols for analyte detection.
Strategically, the array is able to present chemical diversity to respond differentially
to a variety of analytes.
Bunz and Rotello created a sort of sensor array with noncovalent gold nanopar-
ticle (NP)fluorescent polymer conjugates to detect, identify, and quantify protein
12.3 Sensor Array Based on Gold NanoparticleFluorophore Complexes 353
targets [14]. Monolayer-protected gold NPs were polyvalent by design and can be
combined with any type of fluorophore. They covered positively charged gold NPs
with negatively charged conjugated polymers of the poly(para-phenyleneethy-
nylene) (PPE) type and with the likewise anionic green fluorescent protein (GFP).
In both cases, the combination of polyvalent fluorophore with the polyvalent gold
NP generated attractive self-assembled sensors that discerned proteins, bacteria,
and cells by analyte-induced fluorescence turn-on. This work demonstrated the
construction of novel nanomaterial-based protein detector arrays with potential
applications in medical diagnostics.
The combination of ammonium-functionalized 2-nm-core gold NPs with conju-
gated polymers or with GFP in water gave rise to a diverse set of self-assembled
hydrophobic or electrostatic complexes or hybrids. Depending upon the ratio of
conjugated polymer or GFP to gold NPs, the fluorescence of the construct can be
precisely tuned; the higher the concentration of NPs, the lower the emission inten-
sity. While the quenching mechanism was not fully understood, the overlap of the
absorption spectrum of the gold NP with the emission spectra of the fluorophores
suggested efficient energy-transfer quenching. The formation of these nonfluores-
cent constructs was driven by electrostatic interactions, so that the ammonium
head groups can carry a significant number of different substituents. The chemical
nature of the used ammonium groups somewhat modulated the fluorescence quench-
ing through the binding or association constants (Ka) between particle and fluoro-
phore. However, regardless of the NP used, Ka is always high and averages between
107 and 1011 in water, with ammonium salts sporting aromatic head groups display-
ing the highest binding. The high Ka values allowed for the efficient assembly of the
complexes at relatively low analytically and diagnostically relevant concentrations,
with fluorophores in high-nanomolar (100500nm) concentrations. The interaction
of the self-assembled hybrids with (negatively charged) biological analytes such as
proteins in water or in serum, inorganic ions such as PPi, and bacterial or mamma-
lian cells led to a fluorescence modulation of the constructs; in most cases, fluores-
cence turn-on results.
12.3.1Detection of Proteins
and the efficient quenching of fluorophores by the metallic core to impart efficient
transduction of the binding event. Through application of LDA, they were able to
use these fluorescence changes to identify and quantify proteins in a rapid, efficient,
and general fashion. The robust characteristics of the NP and polymer components,
coupled with the diversity of surface functionality that can be readily obtained using
NPs, made this array approach a promising technique for biomedical diagnostics.
The discrimination of proteins by the nonbiological NPPPE constructs is
powerful, perhaps because each NPPPE construct has an analog response toward
each protein. The sensing of single proteins in water, albeit of fundamental interest,
is not sufficient to use these types of constructs to determine analytes in complex
matrices such as serum, sperm, urine, or sweat. The most diagnostically important
biological matrix is human serum, the proteinacious solution that remains when
blood is freed from white and red blood cells.
Medicinal diagnostics of serum samples is generally done by simple electro-
phoresis to determine large-scale protein imbalances in serum that arise for liver
malfunction and other disease states, while for the detection of proteins such as
troponin, which are present in trace amounts, antibody assays are used. LDA
demonstrates that the proteins, with the exception of IgG and antitrypsin, are well
resolved in two dimensions, forming nonoverlapping patterns. Further experiments
indicated that mixtures of different proteins and the addition of one protein in
different concentrations also led to a specific and reproducible change in the LDA-
based patterns. Mixtures of proteins spiked into the serum could be detected and
gave specific responses.
Miranda etal. developed an enzyme-NP sensor array where the sensitivity was
amplified through enzymatic catalysis [17]. In this approach cationic gold NPs were
electrostatically bound to an enzyme (beta-galactosidase, beta-Gal), inhibiting
enzyme activity. Analyte proteins released the beta-Gal, restoring activity and
providing an amplified readout of the binding event. Using this strategy, we have
been able to identify proteins in buffer at a concentration of 1nM, which was lower
than current strategies for array-based protein sensing.
To minimize nonspecific interactions between the fluorophore and the serum
proteins, GFP was selected as the fluorophore in the sensing of serum proteins, as
PPEs can display nonspecific interactions with different proteins. Furthermore, GFP
has a defined size and molecular weight, and its fluorophore core is embedded in a
barrel-shaped protein, thus significantly reducing aggregation-induced quenching
and excimer formation, which can occur with conjugated polymers.
There is a direct correlation between protein levels and disease states in human
serum, which makes it an attractive target for sensors and diagnostics. However, this
is challenging because serum features more than 20,000 proteins, with an overall
protein content greater than 1mM. Rotello etal. reported a sensor based on a hybrid
synthetic biomolecule that used arrays of GFP and NPs to detect proteins at biorel-
evant concentrations in both buffer and human serum [18]. Distinct and reproduc-
ible fluorescence-response patterns were obtained from five serum proteins (human
serum albumin, immunoglobulin G, transferrin, fibrinogen, and alpha-antitrypsin),
both in buffer and when spiked into human serum. Using LDA, they identified these
12.3 Sensor Array Based on Gold NanoparticleFluorophore Complexes 355
proteins with an identification accuracy of 100% in buffer and 97% in human serum.
The arrays were also able to discriminate between different concentrations of the
same protein, as well as a mixture of different proteins in human serum.
These results suggested that simple libraries made from differently functional-
ized gold NPs and either biofluorophores such as GFP or conjugated polymers
recognized and detected proteins and protein imbalances in serum as a complex
matrix. This is a significant achievement for these fairly simple constructs, which
only employ electrostatic and hydrophobic interactions for the facile detection and
quantification of proteins in water, buffer, and serum.
metastatic cells. A successful differentiation would generate potential tools for the
development of simple assays for the early diagnosis of neoplastic growth and its
classification into metastatic or nonmetastatic cells. These three NPs in combination
with PPE generated the largest responses toward mammalian cells and were obtained
from the library; other NPs were tested but did not show significant responses when
exposed to mammalian cells. The three NPPPE constructs are able to differentiate
among four different human cancer cell lines but are also capable of discerning
breast tissue that is normal, cancerous, or metastatic.
The first report to study the CTL emission of organic vapors on nanosized materials
was published by Zhang and coworkers [23]. Seven nanosized materials in their
work were investigated, and CTL was detected on six of them, including MgO
(28nm), TiO2 (20nm), Al2O3 (18nm), Y2O3 (90nm), LaCoO3:Sr2+ (50nm), and
SrCO3 (25nm) [ZnO (68nm) did not emit CTL] while organic vapor passed through.
12.4 Catalytic Nanomaterial-Based Optical Sensor and Sensor Array 357
They applied this phenomenon to the development of several sensitive, stable gas
sensors. To demonstrate their original idea, a nanosized TiO2 was chosen as the
sensing material for preparing the gas sensor. Strong CTL emission was generated
by the catalytic oxidation of organic molecules on the surface of TiO2 NPs. The
analytical characteristics were evaluated by the examination of CTL emission from
trace ethanol and acetone on the sensor. The results indicated that this new sensing
mode could be developed by utilizing CTL on nanosized materials.
They also developed a catalytic chemiluminescent trimethylamine (TMA) sen-
sor [24]. Intensive CTL was detected when TMA was introduced over the surface of
nanosized catalysts and subsequently catalytically oxidized by O2 from the air, and
four catalysts were investigated, with the strongest CTL intensity obtained on nano-
sized Y2O3. This effect was utilized to develop a novel nanosized Y2O3-based cata-
lytic CTL sensor for TMA, which under optimal conditions exhibited a wide linear
range of 6042,000ppm and a detection limit of 10ppm. An attractive advantage of
this novel CTL sensor is its high selectivity to TMA with negligible responses to
many other gases such as NH3 and organic vapors. This CTL sensor had a short
response time of less than 3s, and showed good stability when examined by con-
tinual introduction of TMA into the sensor for 96h. The applicability of this sensor
to actual fish samples was also demonstrated in Zhang and coworkers report.
The released energy during a catalytic reaction can also be transformed into rare
earth metal ions to generate emission instead of excited intermediates. Zhang etal.
reported the observation of an energy-transfer process between excited intermedi-
ates and the nanosized catalysts [25]. The CTL was quenched when Ho3+, Co2+, and
Cu2+ were introduced into the catalyst, while new intensive CTL peaks appeared
when the catalyst was doped with Eu3+ or Tb3+. Further study indicated that the new
CTL peak on Eu3+- or Tb3+-doped catalyst originated from the luminescence of the
doped ions, excited by the energy transferred from excited intermediates produced
during the reaction. An ethanol sensor was developed with Eu3+-doped nanosized
ZrO2 that was linearly response to ethanol concentrations from 45 to 550 ppm.
Theenergy transfer led to 72 times higher sensitivity than the CTL from excited
intermediates in the sensor. High selectivity and stability were also obtained for this
sensor. The results indicated that the main factor limiting the sensitivity of a CTL
sensor on pure catalyst may be the inevitable energy quenching of excited inter
mediates with the catalyst, which was artfully utilized in the work by the intro
duction of Eu3+ that effectively absorbed this part of energy and transferred it into
light energy.
Hu etal. reported a gas sensor by using the CTL emission from the oxidation of
ethyl ether by oxygen in the air on the surface of borate glass [26]. Theoretical
calculations, together with experimental investigations, revealed that ethyl ether
was first oxidized to acetaldehyde and then to acetic acid, during which the main
luminous intermediates such as CH3CO were generated and emitted light with a
peak at 493nm. At a reaction temperature of 245C, the overall maximal emission
was found around 460nm. Interference from foreign substances, including alcohol
(methanol, ethanol and isopropanol), acetone, ethyl acetate, n-hexane, cyclo-
hexane, dichloromethane, or ether (n-butyl ether, tetrahydrofuran, propylene oxide,
358 12 Assembly of Nanostructures for Taste Sensing
isopropyl ether, and methyl tert-butyl ether), was not significant except for a minimal
signal from n-butyl ether (<2%). The proposed ethyl ether gas sensor offered the
following distinct advantages: (1) Compared with GC and GC/MS, it is a simple
and cost-effective sensor; (2) it is a greener sensor, since no toxic reagent/solvent
is needed, and the exhaust is of low toxicity and at low concentrations; and (3) it
shows much better sensitivity and selectivity, and faster response than many com-
mon VOC sensors.
Yang etal. described the development of a novel CTL sensor coupled with ionic
liquids (ILs)based headspace solid-phase microextraction (HS-SPME) technolo-
gies for the quantification of human plasma acetone levels associated with diabetic
disease ex vivo [27]. The unique properties of ILs, such as their nonvolatile and
nonflammable nature, coupled with their high thermal stability allowed ILs to be
conveniently adopted as pseudo-solid carriers for direct loading of acetone into a
CTL sensor without matrix interference. Acetone from diabetic patient plasma and
plasma samples spiked with acetone along with methanol, ethanol, and formalde-
hyde was conveniently and rapidly extracted and enriched in 3mL of IL and then
rapidly quantified by a CTL sensor. The presence of plasma alone or spiked plasma
containing methanol, ethanol, or formaldehyde did not interfere with acetone mea-
surements. HS-SPME-CTL provided higher enrichment efficiency than headspace
single-drop microextraction-based CTL (HS-SDMECTL) methods, possibly due to
the fact that the thin film formed in HS-SPME instead of the single IL drop in
HS-SDME increased the exchange area for extracted acetone. The enrichment effi-
ciency by HS-SPME-CTL was almost 80-fold higher than that with direct injection
using the same volume of aqueous samples and more than sixfold higher than that
using HS-SDME-CTL. Considering that ILs can be easily prepared from inexpen-
sive materials and tuned by the combination of different anions and cations for the
extraction of specific analytes from various solvent media, this proposed technology
raises an exciting possibility by employing HS-SPME-CTL for the fast determina-
tion of specific targets in many fields.
The different organic vapors can be discriminated from the different CTL responses
in the presence of the different nanosized materials. These materials are potentially
suitable for processing as a chip-mounted sensor array.
Na et al. found that luminescent efficiencies of the CTL are different for a
compound on different nanomaterials [28]. As shown in Fig.12.1a, ethanol gave the
strongest emission signal on ZrO2:Tb3+ (ZrO2 doped with 5% Tb3+), while giving no
signal on nanosized WO3 and Fe2O3. Hydrogen sulfide generated an obvious emis-
sion on WO3, Fe2O3, and Y2O3, but no emission on others. TMA produced strong
signals on ZrO2:Eu3+ (ZrO2 doped with 5% Eu3+), Al2O3, and ZrO2:Tb3+, but only
weak signals on others. Similarly, the same nanomaterial exhibited different CTL
12.4 Catalytic Nanomaterial-Based Optical Sensor and Sensor Array 359
Fig.12.2 Schematic diagrams of the CTL sensor array and the recorded images upon exposure to
various samples. (a) Schematic diagrams of the CTL sensor array; (b) images obtained by the sen-
sor array after exposure to air for 1min (a) without sample, (b) with ethanol vapor, (c) hydrogen
sulfide, and (d) TMA vapor. (c) Images obtained by the sensor array upon exposure to four alcohol
vapors: (a) methanol; (b) ethanol; (c) n-propanol; and (d) n-butanol. Reprinted with permission
from Na etal. [28]. 2006, American Chemical Society
The images of the sensor array can be adjusted by changing the working
temperature, because the route and rate of catalytic reaction are dependent on
temperature, which leads to different luminescent efficiencies and spectral shapes.
Their results indicated that even if similar images were recorded with the sensor
array for two analytes at one temperature, they may be differentiated according to
the images at another temperature.
The sensor array can also be used to fulfill quantification of a given analyte
by the CTL emission intensity, because the CTL intensities vary linearly with
the change in the analyte concentration. The linear range for the determination
of ethanol was 45550 ppm with a detection limit of 15 ppm on the spot of
ZrO2:Eu3+; for hydrogen sulfide on Fe2O3 it was 8.02,000ppm with a detection
limit of 3.0ppm; while for TMA on Y2O3, it was 6042,000ppm with a detec-
tion limit of 10ppm. It should be pointed out that the linear range and detection
limit for each analyte vary significantly from different catalytic nanomaterials.
12.4 Catalytic Nanomaterial-Based Optical Sensor and Sensor Array 361
The reversible response and relative long-term stability of the sensor array
indicated its perspective in real applications.
This sensor array was applied for the discrimination and identification of flavors
in cigarettes [29]. Twenty-one nanomaterials, including metal oxides, metal oxides
deposited on CNTs, gold NPs deposited on metal oxides, and carbonate, have been
carefully selected as sensing elements of the array. Eleven flavors commonly used
in the cigarette industry were examined, including ethyl acetate, butyl acetate,
benzyl benzoate, benzyl alcohol, phenylethyl alcohol, furfural, eugenol, butane-
dione, anisaldehyde, benzaldehyde, and iso-valeric acid. The unique patterns of the
11 flavors were obtained individually on the sensor array, indicating the different
CTL properties of each flavor on the catalytic nanomaterials. The results were given
numerically, which functioned as fingerprints of each flavor and gave us the
intuitionistic information of the differences among flavors. With these results, facile
discrimination of one flavor from the others can be achieved by the naked eye from
their unique patterns. Hierarchical cluster analysis (HCA) and LDA were used to
analyze the patterns. The obtained CTL patterns were temperature-dependent; thus,
additional discrimination power could be provided by changing the working
temperature of the array. Quantification of the flavors has been performed according
to the emission intensity on the specific sensing element. The linear range of the
sensor array for the flavors was 202,000ppmv, with the limits of detection below
10ppmv, which varied with the kinds of flavors.
Six brands of cigarettes were analyzed in order to demonstrate the potential of
the sensor array for complex compounds. The tobacco was heated at 80C for
15min to evaporate the flavors, and the vapors were injected into the sensor array
by the carrier gas for the detection. Six brands of cigarettes were discerned by their
CTL patterns obtained with the present sensor array. The results demonstrated the
potential analytical applications of a catalytic nanomaterial-based CTL sensor array
for the discrimination and identification of flavors. The robust and reversible
response of this array, combined with its simple instrumentation, indicated the
promise of this array to real-world applications.
The sensor array based on nanomaterials has been extended to the discrimination of
samples in solution, since amino acids, saccharides, and steroid pharmaceuticals
can produce CL emission on the surface of nanomaterials.
A novel aerosol CTL detector based on porous alumina was developed according
to the generated CTL emission from catalytic oxidation of the analytes [30]. This
aerosol CTL-based detector has three main processes: nebulization of solution; CTL
emission on surface of porous alumina material; and optical detection. To demon-
strate the utility of the aerosol CTL detector, some compounds such as saccharides,
poly(ethylene glycol)s, amino acids, and steroid pharmaceuticals were determined
362 12 Assembly of Nanostructures for Taste Sensing
12.5Conclusions
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Chapter 13
Nanostructured Biosensing for Detection of
Insecticides
13.1Introduction
Great efforts have been devoted to develop highly sensitive methods for the deter-
mination of pesticide residues in food and water. The identification and quantifica-
tion of pesticides are generally based on chromatographic methods, such as gas
chromatography (GC) or high-performance liquid chromatography (HPLC) cou-
pled with mass spectroscopy (MS). These analytical techniques have been described
and reviewed extensively in the literature [810].
These methods are highly efficient and allow for multiresidue analysis; how-
ever, they require tedious sample pretreatments, highly qualified technicians, and
OPs have been detected based on various principles, including spectroscopy, chro-
matography, microgravimetry, and electrical and electrochemical techniques.
Among the devices used for detecting OPs, electrochemical devices such as modi-
fied electrodes are relatively inexpensive, small in size, and easy to operate [2022].
One of the characteristic features of electrochemical devices is that the function and
performance of electrodes can arbitrarily be regulated by modifying the surface
with functional molecules, proteins, DNA, etc. [2325]. Thus, electrochemical
devices can be developed for detecting chemicals in sample solutions and in a gas
phase. The modified electrode with a surface modified with proteins and other bio-
logical molecules is often called a biosensor. For this reason, modified electrodes or
biosensors have been widely used for detecting OP agents [2628].
It is well established that electrochemical techniques are useful for determining
ions and molecules in solution and in a gas phase. Oxidizable and reducible chemical
species dissolved in solution can be detected by measuring oxidation or reduction
current that is produced upon electrolysis of the species on the surface of electrodes.
Usually, the output current of the system depends on the concentration of species in
the solution. Thus, one can quantify the concentration of the analyte. Another merit
of electrochemical techniques is the easy identification of analyte by recording the
13.2 Enzymes Used in Pesticide Biosensors 367
redox potential of the analyte, because the redox species can be characterized by its
own redox potential. Electrochemical biosensors are devices that are fabricated by
combining proteins or other biological molecules and electrodes. In recent years,
electrochemical biosensors with high sensitivity, long-term stability, and low-cost
detection of specific biological binding events have extensively reduced sampling
and testing times in pesticide determinations [29]. Satisfactory results were obtained
when AChE was immobilized on modified electrode matrices. However, the enzyme-
immobilization technique remains rather complicated and often involves very com-
plex matrices [15]. Further, the incorporation of AChE into certain mediator matrices
also lowered the stability of the enzyme [30], and the reproducibility after pesticide
inhibition was also poor in such AChE sensors. In recent years, AChE has been
immobilized onto various nanomaterial surfaces to improve the response and stabil-
ity in trace pesticide detection. These nanomaterial matrices include carbon nano-
tubes (CNTs) [1722], gold nanoparticles (Au NPs), and others [2326]. These
matrices significantly promote the stability, sensitivity, and detection limit in OP
determination in the picomolar (pM) to nanomolar (nM) concentration range.
The electrochemical biosensors used for detecting OP compounds can be divided into
three types depending on the type of enzymes used for constructing biosensors: (1)
(ChE)-choline oxidase (ChO) or bienzyme-modified biosensors; (2) ChE-modified
biosensors; and (3) OPH-modified biosensors.
The most general approach for the determination of OPs is based on their inhibition
of the activity of ChE. The very interesting method for determination of choline
esterase activity is based on coupling two consecutive reactions catalyzed by AChE
and choline oxidase (ChO) enzymes.
The primary objective of ChE-ChO bienzyme biosensors is to determine
neurotransmitter ACh in biological samples [31]. The redox potential of ACh is too
high to be determined directly using electrochemical reaction. For this purpose,
AChE is employed as ChE. AChE and ChO are immobilized on the surface of the
electrode to perform electrochemical analysis. These sensors are based on the
enzymatic reaction of ChO to detect ACh or the use of AChE with ChO to measure
ACh, shown as follows:
ChO sensor:
Choline + O 2
ChO
Betaine aldehyde + H 2 O 2 (13.1)
368 13 Nanostructured Biosensing for Detection of Insecticides
Acetylcholine + H 2 O
AChE
Choline + acetate (13.2)
Choline + O 2
ChO
Betaine aldehyde + H 2 O 2 (13.3)
2Thiocholine(red) Thiocholine(ox)(dimeric) + 2H + + 2e -
With the signal amplification of Au NPs, the specific interactions between the Au
NP-labeled carbamate inhibitors (ALC1 and ALC2) and the immobilized AChE on
the sensor chip surface were readily examined. The association/dissociation con-
stants for the binding interaction between carbamate inhibitors and AChE were
reported for the first time. This Au NP labeling strategy is versatile and may be
applicable for the direct or competitive SPR kinetic assay of the interaction between
small-molecule inhibitors and their target proteins with high sensitivity.
The key aspect in the construction of this kind of biosensor is the immobilization
of AChE on the solid electrode surface with a high electron-transfer rate and bioac-
tivity. In order to settle it, a variety of matrix materials have been employed, such as
cobalt phthalocyanine (CoPc) [36], Au NPs [37, 38], CdTe QDs [39], multiwalled
carbon nanotubes (MWNTs) [21], Al2O3 [40], Prussian blue [41], polyaniline [42],
chitosan [43, 44], and so on.
Despite the significant focus of scientific research dedicated to AChE biosen-
sors, little success has been realized through real practical applications and com-
mercialization of these devices for solving real-world problems. A major limitation
of existing AChE biosensors is related to their inability to correctly differentiate
and identify particular analytes, so the selectivity for measuring AChE inhibitors is
very poor [45, 46]. In general, all organophosphorous and carbamate pesticides
and heavy metals inhibit the AChE activity with a different degree of inhibition,
which makes proper identification difficult. In other cases, the inhibition power
registered with AChE biosensors does not follow the same pattern as obtained with
the enzyme in solution. This has been attributed to the matrix used to immobilize
the enzyme [4749]. Due to this important limitation, AChE biosensors are mainly
attractive for measuring the total toxicity of the sample, rather than measuring a
13.2 Enzymes Used in Pesticide Biosensors 371
specific inhibitor. This will reflect the sum of all AChE inhibiting species present
in the sample. The most important challenge in the development of AChE biosen-
sors for practical applications is the transfer of these devices from pristine research
laboratory conditions to real-life and commercial applications. In this direction,
some critical parameters such as enzyme stability, reliability, and selectivity still
have to be improved.
The detection of OPs using biosensors modified with ChE-ChO or ChE enzymes
relies on an inhibition of catalytic activity of ChE enzymes by OPs. Therefore, it is
a drawback of the sensors that the magnitude of the output signal correlates inversely
with the concentration of OPs in the sample. The protocol in measurements is some-
what complicated because the substrate of ChE has to be added in the sample solu-
tion before measurements, resulting in multiple steps needed for OP detection. In
addition, the inhibition of ChE by OPs is usually irreversible, and thus reactivation
of ChE activity is required for repeated use of the sensors, although this problem
can be circumvented by using disposable sensor tips.
In contrast to the inhibition mode of detection in the above biosensors, an
alternative direct biosensing route for the detection of OPs has been proposed by the
use of OPH [50], which can be used for continuous monitoring of OPs in the envi-
ronment. The foundation of these devices is the hydrolysis of OPs, producing two
protons as a result of the cleavage of the PO, PF, PS, or PCN bonds and an
alcohol, which in many cases is chromophoric and/or electroactive, catalyzed in a
highly specific manner by OPH [5153].
Because the organophosphate is the substrate for OPH, this process leads to
a direct determination of analyte, as the rate of signal generation is directly
proportional to the concentration of organophosphate. Among a variety of bio-
logical methods based on the biocatalytic activity of OPH, amperometric, poten-
tiometric, and optical biosensing devices have been developed for detecting OPs
[54]. Electrochemical biosensors in particular have been widely investigated to
monitor various pesticides, including OP compounds such as paraoxon [55],
parathion [56], sarin [57], and soman [58], via an enzyme-catalyzed hydrolysis
reaction by OPH due to their fast speed, high efficiency, low cost, and small
sample size [55].
Recently, Deo etal. [59] described an amperometric biosensor for organophosphate
pesticides based on a CNTmodified transducer and OPH biocatalyst. Chen et al.
developed OPH amperometric biosensors with carbon electrodes for monitoring OPs
[57]. The widespread interest in OPH-based electrochemical biosensors stems from
their simplicity, directness, and speed of determining OPs. The detection limit of OPH-
based biosensors could be further improved by either lowering the enzyme Michaelis
constant (Km) or increasing the bimolecular rate constant.
372 13 Nanostructured Biosensing for Detection of Insecticides
Despite the fact that the above biosensors provide great advantages, their emergence
from the research laboratory to the marketplace has been slow. The obstacles to
exploitation have been fundamentally related to the presence of biomaterial in the
biosensor (immobilization of biomolecules on transducers, stability and selectivity
of enzymes and antibodies), the development of the sensor device (sensitivity and
reproducibility issues), and the integration of biosensors into complete systems.
Major fundamental and technological advances should be made in the near future
toward enhancing the sensitivity, selectivity, and reliability of OP biosensing
devices, and the emergence of nanotechnology may open new horizons and satisfy
the above targets [60].
In recent years, nanomaterials have been widely employed in electrode modifica-
tion and electrochemical sensor development. The nanomaterials and their applica-
tions have been the subject of many reviews [61]. The advantages of application of
nanotechnology in the field of biosensors are multiple. First, their extremely small
sizes offer high surface-to-volume ratios, leading to loading of more enzyme as well
as high interface sensitivity. Second, due to the unique nanosize effect, nanomaterials
such as metal and semiconductor nanoparticles have excellent optical or electrical
properties, which can be used as the highly sensitive transducer signals in biosen-
sors. Finally, enzyme engineering at the nanoscale may be one of the most promis-
ing techniques with respect to the design of more sensitive and selective AChE and
OPH variants. Altogether, nanotechnology can be expected to be an effective
approach to develop more sophisticated multianalyte detection systems with a low
cost.
Since their discovery in 1991 [62], CNTs have been the target of numerous
investigations, due to their extraordinary conductivity, large surface areas, excellent
biocompatibility, and easy chemical functionalities, which make them ideal for the
development of compound-specific biosensors [63]. Recent studies have demon-
strated that CNTs are promising materials for electrochemical biosensors and show
great potential for use in the next generation of biosensors [18]. CNTs have been
considered potential electrode materials of electrochemical biosensors due to their
highly accessible surface area, electronic conductivity, stability, and capacity to
immobilize enzymes [20].
Most reported CNTs-based biosensors for detecting OPs are enzyme-modified
electrodes. These types of sensors take advantage of enzymatic reaction-generated
electroactive products, which can be sensitively detected on CNTs-modified elec-
trodes. Here, the CNTs on the electrode surface functions as both an electrode for
attaching enzymes and a transducer for enhancing the electrochemical response.
Moreover, the CNTs can promote the electron-transfer reaction between the signaling
molecules and the electrode.
13.3 Nanostructured Biosensor Design for Pesticide Analysis 373
Fig.13.2 Schematic
representation of layer-by-
layer electrostatic self-
assembly of AChE on
MWCNTs: (a) assembling
positively charged PDDA on
negatively charged
MWCNTs; (b) assembling
negatively charged AChE; (c)
assembling the second PDDA
layer. Reprinted with
permission from Liu and Lin
[19]. 2006, American
Chemical Society
GCE surface and dried. More negative charges were introduced above this
MWCNT-modified GCE by dipping in 1M NaOH solution for 5min. This nega-
tively charged MWCNT/GCE was dipped into an aqueous solution of 1mg/mL
PDDA containing 0.5M NaCl for 20min, which led to the adsorption of a posi-
tively charged polycation layer (Fig.13.2a). Above this polycation layer, a nega-
tively charged AChE layer was adsorbed by dipping the PDDA/MWCNTs/GCE
in 0.2unit/mL of AChE in TrisHCl buffer solution pH 8.0 (Fig.13.2b). Finally,
in order to prevent the leakage of AChE from the electrodes surface, another
PDDA layer was adsorbed above this AChE layer (Fig.13.2c). This results in a
sandwich-like structure of PDDA/AChE/PDDA on the MWCNTs surface.
Transmission electron microscopy (TEM) results confirm the formation of LBL
nanostructures on MWCNTs. The flow-injection analysis (FIA) results show that
PDDA/AChE/PDDA/MWCNT/GCE exhibits good reproducibility and stability,
with no surface fouling effect. The relative inhibition of AChE activity was linear
with-log [paraoxon] at the concentration range 110120.1109M. The detec-
tion limit was reported as 0.41012M.
Similarly, Joshi etal. reported nM paraoxon detection at AChE-functionalized
MWCNT-modified screen-printed electrode (SPE) [17]. They immobilized AChE
by physical adsorption without any mediators. Further, Du et al. reported that
incorporation of silica solgel (SiSG) into an MWCNT matrix improved triazo-
phos detection [21]. The MWCNT-incorporated solgel matrix provided a bio-
compatible microenvironment around the enzyme, which efficiently prevented
the leakage of the enzyme from the film. Under optimum experimental condi-
tions, the inhibition of triazophos was proportional to its concentration, from 20
to 1,000 nM and 530 mM, respectively. The detection limit was 5 nM. The
determination of triazophos in garlic samples showed an acceptable accuracy.
The fabrication reproducibility of this sensor was good, and the stability was also
acceptable. The same group developed MWCNT as a new sorbent for the solid-
phase extraction (SPE) of OPs [64]. A combination of SPE with square-wave
voltammetric (SWV) analysis resulted in a fast, sensitive, and selective electro-
chemical method for the determination of OPs using methyl parathion (MP) as a
representative. Because of the strong affinity of MWCNT for phosphoric group,
nitroaromatic OP compounds can strongly bind to the MWCNT surface. The
macroporosity and heterogeneity of MWCNT allow a large amount of MP to be
extracted in less than 5min. The stripping response was highly linear over the
MP range of 0.052.0mg/mL, with a detection limit of 5ng/mL. The determina-
tion of MP in garlic samples showed an acceptable accuracy. The fast extraction
ability of MWCNT makes it a promising sorbent for various solid-phase
extractions.
Jus group reported a novel method for the immobilization of AChE by binding
covalently to a cross-linked chitosanMWCNT composite [65]. As shown in
Fig.13.3, the MWNTs were homogeneously distributed in the chitosan membrane,
which showed a homogeneous porous structure. The immobilized AChE could
catalyze the hydrolysis of ATCl with a Km of 177mm to form thiocholine, which
was then oxidized to produce a detectable signal in a linear range of 1.0500mm
13.3 Nanostructured Biosensor Design for Pesticide Analysis 375
Au NPs have also been widely used in biosensors to immobilize biomolecules. The
ability of Au NPs to provide a stable immobilization of biomolecules retaining their
bioactivity is a major advantage for the preparation of biosensors. Furthermore, Au
NPs permit the direct electron transfer between redox proteins and bulk electrode
materials, thus allowing electrochemical sensing to be performed with no need for
electron-transfer mediators. Characteristics of gold nanoparticles, such as high
surface-to-volume ratio, high surface energy, ability to decrease the proteinmetal
particle distance, and functioning as electron-conducting pathways between pros-
thetic groups and the electrode surface, have been claimed as reasons to facilitate
electron transfer between redox proteins and electrode surfaces [66, 67]. With the
use of Au NPs, the efficiency and stability of the pesticide sensor have been greatly
amplified. Moreover, the nanoparticle matrix offers a very friendly environment to
the immobilized enzyme.
A simple strategy for the design of an electrochemical AChE sensor based on the
enzyme-induced growth of Au NPs without adding gold nanoseeds was proposed [24].
376 13 Nanostructured Biosensing for Detection of Insecticides
In this method, immobilized AChE mediated hydrolysis of ATCl and yielded a reducing
agent, thiocholine. In the presence of AuCl4, Au NPs were formed without the addi-
tion of any metal nanoparticles. Atomic force microscopy (AFM) images proved that
small spherical nanoparticles could grow on the enzyme-modified surface after incu-
bation in HAuCl4 and ATCl solution. An [Fe(CN)6]3/4 redox system was used as
a probe to indicate the process of electron transfer across the interface and also to
analyze the enzyme inhibitor quantitatively. The inhibition effect of malathion on
AChE was proportional to its concentration in the range 0.1500ng/mL, with detec-
tion limit of 0.03ng/mL.
Fig.13.4 Assembly of the CdS nanoparticle/AChE hybrid system used for the photoelectrochem-
ical detection of the enzyme activity. Reprinted with permission from Pardo-Yissar et al. [73].
2003, American Chemical Society
13.4Pesticide Immunosensors
Electrochemical transducers are the oldest and most common methods used in
biosensors. The principle is based on the electrical properties of the electrode or
buffer that is affected by AbAg interaction. They can determine the level of pesti-
cides by measuring the change of potential, current, conductance, or impedance
378 13 Nanostructured Biosensing for Detection of Insecticides
activity levels will not identify the specific inhibitory OP agent. Many animals,
including pig and rodent species, have abundant carboxylesterase (CE) in plasma. In
humans, CE is not free in plasma, but can be purified from a membrane-bound form
on monocytes [85]. While perhaps not an ideal biomarker, CE can be inhibited by an
array of OPs. Due to its freely soluble presence in plasma, BChE has been a popular
target for biomarker research. Newer methods have been developed that can identify
and quantify OP exposure based on BChE modification. MS detection is a major
advance in diagnosing OP exposure.
A disadvantage of AChE or BChE as biomarkers is that the OP residues are
prone to aging, in which the O-alkyl group is cleaved, thus losing key structural
information; this occurs particularly rapidly with soman. Also, the entire OP
moiety may be partially displaced from nonaged residues if therapeutic oximes
have been administered. Albumin adducts, which are much more stable with
regard to aging or oxime displacement, have been identified in experimental
animals but appear to be valuable biomarkers at higher exposure levels in com-
parison to BChE adducts.
Fig. 13.7 Schematic illustration of OP-AChE formation and AChE regeneration process by
reactivator. Reprinted with permission from Du etal. [121]. 2009, American Chemical Society
Motivated by this idea, Du, Lin, and colleagues [121] developed a portable,
rapid, and sensitive assessment of subclinical OP exposure based on the reactivation
of cholinesterase (ChE) from OP-inhibited ChE using rat saliva (invitro) using an
electrochemical sensor coupled with a microflow-injection system (Fig.13.7). The
sensor was based on a CNTmodified screen-printed carbon electrode (SPE), which
was integrated into a flow cell. Due to the extent of interindividual ChE activity
variability, ChE biomonitoring often requires an initial baseline determination
(noninhibited) of enzyme activity, which is then directly compared with activity
after OP exposure. They describe an alternative strategy where the reactivation of
the phosphorylated enzyme was exploited to enable the measurement of both inhib-
ited and baseline ChE activity in the same sample. Paraoxon was selected as a model
OP compound for invitro inhibition studies. A level of 9295% ChE reactivation
can be achieved over a broad range of ChE inhibition (594%) with paraoxon. The
extent of enzyme inhibition using this electrochemical sensor correlates well with
conventional enzyme-activity measurements. Based on the double determinations
of enzyme activity, this flow-injection device has been successfully used to detect
paraoxon inhibition efficiency in saliva samples, which demonstrated its promise as
a sensitive monitor of OP exposure in biological fluids.
13.6Conclusions
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wwwwwwwwwwwww
Chapter 14
Carbohydrate Detection Using Nanostructured
Biosensing
14.1Introduction
The basic structural unit of carbohydrate, which cannot be hydrolyzed into a simple
unit, is a monosaccharide. There are two types of monosaccharides: aldoses and
ketoses. A monosaccharide with an aldehyde group at the end of the carbon chain is
called an aldose, whereas that with a ketone group at an inner carbon of the carbon
chain is called a ketose. Free monosaccharides can exist in open chain or ring forms
(Fig. 14.1). Ring forms of the monosaccharides are the rule in oligosaccharides,
which are branched or linear chains of monosaccharides attached to one another via
glycosidic linkages. The ring form of a monosaccharide generates a chiral center at
C-1 for aldo sugars or at C-2 for keto sugars. A glycosidic linkage involves the
attachment of a monosaccharide to another residue, typically via the hydroxyl group
of this chiral center, which can be a or b linkages. These two types of linkages lead
to very different structural properties and biological functions upon sequences [5].
14.2.2Glycoconjugate
Fig.14.2 Schematic
representation of the
Thy-1-glycoprotein,
including the three N-glycans
and a glycophospholipid
(GPI-glycan) anchor whose
acyl chains would normally
be embedded in the
membrane bilayer
or galactose to the terminal primary hydroxyl group of the lipid moiety ceramide,
which is composed of a long chain base and a fatty acid. These are only the most
common classes of glycans reported in eukaryotic cells. These are several other less
common types found on both sides of the cell membrane in animal cells [912].
Unlike oligonucleotides and proteins, glycan chains are rarely expressed in a linear,
unbranched fashion, and even when they are, such chains are often subject to various
modifications [23, 24]. Changes in expression of glycans are also found in the set-
ting of transformation and progression to malignancy. The evolution of glycosyla-
tion is clearly shared, although there are unique features of glycosylation in different
animals, and an increasing complexity is often seen in higher forms. Intra- and
interspecies variations in gycosylation are also relatively common [1217].
Whenever a new probe (e.g., antibody or lectin), specific for a particular glycan, is
developed for fully understanding the biology of glycoconjugates, it requires
in-depth knowledge of the primary sequence and three-dimensional structure of
carbohydrate chains in relation to cellular activation, embryonic development,
organogenesis, and differentiation. These spatially and temporally controlled
patterns of glycan expression imply the mechanistic involvement of the glycan in
many processes. However, the biological consequences of altering glycosylation in
various systems seem to be highly variable and unpredictable. A given glycan can
also have different roles in different tissues or at different times in development.
Such unusual glycans or modifications are also more likely to be targets for
pathogens and toxins. Finally, since genetic defects in glycosylation are easily
obtained in cultured cells, but often have limited consequences, many major func-
tions of glycans may be imperative only within an intact organism [21, 22, 25].
14.5ProteinGlycan Interactions
For most carbohydrate, the secondary and higher-order structures in solution are not
readily defined, due to their inherent flexibility. The structural diversity of naturally
occurring glycans is great, making it difficult to define a universal protocol for their
analysis. The building blocks of complex carbohydrates (i.e., the monosaccharides)
exhibit very similar structures; thus, their differentiation is more involved than that
of amino acids. The structural analysis of carbohydrates requires a flexible approach,
which all but precludes full automation of the process. Only within a particular sub-
class of carbohydrate structures (e.g., for N- and O-glycans commonly found in gly-
coproteins) can some general techniques be applied universally. At any given juncture
in the procedure, the choice of methodology is dictated by the amount and purity of
carbohydrate material available. If pure carbohydrate material is abundantly
available, the goals for structural characterization can be set as high as possible:
14.6 Techniques Used to Identify a Carbohydrate and Its Derivates 399
14.6.2Detection of Carbohydrates
14.6.3Linkage Analysis
14.6.3.2Determination of Chirality
14.6.3.3Sequence Analysis
NMR spectroscopy is the only technique that has the potential for full structural
characterization of a glycan with little or no assistance from other methods. A com-
plete structural investigation requires both the 1H and 13C NMR spectra of the oligo-
saccharide by a combination of two-dimensional NMR techniques such as correlated
spectroscopy (COSY) and total correlation spectroscopy (TOCSY) for 1H, and then
heteronuclear single-quantum coherence (HSQC) for 13C. However, in instances of
inadequate sample for these two-dimensional 1H13C NMR experiments, data pro-
vided by other techniques are required. A less rigorous NMR approach for glycan
sequencing relies on two-dimensional 1H NMR spectroscopy, using through-space
effects (nuclear Overhauser effects [NOEs]) as the sole source of evidence for link-
ing position and sequence. The sequence information of intact derivatized oligosac-
charides was provided by various MS approaches, such as GLC-MS, direct-inlet
EI-MS, fast atom bombardment (FAB-MS), and liquid secondary ion-mass spec-
trometry (LSIMS). These techniques yield molecular ions and only some fragmen-
tation. The spectra can be interpreted to obtain some sequence information of
carbohydrates much larger than those amenable to EI- and chemical ionization
(CI)-MS. In some instances, the sequence information of native glycoconjugate
molecules can be obtained because their physical properties make them good pro-
ducers of ions on the surface of the matrix used for the analysis. These MS tech-
niques are capable of analyzing mixtures of oligosaccharides and provide (1) the
molecular weight (MW) of the oligosaccharides, (2) the heterogeneity of the sam-
ple, (3) structural information, for example, on branching, and (4) structural infor-
mation on individual components (using MS/MS). Newer ionization approaches
introduced in the 1990s include electrospray ionization (ESI) and matrix-assisted
laser-desorption ionization (MALDI). Today, ESI-MS has mostly replaced FAB-MS
for peptide- and glycopeptide-mapping purposes. Although MALDI-time of flight
[TOF]-MS still lags behind other MS approaches in terms of resolution and mass
accuracy, its results are far better than those obtained by SDS-PAGE or SEC.
If each of the glycans isolated from a glycoconjugate is analyzed using the appro-
priate combination of approaches, a complete picture of the glycoconjugate can be
achieved. The ultimate level of glycoprotein primary structure analysis involves
the detailed determination of the particular glycans present at each glycosylation
site. This could be accomplished, for instance, by the production and mapping of
peptides, the identification and preparative isolation of those peptides containing
402 14 Carbohydrate Detection Using Nanostructured Biosensing
sugars, and the study of each peak using the methods described above. Alternatively,
current state-of-the-art direct MS analysis makes it possible to accomplish this
task at the level of the intact glycoconjugate.
14.7Nanotechnology
Fig. 14.4 The structure of hesperidin (top) and below the proposed ECirrevE mechanism to
explain the observed voltammetry in aqueous solution. Reprinted with permission from Sims etal.
[66]. 2009, Elsevier
Fig.14.5 (a) Mixed SAM on the gold substrate; (b) covalent immobilization of the lectin; (c)
addition of the tagged and untagged sugars; (d) dissolution of the captured nanocrystals, followed
by their stripping voltammetric detection at a mercury-coated glassy carbon electrode. Reprinted
with permission from Dai etal. [75]. 2006, American Chemical Society
on cell surfaces [35]. Two recent studies have described the use of lectin arrays to
analyze the carbohydrate motifs expressed on glycoproteins [36, 37].
The protocol of electrochemical recognition of sugars was established by com-
petitions of target sugars and the quantum dotlabeled sugars. As shown in Fig.14.5,
the bioassay process involves the immobilization of the lectin onto the gold surface,
competition between a nanocrystal (CdS)-labeled sugar and the target sugar for the
carbohydrate-binding sites on lectins, and monitoring the extent of competition
through highly sensitive electrochemical stripping detection of the captured nano-
crystal. EDAC/NHS coupling was also used for conjugating the CdS tracer to the
4-aminophenyl-a-d-galactopyranoside sugar. The protocol relies on a one-step
competitive assay, which is more suitable for monitoring small sugar molecules and
lectinsugar interactions. The lectinsugar recognition event thus yields a distinct
cadmium-stripping voltammetric current peak, whose size decreases upon increas-
ing the level and affinity of the target glycan [75].
Based on integrating the specific carbohydrate recognition and enzymatic signal
amplification of proteins on Au nanoparticles, a dual-functionalized nanoprobe was
designed for highly sensitive and selective in situ evaluation of carbohydrates on
living cells. As shown in Fig. 14.6, a nano-scaffold of nanohorns functionalized
with arginine-glycine-aspartic acid-serine tetrapeptide was immobilized on the sur-
face of an electrode for capturing cells and enhancing the electrical connectivity.
Combined with the nanoprobe and peptide-nanohorns, a highly sensitive electro-
chemical strategy was developed for cytosensing, which showed a detection limit
down to 15 cells, broad dynamic range, acceptable rapidity, and low cost. The pro-
posed method was further used for monitoring the dynamic variation of carbohy-
drate expression on cancer cells in response to drugs, which obviated the destruction
or labeling of cells and the covalent tagging of lectin and enzyme. The one-pot con-
jugation of three components was very convenient and could be extended for prepa-
ration of other lectin-functionalized nanoprobes. Further development of this
technique would contribute considerably to meeting the challenges in a comprehen-
sive understanding of the glycomic codes [76].
Nanoparticle-based probes are emerging as alternatives to molecular probes due
to their various advantages, such as bright and tunable optical property, enhanced
406 14 Carbohydrate Detection Using Nanostructured Biosensing
Fig. 14.6 Scheme of nanoprobe assembly and electrochemical strategy for in situ detection of
mannose groups on living cells. Reprinted with permission from Ding etal. [76]. 2010, American
Chemical Society
Fig.14.8 Con A-induced nanoparticle aggregation of (a) dextran1K-Ag, (b) dextran 6K-Fe3O4,
and (c) dextran 40K-QD. In each sample, Con A was added with a final concentration of 10mM,
and the absorption spectrum was obtained after 0min (black line), 30min (blue line), 1h (green
line), and 2h (red line). A magnet is shown to remove the aggregated Fe3O4 from the solution in
(b). Reprinted with permission from Earhart etal. [77]. 2008, American Chemical Society
from the solution. Complete precipitation of all nanoparticle systems was observed
within 4h. This precipitation process was associated with a significant red-shifting
of the plasmon absorption band and reduced peak absorbance for the Ag nanopar-
ticles (Fig. 14.8a). No significant red shift was observed with Con Adextran
ZnSCdSe aggregation, but both the absorbance and fluorescence were reduced
with time as the particle precipitated from the solution (Fig.14.8c). Con Adextran
Fe3O4 aggregates were removed from the solution over time using a magnetic field,
resulting in a decrease in absorbance (Fig. 14.8b). The dextranFe3O4 particles
could not be separated by a magnetic field unless Con A was added. This approach
is simple, and widely applicable to different types of nanoparticles and dextran of
different MWs [77].
A simple and easy-to-perform detection assay for lectins using a two-step-
synthesized, water-soluble, carbohydrate-conjugated core-shell CdSe ZnS QDs was
developed. As shown in Fig.14.9, several neoglycoconjugates with a reactive thiol
group were synthesized for the solubilization and stabilization of CdSe-ZnS QDs in
aqueous solution. Three different sizes of QDs with lactose, melibiose, and maltotriose
on their surface were utilized for the first time for lectin detection through agglutina-
tion assay. The sugarQDs were characterized by TEM, fluorescence, and absorption
spectroscopy. Agglutination of sugarQDs by three different lectins occurred through
specific multivalent carbohydratelectin interactions and was studied extensively by
monitoring the scattered light at 600nm. At first, soluble smaller sugarQD lectin
aggregates are formed through the specific interaction of carbohydrates and multiva-
lent lectins, which leads to an increase in turbidity and consequently an increase in
the scattering. Further association of the smaller aggregates into much larger aggre-
gates induces the precipitation and subsequent decrease in the absorbance at longer
time scales as observed experimentally (Fig.14.10). The detection sensitivity of the
maltotrioseQD was as little as 100nM of the lectin using light scattering. Further
enhancement of the detection sensitivity of the protocol could be considerable from
the fluorescence properties of QDs. This agglutination process seems to occur
through formation of smaller soluble aggregates, which further associate to form
larger aggregates [78].
408 14 Carbohydrate Detection Using Nanostructured Biosensing
Fig.14.11 Comparative analysis of lectin microarray and lectin nanoprobe array. Error bars in
the graph represent standard error of the mean (N=5). Reprinted with permission from Nagaraj
etal. [79]. 2008, Elsevier
dye and used for probing the lectin array either by flooding the array (Lectin
Microarray) or by piezoelectric liquid dispensing (Lectin NanoProbeArray). The
reactivity of lectins (SNA, MAA, and DSA) on the array with a range of concentra-
tions for ASF and 6SF were analyzed. A sample image for one block on the array
was shown with the graph. Each block consisted of five identical rows of six spots
with alternate spots of lectins and PBS buffer (control). Sensitivity levels compa-
rable to lectin arrays that use evanescent-field scanners were achieved along with a
several orders of magnitude reduction in the amount of probe required for glycosy-
lation analysis [79].
A strategy for profiling cell surface carbohydrate expression patterns by
evaluating cell-binding patterns on lectin arrays was present using an inverted
optical microscope. The approach was demonstrated using a small array of six com-
mercially available lectins with well-characterized binding specificities. The lectin
array was fabricated on gold thin-film substrates functionalized with NHS ester
alkyl disulfide (Fig.14.12). Different lectins were spotted onto the substrate to react
for 3h and the chip was then incubated in bovine serum albumin (BSA) to minimize
nonspecific binding. The lectin arrays were used to study cell surface carbohydrate
expression. The cells employed were BHK-21. Lectin arrays were placed in the
wells of a 6-well tissue culture plate, and 2mL of cell suspension was added to each
410 14 Carbohydrate Detection Using Nanostructured Biosensing
Fig.14.13 BHK-21 cells and Caco-2 cells showed distinct binding patterns to the lectin array. The
lectins were spotted according to the pattern in (c). (a) BHK-21 cells bound to three of the lectins,
while (b) Caco-2 cells bound to every lectin except GSL-II. Reprinted with permission from Zheng
etal. [80]. 2005, American Chemical Society
well to cover the lectin chips. After washes, the chips were observed with an optical
microscope. The two cell lines exhibited distinct patterns of binding to the lectin
arrays; BHK-21 cells bound to only three of the lectins, ConA, WGA, and RCA,
while Caco-2 cells bound to every lectin except GSL-II (Fig.14.13). These results
indicated that BHK-21 cells have mannose, galactose, and GlcNAc expressed on
their surfaces, and no detectable expression [80]. The work provides a proof-of-
principle demonstration of the direct qualitative profiling of cell surface carbohy-
drate expression patterns by simple microscopic observation of cell binding to lectin
arrays, and may readily be expanded to encompass a wider variety of carbohydrate
structural motifs by increasing the number of lectins or other specific ligands pres-
ent in the array.
14.7 Nanotechnology 411
Fig.14.14 LDI-TOF mass spectra of (a) 2a deposited on the surface of the gold-coated MALDI
plate and (b) 2a on the GCNPs. Reprinted with permission from Noriko and Shin-Ichiro [81].
2006, Wiley-VCH
Fig.14.15 Gold colloid solutions (a) before and (b) after Mal-C12S modification; (c) MalC12S-
modified gold nanoparticle solution after adding Con A (2.6mM); (d) unmodified gold nanoparti-
cle solution after adding Con A (2.6mM). Models of each step are also shown. Reprinted with
permission from Sato etal. [82]. 2008, Springer
alkanethiols are first prepared. The functioned gold nanoparticles aggregate in the
presence of corresponding proteins (lectins) that have specific recognition for cer-
tain carbohydrates. Therefore, the corresponding lectins in sample solutions can be
estimated. As shown in Fig.14.15, maltoside alkanethiol, which has a disaccharide
group and a flexible long alkyl chain, is selected as an effective sensing modifier.
The surface modification of gold nanoparticles with maltoside alkanethiol results in
a shift and broadening (from 520 to 610nm) of the absorption peak. Monodispersed
maltoside-adsorbed gold nanoparticles aggregate in the presence of Con A, which
can be used to detect the presence of Con A in sample solutions. The precipitate of
the maltosidegold nanoparticleCon A mixture can be further redispersed by the
addition of methyl a-d-mannopyranoside, whose adsorption coefficient is larger
than that of maltoside with Con A [82].
Cell surface glycoproteins are shown to play important roles as receptors for hor-
mones and as mediators of immunological specificity in intracellular recognition
and adhesion, and in cell development and differentiation [47, 83]. Therefore, in
order to develop a novel high-throughput tool for monitoring carbohydrate-protein
interactions, a series of carbohydrate or glycoprotein microarrays were prepared by
14.7 Nanotechnology 413
Fig.14.16 Schematic representation of one spot on the microarray. From left to right, the binding
of a biotin-modified lectin to the (a) spotted monosaccharide and (b) glycoprotein, then labeled by
avidin-FITC followed by the attachment of peptide stabilized gold nanoparticles, the silver
enhancement step, and reading by detection of resonant light scattering. Reprinted with permission
from Gao etal. [84]. 2008, American Chemical Society
Fig.14.17 (a) Schematic illustration of the interactions of carbohydrateAu NPs and Con A on
the biosensor chip used in the competition assays. (b) Five different carbohydrates (sugar-R)
encapsulated on two different sizes of gold nanoparticles are summarized. Specifically, gluco- and
galatopyranoside are encapsulated on nanoparticles of 6 nm and abbreviated as 6-g-AuNP and
6-t-AuNP, respectively. Mannopyranoside clustered on nanoparticles of 6 and 20nm in diameter
are abbreviated as 6-m-AuNP and 20-m-AuNP, respectively. Mannopyranoside with short or long
linker lengths are encapsulated on 20-nm nanoparticles to form s-20-m-AuNP and l-20-m-AuNP,
respectively. Reprinted with permission from Lin etal. [85]. 2003, Royal Society of Chemistry
Fig.14.18 Schematic representation of the self-assembled multilayer system for the investigation
of biomolecular binding. Reprinted with permission from Guo etal. [86]. 2007, Elsevier
Fig.14.19 Synthesis of m-AuNP. (a) Ac2O, pyr. DMAP, 90%; (b) HBr/HOAc, 80%; (c) 4-pentenyl
alcohol, Hg(CN)2, 88%; (d) HSAc, AIBN, dioxane, 80%; (e) NaOMe (cat.), MeOH, 97%;
(f)HAuCl4, NaBH4. Reprinted with permission from Lin etal. [87]. 2002, American Chemical
Society
Fig.14.20 Typical TEM images of sectioned areas of (a) pili of the Escherichia coli ORN178
strain bound with m-AuNP; (b) the E. coli ORN208 strain deficient of the fimH gene without
m-AuNP binding. The experiments were performed in LB at room temperature. Scale bar: 100nm.
Reprinted with permission from Lin etal. [87]. 2002, American Chemical Society
binding of m-AuNP to bacterial pili in each condition was examined by TEM. The
TEM results showed that m-AuNP selectively bound the pili of the ORN178 strain
but not those of the ORN208 strain (Fig.14.20), demonstrating specific binding of
m-AuNP to FimH. This work further demonstrates that carbohydrate-attached gold
nanoparticles can be used as an efficient labeling probe and multiligand carrier in a
biological system [87].
A novel approach to the optical detection of viruses using gold nanoparticles was
proposed. Although inorganic nanostructures prepared with viruses as templates
have often been reported recently, there are no reports on the use of their inherent
molecular recognitions for the spatially ordered arrangement of metal nanoparticles,
which can be applied to virus detection. Since viruses have multiple recognition sites
on their surface for ligands, the ligand-displaying nanoparticles could be specifically
14.7 Nanotechnology 417
Fig.14.21 Schematic representation of the detection of virus particles. (a) The formation of the
sialylAu particle array based on virus-like particles (VLPs) through viral ligand recognition;
(b)structures of the sialic acid-linked and carboxyl lipids used in the preparation of sialylAu and
carboxylAu particles. Reprinted with permission from Niikura et al. [88]. 2009, American
Chemical Society
arranged on the viral surface (Fig.14.21a). Therefore, viruses can be detected from
the plasmon shift derived from the assembled gold nanoparticles on the virus surface
rather than the analyte-induced aggregation of metallic nanoparticles. The surface
plasmon resonance shift strongly depends on both the interparticle distance and the
number of coupled particles. The plasmon shift for multiple (more than two) nano-
particles in a linear arrangement is larger than that for two nanoparticles; thus, the
multiple coupling of nanoparticles on the virus is expected to cause a large shift of
the plasmon band. In addition, the assembly of metallic nanoparticles into a shell-
shaped structure is expected to lead to a large plasmon shift. Metallic clusters formed
on a spherical body, such as silica particles (diameter~100nm) or liposomes (diam-
eter~50 nm), show a large red shift (~200 nm) of the plasmon band. The virus-
templated assembly of metallic nanoparticles in a shell-shaped structure is thought
to contribute to this large plasmon shift [88].
A single-walled carbon nanotube (SWNT) is a pseudo-one-dimensional nano-
structure capable of carrying/displaying a large number of bioactive molecules and
species in aqueous solution. A series of dendritic b-d-galactopyranosides and a-d-
mannopyranosides with a terminal amino group have been synthesized and used for
the functionalization of SWNTs, which targeted the defect-derived carboxylic acid
moieties on the nanotube surface (Fig.14.22a). The higher-order sugar dendrons
were more effective in the solubilization of SWNTs, with the corresponding func-
tionalized nanotube samples of improved aqueous solubility characteristics. Through
the functionalization, the nanotube apparently serves as a unique scaffold for dis-
playing multiple copies of the sugar molecules in pairs or quartets. Results from the
biological evaluation of these sugar-functionalized SWNTs in binding assays with
pathogenic E. coli and with Bacillus subtilis are shown in Fig.14.22b. Obviously,
418 14 Carbohydrate Detection Using Nanostructured Biosensing
14.7 Nanotechnology 419
Fig.14.22 (a) Carbon nanotubes functionalized with Gal-, Man-, or their dendrons. (b) Fluorescence
microscopy images (10 mm for all scale bars) on the agglutination of E. coli O157:H7 (GFP-
expressing) cells by (a) Gal2SWNT, (b) GalSWNT, and (c) the control (cells only). (d) A visual
comparison on the amount of precipitates (in centrifuge tubes) associated with the agglutination of E.
coli O157:H7 cells (top, 5107cells/mL; bottom, 108cells/mL) by Gal2SWNT (left) and Gal
SWNT (right). Reprinted with permission from Gu etal. [89]. 2008, American Chemical Society
420 14 Carbohydrate Detection Using Nanostructured Biosensing
acid layer was confirmed by electrochemistry and atomic force microscopy imaging.
The intrinsic oxidation signal of the captured Ab(140) and (142) peptides, con-
taining a single tyrosine (Tyr) residue, was monitored at a peak potential of 0.6V
(vs. Ag/AgCl within this sensor) in connection with differential pulse voltammetry.
The peak current intensities were concentration-dependent. The proposed process
provides new routes for the analysis of saccharideprotein interactions and for elec-
trochemical biosensor development [90].
14.8Conclusions
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Chapter 15
Nanomaterials for Immunosensors and
Immunoassays
15.1Introduction
Many different types of antibodies exist in the serum of animals immunized with
specific antigens. The mixture of these different types of antibodies is a so-called
polyclonal antibody. Because these antibodies arise from the clones of a number of
separate B cells, they are heterogeneous, and different antibodies in this mixture
react with different antigenic determinants. With the ever-increasing sophisticated
genetic techniques, the production and use of monoclonal antibodies have attracted
more interest since this technique was first developed by Khler and Milstein in
1975, who won the Nobel Prize in 1984 [4]. Monoclonal antibodies are produced by
the fusion of myeloma (tumor) cells cultivated in vitro with mouse spleen
B-lymphocytes immunized with a specific antigen. The fusion called a hybridoma
is cultivated and screened. Once we are sure that a certain hybridoma is producing
the right antibody, we can culture that hybridoma indefinitely and harvest monoclo-
nal antibody from it. Because a monoclonal antibody reacts only with one specific
antigenic determinant, it shows a higher sensitivity and better specificity than the
conventional polyclonal antibodies for immunoassays.
The principle of antigenantibody interaction lies in the specific combination of
the antigen determinant with the Fab fragment of the antibody. The specific binding
between antigen and antibody is a collection of noncovalent forces, including
electrostatic forces, hydrophobic attractions, hydrogen bonding, and van der Waals
interactions. The interaction between antigen and antibody is quite strong, as
indicated by the large association constant of 1051012 M1 [5]. Therefore, the
antibodyantigen complex does not dissociate so readily unless some harsh solu-
tions such as buffers at pH higher than 10 or lower than 3, organic solvents, and
saline solutions at high concentration are used to regenerate it [6].
Fig. 15.3 Preparation and detection procedures of the CNF-based CA 125 immunosensor.
Reprinted with permission from Wu etal. [23]. 2007, Elsevier
Fig.15.4 Schematic representation of the preparation and detection procedure of MC-LR immu-
nosensor. Reprinted with permission from Zhang etal. [24]. 2010, American Chemical Society
Fig.15.5 Schematic diagram for biofunctionalization of 3D nanoporous SiO2 film and one-step
sandwich immunoassay procedure for CA 125. Reprinted with permission from Yang etal. [27].
2008, Wiley
Fig. 15.6 Schematic representation of the preparation of immunosensor array and analytical
p rocedure for the simultaneous detection of human IgG and goat IgG. Reprinted with permission
from Leng etal. [45]. 2010, Elsevier
Duan etal. [44] found that Au NPs surrounded by protein could trigger the CL
reaction between AgNO3 and luminol. Based on this finding, this novel CL system,
that is, luminolAgNO3Au NP system, was utilized to develop a novel nonstrip-
ping CL immunoassay protocol. Besides the application in optical immunosensors,
Au NPs have also been utilized as signal probes for immunoassays with electro-
chemical analysis approaches, such as amperometric, potentiometric, stripping
voltammetric, and piezoelectric methods. For example, Jus group [45] proposed a
simple, sensitive, and low-cost, inherently cross-talk-free multiplexed immunoas-
say by combining a disposable chip with Au NP as an electrochemical label.
As shown in Fig.15.6, the immunosensor array was first prepared by immobilizing
capture antibodies on different SPCEs by passive adsorption. Following a sandwich
immunoassay format, the Au NP-labeled antibodies were conjugated on the immu-
nosensors surface. The analytes were detected by electrooxidization of Au NPs in
0.1 M HCl. This method eliminated electrochemical cross-talk between adjacent
immunosensors due to the strong adsorption of the AuCl4 on the printed carbon
surfaces. Since each Au NP contains thousands of atoms, the immunoassay pos-
sesses a relatively high sensitivity. Using human IgG and goat IgG as model targets,
under optimal conditions this method achieved linear ranges from 5.0 to 500 and
5.0400ng/mL with limits of detection of 1.1 and 1.6ng/mL, respectively.
Selvaraju etal. [46] developed a nanocatalyst-based electrochemical immunoas-
say using Au NPs. Au NP tags of the immunocomplexes on the electrode produced
15.3 Immunosensors Based on Biocompatible Nanomaterials 439
a competitive immunoassay, and the linear range for AFP was 0.0166.8mg/L [58].
A similar mass spectrometric immunoassay strategy also has been proposed by the
same group to detect human IgG in immunomicroarray mode with a detection limit
of 0.012ng/mL [59].
Xie etal. [60] first presented a highly sensitive immunoassay method based on a
single Au NP counter in solution. This strategy was based on the photon burst
counting in a small detection volume by utilizing strong resonance scattering and
Brownian motion of a single Au NP. The relationship between the photon burst
counts and the Au NP amount showed an excellent linearity. Based on this phenom-
enon, this group developed an ultrasensitive and highly selective detection platform
for homogeneous immunoassay, which was two to five orders of magnitude more
sensitive than the current homogeneous immunoassays. Moreover, the detection
volume was less than 1fL, and the sample requirement can be easily reduced to the
nanoliter level by using a droplets array. Thus, this method had the potential to
evolve a high-throughput detection platform similar to currently used microarray
immunochips.
QDs, also known as nanocrystals, are the most eye-catching fluorophores devel-
oped for fluorescent images and bioconjugates in the past two decades. They exhibit
some important differences compared to traditional fluorophores, such as organic
fluorescent dyes and naturally fluorescent proteins. QDs are nanometer-scale atom
clusters, containing from a few hundred to a few thousand atoms of semiconductor
material such as CaSe or CaTe, which sometimes have been coated with an addi-
tional semiconductor shell such as ZnS to improve their optical properties. Besides
their excellent quantum efficiency, QDs also show several other advantages over
conventional organic dyes. Their emission spectra are symmetric, narrow, and tun-
able according to their size and chemical composition, permitting closer spacing of
different probes without substantial spectral overlap. They exhibit excellent photo-
stability, tolerating long-time excitation. QDs also display broad absorption spectra,
making it possible to minimize sample autofluorescence by choosing an appropriate
excitation wavelength. Thus, QDs have attracted increasing interest as tags for
immunoassays besides their application in bioimage in the past decade.
Vinayaka etal. [61] developed a reliable and rapid fluoroimmunoassay method
for analysis of 2,4-dichlorophenoxyacetic acid by using CdTe QDs to tag 2,4-dichlo-
rophenoxyacetic acid. Antibodies were immobilized in an immunoreactor column
using Sepharose CL-4B as an inert matrix. The detection of 2,4-dichlorophenoxya-
cetic acid was carried out by competitive binding between conjugated 2,4-dichloro-
phenoxyacetic acidCdTe and free 2,4-dichlorophenoxyacetic acid with immobilized
antibodies in an immunoreactor column. 2,4-Dichlorophenoxyacetic acid can be
detected in the range of 250pg/mL to 1,000ng/mL. Another fluorescent sandwich
immunoassay using high-affinity antibodies and QD optical reporters have been
developed for the detection of botulinum neurotoxin serotype A using a renewable
surface column and 96-well plate formats. Using QDs with a maximum emission of
455nm, detection limits of 31 and 5pM were obtained for the two formats, respec-
tively [62]. Chen etal. [63] described a rapid and ultrasensitive detection method
using a microfluidic chip for analyzing 7-aminoclonazepam residues in human urine.
15.3 Immunosensors Based on Biocompatible Nanomaterials 443
As mentioned earlier, one of the most efficient strategies to improve the label
amount for antibodies or antigens is to prepare nanosized tags, that is, to prepare
nanoparticles composed of a large amount of signal molecules. Unfortunately, the
preparation of nanosized tags is sometimes challenging and difficult. Up to now,
only several of the usual tags have been prepared in a nano size and used for
labeling biomolecules. An alternative approach for the same purpose is the use of
currently existing nanomaterials as nanovehicles to load a large number of signal
molecules for highly sensitive immunoassays.
Si NPs have shown many advantages in the development of carrier vehicles
for the signal probe, such as ease of fabrication and functionalization, and high
stability in a variety of environments. Different strategies, including covalent
binding, entrapment, and electrostatic interaction, have already been adopted to
prepare functionalized Si NPs for bioconjugation. With the inverse microemul-
sion polymerization protocol, Ru(bpy)3Cl2 was doped into Si NPs by Hun and
Zhang [75]. Then core-shell structured fluorescent Si NP-labeled anti-TNF-a
monoclonal antibodies were prepared and used for the fluoroimmunoassay of
TNF-a in human serum samples, with a detection limit of 0.1 pg/mL. With a
similar fluoroimmunoassay strategy, some other analytes such as staphylococcal
enterotoxin C1 [76], and IL-6 [77] have also been detected by using the nanopar-
ticles with a core of Ru(bpy)3Cl2 and a shell of silica.
Luminescent europium(III) and terbium(III) chelates can be covalently immobi-
lized on the surface of prepared Si NPs to which reporter antibodies or bridging
proteins for antibody binding are conjugated. Xu and Li [78] utilized the resulting
conjugates in time-resolved fluorescent immunoassays for hepatitis B surface
antigen and hepatitis B antigen, both individually and simultaneously. The prepared
nanoparticle conjugates were homogeneous in size at 55nm in diameter, and stable
for long-term storage (>2 years). The proposed method showed good sensitivity and
repeatability. The europium chelate-loaded Si NPs were also successfully adopted
15.3 Immunosensors Based on Biocompatible Nanomaterials 445
to prepare a portable lateral flow immunoassay strip with better sensitivity than the
conventional strip using Au NP tags [79].
Wang et al. [80] prepared an electroactive Si NP in which poly(guanine) was
used to functionalize Si NPs. The Si NPs loading poly(guanine) could serve as a
biological tag for a sensitive electrochemical immunoassay. It was found that there
were ca. 60 strands of poly(guanine)20 per Si NP, which meant that the average
surface coverage of poly(guanine)20 on an Si NP was ca. 8.51012 molecules/cm2.
The detection limit for TNF-a was found to be 5.01011g/mL (2.0pM) for an
immunosensor using the nanoparticles as tags, which corresponds to 60 aM of
TNF-a in 30mL of sample. This immunosensor based on the poly(guanine)-func-
tionalized Si NP tags offered great promise for the rapid, simple, cost-effective
analysis of biological samples.
Au NPs can be used as a probe carrier for their excellent adsorption ability. They
are generally used for loading enzymes or enzyme-labeled antibodies by the adsorp-
tion of these biomolecules on their surface. Due to their excellent biocompatibility,
the activity of the adsorbed enzymes can be well retained to generate a strong
signal. Zhou etal. [81] described a novel immunoassay strategy for the amplified
detection of AFP using DNAzyme-functionalized Au NPs as catalytic tags. Since
the Au NPs carried a large number of DNAzyme units per protein-binding event,
there was substantial amplification. A chromogenic reagent ABTS showed a green
color in the presence of DNAzyme catalysis. This method resulted in a linear
working curve in the concentration range of 0.220 ng/mL. Wang et al. [82]
described a very simple and easily operated colorimetric multiplexed immunoassay
method for the sequential detection of tumor markers. Magnetic microparticles con-
jugated with antibodies were used to capture antigens. Through different enzymatic
reactions of 3,3,5,5-tetramethylbenzidine and o-phenylenediamine catalyzed by
HRP molecules that were loaded on the surfaces of Au NPs, two antigens, CEA and
AFP, can be detected even with the naked eye. The detection limit obtained from the
spectrophotometric measurements is as low as 0.02 ng/mL. Ambrosi et al. [83]
developed an optical ELISA for the analysis of the CA15-3 antigen. Amplification
of the optical signal was achieved by using Au NPs as carriers of the signaling anti-
bodies anti-CA15-3-HRP. In the range of 060U/mL, the method using Au NPs as
enhancers resulted in a higher sensitivity and shorter assay time when compared to
classical ELISA procedures. Tang etal. [84] proposed a new signal amplification
strategy based on thionine-doped magnetic Au NPs as tags and HRP as enhancer for
the immunoassay of CEA. This immunoassay system was performed on a carbon
fiber microelectrode covered with a well-ordered anti-CEAprotein Ananogold
architecture. The reverse-micelle method was applied for the preparation of thionine-
doped magnetic Au NPs, and the nanoparticles were then used to load HRP-bound
anti-CEA. A sandwich-type protocol was successfully introduced to develop a high-
efficiency electrochemical immunoassay with the labeled bionanospheres toward
the reduction of H2O2. CEA can be detected in the range 0.01160ng/mL by using
the Au NP-based tags.
Polystyrene nanoparticles (PNPs) is a polymer mainly used for loading
europium(III) chelate, a time-resolved fluorescent probe. Europium(III) chelate-dyed
446 15 Nanomaterials for Immunosensors and Immunoassays
into 24 subunits at a low pH of 2.0 and reconstituted at a high pH of 8.5. Due to this
unique property, it is possible to pack different small-molecule probes into
apoferritin cages by adjusting the pH to form nanosized tags for immunoassays.
Hexacyanoferrate (electroactive probe) and fluorescein (fluorescence probe) were
chosen as model probes for loading into the apoferritin cages to develop versatile
nanosized tags by Liu etal. [93]. The process for preparing probe-loaded apoferritin
nanocages is briefly depicted in Fig.15.8. Apoferritin was dissociated into subunits
at a low pH and then reconstituted at a high pH, thereby packing the probes in solu-
tion within its interior. The protein shell of the apoferritin remained a substantially
stable structure and sustained no obvious alteration during the process, which ascer-
tained the formation of shell-core structure nanoparticles containing a large number
of probe molecules. These nanocage-based tags enabled the immunoassay of IgG
with detection limits of 0.39 and 0.52 pM by hexacyanoferrate and fluorescein,
respectively. The same authors also proposed a simple and facile apoferritin-
templated synthesis of metallic phosphate nanoparticle tags for immunoassay. In
this strategy, metal ions diffused into the cavity of apoferritin and precipitated in the
presence of phosphate. The protein shells of the resulting metal phosphate particles
were then surface-biotinylated to form anodic stripping voltammetric immunoassay
tags. The release of metal ions from apoferritin nanocages in an acetate buffer at pH
4.6 avoided the harsh conditions in the traditional metallic nanoparticles dissolu-
tion. This assay method was ultrasensitive, and its detection limit for biomarkers
was as low as 77fM [94].
15.4Conclusions
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Chapter 16
Nanostructured Biosensing and Biochips
for DNA Analysis
16.1Introduction
Nucleic acids, including deoxyribonucleic acid (DNA) and ribonucleic acid (RNA),
are required for the storage and expression of genetic information. DNA is present
not only in chromosomes in the nucleus of eukaryotic organism, but also in mito-
chondria and in the chloroplasts of plants. Prokaryotic cells, which lack nuclei, have
a single chromosome but also contain nonchromosomal DNA in the form of plas-
mids. The DNA contained in a fertilized egg encodes the information that directs
the development of an organism. This development may involve the production of
billions of cells. Each of these cells is specialized, expressing only those functions
that are required for it to perform its role in maintaining the organism. Therefore,
the DNA must be able not only to replicate precisely each time a cell divides, but
also to have the information that it contains be selectively expressed. RNA partici-
pates in the expression of the genetic information stored in the DNA [1].
DNA-based tests, known as genetic tests, allow the genetic diagnosis of
vulnerabilities to inherit diseases and can also be used to determine a childs pater-
nity (genetic father) or a persons ancestry. Normally, every person carries two
copies of every gene, one inherited from the mother, and another inherited from the
father. The human genome is believed to contain around 20,00025,000 genes. In
addition to studying chromosomes to the level of individual genes, genetic testing
in a broader sense includes biochemical tests for the possible presence of genetic
diseases, or mutant forms of genes associated with increased risk of developing
genetic disorders. Genetic testing identifies changes in chromosomes, genes, or
proteins [2]. Most of the time, testing is used to find changes that are associated with
inherited disorders. The results of a genetic test can confirm or rule out a suspected
genetic condition or help determine a persons chance of developing or passing on
a genetic disorder. Several hundred genetic tests are currently in use, and more are
being developed [3]. Available types of testing include the following [4]:
1. Newborn screening: Newborn screening is used just after birth to identify genetic
disorders that can be treated early in life. Many countries currently test infants
for phenylketonuria (a genetic disorder that causes mental illness if left untreated)
and congenital hypothyroidism (a disorder of the thyroid gland).
2. Diagnostic testing: Diagnostic testing is used to diagnose or rule out a specific
genetic or chromosomal condition. In many cases, genetic testing is used to
confirm a diagnosis when a particular condition is suspected based on physical
mutations and symptoms.
3. Carrier testing: Carrier testing is used to identify people who carry one copy of a
gene mutation that, when present in two copies, causes a genetic disorder. This type
of testing is offered to individuals who have a family history of a genetic disorder
and to people in ethnic groups with an increased risk of specific genetic conditions.
4. Prenatal testing: Prenatal testing is used to detect changes in a fetuss genes or
chromosomes before birth. This type of testing is offered to couples with an
increased risk of having a baby with a genetic or chromosomal disorder.
5. Preimplantation genetic diagnosis: These are genetic testing procedures that are
performed on human embryos prior to the implantation as part of an in vitro
fertilization procedure.
6. Forensic testing: Forensic testing uses DNA sequences to identify an individual
for legal purposes. This type of testing can identify crime or catastrophe victims,
rule out or implicate a crime suspect, or establish biological relationships between
people (e.g., paternity).
7. Research testing: Research testing includes finding unknown genes, learning
how genes work, and advancing our understanding of genetic conditions.
DNA analysis has a particular interest in genetics, molecular diagnosis, pathology,
food safety, biothreat detection, criminology, environmental protection, and so on.
The main principle of DNA biosensors is immobilizing one single-stranded DNA (or
PNA), a probe-target DNA, onto a transducer surface, and these DNA sequences have
the ability to precisely recognize its partner of complementary base sequence, target-
probe DNA. Consequently, a DNA biosensor conveys this recognition event into a
useful readable signal. In the early stage of DNA analysis, scientists used different
tags to label specific DNA or oligonucleotides for specific recognition, including
radioactive-tag [5], molecular beacons [6], fluoresceins [7], dyes, metal complexes,
biomolecules (enzyme, etc.), pharmic molecules [8], and so on. Nowadays, the nano-
technology and hybridization indication techniques are playing important roles in
developing sensitive, selective, and miniaturized DNA biosensors for fast and reliable
DNA sequence analysis in many applications, such as newborn screening, diagnostic
testing, presymptomatic testing, and forensic testing [9]. Due to their huge surface
area, nanoscale, excellent optical/electrochemical/magnetic properties, and flexible
surface chemistry, nanostructures have received considerable attention in the DNA
biosensing field. The large surface area of nanostructures can increase the attached
DNA amount on the nanomaterial substrates surface, and concentrate a large number
of enzyme molecules or different nanoparticles to indicate DNA hybridization.
16.2 Nanostructures in DNA Biosensing 455
In this chapter, we discuss recent advances in DNA analysis and DNA biochips
based on nanomaterials and nanostructures, and summarize the nanostructures for
DNA-labeling or label-free techniques, various detection methods, and DNA-
sensing strategies.
Over the past two decades, many important nanomaterials and technologies have
provided us with the key tools to design novel DNA-sensing methods and devices,
which have led to enormous improvements in sensitivity, selectivity, multiplexing
capacity, and simplicity. Many studies still focus on various combinations of DNA
with different types of transducers. Electrical/electrochemical, optical, and acoustic
sensing techniques have emerged as some of the most promising DNA-biosensing
technologies.
Moreover, different types of nanostructures can be associated together to achieve
versatile sensing units and/or microarrays, to meet the demands of fast, simple, and
inexpensive methods for specific DNA analysis.
Fig. 16.3 Schematic representation of the analytical protocol: (a) Capture of the ALP-loaded
CNT tags to the streptavidin-modified magnetic beads by (a) a sandwich DNA hybridization or (b)
AbAgAb interaction. (b) Enzymatic reaction. (c) Electrochemical detection of the product of the
enzymatic reaction at the CNT-modified glassy carbon electrode. MB, magnetic beads; P, DNA
probe 1; T, DNA target; P2, DNA probe 2; Ab1, first antibody; Ag, antigen; Ab2, secondary anti-
body; S and P, substrate and product, respectively, of the enzymatic reaction; GC, glassy carbon
electrode; CNT, carbon nanotube layer. Reprinted with permission from Wang etal. [17], 2004,
American Chemical Society
Fig.16.4 TEM images of the magnetic beads-DNA-CNT assembly produced following a 20-min
hybridization with the (a) 10- and (b) 0- pg/mL target sample. Reprinted with permission from
Wang etal. [17], 2004, American Chemical Society
due to the strong pp stacking interaction between Fc and SWNT (Fig.16.6). In this
case, SWNT played a dual role in both the recognition and transduction events. Using
the electrocatalytic properties of Fc/SWNT toward H2O2, they succeeded in designing
a sandwich-type gene-detection system that recognized target DNA. The different
responses toward c-DNA and n-DNA sequences, combined with the Fc/SWNT stabil-
ity, led to a useful approach for the preparation of CNT-based DNA biosensors.
Jiaos group reported a series of nanoparticleCNT complexes for developing
electrochemical DNA hybridization sensors [19, 20], which yielded excellent sensi-
tivities and considerable wide ranges for target DNA concentration from 1.01011
1.0106 mol/L, with a detection limit of 2.81012 mol/L, and from 1.0107
1.01012mol/L, with a detection limit of 2.81013mol/L, respectively.
16.2 Nanostructures in DNA Biosensing 459
Fig.16.5 Chronopotentiometric signals for increasing levels of the DNA target: (a) 0.01, (b) 0.1,
(c) 1, (d) 50, (e) 100pg/mL. Also shown (inset) are (A) the resulting calibration plot, and (B) the
response for 5 fg/mL-target DNA and (C) 10-ng/mL noncomplementary (NC) oligonucleotide.
Sample volume, 25ml (B) and 50ml (C). Reprinted with permission from Wang etal. [17], 2004,
American Chemical Society.
Fig.16.6 Schematic illustration of the electrochemical gene sensing system based on the forma-
tion of a complementary sandwich-type complex. Reprinted with permission from Yang etal. [18],
2007, Springer
460 16 Nanostructured Biosensing and Biochips for DNA Analysis
Fig.16.7 (a) Optical image of the central region of a single sensor chip with four SWNT devices
(scale bar 200mm). Electrodes extending out of the liquid cell (dashed circle) connect to large
wire-bonding pads. (b) Schematic illustration of a single device during electrical measurement.
Complementary ssDNA oligos hybridize to thiolated ssDNA coimmobilized with MCH on the
gold electrodes. Real-time monitoring of (c) 15-mer and (d) 30-mer DNA hybridization in PBS,
pH 7.4, is shown. Two liquid cells were used in parallel for simultaneous drop, adding 5mL of
complementary and mismatched target oligo solutions to 500mL of buffer. Reprinted with permis-
sion from Tang etal. [22], 2006, American Chemical Society
Label-free assay: Ye and Ju [21] reported a method for the rapid sensitive detection
of DNA or RNA using a screen-printed carbon electrode modified with multiwalled
carbon nanotubes (MWNTs), where the MWNTs displayed catalytic characteristics
for the direct electrochemical oxidation of guanine or adenine residues of ssDNA
and adenine residues of RNA, leading to an indicator-free detection of ssDNA and
RNA concentrations.
Tang etal. fabricated virtually two terminal SWNT-DNA sensor arrays (Fig.16.7)
and the simple and yet generic protocol for direct label-free detection of DNA
hybridization [22]. They also carried out a systematic study of sensing mechanism,
involving XPS, quartz crystal microbalance (QCM) (Fig.16.8), and fluorescence
measurements (Fig.16.9).
Recently, Mao described a method for label-free and sequence-specific DNA
detection with a low detection limit by using CNTs as support to increase the surface
loading of probe DNA. Figure16.10 shows the preparation of the label-free DNA
16.2 Nanostructures in DNA Biosensing 461
Fig.16.8 QCM frequency shift (F3) versus time curves showing that (a) thiolated 15-mer ssDNA
(p15) absorbs onto SWNT sidewall spontaneously and (b) no further binding to mismatched
(MM15) and complementary (CM15) ssDNAs. (c) Phosphilipid-PEG maleimide (PL-PEG-M)
self-assembles onto SWNTs and allow effective linkage to p15. (d) The SWNT-PL-PEG-M linked
ssDNA probes selectively hybridize to complementary strands. The inserts are schematic drawings
of the SWNTssDNA complexes. Reprinted with permission from Tang et al. [22], 2006,
American Chemical Society
Fig. 16.9 Fluorescence images showing DNA hybridization on patterned (circle) SWNT film.
Thiolated ssDNA (probe) was conjugated to phospholipid-maleimide wrapped on an SWNT side-
wall. Fluorescence intensity from the cy3-labeled complementary target (b) is about five times
higher than the mismatched target (a). Insert is the atomic force microscopy image of one circle
region (scale bar 5 mm). Reprinted with permission from Tang et al. [22], 2006, American
Chemical Society
462 16 Nanostructured Biosensing and Biochips for DNA Analysis
Fig.16.10 Schematic of label-free and sequence-specific DNA detection with CNTs as support
for probe DNA. Reprinted with permission from Zhu etal. [23], 2009, Elsevier
Fig.16.11 Aligned carbon nanotube thin films obtained by CVD. (a) SEM image of the CNT film
grown on a SiO2 substrate (CNT/Ni/SiO2); (b) SEM image of the CNT film grown on an Al/SiO2
substrate (CNT/Ni/Al/SiO2); (c) scheme of CNT/Ni/SiO2; (d) scheme of CNT/Ni/Al/SiO2.
Reprinted with permission from Berti etal. [24], 2009, Elsevier
Fig.16.12 Schematic diagram illustrating the DNA damage induced by the E-Fenton reaction and
the detection method. Reprinted with permission from Wang and Jiao [25], 2010, Elsevier
The DNA damage in situ induced by the E-Fenton reaction can be detected by
differential pulse voltammetry (DPV) with 20mM Co(phen)33+. Due to the interca-
lative binding of Co(phen)33+ to dsDNA, Co(phen)33+ binds more strongly to intact
dsDNA than the damaged DNA. After the sensor is treated by E-Fenton reaction for
464 16 Nanostructured Biosensing and Biochips for DNA Analysis
a given time, the DPV response of Co(phen)33+ on the biosensor is obtained and the
results are shown in Fig.16.13(a).
The DNA damage can also be detected using another electrochemical indicator,
Ru(NH3)63+. After the biosensor is treated by the E-Fenton reaction, an increasing DPV
peak current is obtained with an extended reaction time, as shown in Fig.16.13(b).
16.2.2.1Gold Nanoparticles
The excellent physical and chemical properties of Au NPs (colloidal gold) offer
excellent prospects for a wide range of biosensing applications, especially toward
signal amplications in biorecognition systems. Wittenberg and Haynes [26]
discussed different detection methods of Au NP-enhanced sensing, including
extinction/scattering/reflectivity (localized surface plasmon resonance, LSPR;
surface-enhanced Raman scattering, SERS), electricity/electrochemistry, gravimetry,
and so on. Wang reviewed the recent developments of nanoparticle-based biosensing
methods [2729], where Au nanoparticles were used for nanoparticle-amplified
optical bioaffinity assays [27], tags for electrochemical detection of proteins and
DNAs, ultrasensitive particle-based bioassay-based multiple-amplification schemes
[28], and amplified microgravimetric monitoring of bioaffinity events [27]. The
aspect of electrochemical biosensing also was emphasized [29]. Liu and Lin also
introduced the application of colloidal gold as nanomaterial labels in immunosensors
and immunoassays [30].
Au NPs can play multiple roles in DNA sensing for immobilization, signal
amplification, and labeling. These aspects of Au NPs are discussed in Chap. 2 in
detail. Table 16.1 summarizes some aspects of the applications of Au NP-based
DNA-biosensing amplifying strategies and techniques.
16.2 Nanostructures in DNA Biosensing 465
Recent studies have demonstrated that metal nanoparticles such as silver can be
readily assembled on DNA via the chemical or photoinduced reduction of DNA-
complexed metal ions in aqueous media. Dongs team presented the studies on
fabricating hybrid nanobiomaterials containing thin films, where multilayer films
were utilized as nanoreactors to prepare DNA-silver nanohybrids in situ [44].
Zhang etal. proposed a strategy of using copper(II) complex of Luteolin C30H18CuO12
(CuL2) as an electroactive indicator based on silver nanoparticle- and multiwalled
carbon nanotubs (Ag/MWCNT) -modified glassy carbon electrode (GCE). The
proposed method dramatically increased the DNA attachment quantity and
complementary ssDNA detection sensitivity for its large surface area and good
charge-transport characteristics [45].
Table 16.2 summarizes different metal nanoparticles used in DNA-related
biosensing.
466
Table16.2 Applications of sensing strategies and detection methods of metal nanoparticle-based DNA biosensoring.
Sensing
strategies Methods Signal Immobilization method Label Nanostructure Targets DL Ref.
+
Optical UV-Vis, SERS, DNA-Ag ITO-PDDA immerse SERS: Rhodamine Ag NPs [44]
XPS DNA-Ag+ 6G, 4-aminothio
phenol
Fluorescence EB-DNA Ag NPMWCNT Cu-Luteolin Ag NPs DNA 65pM [45]
spectroscopy modified GCE complex
Colorimetric Pt-Aptamer Pt NPs Thrombin [46]
detection
Electro CV, DPV, EIS CuL2 Ag NPMWCNT Cu-Luteolin (CuL2) Ag NPs DNA 0.65pM [45]
chemical modified GCE complex
CV, DPV Ag-oxidation, Passive adsorption Ag Ag NPs DNA [47]
Guanine using amino linked
DNA onto Ag NPs
CV, i-t AgNPs-DNA Electrodeposition H2O2, glucose [48]
CV Ag+ reduction Thiolated DoxorubicinAg Ag NPs DNA of avian 1:00 PM [49]
oligonucleotide NPs flu virus
probe on Au H5N1
electrode
CV, DPV Co(phen)33+ DNA-PDDA-Fe@ Fe@Fe2O3, DNA damage 0.16mg [25]
Ru(NH3)63+ Fe2O3-MWCNT- MWCNTs
PDDA-GCE
CV, DPV Co(phen)33+ DNA-PDDA-Fe@ Fe@Fe2O3 DNA damage 0.4mg [50]
Ru(NH3)63+ Fe2O3-AuNP- nanonecklace,
PDDA-GCE Au NPs
16 Nanostructured Biosensing and Biochips for DNA Analysis
16.2 Nanostructures in DNA Biosensing 467
Table16.3 (continued)
Sensing strategies Methods Indicator/signal Immobilization method Label Nanostructure Targets DOL Ref.
Optical UV-Vis, FTIR, ZnO NPs ZnODNA [61]
XPS, AFM interaction
Photoluminescence ThiolDNAMTPS Label-free GaN nanowires [54]
spectroscopy GaNNWs
Time-resolved Europium DNA covalently immobi- Silica NPs Silica NPs DNA [62]
fluorescence complex lized on the common
glass slides surface
SPR, CV, CdS Nps, Pt AptamerNPscocaine CdS Nps, Pt NPs, Cocaine [63]
photocurrent NPs, Au NPs Au NPs
Fluorescence CdTe CdTemolecular beacon CdTe, graphene DNA 12nM [52]
oxide
Gravity QCM Absorption Label-free ZnO nanorod 60-fold, [64]
mercaptohexanol 30-fold
for DNA
16 Nanostructured Biosensing and Biochips for DNA Analysis
16.3 Nanostructures for DNA Biochips 471
Fig.16.16 Nanomechanical detection of DNA hybridization. Schematic of the (a) experiment and
(b) stiffness map. After hybridization, the surface of the array spot is scanned with the AFM to
generate the stiffness map. Scan size, 3mm. Hybridized molecules are measured to be less stiff and
appear as dark brown spots. dsDNA, double-stranded DNA; ssDNA, single-stranded DNA.
(c)stiffness at each pixel in (b) is calculated from forcedistance curves. Reprinted with permis-
sion from Husale etal. [92], 2009, Nature
information about the system. Thus, the choice of the detection technique heavily
depends on what one is interested in and the system under investigation.
Husale and coworkers [92] used a label-free atomic force microscope (AFM) tech-
nique to investigate the stiffness of single- and double-stranded DNA molecules, as
illustrated in Fig.16.16. The resulting stiffness maps show hybridized DNA molecules
as dark brown spots. This identification is verified by a series of experiments with
noncomplementary targets and complementary targets with varying concentrations.
Golovlev etal. [93] developed two computational models to study the ionic inter-
action of colloidal particles and biopolymer molecules on a microarray surface. The
theoretical considerations are in qualitative agreement with the experimental results
for detecting large target DNA and a 50-base-long synthetic oligonucleotide on the
amino-modified microarray substrate. The experimental and theoretical results of
this study allowed further development of the label-free microarray technology
demonstrated previously for gene expression analysis that is free of reverse tran-
scription and dye labeling [93].
Tamiya etal. reported an Au-coated nanostructured biochip with functional-
ized thiolated primers on its surface for the label-free and real-time optical detec-
tion of polymerase chain reaction (PCR) [94]. The immobilization of 5-end
474 16 Nanostructured Biosensing and Biochips for DNA Analysis
The advantage of fully electrical chips is the intrinsic high spatial resolution and
direct signal coupling of the biosensing element and the transducer. Numerous stud-
ies have been devoted to the construction of miniaturized biosensing devices
enabling the electrical monitoring of metabolites as well as the evaluation of biomo-
lecular interactions [95].
The use of metal nanoparticles in bioanalytical applications has recently attracted
increased attention. Besides using optical properties of nanoparticles, such as
changes in light scattering due to the induced aggregation of particles by interac-
tions between biomolecules, colorimetric measurements [78, 79, 89], electrochemi-
cal detection, and quantification principles have been successfully adapted for
biochip analysis, including stripping voltammetry [75, 95, 96], cyclic voltammetry
[94, 95, 97], amperometry [98], electrical/FET [77, 79, 81, 83, 88, 91], impedance
spectra [97, 99], and resistance [96].
Label-based detection methods: Signal-enhancement strategies such as silver
deposition on gold particles can further increase the sensitivity and facilitate the
use of nanoparticles in highly sensitive biomolecule detection [75]. Hsing and
coworkers presented a complete DNA-based assay in a single siliconglass micro-
chamber for multiple pathogen (Escherichia coli and Bacillus subtilis) detection.
The assay involves the following steps: (1) sample preparation using thermal cell
lysis and magnetic particle-based target genome isolation; (2) target DNA amplifi-
cation by the PCR; (3) hybridization of the amplicons to their complementary
oligonucleotide capture probes immobilized onto individual detection electrode
surfaces; and (4) electrochemical transduction of the recognition event via gold
nanoparticles and silver enhanced with signal amplification using electrocataytic
silver deposition [75].
Chens team designed and developed an array-based CMOS biochip to replace
the conventional DNA microarray for DNA identification by electrical detection
[77]. They used a self-assembly gold nanoparticle multilayer to conduct and amplify
electrical signals. The electric current passing through the gold nanoparticle multi-
layer, which is formed by complementary target oligonucleotide strands, exceeded
that of the gold nanoparticle monolayer by three orders of magnitude. Chen also
demonstrated an electrical detection method on a DNA biochip that employed an
approach for the ultrasensitive detection of DNA using self-assembled gold nano-
particles and bio-bar-code-based amplification (BCA) DNA [81]. The detective
16.4 Conclusions 475
concentration of target DNA with electrical DNA biosensor was as low as 1fM for
the analysis of current-voltage curves.
Schuler etal. demonstrated that it was possible to utilize screen-printed electrode
structures for a chip-based electrical DNA detection, along with silver nanoparticles
as amplification agents [83]. The screen printing of electrode structures for DNA-
chip with electrical detection offered an interesting and cost-efficient possibility to
produce DNA chips with microstructured electrodes.
Label-free detection methods: Wang and coworkers showed that the nanopore alu-
minum anodized oxide (AAO) membrane could be employed to immobilize specific
ss-probes by aminosilanes and glutaraldehyde linker, and then used for the label-
free electrical detection of complementary target bacteria DNA without additional
modification [97]. This AAO membrane-based biosensor provided a detection limit
of 0.5nM for complementary target DNA.
Shiigi etal. fabricated a highly controlled nanogap electrode, consisting of an Au
nanoparticlealkylchainAu nanoparticle repeated sequence, in which the gap
between particles was precisely controlled with the binder molecule [96]. Combining
this with the conductive DNA enabled a label-free measurement of the very small
electrical property change in DNA with a high S/N ratio (>30). Upon adding a
complementary oligonucleotide on the nanoparticle film chip, an immediate
decrease in the film resistance (ca. 1.4W) due to a hybridization event occurred in
a reproducible manner with this simple setup.
Table 16.4 summarizes the applications of nanostructures in DNA biochips/
microarrays.
16.4Conclusions
A variety of nanostructures have been introduced into DNA biosensing and DNA
microarrays for investigating DNA hybridizations, DNA damage, interactions
between DNA and other molecules, and so on. This chapter overviews a few
strategies using nanostructures for the preparation of DNA biosensors and microar-
rays, which may contribute to improving DNA biosensing, including a simple and
low-cost design, and sensitivity enhancement. These nanostructures are believed to
be very promising in the stages such as fictionalization, specific binding, and biorec-
ognition. Moreover, multifunctional nanostructures can be composited together to
achieve versatile sensing units, to meet the demands of fast, simple, and inexpensive
methods for DNA biosensing.
DNA microarrays clearly demonstrate the value of miniaturization and automa-
tion. More and more researchers and commercial organizations have started apply-
ing techniques to manipulate from micro- to nano- even picoliter sample volumes
into micro-, even nano-space automated biochip systems. Such microdevices will
give various science researchers and even ordinary consumers significant benefits,
including automation, cost savings, high throughput, and ease of use.
Table16.4 Using nanostructures binding DNA biochip technology
476
(continued)
Table16.4 (continued)
478
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Chapter 17
Cytosensing and Cell Surface Carbohydrate
Assay by Assembly of Nanoparticles
17.1Introduction
Cells are the basic units of life. All organisms are made up of cells. Thus, developing
sensing platforms for probing the chemistry and physics in or at a living cell is one
of the basic goals in understanding the intricate processes that ultimately contribute
to life and life processes. Moreover, in the field of medicine, each cell type has a
unique molecular signature that distinguishes between healthy and diseased tissues
[1]. Cancerous cells are differentiated from noncancerous ones on the basis of
intracellular or extracellular (cell surface) biomarkers. In most cases, the distinc-
tions between normal vs. tumor and benign vs. metastatic cells are often subtle.
Thus, the identification of cellular signatures for early cancer cell detection is a
major hurdle for cancer therapy. The earlier these signatures can be established, the
more effectively they can be treated [2]. In this regard, technological systems that
provide sensors with high sensitivity, selectivity, and stability are in high demand
to identify the unique cellular characteristics such as proteins and nucleic acids in
and on living cells at early disease states, providing the prospects of better health
and more effective therapy [3].
In the context of cytosensing, researchers require the ability to track molecules
within their native environs. Thus, the questions relating quantity, timing, and loca-
tion become as important as knowing the molecular targets of enzymes or receptors.
The efficiency of sensing systems consists of recognition elements and the trans-
duction process and is critically related to the outcome of the detection process in
terms of the response time, signal-to-noise (S/N) characteristics, sensitivity, and
selectivity of the system.
Current methods for cell interrogation are mainly based on antibody-, nuclear
acid-, and aptamer-based recognition techniques to introduce a unique probe into
the cellular components under investigation. This is due to the fact that few biomol-
ecules are naturally endowed with features that permit their direct detection in
complex milieus. Due to the flexibility in implementation, noninvasiveness, and
high sensitivity, fluorescence microscopes and flow cytometers have been considered
the key tools for probing cells. Although notable progress has been made in this
field, challenges in improving the recognition process as well as designing new
signal-transduction mechanisms still exist.
To achieve ultrasensitive detection and imaging for cytosensing, the combination
of nanotechnology with chemistry, biology, physics, engineering, and medicine has
emerged as a cornerstone solution. Nanoscale materials are typically smaller than
several hundred nanometers and are comparable to the size of large biological
molecules such as enzymes, receptors, and antibodies. With sizes about 10010,000
times smaller than those of human cells, these nanomaterials can offer unprece-
dented interactions with biomolecules both on the surface of and inside cells [4].
The most well-studied nanomaterials include metal nanoparticles (NPs) [5, 6],
quantum dots (QDs) [7, 8], carbon nanomaterials [9, 10], silica nanomaterials [11],
and paramagnetic NPs [12]. In addition to the large surface-to-volume ratio that
favors miniaturization, NPs possess unique optical, electronic, and magnetic prop-
erties depending on their core materials. Furthermore, these properties of the nano-
materials depend on their size and shape and vary with their surrounding chemical
environment. In addition, NPs can be fashioned with a wide range of small organic
ligands and large biomacromolecules by using tools and techniques of surface
modification [3]. These distinctive physical and chemical properties of nanomateri-
als make them a promising scaffold for the pursuit of new recognition and transduc-
tion processes, as well as increasing the sensitivity, selectivity, reliability, and
practicality of detection [13]. The progress may revolutionize research on cell adhe-
sion, glycobiology, cell trafficking, and signal transduction along with structural
biology, transcriptional regulation, and disease diagnosis and treatment.
This chapter describes recent advances in research involving the use of NPs in
the detection of cells and diagnosis of analytes at or within a cell. A significant
number of modern technologies and instrumentations involved in the progression of
cytosensing are reviewed here. Because cell surface carbohydrates, in the form of
glycopeptides, glycolipids, glycosaminoglycans, proteoglycans, or other glycocon-
jugates, have long been known to participate in the plethora of biological processes,
this chapter will also give a brief introduction to the application of nanomaterials in
cell surface carbohydrate assay.
The surface coating of nanomaterials can be readily adjusted to achieve not only
nontoxic and biocompatible but also specific localization on a living cell. The nature
of surface coatings and their subsequent geometric arrangement on the NPs deter-
mine the overall size of the colloid, which plays a significant role in the biokinetics
and biodistribution of NPs in the body. Nevertheless, the biological or molecular
coating acts as a bioactive and selective interface. Moreover, multiple, potentially
different targeting ligands on the NPs can provide enhanced binding affinity and
specificity. The types of specific coatings or derivatization of these NPs depend on
the end application and should be chosen by keeping a particular application in
mind [15]. This will ultimately lead to further improvements in early cancer diagno-
sis and treatment.
Nanomaterials can be designed to integrate multiple functions in a very predict-
able manner to meet the needs of specific applications. There are two strategies to
fabricate multifunctional nanostructures. The first, molecular functionalization,
involves attaching antibodies, proteins, and dyes to the NPs. The second method
integrates one NP with other functional nanocomponents, such as encapsulating
magnetic NPs in QDs or metallic NPs. Because they can exhibit several features
synergistically and deliver more than one function simultaneously, such multifunc-
tional nanomaterials can have unique advantages in real-time cytosensing [12].
NPs hold immense promise as versatile amplifying labels for cytosensing. There
are two central and related concepts that have been used for the amplified transduc-
tion of biorecognition events. One is the simultaneous immobilization of the recog-
nizing component and reporter tag on nanomaterials; the latter is often species with
catalytic ability, such as enzymes. The other is the multiple loading of the reporter
tags or a combination of tags on the surface of nanomaterials or by entrapping them
within the nanomaterials, which provides the opportunity for dramatic cascade
signal amplification.
The nanomaterial-based techniques, combined with nanofluidics, single-
molecule detection, and multiplexing methodologies, provide exciting new possi-
bilities for ultrasensitive cytosensing. In particular, benefiting significantly from
technological advancements in nanotechnology, emerging biomedical sciences and
biotechnology will inevitably shed new light on acquiring a deep understanding of
biochemical processes of life and improving disease diagnosis and treatment.
since longitudinal studies can be performed in the same animals, aids in early lesion
detection in patients and patient stratification, and helps in individualized treatment
monitoring and dose optimization [16]. Cytosensing modalities include optical
fluorescence, molecular magnetic resonance imaging (MRI), surface-enhanced
Raman scattering (SERS), electrochemistry, and colorimetry. Many hybrid systems
that combine two or more of these modalities are already commercially available,
and a few others are under active development [12].
Fig. 17.1 (a) Model structure of a silica-shelled single QD micelle; (b) intracellular delivery
(fluorescence confocal microscopic image) of the micelles in viable HeLa cells. Reprinted with
permission from Zhelev etal. [24]. 2006, American Chemical Society
17.3.1.1Peptide-Conjugated Nanoparticles
Although aptamer-conjugated NPs have been ideal candidates for cell surface pro-
tein recognition and therefore cancer cell targeting [25], the targeting of QD conju-
gates in living subjects was first reported using peptide-coated ZnS-capped CdSe
QDs to bind the lung, blood vessels, and the lymph, respectively [26]. In this work,
polyethylene glycol (PEG) was used to eliminate the nonspecific uptake of the con-
jugate by the reticuloendothelial system, which opened up a new field of QD-based
nanoplatforms for cellular specific targeting. A variety of targeting peptides have
been explored to label QDs for improving the stability and target ability during
invitro studies, such as cellular nuclear localizing sequence (NLS) (Pro-Pro-Lys-
Lys-Lys-Arg-Lys-Val), cell-penetrating Tat peptides (Arg-Arg-Arg-Gln-Arg-Arg-
Lys-Lys-Arg-Gly-Tyr), neuropeptide AST1 (Ala-Pro-Ser-Gly-Ala -Gln-Arg-Leu-T
yr-Gly-Phe-Gly-Leu-NH2), and neurotoxins such as chlorotoxin (CTX) and
490 17 Cytosensing and Cell Surface Carbohydrate Assay by Assembly of Nanoparticles
Fig. 17.2 In vitro staining of human glioblastoma U87MG cells using 1 nM QD705-RGD.
Staining of U87MG cells with 1nM QD705 (denoted U87MG+QD705) or 1nM QD705-RGD
in the presence of 2mM c(RGDyK) (denoted U87MG+Block) are also shown. Reprinted with
permission from Cai etal. [29]. 2006, American Chemical Society
QDs showed great potential as nanoprobes for detecting tumor vasculature invivo
for most cancer types (Fig.17.2). More recently, Choi etal. fabricated zwitterionic
QDs with an overall diameter of 5.5nm coupled with RGD [30]. Those particles
17.3 Cytosensing by Assembly of Nanomaterials 491
demonstrated good tumor targeting and, because of their extremely compact size,
showed renal clearance by the kidneys within 4h postinjection. The results of this
study suggested a set of design rules for tumor-targeted NPs that could be elimi-
nated through renal clearance.
17.3.1.2Antibody-Conjugated Nanoparticles
QDs functionalized with antibody have also fulfilled their promise as nano-
probes for molecular imaging. Gao et al. conjugated CdSe@ZnS QDs with
prostate-specific antigen (PSA)specific monoclonal antibodies for prostate
cancer targeting and imaging in mice [31]. The QD probes were accumulated at
tumors both by the enhanced permeability and retention of tumor sites and by
antibody binding to cancer-specific cell surface biomarkers (Fig.17.3). These
results raised new possibilities for the ultrasensitive and multiplexed imaging of
molecular targets invivo.
In a recent study, by incorporating antibody-coated QDs within biodegradable
polymeric nanospheres, a hybrid nanocomposite (QDNC) was developed and a
noninvasive method to image subcellular structures in living cells was designed
[32]. The bioresponsive delivery system underwent endolysosomal to cytosolic
translocation via the pH-dependent reversal of nanocomposite surface charge polar-
ity (Fig. 17.4). Upon entering the cytosol, the polymer nanospheres underwent
hydrolysis, thus releasing the QD bioconjugates. This approach facilitated multi-
plexed labeling of subcellular structures inside live cells without the requirement of
cell fixation or membrane permeabilization. More importantly, this work demon-
strated an important rational strategy for the design of a multifunctional nanosystem
for biological applications.
492 17 Cytosensing and Cell Surface Carbohydrate Assay by Assembly of Nanoparticles
Fig.17.4 Mechanism of cytosolic delivery and subcellular targeting of QDNCs. Schematic repre-
sentation depicting QDNC escape from the endolysosomal compartment upon cellular internaliza-
tion with cytosolic release of the encapsulated cargo. Antibody-conjugated QDs can be delivered
in this manner to allow the labeling of subcellular organelles or other molecular targets. Reprinted
with permission from Kim etal. [32]. 2008, American Chemical Society
17.3.1.3Aptamer-Conjugated Nanomaterials
As is well known, the early detection of cancer greatly increases the chances for
successful treatment. Consequently, research on accurate and sensitive recognition
and detection of cancer cells is extremely important for cancer diagnosis and ther-
apy. Cancer cells, especially those in the early stages of disease development, may
have a very low density of the target on the cell surface. Recent research discovered
that aptamers represent an attractive alternative to the routinely used antibodies to
recognize these targets because of their small sizes, chemical robustness, and ease
of synthesis [33]. As the DNA molecules with predicable structures and easy site-
specific chemical modification, aptamers can readily link to advanced signaling
mechanisms [33, 34].
Bagalkot et al. developed a QD-aptamer (Apt)doxorubicin (Dox) conjugate
[QD-Apt(Dox)] for a targeted cancer imaging, therapy, and sensing system [35].
The A10 RNA aptamers used were specific for prostate-specific membrane anti-
gen (PSMA) expressing on the LNCaP cell surface, and the Dox, a fluorescent
17.3 Cytosensing by Assembly of Nanomaterials 493
Fig.17.5 Schematic illustration of (a) QD-Apt(Dox) Bi-FRET system and (b) specific uptake of
QD-Apt(Dox) conjugates into target cancer cell through PSMA-mediated endocytosis. Reprinted
with permission from Bagalkot etal. [35]. 2007, American Chemical Society
anthracycline drug emitting at 520640 nm, was then intercalated into the
double-stranded CG sequences in the PSMA aptamer. As shown in Fig.17.5,
the formation of this nanostructure resulted in quenching of both QDs and Dox
based on a bifluorescence resonance energy-transfer (Bi-FRET) mechanism. After
incubating the aptamerQDDox conjugates with target cells, Dox was released
from the conjugates, resulting in fluorescence recovery of both QDs and Dox in
cells. This strategy was sensitive to detecting cancer at a single-cell level since both
QDs and Dox gave sharp images of cancer cells with low background noise.
To increase the signal and enhance the binding affinity, multivalent binding,
instead of single-aptamer binding, is usually considered to be an effective approach.
Nanomaterials are thus considered to be particularly advantageous as multivalent
ligand scaffolds by virtue of their large surface area and variable sizes. Tans group
synthesized AuAg nanorods (NRs) as nanoplatforms for multivalent binding by
multiple aptamers on the rods to increase both the signal and binding strengths of
these aptamers in cancer cell recognition [36]. Up to 80 fluorophore-labeled aptam-
ers could be attached on one Nile red (NR), leading to a 26-fold higher affinity and
over 300-fold higher fluorescence signal. The molecular assembly of aptamers on
nanomaterials achieved multivalency, and showed the potential application for the
elucidation of cells with low density of binding sites. This also illustrated how
molecular assemblies could integrate the properties of aptamers and nanomaterials
to improve cancer cell targeting.
494 17 Cytosensing and Cell Surface Carbohydrate Assay by Assembly of Nanoparticles
17.3.1.4Streptavidin-Conjugated Nanoparticles
17.3.1.5Carbohydrate-Conjugated Nanomaterials
The cell membrane surface consists primarily of a thin layer of amphipathic phos-
pholipids, carbohydrates, and many integral membrane proteins. Their amount and
types differ between species according to the function of cells [1]. Current detection
methods for the specific recognition of intracellular/extracellular biomarkers (e.g.,
DNA/RNA/proteins) require previous knowledge of specific mutations in genome
or changes in protein biomarkers. However, there has been no single marker or a
combination of biomarkers that has sufficient sensitivity and specificity to differen-
tiate among normal, cancerous, and metastatic cell types [41].
An NP-conjugated polymer sensor array, which relies on selective interactions
between multiple reporter elements and the target cell, represents a new method
for diagnostic, biophysical, and surface science processes involving cell surfaces
[1, 42]. The multivalent capabilities and sensitivity of conjugated polymers to
minor conformational or environmental changes make them ideal candidates for
biosensing applications. Combining polymer-conjugated NPs with array technol-
ogy, this platform holds particularly promise for cell sensing, such as bacteria
identification [42] and mammalian cancer cell detection [1].
For example, a multivalent fluorescent polymer (PPECO2) conjugated differentially
with functionalized gold NPs has been subjected to incubation with cell sample. The
quenched fluorescent polymer can be displaced by cells with the concomitant restora-
tion of fluorescence. Through detection of the fluorescent pattern, (1) different cell
types, (2) normal, cancerous, and metastatic human breast cells, and (3) isogenic
normal, cancerous, and metastatic murine epithelial cell lines can be rapidly and effec-
tively distinguished [1]. A similar method has also been developed for bacterial sensing
with a conjugated polymer (Sw-CO2) featuring a branched oligo(ethylene glycol) side
chain to suppress nonspecific polymer-microorganism interactions (Fig.17.6) [42].
Unlike small-molecule fluorophores, fluorescent polymers feature a molecular-
wire effect and polyvalent modes of interactions that can enhance signal generation
[43, 44]. Moreover, conjugated polymer chains with multiple recognition elements
can bind to one analyte molecule, thereby increasing both the binding efficiency and
recognition selectivity for specific analytes [44].
Fig.17.6 Design of the NPconjugated polymer sensor array. (a) Schematic representation of the
displacement of anionic conjugated polymers from cationic NPs by negatively charged bacterial
surfaces. (b) Schematic illustration of fluorescence pattern generation on a microplate. The fluo-
rescence response is dependent upon the level of displacement determined by the relative NP-PPE
binding strength and bacteriaNP interactions. In the diagram, AG on the microplate represents
bacteria of different types, and codes 14 represent the different PPENP constructs. Reprinted
with permission from Phillips etal. [42]. 2008, Wiley
Fig.17.7 In vitro and invivo fluorescence imaging with LPSi NPs. (a) In vitro cellular imaging
with LPSiNPs. Red and blue indicate LPSi NPs and cell nuclei, respectively. The scale bar is
20mm. (b) In vivo fluorescence image of LPSi NPs injected subcutaneously and intramuscularly
on each flank of a mouse. Reprinted with permission from Park etal. [48]. 2009, Nature
Hydrogel NPs (nanogels), colloidally stable NPs made from hydrogels, are appealing
probes for use in chemical and biochemical sensing because of their stability, bio-
compatibility, and softness [49]. Nanogels have been employed to develop detection
technology for protein [50] and carbohydrates [51]. Recently, fluorescent nanogels
were reported to be capable of sensing temperature and pH in the cytoplasm of liv-
ing cells [52]. The nanogel was fabricated by the combination of a thermorespon-
sive polyNIPAM unit with a water-sensitive fluorophore, which could transduce
volume changes into a change in fluorescence intensity (FI) [5].
In a later work by Peng et al., a ratiometric fluorescent nanogel was prepared
rather simply from an inert but biocompatible polyurethane polymer that was
made pH sensitive by loading it with the pH indicator Bromothymol blue (BTB)
[53]. Furthermore, efficient fluorescence resonance energy transfer (FRET)
inside the nanogel was achieved by the addition of two standard fluorophores,
coumarin 6 (C6) and NR, and constituted the sensing mechanism. This nanogel
could be incorporated by living epithelial normal rat kidney (NRK) cells by
vesicular uptake mechanisms and was capable of intracellularly sensing pH val-
ues in the physiological range. By replacing the respective indicator dyes (probes)
by indicators for other ions, various other kinds of sensing nanogels could be
constructed and were likely to have a wide scope in terms of intracellular chemical
sensing.
498 17 Cytosensing and Cell Surface Carbohydrate Assay by Assembly of Nanoparticles
Fig.17.8 (a) In vitro NIR photoluminescence images of human malignant glioma cells (U87 MG)
treated with exchange-SWNT/RGD conjugates. Arginineglycineaspartic acid (RGD) peptide
ligand binds to avb3-integrin overexpressed on the cells. (b) Human breast cancer cell line
(MDA-MB-468) is used as a negative. Cells treated with exchange-SWNT/RGD show a high posi-
tive signal with very low nonspecific binding to the negative cell line. Reprinted with permission
from Welsher etal. [57]. 2009, Nature
In order to overcome the issue, Welsher et al. has shown that sonicating SWNTs
with sodium cholate, followed by surfactant exchange to form PL-PEG-coated nan-
otubes, produces in vivo imaging agents that are both bright and biocompatible
[57]. Using the obtained nanoprobes, targeted cell imaging has been carried out in
the 1,1001,700-nm range using the intrinsic NIR photoluminescence of the
exchange-SWNTs (Fig.17.8), and the high-resolution intravital microscopy imag-
ing of tumor vessels beneath thick skin is also achieved.
The iron oxide NPs targeted to a cell surface molecule can remain bound to their
target, thus being useful as molecular MRI probes [62]. To target endothelial markers
of inflammation, anti-VCAM-1 antibodies have been conjugated to microparticles of
iron oxide, allowing specific and quantitative binding to activated endothelial cells.
The nanoprobes can be used for invivo detection of vascular cell adhesion mole-
cule-1 (VCAM-1) expression in a mouse model of acute brain inflammation [63].
In order to improve bioavailability for molecular imaging of extravascular targets,
extremely small but potent NPs open up possibilities to develop targeted MRI probes.
In a recent work, a strongly positive charged peptide, protamine, was used to coat
citrate-stabilized iron oxide NPs by electrostatic attraction [64]. The protamine
allowed the chemical decoration of the NPs by Annexin A5, a cell apoptosis-
detection marker. The nanoprobes with a hydrodynamic diameter of 15nm have been
successfully tested on apoptotic human lymphoblastic T-cell (Jurkat) cultures. The sub-
stantially reduced hydrodynamic diameter is interesting for invivo applications since
it facilitates extravasation, thereby enabling MRI of extravascular targets.
Although various types of cancer cells can be detected both invitro and invivo
by using antibody-immobilized magnetic NPs, there is still the need to develop
other recognition element-based nanoprobes, considering the fact that particular
antigenic structures may be absent in some types of cancer cells. An attractive
ligand for receptor-mediated interaction is carbohydrates and, in particular, glyco-
conjugates, which play important roles in cancer development and metastasis [65].
17.3 Cytosensing by Assembly of Nanomaterials 501
The targeting of cell surface molecules with adequately modified iron oxide
particles has allowed cellular delivery of the magnetic tag and MR imaging of
specific cell types [62]. For cell surface receptor targeting, transferrin, lactoferrin,
transforming growth factor-a (TGF-a), insulin, nerve growth factor (NGF),
ceruloplasmin, pullulan, elastin, albumin, RGD peptide, and folic acid etc. have
been used to modify magnetic NPs for various biomedical applications. The
receptors for these ligands are frequently overexpressed on the surface of mam-
malian cells [15]. These receptors are not only cellular markers, but also have
been shown to efficiently internalize the NPs targeted to these receptors via receptor-
mediated endocytosis [15].
Integrin avb3 is the one of the most well-studied targets for molecular MRI [4].
The avb3-integrin is the receptor for a variety of extracellular matrix proteins
containing an RGD sequence. The avb3-integrin plays a key role in angiogenesis
and is an attractive target for the treatment of certain tumor types (melanoma,
glioblastoma, ovarian, and breast cancer) [69].
In a recent study, cyclic peptide c(RGDyK), with high selectivity and strongly
enhanced binding activity, was employed to modify Fe3O4 NPs using a new ligand
4-methylcatechol (4-MC) for coating NPs and conjugating the peptide [70]. The
c(RGDyK)-conjugated NPs showed good binding ability to integrin on U87MG
human glioblastoma cell lines. The enhanced binding, compared with monomeric
RGD peptide, was due to the multivalent effect. The Fe3O4 NPs synthesized in this
work were ultrasmall, with a 4.5-nm core size, and the obtained c(RGDyK)-MC-
Fe3O4 NPs had an overall diameter of ~8.4nm. This smaller hydrodynamic size was
desirable for overcoming the nonspecific uptake problem and enhancing the
extravascular ability.
Tumor cells expressing the membrane-bound matrix metalloproteinase MMP-2
can also be labeled with MRI nanoprobes with a high level of specificity both
invitro and invivo using a 9L gliosarcoma cell line and mice bearing xenografts
as models [61]. The nanoprobe is composed of an iron oxide core coated by PEG
and functionalized by a 36-amino-acid peptide, CTX. The CTX is of attractive
interest due to its specific binding capability to glioma, medulloblastoma, prostate
cancer, sarcoma, and intestinal cancer. The nanoprobes show a profound improve-
ment in tumor labeling efficiency compared with nontargeting nanoprobes, which
may be attributed to the ligandreceptor-mediated nanoprobe internalization by
target cells.
Besides decreasing the NP size, an alternative strategy for improving the
MRI sensitivity is controlling the NP shape [63]. The mononuclear phagocytic
system (MPS) is one of the most important pathways for clearance of NPs. The
design of elongated NPs offers a new way to prolong blood half-life due to the
low MPS uptake. Another approach to enhance MRI detection is the develop-
ment of new magnetic core materials [71]. For example, a better sensitivity of
MnFe2O4 than Fe3O4 in MRI applications has recently been demonstrated for
individual NPs [72].
17.3 Cytosensing by Assembly of Nanomaterials 503
Fig.17.10 Schematic illustration for the preparation of bimodal imaging nanoprobes having both
19F-based multispectral magnetic resonance and QD-based multicolor optical imaging capabili-
ties. Reprinted with permission from Lim etal. [73]. 2009, American Chemical Society
Fig. 17.11 In vivo accumulation of nanoparticles at tumor site. (a) In vivo T2-weighted MR
images (upper) and color-mapped (lower) images of tumor site before and 3h after intravenous
injection of the NPs (arrows indicate tumor site). (b) Confocal laser scanning microscopic images
of sectioned tumor tissue harvested 24 h after injection. Left: Red fluorescence showing
NP-internalized cells. Right: Merged image with DAPI-stained nuclei (blue) (scale bar 10mm).
Reprinted with permission from Lee etal. [74]. 2010, American Chemical Society
various sizes and chemical nature, designated for specific goals. As a proof of
concept, colloidally stable trifunctional structures have been assembled by binding
together magnetic particles, QDs, and antibodies using barnase and barstar. The
specific interaction of such superstructures with human ovarian carcinoma SKOV-3
cells, which overexpress the HER2/neu receptor on their surface, resulted in fluores-
cent labeling of the cells and their responsiveness to magnetic field.
This new generic method, protein-assisted nanoassembly, can be regarded not as a
substitution of other most general approaches, (strept)avidinbiotin system and
complementary DNA-assembled scaffolds, but as an alternative that broadens the range
of available nanoagents. It can be employed either on its own or in combination with
other techniques allowing the synergistic use of inorganic moieties, organic particles,
and single biomolecules for self-assembly of a wide variety of superstructures with
506 17 Cytosensing and Cell Surface Carbohydrate Assay by Assembly of Nanoparticles
Fig.17.12 Schematic of pure and functionalized QD- and SPIO-nanosomes. Nanosomes were
generated from lipids extracted from human triglyceride-rich lipoproteins. QD- and SPIO-
nanosomes provide information on the location and distribution of the lipoproteins within the cells
or tissues. Radiolabeled SPIO-nanosomes allow quantification of cell uptake and organ distribu-
tion. Functionalizing the nanosomes with apolipoprotein E and lipoprotein lipase allows specific
targeting to the liver lipoprotein receptors. Reprinted with permission from Bruns etal. [76].
2009, Nature
Fig.17.13 Possible variants for assembling of three particles into a superstructure by the protein-
assisted nanoassembler method. The correspondence of different combinations of BBS proteins
conjugated with the green and gray particles and the structure number (ac) are shown in the table.
Reprinted with permission from Nikitin etal. [78]. 2010, National Academy of Sciences
metal nanostructure [80]. Since the first reported SERS on silver and gold colloids
in 1979 [81], they have become the most commonly used metal substrates for SERS.
Colloidal gold has been found to amplify the efficiency of Raman scattering by
1415 orders of magnitude [82]. These nanostructures must satisfy certain condi-
tions to provide a strong Raman signal. First of all, the roughness of the metal
surface should be smaller than the wavelength of the excitation field. Second, the
wavelength of the excitation laser should correspond to the plasmon resonance
wavelength of the SERS substrate. The latter can be controlled by changing the size,
shape, and material of the metallic structures [83]. SWNTs also show an intense
Raman peak produced by the strong electronphoton coupling that causes efficient
excitation of tangential vibration in the nanotubes quasi-one-dimensional structure
upon light exposure. In a recent work reported by Keren et al., SWNTs were used to
demonstrate whole-body Raman imaging, NP pharmacokinetics, multiplexing, and
invivo tumor targeting [84].
Besides substrates, SERS labels and probes need reporter molecules to attach
substrate, which provide the Raman signature of the analyte. These reporter mole-
cules are in many cases fluorescent dyes. In practice, it is necessary to distinguish
two types of SERS labels. In the first type, the substrates are enwrapped in mono-
layers of reporter molecules, or further embedded in a protective coating of glass or
508 17 Cytosensing and Cell Surface Carbohydrate Assay by Assembly of Nanoparticles
PEG [80]. These SERS labels are physically robust and immune to their biological
and chemical environment. Based on the optical signature of the reporter, biological
structures can be highlighted and imaged. The second type does not use a protective
cover, and the surface-enhanced Raman signal generated by other molecules present
in the local optical fields can also be obtained [80]. The main advantage of these
SERS probes lies in the ability to obtain spectra from target analytes present in
subcellular compartments. In order to deliver the SERS labels into cells and control
their intracellular aggregation and distribution, they can be functionalized with
specific antibodies, peptides, and DNA [85].
The high sensitivity of SERS is due to the electromagnetic field coupling on
roughened metal surfaces and junctions, which can cause the scattering signal of the
molecule to increase by several orders of magnitude compared to fluorescence [86].
Moreover, in SERS, excitation can be out of resonance with electronic transitions of a
reporter molecule. Thus, SERS detection is considerably less sensitive to photobleach-
ing, ensuring the stability of SERS labels. The characteristic Raman bands are up to
two orders of magnitude narrower than fluorescence bands [86]. The fingerprint-like
nature of the sharp and distinguishable vibrational spectrum provides an enormous
multiplexing platform for invivo SERS imaging even under stringent physiological
conditions [87]. SERS probes fulfill the requirements of dynamic invivo systems
the use of very low laser powers and very short data acquisition times. Additionally, it
provides the free choice of excitation wavelength (and therefore, also detection range)
as yet another significant advantage over fluorescence labels [80].
Capitalizing on these unique features, recent studies have focused on the investiga-
tion of molecular phenomena inside living cells. These experiments are performed
by first introducing SERS probes into organisms and attaching them to the surface
of cells as targeting agents of specific surface markers, and then detecting the SERS
spectral signature of the reporter molecules [80].
A class of biocompatible and nontoxic pegylated gold NPs (Au NPs coated with
a protective layer of PEG) has been developed for in vivo tumor targeting and
surface-enhanced Raman detection with large optical enhancements [82]. When
conjugated to tumor-targeting ligands such as single-chain variable fragment (ScFv)
antibodies, the conjugated NPs are able to target tumor biomarkers such as epider-
mal growth factor (EGF) receptors on human cancer cells and in xenograft tumor
models (Fig.17.14). The advantage of this NP platform is the facile conjugation of
tumor-targeting ligands to heterofunctional PEG linkers.
The actual mechanism of particle uptake can be investigated by SERS. Using
gold nanoshells as a surface-enhanced Raman platform for intracellular sensing in
NIH-3T3 fibroblast cells, the voluntary, controllable cellular uptake of single gold
nanoshells has been studied by using a NIR Raman system [79]. This work demon-
strates that the uptake is independent of the known endocytotic mechanisms, and
shows that nanoshells can be used as a viable and versatile platform for intracellular
SERS nanosensors.
17.3 Cytosensing by Assembly of Nanomaterials 509
Fig.17.14 Cancer cell targeting by using antibody-conjugated SERS NPs comprising SH-PEG
shell and a heterofunctional PEG. Reprinted with permission from Qian et al. [82]. 2008,
Nature
The greatest potential of SERS labels and probes lies in the efficiency provided by mul-
tivariate methods for their fingerprint-based imaging. In several cellular processes, the
simultaneous investigation of different locations is favorable. To achieve this, it is useful
to introduce with each probe type a probe-specific signature that allows identification.
Zavaleta et al. demonstrated the ability of Raman spectroscopy to separate the
spectral fingerprints of up to ten different types of SERS NPs in a living mouse [88].
Based on the results, they simultaneously injected the five most intense and spec-
trally unique SERS NPs to image their natural accumulation in the liver (Fig.17.15).
This work combined the ultrasensitive properties of Raman spectroscopy with the
multiplexing capabilities of SERS NPs, allowing better detection of multiple
biomarkers associated with a specific disease.
More recent efforts have been reported on the biocompatibility and localization
dynamics of NPs in zebrafish embryos. Zebrafish is chosen as a model for sensor
510 17 Cytosensing and Cell Surface Carbohydrate Assay by Assembly of Nanoparticles
platform development because of its unique optical clarity, rapid growth and
development, high fecundity, and availability of large numbers of genetic mutant
zebrafish lines [86]. Through microinjected SERS NPs in vivo in developing
zebrafish embryos, their localization as the embryos develop from the one-cell stage
into desirable stages as organs or tissues form has been detected and imaged [86].
The NPs appear to be spread throughout the cytoplasmic bridges that connect all
zebrafish blastomeres, and do not affect the development from 4h (40% epibobly
stage) to 7 days postfertilization. This is the first report on multiplex SERS imaging
of zebrafish embryos to study its localization during development in conjunction
with toxicity and gene expression studies. This research can be further applied to
track the fate of targeted NPs in dividing cells as they develop into tissues.
17.3 Cytosensing by Assembly of Nanomaterials 511
Fig. 17.16 (a) Schematic representation of the ACGNP-based colorimetric assay; (b) plots
depicting the absorption spectra obtained for various samples analyzed using ACGNPs. The
spectra illustrate the differences in spectral characteristics observed after the ACGNPs bind to
the target cells. Reprinted with permission from Medley et al. [90]. 2008, American
Chemical Society
Fig.17.17 EIS
measurements of K562 cells
proliferated on Au NP-CS
gel/GCE after cell incubation
for (a) 24h, (b) 48h, (c)
72h, and (d) 96h. Inset:
relationship between
electron-transfer resistance
and proliferation time of
K562 cells on Au NP-CS
gel/GCE. Reprinted with
permission from Ding etal.
[99]. 2007, American
Chemical Society
Fig.17.18 Schematic representation of a microfabricated flow cell with an adjustable gap elec-
trode. Reprinted with permission from Yan etal. [103]. 2007, IOP
In recent years, strategies for the diagnosis, therapy, and treatment of neural disor-
ders have increasingly relied on electrical stimulation techniques [109]. For the
effective transmission of charge from electrodes to cells, the interface for cell adhe-
sion must have a low electrical impedance [110]. Inert metals, including platinum
and iridium oxide, and organic conducting polymers have been widely explored for
electrodecellular interfaces. Nanostructuring such electrodes can improve perfor-
mance by reducing impedance and influencing cell compatibility. Beginning in
2006, nanostructured carbons, such as CNTs and graphene, emerged as novel elec-
trode structures, providing an inherently electrochemically stable organic interface.
CNT-based interfaces produce electrodes with high capacitance and low imped-
ance, which make them one of the most promising materials for applications in
neural biosensing [110]. Interactions between various cell lines and CNTs have
been previously reported and have included the adhesion, growth, and differentia-
tion of neuronal cells on CNT-based substrates [92]. Providing electrical stimula-
tion to neurons via a CNTbased platform has led to the formation of a functional
neural network of neural stem cells (NSCs).
17.4 Cell Surface Carbohydrate Assay by Assembly of Nanoparticles 517
Keefer etal. used CNTs to coat conventional tungsten and stainless steel wire
electrodes, leading to enhanced recording and electrical stimulation of neurons in
culture, rats, and monkeys [111]. Another significant advance is the work by Kam
et al., in which layer-by-layer-assembled composites from SWNTs and laminin
were fabricated and found to be conducive to NSC differentiation and suitable for
their successful excitation [109]. These results indicate that the proteinSWNT
composites can serve as the material foundation of neural electrodes with a chemi-
cal structure better adapted with long-term integration with the neural tissue.
and little GalNAc residues on the K562 cell surface, which were in good agreement
with flow-cytometric results. The strategy was further used for effective monitoring
of the dynamic variation of glycans on cancer cell surfaces during both drug induce-
ment and erythroid differentiation of K562 cells.
For cellular viability and bodily function, glycosylation are in the context of
glycoproteins, glycolipids, glycosylphosphatidylinositol anchors, glycosaminogly-
cans, and polysaccharides. The overproduction of certain glycoproteins is a common
feature of tumors. For example, an energy-dependent transport protein named
P-glycoprotein (P-gp), which is often overexpressed at the tumor cell, is closely
related to multidrug resistance phenomenon [126]. Accordingly, the ability to
characterize the cell surface glycoprotein expression status is critical to advance
chemotherapy of the malignant tumor.
A strategy to detect P-gp on K562/ADM cell and quantify the cell number has
been developed by Jus group using electrochemical immunoassay combined with
the immobilization of cells on a highly hydrophilic interface [127]. The interface
was constructed by adsorption of Au NPs on a methoxysilyl-terminated butyrylchi-
tosan modified glassy carbon electrode (Au-CS/GCE). The incubation with P-gp
monoclonal antibody and then the secondary alkaline phosphatase (AP)conjugated
antibody introduced AP onto the electrode-immobilized cells. The bound AP led to
an amperometric response of 1-naphthyl phosphate, which was proportional to the
logarithm of cell concentration in the range from 5.0104 to 1.0107 cells/mL,
with a detection limit of 1.0104 cells/mL. The results were comparable to the
flow-cytometric analysis of P-gp expression. Later, some groups expanded this
strategy to investigate the expression extent of P-gp on HeLa cells [126] and BGC
823 cells [128].
An alternative method for cell surface carbohydrate assay has been developed
based on the comparison of cell-binding extents to different lectin-modified
electrodes, which could be monitored conveniently by EIS and ECL. These strate-
gies obviate the cell immobilization step and cell labeling, and enable the in situ
investigation of cell surface glycan without disturbing cellular nature.
For example, label-free strategies for facile and specific electrochemical analysis
of cell surface glycan expression have been developed by combining rapid and
sensitive EIS measurement with single-walled carbon nanohorns (SWNHs) [123]
or MWNTs [124] and using lectins as recognition units. An effective three-
dimensional (3D) recognition interface toward cell surface glycan motifs was
constructed on glassy carbon electrode through stable immobilization of lectins
on uniquely sphere-structured SWNHs [123] and poly(diallyldimethylammonium)
functionalized MWNTs [124]. Using unprocessed mammalian living tumor cells
as a model, the glycan expression on cell surfaces was monitored through evalu-
ation of the cell-capturing ability of the recognition interface. When more of
certain glycans were expressed on the cell surface, a larger increase of Rct could
be obtained during EIS detection, resulting from more cells being captured by
the recognition interface.
In order to adopt the above strategy into ECL-based detection, the SWNHs were
replaced by CdSe QDs, which could act as ECL-emitting species and functionalized
by various kind of lectins [125]. These lectins included Con A, dolichos bifows
520 17 Cytosensing and Cell Surface Carbohydrate Assay by Assembly of Nanoparticles
Fig.17.19 Schematic representation of ECL cytosensor for monitoring cell surface carbo-
hydrate expression. Reprinted with permission from Han etal. [125]. 2010, Royal Society
of Chemistry
agglutinin (DBA), peanut agglutinin (PNA), and wheat germ agglutinin (WGA).
As shown in Fig.17.19, the specific capture of cells on the modified electrode led to
a decrease in the ECL intensity, the amplitude of which could be used to investigate
the expression extent of carbohydrates. The above two approaches could both be
used for the sensitive dynamic monitoring of carbohydrate expression on living
cells in response to drugs.
NPs have been extensively employed to couple with biomolecules for the
development of biological nanoprobes [5, 129, 130]. The widespread use of these
hybrid bionanostructures in bioassays and diagnostics is due to an elegant combi-
nation of the superior physical and chemical features of nanomaterials with the
highly selective catalytic and recognition properties of biomolecules. Indeed,
these nanoprobes have been used to develop various sensing and imaging platforms
[131133] and have contributed significantly to applications of bioanalytical
technology in various areas, including medical diagnostics, environmental moni-
toring, and antiterrorism [134].
17.4 Cell Surface Carbohydrate Assay by Assembly of Nanoparticles 521
17.4.2.1Electrochemical Strategies
Fig. 17.20 Schematic representation of the monolayer fabrication and the competitive assay.
Reprinted with permission from Ding etal. [135]. 2008, American Chemical Society
17.4.2.2Fluorescent Strategies
used for drug delivery, magnetic treatment, and magnetic separation due to their
small size, single magnetic domain structures, and superparamagnetism [147].
However, in many biomedical and clinical diagnostic fields, there has always been
some need for materials with the functions of both magnetic separation and
fluorescence tracking.
A type of trifunctional nanospheres with excellent fluorescence, magnetism, and
recognition of cancer cells surface-expressed with sialic acid and N-acetylglucosamine
has been developed [138]. The nanoprobes are fabricated by the encapsulation of
QDs and nano-g-Fe2O3 in poly(styrene/acrylamide) copolymer and then biofunc-
tionalized by WGA. After incubating the nanoprobes with cells for 30min, the cells
captured by the nanoprobes can exhibit obvious orange fluorescence. The success in
capturing DU-145 cells by the nanoprobes has opened up a new field of visualizable
and recognizable manipulation and sorting of cells.
In a later work reported by the same group, different types of lectins WGA,
PNA, and DBA were used to functionalize fluorescent-magnetic nanospheres
[137]. The obtained nanoprobes could be used for qualitative and quantitative
analysis of the glycoconjugates on A549 cell surface. These lectin-modified
trifunctional nanoprobes not only could quantify the different glycoconjugates on
the A549 cell surface, but also could recognize and isolate A549 cells. Using 0.5mg
of WGA-modified fluorescent-magnetic trifunctional nanobiosensors allowed the
capture of 7.0104 A549 cells. Therefore, these nanoprobes might be applied in
mapping the glycoconjugates on cell surfaces and for recognition and isolation of
targeted cells.
The major drawbacks of fluorescence microscopic strategies for cell surface
glycan assay lie in the indirect quantification method and interference by autofluo-
rescence. To solve the problems, the mercaptopropionic acid (MPA)capped CdTe
QDs have been linked to Con A for the fabrication of mannose-specific nanoprobes
[139]. The prepared QDlectin nanoprobe can bind to cells in cell suspensions by
the specific recognition of Con A for cell surface mannosyl groups using leukemic
K562 cells as a model. After homogeneous incubation of cells with the nanoprobe
solution and subsequent removal of the nanoprobe-bound cells, the decrease in FI of
the nanoprobe solution is related to the cell amount and the expression extent of the
mannosyl groups on the K562 cells. This method has further been applied for
dynamically monitoring the change in carbohydrate expression on cancer cells in
the presence of a drug.
17.4.2.3Flow-Cytometric Strategies
Fig.17.21 Scheme of the scanometric strategy for in situ detection of mannose groups on living
cells. Reprinted with permission from Ding etal. [140]. 2010, American Chemical Society
for distinguishing cancer cells from normal ones was based on the ability of some
lectins to interact with specific target oligosaccharides on the cancer cell surface and
to recognize cancer cells from normal ones. These lectin characteristics made them
versatile primary detection reagents in the histochemical and flow-cytometric detec-
tion of cancer cells. The results demonstrate that QD-SBA and QD-DBA conjugates
are appropriate fluorescent markers for the identification of several leukemia cell
lines (especially Jurkat, MOLT-4, Raji, and Daudi cells).
17.4.2.4Scanometric Strategies
17.4.2.5Electrochemiluminescent Strategies
Due to the low background noise and high sensitivity of the ECL technique, the
combination of nanoprobes with this technique can greatly improve in situ assay of
cell surface carbohydrate expression. A facile ECL strategy for label-free monitor-
ing of carbohydrate expression on living cells has been designed based on carbohy-
drate-functionalized CdS QDs/CNT nanocomposites, which act as ECL-emitting
species [141]. The ECL biosensor was fabricated by combining CNTs and mercap-
topropionic acidcapped CdS QDs via a layer-by-layer method, and mannan was
then coupled to QDs. The carbohydrate-functionalized CdS nanocomposites showed
high ECL sensitivity and good stability, and provided an effective 3D architecture
for Con A recognition, which resulted in a decrease in ECL intensity. When BGC
cells, whose surfaces have abundant mannose moieties, were used for competition
with the mannan-derivatized electrode to bind the Con A, the ECL intensity of the
electrode increased. The increase in magnitude depended on both the cell amount
and the expression level of cell surface carbohydrate. This ECL strategy for
evaluating cell surface carbohydrate expression was demonstrated to possess high
sensitivity, perfect stability, and acceptable reproducibility, and the average amount
of mannosyl groups on the cell surface could be obtained by this protocol. This
method obviated cell and lectin labeling; thus, the biological activity of cell and
protein could be maintained to the largest extent. Thus, this method could become a
powerful tool to decode the complex mechanisms underlying carbohydrate-related
biological processes.
Fig. 17.22 (a) Scheme of SECM imaging for cell surface glycan expression. (b) Single-cell
SECM images in situ recorded in PBS containing ferrocenylmethanol (FMA) and H2O2 for BGC
cells after incubation with (a) HRP-WGA, (b) HRP-ConA, (c) HRP-PNA, and (d) HRP-DBA.
Reprinted with permission from Xue etal. [142]. 2010, American Chemical Society
noninvasive manner [152]. Therefore, it has been successfully used for real-time
evaluation of cell viability at the single-cell level by using oxygen as an endogenous
indicator [153]. The nanoscale height change of a single cell has been monitored in
a physiological environment with a novel Pt nanodisk electrode and a newly
designed step-approaching strategy. Most recently, the SECM method has been
employed for in situ electrochemical imaging of different surface glycan motifs on
adherent cells at the single-cell level using HRP-tagged lectins coupled with cell
micropatterning [142]. In this work, these adherent cells were first micropatterned
in the microwell of poly(dimethylsiloxane) membrane for precisely controlling the
localized surface interaction, and the membrane glycans were then specifically
17.5 Conclusions 527
r ecognized with corresponding lectins labeled with HRP. The glycan expression
level was obtained by detecting enzymatic catalysis product at the SECM tip. The
cell surface glycans could thus be in situ imaged by SECM at a single-cell level
without peeling the cells from the culture dish. Four types of membrane glycan
motifs showed statistically distinguishable expression levels (Fig.17.22). Thus, this
work afforded a promising protocol for the study of cell biology related to cell
surface carbohydrate expression of single living cells, and could be applied in cell
biology study based on cell surface carbohydrate expression.
17.5Conclusions
In this chapter, we comprehensively reviewed the current research regarding the use
of nanomaterials for applications of cytosensing and cell surface carbohydrate assay.
Various types of functionalized nanomaterials and several related techniques have
been discussed. NPs are on the threshold of widespread use and almost touch upon
every single modality of the cellular sensing arena. For many sensor applications,
NPs exhibit distinctive attributes that can increase the sensitivity and selectivity of
assays relative to conventional diagnostic techniques. Advanced nano-diagnostic
techniques have opened a promising avenue to provide rapid, low-cost, easy, and
multiplexed cytosensing strategies.
The further development of cytosensing will lie in the fabrication of novel
nanoprobes for both recognition and signal transduction. Surface functionalization
is the most essential and fundamental factor as far as cytosensing is concerned. The
newer conjugation strategies, such as a barnase and barstar noncovalent binding
system or a click chemistrybased covalent linker, will greatly accelerate the devel-
opment of this field. The optimization of the surface composition of nanomaterials
will also enable the development of efficient sensing strategies with the ability to
detect analytes in complex biological fluids like blood, urine, serum, and so forth.
Although tremendous success has been made, the concern of long-term toxicity of
nanomaterials is still an important issue to be addressed.
Moreover, there is tremendous incentive for developing technologies that detect
cancer at its earliest stages. In most cases, the detection of stage 1 cancers is associ-
ated with a greater than 90% 5-year survival rate. Conventional anatomic imaging
techniques typically detect cancers when they are a centimeter or greater in
diameter, at which point they already consist of more than 109 cells [154]. Thus,
cytosensing, especially cell surface assay, which can allow the sensitive and spe-
cific monitoring of the development of diseased cells and the expression of key
biomarkers on the cell surface, is expected to play an important role in biological,
medical, and clinical research. Given the remarkable parallel progress in nanotech-
nology, chemical biology, organic chemistry, and molecular biology, multifunc-
tional nanoprobes for both sensitive diagnostics and delivery of drugs without
systemic toxicity will bring unprecedented opportunities for the future of cancer
diagnosis and therapy.
528 17 Cytosensing and Cell Surface Carbohydrate Assay by Assembly of Nanoparticles
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534 17 Cytosensing and Cell Surface Carbohydrate Assay by Assembly of Nanoparticles
18.1Introduction
Technological platforms that provide the reliable, rapid, quantitative, cheap, and
high-throughput identification of biomolecules play a significant role in the clinical
deployment of personalized treatment [1]. A biosensor is a small device employing
biochemical molecular-recognition properties as the basis for a selective analysis
[2]. Three basic parts are involved in any biosensor system: biosensing, signal trans-
duction, and signal readout. The biosensing element is capable of recognizing the
presence, activity, or concentration of a specific analyte; it could be either a binding
process (affinity ligand-based biosensor with the recognition element of a protein,
peptide, DNA, RNA, whole cell, or tissue) or a biocatalytic reaction (enzyme-based
biosensor). Over the past decades, due to their advantages of specificity, speed,
portability, and low cost, we have witnessed a tremendous amount of activity in the
area of biosensors as well as their clinical applications [3], especially for cancer
diagnosis [410].
The early diagnosis of cancer is crucial for the successful treatment of the disease.
Highly sensitive methods are urgently needed for measuring cancer diagnosis markers
present at ultralow levels during early stages of the disease. Moreover, small, fast,
and high-throughput devices are also highly desired for replacing time-consuming
laboratory analyses to be able to screen large populations for signs of precancerous
developments or the presence of early malignant lesions. Such methods should
facilitate early detection and an adequate selection of treatment options and should
lead to increased patient survival rates. However, existing diagnostic biosensors are
neither sensitive enough to detect biomolecules at levels corresponding to advanced
stages of the disease nor integrated for the fast, automatic, and multiplexed detection
of diverse biomarkers [11].
The emergence of nanotechnology the creation and utilization of materials,
devices, and systems through the control of matter on the nanometer scale is open-
ing new horizons for the development of nanoprobes, nanosensors, and nanosystems
18.2Nanotechnologies in Biosensing
To date, modern material science has reached a high degree of sophistication. A wide
variety of nanoscale materials with different sizes, shapes, and compositions are
now available [17]. Due to the novel properties of their small size (1100nm) and
correspondingly large surface-to-volume ratio, tailorable chemical and physical
properties, unusual target binding properties, and overall structural robustness,
nanomaterials are being gradually applied to biosensors for markedly enhancing the
sensitivity and specificity of detection [18, 19].
18.2.1.1Nanoparticles
Nanoparticles (NPs) are generally defined as isolable particles between 1 and 50nm
in size and are prevented from agglomerating by protective shells. In recent years,
NPs of different compositions and dimensions have been widely used as versatile
and sensitive tracers in a broad spectrum of highly innovative approaches for assays
of small molecules, proteins, and nucleic acid biomarkers [1926]. The most fre-
quently used NPs for biological detection are gold (Au) NPs, quantum dots (QDs),
and magnetic NPs.
Au NPs are particularly attractive labels for sensors because a variety of analytical
techniques can be used to detect them, including optical absorption, fluorescence,
Raman scattering, atomic and magnetic force, and electrical conductivity. The intro-
duction of Au NPs into some well-studied bioassays can lead to improved sensitivity
and specificity. For example, in a real-time DNA surface plasmon resonance detec-
tion system [27], a 1,000-fold lower detection limit is achieved with the use of an
Au NP-labeled oligonucleotide [28]. Moreover, the enormous signal enhancement
associated with the use of NP-amplifying labels and with the formation of
NPbiomolecule assemblies, such as bio-bar-code assay (Fig.18.1), provides the
18.2 Nanotechnologies in Biosensing 537
Fig.18.1 General bio-bar-code assay scheme. (a) Nanoparticle and magnetic microparticle probe
preparation; (b) nanoparticle-based PCR-less DNA amplification scheme. The target can be DNA,
RNA, or protein. Reprinted with permission from Nam etal. [32]. 2004, American Chemical
Society
basis for ultrasensitive optical and electrical detection with polymerase chain
reaction (PCR) or fluorescent taglike sensitivity [2934].
QDs are inorganic fluorophores. With advantages of high sensitivity, broad exci-
tation spectra, sharp emission spectra, easily tunable emission properties, and no
need for lasers, QDs have been used as possible alternatives to the conventional fluo-
rescent markers for molecular diagnostics and genotyping. A major challenge is that
QDs have an oily surface, whereas the cellular environment is water-based. Attempts
are being made to produce biocompatible water-soluble QDs so that they can act as
538 18 Nanobiosensing for Clinical Diagnosis
18.2.1.2Nanotubes
Fig.18.2 Schematic illustration of the method for labeling specific DNA sites and detection with
SWNT AFM probes. Labeled DNA molecules are deposited on freshly cleaved mica and imaged
by AFM using SWNT probes. The presence and locations of the sequence-specific tags ( and )
can be readily observed in the AFM image. Reprinted with permission from Woolley etal. [50].
2000, Nature
CNTs have also been used to develop highly specific electronic biomolecule
detectors for investigating surfaceprotein and proteinprotein binding [51].
Nonspecific binding on nanotubes, a phenomenon found with a wide range of proteins,
is overcome by immobilization of polyethylene oxide chains. A general approach is
then advanced to enable the selective recognition and binding of target proteins by
conjugation of their specific receptors to polyethylene oxide-functionalized nano-
tubes. These arrays are attractive because no labeling is required and the entire
assay can be done in the solution phase. This scheme, combined with the sensitivity
of nanotube electronic devices, enables highly specific electronic sensors for detecting
clinically important biomolecules such as antibodies associated with human auto-
immune diseases.
18.2.1.3Nanowires
Fig.18.4 Motion-based nucleic acid detection. (a) Hybridization of the target and capture of the
Ag nanoparticletagged detector probe in a typical sandwich assay on the ternary SH-CP/
DTT+MCH surface, including washing of unbound SHDPAg NPs. (b) Dissolution of silver
nanoparticle tags in the peroxide fuel, leading to Ag+-enriched fuel. (c) Visual detection of the
motion of the catalytic nanowire motors in the resulting Ag+-enriched fuel. C1, C2, and C3 repre-
sent hypothetical and increasing target nucleic acid concentrations. Reprinted with permission
from Wu etal. [61]. 2010, Nature
18.2.1.4Dendrimers
18.2.1.5Fullerenes
The unique feature of fullerene molecules is that they have numerous points of
attachment, allowing for the precise grafting of active chemical groups in three-
dimensional orientations. This attribute, the hallmark of rational drug design, allows
positional control in matching fullerene compounds to biological targets. Together
with other attributes, such as the size of the fullerene molecules, the redox potential,
and the relative inertness in biological systems, it is possible to tailor requisite phar-
macokinetic characteristics to fullerene-based compounds and to optimize their
therapeutic effect [72].
18.2 Nanotechnologies in Biosensing 543
18.2.1.6Nanobiological Molecules
These antibody fragments have several remarkable features. For example, they
are small and stable and can bind antigen with nanomolar affinity. They have high
target specificity and low inherent toxicity. They can address therapeutic targets not
easily recognized by conventional antibodies (e.g., active sites of enzyme). They
have an extremely low immunogenic potential. Particularly appealing is their ability
to simultaneously inhibit various crucial growth-factor receptors or their ligands
with a single molecule. In addition, they are easy to clone and express on the tip of
filamentous phage, which opens the possibility to select for nanobodies inducing
particular biological effects. The unique and well-characterized properties enable
nanobodies to excel as conventional therapeutic antibodies, making them ideal can-
didates as next-generation cancer therapeutics and for use in diagnostics, for dis-
eases such as cancer [74, 7678]. It is reported that the successful use of untagged
nanobodies for the in vivo treatment of solid tumors [79]. Based on functional
phage-antibody selection using competitive elution with the ligand epidermal
growth factor (EGF), antagonistic anti-EGFR nanobodies for cancer therapy have
been developed. The selected antibody fragments are found to efficiently inhibit
EGF binding to the EGF receptor without acting as receptor agonists themselves. In
addition, they can also block EGF-mediated signaling and EGF-induced cell prolif-
eration. These nanobodies are effective in delaying the outgrowth of A431-derived
solid tumors in an invivo model for solid tumors.
18.2.2Nanofabrication
18.2.2.1Nanoarrays
Currently, the microarray technologies are of great interest for providing a platform
for multiplexed DNA and protein detection in small areas with small amounts of
sample and reagent, hence offering a variety of applications in biomedical diagnostics
and proteomics. Nanoarrays are the next stage in the evolution of the miniaturization
of microarrays because they would allow for orders of magnitude more massively
paralleled multiplexed detection in the same array area as a microarray and poten-
tially improved detection limits resulting from the smaller analyte capture area [19, 80].
Various methods have been developed to fabricate nanoarrays, such as dip-pen
nanolithography (DPN) [81], nanografting [82], scanning probe lithography [83],
and other lithography strategies.
DPN is a direct-write scanning probe-based lithography in which an AFM tip is
used to deliver chemical reagents directly to nanoscopic regions of a target substrate.
18.2 Nanotechnologies in Biosensing 545
Fig.18.5 Direct patterning of multiple-DNA inks by DPN. (a) Combined red-green epifluores-
cence image of two different fluorophore-labeled sequences (Oregon Green 488-X and Texas Red-
X) simultaneously hybridized to a two-sequence array deposited on an SiOx substrate by DPN. (b)
Tapping-mode AFM image of 5- (dark) and 13- (light) nm-diameter gold nanoparticles (Au NPs)
assembled on the same pattern after dehybridization of the fluorophore-labeled DNA. Reprinted
with permission from Demers etal. [84]. 2002, Science
Fig.18.6 Schematic representation of the sandwich immunoassay format used to detect HIV-1 p24
antigen with an anti-p24 antibody nanoarray made by DPN. The HIV-1 p24 antigen was sand-
wiched between anti-p24 antibody bound to the MHA patterned surface and Au NP probes coated
with anti-p24 antibody. The change in height due to the nanoparticle binding event could be detected
by AFM. Reprinted with permission from Lee etal. [86]. 2004, American Chemical Society
18.2.2.2Nanopore Technology
18.2.2.3Nanofluidic Channels
Future molecular diagnostic tools should possess the ability to separate, isolate, and
investigate a small number of molecules in a nanoscale-confined environment. With
recent advances in nanoscience and nanotechnology, the use of nanoscale channels in
place of microfluidic channels for fluid flow have brought this goal closer to reality.
Nanofluidic systems enable the study of physical principles, such as kinetic profiling,
and biomolecular interactions in a controllable fluidic environment at the nanoscale.
In addition, nanofluidic channels are important elements for lab-on-a-chip devices,
which have promise for achieving absolute single-molecule sensitivity combined with
high selectivity along with the advantages of high throughput, low access times, and
extremely small sample volumes (pL to fL range). Nanofluidic devices allow for many
applications, including biomolecule separation, concentration, reaction/hybridization,
sequencing (in the case of DNA), and detection [92]. At the time of this writing, the
main application of nanofluidic devices is biomolecular separation, which can occur
at the nanoscale due to many different phenomena, including steric, hydrodynamic,
entropic, electrical, and ratcheting. Craigheads group designed a nanofluidic channel
device, consisting of many entropic traps, for the separation of long DNA molecules
[93]. The channel comprises narrow constrictions and wider regions that cause size-
dependent trapping of DNA at the onset of a constriction. This process creates elec-
trophoretic mobility differences, thus enabling the efficient separation of long DNA
molecules (5,000 to ~160,000 base pairs) in 15-mm-long channels. This device can be
modified to separate smaller or larger DNA molecules as well as various proteins and
other polymers. Moreover, multiple-channel devices, integrating several separation
channels optimized for different length ranges of DNA in parallel, can realize single-run
sorting and analysis of various DNA samples. Recently, a simple nanofluidic device
was developed to perform free-solution hybridization and separation of single-stranded
and double-stranded oligonucleotides [94]. The device can identify target DNA
sequences and quantitatively measure hybridization kinetics through the electrokinetic
separation of hybridized and unhybridized probe DNA in a nanochannel. In this
device, on-chip fluid control is achieved using only electric fields, no sample labeling
is required, and no sieving matrix is needed. These characteristics make the device
well suited for integration into microfabricated lab-on-a-chip systems.
18.2.3Nanodevices
pathogen detection. Nanobiosensors are nanosensors used for the selective detection
of biological materials. Nanobiosensors adopt inexpensive low-voltage measurement
schemes for the direct detection of a binding event and can be electronically gated
to respond the single molecule-binding event. There is no need for costly, compli-
cated, and time-consuming labeling chemistries such as fluorescent dyes or the use
of bulky and expensive optical detection systems. Nanobiosensors provide great
possible to develop implantable detection and monitoring devices.
18.2.3.1Electrical Nanobiosensors
Fig.18.8 (a) The upper figure represents an arbitrarily shaped ssDNA molecule, which is stretched
and attached to a pair of functionalized SWNT electrodes in the presence of a DEP field. The lower
figure depicts a covalently attached dsDNA molecule, which has a definite conformation. The
nanoelectrodes are separated by a gap of 272nm (the contour length of the 80-bp DNA molecule
is ~27nm). The bridging DNA molecule suspends over a trench without touching the silicon diox-
ide surface. (b, c) Current flow at room temperature through single ssDNA and dsDNA molecules,
respectively, in ambient and vacuum conditions. Reprinted with permission from Roy etal. [97].
2008, American Chemical Society
the 68-nm gaps between two gold electrodes. As a model system, the specific binding
of the blood clotting factor human thrombin with different concentrations to its
ribonucleic acid (RNA) alpha-thrombin aptamer, as well as the immobilization pro-
cess of the RNA-aptamer, have been detected in real time.
18.2.3.2Nanowire Nanobiosensors
As the surface of nanowires can be easily modified, nanowires have been used to
decorate with a different potential chemical or biological molecular recognition
unit, making the wires themselves analyte-independent. Nanowire sensors operate
on the basis that the change in chemical potential accompanying a target/analyte-
binding event can act as a field-effect gate upon the nanowire, thereby changing its
conductance. Such a detection format is similar to how a field-effect transistor oper-
ates. The ideal nanowire sensor is a lightly doped, high-aspect ratio, single-crystal
nanowire with a diameter between 10 and 20nm. This is because the nanowire sensor
is too noisy with much smaller wires, and unsensitive with much larger wires.
As the nanomaterials transduce the biological binding event in an extremely sensitive,
real-time, and quantitative fashion, nanowire sensors have great potential for both
biological research and clinical applications. For example, Lieber and coworkers
demonstrated that silicon nanowires functionalized with PNA could be used for
real-time, label-free detection of DNA [54]. In their assay, the conductance of a
PNA-functionalized silicon nanowire bridging two electrodes was measured in the
presence of target DNA and mutant DNA with three consecutive base deletions.
The introduction of target DNA into the assay caused a rapid and immediate change
in conductance, while the effect of mutant DNA was negligible. The concentration-
dependent measurements showed the detection of DNA could be carried out to at
least the tens of femtomolar range. Furthermore, Liebers group also functionalized
18.2 Nanotechnologies in Biosensing 551
Fig. 18.9 Nanowire-based detection of single viruses. (Left) Schematic shows two nanowire
devices, 1 and 2, where the nanowires are modified with different antibody receptors. Specific
binding of a single virus to the receptors on nanowire 2 produces a conductance change (right)
characteristic of the surface charge of the virus only in nanowire 2. When the virus unbinds from
the surface, the conductance returns to the baseline value. Reprinted with permission from Patolsky
etal. [55]. 2004, National Academy of Sciences
the interface of nanowires with antibodies specific for influenza A virus particles
and used a microfluidic sampling system to demonstrate that single-virus/nanowire
recognition events could be detected by measuring real-time changes in nanowire
conductivity [55]. As shown in Fig.18.9, when a virus particle binds to the antibody
receptor on a nanowire device, the conductance of the device changes from the
baseline value. In contrast, when the virus drops off the nanowire surface, the con-
ductance returns to the baseline value. The conductance of a second nanowire device
at which binding does not occur during the same time period showed no change and
can serve as an internal control. One compelling advantage of nanowire sensors is
that the number and density of the sensor elements are limited only by the ability to
electronically address individual nanowires. Very dense nanowire sensor circuits,
with modification of different nanowires within the array with receptors specific for
different analytes, may be addressed. Thus, integrated circuits can be constructed
552 18 Nanobiosensing for Clinical Diagnosis
18.2.3.4Cantilever Nanobiosensors
Recently, it has been demonstrated that molecular adsorption also results in measur-
able mechanical forces, which can be detected by micro- and nanoelectromechanical
systems. Detecting biomolecular interactions by measuring nanomechanical forces
18.2 Nanotechnologies in Biosensing 553
Fig.18.10 Specific biomolecular interactions between target and probe molecules alter intermolecular
interactions within a self-assembled monolayer on one side of a cantilever beam. This can produce a
sufficiently large force to bend the cantilever beam and generate motion. The origin of this nanome-
chanical motion lies in the interplay between changes in the configurational entropy and intermolecular
energetics. Reprinted with permission from Wu etal. [101]. 2001, National Academy of Sciences
offers an exciting opportunity for the development of highly sensitive, miniature, and
label-free biological sensors. Microcantilevers with nanoscale thickness allow the
detection of biomolecules and microorganisms due to surface stresses created by
molecular adsorption, in which the resonance frequency was used as a function of
target binding (Fig. 18.10) [101]. A microfabricated cantilever with an internal
piezoresistive component has been sensitized with thiolated ssDNA strands and uti-
lized to investigate DNA hybridization. The generation of a differential surface stress
onto the functionalized cantilever surface upon target recognition has allowed the
nanomechanical identification of 12-nucleotide complementary DNA probes with
single-base-mismatch discrimination and a low sensitivity of 0.2 mM [102]. For
amplification, microcantilevers have been used to detect DNA strands modified with
Au NPs [103]. The gold labels provide a site for silver ion reduction, which increases
the mass on the cantilever and results in a detectable frequency shift that signals the
binding events. This strategy can detect target DNA as low as 0.05nM. The detection
of proteins, such as cancer biomarkers, viruses, and bacteria is also possible using
cantilever nanobiosensors. For example, prostate-specific antigen (PSA) has been
successfully detected on cantilevers using immobilized specific antibody [104].
Craighead and coworkers modified the microcantilevers with antibodies specific for
either viruses or bacteria to detect their corresponding target [105, 106].
With the development of lithographic technologies and micro- and nano-fabrication
techniques, multitarget sensor arrays involving tens of cantilevers and analog pro-
cessing on a single chip can be manufactured. Increasing the number of sensing
elements in an array can lower noise, increase selectivity, and enhance robustness.
Due to the advantages of simplicity, low power consumption, low cost, inherent
compatibility with array designs, and label-free detection, cantilever nanobiosensors
are very attractive for future clinical applications.
554 18 Nanobiosensing for Clinical Diagnosis
Fig. 18.11 Schematic of the ion-channel switch biosensor with two antibody Fab fragments
r eacting with the target antigen. The binding of the target antigen (#) to the antibody fragments (*)
causes the conformation of gramicidin A to shift from conductive dimers to nonconductive mono-
mers (ac). This causes a loss of conduction of ions across the membrane and a change in the
electrical output of the reader. Reprinted with permission from Oh etal. [108]. 2008, Elsevier
18.3.1Glucose Detection
Glucose plays a critical role in the bodys metabolism. The dysfunction of glucose
handling from insulin deficiency or resistance can lead to diabetes. Recently, with
the development of nanoscience, it is possible to engineer micro/nanoscale devices
for invivo glucose sensing, which opens the way to monitor the glucose of diabetes
patients in real time. A new type of glucose nanosensor has been fabricated by using
SWCNTs to modulate their emission in response to the adsorption of specific
biomolecules [109]. The signal transduction includes two distinct mechanisms:
fluorescence quenching and charge transfer. Glucose oxidase is coated on CNTs;
then ferricyanide is sprinkled onto the surface of nanotubes. Ferricyanide draws
electrons from the nanotubes, quenching their capacity to glow when excited by
infrared light. When glucose is present, it reacts with the oxidase, producing hydrogen
peroxide, which can react with ferricyanide in a way that reduces that molecules
hunger for electrons. The higher the glucose level, the greater is the infrared fluores-
cence of nanotubes. To test the feasibility of implanting the sensors in the body, the
nanotubes, modified with both glucose oxidase and ferricyanide, are placed inside a
sealed glass tube 1cm long and 200mm thick (Fig.18.12). The tube is riddled with
pores large enough to let glucose enter but small enough to keep the nanotubes
inside. The tube could implant in a sample of human skin and the sensor can be
excited with infrared light to detect fluorescence.
Probes encapsulated by biologically localized embedding (PEBBLEs) nanosensors,
with a size of 45nm in diameter, have been fabricated for real-time glucose imaging
in living cells [110]. The nanosensors incorporate glucose oxidase, an oxygen-
sensitive fluorescent indicator, and an oxygen-insensitive fluorescent dye, as a refer-
ence for ratiometric intensity measurements. The enzymatic oxidation of glucose to
gluconic acid results in the local depletion of oxygen, which is measured by the oxy-
gen-sensitive dye. Due to the PEBBLE matrix, this kind of nanosensor is insensitive
to interference from proteins in cells, enabling reliable invivo chemical analysis.
The matrix also significantly reduces the toxicity of the indicator and reference dyes
to the cells, so that a larger variety of dyes can be used in an optimal fashion.
Furthermore, it also enables a synergistic approach in which there is a steady state
of local oxygen consumption, and this cannot be achieved by separately introducing
free enzyme and dyes into a cell.
556 18 Nanobiosensing for Clinical Diagnosis
Fig.18.12 Glucose detection using a carbon-nanotube optical sensor. (a) Reaction at the enzyme
converts glucose to gluconolactone, with the hydrogen peroxide coproduct detected by interaction
with the Fe(CN)63 functional groups on the exposed nanotube surface between enzyme mono-
mers. (b) A 200-mm1-cm, 13-kDa microdialysis capillary, shown to size on a human finger, is
loaded with the nanotube solution allowing glucose to diffuse through the membrane with the
containment of the sensing medium. On placing the capillary beneath a human epidermal tissue
sample, we can clearly map the nanotube fluorescence from the capillary, as seen in this two-
dimensional profile. The dotted square in the picture of the tissue sample symbolizes the area used
for the fluorescence map. Reprinted with permission from Barone etal. [109]. 2005, Nature
As is well known, early detection is particularly important in the case of cancer and
other pathologies, because the early stages of disease are typically treated with the
greatest probability of success. As the levels of the biomarkers, such as tumor-related
antigens, in serum are associated with the stages of tumors, the requirements of
technology platforms that provide the reliable, rapid, quantitative, low-cost, and
multiplexed identification of biomarkers are raising. Nanotechnology is helpful in
this effort, as shown by the following examples.
Nanomaterials are widely used for signal amplifications. For example, Mirkins
group developed a nanoparticle-based bio-barcode amplification strategy for detecting
PSA with a detection limit of 30aM [29]. Rusling etal. used CNTs, modified with
both enzymes and secondary antibodies at a high enzyme/antibody ratio, to fabricate
18.3 Nanobiosensing for Clinical Diagnosis 557
Fig. 18.13 (a) Schematic illustration and (b) photograph of disposable electrochemical immu-
nosensor diagnosis device. Reprinted with permission from Liu etal. [111]. 2007, American
Chemical Society
Fig. 18.14 (a) Optical image (top) of a nanowire device array. The schematic (bottom) shows
details of metal electrodes (golden lines) connecting nanowires (blue lines) in this region with
orientation rotated 90 relative to the red rectangle. (b) Schematic illustrating multiplexed protein
detection by three silicon-nanowire devices in an array. Devices 1, 2, and 3 are fabricated from
similar nanowires and then differentiated with distinct mAb receptors (1 red; 2 green; 3 blue)
specific to three different cancer markers. Reprinted with permission from Zheng etal. [112].
2005, Nature
18.3.3DNA Detection
The recent discovery and sequencing of the human genome has provided valuable
insight into understanding how genetic factors contribute to the development of disease.
Specifically, the sensitive detection of DNA sequence variations plays an important role
in the diagnosis of genetic-related disease and conditions, especially for early-stage
treatment and monitoring. Nanoscale materials offer excellent prospects for designing
highly sensitive and selective bioassays of nucleic acid. For example, relying on nano-
particle-based amplification, Mullers group presented a microarray-based method for
multiplex single nucleotide polymorphism (SNP) genotyping in total human genomic
DNA without the need for target amplification or complexity reduction [113]. Specificity
is derived from two sequential oligonucleotide hybridizations to the target DNA SNP
segments by allele-specific surface-immobilized capture probes and gene-specific
oligonucleotide-functionalized Au NPs. The assay format is simple, rapid, and robust
pointing to its suitability for multiplex SNP profiling at the point of care.
Using QDs, Wangs group has developed an ultrasensitive and reliable nanotech-
nology assay for the detection and quantification of DNA methylation, which
contributes to carcinogenesis by silencing key tumor-suppressor genes [114].
Bisulfite-modified DNA is subjected to PCR amplification with primers that would
differentiate between methylated and unmethylated DNA. QDs are then used to cap-
ture PCR amplicons and determine the methylation status via fluorescence resonance
energy transfer (Fig.18.15). This approach detects as little as 15pg of methylated
DNA in the presence of a 10,000-fold excess of unmethylated alleles, enables reduced
use of PCR (as low as eight cycles), and allows for multiplexed analyses. The high
sensitivity of this method enables one-step detection of methylation at PYCARD,
CDKN2B, and CDKN2A genes in patient sputum samples that contain low concen-
trations of methylated DNA, which normally would require a nested PCR approach.
Nanostructured materials also constitute new platforms for biomolecular sensing
that may provide increased sensitivity and amenability to miniaturization. Tans
18.3 Nanobiosensing for Clinical Diagnosis 559
Fig. 18.15 Principle of MS-qFRET for detection of DNA methylation. In step 1, extracted
genomic DNA is subject to sodium bisufite conversion, wherein unmethylated cytosines are con-
verted to uracil while methylated cytosines remain unaffected. In step 2, DNA is amplified using
PCR wherein the forward and reverse primers are labeled with a biotin (black dot) and a fluoro-
phore (red dot), respectively. In step 3, the resulting labeled PCR product is captured by streptavi-
din-functionalized QDs through streptavidinbiotin affinity. In step 4, upon suitably exciting the
QD, the nanoassembly formed allows for FRET to occur between the QD donor and the fluoro-
phore acceptor. Consequently, the labeled PCR products are detected by emissions of fluorophores
accompanied by quenching of QDs to reveal the status of DNA methylation. Reprinted with per-
mission from Bailey etal. [114]. 2009, Cold Spring Harbor Laboratory Press
monitoring and confirmation of the collected gene products. Kelleys group fabri-
cated gold nanoelectrode ensembles (NEEs) for ultrasensitive biosensing of DNA
probe, oligonucleotide sequences that correspond to a portion of the 23S rRNA gene
from Helicobacter pylori (a pathogen implicated in gastric ulcers and cancer), with
an attomole-level detection limit [116]. The label-free system reports on the binding
of a target DNA sequence to an probe oligonucleotide, immobilized on Au NEEs,
using a catalytic reaction between two transition-metal ions, Ru(NH3)63+ and
Fe(CN)63. The Ru(III) electron acceptor is reduced at the electrode surface and then
reoxidized by excess Fe(III), making the electrochemical catalytic process. The
increased concentration of anionic phosphates at the electrode surface that accompa-
nies DNA hybridization increases the local concentration of Ru(NH3)63+, and there-
fore produces large changes in the electrocatalytic signal.
18.3.4Virus Detection
Fig.18.17 Images of bacterial cells. (a) Scanning electron microscope image of Escherichia coli
O157:H7 cell incubated with antibody-conjugated nanoparticles; (b) scanning electron microscope
image of E. coli DH5a cell (negative control) incubated with nanoparticles conjugated with anti-
body for E. coli O157:H7; (c) fluorescence image of E. coli O157:H7 after incubation with anti-
body-conjugated nanoparticles. The fluorescence intensity is strong, enabling single-bacterium
cell identification in aqueous solution. Reprinted with permission from Zhao etal. [118]. 2004,
National Academy of Sciences
spatial and 8-ms temporal resolution, they observed sliding and tumbling motions
during rapid lateral diffusion on supported lipid bilayers, and repeated back-and-
forth rocking between nanoscopic regions separated by 9nm. These findings suggest
the recurrent swap of receptors and viral pentamers as well as receptor aggregation
in nanodomains.
18.3.5Bacteria Detection
The early diagnosis of cancer is the critical element in successful treatment and long-
term favorable patient prognosis. Late diagnosis is often associated with the lack of
timely sensitive imaging modalities. The promise of nanotechnology is presently
562 18 Nanobiosensing for Clinical Diagnosis
limited by the inability to simultaneously seek, treat, and image cancerous lesions.
Adairs group synthesized fluorescent calcium phosphosilicate nanocomposite par-
ticles (CPNPs) for systemically targeting breast and pancreatic cancer lesions [119].
The CPNPs with a diameter of 20 nm were composed of an amorphous calcium
phosphate matrix doped with silicate in which a near-infrared imaging agent was
embedded. The conjugation of biotinylated human holotransferrin (diferric transfer-
rin) and biotinylated anti-CD71 antibody (anti-transferrin receptor antibody) to
avidin-conjugated CPNPs (Avidin-CPNPs) permits targeting of transferrin receptors,
which are highly expressed on breast cancer cells. Similarly, the conjugation of bioti-
nylated pentagastrin to Avidin-CPNPs and decagastrin (gastrin-10) to PEG-CPNPs
via PEG-maleimide coupling permits targeting of gastrin receptors, which are over-
expressed in pancreatic cancer lesions. These results showed that the bioconjugated
CPNPs could perform as a theranostic modality, simultaneously enhancing drug
delivery, targeting, and imaging of breast and pancreatic cancer tumors.
QDs have the potential to become a new class of fluorescent probes for many
biological and biomedical applications, especially cellular imaging. Wus group
used QDs linked to immunoglobulin G and streptavidin to label the breast cancer
marker Her2 on the surface of fixed and live cancer cells, to stain actin and microtu-
bule fibers in the cytoplasm, and to detect nuclear antigens inside the nucleus [120].
The results showed all labeling signals from QDs were specific for the intended
targets and were brighter and considerably more photostable than comparable
organic dyes. They also realized simultaneously the detection of two cellular targets
with one excitation wavelength by using QDs with different emission spectra con-
jugated to IgG and streptavidin.
18.4Conclusions
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wwwwwwwwwwwww
Index
V
T VACNF. see Vertically aligned carbon
Taste sensing nanofiber
carbon nanotube polymer composite, 352 Vertically aligned carbon nanofiber
catalytic nanomaterial-based optical (VACNF)
sensor covalent functionalization, 164
chemiluminescence (CTL), 356358 CuAAC reaction, 164
chemosensor array, 358361 electron microscope image scanning,
organic compounds recognition, CTL, 162, 163
361362 ferrocene attachment, 165, 166
gold nanoparticle-fluorophore complexes molecules detection, 162, 164
bacteria and mammalian cell detection, photochemical grafting and CuAAC
355356 reaction, 164
chemical nose/tongue approach, 352 photoresist-based blocking, 165167
conjugated polymer/GFP, 353
protein detection, 353355
nanoassembled conducting polymers X
electronic tongue, 351 X-ray diffraction (XRD), 125126