Electrochemical Biosensors For Point-Of-Care Medical Diagnostics

UNIVERSITY OF CALIFORNIA
Santa Barbara
Electrochemical Biosensors for Point-of-Care Medical Diagnostics
A Dissertation submitted in partial satisfaction of the
requirements for the degree Doctor of Philosophy
in Chemistry and Biochemistry
by
Aaron Alexander Rowe
Committee in charge:
Kevin Plaxco, Chair
Professor Alison Butler
Professor Deborah Fygenson
Professor Stanley Parsons
June 2011
UMI Number: 3473790
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____________________________________________
Deborah Fygenson
____________________________________________
Alison Butler
____________________________________________
Stanley Parsons
____________________________________________
Kevin Plaxco, Committee Chair
May 2011
Copyright © 2011
by
iii
ACKNOWLEDGEMENTS
None of this work would have been possible without the constant support of my
colleagues. Ryan White, Rebecca Lai, Brian Baker, and Francesco Ricci have taught
me a great deal about electrochemistry. Hershel Watkins and Alexis Vallée-Bélisle
have helped me think about the physics of my sensors, and offered tremendous
feedback about my manuscripts and presentations. Andrew Bonham has been helpful
in countless ways. My officemate at Lawrence Livermore, Richard Henrikson
greatly broadened my knowledge of molecular diagnostics and techniques for
keeping current with the scientific literature. Nicholas Fisher was a tremendously
patient mentor while teaching me in vitro selection techniques. Yi Xiao has been a
tremendous role model. Her creativity and ability to engineer phenomena at the
molecular scale is a true inspiration.
My mentor, Professor Kevin Plaxco, has shown me how to structure successful
research projects, earn fellowships, prepare grant proposals, make good figures and
pick research papers apart. He has also taught me a great deal about scientific
writing, which will undoubtedly be useful throughout my career. I am particularly
grateful that he creates a lab environment in which his students can work
independently, try risky ideas, and shape the trajectory of their projects.
Professor Gagik Melikyan, my Master’s Thesis adviser, laid the foundation for
my success in a doctoral program. He taught me how to conduct experiments
gracefully, keep a proper lab notebook, give a good presentation, and agonize over
iv
the details of a paper. Most of all, he instilled in me the sort of work ethic that is
necessary for all competitive scientists.
In the purchasing office and stockroom, Cabe Fletcher and Skip Touponce have
worked tirelessly to make sure that my orders arrive quickly. Without their
tremendous help, my projects would have progressed very slowly. On the
administrative side, Erica James and Judy Purcell have been phenomenally helpful.
They can make the greatest of hassles vanish with their expert touch.
My parents have been tremendously supportive of my education. I am
particularly grateful that they exposed me to many science magazines, museums, and
chemistry sets at an early age. My girlfriend, Iris Ahronowitz, has kept my
tremendously busy life in balance, and brought me happiness in a way that no one
else can.
v
VITA OF AARON ALEXANDER ROWE
May 2011
EDUCATION
Bachelor of Science in Materials Science and Engineering, University of Illinois,

Urbana-Champaign, December 2002
Master of Science in Chemistry, California State University, Northridge, December

2005
Doctor of Philosophy in Chemistry and Biochemistry, University of California,

Santa Barbara, June 2011 (expected)
PROFESSIONAL EMPLOYMENT
Spring 2000: Research Assistant, Carolyn Dry Research Group, University of

Illinois, Urbana-Champaign
Fall 2001: Teaching Assistant, Department of Materials Science, University of

Illinois, Urbana-Champaign
Summer 2011: Contributing Editor, Chemical and Engineering News, Science and
New Media Divisions, West Coast News Bureau
PUBLICATIONS
Gagik G. Melikyan, Ferdinand Villena, Arthur Florut, Steve Sepanian, Hagop

Sarkissian, Aaron Rowe, Pogban Toure, Dutt Mehta, Nolan Christian, Steven Myer,
David Miller, Stephanie Scanlon, and Maria Porazik, Tetrahydrofuran-Mediated
Stereoselective Radical C-C Bond Formation in Dicobalthexacarbonyl-
Propargyl Complexes, 2006, Organometallics, 25 (19), 4680 -4690.
Yi Xiao, Aaron A. Rowe, and Kevin W. Plaxco, Electrochemical Detection of

Parts-Per-Billion Lead via an Electrode-Bound DNAzyme Assembly, 2007, J.
Am. Chem. Soc., 129, 262-263.
Gagik G. Melikyan, Ruth Sepanian, Ryan Spencer, Aaron Rowe and Pogban Toure,
Cobalt-Complexed Propargyl Cations: Generation under Neutral Conditions
vi
and Spontaneous, High-Temperature Conversion to Propargyl Radicals,
Organometallics, 2009, 28 (18), pp 5541–5549.
Aaron A Rowe, Erin A Miller, Kevin W Plaxco, Reagentless Measurement of

Aminoglycoside Antibiotics in Blood Serum via an Electrochemical, Ribonucleic
Acid Aptamer-Based Biosensor, 2010, Analytical Chemistry, 82 (17), 7090–7095.
Ryan J. White, Aaron A. Rowe, Kevin W Plaxco, Re-engineering aptamers to

support reagentless, self-reporting electrochemical sensors, 2010, Analyst, 135
(3), 589-594.
Gagik G. Melikyan, Ryan Spencer, Aaron Rowe 1,3-Steric Induction in

Intermolecular Radical Reactions Mediated by a Co2(CO)6−Metal Core,
Organometallics, 2010, 29 (16), 3556–3562.
Aaron A. Rowe, Andrew J. Bonham, Ryan J. White, Kevin W. Plaxco Fabrication

of Electrochemical-DNA Biosensors for the Reagentless Detection of Nucleic
Acids, Proteins and Small Molecules, 2011, J Vis Exp., 52, 2922
Aaron A. Rowe, Kelly N. Chuh, Arica A. Lubin, Erin A Miller, Brett Cook, Daniel
Hollis, and Kevin W. Plaxco Reusable Electrochemical Biosensors for High Gain
DNA Detection Employing an Internal Electrode Attachment Site, 2011, In
Preparation
Aaron A Rowe, Andrew J Bonham, Ryan J White, Michael P Zimmer, Ramsin J

Yadgar, Tony M Hobza, Jim W Honea, Ilan Ben-Yaacov, Kevin W Plaxco
CheapStat: an open-source, “do-it-yourself” potentiostat for analytical and
educational applications, 2011, Submitted to PLoS One
AWARDS
Teaching Assistant of the Year, Department of Chemistry, California State

University Northridge, 2005
Fellow of the Center for Nanoscience in Society, 2006
Outstanding Teaching Assistant, Department of Chemistry and Biochemistry,

University of California, Santa Barbara, 2006
Student Employee Graduate Research Fellowship, Lawrence Livermore National

Laboratory, 2006
Lawrence Scholar, Lawrence Livermore National Laboratory, 2008
vii
ABSTRACT
by
Electrochemical biosensors could become the key components of many point-of-
care medical diagnostics. These reusable devices can make rapid measurements in
untreated samples, providing information that could allow doctors to make better
decisions or change their course of action to avert clinical mishaps. This dissertation
describes a variety of molecular architectures, and a device for using them to make
measurements in the field. In short, we have developed a sensor that can rapidly test
for aminoglycoside antibiotic overdoses in whole blood. Another sensor that was
developed during the course of this dissertation can detect single-stranded DNA
within two minutes. Finally, our laboratory has developed an inexpensive handheld
potentiostat that can be used in conjunction with the aforementioned biosensors.
viii
TABLE OF CONTENTS
I. Point-of-Care Medical Diagnostics .......................................................................... 1
1.1 Background .......................................................................................... 1
1.2 Areas of Need for Point-of-Care Testing ........................................... 3
1.3 Features Required to Support Point-of-Care Applications .................. 7
1.4 Laboratory Based Diagnostic Methods .............................................. 10
1.5 Emerging Point-of-Care Technologies ............................................. 13
1.6 Reagentless, Reusable Electrochemical Biosensors ....................... 32
1.7 Conclusion.......................................................................................... 42
1.8 References ....................................................................................... 43
II. Rapid Analysis of Antibiotics ............................................................................... 56
2.1 Motivation ......................................................................................... 56
2.2 Results and Discussion ....................................................................... 58
2.3 Conclusions ........................................................................................ 69
2.4 Experimental Methods ...................................................................... 70
2.5 Acknowledgements .......................................................................... 75
2.6 References .......................................................................................... 75
III. Signal-On E-DNA Biosensors ............................................................................. 80
3.1 Motivation ......................................................................................... 80
3.3 Conclusions ........................................................................................ 88
ix
3.4 Experimental Methods ...................................................................... 89
3.5 Acknowledgements .......................................................................... 92
3.6 References .......................................................................................... 92
IV. An Inexpensive Potentiostat ............................................................................... 96
4.1 Motivation ......................................................................................... 96
4.3 Conclusions ...................................................................................... 105
4.4 Experimental Methods .................................................................... 105
4.5 Acknowledgements ........................................................................ 111
4.6 References ........................................................................................ 111
x
LIST OF FIGURES
Figure 1.1 A lateral flow immunoassay for influenza. .........................................................................4
Figure 1.2 The general workflow of chemiluminescence and electrochemiluminescence assays. .......14
Figure 1.3 The LavaAmp, a convection-based PCR thermalcycler ....................................................16
Figure 1.4 Centrifuge tubes that contain a positive test for Trypanosoma brucei ............................... 17
Figure 1.5 Silver amplification greatly increases the sensitivity of sandwich assays. .........................20
Figure 1.6 Schematic of a lateral flow immunoassay. ........................................................................23
Figure 1.7 A sandwich assay that makes use of magnetic nanoparticles.............................................25
Figure 1.8 Solution-phase magnetic nanoparticle assays, using T2 relation time................................ 26
Figure 1.9 An electrochemical sandwich assay. .................................................................................30
Figure 1.10 Schematic Representation of E-DNA and E-AB biosensors. ...........................................33
Figure 1.1 E-AB sensor that can detect several aminoglycoside antibiotics. ......................................34
Figure 1.12 Inexpensive potentiostat that is compatible with E-DNA sensors. ...................................35
Figure 1.13 An E-AB sensor for the detection of aminoglycoside antibiotics. ...................................39
Figure 1.14 Inexpensive potentiostat that is compatible with E-DNA sensors. ...................................41
Figure 2.1 An illustration of the sensor in which the RNA aptamer is affixed to a gold electrode ......57
Figure 2.2 Circular dichroism a conformational change ....................................................................59
Figure 2.3 The E-AB sensor robustly responds to aminoglycoside antibiotics ...................................60
Figure 2.4 Sensors constructed with an RNA probe decompose rapidly in serum .............................. 62
Figure 2.5 A stability comparison. ....................................................................................................64
Figure 2.6 Modified probes also bind to aminoglycosides .................................................................66
Figure 2.7 The sensor responds well to gentamicin that was present in spiked serum samples. ..........67
Figure 2.8 A series of measurements made with the E-AB aminoglycoside sensor in a live rat. .........68
Figure 3.1 A fully covalent, high-gain EDNA architecture employing an asymmetric hairpin probe..83
Figure 3.2 The new sensor architecture is sensitive enough to detect low picomolar levels................84
xi
Figure 3.3 The sensor rapidly responds to its complementary target. .................................................86
Figure 3.4 Mismatched target DNA causes a reduced increase in the sensor signal, ..........................87
Figure 4.1 An inexpensive, “do-it-yourself” potentiostat ...................................................................93
Figure 4.2 The CheapStat supports cyclic voltammetry. ................................................................ 100
Figure 4.3 Analysis of the acetaminophen content of an over-the-counter painkiller........................ 102
Figure 4.4 The electrochemical detection of a specific DNA sequence directly in PCR mix ............ 103
Figure 4.5 Analysis of the arsenic content in three acidified lake water solutions. ......................... 104
xii
____________________________________________________________________
1. Introduction: Molecular Diagnostics for the Point-of-Care
____________________________________________________________________
1.1 Background
Point-of-Care diagnostics are medical tests that have short turnaround times
and can be performed near patients, rather than in central pathology laboratories1,2.
By quickly guiding the decisions of clinicians such systems can improve the
outcomes of medical procedures and prevent mishaps such adverse drug reactions.
The aim of my research is to develop inexpensive biosensors capable of fulfilling the
important, but as yet largely unmet, need for quantitative point-of-care molecular
diagnostics.
The current generation of point-of-care diagnostic devices are used to answer
individual questions. For instance, lateral flow immunoassays and dipsticks are used
to test for conditions ranging from bladder cancer 3 and sexually transmitted disease4,5
to pregnancy and malaria6. Handheld electronic devices are used to monitor
cardiovascular markers, drug dosing, and diabetes. Quite recently, some hospitals
have adopted chemiluminescence-based tests for cardiac troponin, which are used to
confirm damage to heart muscle7,8. A recent evaluation of these instruments showed
that their sensitivity is almost on par with laboratory-based systems. The same is true
for point-of-care tests that are used to rule out deep vein thrombosis9,10 by measuring
the level of the protein D-Dimer, a byproduct of clot degradation. Unfortunately, a
1
single bedside test that can simultaneously measure multiple parameters, such as a
panel of cardiac markers, is not yet available.
Currently available molecular point-of-care tests have several additional
shortcomings: most are incapable of making quantitative measurements, and the few
assays that do provide numerical results cannot measure a large number of different
analytes in parallel10. Even worse, several quantitative point-of-care diagnostics have
been cited for poor clinical consistency. In contrast, new electrochemical biosensors,
such as the devices that we have developed in our laboratory, can quickly make
quantitative measurements in complex mixtures and could be arrayed and adapted to
measure many analytes at once.
To create a point of comparison for my own work, in this introduction I
overview the many previously developed tools that have been used to measure the
concentrations of drugs, biomolecules and metabolites in biological fluids. Portable
imaging devices, cell counters, volatile biomarker detectors, wireless cardiovascular
monitors, and miniature microscopes have also entered the clinic in point-of-care
applications, but they are beyond the scope of this review.
Prior to entering into a review of the field, it is first worth noting that, in the
context of point-of-care diagnostics, the term “sensitivity” is, confusingly, often used
to define two different concepts. In a clinical context, it refers to the percentage of
positive samples that were correctly identified as positive. From an engineering
standpoint, “sensitivity” is used to describe the steepness of the input-output curve; in
this context, a more sensitive sensor is one in which a small change in the
2
concentration of the target molecule causes a large change in the output signal. Here I
use “clinical sensitivity,” and “sensitivity” respectively to describe these two distinct
concepts.
1.2 Areas of Need for Point-of-Care Testing
The central concept of point-of-care testing is that shorter test turnaround
times lead to faster, more appropriate medical interventions, leading in turn to better
medical outcomes. This is clearly illustrated in several scenarios from the treatment
of infectious disease. For instance, the gold standard test for Candida fungal
infections is still cell culture, which has a turnaround time of several days. This slow
turnaround time contributes to the alarming mortality rate for bloodstream Candida
infections, which hovers around 40 percent. However, when Candida is treated
properly on the first day in which symptoms are present, the mortality rate drops to 15
percent11. The same holds true for bacterial sepsis treatment. A test that could identify
the specific pathogen, and the drugs that it is resistant to, would likely lower the
mortality rate from its current level of 37 percent 12,13. At present, the most
sophisticated clinical sepsis assays require an abbreviated period of bacterial culture
followed by PCR, allowing a 24-hour turnaround time, and cannot be performed at
the point-of-care14.
Rapid tests for influenza are widespread, but their clinical sensitivity is
abysmal and decreases with each passing flu season15,16,17. Most of them are lateral
flow immunoassays, and the antibodies that they employ cannot recognize the surface
3
antigens of rapidly-changing viruses (Figure 1.1). Some of these tests have clinical
sensitivities of only 30 percent, meaning that far more than half of all positive
individuals who take the test will receive a false negative result 15. Better clinical
sensitivity would be achieved by flipping a coin.
Figure 1.1 A lateral flow immunoassay for influenza. Courtesy of Clint Sever, Ruubix
Point of care molecular diagnostics could also vastly improve patient
compliance with infectious disease treatments. This is perhaps nowhere more true
than in the arena of sexually transmitted diseases (STDs)18. When individuals
undergo slow-turnaround STD tests, often they do not return to the testing center to
receive their results. In one study, only 57.6 percent of sex workers who went to a
4
clinic for STD testing returned19. To obviate the need for return visits, lateral flow
immunoassays are often used. And while the sensitivity of lateral flow assays for HIV
are spectacular, tests for other infections are quite poor. For instance, an
immunoassay for detecting Chlamydia has sensitivity of only 52 percent5. Similarly, a
lateral flow test for trichomoniasis had a 22 percent false negative rate 20.
The poor clinical sensitivity of many point-of-care tests is not confined to
infectious disease. Many point-of-care devices that are currently on the market suffer
from poor accuracy and consistency. In a recent evaluation of eight systems for
monitoring Hemoglobin A1c, a marker of diabetes, six of the evaluated commercial
systems gave readings that were unacceptably inconsistent21. Some of those
instruments displayed imprecision greater than 6 percent, which is twice the clinically
acceptable level.
Another source of great morbidity and mortality that could be ameliorated by
point of care diagnostics is medication overdoses. The few diagnostics that can be
used at the bedside for this purpose are tremendously useful. For instance, overdoses
of the blood thinner Warfarin send nearly 50,000 patients to the emergency room
each year with bouts of internal bleeding22. Several years ago, Roche Medical
Diagnostics introduced a handheld diagnostic device that can monitor blood
coagulation time. When a large cohort of European patients used this device at home,
they experienced far fewer overdoses than individuals who did not monitor
themselves23.
5
Genetic variation in drug metabolism is an often-overlooked cause of
incorrect drug dosing or inappropriate prescriptions. Currently, there are no point-of-
care tests that can quickly check the genetic makeup of a patient before drugs are
prescribed. Such a test could prevent adverse reactions that are forewarned by their
genotype. For instance, individuals with a polymorphism in their human leukocyte
antigen (HLA)-B gene, a member of the major histocompatibility complex, have an
80-fold higher risk of liver injury when they take the antibiotic floxacillin 24. Several
different defects in the glucose-6-phosphate dehydrogenase gene put people at risk of
hemolytic anemia if they take the antimalarial drug primaquine 25. In other cases,
genetic tests can predict the effectiveness of a medication. For example, patients with
certain variations in their CYP2C19 gene, a P450 liver enzyme, may not benefit from
clopidrogel, a medication prescribed to prevent the formation of dangerous blood
clots26. For these reasons, many future point-of-care diagnostics will be focused on
rapidly genotyping patients before drugs are administered.
An area of tremendous utility for Point-of-Care diagnostic tests is emergency
medicine, where several point-of-care tests that measure the level of cardiac troponin
are used to rule out the possibility that someone is having a heart attack 8. Similarly, a
point-of-care device that measures the level of D-Dimer, a fibrin degradation product,
can be used to rule out the possibility that a patient is experiencing a deep vein
thrombosis9,10. In a recent study of 4886 emergency room records, the average delay
before treatment was reduced by 26 minutes when these point-of-care diagnostics
6
were used1. However, the inability to measure many analytes at once greatly limits
the utility of available diagnostics.
Finally, it is important to note that slow turnaround times can decrease the
accuracy of a test. When biological fluid samples are stored or transported, the
apparent levels of biomarkers in those solutions decreases, which may yield
misleading results. Tests that can be conducted at the point-of-care will more
accurately reflect the amount of each analyte in the samples at the time they were
collected.
1.3 Features Required to Support Point-of-Care Applications
In this section I outline the demanding characteristics required to support
realistic point-of-care clinical testing.
Given the very short duration of a typical doctor-patient visit, it is imperative
that point-of-care tests can be performed rapidly. Tests that can be completed during a
single visit would allow the physician to select a medication, or conduct another
intervention, without scheduling a followup. However, doctor-patient interactions,
average under 15 minutes in outpatient settings 27, and the duration of visits during
inpatient surgical rounds are often well under 12 minutes28. In those short timeframes,
a point-of-care test must overcome many obstacles.
The sample collection procedure for an ideal point-of-care test should be so
simple that it can be done without the assistance of a trained healthcare worker.
Indeed, ideally such tests could be performed by patients at home, in retail clinics, or
7
in the developing world. This precludes tests that require drawing blood from a vein,
which requires trained personnel and always carries a risk of infection, embolism, or
intractable bleeding. An additional consideration for deployment in the developing
world is that venous blood draws carry the risk that syringes will be reused, posing
serious risks of infectious disease transmission. Thus, instead, the ideal point-of-care
technology will work on lancet blood or, better, using saliva as its test material. Each
of these sample media, however, is associated with its own complications. Finger
lancet volumes are quite small (~25 µL), and while the salivary proteome largely
parallels that found in blood serum29, saliva is a highly variable sample matrix and
often suffers from very low concentrations of medically relevant proteins.
Consistent with the above arguments a successful point-of-care test cannot be
reliant upon laborious sample preparation procedures. Unfortunately, however, many
sensors, recognition elements, and enzymatic architectures are incapable of
functioning in complex mixtures: Fouling, turbidity and enzymatic degradation get in
the way. In the clinical laboratory, the usual workaround is a lengthy analyte
extraction process, but those are generally far too labor intensive and time consuming
to be recapitulated at the point of care. Moreover, these extraction processes are also
less than quantitative, degrading the accuracy of the final test results. Finally,
elaborate sample processing steps often drive up the cost of a diagnostic test, due to
labor costs and the many consumables, ranging from spin columns to high-purity
reagents, that must be used.
8
For many applications, a point-of-care test must return quantitative results in
order to be useful. In many instances, the reason that numerical results are necessary
is obvious: even healthy individuals have a non-zero LDL cholesterol level –the
medical indication is not qualitative (i.e., whether or not someone has LDL in their
blood) but quantitative (how much do they have)? A numerical decrease in LDL
cholesterol, for example, is indicative of successful treatment with statins, illustrating
how quantitative information is absolutely critical in many medical applications.
Even in infectious disease, numbers are important, despite the fact that infection is a
binary yes/no condition! Without numbers, it is difficult to evaluate the effectiveness
of a treatment. For instance, a quantitative test for HIV viral load would reflect
whether a patient’s infection is becoming resistant to their drug regimen.
The physical manifestations of disease can be misleading, causing doctors to
order the wrong tests and treatments. Multiplexed diagnostics could mitigate this
problem. In a recent study of 3359 children with respiratory infections, doctors
correctly diagnosed the ones who were infected with influenza only 17 percent of the
time30. Among children who were hospitalized with severe respiratory infections,
influenza was correctly identified as the culprit in only 28 percent of cases. For this
reason, infectious disease diagnostics should test for a very broad range of pathogens,
or at least the most common ones.
9
1.4 Laboratory Based Diagnostic Methods
Many diagnostic techniques are used in central laboratories, but few have
been successfully adapted for the point-of-care. In this section, I outline current state
of the art laboratory-based molecular diagnostics that are the targets of efforts to
build point-of-care devices.
Polymerase Chain Reaction
Although it is slow and cumbersome, the polymerase reaction is highly
sensitive and can provide quantitative information, such as viral loads or gene
expression levels. Often used as the benchmark for evaluation of other assays, PCR
works by enzymatically building new DNA molecules, using the analyte DNA as a
template and synthetic oligonucleotides (primers) as starting points. By itself, PCR is
simply a means of producing more analyte that can be measured with another
technique. In most cases, this amplification product is then detected optically. For
that to work, the sample solution must have good transparency. And thus the samples
must be rigorously purified. This, however, is not the only reason for careful
purification. Proteases, nucleases, and other proteins in blood can interfere with the
integrity of the polymerase chain reaction. For these reasons, a lengthy series of
sample preparation procedures must be performed in advance of the amplification
process. Some of the fastest real-time PCR assays take 30 minutes, require a lot of
hands-on work, and require expert interpretation. Polymerase chain reactions can be
multiplexed, but their capacity for multiplexing is limited to tens of analytes at best.
10
Fluorescence Polarization
When a patient is taking a drug with a narrow therapeutic index, a drug for
which the window between toxic levels and effective doses is quite narrow, the serum
levels of that medication must be monitored closely31. Fluorescence polarization
immunoassays are a widely-used technique for measuring the serum concentrations of
those drugs. In this approach, fluorescently-tagged drug analogs are added to a
highly-processed sample along with an antibody or receptor enzyme that binds to
them, and then a series of fluorescence measurements are made 32,33. In each
measurement, a set of polarizing filters are placed in alternate configurations. The net
result of these measurements is a number that reflects the rate of rotation of the
fluorescent drug molecules. If the fluorescent analogs have been displaced from their
receptors by unlabeled drug molecules from a serum sample, their rotation rate
increases. This change in rotation rate is reflected by the anisotropy measurements.
Thus, the fluorescence anisotropy measurements reveal the concentration of the
medication.
Enzyme Linked Assays
Enzyme-linked immunosorbent assays (ELISA) and enzyme-linked
oligonucleotide assays (ELONA), are used to quantify the levels of proteins in
biological fluids34. In most of those assays, a capture molecule is immobilized onto
the bottom of plastic plates or the surfaces of microscopic beads. When the sample
solution is introduced, the analyte is captured onto the surface, and then the surface is
11
washed. After the washing procedure, a second recognition molecule is introduced
and it binds to the analyte, forming a sandwich. This analyte is affixed to an enzyme
such as horseradish peroxidase, which produces a measurable optical change.
Although their sensitivity was once thought to be laudable, it has been surpassed by
other methods. Furthermore, the lengthy incubation and washing steps make these
tests quite slow. Many new sensor architectures, such as the devices that employ
magnetic nanoparticles, are faster and more sensitive by several orders of magnitude.
Microarrays
Microarrays are generally comprised of oligonucleotide capture probes
immobilized onto small spots arrayed onto glass surfaces. When analyte DNA
hybridizes to these spots, they are lit up by secondary recognition elements, and then
imaged with a CCD camera or a similar optical reader. Several labs have developed
microarrays that can identify every documented virus plus a wide variety of other
pathogens35,36. Other groups have developed tests that can simultaneously evaluate
more than 550,000 single nucleotide polymorphisms in human genes, offering a
means to predict drug response and screen for genetic diseases37. At first glance, these
systems seem magnificent, but they have many drawbacks. All of them make use of
an optical readout mechanism, which means that the samples must be aggressively
processed to remove optical interferants. Furthermore, they are dependent upon a
lengthy amplification period, followed by an even longer hybridization protocol. For
this reason, their turnaround times are invariably on the order of hours or days.
12
1.5 Emerging Point-of-Care Technologies
A wide variety of new diagnostic strategies have been reported, but they have
not yet reached the market. Many of these devices possess some of the attributes of an
ideal point-of-care diagnostic tool, but few can claim all of those virtues. Despite
their drawbacks, many of these tools may prove clinically and commercially viable.
Chemiluminescence and Electrochemiluminescence
Many successful diagnostics rely upon fluorescent dyes and complicated
optics. In the past decade, it has become clear that chemiluminescence and
electrochemiluminescence may be superior. In each of those approaches, a
recognition element such as an antibody is tagged with a substance that produces light
when combined with other chemicals or stimulated by an electrical current (Figure
1.2). In the absence of an analyte, no light is produced. Since there is no need for an
excitation light source, the optical hardware can be very simple. For the same reason,
the background signals in these measurements can be phenomenally low, giving them
exceptionally high sensitivity. A commercial point-of-care assay, called PathFast,
takes advantage of those characteristics. It has proven itself reliable as a tool for
measuring cardiac markers such as D-Dimer and troponin9,10. However, pathogen
detection tests that are based upon chemiluminescence and electrochemiluminescence
remain experimental.
13
Figure 1.2 The general workflow of chemiluminescence and electrochemiluminescence assays.
A microarray that uses a chemiluminescent readout can identify pathogens in
under four hours38. This procedure has three key elements. First, an abbreviated PCR
reaction is run for only 30 cycles instead of a more typical 45 to 55 rounds of
amplification. Second, the PCR product is tagged with digoxigenin and then
hybridized onto a microarray in a buffer that is constantly flowing back and forth
across the capture probe surface. Finally, HRP-tagged antibodies that bind
digoxigenin are introduced to the system along with hydrogen peroxide and luminol.
This creates a chemiluminescent signal, which illuminates each spot on the
microarray where nucleic acids were hybridized. However, the turnaround time for
this assay is just under four hours, which presents no advantage over a standard PCR
experiment. Furthermore, this system has not been proven to function in realistic
clinical specimens (which, given the ready availability of such, seems a significant
omission from the literature). So far, it has only been tested with water that was
14
spiked with pathogen cells at high concentrations. To adapt this assay for clinical use,
lengthy sample processing would likely be necessary.
Automation and Acceleration of the Polymerase Chain Reaction
The chief drawbacks of PCR are its slow turnaround time and high demand
for labor-intensive sample handling. To remedy this, many research groups have
developed miniature devices that can amplify DNA via the polymerase chain reaction
and then analyze the product of that reaction. For instance, the DxBox is an
extremely compact system that handles sample preparation, amplification, and
detection of the PCR product39,40.
Rheonix has developed a fully automated system for the analysis of sexually
transmitted diseases. First, it conducts a multiplexed PCR reaction, meaning that
many different primers are present in the same solution in order to amplify multiple
target sequences in a single reaction mixture. All of those primers are tagged with
biotin. Second, the PCR products are denatured and then hybridized onto a low-
density microarray. Finally, a conjugate of streptavidin and horseradish peroxidase is
added to the array, along with tetramethylbenzidine. This develops the spots where a
PCR product has hybridized. (Spizz et al. Submitted)
Other groups are focused upon decreasing the cost of the polymerase chain
reaction by eliminating the need for bulky thermal cycler hardware. In particular,
Victor Ugaz and Nitin Agrawal at Texas A&M University have developed several
systems that use heat blocks at fixed temperatures rather than Peltier chips to drive
15
the reaction41,42,43. In each of their designs, the PCR mix is loaded into a ring or
chamber where it is mixed by convection (Figure 1.3). These systems can generate a
significant amount of PCR product in minutes, rather than hours, because they do not
require cooling cycles, which are a bottleneck in traditional PCR processes. While
this rapid convection-based amplification system is promising, it has not yet been
coupled to a detection system other than (slow, manual) gel electrophoresis.
Figure 1.3 The LavaAmp, a convection-based PCR thermalcycler based upon the work of Victor
Ugaz and Nitin Agrawal. Photo by Aaron Rowe
16
Alternatives to the Polymerase Chain Reaction
Although the polymerase chain reaction is well understood, it suffers from
many drawbacks that limit its utility in point-of-care applications. Most notably, it
requires a device that can rapidly cycle between three high temperatures. As an
alternative, many researchers have turned toward loop-mediated isothermal
amplification (LAMP) or the exponential amplification reaction (EXPAR),
approaches that require only one fixed temperature44. This eliminates the need for
traditional thermalcycler hardware, which usually includes costly thermoelectric
chips and bulky cooling systems. Furthermore, isothermal methods can give either
qualitative results that are visible to the naked eye, or quantitative results, which
require additional equipment.
Figure 1.4 – Centrifuge tubes that contain a positive test for Trypanosoma brucei (green, left)
alongside negative tests (Orange, right). (Nijiru, 2007, PLoS Neglected Tropical Diseases)
Isothermal amplification techniques are regarded as a promising diagnostic
approach for use in the developing world. For instance, a LAMP-based test for the
17
cause of sleeping sickness, Trypanosoma brucei gambiense, was conducted using
little more than a 62ºC water bath, and had higher sensitivity than a PCR assay
(Figure 1.4). It was able to detect as little as one trypanosome per milliliter of blood,
and had a turnaround time in the regime of 40 minutes 45. Similarly, an isothermal
assay for Dengue fever outperformed the sensitivity of a PCR assay, detecting the
virus with a 30 minute turnaround time46. Another LAMP-based test, which was
meant to identify a wider variety of trypanosomes, correctly identified the infection in
90% of infected blood samples47,45,48. A LAMP-based assay for Plasmodium
falciparum, a parasite that causes malaria, had a clinical sensitivity of 96.7% and a
turnaround time of just over an hour49.
A simple but fully-integrated microfluidic device that uses LAMP can detect
influenza with high sensitivity50. In short, a set of channels and reaction wells were
formed in a polydimethylsiloxane film, lyophilized LAMP reagents were loaded into
the channels, and they were sealed between a pair of glass plates. On one of the glass
plates, a layer of indium tin oxide served as a heater. The entire cartridge was
sandwiched between a set of photodiodes, attached to a netbook computer. As the
amplification reaction progressed, there was an increase in the fluorescence of Sybr
Green 1, an intercalating dye that was included in the reaction mixture. Such a test
has allowed the detection of influenza with 91 percent sensitivity in less than 30
minutes with minimum sample handling.
An even faster test has recently been developed for herpes simplex virus51. It
employs EXPAR, an isothermal DNA amplification technique, and then detects the
18
product of that reaction via the aggregation of gold nanoparticles. The entire process
takes under ten minutes. This system is an outstanding source of qualitative results,
but it may not be capable of providing quantitative information. Furthermore, its
potential for multiplexing, testing for many pathogens simultaneously, may be quite
limited.
Silver Amplification Coupled Assays
When gold nanoparticles are immersed in silver nitrate, along with a reducing
agent such as hydroquinone, a large amount of metallic silver plates out of the
solution (Figure 1.5). Many research groups have taken advantage of this reaction as
an alternative to enzymatic amplification. In general, a recognition element is tagged
with a gold nanoparticle and it forms a sandwich with the analyte and an immobilized
capture probe. After a washing step, silver nitrate and a reducing agent are introduced
into the system, and the thickness of the resulting silver film is measured optically.
The first assays that made use of silver amplification reported DNA at
femtomolar levels. This put extracted human genomic DNA (for personalized
medicine applications, for example) within the reach of detection without PCR
amplification. It is not low enough, however, to support the detection of clinically
relevant levels of pathogen nucleic acids, which can span the range from 100 to
100,000 copies per mL52,53. Later, a fully-integrated system called POCKET proved
capable of detecting antibodies in the low femtomolar regime 54. Over time, the
sensitivity of these tests has been optimized, and they are now capable of detecting a
19
wide variety of microbes, including influenza, HIV and syphilis, at clinically relevant
levels55.
Figure 1.5 Silver amplification greatly increases the sensitivity of sandwich assays.
Silver amplification technology has been commercialized by Nanosphere
Incorporated, who sell a kit to check for Factor V Leiden, a blood clotting disorder,
and for several genotypes that are predictive of drug metabolism and response 56. A
similar system was developed by Claros Diagnostics, and is approved in the European
Union as a quantitative test for prostate specific antigen. However, although this
technology has been developed commercially, its prospects for large scale
multiplexing remain questionable. To achieve quantitative results, several surfaces
with different densities of capture reagents are necessary. Thus, arraying this system
would be inherently inconvenient.
Field Effect Transistors
Many groups have built transistors that are switched by biological activity.
Most of these devices have three key components: a source, a drain and a gate. In
each of these devices, a biological phenomenon acts as the gate, increasing the
20
current between the source and drain. The variety of devices in this area of study is
quite broad.
Arrays of highly ordered silicon nanowires can act as transistors. When they
are decorated with peptide nucleic acid probes, these devices can detect DNA at
levels down to 10 femtomolar57. When the negatively charged DNA hybridizes with
the PNA capture probes, it creates a field effect within the nanowires, increasing the
flow of current.
One class of ion-sensitive field effect transistors can measure the release of
protons that takes place during polymerase-catalyzed DNA synthesis. This
technology is already in use in desktop DNA sequencers sold by Ion Torrent, a
division of Life Technologies58. A significant limitation of such systems is that they
require a five hour sample preparation procedure. This is necessary because the
ultrasensitive sequencing hardware must be free from any molecules that could foul
the sensor surface, liberate interfering protons, or interfere with the sequencing
reaction. Furthermore, sequencing is an inefficient means of detecting pathogens in
body fluids because the amount of human genomic DNA and symbiotic microbial
DNA greatly outweighs the microbial genetic material.
Enzymes can also be immobilized onto the alumina gates of field effect
transistors, allowing, in some cases, the detection of their substrates. In one report,
this technique was used to detect urea, glucose, acetylcholine and N-acetyl-L-
tyrosine59. In another paper, the same group immobilized lactate dehydrogenase onto
a transistor and was able to detect 100µM lactate in seconds 60. Another group
21
immobilized a lipase onto magnetic nickelferrite (NiFe2O4) nanoparticles and used a
permanent magnet to draw them onto the gate of a transistor 61. This allowed them to
detect triglycerides.
Next-Generation Lateral Flow Immunoassays
As described earlier, lateral flow immunoassays are the current gold standard
in point-of-care diagnostics. For some applications, such as HIV diagnosis, these tests
are fully adequate because they use well-established recognition elements that bind to
analytes which are almost universally present in infected patients. However, the
clinical sensitivity of other lateral flow assays can be quite poor15,5,6. At best, most
lateral-flow tests detect two analytes. In the case of acute infectious disease, this is
simply not enough: pathogens are so diverse, and evolve so quickly, that no single
recognition element is adequate for the diagnosis of a disease.
The operating principles of a lateral flow immunoassay are quite simple.
Antibodies travel along a paper strip, carried by the wicking motion of a fluid sample.
If the test is positive, the colored antibodies stop at a boundary line and change its
color. A classic example of this: home pregnancy tests, which use a pair of anti-HCG
antibodies dried onto a strip of nitrocellulose (human chorionic gonadotropin is a
marker of pregnancy, and athletic doping). One of those antibodies is labeled with
biotin, and the other is tagged with a bright optical reporter, such as gold or latex
nanoparticles. Urine samples from pregnant women contain the HCG protein. When
the test is wet with urine, capillary forces pull the liquid toward a test strip where
22
streptavidin sits. If the urine sample is positive for HCG, a sandwich will form
between the protein and two antibodies on this line, and the color of the line will
change. The remainder of the colored antibodies flow past this line to a control area,
where they are captured by a secondary antibody (Figure 1.6).
Figure 1.6 Schematic of a lateral flow immunoassay.
Rapid pathogen tests are often directed at the most common cause of an
illness, and provide poor coverage of related species. For instance, the FalciVax rapid
malaria test has excellent sensitivity and specificity for detecting Plasmodium
falciparum, (91.8%) but its ability to detect Plasmodium vivax, another malarial
pathogen, is quite poor (76.6%)6. This assay checks for two markers of malaria: P.
falciparum histidine-rich protein-2 and P. vivax lactate dehydrogenase. The former is
an excellent marker, and the latter is not. The shortcomings of this test underscore the
need for both selecting markers of disease to monitor, and recognition elements that
reliably detect them.
New materials, such as lanthanide complexes, can radically improve the
performance of immunoassays. A new lateral flow test for the detection of the
hepatitis B virus has far lower detection limits than earlier tests. It can detect a viral
23
surface antigen down to 0.03µg/L, which is 100 times better than standard lateral
flow tests62. The key ingredient is a new europium-based complex that is loaded onto
silica nanoparticles, which act as tags on each recognition antibody63. When used in
conjunction with a digital camera, this assay can be used to provide quantitative
results.
In recent years, funding from the Gates Foundation has re-invigorated lateral
flow assay research, and as a result several groups have found ways to overcome
several shortcomings of paper-based devices. Some of these systems use wax or tape
to create separate channels on the paper, so that several different analytes can be
measured at once64,65,66,67,68. Although these devices are purportedly meant for use in
the developing world, so far, not many of them actually detect analytes that are
relevant to tropical medicine.
Magnetic Nanoparticle Systems
Biological samples invariably contain fluorescent material, rendering optical
approaches difficult. But the immeasurable “magnetic” background of such samples
is generally negligible. Furthermore, magnetic nanoparticles have a tremendously
high surface area to volume ratio, which allows each particle to capture a
comparatively large mass of the analyte. Finally, they can be easily manipulated by
strong magnets during the target capture and washing steps of an assay. For these
reasons, assays that employ magnetic nanoparticles in their detection systems can be
exquisitely sensitive.
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Figure 1.7 A sandwich assay that makes use of magnetic nanoparticles.
A number of research groups have developed sensors that use the principle of
giant magnetoresistance to detect cancer biomarkers with extremely low detection
limits. They are able to detect cancer markers such as embryonic antigen, eotaxin,
granulocyte colony stimulating factor, and five other substances at low femtomolar
levels69. This is accomplished by immobilizing a primary capture antibody onto the
magnetic sensor surface and then sandwiching the analytes with a biotinylated
secondary antibody. After several rinses, streptavidin coated iron oxide nanoparticles
were added to the system and magnetic measurements were made.
A smaller company, MagArray (magarray.com), is trying to commercialize
this technology. However, it will primarily be used for research purposes for the next
few years. The key drawback of these systems is their high cost. In order to detect the
magnetic nanoparticles, giant magnetoresistance sensors must be very close to the
analyte. This means that they must be located within the disposable test cartridge
itself, rather than in the cartridge reader device.
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Another fully-integrated device uses frustrated total internal reflection to
quantify the number of iron oxide nanoparticles that have formed sandwiches. It was
able to measure the level of cardiac troponin from a fingerprick sample of blood at
concentrations down to 0.03 ng/mL in under five minutes70. The same device was
used to measure the level of parathyroid hormone in serum samples taken from
patients who elected to have a hyperactive thyroid gland removed 71. The test was
capable of detecting the hormone at picomolar levels within ten minutes. The optics
in this system are similar to those that can be found in a DVD player, and
coincidentally were developed by the same group at Philips electronics that
developed CD and DVD optical information storage technology. Unfortunately, they
function in a signal-off fashion, meaning that the amount of light that is internally
reflected by a waveguide decreases with increasing analyte concentrations. This
means that the overall signal gain is quite subtle, and the devices could theoretically
suffer from false positives due to fouling.
Figure 1.8 Solution-phase magnetic nanoparticle assays, using T2 relation time.
26
In Massachusetts, T2 Biosystems (t2biosystems.com) has developed a novel
approach to quantifying analytes by using magnetic nanoparticles and a desktop
NMR spectrometer (Figure 1.8). With that system, they were able to identify Candida
yeast species that had been spiked into blood samples. The central concept of this test
is that antibody-tagged magnetic nanoparticles will bind to and aggregate around
analyte proteins. This causes a change in the T2 relaxation time of the water in each
sample. When the nanoparticles are widely dispersed, the decay constant decreases,
as most of the water molecules are influenced by the magnetic fields of the
nanoparticles. If they aggregate, the decay constant is on par with normal water. From
start to finish, the entire workflow for this procedure is under two hours and it takes
place in unfiltered samples, representing a very significant improvement over
previously developed techniques.
Optical Waveguide Biosensors
Many optical waveguide biosensors have been reported in the
literature and most of them have similar operating principles. Capture molecules are
immobilized onto the surface of a waveguide, a material that transmits light. The
binding of analyte molecules then causes a change in the refractive index at the
surface of the waveguide. The amount of analyte is proportional to the photocurrent
that is passing through the waveguide, parallel to the sensor surface. While these
systems are quite sensitive, many of them require the use of consumable materials
that were fabricated in cleanrooms, so the cost per test would be very high.
27
Furthermore, they are tremendously sensitive to nonspecific adsorption, and thus
require aggressive sample preparation or washing steps.
Local evanescent array coupled biosensors are made from semiconductor
material, such as silicon nitride. Antigens or antibodies can be immobilized onto the
nitride surfaces by a microarray printer, and then the sensor array can be placed above
a set of photodetectors. This system was used to detect anti-tuberculosis antibodies. It
had a limit of detection around 100 picomolar72. It is not clear, however, how well
these systems perform in clinical samples; the non-specific adsorption of
contaminants to the sensor surface will also cause a change in refractive index, which
has generally rendered approaches like this poorly suited for clinical applications.
A second example of a waveguide biosensor was constructed by Rong and co-
workers from porous silicon and decorated with DNA capture probes. This sensor
achieves a detection limit of 50nM for its target DNA sequence in buffer. And it was
capable of distinguishing between complementary and non-complementary DNA,
though it was not able to identify single nucleotide polymorphisms or other subtle
mismatches73. Again, however, this sensor was only tested in HEPES buffer. No
data are presented demonstrating the devices performance in realistically complex
samples, which is likely poor.
Total internal reflectance has been widely used by research labs to analyze
protein biomarkers, and recently several compact devices have been developed. In
one such system, capture probes were affixed to a tantalum pentoxide coated glass
surface, which was sealed within a flow cell and imaged with a CCD camera. In the
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automated system, biomarkers were sandwiched between the capture probe and a
relevant antibody. It was capable of detecting C-reactive protein, interleukin-6 and
procalcitonin in clinical serum samples74 thanks to a set of antifouling coatings on the
waveguide surface, further blocking of the surface with a surfactant, and aggressive
automated washing of the surface prior to measurement. The entire procedure takes
under 35 minutes.
At Los Alamos National Laboratory, the group of Basil Swanson has
developed waveguide biosensors with 10 minute turnaround times and sensitivity that
is comparable with ELISA assays. A sandwich assay built atop a planar waveguide
was capable of detecting carcinoembryonic antigen in serum samples at levels down
to 0.5 picomolar, well below the 39 picomolar level that is considered the upper
threshold for a normal test result75. This assay could be completed in fifteen minutes.
A similar sensor developed by the same research group achieved Anthrax-related
antigen detection in a similar timeframe 76.
Electrochemical Assays
Electrochemical techniques have two key advantages: They are largely
capable of functioning in complex mixtures, and the necessary electronic hardware
that is compact, convenient, and inexpensive.
Simple electrochemical sandwich assays, analogous to the optical sandwich
assays described above, have achieve picomolar detection levels, directly in
biological fluids such as urine77,78,79,80. Although this is not sensitive enough to detect
29
genomic DNA (much less pathogen DNA at clinically relevant levels), the high
specificity of these systems allows them to detect amplicons directly in reaction
mixtures. Perhaps the greatest drawback of these systems is that the sensors are not
reusable, and they require several consumables including horseradish peroxidase
tagged oligonucleotides and tetramethylbenzidine, a small redox-active molecule that
shuttles electrons to the electrode surface.
Many electrochemical assays are comprised of DNA and mercaptohexanol
affixed to gold electrode surfaces (Figure 1.9). The performance of these tests is
improved when they are conducted on surfaces that have been passivated with a
mixed monolayer containing mercaptohexanol and a dithiol, such as 1,6-
hexanedithiol. It is believed that these passivating agents reduce fouling on the sensor
surface and thus increase the signal to noise ratio. Such systems have been used to
detect synthetic oligonucleotides bearing a sequence from human papillomavirus.
Figure 1.9 An electrochemical sandwich assay.
30
In a similar endeavor, the company Gene Fluidics has developed a fully
integrated cartridge that uses an array of gold electrodes on a plastic substrate. It
makes use of a 35-nucleotide capture probes that are tagged with biotin, and 35-
nucleotide detection probes that are tagged with horseradish peroxidase 81. Those
probes hybridize with rRNA from the pathogen, and are detected via amperometry
thanks to a tetramethylbenzidine-mediated reaction. The entire procedure takes under
45 minutes and can identify a wide variety of pathogens in clinical urine specimens 82.
Although this is a substantial improvement over PCR-based nucleic acid detection, it
is still not fast enough to be used during the 15-minute window offered by a standard
doctor’s office visit.
A number of electrochemical sandwich or sandwich-like methods have been
described by which PCR products can be detected after amplification is complete. In
one example, a sandwich assay that employed alkaline phosphatase was used to
detect Legionella infection83. Once the sandwich formed, the enzyme would convert
1-naphthyl phosphate into 1-naphthol, which could be electrochemically detected.
Electrochemical methods have also been used to monitor real-time PCR reactions.
For instance, dUTP building blocks can be coupled with ferrocene and integrated into
a reaction mixture84. During the reaction, electrochemical measurements can be
performed after each annealing cycle.
Electrochemical sandwich assays have also been used to detect proteins in
biological fluids, but the workflows of those tests are lengthy and they often require
31
enzyme or nanoparticle-labeled recognition elements. For instance, a sandwich assay
for the detection of the inflammatory marker C-Reactive Protein was capable of
detecting the important biomarker in serum, but it required several 15 minute
incubation and washing steps85. Quite recently, Das and Kelley developed a system
that does not require a multi-step detection process, labeled probes, or chemical
amplification. It makes use of a single set of unlabeled antibodies immobilized onto
gold electrodes and interrogated with differential pulse voltammetry. In theory,
binding to the target increases the amount of crowding on the sensor surface,
changing the rate of an electrochemical signal caused by an added mediator
(Fe(CN)6). This technique allowed for the detection of CA-125, an ovarian cancer
biomarker, in both human serum and whole blood at 0.1 units per milliliter 86. For this
technique to be successful, the gold electrodes must have a fine-grained
microsctructure. Scanning electron microscope studies showed that the best
electrodes were comprised of 8µM gold surfaces. Since this sensor design was
published recently, it is not yet clear how versatile or reproducible the results will be.
One key drawback is that it makes use of a consumable reagent Fe(CN)6, which is
added to the sample solution rather than immobilized onto the capture probes.
1.6 Reagentless, Reusable Electrochemical Biosensors
This section is an introduction to the specific topics that will be covered in the
remainder of my thesis. It is meant to give a brief overview of the projects that
comprise my dissertation research.
32
Electrochemical DNA biosensors (E-DNA) have a uniquely simple design.
Their recognition elements are modified oligonucleotides, affixed to the surfaces of
gold electrodes (Figure 1.10). At the end of each oligonucleotide is a redox-active dye
molecule such as methylene blue, which transfers electrons through the sensor circuit.
When a complementary molecule binds to the recognition element, the rate of
electron transfer changes. This change in current is proportional to the analyte
concentration.
Figure 1.10 Schematic of an E-DNA biosensor. Hybridization of target DNA to the probe
molecules restricts the movement of a methylene blue redox probe, causing a decrease in the Faradaic
current that passes through the sensor circuit.
In comparison to the other diagnostic approaches described earlier, E-DNA
devices offer a wide variety of advantages. They are reusable, require few
consumables, and have short response times, and can be arrayed or integrated with
33
microfluidics. Perhaps their most remarkable attribute is their high selectivity. They
are capable of functioning in PCR mix, LAMP reaction mix, or blood serum. For this
reason, they could be used to make measurements in samples that have not undergone
a lengthy preparation procedure. They come in a wide variety of architectures (Figure
1.11), ranging from linear probes, to stem-loops, to inverted hairpins and
pseudoknots.
Figure 1.11 Schematic Representation of some of the probe architectures employed in the E-DNA
class of reagentless, electrochemical biosensors.
In clinical samples, pathogen nucleic acids are often found at the low
femtomolar to attomolar range. At present, even the most sensitive E-DNA
architectures are not capable of operating in that regime. To detect genetic material at
those extremely low concentrations, E-DNA can be seamlessly integrated into
microfluidic devices and coupled with amplification systems. This strategy has been
validated on several occasions for amplification and detection of pathogen DNA on a
single chip. For instance, Ferguson et al. were able to detect Salmonella DNA at 10
34
attomolar levels87. More recently, Patterson et al. have been able to make reliable
measurements in a 1:10 blend of blood serum and the LAMP reaction mixture. By
comparison, fluorescence-based assays such as quantitative PCR require stringent
sample preparation.
To further increase the sensitivity of E-DNA biosensors, our laboratory has
developed a signal-on biosensor that exhibits remarkably high gains. Unlike every
other E-DNA sensor reported to date, this architecture is immobilized via an internal
thiol in the midsection of the probe DNA molecule (Figure 1.12). Most other DNA
biosensors are immobilized by a thiol group at their 3’- or 5'-ends. This unique design
gives it excellent sensitivity and stability. When the sensor is exposed to a high
analyte concentration, the signal increases eightfold from its baseline level. This high
gain also allows it to detect low picomolar levels of DNA targets.
Figure 1.12 A reusable E-DNA sensor that exhibits high signal gain. (Rowe, 2011)
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The predecessor of this signal-on sensor was developed by Xiao et al88. It
demonstrated comparable signal gain, and a remarkable detection limit. However, its
double-stranded probe was held together by hybridization alone, which prevented
sensor re-use or storage. By comparison, the new sensor architecture is remarkably
stable. Since the entire architecture is held together by covalent bonds, it can be re-
used repeatedly (>5X) after a brief rinse with dimethyl sulfoxide.
The initial design of this signal-on E-DNA sensor detects a sequence from
Salmonella, but there is a greater demand for the detection of other pathogens. As a
result, our group has recently designed a new sensor with the same architecture. It
bears a probe sequence that has been carefully validated as capable of detecting
Mycobacterium tuberculosis in clinical isolates. The development and use of the
Salmonella sensor system is the focus of my third thesis chapter.
Electrochemical Aptamer-Based Biosensors
While E-DNA biosensors are a promising means of detecting pathogens or
genotyping patients, they are not capable of analyzing drugs, proteins, or metabolites.
To fill that void, our laboratory has developed electrochemical aptamer biosensors (E-
AB). Aptamers are oligonucleotides that were selected in vitro for their ability to bind
to a specific analyte, often with high affinity and specificity89. They have been touted
as potential replacements for antibodies. Since they can be synthesized with
automated machinery, and modified very easily, they have become popular affinity
reagents in a wide variety of biosensor architectures.
36
With the aim of developing point-of-care devices that are versatile enough to
detect a broad range of analytes, our lab has adapted the (E-DNA) signaling concept
to make use of aptamers as recognition elements. In each device, a binding event
causes a change in the conformation of the aptamer, and this changes the rate at
which a redox reporter linked to the aptamer’s free end collides with the sensor
surface. Since the sensor signal is tied to a binding-induced conformational change, it
is not affected by nonspecific adsorption. The resulting electrochemical aptamer
biosensors (E-AB) can detect proteins directly in urine, serum, or whole blood. This
remarkable selectivity stems from their unique signaling mechanism.
Many different aptamers, adopting a wide range of structures, have been
adapted into the E-AB platform. The first aptamer employed in an E-AB sensor, the
thrombin-biding aptamer of Bock90 adopts a G-quadruplex structure. When it is
labeled with a methylene blue reporter at its 3’-end, and immobilized onto a gold
electrode surface at its 5’-end, it is capable of detecting thrombin directly in blood
serum. An aptamer that binds to platelet-derived growth factor folding into a three-
way junction structure, that, in the E-AB platform, supports the detection of this
cancer biomarker at picomolar levels again, directly in blood serum91. Finally, an
aptamer that binds to immunoglobulin E has a simple stem-loop structure. It is
capable of detecting the antibody at low nanomolar levels92.
E-AB sensors are also suitable for the detection of small molecule analytes.
An aptamer-based sensor that our laboratory has developed, for example, can be used
to analyze cocaine in whole blood or undiluted serum samples93. The first generation
37
of cocaine sensors were based on an aptamer discovered by the Stojanovic group. The
aptamer is a DNA molecule with a three-way junction structure. To convert this
structure into a sensor, it was anchored to a gold electrode at its 5’-end and tagged
with methylene blue at its 3’-end. Upon binding to cocaine, the sensor undergoes a
conformational change, which allows the methylene blue to more efficiently carry
electrons through the sensor circuit.
Given its ability to detect specific small molecules in blood serum, a potential
application of the E-AB platform would be the monitoring the serum levels of
medications with narrow therapeutic indices. Aminoglycoside antibiotics, for
example, are widely used to treat gram-negative bacterial infections, but the dose that
can kill bacteria is not far below the toxic level, which can cause hearing loss or
kidney damage. For this reason, patients who are taking these medications must be
monitored on a daily basis. The standard tests for measuring aminoglycosides in
serum are fluorescence anistotropy or turbidimetry, which require lengthy sample
preparation procedures. Using the DNA equivalent of an aptamer developed by Wang
and Rando94,95, we were able to construct an E-AB sensor that can function in whole
blood96. It responds to the antibiotic within seconds, allowing measurements to be
made in real time within the vasculature of live animals.
38
Figure 1.13 An E-AB sensor for the detection of aminoglycoside antibiotics. (Rowe, 2010)
Our intention was to measure the levels of aminoglycosides in serum or whole
blood, and an RNA sensing element is not stable enough to accomplish such a task.
During the course of this project, we tested the stability of RNA aptamers that had
been modified with a variety of stabilizing groups. This included F- and OMe-
substitutions on its vulnerable 2’-OH groups. Although these modifications did
increase the stability of the sensor, none had as pronounced a stabilizing effect as
removing the 2’-OH groups entirely. This effectively converted our RNA aptamer
into a DNA recognition element. Surprisingly, it retained much of its affinity for the
aminoglycosides. A close examination of the solution NMR structure of the RNA
39
aptamer showed that none of the 2’-OH groups were within hydrogen bonding
distance from a bound antibiotic molecule.
With the unmodified RNA aptamer, we were able to detect gentamicin in
filtered serum samples at clinically relevant concentrations. Using the
aminoglycoside-binding DNA aptamer, we have successfully monitored the
concentrations of gentamicin in live animals. The development and use of this sensor
system is the focus of the second chapter of this thesis.
Electronics for Inexpensive Point-of-Care Diagnostics
Our laboratory has developed a handheld potentiostat that is capable of
making measurements with several different E-DNA architectures. The total cost of
this device is under $80 when it is constructed with parts that were sold at retail
prices. Currently, the signal to noise ratio is such that it can detect nanomolar target
DNA, but it is not yet capable of reaching the picomolar detection limits that have
been seen with research-grade instruments.
40
Figure 1.14 Inexpensive potentiostat that is compatible with E-DNA sensors. (Rowe, 2011)
The need for such an instrument is illustrated by a recent paper by the Nie
research group68. They re-purposed a blood glucose meter in order to conduct other
electrochemical assays. Since blood glucose meters are proprietary, they were not
able to re-program that device, and thus were limited to making measurements with a
single type of waveform. To analyze their data, they had to manually convert the
output of the meter into other units. By comparison, our new potentiostat is
completely open source, meaning that the schematics and source code are publicly
available and that anyone may build the device without paying a licensing fee. With
some further refinement, this potentiostat could be used in conjunction with a
portable thermalcycler to analyze PCR, LAMP, or EXPAR products in the field.
41
In addition to its utility as a diagnostic tool, the instrument we have developed
could be used for educational purposes or environmental monitoring. It is capable of
performing square wave, linear sweep, alternating current and stripping voltammetry.
This allowed us to reproduce a set of classic educational exercises, including the
measurement of ascorbic acid in orange juice and the acetaminophen content of a
Tylenol tablet. We also analyzed the arsenic content of water samples taken from two
nearby sources (Lake Cachuma and Cold Spring Creek, Santa Barbara, CA). This
may make it feasible to teach classic electrochemistry techniques as part of large
undergraduate laboratory courses.
The development and use of this instrument is the focus of the fourth chapter
in this thesis.
1.8 Conclusions
As outlined above, there is an unmet need for inexpensive instruments that
can make quantitative measurements at the point-of-care. The current generation of
lateral flow immunoassays do not provide numerical results, and in some instances
their clinical sensitivity is abysmal. Bulky chemiluminescence instruments, such as
the PathFast system, can make reliable quantitative measurements in the clinic but
they have a turnaround time greater than 17 minutes and can analyze only one species
at a time.
The electrochemical biosensors described in this dissertation offer many of the
features that are missing from the aforementioned point-of-care tests. For rapid
42
genotyping or the diagnosis of infectious disease, E-DNA sensors have a key set of
advantages over other point-of-care molecular assays. They can function directly in
isothermal amplification mixes, are extremely rapid, can be arrayed to allow multiple
simultaneous measurements, and are compatible with inexpensive handheld
electronics. Along the same lines, E-AB devices have recently shown their potential
as instruments for the continuous monitoring of drug dosing.
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____________________________________________________________________
2. Rapid Monitoring of Aminoglycoside Antibiotics

____________________________________________________________________
2.1 Motivation
Unintentional drug overdoses are among the most common and costly medical
crises. For example, the narrow therapeutic indices of Coumadin (Warfarin) and other
blood thinners cause uncontrolled bleeding events that send 58,000 Americans per
year to the emergency room1. Aminoglycoside antibiotics, likewise can cause hearing
loss, tinnitus, and kidney failure2,3. To administer these drugs properly, physicians
must monitor their patients between doses by drawing blood for fluorescence
polarization immunoassay, or turbidimetry analysis. 4,5 However, while these tests can
achieve a turnaround time of under one hour, they require dedicated instruments that
are largely confined to clinical laboratories. A point-of-care device may thus better
serve both patients and physicians by eliminating the follow-up visits that are often
necessary in order to achieve proper dosing.
In response to the largely unmet need for rapid, point-of-care diagnostics our
laboratory6,7 and others8 have developed a reagentless, electrochemical platform
capable of quantifying a range of molecular analytes in blood, urine and other
complex media9,10. These electrochemical, aptamer-based sensors are rapid,
sensitive, reagentless, reusable, and convenient6. They are also versatile, with E-AB
sensors having been reported against targets ranging from proteins11,12 to inorganic
ions13,14 to small molecules8,6,15. With a single exception15,16,17 all of these
electrochemical, aptamer-based (E-AB) sensors employed DNA aptamers as their
56
recognition elements, presumably because of the significantly greater stability of this
probe. Unfortunately, however, the only oligonucleotides reported to date that bind
aminoglycoside antibiotics are composed of RNA, which renders them vulnerable to
degradation by nucleases in blood serum samples and thus would likely limit the
utility of E-AB sensors fabricated using them. With the aim of solving this problem
we have explored several strategies for protecting RNA probes from degradation. In
doing so we have developed a reagentless, electrochemical approach that can be used
to measure the concentrations of aminoglycoside antibiotics in serum 18.
Figure 2.1 An illustration of the sensor in which the RNA aptamer is affixed to a gold electrode and
the binding of tobramycin draws a methylene blue reporter molecule farther from the electrode surface.
57
2.2 Results and Discussion
E-AB sensors are made by affixing an aptamer capped at one end by a redox-
active reporter group and attached via its other end to the surface of an interrogating
electrode (Fig. 2.1). In this set of experiments, our aptamers were bound to gold
working electrodes via a thiol modification at their 5’-ends and labeled at their 3’-
ends with the redox-active reporter methylene blue. For the first generation sensors
fabricated here, we employed the 26-base RNA aptamer of Wang and Rando19,20,
which adopts a stem-loop configuration that binds aminoglycosides in its loop
region21. Circular dichroism studies indicate that this aptamer undergoes a significant
conformational change upon target binding (Fig. 2.2); specifically, when 4 mM
tobramycin is added to a solution of the aptamer the intensity of the positive
ellipticity band at 260 nm increases significantly. This band is often attributed to the
formation of A-RNA structure, and thus the observed signal change may be linked to
an increase in the helical content of the aptamer. Solution NMR studies indicate,
however, that the loop in the stem-loop aptamer wraps around the tobramycin
molecule21, suggesting that the observed change in ellipticity may signal a more
complex structural transition. Irrespective of the precise nature of this conformational
change, it is sufficient to support E-AB signaling: the binding of aminoglycosides to
our electrode-bound aptamer causes a large, readily measurable decrease in Faradaic
current from the attached redox tag (Fig. 2.3).
58
Figure 2.2 Circular dichroism spectra indicate that the anti-aminoglycoside aptamer undergoes a
substantial conformational change upon binding to tobramycin. Specifically, a positive band at 260nm
that is often attributed to the formation of A-RNA structure22 increases substantially upon drug
binding.
The aminoglycosides comprise a large family of closely related antibiotics.
For example, gentamicin, tobramycin and kanamycin share similar, hydroxyl- and
primary amine-rich structures that form hydrogen bonds with compatible groups on
the nucleobases of their target RNA (Fig. 2.1). Indeed, tobramycin differs from
kanamycin A by the addition of one hydroxyl group, the replacement of a second
with a primary amine, and the absolute configuration of a single stereocenter. Thanks
59
to these structural similarities, the aptamer that we have employed binds all three of
these aminoglycosides with similar affinities 19,20. Consistent with this, we find that
our sensor responds robustly to all three of the aforementioned antibiotics, achieving
signal suppressions of ~60% at saturating drug concentrations (Fig. 2.3). Of note,
while this cross reactivity could confound the measurement of samples taken from
patients who are on more than one member of this family of drugs, the
aminoglycosides, are relatively toxic and exhibit very similar spectra of coverage, and
thus the simultaneous administration of multiple aminoglycosides is medically
contraindicated.
Figure 2.3 The E-AB sensor robustly responds to aminoglycoside antibiotics via the loss in the
faradaic current observed at the -0.35V potential characteristic of the reduction of methylene blue.
(right) Saturation curves indicate that the biosensor has nearly equal affinity for kanamycin and
tobramycin and slightly greater affinity for gentamicin. The error bars on this and the following figures
reflect the standard error of triplicate measurements taken with independently fabricated and measured
sensors.
60
Our sensors are capable of monitoring aminoglycoside concentrations over the
therapeutically relevant23, 2-6 µM (4-10 µg/mL) range. Two of the three antibiotics
we have investigated, tobramycin and kanamycin, produce the hyperbolic saturation
curves expected for single-site binding and yield dissociation constants of 319 and
281 µM. The latter values are an order of magnitude higher than solution-phase
values reported (12 nM to 13.2 µM) for longer aptamers bearing the same core
sequence19,20 These previous reports, however, employed a pyrene-modified
tobramycin molecule –and not tobramycin itself- as a reporter this, and thus this
discrepancy is perhaps not unexpected. In contrast to tobramycin and kanamycin,
gentamicin produces a biphasic binding curve in which the biosensor current
decreases at low concentrations and then increases at higher concentrations. The
origins of this biphasic behavior are unclear, but it is possible that, at high
concentrations, gentamicin causes the hairpin to denature, liberating the redox tag to
collide rapidly with the sensor surface and thus producing the observed increase in
current at very high drug concentrations. Alternatively, at high concentrations the
binding of additional drug molecules may force the aptamer to undergo additional
conformational changes that bring that tag into proximity with the sensor surface.
Because RNA is relatively unstable against chemical and enzymatic
degradation, our first-generation, RNA-based sensor is far less stable than E-AB
sensors fabricated with more robust DNA probes. In tris buffer, for example, the
RNA-based sensor deteriorates significantly within a few days. The problem of RNA
stability is particularly acute when the sensor is deployed in blood serum (Fig. 2.4).
61
Indeed, even in RNase inhibitor-treated serum the sensor’s signal diminishes to
unacceptably low levels within an hour of immersion and the sensor cannot be
regenerated. These deficiencies contrast sharply with the attributes of previously
reported sensors comprised of DNA-probes, which are stable in buffered saline for
months at room temperature, and even withstand more than a week of storage in
blood serum under the same conditions24.
Figure 2.4 Sensors constructed with an RNA probe can endure hours of exposure in buffered saline,
but decompose rapidly in (untreated), room temperature newborn calf serum. Ultrafiltration of the
serum through a 3,000 Da molecular weight cutoff membrane, however, removes the nucleases
rendering it possible to measure aminoglycoside concentrations in realistic clinical samples.
62
The problem of serum nucleases digesting unprotected, RNA-based probes is
a general one, thus motivating our interest in protecting these delicate sensing
elements without altering their ability to bind their specific targets.
To this end we have explored several probes lacking the reactive 2’-hydroxyl
group of RNA. As a first approach, we used the published solution NMR structure of
the aptamer as a guide while replacing the all the aptamer’s 2’-hydroxyl groups with
2’-methoxy groups save for five positions that are closest to the aptamer’s drug-
binding site. We find, however, that while this modified aptamer retains the high
affinity of its parent RNA aptamer (Fig. 2.5), it fails to offer significant protection
against nucleases when deployed in blood serum. Likewise we explored aptamers in
which all of the 2’ positions were replaced with fluorines. This modification,
however, significantly degrades the aptamer’s affinity and coveys only modest
protection in serum.
63
Figure 2.5 A stability comparison. Sensors protected by four different strategies were immersed in
serum and interrogated every hour.
Moving forward, we decided to attempt an experiment in which we simply
removed all 26 of the 2’-hydroxyl groups from the RNA aptamer; that is, we
converted the RNA aptamer into the equivalent DNA sequence. While this is perhaps
naïve – converting an RNA sequence into the equivalent DNA sequence is clearly a
major alteration of the chemistry of the recognition probe -- several arguments
suggested that this approach was at least worth attempting. First, the RNA aptamer
adopts a simple hairpin structure that does not require the formation of complex,
presumably RNA-specific tertiary structure. Second, the aptamer’s drug binding
64
pocket is located in its major groove, far from the 2’ hydroxyls, which are uniformly
oriented away from the bound aminoglycoside. Indeed, the nearest 2’-hydroxyl,
located on base G14, is more than 4 Å from the nearest hydrogen bond acceptor on
the drug. Furthermore, the conversion of RNA aptamers to DNA is not without
precedent. Walsh et al. found that a DNA translation of a dopamine binding
aptamer25,26 retains the ability to bind this target. Finally, Szostak et al. have shown
that the DNA translation of a flavin-binding RNA aptamer binds flavin with just over
a tenfold reduction in affinity27 (albeit this aptamer forms a G-quadruplex that may be
more readily formed from DNA). Consistent with these simple arguments, surface
plasmon resonance experiments indicate that a DNA probe with the same sequence as
the original RNA aptamer readily binds a tobramycin-coated sensor surface, an
observation that further encouraged us to pursue this approach. In doing so we find
that sensors fabricated using this DNA probe respond to our target aminoglycosides,
albeit with affinities against tobramycin, kanamycin and gentamicin that are reduced
4, 2 and 3-fold respectively relative to those of the equivalent RNA aptamer (Fig.
2.6). In contrast, control sensors fabricated using an unrelated DNA stem-loop do not
exhibit any evidence of binding to the aminoglycoside. Unfortunately, however,
while these DNA-based sensors are significantly more stable than our first generation
RNA-based sensors, their reduced affinities impede their ability to detect
aminoglycosides at medically relevant concentrations.
65
Figure 2.6 Probes comprised of deoxyribose or 2’-OMethyl modified ribose also bind
aminoglycosides. All three of the aminoglycosides we have investigated, however, bind both RNA and
2’-OMethyl-RNA probes (left and right bars, respectively) with greater affinity than they bind the
equivalent DNA probe (central bars).
Due to our failure to identify modified probes that are of sufficiently high
affinity and yet remain stable under clinically relevant conditions, we have adopted
an alternative approach for the use of RNA-based sensors with clinically relevant
samples. Because the aminoglycosides are of low molecular weight (and generally do
not form complexes with high-molecular weight serum proteins), they readily pass
through molecular weight cutoff membranes. Ultrafiltration of serum through a 3,000
66
Da cutoff spin column thus effectively separates the antibiotic from serum nucleases.
When immersed in the clear ultrafiltrate obtained from newborn calf serum (a safe
and convenient proxy for human samples), sensors fabricated using the unmodified
RNA aptamer remain quite stable (Fig. 2.4). Moreover, under these conditions we
can readily quantify gentamicin over the therapeutic range both when spiked into
newborn calf serum and as present in commercial human serum calibration samples
(Fig. 2.7). Although this sample protocol is effective, however, it is slow; the need for
ultrafiltration adds more than 30 minutes onto an otherwise short workflow. For that
reason, we believe that the advantages of this RNA biosensor would best be realized
in an integrated microfluidic system that rapidly prepares the serum sample before
feeding it onto the E-AB sensor.
67
Figure 2.7 The sensor responds well to gentamicin that was present in spiked serum samples. Each
measurement was made four minutes after the sensor was immersed in the ultrafiltrate from samples
that were intended for calibrating the Innofluor system, a widely used clinical assay.
The reported E-AB sensor has many attributes that would make it an ideal
component within a point of care medical diagnostic device. It is reusable, reagentless
and compact. It is also rapid: when aminoglycosides are introduced to a sensor, the
resulting current change equilibrates within the 10 s dead time of a single
voltammetric scan. Similarly swift results have been observed with aptamer-based
devices meant to detect cocaine, adenosine triphosphate and theophylline6,15. Because
all of the sensor components are firmly adsorbed onto the electrode surface (Fig. 2.1),
the sensor is readily regenerable: a simple rinse with deionized water disrupts the
target-aptamer complex allowing for the sensor’s reuse. Finally, our sensors are
fabricated on sub-millimeter scale electrodes and driven by simple, desktop
electronics, further speaking to their compatibility with microfluidics or a handheld
device format.
Most recently, White et al. have used this sensor to measure the concentration
of kanamycin injected into live rats (Figure 2.8). A microscale sensor was fabricated
and implanted each animals' jugular vein. Square wave voltammetry measurements
were made every three seconds, and over the course of 50 minutes the animal was
then dosed with increasing concentrations of kanamycin. This proof-of-concept
shows that E-AB sensors are a viable tool for continuous in vivo applications.
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However, fouling of the sensor surface, and mechanical occlusion, remain a problem.
Experiments to ameliorate these problems are ongoing.
Figure 2.8 A series of measurements made with the E-AB aminoglycoside sensor in a live rat.
2.3 Conclusion
Here we have demonstrated a reusable RNA aptamer-based electrochemical
biosensor that supports the rapid (<10 s) measurement of aminoglycoside
concentrations over the therapeutically relevant, low-micromolar range.
69
Unfortunately, it is quite vulnerable to degradation by nucleases. Our first effort to
improve the sensor’s stability, which involved modification of selected nucleotides
with 2’-methoxy groups, did not increase the stability of the aptamer in newborn calf
serum and thus does not appear a promising solution to this problem. Likewise,
replacement of the RNA probe with an equivalent DNA sequence yields a sensor that
is stable in newborn calf serum, but that transformation significantly reduces the
affinity of our probe. And thus, while the substitution of deoxyribose for ribose may
prove useful in some applications, this obviously does not represent a general solution
to the problem of stabilizing RNA aptamers. The approach we finally adopted,
ultrafiltration, is an effective means of removing nucleases from serum samples. It
allows us to make reliable measurements in filtered serum calibrators and may prove
a general approach for the use of RNA probes for the detection of at least low
molecular weight targets. And while this pre-treatment slows the overall workflow of
the sensor, the process is complete in less than 30 minutes and requires only an
inexpensive, desktop centrifuge.
2.4 Experimental Methods
Probe Sequences
RNA: 5’-HS- GGGACUUGGUUUAGGUAAUGAGUCCC-NH-MethyleneBlue-3’
DNA: 5’-HS-GGGACTTGGTTTAGGTAATGAGTCCC-NH-MethyleneBlue-3’
Control DNA: 5’-HS-AGCCCATTTATCCGTTCCTCCTAGTGGTGGGC-NH-MethyleneBlue-3’
OMe1: 5’-HS-(G)(G)(G)(A)(C)(U)UGGU(U)(U)(A)(G)G(U)(A)(A)(U)(G)(A)(G)(U)(C)(C)(C)-NH-
MB-3’
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2’F-RNA: 5’-HS- GGGACUUGGUUUAGGUAAUGAGUCCC-NH-MethyleneBlue-3’
Tris Buffer: 20 mM Tris, 100 mM NaCl 5 mM MgCl2, pH 7.5
All of the probes were synthesized, modified, and purified by BioSearch
Technologies (Novato, CA) and used as received. An unmodified version of the RNA
probe was synthesized by Sigma Genosys (Saint Louis, MO) and purified by high
performance liquid chromatography. Tris-2-carboxyethyl-phosphine, TCEP, was
obtained from Invitrogen (Carlsbad, CA). Tobramycin, Gentamicin, 6-mercapto-1-
hexanol, and urea were obtained from Sigma Aldrich (Saint Louis, MO). Kanamycin
was obtained from Fischer Scientific (Waltham, MA). Newborn calf serum was
obtained from Sigma Aldrich (Saint Louis, MO). Millipore YH-3 spin columns, with
a 3,000 Da cutoff, were obtained from Fisher Scientific (Waltham, MA). Seradyn
calibrator kits containing gentamicin in human serum were obtained from Polymedco
(Chicago, IL). All reagents, unless otherwise mentioned, were used as received.
Multiplex sensor chips were obtained from Osmetech (Pasadena, CA), and modified
according to the following procedure.
Sensor Fabrication
All probe molecules were hydrated with Tris buffer to a final concentration of
200 µM and stored at -20º C until use. To reduce the disulfide bond on each probe
molecule, a 2 µl aliquot of the stock was combined with 8 µL of 10mM TCEP
solution, mixed gently, and stored at 4º C for one hour. Following that reaction, the
probe was diluted to 200 nM and a 200 µl droplet was placed over each set of
71
electrodes on a sensor chip, which was stored under vacuum with minimal handling
up until this point. The chips were allowed to incubate in the dark for one hour at
room temperature, and then rinsed with deionized water. The bottom side and edges
of each chip were dried with a folded kimwipe, and 200 µl of a 3 mM 6-mercapto-1-
hexanol solution was applied for another hour. After that passivation procedure, the
chips were immersed in deionized water and stored in the dark overnight.
Electrochemical Measurements
Sensor experiments were performed with a CHI 730C Electrochemical
Workstation (CH Instruments, Austin, TX), and a 32-channel multiplexer made by
the same manufacturer. Each square wave voltammogram was acquired at 60 Hz,
over a potential range of -0.1 to -0.5 V. In serum, that window was extended to -0.6
V. The sensor chips contained an integrated gold counter electrode and an Ag/AgCl
reference electrode. Measurements were conducted in 1mL of buffer (20 mM Tris,
100 mM NaCl 5 mM MgCl2, pH 7.5), newborn calf serum, or ultrafiltered newborn
calf serum. Prior to each experiment, the chips were immersed in 1 mL of Tris buffer
and subjected to square wave test measurements. All experimental measurements
were made no less than four minutes after the addition of the analyte. Upon
completion of an experiment, the sensors were rinsed and stored in deionized water
for later reuse. After storage but before re-use, re-used sensors were subjected to an
hour of test scans in order to re-equilibrate their baseline signals.
72
Human Serum Measurements and Ultrafiltration
Gentamicin calibrator kits obtained from Polymedco (polymedco.com)
contain six 1mL human serum samples, at concentrations ranging from 0 to 10 mg/L
with <0.1% sodium azide added as a preservative. Since they are of low molecular
weight (Tobramycin, 467.5 Da; Kanamycin B, 483.5 Da; Gentamicin C, 477.6 Da)
and generally do not adhere to serum proteins, the aminoglycosides easily passed
through a 3,000 Da molecular weight cutoff membrane. For each experiment, two
samples of a given concentration were pooled and added to a Millipore YM-3
Centriprep spin column, and then spun at 5,100 RPM for 30 minutes in a Fisher
Centrific 225 centrifuge (Waltham, MA). After spinning, the exterior of each
ultrafiltrate reservoir was carefully dried with a kimwipe before its contents were
transferred to a centrifuge tube for storage. The ultrafiltrate was a clear liquid, and
400 µL was used for each electrochemical measurement. The sensor was immersed in
those solutions and allowed to equilibrate for 4 minutes in advance of each recording.
Sensor Degradation Experiments
The surface of each chip was mounted onto the bottom of a 3ml polycarbonate
well. The well was filled with 1mL of buffer. Square wave measurements were made
on eight channels, and the channel with the flattest baseline was selected for repetitive
measurements. The plastic reservoir was drained, and refilled with buffer or serum.
Immediately upon addition of the fresh liquid, square wave voltammograms were
acquired every minute for 30 minutes.
73
Circular Dichroism
The spectrum of a 2 mM RNA aptamer solution was measured before and
after the addition of tobramycin. Background signals of buffer and buffer with 4mM
tobramycin were subtracted from the spectra of their corresponding RNA solutions to
remove any background circular dichroism caused by the drug itself 28. All
measurements were made from 220 nm to 330 nm on an Aviv 202 spectrometer, at
room temperature, in a 3 ml quartz cuvette. In this, and many other CD studies of
aptamers [data not shown] we have noticed that it is necessary to increase the analyte
concentration up well above the reported dissociation constant (here 13.2 µM) in
order to obtain a pronounced signal change. This is necessary because CD
measurements make use of aptamers at very high concentrations, and it is impossible
to saturate them without reaching comparably high concentrations of analyte.
Surface Plasmon Resonance
Tobramycin was coupled to a Biacore CM 5 chip using a Biacore EDC
coupling kit (Piscataway, NJ). Oligonucleotides were injected over the sample surface
with a flow rate of 40 µl per minute at a series of concentrations. Measurements were
conducted in 20 mM Tris buffer (100 mM NaCl, 5mM MgCl2, pH 7.5).
74
2.5 Acknowledgements
This work was supported by the NIH through Grant GM062958-01 and by the
Institute for Collaborative Biotechnologies through Grant DAAD19-03-D-0004 from
the U.S. This work performed under the auspices of the U.S. Department of Energy
by Lawrence Livermore National Laboratory under Contract DE-AC52-07NA27344.
We are grateful to Osmetech (now Genmark Diagnostics, genmarkdx.com) for
providing us with several batches of their electrode arrays.
2.6. References
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Strategy Using Pharmacogenetics. Clin Pharmacol Ther 84, 301-303 (2008).
2. Lu, C.M., James, S.H. & Lien, Y.H. Acute massive gentamicin intoxication in
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3. Black, R.E., Lau, W.K., Weinstein, R.J., Young, L.S. & Hewitt, W.L.
Ototoxicity of Amikacin. Antimicrob. Agents Chemother. 9, 956-961 (1976).
4. Stead, D.A. & Richards, R.M.E. Sensitive fluorimetric determination of
gentamicin sulfate in biological matrices using solid-phase extraction, pre-column
derivatization with 9-fluorenylmethyl chloroformate and reversed-phase high-
performance liquid chromatography. Journal of Chromatography B: Biomedical
Sciences and Applications 675, 295-302 (1996).
5. Begg, E.J., Barclay, M.L. & Kirkpatrick, C.M.J. The therapeutic monitoring
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6. Baker, B.R. et al. An Electronic, Aptamer-Based Small-Molecule Sensor for
the Rapid, Label-Free Detection of Cocaine in Adulterated Samples and Biological
Fluids. Journal of the American Chemical Society 128, 3138-3139 (2006).
7. Cash, K.J., Ricci, F. & Plaxco, K.W. An Electrochemical Sensor for the
Detection of Protein−Small Molecule Interactions Directly in Serum and Other
Complex Matrices. Journal of the American Chemical Society 131, 6955-6957
(2009).
8. Zuo, X. et al. A target-responsive electrochemical aptamer switch (TREAS)
for reagentless detection of nanomolar ATP. J. Am. Chem. Soc 129, 1042-1043
(2007).
9. Functional Nucleic Acids for Analytical Applications. (Springer New York:
New York, NY, 2009).at <http://www.springerlink.com/content/k4j41u2570764x51/>
10. Lubin, A.A., Lai, R.Y., Baker, B.R., Heeger, A.J. & Plaxco, K.W. Sequence-
Specific, Electronic Detection of Oligonucleotides in Blood, Soil, and Foodstuffs
with the Reagentless, Reusable E-DNA Sensor. Analytical Chemistry 78, 5671-5677
(2006).
11. Lai, R.Y., Plaxco, K.W. & Heeger, A.J. Aptamer-Based Electrochemical
Detection of Picomolar Platelet-Derived Growth Factor Directly in Blood Serum.
12. Xiao, Y., Piorek, B.D., Plaxco, K.W. & Heeger, A.J. A Reagentless Signal-On
Architecture for Electronic, Aptamer-Based Sensors via Target-Induced Strand
Displacement. Journal of the American Chemical Society 127, 17990-17991 (2005).
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13. Radi, A.-E. & O’Sullivan, C.K. Aptamer conformational switch as sensitive
electrochemical biosensor for potassium ion recognition. Chem. Commun. 3432
(2006).
14. Xiao, Y., Rowe, A.A. & Plaxco, K.W. Electrochemical Detection of Parts-
Per-Billion Lead via an Electrode-Bound DNAzyme Assembly. Journal of the
American Chemical Society 129, 262-263 (2007).
15. Ferapontova, E.E., Olsen, E.M. & Gothelf, K.V. An RNA Aptamer-Based
Electrochemical Biosensor for Detection of Theophylline in Serum. Journal of the
American Chemical Society 130, 4256-4258 (2008).
16. Ferapontova, E.E. & Gothelf, K.V. Effect of Serum on an RNA Aptamer-
Based Electrochemical Sensor for Theophylline. Langmuir 25, 4279-4283 (2009).
17. Ferapontova, E.E. & Gothelf, K.V. Optimization of the Electrochemical
RNAAptamer Based Biosensor for Theophylline by Using a Methylene Blue Redox
Label. Electroanalysis 21, 1261-1266 (2009).
19. Wang, Y., Killian, J., Hamasaki, K. & Rando, R.R. RNA Molecules That
Specifically and Stoichiometrically Bind Aminoglycoside Antibiotics with High
Affinities†. Biochemistry 35, 12338-12346 (1996).
20. Wang, Y. Specific binding of aminoglycoside antibiotics to RNA. Chemistry
& Biology 2, 281-290 (1995).
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21. Jiang, L. & Patel, D.J. Solution structure of the tobramycin-RNA aptamer
complex. Nat Struct Mol Biol 5, 769-774 (1998).
22. Clark, C.L., Cecil, P.K., Singh, D. & Gray, D.M. CD, absorption and
thermodynamic analysis of repeating dinucleotide DNA, RNA and hybrid duplexes
[d/r(AC)]12·[d/r(GT/U)]12 and the influence of phosphorothioate substitution.
Nucleic Acids Research 25, 4098 -4105 (1997).
23. Setia, U. & Gross, P.A. Administration of Tobramycin and Gentamicin by the
Intravenous Route Every 6 Hr in Patients with Normal Renal Function. Journal of
Infectious Diseases 134, S125-S129 (1976).
24. Swensen, J.S. et al. Continuous, Real-Time Monitoring of Cocaine in
Undiluted Blood Serum via a Microfluidic, Electrochemical Aptamer-Based Sensor.
Journal of the American Chemical Society 131, 4262-4266 (2009).
25. Walsh, R. & DeRosa, M.C. Retention of function in the DNA homolog of the
RNA dopamine aptamer. Biochemical and Biophysical Research Communications
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26. Mannironi, C., Di Nardo, A., Fruscoloni, P. & Tocchini-Valentini, G.P. In
Vitro Selection of Dopamine RNA Ligands†. Biochemistry 36, 9726-9734 (1997).
27. Lauhon, C.T. & Szostak, J.W. RNA aptamers that bind flavin and
nicotinamide redox cofactors. Journal of the American Chemical Society 117, 1246-
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28. Jezowska-Bojczuk, M., Karaczyn, A. & Bal, W. Copper(II) binding to
geneticin, a gentamycin analog. Journal of Inorganic Biochemistry 71, 129-134
(1998).
This chapter was adapted in part from:
Aaron A Rowe, Erin A Miller, Kevin W Plaxco, Reagentless Measurement of

Acid Aptamer-Based Biosensor, 2010, Analytical Chemistry, 82 (17), 7090–7095.
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__________________________________________________________________
3. Reusable Sensors that Detect DNA with a High Signal Gain
____________________________________________________________________
3.1 Motivation
Electrochemical DNA biosensors (E-DNA) are a promising alternative to
optical sensors for the specific detection of oligonucleotide sequences 1,2,3. These
devices are composed of a redox-reporter-modified DNA probe immobilized on a
gold electrode. Hybridization-linked changes in the flexibility (due to conformational
changes1,4,5 or due to an increase or decrease in the amount of double-stranded DNA6
of this probe produce a change in the rate of electron transfer from the redox reporter.
This, in turn is readily detectable as a change in Faradaic current when interrogated
using alternating current or square wave voltammetry7,8,, signaling the presence of the
target oligonucleotide. As reagentless, electrochemical devices E-DNA sensors are
readily integrated into microfluidic devices, are driven by inexpensive, hand-held
electronics, and are easily multiplexed 9,10,11. Moreover, because their signaling is
predicated on a binding-induced change in the physical properties of the probe DNA
– and not simply to adsorption of analytes to the sensor surface – E-DNA sensors are
not easily spoofed by the non-specific adsorption of interferants and are selective
enough to deploy directly in complex clinical and environmental samples, such as
blood or soil extracts12,13. E-DNA sensors thus appear well suited for point of care
medical diagnostics, as well as portable analysis systems for forensics and food
quality control12.
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Although the E-DNA platform features a number of positive attributes, many
of the diverse E-DNA probe architectures described to date operate in a signal-off
fashion, meaning that the measured current decreases as the concentration of analyte
DNA increases14. For example, first generation E-DNA sensors employ a stem-loop
architecture1,14 such that, when a complementary oligonucleotide is introduced,
hybridization draws the attached redox reporter away from the electrode, reducing the
efficiency with which it transfers electrons to the electrode surface. As more target is
introduced, the signal current continues to decrease. This signal-off mechanism
significantly limits the gain of the sensor: the maximum possible signal change (the
change in current between no added target and saturating target concentrations) can
never be more than a 100% decrease -- total signal suppression. Furthermore, sensor
degradation can cause false positive results. Signal-on sensors, in contrast, can exhibit
arbitrarily high gain; as the background current observed in the absence of target is
reduced, the gain of such a sensor, at least in theory, increases without limit 15 and
false positives cannot be caused by sensor degradation. Thus motivated, we and
others have explored a number of signal-on E-DNA architectures, including a DNA
psuedoknot5, a hybridization-based forked sensor13, a triblock structure16, an inverted
stem-loop17, and a traditional E-DNA sensor probed at new frequencies7. But each of
those devices has at least one drawback that would limit its implementation in
inexpensive medical diagnostics, including poor stability and lack of reusability 13,
limited gain4,5 and/or poor signal-to-noise7.
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Among the highest gain E-DNA sensors reported to date is a signal-on
architecture developed by Xiao et al. that employs a strand-displacement signaling
mechanism13. This architecture makes use of a double-stranded probe comprising a
capture oligonucleotide affixed to a gold electrode, and a longer MB-labeled
signaling DNA strand hybridized to that molecule. Hybridization of the target
oligonucleotide displaces the methylene-blue modified terminus of the signaling
strand, allowing the redox tag to approach the electrode where it can transfer
electrons. This leads to a strongly enhanced signaling current upon target binding:
the sensor exhibits nearly 800% signal gain at saturating target concentrations 13.
Despite these advantages, however, the Xiao architecture suffers from a potentially
important drawback: because its sensing probe is held together by hybridization, the
signaling strand is lost when the sensor is washed or stored for long durations. At the
time, covalent modifications that could join double-stranded DNA probes together,
and affix them onto gold electrodes were not yet readily available. Here we describe a
new linker supporting such attachment, and use it to fabricate a fully covalent probe
that, while similar to this earlier signal-on architecture, is, in contrast, both reusable
and far more stable than the original design.
Our objective was to develop a reusable, high gain, fully covalent E-DNA
sensor. To do so, we designed an asymmetric, double-stranded hairpin probe, the
shorter arm of which (the “signaling strand” is modified at its 5’-end with a
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methylene blue redox reporter (Fig. 3.1). The two arms of the hairpin are joined
together by a “T-linker,” a thymidine nucleotide modified at its C5 position with a
long alkane chain that terminates in a thiol (Fig. 3.1a). This thiol anchors the entire
hairpin-shaped assembly onto a gold electrode via the middle of its turn region.
When deposited onto bare gold with mercaptohexanol, which is used as a dilutent to
control the density with which the probes pack on the surface of the electrode and to
passivate any bare gold, the probe forms a self-assembled monolayer (SAM). In the
absence of a complementary target, the two strands of this SAM-bound probe form
rigid, double-helical DNA, which limits the extent to which the attached methylene
blue can approach the surface of the SAM and transfer electrons. The hybridization of
a target oligonucleotide to the longer “capture” strand displaces part of the signaling
strand, producing a much more flexible single-stranded element (Fig. 1b).
Figure 3.1 A) The structure of a T-linker that allows the DNA probe to be immobilized onto a gold
electrode via its midsection, rather than its ends. B) Here we describe a fully covalent, high-gain
EDNA architecture employing an asymmetric hairpin probe and an internal electrode attachment site.
83
The sensor operates by a strand displacement mechanism in which, when target DNA hybridizes with
the longer arm of a probe molecule, a shorter arm is liberated allowing an attached methylene blue
reporter the conformational freedom to collide with the electrode surface. This results in an increase in
the current through the sensor circuit. Vacant areas between the probe DNAs are back-filled with
mercaptohexanol, producing a stable, continuous self-assembled monolayer.
Target hybridization increases the efficiency with which the methylene blue
approaches the surface of the SAM and exchanges electrons with the electrode,
giving rise to a signal gain that can exceed 800% and sensitivity that can reach low
picomolar levels (Fig. 3.2).
84
Figure 3.2 The new sensor architecture is sensitive enough to detect low picomolar levels of its
target DNA oligonucleotide in less than an hour. The above data come from a single representative
electrode, interrogated by square wave voltammetry one hour after the addition of the target analyte.
The new sensing architecture achieves exceptionally high signal gain,
producing an 800% increase in signal in the presence of saturating target
oligonucleotide (200nM). Under these conditions, the sensor approaches saturation
within ten minutes (Fig. 3.3). Consistent with its fully covalent nature, the sensor is
also quite stable and can be regenerated and reused multiple times by rinsing with a
stream of dimethyl sulfoxide, a known DNA denaturant 18. This regeneration process
takes less than three minutes, and can be repeated at least five times without
observing significant (<1%) signal loss. It is unlikely that test strips or cartridges
containing these sensors would be reused to analyze blood, urine, or saliva samples
from more than one patient. But the reusability of these devices offers an enormous
advantage. It allows for replicate measurements, and controls can be run on the same
sensing element that measures the samples.
85
Figure 3.3. The sensor rapidly responds to its complementary target. In this case,
three separate sensors were immersed into a 15mL solution of magnesium-enriched
PBS and then a fixed amount of complementary DNA was added. Square wave
voltammograms were recorded every minute for ten minutes.
This new detection architecture is specific, and, at low target concentrations,
readily distinguishes between perfectly matched DNA analytes and analytes with
single nucleotide polymorphisms (Fig. 3.4). Specifically, single nucleotide
polymorphisms and three-base mismatches that appear in the double-stranded region
of the probe are easily identified because their thermodynamic incentive to hybridize
with the capture strand and displace the target strand is lower. Mismatches that occur
in the single-stranded region of the capture probe are also identifiable, although the
86
difference in signaling is less pronounced. As is always true for sensors based on a
single capture probe, mismatched DNA analytes are easily misinterpreted as being
fully complementary oligonucleotides at a lower concentration. For this reason, such
sensors are better employed in an array format 9,10, with related but slightly
mismatched capture probes serving as controls, when high levels of specificity are
required.
Figure 3.4 Mismatched target DNA causes an increase in the sensor signal, but that increase is not
as great as the changes caused by perfectly matched analytes. The sensor can easily distinguish
between mismatches that occur near the T-linker, and has less specificity at its distal 3’-end. Error
87
bars were calculated by dividing the standard deviation of three separate sensor currents by their
average value.
Given the extremely low limits of detection that are necessary to identify
pathogens in biological samples, sensors such as those described here invariably need
to be coupled to an amplification system. Doing so is straightforward, as this sensor
architecture performs well even when employed directly in PCR mix. In our future
work, we hope to integrate these new sensors with an inexpensive amplification
device.
3.3 Conclusion
By incorporating an internal thiol modifier, which can immobilize probes onto
gold electrodes via positions distant from their termini, we have succeeded in
developing a fully-covalent, high-gain E-DNA sensor that can detect DNA at
picomolar levels, identify single nucleotide polymorphisms and operate directly in
PCR mix. This architecture was rationally designed, and then its performance was
improved empirically. With further development, it may become a key component in
microfluidic devices for medical diagnostics, environmental management, and food
quality assurance.
88
Key Materials
All of the oligonucleotides were synthesized by Biosearch Technologies
(Novato, CA), where they were purified by HPLC and used as received. The forked
probe molecule employed in our sensors were stored dry at -20° C until the day of
their use. Supermix iQ PCR mix was obtained from Bio-Rad (Hercules, California)
and stored at -20° C until each experiment.
ForkedThiol17
5'MB-AATAAAACGCCGATCCA-3'-T(C12-SH)-5'-TGGATCGGCGTTTTATTCTTGTTCAGATATTCAA-3'
ForkedThiol15
5'MB-TAAAACGCCGATCCA-3'-T(C12-SH)-5'-TGGATCGGCGTTTTATTCTTGTTCAGATATTCAA-3'
Perfectly matched 34 nucleotide target DNA sequence

5’-TTGAATATCTGAACAAGAATAAAACGCCGATCCA-3’
Unlabeled nucleotides were prepared by Sigma Aldrich (St Louis, MO) and
isolated by reverse phase cartridge purification. They were dissolved in phosphate
buffered saline, pH 7.4, and their concentrations verified with a NanoDrop
spectrophotometer. Stock solutions were stored at 100 µM in their original packaging.
Sensor Preparation
Gold disk electrodes were polished using a slurry of 0.05 micron alumina in
water on a standard polishing cloth. They were then subjected to a series of
89
electrochemical cleaning steps as described previously2. While the electrochemical
cleaning procedure was running, the probe DNA was reduced with four equivalents
of 10 mM TCEP and stored in the dark at 4°C for at least one hour. The probe DNA
was diluted with phosphate buffered saline (PBS), to either 100 nM, 250nM, or 1 µM,
and then each electrode was rinsed with deionized water, dried, and immersed in 200
µL of the resulting solution for five minutes. Immediately following this, the
electrodes were rinsed, dried and stored overnight in an aqueous solution of 2 mM
mercaptohexanol. For further details we recommend several papers previously
published by our group2,19,20,21.
In order to fabricate well-behaved sensors, it is imperative to use DNA probes
freshly dissolved in buffer: gel electrophoresis suggests that aged probe molecules
aggregate into dimers, trimers, and higher order clusters that negatively impact sensor
performance. Specifically, sensors fabricated using significantly aged probe solutions,
such as probe DNA that was reconstituted and then stored in a freezer for two weeks
or more before the sensor preparation, exhibit little or no change in signal when target
is added.
Electrochemical Measurements
The reported electrochemical experiments were conducted using square wave
voltammetry on CH Instruments 630B, 650C, and 660D potentiostats (Austin, TX).
Each of those instruments was connected to a CH Instruments 684 multiplexer.
Unless otherwise stated, square wave voltammetry measurements were made at 60Hz
90
with an amplitude of 25 mV and a step width of 4 mV. All of the experiments were
conducted with a platinum wire or platinum disk counter electrode and an Ag/AgCl
reference in phosphate buffered saline, pH 7.4 with 200 µM MgCl2 at room
temperature. With our electrochemical instrumentation we observe optimal signal to
noise ratios at a square wave frequency of 60 Hz. The observed signal gain, however,
can be twice as great at higher frequencies and thus an instrument with improved high
frequency filters may benefit from making measurements in the vicinity of 200 to 600
Hz.
Sensor Tests with Synthetic Targets
A set of three electrodes was immersed in a shot glass with either 15 mL of
phosphate buffered saline or 2 mL of PCR mix. Before data acquisition, at least six
square wave test scans of 60 Hz were performed. To acquire data, a macro
experiment was set to record 60 Hz square wave measurements every minute for ten
minutes. After the first set of data was collected, the target DNA was added, and the
solution was gently mixed by pipetting.
Sensor Tests below 5 nM Analyte
Three gold disk electrodes were immersed in 750 mL of phosphate buffered
saline enriched with 0.2 mM MgCl2. Synthetic target DNA was added after the first
measurement, and on the hour every hour thereafter for three hours. The mixture was
stirred continuously with a magnetic bar throughout the three hour long measurement
91
period. For these experiments, the sensors had a particularly low surface coverage of
probe DNA molecules that is achieved by immersing the freshly prepared gold
surfaces in a 100 nM probe solution for only five minutes.
Safety Considerations
The primary hazards of this work were concentrated sulfuric acid and
dimethyl sulfoxide. All experiments with these reagents were performed in a fume
hood. Suitable protective gear should be worn when handling these substances.
This work was performed in part under the auspices of the U.S. Department of
Energy by Lawrence Livermore National Laboratory under Contract DE-AC52-
07NA27344. It was also funded by a grant, OPP1015402, from the Bill and Melinda
Gates Foundation through the Grand Challenges Explorations Initiative, and by the
NIH through grants GM062958-01 and 2R01EB002046.
3.6. References
1. Fan, C., Plaxco, K.W. & Heeger, A.J. Electrochemical interrogation of
conformational changes as a reagentless method for the sequence-specific detection
of DNA. Proceedings of the National Academy of Sciences of the United States of
America 100, 9134 -9137 (2003).
92
3. Xiao, Y., Lai, R.Y. & Plaxco, K.W. Preparation of electrode-immobilized,
redox-modified oligonucleotides for electrochemical DNA and aptamer-based
sensing. Nat. Protocols 2, 2875-2880 (2007).
3. Lubin, A.A. & Plaxco, K.W. Folding-Based Electrochemical Biosensors: The
Case for Responsive Nucleic Acid Architectures. Accounts of Chemical Research 43,
496-505 (2010).
4. Xiao, Y., Qu, X., Plaxco, K.W. & Heeger, A.J. Label-Free Electrochemical
Detection of DNA in Blood Serum via Target-Induced Resolution of an Electrode-
Bound DNA Pseudoknot. Journal of the American Chemical Society 129, 11896-
11897 (2007).
5. Cash, K.J., Heeger, A.J., Plaxco, K.W. & Xiao, Y. Optimization of a
Reusable, DNA Pseudoknot-Based Electrochemical Sensor for Sequence-Specific
DNA Detection in Blood Serum. Analytical Chemistry 81, 656-661 (2009).
6. Ricci, F., Lai, R.Y. & Plaxco, K.W. Linear, redox modified DNA probes as
electrochemical DNA sensors. Chem. Commun. (Camb.) 3768-3770 (2007).
7. White, R.J. & Plaxco, K.W. Exploiting Binding-Induced Changes in Probe
Flexibility for the Optimization of Electrochemical Biosensors. Analytical Chemistry
82, 73-76 (2010).
8. Ricci, F. & Kevin, P. E-DNA sensors for convenient, label-free
electrochemical detection of hybridization. Microchimica Acta 163, 147-236
93
9. Lai, R.Y., Lee, S.-ho, Soh, H.T., Plaxco, K.W. & Heeger, A.J. Differential
Labeling of Closely Spaced Biosensor Electrodes via Electrochemical Lithography.
Langmuir 22, 1932-1936 (2006).
10. Pavlovic, E. et al. Microfluidic Device Architecture for Electrochemical
Patterning and Detection of Multiple DNA Sequences. Langmuir 24, 1102-1107
(2008).
11. Ferguson, B.S. et al. Integrated Microfluidic Electrochemical DNA Sensor.
(2006).
13. Xiao, Y., Lubin, A.A., Baker, B.R., Plaxco, K.W. & Heeger, A.J. Single-step
electronic detection of femtomolar DNA by target-induced strand displacement in an
electrode-bound duplex. Proceedings of the National Academy of Sciences 103,
16677 -16680 (2006).
14. Fan, C. Electrochemical interrogation of conformational changes as a
reagentless method for the sequence-specific detection of DNA. Proceedings of the
National Academy of Sciences 100, 9134-9137 (2003).
15. Xiao, Y., Piorek, B.D., Plaxco, K.W. & Heeger, A.J. A Reagentless Signal-On
Architecture for Electronic, Aptamer-Based Sensors via Target-Induced Strand
Displacement. Journal of the American Chemical Society 127, 17990-17991 (2005).
94
16. Immoos, C.E., Lee, S.J. & Grinstaff, M.W. DNA-PEG-DNA Triblock
Macromolecules for Reagentless DNA Detection. Journal of the American Chemical
Society 126, 10814-10815 (2004).
17. Farjami, E., Clima, L., Gothelf, K. & Ferapontova, E.E. “Off−On”
Electrochemical Hairpin-DNA-Based Genosensor for Cancer Diagnostics. Analytical
Chemistry (2011)
18. Chakrabarti, R. & Schutt, C.E. The enhancement of PCR amplification by low
molecular-weight sulfones. Gene 274, 293-298 (2001).
19. Ricci, F., Lai, R.Y., Heeger, A.J., Plaxco, K.W. & Sumner, J.J. Effect of
Molecular Crowding on the Response of an Electrochemical DNA Sensor. Langmuir
23, 6827-6834 (2007).
20. Lubin, A.A., Vander Stoep Hunt, B., White, R.J. & Plaxco, K.W. Effects of
Probe Length, Probe Geometry, and Redox-Tag Placement on the Performance of the
Electrochemical E-DNA Sensor. Analytical Chemistry 81, 2150-2158 (2009).
21. White, R.J., Phares, N., Lubin, A.A., Xiao, Y. & Plaxco, K.W. Optimization
of Electrochemical Aptamer-Based Sensors via Optimization of Probe Packing
Density and Surface Chemistry. Langmuir 24, 10513-10518 (2008).
95
____________________________________________________________________
4. Rapid Monitoring of Aminoglycoside Antibiotics

____________________________________________________________________
4.1 Motivation
Potentiostats, the cornerstone of electrochemistry research, have seen little
adoption in resource-poor environments such as the developing world and teaching
laboratories. An important factor in this is cost, with academic labs and industrial
groups typically paying upwards of US $5,000 for a general-purpose, research quality
workstation. Indeed, even the least expensive commercially available laboratory
potentiostats (e.g., Dagan Chem-Clamp, DropSens µSTAT 200) sell for more than a
thousand dollars and achieve only limited functionality. An inexpensive instrument
that is versatile enough to generate standard waveforms could thus significantly
broaden the use of electrochemistry in resource-poor environments. Such a device
would enable, for example, quantitative measurements in food and drug quality
control1, analysis of trace metals2, environmental monitoring3, and allow construction
of simple biosensors4567 in the developing world.
While the quality, and capabilities, of research-grade potentiostats cannot be
rivaled by a do-it-yourself device, many of their features are unnecessary for
environmental or public health applications. Supporting this argument, simple
potentiostats such as home glucose meters can make reasonably precise
electrochemical measurements, and they cost less than US $100. The software and
hardware designs underlying these devices, however, are proprietary and thus are not
96
easily modified to support other applications. Given these arguments, it is perhaps not
surprising that a number of open-source potentiostats have been described in the
scientific literature over the last four decades 8,9,10,11. For example, in 1980 Bond and
Norris published a description of a waveform generator that employed inexpensive
integrated circuits12, followed by Brown who, in 1982, described a more sophisticated
computer-controlled instrument13. More recent open-source potentiostat
development, however, has focused largely on miniature potentiostats, often with
implantation in mind14,15,16,17,18,19. These devices, however, typically do not offer a
wide variety of waveforms, nor do they employ modern computer interfaces. Other
recently described open-source potentiostats are based on custom made
microelectronics, rendering them ill suited for use in resource limited-
applications20,21,22. Thus, despite this literature precedent, there remains an unmet
need for an inexpensive, easily built potentiostat supporting the commonly employed
electrochemical waveforms. Indeed, the desire for a low-cost, open-source, general-
use potentiostat is highlighted by a recent academic attempt to “re-purpose” off-the-
shelf glucose meters to support new analytical applications23.
In response we present here the CheapStat (Figure 4.1), an open-source
potentiostat easily constructed by anyone proficient at assembling circuits. We
believe this device may prove of value in undergraduate chemistry labs, the
developing world and other environments where resources are limited. In support of
this, we have demonstrated the device’s utility in applications including food and
drug testing, environmental monitoring, education, and biosensing.
97
Figure 4.1 The CheapStat, an inexpensive, “do-it-yourself” potentiostat that can be built for under
$80. The device supports cyclic, square wave, linear sweep and stripping voltammetry over the
potential range -990 to +990 mV and over frequencies from 1 to 1000 Hz. The device supports a range
of environmental, food and drug quality control, and educational applications. The reverse of the
circuit board supports a three-line LCD display and a joystick, which is used to select an experiment
protocol, change its parameters (frequency, starting voltage, end voltage, scan rate), and initiate the
experiment.
The heart of the CheapStat is a closed-loop analog control circuit, capable of
regulating electrode voltage with sub-millivolt precision as it measures electrode
98
current with nanoamp precision. This control circuit is driven by an Atmel XMEGA
microcontroller containing a Digital to Analog Converter and an Analog to Digital
Converter of sufficient precision to support the relevant voltage waveform generation
and current quantification. Coupled to this microcontroller a Universal Asynchronous
Receiver-Transmitter to Universal Serial Bus (USB) chip provides a convenient
interface between this microcontroller and a data analysis computer via a USB port.
An operational amplifier feedback system sets the voltage across the electrochemical
cell and supplies the current needed to drive the electrochemical reaction. We
employed Texas Instruments TLC2262CP operational amplifiers (Mouser
Electronics) in the CheapStat because they are low power and require little input
current. Specifically, these amplifiers have an input bias current of only 1pA, which
permits reliable sensing of low nanoamp currents.
Inherent in the specific device choices described above is the overarching
design theme of the CheapStat: ease of fabrication, modification and use. For
example, using the above-described components and an inexpensive, custom-built
(and easily ordered) PC board we have hand fabricated a number of CheapStats
without the use of a reflow oven, wave soldering, or other sophisticated circuit
assembly tools. The CheapStat firmware is likewise easily updated with the aid of a
simple microcontroller programming kit. Finally, the designs of the CheapStat are
available under a Creative Commons license, and the attached documentation is
detailed enough to support efforts to build the CheapStat or expand upon its design.
99
As currently implemented, the CheapStat supports square wave, linear sweep,
stripping, and cyclic voltammetry over potentials from -990mV to 990mV,
frequencies from 1 to 1000Hz, and currents from ~100 nA to 50 µA. These operation
ranges can be expanded with simple hardware adjustments. In the event that a given
experiment produces currents greater than 50 µA, for example, a simple resistor can
be added in series with the working electrode to bring the current back into the useful
range of the device. The CheapStat is easy to use: it includes a three-line LCD display
and a joystick by which the operator selects the specific protocol, defines its
parameters (frequency, starting voltage, end voltage, scan rate), and initiates the
experiment. Copying data from the CheapStat onto a computer is similarly easy,
requiring only a single mouse click. Finally, the CheapStat is hand-held, weighs only
115 grams, is powered via its USB port, and can be controlled by a simple laptop or
netbook computer, rendering the device portable and field ready.
100
Figure 4.2 The CheapStat supports cyclic voltammetry. Shown is the CV-based measurement of
ascorbic acid (Vitamin C) in orange juice, using the method of standard additions. (Left) To do so, 0,
5, 10, and 15 mL of an ascorbic acid standard solution (at 0, 0.1, 0.2, and 0.3M in orange juice ) were
added to a 25 mL sample of name-brand orange juice and interrogated via cyclic voltammetry
performed using an inexpensive pencil “lead” as the working electrode. (Right) A linear extrapolation
of the oxidation current observed at 600 mV was used to determine the initial ascorbate concentration,
which at 1.95 ± 0.11mM is quite close to the “60 mg per serving” (2 mM) indicated on the
manufacturer website.
To demonstrate the utility of the CheapStat we have conducted a set of simple
experiments spanning a range of representative applications. These include the use of
cyclic voltammetry and an inexpensive, pencil “lead” working electrode to measure
vitamin C (ascorbic acid) in orange juice (Figure 4.2), and linear sweep voltammetry
to determine the acetaminophen (paracetamol) content of an over-the-counter
painkiller (Figure 4.3) – experiments typical of those found in undergraduate
laboratory courses. To illustrate the ability of the CheapStat to support more complex
analytical applications, we have also demonstrated the use of square wave
voltammetry to measure the concentration of a specific DNA sequence directly in
PCR mix using a simple electrochemical DNA (E-DNA) biosensor (Fig 4.4), and
anodic stripping square wave voltammetry to analyze the arsenic content in several
spiked lake water solutions (Fig. 4.5).
101
Figure 4.3 Analysis of the acetaminophen content of an over-the-counter painkiller using linear sweep
voltammetry and a gold wire working electrode. An acetaminophen tablet was crushed and dissolved
in sulfuric acid. Standards were added to three solutions, and one was left untouched. (Left) Linear
sweep voltammetry was used to measure the acetaminophen concentration by the method of standard
additions. (Right) A linear extrapolation of the oxidation current observed at 850 mV was used to
determine the initial acetaminophen concentration in a 250 mL solution containing one crushed tablet,
which at 12 mM is close to the 500 mg per tablet (472 ± 63 mg) reported by the manufacturer.
The CheapStat may prove of particular value as an educational tool. In each of
the above experiments, for example, we obtained results of sufficient quality and
reproducibility for facile interpretation by people who have little experience with
chemical benchwork. Moreover, from an educational standpoint, the difficulty of
using the CheapStat is quite low, rendering it quite suitable for college freshmen, or
even high school students. With the increasingly significant role that electrochemistry
plays in alternative energy and analytical technologies, the importance of easy access
to electrochemistry continues to grow. Electrochemistry likewise provides a
102
powerful vehicle for teaching students about a broad range of topics within chemistry,
including thermodynamics, redox reactions, reaction kinetics and titrations. Given
these observations, we believe the availability of an inexpensive potentiostat such as
that described here could have a positive impact on chemical education.
Figure 4.4 The electrochemical detection of a specific DNA sequence directly in PCR mix using an E-
DNA sensor (Left) interrogated via square wave voltammetry. Scanning the E-DNA biosensor at
100Hz before and after the addition of complementary target (20, 60, and 200nM in PCR mix) clearly
indicates the presence of the target DNA: over the course of an hour, hybridization with the analyte (at
200 nM) caused a 61% decrease in the peak current.
Beyond educational use, the CheapStat may also prove useful for analytical
applications in the developing world. As we have shown, for example, the CheapStat
supports the determination of arsenic in lake water 2, an all too common
environmental contaminant across broad areas of South Asia, where groundwater
103
contamination is a pressing problem 24,25,26. Indeed, while the development of low-
cost approaches to remove arsenic from drinking water are well established,
quantitative assays to determine the effectiveness of such treatment are cumbersome,
requiring bulky instruments that cannot be brought into the field 27. The CheapStat
can analyze arsenic in minutes at concentrations well below the 10ppb limit set by the
US EPA and World Health Organization.
Figure 4.5 Analysis of the arsenic content in three acidified lake water solutions. Samples were
collected from Lake Cachuma (Santa Barbara, CA) and mixed in an 11:1 ratio with concentrated HCl.
An arsenic standard was spiked into two of the samples (top 20ppm, middle 5ppm), and one was left as
a blank (bottom). The dissolved arsenate in each solution was reduced at -0.5V and then analyzed by
square wave voltammetry at 40Hz.
104
4.3 Conclusion
The CheapStat represents one example of a growing trend towards low-cost,
easily fabricated analytical devices. Recent years have seen, for example, the
development of inexpensive microfluidic devices made from polystyrene
sheets27,28,29,30, paper23,31,32,33,34,35 and even gelatin36. Agrawal and Ugaz37,38,39 have
even developed an extremely inexpensive and portable PCR machine, which makes
use of a ring shaped reaction vessel and three heating zones set at fixed temperatures,
rather than an expensive Peltier chip. Finally, inexpensive surface enhanced Raman
spectroscopy substrates40,41 centrifugation equipment42, and spectrometers43,44 have
all been reported in the recent literature. Moving forward, it is easy to imagine that
technologies such as these could be integrated with the CheapStat to increase their
utility in advanced analytical applications, including medical diagnostics.
All components for the CheapStat were obtained from Mouser Electronics
(Mansfield, TX), except for the printed circuit boards, which were custom ordered
from PCBex (Houston, TX). The schematics for this device, and instructions for
building it, are available on our website and released under a Creative Commons
105
license. The necessary software (runtime and source code), and other updates, can be
found on our website (http://www.chem.ucsb.edu/~kwp/cheapstat/).
Measurements of Ascorbic Acid in Orange Juice
Because ascorbic acid is redox active its level in orange juice can be measured
via cyclic voltammetry (Van Benschoten et al. 1983) using the method of standard
additions. Doing so represents a safe, simple experiment suitable for a general
chemistry or even high school laboratory course.
In this experiment, four orange juice samples were prepared, one of which
was unmodified and the remaining three of which were modified via the addition of
exogenous ascorbic acid at 0.1, 0.2 or 0.3M. To increase their conductivity KCl was
added to each of these to 1M. A graphite pencil “lead” taken from a mechanical
pencil was used as the working electrode. A standard Ag/AgCl reference and Pt
counterelectrode were used. Measurements were made in 5mL of the various orange
juice samples (Minute Maid Original). Cyclic voltammetry measurements were taken
from 200 to 900mV, and the current at 550mV was used for quantification of the
orange juice.
Although it is the most abundant redox active species in orange juice,
ascorbate is not the only redox active compound present. Specifically, other
substances interfere with these measurements at potentials above 600mV and thus
ascorbate should not be determined using readings collected above 600mV.
106
To determine the initial ascorbate, we make a linear plot of current at 550 mV
versus added ascorbate (Fig. 4.2). Extrapolation of this line to -0.39V indicates that
our orange juice serving contained 60mg of ascorbic acid (1.95 ± 0.11 mM), a very
convincing figure given the amount listed on the manufacturer’s website.
Analysis of Acetaminophen Content in over-the-counter pain medication using
linear sweep voltammetry.
Acetaminophen is a redox active1, anti-inflammatory medication that can be
quantified by linear sweep voltammetry. Here we analyzed the acetaminophen
content of an over-the-counter pain reliever using the method of standard additions.
In this experiment we dissolved a commercial pain reliever (stated to contain
500 mg acetaminophen) in 250 mL of 2M sulfuric acid to a final theoretical
concentration of 13.33 mM. Splitting this into four samples we adulterated three with
increasing amounts of exogenous, pure acetaminophen at 0.002, 0.005 and 0.01M. As
our working electrode we employed a Teflon-coated gold wire of diameter 0.08 mm,
a 1 cm length of which was heated to exposed the bare gold surface. A standard
Ag/AgCl electrode and a length of platinum wire were employed as the reference and
counterelectrode respectively. We conducted linear sweep voltammetry on these
samples over the potential range from 500 to 1000mV at a rate of 10mV/sec.
107
To calculate the acetaminophen concentration in our unadulterated sample we
linearly extrapolate the current observed at 850 mV (Fig. 4.3). Extrapolation and
conversion to total quantity shows that each tablet contained 472 ± 63 mg of
acetaminophen, which is in close agreement with the amount of acetaminophen
reported on the label of the commercial product we employed.
Measurement of Arsenic using Anodic Stripping Square Wave Voltammetry
In some rural parts of India and Bangladesh, arsenic in drinking water is a
major health concern. We have used the CheapStat to measure arsenic45 levels in lake
water. In all areas of the world, the recommended maximum level of arsenic in
drinking water is 10 ppb.
Samples of lake water were collected from Lake Cachuma (Santa Barbara,
CA) and stored in Nalgene polyethylene bottles. The lake water was acidified by
mixing it with concentrated hydrochloric acid in an 11 to 1 ratio.
Gold disk electrodes (CH Instruments, Austin, TX) were polished in a slurry
of 0.5 micron alumina on a polishing cloth and then rinsed thoroughly with deionized
water.
A sealed ampoule of arsenic oxide (Fluka Product #38150, via Sigma Aldrich,
St Louis, Mo) was diluted 1000 fold with acidified lake water. Aliquots of the diluted
standard were added to 5mL samples of acidified lake water. The concentration of
added arsenic in the samples was 5ppb and 20ppb.
108
In each lake water sample, a gold disk, platinum counter, and silver/silver
chloride reference electrode were immersed. The current at the working electrode was
held at -0.500 mV for 120 seconds while the solution was mixed by pipetting. And
then a square wave measurement at 40 Hz was run from -270 mV to 600 mV.
The CheapStat was easily able to make measurements at spiked 5 and 20 ppb
arsenic concentrations. There was no detectable arsenic in the lake water.
Construction of a Simple E-DNA Biosensor and its interrogation using square
wave voltammetry
Electrochemical DNA (E-DNA) biosensors are inexpensive, reusable DNA
detectors46. They can make quantitative measurements in complex mixtures such as
blood, urine, saliva, and PCR reaction products. In this experiment, we have used an
E-DNA sensor to detect a synthetic DNA oligonucleotide with a sequence found
within the salmonella genome.
The sensing element in these devices is a synthetic 17-base oligonucleotide
commercially synthesized with a thiol on its 5’-terminus and a methylene blue
reporter on the opposite terminus. The thiol readily anchors the oligonucleotide to a
gold electrode (via very facile self assembled monolayer chemistry), leaving the other
end free to move (Fig. 4.4). When the methylene blue approaches the electrode, it
often transfers a pair of electrons. If a target molecule hybridizes with this system,
109
the efficiency with which the methylene blue collides with –and thus transfers
electrons to- the electrode surface decreases, decreasing the observed rate of electron
transfer47. As square wave voltammetry is very sensitive to changes in electron
transfer kinetics, it is ideally suited to monitor biosensors in this class 48,49.
Materials and Preparation:
The probe was synthesized by Biosearch Technologies (Novato, CA) and the target
by Sigma Genosys, a division of Sigma Aldrich (St Louis, MO).
LinearSensor : 5′-HS−(CH2)6−TGGATCGGCGTTTTATT−(CH2)7−NH−MB-3′
TargetDNA: 5’-TTGAATATCTGAACAAGAATAAAACGCCGATCCA-3’
To fabricate the sensor we polished gold disk electrodes using 0.05 micron
alumina before cleaning them electrochemically in 0.5M H2SO4 and 0.5 M H2SO4
with 0.01M KCl with a series of cyclic voltammetry scans. Each electrode was then
rinsed in deionized water before immersion in a 200nM solution of the relevant DNA
probe in pH 7.4 phosphate buffered saline. After an hour the electrodes were rinsed
with deionized water and then stored in 2 mM mercaptohexanol (to complete the
formation of the self-assembled monolayer) overnight. Before use, the sensors were
rinsed in deionized water.
Testing:
110
The sensors were immersed in 5mL of phosphate buffered saline. An initial
square wave voltammetry scan was made from 0 to -0.5 V at 100 Hz. We then
challenged the sensor by adding 100 µL of a 10 µM stock of a 34-base analyte DNA.
At 30 min and 60 min after mixing we recorded new square wave scans, observing
the expected 60% decrease in signal after 1 hour.
Our project was funded by a grant (OPP1015402) from the Bill and Melinda
Gates Foundation through the Grand Challenges Explorations Initiative. This work
was performed in part under the auspices of the U.S. Department of Energy by
Lawrence Livermore National Laboratory under Contract DE-AC52-07NA27344.
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117

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UNIVERSITY OF CALIFORNIA

Electrochemical Biosensors for Point-of-Care Medical Diagnostics

A Dissertation submitted in partial satisfaction of the

requirements for the degree Doctor of Philosophy

in Chemistry and Biochemistry

Aaron Alexander Rowe

Kevin Plaxco, Chair

Professor Alison Butler

Professor Deborah Fygenson

Professor Stanley Parsons

All rights reserved

INFORMATION TO ALL USERS

Aaron Alexander Rowe

me a great deal about electrochemistry. Hershel Watkins and Alexis Vallée-Bélisle

in countless ways. My officemate at Lawrence Livermore, Richard Henrikson

greatly broadened my knowledge of molecular diagnostics and techniques for

molecular scale is a true inspiration.

My mentor, Professor Kevin Plaxco, has shown me how to structure successful

writing, which will undoubtedly be useful throughout my career. I am particularly

my success in a doctoral program. He taught me how to conduct experiments

necessary for all competitive scientists.

tremendous help, my projects would have progressed very slowly. On the

My parents have been tremendously supportive of my education. I am

chemistry sets at an early age. My girlfriend, Iris Ahronowitz, has kept my

Bachelor of Science in Materials Science and Engineering, University of Illinois,

Master of Science in Chemistry, California State University, Northridge, December

Doctor of Philosophy in Chemistry and Biochemistry, University of California,

Spring 2000: Research Assistant, Carolyn Dry Research Group, University of

Fall 2001: Teaching Assistant, Department of Materials Science, University of

Gagik G. Melikyan, Ferdinand Villena, Arthur Florut, Steve Sepanian, Hagop

Yi Xiao, Aaron A. Rowe, and Kevin W. Plaxco, Electrochemical Detection of

Aaron A Rowe, Erin A Miller, Kevin W Plaxco, Reagentless Measurement of

Ryan J. White, Aaron A. Rowe, Kevin W Plaxco, Re-engineering aptamers to

Gagik G. Melikyan, Ryan Spencer, Aaron Rowe 1,3-Steric Induction in

Aaron A. Rowe, Andrew J. Bonham, Ryan J. White, Kevin W. Plaxco Fabrication

Aaron A Rowe, Andrew J Bonham, Ryan J White, Michael P Zimmer, Ramsin J

Teaching Assistant of the Year, Department of Chemistry, California State

Fellow of the Center for Nanoscience in Society, 2006

Outstanding Teaching Assistant, Department of Chemistry and Biochemistry,

Student Employee Graduate Research Fellowship, Lawrence Livermore National

Lawrence Scholar, Lawrence Livermore National Laboratory, 2008

Electrochemical Biosensors for Point-of-Care Medical Diagnostics

Aaron Alexander Rowe

Electrochemical biosensors could become the key components of many point-of-

potentiostat that can be used in conjunction with the aforementioned biosensors.

I. Point-of-Care Medical Diagnostics .......................................................................... 1

1.1 Background .......................................................................................... 1

1.2 Areas of Need for Point-of-Care Testing ........................................... 3

1.3 Features Required to Support Point-of-Care Applications .................. 7

1.4 Laboratory Based Diagnostic Methods .............................................. 10

1.5 Emerging Point-of-Care Technologies ............................................. 13

1.6 Reagentless, Reusable Electrochemical Biosensors ....................... 32

1.8 References ....................................................................................... 43

II. Rapid Analysis of Antibiotics ............................................................................... 56

2.1 Motivation ......................................................................................... 56

2.2 Results and Discussion ....................................................................... 58

2.3 Conclusions ........................................................................................ 69

2.4 Experimental Methods ...................................................................... 70

2.5 Acknowledgements .......................................................................... 75