2D Materials For Biomedical Applications
2D Materials For Biomedical Applications
2D Materials For Biomedical Applications
2D Materials
for Biomedical Applications
Edited by
Minas M. Stylianakis and Athanasios Skouras
mdpi.com/journal/molecules
2D Materials for Biomedical
Applications
2D Materials for Biomedical
Applications
Editors
Minas M. Stylianakis
Athanasios Skouras
Editorial Office
MDPI
St. Alban-Anlage 66
4052 Basel, Switzerland
This is a reprint of articles from the Special Issue published online in the open access journal
Molecules (ISSN 1420-3049) (available at: www.mdpi.com/journal/molecules/special issues/2D
materials biomedical).
For citation purposes, cite each article independently as indicated on the article page online and as
indicated below:
Lastname, A.A.; Lastname, B.B. Article Title. Journal Name Year, Volume Number, Page Range.
© 2023 by the authors. Articles in this book are Open Access and distributed under the Creative
Commons Attribution (CC BY) license. The book as a whole is distributed by MDPI under the terms
and conditions of the Creative Commons Attribution-NonCommercial-NoDerivs (CC BY-NC-ND)
license.
Contents
Preface . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . ix
Pengyu Gong, Yi Zhou, Hui Li, Jie Zhang, Yuying Wu and Peiru Zheng et al.
Theoretical Study on the Aggregation and Adsorption Behaviors of Anticancer Drug Molecules
on Graphene/Graphene Oxide Surface
Reprinted from: Molecules 2022, 27, 6742, doi:10.3390/molecules27196742 . . . . . . . . . . . . . . 10
Mohammad Oves, Mohammad Omaish Ansari, Mohammad Shahnawaze Ansari and Adnan
Memić
Graphene@Curcumin-Copper Paintable Coatings for the Prevention of Nosocomial Microbial
Infection
Reprinted from: Molecules 2023, 28, 2814, doi:10.3390/molecules28062814 . . . . . . . . . . . . . . 22
Nayara Balaba, Silvia Jaerger, Dienifer F. L. Horsth, Julia de O. Primo, Jamille de S. Correa
and Carla Bittencourt et al.
Polysaccharides as Green Fuels for the Synthesis of MgO: Characterization and Evaluation of
Antimicrobial Activities
Reprinted from: Molecules 2022, 28, 142, doi:10.3390/molecules28010142 . . . . . . . . . . . . . . 36
Kanae Suzuki, Misato Iwatsu, Takayuki Mokudai, Maiko Furuya, Kotone Yokota and
Hiroyasu Kanetaka et al.
Visible-Light-Enhanced Antibacterial Activity of Silver and Copper Co-Doped Titania Formed
on Titanium via Chemical and Thermal Treatments
Reprinted from: Molecules 2023, 28, 650, doi:10.3390/molecules28020650 . . . . . . . . . . . . . . 50
Michele Massa, Mirko Rivara, Thelma A. Pertinhez, Carlotta Compari, Gaetano Donofrio
and Luigi Cristofolini et al.
Chemico-Physical Properties of Some 1,1′ -Bis-alkyl-2,2′ -hexane-1,6-diyl-bispyridinium
Chlorides Hydrogenated and Partially Fluorinated for Gene Delivery
Reprinted from: Molecules 2023, 28, 3585, doi:10.3390/molecules28083585 . . . . . . . . . . . . . . 78
Sergey Lazarev, Sofya Uzhviyuk, Mikhail Rayev, Valeria Timganova, Maria Bochkova and
Olga Khaziakhmatova et al.
Interaction of Graphene Oxide Nanoparticles with Human Mesenchymal Stem Cells Visualized
in the Cell-IQ System
Reprinted from: Molecules 2023, 28, 4148, doi:10.3390/molecules28104148 . . . . . . . . . . . . . . 95
Qi Zhang, Fengjiao Xu, Pei Lu, Di Zhu, Lihui Yuwen and Lianhui Wang
Efficient Preparation of Small-Sized Transition Metal Dichalcogenide Nanosheets by
Polymer-Assisted Ball Milling
Reprinted from: Molecules 2022, 27, 7810, doi:10.3390/molecules27227810 . . . . . . . . . . . . . . 110
v
Faten Bashar Kamal Eddin and Yap Wing Fen
The Principle of Nanomaterials Based Surface Plasmon Resonance Biosensors and Its
Potential for Dopamine Detection
Reprinted from: Molecules 2020, 25, 2769, doi:10.3390/molecules25122769 . . . . . . . . . . . . . . 122
vi
About the Editors
Minas M. Stylianakis
Minas M. Stylianakis is an assistant professor at the Department of Nursing of the Hellenic
Mediterranean University and a member of the Hybrid Nanostructures group of the Institute of
Electronic Structure and Laser (IESL) of FORTH. He received his Ph.D. degree in Chemistry from
the University of Crete in 2015. His expertise lies in the synthesis, solution processing, and
characterization of universal carbon- and graphene-based materials, 2D materials, small molecules,
and polymers. His research interests include the development of biomedical and environmental
applications, focusing on drug delivery systems and implants design/development, self-healing, and
antimicrobial coatings, water treatment technologies, and energy production.
Athanasios Skouras
Athanasios Skouras is an external collaborator of the Department of Nursing of the Hellenic
Mediterranean University. He received his Ph.D. in Pharmacy from the University of Patras in 2017.
His expertise lies in Pharmaceutical Technology and more specifically in drug delivery systems. His
research interests mainly include the formulation of novel theranostic systems utilizing lipids and the
synthesis and applications of biodegradable nanoparticles.
vii
Preface
Two-dimensional structured materials and metallic NPs exhibit extraordinarily distinctive
optical, thermal, electronic, and mechanical properties. In addition, thanks to their biocompatibility,
low-toxicity, and antimicrobial property, they have been extensively applied in several biomedical
applications such as biosensing, cell labeling, tissue engineering, drug and gene delivery systems,
and mesenchymal stem cells differentiation. Motivated by these achievements, we demonstrate the
present Special Issue, which focuses on the use of 2D materials and other nanoparticles in several
biomedical applications. It contains ten original research contributions (two theoretical studies, seven
research articles and one review article) providing a broad coverage of recent research progress and
insights in the field of nanotechnology induced biomedicine, including studies on the development of
bionanocomposites, biosensing, drug and gene delivery systems, as well as on the evaluation of the
antimicrobial property of graphene derivatives and metallic nanoparticles. This themed collection is
addressed to a broad research audience of chemists, biologists, pharmacologists, materials scientists,
and researchers in the field of nanomedicine. We really appreciate the great efforts of all involved
authors to prepare and submit their excellent manuscripts.
ix
molecules
Article
Nanoindentation of Graphene/Phospholipid Nanocomposite:
A Molecular Dynamics Study
Vladislav V. Shunaev 1 and Olga E. Glukhova 1,2, *
Abstract: Graphene and phospholipids are widely used in biosensing and drug delivery. This paper
studies the mechanical and electronic properties of a composite based on two graphene flakes
and dipalmitoylphosphatidylcholine (DPPC) phospholipid molecules located between them via
combination of various mathematical modeling methods. Molecular dynamics simulation showed
that an adhesion between bilayer graphene and DPCC increases during nanoindentation of the
composite by a carbon nanotube (CNT). Herewith, the DPPC molecule located under a nanotip
takes the form of graphene and is not destroyed. By the Mulliken procedure, it was shown that
the phospholipid molecules act as a “buffer” of charge between two graphene sheets and CNT.
The highest values of electron transfer in the graphene/DPPC system were observed at the lower
indentation point, when the deflection reached its maximum value.
1
Molecules 2021, 26, 346
viously, the authors found the configurations of such composites that provide optimal
current–voltage characteristics and electron transfer values [27]. The aim of this work is
to simulate nanoindentation of the bilayer graphene/DPPC composite with subsequent
evaluation of the mechanical and electronic properties during deflection.
2. Results
2.1. Atomistic Model
At the initial stage, two DPPC molecules containing 1 phosphorus atom, 1 nitrogen
atom, 8 oxygen atoms, 40 carbon atoms, and 80 hydrogen atoms were placed between two
graphene monolayer flakes with dimensions Lx × Ly = 25.5 Å × 35.51 Å. This composite
structure acted as the supercell with translation vectors Lx , Ly (Figure 1a). This supercell
contained 878 atoms, 618 of which belonged to graphene, and 260 to the DPPC molecules.
The geometric center of each DPPC molecule was located at a distance of 6.23 Å from
each graphene sheet. The atomic structure of the designed
− supercell
− and the values of
the translation vectors were optimized by the self-consistent charge density functional
tight-binding (SCC DFTB) 2 method.
Figure 1. Atomic structure of the graphene/dipalmitoylphosphatidylcholine (DPPC) composite: (a) before the optimization;
(b) type I after the optimization; and (c) type II after the optimization. The supercell obtained by the self-consistent
charge density functional tight-binding (SCC DFTB) 2 method is highlighted by the red box. Graphene atoms are grey,
phosphorous—yellow, nitrogen—blue, oxygen—red, carbon in phospholipid—black, and hydrogen—blue.
The fragment of a graphene/DPPC composite film containing 7902 atoms was built
from nine optimized supercells. The fragment sizes were 90.46 Å × 80.56 Å, and the initial
structure is shown in Figure 1a. As the search for the ground state of such a polyatomic
fragment by the SCC DFTB 2 quantum mechanical method is practically impossible,
the AMBER empirical method was applied. It should be noted that the fragment is not
a supercell, but a finite structure. From the result of 20 numerical experiments of the
graphene/DPPC composite optimization, two types of object’s topology were identified.
In the type I topology, 12 DPPC molecules formed a disordered bundle (Figure 1b). If an
additional number of DPPC molecules are added to the composite, double and triple layers
of lipids can be formed in this structure [5]. In the type II topology, the phospholipids were
arranged in a shape vaguely resembling a spiral; part of one DPPC even left the pores
between the graphene sheets (Figure 1c). During optimization, the total energy of the
system decreased from 17.54, to 3.967 Mcal/mol and the adhesion energy between graphene
flakes and DPPC molecules dropped from −896.115 to −4124.72 kcal/mol. Because, in this
structure, two isolated DPPC molecules located in the center of the composite were clearly
distinguished, it was chosen as the object of nanotip indentation.
2.2. Nanoindentation
At the next stage, the carbon nanotube (CNT) (16,0) with a closed edge was placed at
a distance of 4.1 Å from the surface of the upper graphene sheet (Figure 2a). The length of
2
Molecules 2021, 26, 346
the CNT was 50.62 Å. To perform the process of the composite material indentation, the
nanotip approached the composite surface with a step of 0.5 Å. At each step, the process of
relaxation scanning was started, the values of local stresses on the atoms were determined,
and the adhesion energy between bilayer graphene and phospholipids was calculated.
Based on the analysis of the above-mentioned characteristics, the so-called “key points” of
indentation were identified (Table 1).
(a)
(b) (c)
Figure 2. Nanoindentation of the composite graphene/DPPC by the carbon nanotube (CNT) with a closed edge: (a) initial
(left) and last (right) point of indentation F; (b) the atomic structure of the central DPPC molecule at point F; and (c) the
dependence of the system’s total energy on the CNT shift. The solid line corresponds to the forward stroke (FS) and the
dotted line corresponds to the reverse. At the initial point, the edge of the CNT had the coordinate Y = 0, and the atoms of
−
the upper graphene layer had the coordinate Y = −4.1 Å.
Table 1. Key points of the nanoindentation with corresponding Y coordinates; the values of the
adhesion energy between bilayer graphene and dipalmitoylphosphatidylcholine (DPPC) molecules
and maximum local stresses (MLSs) in these points. The abbreviation FS corresponds to forward
stroke and RS corresponds to reverse stroke.
3
Molecules 2021, 26, 346
The dependence of the system’s total energy on the CNT shift is shown in Figure 2c.
A local minimum was observed at point A, indicating the most energetically favorable
location between the CNT and the composite owing to the van der Waals (vdW) interac-
tion. Further, the energy grew steadily until it reached point C. Between points C and
D, there was a slight drop in the total energy of the system. In this region, the atomic
structure of the DPPC molecule was rearranged and it started to take the form of graphene
(Figure 2b). Note that, during the forward stroke, the adhesion energy between graphene
and DPPC molecules reached its maximum value precisely at point D (Table 1). The posi-
tion of CNTs at the lower indentation point F is shown in Figure 2a on the right. As the
CNT left the trough of the composite (reverse stroke), the energy decreased greatly (from
F to H). At this interval, the CNT tip stopped to pressurize the composite and began
to relax. The point H corresponds to the moment of the strongest vdW interaction be-
tween objects during reverse stroke, so the local minimum of the total energy at this
point is observed. Then, the vdW interaction became weaker and, from a certain moment
(point I), the total energy practically did not change. Note that the trough created by
the nanotip in the composite remained even after the CNT returned to its initial posi-
tion. Herewith, the remaining 16 DPPC molecules did not leave the space between the
graphene sheets (Figure 2a). The value of the adhesion energy between graphene and
DPPC molecules at point I and further remained at the maximum for the entire time. Thus,
despite the fact that no chemical bonds between graphene and DPPC molecules were
formed during indentation, the bonds between graphene sheets and DPPC molecules were
significantly strengthened.
4
Molecules 2021, 26, 346
Figure 3. The map of the local stress (LS) in the central area of the graphene sheets at the key points of indentation: (a) point
A; (b) point B (upper layer); (c) point C (upper layer); (d) point D (upper layer); (e) point E (upper layer); (f) point F (upper
layer); (g) point G (lower layer); (h) point H (lower layer); and (i) the dependence of the maximum local stress (MLS)
in graphene sheet atoms on the indentation step (the solid line corresponds to the forward stroke and the dotted line
corresponds to the reverse).
5
Molecules 2021, 26, 346
(a) (b)
(c) (d)
Figure 4. The analysis of the electron transfer in the CNT/graphene/DPPC system during nanoindentation (a) The fragment
of the atomic structure graphene/DPPC with CNT at point F; (b) the distribution of Mulliken charges on atoms in the point
for the upper (left) and the lower (right) graphene sheets; (c) for CNT; and (d) for DPPC.
The charge distributions between the atoms at the initial moment of time, at the lower
point of the forward stroke F, and at the end of the reverse stroke I are shown in Table 2 At
the initial moment of time, the CNT was electrically neutral. In the composite, the DPPC
molecules acted as a donor and transferred the charge of 6.09 e to graphene sheets; the
largest part of the charge was lost by two P-atoms (1.61 and 1.65 e), which is consistent with
the results obtained earlier [26]. The charge between the graphene sheets was distributed
unevenly because of the orientation of the DPPC molecules after optimization—the phos-
phorus molecules that tend to give charge and act as donors were located closer to the
upper graphene sheet. During the indentation, the CNT gradually transferred the charge
to the composite; at the lower point, its value was 0.16 e (Figure 4c). It can be seen that
the phospholipid transferred even more charge to graphene sheets than in the initial state
(7.28 e) (Figure 4d). The charge transferred from DPPC was evenly distributed between the
graphene sheets (Figure 4b). This was caused by the fact that the DPPC molecules have
taken the form of graphene sheets, as shown in Section 2.2, and the P atoms were located
at the same distance from the graphene sheets. As the CNT is removed from the composite,
it recovered the transferred charge and eventually became almost electroneutral again.
Note that, during the reverse stroke, the main part of the charge came from the upper
sheet of graphene that was directly in contact with the CNT. Thus, the DPPC molecules
stopped to act as a “buffer” of charge between the two graphene sheets. The highest values
of electron transfer in the graphene/DPPC system were observed at the lower indentation
point, when the deflection reached its maximum value. Based on the conclusions of [26],
it can be concluded that the deflection strain would significantly affect the current–voltage
characteristics of the considered composite.
6
Molecules 2021, 26, 346
3. Methods
The search for the ground state of the graphene/DPPC composite film, as well as the
study of its atomic structure changes during deflection by a nanotip, was performed by
the AMBER empirical method [28] implemented in the Hyperchem software package [29].
Optimization was performed by the conjugate gradient Fletcher–Reeves method, and the
root mean square (RMS) gradient was 0.1 kcal/(A·mol).
The adhesion energy between the phospholipids and graphene sheets at various steps
was calculated by the following formula (Equation (1)):
where ETOT is the energy of the graphene/DPPC system at this point, and EDPPC , EUP ,
and ELOW are the energies of isolated DPPC, upper, and lower graphene sheets, respectively.
To estimate the strength of graphene sheets during deflection, the previously devel-
oped original method for calculating local stresses of the atomic grid was used [30]. Accord-
ing to this method, the stress σi on each atom is calculated by the formula σi = |wi − w0 |,
where w0 is the energy volume density of the graphene atom before indentation, and wi
is the energy volume density of the graphene atom under external influence. The energy
Ei
volume density of the atom was calculated by the formula wi = V , where Ei is the energy
i
3
of the atom calculated within the AMBER force field, and Vi = 4πr
3 is the volume of the
carbon atom (r = 1.7 Å).
The study of changes in the electronic structure during indentation and electronic
transfer between phospholipid molecules and graphene was performed by Mulliken
population analysis [31]. According to this method, the charge on an atom is calculated
as the difference between the atomic number Z A and GAPA —the sum of the gross orbital
product over all orbitals belonging to atom A: Z = Z A − GAPA . The charges were
calculated by the quantum mechanical self-consistent charge density functional tight-
binding (SCC DFTB) method [32] in the dftb+ software package [33] in the 3ob-3-1 basis.
4. Conclusions
The nanoindentation of the composite on the base of bilayer graphene and 16 DPPC
phospholipid molecules was simulated by the molecular dynamics method. It was noted
that, during indentation, the adhesion between graphene flakes and DPPC molecules
increased and reached a maximum at the end of the reverse stroke. The maximum values
of local stresses (in the region of 2.53 GPA) were observed on the upper graphene layer
at the lower indentation point. At this moment, the DPPC molecule located under the
nanotip took the form of curved graphene, while chemical bonds of the phospholipid
molecules were not destroyed. The pressure of the CNT tip led to the growth of adhesion
energy between graphene sheets and DPPC molecules. It is known that, getting into
blood vessels, drug carriers sense abnormally high shear stresses [34,35], so the discovered
effect of “phospholipid strengthening” by the graphene sheets’ coating can be used in
the field of drug delivery. It was found that the electron transfer between CNT and
graphene/DPPC composites increased during indentation and reached 0.16 e. At this
moment, the phospholipid molecule stopped to act as a “buffer” of charge between the
7
Molecules 2021, 26, 346
two graphene sheets. The observed phenomenon of electronic transfer between graphene
and phospholipid can be applied in biosensorics.
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9
molecules
Article
Theoretical Study on the Aggregation and Adsorption
Behaviors of Anticancer Drug Molecules on
Graphene/Graphene Oxide Surface
Pengyu Gong, Yi Zhou, Hui Li, Jie Zhang, Yuying Wu * , Peiru Zheng * and Yanyan Jiang *
Key Laboratory for Liquid-Solid Structural Evolution and Processing of Materials, Ministry of Education,
Shandong University, Jinan 250061, China
* Correspondence: [email protected] (Y.W.); [email protected] (P.Z.); [email protected] (Y.J.)
Abstract: Graphene and its derivatives are frequently used in cancer therapy, and there has been
widespread interest in improving the therapeutic efficiency of targeted drugs. In this paper, the
geometrical structure and electronic effects of anastrozole(Anas), camptothecin(CPT), gefitinib (Gefi),
and resveratrol (Res) on graphene and graphene oxide(GO) were investigated by density functional
theory (DFT) calculations and molecular dynamics (MD) simulation. Meanwhile, we explored
and compared the adsorption process between graphene/GO and four drug molecules, as well
as the adsorption sites between carriers and payloads. In addition, we calculated the interaction
forces between four drug molecules and graphene. We believe that this work will contribute to
deepening the understanding of the loading behaviors of anticancer drugs onto nanomaterials and
their interaction.
Citation: Gong, P.; Zhou, Y.; Li, H.; Keywords: DFT calculations; MD simulations; adsorption and aggregation; graphene; graphene
Zhang, J.; Wu, Y.; Zheng, P.; Jiang, Y. oxide; anticancer drugs
Theoretical Study on the Aggregation
and Adsorption Behaviors of
Anticancer Drug Molecules on
Graphene/Graphene Oxide Surface. 1. Introduction
Molecules 2022, 27, 6742. https://
Nanomaterials have broad application prospects in the biomedical field because of
doi.org/10.3390/molecules27196742
their unique characteristics. Drug delivery based on nanoparticles has been extensively
Academic Editors: Minas studied to maximize the therapeutic efficacy of drugs [1]. Among the diverse nanomaterials
M. Stylianakis and that have been found, graphene and its derivatives have been demonstrated to provide
Athanasios Skouras efficient drug delivery and are considered as promising and ideal nanocarriers for drug
Received: 29 August 2022
delivery systems, and have been widely studied in the field of cancer treatment [2] due
Accepted: 6 October 2022
to their remarkable physical and chemical properties [2]. Graphene is a two-dimensional
Published: 10 October 2022
(2D) sheet of sp2 hybrid carbon atoms; the carbon atoms are tightly packed in a (2D)
honeycomb lattice, which exhibits excellent properties such as large surface area and good
Publisher’s Note: MDPI stays neutral
biocompatibility, as well as providing a defect-free plane [3]. These pivotal characteristics
with regard to jurisdictional claims in
allow it to interact with drugs through non-covalent interactions such as π-π interaction.
published maps and institutional affil-
As a derivative of graphene, GO is also a promising drug delivery vehicle [4]. Apart from
iations.
features similar to pristine graphene, abundant hydroxyl, epoxy, and carboxyl functional
groups in GO enable it to have a higher adsorption capacity for drug molecules than
pristine graphene [5].
Copyright: © 2022 by the authors.
There are many drugs that could be delivered by graphene and its oxide, such as
Licensee MDPI, Basel, Switzerland. camptothecin, a widely used anticancer drug [6,7]. Its main target in cells is the type I
This article is an open access article DNA topoisomerase, which can inhibit DNA synthesis through chain break, causing cell
distributed under the terms and death during the S phase of the cell cycle, making it an effective inhibitor of leukemia cell
conditions of the Creative Commons growth [8–10]. Based on this mechanism, Liu et al. studied the inhibitory effect of CPT on
Attribution (CC BY) license (https:// the growth of prostate cancer cells, as it can selectively inhibit the androgen-responsive
creativecommons.org/licenses/by/ growth of prostate cancer cells [11]. Therefore, CPT is also a potential candidate drug
4.0/). for the treatment of prostate cancer. Furthermore, resveratrol is a phytoalexin extracted
10
Molecules 2022, 27, 6742
in many edible plants that may play a role in preventing inflammation, atherosclerosis,
cancer, and so forth [12,13]. For example, Kueck et al. found that Res inhibits glucose
metabolism in human ovarian cancer cells. Zhou et al. [14] proposed that Res can induce
apoptosis of pancreatic cancer cells. These studies suggest that Res is an effective cancer
drug. In addition, gefitinib, an oral epidermal growth factor receptor (EGFR) tyrosine
kinase inhibitor, is the first approved targeting drug for the treatment of non-small cell lung
cancer (NSCLC) [15]. It has also been widely studied as a prospective drug for other cancers
besides NSCLC, Li et al. [16] studied the potential role of Gefi in the treatment of pancreatic
cancer and found that it can inhibit the growth, invasion, and colony formation of pancreatic
cancer cells/Kalykaki et al. evaluated the effects of Gefi on circulating tumor cells (CTCs)
in patients with metastatic breast cancer (MBC) [17,18]. Last but not least, anastrozole is a
third generation aromatase inhibitor. As a potent inhibitor of intratumoral estrogen [19],
clinical trials showed that Anas reduced the risk of breast cancer in postmenopausal women
by 53% [20]. In the treatment of advanced breast cancer, it has significant survival benefits
and tolerability advantages compared to other treatment drugs [21]. Therefore, it plays an
important role in the prevention and treatment of breast cancer [22].
In this paper, the adsorption behavior of these drugs on graphene and GO carriers
was investigated in depth using density functional theory (DFT) and molecular dynamics
(MD) simulation, aiming to find the most stable adsorption conformations of different drug
molecules on graphene and GO, and to compare the adsorption performance of the same
carrier for different drugs. We hope that the results of this study can provide significant
value for further design and development of new nanomaterial drug delivery systems,
which we believe will ultimately improve the efficacy of targeted drugs in cancer therapy.
2. Computational Methods
2.1. Quantum Chemistry Calculations
We used quantum chemistry calculations methods to investigate the energetics of
graphene and GO, and the effect of adsorption on drugs. DFT calculation is a quantum
mechanical approach to study electronic systems and is commonly used to calculate the
bind band structure of solids in physics. This method has been used for graphene-related
research [23] All the quantum chemistry calculations were carried out with the Atomistix
ToolKit (ATK) package. Generalized gradient approximation (GGA) [24] with Perdew–
Burke–Ernzerhof (PBE) parametrization [25,26] was used as the exchange-correlation func-
tional. The basis set consists of the double numerical atomic orbitals augmented by polariza-
tion functions, which are comparable to Gaussian 6–31G**. Compared with other methods,
this calculation method is more effective and can meet the accuracy requirements [27,28]. To
avoid neighboring interaction, the distance between the neighboring molecules was larger
than 15 Å. The real-space global cutoff radii were set as 3.7 Å. The convergence criterion
on the energy and electron density was set to be 10−5 hartree. Geometry optimizations
were performed with convergence criteria of 2 × 10−3 hartree/Å on the gradient, and
5 × 10−3 Å on the displacement.
The adsorption energy of CPT on to the studied nanosheets and GO is calculated using
the relation:
Ea = Ecomplex − Enanosheet − Edrug (1)
where Ecomplex , Ecarrier , and Edrug are the total energy of the complex, energy of the carrier
(GRA or GO), and energy of the drug molecule (Res, Ana, Gefi, or CPT).
11
Molecules 2022, 27, 6742
parameters of the drug molecules. All the simulations were carried out by using the
GROMACS 2018 software package. The initial structure of graphene containing 480 carbon
atoms was constructed with the Nanotube Modeler package. To create GO, we randomly
decorate the graphene surface with hydroxyl and epoxy groups. The final oxygen to carbon
(O/C) ratio of GO nanosheets is 1:8. As for the relaxation of drug molecules, a box with
a size of 4 nm × 4 nm × 4 nm was firstly established, small molecules were randomly
inserted into the box, and the steepest descent method was used to optimize the system
to remove close contact and overlapping. Since both sides of the graphene can be used
for drug binding, it was placed in the middle of the box and the drug molecules were
allowed to be randomly distributed on both sides. In all systems, the center of the graphene
sheet was set as the zero point. Each system performed 10 ns NVT relaxation at 298 K and
1 atm, followed by 10 ns NPT-relaxation. After that, 50 ns MD simulation was conducted at
298 K and 1 atm equilibrium, and the integration step was 2.0 fs. The Berendsen thermal
bath method was employed to control the temperatures. The cutoff radius of non-bonding
interaction was set as 1.4 nm. Trajectories were collected every 5000 steps for further
analysis. Visual molecular dynamics (VMD) was used to observe the movement trajectory
of the system.
Figure 1. The electrostatic potential (ESP) distribution of (a) RGO; (b) GO; (c) Gefi; (d) CPT; (e) Anas;
(f) Res.
π
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Molecules 2022, 27, 6742
As shown in Figure 1a, graphene has uniform electron density and abundant π elec-
trons on its surface. According to previous studies, graphene is prone to π-π electron donor
acceptor interactions and van der Waals (vdW) interactions due to its large ring plane
structure [30]. The graphene oxide shown in Figure 1b is plotted in blue at the oxygen
atom. GO has reduced π electron activity to some extent due to the presence of oxygen-
containing functional groups, but it may form hydrogen bonds with other molecules. The
oxygen-containing functional groups of GO possess higher chemical reactivity compared
to graphene. The ESP of Gefi is shown in Figure 1c, with lower ESP at the oxygen atom
position. The ESP distribution of CPT is the same as that of Gefi, as shown in Figure 1d,
with lower ESP near the functional group, which is more nucleophilic compared to the
position of the hydrogen atom. The nitrogen atom position is shown in Figure 1e. The
nitrogen atom position is plotted in blue with lower ESP, as shown in Figure 1f, and the
oxygen atom position is plotted using red, indicating that the point has higher ESP [31].
Figure 2. Optimized geometries of RGO and drug systems; bonds are in Å (the vertical distance
refers to the distance between the centroid of benzene of different drugs to the carbon plane of RGO).
(a) Graphene-Gefi; (b) Graphene-Camptothecin; (c) Graphene-Anas; (d) Graphene-Res.
The vertical distance is the distance from the center of mass of the aromatic ring of
the drug molecule to the plane of the graphene. The vertical distances are between the
graphene and the aromatic rings of Gefi, CPT, Anas, Res are 3.358 Å, 3.462 Å, 4.991 Å, and
2.928 Å. The distance between graphene and different drug molecules follows the order:
Anas > CPT > Gefi > Res; and their adsorption energy is Anas < CPT < Gefi < Res from
small to large, indicating that the greater distance, the weaker the adsorption capacity of
the drug is [32–35].
We used MD simulations to study the effect of drugs adsorption on the graphene and
GO. We employed the root mean square deviation (RMSD), density distribution, radial
distribution function (RDT), hydrogen bond number (H- bond) and mean square displace-
13
Molecules 2022, 27, 6742
ment (MSD) to investigate the dynamics process of the adsorption of drug molecules
on graphene.
Figure 3a shows the RMSD curves for different systems to investigate the equilibrium
state of the simulated system. It can be seen that there are no large fluctuations in the RMSD
curves of the system at the later stages of the simulation, indicating that it is sufficient to
bring the system to equilibrium within the simulation time. As stated above, the main
driving force for the adsorption of different drug molecules by graphene stems from the
π-π interactions. The distribution of drug molecules on both sides of graphene was first
investigated and the results are shown in Figure 3b. Their mass density shows that different
drug molecules are effectively adsorbed on both side of the graphene sheet after the
adsorption has reached equilibrium and that their distribution on both sides is symmetrical.
Their two symmetry peaks are both at a distance of 0.38 nm, which is close to the vdW
radius of the carbon atoms on the graphene sheet. In the range of distance less than 0.5 nm,
all four kinds of drug molecules could appear on both sides of the graphene sheet. No
drug molecule was observed beyond the range of 1 nm after the equilibrium of the system,
because the mass densities of the drug molecules were all close to zero when the distances
greater than 1 nm, which also indicates that their adsorption is relatively tight. Radial
distribution functions (RDF) can be used to study the intermolecular interaction. Figure 3c
shows the interaction between drug molecules and graphene in the simulated system.
There are significant interactions between different drug molecules and graphene. Their
peaks are 0.482 nm for Gefi, 0.461 nm for CPT, 0.644 nm for Anas, and 0.436 nm for Res.
There are only vdW interactions between the four drug molecules and graphene, and the
strength of the interaction between drug molecules and graphene decreases as the distance
increases. Res has the strongest interaction with graphene, while Anas has the weakest
interaction. The adsorption capacities of graphene to the four drug molecules follow
the order Res > CPT > Gefi > Anas. To further investigate the interaction between drug
molecules and graphene, we calculated the probabilistic profiles of the distribution of the
angle between the aromatic ring for drug molecules and the graphene plane in molecular
dynamics. The ability of drug molecules to absorb on the graphene is mainly determined
by the superposition of π-π interactions. Effective interactions between aromatic rings
are considered to occur when the angle α < 30◦ . Figure 3d shows that all angles between
the aromatic rings of four drug molecules and the graphene plane are small during the
simulations. The most observed angles between Gefi, CPT, Anas, Res and the graphene
were approximately 7◦ , 7◦ , 13◦ , and 8◦ , which indicates that their aromatic rings are almost
parallel to the graphene surface. This also demonstrates that the adsorptions between drug
molecules and graphene are stable and that π-π interactions are the main driving force
for this adsorption. The environment will have a strong influence on the motion of the
molecules in the system. Figure 3e shows the MSD results for different drug molecules
adsorbing on graphene. Table 1 show the self-diffusion coefficients of the drug molecules
in different systems. It can be seen that the diffusion coefficient of Anas is the largest,
and the diffusion coefficients of CPT, Gefi, and Res are relatively similar, indicating that
the diffusive motion of Anas is the most active among the four drugs. This result can
be attributed to the weak binding ability of Anas and its low molecule weight (293.73),
which facilitates its diffusion, whereas the other three drugs were strongly absorbed on the
graphene surface and thus diffused more slowly [36,37].
14
Molecules 2022, 27, 6742
Figure 3. (a) The RMSD plots of the system with different drugs adsorbed on graphene as function
of time. (b) Mass density profiles of different drugs. The center of graphene is set as distance = 0.
(c) Radial distribution functions (RDF) of the different drug molecules with graphene. (d) The
probability of the angle between the aromatic rings of the drug molecules and the graphene plane,
(e) The MSD plots of the different drug molecules with graphene.
15
Molecules 2022, 27, 6742
Figure 4. Optimized geometries of GO and drug systems; bonds are in Å (the vertical distance
refers to the distance between the centroid of benzene of different drugs to the carbon plane of
GO). (a) GO-Gefi-2; (b) GO-Gefi-4; (c) GO-Camptothecin-1; (d) GO-Camptothecin-4; (e) GO-Anas-1;
(f) GO-Anas-2; (g) GO-Res-1; (h) GO-Res-2.
As shown in Figure 4a,b, the adsorption energies of stable configurations after Gefi
adsorption on GO with active sites 2 and 4 of Gefi as binding sites are 1.823 eV and
16
Molecules 2022, 27, 6742
1.553 eV, respectively, and the vertical distances are 3.387 Å and 3.451 Å between the
aromatic ring of Gefi and GO, respectively. Thus, the adsorption of GO with Gefi is more
stable when atom number 2 is used as the adsorption site, which is also consistent with
the result of the electrostatic potential of molecule. Active sites 1 and 4 of the CPT were
investigated as binding sites for adsorption onto GO, and their optimized result is shown as
Figure 4c,d. The perpendicular distances between the aromatic ring of the CPT and the GO
are 3.542 Å and 3.781 Å, respectively, and the adsorption energies are 1.326 eV and 1.037 eV,
respectively; therefore, the configuration of the CPT molecules adsorbed on GO in Figure 4c
can be considered as relatively stable. Figure 4e,f show the stable configurations obtained
after adsorption on GO using active sites 1 and 2 of Anas as binding sites. The distances of
the aromatic ring and GO are 6.416 Å and 3.431 Å, respectively. In Figure 4e, although the
Anas molecule can form hydrogen bonds with GO, the vertical distance becomes larger,
thus weakening the π-π interactions and leading to a reduction in the overall adsorption
energy. Therefore, for Anas, the adsorption is more stable when binding at active site 2.
As shown in Figure 1, all three active sites of Res have relatively large in electrostatic
potential values and small differences; therefore, their adsorption with graphene is similar.
Figure 4g,h show the result of the adsorption of active sites 1 and 2 of Res with GO. The
configuration of the adsorption at active site 3 is not shown in the figure because it is
consistent with that of active site 2. It can be seen that the vertical distance and adsorption
energies of Res on GO are relatively close in both cases and, therefore, they are stable as
binding sites for adsorption with GO in both cases. Overall, in the adsorption of four drug
molecules onto GO, the final conformation is more stable when the binding site has higher
electrostatic potential. According to the binding configuration and adsorption energy, the
adsorption capacity of GO for the four drug molecules follows the order: Res > Gefi > CPT
> Anas [38–42]. Comparing Figures 2 and 4, the adsorption capacity of GO for four drug
molecules is generally better than that of graphene. The vertical distance between different
drug molecules and GO increased slightly, which weakened the π-π interaction between
them to some extent, but the formation of hydrogen bonds promoted the binding of the two
and the superposition of the two effects finally promoted the adsorption of drug molecules
on GO.
Figure 5a shows the RMSD result for the adsorption of four drug molecules on GO,
from which it can be seen that all the systems reach the equilibrium state in a short time.
Previous studies have shown that not only π-π interactions, but also some hydrogen
bonds occur during the adsorption of drug molecules on GO. To deeply understand the
strength of the two kinds of interactions during the simulation, we investigated the average
interaction energy between the four drug molecules and the GO sheet. As shown in
Figure 5b. It can be seen that the vdW interaction accounts for the major part of the
potential energy and is much greater than the Coulomb interaction, indicating that vdW
interaction is the dominant force between GO and the drug molecules. This conclusion is
also supported by the results of the radial distribution function of the simulated system [43].
Figure 5c shows the RDFs between different drug molecules and GO sheets. Four drug
molecules had RDFs with GO in the range of less than 0.35 nm, indicating that the hydrogen
bonding between them is relatively weak. Furthermore, the peaks at 0.486 nm, 0.612 nm,
0.662 nm, and 0.455 nm indicate vdW interactions between the four drug molecules and
GO. This result is consistent with the analysis in Figure 5b, which indicates that vdW
interactions between the four drug molecules and GO still play a dominant role, while the
hydrogen bonding is relatively weak.
The distribution of the angle between the aromatic ring of the drug molecule and the
GO plane during kinetic adsorption was also analyzed; the results are shown in Figure 5d.
The probability of the angular distribution between the aromatic rings of four drugs and the
GO planes varied considerably compared to graphene during the simulation. Although the
most probable angle between Gefi, CPT, Anas, and Res and GO remained relatively small,
approximately 7◦ , 10◦ , 14◦ , and 9◦ respectively, the probability of occurrence decreased
and, except for Res, there was a significant increase in the probability of the angle between
17
Molecules 2022, 27, 6742
the other three drug molecules and GO being greater than 30◦ . This also indicates that the
π-π interactions between GO and drug molecules were weakened to some extent. This
result mainly originates from the presence of oxygen-containing functional groups on GO.
Figure 5. (a) The RMSD plots of the system with different drugs adsorbed on graphene as function of
time (b) The average electrostatic, vdW, and total interaction energies of the systems with different
drug molecules and GO. (c) Radial distribution functions (RDF) of the different drug molecules with
GO. (d) The probability of the angle between the aromatic rings of the drug molecules and the GO
plane. (e) Changes of the number of hydrogen bonds between the four kinds of drug molecules and
GO over the time. (f) The MSD plots of the different drug molecules with GO.
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Molecules 2022, 27, 6742
The hydrogen bonding in the system also plays an important role in the overall adsorp-
tion process; therefore, the variation in the number of hydrogen bonds between different
drug molecules and GO during the simulation was investigated, as shown in Figure 5e.
Throughout the adsorption process, it can be seen that the number of hydrogen bonds
formed between all four drug molecules and GO is relatively small, which is also consistent
with the results obtained in Figure 5b. This further confirms that the hydrogen bonding
between the four drug molecules and GO is relatively weak during the adsorption process.
To further understand the movement of molecules in the system, the diffusion coeffi-
cients of the four drug molecules were studied separately, Figure 5f shows the MSD plots of
drug molecules adsorbed on GO. Table 2 shows the diffusion coefficients of drug molecules
in different systems in descending order. Comparing with Table 1, it is found that the
diffusion coefficients of all four drug molecules show different degrees of reduction, which
also indicates that the adsorption of these four drugs on GO is slightly stronger than that
on graphene [39,44,45].
4. Conclusions
In summary, we used DFT methods and MD simulations to investigate the adsorption
processes and interaction mechanisms of graphene and GO with Gefi, CPT, Anas, and
Res. From the result of DFT calculations, it is clear that GO has stronger adsorption
properties than graphene for the four drug molecules, and the adsorption energy follows
the order of Anas < CPT < Gefi < Res for both the graphene and GO systems. Regarding
the adsorption systems of drug molecules with graphene oxide, static calculations further
confirmed the preferential adsorption sites. By utilizing MD simulations, we found the
adsorption mechanisms of different drugs with graphene as well as GO; it was found that
π-π interactions and hydrogen bonding played an important role in the whole adsorption
process. Among the four drug molecules, Res molecules showed the strongest adsorption
capacity on graphene and GO, while Anas showed the weakest adsorption capacity on both
graphene and GO. Furthermore, vdW interactions played a dominant role in the dynamic
adsorption of drug molecules on both graphene and GO. Hydrogen-bonding had only a
small contribution to the adsorption of drug molecules on GO. Taken together, GO has
a stronger ability to adsorb these four drug molecules than graphene. Due to the good
adsorption properties of graphene and GO for the four drug molecules, this study helps
gain insight into the loading behavior of anti-cancer drugs on graphene, and also helps to
provide assistance in the development of carriers of loaded drugs for anti-cancer drugs.
Author Contributions: Conceptualization, P.G. and Y.Z.; methodology, J.Z.; validation, Y.W. and Y.Z.;
formal analysis, P.G.; investigation, H.L.; resources, Y.J.; data curation, P.G.; writing—original draft
preparation, P.G.; writing—review and editing, P.G.; visualization, Y.Z.; supervision, P.Z.; project
administration, Y.W.; funding acquisition, Y.J. All authors have read and agreed to the published
version of the manuscript.
Funding: This research was funded by [the National Natural Science Foundation of China] grant
number [Grant No. 52101287 and U1806219], [the Project of Taishan Scholar Construction Engineering
and the program of the Jinan Science and Technology Bureau] grant number [2020GXRC019] and
[the new material demonstration platform construction project from the Ministry of Industry and
Information Technology] grant number [2020-370104-34-03-043952-01-11].
Institutional Review Board Statement: Not applicable.
Informed Consent Statement: Not applicable.
19
Molecules 2022, 27, 6742
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21
molecules
Article
Graphene@Curcumin-Copper Paintable Coatings for the
Prevention of Nosocomial Microbial Infection
Mohammad Oves 1 , Mohammad Omaish Ansari 2 , Mohammad Shahnawaze Ansari 2 and Adnan Memić 2, *
1 Center of Excellence in Environmental Studies, King Abdulaziz University, Jeddah 21589, Saudi Arabia
2 Center of Nanotechnology, King Abdulaziz University, Jeddah 21589, Saudi Arabia
* Correspondence: [email protected]; Tel.: +966-564975404
Abstract: The rise of antimicrobial resistance has brought into focus the urgent need for the next
generation of antimicrobial coating. Specifically, the coating of suitable antimicrobial nanomaterials
on contact surfaces seems to be an effective method for the disinfection/contact killing of microor-
ganisms. In this study, the antimicrobial coatings of graphene@curcumin-copper (GN@CR-Cu) were
prepared using a chemical synthesis methodology. Thus, the prepared GN@CR-Cu slurry was suc-
cessfully coated on different contact surfaces, and subsequently, the GO in the composite was reduced
to graphene (GN) by low-temperature heating/sunlight exposure. Scanning electron microscopy
was used to characterize the coated GN@CR-Cu for the coating properties, X-ray photon scatter-
ing were used for structural characterization and material confirmation. From the morphological
analysis, it was seen that CR and Cu were uniformly distributed throughout the GN network. The
nanocomposite coating showed antimicrobial properties by contact-killing mechanisms, which was
confirmed by zone inhibition and scanning electron microscopy. The materials showed maximum
antibacterial activity against E. coli (24 ± 0.50 mm) followed by P. aeruginosa (18 ± 0.25 mm) at
25 µg/mL spot inoculation on the solid media plate, and a similar trend was observed in the min-
imum inhibition concentration (80 µg/mL) and bactericidal concentration (160 µg/mL) in liquid
media. The synthesized materials showed excellent activity against E. coli and P. aeruginosa. These
materials, when coated on different contact surfaces such medical devices, might significantly reduce
the risk of nosocomial infection.
Citation: Oves, M.; Ansari, M.O.;
Ansari, M.S.; Memić, A.
Keywords: antimicrobial; coating; graphene; curcumin; copper; Pseudomonas aeruginosa; E. coli
Graphene@Curcumin-Copper
Paintable Coatings for the Prevention
of Nosocomial Microbial Infection.
Molecules 2023, 28, 2814. https://
doi.org/10.3390/molecules28062814 1. Introduction
Infectious diseases that are caused by microorganisms can lead to serious health
Academic Editors: Minas
complications, including death, for both humans and animals. The attachment of microbes
M. Stylianakis and
Athanasios Skouras
to surfaces leading to the formation of biofilms poses a particularly serious threat. Many
industries, ranging from healthcare systems, the food and water industry, as well as the oil
Received: 7 February 2023 and gas industry, suffer huge losses due to complications as a result of biofilm formation [1].
Revised: 2 March 2023 Further challenges arise when considering that microbes adhering to the surfaces can be
Accepted: 7 March 2023 transferred when touched, thereby spreading microbial colonization. During the global
Published: 20 March 2023
COVID-19 outbreak, the development of antimicrobial surfaces attracted the attention
of scientists worldwide. The development of antimicrobial coating could be an effective
strategy to prevent microbial spread in general and specifically biofilm formation. [2]. To
Copyright: © 2023 by the authors.
achieve these goals, several approaches have been proposed [3]. For example, recently, a
Licensee MDPI, Basel, Switzerland. combination of different nanomaterials, such as graphene (GN), metals and metal oxides,
This article is an open access article or natural antibacterial materials, have been proposed as antimicrobial coatings. Such
distributed under the terms and a combination of materials could have exciting synergistic or additional effects when
conditions of the Creative Commons compared to the individual components developed for anti-infective surface deployment.
Attribution (CC BY) license (https:// Some recently designed GN-based nanomaterials are known to exhibit antimicrobial
creativecommons.org/licenses/by/ activity towards bacteria [4], fungi [5], and viruses [6]. The direct physicochemical in-
4.0/). teraction between the GN-based materials and microbes can lead to the damage of the
22
Molecules 2023, 28, 2814
cellular components, principally the proteins, lipids, and nucleic acids [7]. The high affinity
of GN-based materials towards membrane proteoglycans leads to membrane damage.
Another mechanism of action is the further internal leakage of GN-based materials into
cells which can lead to the inhibition of DNA/RNA replication [8]. Therefore, it is proposed
that the mode of action of GN towards bacteria is by physical means as well as by chemical
means [9]. The physical means operate by the direct contact of microorganisms with the
sharp edges of GN. For example, Akhavan et al. [10] showed that the GN-based materials
could potentially encapsulate microorganisms for their isolation but also act as an effec-
tive photothermal agent for the inactivation of the GN-wrapped microorganisms. Pham
et al. [11] similarly showed that the density of GN edges significantly affects its antibacterial
activity. The researchers proposed that GN sheets act as blades cutting through the cell
membrane and induce pores, leading to their rupture leading to bacterial death. On the
other hand, chemical mechanisms involve oxidative stress and the generation of reactive
oxygen species (ROS) responsible for the inactivation of microorganisms [12].
Among metallic particles, many research groups have shown that copper (Cu) and its
oxides have antimicrobial properties [13–20]. The mechanism of killing involves bacterial
cell wall damage leading to the loss of cytoplasmic content [21]. Apart from this, the
reactive oxygen species can induce even greater damage to organelles and even lead to
nucleic acid degradation [22]. Cu has also been found to possess high antiviral properties
as well [23]. Bleichert et al. [24] showed that attenuated vaccine strain vaccinia virus and
virulent MPXV Copenhagen were inhibited within 3 min of exposure to Cu. Similarly,
Noyce et al. [25] showed that the Cu alloys (61–95% Cu) effectively killed E. coli at room
temperature. They also found that samples with a high percentage of Cu possessed the
highest antibacterial properties. Similar results were also reported by Wilks et al. [26], who
showed that a Cu content of 85% or more showed good antibacterial activities. Similar
effects of Cu were found against the vegetative and spore forms of Clostridium difficile and a
significant reduction in survival of the C. difficile vegetative cells and spores was observed
after 24–48 h [27]. From the above discussion, it appears that the amount of Cu can have a
direct effect on the contact killing of microorganisms, including both bacteria and viruses.
Among naturally occurring antimicrobial materials, curcumin (CR) has been widely
used since its antibacterial properties were demonstrated by Schraufstatter and Bernt
in 1949 [28]. CR promotes recombinant protein overexpression, thereby leading to an
apoptosis-like response in bacteria [29]. Several investigations revealed that CR had an-
tibacterial effects on both Gram-positive and Gram-negative bacteria [30]. CR antibacterial
activity includes bacterial membrane rupture, the suppression of virulence factor synthesis
and suppression of biofilm formation, and the activation of oxidative stress [31,32]. Re-
cently, Oves et al. [33] has investigated CR- and ZnO-glazed GN for the successful growth
inhibition of a Methicillin-resistant bacterial strain of Staphylococcus aureus.
Due to the antibacterial properties of both GN and Cu paired with CR, it can be
interpreted that their combination will be highly effective in combating different types of
bacteria via contact killing. Thus, in this work, solutions of Cu particles and CR dispersed
in graphene oxide (GO) gel was prepared. The prepared dispersion was applied on contact
surfaces and its subsequent heat treatment resulted in the reduction of GO into reduced-
graphene oxide (rGO) (Figure 1). The Cu, CR, and reduced-graphene oxide coatings
were thereafter tested for the contact killing of Pseudomonas aeruginosa (P. aeruginosa) and
Escherichia coli (E. coli).
23
Molecules 2023, 28, 2814
Curcumin
Killed bacteria
Cotton sheet
Copper
0.2 M ascorbic CTAB
acid
Copper sulfate
Antibacterial confirmation
24
Molecules 2023, 28, 2814
Figure 2. Low and high magnification (insets) FESEM images of (a) GN; (b) GN@Cu; (c) GN@CR;
and (d) GN@CR-Cu.
Figure 3. Elemental mapping images of (a) GN@CR-Cu; (b) C; (c) O; (d) Cu; and (e) EDS
spectrum of GN@CR-Cu.
25
Molecules 2023, 28, 2814
Figure 4a,b shows low and high magnification HRTEM images of GN@CR-Cu.
Figure 4a reveals that the sample is well-formed and homogeneous with a uniform amal-
gamation of GN, Cu, and CR. All three components, the sheet-like layered structure of
GN, the almost hexagonal/polyhedral-shaped solid surface of Cu, and the fluffy/circular
geometry of CR particles can be easily identified in the image Figure 4b.
Figure 4. (a) Low magnification; (b) High magnification HRTEM images of GN@CR-Cu.
26
Molecules 2023, 28, 2814
– –
E. coli and P. aeruginosa at a 25 µg/mL concentration, and GN@CR showed 17 ± 0.75 and
12 ± 0.5 mm zone inhibition against E. coli and P. aeruginosa at the 25 µg/mL concentrations,
respectively (Figure 6).
± ±
µg ± ±
µg ± ±
Figure 5. XPS spectra of GN@CR-Cu composite: (a) survey scan, (b) C1s,
µg and (c) Cu2p3.
– P. aeruginosa (e–h):
Figure 6. Test bacteria E. coli (a–d), – The zone inhibition by the nanocomposite
material on the bacteria cultivated on nutrient agar media plates.
27 and 160 µg
Molecules 2023, 28, 2814
– –
– The minimum inhibition
Figure 7. Test bacteria E. coli (a–d), P. aeruginosa (e–h): – by the nanocomposite
material on the bacteria cultivated Petri plates.
(a) (b)
Figure 8. The survivability of E. coli (a) and P. aeruginosa (b) in the presence of nanocomposite
material. Image showing excellent dose-response, increasing concentration significantly retards
bacterial growth.
28
° °
Molecules 2023, 28, 2814
Figure 9. Scanning electron microscopy image of E. coli without any treatment as a control without an
effect on cell morphology (a), and E. coli culture treated with GN (b), GN@CR (c), partial cell damage by
the treatment of GN@Cu (d), and complete bacterial cell damage by the treatment of GN@CR-Cu (e).
29
Molecules 2023, 28, 2814
due to the nanoparticle interaction. GN@Cu-CR materials are multifunctional due to the
metal presence and natural CR. In order to prevent microbes from attaching, colonizing,
spreading, and creating biofilms in medical devices, composite materials have the potential
for external uses as antibacterial agents in the surface coatings on a variety of substrates.
Here, the nanocomposite material has been synthesized with a highly stable mate-
rial, i.e., GN which stabilizes both CR and Cu. Due to the high stability of GN-based
materials, the CR and Cu molecules fixed in the GN groves retain their antimicrobial
activity. In our previous reports, the GN-based nanocomposite material with Zinc ox-
ide and CR showed similarly excellent stability and antimicrobial activity against the
multidrug-resistant Staphylococcus aurous bacterial strain [33].
30
Molecules 2023, 28, 2814
3.1.3. Characterization
The morphological and compositional analysis of GN@CR-Cu were conducted by field
emission scanning electron microscopy (FESEM) (JSM-7600F from JEOL, Tokyo, Japan).
The elemental mapping was recorded using the energy dispersive X-ray spectroscope
(EDS) from Oxford Instruments, Oxfordshire, UK equipped with FESEM. For the ele-
mental detection, X-ray photoelectron spectroscopy (XPS) (ESCALAB 250 from Thermo
Fisher Scientific, Warrington, UK) was used at a monochromatized Al Kα X-ray source
λ 1/4 1486.6 eV. The antibacterial studies were performed by studying the surface growth in-
hibition assay, and the minimum inhibitory concentration (MIC) and minimum bactericidal
concentration (MBC) were determined against the Escherichia coli and
Pseudomonas aeruginosa microorganisms.
31
Molecules 2023, 28, 2814
sub-MIC level of an agent, the sensitivity of a bacterial strain could be correlated with the
percentage of cells that survive and form a colony. This ratio of surviving cells to the total
number of plated cells is the plating efficiency. The key to a successful spot-plating assay is
to spot-plate the same number of cells onto each spot. By comparing the surviving cells on
the material containing plates to the control, the sensitivity of the cells to the nanocomposite
concentration by plating efficiency percentage can be determined.
Determination of the optimal sub-MIC level of nanomaterials: The chosen sub-MIC level
nanomaterials should be chosen with care. The concentration should be high enough
to inhibit growth, but not to inhibit all growth. This optimal sub-MIC concentration is
usually just below the MIC of the nanomaterials. For example, the MIC of nanomaterials
and E. coli is 50 µg/mL. The optimal sub-MIC of nanomaterials for the spot plate assay
was found to be 40 µg/mL. Media preparation for bacterial growth with and without
nanomaterials should be made. The bacterial stocks should be grown overnight at their
optimal growth conditions and maintained according to the McFarland standard, culture
optimum incubation at 37 ◦ C, and shaking at 250 rpm. After overnight growth, the culture
should be diluted to a concentration that would allow countable isolated colonies on the
final spots of the LB plates. We recommend dilutions of 1 × 104 CFU/mL. A small volume
(10 µL) of the working stock of bacterial culture should be plated up to six times on each
plate. The number of colonies in each spot should be counted and tallied. The number of
colonies on the antibiotic plates and the control plates are used to calculate.
Nanomaterials stock preparation: The amount of the required nanomaterial concentration
was weighed by analytical balance and then transferred and dissolved with the required
amount of sterile dH2O, before being filtered through a 0.20 µm filter into a sterile 10 mL
falcon tube.
Dilution of bacterial culture to a working concentration: The bacterial culture turbidity
and availability of bacteria count was determined by spectrophotometer at OD600. The
obtained value was multiplying the OD600 of the bacterial culture by the appropriate
conversion ratio of OD600 to CFU/mL (if OD600 = 8 × 108 CFU/mL). Further specific
required dilution of the working concentration was also determined.
Spot plating: First of all, the media plates were prepared and supplemented with and
without nanomaterials of interest at the desired concentrations of 0 to 100 µg. The culture
tubes were inoculated and incubated overnight at 37 ◦ C. The next day, the optical density
of the diluted cultures was determined and converted into CFU/mL using the conversion
factor of the strain, if known (1 OD at 600 equals to 8 × 108 cells/mL). Further, the cultures
(100 µL of culture into 900 µL of fresh growth media) were serially diluted to obtain a 1
× 104 CFU/mL culture. An aseptic pipette was used to separate the spots onto a plate
using 10 µL of 1 × 104 CFU/mL culture. After incubation, the isolated colonies per spot
were counted. The number of colonies on each spot of the nanomaterial’s plates were
also counted.
4. Conclusions
In this study, the nanocomposite material GN@CR-Cu was successfully synthesized
and coated on contact surfaces for studying its antimicrobial activity. The antimicrobial
coatings of GN@CR-Cu were prepared using the chemical synthesis methodology and were
32
Molecules 2023, 28, 2814
further characterized using electron microscopy and X-ray photon spectroscopy. GN@CR-
Cu showed excellent antimicrobial effects against E. coli and P aeruginosa bacterial isolates.
The nanocomposite showed antimicrobial activity, most likely by contact-killing mecha-
nisms, which was suggested by zone inhibition and scanning electron microscopy. The
materials showed maximum antibacterial activity against E. coli (24 ± 0.50 mm) followed by
P. aeruginosa (18 ± 0.25 mm) at 25 µg/mL spot inoculation on the solid media plate, and a
similar trend was observed in the minimum inhibition concentration (80 µg/mL) and bacte-
ricidal concentration (160 µg/mL) in liquid media. According to this proof-of-concept study,
GN@CR-Cu can function as a potent future anti-microbial nanomaterial for the prevention
of nosocomial infection, if coated on medical devices or food preparation instruments.
Supplementary Materials: The following supporting information can be downloaded at: https:
//www.mdpi.com/article/10.3390/molecules28062814/s1, Figure S1: FESEM image of (a) CR and
(b) Cu nanoparticles, Figure S2: XPS survey scan of GN and Cu, Figure S3: Water contact angle
measurement of GN@CR-Cu composite.
Author Contributions: Conceptualization, M.O., M.O.A. and A.M.; data curation, M.S.A. and A.M.;
formal analysis, M.O.A. and M.S.A.; investigation M.S.A. and A.M.; methodology, M.O. and M.O.A.;
supervision, A.M.; validation, M.O. and M.O.A.; writing—original draft, M.O., M.O.A. and M.S.A.;
writing—review and editing, M.S.A. and A.M. All authors have read and agreed to the published
version of the manuscript.
Funding: This research received no external funding.
Institutional Review Board Statement: Not applicable.
Informed Consent Statement: Not applicable.
Data Availability Statement: The data presented in this study are available on request from the
corresponding author.
Conflicts of Interest: The authors declare no conflict of interest.
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35
molecules
Article
Polysaccharides as Green Fuels for the Synthesis of MgO:
Characterization and Evaluation of Antimicrobial Activities
Nayara Balaba 1 , Silvia Jaerger 1 , Dienifer F. L. Horsth 1,2 , Julia de O. Primo 1 , Jamille de S. Correa 1 ,
Carla Bittencourt 2, * , Cristina M. Zanette 3 and Fauze J. Anaissi 1
Abstract: The synthesis of structured MgO is reported using feedstock starch (route I), citrus pectin
(route II), and Aloe vera (route III) leaf, which are suitable for use as green fuels due to their abun-
dance, low cost, and non-toxicity. The oxides formed showed high porosity and were evaluated
as antimicrobial agents. The samples were characterized by energy-dispersive X-ray fluorescence
(EDXRF), X-ray diffraction (XRD), Fourier-transform infrared spectroscopy (FTIR), and scanning
electron microscopy (SEM). The crystalline periclase monophase of the MgO was identified for all
samples. The SEM analyses show that the sample morphology depends on the organic fuel used
during the synthesis. The antibacterial activity of the MgO-St (starch), MgO-CP (citrus pectin), and
MgO-Av (Aloe vera) oxides was evaluated against pathogens Staphylococcus aureus (ATCC 6538P) and
Escherichia coli (ATCC 8739). Antifungal activity was also studied against Candida albicans (ATCC
64548). The studies were carried out using the qualitative agar disk diffusion method and quantitative
minimum inhibitory concentration (MIC) tests. The MIC of each sample showed the same inhibitory
concentration of 400 µg.mL−1 for the studied microorganisms. The formation of inhibition zones
Citation: Balaba, N.; Jaerger, S.;
and the MIC values in the antimicrobial analysis indicate the effective antimicrobial activity of the
Horsth, D.F.L.; Primo, J.d.O.; Correa,
samples against the test microorganisms.
J.d.S.; Bittencourt, C.; Zanette, C.M.;
Anaissi, F.J. Polysaccharides as Green
Keywords: eco-friendly synthesis; Aloe vera; starch; MIC; bacteria; antifungal
Fuels for the Synthesis of MgO:
Characterization and Evaluation of
Antimicrobial Activities. Molecules
2023, 28, 142. https://doi.org/
10.3390/molecules28010142 1. Introduction
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Molecules 2023, 28, 142
reactivity, high combustion power, reduction in the calcination temperature, and action as
a complexing gelling agent [10].
In the present work, starch, citrus pectin, and Aloe vera leaf were used as fuels to obtain
structured MgO by the combustion method. In synthesis route I, starch extracted from
cassava (Manihot esculenta), a high-energy tuber and a low-cost, biodegradable polysac-
charide from renewable raw material sources, consisting of amylose and amylopectin
molecules that are composed of D-glucose units, was used [11]. In route II, citrus pectin, a
polysaccharide derived from the peels of citrus fruits (lemon, orange, etc.) used in the food
and pharmaceutical industries, due to its high gelling capacity, was used [12]. It consists
of α-D-galacturonic acid units joined by glycosidic bonds (α-1,4) and esterified methyl
carboxyl groups [12]. Finally, Aloe vera (Aloe Barbadensis Miller) was used in route III, a
succulent perennial of the family Liliaceae, used for its pharmacological properties [13]. Ac-
cording to Hamman (2008), the Aloe vera leaves have three structural components: the cell
walls, the degenerated organelles, and the viscous liquid contained within the cells. These
components present many compounds, such as proteins, lipids, amino acids, vitamins,
enzymes, inorganic compounds, small organic compounds, and polysaccharides. Among
the polysaccharides, one can find mainly mannose, cellulose, and pectic polysaccharides,
whereas the skin of the leaf contains, in addition, significant quantities of xylose-containing
polysaccharides [13,14]. Describing a reaction mechanism for the synthesis of materials
with Aloe vera is complex due to the different compounds present in the plant extract [15].
However, a sustainable reaction mechanism for the formation of material with organic fuel
is the interaction of metal ions that bind with biomolecules through functional groups and
π electrons by ionic bonds or van der Waals forces; this depends on the concentration of
plant extracts [15]. The synthesized MgO samples were characterized, and their biological
activities were studied.
Antimicrobial resistance (AMR) is a global threat to human health, causing thousands
of deaths annually [16]. One of the leading causes of drug-resistant pathogens is the
excessive exposure of microorganisms to antibiotics as a treatment against infection [17,18].
Another problem caused by microorganisms is biofilms, which are aggregates of cells
embedded in a self-producing matrix of extracellular polymeric substances (EPS), which
adhere to each other and/or to a surface and have greater potential to survive in adverse
conditions and end up generating resistance to antibiotic treatment and the host’s immune
system [19]. Bacteria and diverse fungi show greater susceptibility to antibiotic resistance
due to some strains’ evolutionary and adaptive conditions [17,20]. For example, the
bacterium Staphylococcus aureus, responsible for infections such as postoperative wounds
and prosthetic infections related to endotracheal tubes and other biomaterials, has been
reported to cause nosocomial infections and AMR to several drugs, such as penicillin,
methicillin, quinolone, and vancomycin [17,21]. A fungus that has become resistant to
antibiotics is Candida albicans, an opportunistic pathogen, generally harmless to human
beings, which can be found on the surface of humid mucous, such as in the intestine, vagina,
and oral cavity [17]. The fungus C. albicans shows drug resistance to amphotericin B and
azoles [22], two known antifungal agents. However, colonization, infectious aggravation,
and damage to the structure of cells may occur with the weakening of the host’s immune
system [17]. Therefore, it is necessary to develop alternative strategies to minimize the
problem of AMR, such as materials that inhibit the growth of microorganisms, preventing
contagion with infectious diseases [23]. In this context, some papers present antimicrobial
studies, as Al-Shammar et al. (2021), which uses zein nanoparticles loaded with transition
metal ions to control three Candida species [24], and metal oxide particles have presented
a wide range of biological applications, such as drug and gene delivery and cell, tissue,
and diagnostics engineering, as well as limiting the growth of microorganisms [4]. Reports
on structured magnesium oxide (MgO) describe its efficiency in inhibiting the growth
of food and aquatic pathogen colonies and controlling the proliferation of bacteria and
fungi, attracting significant interest due to its non-toxic nature [4,5,25]. The common
mechanism of the antibacterial activity of MgO is due to the oxygen vacancies, leading
37
Molecules 2023, 28, 142
to the higher production of reactive oxygen species (ROS) (OH− , O2− , and H2 O2 ) on the
surfaces of the particles when oxidative stress occurs on the bacterial cell wall, leading to
cell death [4]. Another possible mechanism is when Mg2+ ions are released from MgO with
irregular morphology and rough edges, coming into contact with the cell membrane of the
microbe [4,8]. The negatively charged cell membrane and Mg2+ attract each other and the
Mg penetrates the cell membrane and damages it, leading to cell death [8]. Therefore, in this
study, three natural polysaccharides were used as precursors to the combustion reaction to
obtain structured MgO, aiming at its application as an antibacterial and antifungal agent.
2. Results
2.1. Characterization of Synthesized MgO Particles
According to the semiquantitative analysis (EDXRF) (Table 1), the sample with the
highest elemental magnesium percentage was MgO-St, with 86.6% atomic, while MgO-Av
presented 64.1%, and MgO-CP 76.2% (Figure S1). This difference can be associated with
the composition of the organic additives: starch contained ~6.6% atomic Mg ions, citrus
pectin presented ~1.7% of magnesium, and Aloe vera gel contained ~0.1% (Figure S2). The
composition of the organic additives varied according to the region from which they were
harvested [26]. The samples’ composition variation indicates that the organic fuel used in
the synthesis influences the amount of metal ions in the material obtained, interfering with
the physicochemical properties and affecting the performance against bacteria and fungi.
Table 1. Elemental chemical compositions of the synthesized MgO samples and precursors used in
the synthesis in element percent (% element) by EDXRF.
Elements (%)
Sample
Mg Ca Zn Cu K Al S P Others
Mg(NO3 )2 .6H2 O 98.0 0.8 - 0.1 - - 1.0 - 0.1
Starch 6.6 26.0 4.0 4.4 22.5 15.2 11.6 4.9 4.8
MgO-St 86.6 2.7 0.7 0.4 1.0 6.3 - 1.7 0.6
Aloe vera 0.1 20.9 1.0 3.4 0.5 - 1.2 - 72.9
MgO-Av 64.1 8.8 0.4 0.7 0.4 - 1.1 0.4 24.1
Citric pectin 1.7 21.8 2.2 2.1 52.6 7.7 4.9 1.4 5.6
MgO-CP 76.2 8.1 1.6 0.5 1.3 7.3 2.6 0.5 1.9
Figure 1 shows the X-ray diffractograms of magnesium oxide samples obtained from
starch (Figure 1a), citrus pectin (Figure 1b), and Aloe Vera (Figure 1c). In all samples, peaks
(111), (200), (220) (113), and (222) of the periclase crystalline phase, the typical phase of the
face-centered cubic MgO (COD: 9000505), were observed [27,28].
A few differences between the diffractograms were observed when using different
fuels. The samples showed different peak intensities, mainly peak (200) at 42.6◦ and
peak (220) at 61.8◦ , and MgO-St was found to have a greater width at the average height
compared to the peaks of the MgO-CP and MgO-Av diffractograms, which would suggest
a smaller crystallite size [28].
The FT-IR spectra of the MgO samples are shown in Figure 2. The IR spectra exhibit a
narrow and intense band at 3700 and 3445 cm−1 , attributed to the vibrational stretching
of the free -OH ions of Mg(OH)2 , generated by the hydration of MgO [29]. The γ(OH)
region shows two bands at 1488 cm−1 for the MgO-St sample (Figure 2a) and 1638 cm−1 ,
which correspond to the O–H bending mode, which is characterized by the bending
vibration of the -OH group of the physiosorbed water molecules [29,30]. The broad bands
at 1427 and 1383 cm−1 for MgO-St, MgO-CP, and MgO-Av (Figure 2b,c) are assigned to
the asymmetrical and symmetrical stretching vibrations of CO2 species chemisorbed onto
the surface of MgO [31,32]. The low-intensity bands at 1110 and 1061 cm−1 observed
for MgO-CP and Mg-Av can be associated with the presence of H ion species as defects
38
Molecules 2023, 28, 142
Figure 1. XRD patterns of samples MgO-St (a), MgO-CP (b), and MgO-Av (c).
γ
− −
Figure 2. FTIR spectra for the samples (a) MgO-St, (b) MgO-CP, and (c) MgO-Av.
39
Molecules 2023, 28, 142
Scanning electron microscopy was used to study the morphology of the synthesized
samples (Figure 3). The morphology of the MgO-St sample (Figure 3a) displayed a structure
composed of non-uniform pores and hole voids, with spongy characteristics in the material,
with an average particle size of approximately 0.99 µm (Figure 3b). The holes may be due
to the large number of gases released during the combustion of the reagents and the starch
used in the synthesis. These voids and pores provide a large surface area, supporting
antimicrobial activity [35]. Figure 3c shows SEM images of the MgO-CP sample, which
had an irregular morphology, forming small pseudo-spheres and hole voids [20], showing
larger particles than the MgO-St sample, with an average size of 1.14 µm (Figure 3d).
Finally, the morphology of the MgO-Av sample (Figure 3e) was irregular and composed of
periodic sheets, and the sample consisted of small particles with an average size of 0.85 µm
(Figure 3f). The different morphologies observed in the samples are attributed to the
different polysaccharides used in each type of synthesis, which will affect the antimicrobial
properties of each material [35–37].
Figure 3. SEM images of synthesized MgO using different natural additives as fuel sources and
average particle sizes of the samples: (a,b) MgO-St, (c,d) MgO-CP, and (e,f) MgO-Av.
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Molecules 2023, 28, 142
Figure 4. Photographs of halos of inhibition formed in tests against C. Albicans using the oxide
samples MgO-St, MgO-CP, and MgO-Av. The oxide MgO-St showed the largest halo of inhibition.
Table 2. Average mean zones of inhibition (in mm) produced by synthesized MgO samples on the
test organisms.
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Molecules 2023, 28, 142
Figure 5. Photographs of halos of inhibition formed in tests against Staphylococcus aureus. *MgO-CP
(labeled as 1,2,3); MgO-Av (labelled as 4,5,6); MgO-St (labeled as 7,8,9).
It has been reported that the antibacterial activity increases with the decreasing size of
nanoparticles due to the bacteria MgO surface interaction, which depends on the surface
area available [20]. Against S. aureus, the oxides synthesized with citric pectin and Aloe
vera fuels were statistically equal; however, against the C. albicans fungi, the oxide MgO-CP
showed significantly higher activity than the MgO-Av sample. The MgO samples were inef-
fective against E. coli, which can be associated with the Gram-negative bacterial composition
of a thin peptidoglycan cell wall and an outer membrane containing lipopolysaccharide,
leading to higher resistance than for Gram-positive bacteria such as S. aureus [20]. Some
studies, such as Umaralikhan and Jaffar, also used Aloe vera leaves as a fuel source for
MgO synthesis and tested them against ≤ S. aureus and E. coli as an antibacterial material
in the disk diffusion method; they obtained considerable inhibition success against both
bacteria [25]. The diffusion of agar disks is a qualitative assay used to test bioactivity in
a sample. However, in this method, the effect of antimicrobial activity is not accurately
estimated [40].Therefore, the synthesized oxides were tested by the minimum inhibitory
concentration (MIC) method to quantify the inhibitory concentration for each strain. The
values for the MIC provide a quantitative evaluation of the antimicrobial action of the sam-
ples against E. coli and S. aureus pathogens. The MIC is defined as the lowest concentration
(µg.mL−1 ) of an antimicrobial agent, which, under strictly in vitro conditions, completely
prevents the growth of the test strain of an organism (EUCAST, 1998) [41]. The MIC of
each sample showed the same inhibitory concentration of 400 µg.mL−1 for both bacteria S.
aureus and E. coli, and also showed a minimum inhibitory concentration for C. albicans of
400 µg.mL−1 . The broth microdilution method allowed the quantification of the minimum
inhibitory concentration; however, the same did not occur in the agar diffusion disk tests.
It can also be observed that, unlike the broth microdilution method, the three samples did
not present inhibitory activity in the disc diffusion tests against E. coli bacteria. The absence
of the inhibition zone does not necessarily indicate that the sample is inactive against the
tested strain but that the diffusion may be incomplete; this occurs especially in compounds
that diffuse more slowly in a solid culture medium or due to the lipophilic characteristics
or/and the chemical nature of the isolated substances [42,43].
The antibacterial efficacy of MgO samples has been explained by a set of factors that
inhibit bacterial growth, such as the production of reactive oxygen species (ROS) due to the
presence of Mg2+ , the interaction between MgO particles and the membrane cell wall, and
the penetration of individual particles into cells [39]. The antibacterial properties may also
be associated with the correlation between the surface area and volume of oxide particles,
which form more active oxygen species outside the bacterial cell, destroying the bacteria’s
cell membranes [8,20].
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Molecules 2023, 28, 142
3. Discussion
The samples synthesized in this work showed differences in their structural, morpho-
logical, and antimicrobial properties, associated with the different polysaccharide sources
used in each type of synthesis. The three MgO samples presented the same crystalline
structure of the face-centered cubic type, characteristic of magnesium oxides; however,
the MgO-St had a smaller crystallite size compared to MgO-CP and MgO-Av. The same
structure was reported by Umaralikhan and Jaffar [25] and El-Shaer et al. [44]. El-Shaer
et al. synthesized MgO using the conventional sol–gel method and annealed it at different
temperatures for 5 h; the samples calcined above 500 ◦ C showed similar antimicrobial activ-
ity results as in this work. According to these authors, the calcination temperature affects
the number of surface defects and therefore the antimicrobial properties [44], justifying
the antimicrobial results presented for the MgO-St, MgO-Av, and MgO-CP samples in the
MIC test.
Some additional FTIR bands were found in the three synthesized oxides that resembled
the FTIR bands found by Karthik et al. (2017) [4] at 3445, 1383, and 1638 nm. According
to the authors, MgO was obtained with the same crystallographic phase by microwave-
assisted green synthesis and calcining at 400 ◦ C for 2 h. The results of the disk diffusion test
for MgO obtained by Karthik et al. [4] were superior to the results reported in this work.
As previously mentioned, the disk diffusion test is a qualitative method as it is limited
by the mobility of the oxide in the disks and does not present the inhibition results as a
concentration value. However, the disk diffusion test is a qualitative method as it is limited
by the mobility of the oxide in the disk, and the inhibition results are not directly connected
to the oxide concentration. Nevertheless, the MgO-St, MgO-CP, and MgO-Av samples in
the MIC test showed higher inhibition against E. coli, S. aureus, and C. albicans than the
samples reported in [45].
The sample morphology varied with the fuel used. MgO-St presented a sponge-
like morphology, while MgO-CP and MgO-Av presented pseudo-spheres and lamellar
morphologies, respectively. According to the literature [2,7], magnesium oxide presents
several possible morphologies, depending on the synthesis method employed or the fuel
used in the synthesis. In the work of Bindhu et al. [20], MgO nanoparticles were obtained
via a chemical precipitation reaction using magnesium nitrate and sodium hydroxide
calcinated at 400 ◦ C for 5 h. The particles were almost spherical in shape, with smooth
surfaces, and this morphology is very similar to that of MgO-CP. The antimicrobial activity
studied by Bindhu et al. [20] was tested against S. aureus using the disk diffusion method,
and the zones of inhibition were similar to those found in this work. However, the MgO
nanoparticles synthesized by Bindhu et al. [20] were not tested against E. coli, but rather
against the Gram-negative pathogen Pseudomonas aeruginosa.
The average particle diameter sizes for the synthesized samples ranged from 0.85 to
1.14 µm. For the MgO-St oxide, the average particle diameter size was 0.99 µm; this
sample was the most porous, justifying its higher antimicrobial activity [36]. According to
Ananda et al. [35], the presence of pores provides a large surface area, which supports high
antimicrobial activity. In addition, this high activity can also be associated with the higher
percentage of Mg ions in this sample, as a possible mechanism of antimicrobial action is
the release of Mg2+ ions, as cited earlier.
Both the antimicrobial activity and the crystalline structures of our samples were
very similar to those found in the literature; however, in this work, we used the low-
est calcination time, only one hour, while other studies have reported calcination times
greater than 5 h using traditional synthesis methods [20,44,45]. However, differences in the
samples’ morphology can be observed, which may explain the differences in the antimi-
crobial activity, associated with the small particle size and high porosity, favoring a larger
contact area.
43
Molecules 2023, 28, 142
44
Molecules 2023, 28, 142
Figure 6. General schematic diagram of synthesis using polysaccharides as gelling complexing fuels.
45
Molecules 2023, 28, 142
Agar using a sterile swab. The sample disks of MgO-St, MgO-CP, and MgO-Av were placed
on the agar surface and then incubated at 36 ◦ C (±1) for 24 h. The tests were performed
in triplicate, and the antibacterial and antifungal activity was evaluated by measuring the
diameters of halos of growth inhibition strains assayed (in mm). The obtained data were
analyzed by one-way analysis of variance (ANOVA) and t-test analysis. A value of p < 0.05
was considered to be statistically significant.
5. Conclusions
MgO samples were successfully obtained using three synthetic routes. The routes are
efficient, reproducible, and low-cost methods that use suitable fuels from renewable sources.
The single-phase periclase was identified for all MgO samples. The MgO-St sample showed
crystallinity and the highest percentage of magnesium (61.7%). These results reflect the
efficiency of controlling the microorganisms Staphylococcus aureus and Candida albicans seen
in the agar disk diffusion method. Moreover, the MIC method showed the concentration of
inhibition of the studied microorganisms. The most satisfactory results were observed for
the antibacterial and antifungal tests with the MgO-St sample. This study presents three
promising and low-cost synthesis methodologies for the preparation of antibacterial and
antifungal MgO materials.
Supplementary Materials: The following supporting information can be downloaded at: https:
//www.mdpi.com/article/10.3390/molecules28010142/s1, Figure S1: Elemental analysis of the MgO
samples MgO-ST, MgO-Av, and MgO-CP. The Identification was performed using the Mg kα line
located at 1.25 keV; Figure S2: Elemental analysis of the organic precursors and magnesium salt. The
percentage of Mg presented by the precursors was 6.6% for starch, 0.1% for Aloe vera, and 1.7% for
citrus pectin. The Mg(NO3 )2 .6H2 O salt used in the three synthetic routes showed purity of 98% of
magnesium. Identification was performed using the Mg kα line located at 1.25 keV.
Author Contributions: Conceptualization, N.B., F.J.A. and J.d.O.P.; methodology, N.B. and J.d.O.P.;
validation, N.B., D.F.L.H. and J.d.S.C.; formal analysis, N.B., J.d.S.C., J.d.O.P., S.J. and C.B.; inves-
tigation, N.B., J.d.S.C. and C.M.Z.; resources, C.B. and F.J.A.; data curation, N.B., J.d.O.P., D.F.L.H.
and J.d.S.C.; writing—original draft preparation, N.B. and J.d.O.P.; writing—review and editing,
C.B., F.J.A. and C.M.Z.; visualization, N.B.; supervision, F.J.A. and C.B.; project administration, F.J.A.;
funding acquisition, F.J.A. and C.B. All authors have read and agreed to the published version of
the manuscript.
Funding: N.B. acknowledges CAPES (grant number 88887.628497/2021-00) for a graduate schol-
arship. F.J.A. is grateful for a CNPq Productivity grant (308625/2019-6) and the grant CNPq
(427127/2018-1). C.B. is a research associate for FR-FNRS, Belgium.
Institutional Review Board Statement: Not applicable.
46
Molecules 2023, 28, 142
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49
molecules
Article
Visible-Light-Enhanced Antibacterial Activity of Silver and
Copper Co-Doped Titania Formed on Titanium via Chemical
and Thermal Treatments
Kanae Suzuki 1 , Misato Iwatsu 2 , Takayuki Mokudai 3 , Maiko Furuya 2 , Kotone Yokota 2 , Hiroyasu Kanetaka 2 ,
Masaya Shimabukuro 4 , Taishi Yokoi 4 and Masakazu Kawashita 4, *
Abstract: Dental implants made of titanium (Ti) are used in dentistry, but peri-implantitis is a serious
associated problem. Antibacterial and osteoconductive Ti dental implants may decrease the risk of
peri-implantitis. In this study, titania (TiO2 ) co-doped with silver (Ag) at 2.5 at.% and copper (Cu) at
4.9 at.% was formed on Ti substrates via chemical and thermal treatments. The Ag and Cu co-doped
TiO2 formed apatite in a simulated body fluid, which suggests osteoconductivity. It also showed
antibacterial activity against Escherichia coli, which was enhanced by visible-light irradiation. This
enhancement might be caused by the synergistic effect of the release of Ag and Cu and the generation
of •OH from the sample. Dental implants with such a Ag and Cu co-doped TiO2 formed on their
surface may reduce the risk of peri-implantitis.
50
Molecules 2023, 28, 650
penetrates the gingiva and activates the photosensitizer within the gingival sulcus to kill
bacteria that reside around the gingival sulcus [25]. Therefore, Ti dental implants with
doped TiO2 on their – surfaces can reduce the risk of peri-implantitis with periodic or on-
demand irradiation of visible light at a dental clinic. We previously prepared N-doped
TiO2 [26–29] and Cu-doped TiO2 [30] on Ti and investigated their surface structure, apatite
formation ability in an SBF, and antibacterial activity. However, it is necessary to improve
the antibacterial activity of N-doped or Cu-doped TiO2 . One possible approach to achieve
this is to increase the N or Cu content, but our method limits the amount of N or Cu that can
be doped into TiO2 to improve photocatalytic antibacterial activity and apatite formation
ability [28,31].
Therefore, in this study, we tried to co-dope silver (Ag) and Cu into TiO2 . The excellent
antibacterial properties of Ag are expected to improve the antibacterial activity of the dental
implants with or without visible-light irradiation. The antibacterial activity of samples is
discussed in terms of their photocatalytic activity and the release of Ag and Cu from the
samples. The present findings will contribute to the development of dental implants with
antibacterial activity to prevent peri-implantitis with and without visible-light irradiation.
AG-CU contained Ag at 2.5 at.% and Cu at 4.9 at.% on its surface, and AG contained
Ag at 6.3 at.% on its surface (Table 1). AG-CU contained almost twice as much Cu as Ag.
The amount of Ag in AG was higher than that in AG-CU, which can be attributed to the
higher concentration of Ag in the silver nitrate (AgNO3 ) solution used for the treatment
of AG (∼
=1 mM) compared to that (∼ =0.5 mM) used for the treatment of AG-CU. Although
the concentrations of Ag and Cu in the AgNO3 -Cu(NO3 )2 mixed solution used for the
treatment of AG-CU were the same (∼ =0.5 mM), AG-CU contained almost twice as much Cu
as Ag on its surface. These results indicate that Ag and Cu can be co-doped into a sample by
using an AgNO3 -Cu(NO3 )2 mixed solution, but the amount of Ag doped into the sample
will not be simply proportional to the Ag concentration of the Ag- and Cu-containing
solution used for treatment.
51
Composition (at.%)
Sample
O Ti Ag Cu C
AG-CU 59.2 ± 0.6 31.0 ± 0.8 2.5 ± 0.2 4.9 −± 0.6 2.3 ± 0.2
− AG 59.0 ± 1.2 31.7 ± 0.7 6.3 ± 1.8 − 3.1 ± 0.3
−: not measured.
3d3/2 2p3/2
Satellite
AG-CU
AG-CU
AG AG
380 376 372 368 364 970 960 950 940 930 920
Binding energy (eV) Binding energy (eV)
Cu 2p3/2 peaks were observed at around 933.0 eV for AG-CU, whereas no Cu 2p3/2
peak was observed for AG. The Cu 2p3/2peak peakaround
around933.0
933.0eV
eV and
and the
the α’
α’ value
value around
around
1850.4 eV suggest that Cu mainly existed as Cu2 O on the surface of AG-CU [37]. These
results indicate that copper was successfully doped into the sample surface by the present
surface treatments. The lack of a TF-XRD peak corresponding to copper compounds for
AG-CU (Figure 1b) and the apparent Cu 2p peak (Figure 2b and Table 1) indicate that the
crystallinity of the doped copper was low for both samples. The formation of Cu2 O with
low crystallinity in AG-CU is interesting, but its mechanism is unclear. This topic is worthy
of further investigation.
The SEM images of the samples after immersion in the SBF (Figure 3) indicate that
apatite uniformly formed on the surface of AG, whereas it partially formed on the surface
of AG-CU. This difference in apatite formation ability between these samples is consistent
with the intensity of the TF-XRD peak of apatite at the 2θ angle of around 32◦ being much
smaller for AG-CU than for AG. The relationship between apatite formation ability in an
SBF and the surface structure of TiO2 formed on Ti [13,31,38–41] or TiO2 gels [42] is not
fully understood; nevertheless, in this study, the higher formation of anatase compared to
that of rutile in AG (Figure 1b) may be responsible for the better apatite formation ability
of AG.
52
θ
Figure 3. (a) SEM images and (b) TF-XRD patterns of samples after immersion in SBF for 7 days.
A slightly higher amount of Ag was released from AG-CU than from AG. The Ag
concentration in PBS reached around 8 µM for 3 days (Figure 4a). However, the Ag
concentration was saturated at around 7 days, which indicates that the release of Ag from
AG-CU almost stopped at around 7 days. In contrast, AG released Ag gradually and
continuously for 28 days. AG-CU slowly released Cu; the Cu concentration reached around
3 µM by day 28. These results indicate that AG-CU preferentially releases Ag over Cu, but
the release of Ag is almost stopped at around 7 days even though AG continuously releases
Ag for 28 days. Figure 4b was obtained by plotting the Ag and Cu concentrations against
the square root of the soaking period. The concentration of Ag released from AG-CU within
3 days and that released from AG within 28 days are proportional to the square root of
the soaking period. This result suggests that Ag was released from both samples via ion
exchange [30,43], although the rate and duration of Ag release were different between the
samples. The concentration of Cu released from AG-CU within 28 days is also proportional
to the square root of the soaking period, which suggests that Cu was released from AG-CU
via ion exchange [44]. However, the mechanism of Ag and Cu release from samples should
be further investigated because Ag and Cu were mainly present as metallic silver with an
oxidized surface and Cu2 O, respectively (Figures 1 and 2, and Table 2), and they are not
likely to be released via ion exchange. The slightly more rapid release of Ag from AG-CU
than from AG and the continuous release of Cu from AG-CU may lead to antibacterial
activity that is somewhat strong at the initial stage of implantation and continues for a
long period.
Figure 4. Ag and Cu ion release behavior from samples in PBS. (a) Accumulated-released amounts of
Ag or Cu vs. soaking period and (b) accumulated-released amounts of Ag or Cu vs. square root of
soaking period.
53
Molecules 2023, 28, 650
AG-CU and AG compared to untreated Ti, and AG-CU showed extremely strong antibacte-
rial activity under visible-light irradiation. The number of viable bacteria on untreated Ti
(control) decreased under visible-light irradiation. Although an LED generates much less
heat than a conventional incandescent bulb, the decrease in the number of viable bacteria
on untreated Ti under visible-light irradiation may be attributed to the heat generated by
the LED light, which was placed only 10 cm−2from the sample and had a high intensity of
250 W·m−2 . Here, we briefly discuss the changes in the oxidation state of copper after
antibacterial activity testing. Although XPS spectra of AG-CU after antibacterial testing
should be measured to clarify the change in oxidation state of copper in the future, it is
possible that Cu2+ is formed from Cu2 O on the surface of AG-CU after antibacterial testing
because the proportion of Cu2+ on the surface of copper metal increases after soaking in a
bacteria-containing solution, and Cu2+ is the most stable chemical state against corrosion
and bacteria [37].
: without visible-light irradiation
: with visible-light irradiation
106
*
Viable bacteria count (CFU·mL−1)
a
105 *
A b
104
c *
B
103
102
101
B
100
Control AG AG-CU
(untreated Ti)
Figure 5. Number of viable bacteria for samples under conditions with and without visible-light irra-
diation. Bars with different letters (lowercase a–c for no
– visible-light irradiation group and uppercase
A and B for visible-light irradiation group) are significantly different (p < 0.01). Asterisk (*) represents
significant differences (p < 0.01) between no visible-light irradiation and visible-light irradiation.
54
Molecules 2023, 28, 650
Table 3. Concentrations of hydrogen peroxide (H2 O2 ) and hydroxyl radical (•OH) for samples.
Concentration (µM)
Sample
H2 O2 •OH
AG-CU 8.0 × 10−2 2.5
AG 8.9 × 10−2 1.3
Control (untreated Ti) 8.9 × 10−2 1.4
55
Molecules 2023, 28, 650
56
Molecules 2023, 28, 650
4. Conclusions
TiO2 co-doped with Cu and Ag was formed on the surface of Ti via NaOH-(Cu(NO3 )2
and AgNO3 ) and heat treatments. The TiO2 co-doped with Cu and Ag formed apatite
on its surface in an SBF and showed higher antibacterial activity than that of TiO2 doped
with only Ag, especially under visible-light irradiation. The excellent antibacterial activity
of TiO2 co-doped with Cu and Ag under visible-light irradiation might be caused by the
synergistic effect of the release of Ag and Cu and the generation of •OH from the sample.
The toxicity of the sample needs to be evaluated in future studies, but dental implants with
such a TiO2 surface layer co-doped with Cu and Ag may reduce the risk of peri-implantitis.
Supplementary Materials: The following supporting information can be downloaded at: https:
//www.mdpi.com/article/10.3390/molecules28020650/s1, Figure S1: Electron spin resonance (ESR)
spectra of control, AG, and AG-CU samples.
Author Contributions: Conceptualization, M.K.; methodology, K.S., T.M., M.I., M.F., K.Y., H.K. and
M.S.; validation, K.S., T.M., H.K., M.S., T.Y. and M.K.; formal analysis, K.S., T.M., M.F., K.Y. and
M.S.; investigation, K.S., T.M. and M.S.; data curation, K.S., T.M., M.F. and K.Y.; writing—original
draft preparation, K.S.; writing—review and editing, M.K.; supervision, M.K.; project administration,
M.K.; funding acquisition, M.K. All authors have read and agreed to the published version of
the manuscript.
57
Molecules 2023, 28, 650
Funding: This research was funded by the Japan Society for the Promotion of Science KAKENHI
[grant number: JP16H03177], the Kazuchika Okura Memorial Foundation, and the Institute of Bioma-
terials and Bioengineering, Tokyo Medical and Dental University [Project “Design and Engineering
by Joint Inverse Innovation for Materials Architecture”] of the Ministry of Education, Culture, Sports,
Science and Technology, Japan.
Institutional Review Board Statement: Not applicable.
Informed Consent Statement: Not applicable.
Data Availability Statement: The data will be available on request.
Conflicts of Interest: The authors declare no conflict of interest.
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59
molecules
Article
Electroanalysis of Ibuprofen and Its Interaction with Bovine
Serum Albumin
Muhammad Dilshad 1 , Afzal Shah 1, * and Shamsa Munir 2
Abstract: The current work presents a sensitive, selective, cost-effective, and environmentally benign
protocol for the detection of ibuprofen (IBP) by an electrochemical probe made of a glassy carbon
electrode modified with Ag-ZnO and MWCNTs. Under optimized conditions, the designed sensing
platform was found to sense IBP up to a 28 nM limit of detection. The interaction of IBP with
bovine serum albumin (BSA) was investigated by differential pulse voltammetry. IBP−BSA binding
parameters such as the binding constant and the stoichiometry of complexation were calculated. The
results revealed that IBP and BSA form a single strong complex with a binding constant value of
8.7 × 1013 . To the best of our knowledge, this is the first example that reports not only IBP detection
but also its BSA complexation.
1. Introduction
Drugs facilitate the prevention and cure of diseases by strengthening the immune
system. The mechanism of drug action is a specific biochemical interaction that results
Citation: Dilshad, M.; Shah, A.; in targeted pharmacological effect. This action includes binding of the drug molecule
Munir, S. Electroanalysis of Ibuprofen to a specific targeted biological species such as enzymes or receptors [1–4]. Overdosage
and Its Interaction with Bovine of drugs can result in adverse short-term or long-term health effects [5]. Drugs affect
Serum Albumin. Molecules 2023, 28, or alter the physiology of living organisms [6]. They stimulate a biological reaction by
49. https://doi.org/10.3390/ targeting macromolecules in the body [7]. As a rule, most drugs impede a particular
molecules28010049 biological response by interfering with the neurological system (particularly the brain).
Academic Editors: Minas
Based on pharmacodynamics, drugs can be classified as depressants, hallucinogens, and
M. Stylianakis, Athanasios Skouras
stimulants. Drugs can also be classified into analgesics and therapeutics. The current work
and Antonella Curulli presents electroanalysis of a non-steroidal anti-inflammatory drug (NSAID), ibuprofen
(Scheme 1), which is the third most popular, prescribed, and sold-over-the-counter drug
Received: 11 November 2022 in the world [8]. The World Health Organization has listed ibuprofen (IBP) as an “essential
Revised: 13 December 2022
drug” [9]. It is extensively used as a pain reliever in conditions such as menstrual cramps,
Accepted: 18 December 2022
headaches, arthritis, and a wide variety of other common aches and pains [10,11]. It plays an
Published: 21 December 2022
anti-inflammatory role by prohibiting the production of pro-inflammatory prostaglandins
through inhibition of the enzyme cyclooxygenase [12].
60
Molecules 2023, 28, 49
IBP enters into the environment due to improper pharmaceutical disposal during
treatment. IBP manufacturing industries are a major contributor to the entrance of this
drug into bodies of water. The sources of IBP water pollution can be seen in Figure S1.
The toxicity of IBP metabolites exceeds that of the parent molecule. After excretion, IBP
makes its way into wastewater treatment plants, sewage treatment plants, rivers, lakes,
groundwater, soil, etc. A number of methods have been proposed for determining the
concentration and effects of IBP in aquatic organisms [13]. To examine the short-term and
long-term effects of IBP exposure, toxicity tests have been conducted on water-dwelling
species. IBP is an emerging organic contaminant as its risk quotient is quite high [14]. Hence,
part of the current work is focused on designing a sensitive electrochemical platform for
the detection of IBP.
The protein–drug binding process involves complexation of a drug with protein.
Protein–drug binding can be intracellular or extracellular. Intracellular binding involves
the drug’s interaction with cell proteins, eliciting a pharmacological response; the receptors
with which the drug interacts are known as primary receptors. Extracellular binding does
not usually result in a pharmacological response, and such drug receptors are known as
secondary or silent receptors. The nature of a drug binding to a protein can be reversible
or irreversible [15]. IBP pharmacokinetically interacts with BSA, which is a natural and
very abundant (59%) plasma protein. BSA has a high affinity for binding with drug ligands
and metabolites [16]. As shown in Figure 1, BSA has three domains (I, II, and III) and two
sub-domains (A and B). The predicted drug-binding site is present in the sub-domains II A
and III A.
ALB
(D β ),
ALB
D 61
ALB + mD ⇔ ALB − mD
Molecules 2023, 28, 49
Consider albumin (ALB) as the host protein that forms a complex with the guest drug
(D). The binding constant (βs ), also known as the affinity constant or association constant,
is determined from the stoichiometry of complexation between ALB and D according to
the following equation [17]:
βs
ALB + mD ⇔ ALB − mD (1)
Here ALB − mD represents the protein drug complex, and the stoichiometric co-
efficient m indicates the number of drug molecules interacting per single molecule of
protein. [FD] is the concentration of the free drug. The square brackets indicate the
concentration of that particular species in Equation (2).
At equilibrium, βs can be obtained by:
[ALB − mD]
βs = (2)
[ALB][FD]m
∆I [ALB − mD]
=
∆Imax − ∆I [ALB]
The right side of the above equation is equal to βs [FD ]m according to Equation (2). Hence,
∆I
= βs [FD]m
∆Imax − ∆I
∆I
Log = Log βs [FD ]m
∆Imax − ∆I
∆I
Log = Log βs + m Log [FD] (6)
∆Imax − ∆I
∆I
A linear relationship between Log ∆Imax −∆I and Log [FD] indicates the formation of
a single drug–protein complex. On the other hand, a non-linear relationship would
suggest
∆I
the formation of multiple complexes with different stoichiometry. If the Φ = Log ∆Imax −∆I
62
Molecules 2023, 28, 49
and Log [FD] plot shows two linear segments, then two types of complexes have been
formed, and this will result in slopes m1 and m2 . When multiple complexes are formed,
then the following equation can be used:
where ∆I is the total decrease of peak current Ip obtained through a current voltage measurement.
∆I
f1 =
[ FD ]
63
Molecules 2023, 28, 49
synthesized material was analyzed using energy dispersive X-ray spectroscopy (EDX). The
different elemental peaks such as Ag, Zn, O, S, and C were obtained as shown in Figure 2C.
According to EDX analysis, the ZnO NPs with Ag doping were predominantly made up
of Zn and O, with some trace amounts of Ag. Minor traces of S emerge from chemical
impurities, whereas the presence of C originates from the carbon tape employed in the
SEM analysis.
Figure 2. (A) X-ray diffractogram of Ag-ZnO, (B) SEM micrograph of Ag-ZnO, and (C) EDX spectrum
of Ag-ZnO nanoparticles.
4 −
Figure 3A shows the cyclic voltammograms for 5 mM [Fe(CN)6 ] obtained for the
modified GCEs in 0.1 M KCl supporting electrolyte. In these voltammograms, − the current
peak at 0.34 V corresponds to the oxidation of [Fe(CN)6 ]4− to [Fe(CN)6 ]3− , while the
current peak at 0.21 V corresponds to the reduction of [Fe(CN) 3− to [Fe(CN) 4−
6] 6 ] . From
− −
these voltammograms, the peak currents were found to be 100, 70, 48, and 25 µA for
− −
64
𝐼 = 2.69 × 10 𝑛 𝐴𝐷 𝑣 𝐶
Molecules 2023, 28, 49
The current values were employed to calculate the electroactive surface area, and the
Randles–Sevcik equation was utilized for both unmodified and modified electrodes [20].
3 1 1
I p = 2.69 × 105 n 2 AD 2 v 2 C (9)
where Ip represents the anodic − peak current in amperes, D represents the diffusion coeffi-
cient of the analyte in cm s , A− is the electroactive surface area in cm2 , v is the scan rate
2 − 1 −
with a potential scan rate of V s , C represents the concentration of the probe in mol cm 3 ,
− 1 −
For [K3 Fe(CN)6 ], D = 7.6 × 10 cm2 s−1 and n = 1. Table S1 demonstrates the elec-
6
Figure 3. (A) Cyclic voltammograms for bare and modified glassy carbon electrodes recorded in
5 mM K3 [Fe(CN6 )] with 0.1 M KCl as supporting electrolyte at a scan rate of 100 mVs−−1 (B) Nyquist
plots of bare and modified GCEs in a solution of 5 mM K3 [Fe (CN)6 ] as a redox probe and 0.1 M KCl
as a supporting electrolyte, and (C) equivalent circuit corresponding to EIS data.
65
Molecules 2023, 28, 49
The method of electrochemical impedance spectroscopy did not impart any damage
to the tested material. Through EIS, charge transfer kinetics for both bare and modified
GCEs were investigated in a 5 mM solution of K3 [Fe(CN)6 ] in a 0.1 M KCl solution [21].
The DC potential at 0 V and 10 mV was set as the peak-to-peak amplitude of the AC
potentials superimposed on the aforementioned DC potential. Figure 3B depicts the
Nyquist plots that were produced at frequencies ranging from 100 kHz to 0.1 Hz with an
amplitude of 10 mV for the bare GCE, Ag-ZnO/GCE, MWCNTs/GCE, and MWCNTs/Ag-
ZnO/GCE. The diameter of the semicircle in the Nyquist plot between the imaginary
impedance (Z′′ ) versus real impedance (Z′ ) represents the resistance to charge transfer
(Rct ), while the linear part in the lower frequency region arises from diffusion limited
processes characterized by Warburg impedance (Wd ) [22,23]. The semicircular section
at a greater frequency corresponds to charge transfer resistance. A semicircle of smaller
diameter represents lower Rct , and vice versa [24]. Table S2 shows Rct values of 8173, 4277,
2627 and 1610 Ω for bare, Ag-ZnO-, MWCNTs- and MWCNTs/Ag-ZnO-modified GCEs,
respectively. It can be seen that Rct values decreased for modified electrodes as the surface
area of the electrodes increased. The increase in the surface area of the electrode owing
to adsorbed molecules of the MWCNTs/Ag-ZnO/GCE and its mediator role between the
transducer GCE and the redox probe caused the reduction in the Rct value. The availability
of the active sites on the GCE increased due to the presence of the Ag-ZnO and MWCNT
molecules. Immobilized molecules on the GCE surface link the analyte molecules to the
electrode [25]. The modifier molecules promote ease of electron transfer between the
analyte and the electrode. In addition, Ag-ZnO and MWCNTs decrease interfacial charge
transfer resistance. Therefore, the selected modifier renders the surface of the GCE an
excellent sensing substrate for detecting the analyte. The EIS-derived parameters in Table
S2 suggest that the impedance parameters have changed because of electrode modification.
This indicates that the electrode surface was successfully fabricated. By modifying the
electrode with the MWCNTs/Ag-ZnO/GCE, there is an increase in the electron transfer
rate between the analyte and the electrode and it decreases the charge transfer resistance.
The CV and EIS data are in good agreement. In CV, the MWCNTs/Ag-ZnO/GCE shows
maximum current, and EIS shows minimum resistance. Therefore, the MWCNTs/Ag-
ZnO/GCE was chosen as a reliable electroanalytical sensing platform for the detection of
the analyte IBP.
66
Molecules 2023, 28, 49
Figure 5. (A) Effect of different scan rates on the anodic peak current of IBP in supporting electrolyte
υ
(0.9 M NaOH), (B) calibration plot of IBP between log Ip vs. log υ, (C) plot of peak current vs. scan rates
of IBP oxidation, and (D) calibration plot of IBP between peak current vs. square root of scan rates.
ν
67
−
Molecules 2023, 28, 49
The slope value of the log of Ip and log of ν plot can be used to deduce the nature of the
redox process. If the value of the slope is 1, the process should be controlled by adsorption;
on the other hand, if the slope value is 0.5 the process should be controlled by diffusion [26].
The slope value of 0.89 as depicted in Figure 5B suggests that the electrochemical oxidation
of IBP is controlled by both processes [27]. The straight-line equation for the graph shown
in Figure 5B is represented by log Ip = 0.89 and log v − 6.57. Since the correlation coefficient
in the plot of oxidation peak current vs. scan rate is higher (Figure 5C) than that of Ip vs.
1
ν 2 (Figure 5D), the process of adsorption works better on the electrode’s surface.
68
Molecules 2023, 28, 49
peak current is seen at the point of saturation, which occurs when the analyte has become
oriented to all of the available active sites. The molecules of the analyte need to be aligned
in the right direction for the best possible deposition. The reduction of drug molecules
occurs when they are accumulated on the electrode surface. These molecules undergo
an oxidation process in the anodic stripping differential pulse voltammograms when the
potential is varied from negative to positive values. At a deposition time of 30 s, maximum
peak current is observed for IBP. The peak current intensity of the analyte is negatively
influenced by further increasing the deposition time, as evidenced in Figure S4B.
Figure 6. (A) Differential pulse voltammograms for different concentrations of IBP using an
−
MWCNTs/Ag-ZnO/GCE under pre-optimized conditions at a scan rate of 10 mVs−1 , (B) calibration
plot obtained by DPV data for various concentrations of IBP.
One particularly interesting aspect of the developed sensor is its ability to function
over a wide linearity range, from about 0.1 μ µM to around μ 90 µM (Figure 6B). It can be
seen from Table 1 that the LOD value of 28 nM is significantly lower than the previously
reported data for the different designed sensors. Therefore, it can be concluded that the
modified electrode is a promising platform for the analytical detection of IBP.
69
Molecules 2023, 28, 49
Measurement LOD
Sensors Linear Range (µM) Ref.
Technique (nM)
Pretreated GCE SWSV 1.45–3.87 960 [28]
SD-MWCNT/GCE FIA-AMP 10–1000 1900 [29]
Polyaniline nanofiber/GCE DPSV 0.96–1.94 480 [30]
P(L-Asp)/GCE SWV 1–150 220 [31]
MWCNT–CPE DPV 2.36–242 9100 [32]
Clay-CPE DPV 1–1000 835 [33]
HKUST-CNF CV 4.84–29.08 100 [34]
Pd-PdO/Mt-CPE DPV 0.01–0.9 28 [35]
AgNPs@Af-GO-MIP/GCE DPV 1–100 8.7 [36]
MWCNTs/Ag-ZnO/GCE DPV 0.1–90 28 This work
Figure 7. (A) DPVs of IBP showing reproducibility of the designed sensor using supporting electrolyte
NaOH (B) DPVs of IBP for the designed sensor at different time intervals.
70
Molecules 2023, 28, 49
71
Molecules 2023, 28, 49
For interaction studies of IBP with BSA, DPV is superior to linear scan voltammetry
because of its sensitivity and ability to reduce the comparatively high background currents
caused by the presence of albumin in solution [39]. For binding constant determination, all
the values for peak current and its punctual difference, free drug concentration, binding
constant, stoichiometry, etc., were repeated multiple times and reported values represent
their means (with RSD±10% of the given values). DPV was carried out for various concen-
trations of IBP in the presence of 0.9 M NaOH in a potential window ranging from 0.4 V to
1.7 V at a scan rate of 10 mV s−1 and step potential of 5 mV (Figure 9A). IBP concentration
was varied, and with decreasing concentrations peak current decreased as expected. The
voltammograms were first obtained in the absence of BSA. Then, BSA in large excess (1 mM)
was added to a solution of the drug, along with 0.9 M NaOH as supporting electrolyte.
Figure 9B depicts the voltammograms obtained in the presence of BSA. The peak current
was significantly decreased compared to the peak current of voltammograms obtained in
the absence of BSA. For example, at 0.23μ µM IBP,μthe addition of BSA decreased the current
from 0.55 µA to 0.09 µA. The decreasing peak current indicates interaction between IBP
and BSA, leaving less free IBP in solution for electrochemical oxidation at the electrode. A
maximum decrease in peak current occurs when the maximum amount of the drug reacts
with protein. Hence, the decrease in peak current is attributed to the interaction between
the drug and BSA.
From the differential pulse, voltammograms recorded in Figure 9A,B show current
values in the absence and presence of BSA; their punctual differences were calculated as
listed in Table S3.
Figure 9. (A) DPV of IBP recorded with a supporting electrolyte of 0.9 M NaOH at different concen-
trations in the absence of BSA (B) DPV of IBP recorded with a supporting electrolyte of 0.9 M NaOH
at different concentrations in the presence of 1 mM BSA.
The voltammetric calibration curve of IBP in 0.9 M NaOH was registered with IBP
concentrations (Cdrug ) in the range of 0.01 to 0.23 μµM (Figure 10A). The resulting linear
plot ITD vs. Cdrug was obtained, where ITD represents current due to the total amount of
the drug (see Figure 10A). In the same way, a voltammetric calibration plot with the same
amounts of the drug (Cdrug ) and a fixed amount of albumin was recorded ([ALB]total),
operated with an albumin concentration of 1 mM. The resulting linear plot IFD vs. Cdrug
was obtained, where IFD represents the current of the free drug. For any point on the
calibration curve, we may calculate the difference between the two values at that point Δ
(∆I −= ITD − IFD ), because the complex is not electroactive under operating conditions.
72
Molecules 2023, 28, 49
Figure 10. (A) Relationship of peak current vs. drug concentration for IBP and no BSA, NaOH 0.9 M, and
5µM BSAμ+ NaOH 0.9 M, and punctual difference between the values of ITD and IFD (∆I = ITDΔ− IFD )
− relationship between Φ andФlog [FD] in the case of IBP with BSA, an example of a formation with
(B)
a single type of complex.
The concentration of free drug [FD] was evaluated by considering the calibration plot
obtained in the absence of BSA with data given in Table S4. By plotting all values of log
[FD] vs Φ,
Ф a linear plot was obtained (Figure 10B) according to equation 6 mentioned in
the introduction. The value of m determined from the slope shows the number of drug
molecules interacting per single molecule of BSA. The binding constant (βs ) withβa value
) of
8.7 × 1013 was evaluated
× 1013 from the antilog of the intercept. A comparison of the binding
constant of IBP−BSA and its−stoichiometry with reported values is given in Table 2. A
reasonably stronger binding of IBP with BSA is required to inhibit the functioning of BSA.
The comparison of binding constant values for various protein–drug complexes shows that
IBP−BSA has the highest− value of binding constant, thus indicating effective inhibition of
BSA by IBP.
Complex
Drug Complexes Binding Constant Ref.
Stoichiometry (m)
Ketoprofen-BSA 3 2.4 × 109 [40]
Ketoprofen-HAS 1 1.4 × 1010 [41]
Lorazepam-OVA 3 2.5 × 1010 [42]
Paroxetine-BSA 4 5.8 × 1018 [43]
Paroxetine-OVA 3 2.6 × 1023 [44]
Ibuprofen-BSA 3 8.7 × 1013 This work
3. Conclusions
A quick-responding, sensitive, and stable electrochemical sensor was developed using
a composite of MWCNTs and Ag-doped ZnO nanoparticles as a modifier of a GCE surface.
The designed sensor demonstrated excellent ability to detect IBP down to 28 nM. The
peak current response of IBP was greatly improved by the components of the recognition
layer in comparison to the bare GCE. Cyclic voltammetric and electrochemical impedance
spectroscopic investigations revealed that the designed sensing scaffold has 4.5 times
greater electroactive surface area and approximately 5 times less interfacial charge transfer
resistance as compared to the bare GCE, which results in the generation of an intense signal
73
Molecules 2023, 28, 49
for IBP oxidation. Voltammetric analysis revealed that the modified electrode possesses
inter-day durability and four individually fabricated electrodes exhibited unaltered effi-
ciency in terms of repeatability. The PG inhibition behavior of IBP was also investigated
by measuring its binding capacity with BSA using DPV, where it was revealed that three
molecules of IBP bind to a single molecule of BSA with a binding constant value of 8.7×1013 .
This strong binding potency of IBP suggests that it can play a significant role in PG inhi-
bition and in turn can be developed as a medicine for reducing pain and inflammation.
Considering the importance of IBP in the pharmaceutical industry and its medical potential,
new and innovative analytical methods with high efficiency are still needed to effectively
control this non-steroidal anti-inflammatory drug in pharmaceutical doses and to detect it
in biological fluids. Moreover, coupling such sensors with industries for the early sensing
of IBP in industrial effluents before their release to freshwater bodies may broaden their
future applicability.
4. Experimental Section
4.1. Materials and Methods
IBP was obtained from a bio-lab pharmaceutical company (Islamabad, Pakistan) and
was used as received. BSA and MWCNTs (purity > 95%) were obtained from Sigma
Aldrich. CH3 COOH, H2 SO4 , NaCl, NaOH, KCl, acetate buffer, Britton–Robinson buffer
(BRB), phosphate-buffered saline (PBS), and KOH were tested as supporting electrolytes.
Zinc acetate dihydrate and silver nitrate were purchased from Sigma Aldrich. PBS was
prepared by dissolving a specified amount of Na2 HPO4 and NaH2 PO4 in distilled water
and using 0.1 M HCl and 0.1 M NaOH for pH adjustment.
The nanomaterials were characterized using X-ray diffraction spectroscopy (X-ray
diffractometer model Analytical 30440/60 X per PRO with copper Kα radiation source, scan
rate of 0.01, and 2θ range from 10◦ –80◦ ) and scanning electron microscopy (JEOL.JAD-2300
module, Tokyo, Japan). A Metrohm multichannel Autolab (M101, PGSTAT302N, Utrecht,
The Netherlands) equipped with NOVA 1.11 software and Gamry Interface 5000E potentio-
stat were used for electrochemical measurements. The electrochemical cell consisted of a
glass cell with two layers of glass walls and a Teflon cover. The cap had five standard taper
ports; three of them were used for the introduction of electrodes (the Ag/AgCl reference
electrode, the working electrode, and the Pt auxiliary electrode), while the other two were
used for the entrance and exit of inert gas purging.
74
Molecules 2023, 28, 49
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77
molecules
Article
Chemico-Physical Properties of Some
1,1′ -Bis-alkyl-2,2′ -hexane-1,6-diyl-bispyridinium Chlorides
Hydrogenated and Partially Fluorinated for Gene Delivery
Michele Massa 1 , Mirko Rivara 2 , Thelma A. Pertinhez 3 , Carlotta Compari 2 , Gaetano Donofrio 4 ,
Luigi Cristofolini 5 , Davide Orsi 5 , Valentina Franceschi 4 and Emilia Fisicaro 2, *
1 Department of Maternal Infantile and Urological Sciences, Sapienza University of Rome, 00165 Rome, Italy;
[email protected]
2 Department Food and Drug, University of Parma, Parco Area delle Scienze, 27/A, 43124 Parma, Italy;
[email protected] (M.R.); [email protected] (C.C.)
3 Department of Medicine and Surgery, University of Parma, Via Volturno, 39, 43125 Parma, Italy;
[email protected]
4 Department of Veterinary Sciences, University of Parma, Via del Taglio, 10, 43126 Parma, Italy;
[email protected] (G.D.); [email protected] (V.F.)
5 Department of Mathematical, Physical and Computer Sciences, University of Parma, Parco Area delle
Scienze 7/a, 43124 Parma, Italy; [email protected] (L.C.); [email protected] (D.O.)
* Correspondence: [email protected]
Abstract: The development of very efficient and safe non-viral vectors, constituted mainly by cationic
lipids bearing multiple charges, is a landmark for in vivo gene-based medicine. To understand the
effect of the hydrophobic chain’s length, we here report the synthesis, and the chemico-physical
and biological characterization, of a new term of the homologous series of hydrogenated gemini
bispyridinium surfactants, the 1,1′ -bis-dodecyl-2,2′ -hexane-1,6-diyl-bispyridinium chloride (GP12_6).
Citation: Massa, M.; Rivara, M.; Moreover, we have collected and compared the thermodynamic micellization parameters (cmc,
Pertinhez, T.A.; Compari, C.;
changes in enthalpy, free energy, and entropy of micellization) obtained by isothermal titration
Donofrio, G.; Cristofolini, L.; Orsi, D.;
calorimetry (ITC) experiments for hydrogenated surfactants GP12_6 and GP16_6, and for the partially
Franceschi, V.; Fisicaro, E.
fluorinated ones, FGPn (where n is the spacer length). The data obtained for GP12_6 by EMSA,
Chemico-Physical Properties of Some
MTT, transient transfection assays, and AFM imaging show that in this class of compounds, the gene
1,1′ -Bis-alkyl-2,2′ -hexane-1,6-diyl-
bispyridinium Chlorides
delivery ability strictly depends on the spacer length but barely on the hydrophobic tail length. CD
Hydrogenated and Partially spectra have been shown to be a useful tool to verify the formation of lipoplexes due to the presence
Fluorinated for Gene Delivery. of a “tail” in the 288–320 nm region attributed to a chiroptical feature named ψ-phase. Ellipsometric
Molecules 2023, 28, 3585. https:// measurements suggest that FGP6 and FGP8 (showing a very interesting gene delivery activity, when
doi.org/10.3390/molecules28083585 formulated with DOPE) act in a very similar way, and dissimilar from FGP4, exactly as in the case
of transfection, and confirm the hypothesis suggested by previously obtained thermodynamic data
Academic Editor: Łukasz
Chrzanowski
about the requirement of a proper length of the spacer to allow the molecule to form a sort of
molecular tong able to intercalate DNA.
Received: 24 March 2023
Revised: 17 April 2023 Keywords: heterocyclic gemini cationic surfactants; non-viral vectors; gene delivery; partially
Accepted: 18 April 2023
fluorinated gemini surfactants; atomic force microscopy on DNA; DNA-surfactant interaction; DNA
Published: 20 April 2023
circular dichroism spectra; ellipsometry
78
Molecules 2023, 28, 3585
delivery has sparked great interest. The internalization of genetic material inside mam-
malian cells is only possible using vectors, both viral and non-viral. Non-viral vectors
are constituted by cationic lipids able to bind and compact the genetic material into soft
nanoparticles of tuneable size, so that it is protected against the action of endo- and ex-
onucleases present in physiological fluids. Very recently, the spreading worldwide of the
SARS-CoV-2 virus has focused scientific and economic efforts on searching for vaccines
to fight the virus. DNA- and RNA-based vaccines, able to express the spike protein when
internalized in cells, are now the most-used around the world [6,7]. Non-viral vectors are
in many cases preferred, particularly when RNA-based, which require only to overcome
the external cell membrane, instead of DNA-based which, needing to reach the nucleus for
expression, must be introduced inside the cells. In fact, viral vectors, notwithstanding their
superior efficiency, could give rise to adverse or immunogenic reactions or replications,
limiting their use. Biomedical and biopharmaceutical applications of cationic gemini am-
phiphiles as gene delivery vectors, together with their chemico-physical and aggregation
properties and the effect on the efficiency of transfection of their chemical structure have
been extensively reviewed [5,8,9]. Non-viral vectors, obtained by synthetic route, have
the advantage of being more reproducible than viral ones, and give us the possibility to
understand the effect of the various moieties constituting the molecule and, eventually, to
add to the vector a chemical group that can be recognized by the target cell. For many years
our research was devoted from a synthetic, thermodynamic, and biomedical point of view
to the study of new cationic gemini surfactants, having as polar head two pyridinium moi-
eties, with the aim to find out structure–activity relationships useful for the optimization
of their gene delivery ability, through the modulation of the lengths of the hydrophobic
chains and spacer, counterion, and other structural modifications [10–18]. Taking advan-
tage of this opportunity, we have considered not only hydrogenated gemini surfactants,
but also the corresponding partially fluorinated (otherwise called “hybrid surfactants”)
with the idea of obtaining non-viral vectors able to protect the genetic material also in
those biological fluids containing endogenous hydrogenated interfering surfactants, as
pulmonary surfactants or bile salts, able to destroy the lipoplexes before they enter inside
the diseased cells [19–21]. Fluorinated surfactants are at the same time highly hydrophobic
and lipophobic due to the structure of the fluorine atoms having larger van der Waals
radii and lower polarizability than the hydrogen atoms, and do not form mixed micelles
with hydrogenated surfactants [22–24]. Fluorination of cationic lipids has been proposed,
among others, in the treatment of cystic fibrosis and cystic fibrosis-associated diseases [20].
Initially, we have studied the solution thermodynamics of new compounds having spacers
of three, four, eight, and twelve carbon atoms, using direct methods and paying attention
to the trends of partial molar volumes, compressibilities, and enthalpies vs. concentra-
tion [10–18]. Particularly, the measurement of their solution enthalpies gives us the key
for understanding their transfection activity. Between the hydrogenated compounds,
those with spacers formed by four carbon atoms shows unexpected enthalpic proper-
ties vs. concentration, which was explained by a conformation change of the molecule
in solution due to stacking interactions between the two pyridinium rings. In this way,
the molecule behaves as a molecular tong, able to grip the DNA bases, giving rise to a
transfection activity comparable to that of the commercial reagent. If the hydrophobic
chain is modified by partial fluorination, a greater length of the spacer is needed to fold the
molecule. The comparison with the hydrogenated analogues reveals a greater ability of
the partially fluorinated compounds to compact DNA, but only in presence of DOPE. In
a recent paper [18], we tried to understand if the transfection ability is limited to a given
value of spacer length, or spans over a range of values, by synthesizing the compounds,
both hydrogenated and partially fluorinated, with spacer constituted by six methylene
groups, namely, 1,1′ -bis-hexadecyl-2,2′ -hexane-1,6-diyl-bis (pyridinium) chloride (GP16_6)
and 1,1′ -bis(3,3,4,4,5,5,6,6,7,7,8,8,8-tridecafluorooctyl)-2,2′ -hexane-1,6-diyl-bis (pyridinium)
chloride (FGP6) to fill the gap between active and non-active compounds. We have outlined
the completely different behaviour between hydrogenated and fluorinated compounds:
79
Molecules 2023, 28, 3585
in the case of hydrogenated compounds, only spacer four can deliver genes inside the
cell, independently of the use of DOPE. On the contrary, fluorinated compounds having a
spacer with six or eight methylene groups give rise to a very high transfection activity only
in the presence of DOPE. This result indicates that the spacer length appropriate for activity
falls in a broader, but always limited, range of values (spacer 12 is inactive for both classes
of compounds). It is known that the critical micelle concentrations (cmc) of the fluorinated
surfactants, determined by the balance of the hydrophobicity and hydrophilicity of the
molecule, are close to those of ordinary surfactants whose hydrocarbon chain lengths are
about 1.5 times longer [3]. This means that the -CF2 group is 1.5 times more hydrophobic
than the -CH2 group. To compare in a correct way the behaviour of FGP6 with the cor-
responding hydrogenated surfactants having a similar hydrophobicity of the alkyl chain,
we synthesized the new compound 1,1′ -bis-dodecyl-2,2′ -hexane-1,6-diyl-bis (pyridinium)
chloride, (GP12_6), similar to GP16_6, but with an alkyl chain 12 carbon atoms long. The
present paper reports the chemico-physical and tensidic properties of the compounds
with spacer 6, together with the synthesis and the biological characterization of the new
compound 1,1′ -bis-dodecyl-2,2′ -hexane-1,6-diyl-bis (pyridinium) chloride (GP12_6), to
evaluate the effect of the hydrophobic chain length.
80
Molecules 2023, 28, 3585
molar enthalpies with reference state of 20 mM. From these curves, the cmc and the mi-
celle formation enthalpy, ∆Hmic, are obtained using a pseudo-phase transition model. The
micellization free energy, ∆Gmic, is calculated from equation:
∆Gmic = RT ln cmc
where the cmc is expressed as mole fraction, without considering the degree of counterion
binding, ββ[28]. Moreover,
−
∆Smic = (∆Gmic − ∆Hmic )/T
Figure 1. Heat rate (µW) vs. time profiles obtained by injecting a 20 mM surfactant solution into a
200 µL reaction cell filled with water and (specified by a single quote mark) the dependence of the
dilution enthalpy change vs. surfactant concentration in the reaction cell for: (a) GP12_6; (b) GP16_6;
(c) FGP6; (d) FGP8.
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Molecules 2023, 28, 3585
The cmc values and the thermodynamic parameters for the micellization process so
obtained are reported in Table 1. Δ Δ Δ
− ∆H mic − mic
∆G ∆Smic
cmc [mM] xcmc 105
−[kJ mol−1 ] [kJ−mol−1 ] [J K−1 mol−1 ]
FGP6 0.89 1.603 − −6.1 −−27.4 71
FGP8 0.77 1.387 − −6.2 −−27.7 72
GP12-6 0.92 1.657 −6.5 −27.3 70
GP16-6 0.41 0.738 −14.0 −29.3 51
Data shown in Table 1 confirm the similarity in hydrophobicity of FPG6 and GP12_6,
allowing us to attribute the different behaviour in gene delivery ability exclusively to the
fluorinated moieties. Moreover, we recall that thermodynamic properties of hydrogenated
and fluorinated surfactants in solution, strictly related to the ability to compact DNA,
depend both on the difference in size between fluorine and hydrogen atoms (van der Waals
radii, F = 1.35 and H = 1.2 Å) and on the difference in electronegativity (Pauling scale,
F = 4.0 and H = 2.1) [24].
Figure 2. MTT tests on RD4 (left) and A549 cells (right) for GP12_6 (upper) and GP16_6 (lower)
from ref. [18]: (a) untreated cells; (b) 40 µM; (c) 20 µM; (d) 10 µM; (e) 5 µM; (f) 2.50 µM; (g) 1.25 µM;
(h) only DOPE. In gray, surfactant alone; in black, surfactant:DOPE = 1:2. Values are the mean ± S.D.
of three independent experiments (n = 8 per treatment, p < 0.05).
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Molecules 2023, 28, 3585
By analogy with similar compounds previously studied [18], we have tested the
newly synthesized compound on the human rhabdomyosarcoma RD4 cell line and on
adenocarcinoma human alveolar basal epithelial cells A549, used as models for the study of
lung cancer and for the development of drug therapies against it. Because this information
is needed for planning transfection experiments, we performed our cytotoxicity assays also
in presence of DOPE, which is essential in transfection experiments. Results in Figure 2
clearly show that the cytotoxicity of GP12_6 is very low on both kinds of cell lines, without
significative differences with and without DOPE and that the cytotoxicity increases with
increasing the hydrophobic chain length. The surfactant with longer tails interacts more
strongly with the cellular membrane, and their complexes with the DNA could penetrate
inside the cell more easily. However, their action is hindered by the increased cytotoxicity
for gene delivery purposes. In fact, it is reported that the biological activity of cationic
αω
surfactants increases with the chain length up to a critical point [30]. In the case of the
homologous series of alkanediyl-α,ω-bis-(dimethylalkylammonium bromide), the member
with 2 alkyl chains of 16 carbon atoms is generally the most biologically active [31].
Information about the ability of the compounds under investigation to interact with
DNA is obtained with EMSA experiments. Figure 3 upper shows that GP12_6 does not
shift the DNA, not even at the highest concentration used, whereas GP16_ shifts the DNA
at a concentration of 200 µM.
Figure 3. Upper: EMSA experiments showing complexation of GP12_6 with circular plasmid pEGFP-
C1 as a function of surfactant concentration (µM), compared with GP16_6 [18]. Only the plasmid
was used as a negative control, which is completely unshifted. Cationic lipid 100 µM corresponds
to N/P ≈ 1. Lower: AFM≈ images of DNA plasmid incubated with the hydrogenated compounds:
(a) plasmid + GP12_6; (b) plasmid + GP16_6; (c) plasmid alone as control. All images were obtained
with supercoiled 0.5 nM pEGFP-C1 plasmid deposited onto mica and with the microscope operating
in tapping mode in the air at N/P ratio = 0.5.
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Molecules 2023, 28, 3585
2.3. CD Spectroscopy
For better understanding the ability of the compounds under study to interact with
DNA, we have undergone CD measurements, which are very sensitive to structural changes
in biological molecules.
The pEGFP-C1 plasmid DNA far UV spectrum (220–320 nm) shows the contribution
of right-handed A- and B-forms: where the positive band is due to base stacking, and
the negative band is due to the polynucleotide right-handed helicity [32] (Figure 4). The
A-form is characterized by an intense positive band around 270 nm and a weak negative
band near 235 nm, and the B-form by a positive band around 280 nm and a negative at
240 nm.
Figure 4. CD spectra of DNA (black), and in the presence of GP12_6 (N/P = 1 red, N/P = 0.5 blue,
N/P = 0.25 green). In the insert: AFM image for N/P = 0.5 (see also Figure 3).
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Molecules 2023, 28, 3585
Upon the addition of surfactant to the DNA solution, different spectra were observed
depending on the surfactant type, the length of the chain, hydrophobicity, and the molar ratio.
A weak interaction was observed adding GP12-6 at all molar ratios, which produced a
shift and decrease in the positive band at 280 nm, attributed to the B-form (Figure 4).
GP16-6 affects the polynucleotide base stacking primarily. The spectra (Figure 5)
show the resolution of the convoluted original positive band of the two positive bands
characteristic of the A- (270 nm) and B- (283 nm) forms, respectively. We interpret this
change in the CD spectrum as the result of a decrease in the A-form and an increase in
the B-form.
Figure 5. CD spectra of DNA (black), and in the presence of GP16_6 (N/P = 1 red, N/P = 0.5 blue,
N/P = 0.25 green). In the insert: AFM image for N/P = 0.5 (see also Figure 3).
Differently, the interaction with FGP8-6 is more complex and depends on the molar
ratio (Figure 6). At a 1:1 molar ratio, the A-form decreases, as suggested by the reduction
of its characteristic bands. At a molar ratio of 1:2, there is an evident shift of the negative
band to the B-form and the convolution of the A- and B-form gives rise to positive bands.
At a higher molar ratio (1:4) a clear increase in the B-form (278 nm) is observed. The
appearance of a positive band at 288 nm wavelength (at 1:2 and 1:4 molar ratio) indicates
DNA compaction [33]. The “tail” in the 288–320 nm region depends on the presence
of FGP6 and it is attributed to a chiroptical feature named ψ-phase. Such a phase is a
consequence of the DNA structural transition from the B-form induced ψ by the surfactant
and reflects the supramolecular structure of the complex [34]. The plasmid is tightly packed
together, forming a highly condensed structure [35,36] as shown in the AFM image reported
in ref. [18]. In Supplementary Materials the deconvolutions of CD spectra (Figure S7) and
the table (Table S1) with the bands obtained using a gaussian model with Origin-Pro 2021
are reported for clarity.
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Molecules 2023, 28, 3585
Figure 6. CD spectra of DNA (black), and in the presence of FGP6 (N/P = 1 red, N/P = 0.5 blue,
N/P = 0.25 green). In the insert: AFM image for N/P = 0.5 from ref. [18].
The small film thickness ensures we are within the Drude approximation. In this ∆ case,
∆
the variation of ∆ is proportional to the film thickness multiplied by a factor proportional
to the difference between the refractive index of the film and that of the subphase (n = 1.33
for water as in the present case). It is, therefore, crucial to know the film’sΔrefractive index.
− Δ ≈ −1.5
Unfortunately, there are no data in the literature as these are newly synthesized molecules,
and it is difficult to predict it a priori.
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Molecules 2023, 28, 3585
∆
Figure 7. The variation of the ellipsometric phase angle ∆ as a function of surfactant concentration,
after 2 h of incubation, for the three surfactant molecules studied. Note the semilogarithmic scale.
A first estimate can be drawn based on the molecular structure: FGPs molecules
∆ of units, fluorinated and hydrogenated. Fluorinated alkyl chains, such
contain two types
as perfluorohexanoic acid (CAS 307-24-4) have n = 1.301, pyridine has n = 1.51, while
alkyl chains have typically n ≃ 1.40 at the relevant wavelength. We can attempt some
rough volumetric considerations, assuming the polar head comprising of pyridine and
alkyl spacer with thickness ∼ 3–4 A, and average refractive index 1.46–1.47 depending
on alkyl spacer length, while the gemini partially fluorinated chains, common to all the
molecules, account for ∼ 2 + 6 A of thickness and refractive index n = 1.40 and n = 1.30 for
the protonated and fluorinated chains, respectively.
𝑛 ≃ 1.40
Basing on such very broad considerations, we can estimate the molecular refractive
index to be not too far from n = 1.37–1.39. With these values, film thickness results to be of
∼ compatible with the formation of a monolayer at the interface. A
the order of 15–20 A, thus
more accurate estimate of film thickness could be made by using X-ray reflectometry, while
∼
neutron reflectometry could provide even more detailed insights into the film structure [37].
The relative weight of the fluorinated part (small refractive index n) in respect to the
hydrogenated part (big refractive index n) is larger in FGP4 than in FGP6 and FGP8. This
suggests that the refractive indexes of FGP4 is the smallest and closest to water, followed by
FGP6, while FGP8 is the largest. This explains, in part, why FGP4 has the smallest variation
of ∆.
At lower concentrations, however, the different molecules exhibit a very different
behaviour, with FGP6 and FGP8 displaying a lower variation of ∆ than FGP4. This cannot
be accounted for by the previous argument on the contribution of fluorinated chains to
the refractive index (which would predict a smaller variation for FGP4) and can only
be rationalized assuming that the films formed by different molecules have different
thicknesses: in films formed by FGP6 and FGP8 at low packing, thanks to the longer alkyl
spacer, the ∆
fluorinated chains may lay oblique and close to the water surface, while on the
contrary in FGP4 even at low coverage the molecules are forced to stand more vertical due
to the shorter alkyl spacer. ∆
Coherently with this explanation, we also find that in this regime of lower concen-
tration, the surface pressure ∏, i.e., the reduction of interfacial tension from its value for
the bare air-water interface γ = 72.6 mN/m, is greater for FGP6 and FGP8 than for FGP4,
especially at the lower concentrations (Figure 8).
87
Π
Molecules 2023, 28, 3585
𝛾 = 72.6 mN/m
Figure 8. Variation of the surface pressure ∏Πas a function of surfactant concentration in the bulk,
after 2 h of incubation, for the 3 surfactant molecules studied (filled symbols); for FGP4 and FGP8,
tensiometry data from ref. [16] have been included (empty symbols). Note the semilogarithmic
scale. The vertical dashed line indicates the maximum concentration considered for the formation of
non-viral gene delivery vectors.
In the same Figure, we also show the limiting pressures that would be reached by the
same surfactants at much higher concentrations, above their respective CMC values.
In the same Figure, we also report previously published data for FGP4 and FGP8
at concentration up to and above cmc, adapted from ref. [16]. In summary, it is also
important to notice that FGP6 and FGP8 act in a very similar way, and dissimilar from
FGP4, exactly as in the case of transfection. This suggests the presence of a dichotomy:
either the spacer is or is not long enough for the flexibility required by the molecule to
accommodate at the interface in the present case, or around a DNA fragment in the case of
interest for transfection.
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Molecules 2023, 28, 3585
′ ′
Figure 9. Chemical
′ structure of the ′compounds under study with spacer six methylene long.
Synthesis of 1,1′ -bis-dodecyl-2,2′ -hexane-1,6-diyl-bispyridinium chloride: 1,6-bis(2-
pyridyl)hexane (1.62 g, 0.007 mol) was dissolved in 10 mL ′ of DMF. The temperature
′ was
raised to 150 ◦ C and dodecyl chloride (14.3 g, 0.07 mol) was slowly added to the solution.
The reaction mixture was stirred for 18 h and the DMF was evaporated under reduced
pressure. The residue was suspended in diethyl ether and crystallized two times from
′
acetonitrile/toluene giving yellow′ crystals. The synthesisδ is reported in Schemeδ 1. Yield
δ 1 δ δ δ δ
2.64 g (58%); H-NMR (400 MHz DMSO-d6 ): δ 0.86 (t, J = 7.9 Hz, 6H); δ 1.23 br s, 40H); δ
δ δ δ 3.09 (m, 4H); δ 4.56 (t, J = 8 Hz, 4H); δ 8.00 (m, 4H); δδ 8.50 (t,
1.72 (m, 4H); δ 1.84 (m, 4H);
J = 8 Hz, 2H); δ 9.03 (d, J = 8 Hz, 2H). 13 C NMR (100 MHz, DMSO-d6 ): δ 158.2, 149.3, 136.7,
ν 121.3, 55.8, 44.2, 34.4, 32.7, 31.7, 29.5, 28.8, 26.7, 24.4, 22.6, 14.4, 13.8. FT-IR: ν = 3479,
123.6,
3432, 2912, 2849, 1638, 1470, 1012, 933, 715 cm–1 . MS-ESI: m/z = 614 [M-Cl]. Anal. Calcd.
For C40 H70 Cl2 N2 (649.91): C 73.92, H 10.86, N 4.31. Found δ δ
C 74.09, H 10.72, N 4.12.
δ δ δ δ δ
δ δ δ
The NMR spectra were recorded using a Bruker 400 Avance (Billerica, MA, USA) spectrometer.
1 H NMR Spectra (400 MHz) chemical shifts (d scale) are reported in parts per million
(ppm) and reported in order: multiplicity and number of protons; signals were character-
ized as s (singlet), d (doublet), t (triplet), m (multiplet), br s (broad signal).
13 C NMR (100 MHz); chemical shifts (d scale) are reported in parts per million (ppm).
′ ′
IR spectra were recorded using an Agilent Technologies Cary 630 FTIR Spectrometer
in the region 700–4000 without KBr support.
Mass spectra were recorded using an Applied Biosystem/MDS SCIEX API-150 EX
instrument (Waltham, MA, USA).
The new compounds were analyzed on a ThermoQuest (Rodano, Italy) FlashEA
1112 Elemental Analyzer, for C, H, N. The percentages recorded were within 0.4% of the
theoretical values.
The NMR, IR and mass spectra can be found in Supplementary Materials (Figures S1–S4).
89
Molecules 2023, 28, 3585
90
Molecules 2023, 28, 3585
F
γ=
l cos(θ)
where l is the wetted perimeter of the Wilhelmy plate and θ is the contact angle between
the liquid phase and the plate. In all the measurements reported here it was found θ = 0,
i.e., complete wetting conditions [38].
3.8. Ellipsometry
Ellipsometry measurements were performed using a multipurpose apparatus (Mul-
tiskop Optrel, Berlin, Germany) with a single wavelength of 632.8 nm. This technique
was employed to evaluate the thickness of the surface layer, always in the Drude approx-
imation [39]. The optical model adopted was that described in detail in ref. [40]. Film
thickness was probed by the variation of the ellipsometric phase-angle δ∆ which, in the
Drude approximation, ref. [39], is linearly proportional to film thickness via a scaling factor
depending on the incidence angle and refractive indexes. This specific apparatus provides
an average thickness value calculated on an area of a few square millimetres.
4. Conclusions
Gene-based medicine is a clinical reality initially thought to fight against acquired
or inherited genetic diseases and, after the pandemic spreading of the SARS-CoV-2 virus,
well-known worldwide due to the use of vaccines based on viral DNA or mRNA. A
great discussion is still open among scientists about the efficacy and the safety of this
medical treatment, and research in this field is increasing more and more. Regardless of the
kind of genetic material to be introduced inside the cells, it must be delivered by means
of vectors, and the development of very efficient and safe non-viral vectors, avoiding
immunogenic reaction and viral replication, is a landmark for in vivo gene-based medicine.
We have devoted many years of our research to the synthesis, and chemico-physical and
biological characterization, of gemini bis-pyridinium surfactants, both hydrogenated and
partially fluorinated, and the results obtained confirm that pyridinium gemini surfactants,
particularly those partially fluorinated, could be valuable tools for gene delivery purposes,
but their performance highly depends on the spacer length and is strictly related to their
structure in solution. To find out structure–activity relationships necessary to optimize
their performance, we are collecting their chemico-physical properties in relation to their
biological behaviour. The synthesis of the new hydrogenated compound having the spacer
six methylene long and the hydrophobic tails of twelve carbon atoms each shows that the
gene delivery ability of the hydrogenated compounds depends on the spacer length but
barely on the hydrophobic tail length. Moreover, the physico-chemical data, particularly
cmc and enthalpy of micellization, confirm that GP12_6 has a hydrophobicity very similar
to FGP6, making their comparison correct. CD spectra have been shown to be useful tools
to verify the formation of lipoplexes. In fact, under lipoplex formation, the CD spectra show
a “tail” in the 288–320 nm region dependent on the presence of FGP6 and attributed to a
chiroptical feature named ψ-phase. Such a phase is a consequence of the DNA structural
transition from the B-form induced by the surfactant and reflects the supramolecular
structure of the complex.
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Molecules 2023, 28, 3585
We have previously shown that the fluorinated compounds with spacer formed by
6 (FGP6) and 8 (FGP8) carbon atoms gives rise to a very interesting gene delivery activity,
superior to that of the commercial reagent, when formulated with DOPE. Ellipsometric
measurements suggest that FGP6 and FGP8 act in a very similar way, and dissimilar from
FGP4, exactly as in the case of transfection, and confirm the hypothesis, suggested by
thermodynamic data, about the requirement of a right length of the spacer for allowing
the molecule to form a sort of molecular tong able to intercalate DNA. It remains to
verify the effect of the hydrophobic chain length on fluorinated compounds, to optimize
their efficiency.
Supplementary Materials: The following supporting information can be downloaded at: https://
www.mdpi.com/article/10.3390/molecules28083585/s1. Figure S1: 13 C-NMR of 1,1′ -bis-hexadecyl-
2,2′ -hexamethylenebispyridinium chloride (GP12_6).; Figure S2. 1 H-NMR of 1,1′ -bis-dodecyl-2,2′ -
hexamethylenebispyridinium chloride (GP12_6). Figure S3. IR spectrum of 1,1′ -bis-dodecyl-2,2′ -
hexamethylenebispyridinium chloride (GP12_6). Figure S4. Mass spectrum of 1,1′ -bis-dodecyl-2,2′ -
hexamethylenebispyridinium chloride (GP12_6). Figure S5. Transfection of RD4 by GP12_6 as a
function of concentration. On the left, phase contrast and, on the right, fluorescence microscope
observation of the transfected cells (as shown by green cells expressing EGFP) are shown. The
experiments were done with the surfactant alone and with surfactant:DOPE = 1:2. Cells are not
transfected with DOPE alone. Figure S6. Transfection of A549 cells (right) by GP12_6 as a function of
concentration. On the left, phase contrast and, on the right, fluorescence microscope observation of
the transfected cells (as shown by green cells expressing EGFP) are shown. The experiments were
done with the surfactant alone and with surfactant:DOPE = 1:2. Cells are not transfected with DOPE.
Figure S7. Deconvolution of the spectra using Origin Pro, gaussian model. Figure S8. Kinetics of
surface layer formation for FGP4 100 µM. It takes about 6 h to reach the equilibrium. Surface tension
is lowered to 26 mN/m. Figure S9. Kinetics of surface layer formation for FGP4 50 µM. After 15 h,
the equilibrium is not reached. Table S1. Deconvolution of the spectra. DNA: A-form yellow, B-form
green. Table S2. Surface tension values of FGP4, FGP6 and FGP8 after 2 h. For each concentration,
the mean and its relative positive and negative error are reported.
Author Contributions: Supervision, E.F.; Conceptualization, M.M. and E.F.; Methodology, M.R., C.C.,
T.A.P., D.O., V.F., G.D. and L.C.; Writing—original draft, M.M., E.F. and M.R.; Writing—review and
editing, C.C., L.C., D.O., V.F., T.A.P. and G.D. All authors have read and agreed to the published
version of the manuscript.
Funding: This research received no external funding.
Informed Consent Statement: Not applicable.
Data Availability Statement: All experimental data are reported in the paper and in Supplemen-
tary Materials.
Acknowledgments: The authors thank the Interdepartmental Centre of Measurements (CIM) of
the University of Parma for allowing the use of the AFM facilities and the SCUSA Department for
ITC instrumentation.
Conflicts of Interest: The authors declare no conflict of interest.
Sample Availability: Samples of the studied compounds are available from the authors.
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94
molecules
Article
Interaction of Graphene Oxide Nanoparticles with Human
Mesenchymal Stem Cells Visualized in the Cell-IQ System
Sergey Lazarev 1,2, * , Sofya Uzhviyuk 1 , Mikhail Rayev 1,2 , Valeria Timganova 1 , Maria Bochkova 1,2 ,
Olga Khaziakhmatova 3 , Vladimir Malashchenko 3 , Larisa Litvinova 3 and Svetlana Zamorina 1,2
1 Institute of Ecology and Genetics of Microorganisms, Ural Branch of the Russian Academy of Sciences-Branch
of Perm Federal Research Center, 614081 Perm, Russia; [email protected] (S.U.);
[email protected] (M.R.); [email protected] (V.T.); [email protected] (M.B.);
[email protected] (S.Z.)
2 Department of Microbiology and Immunology, Faculty of Biology, Perm State University, 614990 Perm, Russia
3 Department of Microbiology and Immunology, Faculty of Biology, Immanuel Kant Baltic Federal University,
236041 Kaliningrad, Russia; [email protected] (O.K.); [email protected] (V.M.);
[email protected] (L.L.)
* Correspondence: [email protected]
Abstract: Graphene oxide is a promising nanomaterial with many potential applications. However,
before it can be widely used in areas such as drug delivery and medical diagnostics, its influence on
various cell populations in the human body must be studied to ensure its safety. We investigated
the interaction of graphene oxide (GO) nanoparticles with human mesenchymal stem cells (hMSCs)
in the Cell-IQ system, evaluating cell viability, mobility, and growth rate. GO nanoparticles of
different sizes coated with linear or branched polyethylene glycol (P or bP, respectively) were used at
concentrations of 5 and 25 µg/mL. Designations were the following: P-GOs (Ø 184 ± 73 nm), bP-GOs
(Ø 287 ± 52 nm), P-GOb (Ø 569 ± 14 nm), and bP-GOb (Ø 1376 ± 48 nm). After incubating the
cells with all types of nanoparticles for 24 h, the internalization of the nanoparticles by the cells was
Citation: Lazarev, S.; Uzhviyuk, S.; observed. We found that all GO nanoparticles used in this study exerted a cytotoxic effect on hMSCs
Rayev, M.; Timganova, V.; Bochkova, when used at a high concentration (25 µg/mL), whereas at a low concentration (5 µg/mL) a cytotoxic
M.; Khaziakhmatova, O.; effect was observed only for bP-GOb particles. We also found that P-GOs particles decreased cell
Malashchenko, V.; Litvinova, L.;
mobility at a concentration of 25 µg/mL, whereas bP-GOb particles increased it. Larger particles (P-
Zamorina, S. Interaction of Graphene
GOb and bP-GOb) increased the rate of movement of hMSCs regardless of concentration. There were
Oxide Nanoparticles with Human
no statistically significant differences in the growth rate of cells compared with the control group.
Mesenchymal Stem Cells Visualized
in the Cell-IQ System. Molecules 2023,
28, 4148. https://doi.org/10.3390/
Keywords: graphene oxide nanoparticles; human mesenchymal stem cells; PEG; cell viability; cell
molecules28104148 growth; Cell-IQ; flow cytometry
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Molecules 2023, 28, 4148
2. Results
2.1. Determination of the Relative Numbers of Live and Dead Cells after 24-h Incubation with
GO Nanoparticles
Data on the number of live, apoptotic, and dead cells after 24-h incubation with GO
nanoparticles, as determined by flow cytometry analysis, are shown in Figure 1. Cultivation
of human mesenchymal stem cells in the presence of graphene oxide nanoparticles of types
P-GOs, bP-GOs, and P-GOb at a concentration of 5 µg/mL did not result in a statistically
significant decrease in the number of viable cells compared with the control group. The
number of viable cells in these groups, as in the control group, was approximately 90%,
which corresponds to the viability of the cells before the start of the experiment. At the
same time, incubation of cells with these types of nanoparticles at a higher concentration
of 25 µg/mL significantly decreased the relative number of viable cells. A decrease in the
number of living cells correlated with an increase in the number of both dead and apoptotic
cells, with a greater increase in the number of dead cells in all cases. The ratio between
the number of dead cells and the number of apoptotic cells (D/A) in these samples was
1.74 ± 0.17, whereas in the control group D/A = 1.1.
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Molecules 2023, 28, 4148
Figure 1. Comparison of the number of viable (A), apoptotic (B), and dead (C) cells after 24 h of
ff
cultivation in the presence of
ff different types of GO nanoparticles (median, 1st and 3rd quartiles are
shown). n = 2. Significant differences compared with control group (<0.05) are marked with *.
The greatest cytotoxicity among the samples studied was exerted by the bP-GOb
nanoparticles. In this sample, cell viability decreased significantly after 24 h of cultivation,
regardless of the concentration of nanoparticles used. Interestingly, the low concentration
of bP-GOb resulted mainly in an increase in the number of dead cells (D/A = 2.54), whereas
at a high concentration the loss of cell viability was mainly due to an increase in the number
of apoptotic cells (D/A = 0.39).
ff
Thus, it was shown that all particles studied had a cytotoxic effect on human mes-
ff
enchymal stem cells at high concentrations. At low concentrations, the cytotoxic effect was
observed only for bP-GOb particles. In all cases, there is a decrease in viability, mainly due
to an increase in the number of dead cells. A decrease in viability due to the predominant
increase in the number of apoptotic cells was observed only for the bP-GOb sample at
high concentration.
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Molecules 2023, 28, 4148
Figure 2. Comparison of the number of cells with high granularity after 24 h of co-cultivation with
ff
samples from GO (median is shown). n = 2. Significant differences compared
ff with the control group
(p < 0.05) are marked with *.
2.3. Evaluation of Cell Growth and Migration Activity in the Cell-IQ System
A total of 188 images were acquired for each well of the cell culture plate (47 images
for each area of visualization) (Figure 3). Adhesion of particles on the cells is clearly
visible in these images (Videos S1–S9). This is particularly noticeable in wells with high
concentrations of samples. In addition, 24 h after the start of cultivation, areas without
nanoparticles can be seen on the images. These correspond to the trajectories of hMSC
movement as the cells have adhered/internalized most of the particles they encountered
along the way.
Figure 3. Photographs of cell culture plate wells taken in the Cell-IQ system. Each pair of images
corresponds to the one area of visualization showing the changes during incubation of hMSCs with
GO nanoparticles.
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Molecules 2023, 28, 4148
To assess the change in the number of cells in the visualization areas, the cells in every
other image were counted. A pivot table was created based on the data obtained (Table S1).
ff in either the increase in cell number after
There were no statistically significant differences
one day of cultivation or in the value of the K1 coefficientfficompared with the control group
(Figure 4). In the evaluation of cell activity (K2), a statistically significant decrease in the
activity of the cells cultured with the P-GOs sample at high concentration was observed
compared to the control group. The opposite was true for the bP-GOb sample. At the
minimum concentration, it apparently enhanced cell activity, resulting in a statistically
significant increase in K2 levels compared to the control group. No statistically significant
ff
differences were found in the other samples compared to the control group. In general,
it appears that low concentrations of GO nanoparticles contributed to an increase in cell
ff
activity, while high concentrations, on the contrary, had an inhibitory effect.
Figure 4. Comparison of (A) cell growth after 24 h of cultivation (mean and 95% confidence intervals
shown); (B) change in cell number—K1 (median, 1st and 3rd quartiles ff shown). n = 12; (C) cell
activity—K2 (median, 1st and 3rd quartiles shown). n = 12. Significant differences compared with
control group (p < 0.05) are marked with *.
In addition, the velocity of cell movement was evaluated (Figure 5). An increase
in the speed of cell movement was observed for all samples and concentrations studied
compared to the control group, while this was statistically significant only for the P-GOb
and bP-GOb samples.
To assess the effect of the samples on the functional properties of the hMSCs, the
change in cell activity (K2) was evaluated. It can be seen that the concentration of the
nanoparticles has a significant effect on the K2 value. The average K2 value for areas of
visualization containing samples at a concentration of 25 µg/mL is lower than that of
the control group, whereas it is higher for areas containing samples at a concentration of
5 µg/mL. Visually, it was observed that isolated cells moved farther and faster, while they
slowed down when they were near other cells.
Overall, it was found that a high concentration of P-GOs particles statistically signifi-
cantly reduced the value of the K2 coefficient, while high concentrations of bP-GOs particles
increased K2. A statistically significant effect of other particle types on the K2 coefficient
was not observed, but an upward trend for the K2 value can be seen for the samples in
the following series: P-GOs → bP-GOs → P-GOb → bP-GOb (Figure 4). Moreover, a
statistically significant increase in the speed of cell movement was observed for P-GOb and
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Molecules 2023, 28, 4148
bP-GOb type particles at both concentrations, while no change in cell speed was registered
for P-GOs and bP-GOs particles compared to the control.
ff
Figure 5. Comparison of the speed of cell movement during cultivation in the presence of different
types of GO nanoparticles. ff Shown are the mean values and 95% confidence intervals of the mean
values. n = 12. Significant differences compared with the control group (p < 0.05) are marked with *.
3. Discussion ff
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Molecules 2023, 28, 4148
increased when the concentration of the bP-GOs sample was low. Visually, a trend of
increasing cell activity with increasing particle size can be observed.
In previous studies on the effects of other types of nanoparticles on MSCs, it has been re-
peatedly shown that these cells are able to internalize small particles (up to 200 nm) [38–40].
However, previous studies have predominantly used spherical polymer or metal parti-
cles. There are far fewer studies investigating the effect of graphene nanoparticles and
their derivatives on MSCs. An important difference between graphene-based nanopar-
ticles and those mentioned above is the two-dimensional structure of graphene-based
materials, which provides a large surface area and the possibility of obtaining particles in
different shapes.
The shape of the particles and surface modifications play an important role in achiev-
ing low cytotoxicity. For example, reduced GO nanoribbons were shown to cause more
extensive DNA damage in hMSCs after a short incubation than nanosheets of similar
size [41]. In another study using nanoparticles of PEG-coated reduced GO, incubation of
cells for an extended period of time (up to 72 h) did not result in a significant decrease in
viability [42]. In this case, particles with a diameter of 1 µm were used at concentrations
of 5, 10, 50, and 100 µg/mL. The results obtained in our study are not consistent with
these data. This could be due to several reasons, including the use of PEG with different
lengths for nanoparticle coating. Syama et al. used PEG with a molecular weight of 1.9 kDa,
whereas our particles were coated with PEG which had a molecular weight of 5 kDa.
The uptake of 2.7 µm-sized capsules by MSCs has previously been associated with a
decrease in cell mobility [43]. The decrease in velocity was shown to be positively correlated
with the concentration of capsules in the medium during incubation. At the same time,
some studies have shown the ability of nanoparticles to accumulate in endosomes, leading
to a change in cellular metabolism, stimulation of cells, and an increase in their mobility [44].
In this study, it was shown that incubation of cells with P-GOb and bP-GOb samples
resulted in an increase in cell velocity, which may indicate that these particles have some
effect on cellular metabolism, although the mechanism of this effect is not clear at present.
In general, there are good prospects for the use of graphene nanoparticles and their
derivatives in diagnostics, therapy, and regenerative medicine. At the same time, the effect
of these particles on human stem cells is still insufficiently studied. We see the need for
further research in this area to gain a more comprehensive understanding of the parameters
that play a key role in the cyto- and genotoxicity of graphene-based nanoparticles with
respect to human stem cells. It is critical to know the consequences of short- or long-term
exposure of human tissues to these materials.
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Molecules 2023, 28, 4148
authors [45]. The following particles were used in the study: P-GOs (Ø 184 ± 73 nm),
bP-GOs (Ø 287 ± 52 nm), P-GOb (Ø 569 ± 14 nm), and bP-GOb (Ø 1376 ± 48 nm). The
information about the types of nanoparticles used in this study is summarized in Table 1.
Further information is available in [45] and Appendix B.
Figure 6. Gating strategy for human mesenchymal stem cells to assess the relative amounts of live,
dead, and apoptotic cells.
102 tt
Molecules 2023, 28, 4148 tt
Evaluation of the dynamics of cell growth and migration activity of cells in the
Cell-IQ system. Cells were counted in every second image acquired with the Cell-IQ
system. Coefficients K1 and K2 were used to evaluate the dynamics of changes in cell
number per area of visualization.
The K1 coefficient reflects changes in the number of cells in the observation area per
unit time. It was calculated as the average value of the difference between the number of
observed cells in each pair of analyzed images divided by the time interval between these
images. In addition to K1, the final change in cell number in each visualization area was
assessed. For this purpose, the ratio between the number of cells at the end of cultivation
(after 24 h) and the number of cells at the beginning of cultivation was calculated for each
area. This ratio reflects the total cell growth that occurred during cultivation.
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The coefficient K2 reflects the activity of cell movement in the area of observation. It
was calculated based on K1, but the absolute value of the difference between the number
of cells observed in each pair of analyzed images was used. While K1 is influenced by
cell growth, K2 is more appropriate to evaluate the migration of cells. If the average
number of cells remains constant, their migration rate to/from the observation area can
still be evaluated.
K2 = Σ (|ki start − ki end\ |/ti ), (2)
5. Conclusions
We studied the interaction of graphene oxide nanoparticles with hMSCs in the Cell-IQ
in vivo monitoring system. We have shown that all the studied GO nanoparticles after
24 h of incubation have a cytotoxic effect on hMSCs at a high concentration (25 µg/mL),
however, only bP-GOb particles were able to reduce cell viability at a low concentration
(5 µg/mL).
We also showed that small particles (P-GOs) at a high concentration (25 µg/mL)
reduced cell motility, while large-sized particles coated with branched PEG (bP-GOb) at a
similar concentration increased this parameter. It was demonstrated that larger particles of
P-GOb and bP-GOb (5 and 25 µg/mL) increased the speed of hMSCs movement.
However, the particles did not affect the changes in cell count during cultivation.
In general, GO nanoparticles can reduce the viability of hMSCs, but increase the rate
of cell movement. Thus, we have demonstrated for the first time the interaction of GO
nanoparticles with hMSCs in the Cell-IQ in vivo monitoring system.
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Molecules 2023, 28, 4148
Author Contributions: Conceptualization and funding acquisition, S.Z.; formal analysis, M.B.;
investigation, S.U., O.K., M.B. and V.M.; project administration, M.R., L.L. and S.Z.; writing—original
draft, S.L.; writing—review and editing, V.T. All authors have read and agreed to the published
version of the manuscript.
Funding: This research was funded by the Russian Science Foundation, grant number 19-15-00244-Π.
The APC was funded by the same organization.
Institutional Review Board Statement: The study was conducted in accordance with the Declaration
of Helsinki and approved by the Institutional Review Board (or Ethics Committee) of Immanuel Kant
Baltic Federal University (Approval No. 1, 28 February 2019).
Informed Consent Statement: Informed consent was obtained from all subjects involved in the study.
Data Availability Statement: The datasets used and/or analyzed during the current study are
available from the corresponding author on request.
Conflicts of Interest: The authors declare no conflict of interest. The funders had no role in the design
of the study; in the collection, analyses, or interpretation of data; in the writing of the manuscript; or
in the decision to publish the results.
Sample Availability: Samples of the GO nanoparticles used in this study are available from the authors.
Appendix A
Cultivation groups:
1. Control—cells in a culture medium without the addition of graphene oxide nanoparti-
cles (3 iterations per plate, 6 iterations in total).
2. Sample 1 (5 µg/mL)—cells in a culture medium with the addition of P-GOs (Ø 184 ± 73 nm)
graphene oxide nanoparticles at a concentration of 5 µg/mL (3 iterations).
3. Sample 1 (25 µg/mL)—cells in a culture medium with the addition of graphene oxide
nanoparticles P-GOs (Ø 184 ± 73 nm) at a concentration of 25 µg/mL (3 iterations).
4. Sample 2 (5 µg/mL)—cells in a culture medium with the addition of graphene oxide
nanoparticles bP-GOs (Ø 287 ± 52 nm) at a concentration of 5 µg/mL (3 iterations)
5. Sample 2 (25 µg/mL)—cells in a culture medium with the addition of graphene oxide
nanoparticles bP-GOs (Ø 287 ± 52 nm) at a concentration of 25 µg/mL (3 iterations)
6. Sample 3 (5 µg/mL)—cells in a culture medium with the addition of graphene oxide
nanoparticles P-GOb (Ø 569 ± 14 nm) at a concentration of 5 µg/mL (3 iterations)
7. Sample 3 (25 µg/mL)—cells in a culture medium with the addition of graphene oxide
nanoparticles P-GOb (Ø 569 ± 14 nm) at a concentration of 25 µg/mL (3 iterations)
8. Sample 4 (5 µg/mL)—cells in a culture medium with the addition of graphene oxide
nanoparticles bP-GOb (Ø 1376 ± 48 nm) at a concentration of 5 µg/mL (3 iterations)
9. Sample 4 (25 µg/mL)—cells in a culture medium with the addition of graphene oxide
nanoparticles bP-GOb (Ø 1376 ± 48 nm) at a concentration of 25 µg/mL (3 iterations)
10. Group 0 control—in the second plate, row D is filled for unstained control. Cells in a
culture medium with the addition of graphene oxide nanoparticles of each sample at
a maximum concentration of 25 µg/mL.
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Molecules 2023, 28, 4148
Table A1. Layout of wells in experimental plates for analysis in the Cell-IQ in vitro imaging system.
Plate No. 1
1 2 3 4 5 6
Control Control Control
A
20,000 cells 20,000 cells 20,000 cells
Sample 1 Sample 1 Sample 1 Sample 2 Sample 2 Sample 2
B 20,000 cells 20,000 cells 20,000 cells 20,000 cells 20,000 cells 20,000 cells
5 µg/mL 5 µg/mL 5 µg/mL 5 µg/mL 5 µg/mL 5 µg/mL
Sample 1 Sample 1 Sample 1 Sample 2 Sample 2 Sample 2
C 20,000 cells 20,000 cells 20,000 cells 20,000 cells 20,000 cells 20,000 cells
25 µg/mL 25 µg/mL 25 µg/mL 25 µg/mL 25 µg/mL 25 µg/mL
D
Plate No. 2
1 2 3 4 5 6
Control Control Control
A
20,000 cells 20,000 cells 20,000 cells
Sample 3 Sample 3 Sample 3 Sample 4 Sample 4 Sample 4
B 20,000 cells 20,000 cells 20,000 cells 20,000 cells 20,000 cells 20,000 cells
5 µg/mL 5 µg/mL 5 µg/mL 5 µg/mL 5 µg/mL 5 µg/mL
Sample 3 Sample 3 Sample 3 Sample 4 Sample 4 Sample 4
C 20,000 cells 20,000 cells 20,000 cells 20,000 cells 20,000 cells 20,000 cells
25 µg/mL 25 µg/mL 25 µg/mL 25 µg/mL 25 µg/mL 25 µg/mL
Sample 1 Sample 2 Sample 3 Sample 4
D 20,000 cells 20,000 cells 20,000 cells 20,000 cells
25 µg/mL 25 µg/mL 25 µg/mL 25 µg/mL
Table A2. Layout of wells in experimental plates for viability analysis by flow cytometry.
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Appendix B
Figure A1. Characterization of P-GO nanoparticles (LnGO = P-GOb, LbGO = bP-GOb, SnGO = P-GOs,
SbGO = bP-GOs). (A)—IR spectra; (B)—Raman spectra; (C)—intensity-weighted size distribution
determined by DLS; (D,E)—SEM images of GO (D) and LbGO (E); (F,G)—TGA/DSC of P-GO. Scale
bars are 1 µm (D) and 500 nm (E).
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109
molecules
Article
Efficient Preparation of Small-Sized Transition Metal
Dichalcogenide Nanosheets by Polymer-Assisted Ball Milling
Qi Zhang, Fengjiao Xu, Pei Lu, Di Zhu, Lihui Yuwen * and Lianhui Wang *
State Key Laboratory of Organic Electronics and Information Displays & Jiangsu Key Laboratory for Biosensors,
Institute of Advanced Materials (IAM), Nanjing University of Posts and Telecommunications, 9 Wenyuan Road,
Nanjing 210023, China
* Correspondence: [email protected] (L.Y.); [email protected] (L.W.);
Tel.: +86-25-85866333 (L.W.); Fax: +86-25-85866396 (L.W.)
Abstract: Two-dimensional (2D) transition metal dichalcogenide nanosheets (TMDC NSs) have
attracted growing interest due to their unique structure and properties. Although various methods
have been developed to prepare TMDC NSs, there is still a great need for a novel strategy combining
simplicity, generality, and high efficiency. In this study, we developed a novel polymer-assisted ball
milling method for the efficient preparation of TMDC NSs with small sizes. The use of polymers
can enhance the interaction of milling balls and TMDC materials, facilitate the exfoliation process,
and prevent the exfoliated nanosheets from aggregating. The WSe2 NSs prepared by carboxymethyl
cellulose sodium (CMC)-assisted ball milling have small lateral sizes (8~40 nm) with a high yield
(~60%). The influence of the experimental conditions (polymer, milling time, and rotation speed) on
the size and yield of the nanosheets was studied. Moreover, the present approach is also effective in
producing other TMDC NSs, such as MoS2 , WS2 , and MoSe2 . This study demonstrates that polymer-
assisted ball milling is a simple, general, and effective method for the preparation of small-sized
TMDC NSs.
Citation: Zhang, Q.; Xu, F.; Lu, P.;
Zhu, D.; Yuwen, L.; Wang, L. Efficient Keywords: high yield; polymer-assisted; ball milling; TMDC nanosheets
Preparation of Small-Sized Transition
Metal Dichalcogenide Nanosheets by
Polymer-Assisted Ball Milling.
Molecules 2022, 27, 7810. https:// 1. Introduction
doi.org/10.3390/molecules27227810
2D TMDCs have a unique layered structure in which the layers interact with each
Academic Editors: Minas M. other by weak van der Waals force rather than by strong chemical bonding, which makes
Stylianakis and Athanasios Skouras them different from other materials [1–4]. 2D TMDCs usually have the general formula
Received: 1 November 2022
of MX2 , in which transition metal (M) atoms and chalcogen (X) atoms form an X-M-X
Accepted: 9 November 2022
‘sandwich’ structure, such as MoS2 , MoSe2 , WS2 , WSe2 , TiS2 , or TiSe2 [1,5]. Since the
Published: 12 November 2022
interaction between TMDC layers is weak, few-layer or single-layer TMDC NSs can be
obtained by physical or chemical exfoliation. These dimension changes in 2D TMDCs cause
Publisher’s Note: MDPI stays neutral
significant variations in their electronic structures and have a great impact on their physical
with regard to jurisdictional claims in
and chemical properties [2,3]. For example, ultrathin MoS2 NSs have high carrier mobility,
published maps and institutional affil-
enhanced photoluminescence, and high catalytic activity [6–9]. The size and aggregation of
iations.
TMDC nanosheets also strongly influence the lubrication property in solid lubricants [10–12].
Moreover, when the lateral size of TMDC NSs is significantly reduced, typically at less than
20 nm, their properties are influenced by quantum confinement effects [13,14], which make
Copyright: © 2022 by the authors.
small TMDC NSs promising nanomaterials for applications in optoelectronics, catalysis,
Licensee MDPI, Basel, Switzerland. energy storage, and biomedicine [3,5,15].
This article is an open access article Over the last few decades, various methods have been developed to control the di-
distributed under the terms and mensions of TMDC NSs [2,4,16]. Bottom-up methods, such as chemical vapor deposition
conditions of the Creative Commons (CVD) and colloidal synthesis, can be used to synthesize TMDC NSs with various com-
Attribution (CC BY) license (https:// positions and structures, for which high temperatures and rigorous reaction conditions
creativecommons.org/licenses/by/ are usually needed [17–19]. As well as bottom-up routes, top-down methods that involve
4.0/). exfoliating bulk TMDCs have also attracted great attention. Novoselov et al. developed a
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Molecules 2022, 27, 7810
micromechanical exfoliation method for TMDC NSs by using Scotch tape [20]. Although
high-quality TMDC NSs can be prepared by using this method, low yield and tedious work
limit its use. Chemical exfoliation by alkaline metal intercalation is an efficient top-down
approach to prepare ultrathin TMDC NSs [21–23]. However, the intercalation reactions
usually need to be carried out in strict oxygen-free and water-free conditions, and the
as-prepared TMDC NSs often have altered crystal structures with the formation of abun-
dant defects. Ultrasonication-assisted liquid phase exfoliation (LPE) is another popular
method that has been extensively explored for the preparation of TMDC NSs due to its
simplicity and generality [24,25]. However, the efficiency of LPE is relatively low, especially
for the preparation of TMDC NSs with small sizes [24–26]. Ball milling is another top-
down method to produce TMDC NSs with the potential for large-scale production [27–30].
Compared with the normal force-dominated LPE method, ball milling utilizes both shear
force and normal force provided by the milling balls to exfoliate and pulverize the layered
TMDCs [31,32]. Similar to the LPE method, the low yield of nanosheets is also a common
issue due to the limited milling ball–material contact interface and the reaggregation of
nanosheets during ball milling. Although sonication-assisted exfoliation has been used
together with ball milling, the improvement of the yield is still limited and the operation
becomes complicated [28,33,34]. Therefore, a novel method for highly efficient preparation
of small TMDC NSs with high yield and simplicity is still needed.
In this report, we develop a polymer-assisted ball milling strategy for the efficient
preparation of TMDC NSs with small sizes (Scheme 1). By using carboxymethyl cellulose
sodium (CMC) as a solid intermedium in the ball milling process, WSe2 NSs with different
average sizes from about 8 nm to 40 nm were successfully prepared with a total yield of
over 60%. The influence of the polymer, rotation speed, and milling time on the size and
yield of WSe2 NSs was studied. This method provides polymer surface modification during
preparation, which gives the as-prepared WSe2 NSs good colloidal stability. Moreover,
this method can also be used to prepare other TMDC NSs, such as MoS2 , MoSe2 , and WS2 .
Our study demonstrates a novel method for the preparation of small-sized TMDC NSs
with high yields by using polymer-assisted ball milling, which is efficient, simple, general,
and scalable.
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Molecules 2022, 27, 7810
the large aggregates, and the supernatant was further centrifuged at different speeds to
obtain TMDC NSs with different sizes.
CMC was first explored to prepare WSe2 NSs by ball milling. As a carboxymethyl
functionalized cellulose, CMC is an abundant, cheap, and environmentally friendly material
and has been extensively used in various industrial applications [35]. After ball milling,
CMC-WSe2 NSs composites can easily form aqueous dispersions due to the electrostatic
repulsion force provided by CMC. After gradient centrifugation at different rotation speeds,
a series of small WSe2 NSs were obtained and named as WSe2 -Low (10,000 rpm), WSe2 -
Medium (16,000 rpm), and WSe2 -High (21,000 rpm), respectively. TEM was used to
investigate the morphology of WSe2 NSs. Figure 1a–c and Figure S1 show that WSe2 NSs
have uniform sheet-like morphology with average sizes of 39.66 ± 13.63 nm (WSe2 -Low),
20.02 ± 6.28 nm (WSe2 -Medium), and 7.90 ± 3.11 nm (WSe2 -High), respectively. As shown
by the HRTEM images in Figure 1d–f, WSe2 NSs have a clear crystalline structure and the
lattice spacing of 2.8 Å can be assigned to the (100) plane of 2H-WSe2 [36]. The six-fold
SAED patterns (Figure 1g–i) indicate that the WSe2 NSs with different sizes have the same
hexagonal symmetry structure, suggesting no significant change in the structure during
the ball milling process.
Figure 1. (a–c) TEM, (d–f) HRTEM, and (g–i) SAED images of WSe2 NSs with different sizes prepared
by CMC–assisted ball milling at different centrifugation speeds: (a,d,g) WSe2 –Low (10,000 rpm for
1 h), (b,e,h) WSe2 –Medium (16,000 rpm for 1.5 h), (c,f,i) WSe2 –High (21,000 rpm for 4 h). Scale bars:
200 nm for (a–c), 2 nm for (d–f), and 5 1/nm for (g–i).
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Molecules 2022, 27, 7810
Atomic force microscopy (AFM) was used to investigate the thickness of WSe2 NSs.
AFM images (Figures 2 and S2) show that the average thickness of WSe2 NSs gradually
decreases from 8–10 nm (WSe2 -Low) to 2–3 nm (WSe2 -High) according to the increase
in centrifugation speed from 10,000 rpm to 21,000 rpm. Hence, small-sized WSe2 NSs
with different thicknesses can be obtained by varying the rotation speed during gradient
centrifugation. Since CMC molecules can still absorb on the surface of WSe2 NSs after
purification, the apparent thickness of WSe2 NSs detected by AFM may be a little higher
than the exact true values.
Figure 2. (a–c) AFM images and (d–f) corresponding height profiles of WSe2 NSs with different sizes
prepared by CMC–assisted ball milling after gradient centrifugation: (a,d) Wse2 –Low, (b,e) Wse2 –
Medium, (c,f) Wse2 –High. Scale bar: 200 nm.
The structure, composition, and properties of the WSe2 NSs were further studied.
As shown by the XRD patterns in Figure 3a, diffraction peaks at 13.6◦ , 31.4◦ , 37.8◦ , and
47.4◦ belong to the (002), (100), (103), and (105) planes according to the standard diffraction
data of WSe2 (JCPDS, 38-1388). The broadening and weakening of these diffraction lines
originate from the size and thickness reduction of WSe2 NSs after ball milling [37,38]. XPS
was used to investigate the chemical state of the as-prepared WSe2 NSs. As shown by the
core-level XPS spectra of W in Figure 3b, the doublet peaks near 33 eV and 35 eV belong to
W4+ 4f7/2 and W4+ 4f5/2 of 2H-WSe2 , while the binding energy peak located at about 38 eV
can be ascribed to W4+ 5p3/2 [39]. The absence of doublet peaks for W6+ between 36 and
38 eV indicates that no WOx formed during ball milling [40]. As illustrated in Figure 3c,
the doublet peaks near 55 eV and 56 eV belong to Se 3d5/2 and Se 3d3/2 of 2H-WSe2 [41].
Similarly, WSe2 NSs with other sizes (WSe2 -Low and WSe2 -High) have the same chemical
states of W and Se as WSe2 -Medium (Supplementary Materials Figure S3), which implies
that WSe2 NSs were prepared by polymer-assisted ball milling mainly through physical
exfoliation and fragmentation rather than the mechanochemical way. Raman spectra of
WSe2 NSs (Figure 3d) show that the characteristic peak located at about 250 cm−1 can
be ascribed to the degenerate A1g (out-of-plane) and E1 2g (in-plane) vibrational modes,
suggesting the few-layer structure of WSe2 NSs [39,41]. UV-vis-NIR spectra of WSe2 NSs
(Figure 3e) indicate that the absorption peaks near 750 nm blueshift with the size and
thickness reduction, similar to previous reports [42,43]. As the FT-IR spectra of WSe2 NSs
depict in Figure 3d, IR absorption bands at 3430 cm−1 and 1630 cm−1 can be assigned to
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Molecules 2022, 27, 7810
the stretching vibration of the hydroxyl group (−OH) and asymmetric vibration of the
carboxyl group (COO−) of CMC [44,45], respectively, which suggests the existence of
CMC molecules on the surface of WSe2 NSs. Due to the hydrophilicity and electrostatic
repulsion of CMC, WSe2 NSs are stable in phosphate buffer saline (PBS) and cell culture
medium (DMEM) (Supplementary Materials Figure S4), and no obvious precipitation
can be observed even after being stored for months in water, which suggests their good
colloidal stability in biological environments.
Figure 3. (a) XRD patterns of WSe2 NSs with different sizes. XPS spectra of WSe2 NSs (WSe2 –Low)
for (b) W 4f and W 5p, and (c) Se 3d. (d) Raman spectra of bulk WSe2 and WSe2 NSs with different
sizes. (e) UV–vis–NIR absorption spectra and photos of WSe2 NSs aqueous dispersions (inset).
(f) FT–IR spectra of WSe2 NSs, bulk WSe2 , and CMC.
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Molecules 2022, 27, 7810
rather than the yield. As shown in Supplementary Materials Figure S7 and Table 1, the
size of most WSe2 NSs decreases from about 60 nm to 30 nm when the rotation speed of
the ball mill increases from 400 rpm to 800 rpm. Meanwhile, the total yield of WSe2 NSs
increases from about 30% to 60% once the rotation speed has exceeded 650 rpm. Therefore,
both the size and yield of WSe2 NSs can be easily adjusted by varying the rotation speed
and milling time during ball milling, suggesting the controllability of this method.
Table 1. Average lateral size and yield of WSe2 NSs prepared by polymer-assisted ball milling under
different experimental conditions.
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Molecules 2022, 27, 7810
Figure 4. (a–c) TEM, (d–f) HRTEM, and (g–i) SAED images of MoS2 , MoSe2 , and WS2 NSs prepared
by CMC–assisted ball milling: MoS2 (a,d,g), MoSe2 (b,e,h), and WS2 (e,f,i). Scale bars: 200 nm for
(a–c), 2 nm for (d–f), and 5 1/nm for (g–i).
Figure 5. AFM images and corresponding height profiles of MoS2 , MoSe2 , and WS2 NSs prepared by
CMC–assisted ball milling: (a,d) MoS2 , (b,e) MoSe2 , (c,f) WS2 . Scale bar: 200 nm.
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Molecules 2022, 27, 7810
Figure 6. (a) XRD patterns of MoS2 , MoSe2 , and WS2 NSs prepared by CMC–assisted ball milling.
XPS spectra of MoS2 NSs for Mo 3d (b) and S 2p (c) core level energy regions. (d) Raman spectra of
MoS2 , MoSe2 , and WS2 NSs. (e) UV–vis–NIR absorption spectra with the photos (inset) and (f) FT–IR
spectra of MoS2 , MoSe2 , and WS2 NSs.
3.2. Characterization
Transmission electron microscopy (TEM) images were obtained by using a HT7700
at 120 kV (Hitachi, Tokyo, Japan). High-resolution transmission electron microscopy
(HRTEM) and selected area electron diffraction (SAED) images were obtained using field
emission electron microscopes (Talos F200x, FEI, 200 kV, Waltham, MA, USA). Atomic force
microscopy (AFM) images were acquired on Nanoscope IIIa (Bruker, Billerica, MA, USA).
X-ray diffraction (XRD) patterns were recorded on an X-ray diffractometer (D8 Advance
A25, Bruker, Billerica, MA, USA) with Cu Kα radiation (λ = 1.54178 Å). X-ray photoelectron
spectroscopy (XPS) was performed on KRATOS Axis Supra (Shimadzu, Kyoto, Japan)
with Al Kα (hν = 1486.6 eV) as the excitation source. Raman characterization was carried
out on a micro-Raman spectroscopy system (inVia, Renishaw, Wotton-under-Edge, UK)
equipped with a 532 nm laser. Ultraviolet-visible-near infrared (UV-vis-NIR) absorption
spectroscopy was performed on a UV-3600 spectrophotometer (Shimadzu, Kyoto, Japan).
Fourier transform infrared (FT-IR) spectra were recorded on a FT-IR spectrometer (Perkin
Elmer, Waltham, MA, USA). The concentration of TMDC NSs was determined by using
an inductively coupled plasma optical emission spectrometer (ICP-OES, Optima 5300DV,
Perkin Elmer, Waltham, MA, USA).
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3.4. WSe2 NSs Prepared by Using Different Polymers during Ball Milling
The preparation of WSe2 NSs by using different polymers, including F127, PVP, and
PEG, has similar experimental conditions to CMC-assisted ball milling. Since the stabi-
lization abilities of different polymers are different, the centrifugation conditions used to
purify WSe2 NSs were adjusted according to the polymer used. For F127, WSe2 NSs were
obtained by gradient centrifugation at 5000 rpm (2124× g) for 20 min (WSe2 -Low), 7500 rpm
(4779× g) for 20 min (WSe2 -Medium), and 10,000 rpm (8497× g) for 30 min (WSe2 -High).
For PVP, WSe2 NSs were obtained by gradient centrifugation at 10,000 rpm (8497× g) for
1 h (WSe2 -Low), 16,000 rpm (21,752× g) for 2 h (WSe2 -Medium), and 21,000 rpm
(37471× g) for 4 h (WSe2 -High). For PEG, WSe2 NSs were obtained by gradient centrifugation
at 5000 rpm (2124× g) for 20 min (WSe2 -Low), 7500 rpm (4779× g) for 20 min (WSe2 -Medium),
and 10,000 rpm (8497× g) for 30 min (WSe2 -High).
4. Conclusions
In summary, we have developed a novel polymer-assisted ball milling method for the
efficient preparation of TMDC NSs with small sizes. The as-prepared WSe2 NSs by using
CMC have small sizes (8–40 nm) with high yield (over 60%), which is not easy to achieve
by using other top-down methods. The high efficiency of this method is attributed to the
enhanced interaction of the 2D TMDCs and the milling balls because of the polymer used
during ball milling. The size and thickness of the WSe2 NSs can be adjusted by changing
the rotation speed and milling time during ball milling and the centrifugation conditions
during purification. Moreover, this polymer-assisted ball milling method can also be used
to prepare other TMDC NSs, such as MoS2 , WS2 , and MoSe2 . The as-prepared TMDC NSs
have good colloidal stability in PBS and cell culture medium. This study provides a highly
efficient, simple, general, and scalable method for the preparation of TMDC NSs with small
sizes, which may also be used for other 2D materials.
Supplementary Materials: The following supporting information can be downloaded at: https://www.
mdpi.com/article/10.3390/molecules27227810/s1, Figure S1: Large-scale TEM images of WSe2 NSs
prepared by CMC-assisted ball milling at 650 rpm for 12 h and centrifuged under different conditions;
Figure S2: Large-scale AFM images of WSe2 NSs prepared by CMC-assisted ball milling at 650 rpm
for 12 h and centrifuged under different conditions; Figure S3: XPS spectra of WSe2 NSs prepared by
CMC-assisted ball milling at 650 rpm for 12 h; Figure S4: Photographs of CMC-WSe2 NSs dispersed
in water, PBS, and DMEM for different times; Figure S5: TEM images of WSe2 NSs prepared by ball
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Molecules 2022, 27, 7810
milling with different polymers under different centrifugal conditions; Figure S6: TEM images of WSe2
NSs prepared by CMC-assisted ball milling for different times after gradient centrifugation; Figure S7:
TEM images of WSe2 NSs prepared by CMC-assisted ball milling with different rotation speeds after
gradient centrifugation; Figure S8: Size statistics of (a) MoS2, (b) MoSe2, and (c) WS2 NSs prepared by
CMC-assisted ball milling; Figure S9: Large-scale AFM images of (a) MoS2, (b) MoSe2, and (c) WS2 NSs
prepared by CMC-assisted ball milling; Figure S10: High-resolution XPS spectra of Mo 3d (a) and Se
3d (b) core level energy regions for MoSe2, and W 4f (c) and S 2p (d) core level energy region for WS2;
Figure S11: FT-IR spectra of bulk MoS2, MoSe2, and WS2.
Author Contributions: Conceptualization, L.Y. and D.Z.; investigation and methodology, Q.Z., F.X.,
D.Z., and P.L.; writing—original draft preparation, Q.Z. and L.Y.; writing—review and editing, L.W.
and L.Y. All authors have read and agreed to the published version of the manuscript.
Funding: The authors are grateful to the Natural Science Foundation of Jiangsu Province (BK20191382),
the Leading-edge Technology Programme of Jiangsu Natural Science Foundation (BK20212012), and
the Open Research Fund of Jiangsu Key Laboratory for Biosensors (JKLB202204).
Institutional Review Board Statement: Not applicable.
Informed Consent Statement: Not applicable.
Data Availability Statement: The raw data will be available from the corresponding authors upon
reasonable request.
Conflicts of Interest: The authors declare no conflict of interest.
Sample Availability: Samples of the TMDC NSs are available from the authors.
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molecules
Review
The Principle of Nanomaterials Based Surface
Plasmon Resonance Biosensors and Its
Potential for Dopamine Detection
Faten Bashar Kamal Eddin 1 and Yap Wing Fen 1,2, *
1 Department of Physics, Faculty of Science, University Putra Malaysia, UPM,
Serdang 43400, Selangor, Malaysia; [email protected]
2 Functional Devices Laboratory, Institute of Advanced Technology, University Putra Malaysia, UPM,
Serdang 43400, Selangor, Malaysia
* Correspondence: [email protected]
1. Introduction
Over the last few decades there has been a great effort towards the development of label-free
optical biosensors. These essential analytical tools offer real-time analysis, detection of chemical and
biological species with high sensitivity and selectivity. The tremendous advances in these biosensors
will have a major impact on our health care. Among these technologies used to analyze the bio-specific
interactions, surface plasmon resonance (SPR) biosensors today belong to the most advanced [1]. It has
proven effective in medical diagnostics [2,3], food quality tests [4], detection of heavy metal ions [5],
and others with respect to environmental protection.
Comparing to the conventional diagnostic tools, SPR biosensors have multiple advantages such
as easy preparation, no requirement of labeling, real-time detection capability, cost- effectiveness,
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and high specificity and sensitivity. However, for the label-free detection of low concentrations of
analytes with small molecular weight its sensitivity is not enough. Therefore, considerable efforts
have been invested to overcome these challenges and improve the sensitivity of the SPR biosensor
with keeping all its advantages. Nanomaterials are promising candidates and have demonstrated
their appropriateness in the biosensing field. All nanomaterials have a general feature, which is the
high specific surface. This enables the immobilization of an enhanced amount of bioreceptor units.
Using the functional nanomaterials significantly enhanced the sensors performances, increased the
sensitivity and selectivity of the sensing platform. The sensing performance is affected by synthetic
procedure of the used nanomaterial, its shape and size. Additionally, the immobilization strategy used
to functionalize the sensor chip is still challenge [6]. The purpose of this concise review is to introduce
SPR concepts, and simplify the mechanism of SPR based sensor from dip to real-time measurements,
explain the important characteristics in SPR sensor performance, mention several clinically related
analytes that have been detected using SPR biosensors efficiently. Additionally, this mini review will
explain the critical role of dopamine (DA) in human body and the potential of nanomaterials based
SPR biosensors to detect it with mentioning its advantages and disadvantages.
2. SPR Phenomena
SPR is a quantum electromagnetic phenomenon that occurs when light interacts with free
electrons at the interface between the metal and dielectric [7,8]. This optical process happens when
monochromatic and p-polarized light beam strikes the surface of metal (typically gold) as shown in
Figure 1.
At a specific incidence angle when light satisfies resonance conditions and the frequency of the
incident light matches the frequency of the surface plasmon wave, the light energy partially transfers to
the electron packages on the metallic surface. After that, the observed reflected light shows a dip in the
intensity as shown in Figure 2a. The electron coherent oscillations that were excited by exponentially
decaying evanescent field of the incident light are called surface plasmons (SPs) and propagate parallel
to the metallic surface. The angle at which the reflected light shows the maximum loss of intensity is
called resonance angle or SPR-dip [9,10].
At the beginning of the twentieth century in 1902, the first observation of SPs was obtained
by Wood, who reported anomalies in the diffraction spectrum of polychromatic light on a metallic
diffraction grating [11]. Then, the connection between these abnormal diffractions and the excitation of
electromagnetic surface waves on the diffraction grating surface was proved by Fano [12]. The clear
explanation of this phenomenon was not complete until 1968, when Otto verified the concept
experimentally and proved that in the attenuated total reflection (ATR) method, the excitation of
SPs led to drop in the reflectivity [13]. Before the end of the same year, Kretschmann and Raether
made some modifications on the configuration of ATR method to observe the excitation of SPs [14].
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These important achievements done by Otto, Kretschmann, and Raether established an appropriate
method to excite SPs and ushered in a promising future in modern optics. There are two categories
of SPs, propagating SPs (PSPs), and localized SPs (LSPs). The excitation of SPs in the first category
occurs on the metallic films. There are several approaches for this type of SPs such as the Kretschmann
and Otto prism coupler, optical waveguides coupler, diffraction gratings, and optical fiber coupler.
While the excitation of LSPs occurs on metallic nanoparticles [15]. The most common approach used in
triggering SPs is prism coupling, which is also known as the ATR mode [16]. In Otto configuration,
there is a certain distance separates the prism and the thin metallic film, the refractive index (RI) of this
dielectric layer is small. The Kretschmann configuration is the easiest one. In this geometry, the prism
is in contact with the metallic layer directly, and the wave vector component of SPs propagating along
the interface is coupled with the wave vector of the incident photon. The practical performance of the
sensor is confined to the resonance angle. The sensitivity of SPR sensors based on prisms is higher
than that of SPR sensors based on grating couplers. Additionally, prism-based SPR sensors using
wavelength interrogation provide the best resolution and a great deal of flexibility in terms of the
analyte refractive index (RI) covered [17]. So, it has become a highly efficient mechanism for optical
sensing of several biological, chemical and environmental changes.
Figure 2. (a) A dip in the intensity of the reflected light after SPs excitation and (b) an angular shift
from a to b due to a refractive index (RI) change on the Au film.
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field detected. When the ligand captures the analyte, the measurable signal rises and this is called
direct label free detection.
label free detection.
Following in time the resonance angle or wave length shift at which the dip is observed produces
the sensogram (Figure 4), then the amount of adsorbed species after injection of the original baseline
buffer can be determined, and a study of the kinetics of the biomolecular interaction can be done.
The sensor surface should be conditioned with a suitable buffer solution to start each measurement.
It is essential to have a stable base line first [18]. After analyte injection, the association phase starts and
the ligands on the surface of the sensor capture the target compounds. Upon injection of the baseline,
the dissociation of target compounds and non-specifically binding molecules from the surface start.
To break the specific binding between the analyte and ligands, regeneration solution should be injected.
This step is vital in order to perform many tests using the same sensor surface.
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The accuracy defines the degree to which the sensor readout value corresponds to the actual value
of the measurand. Sensor’s precision also includes reproducibility, which shows whether entire
measurements can be reproduced in its entirety. Additionally, the repeatability is a way to measure the
sensor precision. It shows the sensor ability to reproduce the same response under the same conditions
over many repetitions. The LOD means the lowest concentration of analyte that can be detected by
the sensor.
The shape of the SPR response curve affects the two important performance parameters, sensitivity
and signal-to-noise ratio (SNR). Sharp SPR-dip is desirable because the narrower the width, the higher
the detection accuracy and the deeper the curve, the higher the sensitivity. The sensor design parameters
such as light polarization modes, light coupling techniques, types of metals influence the width and
depth of the SPR dip. In prism-based SPR sensor, the physical structure of prism used (triangular,
conical, hemispherical, and half cylindrical) and its RI affects the SPR curve [20]. Among the parameters
that play a prominent role in the development of a highly sensitive SPR sensor, the thickness of the
metallic thin films must be mentioned [21]. Until now, the ideal thickness of prepared layers to generate
maximum SPR is within the range of 40 nm to 60 nm [22,23]. Additionally, the used wavelengths
for excitation strongly affect the resonance curve width. Narrowing the reflectance curve necessarily
increases the SPR propagation length [24,25]. Using the excitation wavelength in the IR region results
in an increase in the penetration depth with the consequence that the reflectance minimum will become
more sensitive to dielectric changes relatively far from the metal/dielectric interface; thus, the SPR
signal gets weaker and the surface-sensitive character of SPR becomes less prominent. The usage of
red laser, λ = 633 nm significantly enhances the maximum excitation of SPs and leads to strong SPR
signal, while the excitation wavelength in UV region generates a very weak SPR signal [26].
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testosterone [48,49], gonadotropic hormones and luteinizing hormone [50], pituitary hormones such as
human thyroid-stimulating, growth, follicle-stimulating [51], and insulin [52]. There are other reviews
and articles that focus on the importance and the application of SPR biosensors for the diagnosis of
medically important entities such as viruses, neurotransmitters, proteins, hormones, nucleic acids,
cells, drugs, and disease biomarkers [53–57].
Abnormal concentrations of DA in different biological fluids are associated with various diseases.
Cardiotoxicity and subsequent rapid heart rate, hypertension, and heart failure can be an indicator of the
high levels of DA [62]. While deficiency or practically complete depletion of DA may result in various
neurodegenerative diseases such as Parkinson’s disease (PD) [63,64], Alzheimer’s disease [65,66],
schizophrenia [67–75], and depression [76]. Therefore, the fabrication and development of highly
sensitive and selective sensors for quantitative determination of DA in vivo and in vitro is extremely
necessary in the clinical diagnosis, it can make great contribution monitoring the effectiveness of
the treatment, prevention of diseases [77]. The monitoring of DA levels can be done in different
biological samples including saliva, urine, plasma, serum, platelets, and cerebral spinal fluid [78,79].
DA physiological levels in humans vary in these biofluids. According to the Human Metabolome
Database, DA concentration is less than 130 pM in blood, while its levels in human urine and
cerebrospinal fluid are approximately 5 nM [80]. To date, the development of analytical methods to
measure the concentration of DA directly continues to grow. A variety of these methods demonstrated its
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capability to detect low levels of DA including high performance liquid chromatography (HPLC) [81–83],
capillary electrophoresis [84–87], Fourier transform infrared (FTIR) spectroscopy [88], flow injection [89],
enzymatic methods [90], electrochemical (EC) methods [91–93], mass spectroscopy [94–96], and various
types of optical methods such as colorimetry and spectrophotometry [97], fluorescence [98–101],
electrochemiluminescence (ECL) [102], surface-enhanced Raman spectroscopy (SERS) [103–108],
chemiluminescence (CL) [109], photoelectrochemical (PEC), photoluminescence (PL), solid phase
spectrophotometry (SPS), resonance Rayleigh scattering (RRS), and SPR spectroscopy [110–114].
The interested reader in electrochemical and optical methods is referred to the review paper by Kamal
Eddin and Fen (2020) [115], and references cited therein.
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(BSA) protein (DA–BSA) conjugate on the sensor chip by physical adsorption, the loss of activity of
biomolecules was minimized. The DA–BSA conjugate flowed over the gold surface, the increase in
the resonance angle was an indicator on the saturation of the immobilization onto the sensor surface.
Different concentrations (5–400 µg/mL) of the conjugate were used in the immobilization. The SPR
angle shift reached a plateau around 100 µg/mL of the DA–BSA conjugate. The principle of indirect
competitive inhibition to detect DA was employed in this work. DA-RC was allowed to flow over the
DA–BSA surface, the resonance angle increased due to the binding of the receptor to the DA haptens
present on the sensor surface. After completion of the DA-RC flow, the flow was switched back to
phosphate buffered saline (PBS) buffer and the resonance angle shift remained almost stable due to
the strong affinity interaction between DA-RC and DA–BSA conjugate. The sensor sensograms were
observed for the affinity reaction between immobilized DA–BSA conjugate and DA-RC in the absence
of DA, and in the presence of different concentrations of DA. The shift in the resonance angle decreased
by increasing in the concentration of free DA in solution, this was because the free DA inhibited the
binding interaction of DA-RC with the DA–BSA conjugate. The sensor exhibited a linear detection
range from 0.085 to 700 ng/mL with a lower LOD of 0.085 ng/mL. The results showed that there is no
significant interference species such as ascorbic acid (AA), uric acid (UA), and other DA analogues viz.,
DOPAC and 3-(3,4 dihydroxyphenyl)-alanine (DOPA). The stability of the sensor surface was high
during repeated regeneration and affinity reaction cycles. According to the simplicity and effectiveness
of this biosensor, their study presented an encouraging scope to develop portable detection systems to
measure DA in vitro and in vivo in clinical and medical diagnostics.
The adsorption capability of Ibuprofen and DA on pure gold layer and gold functionalized with
L-cysteine (L-Cys) and L-glutathione (L-GSH) using SPR spectroscopy was investigated by Sebők et al.
(2013) [112]. From the description of DA adsorption by a two-stage isotherm it could be concluded
that first layer of DA was irreversibly bound while the second layer was anchored by physical forces,
so DA adsorption on the gold surface was only partially reversible. The amino groups available
in DA enabled formation of a stronger chemical bond with the cysteine molecule comparing with
ibuprofen. The bounded DA on the gold surface functionalized with glutathione showed perpendicular
orientation to the gold surface, while glutathione orientation was the same as ibuprofen parallel to
the surface. The initial energies of glutathione/ibuprofen interaction were lower comparing with the
glutathione/DA system.
Among two-dimensional materials used to design high performance sensor platform, graphene has
received profound interest due to its remarkable properties. In the work reported by Kamali et al. (2015),
they developed the silver @ graphene oxide (Ag@GO) nanocomposite-based SPR optical sensor to
detect DA and other biomolecules such as AA and UA [130]. The nanocomposite showed an SPR band
at 402 nm due to the Ag NPs. The SPR intensity-based LOD of DA was 49 nM in the DA concentration
linear range (100 nM to 1 µM), and 62 nM in the range (1–2 µM). While the SPR band position-based
LOD of DA was 30 nM. The SPR absorbance peak changes toward UA and AA were not comparable
with DA, which proved its excellent selectivity. The adsorption and sensing ability of the Ag@GO
nanocomposite mainly depended on the nature of the adsorption site and its interaction with the
functional groups of the molecules. The results proved that DA had more affinity with Ag@GO than
UA and DA.
Next, Rithesh Raj et al. (2016) used green synthesized Ag NPs as sensing material to fabricate
SPR based fiber optic sensor for DA measurement [131]. SPR spectra showed decrease in SPR dip
intensity. Additionally, a shift in resonance wavelength towards lower wavelength was observed by
increasing the concentrations of DA, this probably occurred because of the interaction between DA and
Ag clusters which led to formation of Ag NPs. Poly vinyl alcohol (PVA) coated fiber showed negligible
shift in wavelength comparing to green Ag NPs coated fiber, which demonstrated the role of the green
Ag NPs in the sensing enhancement. The sensor response time was 6 min and the corresponding
maximum resonance wavelength shift was 12 nm. The LOD was 2 × 10−7 M enhanced the selectivity
of the proposed sensor in the presence of other biological species.
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and DA did not happen, then the conjugate probe was in its “ON” state. Additionally, the binding
of the conjugate to the cDNA probe was detected strongly. As a result, a big signal response was
generated. In this study, the target DA molecule was acting as a switch to turn “OFF” and “ON” the
DAAPT-AuNP conjugate during the whole process. The proposed assay showed good sensitivity,
reproducibility, and its specificity to DA was high. Using this nanoparticle enhanced SPR aptasensor
has the potential to detect small molecules, proteins, and other analytes, as long as the target has a
corresponding aptamer.
Additionally, in 2018, Sharma used molecular imprinted graphene nanoplatelets (GNP)/tin oxide
(SnO2 ) nanocomposite to fabricate a fiber optic SPR-based DA sensor with high selectivity. The LOD
of this sensor was lower than the LOD values of other DA sensors, it was 0.031 µM [135]. The dipping
time of sensing element over the silver coated probe greater than 20 min produces sensing layer with a
large thickness, which perturbs the intensity of the electric field at the interface and decreases the shift.
By increasing DA concentration, the blue shift with a decrease in the depth of the SPR spectra was
recorded. The change in the real and imaginary parts of the RI of the sensing layer occurred as a result
of the binding of DA molecules with active sites in the sensing layer. The decrease in the real part of
the effective RI led to that observed blue shift.
Recently, Sun et al. (2019) proposed an Au film/graphene D-shaped plastic optical fiber (D-POF)
functionalized with DA binding aptamer (DBA) to detect DA sensitively and selectively in the presence
of three different interferences. They demonstrated that graphene enhanced the sensitivity and played
a role in amplification of the SPR signal by introducing DBA. They demonstrated the potential of this
sensor to detect DA-induced disorders [136]. Graphene improved the sensor sensitivity and amplified
the SPR signal. As RI changed from 1.3330 to 1.3612, the resonant wavelength of the proposed sensor
had a red shift of 43.4 nm, which was larger than that of the sensor covered only by Au film 34.9 nm.
Adding graphene increased the sensor sensitivity from 1238 to 1539 nm/RIU. The SPR dips decreased
as the RI increased due to the higher loss of energy and the increasing in the penetration depth of
evanescent field.
In the same year, Yuan et al. used DA to modify a gold sensing surface and produce self-assembled
monolayers for secondary surface mediated reactions under different environmental conditions
including pH, different buffers, varying DA concentrations in buffer solutions as well as the
immobilization time of DA [137]. The favorable environmental conditions for DA immobilization
onto gold surface were only alkaline (pH 7.6) and mildly alkaline media (pH 8.6 or 9.5) for 1 h.
The immobilization for 2 h appeared to occur only in pH 7.6 or pH 8.6, and the SPR phenomenon did
not exist at pH 9.5. This is because the thickness of the immobilized DA was over a critical reflectivity
value. Using 2 mg/mL DA in pH 8.6 Tris buffer resulted in the optimum reactive on the gold chip.
The critical DA immobilization time for SPR disappearance under the previous favorable conditions
was estimated to be 277 min. These results provide valuable information for using DA for surface
modification in the future studies. The recent works that used SPR phenomenon in DA measurements
are summarized and discussed in Table 1.
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An EC sensor offers real-time detection with simplicity and excellent sensitivity, fast response
time, wide linear concentration range, cost effectiveness, and an ability to be miniaturized. However,
it suffers from limited selectivity in the presence of other biological analytes, large noise and background
signal. In addition to the fouling of the sensor surface and its degradation over time. Although the
sensitivity and selectivity of the SERS-based DA sensors are higher in comparison to other detection
methods. However, there is an obstacle to the availability of these sensors, they require expensive
equipment for analysis. An SPR sensor has several important advantages such as direct label free
detection of diverse molecule sets including small molecules, real-time measurements, high reliability,
very high sensitivity with low detection limit, long-term stability, cost-effectiveness, easy sample
preparation, small consumption of sample and reagent, reproducibility, regeneration of sensor chips,
and specificity to the binding event. On the other hand, the disadvantages of this sensor are non-specific
binding to surfaces needs to be controlled, which requires a meticulous experimental design, mass
transport limitations when the analyte transfer to the ligand is limited, the immobilization effects, and
the ligand can change its orientation after immobilization on the sensor chip and prevents binding
with the analyte [151]. The combination of SERS with SPR has the potential to massively increase
the local electromagnetic field intensity of nanoparticles to a level far exceeding the single-molecule
SERS detection limit but this still lacks versatility [152]. However, in the context of a point-of-care
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testing (POCT) configuration, the bulky nature of the SPR apparatus hinders its field applications. So,
the efforts are mainly made on the miniaturization of SPR devices.
9. Conclusions
In recent decades, the development of biosensors to identify and measure low concentrations
of different NTs with high selectivity, sensitivity, low-cost, and rapid response attracted considerable
attention due to the crucial role that neurotransmitters play in clinical diagnostics and curing mental
disorders such as schizophrenia, Parkinson’s disease, and Alzheimer’s disease. SPR has emerged
as a very suitable technology for clinical analytes detection. It is based on IR variations due to the
mass change at the sensor chip. The SPR sensing platform has many features. It is easy to prepare it,
the basic optics that it needs can be miniaturized to a suitable size in diagnosis, the direct quantification
of the specific bindings occurs with high specificity and sensitivity. This low-cost technology is not
based on labeling of the target molecules and does not alter its binding affinity and kinetics properties.
Rapid and reliable detection of various medically important entities using SPR spectroscopy has been
done over the last years. The reported works on DA sensing using this promising method is still
limited despite the impressive results obtained. This is a strong motivation for researchers to develop
these sensors by modification SPR chips using nanomaterials. Ongoing research employing the unique
capabilities of carbon-based nanomaterials as well as the advantages of polymers and incorporating
them with SPR technology for ultra-sensitive and selective detection of DA may introduce exciting
progress in neuroscience. The high potential of SPR sensors and the continuous efforts to develop them
and overcome their limitations qualify them to have a prominent presence in future developments in
lab-on-a-chip technologies including point-of-care devices.
Author Contributions: Conceptualization, Y.W.F. and F.B.K.E.; Original Draft Preparation, F.B.K.E.; Writing and
Editing, F.B.K.E.; Review, Y.W.F.; Visualization, F.B.K.E.; Software, F.B.K.E.; Resources, F.B.K.E.; Validation, Y.W.F.;
Supervision, Y.W.F. All authors have read and agreed to the published version of the manuscript.
Funding: This research was funded by the Ministry of Education Malaysia through the Fundamental Research
Grant Scheme (FRGS/1/2019/STG02/UPM/02/1).
Acknowledgments: F.B.K.E. acknowledges the support received from OWSD and Sida (Swedish International
Development Cooperation Agency).
Conflicts of Interest: The authors declare no conflict of interest.
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