Low-Molecular-Weight Hydrogels as New Supramolecular Materials

Dec 8, 2016 - Controlling the interface between biological tissues and electrodes remains an important challenge for the development of implantable de...
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Low Molecular Weight hydrogels as new supramolecular materials for bioelectrochemical interfaces Deepak Ramesh Jain, Aleksandar Karajic, Magdalena Murawska, Bertrand Goudeau, Sabrina Bichon, Sébastien Gounel, Nicolas Mano, Alexander Kuhn, and Philippe Barthelemy ACS Appl. Mater. Interfaces, Just Accepted Manuscript • DOI: 10.1021/acsami.6b12890 • Publication Date (Web): 08 Dec 2016 Downloaded from http://pubs.acs.org on December 8, 2016

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Low Molecular Weight hydrogels as new supramolecular materials for bioelectrochemical interfaces

Deepak Jain,†,††, ‡ Aleksandar Karajic, &,#,‡ Magdalena Murawska,&,#,‡ Bertrand Goudeau, #,‡ Sabrina Bichon,&,‡ Sébastien Gounel,&,‡ Nicolas Mano,& Alexander Kuhn#,‡ and Philippe Barthélémy†,††, ‡,*



Inserm U1212, F-33076 Bordeaux, France

††



Université de Bordeaux, 146 rue Léo Saignat, 33076 Bordeaux Cedex, France &

#

CNRS 5320, F-33076 Bordeaux, France

CNRS, CRPP, UPR 8641, 33600 Pessac, France

Bordeaux INP- UMR 5255, CNRS – ENSCBP, 16 Avenue Pey Berland, 33607 Pessac (France)

Corresponding author E-mail: [email protected]

KEYWORDS. Low Molecular Weight Gel, Enzyme Electrodes, Bioelectrochemistry, Porous

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electrodes, Supramolecular Assembly, Fluorocarbon Amphiphile, Nucleoside, Glycosyl.

ABSTRACT. Controlling the interface between biological tissues and electrodes remains an important challenge for the development of implantable devices in terms of electroactivity, biocompatibility and long-term stability. In order to engineer such a biocompatible interface a low molecular weight gel (LMWG) based on a Glycosylated Nucleoside Fluorocarbon amphiphile (GNF) was employed for the first time to wrap gold electrodes via a non-covalent anchoring strategy, i.e., self-assembly of GNF at the electrode surface. Scanning electron microscopy (SEM) studies indicate that the gold surface is coated with the GNF hydrogels. Electrochemical measurements using cyclic voltammetry (CV) clearly show that the electrode properties are not affected by the presence of the hydrogel. This coating layer of 1 to 2 microns does not significantly slow down the mass transport through the hydrogel. Voltammetry experiments with gel coated macroporous enzyme electrodes reveal that during continuous use their current is improved by 100% compared to the non-coated electrode. This demonstrates that the supramolecular hydrogel dramatically increases the stability of the bioelectrochemical interface. Therefore such hybrid electrodes are promising candidates that will both offer the biocompatibility and stability needed for the development of more efficient biosensors and biofuel cells.

1. INTRODUCTION Implantable electrodes provide an efficient means for designing innovative therapeutic approaches, and electroactive devices have been deeply investigated during the past decades.1-5 For example, the monitoring of drugs has been proposed to more precisely adjust the drug dosage according to the pharmacokinetic profiles and a precise quantification of drug concentration after administration.6 Electroanalytical techniques have been found to be efficient for monitoring drugs in different biological fluids.7,8 Cyclic voltammetry techniques have been used to follow several drugs,9 including analgesics, antibiotics, anti-psychotic and hypoglycaemics.10 Also, in parallel to the detection of biologically relevant molecules, electrodes have been used to electrostimulate the delivery of drugs as it was

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previously reported by Zhong et al.11 Last but not least, enzyme modified electrodes are the crucial ingredients for miniaturized biofuel cells that may power implantable medicals devices and will rely on the conversion of molecules present in different body fluids.12-15 However, despite the promising possible biomedical applications, in particular in the field of personalized medicine, the biocompatibility of the electrodes remains one of the major issues. Indeed, most of the systems investigated so far suffer from poor stability of the electroactive device, mainly due to strong biological responses, which are detrimental to short term and long term implantation. In this context, engineering soft electroactive electrode interfaces with a 3D structure for in vivo applications has attracted great attention.16,17 Based on their intrinsic properties, polymeric hydrogels have been intensively studied in the biomedical context, including scaffolds for tissue engineering,18,19 drug delivery carriers,20,21 biological assays or sensors.22 However, polymeric structures often suffer from several drawbacks, including proinflammatory activity, toxicity or the lack of biocompatibility. We suggest here a new approach to the compatibility problem of bioelectrode interfaces by exploiting the favorable biological properties of supramolecular low-molecular-weight-gelators (LMWG). LMWG are currently emerging as novel promising materials compatible with biological environments.23,24 We recently reported a new family of LMWG combining sugar, nucleoside and hydrophobic moieties. These glycosyl-nucleoside-amphiphiles are composed of lipids (GNLs),25 fluorinated hydrophobic chains (GNFs)26 or bola-amphiphilic (GNBA). These hybrid bio-inspired amphiphiles provide nanostructured gels exhibiting a variety of very interesting physical and biological properties for different fields, including the delivery of nucleic acids,27 regenerative medicine28 or the decontamination of nano-waste.29

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Scheme 1. Construction of hybrid LMWG-electrodes investigated in this study. a) Self assembly of GNF amphiphile to form a nanostructured hydrogel, b) TEM image of the obtained gel (scale bar: 100 nm), c) SEM image showing a cross section of a coated gold electrode.

Herein we report the coating of electrodes, including macroporous enzyme modified electrodes, with such a nano-structured gel using the LMWG approach. As an example, Scheme 1 depicts the hybrid LMWG-gold macroporous electrodes investigated in this study.

We assessed the electrochemical

properties of this hybrid material, namely the electrodes coated with LMWG, and demonstrated that the presence of a GNF coating first of all does not alter their general electrochemical behavior, but, most importantly, also significantly improves the lifetime of an electroenzymatic device. This allows envisioning the use of these gels in a general approach to design hybrid gel-electrode materials for biocompatible devices.

2. EXPERIMENTAL 2.1. Preparation of macroporous electrodes

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Macroporous electrodes were prepared according to a procedure that has been described in previous work.30 Briefly, the fabrication of macroporous substrates is based on potentiostatic electrodeposition of a conductive material (in the present work gold) into a highly-organized inorganic template (based on colloidal silica particles, Ø = 600 nm) that has been prepared by the Langmuir-Blodgett technique. The electrochemical infiltration of gold was performed at -660 mV in a conventional three-electrode system (silica template/gold working electrode, hollow Pt cylinder as counter and Ag/AgCl (3M NaCl) as reference electrode) by using a commercial electroplating gold solution (ECF60, Metalor®). In the last step, the silica particles were chemically etched from the silica/gold composite structure by 10 % HF for 10 min, and a macroporous structure has been obtained. This method presents a unique and convenient way to synthesize high-quality macroporous electrodes with a tunable thickness and porosity.31 2.2. Coating of LMWG on gold electrodes The coating of the gold substrates with the LMWG was achieved by dipping the electrodes into the hydrogel. In a typical experiment, a hydrogel of GNF was formed in a 2.5 mL glass tube (1% w/w). Prior to surface modification with GNF based hydrogel and in some cases also with enzymes, gold electrodes were pre-cleaned in Piranha solution (75% v/v conc. H2SO4 (98% wt.) / 25% v/v H2O2 (30% wt.)), thoroughly rinsed with MilliQ water and air-dried. Then the cylindrical gold electrodes were surface modified with hydrogel over a length of 1 cm (Ø (gold electrode) = 250 µm; S geometric = 16 mm2) by dipping in GNF based gel for 4 h at room temperature. After removing the electrode from the hydrogel, it was air dried for 10 min and used for cyclic voltammetry. This last step does not affect the gel integrity as confirmed by fluorescence imaging. 2.3. GNF based hydrogel The GNF-hydrogel was prepared at a concentration of 1 % (w/v) using the GNF powder simply dissolved in water. The GNF-based amphiphile was synthesized in three steps as previously reported by Godeau et al.26 The synthesis follows a first “click” reaction where the starting N-propargylated fluorocarbon

(N-propargyl-2H,2H,3H,3H-perfluoroundecanamide)

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reacted

with

5’azido-2’-

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deoxythymidine in the presence of CuSO4 and sodium ascorbate in a THF/H2O mixture to lead to the expected 2H,2H,3H,3H-perfluoroundecanamide-triazolyl-thymidine intermediate. This fluorocarbon nucleoside intermediate is then treated at room temperature in the presence of potassium carbonate with propargyl bromide to result in the N-propargylated thymidine derivative. The 1-azido-β-Dglucopyranosyl moiety is reacted with the N-propargyl thymidine derivative in the presence of CuSO4 and sodium ascorbate following a second “click” reaction to provide the expected non-ionic GNF. The final product was purified twice by silica gel column chromatography (ethyl acetate/methanol, increasing from 95/5 to 85/15), providing pure GNF characterized by NMR. Spectroscopic data agreed with the literature values. 2.4. Modification with enzymes The macroporous gold electrodes were pre-cleaned in a Piranha solution for 15 minutes and further rinsed with MilliQ water. Bilirubin oxidase (BOD) from Magnaporthe oryzae was produced and purified as already described.32 In the first step of the modification, the cleaned macroporous gold electrodes were immersed in 15µl of an aqueous enzyme solution (concentration 4 mg ml-1) and left for 18h at 40C to allow for the enzyme adsorption. Then, after rinsing the modified electrodes with water to remove unbound enzymes, the electrodes were dipped in an oxygen saturated phosphate buffer pH 7,2 to study the reduction of O2 at a scan rate of 5 mVs-1. In the experiments for which the effect of GNF was studied, after modification with the enzyme, the electrodes were dipped for 10 min in a 1% a GNF aqueous solution. 2.5. Electrochemical measurements The electrochemical measurements of different electrodes were performed using an Autolab PGSTAT 20 potentiostat/galvanostat (EcoChemie) system monitored by PC running with GPES 4.9 software. All experiments were conducted in a standard three-electrode system, consisting of a working electrode (bare Au electrodes, hydrogel-modified electrodes, and hydrogel-modified macroporous gold electrodes, respectively), a hollow Pt cylinder as counter and Ag/AgCl (3 M NaCl) as reference electrode. Cyclic

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voltammograms (CV) were recorded in a potential window from -0.05 V to +0.4 V at a typical scan rate of 5 mVs-1. The study of the scan rate dependence was carried out in the same potential window for scan rates of 5 mVs-1, 10 mVs-1, 20 mVs-1, 30 mVs-1, 40 mVs-1, 50 mVs-1 and 60 mVs-1, respectively. Electrochemical characterization measurements were carried out with a 1 mM aqueous solution of ferrocenemethanol (Fc·MeOH)) in 0.1 M KNO3 as supporting electrolyte under oxygen-free conditions. (All solutions were deaerated by bubbling argon for 10 min prior to the experiments and the electrochemical cell was kept under argon atmosphere throughout the experiments). Enzyme modified electrodes were studied by cyclic voltammetry and chronoamperometry and experiments were performed in a water-jacket electrochemical cell at 37,50C using a bipotentiostat (CH-Instruments, model CHI 842B, Austin, TX, USA) in a standard three-electrode cell with platinum wire as counter electrode and Ag/AgCl (3M KCl) as reference electrode. 2.6. Electron microscopy studies. SEM studies were carried out on GNF hydrogel coated gold electrodes before and after performing cyclic voltammetry. The uncoated gold wire was also observed under the SEM microscopy as control. All SEM experiments were performed on a Hitachi TM-1000 tabletop microscope. Samples containing GNF were obtained from the mixtures of 1 mg/mL in water. Before TEM imaging, the mixture was incubated for 12h hours at room temperature. TEM microscopy experiments were performed on a HITACHI H 7650 (negative staining with Uranyl acetate 1% in water, Ni carbon coated grids). 2.7. Fluorescence microscopy Rhodamine, a fluorescent dye (20 µM), was encapsulated in the GNF hydrogel in order to facilitate its visualization. This fluorescent hydrogel was then coated on macroporous gold wires in order to confirm the presence of the hydrogel coating on the gold surface by fluorescence imaging. Images were obtained with a Leica DM IRE2 confocal microscope at a 543 nm excitation wavelength.

3. RESULTS AND DISCUSSION

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3.1. Construction of LMWG on gold electrode Most of implanted electrodes are likely to generate fibrosis resulting in a loss of their activity within hours or days, depending on the bioelectrode interfaces. Hence, it is highly desirable to create an interface between the electrode and the surrounding medium like body fluid to protect it from harmful factors produced by inflammation and to minimize fibrosis induction. For this purpose, LMWGs have been used to wrap gold electrodes. We selected our recently reported hydrogelator GNF based on its biological and mechanical properties (Scheme 1a).23,29 Indeed, we could previously demonstrate that gels composed of GNF show only moderate inflammatory reaction and faint fibrosis. Several studies have evidenced that material stiffness is a key parameter governing cell fate, including the differentiation towards specialized cells.33 Here, the gels were generated by self-assembly of GNF, resulting in supramolecular complexes in aqueous medium in a temperature-dependent manner. Thanks to its amphiphilic properties the GNF forms a supramolecular assembly, stabilized by various weak interactions like hydrogen bonding, π stacking and van der Waals interactions. Previously, it has been reported that this molecule forms an entangled nanofibre network, which was confirmed by transmission electron microscopy.29 The non-cytotoxicity and biocompatibility of GNF hydrogel was demonstrated in vitro26 as well as in vivo,23 which further motivated the use of GNF LMWG as a bioelectrode interface. The GNF hydrogel was coated on bare and macroporous gold wires by dipping the gold wire in GNF hydrogel at room temperature. The SEM study was carried out to confirm the presence of hydrogel on the gold surface. The top view SEM image of the uncoated gold wire is shown in Figure SI1a, whereas the hydrogel coated gold surface is shown in Figure SI1b, which demonstrates the presence of hydrogel on the gold wire. The coating of GNF hydrogel on gold surface was further confirmed by a cross sectional view in SEM (Figs SI1c and SI1d). The thickness of the GNF hydrogel coating at the surface of the electrode was estimated to be of 1 to 2 micrometers.

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Figure 1. a) Fluorescence microscope image of a GNF/Rhodamine coated macroporous gold wire. b) Cross section fluorescence images of the GNF/Rhodamine coated bare gold wire. Fluorescence microscopy was chosen as an alternative technique to prove the presence of hydrogel on the gold wire. For that purpose, rhodamine was physically entrapped in the GNF hydrogel and coated on gold wires, which were then observed with the fluorescence microscope at 572nm. The fluorescence of rhodamine can be clearly seen everywhere on the gold wire (Fig. 1a). In addition, the cross section image of the modified fiber clearly indicates the presence of the GNF all around the fiber (Fig. 1b). 3.2. Physico-chemical studies. Cyclic-voltammetry. Electrochemical measurements enable us to gain further insight into the microstructure of the coatings. Cyclic voltammogramms (CVs) of bare gold electrodes and hybrid hydrogel-modified Au electrodes are presented in Figure 2. Hybrid electrodes were equilibrated in the test solution (1 mM Fc·MeOH in 0.1 M KNO3) prior to the measurements, since fully hydrated hydrogel coatings provide a more favorable microenvironment for unhindered mass transfer.

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Figure 2. CV of a bare gold wire electrode (green line) and one coated with GNF when cycled in 1mM Fc·MeOH in 0.1 M KNO3 (scan rate 5mV/s). For a bare Au electrode, a couple of well-defined reversible redox peaks can be noticed with a peak-topeak separation (∆Ep) of 70 mV, as it is approximately theoretically expected for a reversible electrochemical system. The self-assembly of GNF hydrogel on the electrode causes slight changes in the shape of the voltammogram. The behavior of the coated electrode is closer to what is typically observed for a microelectrode, namely almost steady state plateau currents. This is most likely due to the porosity of the gel, with the pores giving access to an ensemble of individual electroactive spots with non-overlapping diffusion layers. However as the global current is almost unchanged this does not seem to significantly affect the overall diffusion of electroactive species. Diffusion studies. Cyclic voltammetry has been used as a tool for the qualitative investigation of mass transport phenomena of redox-active species from the bulk solution towards GNF-modified electrodes. Figure 3a presents a set of CVs that have been recorded in a 1 mM Fc·MeOH solution at different scan rates as indicated. The linear dependence (Fig. 3b) of anodic peak current as a function of the square root of scan rate is given by the Randles-Ševčik equation as:

where:

- number of exchanged electrons,

- Faraday constant,

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diffusion coefficient of redox species,

– concentration of redox species,

- scan rate. This suggests

that the dominant mass transport of electroactive species from the solution to the electrode surface is diffusion, seemingly unaffected by the presence of the GNF coating. Furthermore, this result indicates the presence of functional porosity within the hydrogel structure, allowing a good permeation of small molecules through the electrically resistive GNF coating.

Figure 3. a) Set of cyclic voltammograms recorded with GNF coated gold electrodes in 1 mM Fc·MeOH solution (0.1 M KNO3) at different scan rates; b) Linear dependence of the anodic peak current intensity as a function of the square root of scan rate suggesting diffusional control of the electrode process.

To further prove the presence of GNF around the electrode, additional experiments were performed on GNF coated macroporous gold electrodes and uncoated ones. CVs have been first recorded for the uncoated GNF macroporous gold electrode (5 scans) in a 1 mM Fc·MeOH solution (0.1 M KNO3) at a scan rate 5 mV s-1 (results not shown). Then, the electrode was directly (without additional rinsing step to avoid washing out the residual of Fc·MeOH from the pores of the electrode) immersed in a deoxygenated solution of pure supporting electrolyte 0.1 M KNO3 and CVs were recorded (Fig. 4A). Similar experiments have been performed with electrodes modified with GNF and the results are shown in Figure 4B.

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In the first case (Fig. 4A) the difference in signal, in the potential range of the Fc+/Fc couple, between the 1st scan (black line) and the last scan (blue line) is almost unnoticeable, indicating that no ferrocene was trapped in the bare macroporous structure. The situation is different for the macroporous gold electrode covered by GNF. During exposure to the electrolyte containing the electroactive species, Fc·MeOH could be physically trapped in the gel. After the transfer to pure supporting electrolyte there is still a significant ferrocene signal in the first scan (black line, Fig. 4B) which then decreases and becomes undetectable after twelve scans (blue line). This clearly demonstrates the presence of the GNF film on the surface of the gold electrode and its ability to temporarily store Fc·MeOH molecules within its pores. The diffusion of Fc·MeOH from the film towards the bulk solution leads to a decrease of its concentration in the GNF film, resulting in a gradual alteration of the signal for consecutive cyclic voltammograms.

Figure 4. Cyclic voltammograms of residual Fc·MeOH recorded for macroporous gold electrodes without GNF (A) and with GNF (B) in a deoxygenated solution of pure supporting electrolyte 0.1M KNO3. 3.3. GNF coated macroporous enzyme electrodes To investigate the effect of the outer layer of GNF on the diffusion of O2 and on the stability of the enzyme modified electrodes, electrochemical experiments were performed with electrodes with or without GNF. As seen in Figure 5A, the current of electroenzymatic O2 reduction for the GNF modified electrode is slightly lower (red curve) than the current obtained for an electrode without GNF (black

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curve). This difference may be ascribed to a small difference in the geometric surface of the electrodes. However, as illustrated in Figure 5B, after 6h of continuous operation at +0.1V in an O2 phosphate buffer, the remaining current is almost two times higher for the GNF modified electrode (Fig. 5B red line) than for electrodes without GNF (Fig. 5B black line). This indicates that the presence of a GNF film on the modified electrode first of all does not affect the enzyme activity, does not impede the diffusion of O2 and, most importantly, allows for a better stability of the macroporous enzyme electrodes because it slows down the leaking of enzymes out of the macropores into the solution.

Figure 5. (A) Cyclic voltammograms obtained for a BOD-modified macroporous gold electrode without GNF film (black line) and with GNF film (red line) in oxygen saturated phosphate buffer pH 7.2; scan rate 5 mV s-1 (B) Relative change of the bioelectrocatalytic current with respect to the initial current of the modified electrodes without GNF (black line) and with GNF (red line) in oxygen saturated phosphate buffer pH 7.2 during 6h of continuous operation at +0.1V.

CONCLUSION In summary, novel supramolecular hydrogel coated electrodes were elaborated using a simple protocol and the (bio)electrochemical properties of this platform were assessed. The superior properties of the hybrid materials include the following: (1) the coverage of the hydrogel layer maintains the electrochemical performance of the electrode, (2) as revealed by cyclic voltammetry and diffusion studies, a high porosity within the hydrogel structure allows a good permeation of small molecules

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through the GNF coating, (3) importantly, the presence of a GNF film on macroporous enzyme electrodes slows down the current decay so that the final bioelectrocatalytic current after several hours of continuous operation is enhanced by 100% compared to the non-coated electrode. This indicates that the supramolecular hydrogel allows for a better stability of the modified electrodes. Altogether, these results, combined with the excellent biocompatibility of GNF based hydrogels, allow us to conclude that hybrid supramolecular hydrogel coated electrodes hold great potential for the development of biocompatible electrochemical devices, such as biosensors and biofuel cells, thus opening promising perspectives in terms of implantation.

ACKNOWLEDGMENT. This work, realized within the framework of the Laboratory of Excellence AMADEus with the reference ANR-10-LABX-0042-AMADEUS, has benefitted from aid by the state operated “Agence Nationale de la Recherche" under the program ‘’Initiative for Excellence IdEx Bordeaux’’ holding the reference ANR-10-IDEX-0003-02. The work has also been supported by the European project Bioenergy (FP7-PEOPLE-2013-ITN 607793) and by the ANR RATIOCELLS (12BS08-0011-01).The authors thank the BIC Center for technical assistance during TEM observations.

ASSOCIATED CONTENT Supporting Information. Hydrogel photographs, complementary TEM SEM microscopy images. This material is available free of charge via the Internet http://pubs.acs.org REFERENCES (1) Famm, K.; Litt, B.; Tracey, K. J.; Boyden E. S.; and Slaoui, M. Drug discovery: A Jump-start for Electroceuticals Nature 2013, 496, 159-161. (2) Hiemke, C. Clinical Utility of Drug Measurement and Pharmacokinetics – Therapeutic Drug Monitoring in Psychiatry Eur. J. Clin. Pharmacol. 2008, 64, 159-166.

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(3) Wise, K. D.; Sodagar, A. M.; Yao, Y.; Gulari, M. N.; Perlin, G. E.; and Najafi, K. Microelectrodes, Microelectronics, and Implantable Neural Microsystems Proc. IEEE 2008, 96, 11842002. (4) Takashi, D.; Kozai, Y.; Langhals, N. B.; Patel, P. R.; Deng, X.; Zhang, H.; Smith, K. L.; Lahann, J.; Kotov, N. A.; and Kipke. D. R. Ultrasmall Implantable Composite Microelectrodes with Bioactive Surfaces for Chronic Neural Interfaces Nat. Mater. 2012, 11, 1065–1073 (5) Reardon S. Memory-boosting Devices Tested in Humans Nature. 2015, 527, 15-16. (6) Aarnoutse, R. E.; Schapiro, J. M.; Boucher, C.A.B.; Hekster, Y.A.; and Burger, D. M. Therapeutic Drug Monitoring: an Aid to Optimising Response to Antiretroviral Drugs? Drugs, 2003, 63, 741-753. (7) Hajian, R.; Mehrayin, Z.; Mohagheghian, M.; Zafari, M.; Hosseini, P.; Shams N. Fabrication of an Electrochemical Sensor Based on Carbon Nanotubes Modified with Gold Nanoparticles for Determination of Valrubicin as a Chemotherapy Drug: Valrubicin-DNA Interaction Mater. Sci. Eng., C. 2015, 49, 769-775. (8) Heidarimoghadam, R.; Farmany, A. Rapid Determination of Furosemide in Drug and Blood Plasma of Wrestlers by a Carboxyl-MWCNT Sensor Mater. Sci. Eng., C. 2016, 58, 1242-1245. (9) El-Maali, N.A. Voltammetric Analysis of Drugs Bioelectrochemistry, 2004, 64, 99- 107. (10) V.K. Gupta, R. Jain, K. Radhapyari, N. Jadon, and S. Agarwal Voltammetric Techniques for the Assay of Pharmaceuticals-A Review Anal. Biochem., 2011, 408, 179- 196. (11) Zhong, Y.; Bellamkonda, R.V. Controlled Release of Anti-inflammatory Agent α-MSH from Neural Implants J. Controlled Release 2005, 106, 309-318. (12) Rasmussen, M.; Abdellaoui, S.; Minteer, S. D. Enzymatic Biofuel cells: 30 Years of Critical Advancements Biosens. Bioelectron. 2016, 76, 91-102 (13) Leech, D.; Kavanagh, P.; Schuhmann, W. Enzymatic Fuel Cells: Recent Progress Electrochim. Acta. 2012, 84, 223-234 (14) Falk, M.; Narváez Villarrubia, C. W.; Babanova, S.; Atanassov, P.; Shleev, S. Biofuel Cells for Biomedical Applications: Colonizing the Animal Kingdom ChemPhysChem 2013, 14, 2045-2058; (15) Katz, E.; MacVittie, K. Implanted Biofuel Cells Operating in vivo – Methods, Applications and

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c) Electrochemical performance



Supramolecular Hydrogel Coating

Stability of Ez modified electrodes

Gold Electrodes

Table of contents graphic

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