Electrochemical Characterization of Layer-By-Layer Assembled

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Electrochemical Characterization of Layer-by-Layer Assembled Ferrocene-Modified Linear Poly(ethylenimine)/Enzyme Bioanodes for Glucose Sensor and Biofuel Cell Applications Nicholas P. Godman, Jared L. DeLuca, Sean R. Mccollum, David W Schmidtke, and Daniel T. Glatzhofer Langmuir, Just Accepted Manuscript • DOI: 10.1021/acs.langmuir.5b04753 • Publication Date (Web): 21 Mar 2016 Downloaded from http://pubs.acs.org on March 28, 2016

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Electrochemical Characterization of Layer-By-Layer Assembled Ferrocene-Modified Linear Poly(ethylenimine)/Enzyme Bioanodes for Glucose Sensor and Biofuel Cell Applications Nicholas P. Godmanψ, Jared L. DeLucaα,ξ, Sean R. McCollumα,ξ, David W. Schmidtkeα,ξ,β, and Daniel T. Glatzhoferψ,* The University of Oklahoma, 100 East Boyd, Norman, OK 73019 ψ

Department of Chemistry and Biochemistry

α

University of Oklahoma Biomedical Engineering Center

ξ

School of Chemical, Biological and Materials Engineering

β

Current Address: University of Texas at Dallas Department of Bioengineering 800 W. Campbell Rd. Richardson, TX 75083 *

Corresponding author University of Oklahoma, Department of Chemistry and Biochemistry 800 W. Campbell Rd. Norman, OK 73019 E-mail: [email protected] TEL: 405-325-3834, FAX: 405-325-6111


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ABSTRACT: Ferrocenylhexyl- and ferrocenylpropyl- modified linear poly(ethylenimine) (Fc-C6-LPEI, Fc-C3-LPEI) were used with periodate modified glucose oxidase (p-GOX) in the layer-by-layer assembly of enzymatic bioanodes on gold. (Fc-C6-LPEI/p-GOX) and (Fc-C3-LPEI/p-GOX) films of sixteen bilayers were capable of generating up to 381 ± 3 and 1417 ± 63 µA cm-2, respectively, in response to glucose. These responses were greater than those of analogous bioanodes fabricated using conventional cross-linking techniques, and are extremely high for planar, low surface area, single enzyme electrodes. (Fc-C3-LPEI/p-GOX)8 films generated 86 ± 3 µWcm-2 at pH 7.0 and 149 ± 7 µWcm2

at pH 5.0, when poised against an air-breathing platinum cathode in a compartment-less biofuel cell.

An increase in power output with decreasing pH was shown to be a result of increases in the platinum cathode performance, indicating it is the rate-limiting electrode in the biofuel cells. The effect of fabrication wash time on the build-up of material at the electrode’s surface was probed using cyclic voltammetry (CV) and constant potential amperometry. The use of electrochemical techniques as a diagnostic tool for studying the material deposition process is discussed. CV peak separation (∆E), surface coverage of the electroactive ferrocene (ΓFc), and amperometric sensitivity of the enzyme to glucose (Jmax), studied as a function of numbers of bilayers, showed that physisorption of materials onto the surface results from initial patchy deposition, rather than in distinctly uniform layers.

INTRODUCTION Biofuel cells generate electrical currents by capturing electrons from redox processes found in nature. One method by which this is accomplished is through the immobilization of redox active enzymes and harvesting electrical current from these natural catalysts1. Glucose oxidase (GOX) is one such redox enzyme that has been extensively used for enzymatic biofuel cells2,3. There is particular interest in “wiring” GOX together with osmium4,5 or ferrocene6,7 based redox mediators to form


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cohesive bioanode films. Recent work has shown that hydrogels containing ferrocene moieties are exceptionally capable of mediating electron transfer from the GOX active site to the surface of an electrode.8,9 There are many ways to immobilize enzymes on the surface of an electrode: entrapment within a polymer matrix10, adsorption onto an electrode surface11, chemical crosslinking12,13, self-assembled monolayers14, and layer-by-layer assembly15. Due to the fast interaction times between layers, the layerby-layer (LBL) fabrication method is a useful method to quickly assemble redox active films. Electrostatic LBL assembly is based on the adsorption and self-assembly of oppositely charged polyelectrolytes. By alternating layers of polyelectrolytes, thickness-tunable enzyme/polymer multilayers can be quickly generated without the need for extended curing of chemically cross-linked films. The LBL fabrication of molecular bilayers is also useful in that it allows for the construction of systems in a relatively ordered, predetermined fashion. Alternating layers of charge-complementary enzymes and redox polymers allows for material dispersion throughout the entire film, and additional stability can be achieved by covalently binding the enzyme directly to the polymer. Polyamines are one class of polymers that are useful as the cationic layer in the LBL technique. One such polyamine that has been used as a scaffold in LBL assembled biosensors is branched poly(ethylenimine) (BPEI). Sensors have been fabricated that are specific to a variety of targets including: cocaine16, cancer cells17, thrombin and lysozymes18, lactose19,20

,21

, and glucose22. LBL

systems using BPEI have also been fabricated using DNA as a polyelectrolyte23, made for the controlled delivery of drugs24, the oxidation of NADH25, and used to increase the growth of E. coli26. While BPEI has been well established as a polycation for LBL assembled biosensors, there have been relatively few reports using linear poly(ethylenimine) (LPEI) or it’s derivatives. Our group has previously shown that ferrocene-modified LPEI produces significantly higher current densities than analogous ferrocene-


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modified BPEI for solution cast, crosslinked enzymatic bioanodes27, and have recently shown that ferrocene tethered to LPEI by a six carbon spacer (Fc-C6-LPEI) can be used in the LBL assembly of glucose biosensors28. However, these Fc-C6-LPEI LBL electrodes produced ca. 1/3 the current compared to the analogous, conventionally cross-linked Fc-C6-LPEI bioanodes in response to glucose. Based on the results for these systems, it was desirable to further probe the use of other LPEI scaffolds for the LBL technique by varying structural aspects of the redox polymer derived from it (e. g. spacer length), and in optimization of the LBL fabrication and characterization. The characterization of LBL films is typically achieved through the use of ellipsometry29, UVVis spectroscopy30, and quartz crystal microgravimetry31. These methods are especially useful for quantifying the thickness, type, and amount of materials deposited in each layer, and have been used to characterize Fc-C6-LPEI LBL electrodes28. However, these techniques give very limited information about the deposition process itself. Surprisingly, electrochemical methods such as cyclic voltammetry (CV) have been underutilized in the study and characterization of LBL multilayered polymer/enzyme films. Hodak et al. looked at the increase in charge with the addition of ferrocene-modified poly(allylamine) layers7, but most reported data simply deals with the increase in sensor response with the build-up of material. Since the electrochemistry of self-assembled monolayers is well-studied32, the use of electrochemical methods to characterize LBL multilayers to study the LBL assembly process is promising. Herein we describe how the tether length of ferrocene modified linear poly(ethylenimine) (FcCn-LPEI), and the wash time between bilayers, effect the electrochemical and enzymatic response of LBL assembled films. The use of electrochemical methods as a tool for characterization, and the ability to use them to study the build-up of materials during the LBL assembly process, is discussed. Most LBL assembled glucose bioanodes use conductive fillers or high-surface area substrates to enhance


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electrochemical response, whereas our system uses planar, low-surface area electrodes coated with ultrathin conducting films. Nevertheless, the results presented here are among the most highly sensitive LBL assembled glucose bioanodes reported in the literature to date, and are capable of producing some of the highest current densities for LBL assembled glucose biosensors. The high current densities of the biosensors led us to demonstrate for the first time that LBL assembled films based on ferrocene redox polymers can be used as bioanodes in biofuel cells. The power densities obtained compare favorably with other LBL glucose/oxygen biofuel cells reported in the literature, including those with augmented conductive substrates or fillers. EXPERIMENTAL Chemicals and Solutions: Glucose oxidase (GOX) from Aspergillus niger (EC 1.1.3.4, type X-S, 117 units/mg solid, 75% protein), cystamine dihydrochloride, and D-glucose were purchased from Sigma-Aldrich Co. and all chemicals were used as received. The redox polymers Fc-C3-LPEI and Fc-C6-LPEI were synthesized according to a previously published procedure6 from LPEI (avg. MW ca. 86000),33 (3bromopropyl)ferrocene34, and (6-bromohexyl)ferrocene (Sigma-Aldrich). The polymers were found to have 17-20% of the nitrogen atoms in the polymer backbone substituted with ferrocene functional groups using NMR spectroscopy.6 Sodium phosphate buffer (50 mM) was prepared by dissolving NaH2PO4 in Nanopure deionized water and adding sodium hydroxide pellets with stirring to adjust the pH to 7.0. Spectra/Por molecular porous membrane tubing (Mw cutoff 12000–14 000) from Spectrum Labs and a sodium phosphate buffer (20 mM) at pH 7.4 were used for dialysis of the modified enzyme. Stock solutions of 2 M glucose were stored at 4 °C and allowed to mutorotate for 24 hours prior to use. LBL Assembly of Films


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Gold electrodes (2.0 mm diameter, CH instruments, Austin, TX) were polished with 1 and 0.25 µm diamond polishing paste on nylon polishing pads before being polished with 0.05 µm alumina on a microcloth pad. Redox polymer solutions (Fc-C6-LPEI, Fc-C3-LPEI) were prepared by dissolving them in Nanopure water until the final concentration was 10 mg/mL and the pH was adjusted to 5.0 by the addition of small aliquots of concentrated HCl and NaOH. Periodate-modified glucose oxidase (p-GOX) solution (20 µM) was prepared using a previously reported protocol.28 GOX’s peripheral oligosaccharides are oxidized by sodium periodate to install aldehyde functional groups. This allows for convenient sites with which to attach the enzyme to amine moieties. Gold electrodes were modified with cystamine by immersing the electrode in a 20 mM solution of disulfide cystamine dihydrochloride for 20 minutes. Electrodes were removed from solution and washed with Nanopure water to remove excess unbound material. Modified electrodes were immersed in p-GOX solution for five minutes to deposit and covalently attach enzymes to the cystamine layer. Electrodes were washed with Nanopure water by one of two methods: a long wash (6-8 sec.) or short wash (Km). If it is assumed that all the active enzyme molecules in the first bilayer are effectively “wired” to the electrode by the FcLPEI layer, so that electron transport is not rate limiting, the minimum effective enzyme loading (ΓE) can be calculated from: ΓE = Jmax/2Fkcat, where F is Faraday’s constant and kcat is the turnover number for the enzyme (700 sec-1)39. Although not apparent in Figure 4 because of the scale, Jmax values for single bilayer films are 7.2 and 8.0 µA/cm2 for the short wash (Fc-C3-LPEI/p-


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GOX)1 and (Fc-C6-LPEI/p-GOX)1 films, respectively, and 2.4 and 0.9 µA/cm2 for the long wash (FcC3-LPEI/p-GOX)1 and (Fc-C6-LPEI/p-GOX)1 films, respectively. A Jmax value of 8.0 µA/cm2 corresponds to a ΓE of 0.15 µg/cm2. This is in reasonable agreement with a value of ca. 0.8 µg/cm2 found for equilibrium adsorption of GOX onto cysteine coated gold electrodes measured using a quartz crystal microbalance38. It should be noted that ΓE is only a lower limit of total enzyme loading and the lower value may result from loss of material on rinsing, inactive enzyme, inefficient electron transfer to the electrode surface for some enzyme molecules, or an overestimation of kcat. Most literature values of kcat for GOX are lower than 700 sec-1. If a kcat value of 250 sec-1 is used40, a ΓE value of 0.42 µg/cm2 is obtained, much closer to the literature value. The electrochemical response for the first bilayer for both short wash (Fc-C3-LPEI/p-GOX)1 and (Fc-C6-LPEI/p-GOX)1 films are similar, as might be expected since the surface loading of GOX should be nearly the same. It is also clear from Figure 4 that Jmax does not increase proportionally with the number of bilayers, consistent with the model of a non-uniform deposition process discussed earlier. Also as expected, this treatment of the data is consistent with the view that the long wash procedure removes more material, likely including enzyme. Performance of LBL Assembled Anodes in a Biofuel Cell The high current density response of LBL assembled (Fc-C3-LPEI/p-GOX)n films to glucose made them of interest for the construction of compartment-less biofuel cells. Due to their maximal current density and ease of fabrication, (Fc-C3-LPEI/p-GOX)8 films constructed using the short washing method were chosen as the bioanodes. A commercially available, air-breathing platinum electrode was chosen as the cathode in an effort to ensure that it would not be the limiting electrode in the biofuel cell. The (Fc-C3-LPEI/p-GOX)8 bioanode and air breathing platinum cathode were allowed to equilibrate in a solution of 100 mM glucose at open circuit potential for 60 seconds before being tested.


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The resulting power curves are shown in Figure 7A. These fuel cells were able generate 86 ± 3 µWcm-2 at ca. 132 mV with a maximum current density of 776 ± 3 µAcm-2 at pH 7.0 and 25°C. This demonstrated that LBL fabrication provides a quick and facile means to produce redox active bioanodes that can be used in effective biofuel cells. The compartment-less fuel cells were originally run at pH 7.0 to mimic physiological conditions, but because the activity of platinum is known to be pH dependent, the fuel cells were retested at pH 5.0. The results for the fuel cell and the corresponding steady-state glucose response curves are shown in Figures 7. By lowering the pH two units, the power density of the fuel cell increased to 149 ± 7 µWcm-2 at ca. 184 mV, with a maximum current density of 930 ± 34 µAcm-2 at pH 5 and 25°C. When the same bioanodes were run as a biosensor (Figure 7B), Jmax decreased from 773 ± 13 µAcm-2 at pH 7.0 to 355 ± 17 µAcm-2 at pH 5.0. Therefore, the increased biofuel cell power results from the air breathing cathode, and shows it is the rate limiting electrode is spite of its large surface area. Raising the potential of platinum, and having a greater proton concentration that facilitates the reduction of oxygen to water, accounts for the increase in response of the fuel cell at pH 5.0. Biofuel response is expected to improve with the addition of more bilayers to the bioanode, but only if the cathode is improved as well.


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Figure 7. Effect of electrolyte pH on (Fc-C3-LPEI/p-GOX)8 bioanode performance: pH 7.0 (solid), pH 5.0 (dotted). (A) Representative power curves obtained by poising the potential against an air-breathing Pt cathode. Scan rate = 2 mVs-1. (B) Constant potential, steady state glucose calibration curves. E = 0.35 V. The power densities for (Fc-C3-LPEI/p-GOX)8/Pt biofuel cells compare favorably with other glucose/oxygen biofuel cells using LBL surface modified electrodes (Table 2). All previously reported LBL assembled biofuel cells have used nanoparticles (gold41,42 or platinum43), nanotubes (single44 or


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multiwall42,43,45), graphene45, or high surface area electrodes (graphite46, carbon paper43, macroporous gold41) in their fabrication. These materials either artificially increase the electrode surface area or enhance electronic conductivity through the assembled films. The work presented herein employs planar, low-surface area gold electrodes coated with thin conducting films that accurately reflect the geometric surface in the power densities. Fc-C3-LPEI produces current via collisional electron transfer that is not enhanced by the addition of nanoparticles, which makes the obtained results even more favorable in comparison. Table 2. Comparison of glucose/oxygen biofuel cells fabricated using LBL assembly

(AuNP/laccase)5

Pmax (µW/cm2) 178

VPmax (mV) 226

6.0

T (°C) RT

(AuNP/GDH)5

(AuNP/laccase)5

12.6

360

6.0

RT

41

ITO

(MWNT/thinonine/AuNP)8/GDH

(MWNT/PLL/laccase)15

329

470

6.0

RT

42

carbon paper

MWNT/(GOX/Pt-DEN)3

Pt on carbon paper*

17

NR

6.8

RT

43

glassy carbon

(SWNT/NADP+/GDH)1

SWNT/bilirubin oxidase*

23

NR

7.0

RT

44

glassy carbon

MG/(graphene/MWNT)5/GDH

SWNT/laccase*

22.5

480

6.0

RT

45

graphite

(PVI-OsOMe/GOX)2

(PVI-OsCl/laccase)2

103

350

5.0

37

planar gold

(Fc-C3-LPEI/p-GOX)8

air-breathing Pt*

149

184

5.0

RT

46 This work

Electrode

Anode

Cathode

3DOM gold

(AuNP/GDH)5

planar gold

pH

Ref. 41

3DOM = three-dimensional ordered macroporous AuNP = gold nanoparticle GDH = glucose dehydrogenase MWNT = multiwall nanotube ITO = iridium tin oxide PLL = poly-L-lysine Pt-DEN = poly(amidoamine) dendrimer-encapsulated platinum nanoparticles SWNT = single wall nanotube MG = methylene green PVI-OsOMe = methoxypbipyridylosmium-modified poly(vinylimidizole) PVI-OsCl = chloropbipyridylosmium-modified poly(vinylimidizole) NR = not reported RT = room temperature * not constructed using LBL assembly

CONCLUSIONS


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We have shown that the response of layer-by-layer assembled glucose bioanodes can be greatly improved by shortening the tether length of Fc-Cn-LPEI from six carbons by three carbons. We have also demonstrated that the washing procedure employed while processing electrodes is important to the overall response of the films. The combination of Fc-C3-LPEI and the short washing method account for some of the highest current densities thus far reported for LBL assembled glucose biosensors. The use of electrochemical methods to gain a better understanding of how materials initially adsorb, and then build upon the surface of an electrode, has been demonstrated. A simplified model based on the formation of distinctly isolated layers of material was shown to not be the best approximation for the LBL assembly of the redox polymers and glucose oxidase used here, especially for the first several bilayers. Initial “patchy” deposition, followed by “filling in” of the surface in the next several bilayers, was shown to be more consistent with the experimental data. The mediator tether length and the fabrication wash time were shown to effect the packing of material onto the electrode based on the surface coverage and connectivity of polymer/enzyme composites. When poising against at air-breathing Pt cathode, (Fc-C3-LPEI/p-GOX)8 bioanodes (short wash) were able to reach a maximum power density of 86 ± 3 µWcm-2 at ca. 132 mV at pH 7 and 25°C. By lowering the pH to affect the reduction potential at which the Pt electrode reduces oxygen, the maximum power density of the biofuel cells increased to 149 ± 7 µWcm-2 at ca. 132 mV. It is conceivable that the power output could be further increased if larger numbers of bilayers and a truly non-rate limiting cathode were used. The potentials of these fuel cells at maximum power are modest, between 0.1 and 0.2 V. Further work to increase the cell voltage needs to be done by lowering the overpotential of the redox polymer at the bioanode. It was previously reported that methylation of the ferrocene mediator moieties decreases the redox potential by ca. 50 mV.9 As there are nine positions for methylation on FcC3-LPEI, a decrease in its redox potential of 0.45 V may be able to be achieved. This would


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theoretically result in fuel cells with potentials at maximum power between 0.5 and 0.7 V. Pursuit of this strategy is ongoing. The use of the LBL technique with cationic FcLPEI-type polymers on gold electrodes constrains the films to first have a coupling layer (cysteine), followed by a layer of anionically surface charged GOX. The use of carbon electrode materials may obviate this problem as they can be modified to have carboxylic acid or phenolic groups on the surface47, easily giving an anionic surface charge at appropriate pH’s, and allowing the deposition of cationic FcLPEI polymers as the first layer. Carbon electrode materials are available with very high surface areas that can increase polymer/enzyme loading and current outputs, as discussed at the end of the previous section, and are amenable to the LBL technique46. We have used such substrates in previous work with cross-linked FcLPEI polymer films that illustrate increased loading and current outputs48. Studies using the LBL technique applied to FcLPEI-type polymer/enzyme systems on carbonaceous materials are ongoing. Aknowledgements The authors would like to thank the University of Oklahoma and the National Science Foundation (grants CBET 0967988 and DMR-1310407) for support of this work. We thank Mr. Daniel Bamper for some material preparations.

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22. Liu, D.; Hu, N., pH-switchable bioelectrocatalysis based on layer-by-layer films assembled with glucose oxidase and branched poly(ethyleneimine). Sensors and Actuators B: Chemical 2011, 156 (2), 645-650. 23. Kang, Z.; Yan, X.; Zhang, Y.; Pan, J.; Shi, J.; Zhang, X.; Liu, Y.; Choi, J. H.; Porterfield, D. M., Single-Stranded DNA Functionalized Single-Walled Carbon Nanotubes for Microbiosensors via Layerby-Layer Electrostatic Self-Assembly. ACS Applied Materials & Interfaces 2014, 6 (6), 3784-3789. 24. Takahashi, S.; Aikawa, Y.; Kudo, T.; Ono, T.; Kashiwagi, Y.; Anzai, J.-i., Electrochemical decomposition of layer-by-layer thin films composed of TEMPO-modified poly(acrylic acid) and poly(ethyleneimine). Colloid Polym Sci 2014, 292 (3), 771-776. 25. Abe, K.; Horiuchi, T.; Nishibayashi, Y.; Kobayashi, K.; Aburai, N., Development of a novel enzyme-CdS nanoparticle hybrid system for the oxidation of NADH to NAD+. Materials Letters 2014, 134 (0), 20-23. 26. Xia, B.; Shi, J.; Dong, C.; Zhang, W.; Lu, Y.; Guo, P., Covalent assembly of poly(ethyleneimine) via layer-by-layer deposition for enhancing surface density of protein and bacteria attachment. Applied Surface Science 2014, 292 (0), 1040-1044. 27. Merchant, S. A.; Glatzhofer, D. T.; Schmidtke, D. W., Effects of Electrolyte and pH on the Behavior of Cross-Linked Films of Ferrocene-Modified Poly(ethylenimine). Langmuir 2007, 23 (22), 11295-11302. 28. DeLuca, J. L.; Hickey, D. P.; Bamper, D. A.; Glatzhofer, D. T.; Johnson, M. B.; Schmidtke, D. W., Layer-by-Layer Assembly of Ferrocene-Modified Linear Polyethylenimine Redox Polymer Films. ChemPhysChem 2013, 14 (10), 2149-2158. 29. Arwin, H., Ellipsometry on thin organic layers of biological interest: characterization and applications. Thin Solid Films 2000, 377–378 (0), 48-56. 30. Lvov, Y.; Ariga, K.; Ichinose, I.; Kunitake, T., Assembly of Multicomponent Protein Films by Means of Electrostatic Layer-by-Layer Adsorption. Journal of the American Chemical Society 1995, 117 (22), 6117-6123. 31. Marx, K. A., Quartz Crystal Microbalance: A Useful Tool for Studying Thin Polymer Films and Complex Biomolecular Systems at the Solution−Surface Interface. Biomacromolecules 2003, 4 (5), 1099-1120. 32. Eckermann, A. L.; Feld, D. J.; Shaw, J. A.; Meade, T. J., Electrochemistry of redox-active selfassembled monolayers. Coordination Chemistry Reviews 2010, 254 (15–16), 1769-1802. 33. York, S.; Frech, R.; Snow, A.; Glatzhofer, D., A comparative vibrational spectroscopic study of lithium triflate and sodium triflate in linear poly(ethylenimine). Electrochimica Acta 2001, 46 (10–11), 1533-1537. 34. Métay, E.; Duclos, M. C.; Pellet-Rostaing, S.; Lemaire, M.; Schulz, J.; Kannappan, R.; Bucher, C.; Saint-Aman, E.; Chaix, C., Synthesis and Anion-Binding Properties of Novel Redox-Active Calixarene Receptors. European Journal of Organic Chemistry 2008, 2008 (25), 4304-4312. 35. Lillethorup, M.; Shimizu, K.; Plumeré, N.; Pedersen, S. U.; Daasbjerg, K., Surface-Attached Poly(glycidyl methacrylate) as a Versatile Platform for Creating Dual-Functional Polymer Brushes. Macromolecules 2014, 47 (15), 5081-5088. 36. Liang, B.; Guo, X.; Fang, L.; Hu, Y.; Yang, G.; Zhu, Q.; Wei, J.; Ye, X., Study of direct electron transfer and enzyme activity of glucose oxidase on graphene surface. Electrochemistry Communications 2015, 50 (0), 1-5. 37. Zhang, Y.; Li, Y.; Wu, W.; Jiang, Y.; Hu, B., Chitosan coated on the layers’ glucose oxidase immobilized on cysteamine/Au electrode for use as glucose biosensor. Biosensors and Bioelectronics 2014, 60 (0), 271-276.


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38. Calvo, E. J.; Etchenique, R.; Pietrasanta, L.; Wolosiuk, A.; Danilowicz, C., Layer-By-Layer SelfAssembly of Glucose Oxidase and Os(Bpy)2ClPyCH2NH−poly(Allylamine) Bioelectrode. Analytical Chemistry 2001, 73 (6), 1161-1168. 39. Calvo, E.; Wolosiuk, A, Wiring Enzymes in Nanostructures Built with Electrostatically SelfAssembled Thin Films, ChemPhysChem 2005, 6, 43-47. 40. Zink, J. I.; Dunn, B., Sol-Gel Encapsulated Molecules: Optical Probes and Optical Properties, in Sol-gel Optics Processing and Applications, Klein, L. C. Ed., New York: Springer Science, 1994, p. 321. 41. Deng, L.; Wang, F.; Chen, H.; Shang, L.; Wang, L.; Wang, T.; Dong, S., A biofuel cell with enhanced performance by multilayer biocatalyst immobilized on highly ordered macroporous electrode. Biosensors and Bioelectronics 2008, 24 (2), 329-333. 42. Deng, L.; Shang, L.; Wang, Y.; Wang, T.; Chen, H.; Dong, S., Multilayer structured carbon nanotubes/poly-l-lysine/laccase composite cathode for glucose/O2 biofuel cell. Electrochemistry Communications 2008, 10 (7), 1012-1015. 43. Zhang, J.; Zhu, Y.; Chen, C.; Yang, X.; Li, C., Carbon nanotubes coated with platinum nanoparticles as anode of biofuel cell. Particuology 2012, 10 (4), 450-455. 44. Yan, Y.-M.; Yehezkeli, O.; Willner, I., Integrated, Electrically Contacted NAD(P)+-Dependent Enzyme–Carbon Nanotube Electrodes for Biosensors and Biofuel Cell Applications. Chemistry – A European Journal 2007, 13 (36), 10168-10175. 45. Wang, X.; Wang, J.; Cheng, H.; Yu, P.; Ye, J.; Mao, L., Graphene as a Spacer to Layer-by-Layer Assemble Electrochemically Functionalized Nanostructures for Molecular Bioelectronic Devices. Langmuir 2011, 27 (17), 11180-11186. 46. Rengaraj, S.; Mani, V.; Kavanagh, P.; Rusling, J.; Leech, D., A membrane-less enzymatic fuel cell with layer-by-layer assembly of redox polymer and enzyme over graphite electrodes. Chemical Communications 2011, 47 (43), 11861-11863. 47. Tikhomerov, A.S.; Sorokina, N. E.; Avdeev, V. V., Surface Modification of Carbon Fibers with Nitric Acid Solutions, Inorganic Materials, 2011, 47 (6), 684-688. 48. Hickey, D. P.; Halmes, A. J.; Schmidtke, D. W.; Glatzhofer, D. T., Electrochemical Characterization of Glucose Bioanodes Based on Tetramethylferrocene-Modified Linear Poly(ethylenimine), Electrochimica Acta, 2014, 149, 252-257.


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Periodate modification of glucose oxidase and presumed attachment to Fc-Cn-LPEI 137x66mm (300 x 300 DPI)


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Representative CVs for Fc-Cn-LPEI/p-GOX films assembled with increasing numbers of bilayers (1, 2, 4, 8, 12, 16) and various fabrication wash methods. A) Fc-C6-LPEI, long wash. B) Fc-C3-LPEI, long wash. C) FcC6-LPEI, short wash. D) Fc-C3-LPEI, short wash. 177x165mm (300 x 300 DPI)


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Plot of ferrocene surface coverage (ΓFc), obtained by integration of the anodic wave of the CV, against the potential separation between the anodic and cathodic peaks (∆E) of the CV. (A) Comparison of long wash (white triangle) and the short wash (black triangle) fabrication for Fc-C3-LPEI/p-GOX films. (B) Comparison of long wash (white circle) and the short wash (black circle) fabrication for Fc-C6-LPEI/p-GOX films. 78x165mm (300 x 300 DPI)


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Effect of mediator tether length and fabrication wash time on glucose response for Fc-Cn-LPEI/p-GOX films assembled with increasing number of bilayers (1, 2, 4, 8, 12, 16). A) Fc-C6-LPEI, long wash. B) Fc-C3-LPEI, long wash. C) Fc-C6-LPEI, short wash. D) Fc-C3-LPEI, short wash . 177x165mm (300 x 300 DPI)


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Plot of biosensor sensitivity at 5 mM glucose against the number of assembled bilayers. Fc-C3-LPEI, short wash (black triangle). Fc-C3-LPEI, long wash (black circle). Fc-C6-LPEI, short wash (white triangle). Fc-C6LPEI, long wash (white circle). 261x246mm (96 x 96 DPI)


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Effect of mediator tether length and fabrication wash time on K*M for Fc-Cn-LPEI/p-GOX films assembled with increasing number of bilayers (1, 2, 4, 8, 12, 16). Fc-C6-LPEI, long wash (light gray). Fc-C6-LPEI, short wash (dark gray). Fc-C3-LPEI, long wash (white). Fc-C3-LPEI, short wash (black ) 177x50mm (300 x 300 DPI)


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Figure 7. Effect of electrolyte pH on (Fc-C3-LPEI/p-GOX)8 bioanode performance: pH 7.0 (solid), pH 5.0 (dotted). (A) Representative power curves obtained by poising the potential against an air-breathing Pt cathode. Scan rate = 2 mVs-1. (B) Constant potential, steady state glucose calibration curves. E = 0.35 V. 83x165mm (300 x 300 DPI)


Electrochemical Characterization of Layer-By-Layer Assembled

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