Electrical Contacting of Redox Enzymes by Means of Oligoaniline

Aug 12, 2009 - aniline components lead to the effective electrical wiring of the enzyme units ... The electrical contacting of redox proteins with ele...
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Electrical Contacting of Redox Enzymes by Means of Oligoaniline-Cross-Linked Enzyme/Carbon Nanotube Composites† Ilina Baravik, Ran Tel-Vered, Oded Ovits, and Itamar Willner* Institute of Chemistry, The Hebrew University of Jerusalem, Jerusalem 91904, Israel Received June 10, 2009. Revised Manuscript Received July 16, 2009 The electropolymerization of aniline-functionalized carbon nanotubes (CNTs) and thioaniline-modified glucose oxidase (GOx) on a thioaniline monolayer-modified Au electrode yields a CNTs/GOx composite cross-linked by the redox-active bisaniline units. The three-dimensional conductivity of the CNTs matrix and the electron-transfer (relay) properties of the bisaniline components lead to the effective electrical wiring of the enzyme units with the electrode, and to the efficient bioelectrocatalytic oxidation of glucose (turnover rate ca. ket = 1025 s-1). The conditions to synthesize the three-dimensional CNTs/GOx composite and the parameters influencing the bioelectrocatalytic functions of the modified electrode are discussed.

The electrical contacting of redox proteins with electrodes is a basic challenge in modern bioelectrochemistry and is an essential process for designing amperometric biosensors or biofuel cells.1-4 Diffusional electron mediators,5,6 the tethering of redox relay units to the proteins,7,8 and the incorporation of the enzymes in redox polymer matrices9,10 are common practices to electrically wire proteins with surfaces. A major challenge in the electrical wiring of enzymes with electrodes is, however, the generation of high turnover rates between the redox enzymes and the surfaces. These are essential to fabricate sensitive and selective enzyme sensing electrodes, as well as high-power generating biofuel cells. For high electron-transfer turnover rates, it is essential that all enzyme units are oriented in an optimal position in respect to the conductive supports, and that the relay units are located in between the redox site and the conductor, to shorten the electrontransfer distances. The structural alignment of redox enzymes on electrode surfaces and their effective electrical wiring were demonstrated by the application of the reconstitution method.1,11 According to this approach, apo-enzymes were reconstituted on † Part of the “Langmuir 25th Year: Self-assembled monolayers: synthesis, characterization, and applications” special issue. *Corresponding author. Address: Institute of Chemistry, The Hebrew University of Jerusalem, 91904 Jerusalem, Israel. Phone: þ972-2-6585272. Fax: þ972-2-6527715. E-mail: [email protected].

(1) (a) Zayats, M.; Willner, B.; Willner, I. Electroanalysis 2008, 20, 583–601. (b) Willner, I.; Willner, B. Trends Biotechnol. 2001, 19, 222–230. (2) (a) Heller, A. J. Phys. Chem. 1992, 96, 3579–3587. (b) Heller, A. Acc. Chem. Res. 1990, 23, 128–134. (c) Heller, A.; Feldman, B. Chem. Rev. 2008, 108, 2482–2505. (3) Willner, I.; Yan, Y. -M.; Willner, B.; Tel-Vered, R. Fuel Cells 2009, 1, 7–24. (4) Cracknell, J. A.; Vincent, K. A.; Armstrong, F. A. Chem. Rev. 2008, 108, 2439–2461. (5) Bartlett, P. N.; Tebbutt, P.; Whitaker, R. G. Prog. React. Kinet. 1991, 16, 55– 155. (6) Janda, P.; Weber, J. J. Electroanal. Chem. 1991, 300, 119–124. (7) (a) Schuhmann, W.; Ohara, T. J.; Schmidt, H. -L.; Heller, A. J. Am. Chem. Soc. 1991, 113, 1394. (b) Degani, Y.; Heller, A. J. Am. Chem. Soc. 1988, 110, 2615– 2620. (8) Willner, I.; Riklin, A.; Shoham, B.; Rivenson, D.; Katz, E. Adv. Mater. 1993, 5, 912–915. (9) (a) Gregg, B. A.; Heller, A. J. Phys. Chem. 1991, 95, 5970–5975. (b) Mano, N.; Kim, H. -H.; Zhang, Y.; Heller, A. J. Am. Chem. Soc. 2002, 124, 6480–6486. (10) (a) Mano, N.; Kim, H. -H.; Heller, A. J. Phys. Chem. B 2002, 106, 8842– 8848. (b) Mano, N.; Mao, F.; Heller, A. J. Am. Chem. Soc. 2003, 125, 6588–6594. (11) Fruk, L.; Kuo, C. -H.; Torres, E.; Niemeyer, C. M. Angew. Chem., Int. Ed. 2009, 48, 1550–1574. (12) (a) Willner, I.; Heleg-Shabtai, V.; Blonder, R.; Katz, E.; Tao, G.; B€uckmann, A. F.; Heller, A. J. Am. Chem. Soc. 1996, 118, 10321–10322. (b) Katz, E.; Heleg-Shabtai, V.; Willner, B.; Willner, I.; B€uckmann, A. F. Bioelectrochem. Bioenerg. 1997, 42, 95–104.

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relay-cofactor monolayers linked to electrode surfaces.12 This method was further extended by the reconstitution of apoenzymes on cofactor units linked to metal nanoparticles13 or carbon nanotubes (CNTs).14 In these systems, the metal nanoparticles or CNTs acted as charge-transporting nanoconnectors between the redox-active cofactor and the electrode. Although very high electron-transfer turnover rates were observed in these systems, the monolayer configuration of the resulting enzyme electrodes presented a major disadvantage to the method, as a result of the low content of enzyme associated with the surface. Recently, we reported on an effective method to generate threedimensional electrically wired enzyme electrodes exhibiting efficient electron-transfer turnover rates.15 According to this method, the relay-cross-linked enzyme-Au nanoparticle (NP) composite provided a conductive three-dimensional matrix, where the bridging redox-active units mediated the electron transfer between the redox sites of the enzyme units and the electrode. CNTs provide conductive nanoelements, and their coupling to biomolecules, and particularly redox enzymes, attracts growing interest.16 Indeed, the conjugation of redox enzymes to CNTs associated with transistor devices,17 or the integration of redox enzymes with CNTs on electrodes, led to different biosensor devices.18 Electrical contacting of redox enzymes with electrodes was demonstrated using integrated electrodes consisting of relayenzyme-functionalized CNTs, and the systems were used as amperometric biosensors,19 or as bioelectrocatalytic electrodes in biofuel cell elements.20 (13) Xiao, Y.; Patolsky, F.; Katz, E.; Hainfeld, J. F.; Willner, I. Science 2003, 299, 1877–1881. (14) Patolsky, F.; Weizmann, Y.; Willner, I. Angew. Chem., Int. Ed. 2004, 43, 2113–2117. (15) Yehezkeli, O.; Yan, Y.; Baravik, I.; Tel-Vered, R.; Willner, I. Chem.;Eur. J. 2009, 15, 2674–2679. (16) (a) Katz, E.; Willner, I. ChemPhysChem 2004, 5, 1084–1104. (b) Allen, B. L.; Kichambare, P. D.; Star, A. Adv. Mater. 2007, 19, 1439–1451. (17) (a) Star, A.; Gabriel, J. -C. P.; Bradley, K.; Gr€uner, G. Nano Lett. 2003, 3, 459–463. (b) Bradley, K.; Briman, M.; Star, A.; Gr€uner, G. Nano Lett. 2004, 4, 253– 256. (18) Gooding, J. J.; Wibowo, R.; Liu, J.; Yang, W.; Losic, D.; Orbons, S.; Mearns, F. J.; Shapter, J. G.; Hibbert, D. B. J. Am. Chem. Soc. 2003, 125, 9006– 9007. (19) (a) Wang, J. Electroanalysis 2005, 17, 7–14. (b) Xu, J. Z.; Zhu, J. J.; Wu, Q.; Hu, Z.; Chen, H. Y. Electroanalysis 2003, 15, 219–224. (c) Yan, Y.; Baravik, I.; Yehezkeli, O.; Willner, I. J. Phys. Chem. C 2008, 112, 17883–17888. (20) (a) Yan, Y.; Yehezkeli, O.; Willner, I. Chem.;Eur. J. 2007, 13, 10168– 10176. (b) Gao, F.; Yan, Y.; Su, L.; Wang, L.; Mao, L. Electrochem. Commun. 2007, 9, 989–996. (c) Yan, Y.; Zheng, W.; Su, L.; Mao, L. Adv. Mater. 2006, 18, 2639–2643.

Published on Web 08/12/2009

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Scheme 1. (A) Modification of the CNT with the Electropolymerizable Aniline Functionalities and (B) Modification of GOx with the Electropolymerizable Thioaniline Functionalities

Here we report on the modification of CNTs with electropolymerizable aniline units, and on the coelectropolymerization of the modified CNTs with thioaniline-functionalized glucose oxidase (GOx) on electrode surfaces. The resulting three-dimensional CNTs/GOx composite is effectively wired with the electrode, and the bioelectrocatalytic oxidation of glucose is activated. We characterize the CNT/GOx composite and discuss the parameters affecting the bioelectrocatalytic oxidation of glucose.

Results and Discussion Scheme 1 depicts the method to assemble the CNT/GOx composite electrode. The single-walled CNTs were oxidatively cleaved, and the resulting carboxylic acid residues associated with the CNTs were used to covalently couple 2-(4 aminophenyl)ethylamine (1) to the CNTs (Scheme 1A). Thioaniline was covalently tethered to GOx by the primary modification of the lysine residues of GOx with the bifunctional 6-maleimidoN-hydroxysucinimide hexanoic acid ester (2), followed by the substitution of the maleimide residue with thioaniline (3) (Scheme 1B). The activity of the thioaniline-modified enzyme was determined by the O2-mediated oxidation of glucose, and the colorimetric detection of the resulting H2O2. The activity of the modified enzyme corresponded to 96 ( 2% of the native enzyme activity. The aniline-modified CNTs were coelectropolymerized with the thioaniline-tethered GOx onto Au-electrodes modified with a thioaniline monolayer to yield the bis-aniline-bridged CNTs/GOx composite on the electrode. The electropolymerization was carried out by the application of a constant number of electropolymerization cycles, in the potential range of -0.1 to 1.1 V versus saturated calomel electrode (SCE). Figure 1 shows the cyclic voltammograms corresponding to the bioelectrocatalytic anodic currents generated upon the oxidation of variable concentrations of glucose by the CNT/GOx crosslinked composite-modified electrode, prepared by applying 60 polymerization cycles and a CNT:GOx ratio corresponding to 3:1 (w/w). The electrocatalytic anodic currents increase upon elevating the concentration of glucose. The results show that the enzyme exists in the CNT/GOx cross-linked matrix in an electrically contacted configuration with the electrode. The bioelectrocatalytic CNTs composite was further optimized and characterized. The incorporation of the GOx units into the composite is anticipated to insulate the CNTs, and thus, perturb the growth of the conductive CNT network. As a result, the incorporation of a high content of enzyme into the electropolymerized composite is anticipated to interfere with the build-up of the CNTs/enzyme structure. Hence, the ratio of CNTs/GOx used in the electropolymerization process should have an effect on the resulting bioeleectrocatalytic functions of the modified Langmuir 2009, 25(24), 13978–13983

Figure 1. Cyclic voltammograms corresponding to the bioelectrocatalyzed oxidation of glucose by the bis-aniline-cross-linked CNT/GOx composite-modified Au electrode in the presence of variable concentrations of glucose: (a) 0 mM; (b) 20 mM; (c) 40 mM; (d) 60 mM; (e) 80 mM; (f) 100 mM; (g) 120 mM. Scan rate 5 mV 3 s-1. Inset: calibration curve corresponding to the electrocatalytic currents, measured at E = 0.3 V vs SCE for different concentrations of glucose. The electrodes were prepared by the application of 60 cyclic voltammetry scans between -0.1 and 1.1 V vs SCE at 100 mV 3 s-1, using aniline-modified CNTs and thioaniline-modified GOx at a molar ratio of 3:1. All data were recorded in a 0.1 M phosphate buffer solution, pH = 7.4, under N2.

electrode. Figure 2A depicts the intensities of the electrocatalytic anodic currents (at E = 0.3 V vs SCE) generated by different CNT/GOx composites, synthesized by 60 electropolymerization cycles, in the presence of variable ratios of the electropolymerizable GOx and CNTs. The most electrocatalytically efficient composite is obtained at a 1:1 (w/w) ratio of the CNT/GOx. The composite was, then, further optimized by controlling the number of electropolymerization cycles applied to form the composite. Figure 2B shows the electrocatalytic anodic currents generated by CNT/GOx composites synthesized by the application of variable numbers of electropolymerization cycles in the presence of the optimized CNTs/GOx ratio corresponding to 1:1. As can be seen, the electrocatalytic anodic currents are intensified as the number of electropolymerization cycles increases, and they level-off to a saturation value of ca. 15 μA after 80 cycles. The fact that the electrocatalytic currents reach saturation may be attributed to the insulation of the CNTs as the content of the enzyme in the composite increases, and/or to barriers for the penetration of DOI: 10.1021/la902074w

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Figure 2. (A) Electrocatalytic anodic currents, at E = 0.3 V vs SCE, generated by the bis-aniline-cross-linked CNT/GOx compositemodified Au electrodes, prepared by the application of 60 cyclic voltammetry electropolymerization scans between -0.1 and 1.1 V vs SCE, at 100 mV 3 s-1, using variable thioaniline-modified GOx and aniline-modified CNT ratios (the concentration of GOx, 0.125 mg mL-1, was kept constant, and the concentration of the CNTs was varied). Cyclic voltammograms were performed in a 0.1 M phosphate buffer solution (pH = 7.4) that included 60 mM glucose. (B) Electrocatalytic anodic currents, at E = 0.3 V vs SCE, generated by the bis-aniline-cross-linked CNT/ GOx composite-modified Au electrodes, prepared by the application of a variable number of cyclic voltammetry electropolymerization scans between -0.1 and 1.1 V vs SCE, at 100 mV 3 s-1, in the presence of aniline-modified CNTs and thioaniline-modified GOx at a weight ratio of 1:1. Data were recorded in a 0.1 M phosphate buffer solution (pH = 7.4) that included 60 mM glucose. (C) Cyclic voltammograms corresponding to the bioelectrocatalyzed oxidation of glucose by the bis-aniline-cross-linked CNT/GOx composite-modified Au electrode, generated by the application of 60 electropolymerization scans and at a CNTs/GOx ratio of 1:1, in the presence of variable concentrations of glucose: (a) 0 mM; (b) 20 mM; (c) 40 mM; (d) 60 mM; (e) 80 mM; (f) 100 mM; (g) 120 mM. Inset: calibration curve corresponding to the electrocatalytic anodic currents, measured at E = 0.3 V vs SCE for different concentrations of glucose. All data were recorded in a 0.1 M phosphate buffer solution, pH = 7.4, under N2. Scan rate 5 mV 3 s-1.

glucose into inner regions of the composite. Figure 2C depicts the cyclic voltammograms generated by the optimized CNTs/GOx composite-modified electrode (60 electropolymerization cycles, CNTs/GOx ratio 1:1), in the presence of variable concentrations of glucose. Figure 2C, inset, shows the derived calibration curve. Interestingly, the cyclic voltammograms do not show a constant increase in the catalytic currents upon shifting the potential positively. One region of the electrocatalytic anodic current is observed at an onset potential of -0.1 V versus SCE, while a second region, where the electrocatalytic current is intensified, is observed at an onset potential of 0.15 V versus SCE. These two regions of electrocatalytic anodic currents may be attributed to different compositions of the oligoaniline bridging units that exhibit different redox potentials and variable electron mediating properties toward the bioelectrocatalyzed oxidation of glucose. The CNTs/GOx composite was further characterized by microgravimetric quartz crystal microbalance (QCM) measurements. The application of 60 electropolymerization cycles to polymerize the aniline-tethered CNTs on the thioaniline-modified electrode, resulted in a frequency change of 319 Hz that corre13980 DOI: 10.1021/la902074w

sponded to a mass accumulation of 1.74  10-6 gr 3 cm-2. Similarly, applying 60 electropolymerization cycles to generate the CNT/GOx composite (employing both the aniline-modified CNTs and GOx at a 1:1 ratio), resulted in a frequency change that corresponded to 451 Hz. Assuming that the coverage of the CNTs in the CNT/GOx composite is similar to the coverage of the CNTs generated by electropolymerization of the CNTs in the absence of GOx, we estimate that the coverage of GOx in the composite is ca. 7.2  10-7 gr 3 cm-2. This value suggests that the ratio of CNTs/GOx in the composite is ca. 2.5:1 (w/w). The weight of GOx translates to 4.5  10-12 mol 3 cm-2 and to a coverage that corresponds to approximately three random densely packed monolayers (a random densely packed monolayer corresponds to 64% of a closed packed monolayer configuration).21 The topography of the CNTs/GOx composite was further examined by imaging a step region of the electrodeposited biopolymer using atomic force microscopy (AFM) (see Figure S1, Supporting Information). The electropolymerized (21) Berryman, J. G. Phys. Rev. A 1983, 27, 1053–1061.

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Figure 3. Chronoamperometric responses, corresponding to the bioelectrocatalyzed oxidation of glucose by the bis-aniline-crosslinked CNT/GOx composite-modified Au electrode (I) under N2 and (II) under air. The measurements were performed in the presence of variable concentrations of glucose: (a) 0 mM; (b) 5 mM; (c) 10 mM; (d) 15 mM; (e) 20 mM; (f) 25 mM. Constant potential applied 0.3 V vs SCE. Inset: calibration curves corresponding to the electrocatalytic currents (9) under N2 and (0) under air. The electrodes were prepared by the application of 60 cyclic voltammetry scans, using aniline-modified CNTs and thioaniline-modified GOx at a weight ratio of 1:1. All data were recorded in a 0.1 M phosphate buffer solution, pH = 7.4.

CNTs/GOx composite exhibits a thickness of ca. 6 nm, with spikes of electropolymerized material in the height range of 20 to 80 nm. The electropolymerization of the thioaniline-modified GOx might affect the activity of the enzyme, and thus, perturb the bioelectrocatalytic functions of the composite. In order to determine the activity of the enzyme in the CNTs/GOx network, we assayed the composite electrode spectroscopically (see Supporting Information). In this experiment, we probed the amount of H2O2 generated by the enzyme (GOx) associated with the composite, and compared the results to a calibration curve that examined the amount of H2O2, generated by variable concentrations of the native enzyme (Figure S2). Our measurements indicated that the content of GOx associated with the electrode corresponded to a surface coverage of GOx that equaled 1.1  10-12 mole 3 cm-2 (Figure S3), assuming that the activity of the enzyme in the composite is similar to that of native GOx. This surface coverage value is ca. 4 times lower than the surface coverage derived by the QCM measurements, suggesting that the average activity of the enzyme incorporated in the CNT/GOx matrix is ca. 25-30% of a similar content of the native enzyme activity. This might originate from the partial denaturation of the enzyme units as a result of the electropolymerization and/or from the adsorption of the enzyme on the CNTs, leading to the partial deactivation of the enzyme or to inefficient electrical wiring of part of the enzyme units associated with the network. Knowing the saturation current generated by the CNT/GOx composite (ca. 25 μA) and the coverage of the enzyme on the electrode and its activity, we estimated the average apparent turnover rate of electrons between the enzyme units and the electrode to be ca. 1025 s-1. This value should be compared to the turnover rate of electrons between (22) Eisenwiener, H. G.; Schultz, G. V. Naturwissenschaften 1969, 56, 563–564.

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Figure 4. Cyclic voltammograms corresponding to the bioelectrocatalyzed oxidation of glucose by a bis-aniline-monolayer GOxmodified Au electrode. Measurements were performed at variable concentrations of glucose: (a) 0 mM; (b) 20 mM; (c) 40 mM; (d) 60 mM; (e) 80 mM. Scan rate 5 mV 3 s-1. Inset: calibration curve corresponding to the electrocatalytic currents measured at E = 0.3 V vs SCE for different concentrations of glucose. The electrodes were prepared by the application of 60 cyclic voltammetry scans between -0.1 and 1.1 V vs SCE at 100 mV 3 s-1, using 0.125 mg mL-1 thioaniline-modified GOx.

GOx and its native acceptor O2, ca. 700 s-1.22 Thus, the electrical contacting of the active GOx units with the electrode is remarkably efficient, and this is anticipated to yield a GOx sensing electrode that is insensitive to oxygen. Figure 3 shows the chronoamperometric responses of the CNT/GOx-cross-linked electrode under N2 (curve I) and under air (curve II). The responses are within the experimental errors, and they differ by (5%. Figure 3, inset, shows the derived calibration curves for analyzing glucose. Specifically, the calibration curves in the concentration range applicable for analyzing diabetes are linear, suggesting the potential future design of a glucose sensor with a single concentration calibration. The significance of the CNT/GOx composite for analyzing glucose was further characterized by comparing the performance of the CNT/GOx composite electrode to an electrode prepared by the direct electropolymerization of the thioaniline-functionalized GOx on the thioanline monolayer-modified electrode. Figure 4 shows the cyclic voltammograms corresponding to the bioelectrocatalyzed oxidation of different concentrations of glucose by the enzyme monolayer-modified electrode. The electrode was prepared by the application of 60 electropolymerization cycles, and complementary QCM measurements indicated a surface coverage of nearly a single densely packed monolayer on the electrode. We observe electrocatalytic anodic currents that intensify upon elevating the concentration of glucose. These results indicate that the enzyme units linked to the electrode by the bis-aniline bridging components are electrically wired. Nonetheless, comparison of the anodic currents generated by the GOxmonolayer-modified electrode to those generated by the CNT/ GOx composite electrode reveal that the currents associated with the composite are ca. 12 times higher than those formed by the GOx-monolayer electrode. Thus, although the GOx content in the CNT/GOx composite is only 3 times higher than that in the GOx-monolayer-modified electrode, the resulting currents DOI: 10.1021/la902074w

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Scheme 2. (A) Preparation of a Bis-Aniline-Crosslinked GOx Monolayer on a Au Electrode by the Electropolymerization of the ThioanilineModified GOx with the Thioaniline Monolayer-Functionalized Au Electrode;a (B) Preparation of the Three-Dimensional Bis-Aniline-Crosslinked CNT/GOx Composite by the Electropolymerization of the Thioaniline-Modified GOx and the Aniline-Modified CNTs on the Thioaniline Monolayer-Functionalized Au Electrode

a

The arrow indicates a schematic configuration exhibiting an effective electron transfer between the enzyme redox center and a bis-aniline relay unit.

generated by the composite electrode are significantly higher. These results highlight that GOx exhibits improved electrical contacting in the CNT/GOx structure, as compared to the monolayer configuration. The improved electrical wiring of GOx in the CNT composite is schematically depicted in Scheme 2. The electropolymerizable thioaniline units tethered to the GOx yield, upon electropolymerization with the thioaniline-functionalized electrode (in the absence of the aniline-modified CNTs), an enzyme monolayer of random orientations (Scheme 2A). While, in some of the orientations, the bis-aniline electron relay units are close to the active center (e.g., marked with an arrow), which leads to effective electrical contacting, in other enzyme units the electron mediator is in a remote position in respect to the enzyme redox center, leading to inefficient wiring. The observed electrocatalytic currents are, thus, the average of the wiring efficiencies for the different orientations of the enzyme in the monolayer. The conductivity of the three-dimensional CNT/GOx composite transforms the nonoptimized random orientations of the enzyme units into a matrix exhibiting improved electrical wiring by forcing additional relay units and conductive elements to the redox enzyme centers that were not accessible in the monolayer configurations (Scheme 2B).

Conclusions The study has demonstrated the synthesis of an electrically contacted CNTs/GOx composite on electrode surfaces by the electropolymerization of aniline derivative-modified CNTs and a thioaniline-functionalized GOx on a thioaniline monolayer-modified electrode. The enzyme in the composite revealed effective electrical communication with the electrode (turnover rate ca. 1025 s-1). While the CNTs provided the three-dimensional 13982 DOI: 10.1021/la902074w

conductivity of the network, the bis-aniline bridging units acted as electron-mediating components for the electron transfer between the enzyme redox centers and the electrode. The high surface area of the CNTs yielded close proximities between the enzyme redox centers and the electron relay units, and this led to the effective wiring of the enzyme with the electrode. This approach could be adapted to electrically wire other redox enzymes with electrodes, and related enzyme/CNTs composites could lead to other electrochemical biosensor devices.

Experimental Section Modification of the CNTs. Single-walled CNTs were sonicated for 8 h in a 3:1 mixture of concentrated sulfuric and nitric acids. The resulting oxidized CNTs were repeatedly water-washed and centrifuged to a final pH of 7.0. The CNTs were then reacted with a HEPES buffer, 0.01 M, pH = 7.4, containing 2-(4 aminophenyl)-ethylamine, 10 mM. The solution was stirred with 8 mM 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride (EDC) and N-hydroxysulfosuccinimide sodium salt (NHS) for 2 h at room temperature. The resulting modified CNTs were repeatedly washed and centrifuged, four times, using a HEPES buffer, 0.01 M (pH = 7.2). Modification of the Enzyme. GOx (52 mg; EC 1.1.3.4 from Aspergillus niger, 210 000 U 3 g-1, purchased from Sigma), was dissolved in 3 mL of 0.01 M HEPES buffer (pH = 7.2). The solution was then treated with 52 μL of N-(maleimidocapropyloxy) sulfosuccinimide ester (sulfo-EMCS, purchased from PIERCE), 12 mg 3 mL-1. The resulting solution was stirred for 40 min, and was then mixed with 0.8 mL of ethanol that included 4-aminothiophenol (thioaniline, 1.6 mg mL-1). After 2.5 h of reaction, the solution was eluted through a G-25 column Langmuir 2009, 25(24), 13978–13983

Baravik et al. (GE Healthcare) using a phosphate buffer solution (0.1 M, pH = 7.4) as the eluent. The resulting purified, functionalized-GOx solution was then lyophilized to yield a pale-yellow powder that was stored at -20 °C. Modification of the Electrodes. Clean Au wires (0.3 cm2) were reacted for 12 h with a 50 mM thioaniline in ethanol solution. The thioaniline monolayer-functionalized electrodes were then used to electropolymerize the aniline-modified CNTs and the thioanilinemodified GOx, in a 0.1 M phosphate buffer solution (pH = 7.4) using a fixed number of repetitive cyclic voltammetry scans, ranging between -0.1 and 1.1 V versus SCE, at a scan rate of 100 mV 3 s-1. Instrumentation. All electrochemical measurements were carried out using a PC-controlled (Autolab GPES software)

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Article potentiostat/galvanostat (μAutolab, type III). A graphite rod (d = 5 mm) was used as the counter electrode and the reference was a SCE. QCM measurements were performed using a homebuilt instrument linked to a frequency analyzer (Fluke) using Au-quartz crystals (AT-cut 10 MHz).

Acknowledgment. This research is supported by the EC BIOMEDNANO project. Supporting Information Available: The determination of the catalytically active bis-aniline-cross-linked GOx in the composite electrode is described. This material is available free of charge via the Internet at http://pubs.acs.org.

DOI: 10.1021/la902074w

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