Following the Biocatalytic Activities of Glucose Oxidase by

Oct 8, 2008 - E-mail: [email protected]. Phone: +972-2-6585272. Fax: +972-2-6527715., †. The Hebrew University of Jerusalem. , ‡. Universita ...
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Anal. Chem. 2008, 80, 8253–8259

Following the Biocatalytic Activities of Glucose Oxidase by Electrochemically Cross-Linked Enzyme-Pt Nanoparticles Composite Electrodes Lily Bahshi,† Marco Frasconi,‡ Ran Tel-Vered,† Omer Yehezkeli,† and Itamar Willner*,† Institute of Chemistry, The Hebrew University of Jerusalem, Jerusalem 91904, Israel, and Dipartimento di Studi di Chimica e Tecnologia delle Sostanze Biologicamente Attive, Universita “La Sapienza”, Rome, Italy An integrated platinum nanoparticles (NPs)/glucose oxidase (GOx) composite film associated with a Au electrode is used to follow the biocatalytic activities of the enzyme. The film is assembled on a Au electrode by the electropolymerization of thioaniline-functionalized Pt NPs and thioaniline-modified GOx. The resulting enzyme/Pt NPsfunctionalized electrode stimulates the O2 oxidation of glucose to gluconic acid and H2O2. The modified electrode is then implemented to follow the activity of the enzyme by the electrochemical monitoring of the generated H2O2. The effect of the composition of the Pt NPs/GOx crosslinked nanostructures and the optimal conditions for the preparation of the electrodes are discussed. Numerous oxidases catalyze the oxidation of their specific substrates by molecular oxygen with the concomitant generation of H2O2 (e.g., glucose oxidase, lactate oxidase, choline oxidase, or cholesterol oxidase). Accordingly, numerous electrochemical,1 optical,2-4 or chemiluminescent5,6 biosensors for the different substrates were developed by probing the H2O2 generated by the respective biotransformations. Specifically, the electrocatalytic reduction of H2O2 by horseradish peroxidase-functionalized electrodes7-9 or other hemoprotein-modified electrodes10 were used to develop biosensors for H2O2 and for the substrates of different oxidases. Similarly, different metals11,12 or transition * To whom correspondence should be addressed. E-mail: [email protected]. Phone: +972-2-6585272. Fax: +972-2-6527715. † The Hebrew University of Jerusalem. ‡ Universita “La Sapienza”. (1) Wang, J. Chem. Rev. 2008, 108, 814–825. (2) Wu, M.; Lin, Z.; Schaeferling, M.; Duerkop, A.; Wolfbeis, O. S. Anal. Biochem. 2005, 340, 66–73. (3) Wolfbeis, O. S.; Schaeferling, M.; Duerkop, A. Microchim. Acta 2003, 143, 221–227. (4) Gill, R.; Bahshi, L.; Freeman, R.; Willner, I. Angew. Chem., Int. Ed. 2008, 47, 1676–1679. (5) Shi, C. G.; Xu, J. J.; Chen, H. Y. J. Electroanal. Chem. 2007, 610, 186–192. (6) Zhou, G. J.; Wang, G.; Xu, J. J.; Chen, H. Y. Sens. Actuators, B: Chem. 2002, B81, 334–339. (7) Ohara, T. J.; Vreeke, M. S.; Battaglini, F.; Heller, A. Electroanalysis 1993, 5, 825–831. (8) Su, X.; O’Shea, S. J. Anal. Biochem. 2001, 299, 241–246. (9) Asberg, P.; Inganas, O. Biosens. Bioelectron. 2003, 19, 199–207. (10) Narvaez, A.; Dominguez, E.; Katakis, I.; Katz, E.; Ranjit, K. T.; Ben-Dov, I.; Willner, I. J. Electroanal. Chem. 1997, 430, 227–233. (11) Dodevska, T.; Horozova, E.; Dimcheva, N. Anal. Bioanal. Chem. 2006, 386, 1413–1418. (12) Arjsiriwat, S.; Tanticharoen, M.; Kirtikara, K.; Aoki, K.; Somasundrum, M. Electrochem. Commun. 2000, 2, 441–444. 10.1021/ac801398m CCC: $40.75  2008 American Chemical Society Published on Web 10/08/2008

metal complexes13-16 acted as catalysts for the electrocatalytic reduction of H2O2 and for the development of biosensors for different substrates of oxidases. Furthermore, the development of glucose-sensing electrodes employing glucose oxidase as a biocatalyst attracted extensive research efforts. Different enzyme electrodes that monitored the biocatalytically generated H2O2 were used to electrochemically analyze glucose.17 Similarly, electrically contacted glucose oxidase electrodes prepared by the tethering of a redox relay to the protein18 or the incorporation of glucose oxidase in redox-active conductive polymers associated with electrodes19 were implemented for glucose sensing. Nanobiotechnology has introduced new tools for biosensor technologies, and biomolecule-nanoparticle (NP) hybrid systems were widely applied to develop biosensors.20 Au NPs conjugated to redox enzymes were used to electrically contact the redox sites of the biocatalysts with electrodes and to activate their bioelectrocatalytic functions.21,22 The catalytic enlargement of Au NPs associated with electrodes enhanced the conductivity at the enzyme-modified electrode surfaces, and this improved the bioelectrocatalytic functions of the modified electrodes.23 Similarly, different biomolecule-metal NP hybrids were used as catalytic labels for the amplified detection of biorecognition events through the (13) Lin, M. S.; Shih, W. C. Anal. Chim. Acta 1999, 381, 183–189. (14) Wang, J.; Naser, N.; Angnes, L.; Wu, H.; Chen, L. Anal. Chem. 1992, 64, 1285–1288. (15) Wang, J.; Liu, J.; Chen, L.; Lu, F. Anal. Chem. 1994, 66, 3600–3603. (16) Wang, J.; Rivas, G.; Chicharro, M. Electroanalysis 1996, 8, 434–437. (17) (a) Bourdillon, C.; Bourgeois, J. P.; Thomas, D. J. Am. Chem. Soc. 1980, 102, 4231–4235. (b) Wieck, H. J.; Shea, C.; Yacynych, A. M. Anal. Chim. Acta 1982, 142, 277–279. (c) Urban, G.; Jobst, G.; Kohl, F.; Jachimowicz, A.; Olcaytug, F.; Tilado, O.; Goiser, P.; Nauer, G.; Pittner, F.; Schalkhammer, T.; Mann-Buxbaum, E. Biosens. Bioelectron. 1991, 6, 555–562. (d) Laury, J. P.; McAteer, K.; Atrash, S. E.; O’Neill, R. D. J. Chem. Soc., Chem. Commun. 1994, 2483–2484. (e) Alfonta, L.; Katz, E.; Willner, I. Anal. Chem. 2000, 72, 927–935. (18) (a) Degani, Y.; Heller, A. J. Am. Chem. Soc. 1988, 110, 2615–2620. (b) Willner, I.; Riklin, A.; Shoham, B.; Rivenson, D.; Katz, E. Adv. Mater. 1993, 5, 912–915. (19) (a) Heller, A. J. Phys. Chem. B 1992, 96, 3579–3587. (b) De LumleyWoodyear, T.; Rocca, P.; Lindsay, J.; Dror, Y.; Freeman, A.; Heller, A. Anal. Chem. 1995, 67, 1332–1338. (c) Kenausis, G.; Taylor, C.; Katakis, I.; Heller, A. J. Chem. Soc. Faraday Trans. 1996, 20, 4131–4136. (20) Katz, E.; Willner, I. Angew. Chem., Int. Ed. 2004, 43, 6042–6108. (21) Xiao, Y.; Patolsky, F.; Katz, E.; Hainfeld, J. F.; Willner, I. Science 2003, 299, 1877–1881. (22) Zayats, M.; Katz, E.; Baron, R.; Willner, I. J. Am. Chem. Soc. 2005, 127, 12400–12406. (23) Yan, Y.; Tel-Vered, R.; Yehezkeli, O.; Cheglakov, Z.; Willner, I. Adv. Mater. 2008, 20, 2365–2370.

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Scheme 1. (A) Preparation of a Pt NPs Monolayer Assembly through Covalent Binding of Thioaniline-Modified Pt NPs to a Mercaptopropionic Acid-Modified Au Electrode. (B) Synthesis of a 3D Oligoaniline-Cross-Linked Pt NPs Structure by the Electropolymerization of the Thioaniline-Modified Pt Nanoparticles on the Thioaniline-Modified Au Electrodes

electrocatalyzed reduction of H2O224 or the H2O2-mediated generation of chemiluminescence.25 Also, enzyme-carbon nanotube hybrid systems were used to develop glucose-sensing electrodes.26 Alternatively, the catalytic growth of Au NPs by enzyme-generated H2O2 enabled the optical, colorimetric, determination of enzyme activities,27 their substrates,28 or their inhibitors.29 Here we report on a novel method to assemble composite Pt NPs and glucose oxidase (GOx) films on Au electrodes by the electropolymerization of thioaniline-functionalized Pt NPs and thioaniline-modified GOx. The resulting Pt NPs/GOx arrays are cross-linked by oligoaniline bridges during the electropolymerization process. The modified electrode reveals several unique functions that advance biosensor technology: (i) The Pt particles reveal high electrocatalytic activity toward the electrocatalytic reduction of H2O2 generated by the GOx-mediated oxidation of glucose. (ii) The preparation of the functionalized electrode by electropolymerization allows the addressing of a selective working electrode in a miniaturized threeelectrode configuration and the high-throughput preparation of numerous miniaturized cells. (iii) The electropolymerization process enables control over the sensitivity of the resulting bioelectrocatalytic matrixes by regulating the content of the Pt NPs/ enzyme composite. Pt NPs were capped with a mixed monolayer of thioaniline and mercaptoethanesulfonic acid. While the thioaniline provides the electropolymerizable monomer units, the mercaptoethanesulfonate units enhance the stability of the Pt NPs against aggregation and precipitation in aqueous media. In a primary experiment, the functionalized Pt NPs were covalently tethered to Au electrodes that were functionalized with mercaptopropionic acid, Scheme 1A, to form a two-dimensional monolayer of Pt NPs. Figure 1 shows the cyclic voltammograms of the Pt NPs-modified electrode upon addition of H2O2. Evidently, in the presence of (24) Polsky, R.; Gill, R.; Kaganovsky, L.; Willner, I. Anal. Chem. 2006, 78, 2268– 2271. (25) Gill, R.; Polsky, R. I. Willner, Small 2006, 2, 1037–1041. (26) (a) Wang, J.; Musameh, M.; Lin, Y. J. Am. Chem. Soc. 2003, 125, 2408– 2409. (b) Wang, J.; Musameh, M. Analyst 2003, 128, 1382–1385. (c) Lin, Y.; Lu, F.; Tu, Y.; Ren, Z. Nano Lett. 2004, 2, 191–195. (d) Patolsky, F.; Weizmann, Y.; Willner, I. Angew. Chem., Int. Ed. 2004, 43, 2113–2117. (e) Yan, Y.; Yehezkeli, O.; Willner, I. Chem. Eur. J. 2007, 13, 10168–10175. (27) Baron, R.; Zayats, M.; Willner, I. Anal. Chem. 2005, 77, 1566–1571. (28) Zayats, M.; Baron, R.; Popov, I.; Willner, I. Nano Lett. 2005, 5, 21–25. (29) Parlov, V.; Xiao, Y.; Willner, I. Nano Lett. 2005, 5, 649–653.

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H2O2, electrocatalytic cathodic waves are observed (the overpotential for the reduction of H2O2 at the Au electrode is reduced), and as the concentration of H2O2 is elevated, the intensities of the electrocatalytic currents are enhanced. In a control experiment, no electrocatalytic currents could be observed at a bare Au electrode lacking the Pt NPs within this potential range. These results suggest that the Pt NPs, indeed, electrocatalyze the reduction of H2O2. Figure 1, inset, depicts the calibration curve corresponding to the amperometric response of the Pt NPsmodified electrode at E ) -0.35 V versus saturated calomel electrode (SCE), in the presence of variable concentrations of H2O2. In the subsequent experiment, the thioaniline-modified Pt NPs were electropolymerized on thioaniline-functionalized Au electrodes, Scheme 1B. The electropolymerization of a thioaniline monolayer associated with a Au surface was reported previously,30 and the generation of a two-dimensional conductive polymer on the surface was demonstrated. Also, a thioaniline-modified monolayer associated with Au electrode, in a densely packed configuration31 or in a diluted state,32 enabled the electrochemical

Figure 1. Cyclic voltammograms corresponding to the Pt NPs monolayer-modified Au electrode in the presence of different concentrations of hydrogen peroxide: (a) 0.0, (b) 0.4, (c) 0.8, (d) 1.2, (e) 2.0, (f) 3.0, (g) 4.0, (h) 5.0, (i) 6.0, and (j) 7.0 mM. Scan rate 10 mV · s-1. Inset: calibration curve corresponding to the electrocatalytic currents measured at E ) -0.35 V, using variable concentrations of hydrogen peroxide. All measurements were performed in 0.1 M phosphate buffer solution, pH 7.4.

deposition of aniline on the surface. These studies suggested that the thioaniline-functionalized monolayer associated with the electrode could activate the electrochemical deposition of the thioaniline-modified particles. In the present electropolymerization process, the Pt NPs provide the conductivity for the threedimensional polymerization and cross-linking of the particles. The Pt NPs are modified with thioaniline/mercaptoethanesulfonic acid at a ratio of 1:4. The coverage of thiolates on a 2-nm Pt NP was estimated to be ∼172 thiolates per particle,33 and assuming that the composition of the adsorbed mixed monolayer is similar to the molar ratio of the thiolates in solution, we estimate that ∼34 thioaniline units are associated with each Pt NP. The electropolymerization of the thioaniline-functionalized NPs yields redoxactive bridging units characteristic to conductive oligoaniline chains (see Supporting Information, Figure S1). Although we do not know the precise structure of the electroactive bridging units between the Pt NPs, the dilute coverage of the NPs with the electropolymerizable thioaniline units suggests that bisaniline bridges are formed, although higher oligoaniline bridges cannot be excluded. Also, microgravimetric quartz crystal microbalance measurements indicated that the deposition of the Pt NPs on a thioaniline-modified Au/quartz crystal resulted in a mass change corresponding to 9.8 × 10-7 g · cm-2. Assuming a bisaniline bridge structure, and a coverage of 34 thioaniline units per particle, the microgravimetric analysis suggests a particle coverage of 3.8 × 1013 particles · cm-2. Using the dimensions of a single Pt NP as 2.7 nm (Pt NPs core plus thiolated shell), this value translates to ∼3.5 random densely packed monolayers of Pt NPs that constitute the bridged aggregated structure of the film. Figure 2A shows the cyclic voltammograms observed upon analyzing different concentrations of H2O2 by the Pt NPs-crosslinked electrode, produced by 60 electropolymerization cycles. Figure 2A, inset, depicts the derived calibration curve, where the amperometric responses at E ) -0.35 V versus SCE are plotted as a function of the H2O2 concentration in the system. The effectiveness of the 3D Pt NP-functionalized electrode toward the analysis of H2O2 is shown in Figure 2B. The amperometric responses of the cross-linked Pt NPs composite are up to 30-fold higher than the current responses generated by the Pt NPs monolayer-functionalized electrode. The higher currents observed with the Pt NPs composite electrode, as compared to the Pt NPs monolayer-functionalized electrode, may be attributed to the higher content of the Pt NPs in the 3D electropolymerized composite. The success to enhance the sensitivity of analysis toward H2O2 by the Pt NPs-modified electrodes suggested that the electrode could be applied for the analysis of glucose in the presence of glucose oxidase. As GOx oxidizes glucose by O2 to form gluconic acid and H2O2, the generated H2O2 relates to the concentration of glucose, and its electrochemical determination by the electrocatalytic electrode provides a quantitative measure for glucose. Indeed, we find that the electrode modified with the Pt NPs composite can sense H2O2 generated in the presence of different (30) Qing, J. X.; Xiao, L. G.; You, Y. Z.; Man, C. X.; Ming, M. Chin. Chem. Lett. 1999, 10, 63–66. (31) Niu, L.; Kvarnstro ¨m, C.; Ivaska, A. J. Electroanal. Chem. 2007, 600, 95– 102. (32) Abaci, S.; Shannon, C. Electrochim. Acta 2005, 50, 2967–2973. (33) Eklund, S. E.; Cliffel, D. E. Langmuir 2004, 20, 6012–6018.

Figure 2. (A) Cyclic voltammograms corresponding to the oligoaniline-cross-linked Pt NPs-functionalized Au electrode in the presence of variable concentrations of hydrogen peroxide: (a) 0.0, (b) 0.05, (c) 0.1, (d) 0.2, (e) 0.4, (f) 0.8, (g) 1.2, (h) 2.0, (i) 3.0, j) 4.0, k) 5.0, and (l) 6.0 mM. Scan rate 10 mV · s-1. The electrodes were prepared by the application of 60 cyclic voltammetry scans between -0.1 and 1.1 V versus SCE at 100 mV · s-1. Inset: calibration curve corresponding to the electrocatalytic currents measured at E ) -0.35 V at variable concentrations of hydrogen peroxide. (B) Comparison of the catalytic currents obtained at variable concentrations of hydrogen peroxide using (a) the oligoaniline-cross-linked Pt NPs-modified Au electrode and (b) the Pt NPs monolayer-modified Au electrode. All measurements were performed in a 0.1 M phosphate buffer solution, pH 7.4.

concentrations of glucose and GOx as biocatalyst, in a homogeneous aqueous phase (see Supporting Information, Figure S2). For any practical utility, the enzyme and the electrocatalytic Pt NPs should be integrated with the electrode. Toward this goal, GOx was functionalized with electropolymerizable thioaniline units according to Scheme 2A. The primary functionalization of the lysine residues with the bridging maleimide-active ester, 1, units, was followed by the Michael addition of thioaniline to the maleimide residues. The average loading of GOx with thioaniline was estimated to be 7-8 molecules per enzyme. The activity of the resulting thioaniline-functionalized GOx indicated ∼95% of the activity of the native GOx, prior to the modification. The integrated Pt NPs-GOx composite electrode was prepared by the electropolymerization of the thioaniline-modified Pt NPs in the presence of the thioaniline-functionalized GOx, Scheme 2B. The electropolymerized Pt NPs provide the 3D conductivity for the covalent electrochemical attachment of the GOx units. It should be noted Analytical Chemistry, Vol. 80, No. 21, November 1, 2008

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Scheme 2. (A) Modification of Glucose Oxidase with an Electropolymerizable Thioaniline Function. (B) Synthesis of a 3D Oligoaniline-Cross-Linked GOx/Pt NPs Composite by the Electropolymerization of the Thioaniline-Modified GOx and Thioaniline-Modified Pt Nanoparticles, on a Thioaniline-Modified Au Electrode

that in contrast to previous reports that incorporated enzymes into conductive polyaniline films by physical entrapment34 or adsorption,35 the present method presents a new concept of covalently binding the enzyme to the oligoaniline-Pt NPs composite. The resulting Pt NPs/GOx composite was then used to probe the biocatalytic activities of GOx. Figure 3A shows the cyclic voltammograms corresponding to the analysis of glucose through the electrochemical determination of H2O2, formed by the Pt NPs/ GOx composite electrode. In this experiment, the electrode was prepared by the application of 60 electropolymerization cycles, and using a molar ratio of modified Pt NPs and functionalized GOx, in solution, that corresponded to 2.5:1.0 (for the effects of the ratio of Pt NPs/GOx, as well as the number of electropolymerization cycles on the performances of the bioelectrocatalytic electrodes, vide infra). As the concentration of glucose is elevated, the electrocatalytic cathodic currents are enhanced, allowing the analysis of glucose at a 1 mM limit. Figure 3A, inset, shows the derived calibration curve. A linear relation between the current response of the electrode and the content of glucose is observed in the concentration range of 0-140 mM, a broad domain that overlaps the appropriate region for analyzing sugar levels for diabetes. The activity of the cross-linked Pt NPs/GOx composites has been confirmed by two complementary experiments. In one experiment, Figure 3B, it was confirmed that the electrocatalytic reduction wave originates, indeed, from the reduction of GOxgenerated H2O2. Figure 3B, curve b, depicts the cyclic voltammogram corresponding to the Pt NPs-catalyzed reduction of the H2O2, generated by the GOx-mediated oxidation of glucose. Figure 3B, curve c, shows, however, the cyclic voltammogram of the Pt NPs/GOx composite in the presence of glucose, upon the coaddition of catalase to the electrolyte solution. Evidently, the addition of catalase depleted the electrocatalytic cathodic wave, consistent with the fact that catalase decomposes H2O2 through a disproportionation mechanism. Further support that the enzyme (34) Borole, D. D.; Kapadi, U. R.; Mahulikar, P. P.; Hundiwale, D. G. Polym. Adv. Technol. 2004, 15, 306–312. (35) Chaubey, A.; Pande, K. K.; Singh, V. S.; Malhotra, B. C. Anal. Chim. Acta 2000, 407, 907–913.

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GOx exists in a catalytically active configuration was obtained by the activation of the bioelectrocatalytic functions of the enzyme, in the presence of a diffusional electron mediator. The Pt NPs/ GOx-functionalized electrode was reacted with ferrocenemethanol as a diffusional electron mediator. Electrocatalytic anodic currents were observed in the presence of glucose, and as the concentration of glucose increased, the catalytic currents were intensified (see Supporting Information, Figure S3). The onset of the electrocatalytic anodic currents was observed at ∼0.15 V versus SCE, the redox potential of ferrocenemethanol. These currents imply that GOx exists in the Pt NPs/GOx composite in a biocatalytically active structure, in which ferrocenemethanol mediates the oxidation of glucose. One aspect that should be addressed relates, however, to the electropolymerization of the Pt NPs/GOx composite and the effect of the electropolymerizable Pt NPs and GOx units on the bioelectrocatalytic activity of the resulting electrode. While the electropolymerization of the Pt NPs contributes to the 3D conductivity of the matrix, the bioelectrocatalytic functions are controlled by the content of the enzyme in the matrix (the H2O2 generating units) and the coverage of the Pt NPs sites. While at first glance, it seems that high loading of the enzyme during electropolymerization would be an advantage, due to the enhanced biocatalytic generation of H2O2 by GOx in the matrix, the use of a high content of enzyme in the electropolymerization mixture would favor the incorporation of protein units around the particles, and this would insulate the particles and prevent further growth of the NPs/GOx film. Thus, it seems that an appropriate balance between the electropolymerizable Pt NPs and electropolymerizable enzyme should be retained to yield a Pt NPs/GOx composite with optimal bioelectrocatalytic functions. Figure 4A shows the electrocatalytic cathodic currents generated by Pt NPs/GOx composite electrodes, using 60 electropolymerization cycles, while changing the ratio of electropolymerizable Pt NPs and GOx. The concentration of the Pt NPs was kept constant (0.4 mg · mL-1) while the concentration of GOx was varied. In this set of experiments, the concentration of glucose was kept low, 14 mM,

Figure 3. (A) Cyclic voltammograms corresponding to the oligoaniline-cross-linked GOx/Pt NPs composite-modified Au electrode in the presence of variable concentrations of glucose: (a) 0, (b) 3, (c) 7, (d) 10, (e) 14, (f) 28, (g) 42, (h) 56, (i) 64, (j) 82, (k) 96, (l) 110, (m) 134, (n) 152, and (o) 166 mM. The electrode was interacted with the glucose-containing electrolyte solution for a fixed time interval of 4 min prior to the recording of the respective voltammograms. Inset: calibration curve corresponding to the electrocatalytic currents measured at E ) -0.35 V for variable concentrations of glucose. (B) Cyclic voltammograms corresponding to the oligoaniline-cross-linked GOx/ Pt NPs composite-modified Au electrode in the presence of (a) 0 mM glucose; (b) 64 mM glucose; (c) upon the addition of catalase, 2000 units, to a 64 mM glucose solution. Prior to the measurements, the electrode was immersed for a fixed time interval of 4 min in the respective electrolyte solution.

to ensure that the electrocatalytic cathodic currents are significantly below the saturation currents. As expected, by increasing the concentrations of GOx during the electropolymerization stage, the bioelectrocatalytic activity of the electrode increases, and the electrocatalytic cathodic currents reach a peak value at a concentration of glucose oxidase that corresponds to 0.5 mg · mL-1. Beyond this GOx concentration the current drops, and ultimately, the electrocatalytic activity diminishes. This lack of bioelectrocatalytic activity of the electrode generated at high concentrations of GOx may be attributed to the favored electropolymerization of the enzyme film, preventing the incorporation of the Pt NPs, or to the rapid insulation of the electropolymerized NPs by the enzyme, which prevents further electropolymerization and masks the catalytic functions of the Pt NPs. The insulation of the Pt NPs by the electropolymerized GOx seems to be particularly important, since blocking the three-dimensional conductivity prevents, also,

Figure 4. (A) Electrocatalytic currents corresponding to oligoanilinecross-linked GOx/Pt NPs composite-modified Au electrode, prepared by the application of 60 cyclic voltammetry scans between -0.1 and 1.1 V vs SCE at 100 mV · s-1, in the presence of 0.4 mg · mL-1 Pt NPs and variable concentrations of the thioaniline-modified GOx. Measurements were performed in a 0.1 M phosphate buffer solution (pH 7.4) that included 14 mM glucose. The electrodes were interacted for a fixed time interval of 4 min with the glucose-containing electrolyte solution. The currents were extracted from the respective cyclic voltammograms, recorded at a scan rate of 10 mV · s-1, at E ) -0.35 V. (B) Electrocatalytic currents corresponding to oligoaniline-crosslinked GOx/Pt NPs composite-modified Au electrode, prepared by the application of variable number of cyclic voltammetry scans between -0.1 and 1.1 V vs SCE at 100 mV · s-1, in the presence of 0.4 mg · mL-1 Pt NPs and 0.5 mg · mL-1 GOx. Measurements were performed in a 0.1 M phosphate buffer solution (pH 7.4) that included 56 mM glucose. The electrodes were interacted for a fixed time interval of 4 min in the glucose-containing electrolyte solution. The indicated currents were measured at E ) -0.35 V by cyclic voltammetry, performed at 10 mV · s-1.

the accumulation of GOx in the resulting composite associated with the electrode. Further support that electropolymerization at a high GOx concentration, relative to the Pt NPs, indeed leads to an insulation of the electrode and to the blocking of the incorporation of the Pt NPs was obtained by complementary quartz crystal microbalance measurements. We find that electropolymerization at a high GOx concentration leads to a sharp drop in the mass change associated with the functionalized electrode, implying that the formation of the Pt NPs/enzyme composite is, indeed, perturbed. The optimal bioelectrocatalytic functions of the composite electrodes were observed at a molar ratio of electropolyAnalytical Chemistry, Vol. 80, No. 21, November 1, 2008

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merizable Pt NPs:GOx that corresponds to ∼2.5:1.0. Accordingly, all of the previously described Pt NPs/GOx electrodes were prepared using these optimal conditions. Although the optimized ratio of thioaniline-functionalized Pt NPs and thioaniline-modified GOx in the electropolymerization mixture is 2.5:1.0, the question regarding the actual ratio of Pt NPs/GOx in the cross-linked composite film must be addressed. To characterize the composite structure associated with the electrode we determined as a first step the content of the enzyme in the film. For this purpose, we assayed the activity of the GOx in the composite. Having a calibration curve that relates the enzyme activity and the content, we concluded that the content of GOx in the film was 2.4 × 10-7 g · cm-2. This value translates to a coverage of 1.4 × 10-12 mol · cm-2. In the second step, we electropolymerized the functionalized Pt NPs and the thioanilinemodified GOx on Au quartz crystals under the optimal electropolymerization conditions. By subtracting the mass of the enzyme incorporated in the film (using the activity assay), the net weight of the Pt NPs in the composite was estimated to be 3.5 × 10-7 g · cm-2. Knowing the size of the NPs, this value translates to a surface coverage of 6.6 × 10-12 mol · cm-2. Thus, the molar ratio of Pt NPs/GOx in the composite corresponds to ∼4.7:1.0. The bioelectrocatalytic currents are controlled by the number of electropolymerization cycles applied during the generation of the Pt NPs/GOx electrodes in the presence of the optimal Pt NPs/ GOx ratio, Figure 4B. As the number of polymerization cycles increases, the bioelectrocatalytic currents are intensified, and after 60 cycles, the biocatalytic currents level off to a saturation value. The saturation value of the electrocatalytic cathodic current may be attributed to several reasons: (i) As electropolymerization proceeds, the three-dimensional conductivity of the Pt NPs is perturbed by the insulating enzymes, and this eliminates the further electropolymerization of the active components. (ii) As polymerization proceeds, inner Pt NPs and enzyme layers become inaccessible to glucose/H2O2, and thus, the layers do not contribute to the total cathodic currents. Accordingly, 60 electropolymerization cycles were applied to fabricate the electrodes for the different experiments. The bioelectrocatalytic cathodic currents are also controlled by the time interval allowed to interact the functionalized electrode with glucose in the solution to yield H2O2 (see Figure S4, Supporting Information). As the biocatalytic reaction is prolonged, the electrocatalytic cathodic currents increase, until they level off to a saturation value after ∼6 min. This phenomenon is explained by the fact that H2O2 is generated in the thin enzyme film associated with the electrode surface, and it diffuses out to the bulk electrolyte solution, which is considered as an “empty” H2O2 reservoir. After 6 min, an equilibrium is established since the flux of H2O2 diffusing to the bulk electrolyte is identical to the H2O2 flux generated by the enzyme film, thus leading to the saturation of the cathodic current. A final aspect relates to the stability of the cross-linked composite Pt NPs/GOx electrodes toward sensing of glucose. We find that the electrodes lose