Optimized Preparation and Scanning Electrochemical Microscopy

Optimized Preparation and Scanning Electrochemical Microscopy Analysis in Feedback Mode of Glucose Oxidase Layers Grafted onto Conducting Carbon ...
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Langmuir 2008, 24, 9089-9095

9089

Optimized Preparation and Scanning Electrochemical Microscopy Analysis in Feedback Mode of Glucose Oxidase Layers Grafted onto Conducting Carbon Surfaces Marie Pellissier,† Dodzi Zigah,† Fre´de´ric Barrie`re,*,†,‡ and Philippe Hapiot*,†,‡ Sciences Chimiques de Rennes (Equipe MaCSE), and UMR No. 6226, CNRS, UniVersite´ de Rennes 1, Campus de Beaulieu, Bat 10C, 35042 Rennes Cedex, France ReceiVed April 12, 2008 An optimized immobilization procedure based on the electroreduction of aryldiazonium salt followed by covalent attachment of a cross-linked hydrogel was used to graft glucose oxidase on a carbon surface. Scanning electrochemical microscopy (SECM) and cyclic voltammetry were used to follow the construction steps of the modified electrode. By adjusting the compactness of the layer through the electrografting reaction, the penetration of the mediator through the layer can be controlled to allow the monitoring of the enzymatic activity by both cyclic voltammetry and SECM in feedback mode. The enzymatic activity of the film is finally characterized by SECM.

Introduction The increasing demand for miniaturized bioanalytical devices, such as enzyme-based amperometric biosensors, has stimulated the need for easy and clean methods to immobilize biomolecules onto electrode surfaces and reliable methods to analyze their activities. More than 15 years ago, scanning electrochemical microscopy (SECM)1 was shown to be a very promising method to follow enzymatic reactions.2 Since then, the method has been largely used for the characterization and imaging of many enzymatic systems.3 In short, SECM is a scanning probe method that uses an ultramicroelectrode (UME) that is an electrode with a typical dimension in the micrometric range.1 Detection of enzymatic activities may be performed in generation-collection (GC) mode or in feedback (FB) mode. In the GC mode, the UME tip of the scanning electrochemical microscope is basically used as a passive probe. The UME is approached to the modified surface, and the enzymatic activity is monitored by the detection of an electroactive metabolite (see, for example, ref4 and the references therein). The conditions of use of the method are similar to other applications of ultramicroelectrodes, where the UME is used to map the diffusion-reaction profiles of electroactive species * To whom correspondence should be addressed. E-mail: philippe.hapiot@ univ-rennes1.fr or [email protected]. † Sciences Chimiques de Rennes. ‡ UMR No. 6226, CNRS. (1) (a) Scanning Electrochemical Microscopy; Bard, A. J., Mirkin, M. V., Eds.; Marcel Dekker Inc.: New York, 2001. (b) Bard, A. J.; Fan, F.-R. F.; Pierce, D. T.; Unwin, P. R.; Wipf, D. O.; Zhou, F. Science 2001, 254, 68. (2) (a) Bard, A. J.; Mirkin, M. V.; Unwin, P. R.; Wipf, D. O. J. Phys. Chem. 1992, 96, 1861–1868. (b) Pierce, D. T.; Unwin, P. R.; Bard, A. J. Anal. Chem. 1992, 64, 1795–1804. (c) Pierce, D. T.; Bard, A. J. Anal. Chem. 1993, 65, 3598– 3604. (3) (a) For recent reviews about the use of SECM in the characterization of an enzymatic system, see refs 3b–d and references therein. (b) Sun, P.; Laforge, F. O.; Mirkin, M. V. Phys. Chem. Chem. Phys. 2007, 9, 802. (c) Wittstock, G.; Burchard, M.; Pust, S. E.; Shen, Y.; Zhao, C. Angew. Chem., Int. Ed. 2007, 46, 1584. (d) Wittstock, G.; Burchardt, M.; Nunes Kircher, C. In Electrochemical Sensor Analysis; Alegret, S., Merkoc¸oi, A., Eds.; Elsevier: Amsterdam, 2007; pp 907-939. (4) (a) For some recent examples of the use of SECM in GC mode to detect enzymatic activities, see refs 4b–d. (b) Zhao, C.; Wittstock, G. Biosens. Bioelectron. 2005, 20, 1277. (c) Maciejewska, M.; Schafer, D.; Schuhmann, W. Electrochem. Commun. 2006, 8, 1119. (d) Schafer, D.; Maciejewska, M.; Schuhmann, W. Biosens. Bioelectron. 2007, 22, 1887.

produced at a larger electrode.5 The major difficulty is well identified and consists in the minimization of the interaction of the UME with the diffusional profile under investigation. Conditions in transient and stationary modes have been studied in detail, and theoretical treatments have been developed.6 The major interest of the GC mode is a good sensitivity and the possibility of analyzing many different molecules produced by the enzymatic system.3 However, the spatial resolution is governed by the transport of the product at the level of the substrate (diffusion + natural convection). This leads to poor resolution in imaging applications and provides little information about the mechanism or electrochemical processes involved in the modified electrode. It must be emphasized that, because the response depends on the transport properties on the scale of larger objects, the poor resolution is an inherent limitation of the method and is hence rather difficult to improve. In the FB mode, the redox mediator that initiates the enzyme reaction is directly generated at the UME.3 The enzymatic reaction rate is followed through the regeneration of the mediator measured by the increase of the current at the tip electrode as the tip-substrate distance is decreased (approach curves). The advantages of the FB mode are, first, a very good spatial resolution that depends on the size of the UME tip and, second, the possibility to perform detailed mechanistic investigations through the analysis of regeneration kinetics of the redox mediator. Despite these advantages, the number of enzymes that have been successfully studied in feedback mode is relatively limited3,7 because the regeneration rate has to be extracted from the hindered diffusion of the redox mediator to the electrode tip. Moreover, when the enzyme is deposited on a conducting surface, as in the case of a modified electrode (a common situation for scientists working in the design of electrochemical sensors or biofuel cells), the current recorded at the UME depends not only on the catalytic activity of the enzyme but also on the redox mediator reaction (5) Amatore, C.; Knobloch, K.; Thouin, L. J. Electroanal. Chem. 2007, 601, 17. (6) Amatore, C.; Szunerits, S.; Thouin; L.; Warkocz, J. S. Electroanalysis 2001, 13, 646. (7) (a) For some examples of investigations of the enzymatic activity by SECM in the feedback mode (cofactor regeneration), see refs 7b–d. (b) Zaumseil, J.; Wittstock, G.; Bahrs, S.; Steinru¨cke, P. Fresenius’ J. Anal. Chem. 2000, 367, 352–355. (c) Zhao, C.; Wittstock, G. Anal. Chem. 2004, 76, 3145. (d) Burchardt, M.; Wittstock, G. Bioelectrochemistry 2008, 72, 66.

10.1021/la801150c CCC: $40.75  2008 American Chemical Society Published on Web 07/15/2008

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with the conducting substrate. Hence, the detection in FB mode is generally limited to very active enzymatic systems with high turnover rates, and the analysis with the GC mode often remains the only practical possibility on conducting surfaces.3 A possible way to extend the usability of the FB mode is to control the redox mediator penetration inside the enzyme layer by a designed immobilization method. This property was used on carbon electrodes modified with polypyrrole containing glucose oxidase8 (GOx) and on a gold surface with thioalkane linkers9 to immobilize the enzyme. In many cases, the redox mediator was the [Fe(CN)6]3-/[Fe(CN)6]4- couple. Indeed, because this redox couple is highly charged and is electrochemically slow, it rapidly displays slow heterogeneous electron transfer kinetics and very low currents, as soon as the surface of the substrate is partially blocked.10 Hence, the use of ferri/ferrocyanide as redox mediator limits its regeneration reaction at the substrate surface. However, this redox couple is not very efficient for mediating enzyme activity, in particular that of GOx, because of the large net negative charge borne out in its two redox states.11 There is extensive literature that reports on many different procedures for improving the immobilization of the enzymes to achieve the retention of high catalytic properties on electrode surfaces.12,13 A classical method for increasing the stability of the modified surface is first to functionalize the electrode to subsequently graft the catalytic film. The initial modification of the electrode surface may be achieved in many ways. One of the simplest and most versatile procedures involves the reduction of aryldiazonium salts that can be performed on a large variety of conducting substrates.14–16 This approach provides a number of advantages, such as its facile implementation through the in situ generation of the aryldiazonium salt from the corresponding amine followed by direct electrochemical grafting.16 This procedure avoids the isolation of the aryldiazonium salts, which are sensitive to light and heat. Moreover, this approach allows the introduction of several functional groups at the surface and the possibility of subsequent coupling reactions. Another advantage of this method, based on aryldiazonium salt reduction, is the possibility of a fine patterning of the layer that opens routes for the preparation of devices based on this reaction. Indeed, defined chemical patterns have been achieved on carbon surfaces, allowing the formation of immobilized layers with different chemical groups.17 Also relevant to the present study, aryldiazonium salt reduction has been successfully used for the direct covalent binding of enzymes to electrodes.18–22 (8) Kranz, C.; Wittstock, G.; Wohlschla¨ger, H.; Schuhmann, W. Electrochim. Acta 1997, 42, 3105. (9) Kranz, C.; Lo¨tzbeyer, T.; Schmidt, H.-L.; Schuhmann, W. Biosens. Bioelectron. 1997, 12, 257. (10) Amatore, C.; Save´ant, J.-M.; Tessier, D. J. Electroanal. Chem. 1983, 147, 39. (11) To illustrate this fact, one may perform a simple comparative experiment in homogeneous solution containing 0.1 mol · L-1 glucose, 10-3 mol · L-1 redox mediator, and 0.5 mg/mL GOx in 0.1 mol · L-1 phosphate buffer. The plateau current of the bioelectrocatalytic reaction measured by linear scan voltammetry (20 mV · s-1) at a 3 mm diameter glassy carbon electrode was 6 mA with [Fe(CN)6]3-/[Fe(CN)6]4- and 30 mA with FcCH2OH/FcCH2OH+, while the current in the absence of glucose was similar in both cases (ca. 5 mA). (12) Cao, L. Curr. Opin. Chem. Biol. 2005, 9, 217. (13) Bernhardt, P. V. Aust. J. Chem. 2006, 59, 233. (14) Pinson, J.; Podvorica, F. Chem. Soc. ReV. 2005, 34, 429. (15) Downard, A. J. Electroanalysis 2000, 12, 1085. (16) Baranton, S.; Be´langer, D. J. Phys. Chem. B 2005, 109, 24401. (17) (a) Brooksby, P. A.; Downard, A. J. Langmuir 2004, 20, 5038. (b) Brooksby, P. A.; Downard, A. J. Langmuir 2005, 21, 1672. (c) Downard, A. J.; Garret, D. J.; Tan, E. S. Q. Langmuir 2006, 22, 10739. (18) Bourdillon, C.; Delamar, M.; Demaille, C.; Hitmi, R.; Moiroux, J.; Pinson, J. J. Electroanal. Chem. 1992, 336, 113. (19) Wang, J.; Carlisle, J. A. Diamond Relat. Mater. 2006, 15, 279. (20) Liu, G.; Gooding, J. J. Langmuir 2006, 22, 7421. (21) Polsky, R.; Harper, J. C.; Dirk, S. M.; Arango, D. C.; Wheeler, D. R.; Brozik, S. M. Langmuir 2007, 23, 364.

Pellissier et al. Scheme 1. Surface Modification Procedure

Another common procedure for the immobilization of enzyme is based on the embedding of the enzyme in a reticulated hydrogel. The reaction of poly(oxyethylene) bis(diglycidyl ether) (PEGDGE) as a cross-linker followed by its reticulation with the enzyme, yields a three-dimensional network permeable to the diffusion of small molecules and ions.23 Our procedure for GOx immobilization on a glassy carbon surface combines both diazonium electroreduction and hydrogel immobilization (Scheme 1). The first step is the formation of a layer of phenyl-NH2 moieties on the carbon substrate obtained by the electrochemical reduction of in situ generated (p-aminophenyl)diazonium salt. A mixture of enzyme (GOx) and cross-linker (PEGDGE) is then deposited onto the phenyl-NH2-modified electrode surface and allowed to react. This reaction permits the covalent immobilization of the enzyme layer through the reaction of the cross-linker epoxide functions with the grafted surface amine groups and with the pendant amine groups of the protein (in lysine residues).23 As discussed above, a rather blocking surface is required to study an enzyme-modified carbon surface with SECM in feedback mode. For detailed mechanistic investigations, it is desirable to be able to examine the same catalytic system by cyclic voltammetry and SECM. Both techniques are based on the same diffusion laws. While SECM probes the layer from the solution side, the system is examined from the substrate side with cyclic voltammetry. The use of these two complementary techniques with their inherent advantages and limitations provides a more complete picture of the whole system. Indeed, the blocking character of the modified surface could be adjusted to obtain a sufficient transport of the mediator for cyclic voltammetry but not too high for efficient SECM studies. For this purpose, the surface modification was followed by SECM and cyclic voltammetry, allowing a distinct characterization of each step in terms of permeability and transport processes among the solution, the enzyme layer, and the carbon substrate. The catalytic activity of the fully modified surface was then tested in the presence of glucose, the natural substrate of the GOx enzyme. This procedure has permitted the use of an electrochemically fast redox couple and efficient redox mediator for GOx (such as (hydroxymethyl)ferrocene/(hydroxymethyl)ferrocenium, FcCH2OH/FcCH2OH+) for a combined study with SECM in feedback mode and cyclic voltammetry on a conductive substrate.

Experimental Section Chemicals and Materials. Chemicals were purchased from Sigma-Aldrich, glucose oxidase type X-S (Aspergillus niger, 100 000 units/g) PEGDGE (average molar mass ∼526 g/mol), (hydroxym(22) Corgier, B. P.; Laurent, A.; Perriat, P.; Blum, L. J.; Marquette, C. A. Angew. Chem., Int. Ed. 2007, 46, 4108. (23) Ohara, T. J.; Rajagopalan, R.; Heller, A. Anal. Chem. 1993, 65, 3512.

Glucose Oxirane Layers Grafted onto Carbon Surfaces

Figure 1. SECM approach curves recorded on the carbon surface directly modified with the enzyme layer (GOx + cross-linker with no previous aryldiazonium reduction): (O) without glucose, (9) after addition of 30 mol · .L-1 glucose. The solid line is the theoretical curve for a conducting material. The curves were recorded with a gold working UME of 4.5 µm radius, EUME ) 0.5 V vs Ag/AgCl, approach rate 5 µm · s-1.

ethyl)ferrocene, p-phenylenediamine, and D-(+)-glucose, and from Acros Organics, sodium nitrite and potassium ferricyanide. All chemicals were used as supplied. Ultrapure water (resistivity 18.2 MΩ · cm at 25 °C) from an ELGA Purelab Classic source was used in all experiments. Solutions of glucose were prepared at least 24 h before each experiment to allow complete equilibration of the anomers. Preparation of Modified Surfaces. Functionalization of the Electrode Surface with Amino Functional Groups (See Scheme 1) Amino functional groups were introduced on glassy carbon electrodes by reduction of (p-aminophenyl)diazonium salt generated in situ from p-phenylenediamine. The electrolytic solution consisted of 10-2 mol · L-1 arylamine and 0.5 mol · L-1 HCl. The electrolyte was degassed with N2 for 5 min under stirring, prior to the dropwise addition of a chilled concentrated solution of sodium nitrite.16 After 5 min of reaction, electrochemical modification was immediately performed on previously polished and cleaned electrodes. The modification procedure for grafting layers derived from (paminophenyl)diazonium salt entailed five cycles from +0.35 to -0.75 V at 20 mV · s-1. Electrodes were then thoroughly rinsed. CoValent Grafting of the Enzyme Hydrogel. The modification was carried out by deposition of a mixture of glucose oxidase and PEGDGE cross-linker in 0.1 mol · L-1 phosphate buffer, pH 7.4. The mixture consisted of 300 µL of an 8 mg/mL glucose oxidase solution and 120 µL of a 2 mg/mL PEGDGE solution. A 50 µL droplet of this mixture was deposited on the phenyl-NH2-modified electrode. The layers were allowed to react and dry for 24 h at room temperature. The epoxide groups of the cross-linker react with amine groups, allowing both cross-linking with the enzyme lysine residues and covalent attachment of an enzyme layer to the amino functions on the surface.23 Before measurement, the electrodes were soaked in phosphate buffer solutions (pH 7.4) for 1 h to allow the leaching out of material not covalently attached to the surface or cross-linked in the three-dimensional enzyme layer. Estimation of the Glucose Oxidase Surface Concentration The surface concentration was estimated by the following procedure. We first calculated the mass of enzymes contained in the deposit (m1). Then the electrode was soaked in a small volume (2 mL) of phosphate buffer, 0.1 M, pH 7.4. To this volume of buffer, containing the enzyme not covalently linked to the surface, was added an equal volume of ferrocenemethanol, 1 mM, in the same buffer. Next, the catalytic efficiency was measured by cyclic voltammetry in the presence of an excess of glucose. The catalytic efficiency of a number of solutions of enzymes of known mass concentration was measured in the same conditions, allowing the calculation of the mass of enzymes which has leached out (m2). m1 - m2 gives the mass of enzymes covalently linked to the surface and thus allows calculation of the surface concentration. On the basis of the initial deposited quantity of hydrogel and on the geometric surface of the electrode,

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Figure 2. Cyclic voltammograms of the reduction of a 10-2 mol · L-1 solution of in situ generated NH2PhN2+ in a 0.5 mol · L-1 HCl solution at a glassy carbon electrode, scan rate 20 mV · s-1. The five consecutive cycles are shown.

Figure 3. Cyclic voltammograms (scan rate 10 mV · s-1) of 10-3 mol · L-1 (hydroxymethyl)ferrocene in 0.1 mol · L-1 phosphate buffer, pH 7.4, at a glassy carbon electrode before (black) and after (red) the modification by electrografting of PhNH2.

Figure 4. Approach curves on the glassy carbon electrode in phosphate buffer, 0.1 mol · L-1, pH 7.4, before (O) and after (0) electrografting of PhNH2. The redox mediator was (hydroxymethyl)ferrocene at a concentration of 10-3 mol · L-1. Solid lines are the theoretical curves for totally insulating (negative feedback) and totally conducting (positive feedback) substrate cases. EUME ) 0.5 V vs Ag/AgCl, and the approach rate was 5 µm · s-1.

the maximum surface concentration of GOx (after soaking) could be estimated as 5.9 × 10-9 mol · cm-2. Cyclic Voltammetry and SECM Experiments. Cyclic voltammetry measurements were performed with an Autolab electrochemical analyzer (PGSTAT 30 potentiostat/galvanostat from Eco Chemie B.V.) in a three-electrode configuration. The working electrode was a 3 mm diameter disk glassy carbon electrode. All potentials were measured against a Ag/AgCl, aqueous KCl, 3 mol · L-1, reference electrode. The counter electrode was either a Pt

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Scheme 2. Positive and Negative Feedback Processes When the Signal Is Limited by the Transport through the Layer

grid or a glassy carbon rod. The supporting electrolyte was a 0.1 mol · L-1 phosphate buffer, pH 7.4, prepared with deionized water. A typical SECM experiment consists in recording approach curves.1 In this experiment, the UME is slowly moved toward the sample to maintain steady-state conditions.1–3 The applied potential at the UME is chosen as being sufficiently positive to ensure a fast electron transfer at the UME (diffusion plateau of the mediator). SECM data are usually presented with a plot of I ) i/iinf against the normalized distance L ) d/a, where I is the normalized current (the current flowing through the UME divided by the current recorded when the tip is far from the electrode (iinf)), d is the tip-substrate distance, and a is the radius of the UME. This adimentionalization allows an easy comparison between experimental data and theoretical

Figure 5. Cyclic voltammograms (scan rate 5 mV · s-1) of 10-3 mol · L-1 (hydroxymethyl)ferrocene in 0.1 mol · L-1 phosphate buffer, pH 7.4, at a fully modified glassy carbon electrode (phenyl-NH2 + enzyme + cross-linker) in the absence (black) and in the presence (red) of glucose, 0.1 mol · L-1.

Figure 6. Approach curves on the Ph-NH2-modified GC electrode in phosphate buffer, 0.1 mol · L-1, pH 7.4, in the absence of glucose, before (O) and after (9) grafting of the enzyme layer. The redox mediator was (hydroxymethyl)ferrocene at a concentration of 10-3 mol · L-1. The solid line is the theoretical behavior expected for an insulating substrate. The curves were recorded with a gold working UME of 4.5 µm radius, EUME ) 0.5 V vs Ag/AgCl, approach rate 5 µm.s-1.

Figure 7. Approach curves on the fully modified glassy carbon electrode in 0.1 mol · L-1 phosphate buffer, pH 7.4, in the absence and in the presence of glucose at different concentrations. Experimental data from bottom to top for glucose concentrations: (0) 0, (]) 6 × 10-4, (9) 1.3 × 10-3, (∆) 3 × 10-3, (O) 3 × 10-2 mol · L-1. Solid lines are the theoretical curves expected for the totally insulating and totally conducting cases. The redox mediator was (hydroxymethyl)ferrocene at a concentration of 10-3 mol · L-1. The curves were recorded with a gold working UME of 4.5 µm radius, EUME ) 0.5 V vs Ag/AgCl, approach rate 5 µm · s-1. Scheme 3. Origin of the Positive Feedback in the Case of an Immobilized Catalytic System (GOx)

curves.1–3 SECM measurements were performed using the CHI900B instrument from CH-Instruments equipped with an adjustable stage for the tilt angle correction. The electrochemical cell was that purchased with the scanning electrochemical microscope and was used in a typical three-electrode configuration. The carbon substrate was not electrically connected (unbiased experiment configuration). The UME tip was a homemade 4.5 µm radius disk Au ultramicroelectrode with a typical RG ) 10-15 (RG is the ratio of the total electrode radius including the glass insulator to the UME radius). The electrodes were prepared by sealing a gold wire in a glass tube according to an already published procedure.1 The UMEs were characterized by cyclic voltammetry and by typical approach curves recorded on conducting and isolating surfaces. Fittings were performed using the approximate functions following the Bard-Mirkin formalism1 and their recent resolution for different RG values.24 Zero distance scaling was performed by adjustment of the experimental approach curves to the corresponding relevant theoretical limiting cases, i.e., to the purely insulating case for the completely blocked electrodes or to the conducting case for curves recorded in an excess of glucose or without surface modification. Approach curves corresponding to the intermediate experimental situations were adjusted considering the same offset. In all experiments, the redox mediator was the FcCH2OH/FcCH2OH+ couple (E° ) +0.15 V) at a typical concentration of 10-3 mol · L-1. The UME potential, EUME, was set at a potential corresponding to the diffusive plateau of (hydroxymethyl)ferrocene oxidation, i.e., +0.5 V vs Ag/AgCl. (24) Cornut, R.; Lefrou, C. J. Electroanal. Chem. 2007, 604, 91.

Glucose Oxirane Layers Grafted onto Carbon Surfaces

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Figure 8. Approach curves made on two different samples. Variation of icat with the adimentional distance L. On the right, the concentration of mediator (FcCH2OH) is gradually increased by successive addition of mediator in 0.1 mol · L-1 phosphate buffer, pH 7.4. On the left, its concentration is gradually decreased by successive dilutions. The insets show the variations of icat at a constant distance L ) (0) 0.2 or (O) 1 for both samples. The curves were recorded with a gold working UME of 4.5 µm radius, EUME ) 0.5 V vs Ag/AgCl, approach rate 5 µm · s-1.

Figure 9. Approach curves on a fully modified glassy carbon electrode in phosphate buffer, 0.1 mol · L-1, pH 7.4, glucose concentration 50 × 10-3 mol · L-1. The mediator is FcCH2OH at different concentrations: (9) 1 mmol · L-1, (O) 0.05 mmol · L-1, (0) 0.01 mmol · L-1. The solid line is the theoretical curve for the totally conducting case, and the dashed line is the theoretical curve for κ ) 4.5. The curves were recorded with a gold working UME of 4.5 µm radius, EUME ) 0.5 V vs Ag/AgCl, approach rate 5 µm · s-1.

Results and Discussion SECM approach curves were first recorded on the unmodified carbon substrate and then after the hydrogel deposition. As expected, we found that the unmodified carbon surface displays a large increase of the current, when the UME is moved toward the surface, in agreement with the typical behavior expected for a conducting surface (not shown).1 After deposition of the enzyme layer, a similar positive feedback curve was obtained (see Figure 1). These results show that the hydrogel (enzyme + PEGDGE cross-linker) displays almost no blocking properties toward the penetration of FcCH2OH or FcCH2OH+, allowing the redox mediator to reach the carbon surface and undergo electron transfer. This is clearly an advantage, as most of the GOx embedded in the matrix should also be easily reached, which is a prerequisite for a good activity inside the whole layer. Unfortunately, such a catalytic system cannot be studied by SECM in feedback mode, as the signal already behaves as a perfect conducting system. As expected, no appreciable modification of the signal was obtained after addition of glucose (Figure 1). Characterization of the Phenyl-NH2-Modified Surface Aryldiazonium salt reduction leads to an oligophenylene-like multilayer structure on the surface arising from the addition of the continuously electrogenerated p-aminophenyl radicals onto

the already grafted moieties.16 In our experiments, (p-aminophenyl)diazonium salt was generated by in situ reduction of p-phenylenediamine with sodium nitrite.16 Then electrochemical reduction of the aryldiazonium cation on glassy carbon electrodes was performed using repetitive cyclic voltammetry. The voltammogram recorded on a clean electrode showed the expected large irreversible reduction peak on the first cycle (see Figure 2). On the second and following scans, the current progressively decreased and no reduction peak could be detected. This behavior is consistent with the numerous reports on covalent grafting of electrogenerated phenyl radicals onto glassy carbon.16 Further evidence that electrografting has occurred on the surface, and characterization of the modification, was provided by examination of the oxidation of FcCH2OH by cyclic voltammetry and SECM before and after modification of the electrode. As visible in the cyclic voltammograms recorded before and after modification of the electrode (Figure 3), the potential difference between the forward and reverse peaks increases and only a poorly resolved voltammogram is obtained after modification with PhNH2. The same two samples were also investigated by SECM. As observed on the approach curves measured after the aryldiazonium reduction modification (Figure 4), the current now decreases as the UME is approached toward the substrate, corresponding to a “negative feedback” situation.1 It is noticeable that the experimental curve fits well the theoretical behavior expected for a totally insulating curve. This observation is consistent with the absence of reaction of FcCH2OH+ with the modified glassy carbon substrate. The mediator cannot pass through the oligophenylene layer (see Scheme 2). As a final control and as expected, the feedback remains negative after the addition of glucose. Characterization of the Fully Modified Surface (PhenylNH2 + Enzyme + Cross-Linker) The catalytic activity of the electrode modified by deposition of the cross-linked GOx layer onto a phenyl-NH2-modified electrode, as described in the Experimental Section, was first evaluated by cyclic voltammetry using FcCH2OH (10-3 mol · L-1) as a redox mediator. Figure 5 shows the cyclic voltammograms recorded in the absence and presence of a large excess of D-glucose (0.1 mol · L-1). The cyclic voltammogram in the absence of D-glucose presents the typical behavior for a redox couple with a very slow electron transfer, as indicated by the large peak to peak potential difference. This is in agreement with the behavior expected for a partially blocked electrode.10 It is interesting to compare the cyclic voltammograms recorded in the absence of glucose at

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differently modified surfaces. The voltammogram obtained on the phenyl-NH2-only-modified electrode (Figure 3, red) appears as less resolved than the one recorded on the fully modified surface (see Figure 5). This shows that the fully modified surface becomes less blocking toward the oxidation of FcCH2OH. In other words, the addition of a new layer diminished the blocking property of the modification. Examination of these two samples by SECM in the same conditions shows that both surfaces display a negative feedback behavior, Figure 6. However, the current recorded on the fully modified surface is higher than that measured on the phenyl-NH2-only-modified surface. This confirms that the apparent electron transfer kinetics rate is larger in the case of the fully modified surface. Taken together, these results show a decrease of the blocking character of the oligophenylene layer after addition of the hydrogel. This unexpected behavior could be compared with previously published results. Indeed, blocking properties of organic layers are generally ascribed to the compactness of the modifying layers and to their hydrophobic character, as was recently investigated for carbon electrodes.25 For oligophenylene layers on the carbon surface prepared with aryldiazonium reduction, we have also observed that the blocking character of the modification is dependent on the solvent and is more efficient in water.26 The intrinsic properties of the redox mediator itself (charge, size, hydrophobicity, electron transfer rate, etc.) are also an important factor for the resulting blocking vs conducting behavior obtained for a given modified surface.25,27 Detection of Immobilized GOx Activity by SECM in Feedback Mode Figure 7 displays the approach curves recorded in phosphate buffer on the fully modified electrode, i.e., first modified by the electroreduction of the aryldiazonium followed by grafting of the enzyme layer. Measurements were undertaken under argon to avoid interference with dioxygen, the GOx natural electron acceptor. Glucose was introduced by successive aliquots from a concentrated D-glucose solution in phosphate buffer. In the absence of glucose, a negative feedback similar to that of Figure 6 is observed. Upon successive additions of glucose, the recorded approach curves gradually switch from a negative to a positive feedback until the concentration of glucose is no longer the limiting parameter. As previously discussed,2,3 the origin of the positive feedback shown in Figure 7 is due to the occurrence of the catalytic process, as represented in Scheme 3. The interference of the reaction of FcCH2OH+ with the carbon surface, as depicted in Scheme 2, remains negligible, and it is noticeable that glucose concentrations on the order of 10-4 mol · L-1 can be detected (see the difference in the approach curves recorded without glucose and after the first addition of D-glucose, Figure 7). For the largest concentration of glucose, the experimental approach curve fits with the theoretical behavior expected for a conducting substrate. This indicates that in this extreme case (i) the system is only controlled by the diffusion of the mediator from the tip to the enzyme layer and (ii) the enzymatic process is fast and efficient. In the presence of an excess of glucose and for an adimentional distance L around 1, i.e., when the tip is located at a distance from the substrate equal to its radius, the enzymatic activity leads to a 5-6-fold increase of the current, which is comparable with what is obtained with the best detection by SECM in the GC mode imaging.3 This is sufficient for obtaining a good contrast in imaging applications, (25) Cruickshank, A. C.; Tan, E. S. Q.; Brooksby, P. A.; Downard, A. J. Electrochem. Commun. 2007, 9, 1456. (26) Zigah., D.; Pellissier, M.; Barrie`re, F.; Downard, A. J.; Hapiot, P. Electrochem. Commun. 2007, 9, 2387. (27) Pellissier, M.; Barrie`re, F.; Downard, A. J.; Leech,D. Electrochem. Commun. 2008, 10, 835.

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provided that it is possible to maintain a constant substrate-tip distance during the entire SECM imaging experiment. For a more quantitative view of the process, it is interesting to extract from the rough approach curve the contribution of the current due to the enzymatic catalytic activity. Following the approximate treatment proposed by Wittstock et al.,7d this quantity can be evaluated by icat(L) ) (I(L) - Iins(L))iinf, where Iins is the normalized current expected when the UME approaches an insulating surface, i.e., when the mediator does not react with the substrate. The treatment is based on the simplified reaction mechanism for enzymatic reaction of glucose oxidase with the glucose and a one-electron mechanism as for ferrocene derivatives:2b,7d k1

GOxox+glucose {\} [GOxox-glucose]

(1)

k-1

k2

[GOxox-glucose] 98 GOxred + gluconolactone k3

GOxred+2FcCH2OH+ 98 GOxox+2FcCH2OH

(2)

(3)

In the case of a large excess of glucose, when the concentration of glucose is higher than the Michaelis-Menten constant for glucose, this reaction mechanism leads to the following simplified rate law:2b,7d

dcFcCH2OH+ dt

)

2k2ΓGOx KM,FcCH2OH+ cFcCH2OH+

with +1

KM,FcCH2OH+ )

k2 k3 (4)

If cFcCH2OH+ < KM,FcCH2OH+, i.e., if the regeneration of GOxox by the mediator is the limiting factor, apparent first order with respect to the mediator is expected. The obtained law dcFcCH2OH+/ dt ) (2k2ΓGOx/KM,FcCH2OH+)cFcCH2OH+ has the same form as the equation predicted for an irreversible electron transfer occurring at a conducting surface.1 Thus, approach curves can be treated by the same approximation following the Bard-Mirkin formalism and using the approximate working curves calculated for the determination of the rate constant for a heterogeneous electron transfer between the substrate and the mediator. In such a case, the SECM approach curves depend on a single parameter, κ ) keff(a/D) ) (2k2ΓGOx/KM,FcCH2OH+)(a/D). The pseudo-first-order behavior could be evidenced by the variation of icat versus the initial concentration of mediator FcCH2OH in the presence of a large excess of glucose. Such variations are plotted in Figure 8 for two different samples prepared following the same experimental procedures and in the presence of large concentrations of glucose. As seen on both curves, linear variations are observed, meaning that the enzyme layer is not saturated with respect to the mediator.2b,7d Moreover, it is noticeable that both modified surfaces display almost similar enzymatic activities, which shows a good reproducibility in the sample preparation and in the corresponding obtained responses. Considering such behavior, the apparent first-order rate constant keff ) 2k2ΓGOx/KM,FcCH2OH+ can be derived by a fitting of the experimental approach curves with the theoretical ones calculated for an irreversible electron transfer.1,2 As seen in Figure 9, the observed behavior is very close to the one expected for a kinetically fast system with an infinite rate constant keff, i.e., when the system is only limited by the diffusion of the mediator.1 Considering the accuracy on the determination of L, we can

Glucose Oxirane Layers Grafted onto Carbon Surfaces

derive from the maximum value of I at the lowest L28 (ca. 4.0) that κ > 4.5 and thus keff > 0.08 cm · s-1.29 Given that ΓGOx was estimated as 5.9 × 10-9 mol · cm-2 (see the Experimental Section), this leads to a value of 2k2/KM in the range of 1.4 × 107 mol-1 · cm-1 · s-1. The Michaelis-Menten constant for FcCH2OH is reported to be around 1.8 × 10-3 mol · L-1, meaning that the turnover rate 2k2 for the immobilized GOx is much larger than 25 s-1. As observed before, this value is in agreement with reported values for GOx in solution30 and confirms the high activity of (28) (a) Determining the offset from positive feedback curves could be inaccurate and thus leads to erroneous measurement of the rate constant because the curves for rather fast kinetics could be brought to superposition with the limiting case of an infinite reaction rate at the sample by a shift on the L axis. However, the maximum value observed for the adimentional current I, here around 4.0, makes a condition for the lower value of the adimentional parameter k.28b (b) Lefrou, C. J. Electroanal. Chem. 2007, 601, 94. (29) (a) Taking 7.8 × 10-6 cm2 · s-1 for the diffusion coefficient of FcCH2OH.29b (b) Miao, W.; Ding, Z.; Bard, A. J. J. Phys. Chem. B 2002, 106, 1392. (30) (a) Turnover of the GOx (2k2) in solution is reported to be around 600 s-1. See, for example, ref 30b. (b) Kima, J.; Jiab, H.; Wangb, P. Biotechnol. AdV. 2006, 24, 296.

Langmuir, Vol. 24, No. 16, 2008 9095

the layer and the fact that active enzymes remain reachable even after incorporation into the polymer.

Conclusion Step-by-step construction of an enzyme-modified surface with blocking properties toward (hydroxymethyl)ferrocene oxidation was performed by the combination of the “aryldiazonium” and “hydrogel” methods. The procedure can be controlled by the scanning electrochemical microscope, allowing a reproducible adjustment of the blocking character of the electrode. In optimized conditions, the enzymatic catalysis can be followed both by cyclic voltammetry, despite the relatively low electron transfer rate, and by SECM with a good contrast versus the background signal coming from the hindered diffusion. Quantitative treatment of the SECM signal shows that the enzyme maintained a large enzymatic activity after incorporation into the layer. LA801150C