pH-Controllable On−Off Bioelectrocatalysis of Bienzyme Layer-by

The HRP/GOD bienzyme electrode is thus inherently attractive with its ... (16, 19-21) Particularly, pH-responsive on−off bioelectrocatalysis has bee...
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J. Phys. Chem. B 2010, 114, 9926–9933

pH-Controllable On-Off Bioelectrocatalysis of Bienzyme Layer-by-Layer Films Assembled by Concanavalin A and Glucoenzymes with an Electroactive Mediator Huiqin Yao†,‡ and Naifei Hu*,† Departments of Chemistry, Beijing Normal UniVersity, Beijing 100875, and Ningxia Medical UniVersity, Yinchuan 750004, People’s Republic of China ReceiVed: May 13, 2010; ReVised Manuscript ReceiVed: June 21, 2010

Mediated electrochemical biosensors consisting of two enzymes have attracted increasing interest because of their wider applicability. In this work, concanavalin A (Con A) and two glycoenzymes, horseradish peroxidase (HRP) and glucose oxidase (GOD), were assembled into {Con A/HRP/Con A/GOD}n layer-by-layer films on an electrode surface mainly by lectin-sugar biospecific interaction between Con A and glycoenzymes. The cyclic voltammetry (CV) response of Fe(CN)63- at the bienzyme film electrodes was very sensitive to the environmental pH: at pH 4.0, the CV peak currents were quite large and the films were at the “on” state; at pH 8.0, however, the electrochemical response was significantly suppressed and the films were at the “off” state. By switching the film electrodes in solution between pH 4.0 and pH 8.0, the CV peak currents cycled between the on and off states, demonstrating the reversible pH-sensitive on-off switching. The pH-responsive property of the films toward the probe could be used to switch the on-off bioelectrocatalysis of glucose. That is, the electrochemical oxidation of glucose catalyzed by GOD and HRP in the films mediated by Fe(CN)63- in solution could be controlled by changing the surrounding pH, allowing the reversible transition of bioelectrocatalysis between the on and off states. CV, electrochemical impedance spectroscopy, amperometry, and quartz crystal microbalance studies were used to characterize the {Con A/HRP/Con A/GOD}n films. The mechanism of pH-sensitive switchable behavior of the films was further explored by comparative experiments and should be attributed to the different electrostatic interactions between the films and the probes at different pH values. This pH-switchable bioelectrocatalysis based on the smart bienzyme interface may pave the way for designing novel controllable biosensors. Introduction Electrochemical biosensors on the basis of enzymatic reactions are one of the most well-known biosensors and have attracted continuous attention among researchers due to the high specificity and intrinsic sensitivity of the enzymes.1-3 Within this range of biosensors, the immobilization of two or more enzymes on the surface of one electrode is of particular interest not only for analytical applications with improved characteristics but also for better understanding the mechanism of enzymatic reactions in the living systems. The electrocatalytic reactions with coupling of bienzymes or multienzymes can mimic the sequential electron transfer reactions found in many enzyme pathways of living cells and provide a working model for the mechanistic study of multienzyme-catalyzed reactions in real biological systems.4 The bienzyme biosensors coupling horseradish peroxidase (HRP) with glucose oxidase (GOD) were first designed by Kulys et al. to detect glucose5 and have been developed rapidly since then.6-9 The sensing of glucose is of great significance nowadays in several applications including the diagnosis of diabetes in the clinic. The HRP/GOD bienzyme electrode is thus inherently attractive with its unique advantages. In this system, the in situ generated H2O2 from oxidation of glucose catalyzed by GOD in the presence of oxygen acts as the substrate for the second enzyme HRP and is subsequently reduced to water by HRP, which can be electrically connected * To whom correspondence should be addressed. E-mail: hunaifei@ bnu.edu.cn. Phone: (+86) 10-5880-5498. Fax: (+86) 10-5880-2075. † Beijing Normal University. ‡ Ningxia Medical University.

to the underlying electrode by a diffusing electroactive mediator in solution. In this way, the principle of glucose detection switches from an electrochemical oxidation to a reduction process that is happening at substantially milder potentials, and the interference of coexisting electroactive species can be avoided and the sensor selectivity greatly improved. In addition, the high concentration of H2O2, which is harmful to HRP, can be prevented.10 A variety of redox mediators has been introduced totheHRP/GODbienzyme-basedsystem,includingferricyanide,5,11 hydroquinone,7 phenols,12 aromatic amines,12 osmium complex,13 methylene blue,14 thionine,15 and neutral red.9 In recent years, switchable bioelectrocatalysis based on enzymes has aroused great interest.16-18 The reversible and stimulus-controllable bioelectrocatalysis may enable its application in the switchable biosensors and provide the foundation for bioelectronic devices, signal amplification, biofuel cells, and information storage and processing.16,19-21 Particularly, pHresponsive on-off bioelectrocatalysis has been reported.22-26 For example, Katz and co-workers modified poly(4-vinylpyridine) brush films functionalized with an Os complex on an electrode surface and used the films to electrochemically oxidize glucose catalyzed by GOD in solution and mediated by Os complex redox units in the films.22 The films exhibited a pHresponsive structure change and could be employed to reversibly activate/deactivate the bioelectrocatalysis of glucose by changing the surrounding pH. However, to the best of our knowledge, pH-switchable bioelectrocatalysis on the basis of a bienzyme system has not been reported up to now.

10.1021/jp104360q  2010 American Chemical Society Published on Web 07/09/2010

Bioelectrocatalysis of Bienzyme LbL Films Electrochemical biosensors with enzymes usually require the immobilization of enzymes on electrode surfaces. The HRP/ GOD bienzyme electrodes have also been developed with both HRP and GOD immobilized on electrodes with a variety of methods.6,7,9,12,14,27 Among different immobilization approaches, the layer-by-layer (LbL) assembly demonstrates distinguished advantages in its precise control of the film thickness at a nanometer scale and in its extremely simple procedure and high versatility in the assembly.28,29 The building blocks of LbL films were originally polyelectrolytes, but have now been extended to enzymes, proteins, and other species30,31 The driving force of LbL assembly is usually electrostatic interaction between oppositely charged materials, but the nonelectrostatic interactions including hydrogen bonding, hydrophobic interaction, and biospecific affinity have also been applied in the LbL assembly.12,14,28 Among the various biospecific interactions, lectin-sugar affinity is also used to construct multilayer films. Concanavalin A (Con A) is the best known member of the lectin proteins and exists as a tetramer at neutral pH.32 The most characteristic feature of Con A is that each subunit contains a binding site to sugar groups such as glucose and mannose, forming a highly specific 1:4 Con A-sugar complex.32-34 Therefore, the lectin-sugar interaction between Con A and sugar residues of glycoenzymes such as HRP and GOD can be used to construct LbL films.12,34,35 For example, {Con A/HRP}n/ {Con A/GOD}n LbL films assembled by biospecific affinity between Con A and sugar groups intrinsically existing on the surface of HRP and GOD were fabricated on an electrode surface and used to determine glucose, phenolic compounds, and aromatic amines.12,14 In our previous work,24 {Con A/HRP}n LbL films assembled on an electrode surface showed pHswitchable bioelectrocatalytic reduction of H2O2 with Fe(CN)63as the mediator. Recently, stimulus-responsive films that change their properties in response to environmental stimuli have aroused increasing interest, and different external stimuli, including pH, temperature, light, ionic strength, and potential, have been used to control the film response.16,36-39 In particular, by choosing appropriate materials and processing conditions, it is possible to create thin LbL films on electrodes that show on-off switching electrochemical responses toward electroactive probes under different pH conditions.40-42 We thus expected that this type of pH-sensitive LbL films could be employed to control bioelectrocatalysis of glucose with the HRP/GOD bienzyme system. In the present work, {Con A/HRP/Con A/GOD}n LbL films were assembled on pyrolytic graphite (PG) electrodes mainly through the biospecific lectin-sugar interaction between Con A and glycoenzymes. The films demonstrated a pHsensitive on-off property toward Fe(CN)63- in their electrochemical response. At pH 4.0, the cyclic voltammetry (CV) response of the probe was quite large and the films were at the on state; at pH 8.0, however, the CV response of the probe was greatly suppressed and the films were at the off state. This pHsensitive switching property of the films toward Fe(CN)63- was further used to activate/deactivate the electrocatalytic oxidation of glucose catalyzed by GOD and HRP coimmobilized in the films. In addition, the mechanism of the pH-dependent permeability of the films toward the probe was explored with comparative studies and was believed to be mainly attributed to the electrostatic interaction between the bienzyme films and the probe. This is the first report on pH-switchable on-off bioelectrocatalysis based on sequential enzymatic reactions with two enzymes immobilized on an electrode surface. The better understanding of the essence of interactions in this pH-sensitive

J. Phys. Chem. B, Vol. 114, No. 30, 2010 9927 “smart” model interface may provide a new platform to design pH-controllable biosensors based on bioelectrocatalysis with immobilized bienzymes. Experimental Section Reagents. Chitosan (CS; the degree of deacetylation is more than 85%, MW ≈ 200 000), Con A extracted from jack beans (type V, MW ≈ 104 000), HRP (E.C. 1.11.1.7, type II, MW ≈ 44 000, 250 000 units g-1), GOD (E.C. 1.1.3.4, type VII, MW ≈ 160 000, 192 000 units g-1), ferrocenemethanol (FcOH), hexaammineruthenium(III) chloride (Ru(NH3)6Cl3), methylene blue (MB), 1,1′-ferrocenedicarboxylic acid (Fc(COOH)2), 3-mercapto-1-propanesulfonate (MPS; 90%), and tris(hydroxymethyl)aminomethane (Tris) were purchased from Sigma-Aldrich. Potassium ferricyanide (K3Fe(CN)6), potassium ferrocyanide (K4Fe(CN)6), and hydrogen peroxide (H2O2; 30%) were obtained from Beijing Chemical Engineering Plant. The dilute H2O2 aqueous solutions were freshly prepared before being used. Hydroquinone was from Tianjin Bodi Chemical Engineering. All other reagents were of analytical grade. Britton-Robinson buffers at pH 4.0-8.0 containing 0.1 M NaCl were used, and the pH was adjusted to the desired value with dilute HCl or NaOH solutions. 0.1 M Tris-HCl buffers at pH 7.4 containing 0.1 M NaCl, 1 mM MnCl2, and 1 mM CaCl2 were used to prepare Con A solutions.43 The D-glucose stock solutions were allowed to mutarotate at room temperature for 24 h before being used. All solutions were prepared with water purified twice by ion exchange and subsequent distillation. Film Assembly. For electrochemical study, basal plane PG (Advanced Ceramics) disks (geometric area 0.16 cm2) were used as working electrodes. Prior to assembly, PG electrodes were abraded on 320-grit metallographic sandpaper while flushing with water. After being ultrasonicated in water for 30 s and dried in air, the electrodes were first immersed in 1 mg mL-1 CS solutions at pH 5.0 for 30 min, forming a CS precursor layer on the PG surface. The PG/CS electrodes were then sequentially immersed into Con A (1 mg mL-1, pH 7.4), HRP (1 mg mL-1, pH 7.4), Con A, and GOD (1 mg mL-1, pH 7.4) solutions for 30 min each with intermediate water rinsing and air stream drying, forming a Con A/HRP/Con A/GOD four-layer LbL film on the PG/CS surface. This cycle was repeated until the desired number of four layers (n) was obtained, designated as {Con A/HRP/Con A/GOD}n. In the assembly, the drying step is usually necessary and makes the LbL films more stable.29 For quartz crystal microbalance (QCM) study, gold-coated quartz crystal resonator electrodes (International Crystal Manufacturing Co.) were first covered by a few drops of a freshly prepared “piranha” solution (3:7 volume ratio of 30% H2O2 and concentrated H2SO4) on each side for 10 min and then washed thoroughly with water and ethanol successively. Caution: the piranha solution should be handled with extreme care, and only a small Volume should be prepared at any time! The QCM gold electrodes were then immersed in 4 mM MPS/ethanol solutions for 24 h to chemisorb an MPS monolayer on the gold surface by formation of a Au-S bond between Au and MPS, introducing negative charges on the surface. The CS precursor layer and following {Con A/HRP/Con A/GOD}n LbL films were then assembled on the Au/MPS surface in the same way as on the PG electrodes. Apparatus and Procedures. A CHI 660A or 621B electrochemical workstation (CH Instruments) was used for electrochemical measurements. A typical three-electrode cell was used with a saturated calomel electrode (SCE) as the reference, a platinum foil as the counter electrode, and a PG electrode with

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films as the working electrode. For most of the electrochemical studies, the oxygen in solution was not removed since O2 is a necessary reactant in oxidation of glucose catalyzed by GOD. For the oxygen-free experiments, the solutions were purged with high-purity nitrogen for at least 10 min, and then the N2 atmosphere was kept in the whole experiments. Electrochemical impedance spectroscopy (EIS) measurements were performed in 1:1 K4Fe(CN)6/K3Fe(CN)6 mixture solutions with a total concentration of 5 mM, and a sinusoidal potential modulation with an amplitude of (5 mV and a frequency from 105 to 0.1 Hz was superimposed on the formal potential of the Fe(CN)63-/4- redox couple at 0.17 V vs SCE. QCM measurements were carried out with a CHI 420 electrochemical analyzer (CH Instruments). The quartz crystal resonator (AT-cut) has a fundamental resonance frequency of 8 MHz and is covered by thin gold films on both sides (geometric area 0.196 cm2 per side). After each adsorption step, the QCM gold electrodes were washed thoroughly in water for about 30 s and dried under a nitrogen stream, and the frequency change was then measured in air with the QCM. Scanning electron microscopy (SEM) was performed using an S-4800 scanning electron microscope (Hitachi) with an acceleration voltage of 3 kV. The CS/{Con A/HRP/Con A/GOD}3 films assembled on the MPS-modified QCM gold electrodes were used as the sample. Before the SEM imaging, the surface of the samples was coated by thin Pt films with an E-1045 sputtering coater (Hitachi). All experiments were performed at an ambient temperature of 20 ( 2 °C. Results and Discussion Assembly of {Con A/HRP/Con A/GOD}n LbL Films. At pH 5.0, CS is water-soluble and carries positive charges due to its pKa at about 6.544 and can be used as the precursor layer to be adsorbed on the negatively charged PG surface.45 Con A carries net negative surface charges at pH 7.4 with its pI at around 5.046 and thus can be adsorbed on the oppositely charged PG/CS surface by electrostatic attraction. Con A possesses a strong biospecific affinity with sugar residues intrinsically located on the surface of HRP;12,14,34,35 the HRP and Con A can thus be sequentially assembled on the PG/CS/Con A surface. One Con A contains four binding sites to the sugar groups with different directions;32-34 the outermost Con A layer of the films thus has remaining binding sites after being combined with the previous HRP layer and can be further combined with glycoenzyme GOD.12,14 Thus, a Con A/HRP/Con A/GOD four-layer film was formed on the PG/CS surface. By repeating this assembly cycle n times, the assembly of {Con A/HRP/Con A/GOD}n LbL films was realized mainly by the biospecific interaction between Con A and glycoenzymes. The assembly of {Con A/HRP/Con A/GOD}n LbL films was also reported previously in the literature.14 The growth of {Con A/HRP/Con A/GOD}n LbL films on the PG/CS surface was first monitored and confirmed by EIS with Fe(CN)63-/4- as the electroactive probe (Figure 1). For bare PG electrodes and PG/CS films, the EIS response of Fe(CN)63-/ 4- in pH 7.0 buffers exhibited a Warburg line in a very wide frequency range (curves a and b), characteristic of a diffusioncontrolled electrochemical process. After the assembly of one Con A/HRP/Con A/GOD four-layer film on the PG/CS surface, a semicircle in the high-frequency domain was observed (curve d) since the multilayer behaved as a physical barrier and blocked or limited the access of the probe to the electrode surface. With an increase of the number of four layers (n) for {Con A/HRP/

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Figure 1. EIS responses of 5 mM Fe(CN)3-/4- at 0.17 V in pH 7.0 buffers for (a) bare PG, (b) a CS precursor layer on PG, (c) CS/Con A/HRP films on PG, and (d-h) {Con A/HRP/Con A/GOD}n films on the PG/CS surface with n ) 1-5.

Figure 2. QCM frequency shift (-∆F) with assembly step for assembly of (a) {Con A/HRP/Con A/GOD}n and (b) {GOD/HRP}n LbL films on the Au/MPS/CS surface: Con A (0), HRP (blue 2), and GOD (red b) adsorption steps.

Con A/GOD}n films, the diameter of the semicircles in the EIS responses became larger (curves e-h), indicating that the interfacial charge transfer of the probe becomes more difficult. The diameter of the semicircle usually equals the charge transfer resistance (Rct) of the probe in electron transfer.47 For the film system, however, Rct mainly reflects the restricted diffusion of the probe through the film phase and relates directly to the accessibility of the underlying electrode or the film permeability.48,49 The Rct value estimated by using the Randles equivalent circuit model50,51 showed a nearly linear relationship with n for {Con A/HRP/Con A/GOD}n films (Supporting Information, Figure S1), indicating a roughly uniform growth of the films in the assembly. CV with Fe(CN)63- as the electroactive probe was also used to monitor the assembly of {Con A/HRP/Con A/GOD}n films on the PG/CS electrode surface. For the bare PG electrode and PG/CS films, Fe(CN)63- in solution at pH 7.0 displayed a welldefined and nearly reversible CV redox peak pair at about 0.17 V (Supporting Information, Figure S2A). However, when {Con A/HRP/Con A/GOD}n multilayers were fabricated on the PG/ CS surface, the CV response of Fe(CN)63- was severely suppressed, accompanied by an increase of the peak separation (∆Ep), indicating that a barrier was formed on the electrode surface and the probe was hindered from reaching the PG electrodes and then exchanging electrons with the underlying electrodes. With an increase of n for the films, the reduction peak current (Ipc) of Fe(CN)63- decreased and the ∆Ep value increased (Figure S2B), suggesting that the bienzyme multilayer films are successfully fabricated on the PG/CS surface. The growth of {Con A/HRP/Con A/GOD}n LbL films was further confirmed by QCM measurements. The QCM resonance frequency decrease (-∆F) usually reflects the mass increase on QCM gold electrodes. The QCM results showed that the -∆F value had a roughly linear relationship with the adsorption step in the assembly of {Con A/HRP/Con A/GOD}n films (Figure 2a), and the average frequency decrease for each layer was 286 ( 97 Hz for Con A, 121 ( 29 Hz for HRP, and 102

Bioelectrocatalysis of Bienzyme LbL Films

Figure 3. (A) Cyclic voltammograms of 1 mM K3Fe(CN)6 at 0.1 V s-1 for {Con A/HRP/Con A/GOD}3 films in buffers at pH (a) 4.0, (b) 5.0, (c) 6.0, (d) 7.0, and (e) 8.0. (B) Dependence of the CV Ipc of K3Fe(CN)6 on the cycle number when the solution pH switched between pH 4.0 (0) and 8.0 (red 9) for the same {Con A/HRP/Con A/GOD}3 films.

( 37 Hz for GOD. The result indicates that the films are successfully assembled and the buildup of the films is in a regular and reproducible manner. It should be emphasized that Con A plays a key role in the assembly of {Con A/HRP/Con A/GOD}n films. Due to its unique biospecific affinity toward glycoenzymes, Con A was used herein as a cross-linker to combine HRP and GOD together and also as a matrix or platform to immobilize the enzymes. Without Con A, it was difficult to assemble HRP and GOD into an LbL film. For example, in a control experiment, we tried to assemble {GOD/HRP}n LbL films on QCM Au/MPS/CS surfaces but failed (Figure 2b). The pI of GOD is 4.2,52 and the pI of HRP is 8.9.53 Thus, in pH 7.4 buffer solutions, GOD carries net negative surface charges and HRP has net positive charges, and they should have been assembled into {GOD/HRP}n LbL films by electrostatic interaction between them. The unsuccessful assembly of the films indicates that the electrostatic interaction between GOD and HRP is too weak to immobilize large amounts of enzymes on the solid surfaces. In contrast, with the help of Con A, the {Con A/HRP/Con A/GOD}n films could immobilize considerable amounts of HRP and GOD (Figure 2a). These results not only demonstrate the central role of Con A in the assembly but also imply that the biospecific affinity between Con A and glycoenzymes is quite strong. pH-Sensitive On-Off Property of Fe(CN)63- at {Con A/HRP/Con A/GOD}3 Film Electrodes. At {Con A/HRP/Con A/GOD}3 film electrodes with n ) 3, the CV response of Fe(CN)63- in solution was very sensitive to the environmental pH (Figure 3A). When the solution pH was set at 4.0, Fe(CN)63displayed a quite reversible CV peak pair with a relatively large Ipc and very small ∆Ep. Both reduction and oxidation peak currents showed a linear relationship with the square root of the scan rates from 0.01 to 2.0 V s-1 (Supporting Information, Figure S3), suggesting the diffusion-controlled behavior of the probe. However, in solution of pH > 5.0, Ipc decreased drastically with pH, accompanied by an increase of ∆Ep (Supporting Information, Figure S4). Particularly, when pH g 8.0, the CV signal of Fe(CN)63- could hardly be observed. This pH-sensitive CV behavior of Fe(CN)63- should be attributed to the property of the films since the CV behavior of Fe(CN)63- at bare PG electrodes is pH-independent. The pH-dependent CV behavior of Fe(CN)63- for {Con A/HRP/Con A/GOD}3 films was used to study the pH-sensitive on-off property of the films. Herein, two typical pH values, 4.0 and 8.0, were selected. In pH 4.0 buffers, Fe(CN)63- showed a large CV response (Figure 3A, curve a) and the films were at the “on” state; at pH 8.0, however, the CV response of the probe was significantly suppressed (Figure 3A, curve e) and the films were at the “off” state. This pH-sensitive on-off property of

J. Phys. Chem. B, Vol. 114, No. 30, 2010 9929 the system was quite reversible. By switching the solution pH between 4.0 and 8.0, the CV Ipc of Fe(CN)63- at the film electrodes cycled between a relatively high value at pH 4.0 and a very small value at pH 8.0, and this on-off behavior could be cycled many times (Figure 3B). EIS was also performed to investigate the pH-responsive on-off behavior of {Con A/HRP/Con A/GOD}3 films toward Fe(CN)3-/4- (Supporting Information, Figure S5A). In pH 4.0 buffers, the Nyquist EIS response of the probe showed a Warburg line with no obvious semicircle in the high-frequency domain, indicating that the charge transfer resistance (Rct) of the system was very small and the films were at the on state. At pH 8.0, however, a large semicircle was obviously observed, reflecting the considerably high Rct value for the system, and the films were at the off state. This pH-sensitive on-off behavior of the EIS response was also reversible and could be repeated for at least several cycles between pH 4.0 and pH 8.0 (Figure S5B), which is consistent with the CV results (Figure 3B). The pH-sensitive on-off property of the {Con A/HRP/Con A/GOD}3 films toward Fe(CN)63- can be explained by the electrostatic interaction between the films and the probe. At pH 4.0, the films would carry net positive charges since the pI values of Con A, HRP, and GOD are at about 5.0,46 8.9,53 and 4.2,52 respectively. The strong electrostatic attraction between the positively charged films and negatively charged Fe(CN)63- in solution would make the probe diffuse through the films very easily, leading to the quite large CV response of the probe. In contrast, at pH 8.0, the films would carry net negative charges and have a strong electrostatic repulsion with the probe. This might block the probe from going through the films and limit the electron exchange of the probe with underlying electrodes, thus resulting in the very small CV signal. To explore the possible change of the film structure with solution pH, the surface morphology of {Con A/HRP/Con A/GOD}3 films was examined by SEM after the films were treated with pH 4.0 and 8.0 solutions (Supporting Information, Figure S6). The SEM results showed the similar surface morphologies and roughnesses of the films at pH 4.0 and 8.0 with the same magnification, indicating that the solution pH has no substantial influence on the film structure at least with the present magnification. Thus, the pH-sensitive behavior of the films toward Fe(CN)3- should be mainly attributed to the electrostatic interaction between the films and the probe. Influencing Factors. First, the stability of {Con A/HRP/Con A/GOD}3 films was examined by CV with Fe(CN)63- as the probe. The films were stored in pH 4.0 blank buffers for most of the storage time and periodically placed in Fe(CN)63solutions at pH 4.0 for CV testing. After three days of storage, the peak potentials maintained the same position, and the peak currents remained nearly the same as their initial values, suggesting that the films are quite stable in pH 4.0 solutions. At pH 4.0, while Con A, HRP, and GOD were all positively charged and would repel one another, the films could not be disintegrated because the main driving force to combine Con A and the glycoenzymes is the lectin-sugar biospecific interaction, which would overcome the electrostatic repulsion between the similarly charged species. This is one of the advantages of {Con A/HRP/Con A/GOD}n films over other electrostatic LbL films, since the net film charge of the former can be easily modulated by the surrounding pH without losing the film stability. The influence of the thickness or the number of four layers (n) of {Con A/HRP/Con A/GOD}n films on the on-off property of the films toward Fe(CN)63- was also investigated by CV.

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Figure 4. Influence of the number of four layers (n) of {Con A/HRP/ Con A/GOD}n films on the CV Ipc of 1 mM K3Fe(CN)6 in buffers at pH 4.0 (O) and 8.0 (b) at 0.1 V s-1.

For {Con A/HRP/Con A/GOD}1 films with n ) 1, while the CV response of Fe(CN)63- at pH 8.0 was smaller than that at pH 4.0, it could still be obviously observed (Supporting Information, Figure S7), suggesting that the relatively thin films could not be completely “closed” at pH 8.0 toward the probe. This is probably due to the relatively small amounts of negative charges for one Con A/HRP/Con A/GOD four layer at pH 8.0, which limits its electrostatic repulsion with Fe(CN)63-. Moreover, the PG/CS surface may not be completely covered by one four layer; some underlying CS layer with positive charges may be exposed and may tend to attract Fe(CN)63-. At pH 4.0, the thickness of the films had little effect on the CV Ipc of the probe (Figure 4), implying that, at this pH, the permeability of the films toward the probe is quite good even when the films become thicker. However, in pH 8.0 solutions, the Ipc value of the probe decreased dramatically with an increase of n from 1 to 2 and then tended to reach zero when n g 2. This should be mainly ascribed to the larger amounts of negative charges in the thicker films at this pH and the corresponding stronger electrostatic repulsion between the films and the probe. In addition, thicker films would completely cover the electrode surface, and the underlying CS layer would no longer have the opportunity to be exposed. Considering that the {Con A/HRP/Con A/GOD}3 films demonstrated the most pronounced difference in Ipc values between pH 4.0 and pH 8.0, the films with n ) 3 were usually used in the present work. Further studies showed that the outermost layer of the bienzyme films had little influence on the pH-dependent CV behavior of Fe(CN)63-. For example, at pH 4.0, Fe(CN)63demonstrated nearly identical cyclic voltammograms for {Con A/HRP/Con A/GOD}3, {Con A/HRP/Con A/GOD}2/Con A/HRP/ Con A, and {Con A/HRP/Con A/GOD}2/Con A/HRP films with quite large peak heights; at pH 8.0, the three types of films were all at the off state toward the probe (Supporting Information, Figure S8). These results suggest that the interpenetration or intermixing of the neighboring layers of the films may happen to a great extent, which is a common phenomenon in LbL assembly.54 pH-Controlled Bioelectrocatalysis for {Con A/HRP/Con A/GOD}3 Films with Fe(CN)63- as the Mediator. The pHresponsive switching property of {Con A/HRP/Con A/GOD}3 multilayer films toward Fe(CN)63- inspired us to use this system to control or modulate the electrocatalytic oxidation of glucose. When glucose was added into the Fe(CN)63- solution at pH 4.0, in comparison with the system in the absence of glucose (Figure 5A, curve a), the CV reduction peak of Fe(CN)63- for the films increased, accompanied by a decrease of the oxidation peak (curve c). The electrocatalytic reduction peak current (Ipc) increased initially with the concentration of glucose in solution in the range of 0.5-16 mM and then tended to level off (Figure 5B). All these are characteristic of oxidation of glucose catalyzed

Yao and Hu

Figure 5. (A) Cyclic voltammograms of {Con A/HRP/Con A/GOD}3 films at 0.01 V s-1 in pH 4.0 buffers containing 1 mM K3Fe(CN)6 with (a) dissolved oxygen, (b) 16 mM glucose in the absence of oxygen, (c) dissolved oxygen and 16 mM glucose, and (d) 0.05 mM H2O2 in the absence of oxygen. (B) Dependence of the CV Ipc on the concentration of glucose in the presence of oxygen for {Con A/HRP/ Con A/GOD}3 films at 0.01 V s-1 in pH 4.0 buffers containing 1 mM K3Fe(CN)6.

SCHEME 1: Schematic Representation of the Mechanism of Electrocatalysis of Glucose Catalyzed by GOD and HRP and Mediated by Fe(CN)63-

by GOD and HRP and mediated by Fe(CN)63- in electrocatalysis, and the mechanism5 is depicted in Scheme 1. To confirm the above mechanism, some control CV experiments at {Con A/HRP/Con A/GOD}3 film electrodes were carried out. When CV was performed in the oxygen-free solution containing glucose and Fe(CN)63- at pH 4.0, no electrocatalytic response was observed (Figure 5A, curve b), suggesting that O2 is absolutely necessary for realizing electrocatalytic oxidation of glucose by the bienzyme films. In the pH 4.0 nitrogensaturated buffers containing Fe(CN)63- but no glucose, the addition of H2O2 caused an obvious increase of the CV reduction peak and a decrease of oxidation peak (Figure 5A, curve d), indicating the electrocatalytic reduction of H2O2. However, for {Con A/GOD}3 films containing no HRP, the addition of H2O2 under the same conditions did not cause any observable electrocatalysis (data not shown). These results confirm that the electrochemical reduction of H2O2 is catalyzed by HRP (but not GOD) and mediated by Fe(CN)63- and it is the H2O2 generated in situ by GOD-catalyzed oxidation of glucose in the presence of O2 that causes the further electrocatalytic reduction by HRP. When {Con A/HRP/Con A/GOD}3 film electrodes were placed in pH 8.0 buffers containing the same amount of Fe(CN)63- and glucose, the electrocatalytic response became quite small or even could hardly be observed (Figure 6A, curve b). This is because the films become off toward Fe(CN)63- at pH 8.0, resulting in the interruption of the catalytic cycles. Therefore, the bioelectrocatalytic reactions could be switched by the different permeability of the films toward the probe at different pH values. Bioelectrocatalysis is at the on state at pH 4.0 and at the off state at pH 8.0. This pH-sensitive switching bioelectrocatalysis was also reversible, and the on-off behavior for the films could be repeated for at least several cycles between pH 4.0 and pH 8.0 (Figure 6B). In addition, the CV reduction peak current ratio, Ipc4/Ipc8, could be amplified by bioelectrocatalysis, where Ipc4 and Ipc8 represent CV reduction peak currents at pH 4.0 and 8.0 for the same {Con A/HRP/Con A/GOD}3 films, respectively. In solutions containing only

Bioelectrocatalysis of Bienzyme LbL Films

Figure 6. (A) Cyclic voltammograms of {Con A/HRP/Con A/GOD}3 films at 0.01 V s-1 in buffers containing 1 mM K3Fe(CN)6 and 16 mM glucose at pH (a) 4.0 and (b) 8.0. (B) Dependence of the CV electrocatalytic reduction peak current (Ipc) on the solution pH switched between 4.0 (0) and 8.0 (red 9) for the same {Con A/HRP/Con A/GOD}3 films.

Figure 7. Amperometric responses of {Con A/HRP/Con A/GOD}3 films at 0.05 V in buffers containing 1 mM K3Fe(CN)6 upon the successive addition of 5 mM glucose at pH (a) 4.0 and (b) 8.0.

Fe(CN)63-, the Ipc4/Ipc8 ratio was about 10, while in solutions containing both Fe(CN)63- and glucose, the ratio increased to about 15. Theoretically, monosaccharide glucose in solution could also combine Con A through the sugar-lectin biospecific interaction32,33 and thus would compete with glycoenzymes in the {Con A/HRP/Con A/GOD}n films and might lead to the disintegration of the films. However, in practice, the Con A-glycoenzyme LbL films possessed excellent stability; only a very high concentration of glucose (over 100 mM) with a long period of contact time could break the binding of Con A and glycoenzymes.34,35,55 It is known that the binding constant between Con A and glycoenzymes is on the order of 105-107 M-1,56,57 while the constant of Con A with glucose is only about 8 × 102 M-1.58,59 In the present work, since the maximum concentration of glucose was less than 20 mM, much smaller than that for causing breaking of the bonding between Con A and glycoenzymes, the {Con A/HRP/Con A/GOD}3 films were quite stable in bioelectrocatalysis. pH-Sensitive Bioelectrocatalysis of Bienzyme Films Studied by Amperometry. The pH-controlled on-off bioelectrocatalysis with {Con A/HRP/Con A/GOD}3 films was further characterized by amperometry. In pH 4.0 solutions containing Fe(CN)63-, with the successive addition of glucose, the stepped increase of the amperometric reduction currents was observed (Figure 7a). The reduction current reached the steady state within about 8 s, which had a linear relationship with the glucose concentration in the range of 5-40 mM (Supporting Information, Figure S9a). For the same films at pH 8.0, however, no increase of the reduction current was observed after the addition of glucose (Figure 7b). This pH-sensitive on-off behavior in electrocatalysis for the films was in good agreement with that observed by CV (Figure 6A). In pH 4.0 buffers containing Fe(CN)63- and glucose, the number of four layers (n) of {Con A/HRP/Con A/GOD}n films had a considerable influence on the amperometric response

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Figure 8. Amperometric responses of (a) {Con A/HRP/Con A/GOD}3, (b) {Con A/HRP}3/{Con A/GOD}3, (c) {Con A/GOD}3/{Con A/HRP}3, and (d) {Con A/HRP}3 films at 0.05 V in pH 4.0 buffers containing 1 mM K3Fe(CN)6 upon the successive addition of 5 mM glucose.

(Supporting Information, Figure S10). With the same concentration of glucose, the amperometric current at the steady state increased with n when n e 3. This is understandable since larger n values would lead to a larger immobilization amount of both enzymes in the films. However, when n ) 4, the response became smaller than that at n ) 3, probably because the thicker films would increase the diffusion resistance of glucose in the film phase. Thus, the {Con A/HRP/Con A/GOD}3 films with n ) 3 were usually used in the present work. The assembly sequence of bienzyme LbL films or the different arrangements of HRP and GOD in the films also showed a considerable influence on the amperometric response of glucose in pH 4.0 solutions containing Fe(CN)63-. Three types of bienzyme films, including {Con A/HRP/Con A/GOD}3, {Con A/HRP}3/{Con A/GOD}3, and {Con A/GOD}3/{Con A/HRP}3 films, were assembled LbL on PG/CS electrode surfaces, and their amperometric responses to addition of glucose in solution were investigated (Figure 8). The results demonstrated that while the amperometric response for {Con A/HRP}3/{Con A/GOD}3 films could also be observed (curve b), it was smaller than that of {Con A/HRP/Con A/GOD}3 films (curve a) under the same concentration of glucose. This implies that the alternate arrangement of HRP and GOD in the films is beneficial to the electrocatalysis of glucose. For the {Con A/HRP/Con A/GOD}3 films, the H2O2 generated in situ by GOD-catalyzed reaction of glucose and oxygen can be effectively reduced by the adjacent HRP layer. For {Con A/HRP}3/{Con A/GOD}3 films, however, the generated H2O2 in the outer GOD layers has to diffuse into the inner HRP layers to react with HRP, resulting in an increase of the diffusion distance and decrease of the reaction efficiency. The third type of films, {Con A/GOD}3/{Con A/HRP}3, showed no amperometric response toward addition of glucose under the same conditions (curve c). This is understandable because the diffusion paths of the substances are too complicated in electrocatalysis with this kind of films, and the movement direction of glucose and the produced H2O2 has to be opposite in realizing the enzymatic reactions. The calibration curves for the three different types of films showed the same tendency (Supporting Information, Figure S11). In a control experiment, {Con A/HRP}3 films containing no GOD showed no amperometric response at all toward glucose (Figure 8d), confirming again that the mechanism of electrocatalysis (Scheme 1) is correct. Comparative Studies with Other Probes. To further understand the interaction between {Con A/HRP/Con A/GOD}3 films and probes, the CV responses of different electroactive probes with different charges at different pH values were compared (Figure 9). These probes can be divided into three groups: (1) negatively charged Fe(CN)63- and Fc(COOH)2; (2) positively charged Ru(NH3)63+ and MB; (3) neutral FcOH and hydroquinone.

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Figure 9. Dependence of the CV Ipc of different probes at 0.1 V s-1 in solutions at different pH values for {Con A/HRP/Con A/GOD}3 films: (a) 1 mM Fe(CN)63-, (b) 0.5 mM Fc(COOH)2, (c) 1 mM Ru(NH3)63+, (d) 1 mM MB, (e) 0.5 mM FcOH, and (f) 1 mM hydroquinone.

For negatively charged Fc(COOH)2,26 its pH-sensitive CV responses at the film electrodes were very similar to those of Fe(CN)63- (Supporting Information, Figure S12). That is, the films were at the on state at pH 4.0 and at the off state at pH 8.0. For positively charged Ru(NH3)63+ and MB, the pHresponsive CV behavior of the films was also observed, but the on-off direction was opposite that of negatively charged probes (Supporting Information, Figures S13 and S14). For example, at pH 8.0, Ru(NH3)63+ displayed quite large CV peaks and the films were at the on state; at pH 4.0, however, the peaks were greatly suppressed and the films were at the off state (Figure S13). For neutral probes such as FcOH and hydroquinone, essentially no difference in CV peak currents was observed at pH 4.0 and 8.0 (Supporting Information, Figures S15 and S16). All these results support the conclusion that it is the electrostatic interaction between the films and probes rather than the structure change that plays a key role in deciding the pH-responsive on-off property. For example, at pH 8.0, if the films were always at the off state due to the structure change, all six probes should have been rejected by the films. In fact, however, the positively charged and neutral probes displayed quite large CV responses at this pH, the films were at the on state toward these probes, and only the negatively charged probes were rejected. The results in Figure 9 also showed that only the charged probes could demonstrate the pH-switchable behavior at the film electrodes but the neutral probes could not, which also supports the above conclusion. Further experiments showed that while the neutral hydroquinone could act as the mediator in bioelectrocatalysis of glucose for the bienzyme films, it could not display pH-sensitive on-off bioelectrocatalysis. For ferrocene derivatives such as FcOH and Fc(COOH)2, while they could be used as the mediator in the electrochemical oxidation of glucose catalyzed by GOD,25,26 they could not mediate the electrocatalytic reduction of H2O2 by HRP and thus could not function as the mediator for the present bienzyme films because the detection of glucose in this system was essentially realized by detecting H2O2 through electrocatalytic reduction with HRP (Scheme 1). Three other pH-sensitive probes, Fe(CN)63-, Ru(NH3)63+, and MB, demonstrated good mediated behaviors in bioelectrocatalysis of glucose for the films under suitable conditions (Figures 5A and 6A and Supporting Information, Figures S17 and S18). Thus, the pHtriggered on-off bioelectrocatalysis of glucose can be realized at the bienzyme LbL film electrodes mediated not only by Fe(CN)63- but also by Ru(NH3)63+ or MB, thus greatly increasing the extent of selecting the probes. Conclusions {Con A/HRP/Con A/GOD}n LbL films are successfully assembled on PG electrodes through biospecific interaction

Yao and Hu between Con A and glycoenzymes. The films exhibit a pHsensitive on-off property toward electroactive probes such as Fe(CN)63- mainly because of the electrostatic interaction between the films and the probes. At pH 4.0, the net charge of the films becomes positive and tends to attract negatively charged Fe(CN)63-, thus resulting in a large CV response. At pH 8.0, the films become negatively charged and would repel the similarly charged probe, leading to a very small CV response. The pH-responsive switching property of the bienzyme films toward Fe(CN)63- can also be used to control or modulate the electrocatalysis of glucose. Herein, Fe(CN)63- not only acts as the electroactive probe but also acts as the mediator in enzymatic electrocatalytic reactions. While pH-switchable electrocatalysis based on one enzyme has been reported in the literature, this work provides the first example of using bienzyme films in pH-sensitive on-off bioelectrocatalysis. This pHresponsive switching behavior of a bienzyme system may provide a general way to develop a new kind of smart interfaces for fabricating pH-controllable electrochemical biosensors. Acknowledgment. Financial support from the National Natural Science Foundation of China (NSFC Grants 20975015 and 20775009) is acknowledged. Supporting Information Available: Eighteen figures showing the dependence of the EIS Rct on n of {Con A/HRP/Con A/GOD}n films, cyclic voltammograms of Fe(CN)63- at {Con A/HRP/Con A/GOD}n film electrodes with different n values, dependence of the CV Ipc and ∆Ep of Fe(CN)63- on n of {Con A/HRP/Con A/GOD}n films, cyclic voltammograms of Fe(CN)63- at {Con A/HRP/Con A/GOD}3 films at different scan rates (V) and the dependence of Ipc and Ipa on V, influence of the solution pH on the CV Ipc and ∆Ep of Fe(CN)63- at {Con A/HRP/Con A/GOD}3 films, EIS responses of {Con A/HRP/ Con A/GOD}3 films at different pH values and dependence of Rct on the solution pH switched between 4.0 and 8.0, SEM top views of {Con A/HRP/Con A/GOD}3 films after treatment at different pH values, cyclic voltammograms of Fe(CN)63- for {Con A/HRP/Con A/GOD}1, {Con A/HRP/Con A/GOD}3, {Con A/HRP/Con A/GOD}2/Con A/HRP/Con A, and {Con A/HRP/Con A/GOD}2/Con A/HRP films in buffers at pH 4.0 and 8.0, dependence of the amperometric currents of {Con A/HRP/Con A/GOD}3 films on the concentration of glucose in pH 4.0 and 8.0 buffers, influence of n of {Con A/HRP/Con A/GOD}n films on the amperometric responses in Fe(CN)63buffers upon the successive addition of glucose, dependence of the amperometric responses on the concentration of glucose for {Con A/HRP/Con A/GOD}3, {Con A/HRP}3/{Con A/GOD}3, and {Con A/GOD}3/{Con A/HRP}3 films in Fe(CN)63- buffers, cyclic voltammograms of Fc(COOH)2, Ru(NH3)63+, MB, Fc(OH), and hydroquinone at {Con A/HRP/Con A/GOD}3 film electrodes in pH 4.0 and 8.0 buffers, cyclic voltammograms of {Con A/HRP/Con A/GOD}3 films in pH 8.0 buffers containing Ru(NH3)63+ or MB in the presence and absence of glucose, and cyclic voltammograms of {Con A/HRP/Con A/GOD}3 films in buffers containing Ru(NH3)63+ or MB in the presence of glucose at pH 4.0 and 8.0. This material is available free of charge via the Internet at http://pubs.acs.org. References and Notes (1) Ronkainen, N. J.; Halsall, H. B.; Heineman, W. R. Chem. Soc. ReV. 2010, 39, 1747. (2) Sarma, A. K.; Vatsyayan, P.; Goswami, P.; Minteer, S. D. Biosens. Bioelectron. 2009, 24, 2313. (3) Wang, J. Chem. ReV. 2008, 108, 814.

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