Dual-Switchable Bioelectrocatalysis Synergistically Controlled by pH

Aug 13, 2010 - Poly(4-vinylpyridine) (P4VP) films were electropolymerized on a pyrolytic graphite (PG) electrode surface. The cyclic voltammetric (CV)...
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J. Phys. Chem. B 2010, 114, 11689–11695

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Dual-Switchable Bioelectrocatalysis Synergistically Controlled by pH and Perchlorate Concentration Based on Poly(4-vinylpyridine) Films Shaoling Song and Naifei Hu* Department of Chemistry, Beijing Normal UniVersity, Beijing 100875, P. R. China ReceiVed: June 23, 2010; ReVised Manuscript ReceiVed: July 29, 2010

Poly(4-vinylpyridine) (P4VP) films were electropolymerized on a pyrolytic graphite (PG) electrode surface. The cyclic voltammetric (CV) response of ferrocenedicarboxylic acid (Fc(COOH)2) at P4VP film electrodes was very sensitive to the pH and perchlorate (ClO4-) concentration in testing solutions. Fc(COOH)2 was at the “on” state with a relatively large CV oxidation peak current for the films at pH 4.0 but showed the “off” state with significantly suppressed CV response at pH 7.0. The reversible ClO4- concentration-sensitive on-off property of P4VP films toward Fc(COOH)2 at pH 4.0 was also observed. In particular, the influence of pH and ClO4- concentration on the on-off behavior of the system is not independent or separate but synergetic or cooperative, and the electrostatic interaction between the films and the probe plays a predominant role in deciding the pH- and/or ClO4- concentration-dependent behavior for the system. The dual-responsive property of the P4VP films toward Fc(COOH)2 could also be used to control the bioelectrocatalysis of glucose by glucose oxidase. This synergetic-triggered bioelectrocatalysis on the basis of the intelligent interface system may establish a foundation for fabricating novel multiple factor-controllable biosensors based on enzymatic electrocatalysis. Introduction Electrochemical biosensors based on enzymes are the most commonly used class of biosensors and have aroused great interest among researchers due to the high specificity and inherent sensitivity of enzymatic reactions.1–4 The catalytic reaction between a specific enzyme and its corresponding substrate can be detected electrochemically either by the direct electron transfer between the enzyme and the electrodes or, in most cases, by the use of an electroactive mediator that shuttles electrons between the enzyme and the electrodes. In recent years, the controllable or switchable bioelectrocatalysis has provided a novel platform for constructing biosensors and may also find its application in triggering biofuel cells and fabricating bioelectronic elements, as well as in signal transduction/amplification and information storage/processing.5–8 The switchable or “signal-triggered” bioelectrocatalytic process based on enzymatic reactions is usually controlled by the activation/deactivation of redox mediator on electrode interface. Typically, the electrode surface is modified by some specific films that show two different surface states: one allows the access of the soluble mediator to electrodes, and the other impedes the access of the mediator. Various external stimuli,9 such as light,10,11 magnetic fields,6,12 pH,13–16 and electric field,17 have been used to realize the tunable bioelectrocatalysis. For example, glucose oxidase (GOD) was chemically modified with the photoisomerizable spiropyran (SP-GOD) by Willner and coworkers, and the SP-GOD was assembled as monolayer onto an Au electrode, which could be alternated in the two photoisomer states by light.10,18 Upon 320 nm < λ < 380 nm, the layer took the SP-GOD state and was permeable to electroactive ferrocenecarboxylic acid (Fc(COOH)). Upon λ > 475 nm, the monolayer took the merocyanine-GOD state and was impermeable to the probe. The bioelectrocatalysis of glucose by GOD * Corresponding author: Professor Naifei Hu. E-mail: [email protected]. Tel.: (+86) 10-5880-5498. Fax: (+86) 10-5880-2075.

with Fc(COOH) as a mediator in solution could thus be reversibly switched between the “on” and the “off” states when the films were irradiated by light of different wavelengths. Lately, our group found that some layer-by-layer (LbL) films exhibited pH-sensitive permeability toward electroactive probes, which could be applied to control bioelectrocatalysis.14–16 For instance, the {PSS/BPEI}n LbL films assembled by poly(styrenesulfonate) (PSS) and branched poly(ethyleneimine) (BPEI) on the electrode surface demonstrated a reversible pH-sensitive on-off property toward ferrocenedicarboxylic acid (Fc(COOH)2), which could be used to switch the electrocatalytic oxidation of glucose by GOD in solution.16 However, up to now, most of the stimuli-responsive bioelectrocatalysis have been limited to the single type of external stimuli, and the development of multiple stimuli-triggered bioelectrocatalysis is still a great challenge. As we know, while the multicontrolled switching behavior of various films toward electroactive probes at electrodes has been reported,19–22 the works on the multiswitchable bioelectrocatalysis have been very limited until now. For example, our group recently reported that, at the electropolymerized poly(N-isopropylacrylamide) film electrodes, the activation/deactivation of bioelectrocatalysis of glucose catalyzed by GOD and mediated by Fc(COOH) could be synergistically controlled by environmental temperature and sulfate concentration in solution.23 In this regard, the further development of multicontrolled bioelectrocatalysis based on enzymatic reactions by simultaneously regulating several external stimuli is still of great interest in both fundamental studies and application researches. The charge situation of poly(4-vinylpyridine) (P4VP) and poly(2-vinylpyridine) (P2VP) is pH-sensitive. The pH-triggered switching behavior of responsive P4VP and P2VP thin films has thus been the subject of a number of publications,24–30 including the pH-switchable bioelectrocatalysis.13 For example, Katz and co-workers reported that the P4VP brush functionalized with Os-complex redox units could be modified onto ITO

10.1021/jp105802m  2010 American Chemical Society Published on Web 08/13/2010

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electrodes, creating a pH-sensitive electrochemical system.13 At pH 4.0, the films were at the swollen state and demonstrated a reversible cyclic voltammetric (CV) response of Os-complex units, while at pH > 6.0, the polymer films became electrochemically inactive because its structure was changed to the shrunken state. This pH-sensitive property of the films was used to tune the bioelectrocatalytic oxidation of glucose catalyzed by GOD in solution and mediated by the Os-complex units in the films through changing external solution pH. It was also reported that the P4VP and P2VP polymers had especially strong interaction with the ClO4- anion.31–33 For example, the CV response of Fe(CN)63- at P2VP film modified electrodes was quite large in pH 3.0 solutions containing Cl-, Br-, NO3-, or SO42- but was significantly suppressed in pH 3.0 solutions containing ClO4-.34 All of these properties of P4VP and P2VP inspired us to use P4VP as the model polymer to modify electrodes and establish a dually switchable bioeletrocatalytic system. To the best of our knowledge, the study of the pH- and ClO4- concentration-sensitive behavior of P4VP films and the corresponding dually controlled bioelectrocatalysis has not been reported until now. In the present work, the P4VP films were synthesized on a pyrolytic graphite (PG) electrode surface by electrochemical polymerization. Herein, the electropolymerization was used because the electropolymerized P4VP or P2VP films had been reported to demonstrate unique advantages over those films prepared by cast or solvent evaporation method.34–36 The CV response of Fc(COOH)2 in solution at the P4VP film electrodes was sensitive to both solution pH and ClO4- concentration. Taking pH-sensitive property as an example, at pH 4.0, the CV oxidation peak current (Ipa) of Fc(COOH)2 was quite large, and the films were at the on state; at pH 7.0, however, the CV Ipa of the probe was greatly suppressed, and the films were at the off state. The pH- and ClO4- concentration-sensitive switching property of the films toward the probe was further employed to dually and synergistically control the electrocatalytic oxidation of glucose by GOD with Fc(COOH)2 as the mediator. The mechanism of the dual-switchable permeability of the films toward the probe was explored by a series of comparative experiments and believed to be mainly attributed to the electrostatic interaction between the films and the probe. The present work provided a novel example to combine the multicontrollable CV behavior of the polymer interface toward the probe with bioelectrocatalysis and established a foundation for constructing the multiswitchable biosensors, which may open a new way to develop the multiple stimuli-responsive biosensors based on enzymatic reactions. Experimental Section 1. Reagents. 4-Vinylpyridine (4VP), glucose oxidase (GOD, E.C. 1.1.3.4, type VII, 192 000 units g-1), 1,1′-ferrocenedicarboxylic acid (Fc(COOH)2), poly(4-vinylpyridine) (P4VP), and ferrocenemethanol (FcOH) were purchased from Sigma Aldrich. Hexaammineruthenium(III) chloride (Ru(NH3)6Cl3), hexaammineruthenium(II) chloride (Ru(NH3)6Cl2), and lithium perchlorate (LiClO4) were purchased from Alfa Aesar. Glucose was purchased from Beijing Yili Fine Chemicals. Sodium perchlorate (NaClO4) was purchased from Beijing Nanshangle Chemical Plant. Potassium ferrocyanide (K4Fe(CN)6), potassium ferricyanide (K3Fe(CN)6), sodium chloride (NaCl), and sodium nitrate (NaNO3) were obtained from Beijing Chemical Engineering Plant. Ammonium perchlorate (NH4ClO4) and sodium persulfate (Na2S2O8) was purchased from Aladdin Reagents. Sodium sulfate (Na2SO4) was purchased from Tianjin Tanggu

Song and Hu Dengzhong Chemical Plant. All other chemicals were of analytical grade. Buffers were 0.05 M citric acid (pH 4.0), 0.1 M sodium acetate (pH 4.5-5.5), or 0.05 M sodium dihydrogen phosphate (pH 6.0-7.0), all containing 0.1 M NaCl. The pH value of buffers was adjusted with dilute HCl or NaOH solutions. Solutions were prepared with water purified twice successively by ion exchange and distillation. 2. Electrochemical Polymerization of P4VP Films. Before being used, basal plane PG (Advanced Ceramics) disks (geometric area 0.16 cm2) were abraded with 320 grit metallographic sandpaper while flushing with water. The PG electrodes were then ultrasonicated in water for 30 s and dried in air. The electrochemical polymerization of P4VP was performed at PG electrodes according to the method reported in the literature34,37 with some modification. In brief, CV scans at 0.05 V s-1 between -0.7 and -2.5 V versus the saturated calomel electrode (SCE) were run at PG electrodes in a nitrogen-saturated 20% methanol/water (v/v) solution containing 0.25 M 4VP, 0.1 M NaClO4, and 0.02 M Na2S2O8 for five cycles. After polymerization, the P4VP modified electrodes were washed thoroughly with water to remove the remaining monomers and dried in air. 3. Apparatus and Procedures. A CHI 660A electrochemical workstation (CH Instruments) was used for all electrochemical measurements. A traditional three-electrode cell system was used with the SCE as the reference, a platinum wire as the counter, and the PG electrode with P4VP films as the working electrode. Before electrochemical measurements, the buffers containing electroactive probes in the cell were purged with high-purity nitrogen for at least 15 min, and a nitrogen atmosphere was then maintained during the whole experiment. Electrochemical impedance spectroscopy (EIS) was performed in 5 mM Fe(CN)64-/3- or 0.2 mM Ru(NH3)62+/3+ solutions, and a sinusoidal potential modulation with an amplitude of 5 mV and a frequency from 105 to 10-2 Hz was superimposed on the formal potential of the redox couple Fe(CN)64-/3- at 0.17 V or Ru(NH3)62+/3+ at -0.25 V. Fourier transform infrared (FT-IR) spectra were obtained with a Bruker IFS 66v/s spectrophotometer with a resolution of 4 cm-1. The spectra of 4VP monomer and commercially obtained P4VP were recorded with the transmission mode. The spectrum of P4VP films electropolymerized on gold-coated glass wafers was recorded with the reflection mode. Scanning electron microscopy (SEM) was performed using an S-4800 scanning electron microscope (Hitachi) with an acceleration voltage of 3 kV. Before the SEM imaging, the surface of samples was coated by thin platinum films with an E-1045 ion sputter (Hitachi). Results and Discussion 1. Electrochemical Polymerization of P4VP Films. In the present work, P4VP films were electropolymerized on the PG electrode surface by CV with the method reported by the literature34,37 with some modification. The formation of P4VP films was confirmed by FT-IR (Supporting Information (SI), Figure S1). The peaks at 1597, 1557, and 1414 cm-1 are attributed to the stretching vibration of pyridine ring, and the peak at 3023 cm-1 is from the stretching vibration of CH groups in pyridine group.38–40 All of these peaks were observed in 4VP monomer (Figure S1A), commercially obtained P4VP sample (Figure S1B), and electropolymerized P4VP films (Figure S1C) due to their common pyridine groups. The peaks for CH2dCH out-of-plane bending at 1857 cm-1 and stretching at 3069 and 2987 cm-141 were observed for 4VP monomer but not for the

Dual-Switchable Bioelectrocatalysis

Figure 1. CVs of 0.2 mM Fc(COOH)2 at 0.1 V s-1 for P4VP films in buffers at pH (a) 4.0, (b) 4.5, (c) 5.0, (d) 6.0, and (e) 7.0. (B) Influence of solution pH on CV Ipa of 0.2 mM Fc(COOH)2 at 0.1 V s-1 for P4VP films. Each data point represents the average of three parallel measurements.

two P4VP samples. In the meantime, the peaks at 2853 and 2926 cm-1 were detected for both P4VP samples but not for 4VP monomer since these peaks are attributed to the symmetric and asymmetric stretching of CH2 group of the main polymer chain.42,43 The electropolymerized P4VP films demonstrated almost the same IR characteristic peaks as the commercially obtained P4VP sample. These results verify that the P4VP films are successfully electropolymerized on the PG electrode surface. 2. pH-Sensitive On-Off Property of P4VP Films toward Fc(COOH)2. The CV response of Fc(COOH)2 in buffers at P4VP film electrodes was very sensitive to the solution pH (Figure 1A). At pH 4.0, Fc(COOH)2 showed a relatively large CV oxidation peak at about 0.58 V. While at pH g 6.0, the CV response was significantly suppressed and even could hardly be observed. The oxidation peak current (Ipa) experienced a dramatic decrease from pH 4.0 to 7.0 (Figure 1B). This pHsensitive CV behavior of Fc(COOH)2 should not be attributed to the property of the probe itself since the CV behavior of Fc(COOH)2 at bare PG electrodes was pH-independent.14,16 The Ipa of Fc(COOH)2 at P4VP film electrodes increased linearly with the square root of scan rate in the range of 0.01-0.50 V s-1 in pH 4.0 buffers (SI, Figure S2), suggesting that the electrode reaction of Fc(COOH)2 is a diffusion-controlled process. As a matter of fact, at various pH in buffers, if the CV response of Fc(COOH)2 could be detected, the probe always showed the diffusion-controlled behavior at the film electrodes. The pH-dependent CV behavior of Fc(COOH)2 for P4VP films could be used to study the on-off switching property of the films, and two typical pH values, pH 4.0 and 7.0, were selected in the present study according to Figure 1. In pH 4.0 buffers, the probe showed a relatively large CV oxidation peak for the films, and the films were at the “on” state. In pH 7.0 solutions, however, the CV response of the probe almost disappeared, and the films were at the “off” state. This pHsensitive “on-off” behavior of the films toward Fc(COOH)2 was quite reversible. By switching the film electrode in buffers between pH 4.0 and 7.0, the CV Ipa changed periodically between a relatively high value at pH 4.0 and a very low value at pH 7.0 (Figure 2). The long-term stability of the P4VP films electropolymerized on the PG electrode surface was also examined by CV with Fc(COOH)2 as the probe. The films were stored in pH 4.0 buffers containing Fc(COOH)2 for most of the storage time, and CV was performed periodically. During the 15 days of storage, the CV Ipa was kept similar to their initial value, and the pH-dependent on-off behavior of the system was maintained and clearly observed (SI, Figure S3). All of these results indicate that the P4VP films are quite stable and can be amenable to different pH environments and repeated CV scans. In contrast, the cast P4VP films made from commercially obtained reagent

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Figure 2. Dependence of CV Ipa of 0.2 mM Fc(COOH)2 at 0.1 V s-1 on solution pH switched between pH (O) 4.0 and (9) 7.0 for the same P4VP films.

Figure 3. (A) Influence of NaClO4 concentration on CV Ipa of 0.2 mM Fc(COOH)2 at 0.1 V s-1 for P4VP films in pH 4.0 buffers. Each data point represents the average of three parallel measurements. (B) CVs of 0.2 mM Fc(COOH)2 at 0.1 V s-1 for P4VP films in pH 4.0 buffers containing (a) 0 and (b) 0.25 M NaClO4.

on PG electrodes were very unstable. After only a few cycles of CV scans in solution, the peeling-off of the films was clearly observed even by naked eyes, and the following CV response of Fc(COOH)2 at the cast P4VP film electrodes became similar to that at bare PG electrodes, demonstrating that the films were quickly removed from the PG surface in solution. The distinguished stability of the electropolymerized P4VP films, particularly in comparison with the cast P4VP films, appears to be linked to the fact that the electrochemical polymerization can lead to a high degree of cross-linking and branching in the films, especially in the presence of ClO4-.32,34,36 Fc(COOH)2 is believed to be partially ionized at pH 4.0 and completely ionized at pH 7.0 in its aqueous solutions.16,44–46 Thus, the probe is always negatively charged when the solution pH is higher than 4.0. With the pKa at about 4.5,47,48 P4VP is positively charged at pH 4.0, and most of the pyridine groups in P4VP are protonated. The electrostatic attraction between the oppositely charged films and probe at this pH may thus make the probe enter the films more easily and show a larger CV response. However, at pH 7.0, all pyridine groups in P4VP are unprotonated carrying no charge, and the films no longer show electrostatic attraction to the probe. In addition, the electronegative nitrogen atoms of pyridine rings in P4VP may even repel the negatively charged probe. This leads to the difficulty of the probe entering the films and the very small CV response at pH 7.0. 3. ClO4- Concentration-Sensitive On-Off Property of P4VP Films toward Fc(COOH)2. The CV response of Fc(COOH)2 at P4VP film electrodes was also sensitive to the concentration of NaClO4 in testing solution (Figure 3A). At pH 4.0, the CV Ipa decreased drastically with NaClO4 concentration from 0 to 0.20 M. When the NaClO4 concentration was larger than 0.20 M, the CV response of the probe became very small. The effect of scan rate on the CV Ipa of Fc(COOH)2 at P4VP film electrodes in the presence of NaClO4 was investigated. The results showed that with a variety of NaClO4 concentration, if the CV response of Fc(COOH)2 could be detected, the probe always displayed the diffusion-controlled behavior.

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Figure 4. Influence of concentration of (a) NaClO4, (b) LiClO4, (c) NH4ClO4, (d) Na2SO4, (e) NaCl, and (f) NaNO3 on CV Ipa of 0.2 mM Fc(COOH)2 at 0.1 V s-1 for P4VP films at pH 4.0. Each data point represents the average of three parallel measurements.

The NaClO4 concentration-dependent CV behavior of Fc(COOH)2 for P4VP films could also be used to study the on-off switching property of the films. In the testing solution containing no NaClO4 at pH 4.0, the probe showed a quite large CV oxidation peak for the films (Figure 3B, curve a), and the films were at the on state. While in the solution containing 0.25 M NaClO4 at the same pH, the CV response was significantly suppressed and even could hardly be observed (curve b) with the films at the off state. This on-off behavior of the films toward Fc(COOH)2 was quite reversible (SI, Figure S4). In control experiments with bare PG electrodes, the CV response of Fc(COOH)2 demonstrated no substantial difference in pH 4.0 buffers containing 0 and 0.25 M NaClO4, indicating that the NaClO4 concentration-sensitive CV behavior of Fc(COOH)2 at P4VP film electrodes has nothing to do with the property of the probe itself but should be related to the P4VP films. To further understand the NaClO4 concentration-sensitive switching function of P4VP films toward Fc(COOH)2, the effect of other salts with different concentrations on the CV response of the probe for the films was also investigated at pH 4.0 (Figure 4). For NaClO4, LiClO4, and NH4ClO4, which have the same ClO4- anion but different cations, the CV Ipa of the probe at P4VP film electrodes demonstrated the similar decreasing trend with the salt concentration (curves a-c). These results imply that, for perchlorate salts, it is the anion (ClO4-) rather than the cation that governs the concentration-sensitive on-off behavior of the system. However, for Na2SO4, NaNO3, and NaCl, which have the same Na+ cation but different anions with ClO4-, the CV Ipa essentially did not change with the salt concentration at least in the range of 0-0.40 M (curves d-f), suggesting that it is ClO4- rather than other anions that possesses the unique strong interaction with P4VP films and demonstrates the concentration-dependent property for the system. It is reported that the ClO4- anion can combine P4VP or its analogues with especially strong interaction, while the interaction of other anions with P4VP is much weaker32–34 because ClO4- is a well-known chaotrope in the Hofmeister series,49,50 which does not form hydrogen bonds with water molecules and can cause an extensive breakdown of the hydrogen-bonded water structure.51 When the positively charged P4VP films are placed in ClO4- solution, the electroneutrality leads the entrance of ClO4- into the polymer films, and water is squeezed out of the polymer phase; the films would become dehydrated and compact.52,53 In the present work, the addition of ClO4- in solution may lead to the strong combination of ClO4- with positively charged pyridine groups of P4VP films at pH 4.0, which would repel the negatively charged Fc(COOH)2 from entering the films and result in the decrease of CV response of the probe. The higher the concentration of ClO4- in solution is, the more ClO4- anions will come into the P4VP films to

Song and Hu combine the pyridine groups of P4VP, and the more difficult it will be for Fc(COOH)2 to enter into the films, thus leading to the smaller CV response. 4. Comparative Studies with Other Electroactive Probes. P4VP brush systems are well-known for their switchable properties upon the change of environmental pH.24–30 The P4VP brushes tethered to solid supports demonstrate structure change from the swollen or coil state to the shrunk or globule state upon charging/discharging their pyridine groups due to the decrease/increase of surrounding pH. However, this pH-sensitive restructuring seems impossible for the present P4VP film system since the present P4VP films are formed by electropolymerization, which have a high degree of cross-linking and branching32–34 and cannot form the brush structure. The pH-sensitive on-off electrochemical property of the P4VP films toward Fc(COOH)2 should thus be mainly ascribed to the electrostatic interaction between the films and the probe. To further support this speculation, other electroactive probes with different charges were examined by CV and EIS for the P4VP films at different pH. The negatively charged Fe(CN)64- demonstrated a very large CV response centered at about +0.18 V at pH 4.0 for the P4VP films, but the signal could even hardly be observed at pH 7.0 (SI, Figure S5A). This pH-triggered on-off behavior of Fe(CN)64- for the films is qualitatively in good agreement with that of Fc(COOH)2 because both Fe(CN)64- and Fc(COOH)2 are negatively charged. However, for positively charged Ru(NH3)63+, its pH-sensitive switching property was completely opposite to that of Fc(COOH)2. At pH 7.0, Ru(NH3)63+ demonstrated a pair of CV quasi-reversible peaks at about -0.25 V with quite large peak heights, but this peak pair was greatly suppressed at pH 4.0 (Figure S5B). Since the sizes of Fe(CN)64and Ru(NH3)63+ are similar to each other,54 the different pHsensitive switching behaviors of the films toward these two probes should be attributed to the different types of charges that they carry but not the pore size of the films. Thus, the opposite direction of pH-sensitive on-off behavior of Fe(CN)64and Ru(NH3)63+ for P4VP films verifies again that it is the electrostatic interaction between the films and the probes rather than the structure change of the films that governs the pHswitchable property of the system. For neutral probe FcOH, a large CV oxidation peak with similar heights was observed at both pH 4.0 and 7.0, but the CV reduction peak was greatly suppressed at pH 4.0 in comparison with that at pH 7.0 (Figure S5C). This is the typical characteristic of an electrochemical rectifier.55 When the pyridine groups of P4VP films are unprotonated at pH 7.0, access of both FcOH and FcOH+ to the electrodes is allowed, resulting in the bidirectional electron transfer. At pH 4.0, however, the protonated pyridine groups of P4VP repulse the positively charged FcOH+ but have no influence on the neutral FcOH, thus leading to the unidirectional electron transfer. This “rectification” effect may also be used to explain the asymmetric CV oxidation-reduction peak pair of Fc(COOH)2 at pH 4.0 (Figure 1A). The different pHdependent on-off properties of the different probes at P4VP film electrodes are summarized in Figure 5. All of these results suggest that it is the electrostatic interaction between P4VP films and probes that controls the pH-sensitive CV on-off property of the system. EIS was also performed to investigate the pH-responsive property of P4VP films with Fe(CN)64-/3- or Ru(NH3)62+/3+ as the redox couples (SI, Figure S6). The results are consistent with those of CV with Fe(CN)64- and Ru(NH3)63+ as the probes, confirming again that the electrostatic interaction between P4VP

Dual-Switchable Bioelectrocatalysis

Figure 5. CV Ipa of (A) 0.2 mM Fc(COOH)2, (B) 0.5 mM Fe(CN)64-, (C) Ipc of 0.2 mM Ru(NH3)63+, and (D) Ipa of 0.5 mM FcOH at 0.1 V s-1 in pH 4.0 (dense) and 7.0 (blank) buffers for P4VP films.

Figure 6. CV Ipa of (A) 0.2 mM Fc(COOH)2, (B) 0.5 mM Fe(CN)64-, (C) Ipc of 0.2 mM Ru(NH3)63+, and (D) Ipa of 0.5 mM FcOH at 0.1 V s-1 in pH 4.0 buffers containing 0 (dense) and 0.25 M (blank) ClO4for P4VP films.

films and probes plays a key role in the pH-sensitive on-off property of the system. The ClO4- concentration-dependent behavior of P4VP films toward different probes was also examined. Toward negatively charged Fe(CN)64-, the P4VP films demonstrated the on state in pH 4.0 buffers containing no ClO4- and the off state in pH 4.0 buffers containing 0.25 M ClO4- (SI, Figure S7A), in good agreement with the ClO4- concentration-sensitive behavior of Fc(COOH)2. However, for positively charged Ru(NH3)63+, its ClO4- concentration-sensitive property was opposite to that of Fc(COOH)2. Ru(NH3)63+ showed a very small CV response at pH 4.0 in the absence of ClO4- but a quite large signal in the presence of ClO4- (Figure S7B). This is understandable since ClO4- can strongly combine pyridine groups of P4VP, which not only compensates the positive charges of P4VP at pH 4.0 but also increases the negative charges of the films, leading to the electrostatic attraction toward positively charged Ru(NH3)63+. As expected, for neutral probe FcOH, its CV Ipa was ClO4concentration-independent (Figure S7C). The ClO4- concentration-dependent on-off properties of the different probes at P4VP film electrodes are summarized in Figure 6. All of these results suggest that it is mainly the electrostatic interaction between P4VP films and probes that governs the ClO4- concentrationsensitive CV on-off property of the system. This conclusion is also supported by EIS results with Fe(CN)64-/3- or Ru(NH3)62+/3+ as the probes (SI, Figure S8). To further investigate the interaction between P4VP films and probes, CVs of Ru(NH3)63+ were performed at the film electrodes in pH 7.0 buffers containing 0 and 0.25 M ClO4-, respectively (SI, Figure S9). No substantial difference in CV response of Ru(NH3)63+ in the presence and absence of ClO4was observed. This is most probably because the pyridine groups of P4VP films are unprotonated and carry no charge at pH 7.0 and thus cannot effectively bind the negatively charged ClO4-. The influence of the ClO4- concentration on the CV response of Ru(NH3)63+ for the films at this pH would therefore be insignificant. These results imply that, while the unique strong binding between ClO4- and pyridine groups of P4VP is not solely electrostatic interaction, as shown in Figure 4, the

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Figure 7. (A) CV Ipa of 0.2 mM Fc(COOH)2 at 0.1 V s-1 with different pH and ClO4- concentrations for P4VP films. (B) CV catalytic oxidation wave current (Ipa) at 0.005 V s-1 in solutions containing 0.2 mM Fc(COOH)2, 1.0 mg mL-1 GOD, and 20.0 mM glucose at different pH and ClO4- concentrations for P4VP films.

electrostatic attraction between oppositely charged ClO4- and protonated pyridine groups of P4VP is the prerequisite of their further and stronger combination. Therefore, the electrostatic interaction must also play a key role in the unique interaction between ClO4- and P4VP. The surface morphology of dry P4VP films was compared by SEM after the films were immersed in buffers at different pH values and ClO4- concentrations (SI, Figure S10). While some topographic difference was observed for the films under different conditions, no substantial structure change was seen. For example, the P4VP films at pH 4.0 did not show better porosity than that at pH 7.0 with the same magnification. 5. On-Off Property of P4VP Films Synergistically Controlled by pH and ClO4- Concentration. The essence of pHsensitive on-off property of P4VP films toward Fc(COOH)2 is electrostatic interaction between the films and the probe. The electrostatic interaction also plays a central role in ClO4concentration-sensitive switching behavior of the system. Thus, the influence of pH and the effect of ClO4- concentration on the on-off CV property of Fc(COOH)2 at P4VP film electrodes can find their common ground and inherent relationship in electrostatic interaction. On the one hand, the decrease of pH would lead to the increase of positive charges in P4VP films due to the increase of protonation degree of pyridine groups, which is beneficial to the electrostatic attraction toward negatively charged Fc(COOH)2. On the other hand, the increase of ClO4- concentration would result in the increase of amounts of ClO4- strongly bound with protonated pyridine groups in P4VP films, which is helpful to the electrostatic repulsion with the negatively charged probe. It is therefore understandable that the influences of pH and ClO4- concentration are not independent or separate but synergetic or cooperative. The on-off state of the system not only depends on the pH but also relies on the ClO4- concentration in solution. This is clearly demonstrated in the three-dimensional diagram (Figure 7A). For example, at pH 4.0, the system was either at the on state or at the off state, depending on the concentration of ClO4-, while with 0.05 M ClO4- in solution, the system showed either the on or off state, depending on the solution pH. 6. Switchable Bioelectrocatalysis Dually Controlled by pH and ClO4- Concentration. The pH- and ClO4- concentrationsensitive switching property of P4VP films toward Fc(COOH)2 could be used to control or modulate the bioelectrocatalytic oxidation of glucose by GOD enzyme. For example, when the P4VP film electrode was placed in the solution containing Fc(COOH)2, GOD and glucose without ClO4- at pH 4.0, the CV oxidation peak increased dramatically, accompanied by the decrease or even disappearance of the reduction peak (SI, Figure S11A). The electrocatalytic wave current (Ipa) increased with the concentration of glucose and then tended to level off (Figure S10B). All of these results are characteristic of electrochemical

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Song and Hu Conclusions

Figure 8. (A) CVs of P4VP films at 0.005 V s-1 in solutions containing 0.2 mM Fc(COOH)2, 20 mM glucose, and 1.0 mg mL-1 GOD (A) at pH (a) 4.0 and (b) 7.0 (inset: CV Ipa measured at 0.7 V on solution pH switched between pH 4.0 and 7.0, and (B) in pH 4.0 solutions with ClO4- concentration of (a) 0 and (b) 0.25 M (inset: CV Ipa measured at 0.7 V on ClO4- concentration switched between 0 and 0.25 M).

oxidation of glucose catalyzed by GOD and mediated by Fc(COOH)2.56–58 However, when P4VP films were placed in pH 7.0 buffers containing the same amount of Fc(COOH)2, GOD, and glucose, the electrocatalytic response became quite small (Figure 8A, curve b), especially in comparison with that at pH 4.0 (curve a). This is because the films become “closed” toward the probe at pH 7.0, resulting in the interruption of the catalytic cycles. Therefore, the bioelectrocatalysis of glucose by GOD could be controlled by the different permeability of P4VP films toward Fc(COOH)2 at different pH. The electrocatalysis was “open” at pH 4.0 and “close” at pH 7.0. In addition, the pH-sensitive on-off bioelectrocatalysis for the P4VP films could be repeated for at least several cycles by switching the same films in the Fc(COOH)2 + GOD + glucose solutions between pH 4.0 and 7.0 (Figure 8A, inset). The bioelectrocatalysis of glucose in the presence of GOD and Fc(COOH)2 for the P4VP films could also be controlled by ClO4- concentration (Figure 8B). In pH 4.0 solutions containing Fc(COOH)2, GOD, and glucose in the absence of ClO4-, the films demonstrated a large catalytic wave. However, in the presence of 0.25 M ClO4-, the CV response was greatly suppressed. This ClO4- concentration-dependent on-off bioelectrocatalysis for the P4VP films was also reversible. By switching the same films in the Fc(COOH)2 + GOD + glucose solution system at pH 4.0 between 0 and 0.25 M ClO4-, the Ipa cycled between a relatively large value and a very small one (Figure 8B, inset). In control experiments with bare PG electrodes, the addition of ClO4- only caused a very small decrease of the bioelectrocatalytic response of glucose in the presence of Fc(COOH)2 and GOD (SI, Figure S12), indicating that the ClO4- concentration-trigged on-off behavior of bioelectrocatalysis originates from the property of P4VP films toward the probe rather than the bioelectrocatalytic reaction itself. In particular, the synergetic effect of pH and ClO4- concentration on the on-off bioelectrocatalysis for the P4VP films in the Fc(COOH)2 + GOD + glucose solution system was investigated (Figure 7B). The results confirm that the pH and ClO4- concentration do not influence the on-off bioelectrocatalysis of the system independently or separately; in contrast, they affect the on-off bioelectrocatalytic behavior synergistically or cooperatively. Since the on-off bioelectrocatalysis of the system (Figure 7B) originates from the on-off CV behavior of the P4VP films toward Fc(COOH)2 (Figure 7A), their changing tendencies with pH and ClO4- concentration are very similar.

P4VP films prepared on the PG electrode surface by electropolymerization demonstrate on-off permeability toward Fc(COOH)2, dependent not only on the surrounding pH but also on the ClO4- concentration in solution. This switching behavior of the system is believed to be mainly attributed to the electrostatic interaction between the films and the probe under different conditions. Meanwhile, the specific and strong interaction between ClO4- and protonated pyridine groups of P4VP films also plays an important role. Since there is a common ground in the mechanism, the influence of pH and ClO4concentration on the on-off property of the system is not independent or separate but synergetic or cooperative. The different permeability of the P4VP films toward Fc(COOH)2 at different pH values and/or ClO4- concentrations can be used to modulate the electrocatalytic oxidation of glucose by the GOD enzyme. Herein, Fc(COOH)2 not only acts as the electroactive probe in testing the permeability of P4VP films but also acts as the electron transfer mediator in bioelectrocatalysis. Perhaps the most exciting point in the present work is the realization of dual-switchable bioelectrocatalysis synergistically controlled by pH and ClO4- concentration. The integration or combination of dual signal-triggered interface and bioelectrocatalysis may meet the requirement of developing novel type of multicontrollable electrochemical biosensors and may potentially be applied in the future as the foundation for biological information storage and transduction and for development of switchable biofuel cells, as well as for other bioelectronic devices based on enzymatic electrocatalysis. Acknowledgment. The financial support from the National Natural Science Foundation of China (NSFC 20975015 and 20775009) is acknowledged. Supporting Information Available: Twelve figures showing FT-IR of 4VP monomer, P4VP for commercial reagent and electropolymerized films, CVs of Fc(COOH)2 at different scan rates and dependence of CV Ipa on the square root of scan rates for P4VP films, CVs of Fc(COOH)2 at P4VP film electrodes with different storage time, dependence of CV Ipa on NaClO4 concentration switched between 0 and 0.25 M for P4VP films, CVs of Fe(CN)64-, Ru(NH3)63+, and FcOH for P4VP films in buffers at pH 4.0 and 7.0, EIS of Fe(CN)64-/3- and Ru(NH3)62+/3+ for P4VP films in buffers at pH 4.0 and 7.0, CVs of Fe(CN)64-, Ru(NH3)63+, and FcOH for P4VP films in buffers at pH 4.0 containing 0 and 0.25 M ClO4-, EIS of Fe(CN)64-/3- and Ru(NH3)62+/3+ for P4VP films in buffers at pH 4.0 containing 0 and 0.25 M ClO4-, CVs of Ru(NH3)63+ for P4VP films in pH 7.0 buffers containing 0 and 0.25 M NaClO4, SEM top views of P4VP films treated with different pHs and ClO4- concentrations, CVs in pH 4.0 buffers containing Fc(COOH)2, GOD, and different concentrations of glucose, dependence of Ipa on concentration of glucose for P4VP films, and CVs at bare PG electrode in pH 4.0 solutions containing Fc(COOH)2, GOD, and glucose with 0 and 0.25 M ClO4-. This material is available free of charge via the Internet at http://pubs.acs.org. References and Notes (1) Scheller, F. W.; Wollenberger, U.; Warsinke, A.; Lisdat, F. Curr. Opin. Biotechnol. 2001, 12, 35. (2) Chaubey, A.; Malhotra, B. D. Biosens. Bioelectron. 2002, 17, 441. (3) Murphy, L. Curr. Opin. Chem. Biol. 2006, 10, 177. (4) Wilson, G. S.; Hu, Y. Chem. ReV. 2000, 100, 2693. (5) Loaiza, O. A.; Laocharoensuk, R.; Burdick, J.; Rodriguez, M. C.; Pingarron, J. M.; Pedrero, M.; Wang, J. Angew. Chem., Int. Ed. 2007, 119, 1530.

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