“On−Off” Switchable Bioelectrocatalysis Synergistically Controlled by

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“On-Off” Switchable Bioelectrocatalysis Synergistically Controlled by Temperature and Sodium Sulfate Concentration Based on Poly(N-isopropylacrylamide) Films Shaoling Song and Naifei Hu* Department of Chemistry, Beijing Normal UniVersity, Beijing 100875, P. R. China ReceiVed: February 1, 2010; ReVised Manuscript ReceiVed: March 26, 2010

In this work, poly(N-isopropylacrylamide) (PNIPAm) films were synthesized on a Au electrode surface through the electrochemically induced free-radical polymerization method. The “coil-to-globule” phase transition of PNIPAm films was sensitive to both environmental temperature and sodium sulfate (Na2SO4) concentration in solution and was detected by cyclic voltammetry (CV) of ferrocenecarboxylic acid (Fc(COOH)) probe. For example, in solutions containing no Na2SO4 at 25 °C, the probe demonstrated a well-defined CV peak pair with large peak currents, showing the “on” state; at 35 °C, the CV response was significantly suppressed, showing the “off” state. By switching the film electrodes in solution between 25 and 35 °C, the CV peak currents cycled between the on and off states, demonstrating the reversible thermosensitive switching function of the films. Similarly, the reversible Na2SO4-concentration-sensitive “on-off” property of PNIPAm films toward Fc(COOH) was also observed. In particular, the influence of temperature and Na2SO4 concentration on the on-off behavior of the films was not independent or separate, but synergetic or cooperative. The dual-responsive property of the films could also be used to switch the on-off bioelectrocatalysis. That is, the electrochemical oxidation of glucose catalyzed by glucose oxidase (GOx) and mediated by Fc(COOH) in solution could be controlled or modulated by changing the surrounding temperature, Na2SO4 concentration, or both. This dual- and synergetic-triggered bioelectrocatalysis based on the “smart” PNIPAm interface system may establish a foundation for fabricating novel multiple factor-controllable biosensors. Introduction Electrochemical biosensors based on enzymatic reactions are one of the most commonly used biosensors and have been extensively studied among researchers. The foundation of this type of biosensors is the bioelectrocatalysis based on the direct or mediated electrochemistry of various enzymes. The high recognition capability of enzymes toward specific substrates and the inherent signal amplification of bioelectrocatalysis make this kind of biosensor very useful in analytical application and thus develop very rapidly.1–5 In recent years, the controllable or “signal-triggered” bioelectrocatalysis has aroused great interest and demonstrated great significance not only in the development of “stimuli-responsive” biosensors, but also in the development of biofuel cells, bioelectronic elements, signal transduction and amplification, information storage and processing, and so on.6–9 Various external stimuli,10 such as light,11,12 magnetic fields,7,13 pH,14 and electric fields,15 have been used to activate/deactivate bioelectrocatalytic reactivity of redox enzymes toward different substrates. For example, Willner and co-workers reported that by alternate positioning of magnets below and above the electrochemical cell, the ferrocene-modified magnetic particles were attracted onto and removed from the electrode surface, respectively, resulting in the activation and deactivation of bioelectrocatalysis of glucose by glucose oxidase (GOx) in solution.16 Recently, our group found that some layer-by-layer (LbL) films exhibited pH-sensitive permeability toward electroactive probes, which could be applied to control bioelectrocatalysis.17–19 For instance, {Con A/Dex}n LbL films assembled by concanavalin A (Con A) and dextran (Dex) on an electrode surface demonstrated a reversible pH-sensitive “on-off” prop* Corresponding author. Phone: (+86) 10-5880-5498. Fax: (+86) 105880-2075. E-mail: [email protected].

erty toward Fe(CN)63- that could be used to switch the electrocatalytic reduction of H2O2 by horseradish peroxidase in solution.17 However, until now, most of the stimuli applied in the switchable bioelectrocatalysis have been limited to light, magnetic fields, environmental pH, and electric fields.10 To the best of our knowledge, no study on the temperature- or salttriggered on-off bioelectrocatalysis has been reported. In particular, the realization of multicontrolled bioelectrocatalysis based on enzymatic reactions by simultaneously regulating several external stimuli is still a great challenge and is also of great interest in both fundamental and application research. Recently, different kinds of thermoresponsive polymers that change their physicochemical properties in response to environmental temperature have aroused considerable attention.20–22 Among these, poly(N-isopropylacrylamide) (PNIPAm) is one of the most studied polymers and is often employed to construct the “smart” and thermotriggered interface because of its unique properties.23–25 PNIPAm exhibits a fully reversible phase transition in water with its phase transition temperature or lower critical solution temperature (LCST) at around 32 °C.26–28 Below the LCST, the polymer takes an expanded coil conformation, mainly due to the hydrogen bonding between its amide groups and water molecules, making the polymer water-soluble or hydrophilic. Above the LCST, the polymer takes a collapsed globule structure, since the hydrogen bonds between PNIPAm and water molecules are broken, making the polymer precipitate after the water molecules are squeezed out from the polymer.26,28 Various films containing PNIPAm have been used to modify an electrode surface, and the thermosensitive on-off behavior of the films toward electroactive probes has been reported.29–33 For example, PNIPAm films fabricated on a Au electrode surface by electropolymerization in aqueous solutions showed

10.1021/jp1009753  2010 American Chemical Society Published on Web 04/09/2010

“On-Off” Switchable Bioelectrocatalysis a reversible temperature-sensitive on-off property toward Fe(CN)63-.30 In addition, numerous studies have evidenced that the LCST of PNIPAm is strongly dependent on both the type and concentration of salts added in the aqueous solutions. The addition of salts often results in a decrease in LCST of PNIPAm.34,35 Thus, the conformation of PNIPAm is not only thermosensitive but also salt-sensitive, showing a critical phase transition concentration for a specific ion at a certain temperature. When the concentration of the ion is increased from below to above the critical concentration, the polymer experiences a phase transition from the expanded coil to the compact globule state.35,36 Since the phase transition of PNIPAm induced by a specific ion is essentially the same as that caused by temperature, this transition and the corresponding responsive properties can be dually controlled by both temperature and salt concentration.34–37 For example, Sukhishvili et al. reported that LbL films containing PNIPAm could be employed to control the release of a pyrene probe.38 The films reversibly swelled/deswelled in response to the variation of temperature and salt-concentration, resulting in the controllable release of pyrene. Li and co-workers found that the electrochemical impedance spectroscopic responses of Fe(CN)63-/4- redox couple at PNIPAm-modified electrodes showed reversible on-off switching behavior when the temperature cycled between 20 and 45 °C or the NaCl concentration in solution changed alternately between 0 and 2.0 M.29 As a matter of fact, the dual factors, temperature and salt concentration, do not affect the phase transition of PNIPAm independently or separately; in contrast, they influence the phase transition simultaneously and cooperatively.34–38 However, until now, the study of the on-off switching behavior of PNIPAm films synergistically controlled by temperature and salt concentration has not been reported. Considering the unique properties of PNIPAm, in the present work, the PNIPAm films were synthesized on a Au electrode surface by an electrochemically induced free-radical polymerization method.29–31 The on-off switching behavior of the films toward the electroactive probe ferrocenecarboxylic acid (Fc(COOH)) was synergistically modulated by temperature and sodium sulfate (Na2SO4) concentration in solution. Because the LCST of PNIPAm usually lies between room and body temperature, the PNIPAm films would be very suitable for the study of bioelectrocatalysis based on enzymatic reactions. Thus, the thermo- and Na2SO4-sensitive switching property of the films toward Fc(COOH) was further employed to control the electrocatalytic oxidation of glucose by GOx with Fc(COOH) as a mediator. Particularly, the on-off switch of bioelectrocatalytic process could be synergistically or cooperatively controlled by temperature and Na2SO4 concentration. The present report provides a novel model to combine temperature- and saltsensitive permeability of the PNIPAm interface toward small probes with bioelectrocatalysis so that the synergetic effect of temperature and Na2SO4 concentration on the phase transition of the polymer films could be successfully applied to the dual signal-triggered bioelectrocatalysis. This may open a new way to develop multiresponsive biosensors based on enzymatic electrocatalysis. Experimental Section 1. Reagents. N-Isopropylacrylamide (NIPAm) was obtained from TCI Chemicals. Ferrocenecarboxylic acid (Fc(COOH)) and glucose oxidase (GOx, E.C. 1.1.3.4, type VII, 192 000 units g-1) were purchased from Sigma-Aldrich. Sodium persulfate

J. Phys. Chem. B, Vol. 114, No. 17, 2010 5941 (Na2S2O8) was purchased from Aladdin Reagents. Glucose was obtained from Beijing Yili Fine Chemicals. Sodium chloride (NaCl), sodium bromide (NaBr), and sodium nitrate (NaNO3) were obtained from the Beijing Chemical Engineering Plant. Sodium sulfate (Na2SO4) was purchased from the Tianjin Tanggu Dengzhong Chemical Plant. The buffer used was 0.05 M sodium dihydrogen phosphate at pH 7.0. Water was purified twice successively by ion exchange and distillation. 2. Preparation of PNIPAm Films on Au Electrodes. The Au disk electrodes (diameter 2 mm, CH Instruments) were polished with 0.05 µm γ-alumina to a mirror-like finish, followed by sequential cleaning for 5 min each with ethanol and water in an ultrasonic bath. The electrochemical polymerization was performed at the Au electrodes in a nitrogensaturated aqueous solution containing 0.8 M NIPAm, 0.15 M NaNO3, and 0.01 M Na2S2O8 with cyclic voltammetric (CV) scans at 0.1 V s-1 between -0.1 and -1.3 V vs saturated calomel electrode (SCE) for a certain number of cycles,30,31 forming PNIPAm films on the Au electrode surface. After polymerization, the PNIPAm-modified electrodes were washed with water to remove the remaining monomers. 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 SCE as the reference electrode, a platinum wire as the counter electrode, and the Au disk electrode with films as the working electrode. Prior to electrochemical measurements, the buffers in the cell were purged with high-purity nitrogen for at least 15 min, and a nitrogen atmosphere was then maintained over the cell during the whole experiment. Fourier transform infrared reflection-absorption spectra (FTIR-RAS) were collected at a spectral resolution of 4 cm-1 with an IFS-66v/S FTIR spectrometer (Bruker) in vacuum at around 1-2 mbar of pressure. FTIR-RAS was performed with p-polarized light incidence at 80° relative to the surface normal using a Bruker accessory. The reflected light was detected with a liquid-nitrogen-cooled mercury cadmium telluride (MCT) detector. The PNIPAm films electropolymerized on gold-coated glass wafers were used as the samples for FTIR-RAS measurements. The temperature of solutions in the cell was controlled by an HH-S thermostatic bath (Zhengzhou Greatwall Scientific Industrial and Trade Co.) with a precision of (0.2 °C. Results and Discussion 1. Electrochemical Polymerization of PNIPAm Films. The electropolymerization of PNIPAm films in this work was performed in the same way as that described in the literature.30,31 When Au electrodes were placed in NaNO3 solutions containing NIPAm and Na2S2O8 and the continuing CV cycles were performed between -0.1 and -1.3 V, a reduction peak at about -0.95 V was observed in the first cycle, and the peak current decreased with the number of scanning cycles, accompanied by the positive shift of peak potential (Supporting Information Figure S1). The peak should be attributed to the reduction of Na2S2O8,31,39 since in NaNO3 solutions, NIPAm did not show any reduction peak but Na2S2O8 demonstrated a reduction peak at the same potential (Supporting Information Figure S2). These results indicate that the in situ electrochemically induced freeradical polymerization of NIPAm is successfully realized with the help of Na2S2O8 initiator. The formation of PNIPAm films on the Au electrode surface acted as a barrier and hindered the Na2S2O8 in solution from reaching the Au electrode surface, resulting in the continuing decrease of the reduction peak current

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Figure 1. (A) CVs of 0.5 mM Fc(COOH) at 0.05 V s-1 in pH 7.0 buffers for PNIPAm films at temperatures (a) 25, (b) 31, (c) 32, (d) 33, and (e) 35 °C. (B) Influence of solution temperatures on CV oxidation peak current (Ipa) of Fc(COOH) in pH 7.0 buffers containing (a) 0 and (b) 0.28 M Na2SO4 for PNIPAm films.

with the CV cycles. In the present work, 20 cycles were used in preparation of the PNIPAm films on the Au electrode surface. FTIR-RAS was used to confirm the formation of PNIPAm films electrochemically polymerized on the Au surface (Supporting Information Figure S3). In the wavenumber ranges of 3600-2600 and 1800-1250 cm-1, the spectrum showed all the characteristic peaks of PNIPAm reported in the literature.39,40 2. Temperature-Controlled On-Off Property of PNIPAm Films. The CV response of Fc(COOH) in pH 7.0 buffers at PNIPAm film electrodes was very sensitive to the temperature of the testing solutions (Figure 1A). When the temperature was below 32 °C, Fc(COOH) displayed a well-defined and nearly reversible CV oxidation-reduction peak pair at about 0.26 V with quite large peak currents; however, when the temperature was higher than 32 °C, the CV signals were greatly suppressed. The phase transition temperature of 32 °C was clearly detected in the curve of the CV oxidation peak current (Ipa) vs temperature (Figure 1B, curve a), which is consistent with the LCST of PNIPAm.26–28 Below the LCST, the PNIPAm films tended to take on a swollen and expanded coil structure in solution, mainly because of the strong hydrogen bonding between its amide groups and water molecules.26,28 The probe would thus diffuse through the films more easily, leading to the larger CV response. When the temperature was above the LCST, the PNIPAm chains would collapse and take on a contracted and compact globule structure in solution, since the hydrogen bonds between PNIPAm and water molecules are broken, allowing attractive inter- and intramolecular hydrophobic interactions within PNIPAm to dominate.41 The contracted structure would retard the probe to transport through the films, resulting in the very small CV signal of Fc(COOH). Herein, the conformational change of PNIPAm films tethered on the Au electrode surface with environmental temperature could be detected by CV very sensitively and rapidly. In addition, the phase transition temperature or LCST of PNIPAm is dependent on the salt identity and concentration.34,35 For example, with 0.28 M Na2SO4 in solution, the phase transition temperature of the PNIPAm films measured by CV with the Fc(COOH) probe was found to shift to about 23 °C (Figure 1, curve b). The temperature-dependent CV behavior of Fc(COOH) at PNIPAm-modified electrodes could be used to study the onoff switching property of the films, and two typical temperatures, 25 and 35 °C, were selected according to Figure 1. At 25 °C, Fc(COOH) showed a quite reversible CV peak pair with relatively large peak currents for the films (Figure 1A, curve a); at 35 °C, the CV response was significantly reduced or even could hardly be observed (Figure 1A, curve e). That is, the CV response was at the on state at 25 °C, and at the off state at 35 °C. This thermosensitive on-off behavior was quite reversible. By switching the temperature of the testing solutions between

Song and Hu

Figure 2. Dependence of CV Ipa of 0.5 mM Fc(COOH) at 0.05 V s-1 on solution temperature switched between (O) 25 and (9) 35 °C for the same PNIPAm films.

Figure 3. (A) CVs of 0.5 mM Fc(COOH) at 0.05 V s-1 in pH 7.0 buffers containing (a) 0, (b) 0.15, (c) 0.20, (d) 0.25, and (e) 0.28 M Na2SO4 for PNIPAm films at 25 °C. (B) Influence of Na2SO4 concentration on CV Ipa of Fc(COOH) for PNIPAm films at (a) 25 and (b) 17 °C.

25 and 35 °C, the oxidation peak current (Ipa) cycled between a relatively high value at 25 °C and a very small value at 35 °C (Figure 2), suggesting that the CV response of Fc(COOH) for PNIPAm films can be switched by changing the environmental temperature. In control experiments, CVs of Fc(COOH) at bare Au electrodes at different temperatures were also performed. The well-defined CV responses of the probe were observed at all studied temperatures, and no phase transition was observed between 17 and 35 °C (Supporting Information Figure S4). The peak currents showed a small, increasing trend when the temperature was raised because the diffusion coefficient of the probe became larger at higher temperature. Thus, the temperature-sensitive on-off CV behavior of Fc(COOH) at PNIPAm film electrodes should not be attributed to the property of the probe itself but to the thermosensitive properties of PNIPAm films. The stability of the PNIPAm films electropolymerized on a Au electrode surface was examined by CV with Fc(COOH) as the probe. The films were stored in pH 7.0 buffers containing Fc(COOH) at room temperature for most of the storage time, and CV was performed periodically at 25 and 35 °C to check the on-off behavior of the system. After 3 weeks of storage, for example, the CV peak potentials and currents remained almost the same as their initial values, and the on-off switching behavior of the system was still clearly observed between 25 and 35 °C (Supporting Information Figure S5), indicating that the PNIPAm films are quite stable and can be amenable to a series of electrochemical testing. 3. Na2SO4 Concentration-Controlled On-Off Property of PNIPAm Films. The CV response of Fc(COOH) at PNIPAm film electrodes was also very sensitive to the concentration of Na2SO4 in the testing solution (Figure 3A). At 25 °C, when the concentration of Na2SO4 in solution was below 0.10 M, the welldefined and nearly reversible CV response of Fc(COOH) was observed with relatively large peak heights. However, the peak currents decreased drastically when the Na2SO4 concentration increased from 0.10 to 0.25 M. When the concentration of

“On-Off” Switchable Bioelectrocatalysis

Figure 4. Dependence of CV Ipa of 0.5 mM Fc(COOH) at 0.05 V s-1 on Na2SO4 concentration in testing solution switched between (O) 0 and (9) 0.28 M for the same PNIPAm films at 25 °C.

Na2SO4 was larger than 0.25 M, the CV signals became very small or even could hardly be observed. According to the relationship between CV oxidation peak current and Na2SO4 concentration (Figure 3B, curve a), the critical phase transition concentration of Na2SO4 was estimated to be about 0.20 M at 25 °C, very close to that reported in the literature.34,35 In addition, the critical concentration of Na2SO4 was dependent on the temperature of the testing solutions. For example, when the solution temperature was set at 17 °C, the critical phase transition concentration of Na2SO4 shifted to ∼0.45 M (Figure 3B, curve b). The essence of the phase transition of PNIPAm films induced by Na2SO4 concentration is the same as that induced by the temperature.34–37 Below the critical concentration of Na2SO4, because of the formation of numerous hydrogen bonds between amide groups of PNIPAm and water molecules, PNIPAm films tended to take on an expanded coil conformation. The Fc(COOH) probe would diffuse through the films easily, leading to the large CV response. When the concentration of Na2SO4 was above the critical one, the ability of PNIPAm to form hydrogen bonds with water molecules was greatly impeded, leading to the contracted globule structure of the PNIPAm films.41 The films would thus block the probe to go through the films, resulting in the very small CV signal. The Na2SO4-concentration-dependent CV behavior of Fc(COOH) for PNIPAm films could also be used to study the on-off switching property of the films. In the testing solution containing no Na2SO4, the PNIPAm film electrodes were at the on state (Figure 3A, curve a); in the solution containing 0.28 M Na2SO4, the films were at the off state (Figure 3A, curve e). This on-off switching behavior of the films toward Fc(COOH) was quite reversible (Figure 4). Herein, the “coil-to-globule” phase transition induced by Na2SO4 concentration was quite fast and sensitive, and only dozens of seconds of immersion in 0 or 0.28 M Na2SO4 solutions would complete the corresponding phase transition. In control experiments at 25 °C, the CV response of Fc(COOH) at bare Au electrodes in testing solutions containing 0 and 0.28 M Na2SO4 demonstrated no substantial difference in peak potentials and currents (Supporting Information Figure S6), indicating that the Na2SO4-concentration-sensitive on-off CV behavior of Fc(COOH) at PNIPAm film electrodes has nothing to do with the property of the probe itself but should be attributed to the PNIPAm films. 4. On-Off Property of PNIPAm Films Synergistically Controlled by Temperature and Na2SO4 Concentration. Both the temperature and the Na2SO4 concentration could effectively influence the CV behavior of Fc(COOH) at PNIPAm film electrodes, and these two factors demonstrated the synergetic effect on the on-off property of the films. For example, the LCST of the films was found at 32 °C in the absence of Na2SO4

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Figure 5. CV Ipa of 0.5 mM Fc(COOH) at 0.05 V s-1 with different temperatures and Na2SO4 concentrations for the same PNIPAm films. Each data represents the average of six parallel measurements.

Figure 6. CVs at 0.01 V s-1 at 25 °C in pH 7.0 buffers containing 0.5 mM Fc(COOH), 1.0 mg mL-1 GOx, and glucose with (a) 0, (b) 1.0, (c) 2.0, and (d) 4.0 mM for PNIPAm films.

(Figure 1B, curve a), but was observed at 23 °C in the presence of 0.28 M Na2SO4 (Figure 1B, curve b). Therefore, in the solution containing 0.28 M Na2SO4, the PNIPAm films would cycle between the on and off states when the temperature was switched between 17 (lower than LCST) and 25 °C (higher than LCST) (Supporting Information Figure S7A). With the same temperature of 25 °C, the films could be at either the on or off state, depending on the concentration of Na2SO4 in the solution. Meanwhile, the critical phase transition concentration of Na2SO4 was also affected by the temperature. For instance, at 25 °C, the critical concentration of Na2SO4 was observed at about 0.20 M (Figure 3B, curve a), but at 17 °C, it shifted to ∼0.45 M (Figure 3B, curve b). Thus, at 17 °C, the PNIPAm film electrodes were switched between the on and off states when the Na2SO4 concentration cycled between 0.28 and 0.60 M (Supporting Information Figure S7B). This synergetic effect of temperature and Na2SO4 concentration on the on-off property of the PNIPAm films toward Fc(COOH) is clearly demonstrated in the three-dimensional diagram (Figure 5). The two factors, temperature and Na2SO4 concentration, do not influence the on-off property of the films independently or separately; in contrast, they affect the switchable behavior of the films synergistically or cooperatively. 5. On-Off Switchable Bioelectrocatalysis Synergistically Controlled by Temperature and Na2SO4 Concentration. The temperature- and Na2SO4-concentration-sensitive on-off switching property of PNIPAm films toward Fc(COOH) could be used to control or modulate the bioelectrocatalytic oxidation of glucose by GOx enzyme. For example, when the PNIPAm film electrode was placed in the solution containing glucose, GOx, and Fc(COOH) with no Na2SO4 at 25 °C, the CV oxidation peak at about 0.26 V increased dramatically in comparison with that in the absence of glucose, accompanied by a decrease or even disappearance of the reduction peak (Figure 6). The Ipa increased initially with the concentration of glucose in solution and then tended to level off (Supporting Information Figure S8). All these results are characteristic of electrochemical oxidation of glucose catalyzed by GOx and mediated by Fc(COOH), and

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Figure 7. (A) CVs at 0.01 V s-1 in pH 7.0 solutions containing 0.5 mM Fc(COOH), 1.0 mg mL-1 GOx, and 5.0 mM glucose at (a) 25 and (b) 35 °C for PNIPAm films. (B) Dependence of CV Ipa on solution temperatures switched between (O) 25 and (9) 35 °C for the same PNIPAm films.

the mechanism can be expressed by the following equations:1,42,43

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Figure 8. CV Ipa of 0.5 mM Fc(COOH) at 0.01 V s-1 in pH 7.0 solutions containing 1.0 mg mL-1 GOx and 5.0 mM glucose at different temperatures and Na2SO4 concentrations for the same PNIPAm films. Each data represents the average of six parallel measurements.

GOx(FAD) + glucose f GOx(FADH2) + gluconolactone GOx(FADH2) + 2Fc(COOH)Ox f GOx(FAD) + 2Fc(COOH)Red Fc(COOH)Red - e- h Fc(COOH)Ox

at electrode

where GOx(FAD) and GOx(FADH2) represent oxidized and reduced forms of glucose oxidase, respectively. However, when the same PNIPAm-modified electrode was transferred in the solution containing the same amount of Fc(COOH), glucose, and GOx at 35 °C, the electrocatalytic response became quite small (Figure 7A, curve b). This is because the films become off for the probe at 35 °C, resulting in the interruption of the catalytic cycles. Therefore, the bioelectrocatalysis of glucose by GOx can be controlled by the different permeability of PNIPAm films toward Fc(COOH) at different temperatures. The bioelectrocatalysis is at the on state at 25 °C and at the off state at 35 °C (Figure 7A). If Ipa25 and Ipa35 were defined as the oxidation peak/wave currents at 25 and 35 °C, respectively, the Ipa25/Ipa35 ratio could be amplified by the electrocatalysis. For the same PNIPAm films in solutions containing only Fc(COOH), the Ipa25/Ipa35 ratio was 7.0, whereas in the presence of Fc(COOH), GOx, and glucose, the ratio increased to about 13.8. The thermo-sensitive on-off bioelectrocatalysis for the system was reversible and could be repeated for at least several cycles by switching the same modified electrodes in the solutions containing Fc(COOH), GOx, and glucose between 25 and 35 °C (Figure 7B). The Na2SO4-concentration-sensitive on-off behavior of PNIPAm films toward Fc(COOH) could also be used to control the bioelectrocatalysis of glucose in the presence of GOx (Supporting Information Figure S9). However, in the control experiments with bare Au electrodes, the addition of Na2SO4 showed little effect on the bioelectrocatalytic response of glucose in the presence of Fc(COOH) and GOx (Supporting Information Figure S10), indicating that the Na2SO4-trigged on-off behavior of bioelectrocatalysis (Figure S9) originates from the saltinduced phase transition of PNIPAm films rather than the salt effect on the bioelectrocatalytic reactions. In particular, the synergetic effect of temperature and Na2SO4 concentration on the on-off bioelectrocatalysis for the PNIPAm films in the solutions containing Fc(COOH), GOx, and glucose is shown in Figure 8. These results confirm that the temperature and Na2SO4 concentration do not influence the on-off bioelectrocatalysis of the PNIPAm film system independently or sepa-

Figure 9. Influence of concentration of (a) Na2SO4, (b) NaCl, and (c) NaBr on CV Ipa of 0.5 mM Fc(COOH) at 0.05 V s-1 for PNIPAm films at 25 °C.

rately. In contrast, they affect the on-off bioelectrocatalytic behavior of the system synergistically or cooperatively. Since the on-off bioelectrocatalysis of the system (Figure 8) originates from the on-off electrochemical behavior of PNIPAm films toward Fc(COOH) (Figure 5), their changing tendencies with temperature and Na2SO4 concentration are very similar. 6. Comparative Study with Other Salts. The on-off switching CV property of PNIPAm films toward Fc(COOH) could be controlled not only by the Na2SO4 concentration but also by the concentration of other salts. For example, at 25 °C, the CV Ipa of the probe at PNIPAm film electrodes with different concentrations of NaCl or NaBr also demonstrated the on-off behavior (Figure 9, curves b and c) with a shape of the Ipa-vsconcentration curve similar to that of Na2SO4 (Figure 9, curve a). Herein, the major difference among different salts lies in the different critical phase transition concentration for the PNIPAm films. At 25 °C, the critical concentrations of NaCl and NaBr were estimated to be about 0.90 and 1.10 M, respectively, much larger than that of Na2SO4 (0.20 M). Anions usually exhibit a stronger influence on the phase transition of PNIPAm than cations.35,41 NaCl, NaBr, and Na2SO4 share the same cation (Na+) but have different anions. The pronounced difference of their critical phase transition concentration (Figure 9) confirms that in the present work, it is the anion rather than the cation of the salts that greatly influences the LCST of the PNIPAm films. The critical concentration of the anions showed the sequence of Br- (1.10 M) > Cl- (0.90 M) > SO42- (0.20 M), in good agreement with the Hofmeister anion series.34,35,44 As a “water structure maker” or kosmotrope,44 SO42- demonstrated an obvious advantage over Br- and Cl- due to its much lower critical phase transition concentration. Na2SO4 was thus more sensitive than the other salts in inducing the on-off property of the PNIPAm films toward the probe. The lower critical concentration for Na2SO4 is of particular importance for studying the on-off bioelectrocatalysis based on enzymes, since the enzymes may alter their conformation and lose their bioactivity in solution with a high concentration of salts,

“On-Off” Switchable Bioelectrocatalysis especially for the salts known as chaotropes or “water structure breakers”, such as Br-.45–48 That is why Na2SO4 was selected as the model salt to control the on-off bioelectrocatalysis in the present work. Conclusions PNIPAm films fabricated on a Au electrode surface by electropolymerization demonstrate a “coil-to-globule” phase transition, which is dependent not only on temperature but also on the Na2SO4 concentration. The PNIPAm films are “opened” toward small molecular probes such as Fc(COOH) when the films are in the expanded coil state, but “closed” when the films are in the contracted globule state. This “open-close” or on-off switching property of the films can be dually and synergistically controlled by external temperature and salt concentration and detected in this work by CV with Fc(COOH) as the electroactive probe. To the best of our knowledge, this is the first report to apply the synergetic effect of temperature and Na2SO4 concentration on phase transition of PNIPAm films to the study of the dual-responsive on-off behavior of the films. Perhaps the most exciting point in the present work is the realization of dualcontrolled on-off bioelectrocatalysis. The different permeabilities of the PNIPAm films toward Fc(COOH) at different temperatures and Na2SO4 concentrations can be used to modulate the electrocatalytic oxidation of glucose by GOx enzyme. Herein, Fc(COOH) not only acts as the electroactive probe in testing the permeability of PNIPAm films but also acts as the electron transfer mediator in the bioelectrocatalytic process. The “smart” PNIPAm-Fc(COOH)-GOx-glucose system may be applied in the future as the foundation for biological information storage and processing, for development of biofuel cells and bioelectronic elements, for amplification of weak electrochemical signals, and for biosensing. This intelligent model system may also provide a general way to realize multiresponsive on-off bioelectrocatalysis and develop the novel type of multicontrollable electrochemical biosensors or bioreactors 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: Ten figures showing multicycled CVs at Au electrodes in NaNO3 solutions containing NIPAm and Na2S2O8; CVs of NIPAm and Na2S2O8 in NaNO3 solutions at Au electrodes; FTIR-RAS of PNIPAm films; CVs of Fc(COOH) at bare Au electrodes at different temperatures; CVs of Fc(COOH) at PNIPAm film electrodes with different storage times; CVs of Fc(COOH) at a bare Au electrode at different Na2SO4 concentrations; CVs of Fc(COOH) at PNIPAm film electrodes at 17 and 25 °C in the presence of 0.28 M Na2SO4; CVs of Fc(COOH) at PNIPAm film electrodes at 17 °C in the presence of 0.28 and 0.60 M Na2SO4; dependence of Ipa on the concentration of glucose for PNIPAm films at 25 °C in solutions containing Fc(COOH), GOx and glucose; CVs of Fc(COOH) at PNIPAm film electrodes in the presence of GOx and glucose at different Na2SO4 concentrations at 25 °C; dependence of Ipa on Na2SO4 concentration switched between 0 and 0.28 M at 25 °C; CVs at bare Au electrode in the presence of Fc(COOH), GOx and glucose at different Na2SO4 concentra-

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