Multiple Stimuli-Switchable Bioelectrocatalysis under Physiological

May 29, 2014 - This multiresponsive CV behavior of the system could be further employed to switch the electrochemical oxidation of glucose catalyzed b...
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Multiple Stimuli-Switchable Bioelectrocatalysis under Physiological Conditions Based on Copolymer Films with Entrapped Enzyme Peng Wang,† Shuang Liu,† and Hongyun Liu*,†,‡ †

Department of Chemistry, Beijing Normal University, Beijing 100875, People’s Republic of China Key Laboratory of Theoretical and Computational Photochemistry, Ministry of Education, College of Chemistry, Beijing Normal University, Beijing 100875, People’s Republic of China

‡

S Supporting Information *

ABSTRACT: In the present work, N,N-diethylacrylamide (DEA) and methyl acrylic acid (MAA) monomers were copolymerized into P(DEA-co-MAA) thin films on the electrode surface with a simple one-step polymerization method at ambient temperature and pressure, and the enzyme glucose oxidase (GOD) was entrapped in the films, designed as P(DEA-co-MAA)-GOD. The cyclic voltammetric (CV) response of ferrocene dicarboxylic acid (Fc(COOH)2) at the film electrodes was very sensitive to environmental stimuli, such as temperature, pH, the identity and concentration of anions, and the concentration of CO2 in solution. This multiresponsive CV behavior of the system could be further employed to switch the electrochemical oxidation of glucose catalyzed by GOD entrapped in the films with Fc(COOH)2 as the mediator in solution, demonstrating the amplification effect. The SEM and stereomicroscopy results showed that the multisensitive behaviors of the system were attributed to the structure change of the copolymer films with the stimuli. Specifically, the synergistic effect of temperature and pH was observed, and the hydrogen bonding between PDEA and PMAA components in the copolymer played a key role for this. The present system could be performed under physiological conditions at 37 °C and pH 7.4, which may offer numerous possibilities not only to design new multiswitchable biosensors based on bioelectrocatalysis but also to establish foundations for controlled drug delivery and other medical applications.

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INTRODUCTION In recent years, considerable attention has been focused on the electrochemical catalysis based on enzymes in the research of biosensors, bioreactors, and other biodevices.1−3 The switchable and tunable bioelectrocatalysis combining enzymatic reactions and stimuli-responsive interfaces is of great significance not only because it may provide a novel avenue to understand the mechanism of enzymatic reactions in real life but also because it may be applied in constructing switchable biosensors, triggering biofuel cells and bioelectronic elements, as well as in realizing transduction/amplification and information storage/processing at molecular or nanometersized levels.4−7 A variety of stimuli-triggered hydrogels and polymers have been used to immobilize redox enzymes on an electrode surface, in which their bioelectrocatalytic activity is able to be controlled or modulated with external stimuli such as pH,8−10 temperature,11 light,12,13 electric potential,14 and magnetic field,15 etc. Compared with the single stimulus-responsive bioelectrocatalysis system, the multiple stimuli-triggered ones 16−19 demonstrate particular interest because new dimensions are added in the studied field and the system becomes more complicated, which are more like physiological and biological systems. The multiple stimuli-responsive hydrogels are usually fabricated by copolymerizing two or more different single stimulus-responsive monomers.17,20,21 Herein, © 2014 American Chemical Society

each copolymer component preserves its own stimulussensitive property and the whole copolymer demonstrates multiresponsive behavior. For example, poly(N,N-diethylacrylamide) (PDEA) is one of the most popular thermosensitive polymers and exhibits reversible temperature-responsive phase transition in aqueous solution at the lower critical solution temperature (LCST) of approximately 31 °C.22−24 Poly(methyl acrylic acid) (PMAA) is a typical pH-sensitive polymer and demonstrates a reversible pH-responsive structure change in solution.25,26 The copolymerization of DEA and MAA monomers thus results in the P(DEA-co-MAA) hydrogels which exhibit not only thermoresponsive behavior but also pH-sensitive property.27,28 However, to the best of our knowledge, there has been no report until now to combine the P(DEA-co-MAA) copolymer with electrochemistry and the corresponding bioelectrocatalysis with embedded enzymes. In this work, the P(DEA-co-MAA) copolymer thin films were prepared on an electrode surface with a simple one-step polymerization method at ambient temperature and pressure and glucose oxidase (GOD) was immobilized in the films in the polymerization process. The films are designed as P(DEA-coReceived: February 14, 2014 Revised: April 22, 2014 Published: May 29, 2014 6653

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MAA)-GOD. At the film electrodes, the electroactive probe ferrocene dicarboxylic acid (Fc(COOH)2) demonstrated temperature-, pH-, anion- and CO2-sensitive cyclic voltammetric (CV) response, and the multiple stimuli-responsive ON−OFF behavior. This switchable CV behavior could be further used to activate/deactivate the electrochemical oxidation of glucose catalyzed by GOD entrapped in the films with Fc(COOH)2 as the mediator in solution. The possible mechanism of the multiswitchable permeability of the films toward the probe, especially the synergistic effect of temperature and pH, was discussed and explored in detail. The probe showed the high permeability through P(DEA-co-MAA)GOD films under the physiological condition of 37 °C and pH 7.4 (ON state) and the very small CV response at the higher temperature or lower pH (OFF state), which is very desirable in controlled drug delivery. Compared with our previous works on multiresponsible bioelectrocatalysis,16−19 the present study provides a novel platform with some unique characteristics. This may offer a foundation not only for building up multiswitchable biosensors based on enzymatic reactions but also for some medical applications.

Apparatus and Procedures. Electrochemical measurements were performed using a CHI 660B electrochemical workstation (CH Instruments). A saturated calomel electrode (SCE) and a Pt wire were used as the reference and counter electrodes, respectively. The PG electrode with films was used as the working electrode. Before electrochemical measurements, high-purity nitrogen was purged into the solution for about 15 min, and the nitrogen atmosphere was maintained during the experiments. A PHSJ-3F pH meter from Shanghai Precision & Scientific Instruments was used to perform the pH measurements. An HH-S thermostatic bath (Zhengzhou Greatwall Scientific) was used to control the temperature of solutions in the cell with precision of ±0.1 °C. A Nexus 670 Fourier transform infrared spectrometer (Nicolet) was used to collect the Fourier transform infrared (FTIR) spectra at a resolution of 4 cm−1. A TU-1901 UV−vis double-beam spectrometer (Beijing Purkinje General Instrument) was used to obtain the UV−vis absorption spectra. A Stereo Discovery V12 stereomicroscope equipped with an Axio Cam digital camera (Zeiss) was used to perform stereomicroscopy and then estimate the thickness of hydrogel films. An S-4800 scanning electron microscope (Hitachi) with an acceleration voltage of 3 kV was used to perform scanning electron microscopy (SEM). To prepare the SEM samples, the films were first treated with the solutions at different pHs, temperatures, or Na2SO4 concentrations, and then transferred into liquid nitrogen immediately to “freeze” the structure. After that, an FD-3 freeze drier (Beijing Boyikang Experimental Instrument) was used to freeze-dry the films for 48 h in order to get rid of all water in the films. Before SEM imaging, thin platinum films were coated on the sample surface with an E-1045 sputtering coater (Hitachi).

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EXPERIMENTAL SECTION Reagents. Glucose oxidase (E.C.1.1.3.4, type VII, MW ≈ 160 000, 192 000 units g−1), N,N′-methylenebis(acrylamide) (BIS) and N,N,N′,N′-tetramethylethylenediamine (TEMED) were purchased from Sigma-Aldrich. N,N-Diethylacrylamide (DEA) and 1,1′-ferrocene dicarboxylic acid (Fc(COOH)2) were purchased from TCI. Methyl acrylic acid (MAA) was bought from Tianjin Bodi Chemical Engineering Plant and was purified by distillation. Sodium persulfate (Na2S2O8) was purchased from Aladdin. Sodium sulfate (Na2SO4) was obtained from Beijing Chemical Engineering Plant. Glucose was obtained from Beijing Yili Fine Chemicals. All other reagents were of analytical grade and used without further purification. Britton-Robinson buffers containing 0.05 M NaCl at pH 4.0−9.0 were usually used, and the pH values were adjusted with dilute HCl or NaOH solutions. The water used was purified by a Millipore laboratory water purification system. Preparation of P(DEA-co-MAA)-GOD Films on the Surface of Electrodes. Before being coated, basal plane pyrolytic graphite (PG; Momentive Performance Materials) disk electrodes (geometric area, 0.16 cm2) were abraded with metallographic sandpapers of 320 grit while flushed with water. The electrodes were then ultrasonicated in water for 30 s and dried in air. The copolymer films were then prepared on PG surface according to the literature29−31 with some modification. In brief, the electrode was placed in a sealed bottle under a high-purity N2 atmosphere for about 5 min. At the same time, the freshly prepared pregel solution was deaerated with N2 for about 5 min. After that, 6.5 μL of the pregel solution was cast on the electrode surface with a microsyringe under N2 atmosphere at ambient temperature. The typical pregel solution contained 0.48 M DEA, 0.023 M MAA, 2.0 mg mL−1 GOD, 1.5 mg mL−1 BIS as a cross-linker, 0.60 mg mL−1 Na2S2O8 as an initiator, and 0.70 mg mL−1 TEMED as an accelerator after optimization. The P(DEA-co-MAA)-GOD films were then formed on the PG surface in about 8 min. The formed film electrode was immersed in water for about 8 min to remove the unreacted chemicals. The other films, such as P(DEA-co-MAA), PMAA and PDEA, were polymerized with a similar procedure. The chemical structure of P(DEA-co-MAA) is illustrated in the Supporting Information Figure S1.

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RESULTS Characterization of P(DEA-co-MAA)-GOD Films. P(DEA-co-MAA) films containing GOD on PG electrodes were prepared by radical cross-linking polymerization method according to the procedure reported in the literature29−31 with some modification. The formation of P(DEA-co-MAA)-GOD films was confirmed by IR spectroscopy (Supporting Information, Figure S2, Table S1, and the related explanations). Two characteristic UV−vis absorption peaks of GOD in solution at 454 and 383 nm32 were also observed for P(DEAco-MAA)-GOD hydrogels with the similar position (Supporting Information, Figure S3), indicating that GOD is successfully entrapped in the P(DEA-co-MAA)-GOD films and retains its native conformation. The formation of P(DEA-co-MAA)-GOD films on PG electrodes could also be supported by CV with Fc(COOH)2 as the electroactive probe. In pH 7.4 buffers at 37 °C, Fc(COOH)2 showed a nearly reversible CV peak pair at about 0.4 V at bare PG electrodes (Supporting Information, Figure S4, curve a). At P(DEA-co-MAA)-GOD film electrodes, the CV response of the probe decreased significantly (Supporting Information, Figure S4, curve b). This is because the films act as a diffusion barrier to restrain some of the probe from reaching the electrode surface. However, for the films with PDEA alone, the CV signal of Fc(COOH)2 could even be hardly detected under the same conditions (Supporting Information, Figure S4, curve c). Thermosensitive Property of P(DEA-co-MAA)-GOD Films. PDEA is a typical thermoresponsive polymer.22−24,33 We thus expected that the copolymer P(DEA-co-MAA) was 6654

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also sensitive to the environmental temperature. The CV response of Fc(COOH)2 at P(DEA-co-MAA)-GOD film electrodes was therefore tested in solution at different temperatures (Figure 1). At pH 7.4, when the solution

Figure 2. Variation of CV Ipa of 1.0 mM Fc(COOH)2 at 0.05 V s−1 in 7.4 buffers with immersion time (t) at temperature switched between 37 and 50 °C for the same P(DEA-co-MAA)-GOD films.

Figure 1. CVs of 1.0 mM Fc(COOH)2 at 0.05 V s−1 in pH 7.4 buffers at (a) 35, (b) 37, (c) 40, (d) 42.5, (e) 45, (f) 47.5, (g) 50, and (h) 56 °C for P(DEA-co-MAA)-GOD films. Inset: dependence of CV Ipa on the solution temperature (T).

temperature was set below 40 °C, the CV response of the probe was relatively large; however, when the temperature was higher than 40 °C, the peak currents decreased tremendously. The critical phase transition temperature (Tc) obtained from the curve of CV oxidation peak current (Ipa) versus temperature was at about 45 °C (Figure 1, inset), different from the LCST of 31 °C for pure PDEA reported in the literature.22−24,26 To further explore the thermoresponsive ON−OFF property of the films, two typical temperatures, 37 and 50 °C, were selected. At 37 °C, Fc(COOH)2 exhibited a pair of well-defined and nearly reversible CV peaks with quite large peak currents for the films in pH 7.4 buffers (Figure 1, curve b), indicating that the films are at the ON state; at 50 °C, however, the CV response of the probe was significantly suppressed (Figure 1, curve g), and the films were at the OFF state. This thermosensitive switching behavior of the system was fairly reversible and could be repeated for many times between 37 and 50 °C with the response time of about 4−6 min (Figure 2). The temperature-responsive PDEA is also sensitive to some anions according to the literature.34 It was therefore reasonable to expect that the copolymer P(DEA-co-MAA) was also sensitive to some salts. The CV response of Fc(COOH)2 at P(DEA-co-MAA)-GOD film electrodes was thus tested in the solutions with different concentrations of some anions, such as SO42− and Cl−. For example, in pH 7.4 buffers containing no SO42−, the probe for the films showed a quite large CV response and that they were at the ON state at 37 °C; however, with 0.4 M SO42− added, the CV response was very small and the films were at the OFF state (Figure 3). The ON−OFF behavior of the films toward SO42− was reversible (Supporting Information, Figure S5), and the critical phase transition concentration was at about 0.15 M (Figure 3, inset curve a). For Cl−, the critical phase transition concentration was at about 0.4 M (Figure 3, inset curve b), larger than that of SO42− and consistent with the Hofmeister series.35

Figure 3. CVs of 1.0 mM Fc(COOH)2 at 0.05 V s−1 in pH 7.4 buffers at 37 °C with Na2SO4 concentrations of (a) 0 and (b) 0.4 M. Inset: dependence of CV Ipa on the concentration (C) of (a) Na2SO4 and (b) NaCl.

pH-Sensitive Property of P(DEA-co-MAA)-GOD Films. PMAA is known to be a pH-sensitive polymer, whose structure is sensitive to solution pH.25,26 The P(DEA-co-MAA) copolymer was thus most probably sensitive to surrounding pH. The CV behavior of Fc(COOH)2 at P(DEA-co-MAA)GOD film electrodes was therefore tested in solutions at different pH (Figure 4). At pH 7.4, a quasi-reversible CV peak pair with large peak currents of the probe was observed at 37

Figure 4. (A) CVs of 1.0 mM Fc(COOH)2 at 0.05 V s−1 and 37 °C for P(DEA-co-MAA)-GOD films in buffers at pH (a) 8.0, (b) 7.4, (c) 7.0, (d) 6.8, (e) 6.6, (f) 6.4, (g) 6.2, and (h) 5.6. (B) Dependence of CV Ipa on the solution pH at 37 °C. 6655

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°C; however, when the pH decreased, the peaks decreased gradually and could even hardly be detected at pH 6.2. The films were at the ON state toward the probe at pH 7.4 and at the OFF state at pH 6.2 with the critical phase transition pH (pHc) at about 6.6 at 37 °C (Figure 4B). This pH-responsive ON−OFF property was quite reversible. By switching the same film electrode in solutions of Fc(COOH)2 between pH 7.4 and 6.2, the corresponding CV Ipa was changed periodically between a relatively high value and a very small one for several times with the response time of 4−6 min (Supporting Information, Figure S6). It should be noticed that the ΔpH between the ON and OFF states for this system was only 1.2 pH units (Figures 4 and S6 of the Supporting Information), much less than that reported in the literature.10,16−19,36 The CV response of Fc(COOH)2 at P(DEA-co-MAA)-GOD film electrodes was also sensitive to the concentration of CO2 in the testing solution. After CO2 was bubbled for 10 min in pH 7.4 buffers at 37 °C, the CV peak currents of the probe decreased remarkably in comparison with the original ones, and the films were at the OFF state (Figure 5, curves a and b). This

Figure 6. (A) Dependence of CV Ipa of 1.0 mM Fc(COOH)2 at 0.05 V s−1 on the solution temperature (T) for P(DEA-co-MAA)-GOD films in solutions at pH (a) 6.2, (b) 6.6, (c) 7.0, and (d) 7.4. (B) Dependence of CV Ipa of 1.0 mM Fc(COOH)2 at 0.05 V s−1 on the solution pH for P(DEA-co-MAA)-GOD films at (a) 22, (b) 37, and (c) 43 °C.

curve d), while at pH 6.2, it shifted to about 33 °C (Figure 6A, curve a). That is, with the same temperature, the films could be either at the ON state or at the OFF state, dependent on the solution pH. The synergistic effect of temperature and pH could also be observed for the relationship between CV Ipa and pH at different temperatures (Figure 6B). When the solution temperature increased from 22 to 43 °C, the pHc of the system shifted from 5.0 to 7.0. In control experiments, Fc(COOH)2 showed no essential difference in CV response at pH 7.4 and pH 6.2 either at bare PG electrodes or at PDEA film electrodes (Supporting Information, Figure S8), and the solution pH had nothing to do with Tc of the probe at PDEA film electrodes (Supporting Information, Figure S9). All these results suggest that it is the PMAA component of P(DEA-co-MAA)-GOD films that results in the dependence of Tc of the system on environmental pH. ON−OFF Behaviors of the System in Bioelectrocatalysis of Glucose. The GOD entrapped in P(DEA-co-MAA)GOD films could retain its enzymatic activity. This was supported by the UV−vis spectroscopic experiments (Supporting Information, Figure S3) and confirmed by the following CV experiments. When glucose was added into the solution containing Fc(COOH)2 at pH 7.4 and 37 °C, the CV oxidation peak of the probe at P(DEA-co-MAA)-GOD film electrodes increased obviously in comparison with that in the absence of glucose, accompanied by the decrease or even disappearance of the reduction peak (Supporting Information, Figure S10). All of these are the characteristics of electrochemical oxidation of glucose catalyzed by GOD embedded in the films with the help of Fc(COOH)2 mediator in solution.37,38 The oxidation peak current (Ipa) increased initially with the concentration of glucose in solution up to 4 mM and then tended to be stable (Supporting Information, Figure S10, inset). It should be noticed that the amount of gluconic acid produced by biocatalysis was relatively limited, and no obvious pH change was observed in the buffer solution during the whole bioelectrocatalysis experiment. The thermo-, anion-, pH-, and CO2-sensitive switching properties of P(DEA-co-MAA)-GOD films toward Fc(COOH)2 inspired us to apply the system to control or modulate the electrochemical oxidation of glucose catalyzed by GOD. For example, in pH 7.4 solutions containing Fc(COOH)2 and glucose at 37 °C, the films demonstrated a large electrocatalytic oxidation peak (Figure 7, curve a). However, when the pH of the solution was changed to 6.2, the electrocatalytic response became quite small (Figure 7,

Figure 5. CVs of 1.0 mM Fc(COOH)2 at 0.05 V s−1 and 37 °C for P(DEA-co-MAA)-GOD films in pH 7.4 buffers (a) with saturated N2, (b) after CO2 was bubbled for 10 min, and (c) after N2 was bubbled again for 10 min.

could be understood since the pH of the solution became 6.3 after the bubbling of CO2. In the following experiment, when N2 was continually bubbled in the solution for about 10 min to remove the CO2, the pH of the solution went back to 7.4, and the CV peaks returned to their original level (Figure 5, curve c). This CO2-dependent ON−OFF behavior of the films toward Fc(COOH)2 was also reversible, and the switching cycle between the ON and OFF states could be repeated for several times with the response time of about 10 min (Supporting Information, Figure S7). Synergistic Effect of Temperature and pH. Outwardly, the two stimuli, temperature and pH, seem to affect the ON− OFF behavior of the probe at P(DEA-co-MAA)-GOD film electrodes separately or independently, since the thermosensitive property of the system originates from the PDEA component of the films, and the pH-responsive behavior came from the PMAA constituent. Actually, however, the effect of temperature and pH on the switching behavior of the system is synergistic or cooperative. This was obviously observed from the relationship between CV Ipa and temperature at different pH for the system (Figure 6A). The Tc of the system shifted toward smaller value when the solution became more acidic. For example, at pH 7.4, the Tc was at about 45 °C (Figure 6A, 6656

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DISCUSSION Mechanism of Thermosensitive Behaviors of the Films. The CV response of Fc(COOH)2 at P(DEA-co-MAA) film electrodes was very sensitive to the environmental temperature and presented reversible ON−OFF behaviors (Figures 1 and 2). This should be attributed to the thermosensitive property of the PDEA component in the films instead of the PMAA constituent, since no temperatureresponsive behavior was observed for PMAA polymers.39 Moreover, the CVs of Fc(COOH)2 at bare PG electrodes showed no temperature-dependent property,17,18 indicating that the probe itself is not thermosensitive. PDEA is a typical thermosensitive polymer which can undergo the phase transition or structure change between the coil and globule states in water at its LCST controlled by the balance between hydrophilic and hydrophobic properties of the polymer.22−24,33 At the temperature below LCST, the hydrophilic force is predominant and the PDEA takes a loose and swollen coil state, mainly because of the formation of hydrogen bonding between amide groups of DEA units and water molecules in solution. When the temperature goes above LCST, the hydrophobic force dominates the system and most of the hydrogen bonds between the polymer chains and water molecules are broken, and the PDEA takes the shrunken and compact globule structure after the water molecules are squeezed out.22,33 The structure change of P(DEA-co-MAA)-GOD films with temperature was confirmed by SEM (Figure 9, panels A and B).

Figure 7. CVs at 0.01 V s−1 for P(DEA-co-MAA)-GOD films in solutions containing 1.0 mM Fc(COOH)2 and 5 mM glucose at (a) pH 7.4 and 37 °C, (b) pH 6.2 and 37 °C, (c) pH 7.4 and 50 °C, (d) pH 7.4 and 37 °C with 0.4 M Na2SO4, (e) pH 7.4 and 37 °C with CO2 bubbling for 10 min, and (f) with bubbling N2 for 10 min following e.

curve b). The bioelectrocatalysis was ON at pH 7.4 and OFF at pH 6.2. It should be emphasized that the enzymatic reaction amplified the difference of CV response between the ON and OFF states. If Ipa(ON) and Ipa(OFF) were defined as the CV oxidation peak current at the ON (pH 7.4) and OFF (pH 6.2) states, respectively, the ratio Ipa(ON)/Ipa(OFF) was about 4 for the system in the absence of glucose, while in the presence of glucose, the ratio increased to over 6. The pH-sensitive ON− OFF bioelectrocatalysis could be repeated for several cycles by switching the same films in the solution containing Fc(COOH)2 and glucose between pH 7.4 and 6.2 (Figure 8, panel A). The temperature-, SO42−-, and CO2-responsive switching bioelectrocatalysis of the system was also observed (Figure 7, curves c, d, and e), and the ON−OFF behavior was quite reversible (Figure 8, panels B, C, and D).

Figure 9. SEM top views of P(DEA-co-MAA)-GOD films assembled on the PG disk surface after the films were treated in buffers with 1.0 mM Fc(COOH)2 for 5 min at (A) pH 7.4 and 37 °C, (B) pH 7.4 and 50 °C, (C) pH 6.2 and 37 °C, and (D) pH 7.4 and 37 °C containing 0.4 M Na2SO4.

The films after being treated with 37 °C in pH 7.4 buffers showed a loose network structure with many large pores or channels, whereas the films after treatment at 50 °C displayed a more compact structure with much smaller pore size. The thermosensitive structure change of the films was also supported by the stereomicroscopic results, where the thickness of the films was estimated with the fixed surface area of underlying disks (Table 1). The average thickness of the films after treatment at 50 °C in pH 7.4 buffers became 179 μm, much smaller than 505 μm at 37 °C, reflecting the change from

Figure 8. Dependence of CV Ipa of 1.0 mM Fc(COOH)2 at 0.01 V s−1 for P(DEA-co-MAA)-GOD films in buffers containing 5 mM glucose (A) on solution pH switched between 6.2 and 7.4 at 37 °C, (B) on solution temperature switched between 37 and 50 °C in pH 7.4 buffers, (C) on Na2SO4 concentration switched between 0 and 0.4 M in pH 7.4 buffers at 37 °C, and (D) on alternative bubbling of CO2 and N2 for 10 min in pH 7.4 buffers at 37 °C. 6657

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structure with much smaller pore size. The pH-sensitive structure change of the films was also supported by the stereomicroscopic results (Table 1). The average thickness of the films after treated with pH 6.2 buffers at 37 °C became 151 μm, much smaller than 505 μm at pH 7.4. Thus, at pH 7.4, the system was at the ON state and Fc(COOH)2 could go through the loose films easily and then transfer electrons with electrodes, showing the large CV response. However, when the solution pH became lower than its pHc at 37 °C such as pH 6.2, it became more difficult for the probe to diffuse through the collapsed films, leading to the much smaller CV response and the OFF state. To get rid of the possibility that the pH-sensitive CV behavior of Fc(COOH)2 at P(DEA-co-MAA)-GOD film electrodes might be mainly controlled by the electrostatic interaction between the films and Fc(COOH)2, the neutral electroactive probe hydroquinone was examined by CV for the films at different pH at 37 °C (Supporting Information, Figure S11). Uncharged hydroquinone showed very large peaks at pH 7.4 and much smaller ones at pH 6.2 at the film electrode and the same pH-sensitive ON−OFF behavior as the negatively charged Fc(COOH)2. These results, combined with those of SEM and stereomicroscopy (Figure 9 and Table 1), suggest that it is the structure change instead of the electrostatic interaction that controls the pH-sensitive CV ON−OFF property of Fc(COOH)2 at P(DEA-co-MAA)-GOD film electrodes. Actually, there are two contrary effects when solution pH is above pHc for the system. On the one hand, the increase of pH results in more ionization of carboxylic acid groups of PMAA and leads to the more swollen structure of P(DEA-co-MAA)GOD films, which makes Fc(COOH)2 diffuse through the films more easily and leads to the increase of its CV response. On the other hand, the increase of -COO− groups in PMAA at higher pH leads to more negative charges in the films, and the increase of negative charges of the films would make the electrostatic repulsion with the similarly charged Fc(COOH)2 become stronger, which would result in the decrease of its CV response. In our situation, since the increase of the solution pH causes the larger CV peaks for the system, we speculate that the first effect would be predominant. In addition, while the absolute amount of negative charges of the films would increase with the increase of solution pH, the relative charge density might decrease with the increase of the film volume, which would benefit the diffusion of the probe through the films.16 The CO2-induced CV ON−OFF behavior of Fc(COOH)2 at P(DEA-co-MAA)-GOD film electrodes should have the same mechanism as the pH-sensitive behavior of the system, since the bubbling/removing CO2 would decrease/increase the solution pH to pH 6.2/7.4. Mechanism of Synergistic Effect of Temperature and pH. The temperature- and pH-sensitive CV behaviors of Fc(COOH)2 at P(DEA-co-MAA)-GOD film electrodes were not independent or separate but rather synergistic or cooperative. This was supported by the result that the Tc of the system systematically shifted toward the larger value when the solution pH was increased (Figure 6A) and the observation that the pHc of the system increased with the increase of surrounding temperature (Figure 6B). The synergistic effect of temperature and pH should be attributed to the interaction between the PDEA and PMAA components in the copolymer, and the key interaction between them is the formation/ destruction of hydrogen bonds between the carboxylic acid

Table 1. Average Thickness of the Films under Different Conditions conditions pH pH pH pH

7.4 7.4 6.2 7.4

and and and and

37 50 37 37

°C °C °C °C with 0.4 M Na2SO4

average thickness/μm 505 179 151 160

± ± ± ±

31 16 17 15

swollen to shrunken structure. Thus, at 37 °C, Fc(COOH)2 probe can diffuse through the loose films smoothly and exchange electrons with underlying electrodes easily, displaying the large CV response and leading to the ON state for the system. However, at 50 °C, above the Tc of 45 °C in pH 7.4 buffers, it becomes more difficult for the probe to go through the compact films, resulting in the much smaller CV signals and the OFF state. The SO42−-responsive property of the P(DEA-co-MAA)GOD films should also be attributed to the structure change of the PDEA component of the films. Since SO42− is a typical kosmotropic anion in the Hofmeister series,35 the addition of SO42− in solution above its critical concentration would induce the transition of the P(DEA-co-MAA)-GOD films from the swollen coil state to the shrunken globule state and finally lead to the tremendous decrease in CV response of Fc(COOH)2. The structure change of the films caused by SO42− was also supported by the results of SEM (Figure 9, panels A and D) and stereomicroscopy (Table 1). Mechanism of pH-Sensitive Properties of the System. The CV response of Fc(COOH)2 at P(DEA-co-MAA) film electrodes was also very sensitive to the solution pH and demonstrated reversible ON−OFF behaviors with very narrow pH difference (Figure 4). This should be attributed to the pHresponsive property of the PMAA constituent in the films rather than the PDEA component because no pH-sensitive behavior was observed for PDEA polymers (Supporting Information, Figure S8A). In addition, the CVs of Fc(COOH)2 at bare PG electrodes showed no pH-dependent property (Supporting Information, Figure S8B), suggesting that the probe itself is not pH-sensitive. PMAA is one of the ionizable polyelectrolytes, which can take the structure change between the swollen and collapsed states in aqueous solution at its pKa mainly controlled by the balance between hydrogen bonding and electrostatic interaction.40,41 At the pH above its pKa, PMAA is negatively charged and the electrostatic repulsion between its negatively charged carboxylic groups (-COO−) makes PMAA take expanded conformation. Moreover, to fulfill the condition of electroneutrality, low molecular weight cations in solution need to move inside the network, which generates Donnan osmotic pressure within PMAA and also tends to expand the hydrogel.42 At the pH lower than pKa, however, large amounts of carboxylic acid groups of PMAA become protonated and noncharged, and the hydrogen bonds between these -COOH groups would become predominant, which makes PMAA take a collapsed structure. In addition, the hydrophobic interaction among unionized PMAA would also tend to form the compact structure.40 The structure change of P(DEA-co-MAA)-GOD films with pH was confirmed by SEM (Figure 9, panels A and C). The films after treatment with pH 7.4 buffers at 37 °C demonstrated a network structure with large pores, whereas the films after treatment with pH 6.2 buffers displayed a more compact 6658

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surface of PG electrodes by a simple one-step polymerization method at room temperature. It is the integration of PDEA and PMAA units in P(DEA-co-MAA)-GOD films that results in the reversible pH-, temperature-, SO42−-, and CO2-sensitive CV behavior of Fc(COOH)2 at the film electrodes. The ON−OFF effect of the system can be amplified by bioelectrocatalysis of glucose through GOD entrapped in the films with mediation of Fc(COOH)2 in solution. The multiple stimuli-responsive behavior of the system originates from the structure change of the copolymer films with the stimuli. Herein, the hydrogen bonding between PDEA and PMAA components in the films plays a central role in the interaction between them, which leads to the synergistic effect of temperature and pH on CV response of the system. It is the synergistic effect that makes the films at the ON state under the physiological conditions (37 °C and pH 7.4). This and the stimuli-responsive change of the structure of the films and the corresponding permeability toward small molecules in solution may provide a model or establish a foundation for controlled small drug delivery. The present work is the first study to combine the P(DEA-co-MAA) copolymer with electrochemistry and bioelectrocatalysis, and to construct a multisensitive switch at the physiological conditions. This provides a new avenue to build up the multiresponsive copolymer film system under the desired condition, so that the multiswitchable biosensors based on bioelectrocatalysis with enzymes can be fabricated.

group of the PMAA component (-COOH) and the amide group of the PDEA constituent (-CON(C2H5)2) under suitable conditions (Scheme 1). This type of hydrogen bond was also reported in the literature for similar systems.43,44 Scheme 1. Illustration of the Influence of the Synergistic Effect of Temperature and pH upon the Intermolecular Hydrogen Bonding between MAA and DEA Units

For example, when the system was at pH 6.2 and 37 °C, the copolymer films were at the OFF state, while after the pH was increased to 7.4 at the same temperature, the system became ON (Figure 6A). This is because the carboxylic acid groups of the PMAA component becomes ionized at pH 7.4, resulting in the breaking of hydrogen bonds not only between the two -COOH groups for PMAA but also between the -COOH group of PMAA and the -CON(C2H5)2 group of PDEA (Scheme 1). The electrostatic repulsion between negatively charged -COO− groups and the corresponding Donnan osmotic pressure of counterions inside the copolymer are beneficial to the expanded structure. In the process, water molecules enter into the films with counterions and the free -CON(C2H5)2 groups of PDEA tend to form hydrogen bonds with H2O, leading to the change of the PDEA component from hydrophobic to hydrophilic state. That is, the originally pH-insensitive PDEA constituent now may take the swollen structure at the same temperature when the pH increases to 7.4. All of these factors lead to the ON state of the system. On the other hand, to make the films return to the OFF state, the temperature may be increased to 55 °C at pH 7.4 (Figure 6A). At this higher temperature, the hydrogen bonding between the -CON(C2H5)2 groups of the PDEA component and the water molecules is destroyed, and the hydrophobic interaction between the PDEA backbones becomes predominant, making the copolymer have a strong tendency to take the compact structure after the water is squeezed out from the films. In the meantime, the available free -CON(C2H5)2 groups of PDEA now has a strong tendency to form hydrogen bonds with the -COOH groups of PMAA. This may induce some of -COO− groups to become protonated even at pH 7.4. In addition, the formation of more -COOH groups may further induce the hydrogen bonding between the two -COOH groups of the PMAA component. All of these would make the films take the shrunken OFF state. That is, the originally temperature-insensitive PMAA constituent now may become sensitive to the surrounding temperature because of its interaction with the PDEA component. Herein, the forming/ breaking of hydrogen bonds between the -CON(C2H5)2 group of PDEA and the -COOH group of PMAA plays a central role in the interaction between PDEA and PMAA components in the copolymer.

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ASSOCIATED CONTENT

S Supporting Information *

Text describing the FTIR characterization of P(DEA-co-MAA)GOD films and accompanying references, table listing the assignment of all characteristic IR absorption peaks of DEA, PDEA, P(DEA-co-MAA), P(DEA-co-MAA)-GOD, MAA, PMAA, and GOD samples, and figures showing the IR spectra of the preceding samples, the chemical structure of P(DEA-coMAA), UV−vis absorption spectra of GOD in pH 7.4 buffers, P(DEA-co-MAA)-GOD, and P(DEA-co-MAA) films, CVs of Fc(COOH)2 at bare PG electrodes, P(DEA-co-MAA)-GOD film electrodes, and PDEA film electrodes at 37 °C in pH 7.4 buffers, variation of CV Ipa of Fc(COOH)2 at 37 °C in pH 7.4 buffers with immersion time at Na2SO4 concentration switched between 0 and 0.4 M, at 37 °C in solution when pH was switched between pH 7.4 and pH 6.2, and at 37 °C in pH 7.4 buffers with alternative bubbling of CO2 and N2 for the same P(DEA-co-MAA)-GOD films, dependence of CV Ipa of Fc(COOH)2 on the solution temperature for PDEA film electrodes in solutions at pH 6.2 and pH 7.4, CVs of Fc(COOH)2 at 37 °C for PDEA film electrodes and bare PG electrodes in buffers at pH 6.2 and 7.4, respectively, CVs of Fc(COOH)2 at 37 °C for P(DEA-co-MAA)-GOD films in pH 7.4 buffers containing different concentrations of glucose, and CVs of hydroquinone at 37 °C for P(DEA-co-MAA)-GOD films in pH 7.4 and 6.2 buffers. This material is available free of charge via the Internet at http://pubs.acs.org.

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AUTHOR INFORMATION

Corresponding Author

*Tel.: (86)-10-58805498. E-mail: [email protected].

CONCLUSIONS In this work, MAA and DEA monomers are copolymerized into P(DEA-co-MAA) hydrogel films with entrapped GOD on the

Notes

The authors declare no competing financial interest. 6659

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ACKNOWLEDGMENTS The financial support from the Natural Science Foundation of China (NSFC, Grant 21105004) and the Major Research Plan of NSFC (Grant 21233003) is acknowledged.

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