Triply Responsive Films in Bioelectrocatalysis with a Binary

ARTICLE pubs.acs.org/JPCB

Triply Responsive Films in Bioelectrocatalysis with a Binary Architecture: Combined Layer-by-Layer Assembly and Hydrogel Polymerization Huiqin Yao†,‡ and Naifei Hu*,† † ‡

Department of Chemistry, Beijing Normal University, Beijing 100875, P. R. China Department of Chemistry, Ningxia Medical University, Yinchuan 750004, P. R. China

bS Supporting Information ABSTRACT: In this work, triply responsive films with a specific binary architecture combining layer-by-layer assembly (LbL) and hydrogel polymerization were successfully prepared. First, concanavalin A (Con A) and dextran (Dex) were assembled into {Con A/Dex}5 LbL layers on electrode surface by the lectin-sugar biospecific interaction between them. The poly (N,N-diethylacrylamide) (PDEA) hydrogels with entrapped horseradish peroxidase (HRP) were then synthesized by polymerization on the surface of LbL inner layers, forming {Con A/Dex}5
(PDEA-HRP) films. The films demonstrated reversible pH-, thermo-, and salt-responsive on
off behavior toward electroactive probe Fe(CN)63
in its cyclic voltammetric responses. This multiple stimuli-responsive films could be further used to realize triply switchable electrochemical reduction of H2O2 catalyzed by HRP immobilized in the films and mediated by Fe(CN)63
in solution. The responsive mechanism of the films was explored and discussed. The pH-sensitive property of the system was attributed to the electrostatic interaction between the {Con A/Dex}5 inner layers and the probe at different pH, and the thermoand salt-responsive behaviors should be ascribed to the structure change of PDEA hydrogels for the PDEA-HRP outermost layers under different conditions. The concept of binary architecture was also used to fabricate {Con A/Dex}5
(PDEA-GOD) films on electrodes, where GOD = glucose oxidase, which was applied to realize the triply switchable bioelectrocatalysis of glucose by GOD in the films with ferrocenedicarboxylic acid as the mediator in solution. This film system with the unique binary architecture may establish a foundation for fabricating a novel type of multicontrollable biosensors based on bioelectrocatalysis with immobilized enzymes.

’ INTRODUCTION In recent years, stimuli-responsive interfaces, also known as “smart” or intelligent surfaces, have attracted a great deal of attention among researchers because of their potential applications in biosensors, drug delivery, microfluid devices, permselective membrane, bioseparation, and so on.1
4 The stimuli include pH, temperature, salt, electric field, light, magnetic field, and specific chemicals.1
7 In this regard, the interfaces that respond to more than one stimulus are particularly interesting owing to their relevance to physiological and biological systems.7,8 In the living processes, different external signals may simultaneously induce the same alteration, such as the change of protein conformation, gating of ions across a cell membrane, changes in the permeability of human skin, and the on
off of biocatalytic reactions involving enzymes. In comparison with the commonly used single stimulus-responsive system, the multistimuli-responsive system adds new dimensions to the field, which not only increases the complexity of the system but also opens up new opportunity in research. There are four types of multiresponsive surfaces according to the properties and constituents of the responsive materials. (i) Some specific single polymers can r 2011 American Chemical Society

change their structure or volume with two different stimuli because of the intrinsic characters of the homopolymers.9
12 For example, poly(N-isopropylacrylamide) (PNIPAm) films fabricated on a Au electrode surface via the electrochemical polymerization demonstrated both thermo- and salt-sensitive structure and permeation alteration.12 (ii) Some block copolymers synthesized by two or more different stimuli-responsive block components can exhibit multiresponsive properties.13
16 For instance, p(NIPAm-co-AA) copolymer electrochemically synthesized by combining temperature-sensitive PNIPAm and pH-responsive poly(acrylic acid) (PAA) displayed the sensitive change of ionic permeability with temperature, ionic strength, and pH in solution.14 (iii) Some interpenetrating polymeric networks (IPN) synthesized with two different stimuli-responsive constituents can show multiresponsive properties.16
20 For example, IPN hydrogels prepared by combination of thermosensitive PNIPAm and pH-sensitive poly(methacrylic acid) Received: January 14, 2011 Revised: April 15, 2011 Published: May 03, 2011 6691

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The Journal of Physical Chemistry B (PMAA) exhibited pH- and temperature-responsive swelling.20 (iv) The binary architecture that combines two different kinds of stimuli-responsive layers on the same solid surface can create the multisensitive interfaces.21 Advincula and co-workers assembled pH-sensitive {PAH/PAA}n layer-by-layer assembly (LbL) films on solid surfaces, where PAH = poly(allylamine hydrochloride) and then polymerized thermo-responsive PNIPAm brushes on the top of the inner multilayers, forming the composite films with binary architecture that showed both pH- and temperatureresponsive ionic permeability.21 Herein, the idea of binary architecture opens a novel and general avenue to fabricate multiresponsive interfaces in a simple and convenient way and with a variety of feasibility. Inspired by the concept of binary architecture proposed by Advincula,21 we intended in this work to construct a triply responsive surface that combined the pH-sensitive {Con A/Dex}n LbL films assembled by concanavalin A (Con A) and dextran (Dex) with the thermo- and salt-responsive poly(N,Ndiethylacrylamide) (PDEA) hydrogel films. The {Con A/Dex}n LbL films reported in our previous work showed pH-sensitive permeability toward ionic probes22 and thus were used in this study as a pH-responsive component. PDEA is a type of N-substituted polyacrylamides (Figure 1) and exhibits a reversible phase transition in aqueous solution at the lower critical solution temperature (LCST) of around 31 °C.23
25 Below the LCST, PDEA takes an expanded coil conformation mainly due to the formation of hydrogen bonds between its amide groups and water molecules, making the polymer become hydrophilic. As the temperature increases above the LCST, the polymer takes a collapsed globule structure since the hydrogen bonds between PDEA and water molecules are disrupted, and the hydrophobic aggregation of PDEA predominates, causing a drastic decrease in its volume. In addition, the PDEA hydrogels are also sensitive to the type and concentration of salts added in solution, and the same phase transition from the extended coil state to the compact globule state can be induced by the specific ions with appropriate concentration.26
28 Compared with the most studied thermoresponsive PNIPAm hydrogels, PDEA demonstrates similar stimuli-responsive behaviors but with better biocompatibility and lower toxicity.27,29,30 Thus, in the present study, PDEA hydrogel layers were used as a dual temperature- and saltresponsive component. We expected that the combination of pH-responsive {Con A/Dex}n LbL films and the thermo- and salt-responsive PDEA hydrogels in the binary architecture would provide a foundation for developing the triply responsive surfaces. The bioelectrocatalysis on the basis of various enzymatic reactions is an important foundation in the development of electrochemical biosensors. In particular, the stimuli-controllable bioelectrocatalysis may enable its application not only in switchable biosensors, biofuel cells, and other bioelectronic devices, but also in signal transduction/amplification and information storage/processing.31
34 Up to now, while the switchable bioelectrocatalysis has been reported, most of them have been restricted to that with the single stimulus.31
34 The works on multiswitchable bioelectrocatalysis have been very limited until now.11,12 For example, in our recent report, poly(4-vinylpyridine) films were electropolymerized on the surface of electrodes, the reversible on
off electrochemical oxidation of glucose catalyzed by glucose oxidase (GOD) and mediated by ferrocenedicarboxylic acid (Fc(COOH)2) could be simultaneously triggered and modulated by pH and perchlorate concentration in solution.11

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Figure 1. Chemical structure of PDEA.

In the present work, {Con A/Dex}5 multilayers were assembled on the surface of pyrolytic graphite (PG) electrodes, PDEA hydrogels containing horseradish peroxidase (HRP) were then polymerized on their surface, forming the {Con A/Dex}5
(PDEA-HRP) films with the binary architecture. Herein, HRP was entrapped in PDEA hydrogel layers so that the enzyme could be immobilized on the electrode surface. It is well-known that the immobilization of enzymes on the surface of electrodes is an important and necessary step to fabricate biosensors. The electroactive probe Fe(CN)63
demonstrated pH-, temperature-, and SO42
-sensitive on
off cyclic voltammetric (CV) response at the {Con A/Dex}5
(PDEA-HRP) film electrodes. This triply switchable CV behavior of the probe for the films could be further employed to control and modulate the electrochemical reduction of H2O2 catalyzed by HRP immobilized in the films with Fe(CN)63
as the mediator in solution. The mechanism of the multiswitchable permeability of the films toward the probe was discussed and explored by a series of comparative experiments. To the best of our knowledge, this is the first report on multicontrollable bioelectrocatalysis on the basis of binary architecture. The present work provided a novel model system to combine the concept of binary architecture and the multiresponsive interfaces, which may establish a foundation for constructing the multiswitchable biosensors based on bioelectrocatalysis. Herein, we have to emphasize that the present study represents only the proof of the concept, a lot of theoretical and experimental works will be needed before the practical application of the system. However, this work did represent the first important step toward the development of a novel type of multiswitchable biosensors based on bioelectrocatalysis.

’ EXPERIMENTAL SECTION 1. Reagents. Chitosan (CS, the degree of deacetylation is more than 85%, Mw ≈ 200 000), concanavalin A extracted from Jack beans (Con A, type V, Mw ≈ 104 000), dextran (Dex, Mw ≈ 200 000), horseradish peroxidase (HRP, E.C. 1.11.1.7, type II, Mw ≈ 44 000, 250 000 units g
1), glucose oxidase (GOD, E.C. 1.1.3.4, type VII, Mw ≈ 160 000, 192 000 units g
1), 1,10 ferrocenedicarboxylic acid (Fc(COOH)2), ferrocenemethanol (FcOH), hexaammineruthenium(III) chloride (Ru(NH3)6Cl3), tris(hydroxymethyl) aminomethane (Tris), N,N0 -methylenebisacrylamide (BIS), and N,N,N0 ,N0 -tetramethylenediamine (TEMED) were obtained from Sigma
Aldrich. N,N-Diethylacrylamide (DEA) was purchased from TCI. Sodium persulphate (Na2S2O8) was purchased from Aladdin. Potassium ferricyanide (K3Fe(CN)6), potassium ferrocyanide (K4Fe(CN)6), sodium sulfate (Na2SO4), sodium nitrate (NaNO3), sodium chloride (NaCl), sodium bromide (NaBr), and hydrogen peroxide (H2O2, 30%) were obtained from Beijing Chemical Engineering Plant. The dilute H2O2 aqueous solutions were freshly prepared before being used. Glucose was obtained from Beijing Yili Fine Chemicals, and the D-glucose stock solutions were allowed to 6692

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The Journal of Physical Chemistry B mutarotate at room temperature for 24 h before being used. All other reagents were of analytical grade. Buffers were usually 0.1 M sodium acetate (pH 4.0
6.0) or 0.05 M potassium dihydrogen phosphate (pH 6.5
8.0), all containing 0.1 M NaCl. The pH of buffers was adjusted to the desired value with dilute HCl or NaOH solutions. 0.1 M Tris-HCl buffers at pH 7.4 containing 0.1 M NaCl, 1 mM MnCl2, and 1 mM CaCl2 were used to prepare Con A solutions.35 All solutions were prepared with water purified twice successively by ion exchange and distillation. 2. Assembly of {Con A/Dex}5 Multilayers. The assembly of {Con A/Dex}5 multilayers on basal plane pyrolytic graphite (PG, Advanced Ceramics, geometric area 0.16 cm2) disk electrodes was the same as in our previous work.35 In brief, the PG electrodes were first immersed in positively charged CS solutions (pKa ≈ 6.5,36 1 mg mL
1, pH 5.0) for 20 min to adsorb CS as the precursor layer. The PG/CS electrodes were then sequentially immersed into negatively charged Con A (pI ≈ 5.0,37 1 mg mL
1, pH 7.4) and neutral Dex (1 mg mL
1, pH 7.4) solutions for 30 min each with intermediate water rinsing and air stream drying, forming a Con A/Dex bilayer mainly by the biospecific binding between them. This cycle was repeated for five times, so that the {Con A/Dex}5 multilayers were formed on the PG/CS surface. 3. Preparation of PDEA-HRP Hydrogel Layers onto {Con A/Dex}5 Multilayers. PDEA hydrogel layers containing HRP, designated as PDEA-HRP, were prepared on the surface of {Con A/Dex}5 layers according to the polymerization procedure reported previously38
40 with some modification. After the {Con A/Dex}5 multilayer electrode was placed in a sealed bottle under a high-purity N2 atmosphere for at least 10 min, 4 μL of the pregel solution was cast on the layer surface via a syringe. Herein, the typical pregel solution containing 0.5 mg mL
1 HRP, 0.5 M DEA monomer, 1.5 mg mL
1 BIS cross-linker, 0.4 mg mL
1 Na2S2O8 initiator, and 0.46 mg mL
1 TEMED accelerator was freshly prepared every time and deaerated with N2 before being cast. The PDEA-HRP outermost layers were then formed on the surface of {Con A/Dex}5 inner layers in about 10 min. During the whole polymerization process, the N2 atmosphere was kept in the sealed bottle. The formed {Con A/Dex}5
(PDEA-HRP) film electrode was then immersed in water for about 10 min to remove the unreacted chemicals. 4. Apparatus and Procedures. A CHI 660A or CHI 621B electrochemical workstation (CH Instruments) was used for electrochemical measurements. A typical three-electrode cell was used with a saturated calomel electrode (SCE) as the reference, a platinum flake as the counter, and the PG disk electrode with films as the working electrode. The solution was purged with high-purity nitrogen for at least 10 min before electrochemical measurements. The nitrogen atmosphere was then kept above the cell for the entire experiment. The pH measurements were performed with PHSJ-3F pHmeter (Shanghai Precision & Scientific Instruments). The temperature of solutions in the cell was controlled by an HH-S thermostatic bath (Zhengzhou Greatwall Scientific Industrial and Trade Co.) with precision of (0.2 °C. The Fourier-transform infrared (FTIR) spectra were recorded at a resolution of 4 cm
1 by Nexus 670 Fourier-transform infrared spectrometer (Nicolet). The UV
vis absorption spectra were collected with a TU-1900 UV
vis double-beam spectrometer (Beijing Purkinje General Instrument). The thickness of hydrogel films was estimated with a Stereo Discovery V12 stereomicroscope

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Figure 2. FTIR spectra of (a) HRP, (b) DEA, (c) PDEA, and (d) PDEA-HRP samples.

equipped with an AxioCam digital camera (Zeiss). The surface SEM of the films was obtained by an S-4800 scanning electron microscope (Hitachi) with an acceleration voltage of 5 kV. The {Con A/Dex}5
(PDEA-HRP) films assembled on PG/CS electrodes were used as the SEM sample. The samples after treated with different pH, temperatures, and Na2SO4 concentrations were transferred into liquid nitrogen immediately to “freeze” the structure, followed by the freeze-drying in a FD-3 freeze drier (Beijing Boyikang Experimental Instrument) for 20 h until all water in the films was sublimed. Before SEM imaging, the sample surface was coated by thin platinum films with an E-1045 sputtering coater (Hitachi).

’ RESULTS AND DISCUSSION 1. Preparation of {Con A/Dex}5
(PDEA-HRP) Films on Electrodes. The driving force of assembly of {Con A/Dex}5 LbL

films on PG/CS surface was mainly the lectin-sugar biospecific interaction between Con A and Dex, and the successful assembly of the LbL films was confirmed by various approaches, including quartz crystal microbalance, electrochemical impedance spectroscopy, and CV in our previous works.22,35 PDEA hydrogel layers containing HRP on the surface of {Con A/Dex}5 inner layers were prepared by free radical polymerization according to the procedure reported in the literature38
40 with some modification. The formation of PDEA-HRP layers was confirmed by FTIR spectroscopy in comparison with pure DEA, PDEA, and HRP samples (Figure 2). All characteristic IR peaks of the pure PDEA sample were observed at the same or similar positions for PDEA-HRP films and consistent with those reported by the literature.24,41,42 The characteristic CdC stretching band at 1609 cm
1 observed for DEA monomer was not detected for PDEA and PDEA-HRP, indicating that the DEA monomer is polymerized to PDEA in PDEA-HRP. The amide II band at 1540 cm
1 for HRP43 was not observed for DEA and PDEA but detected for PDEA-HRP, suggesting that HRP is successfully entrapped in the PDEA-HRP hydrogel layers. The Soret absorption band of heme proteins in UV
vis spectra is a characteristic probe for the existence of HRP in the 6693

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Figure 3. (A) CVs of 1.0 mM K3Fe(CN)6 for {Con A/Dex}5
(PDEA-HRP) films at 0.05 V s
1 and 25 °C in buffers at pH (a) 4.0, (b) 5.0, (c) 5.5, (d) 6.0, (e) 6.5, (f) 6.8, and (g) 7.2. (B) Variation of CV Ipc with immersion time (t) in solution when pH switched between pH 4.0 (9) and 7.2 (red b) for the same films.

synthesized PDEA films. The Soret peak was observed at 403 nm for PDEA-HRP hydrogels (Supporting Information, Figure S1, curve c) with the same position for HRP in solution (curve b), indicating that HRP is successfully entrapped in the PDEA hydrogels and retains its native conformation. CV with Fe(CN)63
as the electroactive probe was used to confirm the attachment of PDEA-HRP layers on the {Con A/Dex}5 multilayer surface (Supporting Information, Figure S2). In pH 4.0 buffers, Fe(CN)63
displayed a well-defined and nearly reversible CV peak pair at about 0.17 V at {Con A/Dex}5 film electrodes. After PDEA-HRP layers were formed on the surface of {Con A/Dex}5 layers, the CV response of the probe decreased obviously. This is because a barrier of PDEAHRP is formed on the multilayer surface and the diffusion of the probe through the whole films becomes more difficult. The CV response time of Fe(CN)63
at {Con A/Dex}5
(PDEA-HRP) film electrodes was greatly influenced by the amount of DEA monomer used in the polymerization. The amount of DEA also reflects the thickness and the surface density of PDEA-HRP layers. The increase of the amount of DEA monomer in the polymerization could result in the thicker hydrogel layers and the higher density of the monomer on the electrode surface. Herein, the response time is defined as the duration between the immersion of the film electrode into Fe(CN)63
solution and the reaching of the steady state of the CV reduction peak current (Ipc) of the probe. The response time increased with the amount of DEA in polymerization (Supporting Information, Figure S3A), implying that the diffusion of the probe through the films becomes more difficult when the hydrogel layers become thicker. Considering that the shorter response time was preferred for the present work, 2 μmol of DEA were usually used in the polymerization in the following study. In this case, the surface density of DEA was about 1.96  10
3 mol cm
2 and the average thickness of the films in water estimated by stereomicroscopy was 220 ( 30 μm at 25 °C. Under this condition, the response time was about 2 min, as demonstrated in continuous CVs at 0.05 V s
1 between
0.2 and 0.6 V (Supporting Information, Figure S3B), where the CV response of Fe(CN)63
initially increased with the cycle number (n) and then reached the steady state when n g 5. The stability of {Con A/Dex}5
(PDEA-HRP) films was examined by CV with Fe(CN)63
as the probe. The films were stored in pH 4.0 blank buffers for most of the storage time, and periodically placed in Fe(CN)63
solutions at pH 4.0 and 25 °C for CV testing. After two weeks of storage, the CV peak potentials and currents of the probe maintained nearly the same as their initial values, suggesting that the films are considerably stable.

2. pH-Sensitive Switching Behavior of {Con A/Dex}5
(PDEA-HRP) Films. At {Con A/Dex}5
(PDEA-HRP) film

electrodes, the CV behavior of Fe(CN)63
in buffers was very sensitive to the solution pH at 25 °C (Figure 3A). At pH 4.0, the CV signal of Fe(CN)63
was quite large, but it decreased drastically when the pH increased from 4.0 to 7.2, accompanied by the increase of peak separation (ΔEp) (Supporting Information, Figure S4). At pH 7.2, the CV response of the probe even could hardly be observed. Thus, the {Con A/Dex}5
(PDEAHRP) films were at the on state at pH 4.0 and at the off state at pH 7.2. This pH-sensitive on
off property of the system was reversible. By switching the film electrode in solution of Fe(CN)63
between pH 4.0 and 7.2, the Ipc was changed periodically between a considerably high value and a very small one, and this on
off cycle could be repeated for many times with the response time of about 2 min (Figure 3B). In addition, both reduction and oxidation peak currents of the probe showed a linear relationship with square root of scan rates from 0.05 to 1.6 V s
1 at pH 4.0 and 25 °C (Supporting Information, Figure S5), suggesting the diffusion-controlled behavior of the probe. In our previous work,22 {Con A/Dex}4 films alone also demonstrated the pH-sensitive CV behavior toward Fe(CN)63
, and the mechanism was ascribed to the electrostatic interaction between the probe and the films at different pH. We thus speculate that the pH-responsive on
off property of {Con A/Dex}5
(PDEA-HRP) films toward Fe(CN)63
should be mainly attributed to the {Con A/Dex}5 inner layers of the films and have little relationship with the PDEA-HRP outermost layers. To support this speculation, the PDEA-HRP films were polymerized directly on the PG electrode surface, and the CV behavior of Fe(CN)63
was tested for the films at pH 4.0 and 7.2, respectively. The results showed that there was no substantial difference in CV response between these two pH values at the PDEA-HRP film electrode (Supporting Information, Figure S6), indicating that the PDEAHRP films are pH-insensitive. To further support our speculation, the surface morphology of {Con A/Dex}5
(PDEA-HRP) films after treated with solutions at different pH was examined and compared by SEM (Figure 4, panels A and B). Since the PDEAHRP layer of the films was located on the film surface, the top-view SEM actually reflected the topography of the PDEA-HRP outermost layer. The films treated with pH 4.0 buffers displayed the surface morphology essentially the same as those treated with pH 7.2 buffers, suggesting that the solution pH has no significant influence on the structure of PDEA-HRP layers at least with the present magnification. Moreover, the PDEA-HRP layers were very porous with a lot of micrometer-sized pores or channels, which would allow the small probe like Fe(CN)63
to go through the 6694

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Figure 4. SEM top views of {Con A/Dex}5
(PDEA-HRP) films assembled on PG/CS surface after the films were treated with 1.0 mM K3Fe(CN)6 buffers for 5 min at (A) pH 4.0 and 25 °C, (B) pH 7.2 and 25 °C, (C) pH 4.0 and 34 °C, and (D) pH 4.0 and 25 °C with 0.26 M Na2SO4.

layers and reach the underneath {Con A/Dex}5 layers very easily. All these results imply that the pH-sensitive behavior of {Con A/ Dex}5
(PDEA-HRP) films toward Fe(CN)63
is actually decided by the {Con A/Dex}5 inner layers, and the mechanism must be the same as that for the simple {Con A/Dex}4 films reported previously.22 The pI value of Con A is at about 5.0,37 and Dex is a neutral polymer and its property is pH-independent. At pH 4.0, the Con A in the films carries net positive surface charges, and would have a strong electrostatic attraction with negatively charged Fe(CN)63
in solution, thus making the probe go through the {Con A/Dex}5 inner layers of the films very easily and leading to the quite large CV response. In contrast, at pH 7.2, the Con A in the films is negatively charged, and would have a strong electrostatic repulsion with Fe(CN)63
. This may hinder the probe from entering the {Con A/Dex}5 inner layers and restrict the electron exchange of the probe with underlying electrodes, thus resulting in the very small CV signal. To further support the mechanism, other electroactive probes with different charges were investigated and compared (Supporting Information, Figure S7). For positively charged Ru(NH3)63þ, its pH-dependent CV behavior for the films was also observed but the direction was opposite to that of Fe(CN)63
. For neutral probe FcOH, no pH-sensitive CV property for the films was observed. It can thus be concluded that the pH-sensitive property of Fe(CN)63
for the {Con A/Dex}5
(PDEA-HRP) films should be mainly attributed to the electrostatic interaction between the Con A component in the films and the probe in solution. 3. Temperature-Sensitive On
Off Property of {Con A/ Dex}5
(PDEA-HRP) Films. The CV response of Fe(CN)63
at {Con A/Dex}5
(PDEA-HRP) film electrodes was also very sensitive to the environmental temperature (Supporting Information, Figure S8). When the solution temperature was set below 31 °C, the CV signal of Fe(CN)63
in pH 4.0 buffers was

Figure 5. CVs of 1.0 mM K3Fe(CN)6 at 0.05 V s
1 in pH 4.0 buffers for {Con A/Dex}5
(PDEA-HRP) films at (a) 25 and (b) 34 °C. Inset: influence of solution temperatures (T) on CV Ipc.

quite large; however, the peak currents decreased greatly when the temperature was higher than 31 °C. The critical phase transition temperature was observed at about 31 °C in the Ipc versus temperature curve (Figure 5, inset), consistent with the LCST of PDEA in aqueous solution.23
25 As a control, the {Con A/Dex}5 films without PDEA-HRP surface layers in pH 4.0 buffers showed no phase transition with temperature in the same range (Supporting Information, Figure S9). All these results demonstrate that the thermo-sensitive CV behavior of {Con A/ Dex}5
(PDEA-HRP) films toward the probe should be attributed to the PDEA-HRP outermost layers instead of {Con A/Dex}5 inner layers of the films. Herein, the PDEA hydrogel component of the films plays a decisive role and the HRP entrapped in the PDEA layers has little influence on the thermo-responsive property of the PDEA layers. To study the thermo-responsive on
off property of {Con A/ Dex}5
(PDEA-HRP) films toward Fe(CN)63
at pH 4.0, two 6695

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The Journal of Physical Chemistry B typical temperatures, 25 and 34 °C, were selected in this work. At 25 °C, Fe(CN)63
showed a well-defined and nearly reversible CV peak pair with quite large peak currents for the films, indicating that the films were at the on state (Figure 5, curve a). At 34 °C, the CV peak currents were significantly suppressed, and the films were at the off state (Figure 5, curve b). This thermosensitive switching behavior of the films was quite reversible and could be repeated for many times, and the corresponding response time was about 2 min when the films were transferred from 25 to 34 °C, and around 3 min from 34 to 25 °C (Supporting Information, Figure S10). PDEA is a typical thermo-sensitive hydrogels and can undergo the phase transition or conformation change between the coil and globule states in water at its LCST.23,26,41,42 This can be used to explain the temperature-responsive switching behavior of the {Con A/Dex}5
(PDEA-HRP) films toward Fe(CN)63
. At 25 °C, which is below the LCST of PDEA, the PDEA component in the films absorbs a great amount of water and takes the swollen and expanded coil state, mainly because of the predominant hydrogen bonding between amide groups of PDEA and water molecules in solution. Thus, the probe can diffuse through the films easily and exchange electrons with underlying electrodes, displaying the large CV response. When the temperature goes above the LCST such as 34 °C, most of the hydrogen bonds between the PDEA and water molecules are broken, and PDEA takes the shrunken and compact globule structure after the water molecules are squeezed out of the films, leading to the difficulty of the probe going through the films and the corresponding very small CV signal. This was also supported by the change of film thickness with temperature in water. With the fixed surface area of electrodes, the average thickness of the films at 34 °C (145 ( 20 μm) was much smaller than that at 25 °C (220 ( 30 μm). The surface structure change of {Con A/Dex}5
(PDEAHRP) films at different temperatures was further confirmed by SEM (Figure 4, panels A and C). The films treated at 25 °C presented the network structure with many microsized pores and channels, whereas the films treated at 34 °C displayed a much more compact and smooth surface with much smaller pore size. The obvious difference in surface morphology of the {Con A/Dex}5
(PDEA-HRP) films treated between 25 and 34 °C actually reflects the structure change of PDEA in the outermost layers. It is noteworthy that the {Con A/Dex}5 inner layers of the films would not influence the thermo-sensitive property of the whole films not only because the {Con A/Dex}5 layers are thermo-independent, but also the pH of the Fe(CN)63
solution is set at 4.0, where the {Con A/Dex}5 inner layers are always at the on state. 4. Salt-Sensitive On
Off Property of {Con A/Dex}5
(PDEA-HRP) Films. The CV response of Fe(CN)63
at {Con A/Dex}5
(PDEA-HRP) film electrodes was also sensitive to the concentration of salt in solution. Taking Na2SO4 as an example, under the condition of 25 °C and pH 4.0, the increase of Na2SO4 concentration in Fe(CN)63
solution would cause the CV peaks of the probe to decrease dramatically and even to disappear (Supporting Information, Figure S11). This Na2SO4-sensitive CV behavior of the films toward the probe could be used to examine the switching property of the system. When the testing solution contained no Na2SO4 at 25 °C and pH 4.0, the probe showed a quite large CV peaks for the films (Figure S11, curve a), and the films were at the on state. When the solution contained 0.26 M Na2SO4 under the same conditions, the CV peaks of the probe even could not be detected, and the films were at the off

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Figure 6. Dependence of CV Ipc of 1.0 mM K3Fe(CN)6 at 0.05 V s
1 in pH 4.0 buffers at 25 °C on salt concentration of (a) Na2SO4, (b) NaCl, (c) NaBr, and (d) NaNO3 for {Con A/Dex}5
(PDEA-HRP) films.

state (Figure S11, curve f). This on
off behavior was reversible and could be repeated for many times with the response time of about 3 min in the transition between 0 and 0.26 M Na2SO4 (Supporting Information, Figure S12). In comparison, for {Con A/Dex}5 films with no PDEA-HRP surface layers, the CV response of Fe(CN)63
demonstrated no essential difference in pH 4.0 buffers with and without 0.26 M Na2SO4 at 25 °C (Supporting Information, Figure S13), indicating that the Na2SO4-sensitive CV behavior of the probe at {Con A/Dex}5
(PDEA-HRP) film electrodes has nothing to do with the {Con A/Dex}5 inner layers and should be ascribed to the PDEA-HRP outermost layers. The mechanism of salt-induced phase transition of PDEA is similar to that induced by temperature,26,27 which can be used to explain the Na2SO4-sensitive CV behavior of the {Con A/Dex}5
(PDEA-HRP) films toward Fe(CN)63
. The addition of Na2SO4 in solution may cause the disruption of the hydrogen bonding between PDEA and water molecules, and induce the transition of PDEA from the swollen coil state to the shrunken globule state, resulting in the block of the probe to go through the films and the corresponding decrease of CV response. The structure change of the films caused by Na2SO4 could also be observed by SEM (Figure 4, panels A and D). Other salts, such as NaCl, NaBr and NaNO3, could also induce the phase transition or structure change of PDEA layers in {Con A/Dex}5
(PDEA-HRP) films, which could be monitored by CV of Fe(CN)63
(Figure 6). Herein, the major difference among these different salts lies in their different critical phase transition concentration. Na2SO4, NaCl, NaBr, and NaNO3 contain the same Naþ cation but different anions, suggesting that the anion is more sensitive than cation in inducing the phase transition of PDEA. The critical phase transition concentration of the anions showed the sequence of SO42
(0.15 M) < Cl
(0.42 M) < Br
(0.86 M) < NO3
(1.10 M), consistent with the Hofmeister series.44,45 Considering that the SO42
showed the lowest critical concentration among these anions and the HRP enzyme in the films might alter its conformation and lose its bioactivity in the presence of high concentration of salt in solution, Na2SO4 was selected as the model salt in this work to investigate the salt-responsive switching property of the system. 5. Bioactivity of HRP in {Con A/Dex}5
(PDEA-HRP) Films. One of our major concerns for the {Con A/Dex}5
(PDEAHRP) films was whether the HRP enzyme immobilized in the films could retain its native structure and bioactivity. From the FTIR (Figure 2) and UV
vis (Supporting Information, Figure S1) spectra, we speculate that the HRP in the films essentially retains its 6696

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1

Figure 7. CVs for {Con A/Dex}5
(PDEA-HRP) films at 0.01 V s in pH 4.0 buffers containing 1.0 mM K3Fe(CN)6 and (a) 0, (b) 0.08, (c) 0.14, (d) 0.18, (e) 0.22, (f) 0.26, and (g) 0.32 mM H2O2 at 25 °C. Inset: dependence of CV Ipc on concentration of H2O2.

Figure 8. CVs of 1.0 mM K3Fe(CN)6 at 0.01 V s
1 for {Con A/ Dex}5
(PDEA-HRP) films in solutions containing 0.32 mM H2O2 at (a) pH 4.0 and 25 °C, (b) pH 7.2 and 25 °C, (c) pH 4.0 and 34 °C, and (d) pH 4.0 and 25 °C with 0.26 M Na2SO4.

original conformation. To further support this point of view, the biocatalytic activity of HRP in the films toward H2O2 was tested by CV with Fe(CN)63
as the electron transfer mediator. When H2O2 was added into the Fe(CN)63
solution at pH 4.0 containing no Na2SO4 at 25 °C, in comparison with the system in the absence of H2O2, the CV reduction peak of Fe(CN)63
for the films increased dramatically, accompanied by the decrease or even disappearance of the oxidation peak (Figure 7). The CV reduction peak (Ipc) increased initially with the concentration of H2O2 in solution in the range of 0.05
0.32 mM and then tended to level off (Figure 7, inset). All these are characteristic of electrochemical reduction of H2O2 catalyzed by HRP immobilized in the films and mediated by Fe(CN)63
in solution, and the mechanism can be expressed by the following equations:46
48 H2 O2 þ HRPðredÞ f H2 O þ HRPðoxÞ

ð1Þ

HRPðoxÞ þ 2FeðCNÞ6 4
f HRPðredÞ þ 2FeðCNÞ6 3
ð2Þ FeðCNÞ6 3
þ e
S FeðCNÞ6 4
at electrode

ð3Þ

where HRP(red) stands for the HRP-Fe(III) form and HRP(ox) usually represents the radical intermediate with oxidation state þ5, known as Compound I. The typical electrocatalytic behavior of the system proves that the HRP immobilized in the films retains its bioactivity. 6. Triply Switchable Electrocatalytical Reduction of H2O2 by {Con A/Dex}5
(PDEA-HRP) Films with Fe(CN)63
as Mediator. The pH-, thermo-, and SO 4 2
-sensitive switching property of {Con A/Dex}5
(PDEA-HRP) films toward Fe(CN)63
inspired us to use this system to control or modulate the electrocatalytic reduction of H2O2 by HRP entrapped in the films with these three stimuli. For example, in pH 4.0 solutions containing Fe(CN)63
and H2O2 in the absence of Na2SO4 at 25 °C, the films demonstrated a large electrocatalytic reduction peak (Figure 8, curve a). However, when the films were placed in pH 7.2 buffers containing the same amount of Fe(CN)63
and H2O2, the electrocatalytic response became quite small (Figure 8, curve b). This is because the films become “closed” to the probe at pH 7.2, resulting in the interruption of the catalytic cycles. The electrocatalysis was “open” at pH 4.0 and “closed” at pH 7.2, and this pH-sensitive on
off bioelectrocatalysis for the system could be repeated for at least several cycles by

Figure 9. Dependence of CV electrocatalytic Ipc at 0.01 V s
1 in solutions containing 1.0 mM K3Fe(CN)6 and 0.32 mM H2O2 on testing step when the system was switched between (A) pH 4.0 and 7.2 at 25 °C, (B) 25 and 34 °C at pH 4.0, and (C) 0 and 0.26 M Na2SO4 at pH 4.0 and 25 °C for {Con A/Dex}5
(PDEA-HRP) films.

switching the same films in the Fe(CN)63
þ H2O2 solutions between pH 4.0 and 7.2 (Figure 9, panel A). The bioelectrocatalysis of H2O2 for the system could also be switched by temperature and SO42
concentration (Figure 8). This on
off bioelectrocatalysis was also reversible and could be repeated for many times (Figure 9, panels B and C). If Ipc/on and Ipc/off were defined as the reduction peak currents at the on and off state, respectively, the Ipc/on/Ipc/off ratio could be amplified by the electrocatalysis. For the same {Con A/Dex}5
(PDEA-HRP) films in solutions containing only Fe(CN)63
, the Ipc/on/Ipc/off ratio was about 7.0, while in the presence of 0.32 mM H2O2, the ratio was usually increased to about 50. 7. Triply Responsive Electrocatalytical Oxidation of Glucose by {Con A/Dex}5
(PDEA-GOD) Films with Fc(COOH)2 as Mediator. The design of binary architecture, which combines LbL and hydrogel polymerization, is the key to develop the triply responsive films in this work. The multisensitive on
off in electrochemical reduction of H2O2 catalyzed by HRP immobilized in 6697

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ARTICLE

Figure 10. (A) CVs of 0.3 mM Fc(COOH)2 at 0.05 V s
1 for {Con A/Dex}5
(PDEA-GOD) films in buffers at (a) pH 4.5 and 25 °C, (b) pH 7.2 and 25 °C, (c) pH 4.5 and 34 °C, and (d) pH 4.5 and 25 °C with 0.26 M Na2SO4. (B) CVs for {Con A/Dex}5
(PDEA-GOD) films at 0.01 V s
1 in solutions containing 0.3 mM Fc(COOH)2 and 11.0 mM glucose at (a) pH 4.5 and 25 °C, (b) pH 7.2 and 25 °C, (c) pH 4.5 and 34 °C, and (d) pH 4.5 and 25 °C with 0.26 M Na2SO4.

{Con A/Dex}5
(PDEA-HRP) films and mediated by Fe(CN)63
in solution was thus realized. This concept not only is novel but also demonstrates some generality, and should be extended to other bioelectrocatalysis system. For example, PDEA hydrogel layers containing GOD designated as PDEA-GOD were synthesized with the same free radical polymerization method on the surface of {Con A/Dex}5 multilayers, forming {Con A/Dex}5
(PDEAGOD) films on PG/CS electrodes. The negatively charged probe Fc(COOH)2 showed pH-, temperature-, and SO42
-sensitive CV on
off behavior at the film electrodes (Figure 10A). The electrochemical oxidation of glucose catalyzed by GOD immobilized in the films and mediated by Fc(COOH)2 in solution was observed, and the corresponding triply switchable bioelectrocatalysis of glucose was realized (Figure 10B). This on
off behavior in bioelectrocatalysis for the system was reversible and could be repeated for many times (Supporting Information, Figure S14).

’ CONCLUSIONS The triply responsive {Con A/Dex}5
(PDEA-HRP) films with the unique binary architecture are successfully prepared on electrodes through the combination of the pH-sensitive {Con A/Dex}5 inner layers and the thermo- and salt-sensitive PDEAHRP hydrogel surface layers. The films exhibit reversible pH-, temperature-, and SO42
-dependent CV on
off behavior toward Fe(CN)63
. The mechanism of the stimuli-responsive behavior of the inner and outermost layers is different. For the {Con A/Dex}5 inner layers, the pH-sensitive property is attributed to the electrostatic interaction between the layers and the probe at different pH; for the PDEA-HRP outermost layers, however, the thermo- and salt-sensitive behavior is ascribed to the structure change of PDEA at different temperatures and sulfate concentrations. This multitriggered switch can be used to realize the triply controllable electrochemical reduction of H2O2 catalyzed by HRP immobilized in the films and mediated by Fe(CN)63
in solution. Similarly prepared {Con A/Dex}5
(PDEA-GOD) films can be applied to realize the triply switchable electrochemical oxidation of glucose catalyzed by GOD in the films with Fc(COOH)2 as the mediator, indicating the generality of this approach in preparation of multiresponsive films, in immobilization of enzymes, and in realization of multiswitchable bioelectrocatalysis. Although there are many papers reporting on the multisignal controlled electrochemistry,11,12,14,21,49,50 the works on multiswitchable bioelectrocatalysis have been very limited up to now.11,12 Bioelectrocatalysis is not only the foundation of fabricating

biosensors based on enzymes, but can also significantly enlarge or magnify the difference of electrochemical responses between the on and off states. While this work represents only the proof of the concept at this stage, some potential applications can be envisaged and speculated. For example, the triply responsive system may be used as a 3-input AND logic gate in chemical or biomolecular computing.51,52 The bioelectrocatalytic response of the system is switched “on” only when all the three input signals are at the “lower” levels (pH 4.0, temperature 25 °C, and sulfate concentration 0 M), which is designated as “1,1,1” in the truth table of the AND gate. Other seven combinations of these three input signals (0,0,0; 0,0,1; 0,1,0; 1,0,0; 0,1,1; 1,1,0; 1,0,1) would lead to the “off” state of bioelectrocatalysis. In conclusion, this intelligent model interface based on the concept of binary architecture provides a simple and convenient way to realize multiswitchable bioelectrocatalysis on the basis of enzymatic reactions, and may establish the foundation for developing novel types of multicontrollable electrochemical biosensors.

’ ASSOCIATED CONTENT

bS Supporting Information. Fourteen figures showing UV
vis absorption spectra of PDEA hydrogels, HRP in water, and PDEA-HRP hydrogels, CVs of Fe(CN)63
at {Con A/Dex}5 and {Con A/Dex}5
(PDEA-HRP) film electrodes in pH 4.0 buffers at 25 °C, influence of the amount of DEA monomer in polymerization on CV response time of Fe(CN)63
for {Con A/Dex}5
(PDEA-HRP) films in pH 4.0 buffers at 25 °C, continuous CVs of Fe(CN)63
for {Con A/Dex}5
(PDEA-HRP) films with 2 μmol of DEA monomer in polymerization in pH 4.0 buffers at 25 °C, influence of solution pH on CV Ipc and ΔEp of Fe(CN)63
for {Con A/Dex}5
(PDEA-HRP) films at 25 °C, CVs of Fe(CN)63
in pH 4.0 buffers at 25 °C for {Con A/Dex}5
(PDEA-HRP) films at different scan rate (v) and the dependence of CV peak currents on v1/2, CVs of Fe(CN)63
at PDEA-HRP film electrodes in pH 4.0 and 7.2 buffers at 25 °C, CVs of Ru(NH3)63þ and Fc(OH) at {Con A/ Dex}5
(PDEA-HRP) film electrodes in pH 4.0 and 7.2 buffers at 25 °C, CVs of Fe(CN)63
for {Con A/Dex}5
(PDEA-HRP) films in pH 4.0 buffers at different temperatures, CVs of Fe(CN)63
at {Con A/Dex}5 film electrodes in pH 4.0 buffers at 25 and 34 °C, dependence of CV Ipc of Fe(CN)63
on the immersion time when the solution temperature switched between 25 and 34 °C in pH 4.0 buffers, CVs of Fe(CN)63
for {Con A/Dex}5
(PDEA-HRP) films at 25 °C in pH 4.0 buffers containing different concentrations of Na2SO4, dependence of 6698

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The Journal of Physical Chemistry B CV Ipc of Fe(CN)63
for {Con A/Dex}5
(PDEA-HRP) films on the immersion time in pH 4.0 buffers at 25 °C when the concentration of Na2SO4 switched between 0 and 0.26 M, CVs of Fe(CN)63
for {Con A/Dex}5 films at 25 °C in pH 4.0 buffers containing 0 and 0.26 M Na2SO4, dependence of CV Ipa of Fc(COOH)2 in the presence of glucose on testing step for {Con A/Dex}5
(PDEA-GOD) films when the system was switched between pH 4.5 and 7.2, 25 and 34 °C, 0 and 0.26 M Na2SO4, respectively. This material is available free of charge via the Internet at http://pubs.acs.org.

’ AUTHOR INFORMATION Corresponding Author

*E-mail: [email protected]. Tel: (þ86) 10-5880-5498. Fax: (þ86) 10-5880-2075.

’ ACKNOWLEDGMENT The financial support from the National Natural Science Foundation of China (NSFC 20975015) is acknowledged. We also thank Mr. Jin Liu of College of Life Sciences in Beijing Normal University for his help in the measurement of film thickness with stereomicroscopy. ’ REFERENCES (1) Mendes, P. M. Chem. Soc. Rev. 2008, 37, 2512. (2) Liu, Y.; Mu, L.; Liu, B.; Kong, J. Chem.—Eur. J. 2005, 11, 2622. (3) Alarcon, C. H.; Pennadam, S.; Alexander, C. Chem. Soc. Rev. 2005, 34, 276. (4) Nandivada, H.; Ross, A. M.; Lahann, J. Prog. Polym. Sci. 2010, 35, 141. (5) Liu, F.; Urban, M. W. Prog. Polym. Sci. 2010, 35, 3. (6) Nath, N.; Chilkoti, A. Adv. Mater. 2002, 14, 1243. (7) Xia, F.; Jiang, L. Adv. Mater. 2008, 20, 2842. (8) Gopishetty, V.; Roiter, Y.; Tokarev, I.; Minko, S. Adv. Mater. 2008, 20, 4588. (9) Jiang, Y. G; Wan, P.; Smet, M.; Wang, Z.; Zhang, X. Adv. Mater. 2008, 20, 1972. (10) Zhang, M.; Liu, L.; Zhao, H.; Yang, Y.; Fu, G.; He, B. J. Colloid Interface Sci. 2006, 301, 85. (11) Song, S.; Hu, N. J. Phys. Chem. B 2010, 114, 11689. (12) Song, S.; Hu, N. J. Phys. Chem. B 2010, 114, 5940. (13) Klaikherd, A.; Nagamani, C.; Thayumanavan, S. J. Am. Chem. Soc. 2009, 131, 4830. (14) Zhou, J.; Wang, G.; Hu, J.; Lu, X.; Li, J. Chem. Commun. 2006, 46, 4820. (15) Xia, F.; Ge, H.; Hou, Y.; Sun, T.; Chen, L.; Zhang, G.; Jiang, L. Adv. Mater. 2007, 19, 2520. (16) Dimitrov, I.; Trzebicka, B.; Muller, A. H. E.; Dworak, A.; Tsvetanov, C. B. Prog. Polym. Sci. 2007, 32, 1275. (17) Bajpai, A. K.; Shukla, S. K.; Bhanu, S.; Kankane, S. Prog. Polym. Sci. 2008, 33, 1088. (18) Zhao, Y.; Kang, J.; Tan, T. Polymer 2006, 47, 7702. (19) Wandera, D.; Wickramasinghe, S. R.; Husson, S. M. J. Membr. Sci. 2010, 357, 6. (20) Zhang, J.; Peppas, N. Macromolecules 2000, 33, 102. (21) Fulghum, T. M.; Estillore, N. C.; Vo, C.-D.; Armes, S. P.; Advincula, R. C. Macromolecules 2008, 41, 429. (22) Yao, H.; Hu, N. J. Phys. Chem. B 2009, 113, 16021. (23) Maeda, Y.; Yamamoto, H.; Ikeda, I. Langmuir 2001, 17, 6855. (24) Chen, J.; Liu, M.; Liu, H.; Ma, L.; Gao, C.; Zhu, S.; Zhang, S. Chem. Eng. J. 2010, 159, 247. (25) Mao, H.; Li, C.; Zhang, Y.; Bergbreiter, D. E.; Cremer, P. S. J. Am. Chem. Soc. 2003, 125, 2850.

ARTICLE

(26) Chen, Y.; Liu, M.; Bian, F.; Wang, B.; Chen, S.; Jin, S. Macromol. Chem. Phys. 2006, 207, 104. (27) Panayiotou, M.; Freitag, R. Polymer 2005, 46, 6777. (28) Liu, T.; Fang, J.; Zhang, Y.; Zeng, Z. Macromol. Res. 2008, 8, 670. (29) Keerl, M.; Richtering, W. Colloid Polym. Sci. 2007, 285, 471. (30) Luo, S.; Ling, C.; Hu, X.; Liu, X.; Chen, S.; Han, M.; Xia, J. J. Colloid Interface Sci. 2011, 353, 76. (31) Loaiza, O. A.; Laocharoensuk, R.; Burdick, J.; Rodriguez, M. C.; Pingarron, J. M.; Pedrero, M.; Wang, J. Angew. Chem., Int. Ed. 2007, 119, 1530. (32) Willner, I.; Katz, E. Angew. Chem., Int. Ed. 2003, 42, 4576. (33) Tam, T. K.; Strack, G.; Pita, M.; Katz, E. J. Am. Chem. Soc. 2009, 131, 11670. (34) Katz, E.; Privman, V. Chem. Soc. Rev. 2010, 39, 1835. (35) Yao, H.; Guo, X.; Hu, N. Electrochim. Acta 2009, 54, 7330. (36) Denuziere, A.; Ferrier, D.; Domard, A. Carbohydr. Polym. 1996, 29, 317. (37) Lvov, Y.; Ariga, K.; Ichinose, I.; Kunitake, T. Thin Solid Films 1996, 284/285, 797. (38) Panayiotou, M.; Freitag, R. Polymer 2005, 46, 615. (39) Ding, X.; Fries, D.; Jun, B. Polymer 2006, 47, 4718. (40) Chen, J.; Liu, M.; Liu, H.; Ma, L. Mater. Sci. Eng., C 2009, 29, 2116. (41) Maeda, Y.; Nakamura, T.; Ikeda, I. Macromolecules 2002, 35, 10172. (42) Chu, L.; Zou, X.; Knoll, W.; Forch, R. Surf. Coat. Technol. 2008, 202, 2047. (43) Jia, N.; Zhou, Q.; Liu, L.; Yan, M.; Jiang, Z. J. Electroanal. Chem. 2005, 580, 213. (44) Zhang, Y.; Furyk, S.; Bergbreiter, D. E.; Cremer, P. S. J. Am. Chem. Soc. 2005, 127, 14505. (45) Lopez-Leon, T.; Jodar-Reyes, A. B.; Bastos-Gonzalez, D.; Ortega-Vinuesa, J. L. J. Phys. Chem. B 2003, 107, 5696. (46) Pan, S.; Arnold, M. A. Anal. Chim. Acta 1993, 283, 663. (47) Li, W.; Yuan, R.; Chai, Y.; Zhou, L.; Chen, S.; Li, N. J. Biochem. Biophys. Methods 2008, 70, 830. (48) Lu, B.; Smyth, M. R.; Quinn, J.; Bogan, D.; O’Kennedy, R. Electroanalysis 1996, 8, 619. (49) Wan, P. B.; Wang, Y. P.; Jiang, Y. G.; Xu, H. P.; Zhang, X. Adv. Mater. 2009, 21, 4362. (50) Takahashi, S.; Anzai, J. Langmuir 2005, 21, 5102. (51) Wang, J.; Katz, E. Anal. Bioanal. Chem. 2010, 398, 1591. (52) De Silva, A. P.; Uchiyama, S. Nat. Nanotechnol. 2007, 2, 399.

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