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Electrochimica Acta 52 (2007) 7425–7431

Direct electrochemistry of horseradish peroxidasein gelatin-hydrophobic ionic liquid gel films

Rui Yan a, Faqiong Zhao a, Jiangwen Li a, Fei Xiao a,Shuangshuang Fan b, Baizhao Zeng a,∗

a Department of Chemistry, Wuhan University, Wuhan 430072, PR Chinab Chengde Teacher’ College for Nationalities, Chengde 067000, PR China

Received 11 February 2007; received in revised form 9 June 2007; accepted 18 June 2007Available online 22 June 2007

bstract

The direct electrochemistry and electrocatalysis of horseradish peroxidase (HRP) immobilized on a gelatin – N, N-dimethylformamide (DMF)hydrophobic ionic liquid (i.e. 1-octyl-3-methylimidazolium hexafluorophsohate) gel film coated glassy carbon electrode has been studied for therst time. The immobilized HRP exhibits a pair of well-defined quasi-reversible peaks in pH 7.0 phosphate buffer solutions, which results from theirect electron transfer between the enzyme and the underlying electrode. In this case there is about 2.7% of the immobilized HRP undergoing thelectrochemical reaction, which corresponds to multi-layer of HRP on the electrode surface. The HRP immobilized has higher thermal stabilityhan in gelatin hydrogel. Experiment results also show that the voltammetric behavior of the enzyme electrode depends on the type of roomemperature ionic liquid (RTIL) used. When a more hydrophobic RTIL is adopted, the resulting enzyme electrode gives better performance. In

he presence of hydrogen peroxide, the enzyme electrode shows sensitive response. The sensitivity of the catalytic peak is up to 1.38 A cm−2 M−1

nd the Michaelis constant is down to 6.84 × 10−5 M, which are superior to that reported elsewhere. In addition, the UV–visible spectra of HRPntrapped in different films and the mass transfer of hydrogen peroxide are discussed as well.

2007 Elsevier Ltd. All rights reserved.

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eywords: Direct electrochemistry; Horseradish peroxidase; Ionic liquid; Gela

. Introduction

The direct electrochemistry of redox proteins has attractedonsiderable attention recently, because it is quite helpful fornderstanding the electron-transfer mechanisms in biologicalystems [1–4]. On the other hand, the direct electrochemistryf redox proteins is the basics for constructing biofuel cells andhe third-generation biosensors [5,6]. However, the electroac-ive centers of proteins are usually embedded in the molecules,hus it is difficult for the direct electron transfer between thelectroactive centers and electrode surface to occur. Hence var-ous modified electrodes were developed for such purpose, and

he immobilization of proteins was widely studied. To immo-ilize proteins well and realize the direct electrochemistry ofedox proteins, the host materials are important. Generally,

∗ Corresponding author. Tel.: +86 2787218704.E-mail address: bzzeng@whu.edu.cn (B. Zeng).

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013-4686/$ – see front matter © 2007 Elsevier Ltd. All rights reserved.oi:10.1016/j.electacta.2007.06.039

ydrogen peroxide

hey should be nontoxic, stable, and biocompatible. In addi-ion, they are able to promote the electron transfer of proteins7–11].

Room temperature ionic liquids (RTILs) are a class ofompound consisting of ions entirely. They possess many dis-inctive properties such as negligible vapor pressure, wideotential window, high thermal stability, high viscosity, andood conductivity. Thus they have great potential applica-ion in various fields. In fact, they have been widely usedn organic synthesis, catalysis, separation, and electrochem-stry fields [12]. So far the RTILs used more extensively arehose comprising 1-alkyl-3-methylimidazolium (CnMIM), N-lkylpyridinium, tetraalkylammonium, tetraalkylphosphonium,exafluorophosphate (PF6), trifluoromethylsulfonate (CF3SO3),is[(trifluoromethyl)sulfonyl]amide (Tf2N), trifluoroethanoate

CF3CO2), acetate (CH3CO2), nitrate, and halide [13,14].

Recently, more attention has been paid on their applicationsn bioelectrochemistry. On the one hand, RTILs are used as elec-rolyte so that the direct electrochemistry and electrocatalysis of

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426 R. Yan et al. / Electrochim

roteins can be conveniently studied in nonaqueous media. Fornstance, Wang et al. investigated the electrochemistry and elec-rocatalysis of a series of heme proteins immobilized by agaroseydrogel films in BMIM·PF6 [15]. They observed the directlectron transfer between the heme proteins and glassy carbonlectrode, and also found that the redox potentials of heme pro-eins were more negative than those in aqueous solutions. On thether hand, RTILs are used as immobilization material, associ-ting with traditional host materials. This also attracts manyesearchers’ interest. Rogers et al. developed a new compos-te material of cellulose and RTILs for the immobilization ofaccase [16,17]. They demonstrated that the enzyme retainedctivity and had application in biocatalytic systems. Other com-osite systems were also reported after that. For example, Lut al. reported a composite system of chitosan–BMIM·BF4, andhe direct electrochemistry of hemoglobin and horseradish per-xidase immobilized in it [18,19]. The potential applications ofuch composite system in biosensors and biocatalysis were dis-ussed. Although hydrophobic RTILs present good performancen maintaining bioactivity of proteins, to our knowledge, theyave not been used in constructing composite system with hydro-el, probably because it is difficult for them to form homogenousolution or colloid.

In the present work, a novel composite system is con-tructed, which contains gelatin, DMF, and hydrophobic RTILi.e. 1-octyl-3-methylimidazolium hexafluorophsohate). BothV–visible spectra and electrochemical results prove that the

omposite system can provide a favorable microenvironmentor HRP to retain its bioactivity. In order to explore the effect ofTILs, four kinds of RTILs, including OMIM·PF6, BMIM·PF6,MIM·BF4, and P6,6,6,14·Tf2N, have been compared in thisork. In addition, the electrocatalytic activity of the resulting

nzyme electrode toward hydrogen peroxide is studied.

. Experimental

.1. Materials and solutions

Gelatin and polyvinyl alcohol (PVA) were purchased frominopharm Chemical Reagent Co. Ltd. (Shanghai, China).hree milligram per milliliter gelatin aqueous solution wasrepared by dissolving 30 mg gelatin in 10 ml boiling waternd stored in a refrigerator. Chitosan was a product of Shang-ai Biotechnology Co. Ltd. (Shanghai, China). Horseradisheroxidase (HRP, >250 u mg−1) came from Beijing Biodeeiochemistry Co. Ltd. (Beijing, China). The stock solutionf HRP (2 mg ml−1) was prepared by dissolving 1 mg HRPn 0.5 ml of 0.5 M pH 7.0 phosphate buffer solution (PBS)nd stored in a refrigerator. 1-Butyl-3-methylimidazoliumetrafluoroborate (BMIM·BF4), 1-butyl-3-methylimidazoliumexafluorophosphate (BMIM·PF6), and trihexyltetradecyl phos-horium bis[(trifluoromethyl)solfonyl]amine (P6,6,6,14·Tf2N)ere obtained from Acros Organics, Ltd. 1-Octyl-3-

ethylimidazolium hexafluorophsohate (OMIM·PF6) was

urchased from Xinnong Chemical Co. (Shanghai, China)., N-dimethylfomamide (DMF) and H2O2 were products ofinopharm Chemical Reagent Co., Ltd. Other reagents used

3

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cta 52 (2007) 7425–7431

ere of reagent or analytical grade. Aqueous solutions wererepared with redistilled water.

.2. Preparation of enzyme electrode

Prior to use, the glassy carbon electrodes (GC, Ø = 2 mm)ere polished on a polishing pad with 0.05 �m alumina slurry,

insed with water and ultrasonicated in redistilled water andthanol, respectively. The effective area of the GCs werestimated to be about 0.0116 cm2 according to the cyclic voltam-ograms of electrochemical probe [Fe (CN)6]3−/[Fe (CN)6]4−

20].The gelatin–RTILs hydrogel was made by mixing 3 mg ml−1

elatin solution, RTILs, and DMF in a 1.5 ml plastic tubeith the aid of ultrasonication. Their ratio was 10:3:5

Vgelatin:VRTILs:VDMF). Then 2 mg ml−1 HRP solution wasdded to the hydrogel (VHRP:Vhydrogel:1:2), and the mixture wasltrasonicated for 5 min. 2.5 �l of the mixture was dropped onhe surface of a clean GC electrode and let it dry for 12 h atoom temperature. Thus an HRP–gelatin–RTILs/GC electrodeas obtained. During drying the electrode was covered with alastic tube so that the solvent can evaporate gently in air and aniform film can be gotten. For comparison, HRP–gelatin/GCwithout RTILs) and HRP/GC (without RTILs and gelatin) werelso prepared through similar procedures. The electrodes weretored at 4 ◦C.

.3. Spectroscopic analysis

UV–vis spectra experiments were carried out on a TU-901 UV spectrophotometer (Purkinje General Instrumento. Ltd., Beijing, China). For spectra measurement, theRP–gelatin–RTILs (i.e. OMIM·PF6, BMIM·PF6, BMIM·BF4,

nd P6,6,6,14·Tf2N) were cast on quartz glass slides and dried inir. The absorption curves of the HRP–gelatin–RTIL films wereecorded.

.4. Electrochemical measurements

Electrochemical experiments were performed with a CHI17 electrochemical workstation (CH Instrument Company,hanghai, China). A conventional three-electrode system wasdopted, including a modified electrode as working electrode,platinum wire as auxiliary electrode, and a saturated calomel

lectrode (SCE) as reference electrode. A 0.1 M pH 7.0 phos-hate buffer solution was used as supporting electrolyte. Theorking solutions were deaerated with nitrogen gas for 30 minefore measurement, and a nitrogen atmosphere was maintainedver the electrochemical cell during the experiment. All exper-ments were performed at room temperature.

. Results and Discussion

.1. Characterization by UV–vis absorption spectra

UV–vis spectrometry is an effective method to probe into theharacteristic structure of proteins [21]. The position of Soret

R. Yan et al. / Electrochimica A

Fig. 1. UV–visible absorption spectra of HRP (a), HRP–gelatin–OMIM·PF6

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tFtheir peak currents and peak potentials are similar. Accordingly,

lm (b), HRP–gelatin–BMIM·PF6 film (c), HRP–gelatin–BMIM·BF4 film (d),nd HRP–gelatin–P6,6,6,14·Tf2N film (e) in 0.10 M pH 7.0 PBS. To make themiscernible, the curves are moved along the A-axis.

bsorption band of heme can provide some information abouthe denaturation of heme proteins and conformational change ofhe heme-group region [22]. Therefore, the absorption spectraf HRP under different conditions are studied first in this work.s shown in Fig. 1, HRP dissolved in pH 7.0 PBS exhibitscharacteristic Soret absorption band at 403 nm, while HRP

mmobilized in different gelatin–RTILs hydrogel films presentsuch absorption band as well. But the absorption peak shows aed shift of 2–4 nm. This can be ascribed to the interaction ofRP with hydrogel. As the shift is very small, it is thought that

he secondary structure of HRP in gelatin–RTILs hydrogel filmseeps almost unchanged and the HRP is not denatured.

.2. Voltammetric behavior ofRP–gelatin–OMIM·PF6/GC

Fig. 2 shows the typical cyclic voltammograms ofRP immobilized in different gels in 0.1 M pH 7.0BS. A pair of well-defined peaks is observed for theRP–gelatin–OMIM·PF6/GC, which results from the direct

ig. 2. Cyclic voltammograms of HRP–gelatin–OMIM·PF6/GC (a),RP–gelatin/GC (b) and HRP/GC (c) in 0.10 M pH 7.0 PBS. Scan rate:.2 V s−1.

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cta 52 (2007) 7425–7431 7427

lectron transfer between the HRP and the underlying elec-rode. The peak separation (�Ep) is 64 mV, and the peakurrents of the anodic and cathodic peaks are almost equal,eaning that the electrochemical process is almost reversible.he HRP–gelatin/GC also exhibits a pair of peaks, but theyre much smaller and not so symmetric. The behavior ofRP/GC is similar to that of HRP–gelatin/GC. As the voltam-etric peaks are related to the direct electron transfer and

he activity of HRP, it can be proposed that the OMIM·PF6ould promote the electron transfer of HRP and improve itsctivity. According to the equation Q = nFAΓ * (where F is Fara-ay constant, Γ * is the surface concentration of electroactiveRP, Q represents the charge amount, A and n stand for the

rea of the electrode and the number of electron transferred,espectively) [23], the surface concentration of electroactiveRP for the HRP–gelatin–OMIM·PF6/GC was estimated toe 1.35 × 10−10 mol cm−2, supposing that the electrochemi-al reaction is 1e transfer process. It is about 31% higherhan that of the HRP–chistan–BMIM·PF4/GC [18]. Accord-ng to the 3D structure of HRP (15.9 × 15.9 × 11.4 nm3), theheoretical value of monolayer coverage of HRP should be.1 × 10−11 mol cm−2, supposing that HRP adopts the orien-ation with the long axis parallel to the electrode surface. As thexperimental value is about 2.65 times as large as the theoret-cal one, multilayer of HRP must participates in the electrodeeaction in this case. The percentage of electroactive HRP onhe electrode surface is ca. 2.76%, which is 2.58 times as larges that of HRP–chistan–BMIM·PF4/GC [18]. This implies thatnly the protein molecules close to the electrode surface and withuitable orientation can exchange electrons with the electrodeurface [15].

To explore the influence of polymer on the direct electronransfer of HRP, two more polymers are tested. As shown inig. 3, they all exhibit a pair of quasi-reversible redox peaks, and

t is thought that the influence of polymer is slight. It is theTIL that enhance the electron transfer rate between HRP andnderlying electrode.

ig. 3. Cyclic voltammograms of different enzyme electrodes in.10 M pH 7.0 PBS. Electrodes: (a) HRP–gelatin–OMIM·PF6/GC, (b)RP–PVA–OMIM·PF6/GC and (c) HRP–chitosan–OMIM·PF6/GC; scan rate:.2 V s−1.

7428 R. Yan et al. / Electrochimica Acta 52 (2007) 7425–7431

Table 1Electrochemical parameters corresponding to the voltammetric peaks of HRP immobilized in different hydrogels (scan rate: 0.2 V s−1)

Gel composition ipa (�A) ipc (�A) Epa (V) Epc (V) �Ep (V) ipa/ipc

Gelatin–OMIM·PF6 0.205 −0.199 −0.281 −0.345 0.064 1.03GGG

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titin basic solutions. The peak potentials of HRP strongly dependon the solution pH. Both the anodic and cathodic peaks shift neg-atively when the pH increases, implying that the electrochemicalprocess involves proton transfer. The formal potential of HRP

elatin–BMIM·PF6 0.189 −0.170elatin–BMIM·BF4 0.150 −0.152elatin–P6,6,6,14·Tf2N 0.207 −0.211

.3. Influence of different RTILs

To study the influence of different RTILs, four kindsf RTIL are tested, including OMIM·PF6, BMIM·PF6,MIM·BF4, and P6,6,6,14·Tf2N. As displayed in Table 1, with

he hydrophobic action of RTIL increasing, for example,MIM·BF4 < BMIM·PF6 < OMIM·PF6 [24], the voltammet-

ic behavior of the HRP immobilized becomes better.his is related to the change of hydrophobic interactionetween HRP and RTIL. For the P6,6,6,14·Tf2N based hydro-el the situation is more complicated. On the one hand,6,6,6,14·Tf2N is quite hydrophobic [24], thus the peak currents ofRP–gelatin–P6,6,6,14·Tf2N/GC are expected to be larger. On thether hand, the Tf2N anion can interact with HRP. Through mod-rately complexing with the metallic ions [25–27], P6,6,6,14·Tf2Nikely makes the activity of HRP decrease. Therefore, the peakurrents of HRP–gelatin–P6,6,6,14·Tf2N/GC are quite close tohat of HRP–gelatin–OMIM·PF6/GC, although P6,6,6,14·Tf2Ns more hydrophobic. OMIM·PF6 is adopted in the followingxperiments.

.4. Influence of scan rate

Fig. 4A reveals the influence of scan rate on the cyclicoltammograms of the HRP–gelatin–OMIM·PF6/GC in 0.1 MH 7.0 PBS. With increasing the scan rate, both anodicnd cathodic peak currents increase, while peak potentialseep almost unchanged. This suggests that the electrochem-cal reaction of HRP almost belong to reversible process.s can be seen, the anodic peak current (ipa) is linear to

can rate from 0.1 to 1.0 V s−1, and the regression equations: ipa = 1.37ν−0.388 (ipa: �A, ν: V s−1, r = 0.993), meaninghat the electrochemical process belongs to surface-confinedlectrochemical process. According to the equation: ipa =2F2νAΓ ∗

0 /4RT = nFQν/4RT [20], the number of electronransferred is calculated to be 1. Fig. 4B shows the varia-ion of peak-to-peak separation (�Ep) with scan rate. Whencan rate is smaller, it keeps almost unchanged. However,hen scan rate exceeds 6 V s−1, the �Ep changes linearly with

t, meaning that the electron-transfer rate is not fast enough.s mentioned above, there are multilayer of HRP undergo-

ng electrochemical reaction, hence the apparent heterogeneouslectron-transfer rate constant (ks) cannot be well establishedy Laviron’s expression [28]. Herein, an expression, �Ep = ν/ks,aised by Huck is adopted [29], which suits for this situation bet-

er. According to the linear relationship of �Ep and scan rate,

Ep = 0.0213ν−0.0393 (�Ep: V, ν: V s−1, r = 0.998), the ks isalculated to be 46.9 s−1, which is much higher than that ofRP–agarose/GC in ionic liquids [15].

Fa0p

−0.288 −0.351 0.063 1.11−0.306 −0.364 0.058 0.986−0.306 −0.368 0.062 0.980

.5. Influence of solution pH

The influence of solution pH on peak current and peak poten-ial is displayed in Fig. 5. With pH rising the peak currentsncrease slightly firstly and then decrease rapidly. This indicateshat the HRP is stable in weak acidic environment, but unstable

ig. 4. Variation of cyclic voltammogram of HRP–gelatin–OMIM·PF6/GC (A)nd the peak separation with scan rate (B). Scan rate (for A): 0.1, 0.2, 0.3, 0.4,.5, 0.6, 0.7, 0.8, 0.9, 1.0 V s−1 (from inner to outer); inset: plot of the anodiceak current vs. scan rate; in 0.10 M pH 7.0 PBS.

R. Yan et al. / Electrochimica A

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stcwrttkArtrange of 1.94–107 �M (Fig. 7), and the regression equation is:

ig. 5. Influence of pH on peak current (a) and peak potential (b) ofRP–gelatin–OMIM·PF6/GC. Scan rate: 0.1 V s−1.

nd pH show a linear relationship. The slope of the regressionurve is −46.8 mV pH−1, which is smaller than the theoreticalalue (i.e. 59 mV pH−1) for one electron coupled with one pro-on. This should be ascribed to the influence of the protonationf HRP and/or the hydration of ferrum ions [20,30]. Therefore,he electrochemical reaction can be expressed as

RP–Fe(III) + e− + H+ = HRP′–Fe (II)

dditionally, when the pH exceeds 9.0 the anodic peak cur-ent of HRP gradually decreases and the cathodic peak currentises, thus the voltammogram becomes asymmetric. However,he cyclic voltammogram can become symmetric again whenhe electrode is immersed in pH 7.0 buffer solutions. Therefore,t is thought that the secondly structure of HRP turns to otherorm in basic solutions, but such transition is reversible overertain pH range.

.6. Influence of electrolyte temperature

Fig. 6 presents the relationship between temperature andeak current of HRP entrapped in gels with or withoutTILs. When the temperature rises, the peak currents of bothRP–gelatin–OMIMPF6/GC and HRP–gelatin/GC increase

ig. 6. Influence of temperature on anodic peak current of HRP. Electrodes: (a)RP–gelatin–OMIM·PF6/GC and (b) HRP–gelatin/GC; scan rate: 0.2 V s−1.

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cta 52 (2007) 7425–7431 7429

nd reach maximum values at around 20 ◦C, and then decrease.owever, there is some difference between them. For theRP–gelatin–OMIM·PF6/GC, the peak current lowers slowly

fter the maximum point; at 50 ◦C it decreases by 40% inomparison with the maximum peak current. The peak cur-ent of the HRP–gelatin/GC decreases more rapidly followinghe maximum point and only 35% of the maximum peak cur-ent is remained when the temperature is 50 ◦C. Therefore,RP in gelatin–OMIM·PF6 film has better tolerance of tem-erature than in the gelatin film. To further investigate thehermal stability of HRP in different films, the supporting elec-rolyte is heated to 80 ◦C and the electrodes are immersedt for 20 min. After it is cooled to room temperature, cyclicoltammograms are recorded. As a result, the peak currentsf HRP–gelatin–OMIM·PF6/GC decrease by about 20%, whilehe peak currents of HRP–gelatin/GC decrease by more than0%, in comparison with those measured before heating. Thislso indicates that the HRP–gelatin–OMIM·PF6 film is moretable.

.7. Electrocatalytic properties

Bioactive HRP immobilized on electrode surfaces usuallyhows good electrocatalytic activity for the reduction of oxygen,richloroacetic acid, NO2

−, H2O2, etc [31]. Here its electro-atalytic properties are also tested. Taking H2O2 for example,hen it is introduced into the solution the cathodic peak cur-

ent of HRP–gelatin–OMIM·PF6/GC increases significantly. Athe same time, the anodic peak current decreases. However,he cyclic voltammograms of gelatin–OMIM·PF6/GC electrodeeeps almost unchanged when H2O2 is added (Fig. 7, inset). This indicates that the immobilized HRP can catalyze the

eduction of H2O2. Further more, the cathodic peak current andhe concentration of H2O2 show a linear relationship in the

pc = −0.101–0.016c (ipc: �A, c: �M, r = 0.997). The sensitiv-ty is 1.38 A cm−2 M−1, which is higher than that reported forRP immobilized in chistan–BMIM·BF4 composite film and

ig. 7. Cyclic voltammograms of HRP–gelatin–OMIMPF6/GC (a–d) andelatin–OMIMPF6/GC (e and f) in 0.10 M pH 7.0 PBS containing 0 �M (and e), 29.1 �M (b and f), 58.2 �M (c), 87.3 �M (d) H2O2. Scan rate: 0.1 V s−1;nset: plot of catalytic peak current vs. concentration of H2O2.

7430 R. Yan et al. / Electrochimica Acta 52 (2007) 7425–7431

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ig. 8. Cyclic voltammograms of HRP–gelatin–OMIMPF6/GC (a and b) andelatin–OMIMPF6/GC (c and d). Solution condition: 0.10 M pH 7.0 PBSeareated (a and c), (a) plus 100 �L of air (b and d); scan rate: 0.1 V s−1.

TIL sol–gel matrix prepared through hydrolysis of tetraethylrthosilicate in BMIM·BF4 solution [18,32].

When the concentration of H2O2 is above 107 �M, the peakurrent tends to stable. Thus the concentration of H2O2 and cat-lytic current follow a Michaelis–Menten response relationshipo some extent. According to the Lineweaver–Burk equation33]:

1

Iss= 1

Imax+ Km

Imaxc

here Iss is the catalytic current, c is the bulk concentrationf the substrate, Imax is the maximum catalytic current, and Kms Michaelis constant. The Imax and Km values are estimated toe 1.63 × 10−6 A and 6.84 × 10−5 M, respectively, based on thelope and the intercept of the 1/Iss versus 1/c plot. The small Kmndicates that the HRP–gelatin–OMIM·PF6/GC electrode hasigh catalytic efficiency to the reduction of H2O2 [32].

It should be mentioned, in this case the catalytic peak poten-ial is quite different from that reported in literatures [34,35],

eaning that the catalytic mechanism is different. Accordingo the postulation of Huang et al. [36], the catalytic reductionf H2O2 is related to O2. To prove this postulation, the cyclicoltammogram of HRP–gelatin–OMIM·PF6/GC electrode isecorded in the presence of O2 (Fig. 8). As can been seen,he electrode exhibits a similar catalytic reduction peak. Thisndicates that O2 may be involved in the catalytic reduction of

2O2. Therefore, the electrocatalytic mechanism is thought toe similar to that of reference [36], as follows:

RP–Fe(III) + H2O2 → CompoundI + H2O

ompoundI + H2O2 → HRP–Fe(III) + O2

trHc

able 2elationship between charge and square root of time in the presence and absence of H

nzyme electrode H2O2 (�M) Linear

RP–gelatin–OMIM·PF6/GC 38.8 Q = 0.80 Q = 0.8

elatin–OMIM·PF6/GC (inset) in 0.10 M pH 7.0 PBS with 38.8 �M

2O2 (a) or 0 �M H2O2 (b). Initial potential: 0.00 V; final (step) potential:0.50 V.

RP–Fe(III) + e− ↔ HRP–Fe(II) (at electrode)

RP–Fe(II) + O2 → HRP–Fe(II)–O2 (fast)

HRP–Fe(II)–O2 + 2e− + 2H+

→ HRP–Fe(II) + H2O2 (at electrode)

owever, we have not obtained more evidences to support thisoint.

Fig. 9 shows the chronocoulometric responses ofRP–gelatin–OMIM·PF6/GC in different solutions. Basedn the curves the plots of charge (Q) versus square root ofime (t1/2) are made and the regression equations are shown inable 2. As the integrated charge (Q) and time (t) should follow

he integrated Cottrell expression [20]:

= (2n FAD1/20 C0t

1/2)

π1/2 + Qdl + nFAΓ0

Thus the apparent D0 of H2O2 can be calculated and its 1.96 × 10−9 cm2 s−1. This is much smaller than the realalue [37], indicating that the diffusion of H2O2 happensn the hydrogel film and just the HRP molecules located inhe inner layer of the film undergo electrochemical reaction

apidly in this case. In addition, the adsorption of H2O2 at theRP–gelatin–OMIM·PF6/GC is negligible because the inter-

ept keeps almost unchanged whether H2O2 is present or not.

2O2

regression equations (Q: �C, t: s) Correlation coefficient (r)

30 + 2.17t1/2 0.99630 + 0.625t1/2 0.997

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R. Yan et al. / Electrochim

.8. Stability of HRP–gelatin–OMIM·PF6/GC

To explore the stability of the HRP–gelatin–OMIM·PF6/GC,he variation of the anodic peak current with time was tested.s a result, the anodic peak current decreased by less than.3% after 12 h storage. The cyclic voltammogram kept almostnchanged after 40 consecutive cyclic scans. This indicates thathe HRP–gelatin–OMIM·PF6/GC is quite stable. The good sta-ility of the HRP–gelatin–OMIM·PF6/GC can be ascribed to thenteractions among gelatin, OMIM·PF6 and HRP, which includeydrogen bond, hydrophobic interactions, charge–charge inter-ctions [18].

. Conclusions

The gelatin–OMIM·PF6 hydrogel film can provide a favor-ble microenvironment for the direct electrochemistry oforseradish peroxidase (HRP) at glassy carbon electrodes. WhenRP is immobilized in the hydrogel, it retains its native sec-ndary structure and bioactivity. The voltammetric behaviorf the resulting HRP–gelatin–OMIM·PF6/GC enzyme elec-rode depends on the RTIL used. When the RTIL is moreydrophobic, the voltammetric peaks are more sensitive. Thenzyme electrode has good catalytic activity to the reductionf hydrogen peroxide. The sensitivity of the catalytic peak isp to 1.38 A cm−2 M−1 and the Michaelis constant is down to.84 × 10−5 M. The enzyme electrode also shows good thermaltability and reproducibility. These are superior to that reported.herefore, this study provides a good strategy for the immobi-

ization of enzymes and fabrication of biosensors.

cknowledgements

The authors appreciate the support of the National Naturalcience Foundation of China (No. 20173040) and the State Keyaboratory of Electroanalytical Chemistry, Changchun Institutef Applied Chemistry, Changchun, China.

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