Journal of Colloid and Interface Science236,166–172 (2001)doi:10.1006/jcis.2000.7381, available online at http://www.idealibrary.com on

Electrochemistry and Electrocatalysis with Myoglobinin Biomembrane-Like DHP–PDDA Polyelectrolyte–

Surfactant Complex Films

Liwen Wang and Naifei Hu1

Department of Chemistry, Beijing Normal University, Beijing, 100875, China

Received August 14, 2000; accepted December 4, 2000

The polyelectrolyte–surfactant complex DHP–PDDA was pre-pared by reacting the anionic surfactant dihexadecylphos-phate (DHP) with polycationic poly(diallyldimethylammonium)(PDDA). Thin films made from DHP–PDDA on solid substratesdemonstrated an ordered multibilayer structure by XRD and DSC.Incorporated myoglobin (Mb) in DHP–PDDA films on pyrolyticgraphite (PG) electrodes showed a pair of well-defined and nearlyreversible cyclic voltammetric peaks for the Mb Fe(III)/Fe(II) cou-ple at about −0.3 V vs SCE in pH 7.0 buffers. Electron transferbetween Mb and PG electrodes was greatly facilitated in the filmmicroenvironment. The positions of the Soret absorption band sug-gest that Mb maintains its secondary structure similar to its nativestate in DHP–PDDA films in the medium pH range. Mb could actas an enzyme-like catalyst in DHP–PDDA films as demonstratedby catalytic reduction of trichloroacetic acid, nitrite, and oxygenwith a decrease in the electrode potentials required. Mb–DHP–PDDA films may thus have potential application as biosensors.C© 2001 Academic Press

Key Words: polyelectrolyte–surfactant complex; myoglobin; di-hexadecylphosphate; poly(diallyldimethylammonium); direct elec-trochemistry; electrocatalysis.

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INTRODUCTION

Bilayers of phospholipids are integral structural units of blogical membranes, which generally exist in a partly fluid,lectively permeable state (1, 2). Some synthetic or naturalfactants can be designed to form ordered films featuring stabilayers on electrodes, which can incorporate redox protand facilitate reversible electron transformation (3). Thus, dielectrochemistry of proteins in these biomembrane-like fican provide a good model for studies of redox processes inlogical systems. Several recent examples have demonstratapplication of an electrochemical method for the studies ofteins using surfactant-modified electrodes (4–10). Among thRusling and co-workers reported that in lamellar surfactant ficast on pyrolytic graphite (PG) electrodes, direct electron tr

1 To whom correspondence should be addressed. E-mail: hunaifei@edu.cn.

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160021-9797/01 $35.00Copyright C© 2001 by Academic PressAll rights of reproduction in any form reserved.

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fer was greatly enhanced for myoglobin (Mb), compared to thon bare PG with the protein in solution (6–9). This may provide a general way to study the direct electrochemistry of redproteins.

Polyelectrolyte–surfactant complex films can be viewed asnew type of biomembrane-like films. Such complexes combinin unique ways the properties of amphiphilic surfactants withose of polymers. The polymeric components can provide mchanical strength and good stability, while the surfactants retatheir tendency to assemble in bilayered structures (11–13). Pteins in this kind of films showed well-behaved electrochemistand good stability (14, 15). Since these films are amenablea variety of electrochemical and other experiments for a longtime than surfactant films, they may have more practical appcations as biosensors or bioreactors.

In previous works (7, 14, 15), polyelectrolyte–surfactancomplexes were usually prepared by reacting cationic surfatant with polyanion. In this work, a new kind of polyioniccomplex, DHP–PDDA, was synthesized by reacting the anionsurfactant dihexadecylphosphate (DHP) with polycationpoly(diallyldimethylammonium) (PDDA). The chemical struc-ture of DHP–PDDA is shown below. As a kind of synthesizephospholipids, DHP was deposited once on PG electrodes aformed multibilayer films, in which incorporated Mb showedgood voltammetric response (16). We thus expect that DHPDDA composite films would have structure similar to that oDHP films alone and can also be applied to the study of Melectrochemistry. X-ray diffraction and differential scannincalorimetry were used to characterize DHP–PDDA films. Thelectrochemistry of Mb incorporated in DHP–PDDA filmson PG electrodes was then studied. Electrocatalytic reductof trichloroacetic acid (TCA), nitrite (NO−2 ), and dissolvedoxygen in solution was also demonstrated on Mb–DHP–PDDfilm electrodes.

EXPERIMENTAL

Chemicals

Horse heart myoglobin (Mb) from Sigma was used as receivwithout further purification. Poly(diallyldimethylammonium

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SCHEME. Chemical structure of DHP–PDDA.

chloride) (PDDA, MW 60,000) was from Aldrich. Dihexadecylphosphate (DHP) was from Sigma. Trichloroacetic a(TCA) was from Beijing Dongjiao Chemicals. Sodium nitriwas from Beijing Sanhuan Chemicals. All other chemicwere reagent grade. The supporting electrolyte was usu0.01 M sodium dihydrogen phosphate buffer at pH 7.0 containg 0.1 M KBr. Other buffers were 0.1 M sodium acetate, 0.05boric acid, or 0.05 M citric acid, all containing 0.1 M KBr. ThpH values were regulated with HCl or KOH solutions. Solutiowere prepared with twice-distilled demineralized water.

Preparation of Mb–DHP–PDDA Films

The polyelectrolyte–surfactant complex DHP–PDDA wprepared by reacting DHP with PDDA. A 10 ml amount8.9 mg ml−1 (about 55 mM of positive charge) PDDA solutioand 50 ml of 6 mg ml−1 (about 11 mM of negative charge) aquous dispersion of DHP were mixed with stirring at room teperature for 30 min until the reaction was completed. A whprecipitate of DHP–PDDA was formed. After the solution wleft to stand at ambient temperature over night, and then bcentrifuged, washed, and dried, the pure and dry DHP–PDpowder was collected. The complex powder of DHP–PDDA wdispersed in water by ultrasonication for about 12 h to obta2 mg ml−1 cloudy suspension. Right before the preparationthe films, the dispersion was ultrasonicated for another 5 m

Prior to coating, basal plane pyrolytic graphite (PG, gifrom Chinese Geologic Academy of Science; geometric a0.28 cm2) electrodes were abraded with metallographic sandper, and then polished on a clean billiard cloth with pure waElectrodes were sonicated in pure water for 30 s after eachishing step.

DHP–PDDA films were prepared by casting 10µl of2 mg ml−1 DHP–PDDA dispersion onto a PG electrode wa microsyringe. Mb–DHP–PDDA films were prepared by dpositing 15µl of a mixture of 1 part of 0.15 mM Mb and 2 partof 2 mg ml−1 DHP–PDDA dispersion onto a PG electrode.small bottle was fit tightly over the electrodes to serve as a cloevaporation chamber so that water was evaporated slowly

more uniform films were formed. The films were then drieovernight.

ROCATALYSIS WITH MYOGLOBIN 167

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Apparatus and Procedures

X-ray diffraction (XRD) studies were done with a D/MAX-RB powder diffractometer (Rigaku) using a CuKα source(1.54 A) at 40 kV and 80 mA. The scan rate was 2◦ min−1.DHP–PDDA films were prepared by depositing a few microliters of 2 mg ml−1 DHP–PDDA dispersion onto glass slideand drying them in air. Mb–DHP–PDDA films were prepareby depositing a 1 : 2 (v/v) mixture of 0.15 mM Mb and 2 mg ml−1

DHP–PDDA dispersion onto glass slides.Differential scanning calorimetry (DSC) was performed wit

a DSC 2010 differential scanning calorimeter (TA). The filmwere prepared by a method similar to that described for XRAbout 1–2 mg of DHP–PDDA and Mb–DHP–PDDA samplewere collected from the films, respectively, and hermeticasealed in aluminum pans after addition of a few drops of puwater. The heating rate was 5◦C min−1.

A CHI 660 electrochemical workstation (CH Instrumentswas used for cyclic voltammetry (CV) and square wave voltammetry (SWV). A conventional three-electrode cell was used wa saturated calomel electrode (SCE) as reference, a platinwire as counter electrode, and a PG disk with films as workielectrode. All experiments were done at a room temperature18± 2◦C.

Voltammetry on electrodes coated with Mb–DHP–PDDfilms was done in buffers containing no Mb. Buffers were purgewith highly purified nitrogen for about 30 min before a seriesexperiments. A nitrogen environment was kept over solutionsthe cell during the experiment. In the experiment with oxygemeasured volumes of air were injected through solutions visyringe in a sealed cell, which had been previously degaswith purified nitrogen.

UV–vis absorption spectroscopy was done with an U250 spectrophotometer (Shimadzu). Composite films of DHPDDA and Mb–DHP–PDDA were prepared by a method similto that described for XRD but on indium tin oxide-coated slide(ITO, from Delta Technologies).

Scanning electron microscopy (SEM) done with an X-65scanning electron microanalyzer (Hitachi) was used to estimthe thickness of dry films. Sample films were prepared on vethin disk PG in the same way as for electrochemistry, and wethen fractured after immersion in liquid nitrogen in order to geclear dividing line between the films and underlying PG. Crossectional views of SEM at room temperature showed thatdry thickness of DHP–PDDA and Mb–DHP–PDDA films waalmost the same and about 7.5µm.

RESULTS AND DISCUSSION

Structure Characterization by XRD and DSC

The lowest reflection angle 2θ of X-ray diffraction forpolyionic complex films can be used to obtain the interlay

dbasal spacing of the films by Bragg’s law. For DHP–PDDAfilms (Fig. 1a), a main XRD peak at 2.62◦ gave a PDDA basal

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FIG. 1. X-ray diffraction of (a) DHP–PDDA films and (b) Mb–DHP–PDDAfilms. The CPS coordinate reflects only relative CPS values.

spacing of 33.7A, while two other much smaller peaks at 5.3◦

(16.6A) and 7.90◦ (11.2A) giving about one-half and one-thirthe Bragg’s spacing of the main peak were identified assecond- and third-order diffraction, respectively. Similar pterns and almost the same peak positions were observeXRD experiments for Mb–DHP–PDDA films (Fig. 1b), indcating that incorporation of Mb essentially did not enlargeinterlayer spacing of DHP–PDDA films. Rather sharp 2θ mainpeaks suggest well-defined layer orders in both films. Crosectional views of DHP–PDDA and Mb–DHP–PDDA films bSEM showed that the dry thicknesses of both films were ab7.5µm. Considering that the basal spacing of the films (orthickness of one PDDA layer) was about 34A, the total numberof basal layer for the films would be about 2200.

DSC may provide information on the phase transition of lipbilayers (2). DHP–PDDA films showed a transition tempeture (Tc) at 72.6◦C (Table 1), very close to the 72◦C for DHPfilms alone (17), indicating that the films undergo a transitfrom a solid-like gel phase to a more fluid liquid crystal phasethis temperature, and DHP in the complex films is arrangea tail-to-tail bilayer structure. Mb–DHP–PDDA films showetwo Tc peaks at 69.7 and 74.0◦C, respectively (Table 1). Thefirst one was very close to the melting point of pure Mb69.0◦C (Table 1), and should be assigned to melting of Min the films. Thus, the second DSC peak of Mb–DHP–PDfilms at 74.0◦C is most probably attributed to the phase trasition of DHP lipid in the films, showing that DHP–PDDA

TABLE 1Gel-to-Liquid Crystal Phase Transition Temperatures Measured

by DSC for Films of Lipids and Mb–Lipids

Film Tc (◦C) Reference

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Mb 69.0 This work

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films essentially maintain the multibilayer structure aftercorporation of Mb. Binding of Mb to the lipid films causea slight change inTc, which was also observed for surfactafilms (6).

The results of XRD and DSC for DHP–PDDA films suport the proposal that surfactant DHP is self-assembled intordered bilayer structure which is complexed with and sawiched by PDDA backbone layers, and thousands of suchlayers were parallel stacked in the films. The length of the hydcarbon chain of DHP was about 19.8A (18). Twice the lengthof the DHP chain is about 39.6A, smaller than the interlayebasal spacing of 33.7A for DHP–PDDA films; even the thick-ness of PDDA backbone itself is ignored. This suggests posstilting or intercalating of hydrocarbon chains of DHP to somextent in the surfactant bilayer region. The size of Mb is ab45× 35× 25 A (19). Among the three dimensions, only thsmallest one (25A) is less than the basal spacing (34A). Thus,Mb intercalated in the films has to have some specific orietion so that it can be fit into the interlayer spacing. Howevother possibilities cannot be ruled out, such as that some oproteins may be on the outer surface of the PDDA backbonepartly buried in the hydrophobic regions of the DHP bilayer.

Direct Electrochemistry Studies by CV

Mb–DHP–PDDA film electrodes in pH 7.0 buffer solutioncontaining no Mb gave a pair of well-defined and nearlyversible CV peaks at about−0.3 V vs SCE after several cyclicscans (Fig. 2b), characteristic of a Mb heme Fe(III)/Fe(II)dox couple (6). In contrast, plain DHP–PDDA films withoMb showed no CV response at all in the same potential wind(Fig. 2a). This indicates direct electrochemistry between Mbthe PG electrode in DHP–PDDA films and much faster electtransfer for Mb in the films than for Mb in solution on bare P(6, 8). Thus, DHP–PDDA films must have a great effect onelectron transfer kinetics and provide a favorable microenvirment for Mb to transfer an electron with underlying PG.

CVs of Mb–DHP–PDDA films had symmetric peak shapand nearly equal heights of their reduction and oxidation pe

FIG. 2. Cyclic voltammograms at 0.2 V s in pH 7.0 buffers for (a) DHP–PDDA films and (b) Mb–DHP–PDDA films.

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ELECTROCHEMISTRY AND ELEC

FIG. 3. Cyclic voltammograms at 0.2 V s−1 for DHP–PDDA films im-mersed in pH 7.0 buffers containing 15 mM Mb for (a) 20 min, (b) 50 m(c) 120 min, (d) 330 min, (e) 19 h, (f) 49 h, and (g) 52 h.

The heights of the reduction peaks were linearly proportioto scan rates from 0.05 to 2 V s−1. These results are charateristic of thin-layer electrochemistry (20), suggesting thatelectroactive MbFe(III) in the films is converted to MbFe(II) othe forward negative scan, with full conversion of MbFe(II) bato MbFe(III) on the reverse positive scan. The total amouncharge (Q) that passed through the electrode for reductionelectroactive MbFe(III) in the films was essentially independof scan rates, and gave the average surface concentration oftroactive Mb (0∗) of about 1.48× 10−10 mol cm−2, according totheQ–0∗ relationship (20). Compared with the total amountMb deposited on the electrode, it accounted for about 6%. Othose myoglobin molecules which are in an ideal orientationclose to the electrode surface would be able to contribute toobserved redox reaction.

To examine the possibility of Mb entering DHP–PDDA filmfrom its solution, a PG electrode coated with DHP–PDDA filmwas placed into a pH 7.0 buffer containing 0.15 mM Mb, and Cwere run at different immersion times. The peaks at about−0.3 Vgrew with immersion time, suggesting that increasing amouof Mb enter the DHP–PDDA films (Fig. 3). CVs representifilms fully loaded with Mb were obtained in about 52 h. Whefully loaded Mb–DHP–PDDA films were removed from Msolutions and transferred to a pH 7.0 buffer containing no Mreproducible CVs were identical to the CVs in Mb solutioBoth casting and immersing methods showed very similar ppositions and currents for Mb–DHP–PDDA films at the steastate, but the former was more convenient and quantitativethus was used in the following studies.

The stability of Mb–DHP–PDDA films was tested by CV.PG electrode coated with Mb–DHP–PDDA films was storeda pH 7.0 buffer all the time, CVs were run periodically, and treduction peak currents were measured with storing time.results showed a very good stability for the films. The CV pehad a tendency of slowly increasing during the first 1 to 2 dof soaking; afterward the peak potentials and currents rema

essentially unchanged for at least 80 days. Since the film etrode was inside the solutions while nitrogen was bubbled

ROCATALYSIS WITH MYOGLOBIN 169

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30 min in buffers before each CV test, we expect the films wouretain stable even under flow conditions.

The polyionic PDDA has positive charges on its backbonwhich combines with negatively charged surfactant DHPCoulombic attraction, forming a neutral precipitate of DHPPDDA. Thus, the driving force for Mb to enter DHP–PDDAfilms would be mainly hydrophobic interaction between macrmolecular Mb and DHP–PDDA films, in which a tail-to-taihydrocarbon chain bilayer constitutes the hydrophobic regiof the films. This hydrophobic interaction would also be mainresponsible for the excellent stability of Mb–DHP–PDDA filmand the retention of Mb in the films.

The role of DHP–PDDA films in enhancing Mb electron transfer is not yet very clear. This is probably because the films inhiadsorption of macromolecular impurities from Mb solution including denatured Mb on the electrodes, which could otherwblock electron transfer to Mb (8). Another possibility is that thorientation of Mb in DHP–PDDA films, or its interaction withsurfactant DHP, may be more favorable for the electron transFurther studies are needed in this respect.

Estimation of Parameters by SWV

SWV, basically as a pulse method, is easier to analyze quatatively and treat theoretically over CV (21), and thus was ushere to estimate the average apparent heterogeneous eletransfer rate constant (ks) and formal potential (E◦′). The proce-dure employed nonlinear regression analysis for SWV forwaand reverse curves, with the combination of the thin-layer SWmodel (22) and theE◦′ dispersion model, as described in detapreviously (9, 16).

Examples of analysis of SWV data for Mb–DHP–PDDA filmshowed goodness of fit onto the models over a range of fquencies (Fig. 4). The average rate constant (ks) obtained fromfitting SWV data at pH 7.0 was 27 s−1, and the averageE◦′

was−0.326 V vs SCE (Table 2). Values obtained by the sam

FIG. 4. Square wave forward and reverse current voltammograms for MDHP–PDDA films in pH 7.0 buffers at different frequencies. Points represethe experimental SWVs from which background has been subtracted. The sline is the best fit by nonlinear regression onto the 5E◦′ dispersion model.

lec-forSWV conditions: pulse height, 60 mV; step height, 4 mV; and frequencies (Hz),(a) 100, (b) 125, (c) 152, and (d) 179.

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TABLE 2Apparent Heterogeneous Electron Transfer Rate Constants and

Formal Potentials for Myoglobin Films on PG Electrodes in pH 7.0Buffers Containing No Myoglobin

Av. E◦′ vs SCE

Filma Av. ks, s−1 CV SWV Ref.b

Mb–DHP–PDDA 27± 3 −0.323 −0.326 TwMb–DDAB 31± 3 −0.228 −0.240 9Mb–DMPC 59± 9 −0.326 −0.342 9Mb–DLPC 50± 8 −0.329 −0.343 9Mb–DDAB-PA 11± 1 −0.231 −0.212 14Mb–DDAB-Nafion 40± 6 −0.193 −0.202 10Mb–AQ 52± 6 −0.362 −0.340 10

a DHP, dihexadecylphosphate; PDDA, poly(diallyldimethylammoniumDDAB, didodecyldimethylammonium bromide; DMPC, dimyristoyl phophatidylcholine; DLPC, dilauroyl phosphatidylcholine; PA, polyacrylate; AEastman AQ38.

b Tw, this work, reporting average values for analysis of eight SWVs atquencies of 100–179 Hz, amplitudes of 60–75 mV, and a step height of 4 m

method as that used for Mb in other films are listed in Tablfor comparison.

Theks value for Mb in DHP–PDDA films was similar to thain DDAB films, but smaller than those in the films of phophatidylcholines (PCs) and polymers, and larger than thosDDAB–PA films (Table 2), though all of them are in the samorder. TheE◦′ value of heme Fe(III)/Fe(II) couple for Mb inDHP–PDDA films was more negative than those in DDAB aother complex films and similar to those in PCs and ionom(Table 2). This confirms a specific influence of the film enviroment onE◦′ for heme proteins which had been reported preously (9, 16). Film components may change potentials viateractions with the protein or by their influence on the electrodouble layer (9, 16).

Influence of pH

CVs of Mb–DHP–PDDA films showed a strong dependenon the pH of external solutions. An increase of the pH insolution led to a negative shift in potential for both reductiand oxidation peaks for the films. In general, all changes inpeak potentials and currents with pH were reversible betwpH 4.5 and 10.0. For example, CVs for a Mb–DHP–PDDA fiin a pH 5.5 buffer were reproduced after immersing the fiin a pH 8.0 buffer and then returning it to the pH 5.5 buffCV data were used to investigate the pH effect on the forpotential (E◦′), which was estimated as the midpoint betweenreduction and oxidation peak potentials for the Mb Fe(III)/Fe(redox couple.E◦′ had a linear relationship with pH from pH 4.to 12.0 with a slope of−49 mV pH−1 (Fig. 5). This slope valueis reasonably close to the theoretical value of−57.7 mV pH−1

at 18◦C for a reversible proton-coupled single electron trans(23, 24), which could be represented by

MbFe(III)+ H+ + e−⇀↽MbFe(II).

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An inflection point appeared in theE◦′–pH plot at pH 4.5(Fig. 5). At pH< 4.5, the variation ofE◦′ values with pH showeda much smaller slope. The position of the break in theE◦′–pHplot suggests that the protonatable site associated with electrreaction has an apparent pKa value of 4.5.

The positions of the Soret absorption band of iron heme prvide information about the possible denaturation of Mb (25The solution spectrum has a Soret band at 408 nm for MbpH 5.5. Both dry films cast from Mb and Mb–DHP–PDDA ontransparent ITO slides showed Soret bands at 408 nm (Figs.6b), suggesting that Mb in dry DHP–PDDA films has a secondary structure nearly the same as that in dry Mb films alonThe position of the Soret band depended on the pH when MDHP–PDDA films were immersed into buffer solutions. At pHvalues between 5.5 and 10.0, the Soret band appeared at 408(Figs. 6d, 6e, 6f), the same as for Mb in solutions at mediupH. Since the Soret band is not very sensitive to changes in

FIG. 6. UV–vis spectra of Mb and Mb–DHP–PDDA films on indium tinoxide (ITO) slides for (a) dry Mb films, (b) dry Mb–DHP–PDDA films, and

Mb–DHP–PDDA films in buffers: (c) pH 4.0, (d) pH 5.5, (e) pH 7.0, and(f) pH 10.0. The absorbance coordinate reflects only relative absorbance.

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ELECTROCHEMISTRY AND ELEC

secondary structure of Mb, the possibility of minor denatution not involving changes in the heme group regions cannoruled out by the Soret band spectra. However, the Soret baindeed sensitive to the conformation change in the heme gregion. If the heme prosthetic group were dissociated frompolypeptides and existed as an independent or “dissolved” fin DHP–PDDA films, its Soret band position would have bevery different from that of native Mb. When the pH was changtoward the more acidic direction, the Soret band showed a sigicant blue shift. For example, at pH 4.0, the Soret band shito 395 nm (Fig. 6c), indicating that Mb in DHP–PDDA filmmight denature to a considerable extent in this more acidic eronment, as observed previously in aqueous solutions (25and in surfactant films (9).

Catalytic Reactivity

Catalytic reduction of oxygen by Mb–DHP–PDDA films wexamined by CV with oxygen present in the external solutiWhen a certain amount of air was passed through a pHbuffer solution by a syringe, a significant increase in the redtion peak at about−0.3 V was observed for Mb–DHP–PDDAfilms (Fig. 7d), compared with the reduction peak for the filmsthe absence of oxygen (Fig. 7b). This increase in the reducpeak was accompanied by the disappearance of the oxidpeak for MbFe(II). The oxidation peak for MbFe(II) did not apear because MbFe(II) had reacted with oxygen. An increin the amount of oxygen in the solution increased the reducpeak current (Fig. 7e). For DHP–PDDA films with no Mb incoporated, the peak reflecting direct reduction of oxygen wasserved at about−0.6 V (Fig. 7c). Catalytic efficiency expresseas the ratio of the reduction peak current of MbFe(III) in tpresence (Ic) and in the absence (Id) of oxygen,Ic/Id, decreasedwith an increase in the scan rate (Fig. 8). All of these resultscharacteristic of reduction of oxygen by electrochemical catsis with Mb in DHP–PDDA films (28, 29). Similar voltamme

FIG. 7. Cyclic voltammograms at 0.2 V s−1 in 5 ml of pH 7.0 buffers:(a) DHP–PDDA films with no oxygen present; (b) Mb–DHP–PDDA films wno oxygen present; (c) DHP–PDDA films after 50 ml of air was injected i

a sealed cell; (d) Mb–DHP–PDDA films after 50 ml of air was injected; a(e) Mb–DHP–PDDA films after 80 ml of air was injected.

ROCATALYSIS WITH MYOGLOBIN 171

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FIG. 8. Influence of scan rate on catalytic efficiency,Ic/Id, for Mb–DHP–PDDA films in pH 7.0 buffers, whereId is the CV reduction peak current inbuffers without oxygen andIc is the CV reduction peak current in 5 ml obuffers with 50 ml of air injected.

ric behaviors were observed previously for Mb in other film(10, 30).

The electrocatalytic reduction of trichloroacetic acid (TCAby Mb–DHP–PDDA films was also tested by CV. When TCwas added to the buffer, the MbFe(III) reduction peak for MDHP–PDDA films increased (Fig. 9c). This was accompanby a decrease and disappearance of the MbFe(II) oxidation pThe reduction peak current for MbFe(III) increased as the ccentration of TCA increased. These results indicate reactioMbFe(II) with TCA in a catalytic cycle, presumably resultinin reductive dechlorination of TCA (31). Compared to CVDHP–PDDA films in TCA solution (Fig. 9b), Mb–DHP–PDDAfilms lowered the overpotential for reduction of TCA by abo0.6 V. Other organohalides, such as trichloroethylene (TCand perchloroethylene (PCE), were also tested with Mb–DHPDDA films for electrocatalysis. However, TCE and PCE dnot show any catalytic behavior at Mb–DHP–PDDA films, idicating that the films may have some selectivity to substraalthough the mechanism is not yet clear by now.

Farmer and co-workers (32) have studied electrocatalreduction of NO−2 with Mb–DDAB films on PG electrodes

FIG. 9. Cyclic voltammograms at 0.04 V s−1 in pH 7.0 buffers: (a) Mb–

ndDHP–PDDA films with no TCA present; (b) DHP–PDDA films with 30 mMTCA present; and (c) Mb–DHP-PDDA films with 30 mM TCA present.

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FIG. 10. Cyclic voltammograms at 0.2 V s−1 in pH 7.0 buffers: (a) DHP–PDDA films with no NaNO2 present; (b) Mb–DHP–PDDA films with no NaNO2

present; (c) DHP–PDDA films with 50 mM NaNO2 present; (d) Mb–DHP–PDDA films with 50 mM NaNO2 present; and (e) Mb–DHP–PDDA films wit120 mM NaNO2 present.

Similar results were found at Mb–DHP–PDDA film electrodWhen NO−2 was added into a pH 7.0 buffer, a catalytic pewas observed at about−0.84 V (Fig. 10d). The peak currenincreased with increasing concentration of NO−2 (Fig. 10e). Di-rect reduction of NO−2 on DHP–PDDA films without Mb wasfound at potentials more negative than−1.3 V (Fig. 10c). Thus,Mb–DHP–PDDA films decreased the reduction overpotenfor NO−2 by at least 0.5 V. Although the mechanism of NO−2 re-duction on Mb–DHP–PDDA films is not yet known and furthstudies are needed, the reduction product at−0.84 V is mostprobably N2O, which was detected previously by mass sptroscopy with Mb–DDAB films on electrolysis at−0.895 V inpH 7 buffers (32).

SUMMARY

Myoglobin in stable biomembrane-like polyelectrolytsurfactant complex films of DHP–PDDA gave direct, reverselectron transfer with underlying pyrolytic graphite eletrodes. Effective electron transfer rates involving the Mb heFe(III)/Fe(II) redox couple were comparable to those for Mbsurfactant and other complex films and much faster than tfor bare PG electrodes in Mb solutions. These ordered mulayer films were presumably stabilized mainly by hydropbic interactions between the protein and film componeMb–DHP–PDDA films on electrodes could catalyze reducof trichloroacetic acid, nitrite, and oxygen, which providedeasily prepared experimental model for a membrane-boun

zyme catalytic reaction. Such films may have potential applition as biosensors and bioreactors.

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ACKNOWLEDGMENTS

The support from the National Natural Science Foundation of China isknowledged.

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