Biosensors and Bioelectronics 20 (2005) 1373–1379

Conducting polymer polypyrrole supported bilayer lipid membranes

Yong Shao, Yongdong Jin, Jianlong Wang, Li Wang, Feng Zhao, Shaojun Dong∗

State Key Laboratory of Electroanalytical Chemistry, Changchun Institute of Applied Chemistry,Chinese Academy of Sciences, Changchun 130022, China

Received 16 April 2004; accepted 3 June 2004Available online 7 July 2004

Abstract

Electrochemically synthesized conducting polymer polypyrrole (PPy) film on gold electrode surface was used as a novel support for bilayerlipid membranes (BLMs). Investigations by surface plasmon resonance (SPR) suggest that dimyristoyl-l-alpha-phosphatidylcholine (DMPC)and dimyristoyl-l-alpha-phosphatidyl-l-serine (DMPS) can form BLMs on PPy film surface but dimyristoyl-l-alpha-phosphatidylglycerol(DMPG) and didodecyldimethylammonium bromide (DDAB) can not do so, indicating the formation of PPy supported bilayer lipid membranes(s-BLMs) is dependent on the chemical structure of the lipids used. The self-assembly of DMPC induces a smoother topography than thePPy layer with rms roughness decreasing from 4.484 to 2.914 nm convinced by atomic force microscopy (AFM). Impedance spectroscopymeasurements confirm that the deposition of BLM substantially increases the resistance of the system indicating a very densely packed BLMstructures. The little change of PPy film in capacitance shows that solvent and electrolyte ions still retain within the porous PPy film afterBLM deposition. Therefore, the PPy supported BLM is to some extent comparable to conventional BLM with aqueous medium retaining atits two sides. As an example and preliminary application, horseradish peroxidase (HRP) reconstituted into the s-BLM shows the expectedprotein activity and can transfer electron from or to the underlying PPy support for its response to electrocatalytic reduction of hydrogenperoxide in solution. Thus the system maybe possesses potential applications to biomimetic membrane studies.© 2004 Elsevier B.V. All rights reserved.

Keywords:Supported bilayer lipid membranes; Surface plasmon resonance; Polypyrrole; Electron transfer; Electrochemistry

1. Introduction

Bilayer lipid membranes (BLMs) have been extensivelyused in recent years as models for biological membranes. Itis often necessary to immobilize lipid bilayers on solid sup-ports for long-term basic studies. BLMs formed on supportsare known as supported BLMs (s-BLMs). Up to now, severaltypes of solids or cushions, for example, metal with nascentsurface (Tien and Salamon, 1989), glass fiber (Nikolelis andPantoulias, 2001; Siontorou et al., 2000), agar (Lu et al.,1996), protein (Schuster et al., 2001), self-assembly mono-layer of alkanethiolates (Lahiri et al., 2000; Raguse et al.,1998; Cornell et al., 1997) and nonconducting polymers(Hillebrandt et al., 1999; Lang et al., 1994; Seitz et al., 2000;Naumann et al., 1995; Sackmann, 1996) have been used assupports.

∗ Corresponding author. Tel.:+86 431 5262101;fax: +86 431 5689711.

E-mail address:dongsj@ns.ciac.jl.cn (S. Dong).

The promising support for BLM with potentially extendedapplications should possess such properties as facilitating,tightly linking to substrates and easily controlling its thick-ness as well as retaining hydrophilicity. For these purposes,however, the previously reported supports usually need com-plex pretreatments and synthesis procedures (Raguse et al.,1998). Additionally, an electric phenomenon is one of thebasic natures of biomembranes in vivo cell. In order to carryout electrochemical studies on the electronic properties ofBLMs, investigators always hope the supports between theelectrode and the BLMs possess electronic conductivitybesides their hydrophilicity. Ideally, electronically conduct-ing polymers could easily meet the requirements. Previousstudies have proven that PPy shows some interesting prop-erties with respect to biomimetic applications. The nega-tively charged multilamellar vesicles in aqueous solutioncan be included into a polypyrrole matrix by eletrophoreticcopolymerization (Belamie et al., 2001). Interestingly, thiselectrically conducting polymer can represent a type ofculture substrate which provides a noninvasive means to

0956-5663/$ – see front matter © 2004 Elsevier B.V. All rights reserved.doi:10.1016/j.bios.2004.06.001

1374 Y. Shao et al. / Biosensors and Bioelectronics 20 (2005) 1373–1379

control the shape and function of mammalian cells byelectrochemically adjusting its redox state (Wong et al.,1994). Additionally, Tien and his colleagues (Kotowski andTien, 1989) have made significative results on conventionalBLMs with polypyrrole as a modifier within BLM. Hianikand his coworkers used the method of spontaneous thinningof cephalin/decane mixtures on polypyrrole (Hianik et al.,1998; Nikolelis et al., 1999) but the usage of organic solventshould make the spontaneously incorporated proteins keepless activity. Up to now, the model of PPy film supportedBLM itself has not been experimentally proved. Herein,we first studied the deposition of BLM onto PPy film sur-face by vesicle fusion and investigated interactions betweenthem with surface plasmon resonance (SPR) combinedwith AFM and electrochemistry. Our work mainly aimedto understand the basal aspects of the BLM supported byconducting polymers.

2. Experimental section

2.1. Reagents and materials

Pyrrole was purified by passage through an activated alu-mina column until it became colorless. 1 mg ml−1 vesiclestocking solution containing dimyristoyl-l-alpha-phosphati-dylcholine (DMPC, Sigma, 99%), dimyristoyl-l-alpha-pho-sphatidylglycerol (DMPG, Sigma, 99%), dimyristoyl-l-al-pha-phosphatidyl-l-serine (DMPS, Sigma, 99%), or didode-cyldimethylammonium bromide (DDAB, Acros, 99%) wasdirectly dissolved in water by sonication after treatment withchloroform according to the procedure (Pierrat et al., 1997).Horseradish peroxidase (HRP) from Sigma was used with-out further purification. Thin (47–50 nm) Au films for SPRwere prepared by the nanoparticle-seeding electroless Auplating on a glass slide substrate according to the procedures(Jin et al., 2001).

2.2. Preparation of conducting polymer film

Electrochemical synthesis of polypyrrole film was car-ried out in a Teflon cuvette with gold film as the workingelectrode, a platinum wire as the counter electrode, andAg/(AgCl, saturated KCl) as the reference electrode. Allpotentials were recorded with respect to this referenceelectrode. The surface area of the working electrode was0.32 cm2, which was calculated from the charge consumedduring the reduction of the surface oxide layer in 0.5 MH2SO4 (Angerstein-Kozlowska et al., 1987). The elec-trodeposition solution containing 0.05 M pyrrole monomerand 0.05 M NaCl was purged by nitrogen 10 min. Polypyr-role films were formed potentiostatically at 0.65 V (versusAg/AgCl) until about 1.05 mC cm−2 was passed, corre-sponding to 5.0 nm thickness of PPy films (Lassalle et al.,2001). After polymerization, the gold film coated with PPywas immediately rinsed twice with ethanol and then with

water thoroughly. The resulted gold electrode was immersedin 0.05 M NaCl solution, and cyclic potential scanning wascarried out several times for equilibrating the film. Theas-prepared PPy film was stored in 0.05 M NaCl solution3 h before SPR measurements and vesicle fusion onto it.

2.3. Vesicles fusion onto conducting polymer film surface

Vesicle stocking solutions were first sonicated for 10 minbefore use and then diluted with a mixture of 1.0 mM KCland 1.0 mM NaCl to a final concentration of 0.50 mg ml−1.The dispersion was contacted over the polymer substrateat 32◦C, during which the kinetics of vesicle fusion wasrecorded by SPR measurements. Vesicles doped with HRPwere prepared by dissolving HRP into the 0.50 mg ml−1

DMPC vesicle solution with a final concentration of2 mg ml−1. If not stated, BLM was obtained from DMPCsolution.

2.4. SPR measurements

The glass slide covered with gold film for the SPRapparatus (SPR 2000, Electronic Institute of ChineseAcademy of Sciences, China) was pressed onto the baseof a half-cylindrical lens (n = 1.61) via an index-matchingoil. Linearly p-polarized light having wavelength of 670 nmfrom a diode laser was directed through the prism onto thegold film in the Kretshmann configuration. The intensity ofthe reflected light was measured as a function of the angleof incidence,θ, using a photodiode with a chopper/lock-inamplifier technique. For SPR combined with electrochem-ical detection, the as-prepared Au/glass substrates weremounted against the Teflon cuvette with 1 ml volume usinga Kalrez O-ring, which provided a liquid-tight seal. TheTeflon cuvette allowed for the simultaneous recording ofthe electrochemical data and the light interrogation fromSPR. The gold film on the glass slide used for the excita-tion of surface plasmon modes also served as the workingelectrode.

2.5. Electrochemical measurements

All electrochemical measurements were carried out with0.05 M NaCl as electrolyte. The complex impedance wasmeasured using Autolab/PG30 electrochemical analyzersystem (ECO Chemie B.V., Netherlands) over a frequencyrange from 0.1 Hz to 1 MHz with ac amplitude of 5 mV.The applied dc potential was 0.20 V, corresponding to theopen circuit potential of the resulted PPy film in order toavoid deviation of the system from its steady state. Equiva-lent circuits for the analysis of RC networks were appliedin order to determine the contributions from the electrolyte,the PPy films, and the BLM to the measured capacitanceand resistance. Cyclic voltammetric experiments (CHI 800,Shanghai Chenhua Instruments Co., China) combined with

Y. Shao et al. / Biosensors and Bioelectronics 20 (2005) 1373–1379 1375

SPR measurements were performed before and after theBLM formation.

2.6. AFM measurements

The samples were imaged by a SPA400 equipped with aSPI-3800 controller (Seiko Instruments Industry Co., Tokyo,Japan) at room temperature in distilled water. The tip typewas SN-AF01 (Seiko Instruments Co.) and the cantileverused was fabricated from Si3N4 with a spring constant of0.02 N m−1. All images were recorded with scan rate of2.0 Hz and repeated several times with different tips.

3. Results and discussion

3.1. Formation of PPy film

The formation of conducting polymer film is usually at-tained by means of electrochemically induced polymeriza-tion of monomer in aqueous solution for biosensor andbiomolecular immobilization (Schuhmann, 1995; Cosnier,1999). The applied electropolymerization potential, togetherwith the temperature, the monomer concentration, the sol-vent used and the nature of the incorporated counter-anion,has an important effect on average length of the resultedpolymer chains and the morphology of the resulted conduct-ing polymer film. To avoid the overoxidation of the PPy film,here the PPy film was potentiostatically formed at+0.65 Vwith Cl− as counter-anion that doped during polymeriza-tion process. As shown inFig. 1, when immediately dippedinto 5 mM potassium ferricyanide, the resulted PPy filmsdramatically reduced its redox peak current, and increasingthe dip time made the redox peak gradually clear, indicat-ing anion exchange and diffusion occurred in the PPy film.These observations demonstrate that the PPy film is porous

Fig. 1. Cyclic voltammograms at bare gold electrode (a) and gold electrodecoated by 5.0 nm PPy immediately dipped (b) or after scanned five cyclesin 0.05 M NaCl containing 5 mM K3Fe(CN)6.

(Bull et al., 1982) but not enough to allow the negativelycharged ferricyanide to be immediately exchanged into thefilm. This is in agreement with the film doped with ClO4

−(Curtin et al., 1988). The SPR signals show very distinct re-sponses to the deposition of PPy film onto gold electrode.It was convinced, fromFig. 2, that after the deposition, theSPR angle shifted to higher values and the shape of the SPRcurve became broader that resulted from the imaginary partof the refractive index of the PPy films. But if the PPy filmsbecame thicker, the reflectance at the SPR angle would in-crease, and the profile of the SPR curve would be dampedmore seriously (Bailey et al., 2002). In order to obtain a con-vincing result for the further self-assembly of lipids onto thePPy film the thickness of the PPy film was controlled notbeyond 5.0 nm. Because after the polymerization potentialwas removed a slow change in PPy film refractive index wasstill observed by previous authors (Bailey et al., 2002), thusthe prepared PPy film was simply stored in 0.05 M NaCl so-lution for 3 h until there was no any change in SPR responsebefore the self-assembly was carried out.

3.2. Deposition of BLM onto PPy surface

SPR is a convenient way to sense the mass change of itssensor surface. After self-assembly of DMPC onto PPy film,the profile of the SPR curve had a slight change and theSPR angle increased about 0.58◦ obtained by the Softwarefitting (Figs. 2 and 3). Considering the headgroup area perPC molecule in the condensed phase (about 50 Å2) (Lahiriet al., 1996), the adsorption of a DMPC monolayer corre-sponds to about 2.4 ng mm−2 of the adsorbed lipid. Using

Fig. 2. SPR responses of bare gold film (a), gold film coated by 5.0 nmPPy film (b), and gold film coated sequentially by 5 nm PPy film andBLM of DMPC (c) with 0.05 M NaCl as electrolyte.

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Fig. 3. Dependence of shift of SPR angle on time. Arrow 1 indicates0.05 M NaCl solution being exchanged by a mixture of 1 mM NaCl and1 mM KCl solution containing 0.5 mg ml−1 lipid vesicle of DMPC (curvea), DMPS (curve b), DDAB (curve c) and DMPG (curve d). Arrow 2corresponds to the solution in cuvette being changed by 0.05 M NaClsolution and arrow 3 by 50% ethanol solution.

the relationship of 0.10◦ shift of SPR angle corresponding to1 ng mm−2 of the adsorbed lipid monolayer (Stenberg et al.,1991) and the refractive index of the lipid (about 1.45, sim-ilar to that of protein (Lang et al., 1994; Hubbard et al.,1998)), we ascertain that the shift of SPR angle correspond-ing to the adsorption of a lipid bilayer is 0.48◦. Therefore,the data obtained indicate the adsorption of a closely packedDMPC bilayer on the PPy film surface. Interestingly, theDMPC bilayer supported on PPy surface was not disturbedby rinsing the electrode with 50% ethanol (curve a,Fig. 3).From Fig. 3, the deposition of DMPS made the SPR an-gle shift about 0.45◦, indicating the bilayer formation. ButDMPG and DDAB did not induce significant shifts of theSPR angle, showing no occurrence of the respective bilayer.These observations suggest a dependence of PPy supportedBLM on chemical structures of the lipid molecules used.

For vesicle spreading onto a surface from solution, the sur-face of the support has to be attractive or the vesicles must be

Fig. 4. Typical AFM images of the bare gold (a), the electropolymerized PPy layer (b) and PPy supported BLM (c). All were obtained in distilled water.

under tension (Bayerl and Bloom, 1990). The bilayer may belinked electrostatically through ion bridges or covalently bylock-and-key forces. Polypyrrole obtained via electrochemi-cal polymerization can be electrochemically cycled betweenits charged and neutral forms. But when the reduction po-tential is removed, the neutral polymer reverts completelyto its oxidized state within 30 s (Wong et al., 1994; Li andQian, 1989). Thus, two types of positively charged groups,which are located at carbon backbone about one positivecharge per four pyrrole units and protonated nitrogen atomper pyrrole unit, respectively, present at the PPy film sur-face. The negatively charged groups of the lipid molecules,therefore, can electrostatically interact with these positivelycharged groups located at PPy film surface even if withoutpositive potential applied to the electrode. Therefore, it isreasonable that negatively charged DMPS can form bilayerbut positively charged DDAB cannot form bilayer on PPysurface. However, it is interesting that DMPC possessing onenegatively charged and one positively charged headgroupscan also exhibit bilayer on PPy surface; DMPG, althoughalso containing two negatively charged and one positivelycharged headgroups as DMPS, cannot do so. Additionally,other factor such as different predominant disperse phasesof lipid in water may have profound effect on the forma-tion of BLM and can be converted by the experimental tem-perature between gel phase and liquid crystalline phase be-low or above the phase transition temperature. But the com-bined facts that the phase transition temperatures of DMPS,DMPC, DMPG, DDAB are 35 (Silvius, 1982), 23 (Lai et al.,2002), 23 (Cajal et al., 2003), 15◦C (Marques et al., 2002)and BLM is formed only for DMPC and DMPS seem todemonstrate that phase transition temperature may have lit-tle effect on the BLM formation here at our adopted tem-perature 32◦C. These phenomena indicate that the processof vesicles fusion to form BLM on PPy surface is very intri-cate. As indicated inFig. 3, DMPC showed a faster kineticdeposition than DMPS.

It has been proved that the PPy film was porous tosolvent and electrolyte ions (Bull et al., 1982). This wasalso convinced by our AFM experiments inFig. 4. Asconvinced by AFM, the gold surface seemed to be totally

Y. Shao et al. / Biosensors and Bioelectronics 20 (2005) 1373–1379 1377

covered by PPy but with some parts growing faster thanothers so that rough surface of PPy film occurred and someporous structures were clearly seen in AFM. These observa-tions accord with the model for pyrrole electropolymeriza-tion proposed byKim et al. (1991). The porous structuresmade the PPy film filled with solvent and electrolyte ions.On the other hand, the self-assembly of DMPC induced asmoother topography than the PPy layer with rms roughnessdecreasing from 4.484 to 2.914 nm (Fig. 4), showing thedeposition of BLM onto the PPy surface across the porousfilm.

The electric properties of the system should be differ-ent before and after vesicle fusion onto PPy film surface.In order to verify the point further and compare with theSPR data, impedance spectroscopy measurements were per-formed before and after the BLM deposition onto PPy filmsurfaces. Impedance techniques offer advantages over CVmethods mainly due to small perturbation of the systemfrom its steady state. The impedance spectroscopy of PPyfilm is very complex, which is seriously dependent on thepreparation method, the redox state, and the nature of thedoped counterion, of the film (Ferloni et al., 1996). In thestudied frequency range, the impedance spectroscopy of thePPy film should theoretically consist of two semicircles. Thefirst semicircle corresponded to the contribution of resis-tance of the polymer phase (R2) in parallel with its capaci-tance (represent with CPE1 due to its nonideal capacitanceproperty) at high frequency. The second semicircle was as-cribed to the resistance of the metal electrode/polymer in-terface in parallel with its double-layer capacitance (CPE2).Due to no apparent redox reaction took place at the inter-face during the applied potential range, the resistance at theelectrode/polymer interface was very big and thus it couldnot be reflected in the total impedance. If BLM deposited,the third semicircle appeared corresponding to the resistance(R3) of the BLM in parallel with its double-layer capacitance(CPE3) (Johnson et al., 1994). Based on literatures (Inzeltand Lang, 1994), we proposed the equivalent circuit (shownin Fig. 5) with constant phase elements (CPE) compensat-ing the non-ideal state of the interfaces (Lindholm-Sethson,1996). The fitting results (shown as solid lines inFig. 5)coincided with the experimental data very well in Nyquistplots (Zim versusZre) at high and intermediate frequency.Apparently, the deposition of BLM made the impedancespectroscopy markedly changed, showing the formation ofdensely packed bilayer. It is predictable that this change re-sults from high resistance of the hydrophobic chains withinthe BLM. The fitting results showed that CPE1 was changedfrom 74 to 75 pF after BLM deposition. This observationand AFM results confirm that solvent and electrolyte ionsstill retain within the porous PPy film when BLM depositionoccurs. Therefore, the PPy supported BLM is to some ex-tent comparable to conventional BLM with aqueous mediumretaining at its two sides. Thus the PPy supported BLMis a simple and specially promising system for biomimeticinvestigations.

Fig. 5. Impedance spectra of PPy film in 0.05 M NaCl solution before(a) and after (b) the vesicle fusion. Solid lines represent the fitting curvesaccording to the inserted equivalent circuit.R1 represents the resistanceof electrolyte solution.

3.3. Preliminary application

To test whether the quality of the s-BLM was sufficientlyhigh to immobilize proteins, we reconstituted HRP into thes-BLM by direct fusion of HRP-doped DMPC vesicles ontoPPy surface. The previous work reported that HRP could re-constitute into lipid bilayer and show its expected protein ac-tivity (Peng et al., 2002). Therefore, in our case, if HRP wasreconstituted into the s-BLM and contacts with the underly-ing PPy layer, we should observe electrochemical responsesof the functionalized enzyme electrode. Cyclic voltammetrywas used to prove the implant of HRP in the s-BLM andto investigate the electrocatalytic responses of the electrodeto H2O2 in solution. Compared to PPy supported BLM, theelectrocatalytic reduction of 1 mM H2O2 by HRP immobi-lized in DMPC bilayer occurred (Fig. 6) and the catalytic

Fig. 6. Cyclic voltammetric responses of the supported BLM to 1 mMH2O2 (curve a) and the supported HRP-doped BLM to 1 mM H2O2 (curveb), 2 mM H2O2 (curve c), 3 mM H2O2 (curve d), 4 mM H2O2 (curve e).

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currents increased with increasing H2O2 concentration. Theresults indicate that HRP can integrate into the PPy sup-ported BLM and still retain its biological activity. So theheme group of HRP in the s-BLM should achieve a favor-able orientation for exchanging electrons with the underly-ing PPy layer (Tatsuma et al., 1992), showing a promisingdesign for biosensors. Although a quantitative comparisonis beyond the scope of this research this time as to the sensi-tivity and life-span between the HRP-doped BLM electrodewith and without the underlying PPy support because theamount of HRP, the HRP-electrode distance and the surfacestructure of the electrode are different between them, our ex-perimental results indicate that PPy layer as support is suit-able for exchanging electron with the protein incorporatedin BLM.

4. Conclusions

We have demonstrated a simple method of assembly ofbilayer lipid membranes deposited onto electronically con-ducting polymer surface by vesicles fusion. The practicabil-ity of vesicles fusion onto the PPy surface to form s-BLMshows a dependence on the chemical structure of the lipidsused. The deposition of BLM markedly changes the elec-tronic properties of the electrode convinced by impedancespectroscopy. Therefore, electrochemically synthesized PPyfilm can be served as a novel support for s-BLM. Comparedwith other BLMs supports, the PPy support has some ad-vantages. Firstly, it can be formed faster than alkanethiolatesupports that usually need more than several hours to ob-tain a densely packed support monolayer. Secondly, it is ex-pected that the as-prepared s-BLM should show lateral flu-idity comparable to conventional BLM, while the tetheredlipid bilayer membranes possess susceptible lateral fluiditybecause the reservoir lipid as a part of the BLM was immo-bilized onto gold surface by gold–sulfur bond (Raguse et al.,1998). Very importantly, the support is electrically conduct-ing, thus it is convenient to carry out electron transfer stud-ies for biomimetic membranes with respect to proteins bysimply electrochemical methods.

Acknowledgements

We gratefully acknowledge the special funds from MajorState Basic Research of China (2002CB713803) and the Na-tional Natural Science Foundation of China (no. 20275036and 20211130506) for supports of this research.

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