Electrochemical and Surface Plasmon Resonance Characterization of

Microscopic Study of Menadione Permeability through a Self-Assembled Monolayer on a Gold Electrode. Céline Cannes, Frédéric Kanoufi, and Allen ...
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Langmuir 1997, 13, 4112-4118

Electrochemical and Surface Plasmon Resonance Characterization of the Step-by-Step Self-Assembly of a Biomimetic Structure onto an Electrode Surface Olivier Pierrat, Nathalie Lechat, Christian Bourdillon, and Jean-Marc Laval* Laboratoire de Technologie Enzymatique, UPRESA 6022, Universite´ de Technologie de Compie` gne, B.P. 20 529, 60205 Compie` gne Ce´ dex, France Received February 10, 1997X A plane gold-supported bilayer was prepared on an electrode by fusion of phospholipid (dimyristoylphosphatidylcholine (DMPC)) vesicles onto an alkanethiol (octadecylmercaptan (OM)) self-assembled monolayer (SAM). Escherichia coli pyruvate oxidase (Pox), a peripheral membrane enzyme, was incorporated into the supported bilayer. This supramolecular assembly was characterized by contact angle goniometry, electrochemical blocking studies, double-layer capacitance, and BIAlite (surface plasmon resonance) measurements. Electrochemistry of ferrocenemethanol at the gold surface was blocked by the well-ordered alkane chains of the OM monolayer. In order to prevent this blocking effect, dibenzyl disulfide (DBDS) was used to produce defect sites in the OM monolayer and to allow the reversibility of ferrocene electrochemistry. BIAlite measurements showed that fusion of DMPC on the OM + DBDS monolayer was not significantly different from the fusion of DMPC on the OM monolayer. Pox incorporation into the (OM + DBDS)/DMPC gold-supported bilayer was detected by BIAlite measurements. The activity of incorporated Pox was detected by the electrocatalytic current produced when substrate and the electron acceptor, ferricinium methanol, were present in solution.

Introduction Self-assembled monolayers (SAMs) of alkanethiols on solid support have been the subject of active research since the past decade (for review see refs 1-3). Such assemblies can provide useful tools for studying various phenomena such as catalysis, corrosion, wettability, adhesion, molecular recognition, and microelectronics. SAMs of alkanethiols are also useful for building up biomimetic models of the biological membranes.4 Previous work from our laboratory has reported the formation of supportedhybrid bilayers on microporous aluminum oxide at gold electrodes: a monolayer of electroactive amphiphile molecules (octadecylferrocene, octadecylviologene) was self-assembled on a hydrophobic supported monolayer of octadecyltrichlorosilane.5,6 Following the formation of this two-dimensional assembly, redox enzyme (i.e., glucose oxidase, hydrogenase) was immobilized on the monolayer of electroactive amphiphiles and enzyme activity was measured by the electrocatalytic current with electroactive amphiphile molecules as the redox mediator between the enzyme and gold surface.5,6 More recently, we replaced artificial amphiphile molecules with phospholipids: a monolayer of phospholipids was self-assembled onto a first monolayer of octadecyltrichlorosilane supported on a microporous aluminum oxide electrode.7 The spatial structure of the microporous aluminum oxide electrodes allowed the measurement of the lateral diffusion of octadecylviologene electroactive probe molecules in the phospholipid monolayer.7 Whereas microporous aluminum oxide electrodes allow the study of the dynamics of the lipid monolayers, plane gold electrodes allow the characterization of the structure of the assembly by electrochemistry (electrochemical blocking, capacitance X

Abstract published in Advance ACS Abstracts, July 1, 1997.

(1) Bain, C. D.; Whitesides, G. M. Angew. Chem. 1989, 101, 522. (2) Xu, J.; Li, H. L. J. Colloid Interface Sci. 1995, 176, 138. (3) Zhong, C. J.; Porter, M. D. Anal. Chem. 1995, 367, 709A. (4) Plant, A. L. Langmuir 1993, 9, 2764. (5) Bourdillon, C.; Majda, M. J. Am. Chem. Soc. 1990, 112, 1795. (6) Parpaleix, T.; Laval, J. M.; Majda, M.; Bourdillon, C. Anal. Chem. 1992, 64, 641. (7) Torchut, E.; Laval, J. M.; Bourdillon, C.; Majda, M. Biophys. J. 1994, 66, 753.

S0743-7463(97)00133-9 CCC: $14.00

measurements) or by surface plasmon resonance (SPR). In the model of membrane presented here (Figure 1), an alkanethiol monolayer was self-assembled on a plane gold electrode and a successive monolayer of phospholipid was fused onto the first hydrophobic alkanethiol monolayer to form the hybrid bilayer. Then, membrane enzyme pyruvate oxidase (Pox) of Escherichia coli (pyruvate: ubiquinone-8 oxidoreductase, E. C. 1.2.2.2) was incorporated into the gold-supported bilayer (Figure 1). Pyruvate oxidase is a tetrameric enzyme that exists in the form of a soluble enzyme or in the form of a peripheral membrane enzyme. Incorporation of the enzyme in the membrane occurs via a small amphipatic R-helix8 and requires the reduction of the enzyme by pyruvate in the presence of thiamin pyrophosphate.9 Such insertion produces an increase in Pox activity by a factor 25.9 The biological electron acceptor for the Pox localized in the membrane is ubiquinone-40.10 In the study presented in this paper, electrochemistry and BIAlite measurements were made to characterize the step-by-step formation of the biomimetic assembly. Catalytic current measurements have demonstrated that the flavoprotein Pox, incorporated into the two-dimensional membrane structure, can use the ferricinium methanol as an electron artificial acceptor, as was the case for the flavoprotein glucose oxidase.11 Experimental Section Dibenzyl disulfide (DBDS) and ferrocenemethanol (FcMe) were from Aldrich. L-R-Dimyristoylphosphatidylcholine (DMPC) was from Sigma. All three products were used without further purification. Octadecylmercaptan (OM) was from Aldrich and was recrystallized in ethanol. Water was purified by a Milli-Q System (Millipore). Escherichia coli pyruvate oxidase (Pox) was purified from a mutant strain YYC 458, generously given by L. P. Hager laboratory (University of Illinois, Champaign-Urbana). (8) Grabau, C.; Chang, Y. Y.; Cronan, J. E. J. Biol. Chem. 1989, 264, 12510. (9) Schrock, H. L.; Gennis, R. B. Biochim. Biophys. Acta 1980, 614, 215. (10) Koland, J.; Miller, M.; Gennis, R. B. Biochemistry 1984, 23, 445. (11) Bourdillon, C.; Demaille, C.; Gueris, J.; Moiroux, J.; Saveant, J. M. J. Am. Chem. Soc. 1993, 115, 12264.

© 1997 American Chemical Society

Step-by-Step Self-Assembly of a Biomimetic Structure

Figure 1. Hypothetic scheme of the biomimetic assembly. For electrochemical measurements, gold substrates on clean glass microscope slides were prepared by vacuum deposition (Edwards Model E306A) of 5 nm of a chromium adhesion layer followed by 200 nm of gold. Prototype sensor chips with bare gold for BIAlite measurements were provided by Biacore AB. Self-Assembled Monolayer (SAM) Formation. The gold surfaces were cleaned by freshly prepared sulfochromic acid for 30 s, rinsed with copious amounts of water (Milli-Q water), and dried in a stream of pure N2, just before the formation of monolayers. SAMs of OM were formed by immersing the gold substrates in 1 mM OM in ethanol/water (4:1) for 10 min to 4 h at room temperature. Previous experiments12 using the same procedure have shown that overnight incubation led to the selfassembly of a very well-packed OM monolayer. Mixed SAMs of OM + DBDS were obtained as follows: gold substrates were immersed in 10 mM DBDS in ethanol/water (4:1) for at least 4 h, rinsed with toluene and water, dried in a stream of pure N2, and immersed again in 1 mM OM in ethanol/water (4:1) for 10 min to 4 h. After self-assembling of OM or OM + DBDS monolayers on gold surfaces, the electrodes were rinsed successively with toluene, water, methanol, and water and dried in a stream of pure nitrogen before measuring the contact angles (θ°/H2O) with a goniometer (G1, Kru¨ss). A simple contact angle measurement was made to estimate the hydrophobicity of the layer: a drop of water was deposited on the layer surface, and the contact angle was determined visually using an optical device; each measurement was repeated at least two times. Bilayer Formation. Vesicles of DMPC were obtained mainly according to ref 13. Briefly: dissolution of lipid in chloroform, chloroform evaporation in a stream of pure N2 and overnight dessication, resuspension of lipid at 10-3 M in 0.1 M sodium phosphate buffer, pH 6, sonication at 40 °C in an ultrasonic cleaner (Branson 2210) to obtain an homogeneous suspension, sonication of the lipid suspension with a titanium rod sonifier to obtain an optically transparent vesicle solution (Ultrasonic desintegrator Branson 250; 70 W, 5 cycles of 2 × 2 min sonication), removal of titanium dust by centrifugation, and finally filtration on 0.2 µm Acrodisc and 0.1 µm Whatman Anotop 10. Hybrid OM/DMPC and (OM + DBDS)/DMPC bilayers were obtained by fusion of DMPC vesicles during 60 min respectively on OM and on OM + DBDS monolayers. In the procedure, the electrodes were simply immersed in a beaker containing a vesicle solution. Just after fusion of DMPC on the monolayer and before electrochemical measurements, the electrode is rinsed in two ways: first, by immersion in a beaker containing the same buffer without lipid and, second, by immersion in the electrochemical cell via a continuous stirring tank reactor (CSTR). In order to transfer the (OM + DBDS)/DMPC electrodes, the bilayer-coated electrodes were covered with a glass slide under water before being removed from the buffer solution. Previous experiments using dipalmitoylphosphatidylcholine (DPPC) have demonstrated that this operation is essential in protecting the integrity and stability of the phospholipid monolayer of a OM/DPPC electrode. Transfer of an unprotected OM/DPPC electrode across the air/water interface always resulted in desorption of the DPPC monolayer.12 Purification of Escherichia coli Pyruvate Oxidase (Pox). The procedure of purification of E. coli Pox followed essentially (12) Laval, J. M.; Majda, M. Thin Solid Films 1994, 244, 836. (13) Reinl, H. M.; Bayerl, T. M. Biochemistry 1994, 33, 14091.

Langmuir, Vol. 13, No. 15, 1997 4113 the one described in refs 14 and 15, with some modifications: we used the French Press (14 000 psi) for breaking the cells, and only one anion-exchange chromatograph was made, with Qsepharose fast-flow (Pharmacia Biotech) as the anionic exchanger. Otherwise, we used the same steps as those described in ref 15: thawing, cell suspension, cell breaking, heating, ammonium sulfate precipitation, dialysis, anionic-exchange chromatography, and concentration/desalting on an Amicon Centriprep-30. The same buffer were used except that Pox was eluted from the chromatographic column by 0.4 M NaCl. Spectrophotometric Assay for Enzyme Activity. The standard ferricyanide assay used was that according to ref 15. Briefly, the Pox is incubated for 10 min at 10 µg/mL in 0.1 M sodium phosphate buffer, pH 6, 200 µM thiamine pyrophosphate (TPP), 20 mM Mg(NO3)2, and 200 mM sodium pyruvate (reaction medium), in the presence of lipid activator (1 mL total volume). The lipid activators of Pox were the vesicles of DMPC, prepared as mentioned above, at a final concentration varying from 4 to 200 µM. Enzymatic reaction is measured by adding 9 mM ferricyanide and by monitoring absorbance diminution at 450 nm by spectrophotometry (Milton Roy spectronic 1201). Incorporation of Pox into the (OM + DBDS)/DMPC Supported Bilayer. The (OM + DBDS)/DMPC electrode is transferred into a beaker containing 10-20 µg/mL of Pox in the same reaction medium as the one used for the spectrophotometric assay. Activation/fixation of Pox in the membrane is obtained after a 30 min incubation period. Then, the modified electrode is rinsed in the reaction medium without pyruvate in order to remove all enzyme not well-incorporated into the bilayer. A layer of water is always maintained between the bilayer assembly and the air. Electrochemical Characterization of the Self-Assemblies. Electrochemical experiments were carried out in an anaerobic electrochemical cell with three electrodes: the working modified gold electrode, a saturated KCl calomel electrode (SCE) as the reference electrode, and a platinum foil auxiliary electrode. Cyclic voltammetry was performed at 30 °C in a 0.1 M sodium phosphate buffer, pH 6, or in a reaction medium of Pox (see above), in the absence or presence of 10-4 or 3 × 10-4 M ferrocenemethanol in solution, using an EG&G potentiostat Model 273 (Princeton Applied Research), at a scan rate of 25 or 50 mV/s. Interfacial capacitance was calculated (for potential ranging from -200 to +100 mV) according to the relation12 ic ) AνC, where ic is the double-layer charging current (in the absence of ferrocenemethanol), A is the electrode surface area (0.54 cm2), ν is the scan rate (V‚s-1), and C is the interfacial capacitance (F‚cm-2). Ferrocenemethanol was added to the electrolyte solution to measure the electrochemical blocking effect of the monolayers or bilayers and thus to evaluate their passivating property. The enzymatic activity of Pox incorporated into the supported bilayer was measured using ferricinium methanol as the electron acceptor and mediator between the enzyme and electrode: cyclic voltammetry was performed at 25 mV/s, 30 °C, in the presence of 10-4 M ferrocenemethanol in the reaction medium of the enzyme containing 21 mM of sodium pyruvate. SPR Characterization of the Self-Assemblies. Surface plasmon resonance is a very sensitive technique for processes occurring at or near interfaces.16 Briefly, upon total internal reflection of a laser beam on a prism base, surface plasmons can be excited at a metal/dielectric interface. Their coupling to the evanescent wave of the incident light is observed at the resonance angle θ. Covering the metal with an organic layer typically shifts the resonance angle, θ, to higher values. The measurement of the shift of θ allows the determination of the thickness of the adsorbed layer, given an appropriate layer refractive index. SPR measurements were made with a BIAlite instrument in which delivery of liquid to the sensor chip surface was made at a continuous flow rate (Biacore AB). In this case,17 the formation of a layer on the gold interface with a refractive index of 1.45 will give an SPR response of 700 RU/nm of film thickness (1000 RU is a change in resonance angle of 0.1°). The BIAlite measure(14) Recny, M. A.; Hager, L. P. J. Biol. Chem. 1982, 257, 12878. (15) Bertagnolli, B. L.; Hager, L. P. J. Biol. Chem. 1991, 266, 10168. (16) Raether, H. In Physics of Thin Films; Mass, G., Eds.; Academic Press: New York, 1977; Vol. 9, p 145. (17) Sigal, G. B.; Bamdad, C.; Barberis, A.; Strominger, J.; Whitesides, G. M. Anal. Chem. 1996, 68, 490.

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Table 1. Contact Angle and Interfacial Capacitance of Gold Electrodes Modified by OM or OM + DBDS Monolayers, as a Function of Self-Assembling Time of OMa type of electrode bare gold DBDS DBDS/OM (10 min) DBDS/OM (30 min) DBDS/OM (60 min) DBDS/OM (4 h) OM (10 min) OM (30 min) OM (60 min) OM (4 h)

contact angle (θ°/H2O) capacitance (µF/cm2) 59 ( 1 69 ( 1 84 ( 1 99 ( 2 103 ( 1 101 ( 1 104 ( 1 106 ( 1 109 ( 1 110 ( 1

20.4 6.3 4.8 3.9 3.3 3.0 3.0 2.8 2.2 1.0

a The reproducibility of the capacitance experiments is around 10%.

ments corresponding to the formation of OM or OM + DBDS monolayers were achieved by comparison of the RU signal of the prototype sensor chip with bare gold (Biacore AB) before and after the self-assembly procedure (0.1 M sodium phosphate buffer, pH 6, 2 µL/min flow rate). The fusion of DMPC vesicles was directly monitored during BIAlite experiments (DMPC vesicles solution, 2 µL/min flow rate). The formation of a multilayer of DMPC was first observed during the fusion. A stable RU signal in agreement with a DMPC monolayer can be obtained by successive rinsings with NaOH solutions (10-40 mM NaOH, 40 µL/min flow rate), in accordance with the procedure of Biacore AB for the fusion of lipid vesicles on sensor chip HPA (BIAtechnology Note 106). The incorporation of the Pox on a modified gold sensor chip, after fusion of DMPC vesicles on the OM + DBDS monolayer, was directly monitored by BIAlite experiments (20 µg/mL Pox in the reaction medium, 2 µL/min flow rate). The modified sensor chips with a self-assembled monolayer were regenerated with a 40 mM n-octyl-β-D-glucopyranoside solution (20 µL/min flow rate).

Results and Discussion Monolayer Formation. The contact angle and capacitance results shown in Table 1 indicate that the amount of OM present on the gold surface can be varied by changing the self-assembling time: the contact angle increased and the capacitance decreased as the selfassembling time of OM was increased. However, OM electrodes showed a contact angle more hydrophobic and a lower capacitance than those of the corresponding OM + DBDS electrodes. The capacitance of 1.0 µF‚cm-2 obtained after self-assembling of OM for 4 h on bare gold correlates well with the capacitance of 1.0-1.5 µF‚cm-2 reported for a densely packed monolayer of alkanethiolates on gold.12,18 Even after 4 h of OM self-assembling, the hydrophobicity of the OM + DBDS electrode did not reach the one obtained for the OM electrode and its capacitance is 3 times greater than that of the latter. Self-assembling of DBDS molecules on the gold electrode was demonstrated by a decrease of the capacitance of the DBDS electrode compared to that of the bare gold electrode. It can be concluded from these results that the presence of DBDS on the gold surface decreased and limited the following adsorption of OM. We assumed that, as the self-assembling time was increased, OM molecules in solution were replacing the DBDS molecules on the electrode although, at the time scale used here, all the DBDS molecules were not exchanged with the OM molecules in solution. Such an exchange of adsorbed organosulfur molecules with those in solution has been previously described in the literature.19,20 Moreover, molecules with short hydrocarbon chain lengths are easily exchanged with (18) Chidsey, C. E. D.; Loiacono, D. N. Langmuir 1990, 6, 682. (19) Biebuyck, H. A.; Whitesides, G. M. Langmuir 1993, 9, 1766. (20) Schlenoff, J. B.; Li, M.; Ly, H. J. Am. Chem. Soc. 1995, 117, 12528.

Table 2. SPR Characterization of the Formation of the Assembliesa monolayer

self-assembly of monolayer

fusion of DMPC vesicles

OM OM + DBDS

2000 RU 1950 RU

2000 RU 1900 RU

a The RU values after fusion of DMPC vesicles are in addition to the values obtained for either OM or OM + DBDS monolayer alone. The reproducibility of the SPR experiments is around 10%.

molecules in solution with longer chain lengths.19 The hydrophobicity of the OM + DBDS monolayer and its lower capacitance (at least for 1 or 4 h of OM self-assembling) compared to a single DBDS monolayer suggest that OM was in large excess compared to DBDS. Therefore, we have created a hydrophobic OM monolayer but with some defects artificially created by the DBDS molecules. The study of OM coverage of OM electrodes and OM + DBDS electrodes was made by BIAlite measurements. The results are summarized in Table 2. We assumed that the refractive indexes for the molecules of OM, DMPC, and Pox are the same, close to 1.45.21,22 Thus, mass coverages can be calculated for OM, DMPC, and Pox molecules, assuming that the SPR response is 700 RU/ nm of thickness for the OM, DMPC, and Pox layers.17 With these assumptions, the 2000 RU observed for the OM layer corresponds to a thickness of 2.8 nm. This OM layer thickness is in the range of 2.4-3.0 nm determined in the literature by ellipsometry or SPR.23-25 The full extended chains of OM molecules have a length of around 2.8 nm23 and long chains of alkanethiol have a tilt angle of around 30° when self-assembled onto the gold surface,23,26-28 which leads to a theoretical thickness value of around 2.3 nm. The small difference between our result and the theoretical value can be due to the roughness of the gold surface or to a refractive index of the OM layer somewhat different from 1.45 (see discussion in ref 23). The SPR response for an OM + DBDS electrode (in which OM was self-assembled for 60 min) was 1950 RU. Considering that the reproducibility of the SPR experiments is around 10%, the formation of OM and OM + DBDS monolayers gave very similar results. Moreover, if we reasonably attribute the SPR response only to the OM molecules, we can say that the BIAlite measurements confirm that DBDS forms minor defects in the hydrophobic OM monolayer. The results obtained in the study of the blocking effect produced by the OM and OM + DBDS monolayers on the ferrocenemethanol electrochemistry on the gold surface are illustrated in the Figure 2. The presence of a small amount of DBDS in the OM monolayer has a dramatic effect on the electrochemistry of ferrocenemethanol in solution. Indeed, all OM + DBDS electrodes showed a separation between the anodic and cathodic peak currents of 60 mV at 25 mV/s (also true at 250 mV/s), indicating a reversible electrochemistry of ferrocenemethanol at the gold surface. However, a 10-15% decrease of the anodic and cathodic current peaks was observed relative to the naked gold surface. In comparison, all OM electrodes (30 (21) Salamon, Z.; Wang, Y.; Tollin, G.; Mc Leod, H. A. Biochim. Biophys. Acta 1994, 1195, 267. (22) Prime, K. L.; Whitesides, G. M. Science 1991, 252, 1164. (23) Porter, M. D.; Bright, T. B.; Allara, D. L.; Chidsey, C. E. D. J. Am. Chem. Soc. 1987, 109, 3559. (24) Offord, D. A.; John, C. M.; Griffin, J. H. Langmuir 1994, 10, 761. (25) Peterlinz, K. A.; Georgiadis, R. Langmuir 1996, 12, 4731. (26) Strong, L.; Whitesides, G. M. Langmuir 1988, 4, 546. (27) Nuzzo, R. G.; Dubois, L. H.; Allara, D. L. J. Am. Chem. Soc. 1990, 122, 558. (28) Amatore, C.; Saveant, J. M.; Teissier, D. J. Electroanal. Chem. 1983, 147, 39.

Step-by-Step Self-Assembly of a Biomimetic Structure

Figure 2. Cyclic voltammetry of 3 × 10-4 M ferrocenemethanol in 0.1 M sodium phosphate buffer, pH 6, on OM (thin line) and OM + DBDS (thick line) electrodes (30 °C, 50 mV/s). In both cases, OM was self-assembled for 10 min (A), 30 min (B), 60 min (C), or 4 h (D).

min to 4 h of OM self-assembling) showed an absence of reversibility and a high passivation. The term “reversibility” is only relative here to the shape of the experimental voltammogram. In other words, the absence of reversibility only means a peak-to-peak current separation greater than 60 mV. The different shapes of voltammograms are mainly due to diffusional effects. For the most passivated electrode (OM, 4 h), diffusion of ferrocenemethanol to the electroactive surface was mainly radial, whereas, for all OM + DBDS electrodes, the system can be assimilated to an array of microelectrodes close enough between each other so that a global linear diffusion of ferrocenemethanol to the electroactive surface can be considered.28-32 In view of these results, it seems that DBDS could be assimilated with a “gate site” molecule, allowing access of ferrocenemethanol in solution to the gold surface.33 This system can be compared to the hydroxythiophenol template molecule used in a hexadecanethiol framework by refs 32 and 34, except that, in our case, DBDS was adsorbed on the gold surface before OM. When 1 mM OM was also present during self-assembling of DBDS molecules onto the gold surface (coadsorption), we obtained OM + DBDS electrodes which showed the same blocking effect of the electrochemistry of ferrocenemethanol as their corresponding pure OM monolayers. We conclude from that result that OM is more preferentially adsorbed onto the gold surface than DBDS. Such a preference of adsorption of alkanethiols versus disulfides onto the gold surface has already been described in the (29) Sabatani, E.; Rubinstein, I. J. Phys. Chem. 1987, 91, 6663. (30) Bilewicz, R.; Majda, M. Langmuir 1991, 7, 2794. (31) Finklea, H. O.; Snider, D. A.; Fedyk, J.; Sabatani, E.; Gafni, Y.; Rubinstein, I. Langmuir 1993, 9, 3660. (32) Chailapakul, O.; Crooks, R. M. Langmuir 1995, 11, 1329. (33) Bilewicz, R.; Majda, M. J. Am. Chem. Soc. 1991, 113, 5464. (34) Chailapakul, O.; Crooks, R. M. Langmuir 1993, 9, 884.

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literature: when monolayers were formed from mixtures of alkanethiol and dialkyl disulfide (of the same chain length), adsorption of the thiol was preferred with a ratio 75:1.35 We assume that DBDS is not favored for adsorption on gold, compared to OM, for two main reasons: first, adsorption of disulfides on gold is disfavored compared to that of the corresponding alkanethiol, and,35 second, we know that short organosulfur molecules, adsorbed on gold surfaces, are very efficiently replaced by long hydrocarbon chain thiol molecules in solution.19 As opposed to ferrocenemethanol, ferricyanide did not show any reversibility of its electrochemistry on gold when OM + DBDS electrodes were used (data not shown). Such a difference between different redox probe molecules in solution has been extensively studied by refs 32 and 34. Ferrocenemethanol, as a neutral molecule, is clearly more efficient in penetrating the OM + DBDS monolayer than the ferricyanide, a negatively charged molecule. In the following experiments, OM was systematically self-assembled for 60 min onto DBDS electrodes. As an alternative to the method described above (which used the time of self-assembling of OM as the varying factor), OM coverage in OM + DBDS electrodes can be controlled by reducing its concentration in the self-assembling solution from 1 to 0.01 mM (data not shown). However, below 0.1 mM and with 60 min of OM self-assembling, the OM + DBDS electrodes were not hydrophobic. Bilayer Formation. Electrochemistry of 10-4 M ferrocenemethanol before and after DMPC self-assembling is presented in Figure 3 at OM + DBDS electrodes. The three different OM + DBDS electrodes were obtained by varying the OM concentration in the self-assembling solution from 0.5 to 0.1 mM and, hence, varying their contact angles from 94° to 90°, respectively. Concerning the OM + DBDS electrodes, obtained by incubation with a high concentration of OM in solution (Figure 3A,B), the results indicate that reversibility of ferrocenemethanol electrochemistry at gold was lost after fusion of DMPC vesicles on the OM + DBDS monolayer. Our findings are in agreement with the literature, which reported that, after fusion of phospholipids on an OM monolayer, the electrochemistry of redox species in solution is even more blocked.4,12,36 However, the OM + DBDS electrode, obtained by incubation with OM at 0.1 mM in solution (Figure 3C), showed a quasi-reversible electrochemistry of ferrocenemethanol at the bilayer-supported gold electrode: a separation between anodic and cathodic peak currents around 70 mV at 25 mV/s was obtained. The fusion of DMPC vesicles on OM + DBDS monolayer was also directly monitored by BIAlite measurements (Table 2), and the data were similar to those (BIAtechnology Note 106) obtained for the fusion of lipid vesicles on a commercial gold chip with OM monolayer (sensor chip HPA). The RU values after fusion of DMPC vesicles (Table 2) are in addition to the values obtained for either OM or OM + DBDS monolayer alone. There was no significant difference between the RU value obtained for the OM/DMPC bilayer (2000 RU) and that obtained for the (OM + DBDS)/DMPC bilayer (1900 RU). These RU values are very close to those previously obtained for the formation of a OM monolayer and are in agreement with our assumption of formation of a DMPC monolayer of 2.8 nm of thickness (see discussion above). In the case of DMPC, the smaller length of the hydrocarbon chain can be compensated by a smaller angle tilt of the chain. For example, the tilt of the chain in the DMPC crystal is only (35) Bain, C. D.; Whitesides, G. M. J. Am. Chem. Soc. 1989, 111, 7164. (36) Plant, A. L.; Gueguetchkeri, M.; Yap, W. Biophys. J. 1994, 67, 1126.

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Figure 4. Activation of Pox at 24 °C by DMPC vesicles. Pox was incubated at 10 µg/mL for 10 min in 0.1 M phosphate buffer, pH 6, 200 µM TPP, 20 mM Mg(NO3)2, and 200 mM sodium pyruvate, in the presence of various concentrations of DMPC ranging from 4 to 200 µM. The enzymatic activity was measured spectrophotometrically by the standard ferricyanide assay. Activity corresponding to a Kcat of 27 s-1 was detected without DMPC.

Figure 3. Cyclic voltammetry of 10-4 M ferrocenemethanol on OM + DBDS electrodes, before (thick line) and after (thin line) self-assembling of DMPC for 60 min (30 °C, 25 mV/s). The electrolyte solution corresponded to the reaction medium of the enzyme (see the Experimental Section). OM concentrations in the self-assembling solution were at 0.5 mM (A), 0.25 mM (B), or 0.1 mM (C).

12° away from the bilayer normal.37 In comparison, a bilayer of phospholipids typically has a thickness of around 40-50 Å.37 The fusion of lipid vesicles onto a hydrophobic surface to form a lipid monolayer has been demonstrated by Kalb et al.38 In our previous studies, radioactive-labeled lipids have been used to examine the fusion of DMPC vesicles onto a hydrophobic octadecyltrichlorosilane monolayer. The lipid coverage was maximum after 30 min of fusion of vesicles and corresponded to a phospholipid monolayer in the solid/liquid coexistence region.7 Spectrophotometric Assay for Enzyme Activity. It was essential to confirm that our purified Pox could interact with the phospholipid membrane in a manner that allowed its incorporation. An easy way was to verify that the enzyme was correctly activated by DMPC. Figure 4 represents the level of Pox activity obtained by increasing the amount of DMPC vesicles in solution. The maximal activation corresponded to a 23 times increase of the rate of ferricyanide reduction by Pox. This maximal activation is in good accordance with the previously reported 25 times increase of Pox activity with DMPC.9 Additional experiments (not shown) have demonstrated that activation of Pox by surfactants (sodium dodecyl sulfate) was also in good agreement with the literature data.39 (37) Gennis, R. B. In Biomembranes: molecular structure and function; Cantor, C. R., Ed.; Springer-Verlag: New York, 1989; Chapter 2. (38) Kalb, E.; Frey, S.; Tamm, L. K. Biochim. Biophys. Acta 1992, 1103, 307. (39) Blake, R.; Hager, L. P. J. Biol. Chem. 1978, 253, 1963.

Figure 5. Detection of Pox activity in the supported bilayer by enzymatic electrocatalysis with ferrocenemethanol: (A) Cyclic voltammetry of 10-4 M ferrocenemethanol on (OM + DBDS)/(DMPC + Pox) electrode, in the absence (thin line) or presence (thick line) of 21 mM pyruvate in solution (30 °C, 25 mV/s). The electrolyte solution corresponded to the reaction medium of the enzyme (see the Experimental Section). (B) Catalytic current as a function of the potential value. At each potential, the catalytic current (icat) was obtained from the difference in Figure 5A between current with pyruvate (thick curve) and current without pyruvate (thin curve).

Detection of Pox Incorporated into the (OM + DBDS)/DMPC Bilayer. Figure 5 illustrates the enzymatic electrocatalysis reactions of the oxidation of pyruvate by Pox in the presence of ferrocenemethanol (FcMe). The catalytic current was obtained by coupling the oxidation of pyruvate by Pox and the electrochemical oxidation of ferrocenemethanol at the gold surface (Figure 6). In our system, the cosubstrate ferricinium methanol (Fc+Me) was only produced at the electrode surface during

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Figure 6. Schematic representation of the catalytic cycle of pyruvate oxidase with ferrocenemethanol in solution as the redox mediator between the enzyme and the electrode surface.

the cyclic voltammetry, and, hence, the enzymatic reaction was controlled by this electrochemical production of Fc+Me. In order to further study Pox kinetics, we were particularly interested in the reversible electrochemistry of ferrocenemethanol for two reasons: (i) the linear diffusion of ferrocenemethanol to the gold surface allowed a more efficient electrocatalytic reaction and (ii) the cosubstrate concentrations at each value of potential can be easily calculated by the Nernst relation.11 In the absence of pyruvate in the electrochemical cell, the peakto-peak separation of 65 mV, at 25 mV/s, indicated a quasireversible electrochemistry of ferrocenemethanol in solution (Figure 5; thin line curve). We verified that the peak currents were proportional to the square root of the scan rate as expected. In the presence of 21 mM sodium pyruvate in solution (Figure 5; thick line curve), the enzymatic activity of Pox was detected by an increase in the anodic current and a decrease in the cathodic current. The catalytic current (icat) was obtained at each potential by subtracting the current without catalysis (in the absence of pyruvate) to the current with catalysis (in the presence of 21 mM pyruvate) (see Figure 5). We verified that icat was independent of the scan rate. The detection of catalytic current demonstrated that the incorporated Pox of Escherichia coli (membrane enzyme) can react with the ferricinium during the catalytic cycle, as glucose oxidase can do.11 Moreover, we noticed that if Pox is inserted into the bilayer in the absence of pyruvate, no catalytic current is detectable. This finding is wellcorrelated to the fact that Pox needs to be reduced by its cosubstrates (pyruvate, TPP) to interact properly with DMPC vesicles.9 In addition, the catalytic current obtained on the supported membrane was not maintained if the overall structure was removed from the solution of the electrochemical cell through the air/water interface. This indicates that the Pox was well-incorporated into the lipid monolayer, and the latter was not stable when passing the air/water interface. In an additional experiment, we tested the stability of the Pox activity as a function of time: the catalytic current decreased by 40% during the first 2 h of measurement and then became relatively stable during the following 5 h (data not shown). After the formation of a DMPC monolayer on the OM + DBDS electrode, the incorporation of both active and inactive Pox in the bilayer was monitored by BIAlite experiment. Figure 7 illustrates the sensorgram of the enzyme incorporation. If Pox was incorporated into the bilayer in the absence of pyruvate, we detected a signal of 700 RU. If additional Pox was then incorporated in the presence of pyruvate, the total signal reached 2200 RU. Thus, the ratio of the total incorporated enzyme on the (OM + DBDS)/DMPC bilayer (2200 RU) in the incubation medium versus the incorporated enzyme without pyruvate (700 RU) in the incubation medium is around 3. In an additional experiment, a Pox solution with pyruvate was directly injected onto the (OM + DBDS)/DMPC monolayer. The RU signal then corresponded to the sum of the RU signal previously observed for the incorporation without

Figure 7. Sensorgram of the enzyme incorporation in the (OM + DBDS)/DMPC gold-supported bilayer. DMPC monolayer was formed on gold sensor-supported OM + DBDS monolayer (see the Experimental Section) directly in the BIAlite, before monitoring the oxidase incorporation. Each medium was injected, at a flow rate of 2 µL/min, in this order: reaction medium without pyruvate (1), Pox solution in reaction medium without pyruvate (2), reaction medium without pyruvate (3), reaction medium with pyruvate (4), Pox solution in reaction medium with pyruvate (5), reaction medium with pyruvate (6). The reaction medium was identical to the one used for Pox activity determination (see the Experimental Section), and Pox was at 20 µg/mL.

and with pyruvate, as shown in the Figure 7. Considering the previous assumptions, 2200 RU corresponded to a Pox film of 3.1 nm. This means a total amount of Pox corresponding to 31% of the theoretical dense monolayer, taking into account a 10 nm diameter for the Pox molecule.40 More precisely, around 10% of the theoretical closely packed monolayer was incorporated without pyruvate and around 21% of the theoretical monolayer was added in the presence of pyruvate. In comparison, when Pox was inserted into the bilayer in the absence of pyruvate, electrochemical analysis of the enzymatic catalysis revealed no detectable catalytic current. As Pox needs to be reduced by pyruvate to interact properly with the biological membrane and hence to be activated,9 our results indicated that incorporation of active Pox in our membrane model occurred in a way similar to that of biological membrane. Conclusion We have managed to characterize each step of the selfassembly of our biomimetic model of membrane onto the gold electrode. This final self-assembly was in good agreement with the predicted model in Figure 1. A mixed SAM on the gold electrode, composed of OM and DBDS molecules, was formed. We found that this mixed monolayer presented some defects compared to a pure OM monolayer. These defects allowed the reversibility of the electrochemistry of ferrocenemethanol. BIAlite measurements revealed that the mixed monolayer still displayed a character consistent with that of an intact OM monolayer, in spite of the presence of DBDS in the monolayer. It was concluded that DBDS forms minor defects in the OM monolayer. This conclusion was also demonstrated by the measurement of wettability by the contact angle technique. As a consequence, fusion of DMPC was as effective on the OM + DBDS electrode as on the OM electrode and resulted in both cases in the formation of a DMPC monolayer on the hydrophobic monolayer. In (40) Raj, T.; Russell, P.; Flygare, W. H.; Gennis, R. B. Biochim. Biophys. Acta 1977, 481, 42.

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this mixed supported bilayer, the membrane enzyme Pox can be incorporated in a way similar to that of lipid vesicles. Pox activity was monitored by catalytic current measurements with a new electron acceptor of the enzyme, i.e., ferricinium methanol. The next step of such an assembly will be to determine the kinetics of Pox in this twodimensional reconstituted membrane system using appropriate electrochemical techniques.11

Pierrat et al.

Acknowledgment. We are grateful to Drs. Bertagnolli and Hager (University of Illinois, ChampaignsUrbana) for the generous gift of the Escherichia coli mutant YYC 458. Special thanks also goes to Biacore AB for providing us with the prototype gold sensor chip. This work was partially supported by a grant from DRET No. 95-159. LA9701337