Effects of Corrosive Chemicals on Solid-Supported Lipid Bilayers As

Because of their wide occurrence, specific functions, and properties, solid-supported phospholipid bilayers are often incorporated in biosensors. The ...
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MATERIALS AND INTERFACES Effects of Corrosive Chemicals on Solid-Supported Lipid Bilayers As Measured by Surface Plasmon Resonance Alexander B. Artyukhin and Pieter Stroeve* Department of Chemical Engineering and Materials Science, University of California, Davis, Davis, California 95616

Because of their wide occurrence, specific functions, and properties, solid-supported phospholipid bilayers are often incorporated in biosensors. The stability of these bilayers in aqueous environments is of utmost importance in sensing. By using surface plasmon resonance, we obtained experimental results for corrosive effects of chemicals on bilayers of 1-palmitoyl-2oleoylphosphatidylcholine (POPC) supported on self-assembled monolayers of thiol on gold. The POPC supported bilayers were treated with aqueous solutions of three surfactants, which were anionic (sodium dodecyl sulfate, SDS), cationic (cetyltrimethylammonium bromide, CTAB), and nonionic (Triton X-100), as well as an alcohol (propanol-2), a strong acid (HCl), and a base (NaOH). For all of the surfactants studied, complete corrosion of the bilayer (i.e., complete bilayer removal) occurred at surfactant concentrations near the critical micelle concentration (cmc). However, onset of bilayer solubilization is observed at surfactant concentrations about an order of magnitude below the cmc. The following values of the corrosive substance concentrations necessary to remove 50% of the lipid bilayer (C1/2, mM) were obtained: 0.18 (Triton X-100), 0.54 (CTAB), 2.4 (NaOH), 3 (SDS), 3000 (HCl), and 3100 (propanol-2). Incorporation of up to 28 mol % cholesterol in the POPC bilayer had no or little effect on the bilayer stability with respect to solubilizing effects of the surfactants Triton X-100 and SDS. The data show that the presence of species such as surfactants and alkali should be taken into account when constructing and using biosensors containing lipid bilayers. Introduction Phospholipids are major constituents of cellular membranes.1 Because of their amphiphilic nature, they tend to form bilayer structures in aqueous solutions where hydrophobic tails are in the core while hydrophilic headgroups are oriented toward water. The bilayer separates the interior of a living cell from the outer medium. Lipids form a matrix of the membrane where other biomolecules such as proteins are embedded. Considerable research has been conducted on model lipid systems to understand the behavior of biomembranes, with vesicles being the most popular one. The reason for this popularity is that the procedure of vesicle preparation is straightforward.2,3 However, there is a disadvantage. Because vesicles are not thermodynamically stable, their properties such as size, number of lamellas, etc., are a function of the preparation procedure. Reproducibility often depends on parameters that are difficult to control. Solid-supported lipid bilayers lack this drawback.4 They are thermodynamically and mechanically stable, they can be prepared on a number of solid substrates, and optical and electrochemical techniques can be used to study their properties. Recently, supported lipid bilayers have been incorporated in a number of biosensors.5-8 Biosensors are * To whom correspondence should be addressed. Tel.: (530) 752-8778. Fax: (530) 752-1031. E-mail: [email protected].

usually not applied in clean laboratory conditions but rather in complex systems, for instance, in the determination of biological and environmental pollutants in wastewater. When designing sensors for such applications, one should keep in mind that there are many chemicals present in the environment that can affect the lipid bilayer used in a biosensor and disturb its integrity. One of the first examples is the earlier work of Sher and Sobotka,9 who studied stripping off of thin films of fatty acids from the surface upon contact with albumin solutions. Therefore, it is necessary to know at what concentrations and under what conditions a chemical may cause corrosion of the supported bilayer. In this work we applied surface plasmon resonance spectroscopy (SPR) to study the effects of various types of lipid-corrosive agents on lipid bilayers supported on thiol monolayers on gold. The SPR technique is a sensitive method to monitor growth and desorption of thin organic layers on noble metal surfaces.10-12 Provided that the refractive index of the adsorbed material is known, one can obtain the amount of material on a surface with angstrom resolution in film thickness. We prepared lipid bilayers on thiol monolayers by vesicle fusion and measured the amount of lipid on the surface by SPR upon flushing it with reactive solutions. Surfactants are known to easily solubilize lipids13-16 and may have an effect on biosensors that employ lipid bilayers. We treated the lipid bilayer with an anionic (sodium dodecyl sulfate, SDS), a cationic (cetyltri-

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methylammonium bromide, CTAB), and a nonionic (Triton X-100) surfactant. We also studied other compounds that might corrode lipid membranes such as organic solvents (propanol-2), strong bases (NaOH), and acids (HCl). The main objective of the work was to find if the treatment of supported lipid bilayers with potentially lipid-corrosive materials can give valuable information on the bilayer stability as measured with SPR. We also determined the conditions where supported lipid bilayers maintain their integrity while exposed to various chemicals. Experimental Section Materials. The following chemicals were used as received: SDS (Fluka), CTAB (Aldrich), Triton X-100 (Sigma), propanol-2 (Mallinckrodt), HCl and NaOH (Fisher), 11-mercaptoundecanoic acid (MUA; Aldrich), and cholesterol (Aldrich). Gold (99.999%) used for deposition was received from Alfa Aesar. The lipid 1-palmitoyl-2-oleoylphosphatidylcholine (POPC), a 10 mg/mL solution in chloroform, was purchased from Avanti Polar Lipids. All solvents and chemicals used were of analytical grade unless otherwise stated. The ethanol used in thiol solution preparation and glass slide cleaning was 200 proof (Gold Shield Chemical Co.). The refractive index matching fluid, sulfur in 1-iodonaphthalene, was obtained from Cargille Laboratories Inc. Water for experiments was purified by a Nanopure Diamond system from Barnstead; the resistivity was 18.2 MΩ‚cm. High refractive index LaSFN9 glass slides from Schott were used for SPR experiments. Gold Deposition. Prior to gold deposition, glass slides were cleaned by sonication at 55 °C first in water, then in a 2% Helmanex II solution (alkaline concentrate for cleaning of cells made of glass and quartz; Hellma GmbH & Co.), and finally in ethanol, for 15 min each with thorough water rinses between the sonications. The slides were dried in a stream of nitrogen (Airgas; 99.997%) and mounted in a holder for deposition. Before gold coating, the slides were baked in a vacuum chamber at 250 °C for 2 h and then cooled to 30 °C. Gold was deposited in a vacuum chamber (Edwards AUTO 306) by thermal evaporation of gold in a current-heated molybdenum boat. During gold evaporation, the vacuum was better than 1 × 10-6 Torr, the rate of deposition was 0.2-0.5 Å/s, and gold was deposited to a final thickness of 500 ( 20 Å, which was measured by a calibrated built-in crystal. The exact thickness of the gold layer was determined in every experiment by fitting an equilibrium SPR angle scan of a gold-coated slide with Fresnel equations using available software.17 Initially, three parameters (the thickness, real part, and imaginary part of the dielectric constant for gold) were fit. Both manual and automatic simulations can be performed; the former usually gives better results with respect to standard deviation and physical meaningfulness of the values obtained and was used in this work. Gold-coated slides were kept in ethanol and used within 10 days after preparation. Preparation of a Thiol Monolayer. When gold is exposed to a thiol solution, a thiolate self-assembled monolayer (SAM) forms spontaneously.18,19 We used a model surface of MUA SAM on gold. A SAM of a thiol can be prepared by two methods. In method 1, the goldcoated glass slide was set up in the SPR flow cell (see below), mounted on the SPR apparatus, and a 5 mM thiol solution in ethanol was injected into the cell. With

SPR, one can monitor in situ the apparent kinetics of thiol chemisorption on gold. In method 2, we immersed the gold slide into a beaker with a 5 mM thiol solution in ethanol and, after the chemisorption was complete, we rinsed the slide with pure ethanol, dried it, and mounted it in the flow cell. In either method, the gold surface was in a contact with a thiol solution for at least 16 h and then rinsed with pure ethanol. The second method was more convenient and gave better SPR results. Despite the well-known chemical inertness of bulk gold,20 its surface is very easily contaminated when a freshly prepared gold film is exposed to an ambient atmosphere.21 Therefore, we exposed the gold-coated slides to as little air as possible. Vesicle Preparation. The lipid bilayer on the SAMmodified gold was obtained by vesicle fusion. We followed the procedure of lipid vesicle preparation described in detail elsewhere22 with some modifications. The protocol used in this work was as follows: A total of 100 µL of a POPC solution in chloroform (10 mg/mL) was put in a vial, and the solvent was evaporated by purging a slow stream of nitrogen. A total of 2 mL of water was then added to the vial, and a lipid emulsion formed upon vortexing. The lipid concentration in the emulsion was about 0.5 mg/mL. The lipid emulsion was preincubated at room temperature (the melting temperature of POPC is about -5 °C)23 for 15 min with a few vortexing periods of 30 s. Sonication of the lipid emulsion was performed four times using a Branson Sonifier 450 with a 1/8 in. tip, 1 min each at a power output of 3, and 1 min intervals between sonications. The sample container was kept in a water-ice bath during sonication to prevent overheating. Titanium particles from the ultrasonic tip were removed from the POPC vesicle solution by centrifugation at 3000 rpm for 2 min. Vesicles were prepared immediately before the experiment and were injected into the flow SPR cell within 15 min after preparation. Flow Cell. The flow cell was designed so that a fully developed laminar flow was achieved prior to a solution entering the SPR measurement area. The cell was fabricated from Teflon with an O-ring (Apple Rubber Products Inc.) for a seal. The flow channel was 14 mm wide and 64 mm long. The gap between the glass slide and the opposite Teflon wall was 0.25 mm. The SPR measurement area was 40 mm from the inlet. The laser beam of the SPR has a cross-sectional area of about 2 mm2. Solutions were pumped using a 100-mL syringe (Popper & Sons Inc.) with a Cole-Palmer 74900 syringe pump. Lipid vesicles were injected into the cell with single-use 3-mL syringes (Sherwood Medical). Connections between the cell and feeding syringes and the output were made from Upchurch Scientific tubes and fittings. The overall volume of the cell and the incoming tube and fittings was 0.8 mL. Surface Plasmon Resonance. Formation of a POPC bilayer and subsequent treatment of the bilayer with corrosive solutions were monitored by a SPR set up in the Kretschmann configuration. The method is based on excitation of the surface plasmons by p-polarized light at the noble metal-dielectric interface. Details of the experimental procedure are described elsewhere.22 Briefly, in a typical experiment, a gold-coated slide modified with a MUA SAM (formed by method 2) was mounted in the flow cell and assembled on the SPR stage. The stage allowed either independent or coordinated rotation of the detector and the sample holder

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along the z axis with a precision better than 0.01°. The cell was rinsed with 20 mL of water at a 50 mL/min flow rate, and a scan of the reflectivity versus the angle of incidence was collected. Kinetic information on the adsorption and desorption of the lipid bilayer was obtained by fixing the angle of incidence about 0.5° below the minimum angle and collecting reflectivity versus time data. When collection of kinetic data was started, POPC vesicles (1.5 mL) were injected into the cell. It resulted in a steep increase in reflectivity due to lipid adsorption on the MUA SAM. Vesicles were kept in contact with the MUA monolayer on gold until equilibrium was reached, usually about 30 min. The cell was then flushed with 20 mL of water at a 50 mL/min flow rate, and an angle scan was run. From the angle spectrum, we were able to calculate the amount of lipid on the surface. If the thickness of the layer was comparable with typical literature values for the lipid bilayer (40-50 Å), the angle of incidence was fixed at the same value as that chosen for the lipid adsorption kinetic measurement and the cell was flushed with 90 mL of a solution containing a lipid-corrosive compound at a flow rate of 10 mL/min. This was enough to reach equilibrium. In most cases, changes occurred during the first minute of flushing. The cell was then rinsed at least twice with 20 mL of water (50 mL/min flow rate) or more until changes in reflectivity upon water rinsing were no longer observed. The angle spectrum was measured again and the remaining amount of lipid on the surface was determined. When less than 5% of the lipid was removed on flushing with a solubilizing solution, the same lipid bilayer was used for another experiment. If the corrosive effect was more than 5%, a new bilayer was prepared. All experiments were carried out at room temperature (24 °C). Results and Discussion Bilayer Formation. We chose POPC as a model lipid because it is one of the major constituents of mammalian cell membranes. The POPC lipid is one of the most studied, and its properties are well-known. Further, this lipid is often used in biosensor applications. Vesicle fusion has been used extensively as an effective method to deposit lipid layers on solid substrates.22,24,25 In this work, POPC layers were deposited on MUAcovered gold substrates by vesicle fusion in pure water. Figure 1 shows typical changes in SPR curves due to deposition of POPC on a MUA monolayer on gold. To calculate the equilibrium thickness of each adsorbed layer from the SPR measurements, the refractive index of the layer is needed. The refractive index values used for lipid layers in the literature vary from 1.45 to 1.50.22,26 A value of 1.493 was calculated for POPC at 36 °C.27 Assuming a temperature dependence of the refractive index for water,28 we calculated a value of 1.495 for POPC at 22 °C that was used in this work. The average thickness of the adsorbed POPC layer measured by SPR is 4.2 ( 0.4 nm (average from 37 experiments), which is in agreement with literature values for the lipid bilayer thickness (5 nm in average). From this result, we can conclude that POPC forms a single bilayer on MUA-covered gold consistent with literature data.26 This is an important observation because it was found previously that POPC formed multilayers when a layer of positively charged polymer poly(diallyldimethylammonium chloride) was first adsorbed on MUA and the lipid was deposited on this

Figure 1. SPR curves measured after deposition of MUA on gold (solid line), POPC bilayer on MUA (dashed line), and after treatment of the lipid bilayer with 5 mM SDS (dotted line) are shown as examples. The curves shift to the right with successive adsorption of the thiol and the lipid bilayer and back to the left upon lipid desorption.

polymer “cushion”.22 The reason for this difference is not yet clear, but it is known that the process of lipid layer formation by vesicle fusion is highly dependent on the substrate nature and roughness.29-32 Surfactants. An equilibrium angle scan performed after POPC treatment with a 5 mM SDS solution and subsequent water rinses to ensure desorption of the surfactant that might take place is also shown in the Figure 1. At this SDS concentration, the lipid is partially desorbed and the SPR curve is still shifted to the right with respect to the initial MUA monolayer (compare the solid line and the dotted line in Figure 1). (When complete desorption of the lipid occurs, the scans measured before lipid deposition and after the lipidcorrosive treatment coincide.) From the angle scan, we calculated the remaining amount of lipid on the surface after treatment with solutions of different surfactant concentrations. The SPR time responses to surfactant solutions in contact with supported lipid bilayers are shown in Figure 2. Also shown is the response to a rinse with water after surfactant treatment. In the case of CTAB (a cationic surfactant) and Triton X-100 (a nonionic surfactant), there are two desorption steps for the kinetic curve: the first one is the lipid solubilization upon contact with the surfactant solution and the second one is the surfactant desorption from the MUA surface upon rinsing with pure water. The surfactant SDS does not adsorb on the MUA SAM, and consequently there is only one drop in the reflectivity versus time curve. The value of the pKa for mercaptopropionic acid confined on the gold surface is near 8.0,33 which is about 3.5 units higher than the pKa measured for similar acids in bulk solutions. The titration curve is much broader (extending from pH 6.0 to 10.0) than those observed for monocarboxylic acids in aqueous solutions.33 Therefore, even in pure water at pH 6-7, the MUA monolayer should possess a negative charge, although dissociation of MUA carboxylic groups may not be complete. Elec-

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Figure 2. SPR kinetic profiles of POPC bilayer desorption upon flushing with surfactants. The angle of incidence was set 0.5° below the minimum angle of MUA on gold (solid line, Figure 1). Only the first water rinse is indicated; subsequent water rinses necessary to reach the steady reflectivity signal are not shown.

trostatic repulsion between partially negatively charged MUA and SDS molecules prevents sufficient adsorption of SDS on the MUA monolayer. Oppositely, electrostatic attraction between cationic CTAB and MUA anionic groups leads to adsorption. There are also van der Waals interactions that govern adsorption in the case of nonionic Triton X-100, and van der Waals forces may play a role with the electrostatic forces in the case of CTAB. Adsorption of CTAB on MUA is reversible below critical micelle concentration (cmc; data not shown). From the SPR data alone, we cannot determine the structure of the surfactant on the surface. According to Tilton et al.,34 CTAB forms a defective bilayer when adsorbed on negatively charged silica from an aqueous solution. This also might be the case for adsorption on negatively charged MUA SAM on gold. From Figure 2 we can only calculate the approximate thickness of the surfactant layer; it is about half of the initial lipid bilayer, about 2 nm. The dependence of the equilibrium amount of lipid still adsorbed on MUA on SDS concentration (after SDS treatment and rinsing with water) is shown in Figure 3. The surface concentration of the lipid is normalized by the initial value before the treatment, with 100% being the unaffected POPC bilayer. SDS completely solubilizes the POPC bilayer near the cmc of the surfactant. We observed gradual solubilization of the bilayer well below cmc when lipid vesicles saturated with the surfactant coexist with mixed micelles of lipid and surfactant. Complete solubilization of the bilayer where only mixed micelles exist occurs at cmc. In our experiments, we did not observe adsorption of surfactants on the lipid bilayer. We studied several representative compounds from different classes that might solubilize lipids and affect the quality of the bilayer. Adsorption of the corrosive reagent on the lipid bilayer was not observed, nor was partitioning into the lipid bilayer. It is a generic property of surfactants to solubilize lipid bilayers near the cmc. The remaining amount of lipid on the surface versus concentration of the surfac-

Figure 3. Equilibrium amount of POPC on MUA SAM on gold after treatment with SDS solutions of different concentrations. The amount of lipid is normalized by the initial amount after deposition. cmc of SDS is indicated. The solid line trend is shown for eye guiding.

Figure 4. Equilibrium amount of POPC on MUA SAM on gold after treatments with surfactants. The amount of lipid is normalized by the initial amount after deposition. The concentration scale is normalized by the cmc of the surfactant.

tant in cmc units is shown in Figure 4 for the three different surfactants. The following values of the surfactant cmc in pure water were used: 8.1 mM for SDS,35 0.9 mM for CTAB,34 and 0.24 mM for Triton X-100.36 When normalized, the data are almost identical for all of the surfactants studied and not dependent on their nature. We also studied the effect of cholesterol on the bilayer stability with respect to corrosive treatment because it is known that cholesterol is present in considerable amounts in many cell membranes and dramatically changes their properties such as permeability and stiffness.37 It was found that when the POPC bilayer was contacted with Triton X-100 solutions, incorporation of 28 mol % cholesterol in the membrane had no effect

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Figure 5. Effect of cholesterol on POPC bilayer treatment with SDS and Triton X-100. Open symbols show lipid without cholesterol, and filled symbols show POPC with 28 mol % cholesterol. The amount of lipid is normalized by the initial amount after deposition. Curves are drawn to aid the eye.

on the concentration dependence of the remaining amount of lipid on the surface (Figure 5). Similarly, in the case of SDS, little difference was observed with bilayers with or without 28% cholesterol. The observation with Triton X-100 is consistent with previous findings that release of a fluorescent probe from POPC vesicles induced by Triton X-100 was insensitive to the presence of cholesterol in the vesicles up to 50 mol % cholesterol.38 However, other authors found for the same system that the presence of cholesterol affected the bilayer permeability and solubilization at an equimolar phospholipid/sterol ratio39 in a rather interesting way. At a 1:1 ratio, solubilization was made more difficult while release of liposomal contents was easier; i.e., it required less surfactant.39 Lundberg et al.40 solubilized egg phosphatidylcholine vesicles with Triton X-100 and measured the solution turbidity. They found that at cholesterol contents up to 25% clear solutions were formed at higher detergent concentrations, while at higher cholesterol molar fractions, the turbidity did not reach low and constant values even at high Triton X-100 concentration.40 From this result they concluded that the solubilization of phosphatidylcholine-cholesterol membranes was highly dependent on the amount of cholesterol in the bilayer. Bahadur et al. investigated interaction of SDS with egg yolk lecithin vesicles by means of fluorescent marker release and determined that in the presence of cholesterol the process was much slower.41 However, the fraction of cholesterol in the membrane was not given, so it is difficult to compare these data with others. Other Corrosive Substances. Concentration dependencies for equilibrium lipid amounts are given in Figures 6 and 7 for the other compounds investigated: NaOH, HCl, and propanol-2. In all cases, we checked that the chemicals did not affect the MUA layer. SPR angle scans of the MUA layer on gold after corrosive treatment of POPC and subsequent removal of all remaining lipid by flushing with ethanol were completely identical to those obtained before lipid bilayer formation. The trend of the results in Figures 6 and 7

Figure 6. Equilibrium amount of POPC on MUA SAM on gold after treatments with different concentrations of NaOH and HCl. The amount of lipid is normalized by the initial amount after deposition.

Figure 7. Equilibrium amount of POPC on MUA SAM on gold after treatments with different concentrations of propanol-2. The amount of lipid is normalized by the initial amount after deposition.

for various compounds is similar, but the mechanism by which they affect the POPC bilayer is different from that of the surfactants. Propanol-2 was found to solubilize the lipid bilayer only at a concentration of 3 M, which is equal to about 25% (v/v), i.e., at a concentration much higher than that observed for the surfactants. At this alcohol concentration, the solubility of the lipid may be sufficient to cause lipid bilayer desorption and dissolution. Supported lipid bilayers are known to have defects.42 The number and nature of these defects may depend on the substrate and the bilayer preparation procedure. Lipid molecules near bilayer defects have a higher chemical potential and can be removed from the surface more easily than molecules in a perfect bilayer.

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Dissolution of lipid molecules from defect areas may be the reason for rapid desorption. Hydrochloric acid did not yield any changes in the POPC bilayer up to 1 M. At a concentration of 5 M, it was found to etch and significantly change optical constants of the gold layer on the glass (data not shown). Some surface changes were found at 2 M, but we believe it is due to partial lipid desorption. Otherwise, if gold were affected at a 2 M HCl concentration, it would mean that all other layers (MUA and POPC) were completely desorbed. However, only 15% of the lipid bilayer was removed from the surface at this HCl concentration. It is impossible to distinguish what layer is primarily affected at concentrations of HCl between 2 and 5 M. We can only definitely say that HCl does not corrode the POPC bilayer at concentrations up to about 1.5 M. Sodium hydroxide was found to remove the POPC bilayer from the surface at much lower concentrations than HCl. In comparison to other kinetics, it took more time for the reflectivity to reach an asymptote when the lipid bilayer was contacted with NaOH solutions. The reproducibility of the final equilibrium lipid thickness was poorer than that for the other compounds studied. It is known that lipids are easily hydrolyzed in an alkaline medium.43-45 This may be an important mechanism of POPC desorption in NaOH solutions. Products of lipid hydrolysis disturb the integrity of the bilayer and may cause its breakage. Conclusions In this work we employed SPR to study the response of POPC bilayers supported on thiol monolayers on gold to treatment with lipid solubilizing reagents. SPR is a powerful technique that permits in situ monitoring of growth and desorption of thin organic films on noble metal surfaces with angstrom thickness accuracy. It was found that surfactants completely destroy the bilayer at concentrations near the cmc independent of the surfactant nature. Onset of lipid removal is observed at much lower surfactant concentrations and may cause severe damage to the bilayer incorporated in biosensors; this must be taken into consideration. Alcohol causes lipid desorption due to an increase in the bulk lipid solubility at relatively high alcohol content in water (about 25% for propanol-2). Alkali results in lipid hydrolysis. In addition, there may be products of hydrolytic decomposition that decrease lipid bilayer stability and yield lipid film breakage at 10-3 M NaOH. On the other hand, HCl does not affect POPC at concentrations up to 1.5 M. Acknowledgment The MRSEC program of the National Science Foundation under Award DMR-9808677 supported this work. Literature Cited (1) Langner, M.; Kubica, K. The Electrostatics of Lipid Surfaces. Chem. Phys. Lipids 1999, 101, 3. (2) Woodle, M. C.; Papahadjopoulos, D. Liposome Preparation and Size Characterization. Methods Enzymol. 1989, 171, 193. (3) Lichtenberg, D.; Barenholz, Y. LiposomessPreparation, Characterization, and Preservation. Methods Biochem. Anal. 1988, 33, 337. (4) Sackmann, E. Supported Membranes: Scientific and Practical Applications. Science 1996, 271, 43.

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Received for review November 19, 2002 Revised manuscript received February 24, 2003 Accepted March 11, 2003 IE0209327