Formation of Tethered Bilayer Lipid Membranes on Gold Surfaces

After 15 and 21 h of deposition the condensed islands disappear and are replaced by lower density moieties in the buffer environment. The apparent dec...
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Langmuir 2007, 23, 7344-7355

Formation of Tethered Bilayer Lipid Membranes on Gold Surfaces: QCM-Z and AFM Study Brian R. Dorvel,† Henk M. Keizer,† Daniel Fine,‡ Jorma Vuorinen,§ Ananth Dodabalapur,‡ and Randolph S. Duran*,† George and Josephine Butler Polymer Laboratory, Department of Chemistry, UniVersity of Florida, GainesVille, Florida 32611, Microelectronics Research Center, UniVersity of Texas at Austin, Austin, Texas, and KSV Instruments Ltd., Ho¨yla¨a¨mo¨tie 7, 00380, Helsinki, Finland ReceiVed April 17, 2006. In Final Form: March 28, 2007 Recently, tethered bilayer lipid membranes (tBLMs) have shown high potential as biomimetic systems due to their high stability and electrical properties, and have been used in applications ranging from membrane protein incorporation to biosensors. However, the kinetics of their formation remains largely uninvestigated. By using quartz crystal microbalance with impedance analysis (QCM-Z), we were able to monitor both the kinetics and viscoelastic properties of tether adsorption and vesicle fusion. Formation of the tether monolayer was shown to follow pseudo-first-order Langmuir kinetics with association and dissociation rate constants of 21.7 M-1 s-1 and 7.43 × 10-6 s-1, respectively. Moreover, the QCM-Z results indicate a rigid layer at the height of deposition, which then undergoes swelling as indicated by AFM. The deposition of vesicles to the tether layer also followed pseudo-first-order Langmuir kinetics with observed rate constants of 5.58 × 10-2 and 2.41 × 10-2 s-1 in water and buffer, respectively. Differential analysis of the QCM-Z data indicated deposition to be the fast kinetic step, with the rate-limiting steps being water release and fusion. Atomic force microscopy pictures taken complement the QCM-Z data, showing the major stages of tether adsorption and vesicle fusion, while providing a road map to successful tBLM formation.

Introduction The cell membrane is regarded as one of the most essential pieces in the puzzle comprising living organisms. A complex mixture of bilayer phospholipids, sterols, and carbohydrates shields the organism from extracellular elements while specialized biomolecules, incorporated into and onto the bilayer, perform tasks ranging from intercellular signaling to molecular transport across the membrane. Recent advances in understanding cell membrane properties, structure, and function have precipitated an increased amount of attention to reproduce this complex system artificially. Currently, increasing the stability of biomimetic systems has been of critical importance with their applicability to such fields as biosensors, drug delivery, biocatalysis, and cellular recognition, which rely on high stability to remain functional in an electronically interfaced environment. The two most stable and commonly used cell membrane biomimetic systems are supported bilayer lipid membranes (sBLMs) and tethered bilayer lipid membranes (tBLMs), with tBLMs being the most recent advance in membrane technology. Supported bilayer lipid membranes are commonly formed on monolayer templates,1,2 hydrophilic substrates,3,4 or polymer cushions5 and are known to maintain the physiological and chemical properties of natural bilayers.6,7 However, the success of sBLMs as a biomimetic system has been limited by their lack of an aqueous reservoir between substrate and membrane. Several * Corresponding author. Telephone: (352) 392-2011. E-mail: duran@ chem.ufl.edu. † University of Florida. ‡ University of Texas at Austin. § KSV Instruments Ltd. (1) Ekeroth, J.; Konradsson, P.; Hoeoek, F. Langmuir 2002, 18, 7923. (2) Keller, C. A.; Kasemo, B. Biophys. J. 1998, 75, 1397. (3) Richter, R. P.; Brisson, A. R. Biophys. J. 2005, 88, 3422. (4) Graneli, A.; Rydstroem, J.; Kasemo, B.; Hoeoek, F. Langmuir 2003, 19, 842. (5) Munro, J. C.; Frank, C. W. Langmuir 2004, 20, 3339. (6) Sackmann, E. Science 1996, 271, 43. (7) Raedler, J.; Strey, H.; Sackmann, E. Langmuir 1995, 11, 4539.

classes of proteins are transporters and require sufficient aqueous media on both sides of the membrane to function; tBLMs do provide these conditions. The construction of sBLMs has been thoroughly studied over time in several aspects,8 while the formation of tBLMs remains largely uninvestigated. Tethered bilayer lipid membranes are often formed using lipids with thiol,9,10 disulfide,11-13 and silane14 modified headgroups, depending on the substrate, and are separated from the substrate by a hydrophilic spacer. The spacer is usually composed of monodisperse oligoethylene oxide units, which allow for an ionic reservoir to exist between the substrate and the lower part of the membrane. The tBLM not only contains the beneficial properties and applications of sBLMs, but also the extra degree of freedom added by the tether allows for a better representation of the bilayer, with the aqueous reservoir acting analogous to the cell’s cytosol.15 Recently, tBLMs have been taken advantage of to sense small ion conducting peptides, such as gramicidin and valinomycin,11,16 as well as macromolecular proteins such as cytochrome c oxidase.17 Moreover, the growing interest in biosensors using ion channels as switches has led to tBLMs becoming one of the choice model systems for this application.18 (8) Reimhult, E.; Hoeoek, F.; Kasemo, B Langmuir 2003, 19, 1681. (9) Plant, A. L. Langmuir 1999, 15, 5128. (10) Plant, A. L. Langmuir 1993, 9, 2764. (11) Cornell, B. A.; Krishna, G.; Osman, P. D.; Pace, R. D.; Wieczorek, L. Biochem. Soc. Trans. 2001, 29, 613. (12) Schiller, S. M.; Naumann, R.; Lovejoy, K.; Kunz, H.; Knoll, W. Angew. Chem. 2003, 42, 208. (13) Burns, C. J.; Field, L. D.; Petteys, B. J.; Ridley, D. D. Aust. J. Chem. 2005, 58, 738. (14) Atanasov, V.; Knorr, N.; Duran, R. S.; Ingebrandt, S.; Offenhaeusser, A.; Knoll, W.; Koeper, I. Biophys. J. 2005, 89, 1780. (15) Krishna, G.; Schulte, J.; Cornell, B. A.; Pace, R. J.; Osman, P. D. Langmuir 2003, 19, 2294. (16) Naumann, R.; Walz, D.; Schiller, S. M.; Knoll, W. J. Electroanal. Chem. 2003, 550-551, 241. (17) Jeuken, L. J. C.; Connell, S. D.; Henderson, P. J. F.; Gennis, R. B.; Evans, S. D.; Bushby, R. J. J. Am. Chem. Soc. 2006, 128, 1711. (18) Cornell, B. A.; Braach-Maksvytis, V. L. B.; King, L. G.; Osman, P. D. J.; Raguse, B.; Wieczorek, L.; Pace, R. J. Nature 1997, 387, 580.

10.1021/la0610396 CCC: $37.00 © 2007 American Chemical Society Published on Web 05/16/2007

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Figure 1. General construction of a microelectrode array (bottom, left) and a corresponding picture of the sensing pad (top, left). The formation of the tBLM onto the microelectrode array begins with the formation of the DPTL tether monolayer on the gold substrate (top, right), followed by DPhPC/DPhPE vesicle fusion forming the tBLM (bottom, right).

In order to simulate an environment for biosensing applications, electronic devices consisting of 100 × 100 µm square grid microelectrode arrays (MEAs) were utilized as the substrate for tBLM formation. The MEAs consisted of 66 pixels per wafer that were formed by evaporation of 3 nm of Ti, 500 nm of 60% Au/40% Pd alloy, and then 200 nm of pure gold. A polyimide resist was then applied and the gold pad size defined using photolithography. Figure 1 shows the MEA and the two-step process for tBLM formation. Briefly, the tBLMs are constructed using Archae analogue thiolipids and phospholipids. In the first step, the thiolipid 2,3-di-O-phytanyl-sn-glycerol-1-tetraethylene glycol-DL-R-lipoic acid ester (DPTL) forms a self-assembled monolayer (SAM) on the gold, creating the tether monolayer.19 Subsequently, phytanyl lipid vesicles are allowed to interact with DPTL and create the tethered lipid bilayer by vesicle fusion.19,20 The phytanyl chains present in the Archae analogues are known for their high thermodynamic stability, with liquid crystallinity ranging from -80 to 120 °C.21 The kinetics of tBLM formation on gold substrates was investigated with quartz crystal microbalance with impedance analysis (QCM-Z) and visualized by atomic force microscopy (AFM). With the QCM-Z technique it is possible to calculate frequency and dissipation changes using direct impedance measurements. The admittance of the crystal, extracted from the impedance, can give crucial information on the viscoelastic properties of a system. Additionally, the versatility of AFM was used in characterizing the tBLM formation. AFM images were (19) Naumann, R.; Schiller, S. M.; Giess, F.; Grohe, B.; Hartman, K. B.; Kaercher, I.; Koeper, I.; Luebben, J.; Vasilev, K.; Knoll, W. Langmuir 2003, 19, 5435. (20) He, L.; Robertson, J. W. F.; Li, J.; Kaercher, I.; Schiller, S. M.; Knoll, W.; Naumann, R. Langmuir 2005, 21, 11666. (21) Hung, W. C.; Chen, F. Y.; Huang, H. W. Biochim. Biophys. Acta 2000, 1467, 198.

taken at different time intervals, illustrating the consecutive steps in the construction of tBLMs, and were then compared with the kinetics determined by QCM-Z. Experimental Section Materials. Diphytanoyl-sn-3-glycerophosphatidylcholine (DPhPC) and diphytanoyl-sn-3-glycerophosphatidylethanolamine (DPhPE) were purchased from Avanti Polar Lipids Inc. (Alabaster, AL) and used as received. The thiolipid DPTL was synthesized through known procedures and stored at -20 °C in ethanol.12 Buffer solutions were made using 5 mM 3-morpholinopropanesulfonic acid (MOPS), 250 mM KCl, and 0.1 mM CaCl2, and were titrated to desired pH using HCl or KOH. Ultrapure water with resistance >18 MΩ cm, obtained by Milli-Q filtration, was used for all preparations of vesicles and buffer solutions. QCM-Z sensor crystals (5 MHz) with a thermally evaporated gold layer were acquired from KSV and used after cleaning as described below. Preparation of Lipid Vesicles. Stock solutions of DPhPC and DPhPE were made in CHCl3 to a concentration of 50 mg/mL. DPhPC and DPhPE solutions were then mixed in a 7:3 molar ratio to 2 mg/mL. The chloroform was removed using a rotary evaporator and stored in a vacuum desiccator overnight to ensure removal of all the solvent. The dried lipid film was resuspended using water and heated to 60 °C, stirring vigorously until a clear solution was obtained. The flask was cooled to room temperature and sonicated for 5 min. The contents were passed through a 450 nm Fisher PTFE filter that resulted in a bimodal distribution of vesicles with mean diameters of 104 and 289 nm, respectively, obtained through dynamic light scattering (see Supporting Information). Quartz Crystal Microbalance with Impedance Analysis (QCMZ). The QCM-Z measurements were acquired on a QCM-Z500 (KSV Instruments, Helsinki, Finland) equipped with a thermoelectric (TE) module (Oven Instruments). The temperature was regulated to 20 ( 0.1 °C using a Peltier element connected to the TE module. The sensor crystals were composed of AT-cut quartz layered with

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thermally evaporated gold, and had a resonant frequency of 5 MHz. The crystals were cleaned on both sides using a known oxidative pretreatment,22-24 which consisted of UV/ozone exposure for 10 min. Initial experiments confirmed the oxidation of the gold by contact angle measurements (40% gauche defects in each chain segment, forming a constant liquid crystalline state.50 Furthermore, high concentrations of ions or ethanol would disrupt the solvent cage around the phytanyl groups, thus allowing even greater conformational freedom for the DPTL molecule. Additionally, both solvents have the chance to interact with the tetraethylene glycol of DPTL and would provide repulsive charges between molecules. The uptake of solution into the monolayer, referred to as swelling, could explain the large decreases in the crystal frequency observed after 5 h deposition time. A change in frequency can be related to the density viscosity product at the crystal interface,25 and a diffusion of solvent into the reservoir would increase both values. AFM has shown regions of thiol monolayers to exhibit structural mobility and rearrangement, and this effect would further contribute to the decrease in DPTL packing density. When the DPTL monolayer forms, there is a solvent concentration gradient, with the TEG reservoir having the lower concentration. If we treat the monolayer as a permeable membrane, the mass transfer is directly proportional to the concentration (C) change from one side of the layer to another. The change in frequency (F) is linear in two areas, thus making the derivatives of frequency and concentration with respect to time at those areas (∂F/∂t and ∂C/∂t) constant. If Fick’s law is followed, the curvature of the concentration (∂2C/∂x2) through the membrane also has to be constant. This would lead to an accumulation of solvent at the beginning of membrane transport, proportional to the square of the distance (x2). The two linear areas in the DPTL QCM-Z results (see Figure 3A) indicate two separate diffusion processes, both of which may follow Fick’s law. The first linear decrease, occurring between 5 and 8 h, has a larger slope with larger ∂C/∂t and ∂2C/∂x2 values than the second decrease, which occurs between 8 and 21 h. As a result, there is a large accumulation of solvent at the interface after the first diffusion process is finished, which then may trigger the second diffusion. We believe both processes to correlate with solvent diffusion into the TEG reservoir, but they are triggered by different conditions in the monolayer. Naumann et al.16,19 have shown (50) Shinoda, W.; Shinoda, K.; Baba, T.; Mikami, M. Biophys. J. 2005, 89, 3195.

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biphasic swelling behavior for DPTL, as mentioned previously. The fast phase of swelling was indicated by FT-IR to be due to a large conformational change of the DPTL TEG chain to a fully extended state. The slower phase was indicated, through mathematical modeling of the electrochemical processes, to be due to a permeation of solvent and ions through the phytanyl layer. The time frame of permeation agrees well with the time frame of the QCM frequency change in our results. The experiments of Naumann et al. were preformed in 0.1 M NaCl instead of ethanol. Since the time frames of each phase are so similar between ethanol and 0.1 M NaCl, the thickness measurements vs time in 0.1 M NaCl mimic the negative of our QCM frequency change, and admittance measurements have shown DPTL to behave similarly in salt solutions and in ethanol, the diffusion processes described by Naumann et al. should hold for our system as well. Based upon observations from AFM, QCM-Z, and DLS, a deposition pathway has been proposed for DPTL in Figure 7A. The deposition pathway begins with the DPTL vesicles contacting the dense ethanol solvent layer above the oxidized Au surface (1). As ethanol is slowly shed from the Au surface by the vesicle’s diffusion, DPTL begins to form Au-S bonds and the consequent interfacial stress causes the vesicles to rupture (2). The result is densely packed areas surrounded by lower density rims. Solvent begins to diffuse to the TEG reservoir and then into the phytanyl layer (3). An accumulation of solution creates a uniformly dense, viscoelastic state in the deposited monolayer (4). Deposition Pathway of Lipid Vesicles in tBLM Formation. The differential analysis of the QCM-Z data showed that the initial deposition rate of vesicles was fast and the same regardless of the aqueous media used. Moreover, there is a phase change which occurs at 20 min in both systems, indicated by an inflection point in the curve. For the water system, deposition is essentially finished at this point and the vesicles begin to fuse, releasing water, which can be seen by an increase in frequency. A Langmuir fit to the increase in frequency gave a kobs of (6.93 ( 0.3) × 10-5 s-1. Unlike water, the buffer system does not show any signs of water release, and continues to decrease in frequency with a kobs of (4.05 ( 0.16) × 10-5 s-1. Since during rinsing no desorption is observed, the rate constant for vesicle desorption (kd) is taken to be ,ka. As a result, the kobs can be directly related to the rate constant of adsorption (kads). In order to determine whether the rate of deposition is limited by either diffusion or adsorption, a quantification comparing the two processes must be made. A dimensionless parameter, R, was defined based on a diffusion-limited model by Rahn and Hallock51 and the adsorption-limited model by Langmuir:

R)

cbxD kobsΓmaxxπτ

(6)

where cb is the bulk concentration (500 µM), D is the vesicle diffusion coefficient (D ) 5.35 × 10-8 cm2 s-1), Γmax is the maximum surface coverage (Γmax ) 3.60 × 10-10 mol cm-2), and τ is the observation time. For R . 1, the rate would be limited by adsorption, while if R , 1 it would be limited by diffusion. Additionally, the equation assumes that both the rate of rupture and the rate of desorption of vesicles are much smaller than the rate of adsorption. In the buffer system, the R for deposition before 20 min was 0.24. For water, the R was 0.11 during the same time frame, making both water and buffer systems diffusion controlled. The magnitude of R for the first 20 min, (51) Rahn, J. R.; Hallock, R. B. Langmuir 1995, 11, 650.

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Figure 7. Proposed deposition pathways for the formation of DPTL monolayers (A) and tBLMs (B). Part A shows the pathway for DPTL monolayer formation. Here vesicles of DPTL diffuse through the solvent (1) and contact the gold surface (2), slowly rupturing into condensed and swollen areas (3), whereby solvent slowly diffuses into the reservoir to form a complete swollen phase (4). Part B shows the pathway for tBLM formation. Similarly, vesicles of DPhPC/DPhPE diffuse through the solvent (1) and contact the DPTL monolayer (2). At the critical coverage point the vesicles rupture, forming supported bilayer patches in water and fused bilayer patches in buffer. The vesicles continue to adsorb and rupture on the surface to fill the bilayer patches, with a larger rate of fusion occurring in the buffer. A tBLM is then formed in buffer (3a), while a supported lipid bilayer is formed in water (3b). The supported lipid bilayer slowly sinks into the DPTL layer to form a tBLM (4).

in both systems, indicates that the diffusion of vesicles to the surface is slower than their adsorption, and the concentration of vesicles adjacent to the surface is smaller than the bulk concentration. The R was not able to be calculated after 20 min since kobs has multiple rate constant contributions, i.e., vesicle adsorption and water release. The kobs for the initial vesicle adsorption in water ((4.86 ( 0.21) × 10-2 s-1) is larger than that in buffer ((2.41 ( 0.13) ×

10-2 s-1). This result is because of the overall stability of the vesicles in solution and, according to DLVO theory, can be related to the net attractive (van der Waals) and repulsive (electrostatic) forces between vesicles and the surface. Zeta (ζ) potential measurements were conducted on the vesicles as a function of ionic strength and showed that the vesicles contained a ζ potential of -20 ( 3 mV in buffer and -1.3 ( 0.5 mV in water. The higher ζ potential in buffer gives the vesicles more stability in

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solution and increases the potential barrier as a function of distance between the vesicles and the surface by electrostatic repulsion, preventing aggregation and flocculation. However, in water the low ζ potential makes electrostatics a small factor and increases the influence of van der Waals forces in the net potential energy function, thus increasing the probability of irreversible vesicle adsorption to the DPTL monolayer. Although density and viscosity can play a great role in the diffusion velocity of the vesicles, we do not believe the density-viscosity differences between water and buffer play a major role in vesicle adsorption. The ratio of the measured density-viscosity product of buffer and water is less than 1% at the QCM measuring temperature of 20 °C, thus minimizing any effect on the vesicle net diffusion. The magnitude of kobs was similar for both water and buffer, even though one is increasing in frequency and one is decreasing. This indicates a very sensitive balance between vesicle deposition and release of water. After the initial deposition process, the rate for release of water from vesicles increases while the rate of vesicle deposition decreases. In the case of the water system, the adsorption of vesicles to the surface is outweighed by water release, shown by an increase in the frequency. In contrast, the buffer system still has a vesicle deposition rate larger than the rate of water release, causing a net decrease in frequency. Overall, the diffusion of vesicles through solution and onto the monolayer was determined as the fast kinetic step in tBLM formation, even though it is diffusion controlled, with the fusion of vesicles being the rate-determining step. When the vesicles contact the DPTL monolayer, they can take two paths: stay intact on the DPTL surface or rupture to form tBLMs; we believe this to be a critical point in the formation of the tBLM. Since both vesicle systems share an inflection point at 20 min, this seems to be a critical coverage point whereby vesicles begin to rupture and form bilayer patches on the DPTL monolayer. AFM has clearly shown that within 3 h vesicles in water are only partially fused, while vesicles in buffer form nearly complete tBLMs. The fusion kinetics is profoundly faster in buffer, when compared to water, and may be attributed to an increased vesicle interfacial energy. The cause of the larger interfacial energy in the buffer is likely due to interactions with Ca2+ in solution,1,48,52,53 along with an established electrochemical gradient between the inside of the vesicle and the bulk solution. However, the initial deposition kinetics are faster in water, and can attributed to the vesicles’ lower ζ potential in water compared to buffer. When we compare the rates of dissipation change in Figure 6, we find a relatively small and narrow peak to identify with the water deposition while a large and broad peak agrees with the buffer deposition. This strongly suggests that the small peak in Figure 6B relates to a supported vesicular layer. The characteristics of the peak may also be due to properties associated with the DPTL layer. Admittance measurements and AFM have shown DPTL to be more rigid in water than in buffer, residing in a densely packed state. As a result, DPTL would need less energy and for a shorter amount of time to counter the force generated by the adsorbing vesicle, which would give us a result similar to our observation. Moreover, the rate of vesicles adsorbing to the monolayer after the critical coverage has been reached is much smaller in water than in buffer, which is indicated by the slow increase in frequency over time instead of the slow decrease observed in buffer. Overall, the water system displays a 75% slower tBLM formation rate than buffer due to a combination of the DPTL monolayer having more rigid properties and the vesicle fusion rate being slower after critical coverage. (52) Morillo, M.; Sagrista, M. L.; de Madariaga, M. A. Lipids 1998, 33, 607. (53) Puu, G.; Gustafson, I. Biochim. Biophys. Acta 1997, 1327, 149.

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Similarly, it has been determined that buffer makes DPTL more viscoelastic and makes vesicles have greater interfacial energies by interactions described previously. If a vesicular mass did rupture and begin to fuse, you would expect to see a steady increase in the dissipation, or a straight line in terms of the rate of dissipation change, and this is observed in Figure 6B. Thus, the steady increase may indeed be due to vesicle fusion into the DPTL layer. As fusion continues, the density near the crystal would increase and thus continue to increase the amount of dissipation. Based on the results, a deposition pathway for the tBLM formation has been proposed in Figure 7B. The vesicles diffuse through solution (1) and then contact the preformed DPTL monolayer (2). When the critical coverage point is reached, the vesicles on the DPTL monolayer rupture into bilayer islands, where they fuse into the DPTL layer in buffer and sit atop the DPTL layer in water. After the initial rupture point, vesicles continue to adsorb to the DPTL layer (with a greater rate in buffer with respect to water) and fuse to form a tBLM in buffer (3a) and a supported bilayer in water (3b). The supported bilayer eventually diffuses into the DPTL layer and forms a tBLM (4). Overall, the rate of vesicle deposition onto DPTL is suggested to be kinetically fast, while both the rupture of vesicles and fusion into the DPTL monolayer contribute rate-determining steps in the process of tBLM formation. However, the exact contribution of each rate to the overall process has not yet been determined.

Conclusions The formation of tBLMs using Archae modified tethers and lipids were investigated by QCM-Z and AFM. The kinetics and energetics of DPTL were studied by QCM-Z and found to follow the Langmuir adsorption model. Moreover, DLS and AFM showed DPTL to form vesicles in solution and are the dominant source for monolayer formation. An overall process of the monolayer formation has been proposed whereby the vesicles rupture, form islands, and eventually swell through solvent diffusion to form the final system. Investigations into the kinetics of bilayer formation suggest most of the vesicles are deposited onto the DPTL layer within the first 20 min and that the process is diffusion controlled. The QCM results indicate a critical coverage point at 20 min whereby adsorbed vesicles begin to rupture and form bilayer islands. When deposition is performed in water, the vesicles rupture forming a supported lipid bilayer on top of a rigid DPTL monolayer. The bilayer then proceeds to slowly fuse with the DPTL monolayer over a period of 5.25 h. The QCM-Z results indicate little vesicle fusion occurring after the critical coverage point. In buffer, the vesicles continue to fuse after the initial rupture and sink into the more viscoelastic DPTL layer, forming a tBLM within 3 h. In comparison, it was determined that the tBLM formation in buffer was 75% faster than that of water. Although the exact kinetics are not yet known, evidence from QCM-Z and AFM shows that vesicle deposition is kinetically fast, while vesicle rupture and fusion both contribute rate-determining steps to the tBLM formation. Through this work we were able to determine the general kinetics of tBLM formation and their physical properties. As a result, we were able to modify MEAs with a tethering molecule, DPTL, and form stable lipid bilayers by vesicle fusion. In the future, we hope to use tBLMs as a biosensor platform in an electronically interfaced environment. Acknowledgment. We gratefully thank DARPA, AFOSR, the Department of Energy (DOE), and NSF REU program for their support and funding.

Formation of tBLMs on Gold Surfaces

Supporting Information Available: DLS data, admittance of the DPTL monolayer, AFM images of the QCM crystal, calibration and accuracy assessment of the QCM-Z, and schematic of the QCM-Z batch

Langmuir, Vol. 23, No. 13, 2007 7355 flow apparatus. This material is available free of charge via the Internet at http://pubs.acs.org. LA0610396