ABSTRACT
DepositionThe substrate upon which these bilayers can be formed is highly doped n-type silicon, n+-Si (111). Clean hydrophilic surfaces are required for LB deposition of uniform monolayers, so the silicon wafers are therefore cleaned in piranha solution prior to monolayer deposition. Briefly, the wafers are immersed sequentially in 2-propanol, acetone, and 2-propanol and sonicated for 15 minutes with each solvent. Finally, the wafers are immersed in piranha etch solution: 30% hydrogen peroxide and 70% sulfuric acid. The wafers are etched for 20 minutes and then rinsed several times in de-ionized water prior to deposition of the bilayer. Lipid bilayers are formed by LB deposition and VF. In the first step, LB deposition is used to form the lower leaflet on the silicon support (Fig. 22.1). The PEG-lipid used to form the polymer
cushion is 1,2-dipalmitoyl-sn-glycero-3-phosphoethanolamine-N-[methoxy(PEG)-2000] (PEG-2k). Lipids, such as 1,2-diphytanoyl-sn-glycero-3-phosphocholine (DPhPC), 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine (POPC), and cholesterol, are premixed in chloroform to achieve a total concentration of 1 mg mL-1, and 25 µL of the lipid solution is spread drop-wise at the air-water interface of the open trough (600 cm2) onto the subphase, 18 MΩ cm water (Fig. 22.1A). After spreading, the chloroform is allowed to evaporate for 30 minutes, and then the monolayer is compressed at a barrier speed of 100 cm2 min-1 to a pressure of 32 mN m-1. The silicon substrates are withdrawn from the trough at 15 mm min-1, while maintaining the pressure constant at 32 mN m-1. This rate is sufficiently slow to ensure that the meniscus formed at the silicon surface remains uniform during withdrawal (Fig. 22.1B). After LB deposition of the monolayer, the silicon wafer is assembled into a three-electrode electrochemical cell. A custom Teflon electrochemical cell is assembled on top of the monolayer with the working electrode area (0.814 cm2) defined by an o-ring. An ohmic contact is formed on the wafer using InGa eutectic after removing the surface oxide by a careful application of 25% HF for one minute. The bilayer is then completed by VF of large unilamellar vesicles (LUVs). LUVs 100 nm in diameter are prepared using standard techniques as follows. First, the chloroform from a stock lipid solution is evaporated under a stream of nitrogen. The lipids are then further dried under vacuum for a minimum of one hour. A buffer solution of 10 mM sodium phosphate and 100 mM potassium chloride is added to yield a lipid vesicle solution with a final concentration of 1 mg mL-1. The solution is vortexed to ensure that all the lipids are suspended in buffer. The vesicles are extruded through a 100 nm polycarbonate membrane; the solution is passed through the membrane a minimum of 10 times. A solution of 450 µL of extruded vesicles is slowly deposited over the PEG-supported lipid monolayer (Fig. 22.1C). The bilayers are completed by incubation of the vesicles in the dark for one hour (Fig. 22.1D). Prior to electrochemical measurements, an additional 10 mL buffer is added to the electrochemical cell. All experiments are conducted at room temperature and in the dark, to avoid photo effects in the silicon.
