The overall goal of this procedure is to describe a method that can be used to study automated lipid bilayer formation using a polydimethylsiloxane thin film. This method can help answer key questions in the nanobiosensor field regarding membrane-protein interactions. The main advantage of this technique is that the lipid bilayer formation is automated and reproducible.
To start, prepare the buffer solution by combining potassium chloride, tris hydrochloride, and EDTA in distilled water. Then, adjust the PH of the solution to 8.0. Then, filter the solution using a 0.2 micron filter, and autoclave the solution at 121 degrees celsius for 15 minutes.
Next, prepare the lipid solution for pre-painting by dissolving 3%of the lipid in a mixture consisting of two parts undecane and eight parts hexadecane by volume. Stir the solution overnight using a rotator. Also, prepare the lipid solution for membrane formation by dissolving 0.1%of the lipid in the same mixture of two parts undecane and eight parts hexadecane by volume.
Stir this solution overnight on a rotator as well. In order to prepare a PDMS thin film, mix nine parts prepolymer with one part curing agent in a mixing cup. Place five grams of this mixture into a petri dish, and spin coat the dish at 800 rpm for 10 seconds to form a film that is 200 to 250 microns thick.
Next, place the petri dish into a vacuum desiccator and pull a vaccum of 100 millitorr for two hours to remove the residual air bubbles. Then, bake the thin film in an oven for five hours at 70 degrees celsius to polymerize the PDMS. Once cooled, cut the polymerized thin film into two centimeter by two centimeter squares, and use a 500 micron diameter punch to make a small aperture in the center of the square.
Then, use a micropipette to prepaint the apertures with one microliter of the 3%lipid solution. To create the black lipid membrane chamber, design two symmetric blocks for the chamber using 3D drawing software. Then, fabricate the chamber using a block of PTFE and a CNC machine.
To assemble the chamber, place the pre-painted PDMS thin film between the two PTFE blocks, such that the aperture on the PDMS thin film is centered with the hole in the chamber. Use cover glass and vacuum grease to seal the outer edges of the chamber. Once sealed, immobilize the assembled chamber using nuts and bolts.
It is important to make sure the chamber's well sealed so that there is no liquid leakage. Using a pipette, deposit 0.5 microliters of the 0.1%lipid solution onto the aperture of the PDMS thin film, assembled with the chamber. Then, place the chamber in a cooled environment below 10 degrees celsius and store it until it is needed.
To form a black lipid membrane with expedited self-assembly, withdraw the chamber from the refrigerator and add two milliliters of the buffer solution into each side of the chamber. Set the chamber aside for 10 minutes until the frozen membrane precursor thaws. Then, place the chamber onto a micro manipulator to precisely control the elevation with respect to the light source and the microscope.
Using a halogen fiber optic illuminator, illuminate one side of the chamber to brighten the aperture of the PDMS thin film. On the other side, place a 20x objective in line with the aperture and observe the center where the color becomes brighter than the annulus. For electrical measurement, prepare silver chloride electrodes by placing a 208 micrometer thick silver wire in a solution of sodium hypochlorite for at least one minute.
Next, connect the electrodes to a microelectrode amplifier. Then, place the silver chloride electrodes into each side of the chamber, so that they are in the buffer solution. Using electrophysiology software, apply a 10 millivolt triangular wave form across the membrane to acquire a square wave.
Record the electrical properties of the membrane by clicking the record button. Continue recording until a uniform square wave is observed. Then, quit recording by clicking on the black square icon.
Two methods can be employed to incorporate gramicidin A into the lipid bilayer. First, gramicidin A can be added directly to the lipid solution before spreading the lipid solution onto the aperture of the PDMS thin film. Second, gramicidin A solution can be added to the chamber when a lipid bilayer forms.
To observe gramicidin A channel activities, apply 100 millivolts across the membrane at a sample rate of 5 kilohertz to measure the holding potential of the membrane. Record the electrical properties as previously done, by clicking the record button. As ion channel activities are shown, keep recording until various current jumps are observed to confirm ion channel incorporation.
Once confirmed, quit the recording by clicking the black square icon. When the recording is finished, filter the data with a low pass bessel filter at 100 hertz using electrophysiology software. Observe the current jumps in the filtered holding potential data.
These jumps represent the dimerization of a gramicidin A ion channel. When the frozen membrane precursor was brought in to room temperature to thaw, the lipid bilayer formation is facilitated due to extraction of hydrophobic solvent into the PDMS thin film. This sequence of images shows the formation of a lipid bilayer, which self assembles when warmed from its frozen form.
Here, the electrical conductance illustrates the incorporation and dimerization of gramicidin A.Upon dimerization, the gramicidin A forms an ion channel and the conductance levels jump to 28 picosiemens, which is consistent with the results of previous reports and shows immediate incorporation of this antibiotic into the bilayer. Our membrane formation technique provides powerful tool for cell membrane and nanochannel studies in contrast to conventional techniques that have limited potentials for practical application. Our system requires no expertise for either membrane formation or ion channeling incorporation making it well suited for drug screening and bio sensing applications.
