Biomembrane Fabrication by the Solvent-assisted Lipid Bilayer (SALB) Method

The overall goal of this procedure is to fabricate supported membranes by the solvent assisted lipid bilayer method. This method can help answer key questions in the membrane biologic field, such as understanding the fundamental properties of lipids and membrane proteins, as well as to enable applications such as drug discoveries and molecular diagnostics. The main advantage of this technique is that it can fabricate support lipids by layer in many cases, which are not possible with the conventional method.

The procedure is also easy to use and require minimal sample preparations. The implications of this technique extend to our therapy and diagnosis because it allows studying important drug targets such as membrane proteins. The method can also be used to fabricate biocompatible substrates for material science applications.

We first had the idea for this Method when we discussed the difficulty of conventional membrane fabrication methods demonstrating the procedure will be mean and SHA to gradual graduate students from our laboratory. Prepare a lipid stock solution of 10 milligrams per milliliter DOPC lipid by dissolving the lipid powder in isopropanol solution. Then dissolve rumine labeled DOPE lipid powder in Isopropanol solution to obtain a lipid stock solution of one milligram per milliliter.

Dilute and mix the stock solutions in Isopropanol to prepare the desired lipid mixture at the final concentration for fluorescence, microscopy and fluorescence recovery after photobleaching or frap experiments, 0.5 weight percent R domine labeled DOPE should be included in the lipid mixture. Inject the lipid mixture in isopropanol into the microfluidic channel until it is filled. Incubate the lipid mixture on the glass surface for about 10 minutes.

The salt exchange should be done at a very low flow rate, otherwise the quality of the bilayer will be low. Gradually replace the lipid solution with water or buffer solution using a peristaltic pump at a very low flow rate. Then rinse the channel thoroughly with excess buffer in order to remove the residual isopropanol for formation of cholesterol enriched supported membranes.

Prepare a stock solution containing DOPC, lipid cholesterol and rho domine, labeled DOPE lipid by first dissolving the respective powders in isopropanol, and then repeating the same protocol as before to perform the membrane fluidity assay. First immerse the glass, slide in 1%sodium ESAL sulfate solution for 10 minutes. Then wash the slides thoroughly with deionized water and rinse with ethanol.

Blow dry the slides using a gentle stream of nitrogen. Then expose the slides to oxygen plasma for 30 seconds at maximum radio frequency power in the oxygen plasma chamber. Next, remove the protective film coating of the bottomless commercial microfluidic chamber using tweezers and attach the glass slide onto the sticky side of the chamber.

Assemble the connectors and tubing into the inlet and outlet positions of the chamber, and place the microfluidic channel on the microscope stage form a fluorescently labeled supported lipid bilayer in the microfluidic channel as before by using the desired lipid composition including a 0.5 weight percent rho domine, labeled DOPE Lipid Inorganic solvent. Next, locate the bilayer plane with a 60 x oil immersion objective. In order to capture images, first, take two preble images before photobleaching.

Warm up the laser until its intensity has become stable. Then photobleach a 30 micron wide circular spot with a 532 nanometer 100 milliwatt laser beam. Immediately after photobleaching.

Capture a series of images every one second for two minutes in order to follow the recovery and fluorescence intensity at the bleach spot to perform cholesterol quantification. First, expose the silicon dioxide coated quartz crystal sensor chip to oxygen plasma for 30 seconds at maximum radio frequency power in the oxygen plasma chamber. Remove the chip from the chamber and immediately mount the chip in the measurement chamber here Q Sense E four A four channel quartz crystal micro balance is used before the experiment.

Acquire the frequency and dissipation of the censorship and air to ensure proper mounting. To do so, run the qof software program. Click acquisition and select setup measurement in the new window, which appears, check the 3 5 7 9 11, and 13 overtones.

Then click find and run. In order to check the resonance spectra, set the temperature at 24 degrees Celsius. Once proper mounting has been achieved, start the peristaltic pump and flow buffer solution into the measurement chamber at a flow rate of 100 microliters per minute in the software program, click acquisition and select restart measurement to record the resonance frequency and energy dissipation signals.

Repeat this step until a stable baseline is obtained. For the frequency and dissipation shifts. Inject the isopropanol without lipid for 10 minutes.

Follow with the injection of the DOPC lipid and cholesterol mixture at the desired molar ratio with the total lipic concentration of 0.5 milligrams per milliliter in isopropanol for 10 minutes. Next, inject buffer for 20 minutes at a flow rate of 100 microliters per minute. Then inject a solution of one millimolar methyl beta cyclodextrin prepared in buffer until the frequency signal reaches a stable value.

Measure the relative positive frequency shifts that are caused by the methyl beta cyclodextrin treatment step and calculate the massive cholesterol and DOPC lipids as described in the text protocol. Then calculate the mole fraction of cholesterol in the supported lipid bilayer, ticking into account the molecular weights of DOPC lipid and cholesterol. Shown here is a representative fluorescence microscopy image of A-D-O-P-C lipid bilayer containing 0.5 weight percent rumine labeled DOPE lipid formed on glass surfaces via the SAL method.

The fluorescent recovery after photobleaching experiment showed almost complete fluorescence recovery indicating the lateral fluidity of the lipid bilayer. Here, representative QCMD curves of frequency and dissipation shifts for supported bilayer formation. Using the SAL method are shown final resonance frequency shifts and dissipation shifts were achieved, which correspond to the formation of a planer bilayer Once mastered.

This technique can be done in 30 minutes if it's performed properly. After watching this video, you should have a good understanding of how to fabricate supported bio membranes by using the solvent assisted lipid bilayer method While attempting this procedure, it's important to remember to prepare fresh lipid solutions before the experiment and to optimize the solvent exchange fluoride depending on your experimental setup following this procedure, other methods like Atomic force microscopy can be performed in order to answer additional questions like formation of membrane domains.