The overall goal of this procedure is to create bio membrane microarrays using a simple preparation method. This is accomplished by first coating silica beads with lipid bilayer membranes. The second step of the procedure is to deposit the lipid coated beads on the silicone microwell substrate.
The third step is to remove beads not in micro wells, using a polymethyl suboxane squeegee. The final step is to image the lipid coated bead arrays with fluorescence microscopy. Ultimately, this method can be used to determine binding constants for toxin lipid interactions using an array imaging approach.
This method can be used to create arrays of spherical supported lipid bilayers and natural membrane particles derived from cells. The main advantage of this technique over existing methods is that apart from fabrication of the microwells, the technique requires no chemical modification of either the substrate or the lipid membranes to create spatially defined bio membrane Arrays. The applications of this technique can be extended to label free detection of lipid protein interactions using surface plasma and resonance based on nano metric metal hole arrays.
Though this method can provide Insight into lipid protein interactions, it can also be applied to other systems such as studies of cellular focal adhesion, Micro well arrays and squeegee preparations are detailed in the text protocol. This video begins with vesicle preparation. First in a small glass vial mix, the component lipids for the vesicles.
A total of half a milligram of lipid is in this solution under vacuum desiccate the mixture for six hours. Next, make a 0.1 molar sodium chloride solution and add half a milliliter to the dried lipids. Allow the mixture to incubate overnight at room temperature.
The next day, vortex the mixture to suspend the vesicles. Then sonicate the mixture for 20 minutes in a bath, sonicate at room temperature. After the sonication, extrude the suspension through a 100 nanometer polycarbonate membrane filter.
Pass the suspension through the extrusion filter a total of 17 times. Then store the extruded vesicles in a glass vial at four degrees Celsius. First using 700 nanometer diameter silicon dioxide beads.
Make a suspension of 15 billion beads in 0.1 molar sodium chloride. Vortex the suspension and then centrifuge it at 1700 Gs for 20 minutes and discard the supernatant. Repeat the wash by resus, suspending the beads in another milliliter of salt solution and spinning them down for another 20 minutes.
Then repeat the process a third time to form the spherical supported lipid bilayers or SSBs. Mix 25 microliters of the silicon dioxide bead suspension with 200 microliters of the vesicle suspension from the previous section. Vortex the mixture and lit it incubate at room temperature for an hour later, centrifuge the mixture at 1700 Gs for 20 minutes.
Discard the supernatant. The pellet is pink to to ruptured vesicles with row DPPE on the beads. Resuspend it in 225 microliters of PBS at PH 7.4.
Repeat the spin and Resus suspension steps twice to remove the unruptured vesicles and complete the creation of the SSBs. Divide wafers of micro well arrays into rectangular pieces with four to six arrays each. Next, vortex, the SSLB suspension from the previous section and transfer 10 microliters to each micro well array.
Wait an hour for the SSBs to settle later. Wash the array chips with PBS gently and submerge the array into PBS using the squeegee glide over the surface of the submerged chips five times. This removes SLPs not inside the micro wells.
Next, grip each array chip with tweezers and gently shake off the PBS. Then transfer it to a fresh PBS bath. The top surfaces of the chips must remain wet.
Remove a chip from the bath and W away. Most of the PBS with a lab wipe. Leave enough PBS to keep the array hydrated.
Now add 200 microliters of BSA solution to the array to block non-specific binding. Allow the array to incubate for an hour. In a humidified box later, remove the BSA with a micro pipette and add 200 microliters of cholera toxin solution.
Then return the array to the humidified chamber. Wait another hour, then wash the array with PBS and wick away excess PBS with a lap wipe. Then place the array chip on a standard microscopy slide and attach a 24 by 40 millimeter square cover slip to the array and proceed with imaging.
Image analysis can be done with the image J.Automated particle analysis functions. Then for each array, summarize the mean intensities of the individual SSBs as histograms. After using the outlined methods, A 100 micron square area on an array shows 936 SSBs.
While occupancy data was calculated and a histogram of SSLB fluorescent intensities was generated after a week in storage. The same array was reanalyzed in the same manner, and there was no change in fluorescent intensities or array occupancy. Sequential deposition of different SSBs with different identifying markers was also possible with the outlined methods.
The second SSBs were deposited at one 10th. The concentration of the first SSBs deposited. An overlay of both markers is shown for an experiment.
Arrays were made with SSBs with various concentrations of GM one and exposed to a fixed concentration of cholera toxin. The inverse was also performed to determine the equilibrium dissociation constant. For GM one.
Cholera toxin binding arrays can also be made from natural bio membranes, such as this array made of myelin particles. It is labeled with lipophilic fluoro four FM 1 43. Lipid rafts are enriched in cholesterol and gangliosides such as GM one.
Thus Alexa 4 88 conjugated cholera toxin bind strongly to lipid raft microarrays by contrast, fluorescently labeled stripped Aden does not bind to these arrays. An array was made by delivering myelin and lipid rafts to the micro wells via a microfluidic chip with 250 micron channels. It was labeled with anti oligodendrocyte IgM and the S sulfide found in myelin was labeled, but the lipid rafts were not.
Not Once the lipid bator coated beads are prepared. This technique can be used to prepare arrays in an hour if performed properly, Following creation of natural membrane particle arrays. Other methods such as nano hole surface plasma and resonance can be used for label-free sensing of biomolecular interactions.
After watching this video, you should have a good understanding of how to create bio membrane arrays using lipid bile layer coated beads and natural membrane particles.
