PIP-on-a-chip：タンパク質 - ホスホイノシチド相互作用のラベルフリー研究

The overall goal of the PIP-on-a-chip assay is to assess protein membrane interactions in a quantitative label-free manner. Protein membrane interactions are the heart of so many processes of the cell and its pathogens, but techniques to study these interactions are few. The advantages of this technique are low simple volume, no ligand or receptor labeling requirements combined with the ability to test membrane interactions in a physiologically relevant manner.

Many therapeutic targets of viruses are membrane proteins, studies of these target proteins are often performed in solution using detergents. Our technique provides a more biologically relevant alternative. While you will see that this assay is capable of monitoring the interactions of proteins with membranes, it's actually quite a versatile assay and can be used to monitor iron membrane, small molecule membrane and even peptide membrane interactions.

To begin, mix polydimethylsiloxane or PDMS prepolymer and the curing agent at a 10:1 ratio, in a large plastic weigh boat. Degas the mixture in a vacuum for one hour with the vacuum strength at 500 tor or less. Place the silicon master which contains multiple replicates of the same SU8 micropattern in a large plastic weigh boat and pour in the degas PDMS, then, cure it in a dry oven at 60 degrees celsius overnight.

The next day, gently peel off the PDMS from the silicon master using one's hands. Mark the boundaries of each micro pattern in rectangles using a surgical scalpel and a ruler. Then, cut the PDMS into blocks, punch 16 holes at both ends of each micro channel with a biopsy punch, to make holes with 1.0 millimeter diameter.

Pipette calculated volumes of phosphatidylcholine, Phosphatidylinositol 4, 5-bisphosphate and pH sensitive fluorescent probe into a single 20 milliliter glass ventilation vial. Dry the mixture in a stream of nitrogen gas inside the chemical fume hood for 10 minutes or until the solvent evaporates and the thin lipid film forms at the bottom of the vial. Then, desiccate the mixture under vacuum for at least three hours at a vacuum strength of 10 millitor to remove any residual organic solvent.

Rehydrate the dried lipid film with five milliliters of running buffer, place the rehydrated lipid in an ultrasonic bath at an operating frequency of 35 kilohertz for 30 minutes at room temperature. Freeze thaw the vesicle suspension with liquid nitrogen and the 40 degree celsius water bath to get unilaminar vesicles. Repeat the freeze thaw 10 times, extrude the vesicle suspension to a 0.1 micron track edge polycarbonate membrane, using a lipid extruder to enrich for small unilaminar vesicles.

Repeat the extrusion 10 times. Test the inlets and outlets of the PDMS block for blockage by squirting de-ionized water through the holes using a water wash bottle, then, dry the PDMS block with nitrogen gas. Next, place the PDMS block and the pre-cleaned cover slip inside the oxygen plasma system sample chamber.

Expose the PDMS block and the cover slip with oxygen plasma for 45 seconds with the power settings at 75 watts, oxygen flow speed at 10 cubic centimeters per minute and the vacuum strength of 200 millitor. Next, place the pattern surface of the PDMS block in the contact with the cover slip immediately after the oxygen plasma treatment. Press gently to remove any air bubbles at contact sites.

Place the device on a level hot plate at 100 degree celsius for three minutes to enhance the bonding. Use a wet lint-free wipe with 100%ethanol to remove any dust particles from the top and the bottom of the device. Then, tape the device on top of a glass microscope slide.

Transfer 100 microliters of the Phosphatidylinositol 4, 5-bisphosphate containing small unilaminar vesicles into a 0.65 milliliter microcentrifuge tube. Adjust the pH of the solution to approximately 3/2 by adding 6.4 microliters of 0.2 normal hydrochloric acid. Pipette 10 microliters of the pH adjusted small unilaminar vesicle solution into each channels through the inlet and apply pressure through the pipette until the solution reaches the outlet.

Detach the tip from the pipette and leave it attached to the device. After repeating this step for each channel, incubate the device for 10 minutes at room temperature, injection of vesicles into micro channels should be performed immediately after the device assembly. Meanwhile, cut sets of inlet and outlet tubing, using tweezers, connect the outlet tubing set to the device and then tape the device onto a microscope stage.

Submerge one end of the inlet tubing set in 25 milliliters of running buffer contained in a conical tube and tape it to make sure that the tubing is secured. Using a lab jack, place the conical tube on a higher ground than the device in order to push the solution through the micro channels via gravity flow. For each inlet tube, use a syringe to draw one milliliter of running buffer from the free end of the tubing.

Remove the pipette tip from the inlet and insert the free end of the inlet tubing into the device. Repeat this process to connect all the inlet tubing pieces to the device. Flowing running buffer through the channels helps to remove excess unruptured vesicles and equilibrate the bilayer to experimental conditions.

Next, open the microscope control software. On the left panel, click on the microscope tab and choose the 10x objective. Click on live and then the Alexa 568 image icons on the toolbar, using the fine and coarse adjustment knobs, focus on the micro channels.

Scan through the device to check through the quality of the SLBs and the channels. Then, click the FL shutter closed image icon on the toolbar, click on the acquisition tab and under basic adjustments, select exposure time. Set the exposure time to 200 milliseconds.

On the left panel, click on multi-dimensional acquisition. Under the filters menu, select the red channel. Then, click on the timelapse menu, set the time interval to five minutes, duration to 30 minutes and lapse menu.

Select the circle tool under the measure tab and draw a circle in any channel. Right click while the circle is selected and choose properties. Under the profile tab, check all T to view the fluorescence intensity as a function of time.

Make sure this curve reaches a plateau which indicates equilibrium before proceeding to the next step, lower the buffer solution to an equal ground as the device, to stop the flow. One at a time, detach each outlet tubing and apply 200 microliters of each protein dilution into the outlet channel using a pipette. Do not apply any pressure, let gravity do the work.

Detach the tip from the pipette and leave it attached to the micro fluidic device. Repeat this process for each channel and make sure that air bubbles are not introduced into the channels during this process. Next, lower the inlet tubing to a ground below the microfluidic device to start flowing the protein through the micro channels.

Tape the free end of the tubing to a waste container. Flow the dilutions of the pleckstrin homology domain for 30 minutes. On the left panel of the software, under the timelapse tab, click on start, to begin imaging again.

Shown here, is a representative view of the Phosphatidylinositol 4, 5-bisphosphate containing SLBs within micro channels. Before, and after, adding the pleckstrin homology domain at the indicated concentrations. Fluorescence intensities from the line scanned across micro channels are plotted as a function of distance and pixels for phosphatidylcholine, Phosphatidylinositol 4 phosphate, and Phosphatidylinositol 4, 5-bisphosphate binding experiments.

Then, normalize binding data from individual experiments, is plotted as a function of phospholipase C delta 1 pleckstrin homology domain concentration and fit to a binding isoterm to extract apparent association constants. Comparison of the apparent association constants, shows that phospholipase C Delta 1 pleckstrin homology domain interacts with Phosphatidylinositol 4, 5-bisphosphate specifically, as expected. After watching this video, you should have a good understanding of how to make small unilaminar vesicles, make microfluidic devices, form supported lipid bilayers inside these microfluidic devices, as your protein membrane binding interactions, using this assay with our PIP-on-a-chip approach.

Once mastered, this technique can be done in three hours if it is performed properly. Following this procedure, other methods like fluorescence recovery after photo bleaching can be performed in order to assess the effect of protein membrane binding on the lateral diffusion of the lipids. This technique paves the way to study protein membrane interactions with the assemble of lipids found in cells instead of one at a time, this approach will therefore broadly impact biochemistry and cell biology of protein membrane interactions.