pH Modulation Assay on Supported Lipid Bilayers to Detect Protein-Phosphoinositide Interactions

Phosphatidylinositol 4,5-bisphosphate, or PIP2, is a minor membrane-bound phosphorylated phospholipid. Phospholipase C, or PLC, with specific PIP2-binding domains, binds to PIP2, generating secondary messengers essential for several cellular processes.

To detect PLC-PIP2 interactions in vitro, begin with a small unilamellar vesicle, SUV, acidic suspension, comprising PIP2 and phospholipid mixture, one of which is conjugated to a pH-sensitive fluorescent dye.

Pipette the solution into a pre-assembled fabricated microfluidic device's inlet and connect outlet tubing. Transfer and secure the device onto a fluorescence microscope stage. Through gravity, SUVs pass through the microchannel floor, made of a hydrophilic glass coverslip.

The SUVs adsorb on the glass surface, the solution's pH reducing electrostatic repulsion between the two. The SUVs interact with the surface, deforming and fusing to form large vesicles.

Following critical surface coverage, the vesicles rupture spontaneously, forming a supported lipid bilayer, SLB. Pass a buffer through the channel, removing unadsorbed vesicles.

Using a suitable fluorescence channel, image the microchannel to visualize and measure the pH-sen