Method and system for determining intracellular mediators' activity

A method for determining the presence of a molecule coupled to the cytoplasmic side of a cellular membrane, wherein the molecule coupled to the cytoplasmic side of a cellular membrane is a protein or multi-protein complex selected from a GαGDP-βγ heterotrimeric G protein, a GαGTP subunit of a G protein, Gβγ subunits of a G protein, and a combination thereof, the method comprising the steps of:

• a) providing a plurality of micrometric polymeric beads, each located in a trap of a multi-trap microfluidic device, wherein the micrometric polymeric beads comprise on their surface a molecule capable of interacting and binding to an extracellular cell-membrane molecule;
• b) loading a plurality of cells into the microfluidic device, wherein the cells adhere to the micrometric polymeric beads through the interaction and binding between the molecule on the surface of the micrometric polymeric beads and the extracellular cell-membrane molecule;
• c) breaking the bead-attached cells to expose cellular plasma membrane fragments on the beads in an inside-out orientation;
• d) loading into the microfluidic device a labelling compound specific for the molecule coupled to the cytoplasmic side of a cellular membrane, wherein the labelling compound comprises a radioactive probe, a chemiluminescent probe, and/or a fluorescent probe;
• and
• e) detecting a radioactive, chemiluminescent, and/or a fluorescent signal associated with the presence of the labelling compound, wherein the signal is indicative of the presence of the molecule coupled to the cytoplasmic side of a cellular membrane;
• wherein the multi-trap microfluidic device comprises a plurality of shifted arrays of traps, each trap being configured to accommodate only one bead, and
• wherein each trap of the multi-trap device is composed of two spaced-apart pillar subunits rotated to create a V shape.