We use a novel calibrated method for titrating intracellular enzymes within single cells. The technique combines the use of plasma membrane permeabilizing agent to increase the membrane permeability and uses a microfluidic system that generates a large number of discrete solution environments around a cell that is translated with high prescision in time (millisecond exchange times). The microfluidic system permeabilizes cells by exposing them to short pulses of permeabilizing agent. Permeabilizing agent creates pores in cell membranes. By, controlling the time the cell is exposed, the number of pores formed can be controlled. The microfluidic system has been described elsewhere, and proven useful for both receptor screening, creation of complex concentration waves and for control of the intracellular environment. The system can be used to monitor changes in intracellular enzymatic activity, and to create dose-response, and dose-inhibition curves, as well as for estimating apparent Km, and Ki values. In addition to the kinetic determinations, this method also offers new possibilities in characterizing effect of temperature on enzyme activity by creating localized thermal gradient through the introduction of IR light.
Figure 1 Experimental setup. Upper part shows the basic experiment setup. Lower part shows schematic of the 16 channels of a micro fluidic device. Fluorescein diphosphate (FDP); digitonin (DIG); fluorescein (Fluro); levamisole(LMS).