A fluorogenic assay using pressure-driven flow on a microchip.

A fluorogenic assay for human T-cell phosphatase (TCPTP) was conducted on an etched glass microchip using pressure driven flow. The TCPTP enzyme catalyzes the removal of a phosphate group from 6,8-difluoro-4-methylumbelliferyl/phosphate (DiFMUP) to produce the fluorogenic product 6,8-difluoro-4-methylumbelliferone (DiFMU). Enzyme assays with real-time on-chip dilution were performed in both low-viscosity (1 cP) buffer and an enzyme solution containing 50% glycerol (6 cP). Single side channels connect a series of reagent wells to a main channel where the fluorescent product of the enzyme reaction passes the detector region. Flow regulation of mixed viscosity fluids requires a pressure control on each arm of the chip contributing to the overall flow. An 8-channel pressure controller was built to regulate the air pressure above all wells feeding channels of the chip, thereby controlling the dilution ratios of buffer, substrate and enzyme. Well pressures maintained a constant concentration of enzyme in the detector channel while adjusting the flow contribution of substrate and buffer. The substrate concentration was stepped over two orders of magnitude while verifying fluid dilutions using marker dyes. The kinetic parameters, Km, Vmax, and Kcat, showed good agreement with the values determined using a standard well plate and fluorometer.