A microfluidic protease activity assay based on the detection of fluorescence polarization

This article describes a fluorescence polarization (FP)-based protease assay on a microfluidic device that is compatible with fast and reproducible analyses of protease activities. The optical systems were arranged for simultaneously measuring fluorescence intensities of vertical and horizontal polarization planes, and the binding of tetramethylrhodamine (TMR) labeled-biotin with streptavidin was utilized for optimizing FP detection in continuously flowing solutions within 74-microm wide, 12-microm deep microchannels of a glass chip. In developing off-chip FP-based assays for proteinase K, trypsin, papain and elastase, TMR conjugated-casein protein (TMR-alpha-casein) was employed as a universal substrate. After optimization of the hydrodynamic flow control to allow complete mixing of TMR-alpha-casein and short proteolysis time as possible, and of buffer composition to minimize protein sticking problems, the developed assay was transferred to the microfluidic chip by monitoring FP changes of TMR-alpha-casein in the main microchannel. The results indicate that the proposed device would serve as an integrated microfluidic platform with automated injection of reacting species, diffusion-controlled mixing, reaction and detection for protease activities without the need to separate the products.