A High-Throughput, Low-Volume Enzyme Assay on Solid Support

A high-throughput enzyme assay is described that uses 1 μL or less of enzyme solution for each test. Enzyme solutions are deposited by robotic handling in a throughput of over 1000 tests/h on the surface of silica gel plates that have been preimpregnated with fluorogenic substrates. The reaction is quantitated by fluorescence. The method is compatible with water-insoluble substrates (lipases), water-soluble substrates (glycosidases), whole-protein substrates (proteases), and enzyme inhibition measurements. Hydrolytically labile umbelliferyl esters can be used to assay lipases in this format without background hydrolysis. High throughput and reproducibility were tested by fingerprint analysis of lipases and esterases against 37 different fluorogenic ester substrates. A set of eight fluorogenic unbelliferyl esters was selected for optimal activity screening of lipases and esterases on silica gel plates.