Date of this Version
We report a new slide based microarray platform for assaying multiple enzyme activities using fluorogenic substrates. The method enables us to achieve the microfluidic requirements for rapid reaction assembly and compartmentalization. We can thus determine enzymatic activities in individually controlled reaction environments containing cofactors, inhibitors and activators. Fluorogenic substrates in glycerol were arrayed onto glass slides with reaction volumes < 5 nL and feature sizes of < 150 μm. Our method allowed rapid multiple sample deliveries onto the slide (< 3 nL/spot) with no cross contamination between array positions. It enabled us to detect the activation of the fibrinolytic and coagulation proteases namely, thrombin, plasmin, factor Xa, tPa and kallikrein in human plasma. Enzyme-substrate-inhibitor assays using ten caspases were also performed. With over 400 spots/cm2, combinatorial substrate libraries with different proteases can now be rapidly profiled. An assay to detect the dose response of a thrombin inhibitor benzamidine was performed. The inhibitor was arrayed in replicates onto selected positions on the chip. After sequential subnanoliter delivery of the reaction components, the result from the array was analyzed. The expected dose response from benzamidine was seen. A CV of 5.26% was achieved for 232 positions on the array not spiked with the inhibitor. Thus, with potentially several thousand compounds per slide, using rapid sub-nanoliter delivery of components and standard equipment, the true potential of the method is in the field of high throughput screening.
microarray, fluorogenic, enzyme, high thoroughput screening
Date Posted: 24 November 2004
This document has been peer reviewed.