Assay development and high-throughput screening of caspases in microfluidic format.
Caspase proteases are familiar targets in drug discovery. A common format for screening to identify caspase inhibitors employs fluorogenic or colorimetric tetra-peptide substrates in 96, 384, or 1536 -well microtiter plates. The primary motivation for increasing the number of wells per plate is to reduce the reagent cost per test and increase the throughput of HTS operations. There are significant challenges, however, to moving into or beyond the 1536-well format, such as submicroliter liquid handling, liquid evaporation, increased surface area-to-volume ratios, and the potential for artifacts and interference from small air-borne particles such as lint. Therefore, HTS scientists remain keenly interested in technologies that offer alternatives to the ever-shrinking microtiter plate well. Microfluidic assay technology represents an attractive option that, in theory, consumes only subnanoliter volumes of reagents per test. We have successfully employed a microfluidic assay technology in fluorogenic screening assays for several caspase isoforms utilizing the Caliper Technologies Labchip  platform. Caspase-3 is used as a representative case to describe microfluidic assay development and initial high-throughput screening results. In addition, microfluidic screening and plate-based screening are compared in terms of reagent consumption, data quality, and ease of operation.
Wu
Ge
G
Amphora Discovery Corp., Los Altos, California 94022, USA. ge_wu@sbcglobal.net
Irvine
Jennifer
J
Luft
Chris
C
Pressley
David
D
Hodge
C Nicholas
CN
Janzen
Bill
B
D061945:Caspase Inhibitors; D020169:Caspases / Q000032:analysis* / Q000378:metabolism; D006801:Humans; D007202:Indicators and Reagents; D008832:Microchemistry / Q000379:methods; D008904:Miniaturization / Q000295:instrumentation / Q000379:methods; D015203:Reproducibility of Results; D013050:Spectrometry, Fluorescence
D061945:Caspase Inhibitors; D007202:Indicators and Reagents; D020169:Caspases