Nanodroplet microarrays for high-throughput enzyme screening.
We describe here a method for the continuous assessment of enzymatic activity using microarrays. By uniformly coating fluorogenic substrates on slides, we generated surfaces capable of detecting enzymatic activity. The enzymes were deposited on the arrays in segregated droplets using standard microarrayers. Surfaces were developed for assessing the activities of both proteases and phosphatases, hence capitalizing on microarray technology to perform miniaturized high-throughput screens for these, as well as potentially any other, classes of enzyme. This offers an unprecedented ability for performing solution-phase enzymatic assays in nanoliter volumes on microarrays, in contrast to microliter volumes typically required in microplate-based assays, thereby reducing the amounts of reagent(s) required by anywhere from a hundred to a thousand-fold. This new approach thus provides a potentially more cost-effective, label-free enzyme screening technique. A single slide is able to accommodate several thousand assays, facilitating the assessment of both dose and time-dependent inhibition parameters in a single run.
Aw
Kang L D
KL
DSO National Laboratories, Defence Medical and Environmental Research Institute, Singapore, Singapore.
Yao
Shao Q
SQ
Uttamchandani
Mahesh
M
D002364:Caseins / Q000737:chemistry; D057075:Enzyme Assays / Q000379:methods*; D004791:Enzyme Inhibitors / Q000138:chemical synthesis / Q000737:chemistry / Q000494:pharmacology; D004798:Enzymes / Q000378:metabolism*; D004852:Epoxy Compounds / Q000737:chemistry; D057166:High-Throughput Screening Assays / Q000379:methods*; D046228:Microarray Analysis / Q000379:methods*; D036103:Nanotechnology / Q000379:methods*; D010447:Peptide Hydrolases / Q000378:metabolism; D010744:Phosphoric Monoester Hydrolases / Q000037:antagonists & inhibitors / Q000378:metabolism; D011327:Printing; D054852:Small Molecule Libraries / Q000138:chemical synthesis / Q000737:chemistry / Q000494:pharmacology
C104678:BODIPY-alpha-casein; D002364:Caseins; D004791:Enzyme Inhibitors; D004798:Enzymes; D004852:Epoxy Compounds; D054852:Small Molecule Libraries; D010744:Phosphoric Monoester Hydrolases; D010447:Peptide Hydrolases