Label-free high-throughput functional lytic assays.
Refractive index-sensitive resonant waveguide grating biosensors are used to assay the label-free enzymatic degradation of biomolecules. These assays provide a robust means of screening for functional lytic modulators. The biomolecular substrates in this study were covalently immobilized through amine groups. Using the Corning Epic System, the digestion signatures for multiple protein substrates on the biosensors are measured. Label-free digestion profiles for these proteins were substrate specific. Similarly, the authors find that the label-free digestion is protease specific. Enzyme-substrate pairs were used to evaluate high- throughput biosensors as tools for screening functional modulators. The lytic inhibitor properties for several proteases and dextranase are determined. The authors find that the IC50 values for the protease inhibitors agree with the reported values for several known inhibitors. The Z' values, using biosensor-based functional lytic screens, were routinely greater than 0.5, making this label-free application feasible for high-throughput screening.
O'Malley
Shawn M
SM
Corning Incorporated, Corning, NY 14831, USA. omalleysm@corning.com
Xie
Xinying
X
Frutos
Anthony G
AG
D000818:Animals; D001681:Biological Assay / Q000379:methods*; D015374:Biosensing Techniques; D050260:Carbohydrate Metabolism / Q000187:drug effects; D002417:Cattle; D003910:Dextranase / Q000378:metabolism; D006801:Humans; D015335:Molecular Probes / Q000378:metabolism*; D010447:Peptide Hydrolases / Q000378:metabolism; D011480:Protease Inhibitors / Q000032:analysis / Q000494:pharmacology; D011499:Protein Processing, Post-Translational; D011506:Proteins / Q000378:metabolism*; D013379:Substrate Specificity; D014357:Trypsin / Q000378:metabolism
D015335:Molecular Probes; D011480:Protease Inhibitors; D011506:Proteins; D003910:Dextranase; D010447:Peptide Hydrolases; D014357:Trypsin