Fluorescent-conjugated polymer superquenching facilitates highly sensitive detection of proteases.
Sensor formats have been developed for detecting the activity of proteolytic enzymes based on fluorescent conjugated polymer superquenching. These sensors employ a reactive peptide sequence within a tether linking a quencher to a biotin. The peptide binds to sensors containing colocated biotin-binding protein and fluorescent polymer by means of biotin-biotin binding protein interactions, resulting in a strong quenching of polymer fluorescence. Enzyme-mediated cleavage of the peptide results in a reversal of the fluorescence quenching. These assays for protease activity are simple, sensitive, fast, and have the specificity required for screening chemical libraries for novel protease inhibitors in a high-throughput screening assay environment. These assays have been demonstrated for enterokinase, caspase-3/7, and beta-secretase.
Kumaraswamy
Sriram
S
QTL Biosystems, 2778 Agua Fria Street, Santa Fe, NM 87507, USA.
Bergstedt
Troy
T
Shi
Xiaobo
X
Rininsland
Frauke
F
Kushon
Stuart
S
Xia
Wensheng
W
Ley
Kevin
K
Achyuthan
Komandoor
K
McBranch
Duncan
D
Whitten
David
D
D016282:Aspartic Acid Endopeptidases / Q000032:analysis*; D020169:Caspases / Q000032:analysis*; D002851:Chromatography, High Pressure Liquid; D003546:Cysteine Endopeptidases / Q000032:analysis*; D004765:Enteropeptidase / Q000032:analysis*; D005453:Fluorescence; D011108:Polymers / Q000378:metabolism*
D011108:Polymers; D004765:Enteropeptidase; D020169:Caspases; D003546:Cysteine Endopeptidases; D016282:Aspartic Acid Endopeptidases