Nanosecond Time‐Resolved Fluorescence Protease Assays

A background reduction of several orders of magnitude in fluorescence protease assays can be achieved by employing time-resolved fluorescence detection with a time gate to eliminate short-lived emission. The use of 2,3-diazabicyclo[2.2.2]oct-2-ene as a hydrophilic and biocompatible fluorophore allows such measurements to be conducted in the nanosecond time domain with a purely organic fluorophore for the first time. The method is applicable to assaying carboxypeptidase A activity on both the laboratory scale and in high-throughput screening.