Label-free, homogeneous, and fluorescence "turn-on" detection of protease using conjugated polyelectrolytes

A label-free, homogeneous, and sensitive fluorescence "turn-on" approach is designed to rapidly detect protease using serine functionalized polythiophene (POWT). The fluorescence of POWT solution containing bovine serum albumin (BSA) as substrate is efficiently quenched by Cu2+ ions through coordinate interaction with serine moieties. Upon adding trypsin to the solution, the BSA is cleaved into amino acid or peptide fragments, which are stronger Cu2+ chelators to form more stable complexes with Cu2+ ions. Thus, the Cu2+ ion is displaced from POWT and the fluorescence of POWT is recovered. By triggering the "turn-on" signal of POWT, it is possible to detect the trypsin in real time. "Turn-on" response as readout signal is able to effectively reduce background noise and increase detection sensitivity. Because of the simplicity, high sensitivity, and rapid response, our new enzyme assay shows great potential for protease detection in the future.