Peptide-induced fluorescence quenching of conjugated polyelectrolyte for label-free, ultrasensitive and selective assay of protease activity

We report here a label-free method for ultrasensitive and selective assay of protease activity based on the peptide-induced fluorescence quenching of conjugated polyelectrolyte (PPESO3). It is very interesting to find that there is a critical length of oligo-polyarginine (i.e., Arg5) below which 1) the quenching efficiency of PPESO3 is sharply decreased, and more importantly, 2) the trypsin-catalyzed hydrolysis is greatly slowed down. This opens good opportunities for not only the ultrasensitive assay of trypsin, but the specific detection of other proteases if carefully designing an appropriate peptide length and the cleavage site. Herein, the enzyme selected as a proof of concept is chymotrypsin. Due to the essence that any cleavage of the designed peptide probes will result in a notable decrease or even a complete loss of their capability to quench the emission of PPESO3, the limits of detection for trypsin and chymotrypsin have been found as low as 0.25 ng/mL (11 pM) and 0.15 ng/mL (6 pM), respectively. Both are superior to those of most previous methods by 1–2 orders or higher.

► A label-free, ultrasensitive and selective assay of protease activity is developed.
► A critical peptide length for PPESO3 quenching and trypsin digestion is demonstrated.
► Proper length of oligo-polyarginine ensures the most efficient fluorescence turn-on.
► Critical peptide length for trypsin digestion enables specific assay of chymotrypsin.
► Detection limit for trypsin and chymotrypsin are as low as 11 and 6 pM, respectively.