A fluorescence turn on trypsin assay based on aqueous polyfluorene

Abstract

A new method based on the electrostatic interaction of a novel anionic water soluble polymer P1 with a positively charged polypeptide Arg₆ was developed for a continuous and real time turn on assay for the enzymatic activity of trypsin under alkaline conditions with a limit of detection of 0.17 nM. This method was also able to screen the inhibitors of trypsin. P1 fluorescence intensity was significantly decreased by the positively charged Arg₆ due to the electrostatic interaction, whereas the enzymatic action recovered P1 fluorescence due to the fragmentation of Arg₆ into small positively charged fragments and these were unable to quench the P1 fluorescence. Therefore, by triggering the fluorescence intensity change, it was possible to assay the enzymatic activity. Use of water soluble conjugated polymer P1 and no labeling on the substrate enhances the utility of this method significantly.