A New Fluorescence Turn-on Assay for Trypsin and Inhibitor Screening Based on Graphene Oxide

In this paper, we describe a new continuous fluorescence turn-on method for trypsin assay and inhibitor screening in situ. This assay is designed based on the following assumptions: (1) It is expected that the fluorescein-labeled peptide composed of six arginine residues (Arg6-FAM) with positive charges will interact with the negatively charged edge of water-soluble graphene oxide (GO) because of electrostatic interactions to form a GO/Arg6-FAM complex. As a result, the fluorescence of fluorescein will be quenched because of the energy transfer from fluorescein to GO. (2) Arg6-FAM can be hydrolyzed into small fragments in the presence of trypsin, and accordingly, the GO/Arg6-FAM complex will be dissociated, gradually leading to fluorescence recovery for the solution. In this way, the trypsin activity can be easily assayed with the ensemble of Arg6-FAM and GO. Additionally, the ensemble can be employed for screening of the inhibitors of trypsin.