High sensitive ratiometric fluorescence analysis of trypsin and dithiothreitol based on WS₂ QDs

In this paper, a simple tungsten disulfide quantum dots (WS₂ QDs)-based ratiometric fluorescence method was established for the detection of trypsin and 1, 4-dithiothreitol. Trypsin can hydrolyze cytochrome c into heme-peptide fragments with peroxidase-like activity. In the presence of hydrogen peroxide, the fragments can generates hydroxyl radicals, which can oxidize o-phenylenediamine (OPD) to form 2,3-diaminophenazine (DAP) with a fluorescence peak at 568 nm. DAP can quench the fluorescence of WS₂ QDs at 448 nm via fluorescence resonance energy transfer (FRET). When 1, 4-dithiothreitol was present, it can react with hydroxyl radicals, and less OPD was oxidized, which accompanied by the fluorescence intensity of WS₂ QDs increased and the fluorescence intensity of DAP decreased. Therefore, the fluorescence intensity ratio (F₅₆₈/F₄₄₈) can be used to monitor trypsin and 1, 4-dithiothreitol. A good linear calibration between fluorescence intensity ratio F₅₆₈/F₄₄₈ versus trypsin activity and1, 4-dithiothreitol concentration were obtained within 0.2-140 μg mL^-1 and 20-200 μmol L^-1, respectively. And the detection limit was 0.09 μg mL^-1 for trypsin and 6.8 μmol L^-1 for 1, 4-dithiothreitol, respectively. Furthermore, the developed ratiometric fluorescence method was successfully applied for trypsin and 1, 4-dithiothreitol assay in human serum samples.