Label-free fluorescence turn-on strategy for trypsin activity based on thiolate-protected gold nanoclusters with bovine serum albumin as the substrate

Abstract

By using bovine serum albumin (BSA) as the enzyme substrate, we propose a label-free, convenient, reliable and highly sensitive fluorescence “turn-on” assay for trypsin activity and inhibitor screening by means of fluorescent gold nanoclusters capped with 11-mercaptoundecanoic acid (namely AuNCs@11-MUA) for the first time. The principle for the assay depends on the following features: (1) Cu2+-triggered fluorescence quenching of AuNCs owing to the binding between Cu2+ and the carboxyl groups, (2) the cleavage of BSA into amino acids/peptide fragments catalyzed by trypsin, and (3) the stronger binding ability between amino acids/peptide fragments and Cu2+ than that between AuNCs and Cu2+, accompanied by the conversion from fluorescence off-state to on-state. Under the optimized conditions, the proposed sensing assay shows a good linear relationship from 0.01 to 2μg/mL and provides an inspiring detection limit of 0.004μg/mL, which possesses adequate sensitivity for practical samples of human urine. In addition, our proposed trypsin bioassay can be expanded into the inhibitor evaluation by using trypsin inhibitor from soybean as a model. Such unprecedented AuNCs-based fluorescence “turn-on” analytical proposal for assessing trypsin activity gives a new sight to develop several other potential bioassays or enzyme activity assays with the help of different kinds of substrates in the near future.