Label-Free Fluorescent Detection of Trypsin Activity Based on DNA-Stabilized Silver Nanocluster-Peptide Conjugates

Abstract

Trypsin is important during the regulation of pancreatic exocrine function. The detection of trypsin activity is currently limited because of the need for the substrate to be labeled with a fluorescent tag. A label-free fluorescent method has been developed to monitor trypsin activity. The designed peptide probe consists of six arginine molecules and a cysteine terminus and can be conjugated to DNA-stabilized silver nanoclusters (DNA-AgNCs) by Ag-S bonding to enhance fluorescence. The peptide probe can also be adsorbed to the surface of graphene oxide (GO), thus resulting in the fluorescence quenching of DNA-AgNCs-peptide conjugate because of Förster resonance energy transfer. Once trypsin had degraded the peptide probe into amino acid residues, the DNA-AgNCs were released from the surface of GO, and the enhanced fluorescence of DNA-AgNCs was restored. Trypsin can be determined with a linear range of 0.0–50.0 ng/mL with a concentration as low as 1 ng/mL. This label-free method is simple and sensitive and has been successfully used for the determination of trypsin in serum. The method can also be modified to detect other proteases.

Detection of Gingipain Activity Using Solid State Nanopore Sensors

“…The LOD for detection of trypsin was 0.005 ng/mL (0.2 pM) and the sensor displayed a dynamic range covering five orders of magnitude (0.005-0.1 µg/mL). For comparison, most of the biosensors discussed in the literature achieve LOD values ranging from 0.1 to 1 ng/mL over a similar dynamic range [28][29][30]. The high performance observed here can primarily be due to the extremely sensitive ionic current measurement using the nanopore system.…”

Sensing of Digestive Enzymes—Diagnosis and Monitoring of Pancreatitis

“…However, since the trypsin concentration in body fluid samples tends to be low, most studies still use specially designed substrates that can be hydrolyzed by trypsin for fluorescence sensing [140]. Specific ideas include the use of protein adsorption to directly affect fluorescence [141] or peptide chain linkage to quench the fluorescence of the substance [142] (Figure 12). First, the aggregation and release of metal nanostructures is the approach used in a large number of reports.…”

A Gold Nanoclusters Film Supported on Polydopamine for Fluorescent Sensing of Free Bilirubin

“…Gold nanoclusters (AuNCs) are a new kind of fluorescent nanomaterial with the advantages of a long lifetime, a large Stokes shift, and biocompatibility. AuNCs have found application in cell imaging, enzyme mimics, biosensors, and photovoltaics [24,25,26,27,28] Gold nanoclusters have been studied extensively in solution. However, only a few studies have discussed the production of an optical AuNCs film.…”