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.
Highlights
Trypsin is one of the most significant digestive protease produced in the pancreas
Fluorescence methods have attracted significant attention because of their simplicity in situ and in real-time detection, e.g., quantum dot (QD) conjugates based on the Förster resonance energy transfer (FRET) between QD and Cy3 [10] (i.e., QD-(peptide-Cy3)n ), the fluorescein-labeled peptide composed of six Arg residues and graphene oxide (GO) [9], and a cationic peptide substrate labeled with p-nitroaniline [11]
We previously found that thiocholine enhanced the fluorescence of DNA-AgNCs and developed a simple and rapid method for the sensitive detection of acetylcholinesterase activity [27]
Summary
Trypsin is one of the most significant digestive protease produced in the pancreas. It cleaves proteins or peptides at the carboxyl terminal of arginine (Arg) or lysine (Lys) residues and plays an important role in the regulation of pancreatic exocrine function [1,2]. Several strategies for trypsin assay have been reported, such as radioimmunoassay [5], colorimetry [6,7], electrochemical methods [8], and fluorescence methods [9,10,11] Among these methods, fluorescence methods have attracted significant attention because of their simplicity in situ and in real-time detection, e.g., quantum dot (QD) conjugates based on the Förster resonance energy transfer (FRET) between QD and Cy3 [10] (i.e., QD-(peptide-Cy3)n ), the fluorescein-labeled peptide composed of six Arg residues and graphene oxide (GO) [9], and a cationic peptide substrate labeled with p-nitroaniline [11]. The peptide consists of a trypsin-cleavable peptide sequence and a cysteine terminus
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