Trypsin, as one of important proteases, is specific for catalyzing the hydrolysis of peptide and ester bonds containing lysine and arginine residues at the C-terminus. The level of trypsin in biological fluids can serve as a reliable and specific diagnostic biomarker for pancreatic function and its pathological changes. Herein, we demonstrate the application of phosphorescent Cu NCs for trypsin detection for the first time depending on the electron transfer between Cu NCs and cyt c. Cyt c and Cu NCs were selected as the quencher and the fluorophore, respectively. Cu NCs could bind to the positively charged cyt c through electrostatic and hydrophobic interactions, and the phosphorescence of Cu NCs was efficiently quenched by the metal-containing heme of cyt c. In the presence of trypsin, cyt c was digested, thus phosphorescence of Cu NCs remained. Therefore, a new and continuous phosphorescence assay for the detection of trypsin activity and its inhibitor screening was established. The plot of relative fluorescence versus trypsin concentration obtains a good linear detection range from 0 to 20 ng/mL (R2 = 0.9657), and a detection limit of 2 ng/mL, which is much lower than 20 ng/mL of the sensor in buffer solution because of urine amplifying the phosphorescence signal of Cu NCs based on the FRET strategy. This assay still has been successfully applied to trypsin inhibitor screening, demonstrating its potential application in drug discovery.
Keywords: Copper nanocluster; Electron transfer; Inhibitor screening; Phosphorescence; Trypsin.
Published by Elsevier B.V.