Ultrasensitive Determination of Trypsin in Human Urine Based on Amplified Fluorescence Response

Abstract

The determination of trypsin activity in human urine is important for evaluating pancreatic disease. We designed an effective fluorescence sensing strategy based on a self-assembled amphiphilic pyrene/protamine complex system that provides an amplified fluorescence response for highly sensitive and selective detection of trypsin. In aqueous solution, the functionalized pyrene formed fluorescent, π-extended aggregates inside micelles, which were effectively quenched by protamine (a trypsin substrate). However, this quenched fluorescence was very sensitively recovered by the trypsin’s enzymatic reaction, and this was attributed to a marked reduction in enhanced exciton migration caused by protamine in π-delocalized pyrene aggregates. The devised sensing platform was successfully utilized to selectively and sensitively detect trypsin at very low concentrations (0.03–0.5 μg mL–1) in non-pretreated human urine and to screen for trypsin inhibitors at concentrations of 0.1–5.0 μg mL–1.

Sensing of Digestive Enzymes—Diagnosis and Monitoring of Pancreatitis

“…Sun et al quenched the anionic fluorochrome Eosin Y using fish sperm protein, achieving a trypsin LOD of 0.21 ng/mL and a linear detection range of 0.4-56 ng/mL [159]. The detection of trypsin in unpretreated urine samples was also achieved by Park et al by utilizing fisetin [160]. They also analyzed the enhancement of exciton migration induced by fisetin as the reason why fluorescence was quenched with high sensitivity.…”

Micelle-based fluorogenic sensing of trypsin: a sensitive method in pancreatic disease diagnosis

“…The aggregation of compound 1 is attributed to the effective interactions between carboxylate groups and cationic guanidinium groups of arginine residues in protamine, along with the water resistance of the central TPE. 25 Interestingly, DLS results revealed granules of similar sizes for compound 1 in the presence of protamine. This observation clarifies that the possibility of intergranular aggregation induced by protamine is excluded.…”

“…Recently, our group reported that the micellar fluorescence system offers an excellent sensing platform with superior photophysical properties compared to known fluorescence-based sensors. [21][22][23][24][25][26] In this system, the outer water-soluble functional groups of the micelle interact efficiently with water molecules, while the fluorophores located inside the micelle have limited structural contact with water. This structural characteristic results in micelles displaying a high fluorescence quantum yield by reducing non-radiative decay caused by contact with highly polar solvents.…”

Tunable Fluorescence via Self-Assembled Switching of AIE-Active Micelle-like Nanoaggregates

“…Recently, fluorescent micelles that are emissive unimolecular assemblies have offered a clear advantage in resolving the problem above. It may permit the enclosure of additional hydrophobic molecules in the remaining core or construct a fluorescent micelle with a fluorescent surfactant [ 21 , 22 , 23 ]. Thus, much effort has been devoted to the development of luminescent micelles to achieve both carrier ability and emissive features.…”