Label-free Fluorescence Turn on Trypsin Assay Based on Gemini Surfactant/heparin/Nile Red Supramolecular Assembly

In this research, we designed a label-free fluorometric turn-on assay for trypsin and inhibitor screening, based on a spherical cationic gemini surfactant ethylene-bis (dodecyl dimethyl ammonium bromide) (EDAB)/heparin/Nile red (NR) supramolecular assembly system. The introduction of gemini surfactant EDAB as template greatly enhanced its salt resistance and resulted in the supramolecular assemblies with diameters ranging from 20 to 100 nm. The fluorometric assay for trypsin was performed by firstly disassembling with protamine (a heparin-binding protein) and then re-assembling through hydrolysis of protamine. The disassembly and reassembly of the system resulted in a turn-off first and then a turn-on behavior of the corresponding fluorescence. The overall processes were characterized by fluorescence spectra, TEM measurements and zeta potential tests. The detection level of this assembly system for trypsin was as low as 4.2 ng mL^-1. Also, the EDAB/heparin/NR assembly could be used to screen the trypsin inhibitors. The assembly system was easily-fabricated and cost-effective, but also exhibited good salt tolerance in NaCl solution at the concentration of 0-500 mM. At last, the supramolecular assembly was successfully applied to detect trypsin in human urine, demonstrating its great potential on clinical diagnosis applications.