Fluorescent Detection Of Trypsin On The Basis Of Surfactant/Polyelectrolyte Supramolecular Assembly

As fluorescent analysis with the advantages of high sensitivity,convenient operation,non-invasion to the samples and fast detection,so the detection based on fluorescence analysis had attracted more and more attention and application.Supramolecular assembly was formed by non-covalent bond with a certain structure and function of the multi-molecular group,This article combined supramolecular assembly formed by the electrostatic and hydrophobic interaction of surfactant and polyelectrolyte with fluorescent analysis.Fluorescent analysis on the basis of supramolecular assembly had significant advantages,which was connected by the recognition portion and the fluorescent reporter portion of the fluorescence sensor in the way of non-covalent effect,avoiding the organic synthesis,easy to prepare and helpful to system optimization.Based on the principle of supramolecular assembly and disassembly,three kinds of supramolecular assemblies were designed for fluorescence detection of trypsin by selecting different surfactants and polyelectrolytes.The details were as follows:1.With Nile red as fluorescent probe,the supramolecular assembly of detecting trypsin and enzyme inhibitors was constructed by the electrostatic and hydrophobic interaction between negatively charged surfactant Sodium dodecyl sulfate(SDS)and positively charged protamine.The supramolecular assembly with high fluorescence emission was formed by electrostatic and hydrophobic interaction with protamine and SDS at a level lower than the critical micelle concentration(CMC),which could be confirmed by fluorescence spectroscopy and TEM images.Due to trypsin on the hydrolysis of protamine into short peptides,the assembly disassembled,and the fluorescence intensity reduced obviously as hydrophobic fluorescent probe Nile red was released,which could be proved by TEM images.The detection limit of this system was as low as 0.044 ng · mL-1,which is at or above the present level of trypsin detection.The IC50 of the enzyme inhibitor was 0.02 mg · mL-1.Substrate and fluorescent probe had a certain effect on the detection.Arg8 was a less efficient substrate than protamine because of the shorter chain of the former than the later peptide.Coumarin 6 and Pyrene were inferior to NR as the probe.2.With Nile red as fluorescent probe,the supramolecular assembly of detecting trypsin in human serum was established by the electrostatic and hydrophobic interaction between negatively charged Gemini surfactant Propane-1,3-bis(dodecyl propane sulfonate)(C3C12C3(SO3)2)and bovine serum albumin(BSA).The supramolecular assembly with high fluorescence intensity was formed by electrostatic and hydrophobic interaction with BSA and C3C12C3(SO3)2 at a level lower than the critical micelle concentration(CMC),which could be confirmed by fluorescence spectroscopy and TEM images.Due to trypsin on the hydrolysis of BSA,the assembly disassembled,and the fluorescence intensity reduced as NR was released.The detection limit of trypsin was as low as 1.6 ng · mL-1 with a good selectivity.It could also be used to detect trypsin inhibitors,IC50 of the enzyme inhibitors was 0.23 mg · mL-1.The salt resistance test showed that the C3C12C3(SO3)2/BSA assembly had good salt resistance and could be used for the detection of trypsin in the actual serum.The recovery experiment showed that the C3C12C3(SO3)2/BSA/Nile red detection system had good accuracy and reliability.3.With Nile red as the fluorescent probe,through the electrostatic and hydrophobic interaction between positively charged Gemini surfactant ethylene-bis(dodecyl dimethyl ammonium bromide)(EDAB)and negatively charged heparin,EDAB/heparin/Nile red supermolecular assembly was constructed,with protamine as trypsin detection substrate,to further build a "turn-on" detection system for the actual detection of trypsin in human serum.Adding protamine in the EDAB/Herapin/Nile red assembly,since the specific effect of protamine on heparin sodium was greater than EDAB,the assembly disassembled,which was confirmed by fluorescence spectroscopy and TEM images,and the fluorescence intensity was decreased.Due to trypsin on the hydrolysis of protamine,EDAB/ Herapin/Nile red assembly assembled again and fluorescence intensity increased.The detection limit of trypsin was as low as 4.2 ng · mL-1 with a good selectivity.It can also be used to detect trypsin inhibitors,IC50 of the enzyme inhibitors was 0.41 mg · mL-1.The salt resistance test showed that the system had good salt tolerance and could be used to detect trypsin in human serum.The recovery experiment showed that EDAB/heparin/Nile red detection system had good accuracy and reliability.