Design And Application Of Organic Fluorescence Probe For Chymotrypsin And Hydrogen Sulfide Detection

The proliferation,differentiation and apoptosis of cells,and the normal physiological metabolism of organisms cannot be separated from the regulation of biological enzymes and small molecules.The stability of cell microenvironment is the premise of the normal operation of the body.Therefore,the trace detection of biological enzymes and small molecules in organisms will become an important way for early disease diagnosis and drug release control.Due to the advantages of high selectivity,fast response and non-invasive real-time imaging in vivo,organic fluorescent probes have gradually become the main means of biological detection in recent years,and are widely used in tissue imaging,drug tracking and environmental monitoring.However,due to the complex internal environment of organisms,it is urgent to develop new fluorescent probes with excellent selectivity,good biocompatibility,high fluorescence quantum yield,high sensitivity and good stability.This paper selects chymotrypsin and hydrogen sulfide,which are common biological enzymes and small molecular substances in organisms,as the target detection substances.According to the existing reports of relevant organic fluorescent probes,two types of fluorescent probes with excellent optical properties are designed based on the mechanism of chymotrypsin and hydrogen sulfide correlation recognition.Combined with experimental analysis,the probe response mechanism is verified,and it is applied to biological detection and related tissue fluorescence imaging.The main work includes the following two parts:1.With the benzopyran fluorescent dye NBD as the luminescent group,and bromoacetyl chloride,3-bromopropionyl chloride,4-bromobutyryl chloride as the recognition group,the strong red light opening fluorescent probes NBD-1,NBD-2,NBD-3 with specific response to chymotrypsin were synthesized,and the effect of the length of the carbon chain of the recognition group on the sensitivity of the probe to recognize chymotrypsin activity was investigated by density functional theory（DFT）calculation,and the best fluorescent probe NBD-3 was selected.NBD-3 can respond to chymotrypsin within 20 min,emit strong red fluorescence at 634 nm,have a low detection limit（15 ng/m L）and low biological toxicity.It is applied to the fluorescence imaging of endogenous chymotrypsin in He La cells,fluorescence imaging in mice and in vitro imaging of main organs,and the presence of chymotrypsin is detected in the liver of mice for the first time.The probe has great development potential in chymotrypsin drug tracking and toxicological research.2.With 4’-（diethylamino）-3-hydroxyflavone as the luminescent group and 2-（2-pyridyldithio）benzoic acid as the recognition group,a double emission ratio fluorescent probe DH-1 with specific response to hydrogen sulfide was synthesized.The probe has the characteristics of high quantum yield,long emission spectrum wavelength,good biocompatibility and p H insensitivity,and the detection limit can reach 91 n M.Probe DH-1 shows excellent selectivity in the emission ratio of hydrogen sulfide to other biological mercaptans(I_{525 nm}/I_{495 nm}).Only in the presence of hydrogen sulfide,probe DH-1 changes from blue fluorescence to green fluorescence.In addition,the probe is also used for fluorescence imaging of exogenous and endogenous hydrogen sulfide in He La cells,which is a potential real-time monitoring method of hydrogen sulfide activity in vivo.