Detection Of Bioligical Enzymes By Small Molecule Fluorescent Probe And Its Application In Biochemical Analysis

The World Health Organization estimates that diseases such as cancer kill millions of people every year worldwide and seriously endanger human health.Therefore,the effective diagnosis and treatment of related disea ses has become a major research problem facing human beings.Most of the biosensors reported so far have complicated preparation processes and high detection background and are susceptible to interference.In order to improve the accuracy and reliability o f clinical diagnosis,this article provides three types of fluorescent probes that are simple to prepare and respond quickly to disease-related biological enzymes The determination was carried out,and the specific research contents are as follows:1.In this work,a phosphorylated 2-（2’-hydroxyphenyl）-benzothiazole derivative was used to construct a ratiometric fluorescent probe（HBTP-mito）based on excited intramolecular proton transfer（ESIPT）.Due to the blockage of ESIPT,the probe emits green fluorescence in the aqueous solution.After the introduction of alkaline phosphatase（ALP）,the phosphate ester bond of the probe is hydrolyzed and the ESIPT process is restored,which reduces the fluorescence emission at 514 nm（enol）,and the emission at 650 nm（keto）“turns on”,achieving the effect of ratiometric detection and targeting mitochondrial organelles.This sensitive detection scheme shows a relatively low detection limit（LOD=0.072 mU/mL）,which can be used not only to detect ALP activity in biological samples,but also to visualize ALP concentration levels in cancer cel s.2.Acetylcholinesterase（AChE）is an important enzyme in the central nervous system,and its expression level is closely related to the development of Alzheimer’s disease.AChE can specifically catalyze the reaction of acetylcholine（ATC h）to produce thiocholine（TCh）.In this work,a fluorescent probe HBTP with ESIPT properties is constructed,which quenches fluorescence when it is coordinated with Cu²⁺.The generated TC h competes with C u²⁺in the HBTP-Cu²⁺complex,and destroys the structure of the complex,so that HBTP is released and fluorescence is restored.This“turn on”fluorescence assay shows high detection sensitivity.In addition,the cytotoxicity produced by this composite probe is low,and it shows good performance as a viable biosensor platform for imaging of cellular AChE activity.3.Butyrylcholinesterase（BChE）as another human cholinesterase,its abnormal concentration expression is not only related to various human diseases,such as liver injury,diabetes and Alzheimer’s disease,but also as organophosphate poisoning diagnostic and predictive indicators.In this experiment,a zero-background biosensor analysis method for detecting BC hE was developed using cyclopropylformyl ester as the recognition site of butyrylcholinesterase（BC hE）.This detection scheme has high selectivity and sensitivity for BChE.The detection scheme can also be used for imaging at the slice and biological level,and provide theoretical guidance for the research on the mechanism of mental il ness h.