Detection And Imaging Analysis Of Biological Enzymes By Organic Small Molecular Fluorecent Probes

Butyrylcholinesterase（BCh E）is an important member of human cholinesterase,widely distributed in plasma,liver,muscle and brain tissues.It is an esterase synthesized by the liver.Serum BCh E levels are typically lower in patients with liver diseases,including chronic liver disease,acute hepatitis,cirrhosis,and liver cancer.In this thesis,three simple and fast-reacting fluorescent probes were provided to detect butyrylcholinesterase activity,respectively.The specific research contents are as follows:（1）Butyrylcholinesterase（BCh E）is an esterase expressed by the liver and is immediately released into the plasma.The activity of BCh E in serum is a sensitive indicator for judging the protein synthesis function of hepatocytes.Based on the ingenious combination of specific BCh E enzymatic reactions,this work establishes a ratiometric fluorescent probe that can easily achieve high-sensitivity determination of BCh E with an LOD calculation as low as 0.077 μg/m L.The probe also exhibits excellent selectivity for BCh E without interference from other biological species or even acetylcholinesterase（ACh E）,which makes it a great potential for sensing in biological samples.Also detects the accurate IC50（0.195 m M）of iso-OMPA,a specific inhibitor of BCh E.This assay can also be used for imaging at the cellular and slice levels.（2）In this experiment,cyclopropylformyl ester was used as the recognition site of butyrylcholinesterase（BCh E）,and heptamethine was used as the fluorophore.propyl to prepare probe W2.The developed organic small molecule probe is a near-infrared fluorescent probe for the detection of BCh E activity,and this detection scheme has a relatively low limit of detection（LOD）of 0.0025 μg/m L and good selectivity.When the probe encounters BCh E,spontaneous substitution occurs and the cyclopropyl group reacts to form a hydroxyl group,resulting in quenching of the probe’s green fluorescence around 798 nm.This detection scheme can also be used for imaging at the slice and organism level.（3）A near-infrared ratiometric fluorescent probe Cy-O-BCh E based on heptacyanine was designed and synthesized for the detection of butyrylcholinesterase（BCh E）activity.When BCh E acts on the probe Cy-O-BCh E,the cyclopropyl formate on the probe is recognized,accompanied by the weakening of the near-infrared emission at 816 nm and the emergence of new red fluorescence at 637 nm,establishing the ratio fluorescent probes.The fluorescent probe has a good signal-to-noise ratio and high sensitivity to BCh E activity.In addition,the probe has been successfully applied to the imaging of living cells,and the results show that the probe Cy-O-BCh E performs well at the cellular level.