| Enzymes are a very critical type of biocatalyst that is essential for the normal development and metabolism of organisms.Many biochemical reactions that require harsher reaction conditions are catalyzed by enzymes and only require a very mild environment in the body to achieve high efficiency.And it happens specifically.The detection of enzyme activity is of great significance in modern medicine,physiology and other disciplines.At present,there have been many reports on methods for enzyme activity detection.The fluorescence sensor detection method is simple and convenient,fast response,high sensitivity and high choice.Many advantages such as sex and realtime imaging stand out among various detection methods,and have been widely used in a series of scientific research fields such as immunoassay,biochemical sensing,medical diagnostic imaging,food environment detection,etc.Therefore,how to design and synthesize a new type of enzyme activity fluorescent sensor that is more concise,convenient,low-loss,large-scale use,accurate and quicker,and has multiple effects is a subject of extremely important significance in scientific research today!This paper starts from the perspective of detecting the activity of multiple types of hydrolases,referring to the design and detection ideas of various fluorescent sensors reported in the past,designing and optimizing the experimental program for the detection of enzyme activity,using different luminophores and The detection mechanism has been designed and prepared with a specific organic small molecule fluorescence sensor corresponding to each enzyme.The fluorescence sensors obtained in this article have excellent specificity and detection performance,and are simple to prepare and easy to use,and can perform detection and imaging operations in cells and living bodies.This article is mainly divided into the following four parts:1.The mitochondrial-targeted butyrylcholinesterase(BChEfluorescent chemical sensor(BCh E-NBD)was synthesized and the imaging study was carried out in mice for the first time.BCh E-NBD has a long absorption(580 nm)emission(628 nm)wavelength,and is very specific in detection without interference from other analytes(especially acetylcholinesterase),and has very low biological toxicity.BCh E-NBD performed well in the detection and imaging experiments of mitochondrial BCh E in various types of cells.Further,the distribution of BCh E in mice was imaged and confirmed the widespread existence of the enzyme in liver tissue.2.Designed the first activity-based ratio fluorescence sensor DCDF for detecting neutrophil elastase(NE).DCDF chose to use pentafluoropropionic acid anhydride as the recognition group.After NE was added,the emission wavelength showed a significant spectral red shift and its Stokes shift was almost 60 nm.DCDF has a strong selectivity and a very sensitive reaction with the analyte,and it can perform endogenous recognition imaging in A549 and He La cells without interference from other enzymes.3.Designed and synthesized the first ratio fluorescence sensor DP-1 that specifically detects pyroglutamate aminopeptidase-1(PGP-1)in vivo and in vitro with a long emission wavelength.DP-1 selects DCD-NH2 to act as a luminescent group and uses pyroglutamate as a recognition group.It interacts with PGP-1 to hydrolyze and break the amide bond,and the UV-visible absorption peak of the solution shifts from420 nm to 520 nm.At the same time,it is accompanied by a significant red shift of the maximum emission peak of the fluorescence spectrum from about 564 nm to about 616 nm.In-depth analysis of the imaging conditions in Hep G2 and RAW264 cells in the presence of inflammation-inducing drugs and studying the imaging of mouse tumor models showed for the first time that the expression of PGP-1 is inseparable from inflammation and certain tumor diseases.Associated.4.Designed a near-infrared ratiometric fluorescence sensor TP-1 for the specific detection of pyroglutamate aminopeptidase-1.The sensor has a Stokes shift of about164 nm and a very low detection limit of PGP-1 of 0.14 ng/m L.After TP-1 reacts with the enzyme,the emission spectrum has a red shift of about 80 nm and the color of the solution changes from yellow to red.TP-1 also studied the imaging of Hep G2 and RAW264.7 cells and the imaging of mice and organs under the stimulation of immune enhancers.Combined with the previous work,it further confirmed the relationship between PGP-1 and inflammation and liver diseases in organisms.close.In addition,this work reveals for the first time that the expression of PGP-1 in tumor model mice far exceeds that of mice under normal conditions.The exploration of fluorescent sensors based on enzyme activity is still a hot direction in scientific research.Through the research experiments on these enzyme sensors,this article provides new detection methods and research ideas for chemistry,biology,medicine,and clinics.Laid the foundation for further research in the future. |
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