The Design,Synthesis And Application Of Fluorescent Probes For NADH

Reduced nicotinamide adenine dinucleotide?NADH?and its phosphate NADPH play important roles as coenzymes in many biocatalytic processes.So far,many efforts have been made to detect the level of NAD?P?H in organisms to understand the role of NAD?P?H in biological systems.Among many biological detection techniques,fluorescence has attracted much attention in environmental and biological imaging applications,because its advantages including simple operation,easy visualization,excellent sensitivity and selectivity,non-invasive and spatial resolution.Moreover the sensitivity,selectivity and dynamic range for the ratio fluorescence probe are greatly improved,because it is not affected by the intensity of the light source and the sensitivity of the instrument.In brief,current design strategies are mainly based on the redox reaction between NAD?P?H and the probe,accompanied by changes of fluorescence intensity or wavelength of the probe.Therefore,based on these strategies,this paper firstly reported a ratiometric oganic small molecular fluorescent probe for detecting NAD?P?H,and the probe was successfully applied to the ratio fluorescence imaging.The definite study contents of this paper are as follows:Firstly,we designed a novel ratio fluorescent probe?Cou-In?based on coumarin and hemicyanine dyes,according to the strong reducibility of NAD?P?H,which can selective nucleophilic addition to a specific hemicyanine derivatives.Then we performed a series of spectral performance tests for the probe.We found a good linear relationship between the change in the ratio of the fluorescence intensity of the probe and the concentration of NADH:I₅₅₀/I₆₇₅=0.22027×[NADH]+1.24914,the linear correlation coefficient was 0.9984,and the detection limit was 60 nM.The results showed that the probe has the advantages of good selectivity and high sensitivity.Secondly,to explore the probe biological value,we combined the fluorescence confocal microscopy imaging technique to successfully apply the probe Cou-In to the visual ratio fluorescence imaging of NAD?P?H in living cells.At the same time,we found that other reductive substances,metal ions,etc,present in living cells did not interfere with the detection of NAD?P?H.Finally,in order to investigate whether the probe can be used to detect the reaction of the enzyme,we choose alcohol dehydrogenase as the analyte,and the experimental results showed that the probe can monitor enzyme reaction in real time.