The Fluorescence Analytical Methods Used In Determining·OH And O₂·^-through Coumarin-7-Boronate Fluorescent Probe

Objective:This paper aims to establish a new fluorescence analytical method for probing the content of·OH,O2· by using7-boronic acid pinacol cyclic ester coumarin as fluorescent probe.Methods:The single factor analytical assay and the response surface method (RSM) were employed to optimize the detecting condition (pH, temperature, and time) regarding the measuring of·OH and O2· by using7-boronic acid pinacol cyclic ester coumarin as the flurescence probe. Under the optimal conditions, the linear relation between7-boronic acid pinacol cyclic ester coumarin and produced·OH by the Fenton system, as well as the relation between7-boronic acid pinacol cyclic ester coumarin and produced O2· by disoving KO2in dimethyl sulfoxide. The accurancy, sensitivity, the reproducibility, and the recovery rate of the new method were determined. Finally, the feasibility was validated by determining the antioxidation ability of several kinds of fungal polysaccharide and some purple fruits and vegetables.Results:The single factor experiment and the response surface optimization experiment results showed that the optimum experiment conditions for probing·OH by7-boronic acid pinacol cyclic ester coumarin were pH7.5carbonate buffer solution,35℃,2.25hours response time,330nm maximum wavelength of exciting light,453nm maximum wavelength of fluorescence. The precision experiment RSD was less than2.41%; the recoveries ranged from96.35%to101.32%, RSD was less than1.25%, the reproducibility experiment RSD was less than1.02%. Compared with the spectrophotometry, this method had a good linear relationship in10-80μmol/L, R2=0.9921.The single factor experiment and the response surface optimization experiment results showed that the optimum experiment conditions for probing O2· by7-boronic acid pinacol cyclic ester coumarin were pH7.0phosphate buffer solution,40℃,3hours response time,330nm maximum wavelength of exciting light,453nm maximum wavelength of fluorescence. The precision experiment RSD was less than2.22%; the recoveries ranged from94.50%to99.50%, RSD was less than1.80%; reproducibility experiment RSD was less than1.64%. Compared with the spectrophotometry, this method had a good linear relationship in5-45μmol/L, R2=0.9892.The new method has advantages compared with spectrophotometry, it could detect the clearance rate of·OH and O2· at low concertration, and could overcome the interference of back-ground noisy singals which often existed in the spectropho-tometry analysis.Conclusion:The study optimized the detecting condition for the reaction between7-boronic acid pinacol cyclic ester coumarin and·OH and O2· and estabolished a new fluorescence analytical method. The method has advantages in sensitivity, accuracy, stability, and handy. It could determine the clearance rate of·OH and O2· by antioxidant at low concertration and could overcome the interference of colored matters in spectrophotometry.