Construction Of Quantum Dots-Laccase Biosensor And Its Application In Polyphenol Detection

Many phenolic compounds are closely related to people's lives.Take dopamine as an example,as a very important neurotransmitter in human body,its content is directly related to many diseases,such as Parkinson's disease.Therefore,it is very necessary for the detection of dopamine content.Traditional phenolic compounds are usually detected by UV-vis method or HPLC method.These methods usually have the disadvantages of high detection limit,time-consuming and laborious.Compared with traditional detection methods,quantum dot-based fluorescence sensors have the advantages of high sensitivity,low detection limit,and relatively simple operation.In this study,a kind of polyphenol oxidase laccase and cadmium sulfide quantum dots were used to prepare the fluorescence sensor.The phenol compound dopamine was used as the detection material,and a good detection effect was achieved,ie lower detection limit and good recovery.The proposed fluorescence sensor achieved a convenient and rapid detection of low concentrations of dopamine.In this study,a method for detecting a phenolic compound dopamine was developed.Using a polyphenol oxidase laccase and a biosynthesized cadmium sulfide quantum dot,a fluorescent sensor capable of detecting dopamine concentration was prepared.Compared to the traditional ultraviolet method and high-performance liquid chromatography detection method,this method has the advantages of lower detection limit,more time-saving and convenient.The main conclusions are as follows:(1)Using the white rot fungus Trametes versicolor 50001,the maximum enzyme activity was of 1071±21 U/L after 9 days of fermentation with 1 mM copper ion as the inducer in the liquid fermentation medium.The fermented crude enzyme solution was fractionated by 30%-80%ammonium sulfate precipitation,dialyzed,and purified by DEAE-Sepharose FF column chromatography and purified 31-fold,and the specific activity reached 165.5 U/mg.The good result was obtained by SDS-PAGE.Pure laccase has a molecular weight of about 60 kDa.The purified laccase was further characterized by conventional enzymatic properties.The results showed that the optimum pH was 2and the optimum temperature was 35°C.The enzyme activity was stable under normal temperature and acidic conditions.(2)Using the property of white-rot fungus Trametes versicolor 50001 to adsorb heavy metal and self-assembled QDs,first,the Trametes versicolor 50001 were cultured in Kirk medium to form mycelium spheres.Then 60 mg of cadmium nitrate,5mL of 0.07 mg/L thioacetamide and 0.5 mL of thioglycolic acid were added.Then the pH of the Kirk medium was adjusted to 10,and the fluorescent nanomaterial CdS QDs were obtained after 24 hours of reaction.Then using scanning electron microscopy(SEM),ultraviolet-visible(UV-vis)and photoluminescence(PL)spectroscopy,Fourier transform infrared spectroscopy(FTIR),thermogravimetry(TG),transmission electron microscopy(TEM)X-ray diffraction(XRD)confirmed the surface morphology,optical and thermal properties,crystal properties,size and shape distribution of nanoparticles.(3)The sensor system was constructedas described using the laccases and quantum dots obtained.The calibration curve of P0/P vs.[DA]was made in the range of 7-100?M,and the correlation coefficient(R~2)was 0.9904.The limit of detection(LOD)was 7?M.The dopamine of known concentration was recovered and the standard deviation of recovery was within 6%.Laccase-QDs biosensors for the detection of dopamine were successfully constructed.