Establishment Of Colorimetric And Fluorescent Methods Based On Copper Nanoclusters For Evaluation Of The Freshness Of Aquatic Products

Aquatic products are prone to spoilage because of their fragile tissue and high soluble protein content.Freshness has an important influence on the sales and storage of aquatic products and the further processing of raw materials of aquatic products.It is of great significance to study the fast and sensitive detection method for freshness to ensure the safety of aquatic products.Hypoxanthine（Hx）is a major product of the decomposition of adenosine triphosphate（ATP）after the death of aquatic products.Its content plays an important role in reflecting the initial freshness of aquatic products.In this thesis,cysteine（Cys）is used as a template to synthesize copper nanoclusters（Cys-Cu NCs）.Cys-Cu NCs owns peroxidase-like activity to catalyze H₂O₂ to oxidize 3,3’,5,5’-Tetramethylbenzidine（TMB）to produce chromogenic reaction,as well as the fluorescence suppression effect by H₂O₂.Combined with the reaction of Hx to produce H₂O₂ under the catalysis of Xanthine oxidase（XOD）,the colorimetric method and fluorescence analysis method were established to detect Hx.The method was successfully applied to the determination of Hx content in aquatic products,and the freshness evaluation of aquatic products was realized by combining with total volatile basalt nitrogen（TVB-N）.The thesis is divided into three parts:1.The properties,synthesis and applications of Cu NCs in sensing,bioimaging and bacteriostatic applications are briefly introduced.After that,the research progress of the freshness of aquatic product and Hx detection was briefly introduced.2.A colorimetric method based on Cys-Cu NCs was established to detect Hx.Cys-Cu NCs was synthesized using Cys as a template at room temperature,and the morphology,element composition and surface functional groups of Cys-Cu NCs were characterized.Experimental results indicated that Cys-Cu NCs own good peroxidase-like activity.They can catalyze the oxidation of TMB by H₂O₂ and induce the reaction system from colorless to blue.Combined with XOD to catalyze the reaction of Hx to produce H₂O₂,a colorimetric analysis for Hx was established.Under the optimal conditions,the Hx concentration within 4-100μmol/L has a good linear relationship with the absorbance value of the mimic enzyme system at 652 nm,and the detection limit is 1.2μmol/L.This method is used to determine the Hx content in grass carp,and combined with TVB-N results to realize the judgment of the freshness of grass carp.3.A fluorescence method was established based on Cys-Cu NCs for the detection of Hx.The optical properties of Cys-Cu NCs were characterized,and the results showed that the maximum excitation wavelength of Cys-Cu NCs is 370 nm and the maximum emission wavelength is 495 nm.Experimental results have found that H₂O₂ has an obvious fluorescence quenching effect on Cys-Cu NCs.Based on this effect and the reaction of Hx to generate H₂O₂under XOD catalysis,a fluorescence method based on Cys-Cu NCs was established to detect Hx.Under the optimal conditions,the Hx concentration in the range of 8-400μmol/L has a good linear relationship with the fluorescence quenching efficiency,and the detection limit is 0.7μmol/L.Finally,the as-established method was successfully applied to the determination of Hx content in carp,combined with TVB-N results,the proposed method was successfully in the evaluation of carp freshness.