Purification Of Phycocyanin From Spirulina,Development Of Fluorescent Probes And Its Application In Rapid Detection

Phycocyanin is a natural pigment protein,and has high theoretical research value and development and utilization value,phycocyanin can be used as a natural blue pigment in food,health care products,cosmetics and other fields,and it has anti-oxidation,anti-inflammatory,anti-tumor and enhance Immunity and other physiological functions,which make it widely used in medicine and health care.At the same time,phycocyanin can be used as fluorescent probes and photosensitizers in medical diagnosis,immunology,bioengineering and other research fields because of its strong fluorescence properties.In this article,the extraction,separation,purification and stability of phycocyanin were studied,which used spirulina as raw material,and a preliminary exploration was made on the application of phycocyanin fluorescent probes in the immunochromatography rapid test strip.Phycocyanin was extracted by oscillametric method,and the extraction conditions of phycocyanin was optimized by single factor experiment and response surface method.The optimization result were:extraction temperature 32°C,extraction time 2.38 h,and ratio of liquid to solid 1:32.7(g/mL),and then the extraction rate reached 76.01%.The separation and purification of phycocyanin mainly includes four steps:membrane separation,two-step ammonium sulfate salting-out,DEAE-Sepharose FF ion exchange chromatography and Sephadex G-150 gel chromatography.After membrane separation,the purity of phycocyanin crude extract increased from 0.42 to 2.75,reached food grade(A620nm/A280nm>0.7),and the recovery rate was 91.88%.When using the two-step ammonium sulfate salting out for further purification,the purity can be increased to 3.65,reaching the pharmaceutical grade(A620nm/A280nm>3.0),and the recovery rate is 74.79%.After purification by ion exchange chromatography and Sephadex G-150 gel chromatography,the purity of phycocyanin was 5.04,which could reach the reagent level(A620nm/A280nm>4.0),and the recovery rate was 35.50%.Analysis by SDS-PAGE showed that phycocyanin obtained from Spirulina contained a and ? subunits,with molecular weights of 16.6 and 21.2 kDa,respectively.At the same time,using high performance size exclusion chromatography(HPSEC)to analyze the molecular weight of phycocyanin,which showed that the phycocyanin of Spirulina is mainly existed in the form of(??)3 with a molecular weight of approximately 91 kDa at a pH value of 7.0,accounting for 95.46%.The stability of phycocyanin shows that the stability of phycocyanin is favored under the condition of low temperature below 30?,neutral pH and avoiding ultraviolet light.Based on the fluorescence characteristics of phycocyanin,a phycocyanin fluorescent probe was prepared and used to establish a rapid detection method for aflatoxin B1 with competitive immunochromatography technology.The results showed that the sensitivity of the aflatoxin B1 immunochromatographic rapid test strip was 20 ng/mL.The method is simple and quick,suitable for rapid detection of large quantities of samples on site.