A Method For Detecting Heavy Metals Based On Phycocyanin As Fluorescent Probe And Its Application

As one of the phycobiliproteins,C-phycocyanin is widespread in marine algae.C-phycocyanin is a natural fluorescent agent and its isolation and purification methods are simple,low cost and easy to obtain.Based on the fluorescence characteristics of C-phycocyanin,it has been developed as an efficient fluorescent probe,which is widely used in medical diagnosis,clinical medicine,environmental monitoring and other fields.With the development of our country's industrialization,heavy metal pollution in seafood has become increasingly prominent,and poses a threat to human health.Therefore,the detection of heavy metal ions in marine products is urgent.The traditional detection methods involve precise instruments and complicated operation procedures which limited their application.It is not enough to meet the needs of rapid on-site detection of heavy metals.Therefore,it is necessary to build a simple,economical and rapid detection technique of heavy metals on the spot.Based on the C-phycocyanin's fluorescence properties,this paper investigated the application of rapid detection of heavy metals and the main contents were as follows:C-phycocyanin from Spirulina maxima was separated by cell disruption,salting out,dialysis and chromatography.The cells were broken by repeated freezing and thawing combined with ultrasonic and the purity(A620/A280)of C-phycocyanin was 0.7 after salting out with 70 % saturated ammonium sulfate.Then the purity of C-phycocyanin reached 4.1 after column chromatography of hydroxy apatite.The UV-Vis absorption spectra of C-phycocyanin showed that the maximum absorption peak at 620 nm with two absorption peaks at 280 nm and 360 nm;SDS-PAGE results illustrated that the molecular weights of C-phycocyanin's two subunits were 19.0 kDa and 21.0 kDa,respectively;the fluorescence spectrum of C-phycocyanin demonstrated that there was an excitation peak between 580~600 nm and the emission peak at 650 nm.These results provided a theoretical basis for the study of C-phycocyanin's application in the next stage.The fluorescence response of C-phycocyanin to 15 kinds heavy metal ions was investigated and it was found that C-phycocyanin could respond selectively and specifically to mercury ions.The mechanism of quenching was studied by fluorescence microscope and it was discovered that the addition of mercury ions could cause the C-phycocyanin's aggregation,which may be the reason of mercury ions caused C-phycocyanin's fluorescence quenching.After optimization,the optimum conditions for the detection of mercury ion by C-phycocyanin fluorescence probe were established: 0.05 mg/m L C-phycocyanin at 30 ? for reaction time10 min in pH 7.0.According to the fluorescence quenching effect between different concentrations of mercury ions and C-phycocyanin,the standard curve was set up and the limit of detection(LOD)was 0.008 ?M.Additionally,the method was successfully applied to detect mercury ions in oysters and Pampus argenteus samples,showing the appropriate rates of recovery between 93.6 % and 107.2 %In this study,C-phycocyanin as protective agent,AgNO3 as raw material and NaBH4 as reducing agent synthesized phycocyanin-Ag nanoparticles(PC-AgNPs).The synthesis conditions of PC-AgNPs were determined by optimization: 0.20 mg/mL C-phycocyanin was added to 0.35 mM AgNO3 and 0.40 mg/m L NaBH4 at pH 7,with a temperature of 30 ? and stirring for 2 hours.The maximum UV absorption peak of PC-AgNPs at 400 nm.The fluorescence excitation wavelength was 580 nm and the emission wavelength was 625 nm.PC-AgNPs was spherical in transmission electron microscope and the particles sizes were about 10~25 nm.In addition,fluorescence quenching was observed after adding copper ions to PC-AgNPs.In this research,a high purity C-phycocyanin was obtained through a series of extraction and purification processes.Based on its fluorescence characteristics,a rapid detection technique for mercury ions was developed.C-phycocyanin fluorescent probe has the advantages of short detection time and high sensitivity.Moreover,it is pure natural,not contaminated samples and has huge potential applications for detecting mercury ions in seafood.Using C-phycocyanin as a protective agent to synthesize PC-AgNPs,and copper ions had obvious quenching effect on the fluorescence of PC-AgNPs,which indicated that PC-AgNPs has potential applications in the detection of copper ions.