The Structural Modification,fluoresc-Ence Detection,and Toxicological/Toxicity Analysis Of Microcystin-LR

Since the 19 th century,numerous outbreaks of cyanobacteria have occurred in many parts of the world.The death of aquatic organisms caused by cyanobacteria bloom has attracted considerable attention.Cyanobacteria could release toxins produced by their own metabolism into the water in the process of growth,reproduction and death decomposition.The most typical toxin in cyanobacteria bloom is microcystins,especially microcystin-LR(MC-LR).All human activities cannot leave water for a long time,which greatly increases the risk of MC-LR exposure and harm to humans.MCLR can enter the human body through drinking polluted water,food chain transmission or other ways.Therefore,fast,sensitive and accurate determination of MC-LR plays an important role in human and animal health.Fluorescent probes have been widely used in the field of detection.However,due to the particularity chemical structure of MCLR and it located in complex environment,the fluorescence detection of MC-LR has not been reported in the application of fluorescent probes.The functional modification of MC-LR has brought great progress to the research of MC-LR,but they rarely directly modify the fluorescent groups into the structure of MC-LR.We believe that the functionalized MC-LR connecting fluorescent groups will provide important help for the study of the distribution,migration and transformation of MC-LR in vivo.In order to develop fluorescent probes that can detect MC-LR,explore the fluorescent functional modification methods of MC-LR and deeply understand the toxicological/ toxicity of MC-LR,this study mainly includes the following four aspects:1.We designed and synthesized six fluorescent probes.MC-Yd TPA,MC-Yd TPE,MC-Rd TPA and MC-Rd TPE can react clearly respond to MC-LR in pure water.However,Yd Rd and Rd Md did not respond to MC-LR.The MC-LR standard recovery experiment was also tested in natural lake water samples by using three probes,but these probes cannot accurately recognize and respond to MC-LR from the complex water samples,which need further improved.Based on the summary of in vitro experiments,we explored the possible mechanism of the response between fluorescent probes and MC-LR.The probes may recognize the structure of MC-LR and the p H changes caused by MC-LR itself.These works laid a foundation for the development of fluorescent probes for specifically recognize MC-LR.2.We designed and carried out the experiment of MC-LR functionalized modification.We try to used NHS activated coumarin ester(NHS-CM)to modify the structure of MC-LR,and determined the formation of functionalized MC-LR(MC-LRCM)by mass spectrometry.This indicates that the modification of MC-LR from arginine is feasible.We believe that this exploration will make important technical accumulation for the subsequent functional transformation of MC-LR.3.Imaging experiments in MCF-7 cells were carried by using probes and MC-LR.The results showed that MC-Yd TPA,MC-Yd TPE and MC-Rd TPA could respond to MC-LR in cells,and the response effects of MC-Yd TPE and MC-Rd TPA were the most significant.Their fluorescence intensity increased significantly in MC-LR treated cells.These probes had the ability to imaging MC-LR in cells and even tissues.Furthermore,we imaged two raw materials(Rd and BJQ)for synthesis MC-Rd TPA with MC-LR in cells,while only BJQ showed weak response to MC-LR in cells,indicating that the polyphenyl ring structure may be the important basis for the MC-LR recognition.We believe that the probes based on the polyphenyl ring structure may be an important breakthrough for developing MC-LR probes.4.We developed fluorescent probe Bptp-MR to image cells and zebrafish incubated by MC-LR for further toxicological/toxicity analysis.It was found that in the presence of MC-LR,the weakly acidic region in the cell environment was no longer limited to the lysosome.This indicates that MC-LR can exert toxicology effects on lysosomal regions.By comparing the fluorescence images of zebrafish cultured with different concentrations of MC-LR,it was found that MC-LR could accumulate in the eyes of zebrafish,and the amount of accumulation might be proportional with the concentration of MC-LR.Therefore,more attention should be paid to the toxic effects of MC-LR on the eyes of animals in future studies.This research attempts to establish a fluorescence analysis method for MC-LR detection and provide a new effective tool for the toxicological/ toxicity study of MCLR.Hoping our research could bring some new ideas for the MC-LR study.