Toxicity Assessment Of Deoxynivalenol Based On Cell Imaging Techniques Employing Fluorescent Nanoprobe

Deoxynivalenol(DON),known as vomitoxin,is a metabolite produced by the pathogen Fusarium graminearum of wheat scab.It can contaminate the main agricultural crops including wheat,barley,oats,rye and corn,as well as processed foods such as noodles and beer.The DON with high incidence,stability,and multiple toxicities posed a great threat to human and animal health while passing through the food chain.Therefore,it is of great significance to study the toxic mechanism and to evaluate the dose-effect relationship between DON and toxicity via monitoring the biomarkers changes under DON stimulation in cytotoxicity experiments.Fluorescence imaging technology enables the in situ and real-time visualization of biomarker in living cells with its high sensitivity,low cost,and low radioactivity.In this work,two novel fluorescent nanoprobes were prepared to evaluate the cytotoxicity through cell imaging techniques by monitoring the biomarkers,which were induced by the activation of mitochondrial signal transduction pathways regulated by DON-induced apoptosis.The approach provides references for its toxicity mechanism and the real time monitor of the response between DON and DON-induced apoptosis.The specific research contents of this study are as follows:1.Real-time and in situ monitoring of DON-induced cell apoptosis using a fluorescent molecularly imprinted probe.A molecularly imprinted polymer(Cd Te@MIP)with high selectivity of MIP recognition and high sensitivity of fluorescence detection was prepared.The molecularly imprinted polymers and Cd Te quantum dots were regarded as a recognition element and a sensing element,respectively.The fluorescence of Cd Te@MIP was quenched when it reacted with Cyt c due to the photoinduced electron transfer.A good linear relationship between the fluorescence intensity and Cyt c concentration was obtained with the linear equation of y=0.07165x+0.8923,R²=0.9897.With the increase of DON concentration(0-10?M)and incubation time(0-7.5 h),intracellular Cyt c induced by DON was gradually increased.The results of laser confocal microscopy showed that the fluorescence intensity was gradually quenched after the specific binding of Cd Te@MIP and gradually increased Cyt c,successfully achieving the real-time monitoring of intracellular Cyt c.The results were consistent with the conventional methods of CCK and flow cytometry,proving that the cell imaging technology based on Cd Te@MIP probe was consistent with the prior toxicological results.Thus,it can pave a new avenue to provide the guidance for the establishment of an early warning system of food mycotoxins.2.Simultaneous imaging of DON and Cyt c was carried out by employing the dual recognition of ZIF-8 base-aptamer probe.A ZIF-8 base-aptamer probe was prepared to realize the dual recognition of DON and Cyt c in living cells.The aptamer and organic fluorescent dye were regarded as a recognition element and a sensing element,respectively.The probe combined the specificity of aptamer and the ability of ZIF-8-based porous carbon to load a large number of fluorescent dyes.Rhodamine 6G(Rh6G)and methylene blue(MB)were loaded into the pores of ZIF-8(ZIF-NC)carbonized in N₂ atmosphere at 700°C,and the pores were gated with Cyt c and DON aptamer,resulting in the fluorescence quenching.In the presence of Cyt c and DON,the aptamer was released from the porous surface due to the specific binding with the corresponding targets.At the same time,the gate was opened to release Rh6G and MB,and the fluorescence intensity of the corresponding organic dyes was recovered.A good linear relationship between the fluorescence intensity and the concentration of Cyt c and DON was obtained.The linear equation of ZIF-NC@MB@D-Apt fluorescence intensity and DON concentration was y=74.22lg C+219.97,R²=0.9987,and the linear equation of ZIF-NC@Rh6G@C-Apt fluorescence intensity and Cyt c concentration was y=58.41lg C+155.75,R²=0.9957.The imaging results of the confocal laser microscope showed that two probes loaded into the cells respectively reacted with DON and Cyt c induced by DON under the induction of DON(10?M).The fluorescence of ZIF-NC@MB@D-Apt and ZIF-NC@Rh6G@C-Apt reached its stability within 6 h,respectively,successfully monitoring the exposure of DON and the biological process of the Cyt c discharge triggered by the activation of the DON-induced apoptosis pathway.Besides,it explained the temporal and spatial characteristics and the response between DON and Cyt c during the apoptosis process,which is of great significance for the comprehensive and systematic development of the cytotoxicity of mycotoxins.