Development On Cell Sensing Detection Methods Based On T-2 Toxin Target Toxicity In Food

Food safety is fundamental to protect the people's lives,however,mycotoxins are widely contaminated in food and cause great harm to the human body through the food chain.T-2 toxin,as one of the mycotoxins,causes drastic toxic effects in the body.Traditional cell and animal model toxicity assays suffer from the disadvantages of long cycle length,insensitivity,and inability to reflect cellular heterogeneity.To develop efficient,real-time,and intuitive T-2 toxin toxicity assays,in this paper,T-2 toxin was taken as the main research object,and cells sensitive to oxidative stress were taken as biorecognition elements.Based on the target genes and signal pathways derived from cell transcriptomics,surrounding the cytotoxicity studies of T-2 toxins in foods,a fluorescent cell sensing model and a single-cell electrochemical sensing model were established from multiple perspectives.A summary of our work is as follows:1.Development of cytotoxicity detection at the cellular and ion level and screening sensitive cell lines based on the main toxin target cells of T-2 toxin.For cytotoxic studies of T-2 toxins,mouse microglioma cells BV2,human hepatoma cells HepG2,mouse Leydig cells TM3,and human colon carcinoma cells HT-29 were analyzed for their proliferative activity,intracellular reactive oxygen species,calcium ion levels,and apoptosis according to the target organ of T-2 toxin.The IC₅₀ values of four cells were BV2<HepG2<TM3<HT-29.The results showed that T-2 toxin could significantly increase the concentration of reactive oxygen species and the level of calcium ion in cells,leading to apoptosis.BV2 cells and HepG2 cells that are more sensitive to T-2 toxin were screened for analysis.2.Analysis of key cytotoxic target genes and signaling pathways of T-2 toxin based on cellular transcriptomics.GO and KEGG enrichment analysis of differentially expressed genes were performed by transcriptomic studies of BV2 cells,HepG2 cells before and after T-2 toxin exposure.In BV2 cells,factors such as AP-1 and IL-6 involved in TNF signal pathway and MAPK signal pathway were upregulated,involved in cellular inflammatory response.In HepG2 cells,the PI3K/Akt signal pathway and PERK signal pathway activate Akt and SIRT1signaling factors,which in turn promote the expression of the pro apoptotic factors.And SOD is significantly upregulated.Multiple pathways trigger oxidative stress to cause apoptosis.RT-PCR verified that AP-1,IL-6,Bax,SOD1,and other factors were significantly upregulated,and the results were consistent with the transcriptomic data.3.Development of T-2 toxin toxicity detection technology based on the key signal pathway HEK293 cell fluorescent sensor.To achieve toxicity detection intuitivity and fluorescence response specificity,based on the up-regulation of AP-1 gene in the transcriptome results,a signal cascade sequence of AP-1 site-m Cherry was constructed,HEK293 cells were used as a marker for transfection and a fluorescence cell sensing model was established.The dose effect relationship induced by T-2 toxin in the fluorescent cell model was obtained by constructing pc DNA3.1-TRE-m Cherry fluorescent plasmid.The concentration of T-2 toxin is linearly related to the fluorescence intensity in the range of 1 ng/mL to 25 ng/mL,R²=0.969,the EC₅₀ was 16.27 ng/mL,and LOD is 0.691 ng/mL.The average spike recovery rate of real sample spiking experiment was 86.13%?126.20%.The toxicity detection of HT-2,the major metabolite of T-2 toxin,gave an EC₅₀ of 27.65 ng/mL.4.Development of T-2 toxin toxicity evaluation model based on single-cell electrochemical sensor.In order to further reduce the toxicity detection limit and realize cell heterogeneity monitoring,based on the cellular oxidative stress caused by the up-regulation of the SIRT1 and SOD in the transcriptome results,a liver cancer cell line HepG2 was established to construct a single-cell electrochemical sensing model.The working electrode is gold-plated nanoparticles and Prussian blue nano-probes.The concentration of H₂O₂ produced by stimulating HepG2 cells in the concentration range of 1?1000 ng/mL for T-2 toxin showed a linear correlation,R²=0.991,and the LOD was 0.138 ng/mL.The average spike recovery rate of real sample spiking experiment was 81.19%?130.17%.Comparing the current difference between the growth of a single cell and that of a single cell in a cell population,the peak current of the former is significantly higher than that of the latter.HepG2 cells were exposed to four toxins,T-2,AFB₁,DON and ZEN,respectively.Compared with the control group,the peak currents of T-2 toxin and AFB₁ toxin were significantly different.In conclusion,we investigated the mechanism by which T-2 toxins cause inflammation and oxidative stress in cells leading to apoptosis and found that key target factors,AP-1,SIRT1,and SOD were upregulated.Herein,two methods for sensing and detecting toxicity cells are established,which will provide new methods and ideas for realizing the efficient and intuitive toxicity primary screening of T-2 toxins under nano environment.