Study On Liver Organ Chip Technology Based On Electrochemical Method To Detect The Toxicity Of Environmental Pollutants

Environmental pollutants seriously threaten human health and cause a variety of diseases.Nowadays,the treatment technology of environmental pollutants is booming,and different environmental pollutants have corresponding treatment methods.However,due to the complex composition and incomplete degradation of environmental pollutants,many environmental pollutants produce highly toxic intermediates in the degradation process.After these substances enter the environment and enter the human body through food chain enrichment,they will lead to acute or chronic diseases,disrupt the normal physiological function of the human body,and even cause death.Therefore,rapid and effective evaluation of the toxicity of environmental pollutants to human body before and after degradation is helpful to improve the environmental treatment process and prevent the occurrence of public health events.In this study,semiconductor technology and biomedical technology are combined and applied to environmental pollution monitoring.This method can realize non-invasive,in-situ and continuous monitoring of the acute biological toxicity of environmental pollutants,and provides a new idea and method for the evaluation of the toxicity of environmental pollutants.The main research contents and results are summarized as follows:（1）In this study,a liver organ chip integrated with microelectrode layer was designed and fabricated.The chip consists of a microfluidic unit made of polymethylmethacrylate（PMMA）and a microarray electrode.The upper microfluidic unit contains a cell culture hole for culturing three-dimensional human liver cancer cell clusters to simulate human liver tissue;An interdigital microelectrode array is designed on the integrated microelectrode layer below.The array corresponds to the cell culture hole of the microfluidic unit.Combined with electrochemical impedance spectroscopy（EIS）,it can realize non-invasive,in-situ and continuous monitoring of three-dimensional human liver cancer cell clusters affected by environmental pollutants.At the same time,a polydimethylsiloxane（PDMS）liver organ chip without integrated microarray electrode was constructed,which was mainly used in the pre experiment of three-dimensional human liver cancer cell cluster culture.The chip is mainly used to verify the feasibility of cell clustering method combined with confinement method and matrix glue method,and explore the growth cycle and other parameters of three-dimensional human liver cancer cell cluster culture.（2）Through the culture of three-dimensional human hepatoma cell clusters in PDMS liver organ chip,it is found that the cell clusters can achieve preliminary aggregation and growth in 24 h under the action of cell culture pore confinement and Matrigel matrix glue.Then,the three-dimensional human hepatoma cell clusters have been basically formed when cultured to 48 h.After cultured to 72 h,the cell clusters are in good condition,and their shape and size have been stable,with little change compared with 48 h.The combination of human liver cancer cells into three-dimensional stable matrix can be proved by the method of limiting the growth of human liver cancer cells into three-dimensional matrix.At the same time,three-dimensional human hepatoma cell clusters were cultured on the liver organ chip integrated with microelectrode layer.The results showed that the microelectrode layer had no effect on cell clustering and growth,and the cell clustering cycle and growth cycle were basically consistent with the results of PDMS liver organ chip.（3）The biological toxicity of typical azo dyes before and after biological treatment was further studied.Three samples of typical azo dye Reactive Black 5（RB5）dye stock solution,RB5supernatant（RB5 + YE + FRU）after 48 hours of ddmz1 flora and ddmz1 flora culture medium（YE + FRU）were selected for biological toxicity evaluation through liver organ chip.During the study,the optimal detection frequency of the chip was determined to be 10 k Hz by the difference△CI between the CI values of the cell group and the cell-free group.According to the biological toxicity test results of three samples in the chip,RB5 sample is less toxic,and has little effect on the activity of three-dimensional human hepatoma cell clusters;RB5 + YE + FRU samples were the most toxic;YE + FRU samples are non-toxic to cells.That is,the biological toxicity of RB5 azo dye itself is small,but its biological toxicity increases after microbial action,which may be due to the incomplete degradation of the dye and the production of some toxic intermediates.（4）In this study,the above conclusions were verified by immunofluorescence staining and CCK-8 cell activity detection kit.The results of immunofluorescence staining showed the effects of RB5,RB5 + YE + FRU and YE + FRU on the expression of ZO-1 and ALB in three-dimensional human hepatoma cell cluster.The expression of ZO-1 and ALB was related to the activity of hepatocytes.The results further confirmed the results of liver organ chip combined with EIS to evaluate the toxicity of RB5 in simulated dye waste water before and after treatment by DDMZ1 flora.It proves that the liver organ chip designed in this study combined with EIS method is feasible to evaluate the toxicity of typical azo dye RB5.In addition,after normalizing the cell index △CI again by statistical method,the change trend diagram of cell activity transformed by impedance data is obtained.The results are highly consistent with the experimental results of CCK-8.However,the three-dimensional human hepatoma cell cluster has been added to the sample for a longer time.The results further show that the three-dimensional human hepatoma cell cluster is more resistant to toxic substances than flat cells,so its toxicity evaluation results will be closer to the real situation of human body.