▎ 摘　　要

NOVELTY - Method for evaluating pesticide immunotoxicity based on three-dimensional cell electrochemical sensor involves (a) preparing ferric meso-tetra(4-carboxyphenyl)porphine chloride-reduced graphene oxide, (b) preparing gelatin methacryloyl (GelMA) precursor, (c) constructing three-dimensional cellular electrochemical sensor, (d) studying nitric oxide detection ability of the three-dimensional cellular electrochemical sensor obtained in step (c) under lipopolysaccharide stimulation and (e) detecting pesticide immunotoxicity. USE - The method is useful for evaluating pesticide immunotoxicity based on three-dimensional cell electrochemical sensor. ADVANTAGE - The method prepares electrochemical cell sensor, which has rapid action, is sensitive and reliable, provides three-dimensional culture of cells which is closer to the physiological conditions in vivo. DETAILED DESCRIPTION - Method for evaluating pesticide immunotoxicity based on three-dimensional cell electrochemical sensor involves (a) self-assembling ferric meso-tetra(4-carboxyphenyl)porphine chloride (FeTCP) onto graphene oxide through hydrophobic interaction and pi-pi stacking, reducing using hydrazine hydrate, washing with ultrapure water, and then dispersing in ultrapure water to obtain FeTCP-reduced graphene oxide (RGO) solution, (b) using methacrylic anhydride to react with gelatin, filtering the product, performing dialysis and then freeze-drying to obtain gelatin methacryloyl (GelMA) precursor, (c) taking a cell culture tank with a hollow structure, aligning the hollow portion of the cell culture tank with the working electrode area of the screen-printed carbon electrode, fixing on its surface for cell culture, then taking the FeTCP-RGO solution obtained in step (a), dripping on the working electrode of screen-printed carbon electrode (SPCE), completely drying the solution, dripping the perfluoroalkane acid solution, drying at room temperature, dissolving the GelMA precursor obtained in step (b) in phosphate-buffered saline (PBS) buffer, adding photoinitiator, incubating in a warm bath to obtain a clear solution, namely a photosensitive GelMA solution, filtering the photosensitive GelMA solution with a filter membrane for sterilization, uniformly mixing the filtered liquid with the cells to obtain a cell-GelMA solution, adding suitable amount of certain volume of cell-GelMA solution to the cell culture tank on the electrode, curing with UV light, adding cell culture medium into the culture tank to obtain a three-dimensional cell culture electrode and connecting the electrode to an electrochemical workstation to obtain a three-dimensional cell electrochemical sensor, (d) adding different concentrations of lipoploysaccharide (LPS) solutions prepared with DMEM medium into each culture pool, where one concentration of LPS solution corresponds to one three-dimensional cell culture electrode, and the two have a one-to-one correspondence, incubating for a period of time, detecting nitric oxide (NO) current signal released by the cells by differential pulse voltammetry (DPV) method, obtaining the peak current value of NO oxidation, establishing then a standard curve for detecting LPS with the logarithm of the LPS concentration and the peak current value and calculating detection limit that reflects ability of sensor to detect NO released by cells and (e) selecting a certain LPS concentration and incubation time based on the LPS standard curve obtained in step (d) to study immunomodulatory effect of atrazine (AT) and its metabolites deethylatrazine (DEA), deisopropylatrazine (DIA), deethyldeisopropylatrazine (DACT), adding different concentrations of AT or its metabolites DEA, DIA, and DACT prepared with Dulbecco's modified Eagle medium (DMEM) into the cell culture tank, incubating for a period of time, performing DPV method to detect the peak current value of NO released by cells, which is recorded as I pesticide, adding certain concentration of LPS solution into the cell culture tank, incubating for a period of time, detecting the peak current value of NO released by the cells by DPV method, which is denoted as I LPS, then adding only DMEM medium into the cell culture tank, incubating for the same time as the pesticide group I, detecting the peak current value of NO released by the cells by the DPV method, which is recorded as the I control, calculating the inhibition rate as follows: inhibition rate (%) = 100 ((I LPS -I pesticide)/(I LPS -I control ))(I), where I LPS is the peak current value of NO released by cells treated with a certain concentration of LPS, I pesticide is the peak current value of NO released by cells treated with LPS solution after pesticide pretreatment, and I control is the release of cells without any treatment NO peak current value, quantitatively determining the degree of immunosuppression of the cells by the pesticide by the calculation of the inhibition rate, which can reflect the immunotoxicity of the pesticide, where the greater the inhibition rate, the greater the immunotoxicity of the pesticide.