测定化学物对乙酰胆碱酯酶抑制毒性的微板吸光法研究

Microplate Absorbance-Based Toxicity Bioassay by Analysis of in Vitro Inhibition of Acetylcholinesterase

随着化学污染物种类与数量的日益增多,高通量测试方法显得越来越重要。基于乙酰胆碱酯酶(ACh E)抑制法原理,以多功能酶标仪为测试设备,96孔微板为暴露反应载体,建立了测定化学物对ACh E抑制毒性的微板吸光法。系统研究了ACh E浓度、碘化硫代乙酰胆碱(ATCI)浓度、二硫代二硝基苯甲酸(DTNB)浓度对显色吸光度、吸光度变化速率、化学物测试毒性的影响。结果表明,随着ACh E、DTNB、ATCI的浓度增加,吸光度均增加;吸光度变化速率与ACh E浓度呈正相关,与DTNB浓度基本无关,与ATCI浓度呈现先增后减的双相关系;随着ATCI浓度增加,灭多威剂量-效应曲线(DRC)向右移动。最终建立的优化测试条件为DTNB 0.2 g·L^(-1)、ATCI 0.2 g·L^(-1)、ACh E 0.04 U·m L^(-1)、p H 6.8、反应温度29℃及暴露时间15 min;并与国标方法进行了对比与验证,发现试剂加样顺序对毒性测定结果有显著影响。基于ACh E的微板毒性测试结果表明,灭多威的DRC呈现良好的S型曲线,可用Weibull函数有效表征,拟合决定系数R2大于0.97,空白变异控制在了±10%以内。此方法可用于化学污染物的高通量毒性检测。

Chemical pollutants assessment requires a broader-spectrum and high-throughput analysis to guarantee compliance with established standards. Nowadays, the increasing number of pollutants and their important effects lead to the development of general toxicity bioassays. Based on the in vitro inhibition of acetylcholinesterase（ACh E）, this work established a microplate absorbance technology to measure the toxicity of chemicals. The ACh E concentration, acetylthiocholine iodide（ATCI） concentration and 5,5 ＇-dithiobis-2-nitrobenzoic acid（DTNB） con-centration were optimized, and their effects on absorbance, absorbance change rate and chemical test toxicity were investigated. The optimized conditions for ACh E microplate toxicity analysis were DTNB 0.2 g·L^（-1）, ATCI 0.2 g·L^（-1）, ACh E 0.04 U·m L^（-1）, p H 6.8, reaction temperature 29 ℃ and exposure time 15 min. The results showed that the concentrations of ACh E, DTNB, and ATCI increased the absorbance. The concentration of ACh E was positively correlated with the absorbance change rate, the concentrations of DTNB was unrelated with the absorbance change rate, and the concentrations of ATCI was biphasic-curved relationship with the absorbance change rate. The doseresponse curve（DRC） of methomyl moved to the right with increasing ATCI concentration. By comparing with the national standard method, the reagent injection order had the significant impact on the toxicity measurement. The microplate absorbance-based toxicity bioassay was further validated to show a good S-type DRC with methomyl toxicity. This DRC can be well characterized by the Weibull function, and the fitting coefficient of determination R2 is〉 0.97, and the blank variation is ± 10%. This method can be used for high-throughput toxicity assays of the chemical pollutants.