陶珊珊^1,2,
葛会林¹,
袁宏球¹,
阳辛凤¹
1. 中国热带农业科学院分析测试中心 海南省热带果蔬产品质量安全重点实验室, 海口 571101;
2. 华中农业大学植物科学技术学院, 武汉 430070

作者简介: 陶珊珊(1994-),女,硕士研究生,研究方向为化学混合物毒理,E-mail:sarah133@126.com.

基金项目: 国家自然科学基金(21607171)；海南省应用技术研发与示范推广专项(ZDXM2014047,ZDXM20130043)；海南省自然科学基金(213027)


中图分类号: X171.5

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

Tao Shanshan^1,2,
Ge Huilin¹,
Yuan Hongqiu¹,
Yang Xinfeng¹
1. Hainan Key Laboratory of Tropical Fruit and Vegetable Products Quality and Safety, Analysis and Testing Center, Chinese Academy of Tropical Agricultural Sciences, Haikou 571101, China;
2. College of Plant Science & Technology, Huazhong Agricultural University, Wuhan 430070, China

CLC number: X171.5

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摘要: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(AChE)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AChE抑制毒性的微板吸光法。系统研究了AChE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AChE、DTNB、ATCI的浓度增加，吸光度均增加；吸光度变化速率与AChE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、AChE 0.04 U·mL^-1、pH 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AChE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。
关键词: 灭多威/
乙酰胆碱酯酶/
剂量-效应曲线/
微板吸光法/
双相关系

Abstract: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 (AChE), this work established a microplate absorbance technology to measure the toxicity of chemicals. The AChE concentration, acetylthiocholine iodide (ATCI) concentration and 5,5'-dithiobis-2-nitrobenzoic acid (DTNB) concentration were optimized, and their effects on absorbance, absorbance change rate and chemical test toxicity were investigated. The optimized conditions for AChE microplate toxicity analysis were DTNB 0.2 g·L^-1, ATCI 0.2 g·L^-1, AChE 0.04 U·mL^-1, pH 6.8, reaction temperature 29 ℃ and exposure time 15 min. The results showed that the concentrations of AChE, DTNB, and ATCI increased the absorbance. The concentration of AChE 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 dose-response 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.
Key words:methomyl/
acetylcholinesterase/
dose-response curve/
microplate absorbance method/
biphasic relationship.