水果及环境水样中苯醚甲环唑的高灵敏免疫分析方法研究  

Detection of Difenoconazole in Fruit and Environmental Water Samples by a Highly Sensitive Immunoassay Method

作　　者：陈梓豪 张咏仪 杨金易[1] 曾道平 贺颖 徐振林[1] 王弘[1] 孙远明[1] 沈玉栋[1] CHEN Zi-hao;ZHANG Yong-yi;YANG Jin-yi;ZENG Dao-ping;HE Ying;XU Zhen-lin;WANG Hong;SUN Yuan-ming;SHEN Yu-dong(Guangdong Provincial Key Laboratory of Food Quality and Safety,College of Food Science,South China Agricultural University,Guangzhou 510642,China;Wens Institute,Wens Foodstuff Groups Co.Ltd.,Yunfu 527499,China)

机构地区：[1]华南农业大学食品学院广东省食品质量安全重点实验室,广东广州510642 [2]温氏食品集团股份有限公司研究院,广东云浮527499

出　　处：《分析测试学报》2023年第6期768-774,共7页Journal of Instrumental Analysis

基　　金：国家自然科学基金项目(32272400);广东省基础与应用基础研究项目(2019B1515210025,2018B030314005);广州市科技计划项目(202206010146)。

摘　　要：采用辣根过氧化物酶(HRP)催化氧化四甲基联苯胺(TMB)显色为信号输出系统,结合间接竞争反应模式建立了水果及环境水样中苯醚甲环唑的间接竞争酶联免疫分析方法(ic ELISA)。基于高特异性单克隆抗体,通过逐步优化策略,确定最佳免疫分析条件为:包被原与抗体质量浓度组合为62.5 ng/mL和0.318μg/mL,一抗竞争反应缓冲溶液为含0.05%Tween-20的0.01 mol/L磷酸盐缓冲液(PBST,pH 7.4),一抗竞争反应时间为20 min,酶标二抗稀释液为0.01 mol/L磷酸盐缓冲液(PBS,pH 7.4),酶标二抗反应时间为30 min。在该条件下,苯醚甲环唑的线性检测范围(IC_(20)~IC_(80))为0.49~3.90 ng/mL,半数抑制浓度(IC_(50))为1.38 ng/mL,检出限(LOD)为0.33 ng/mL。在柑橘、葡萄、香蕉、草莓田水、湖水和河水样品中的平均加标回收率为72.3%~132%,相对标准偏差(RSD)不大于13%,对10种结构和功能类似物的交叉反应率(CR)均小于0.2%。该方法准确可靠,可用于水果及环境水样中苯醚甲环唑的快速筛查和残留检测。An indirect competitive enzyme-linked immunosorbent method(icELISA)was developed for the detection of difenoconazole in food and environmental water in this paper,with the coloration from horseradish peroxidase(HRP)catalytically oxidizing tetramethylbenzidine(TMB)as signal out⁃put system.Based on the highly specific monoclonal antibody,through the gradual optimization strategy,the optimal immunoassay conditions were determined as follows:the optimal combinations of coating antigen and antibody concentration were 62.5 ng/mL and 0.318μg/mL,the competitive reaction buffer solution was 0.01 mol/L phosphatic buffer solution containing 0.05%Tween-20(PBST,pH 7.4),the competitive reaction time of the first antibody was 20 min,the diluent of the enzyme-labeled secondary antibody was 0.01 mol/L phosphatic buffer solution(PBS,pH 7.4),and the reaction time of the enzyme-labeled secondary antibody was 30 min.The icELISA for the detec⁃tion of difenoconazole established under this condition had a linear detection range(IC_(20)-IC_(80))of 0.49-3.90 ng/mL,a half-inhibition concentration(IC_(50))of 1.38 ng/mL,and a limit of detection(LOD)of 0.33 ng/mL.The average spiked recoveries for citrus,grape,banana,strawberry field water,lake water and river water samples ranged from 72.3%to 132%,with the relative standard de⁃viations(RSDs)not more than 13%.The cross-reactivities(CRs)against the ten structural and functional analogues were less than 0.2%.The method was accurate and reliable,and it was applicable for rapid screening of difenoconazole in fruit and environmental water samples.

关 键 词：苯醚甲环唑 单克隆抗体 酶联免疫分析 水果 水样 

分 类 号：O657[理学—分析化学] TS207.3[理学—化学]