基于Eu^(3+)荧光微球的丁香酚时间分辨荧光免疫层析技术的建立  

Establishment of Time-resolved Fluorescence Immunochromatography of Eugenol Based on Eu^(3+)Fluorescent Microspheres

作　　者：宋妍灵 翟璐 李因来 陈青舟 廖杰 金仁耀 SONG Yan-Ling;ZHAI Lu;LI Yin-Lai;CHEN Qing-Zhou;LIAO Jie;JIN Ren-Yao(Institute of Sea Food,Zhejiang Gongshang University,Hangzhou 310012,China;Hangzhou Bioeast Biotech Limited Company,Hangzhou 310018,China;Hangzhou Nankai Biotechnology Limited Company,Hangzhou 310000,China;Zhejiang Huacai Testing Technology Limited Company,Shaoxing 311800,China)

机构地区：[1]浙江工商大学海洋食品研究院,杭州310012 [2]杭州博岳生物技术有限公司,杭州310018 [3]杭州南开日新生物技术有限公司,杭州310000 [4]浙江华才检测技术有限公司,绍兴311800

出　　处：《农业生物技术学报》2024年第9期2190-2202,共13页Journal of Agricultural Biotechnology

基　　金：浙江省科技厅公益性研究项目(LGN22C200028)。

摘　　要：丁香酚具有来源广、成本低、效果好且残留期短等优点,被广泛应用于渔业麻醉,但国际上对于其安全性存在争议,认为丁香酚类化合物对啮齿动物是致癌物或潜在的致癌物。为提供一种高效、简便适用于现场快速检测水产品中丁香酚残留的方法,本研究建立了丁香酚时间分辨荧光免疫层析技术并制备免疫层析试纸条,利用Eu^(3+)标记的荧光微球和抗丁香酚单克隆抗体进行标记,先后对微球活化时间、荧光标记缓冲液pH值、标记抗体量、荧光微球用量及T线和C线包被浓度等参数进行优化,并在最优条件下制备免疫层析试纸条并对其性能进行评价。结果表明,在最优条件下制成的试纸条在0.05~0.40μg/mL之间丁香酚标样浓度的对数值与T/C值具有良好的线性关系,标准曲线方程:y=-2.415lnx+2.589 4,R^(2)=0.994,仪器检出限为0.1μg/mL,表明试纸条具有良好的灵敏度。样品添加回收率试验表明,回收率在94.16%~109.27%范围内,批内和批间变异系数分别为2.00%~7.33%和2.73%~6.97%。利用新研发试纸条对丁香酚标样检测与传统的高效液相色谱检测方法相比结果一致。稳定性试验证明,本研究制备的试纸条的有效保质期至少为1年。本研究所建立的方法适用于水产品中丁香酚残留的快速检测,同时为开发丁香酚快速检测技术提供依据。Eugenol has been widely used in fishery anesthesia because of its wide source,low cost,good effect and short residual period,but its safety is still controversial over the world,and it is considered that eugenol compounds are carcinogens or potential carcinogens for rodents.In order to provide an efficient and simple method for the on-site rapid detection of eugenol residues in aquatic products,in this study,a timeresolved fluorescence immunochromatographic technique for eugenol and prepared immunochromatographic test strips were established.The fluorescent microspheres and anti-eugenol monoclonal antibody were labeled with Eu3+,and parameters such as microsphere activation time,fluorescent labeling buffer pH,amount of labeled antibody,amount of fluorescent microspheres and T-and C-line encapsulation concentration were optimized,and the immunochromatographic test strips were prepared and evaluated under the optimal conditions.The results showed that the test strips made under the optimal conditions had a good linear relationship between 0.05~0.40μg/mL with the standard curve equation:y=-2.415lnx+2.5894,R^(2)=0.994,and the instrumental limit of detection(LOD)of 0.1μg/mL,which indicated that the strips had a good sensitivity.The recoveries of the spiked samples were betweeen 94.16% and 109.27%,and the coefficients of variation(CV)of intra-batch and inter-batch were from 2.00%to 7.33% and from 2.73%to 6.97%,respectively.The results of the eugenol specimen detection by the newly developed test strips were consistent with those of the traditional high-performance liquid chromatography(HPLC)method.The stability test proved that the test strips prepared in this study had an effective shelf life of at least 1 year.This research is suitable for the rapid detection of eugenol residues in aquatic products and provides a reference for the development of eugenol rapid detection technology.

关 键 词：丁香酚 时间分辨荧光 免疫层析 试纸条 

分 类 号：S981.1[农业科学—捕捞与储运]