Abstract：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 time-resolved 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 0.05~0.40 μg/mL with the standard curve equation: y=-2.415lnx+2.589 4, R2=0.994, and the instrumental limit of detection (LOD) of 0.1 μg/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.
[1] 杜凯, 张卓玲, 李婷华, 等. 2018. 抗体固定化方法研究进展[J]. 中国生物工程杂志, 38(4): 78-89. (Du K,Zhang Z L, Li T H, et al.2018. The research progress of antibody immobilization[J]. Chinese Journal of Biological Engineering, 38(4): 78-89.) [2] 段必成. 2020. 丁香酚对斑马鱼胚胎发育的影响及其作用机制研究[D]. 硕士学位论文, 西南大学, 导师: 李云, pp. 1-3. (Duan B C.Effects of eugenol on zebrafish embryonic development and its mechanism[D]. Thiesis for M.S., Southwest University, Suppervisor: Li Y, pp. 1-3.) [3] 方荣谦, 林伟琦. 2022. 鱼类及暂养水中丁香酚等3种兽药残留量的检测与分析[J]. 现代食品, 28(24): 168-170. (Fang R Q, Lin W Q.2022. Detection and analysis of three veterinary drug residues in fish and transient water including eugenol[J]. Modern Food, 28(24): 168-170.) [4] 顾建华, 严艺琳, 杨婷婷, 等. 2021. 玉米赤霉烯酮时间分辨荧光试纸条的制备及特性研究[J]. 粮油食品科技, 2021, 29(2): 59-65. (Gu J H, Yan Y L, Yang T T, et al.2021. Preparation and properties of time-resolved fluorescent strip for zearalenone detection[J]. Cereal, Oil and Food Science and Technology, 29(2): 59-65.) [5] 郭建军. 2020. 喹乙醇化学发光免疫技术及时间分辨荧光免疫层析技术研究[D]. 硕士学位论文, 浙江工商大学, 导师: 金仁耀, pp. 45-46. (Guo J J.2020. Studies on chemiluminescence immunoassay and time-r-esolved fluorescence immunochro-matography based on olaquindox [D]. Thiesis for M.S., Zhejiang University of Technology and Industry, Suppervisor: Jin R Y, pp. 45-46.) [6] 黄先敏, 伍文聪, 吴银梅, 等. 2010. 高效毛细管电泳法测定柴胡中丁香酚的含量[J]. 昭通师范高等专科学校学报, 32(05): 27-29. (Huang X M, Wu W C, Wu Y M, et al.2010. Determination of eugenol in Chaihu by high performance capillary electrophoresis[J]. Journal of Zhaotong Normal College of Higher Education, 32(05): 27-29.) [7] 赖科洋, 陈媛. 2018. 时间分辨荧光微球免疫层析法定量检测克伦特罗[J]. 江西科学, 36(2): 244-247. (Lai K Y, Chen Y.2018. Quantitative detection of clenbuterol by time-resolved fluorescent microsphere immunochromatography[J]. Jiangxi Science, 36(2): 244-247.) [8] 李斌, 石松, 谢俊平, 等. 2018. 一种呋喃类代谢物半抗原, 人工抗原及其在荧光定量免疫层析中的应用. 中国, CN201810456832.2[P]. (Li B, Shi S, Xie J P, et al.2018. A furan metabolite semi-antigen, artificial antig-en and its application in fluorescence quantitative immunochromatography. China, CN201810456832.2[P]. [9] 李晋成, 刘欢, 王群, 等. 2014. 水产品中丁香酚的残留风险评估研究进展[J]. 食品安全质量检测学报, 5(11): 3598-3602. (Li J C, Liu H, Wang Q, et al.2014. Advances in risk assessment of residues of eugenol in aquatic products[J]. Journal of Food Safety and Quality Inspection, 5(11): 3598-3602.) [10] 刘妍, 李晓璐. 2023. 荧光微球免疫层析法快速检测基围虾中环丙沙星[J]. 食品安全导刊, (31): 87-93. (Liu Y, Li X L. 2023. Rapid determination of ciprofloxacin in shrimps by fluorescent microsphere immunochromatography[J]. Food Safety Journal, (31): 87-93.) [11] 吕海燕, 王群, 刘欢, 等. 2013. 鱼用麻醉剂安全性研究进展[J]. 中国渔业质量与标准, 3(02): 24-28. (Lv H Y, Wang Q, Liu H, et al.2013. Research progress on the safety of fish anesthetics[J]. China Fisheries Quality and Standards, 3(02): 24-28.) [12] 童跃聪. 2022. 水产品及其暂养水中丁香酚类化合物残留的检测与分析[J]. 现代食品, 28(22): 174-176, 181. (Tong Y C.2022. Detection and analysis of eugenol drug residues in aquatic products and their corresponding temporary aquaculture water[J]. Modern Food, 28(22): 174-176, 181.) [13] 王彩霞, 熊光权, 白婵, 等. 2019. 高效液相色谱法检测鮰鱼体内丁香酚的残留[J]. 食品安全质量检测学报, 10(8): 2195-2200. (Wang C X, Xiong G Q, Bai C, et al.2019. Determination of eugenol residues in channel catfish by high performance liquid chromatography[J]. Journal of Food Safety and Quality Testing, 10(8): 2195-2200.) [14] 王强, 王旭峰, 赵东豪, 等. 2022. 化学发光酶免疫分析法检测水产品中残留的麻醉剂丁香酚[J]. 分析测试学报, 41(03): 354-360. (Wang Q, Wang X F, Zhao D H, et al.2022. Detection of narcotic residues of eugenol in aquatic products by chemiluminescent enzyme immunoassay[J]. Journal of Analytical Testing, 41(03): 354-360.) [15] 魏东, 刘英. 2012. 氟喹诺酮类药物残留多元快速检测试纸条的研制[J]. 核农学报, 26(9): 1278-1283. (Wei D, Liu Y.2012. Development of a rapid colloidal gold test strip for detecting fluoroquinolones residues[J]. Journal of Nuclear Agriculture, 26(9): 1278-1283.) [16] 解超男, 李芹, 韩刚, 等. 2019. 基于单克隆抗体的胶体金免疫层析法快速检测丁香酚[J]. 食品质量安全检测学报, 10(20): 6938-6943. (Xie C N, Li Q, Han G, et al.2019. Rapid detection of eugenol by mo-noclonal antibody based colloidal gold immunochromatography[J]. Journal of Food Quality and Safety Testing, 10(20): 6938-6943.) [17] 詹屋强, 王弘. 2015. 小分子物质酶联免疫分析方法的研究进展[J]. 食品安全质量检测学报, 6(03): 857-862. (Zhan W Q, Wang H.2015. Research progress of enzyme immunoassay methods for small molecules[J]. Journal of Food Safety and Quality Testing, 6(03): 857-862.) [18] 张博文, 李雪银, 江改青, 等. 2022. 水产品中常用麻醉剂及其检测方法的研究进展[J]. 食品安全导刊, (15): 180-184. (Zhang B W, Li X Y, Jiang G Q, et al. 2022. Research progress on the common anesthetics and inspection methods in aquatic products[J]. Journal of Food Safety, (15): 180-184.) [19] 张少波, 邓常继, 张海涛, 等. 2021. 时间分辨荧光免疫层析法测定粮食中的黄曲霉毒素B-1[J]. 粮食科技与经济, 46(6): 80-82, 110. (Zhang S B, Deng C J, Zhang H T, et al.2021. Detection of AFB1 in grain by the time-resolved fluoroimmunoassay[J]. Grain Science and Technology and Economics, 46(6): 80-82, 110.) [20] 赵义良, 李云, 桑丽雅, 等. 2018. 呋喃唑酮代谢物时间分辨荧光免疫快速检测试纸卡的研制及应用[J]. 食品安全质量检测学报, 9(19): 5187-5194. (Zhao Y L, Li Y, Sang L Y, et al.2018. Development and application of a time-resolved fluorescence immunoassay test card for the rapid detection of furazolidone metabolites[J]. Journal of Food Safety and Quality Testing, 9(19): 5187-5194.) [21] 周宝珠, 余小姚, 邝翠琼, 等. 2020. 紫外分光光度法测定丁香油滴丸丁香酚含量的方法学研究[J]. 佛山科学技术学院学报(自然科学版), 38(04): 15-18. (Zhou B Z, Yu X Y, Kuang C Q, et al.2020. A methodological study on the determination of eugenol in clove oil drops by UV spectrophotometry[J]. Journal of Foshan Institute of Science and Technology (Natural Science Edition), 38(04): 15-18.) [22] Bahadir E, Sezginturk B, Kemal M.2016. Lateral flow assays: Principles, designs and labels[J]. TrAC Trends in Analytical Chemistry, 82: 286-306. [23] Beggs M, Novotny M, Sampedro S.1990. A self performing chromatographic immunoassay for the qualitative determination of human chorionic gonadotrophin (HCG) in urine and serum[J]. Clinical Chemistry, 36(6): 1084-1085. [24] Huang Y X, Li Q, Zhang Y L, et al.2021. Determination of six eugenol residues in aquatic products by gas chromatographyorbitrap mass spectrometry[J]. Journal of Food Quality, 021: 1-9. [25] Jia B, Liao X, Sun C, et al.2021. Development of a quantum dot nanobead-based fluorescent strip immunosensor for onsite detection of aflatoxin B1 in lotus seeds[J]. Food Chemistry, 356: 129614. [26] Li C F, He X L, Yang Y, et al.2020. Rapid and visual detection of African swine fever virus antibody by using fluorescent immunochromatography test strip[J]. Talanta, 219: 121284. [27] Li J C, Zhang J, Liu Y.2015. Optimization of solid-phase-extraction cleanup and validation of quantitative determination of eugenol in fish samples by gas chromatography-tandem mass spectrometry[J]. Anal Bi-o Chem, 407(21): 6563-6568. [28] Li M, Wang H M, Sun J D, et al.2021. Rapid, onsite, and sensitive detection of aflatoxin M1 in milk products by using time-resolved fluorescence microsphere test strip[J]. Food Control, 121: 107616. [29] Shen X Y, Wu X L, Liu L Q, et al.2019. Development of a colloidal gold immunoassay for the detection of four eugenol compounds in water[J]. Food and Agricultural Immunology, 30(1): 1318-1331. [30] Shirshahi V, Liu G.2021. Enhancing the analytical performance of paper lateral flow assays: From chemistry to engineering[J]. TRAC Trends in Analytical Chemistry, 136: 116200. [31] Tang X Q, Li P W, Zhang Q, et al.2017. Time-resolved fluorescence immunochromatographic assay developed using two idiotypic nanobodies for rapid, quantitative, and simultaneous detection of aflatoxin and zearalenone in maize and its products[J]. Analytical Chemistry, 89(21): 11520-11528. [32] Wang J J, Liu B H, Hsu Y T, et al.2011. Sensitive competitive direct enzyme-linked immunosorbent assay and gold nanoparticle immunochromatographic strip for detecting aflatoxin M1 in milk[J]. Food Control, 22(6): 964-969. [33] Wang L Z, Sun J D, Ye J, et al.2022. One-step extraction and simultaneous quantitative fluorescence immunochromatography strip for AFB1 and Cd detection in grain[J]. Food Chemistry, 374: 131684. [34] Wang X Q, Xue S F, Chen Z H, et al.2017. Dual lanthanide-doped complexes: The development of a time-resolved ratiometric fluorescent probe for anthrax biomarker and a paper-based visual sensor[J]. Biosensors and Bioelectronics, 94: 388-393. [35] Wu Y H, Sun J Y, Huang X L, et al.2021. Ensuring food safety using fluorescent nanoparticles-based immunochromatographic test strips[J]. Trends in Food Science & Technology, 118(A): 658-678.
