基于胶体金的8-羟基-2′-脱氧鸟苷免疫层析试纸条  

Development of a colloidal gold based immunochromatographic strip for 8-OHdG detection

作　　者：叶伟伟 王丽文 张宇[2] 李潮锋 钱天润 付贤树 张明洲 单继宏[1] YE Weiwei;WANG Liwen;ZHANG Yu;LI Chaofeng;QIAN Tianrun;FU Xianshu;ZHANG Mingzhou;SHAN Jihong(College of Mechanical Engineering,Zhejiang University of Technology,Hangzhou 310014,Zhejiang,China;College of Food Science and Technology,Zhejiang University of Technology,Hangzhou 310014,Zhejiang,China;College of Life Sciences,China Jiliang University,Hangzhou 310018,Zhejiang,China;Zhejiang Provincial Key Laboratory of Biometrology and Inspection&Quarantine,Hangzhou 310018,Zhejiang,China)

机构地区：[1]浙江工业大学机械工程学院,浙江杭州310014 [2]浙江工业大学食品科学与工程学院,浙江杭州310014 [3]中国计量大学生命科学学院,浙江杭州310018 [4]浙江省生物计量及检验检疫技术重点实验室,浙江杭州310018

出　　处：《生物工程学报》2022年第3期1197-1208,共12页Chinese Journal of Biotechnology

基　　金：浙江省自然科学基金(LY20D060008);浙江省科技计划项目-公益技术(LGG19F010008);宁波市公益类科技计划项目(20211JCGY020387)。

摘　　要：8-羟基-2′-脱氧鸟苷(8-hydroxy-2′-deoxyguanosine,8-OHdG)是评价DNA氧化损伤较灵敏和稳定的生物标志物。文中采用竞争法建立一种快速、灵敏检测8-OHdG的胶体金免疫层析试纸条。将样品垫(玻璃纤维素膜)、结合垫(玻璃纤维素膜)、硝酸纤维素膜和吸水垫依此粘贴在聚氯乙烯(polyvinyl chloride,PVC)底板上,构建试纸条。通过柠檬酸钠还原三水合四氯金酸制备胶体金(gold nanoparticles,AuNPs),8-OHdG配对的抗体(antibody,Ab)包被于AuNPs的外层(Ab coated AuNPs,Ab@AuNPs)作为探针。牛血清蛋白(bovine serum protein,BSA)与8-OHdG用碳二亚胺盐酸盐偶联制备人工抗原,作为检测线的包被抗原。羊抗鼠多抗(imunoglobulin G,IgG)作为质控线的包被抗体。对试纸条的硝酸纤维素膜、上样液的配方、金标抗体喷涂量等实验参数进行了优化。结果表明,硝酸纤维素膜(nitrocellulose film,NC)膜采用CN 95,上样液的最优配方为1%BSA+3%吐温-20+3%蔗糖+0.9%NaCl溶液,最适金标抗体喷涂量为4μL。利用试纸条在可见光下检测8-OHdG,根据检测线(test line,T线)和质控线(control line,C线)的显色强度对比,可初步判断尿液中8-OHdG的含量水平。并通过T线的灰度值计算尿液中的8-OHdG的浓度,检测限为2.55μg/L。该方法简单、快速且有较好的特异性,可检测人体尿液中的8-OHdG含量,以初步评价人体的健康状态。8-hydroxy-2′-deoxyguanosine(8-OHdG)is a sensitive and stable biomarker for evaluating DNA oxidative damage.A rapid and sensitive colloidal gold immunochromatographic strip was developed for 8-OHdG detection by a competitive method.The sample pad(glass cellulose film),bonding pad(glass cellulose film),nitrocellulose film and absorbent pad were pasted on the polyvinyl chloride(PVC)base plate to construct the test strip.Colloidal gold(AuNPs)was prepared by the reduction of chloroauric acid with sodium citrate.8-OHdG antibody(Ab)was coated on the outer layer of AuNPs to form Ab@AuNPs as a probe.Bovine serum albumin(BSA)and 8-OHdG were conjugated with carbodiimide hydrochloride to prepare an artificial antigen,which was used as the coating antigen of detection line.Goat anti mouse polyclonal antibody IgG was used as the coating antibody of control line.The experimental parameters were optimized including the type of nitrocellulose membrane,the formula of loading solution,and the spraying amount of gold labeled antibody.The results showed that the appropriate nitrocellulose membrane was CN 95.The optimal loading solution included BSA(1%),Tween-20(3%),sucrose(3%)and NaCl(0.9%).The optimal spraying amount of gold labeled antibody was 4μL.8-OHdG can be detected by the strip under visible light,and the level of 8-OHdG in urine can be preliminarily determined by comparing the color intensity of T line and C line.The 8-OHdG concentration in urine was further calculated by the gray value of T line and the threshold of detection was 2.55μg/L.This colloidal gold immunochromatographic strip is simple,rapid and specific for detecting 8-OHdG in human urine to preliminarily evaluate the human status.

关 键 词：免疫层析 胶体金 8-羟基-2′-脱氧鸟苷 试纸 

分 类 号：R446.61[医药卫生—诊断学]