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.
Sheng Wu Gong Cheng Xue Bao. 2022 Mar 25;38(3):1197-1208. doi: 10.13345/j.cjb.210384.
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 4 μL. 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 2.55 μg/L. This colloidal gold immunochromatographic strip is simple, rapid and specific for detecting 8-OHdG in human urine to preliminarily evaluate the human status.
8-羟基-2'-脱氧鸟苷（8-OHdG）是评估DNA氧化损伤的一种灵敏且稳定的生物标志物。通过竞争法开发了一种用于检测8-OHdG的快速灵敏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粘贴在聚氯乙烯（PVC）底板上构建试纸条。用柠檬酸钠还原氯金酸制备胶体金（AuNPs）。将8-OHdG抗体（Ab）包被在AuNPs外层形成Ab@AuNPs作为探针。牛血清白蛋白（BSA）和8-OHdG与盐酸碳二亚胺偶联制备人工抗原，用作检测线的包被抗原。山羊抗小鼠多克隆抗体IgG用作对照线的包被抗体。对实验参数进行了优化，包括硝酸纤维素膜类型、加样液配方以及金标抗体喷量。结果表明，合适的硝酸纤维素膜为CN 95。最佳加样液包括1% BSA、3%吐温-20、3%蔗糖和0.9% NaCl。金标抗体的最佳喷量为4 μL。通过试纸条在可见光下可检测8-OHdG，通过比较T线和C线颜色深浅可初步确定尿液中8-OHdG水平。根据T线灰度值进一步计算尿液中8-OHdG浓度，检测限为2.55 μg/L。这种胶体金免疫层析试纸条检测人尿液中8-OHdG简单、快速且具有特异性，可初步评估人体状况。
