目的: 基于培养滤液蛋白10（CFP10）核酸适体（aptamer）和金纳米簇（AuNCs）建立可视化生物传感器检测结核分枝杆菌CFP10,为辅助诊断结核病提供新策略。方法: 采用谷胱甘肽还原法制备AuNCs,TMB（3,3'5,5'-四甲基联苯胺）显色法验证AuNCs的类过氧化物酶催化活性;生物素-亲和素-HRP(辣根过氧化酶)系统验证aptamer与CFP10的结合能力;将巯基修饰的CFP10 aptamer与AuNCs稳定偶联形成aptamer-AuNCs;用CFP10多克隆抗体包被的微孔板捕获CFP10,继而捕获aptamer-AuNCs,构建检测CPF10的AuNCs可视化生物传感器,评价其敏感度、特异性、阴性预测值、阳性预测值和诊断效率。结果: 合成的AuNCs具有类过氧化物酶活性,偶联核酸适体并不影响其活性。与对照组相比,AuNCs可视化传感器在CFP10存在时引起溶液中AuNPs颜色变为蓝色,随着CFP10的浓度增加,肉眼可明显观测到这一变化过程。该传感器检测血清CFP10诊断结核分枝杆菌感染的敏感度、特异性、阳性预测值、阴性预测值及诊断效率分别为94.74%、75.86%、83.72%、91.67%和86.56%。结论: 基于Aptamer和AuNCs的可视化生物传感器可检测CFP10而辅助诊断结核分枝杆菌感染。
Abstract
Objective A visual biosensor based on aptamer of CFP10 and AuNCs mimetic enzyme was established to detect Mycobacterium tuberculosis CFP10 in serum of tuberculosis patients and provide a new strategy for assisting tuberculosis diagnosis. Methods The colloidal solution of AuNCs was prepared by glutathione reduction method and the peroxidase like catalytic activity of AuNCs was confirmed by the TMB colorimetric method; The binding ability of CFP10 aptamer modified by sulfhydryl to AuNCs was verified by using the biotin-avidin-HRP system; The CFP10 was captured by the microwell plate coated with the CFP10 polyclonal antibody and then capture aptamer-AuNCs to construct a gold nanocluster visual biosensor for detecting CPF10; The sensitivity, specificity, negative predictive value, positive predictive value and diagnostic efficiency of the visual biosensor were evaluated. Results The synthesized AuNCs exhibit peroxidase like activity and does not affect their activity when coupled with aptamers. Compared with the control group, the presence of CFP10 caused the color of GNPs to turn blue. As the concentration of CFP10 increases, this change process can be clearly observed with the naked eye. The sensitivity, specificity, positive predictive value, negative predictive value, and diagnostic efficiency of the visual biosensor for detecting serum CFP10 in diagnosing Mycobacterium tuberculosis infection were 94.74%, 75.86%, 83.72%, 91.67%, and 86.56%, respectively. Conclusion The Visual biosensors based on Aptamer and AuNCs can detect CFP10 and assist the diagnosis of Mycobacterium tuberculosis infection.
关键词
结核分枝杆菌 /
核酸适体 /
金纳米簇 /
培养滤液蛋白10
Key words
mycobacterium tuberculosis /
aptamer /
Au nanoparticles /
culture filtrate protein 10
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基金
湖南省自然科学基金“基于发夹型核酸探针和DNA-AgNCs的卵巢癌外泌体miRNA生物传感器研究”（2021JJ40369）; 长沙市自然科学基金“基于核酸适体的金纳米簇可视化免疫传感技术诊断潜伏性结核感染”（kq2208173）
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