张一平,崔猛,曲林姣,方凯,姜磊,刘涵云,杨丽敏.化学通报,2022,85(9):1105-1112.
DNA调节纳米金模拟酶活性的癌胚抗原比色检测
Colorimetric detection of carcinoembryonic antigen by DNA-regulated mimetic enzyme activity of gold nanoparticles
投稿时间:2022-02-10  修订日期:2022-03-14
DOI:
中文关键词:  核酸适配体  纳米金颗粒  过氧化物酶模拟酶活性  癌胚抗原  比色检测
英文关键词:Aptamers, gold nanoparticles, peroxidase-mimicking enzyme activity, carcinoembryonic antigen, colorimetric detection
基金项目:国家自然科学基金项目(31871878)资助
作者单位E-mail
张一平 中国石油大学华东化学化工学院生物与能源化工系 2944606236@qq.com 
崔猛 中国石油大学华东化学化工学院生物与能源化工系  
曲林姣 中国石油大学华东化学化工学院生物与能源化工系  
方凯 中国石油大学华东化学化工学院生物与能源化工系  
姜磊 a:中国石油大学华东化学化工学院生物与能源化工系  
刘涵云 青岛大学附属医院  
杨丽敏 中国石油大学华东化学化工学院生物与能源化工系 yanglimin@upc.edu.cn 
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中文摘要:
      本文构建了一个DNA调节纳米金颗粒(AuNPs)过氧化物酶模拟酶活性的比色检测方法,用于癌胚抗原的检测。将癌胚抗原的核酸适配体及其互补链通过碱基互补配对构成双链DNA,修饰在磁性微球负载的纳米金颗粒上,制备出具有可调节过氧化物酶模拟酶活性的生物探针。癌胚抗原被生物探针上的核酸适配体捕获后,在AuNPs表面形成空间位阻效应屏蔽底物,从而抑制了AuNPs的酶活性。且为了指示纳米金颗粒的酶活性,用生物探针催化氧化色源底物3,3′,5,5′-四甲基联苯胺(TMB)显色。TMB颜色随着癌胚抗原浓度的增加而变浅,根据体系650nm处的吸光度与癌胚抗原浓度之间的反比关系实现了对癌胚抗原的测定,线性范围为2~18 ng/mL,检测限达0.375 ng/mL。此外,癌胚抗原浓度超过4.8 ng/mL时,颜色出现了可直接用肉眼判断的显著变化。为使检测更加便携,本文同时设计了倒置磁分离检测管,在管中就能完成纳米探针捕获癌胚抗原、磁分离、洗涤。最优条件下,比色检测体系回收率为99%~100%,与临床检验差异显著性分析表明,t检验低于3.182,无明显差异。
英文摘要:
      In this paper, a colorimetric assay with DNA-regulated peroxidase-mimicking enzyme activity of gold nanoparticles (AuNPs) was constructed for the detection of carcinoembryonic antigen. The aptamer of carcinoembryonic antigen and its complementary strand were formed into double-stranded DNA by base complementary pairing and modified on magnetic microsphere-loaded AuNPs to prepare a bioprobe with modifiable peroxidase-mimicking enzyme activity. The carcinoembryonic antigen was captured by the aptamer on the bioprobe, which resulted in a spatial site blocking effect on the surface of AuNPs to shield the substrate, thus inhibiting the enzymatic activity of AuNPs. To indicate the enzymatic activity of AuNPs, the chromogentic reaction was performed using the probe-catalyzed oxidation of 3,3′,5,5′-tetramethylbenzidine (TMB). The color of TMB became lighter with increasing concentration of carcinoembryonic antigen, and the linear range was 2~18 ng/mL with a detection limit of 0.375 ng/mL based on the inverse relationship between the absorbance at 650 nm and the concentration of carcinoembryonic antigen. In addition, a significant color change that can be directly judged by the naked eye was observed for carcinoembryonic antigen concentrations above 4.8 ng/mL. To make the assay more portable, an inverted magnetic separation assay tube was also designed, in which the capture of carcinoembryonic antigen, magnetic separation, and washing could be done in the tube. Under the optimal conditions, the recovery was 99%~100%, and the significance analysis of the difference with the clinical test showed that the t-test was lower than 3.182, which was not significantly different.
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