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

本文构建了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AuNPs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。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0.375ng/mL。此外,癌胚抗原浓度超过4.8ng/mL时,颜色出现了可直接用肉眼判断的显著变化。为使检测更加便捷,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。优化条件下,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。

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.According to the inverse relationship between the absorbance at 650 nm of the system and the concentration of carcinoembryonic antigen in the range of 2~18 ng/mL,the determination of carcinoembryonic antigen is realized with a detection limit of 0.375 ng/mL.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.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.