基于砷和汞离子标记 SiO2@Au 纳米探针的超敏多元免疫分析方法研究

摘要

构建了一种基于 As3+和 Hg2+标记 SiO2@ Au 复合纳米探针(NPs)，以及氢化物发生-原子荧光(HG-AFS)同时检测癌胚抗原(CEA)和糖蛋白19-9(CA 19-9)的超灵敏多元免疫分析方法。采用氨基化 SiO2(核)@ Au NPs(壳)分别吸附 As3+和 Hg2+，并标记 CEA 和 CA 19-9的第二抗体(Ab2)，由此获得了富 As(或 Hg)型信号探针(As (Hg)-SiO2@ Au-Ab2)。基于夹心免疫分析方法，将两种信号探针和对应的抗原以及96孔板上一抗，在板底形成两种免疫复合物(Ab 1/ Ag/ As(Hg)-SiO2@ Au-Ab2)，利用 HG-AFS 同时检测 As3+和 Hg2+，其与 CEA 和 CA 19-9的含量对数值成正比，可用于定量分析。此类探针不仅具有良好的分散性、还具有出色的信号放大效果。优化了探针合成和最佳反应条件，并对探针制备过程进行了表征。本方法与 CEA 和 CA 19-9分别在0.001~100μg/ L 和0.01~80 U/ mL 范围内呈线性关系，检出限分别为0.5 ng/ L 和0.005 U/ mL (3σ)，低于标准 ELISA 方法3个数量级。用于血清样品中 CEA 和 CA 19-9同时检测，并与标准 ELISA 方法对照，结果一致。此免疫分析方法灵敏、简便、一次可实现多个肿瘤标志物检测，适合于基层卫生部门进行恶性肿瘤早期筛查。%An ultrasensitive immunoassay was developed based on As3+ and Hg2+ labeled SiO2 @ Au nanoparticles signal tags and hydride generation-atomic fluorescence spectrometry (HG-AFS) for the detection of carcinoembryonic antigen(CEA) and carbohydrate antigen 19-9 (CA 19-9) respectively. Firstly, amino SiO2@ Au NPs were synthesized for selective absorption of As3+ and Hg2+ ions respectively. Subsequently,the secondary antibody (Ab2) of CEA and CA 19-9 was respectively labeled on As3+ or Hg2+-SiO2 @ Au NPs to prepare the corresponding signal tags for CEA and CA 19-9. Based on the sandwich immunoassay scheme, the tags, two antigen and corresponding first antibodies were bio-conjugated on the bottom of 96-well plate at room temperature to form the immunocomplex. After it was dissolved in alkali solution, As3+ and Hg2+ ions were released in solution and detected by HG-AFS, which concentration was proportional with logarithms of CEA and CA 19-9. The reaction conditions were optimized and the tags were characterized. This assay was based on determination of the concentration of As3+ and Hg2+ for quantization of the corresponding CEA and CA 19-9 antigen. The assay showed a wide linear range from 0. 001 to 100. 0 μg / L for CEA and 0. 01-80 U/ mL for CA 19-9, and a lower detection limit of 0. 5 ng / L and 0. 005 U/ mL respectively. This proposed method was used in real serums samples, the results were consistence with that by ELISA. The immunoassay showed three orders of magnitude of sensitivity lower than that of ELISA, which provides a promising simultaneous immunoassay for the early diagnosis of cancer .