Xu Shuping, Ji Xiaohui, Xu Weiqing, Li Xiaoling, Wang Lianying, Bai Yubai, Zhao Bing, Ozaki Yukihiro
Key Laboratory for Supramolecular Structure and Material of Ministry of Education, Jilin University, Changchun 130021, P. R. China.
Analyst. 2004 Jan;129(1):63-8. doi: 10.1039/b313094k. Epub 2003 Dec 11.
This paper reports a novel immunoassay based on surface-enhanced Raman scattering (SERS) and immunogold labelling with silver staining enhancement. Immunoreactions between immunogold colloids modified by a Raman-active probe molecule (e.g., 4-mercaptobenzoic acid) and antigens, which were captured by antibody-assembled chips such as silicon or quartz, were detected via SERS signals of Raman-active probe molecule. All the self-assembled steps were subjected to the measurements of ultraviolet-visible (UV-vis) spectra to monitor the formation of a sandwich structure onto a substrate. The immunoassay was performed by a sandwich structure consisting of three layers. The first layer was composed of immobilized antibody molecules of mouse polyclonal antibody against Hepatitis B virus surface antigen (PAb) on a silicon or quartz substrate. The second layer was the complementary Hepatitis B virus surface antigen (Antigen) molecules captured by PAb on the substrate. The third layer was composed of the probe-labelling immunogold nanoparticles, which were modified by mouse monoclonal antibody against Hepatitis B virus surface antigen (MAb) and 4-mercaptobenzoic acid (MBA) as the Raman-active probe on the surface of gold colloids. After silver staining enhancement, the antigen is identified by a SERS spectrum of MBA. A working curve of the intensity of a SERS signal at 1585 cm(-1) due to the small nu aromatic ring vibration of MBA versus the concentration of analyte (Antigen) was obtained and the non-optimized detection limit for the Hepatitis B virus surface antigen was found to be as low as 0.5 [micro sign]g mL(-1).
本文报道了一种基于表面增强拉曼散射（SERS）和免疫金标记及银染增强的新型免疫分析方法。通过拉曼活性探针分子（如4-巯基苯甲酸）修饰的免疫金胶体与抗原之间的免疫反应，抗原被硅或石英等抗体组装芯片捕获，通过拉曼活性探针分子的SERS信号进行检测。所有自组装步骤均进行紫外可见（UV-vis）光谱测量，以监测在基底上三明治结构的形成。免疫分析通过由三层组成的三明治结构进行。第一层由固定在硅或石英基底上的抗乙肝病毒表面抗原小鼠多克隆抗体（PAb）的抗体分子组成。第二层是基底上PAb捕获的互补乙肝病毒表面抗原（抗原）分子。第三层由探针标记的免疫金纳米颗粒组成，其在金胶体表面通过抗乙肝病毒表面抗原小鼠单克隆抗体（MAb）和作为拉曼活性探针的4-巯基苯甲酸（MBA）进行修饰。银染增强后，通过MBA的SERS光谱鉴定抗原。获得了由于MBA的小ν芳环振动在1585 cm(-1)处的SERS信号强度与分析物（抗原）浓度的工作曲线，发现乙肝病毒表面抗原的非优化检测限低至0.5 [微克]g mL(-1)。
