基于荧光抗体修饰介孔SBA-15的信号放大型免疫探针的制备

Preparation of Signal-amplifying Fluorescent Immune Probe Based on Antibody-modified Mesoporous SBA-15

研制了一种基于异硫氰酸荧光素(FITC)标记荧光抗体修饰介孔SiO2的超灵敏标记探针,并成功用于检测人血清中甲胎蛋白(AFP)的含量。通过EDC/NHS交联剂将FITC标记抗体固定在氨基功能化的介孔SBA-15上,制备得到抗体修饰介孔SBA-15的信号放大型荧光免疫探针(FITC-Ab*/SBA-15)。将AFP单克隆抗体固定在纳米金/介孔SBA-15修饰的玻璃片上,然后植入流动注射体系,构建免疫检测流通池。基于夹心式免疫反应模式,结合流动注射分析技术,待测抗原与FITC-Ab*/SBA-15顺序注射到免疫流通池。免疫反应结束后,FITC-Ab*/SBA-15被引入到流通池的玻璃载体表面,进行荧光检测。介孔SBA-15纳米粒子由于具有较大的比表面积和交错的多维孔道,可以大大提高生物分子的的负载量,使得在每个微球上能够负载更多的FITC标记二抗,使荧光信号得到放大,拓宽了检测的线性范围。

A novel ultra-sensitive immunoassay probe was proposed based on fluorescein isothiocyanate（FITC） labeled antibody modified mesoporous materials.The prepared signal amplification label has been successfully used to detect the level of α-fetoprotein（AFP） in human serum samples.The sensitive fluorescent antibody modified mesoporous SBA-15（FITC-Ab＊/SBA-15） was prepared by covalent-linking FITC-labeled antibody onto the mesoporous SBA-15 surface by the EDC/NHS.AFP monoclonal antibodies were embedded into the glass sheet modified with Au-NP /mesoporous SBA-15.Based on a sandwich immunoassay format combined with flow injection analysis,two mixtures of the sample antigens and corresponding FITC-Ab＊/SBA-15 were introduced into the flow cell for on-line incubation.After the sandwich immunoassay,FITC-Ab＊/SBA-15 was retained in the flow cell,and the fluorescent signal was recorded.More FITC-labeled antibody can be loaded based on the high surface area and large pore volume of mesoporous SBA-15 nanoparticles.Thus,the prepared amplification label can greatly improve the fluorescent response.The prepared amplification label possesses good bioactivity,comparable detection limit and linear range,and acceptable storage stability.