化学学报 ›› 2012, Vol. 70 ›› Issue (22): 2372-2376.DOI: 10.6023/A12080509 上一篇    

研究论文

基于氧化石墨烯信号放大的化学发光法联合检测甲胎蛋白和癌胚抗原

林洁华, 张慧慧, 楚鹏飞   

  1. 青岛科技大学化学与分子工程学院 青岛 266042
  • 收稿日期:2012-08-05 出版日期:2012-11-28 发布日期:2012-10-25
  • 通讯作者: 林洁华 E-mail:linjiehua@qust.edu.cn
  • 基金资助:
    项目受国家自然科学基金(No. 20905041)和山东省博士基金(No. BS2011SW008)资助.

A New Dual Immunoassay for the Determination of α-Fetoprotein and Carcinoembryonic Antigen Based on Chemiluminescence Signal Amplification by Functional Graphite Oxide

Lin Jiehua, Zhang Huihui, Chu Pengfei   

  1. College of Chemistry and Molecular Engineering, Qingdao University of Science and Technology, Qingdao 266042
  • Received:2012-08-05 Online:2012-11-28 Published:2012-10-25
  • Supported by:
    Project supported by the National Natural Science Foundation of China (No. 20905041), and the Doctoral Foundation of Shandong Province (No. BS2011SW008).

提出了一种基于信号放大技术的流动注射化学发光免疫传感器, 用于联合检测人血清中甲胎蛋白(AFP)和癌胚抗原(CEA)的含量. 该传感器首先用表面接枝羧基的氧化石墨烯来吸附固定辣根过氧化物酶(HRP)标记的二抗, 制得信号放大型免疫探针. 然后用合成的磁性介孔材料Fe3O4/SiO2来吸附固定AFP和CEA的单克隆抗体, 用磁铁将其吸附在两个检测池内壁. 当待检样品和信号放大型免疫探针陆续注射进入相应检测池, 发生夹心式免疫反应, 形成的免疫复合物被磁条吸附在检测池内壁. 随后加入鲁米诺和H2O2发光底物, 与酶标记物发生酶促增强的化学发光反应, 并通过控制设在两个管路上的开关来控制鲁米诺和H2O2的流通, 相继检测两个检测池中的化学发光信号, 从而实现两种待测物的同时检测.

关键词: 化学发光免疫分析, 流动注射, 氧化石墨烯, 辣根过氧化物酶, 信号放大

A sensitive dual immunoassay was proposed for the determination of carcinoembryonic antigen (CEA) and α-fetoprotein (AFP) based on signal amplification. Monoclonal antibodies of AFP and CEA were immobilized on magnetic mesoporous silica particles (Fe3O4/SiO2) respectively and prepared as the primary probe (AFP-Ab1/Fe3O4/SiO2 and CEA-Ab1/Fe3O4/SiO2). In order to improve the detection sensitivity, the current work was focused on the signal amplification by using increased amount of horseradish peroxidase (HRP). Graphite oxide (GO) with functioned carboxyl groups were used to immobilize the HRP labeled antibodies to prepare the secondary probes (AFP-HRP-Ab2/GO and CEA-HRP-Ab2/GO). The enhanced signals were attributed to a high HRP-tag molar ratio on the surface of GO. Two subminiature quartz flow cells were set upon the photomultiplier (PMT) in parallel. As soon as CEA-Ab1/Fe3O4/SiO2 and AFP-Ab1/Fe3O4/SiO2 were injected into the corresponding flow cell, they were adsorbed on the inside walls by the bar magnet. Then, the mixed solutions of CEA, AFP, AFP-HRP-Ab2/GO and CEA-HRP-Ab2/GO were injected into the two flow cells and stopped for 30 min. Based on a sandwich immunoassay format, the HRP tags were retained in the flow cells. Finally, the CL substrates of luminol and H2O2 were controlled to the two parallel flow cells, so the sequential determination of two tumor markers was achieved. Due to the increased amount of HRP on the surface of GO and the increased amount of monoclonal antibodies on Fe3O4/SiO2, the signals were largely amplified. The effects on the dual immunoassay, such as the concentrations of luminol and H2O2, the incubation time, and the pH value of the buffer solution, were investigated in the current work. Under the optimal conditions, typical flow injection CL signals have been obtained with the sandwich multiplexed immunoassay for CEA and AFP. AFP could be detected in the linear ranges 0.005~0.5 and 0.5~100 ng/mL. CEA could be detected in the linear ranges of 0.0025~1.0 and 1.0~80 ng/mL. The detection limits of AFP and CEA were 5.0 and 2.5 pg/mL, respectively. The proposed method was in favor of reducing sample consumption and facilitating the operation.

Key words: chemiluminescent immunoassay, flow injection analysis, graphite oxide, horseradish peroxidase, signal amplification