超支化聚合物作为放大信号的纳米磁性微球电化学免疫分析法

Magnetic Nanospheres-based Electrochemical Immunoassay Amplified by Hyperbranched Polymer

提出了一种基于超支化聚合物(HBP)固化酶标二抗作为放大信号和纳米磁球相结合的超灵敏的免疫分析新方法。首先羧基纳米磁性微球共价键合乙肝抗体(HBsAb)形成免疫磁性微球,然后与待测乙肝表面抗原(HBsAg)发生特异性结合,加入HBP标记的酶标二抗(HBPS)与结合的抗原结合发生夹心反应。在外加磁场的作用下,抗体抗原免疫复合物易从样品溶液中分离,在含有邻氨基苯酚和H2O2的底液中,快速生成具有电活性的化合物3-氨基吩呃嗪,用示差脉冲伏安法(DPV)测定响应电流,电流强度(I)与乙肝表面抗原浓度(c)在0.05～10.0μg/L范围内呈线性关系,线性回归方程为I(μA)=0.140+16.80 c(μg/L),相关系数r=0.9995,检出限达0.008μg/L,并用于实际样品的测定。

A novel ultra-sensitive immunoassay for Hepatitis B surface antigen（HBsAg） was proposed.Magnetic nanosphere functionalized with carboxyl group was activated with 1-ethyl-3-（3-dimethylamino-propyl） carbodiimide hydrochloride/N-hydroxy-succinimimide（EDC/NHS）,and then Hepatitis B surface antibody（HBsAb） was covalently immobilized on the surface.Hepatitis B secondary antibody labeled with horseradish peroxide（HRP） was covalently linked to the hyperbranched polymer（HBP）.The antibody could capture the HBsAg when the antibody-modified magnetic nanosphere was incubated with HBsAg.Then the HBP conjugate was added,and a sandwich immunocomplex formed on the surface of magnetic nanospheres.The nanosphere with sandwich complex was separated magnetically from sample solution,and then incubated in the buffer solution containing 2-amino hydroxy benzene and hydrogen peroxide.The HRP could catalyze the reaction between 2-amino hydroxy benzene and hydrogen peroxide to produce electroactive product 2-hydroxy-3-amino phenoxazine.When using differential pulse voltammetry.the peak current was linear with the concentration of HBsAg in the range of 0.05 to 10 μg/L under the optimum conditions.The detection limit was found to be 0.008 μg/L,and the linear regression equation was I（μA）=0.140＋16.80C（μg/L） with r=0.9995.This method was applied to analyze real samples.