Magnetic Nanospheres-based Electrochemical Immunoassay Amplified by Hyperbranched Polymer Conjugate

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