Horseradish peroxidase-loaded nanospheres attached to hollow gold nanoparticles as signal enhancers in an ultrasensitive immunoassay for alpha-fetoprotein

We report on a novel electrochemical signal amplification strategy for use in immunoassays. The highly responsive immunoelectrode was constructed in the following way: (1) The surface of a gold electrode was covered with a layer single-walled carbon nanotubes dispersed in chitosane functionalized with L-cysteine; (2) Gold nanoparticles containing protein A and anti-alpha-fetoprotein (anti-AFP) were then covalently attached to the surface via the thiol groups of the chitosane. The electrode is then exposed to the analyte (AFP) which then is bound by the antibody. In the next step, a gold-conjugated secondary antibody is added that was prepared in the following way: (1) Horseradish peroxidase was crosslinked and the resulting spheres were coated with hollow gold nanoparticles (hollow Au-NPs) to give nanospheres of ~100 nm in diameter. (2) These were the coated with thionine and, in a last step, with secondary antibody. The use of these materials has several attractive features: The HRP-NPs functionalized with hollow Au-NPs possess a large surface area that can load the large amount of secondary antibody. Thionine (Thi) is highly redox active and improves the intensity of the signal. Carbon nanotubes were used because they possess an excellent electron transfer rate and large surface area. Following incubation of the modified electrode (a) with a sample containing AFP, (b) then with the secondary antibody, and (c) with washing buffer, the electrode is placed in a solution containing H₂O₂. The HRP in the smart secondary antibody causes the catalytic decomposition of H₂O₂ and the results in an electrical current that is linearly related to the concentration of AFP in the 0.025 to 5.0 ng mL^?1 concentration range. The detection limit for AFP is as low as 8.3 pg mL^?1. We believe that this novel kind of immunoassay represents a promising tool for use in sensitive clinical assays.