Characterization of multienzyme-antibody-carbon nanotube bioconjugates for immunosensors

Characterization studies of a multi-enzyme-antibody-carbon nanotube bioconjugate designed for the amplification of electrochemical immunosensing are described. Secondary antibodies for prostate specific antigen (PSA) were covalently linked to highly carboxylated multi-walled carbon nanotube (CNT) along with multiple horseradish peroxidase (HRP) enzyme labels. These bioconjugates provide ultra-sensitive amperometric detection of PSA on a single-wall carbon nanotube forest sandwich immunosensor platform. A single layer of HRP on the surface of the CNT was suggested by images from atomic force microscopy (AFM) and transmission electron microscopy (TEM). HRP on the bioconjugate surface was visualized by confocal microscopy using in-situ HRP-catalyzed polymerization yielding a fluorescent product, and HRP activity was estimated in a conventional assay. Binding of quantum-dot labeled PSA to antibodies on the bioconjugate was used for visualization by TEM. Combining TEM and enzyme activity results gave estimates of approximately 82 HRPs and 30 +/- 15 secondary antibodies per 100 nm of antibody-HRP-CNT bioconjugate.