Document Type: Reasearch Paper
Laboratory of Microanalysis, Institute of Biochemistry & Biophysics, University of Tehran, Tehran, Iran.
Biotechnology Research Center, Research Institute of Petroleum Industry (RIPI), Tehran, Iran.
A new signal amplification strategy based on simultaneous application of gold nanoparticles (AuNPs) and horseradish peroxidase (HRP) was employed to improve the sensitivity of an electrochemical immunoassay for detection of human IgG (hIgG), as a model antigenic protein. This immunoassay system was fabricated on magnetic carboxyl-functionalized multi-walled carbon nanotubes (COOH-MWCNT/Fe3O4 nanocomposite). The COOH-MWCNT/Fe3O4 nanocomposite was constructed by chemical co-precipitation of Fe2+ and Fe3+ in alkaline solution in the presence of the COOH-MWCNT. Goat anti-human IgG (anti-hIgG) was covalently bound to the carbon nanotubes and the resulting anti-hIgG bearing nanocomposite was immobilized on the surface of a gold electrode using a permanent magnet. Human IgG (hIgG) as an antigen was detected electrochemically using a secondary HRP-conjugated anti-hIgG immobilized noncovalently on the surface of AuNPs. Electrochemical detection of hIgG was performed in the presence of H2O2 and KI as substrates of HRP. When the concentration of antigen was 200ng/ml, the sandwich arrangement with AuNPs amplified electrochemical response 56 µA more than a same sandwich arrangement without AuNPs. Binding of HRP-conjugated anti-hIgG to AuNPs was studied by UV–Visible and ﬂuorescence spectrophotometry. The spectrophotometric data showed that binding of antibody molecules to AuNPs occurred without any significant structural changes.