Increased Electrocatalyzed Performance through Dendrimer-Encapsulated Gold Nanoparticles and Carbon Nanotube-Assisted Multiple Bienzymatic Labels: Highly Sensitive Electrochemical Immunosensor for Protein Detection

A highly sensitive electrochemical carcinoembryonic antigen (CEA) immunosensor was fabricated by covalently immobilizing a monoclonal CEA antibody (anti-CEA, Ab(1)) and a mediator (thionine, Th) on a gold nanoparticle (AuNP)-encapsulated dendrimer (Den/AuNP). Multiwalled carbon nanotube (MWCNT)-supported secondary antibody (Ab(2))-conjugated multiple bienzymes, glucose oxidase (GOx), and horseradish peroxidase (HRP) (Ab(2)/MWCNT/GOx/HRP) were used as electrochemical labels. The highly sensitive detection was achieved by the increased HRP-electrocatalyzed reduction of hydrogen peroxide, which was locally generated by the enzyme GOx. The immunosensor surface was characterized using electrochemical impedance spectroscopy, atomic force microscopy, and quartz crystal microbalance techniques. The Den/AuNP and Ab(2)/MWCNT/GO(x)/HRP bioconjugates were characterized using high-resolution transmission electron microscopy, scanning electron microscopy, and X-ray photoelectron spectroscopy. Cyclic voltammetry and square wave voltammetry techniques were used to monitor the increased electrocatalyzed reduction of hydrogen peroxide by HRP. The linear dynamic range and the detection limit were determined to be 10.0 pg/mL to 50.0 ng/mL and 4.4 +/- 0.1 pg/mL, respectively. The validity of the immunosensor response was tested in various CEA-spiked human serum samples, and the results were compared to those of an enzyme-linked immunosorbent assay method.