Functional fusion proteins and prevention of electrode fouling for a sensitive electrochemical immunosensor

Abstract A highly sensitive electrochemical immunosensor was developed by preventing electrode fouling and using a novel fusion protein of silica binding polypeptides (SBP)-protein G (ProG) created by recombinant DNA technology as a functional crosslinker for rapid and self-oriented immobilization of antibodies onto silica nanoparticles (SiNPs). Antibody immobilization onto the SiNPs by the SBP-ProG could rapidly be achieved without any chemical treatment. The immunosensor was fabricated through bonding of a partially gold-deposited cyclic olefin copolymer (COC) (top substrate) and gold patterned interdigitated array COC electrode (bottom substrate). To prevent electrode fouling, human immunoglobulin G (hIgG) was immobilized onto the ceiling inside the microchannel, instead of the bottom electrode. Alkaline phosphatase (AP)-labeled anti-hIgG was allowed to immunoreact with hIgG on the ceiling, followed by addition of an enzyme to generate an oxidative peak current. A three-fold increase in current was observed from the immunosensor without any electrode fouling compared with a control with the protein functionalized electrode. Also, the SiNPs facilely coated with AP-anti-hIgG via the SBP-ProG could increase the electrochemical signal up to 20% larger than that of the AP-anti-hIgG alone. Furthermore, this immunosensor was ultrasensitive with a detection limit of 0.68 pg/mL of a biomarker associated with prostate cancer.