A reusable electrochemical immunosensor for carcinoembryonic antigenvia molecular recognition of glycoproteinantibody by phenylboronic acid self-assembly layer on gold

Abstract

A reusable amperometric immunosensor based on the reversible boronic acid–sugar interaction is proposed. The immunosensor was prepared by self-assembling a thiol-mixed monolayer comprised of conjugates of 3-aminophenylboronic acid with 11-mercaptoundecanoic acid (APBA-MUA) and 11-mercapto-1-undecanol (MU) on gold. The resulting boronic acid coating layer can specifically bind with the glycoproteinantibody, enzyme conjugated carcinoembryonic antibody (HRP-anti-CEA). Voltammetric and electrochemical impedance spectroscopic (EIS) studies and surface plasmon resonance (SPR) measurements show that the binding of HRP-anti-CEA to the APBA interface is reversible and the HRP-anti-CEA can be removed with an acidic buffer or a solution containing sorbitol. The bound enzyme-conjugated antibody can retain its enzymecatalytic activity to the reduction of hydrogen peroxide (H₂O₂) and its immunoactivity while binding with CEA to form an immunocomplex. After the formation of the immunocomplex, the access of the active center of HRP to thionine was partially inhibited. This leads to a linear decrease in the electrocatalytic response of HRP-anti-CEA-modified electrode over a CEA concentration range of 2.5 to 40.0 ng mL⁻¹. After monitoring the immunoreaction signals, the immunocomplex can be easily removed from the APBA interface with a regeneration solution. This regenerated APBA interface can rebound with HRP-anti-CEA and be recognized by the antigen, through which a reusable immunosensor with an RSD of 7.1% for four cycles can be obtained. Under optimal conditions, the detection limit for the CEA immunoassay is 1.1 ng mL⁻¹, at three times background noise. Serum CEA determination results, obtained with the proposed method, shows that the immunosensor has an acceptable accuracy