Studies On Reusable Electrochemical Immunosensor For Tumor Markers Based On Molecular Recognition Of Glycoprotein Antibody By Phenylboronic Acid Self-assembly Layer On Gold

Electrochemical immunosensor is one kind of labeled immunosensor, based on the combination of immunoassay and electrochemical detect technology. Through various kinds of methods, first, mount different antibody or antigen on the electrodes. The immuno-reaction between the mounting molecule indentifying mark and analyte or markers makes the electrochemical immunosensor surface changed. Then the change signal was transfer into electrochemical signal (electric current or electric pressure) via electrochemical immunosensor. So, the electrochemical immunosensor takes the advantage of both sensor technology and immunoreaction. It turns to be high sensitive, good specificity with a wild scope of application.Electrochemical immunosensor has been wildly applied in clinical diagnosis via detecting the concentration of tumor markers. Considering the detecting cost and efficiency, the electrochemical immunosensor become more and more minialurized, reusable and commercialized.In this article, two 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 glycoprotein antibody, enzyme conjugated carcinoembryonic antibody (HRP-anti-CEA) or prostate specific antigen (HRP-anti-PSA). Voltammetric and electrochemical impedance spectroscopic (EIS) studies and surface plasmon resonance (SPR) measurements show that the binding of HRP-anti-CEA or HRP-anti-PSA to the APBA interface is reversible and the HRP-anti-CEA, HRP-anti-PSA can be removed with an acidic buffer or a solution containing sorbitol. The bound enzyme-conjugated antibody can retain its enzyme catalytic activity to the reduction of hydrogen peroxide (H2O2) and its immunoactivity while binding with CEA or PSA to form an immuno complex. 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 electro- catalytic response of HRP-anti-CEA modified electrode over a CEA concentration range of 2.5 to 40.0 ng mL?1, HRP-anti-PSA modified electrode over a PSA concentration range of 2.0 to 45ng 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 or HRP-anti-PSA and be recognized by their antigen, through which a reusable immunosensor for CEA with an RSD of 7.1% for four cycles, for PSA with an RSD of 3.3% for ten cycles can be obtained. Under optimal conditions, the detection limit for the CEA, PSA immunoassay is 1.1 ng mL?1, 3.3 ng mL?1, separately(S/N=3). Serum CEA and PSA determination results, obtained with the proposed method, shows that the immunosensor has an acceptable accuracy.