Development of Amperometric Immunosensor Using Boron-Doped Diamond with Poly(o-aminobenzoic acid)

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Abstract
An alternative method of a protein immunosensor has been developed at boron-doped diamond (BDD) electrode material. In order to construct the base of the immunosensor, o-aminobenzoic acid (o-ABA) was electropolymerized at an electrode by cyclic voltammetry. The poly-o-ABA-modified BDD was characterized by scanning electron microscopy (SEM) and X-ray photoelectron spectroscopy (XPS). The XPS result found that carboxyl groups were formed at the electrode surface. The carboxyl groups were then used to covalently attach protein probes. The amperometric sensing of mouse IgG (MIgG) was selected as the model at the poly-o-ABA-modified BDD to compare to the poly-o-ABA-modified glassy carbon (GC) at the same condition. An antimouse IgG from goat (GaMIgG) was covalently immobilized at a poly-o-ABA-modified BDD electrode which used a sandwich-type alkaline phosphatase (ALP) catalyzing amperometric immunoassay with 2-phospho-l-ascorbic acid (AAP) as substrate. The ALP enzyme conjugated at the immunosensor can generate AAP to the electroactive species of ascorbic acid (AA), which can be determined by amperometric detection. The signal was found to be proportional with the quantity of MIgG. The limits of detection (LODs) of 0.30 (3 SD) and 3.50 ng mL-1 (3 SD) for MIgG at BDD and GC electrodes were obtained. It also was found that the dynamic range of 3 orders of magnitude (1−1000 ng mL-1) was obtained at BDD, whereas at GC, the dynamic range was more narrow (10−500 ng mL-1). The method was applied to a real mouse serum sample that contains MIgG.

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