A new chemically amplified electrochemical system for the detection of biological affinity reactions: direct and competitive biotin assay

Abstract

Quantitation of biological affinity reactions by a newly developed chemically amplified electrochemical detection method was demonstrated with the biotin-avidin binding pair. In the method, ruthenium tris(2,2′-bipyridine) (Ru-bipy) was used as an electrochemical signal-generating tag. Its oxidation current on an indium tin oxide (ITO) electrode was amplified with a sacrificial electron donor, oxalate. Because oxalate itself produced negligible current on the electrode, the signal-to-background ratio was greatly enhanced in comparison with other chemical amplification systems. Although the Ru-bipy/oxalate redox couple has been employed previously in electrochemiluminescent and photoelectrochemical detection, its use in a catalytic amperometric detection of biological binding assays has not been reported. To implement the method in the detection of biotin–avidin recognition, avidin was immobilized on an ITO electrode, and was reacted with biotin in solution. Immobilization of avidin by passive adsorption was found to be relatively stable under the condition of the affinity reaction. In the direct assay, biotin labelled with Ru-bipy was recognized by avidin and accumulated on the electrode surface, which was then detected electrochemically in the presence of oxalate. A linear relationship between electrochemical current and biotin concentration was obtained in the range of 1–300 ng mL⁻¹. In the competitive assay, a mixed solution of unlabelled biotin (the analyte) of various concentrations and 100 ng mL⁻¹ labelled biotin was reacted with avidin on the surface. As the concentration of the unlabelled biotin increased, less labelled biotin bound to avidin, leading to a reduction in the electro-catalytical response of Ru-bipy. A detection limit of 1 ng mL⁻¹ biotin was obtained in the competitive assay, which is close to the sensitivity of some enzyme-labelled amperometric assays.