• antigens;
  • biosensors;
  • conjugation;
  • electrochemistry;
  • immunoassays


A homogeneous electrochemical immunoassay is based on the interaction of osmium–antigen conjugate with its antibody. The novelty presented herein is the direct conjugation of the osmium complex to a small antigen and the application of the quantitative analysis of the antigen and its antibody as the electrical signal for homogeneous immunoassay. The small antigen chosen is hippuric acid (HA), a major urinary metabolite in toluene-exposed humans. As a redox mediator, [Os(4,4′-dimethoxy-2,2′-bipyridine)2(4-aminomethylpyridine-HA)Cl]+/2+ (Os-HA antigen) has been synthesized and characterized on screen-printed carbon electrodes. The synthesized Os-HA antigen shows reversible redox peaks at E1/2=0.056 V versus Ag/AgCl. The homogeneous competitive immunoassay relies on the interaction between Os-HA antigen conjugate and free antigen to its antibody, which can generate electrical signals linearly proportional to the free antigen monitored by cyclic voltammetry and differential pulse voltammetry in the range of 10 μg mL−1 to 5.12 mg mL−1. The cutoff concentration of HA in urine samples is 2.0 mg mL−1, so the method can be used to develop a HA immunosensor. Moreover, the proposed homogeneous electrochemical immunoassay method can be applied to detect low concentrations of small antigens found in the healthcare area.