A highly sensitive automated flow immunosensor based on kinetic exclusion analysis for determination of the cancer marker 8-hydroxy-2′-deoxyguanosine in urine

Abstract

A highly sensitive automated flow immunosensor has been developed and validated for the measurement of the cancer marker 8-hydroxy-2′-deoxyguanosine (8-OHdG) in urine. The sensor employed the kinetic-exclusion analytical technology using the KinExA™ 3200 instrument. Various concentrations of 8-OHdG were incubated with a fixed amount of mouse anti-8-OHdG monoclonal antibody until the binding reaction reached equilibrium. These solutions were then passed rapidly over a bovine serum albumin conjugate of 8-hydroxyguanosine (8-OHG–BSA) coated onto polymethylmethacrylate beads contained in the observation cell of the KinExA instrument. The free anti-8-OHdG antibody was bound to the immobilized 8-OHG–BSA, however the unbound reagents were removed from the beads bed by flushing the system with phosphate-buffered saline. Fluorescent-labeled secondary antibody was passed rapidly over the beads bed, and the fluorescence was recorded during the flow of the secondary antibody through the beads. A calibration curve was generated by plotting the fluorescence signals that were retained on the beads as a function of 8-OHdG concentrations. The assay limit of detection was 0.05 ng ml⁻¹ and the working range of the assay was 0.05–5 ng ml⁻¹. The analytical recovery of urine-spiked 8-OHdG was 96.05–106.55 ± 3.00–6.40%. The precision of the sensor was satisfactory; relative standard deviation values were 3.28–4.89 and 4.40–6.89% for the intra- and inter-assay precision, respectively. The analytical performance of the proposed sensor was found to be superior to the conventional enzyme-linked immunosorbent assay for 8-OHdG. The proposed sensor is anticipated to have a great value in measurement of 8-OHdG where a more confident result is needed.