Abstract
A novel automated flow-based immunosensor has been developed and validated for the measurement of the breast cancer prognostic marker 2'-deoxycytidine (dCyd) in plasma. The sensor employed the kinetic-exclusion analytical technology using the KinExA (TM) 3200 instrument. Various concentrations of dCyd were incubated with a fixed amount of mouse anti-dCyd monoclonal antibody until binding reaction reached equilibrium. These solutions were then passed rapidly over dCyd-bovine serum albumin conjugate (dCyd-BSA) coated onto polymethylmethacrylate beads contained in the observation cell of the KinExA instrument. The free anti-dCyd antibody was bound to the immobilized dCyd-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. The calibration curve was generated by plotting the fluorescence signals that were retained on the beads as a function of dCyd concentrations. The assay limit of detection was 20nM, and the working range of the assay was 20-2200 nM. The analytical recovery of plasma-spiked dCyd was 94.8-107.4 +/- 2.5-8.4%. The precision of the sensor was satisfactory; RSD was 3.6-6.2 and 5.2-7.5% 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 dCyd. The proposed sensor is anticipated to have a great value in measurement of dCyd where a more confident result is needed.