Miniaturization of a Homogeneous Fluorescence Immunoassay Based on Energy Transfer Using Nanotiter Plates as High-Density Sample Carriers

The miniaturization of a homogeneous competitive immunoassay to a final assay volume of 70 nL is described. As the sample carrier, disposable plastic nanotiter plates (NTP) with dimensions of 2 × 2 cm2 containing 25 × 25 wells, corresponding to ~15 000 wells on a traditional 96-well microtiter plate footprint, were used. Sample handling was accomplished by a piezoelectrically actuated micropipet. To reduce evaporation while pipetting the assays, the NTP was handled in a closed humid chamber and cooled to the point of condensation. To avoid washing steps, a homogeneous assay was developed that was based on energy-transfer (ET). As a model system, an antibody-based assay for the detection of the environmentally relevant compound, simazine, in drinking water was chosen. Antibodies were labeled with the long-wavelength-excitable sulfoindocyanine dye Cy5 (donor), and a tracer was synthesized by labeling BSA with a triazine derivative and the acceptor dye Cy5.5. At low analyte concentrations, the tracer was preferably bound to the antibody binding sites. As a result of the close proximity of Cy5.5 and Cy5, an efficient quenching of the Cy5 fluorescence occurred. Higher analyte concentrations led to a progressive binding of the analyte to the antibody binding sites. The increased Cy5 fluorescence was determined by using a scanning laser-induced fluorescence detector. The limit of detection (LOD), using an antibody concentration of 20 nM, was 0.32 mug/L, or 1.11 × 10-16 mol of simazine. In comparison, the LOD of the 96-well microtiter-plate-based ET immunoassay (micro-ETIA) was 0.15 mug/L, or 1.87 × 10-13 mol. The LOD of the optimized micro-ETIA at 1 nM IgG, was 0.01 mug/L.