Facile preparation of streptavidin-coated sephadex beads and their application to chemiluminescence detection of a target DNA

摘要

We report on an efficient strategy for the preparation of streptavidin (SA)-coated sephadex beads by the reductive amination of the aldehyde groups on the oxidized sephadex beads that were coupled to the free amino groups of SA. The resulting beads have a stable spherical shape with an average diameter of 20-100 mu m. The capacity of SA conjugated to 1 mg of these beads is, respectively, similar to 24 and similar to 5 times higher than that of commercially available SA-coated polystyrene and sepharose beads. The new beads were successfully applied to the chemiluminescence (CL) detection of telomer DNA via a sandwich-type hybridization assay that involves the following steps: (a) Target DNA is first hybridized with biotinylated capture cDNA immobilized on the SA-coated sephadex beads; (b) the hybrid is then sandwiched with a guanine-rich polymeric probe which (c), is sensitively detected by CL after mixing with a 3,4,5-trimethoxy-phenylglyoxal reagent at room temperature for 10 s. The entire assay can be completed within 2-3 h and has a linear range that extends from 5 to 200 nM concentrations of the target DNA. The lower detection limit is approximately 0.75 nM (1.3 ng center dot 100 mu L-1) in a test tube. Compared to other methods, this one has a wider linear range, a lower detection limit, and is faster. We presume that the new method and materials will be readily applicable to the sensitive detection of target DNA, and also in bead-based CL immunoassays.