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

Abstract

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 μm. The capacity of SA conjugated to 1 mg of these beads is, respectively, ~24 and ~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 · 100 μL−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.

We describe a facile strategy for the preparation of streptavidin (SA)-coated sephadex beads with high capacity of SA. The prepared beads were applied to the chemiluminescence detection of a target DNA via a sandwich-type DNA hybridization assay. This assay exhibited high sensitivity and good specificity for the target DNA having a telomere sequence.

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Acknowledgments

This work was supported in part by Grants-in-Aid for Scientific Research from the Ministry of Education, Culture, Sports, Science, and Technology, Japan, and also supported partially by the Global Center of Excellence Program at Nagasaki University.

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Correspondence to Masaaki Kai.

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El-Mahdy, A.F.M., Ejupi, V., Shibata, T. et al. Facile preparation of streptavidin-coated sephadex beads and their application to chemiluminescence detection of a target DNA. Microchim Acta 182, 495–503 (2015). https://doi.org/10.1007/s00604-014-1348-9

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Keywords

  • Streptavidin-coated sephadex beads
  • Immobilization
  • Chemiluminescence detection
  • Hybridization
  • Target DNA