Triple Amplification Strategy for the Improved Efficiency of a Microplate-Based Assay for the Chemiluminescent Detection of DNA

Significant research efforts are currently focused on advancing DNA detection methodology. Various nanoparticles (NPs), which are currently the most widely used solid-phase carriers for nucleic acid assays, have a number of essential drawbacks. Microtiter plates provide a simple and economical alternative to the NPs. This article reports the development of a sandwich assay for DNA detection using a microtiter plate as the solid carrier. A capture oligonucleotide modified with fluorescein was bound to the anti-fluorescein antibody adsorbed on the polystyrene microplate surface. A hepatitis B virus DNA fragment was used as the model analyte. To improve the assay sensitivity, the biotinylated reporter oligonucleotide and streptavidin-horseradish polyperoxidase (polyHRP) conjugate were used to provide an amplified detection system. Additional amplification was achieved because the peroxidase activity was measured by a chemiluminescent method using the 3-(10′-phenothiazinyl)propane-1-sulfonate/N-morpholinopyridine pair as an enhancing reagent. The detection limit of the developed assay was 0.9?pM with a linear dynamic range from 0.9 to 100?pM. This method may be used as a platform for the development of sensitive DNA assays.