Triple Amplification Strategy for the Improved Efficiency of a Microplate-Based Assay for the Chemiluminescent Detection of DNAстатья
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Дата последнего поиска статьи во внешних источниках: 29 мая 2019 г.
Аннотация:Nowadays, considerable research efforts are focused on advancing DNA detection
methodology. Various nanoparticles (NPs), which are currently the most widely employed
solid-phase carriers for nucleic acid assays, have a number of essential drawbacks.
Microtiter plates provide a simple and economical alternative to the NPs. The present paper
reports the development of sandwich assay for DNA detection using microtiter plate as a
solid carrier. Capture oligonucleotide modified with fluorescein was bound to the antifluorescein
antibody adsorbed on the polystyrene microplate surface. Hepatitis B virus
(HBV) DNA fragment was used as a model analyte. To improve the assay sensitivity, the
biotinylated reporter oligonucleotide and streptavidin-horseradish polyperoxidase
(polyHRP) conjugate were used as an amplified detection system. Additional amplification
was achieved due to the fact that peroxidase activity was measured by chemiluminescent
method using 3-(10’-phenothiazinyl)propane-1-sulfonate/N-morpholinopyridine pair as a
enhancer. Detection limit of the developed assay was 0.9 pM, a linear range – from 0.9 to
100 pM. This method can be used as a platform for the development of sensitive
bio(DNA/apta)assays.