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Microplate Chemiluminescent Assay for DNA Detection Using Apoperoxidase-Oligonucleotide as Capture Conjugate and HRP-Streptavidin Signaling System

Department of Chemistry, Lomonosov Moscow State University, 119991 Moscow, Russia
Sensors 2018, 18(4), 1289; https://doi.org/10.3390/s18041289
Received: 15 March 2018 / Revised: 19 April 2018 / Accepted: 19 April 2018 / Published: 23 April 2018
(This article belongs to the Special Issue Recent Advances in Nucleic Acid Sensors)
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Abstract

A covalent conjugate of horseradish apoperoxidase and amino-containing oligonucleotide was synthesized for the first time. Using the obtained conjugate as a capture reagent chemiluminescent microtiter plate-based assay for detection of 35-mer fragment of hepatitis B virus (HBV) DNA (proof-of-concept analyte) was developed. To detect the target DNA, a signaling system consisted of biotinylated reporter oligonucleotide and HRP-streptavidin conjugate was used. The high sensitivity of the assay was due to the enhanced chemiluminescence reaction, where 3-(10′-phenothiazinyl)propane-1-sulfonate/N-morpholinopyridine pair was used as an enhancer. Under the optimized conditions the limit of detection and a working range of the assay were 3 pM and 6–100 pM, respectively. The assay sensitivity was 1.6 × 105 RLU/pM of target. The coefficient of variation (CV) for determination of HBV DNA within the working range was lower than 6%. View Full-Text
Keywords: DNA; enzyme-linked assay; capture conjugate; peroxidase; chemiluminescence DNA; enzyme-linked assay; capture conjugate; peroxidase; chemiluminescence
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This is an open access article distributed under the Creative Commons Attribution License which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited (CC BY 4.0).

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Sakharov, I. Microplate Chemiluminescent Assay for DNA Detection Using Apoperoxidase-Oligonucleotide as Capture Conjugate and HRP-Streptavidin Signaling System. Sensors 2018, 18, 1289.

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