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

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 × 10(5) RLU/pM of target. The coefficient of variation (CV) for determination of HBV DNA within the working range was lower than 6%.