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

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%.

Improving the Sensitivity of the miRNA Assay Coupled with the Mismatched Catalytic Hairpin Assembly Reaction by Optimization of Hairpin Annealing Conditions

“…The effect of the NaCl concentration in HP solutions during their annealing on the signal and background in mCHA is presented in Figure a,b. In the case of HP1, the addition of the NaCl concentration to 10 mM Tris, pH 7.2, which is commonly used as annealing buffer, ,, up to 300 mM resulted in a ∼2-fold decrease of the signal in the mCHA-based assay (Figure a). A further increase in the NaCl concentration up to 900 mM did not affect the signal intensity.…”