Department of Chemistry, Lomonosov Moscow State University, 119991 Moscow, Russia.
Sensors (Basel). 2018 Apr 23;18(4):1289. doi: 10.3390/s18041289.
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/-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%.
首次合成了辣根过氧化物酶与含氨基寡核苷酸的共价缀合物。利用所得缀合物作为捕获试剂，建立了基于化学发光微滴定板的检测乙型肝炎病毒（HBV）DNA 35 -mer 片段的方法（概念验证分析物）。为了检测靶 DNA，使用了由生物素化报告寡核苷酸和 HRP-链霉亲和素缀合物组成的信号系统。该测定方法具有较高的灵敏度，这归因于增强的化学发光反应，其中 3-(10′-吩噻嗪基)丙烷-1-磺酸/-吗啉代吡啶对被用作增强剂。在优化条件下，该测定方法的检测限和工作范围分别为 3 pM 和 6-100 pM。该测定方法对靶的灵敏度为 1.6 × 10⁵ RLU/pM。在工作范围内测定 HBV DNA 的变异系数（CV）低于 6%。
