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One-pot microplate-based chemiluminescent assay coupled with catalytic hairpin assembly amplification for DNA detection

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Abstract

Nowadays, considerable efforts are focused on advancing DNA detection methods, which are extremely important in clinical diagnostics, pathogen determination, gene therapy, and forensic analysis. A one-pot sensitive microplate-based chemiluminescent assay coupled with catalytic hairpin assembly (CHA) amplification for detection of a 35-mer DNA oligonucleotide was developed. To improve the assay sensitivity, a triple amplification strategy based on the application of CHA (1), streptavidin-polyperoxidase conjugate (Stp-polyHRP) (2), and an enhanced chemiluminescent reaction (3) was used. The one-pot format of the assay, where all steps of the DNA determination are performed in the same well without transfer of samples from one test tube to another, increased its precision. The proposed assay detected the target DNA in the fM range and distinguished the target DNA from related DNAs, demonstrating its high sensitivity and high selectivity. Moreover, the assay was applied successfully for the quantitative determination of the target in spiked samples of human plasma. A microplate format of the assay was convenient for the analysis of a large number of samples. This study provides a prospective tool for DNA detection.

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Funding

This investigation was financially supported by the Russian Science Foundation (Grant No. 17-14-01042).

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Authors and Affiliations

  1. Department of Chemistry, Lomonosov Moscow State University, Leninskie Gory, Bldg. 1, Moscow, 119991, Russia

    Oleg L. Bodulev, Konstantin M. Burkin & Ivan Yu. Sakharov

  2. Russian Cardiology Research Center, Moscow, 121552, Russia

    Eugene E. Efremov

Authors
  1. Oleg L. Bodulev
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  2. Konstantin M. Burkin
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  3. Eugene E. Efremov
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  4. Ivan Yu. Sakharov
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Corresponding author

Correspondence to Ivan Yu. Sakharov.

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The research was approved by the Ethical Committee of the Russian Cardiology Research Center (Moscow, Russia). Informed consent was obtained from all individual participants included in the study.

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The authors declare that they have no conflict of interest.

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Published in the topical collection Euroanalysis XX with guest editor Sibel A. Ozkan.

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Bodulev, O.L., Burkin, K.M., Efremov, E.E. et al. One-pot microplate-based chemiluminescent assay coupled with catalytic hairpin assembly amplification for DNA detection. Anal Bioanal Chem 412, 5105–5111 (2020). https://doi.org/10.1007/s00216-020-02438-6

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  • DOI: https://doi.org/10.1007/s00216-020-02438-6

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