Sensitive and selective DNA detecting electrochemical sensor via double cleaving CRISPR Cas12a and dual polymerization on hyperbranched rolling circle amplification

Juneseok You, Hyunjun Park, Hakbeom Lee, Kuewhan Jang, Jinsung Park, Sungsoo Na

Research output: Contribution to journalArticlepeer-review

18 Citations (Scopus)

Abstract

Electrochemical sensors are widely used for nucleic acid detection. However, they exhibit low sensitivity and specificity. To overcome these limitations, DNA amplification method is necessary. In this study, we introduced CRISPR (Clustered regularly interspaced short palindromic repeats) Cas12a-dependent hyperbranched rolling circle amplification (HRCA) into an electrochemical sensor platform. By resolving the existing false-positive issue of HRCA, CRISPR Cas12a determines the real positive amplification that able to enhance its sensitivity for extremely low concentrations of nucleic acids and specificity for single-point mutations. In detail, CRISPR Cas12a, which activates the nucleic acid amplification reaction, was used for both trans and cis cleavage for the first time. Finally, selectively amplified DNA was detected using a screen-printed electrode. Using the change in surface coverage by DNA, the electrochemical sensor detected a decrease in the redox signal. In summary, combining a novel DNA amplification method and electrochemical sensor platform, our proposed method compensates for the shortcomings of existing RCA and hyperbranched RCA, secures a high sensitivity of 10 aM, and overcomes false-positivity problems. Moreover, such creative applications of CRISPR Cas12a may lead to the expansion of its applications to other nucleic acid amplification methods.

Original languageEnglish
Article number115078
JournalBiosensors and Bioelectronics
Volume224
DOIs
Publication statusPublished - 2023 Mar 15

Bibliographical note

Funding Information:
This study was supported by the National Research Foundation of Korea (NRF) , under grant numbers NRF-2016R1A5A1010148 and NRF-2020R1F1A1075581 , and a Korea University Grant.

Publisher Copyright:
© 2023 Elsevier B.V.

Keywords

  • CRISPR (Clustered regularly interspaced short palindromic repeats) Cas12a
  • Electrochemical sensor
  • Hyperbranched rolling circle amplification (HRCA)

ASJC Scopus subject areas

  • Biotechnology
  • Biophysics
  • Biomedical Engineering
  • Electrochemistry

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