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Label-free and highly sensitive APE1 detection based on rolling circle amplification combined with G-quadruplex.

Authors
  • Liu, Birong1
  • Yang, Zizhong1
  • Huang, Ting1
  • Li, Min-Min2
  • Duan, Wenjun1
  • Xie, Baoping3
  • Chen, Jin-Xiang4
  • Dai, Zong5
  • Chen, Jun6
  • 1 NMPA Key Laboratory for Research and Evaluation of Drug Metabolism, Guangdong Provincial Key Laboratory of New Drug Screening, School of Pharmaceutical Sciences, Southern Medical University, Guangzhou, 510515, China. , (China)
  • 2 Center of Clinical Laboratory, The First Affiliated Hospital of Jinan University, Guangzhou, 510630, PR China. , (China)
  • 3 NMPA Key Laboratory for Research and Evaluation of Drug Metabolism, Guangdong Provincial Key Laboratory of New Drug Screening, School of Pharmaceutical Sciences, Southern Medical University, Guangzhou, 510515, China. Electronic address: [email protected] , (China)
  • 4 NMPA Key Laboratory for Research and Evaluation of Drug Metabolism, Guangdong Provincial Key Laboratory of New Drug Screening, School of Pharmaceutical Sciences, Southern Medical University, Guangzhou, 510515, China. Electronic address: [email protected] , (China)
  • 5 Key Laboratory of Sensing Technology and Biomedical Instrument of Guangdong Province, School of Biomedical Engineering, Sun Yat-Sen University, Guangzhou, 510006, PR China. , (China)
  • 6 NMPA Key Laboratory for Research and Evaluation of Drug Metabolism, Guangdong Provincial Key Laboratory of New Drug Screening, School of Pharmaceutical Sciences, Southern Medical University, Guangzhou, 510515, China. Electronic address: [email protected] , (China)
Type
Published Article
Journal
Talanta
Publication Date
Mar 24, 2022
Volume
244
Pages
123404–123404
Identifiers
DOI: 10.1016/j.talanta.2022.123404
PMID: 35349840
Source
Medline
Keywords
Language
English
License
Unknown

Abstract

The highly sensitive detection of low-abundant apurinic/apyrimidinic endonuclease 1 (APE1) activity is of great significance for early diagnosis of disease and pathological research. Many methods for detecting APE1 based on isothermal nucleic acids amplification have been developed for improving its sensitivity. However, some of these methods have certain limitations, such as multiple reaction steps, narrow linear range, and complicated processes for fluorescent labeling. Herein, we develop a highly sensitive and label-free APE1 fluorescence detection method based on rolling circle amplification combined with G-quadruplex (RCA-G4). A hairpin probe (HP) labeled with the AP site can be recognized and cleaved by APE1, leading to the release of the primer sequence, which triggered RCA to produce long chain amplification products with a great amount of repeated sequences. The formed amplicon contains a series G-quadruplex structure, which can be combined with Thioflavin T (ThT) to produce fluorescence and achieve high sensitivity label-free detection of APE1. Benefit from the high amplification efficiency of RCA and the high fluorescence quantum yield of G-quadruplex/ThT, a detection limit as low as 1.52 × 10-6 U/mL and the linear range from 2 × 10-6 to 10 U/mL were obtained. The developed RCA-G4 method can be successfully used to detect APE1 in serum samples with a recovery from 96.3% to 105.7%. We believe that this approach is expected to play an important role in APE1-related disease research and drug development. Copyright © 2022 Elsevier B.V. All rights reserved.

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