ORIGINAL RESEARCH article
Front. Chem.
Sec. Analytical Chemistry
Volume 13 - 2025 | doi: 10.3389/fchem.2025.1631086
This article is part of the Research TopicEmerging Trends in Biosensors: Bridging Chemistry and Practical ApplicationsView all articles
A Naked-eye Biosensing System based on One-Pot RPA-CRISPR/Cas12a Driver G4-hemin Self-Assembly for Mycobacterium Tuberculosis
Provisionally accepted
- 1Zunyi Medical University, Zunyi, China
- 2Affiliated Hospital of Zunyi Medical University, Zunyi, China
- 3Chongqing Medical University, Chongqing, China
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Introduction：The rapid and accurate identification of Mycobacterium tuberculosis (MTB) is essential for effective tuberculosis (TB) control. However, conventional diagnostic methods for MTB suffer from limitations such as low sensitivity, poor specificity, high cost, reliance on specialized instruments, and complex, timeconsuming procedures. To address these challenges, there is an urgent need for a simple, rapid, and highly sensitive detection method that can be deployed in point-of-care settings.2 Methods ： We developed a one-pot biosensing system combining recombinase polymerase amplification (RPA) and CRISPR/Cas12a-driven G4-hemin self-assembly for the colorimetric detection of MTB. Glycerol was employed as a phase-separation barrier to prevent interference between RPA amplification and CRISPR/Cas12a transcleavage. A single-stranded DNA (ssDNA) probe, designed to self-assemble with ssDNA-hemin into G4-hemin nanozymes upon CRISPR/Cas12a-mediated cleavage, served as the reaction substrate. The ssDNA-hemin further enhanced the catalytic activity of the generated G4-hemin DNAzyme. The entire assay was completed in a single step within 60 minutes without requiring complex instrumentation.Results and Discussion：Under optimized conditions, the biosensing system achieved ultrasensitive naked-eye detection of MTB with a limit of detection (LOD) of 10 copies/μL, comparable to traditional four-step fluorescent assays. Clinical validation using 104 patient samples demonstrated high concordance with standard diagnostic methods. This approach combines the advantages of rapid amplification (RPA), CRISPR/Cas12a specificity, and G4-hemin DNAzyme-based colorimetric signal amplification, enabling simple, equipment-free visual detection. Given its speed, sensitivity, and ease of use, this biosensing system holds significant promise for pointof-care MTB nucleic acid testing in resource-limited settings.
Keywords: RPA-CRISPR/Cas12a, G4-hemin DNAzyme, naked-eye biosensing System, One-pot, Mycobacterium tuberculosis
Received: 22 May 2025; Accepted: 28 Jul 2025.
Copyright: © 2025 Yuan, Yuan, Huang, Li, Cai, Yang, Li, Chen and Min. This is an open-access article distributed under the terms of the Creative Commons Attribution License (CC BY). The use, distribution or reproduction in other forums is permitted, provided the original author(s) or licensor are credited and that the original publication in this journal is cited, in accordance with accepted academic practice. No use, distribution or reproduction is permitted which does not comply with these terms.
* Correspondence:
Rui Chen, Chongqing Medical University, Chongqing, China
Xun Min, Zunyi Medical University, Zunyi, China
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