{"status":"ok","message-type":"work","message-version":"1.0.0","message":{"indexed":{"date-parts":[[2026,9,28]],"date-time":"2026-09-28T19:20:28Z","timestamp":1790623228759,"version":"4.1.0"},"reference-count":41,"publisher":"Frontiers Media SA","license":[{"start":{"date-parts":[[2025,8,7]],"date-time":"2025-08-07T00:00:00Z","timestamp":1754524800000},"content-version":"vor","delay-in-days":0,"URL":"https:\/\/creativecommons.org\/licenses\/by\/4.0\/"}],"content-domain":{"domain":["frontiersin.org"],"crossmark-restriction":true},"short-container-title":["Front. Chem."],"abstract":"<jats:sec><jats:title>Introduction<\/jats:title><jats:p>The rapid and accurate identification of <jats:italic>Mycobacterium tuberculosis<\/jats:italic> (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, time-consuming 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.<\/jats:p><\/jats:sec><jats:sec><jats:title>Methods<\/jats:title><jats:p>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 trans-cleavage. 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\u00a0min without requiring complex instrumentation.<\/jats:p><\/jats:sec><jats:sec><jats:title>Results and Discussion<\/jats:title><jats:p>Under optimized conditions, the biosensing system achieved ultrasensitive naked-eye detection of MTB with a limit of detection (LOD) of 10 copies\/\u03bcL, 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 recombinase polymerase 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 point-of-care MTB nucleic acid testing in resource-limited settings.<\/jats:p><\/jats:sec>","DOI":"10.3389\/fchem.2025.1631086","type":"journal-article","created":{"date-parts":[[2025,8,7]],"date-time":"2025-08-07T05:29:42Z","timestamp":1754544582000},"update-policy":"https:\/\/doi.org\/10.3389\/crossmark-policy","source":"Crossref","is-referenced-by-count":4,"title":["A naked-eye biosensing system based on one-pot RPA-CRISPR\/Cas12a driver G4-hemin self-assembly for Mycobacterium tuberculosis"],"prefix":"10.3389","volume":"13","author":[{"given":"Ting","family":"Yuan","sequence":"first","affiliation":[],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Jianbo","family":"Yuan","sequence":"additional","affiliation":[],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Jian","family":"Huang","sequence":"additional","affiliation":[],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Nana","family":"Li","sequence":"additional","affiliation":[],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Huan","family":"Cai","sequence":"additional","affiliation":[],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Yan","family":"Yang","sequence":"additional","affiliation":[],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Juan","family":"Li","sequence":"additional","affiliation":[],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Rui","family":"Chen","sequence":"additional","affiliation":[],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Xun","family":"Min","sequence":"additional","affiliation":[],"role":[{"vocabulary":"crossref","role":"author"}]}],"member":"1965","published-online":{"date-parts":[[2025,8,7]]},"reference":[{"key":"B1","doi-asserted-by":"publisher","first-page":"742370","DOI":"10.3389\/fimmu.2021.742370","article-title":"Mycobacterium tuberculosis exploits focal adhesion kinase to induce necrotic cell death and inhibit reactive oxygen species production","volume":"12","author":"Afriyie-Asante","year":"2021","journal-title":"Front. 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