A naked-eye biosensing system based on one-pot RPA-CRISPR/Cas12a driver G4-hemin self-assembly for Mycobacterium tuberculosis
IntroductionThe 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 instrumen...
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Frontiers Media S.A.
2025-08-01
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| Series: | Frontiers in Chemistry |
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| Online Access: | https://www.frontiersin.org/articles/10.3389/fchem.2025.1631086/full |
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| author | Ting Yuan Ting Yuan Jianbo Yuan Jianbo Yuan Jian Huang Jian Huang Nana Li Nana Li Huan Cai Yan Yang Juan Li Rui Chen Xun Min Xun Min |
| author_facet | Ting Yuan Ting Yuan Jianbo Yuan Jianbo Yuan Jian Huang Jian Huang Nana Li Nana Li Huan Cai Yan Yang Juan Li Rui Chen Xun Min Xun Min |
| author_sort | Ting Yuan |
| collection | DOAJ |
| description | IntroductionThe 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, 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.MethodsWe 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 min without requiring complex instrumentation.Results and DiscussionUnder 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 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. |
| format | Article |
| id | doaj-art-f9fa512ec9ad4a7bb64bf0e3bc375bfd |
| institution | Kabale University |
| issn | 2296-2646 |
| language | English |
| publishDate | 2025-08-01 |
| publisher | Frontiers Media S.A. |
| record_format | Article |
| series | Frontiers in Chemistry |
| spelling | doaj-art-f9fa512ec9ad4a7bb64bf0e3bc375bfd2025-08-20T03:40:46ZengFrontiers Media S.A.Frontiers in Chemistry2296-26462025-08-011310.3389/fchem.2025.16310861631086A naked-eye biosensing system based on one-pot RPA-CRISPR/Cas12a driver G4-hemin self-assembly for Mycobacterium tuberculosisTing Yuan0Ting Yuan1Jianbo Yuan2Jianbo Yuan3Jian Huang4Jian Huang5Nana Li6Nana Li7Huan Cai8Yan Yang9Juan Li10Rui Chen11Xun Min12Xun Min13Department of Laboratory Medicine, Affiliated Hospital of Zunyi Medical University, Zunyi, ChinaSchool of Laboratory Medicine, Zunyi Medical University, Zunyi, ChinaDepartment of Laboratory Medicine, Affiliated Hospital of Zunyi Medical University, Zunyi, ChinaSchool of Laboratory Medicine, Zunyi Medical University, Zunyi, ChinaDepartment of Laboratory Medicine, Affiliated Hospital of Zunyi Medical University, Zunyi, ChinaSchool of Laboratory Medicine, Zunyi Medical University, Zunyi, ChinaDepartment of Laboratory Medicine, Affiliated Hospital of Zunyi Medical University, Zunyi, ChinaSchool of Laboratory Medicine, Zunyi Medical University, Zunyi, ChinaKey Laboratory of Clinical Laboratory Diagnostics (Ministry of Education), College of Laboratory Medicine, Chongqing Medical University, Chongqing, ChinaDepartment of endocrinology, Affiliated Hospital of Zunyi Medical University, Zunyi, ChinaKey Laboratory of Clinical Laboratory Diagnostics (Ministry of Education), College of Laboratory Medicine, Chongqing Medical University, Chongqing, ChinaKey Laboratory of Clinical Laboratory Diagnostics (Ministry of Education), College of Laboratory Medicine, Chongqing Medical University, Chongqing, ChinaDepartment of Laboratory Medicine, Affiliated Hospital of Zunyi Medical University, Zunyi, ChinaSchool of Laboratory Medicine, Zunyi Medical University, Zunyi, ChinaIntroductionThe 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, 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.MethodsWe 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 min without requiring complex instrumentation.Results and DiscussionUnder 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 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.https://www.frontiersin.org/articles/10.3389/fchem.2025.1631086/fullRPA-CRISPR/Cas12aG4-hemin DNAzymenaked-eye biosensing systemone-potMycobacterium tuberculosis |
| spellingShingle | Ting Yuan Ting Yuan Jianbo Yuan Jianbo Yuan Jian Huang Jian Huang Nana Li Nana Li Huan Cai Yan Yang Juan Li Rui Chen Xun Min Xun Min A naked-eye biosensing system based on one-pot RPA-CRISPR/Cas12a driver G4-hemin self-assembly for Mycobacterium tuberculosis Frontiers in Chemistry RPA-CRISPR/Cas12a G4-hemin DNAzyme naked-eye biosensing system one-pot Mycobacterium tuberculosis |
| title | A naked-eye biosensing system based on one-pot RPA-CRISPR/Cas12a driver G4-hemin self-assembly for Mycobacterium tuberculosis |
| title_full | A naked-eye biosensing system based on one-pot RPA-CRISPR/Cas12a driver G4-hemin self-assembly for Mycobacterium tuberculosis |
| title_fullStr | A naked-eye biosensing system based on one-pot RPA-CRISPR/Cas12a driver G4-hemin self-assembly for Mycobacterium tuberculosis |
| title_full_unstemmed | A naked-eye biosensing system based on one-pot RPA-CRISPR/Cas12a driver G4-hemin self-assembly for Mycobacterium tuberculosis |
| title_short | A naked-eye biosensing system based on one-pot RPA-CRISPR/Cas12a driver G4-hemin self-assembly for Mycobacterium tuberculosis |
| title_sort | naked eye biosensing system based on one pot rpa crispr cas12a driver g4 hemin self assembly for mycobacterium tuberculosis |
| topic | RPA-CRISPR/Cas12a G4-hemin DNAzyme naked-eye biosensing system one-pot Mycobacterium tuberculosis |
| url | https://www.frontiersin.org/articles/10.3389/fchem.2025.1631086/full |
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