High sensitivity detection of Hepatitis B virus RNA based on 3D-DNA nanomachine and protein nanopore sensing
Abstract Serum Hepatitis B virus (HBV) RNA serves as a non-invasive biomarker for monitoring chronic hepatitis B and guiding treatment decisions. However, current diagnostic tests relying on Reverse Transcription Quantitative Polymerase Chain Reaction (RT-qPCR) require centralized facilities and ted...
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2025-08-01
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| Series: | Molecular Biomedicine |
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| Online Access: | https://doi.org/10.1186/s43556-025-00282-7 |
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| author | Shixin Yan Chuipeng Kong Jiazhe Cheng Zhuoyun Tang Ke Sun Shanchuan Chen Minghan Li Chengyan Tao Yue Li Yanhua Zhao Chuanmin Tao Jia Geng Feng Li |
| author_facet | Shixin Yan Chuipeng Kong Jiazhe Cheng Zhuoyun Tang Ke Sun Shanchuan Chen Minghan Li Chengyan Tao Yue Li Yanhua Zhao Chuanmin Tao Jia Geng Feng Li |
| author_sort | Shixin Yan |
| collection | DOAJ |
| description | Abstract Serum Hepatitis B virus (HBV) RNA serves as a non-invasive biomarker for monitoring chronic hepatitis B and guiding treatment decisions. However, current diagnostic tests relying on Reverse Transcription Quantitative Polymerase Chain Reaction (RT-qPCR) require centralized facilities and tedious operational steps, and there is currently no standardized HBV RNA detection method. Herein, we describe the rapid amplification-free detection of HBV RNA in clinical serum samples via a nanopore sensing method. Our method involves the design of a DNA nanomachine capable of translation of each target HBV RNA into a large amount of 7-nt single-strand DNA (ssDNA) as signal reporters that produce blockage features when passing through an alpha-hemolysin (α-HL) nanopore, the target can be quantified by identifying the frequency of this blockage occurrence. We demonstrate that this unique nanopore sensing feature not only distinguishes Signal Report (SR) DNA from interfering nucleic acids in clinical serum samples but also ensures a high assay sensitivity with a limit-of-detection (LOD) at 12.5 fM. The clinical potential of this method was validated through testing serum samples from 26 HBV-infected-patients and 7 healthy-participants. When compared with the RT-qPCR method, it showed only one false result, achieving an accuracy rate of 97.0%. Additionally, testing results for 18 cases during the treatment period demonstrated that our method’s conclusions on continued treatment and drug discontinuation were highly consistent with clinical diagnoses, with an accuracy rate of 100%. Therefore, the method developed in this study expands the clinical applications of 3D DNA nanomachine and nanopore sensing technology in clinical detection. |
| format | Article |
| id | doaj-art-31ef5f32aefb44ebb5c2e9fd3e2989d8 |
| institution | Kabale University |
| issn | 2662-8651 |
| language | English |
| publishDate | 2025-08-01 |
| publisher | Springer |
| record_format | Article |
| series | Molecular Biomedicine |
| spelling | doaj-art-31ef5f32aefb44ebb5c2e9fd3e2989d82025-08-20T04:01:42ZengSpringerMolecular Biomedicine2662-86512025-08-016111610.1186/s43556-025-00282-7High sensitivity detection of Hepatitis B virus RNA based on 3D-DNA nanomachine and protein nanopore sensingShixin Yan0Chuipeng Kong1Jiazhe Cheng2Zhuoyun Tang3Ke Sun4Shanchuan Chen5Minghan Li6Chengyan Tao7Yue Li8Yanhua Zhao9Chuanmin Tao10Jia Geng11Feng Li12Department of Laboratory Medicine, State Key Laboratory of Biotherapy and Cancer Center, West China Hospital, Sichuan University and Collaborative Innovation CenterKey Laboratory of Green Chemistry & Technology of Ministry of Education, College of Chemistry, Sichuan UniversityDepartment of Laboratory Medicine, State Key Laboratory of Biotherapy and Cancer Center, West China Hospital, Sichuan University and Collaborative Innovation CenterDepartment of Laboratory Medicine, West China Hospital, Sichuan UniversityDepartment of Laboratory Medicine, State Key Laboratory of Biotherapy and Cancer Center, West China Hospital, Sichuan University and Collaborative Innovation CenterDepartment of Laboratory Medicine, State Key Laboratory of Biotherapy and Cancer Center, West China Hospital, Sichuan University and Collaborative Innovation CenterDepartment of Laboratory Medicine, State Key Laboratory of Biotherapy and Cancer Center, West China Hospital, Sichuan University and Collaborative Innovation CenterDepartment of Laboratory Medicine, State Key Laboratory of Biotherapy and Cancer Center, West China Hospital, Sichuan University and Collaborative Innovation CenterDepartment of Laboratory Medicine, State Key Laboratory of Biotherapy and Cancer Center, West China Hospital, Sichuan University and Collaborative Innovation CenterDepartment of Laboratory Medicine, West China Hospital, Sichuan UniversityDepartment of Laboratory Medicine, West China Hospital, Sichuan UniversityDepartment of Laboratory Medicine, State Key Laboratory of Biotherapy and Cancer Center, West China Hospital, Sichuan University and Collaborative Innovation CenterKey Laboratory of Green Chemistry & Technology of Ministry of Education, College of Chemistry, Sichuan UniversityAbstract Serum Hepatitis B virus (HBV) RNA serves as a non-invasive biomarker for monitoring chronic hepatitis B and guiding treatment decisions. However, current diagnostic tests relying on Reverse Transcription Quantitative Polymerase Chain Reaction (RT-qPCR) require centralized facilities and tedious operational steps, and there is currently no standardized HBV RNA detection method. Herein, we describe the rapid amplification-free detection of HBV RNA in clinical serum samples via a nanopore sensing method. Our method involves the design of a DNA nanomachine capable of translation of each target HBV RNA into a large amount of 7-nt single-strand DNA (ssDNA) as signal reporters that produce blockage features when passing through an alpha-hemolysin (α-HL) nanopore, the target can be quantified by identifying the frequency of this blockage occurrence. We demonstrate that this unique nanopore sensing feature not only distinguishes Signal Report (SR) DNA from interfering nucleic acids in clinical serum samples but also ensures a high assay sensitivity with a limit-of-detection (LOD) at 12.5 fM. The clinical potential of this method was validated through testing serum samples from 26 HBV-infected-patients and 7 healthy-participants. When compared with the RT-qPCR method, it showed only one false result, achieving an accuracy rate of 97.0%. Additionally, testing results for 18 cases during the treatment period demonstrated that our method’s conclusions on continued treatment and drug discontinuation were highly consistent with clinical diagnoses, with an accuracy rate of 100%. Therefore, the method developed in this study expands the clinical applications of 3D DNA nanomachine and nanopore sensing technology in clinical detection.https://doi.org/10.1186/s43556-025-00282-73D-DNA nanomachineNanopore sensingHepatitis B virus RNAssDNAClinical detection |
| spellingShingle | Shixin Yan Chuipeng Kong Jiazhe Cheng Zhuoyun Tang Ke Sun Shanchuan Chen Minghan Li Chengyan Tao Yue Li Yanhua Zhao Chuanmin Tao Jia Geng Feng Li High sensitivity detection of Hepatitis B virus RNA based on 3D-DNA nanomachine and protein nanopore sensing Molecular Biomedicine 3D-DNA nanomachine Nanopore sensing Hepatitis B virus RNA ssDNA Clinical detection |
| title | High sensitivity detection of Hepatitis B virus RNA based on 3D-DNA nanomachine and protein nanopore sensing |
| title_full | High sensitivity detection of Hepatitis B virus RNA based on 3D-DNA nanomachine and protein nanopore sensing |
| title_fullStr | High sensitivity detection of Hepatitis B virus RNA based on 3D-DNA nanomachine and protein nanopore sensing |
| title_full_unstemmed | High sensitivity detection of Hepatitis B virus RNA based on 3D-DNA nanomachine and protein nanopore sensing |
| title_short | High sensitivity detection of Hepatitis B virus RNA based on 3D-DNA nanomachine and protein nanopore sensing |
| title_sort | high sensitivity detection of hepatitis b virus rna based on 3d dna nanomachine and protein nanopore sensing |
| topic | 3D-DNA nanomachine Nanopore sensing Hepatitis B virus RNA ssDNA Clinical detection |
| url | https://doi.org/10.1186/s43556-025-00282-7 |
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