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Qi Y, Yu J, Lou M, Yu Y, Li R, Zhang Z, Dai Y, Lao K, Cao M, Gou X. Lab on a single microbead: An enzyme-free strategy for the sensitive detection of microRNA via efficient localized catalytic hairpin assembly. Anal Chim Acta 2025; 1340:343659. [PMID: 39863312 DOI: 10.1016/j.aca.2025.343659] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 12/03/2024] [Accepted: 01/11/2025] [Indexed: 01/27/2025]
Abstract
BACKGROUND Accurate quantification of microRNA (miRNA) is of great significance because it provides opportunities for the accurate early diagnosis of a series of human diseases including cancers. Currently, complicated nucleic acid amplification technologies are always required for the highly sensitive miRNA detection. The introduction of nucleic acid signal amplification coupled with various enzymes will inevitably lead to tedious work and increase the complexity of the analysis process. It is still urgently desired to develop enzyme-free yet sensitive assays that enable the sensitive analysis of miRNA in complicated biological samples. RESULTS A single microbead (MB)-based localized catalytic hairpin assembly (CHA) strategy is proposed for the sensitive analysis of microRNA (miRNA). This rationally designed CHA strategy allows target miRNA to walk only on a single MB which can create a micro-amplification zone, initiating a highly efficient localized CHA reaction, generating a large number of fluorescent DNA duplexes on the surface of single MB. The efficient localized CHA on single MB can not only greatly suppress the nonspecific reaction between two hairpin probes, thus decreasing the background signal, but also greatly enhance the brightness of MB owing to the highly-concentrated fluorescence enrichment on only one MB. Therefore, highly sensitive quantification of miRNA has been achieved by measuring the fluorescence signal on MB using a confocal fluorescence microscope. This new strategy exhibits a detection limit of 1.09 pM for let-7a detection, and enables high specificity of distinguishing homologous miRNA family members. SIGNIFICANCE This is the first report by only using one single MB as a carrier to conduct localized CHA, rendering highly-concentrated fluorescence enrichment on only one MB and a dramatic increase in sensitivity. This single MB-based localized CHA strategy has been successfully applied to the accurate analysis of miRNA target in complex biological sample.
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Affiliation(s)
- Yan Qi
- Institute of Basic and Translational Medicine & Shaanxi Key Laboratory of Brain Disorders, Xi'an Medical University, Xi'an, 710021, Shaanxi Province, PR China; Engineering Research Center of Brain Diseases Drug Development, Universities of Shaanxi Province, Xi'an Medical University, Xi'an, 710021, Shaanxi Province, PR China
| | - Jiangtao Yu
- Institute of Basic and Translational Medicine & Shaanxi Key Laboratory of Brain Disorders, Xi'an Medical University, Xi'an, 710021, Shaanxi Province, PR China; Engineering Research Center of Brain Diseases Drug Development, Universities of Shaanxi Province, Xi'an Medical University, Xi'an, 710021, Shaanxi Province, PR China
| | - Ming Lou
- Stomatology College of Xi'an Medical University, Xi'an Medical University, Xi'an, 710021, Shaanxi Province, PR China
| | - Yameng Yu
- Institute of Basic and Translational Medicine & Shaanxi Key Laboratory of Brain Disorders, Xi'an Medical University, Xi'an, 710021, Shaanxi Province, PR China; Engineering Research Center of Brain Diseases Drug Development, Universities of Shaanxi Province, Xi'an Medical University, Xi'an, 710021, Shaanxi Province, PR China
| | - Ruohan Li
- Yantai Yongqi Technical Consulting Service Co., Ltd., Yantai, 264003, Shandong Province, PR China
| | - Zhenhao Zhang
- Institute of Basic and Translational Medicine & Shaanxi Key Laboratory of Brain Disorders, Xi'an Medical University, Xi'an, 710021, Shaanxi Province, PR China; Engineering Research Center of Brain Diseases Drug Development, Universities of Shaanxi Province, Xi'an Medical University, Xi'an, 710021, Shaanxi Province, PR China
| | - Yuxuan Dai
- Institute of Basic and Translational Medicine & Shaanxi Key Laboratory of Brain Disorders, Xi'an Medical University, Xi'an, 710021, Shaanxi Province, PR China; Engineering Research Center of Brain Diseases Drug Development, Universities of Shaanxi Province, Xi'an Medical University, Xi'an, 710021, Shaanxi Province, PR China
| | - Kejing Lao
- Institute of Basic and Translational Medicine & Shaanxi Key Laboratory of Brain Disorders, Xi'an Medical University, Xi'an, 710021, Shaanxi Province, PR China; Engineering Research Center of Brain Diseases Drug Development, Universities of Shaanxi Province, Xi'an Medical University, Xi'an, 710021, Shaanxi Province, PR China
| | - Meng Cao
- Institute of Basic and Translational Medicine & Shaanxi Key Laboratory of Brain Disorders, Xi'an Medical University, Xi'an, 710021, Shaanxi Province, PR China; Engineering Research Center of Brain Diseases Drug Development, Universities of Shaanxi Province, Xi'an Medical University, Xi'an, 710021, Shaanxi Province, PR China
| | - Xingchun Gou
- Institute of Basic and Translational Medicine & Shaanxi Key Laboratory of Brain Disorders, Xi'an Medical University, Xi'an, 710021, Shaanxi Province, PR China; Engineering Research Center of Brain Diseases Drug Development, Universities of Shaanxi Province, Xi'an Medical University, Xi'an, 710021, Shaanxi Province, PR China.
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Wang J, Cui X, Wang W, Wang J, Zhang Q, Guo X, Liang Y, Lin S, Chu B, Cui D. Microfluidic-based electrically driven particle manipulation techniques for biomedical applications. RSC Adv 2025; 15:167-198. [PMID: 39758908 PMCID: PMC11697266 DOI: 10.1039/d4ra05571c] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [Grants] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 08/01/2024] [Accepted: 09/29/2024] [Indexed: 01/07/2025] Open
Abstract
Microfluidic chips exhibit unique advantages in both economy and rapidity, particularly for the separation and detection of biomolecules. In this review, we first introduced the mechanisms of several electrically driven methods, such as electrophoresis, dielectrophoresis, electro-wetting and electro-rotation. We then discussed in detail the application of these methods in nucleic acid analysis, protein manipulation and cell treatment. In addition, we outlined the considerations for material selection, manufacturing processes and structural design of microfluidic chips based on electrically driven mechanisms.
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Affiliation(s)
- Jiulin Wang
- Institute of Nano Biomedicine and Engineering, School of Sensing Science and Engineering, School of Electronic Information and Electrical Engineering, Shanghai JiaoTong University Shanghai 200240 PR China
| | - Xinyuan Cui
- Department of Radiology, Ruijin Hospital, Shanghai Jiao Tong University School of Medicine Shanghai 200025 PR China
| | - Wei Wang
- Institute of Nano Biomedicine and Engineering, School of Sensing Science and Engineering, School of Electronic Information and Electrical Engineering, Shanghai JiaoTong University Shanghai 200240 PR China
| | - Junhao Wang
- Institute of Nano Biomedicine and Engineering, School of Sensing Science and Engineering, School of Electronic Information and Electrical Engineering, Shanghai JiaoTong University Shanghai 200240 PR China
| | - Quili Zhang
- First Affiliated Hospital, Henan University School of Medicine Kaifeng 475000 PR China
| | - Xiaonan Guo
- First Affiliated Hospital, Henan University School of Medicine Kaifeng 475000 PR China
| | - Yanfeng Liang
- First Affiliated Hospital, Henan University School of Medicine Kaifeng 475000 PR China
| | - Shujin Lin
- Institute of Nano Biomedicine and Engineering, School of Sensing Science and Engineering, School of Electronic Information and Electrical Engineering, Shanghai JiaoTong University Shanghai 200240 PR China
| | - Bingfeng Chu
- Department of Stomatology, The First Medical Centre, Chinese PLA General Hospital Beijing 100853 PR China
| | - Daxiang Cui
- Institute of Nano Biomedicine and Engineering, School of Sensing Science and Engineering, School of Electronic Information and Electrical Engineering, Shanghai JiaoTong University Shanghai 200240 PR China
- First Affiliated Hospital, Henan University School of Medicine Kaifeng 475000 PR China
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3
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Chai H, Shi J, Zhuang Y, Miao P. Assembly of ligation chain reaction and DNA triangular prism for miRNA diagnostics. Biosens Bioelectron 2024; 262:116551. [PMID: 38971039 DOI: 10.1016/j.bios.2024.116551] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 12/06/2023] [Revised: 05/21/2024] [Accepted: 07/03/2024] [Indexed: 07/08/2024]
Abstract
Controllable assembly of DNA nanostructure provides a powerful way for quantitative analysis of various targets including nucleic acid molecules. In this study, we have designed detachable DNA nanostructures at electrochemical sensing interface and constructed a ligation chain reaction (LCR) strategy for amplified detection of miRNA. A three-dimensional DNA triangular prism nanostructure is fabricated to provide suitable molecule recognition environment, which can be further regenerated for additional tests via convenient pH adjustment. Target triggered LCR is highly efficient and specific towards target miRNA. Under optimal experimental conditions, this approach enables ultrasensitive exploration in a wide linear range with a single-base resolution. Moreover, it shows excellent performances for the analysis of cell samples and clinical serum samples.
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Affiliation(s)
- Hua Chai
- Suzhou Institute of Biomedical Engineering and Technology, Chinese Academy of Sciences, Suzhou, 215163, China
| | - Jiayue Shi
- Suzhou Institute of Biomedical Engineering and Technology, Chinese Academy of Sciences, Suzhou, 215163, China
| | - Yuan Zhuang
- Affiliated Hospital of Nantong University, Nantong, 226001, China.
| | - Peng Miao
- Suzhou Institute of Biomedical Engineering and Technology, Chinese Academy of Sciences, Suzhou, 215163, China.
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Qi Y, Sun J, Wang S, Ren W, Liu C. Mix-and-Read Digital MicroRNA Analysis Based on Flow Cytometric Counting of Target-Clicked Nanobead Dimer. Anal Chem 2024; 96:13734-13741. [PMID: 39114879 DOI: 10.1021/acs.analchem.4c03092] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 08/21/2024]
Abstract
A one-step, enzyme-free, and highly sensitive digital microRNA (miRNA) assay is rationally devised based on flow cytometric counting of target miRNA-clicked nanobead dimers via a facile mix-and-read manner. In this strategy, highly efficient miRNA-sandwiched click chemical ligation of two DNA probes may remarkably stabilize and boost the dimer formation between two kinds of fluorescence-coded nanobeads, and the number of as-produced bead dimers will be target dose-responsive, particularly when the trace number of miRNA is much less than that of employed nanobeads. Finally, each fluorescence-coded bead dimer can be easily identified and digitally counted by a powerful flow cytometer (FCM) and accordingly, the amount of target miRNA can be accurately quantified in a digital way. This new digital miRNA assay can be accomplished with a facile mix-and-read operation just by simply mixing the target miRNA with two kinds of preprepared DNA probe-functionalized nanobeads, which do not require any nucleic acid amplification, purification, and complex operation procedures. In spite of the extremely simple one-step operation, benefiting from the low-background but high target-mediated click ligation efficiency, and the powerfully digital statistical capability of FCM, this strategy achieves high sensitivity with a quite low detection limit of 5.2 fM target miRNA as well as high specificity and good generality for miRNA analysis, pioneering a new direction for fabricating digital bioassays.
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Affiliation(s)
- Yan Qi
- Institute of Basic and Translational Medicine & Shaanxi Key Laboratory of Brain Disorders, Xi'an Medical University, Xi'an 710021, Shaanxi Province, P. R. China
- Engineering Research Center of Brain Diseases Drug Development, Universities of Shaanxi Province, Xi'an Medical University, Xi'an 710021, Shaanxi Province, P. R. China
| | - Junyue Sun
- Key Laboratory of Applied Surface and Colloid Chemistry, Ministry of Education; Key Laboratory of Analytical Chemistry for Life Science of Shaanxi Province; School of Chemistry & Chemical Engineering, Shaanxi Normal University, Xi'an 710119, Shaanxi Province, P. R. China
| | - Shan Wang
- Institute of Basic and Translational Medicine & Shaanxi Key Laboratory of Brain Disorders, Xi'an Medical University, Xi'an 710021, Shaanxi Province, P. R. China
- Engineering Research Center of Brain Diseases Drug Development, Universities of Shaanxi Province, Xi'an Medical University, Xi'an 710021, Shaanxi Province, P. R. China
| | - Wei Ren
- Key Laboratory of Applied Surface and Colloid Chemistry, Ministry of Education; Key Laboratory of Analytical Chemistry for Life Science of Shaanxi Province; School of Chemistry & Chemical Engineering, Shaanxi Normal University, Xi'an 710119, Shaanxi Province, P. R. China
| | - Chenghui Liu
- Key Laboratory of Applied Surface and Colloid Chemistry, Ministry of Education; Key Laboratory of Analytical Chemistry for Life Science of Shaanxi Province; School of Chemistry & Chemical Engineering, Shaanxi Normal University, Xi'an 710119, Shaanxi Province, P. R. China
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5
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Zhang Z, Liu T, Dong M, Ahamed MA, Guan W. Sample-to-answer salivary miRNA testing: New frontiers in point-of-care diagnostic technologies. WILEY INTERDISCIPLINARY REVIEWS. NANOMEDICINE AND NANOBIOTECHNOLOGY 2024; 16:e1969. [PMID: 38783564 PMCID: PMC11141732 DOI: 10.1002/wnan.1969] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Grants] [Track Full Text] [Subscribe] [Scholar Register] [Received: 12/22/2023] [Revised: 03/10/2024] [Accepted: 05/02/2024] [Indexed: 05/25/2024]
Abstract
MicroRNA (miRNA), crucial non-coding RNAs, have emerged as key biomarkers in molecular diagnostics, prognosis, and personalized medicine due to their significant role in gene expression regulation. Salivary miRNA, in particular, stands out for its non-invasive collection method and ease of accessibility, offering promising avenues for the development of point-of-care diagnostics for a spectrum of diseases, including cancer, neurodegenerative disorders, and infectious diseases. Such development promises rapid and precise diagnosis, enabling timely treatment. Despite significant advancements in salivary miRNA-based testing, challenges persist in the quantification, multiplexing, sensitivity, and specificity, particularly for miRNA at low concentrations in complex biological mixtures. This work delves into these challenges, focusing on the development and application of salivary miRNA tests for point-of-care use. We explore the biogenesis of salivary miRNA and analyze their quantitative expression and their disease relevance in cancer, infection, and neurodegenerative disorders. We also examined recent progress in miRNA extraction, amplification, and multiplexed detection methods. This study offers a comprehensive view of the development of salivary miRNA-based point-of-care testing (POCT). Its successful advancement could revolutionize the early detection, monitoring, and management of various conditions, enhancing healthcare outcomes. This article is categorized under: Diagnostic Tools > Biosensing Diagnostic Tools > Diagnostic Nanodevices.
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Affiliation(s)
- Zhikun Zhang
- Department of Electrical Engineering, Pennsylvania State University, University Park 16802, USA
- Department of Biomedical Engineering, Pennsylvania State University, University Park 16802, USA
| | - Tianyi Liu
- Department of Electrical Engineering, Pennsylvania State University, University Park 16802, USA
| | - Ming Dong
- Department of Electrical Engineering, Pennsylvania State University, University Park 16802, USA
| | - Md. Ahasan Ahamed
- Department of Electrical Engineering, Pennsylvania State University, University Park 16802, USA
| | - Weihua Guan
- Department of Electrical Engineering, Pennsylvania State University, University Park 16802, USA
- Department of Biomedical Engineering, Pennsylvania State University, University Park 16802, USA
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Liang Y, Chen D, Wang H, Pian H, Liu W, Wang F, Wang H, Li Z. Single-microbead space-confined digital quantification strategy (SMSDQ) for counting microRNAs at the single-molecule level. Biosens Bioelectron 2023; 238:115578. [PMID: 37573644 DOI: 10.1016/j.bios.2023.115578] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 06/27/2023] [Revised: 08/03/2023] [Accepted: 08/05/2023] [Indexed: 08/15/2023]
Abstract
Quantification of microRNAs (miRNAs) at the single-molecule level is of great significance for clinical diagnostics and biomedical research. The challenges lie in the limits to transforming single-molecule measurements into quantitative signals. To address these limits, here, we report a new approach called a Single Microbead-based Space-confined Digital Quantification (SMSDQ) to measure individual miRNA molecules by counting gold nanoparticles (AuNPs) with localized surface plasmon resonance (LSPR) light-scattering imaging. One miRNA target hybridizes with the alkynyl-modified capture DNA probe immobilized on a microbead (60 μm) and the azide-modified report DNA probe anchored on AuNP (50 nm), respectively. Through the click reaction between the alkynyl and azide group, a single microbead can covalently link the AuNPs in the confined space within the view of the microscope. By digitally counting the light-scattering spots of AuNPs, we demonstrated the proposed approach with single-molecule detection sensitivity and high specificity of single-base discrimination. Taking the advantages of ultrahigh sensitivity, specificity, and the digital detection manner, the approach is suitable for evaluating cell heterogeneity and small variations of miRNA expression and has been successfully applied to direct quantification of miRNAs in one-tenth single-cell lysates and serum samples without RNA-isolated and nucleic acid amplification steps.
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Affiliation(s)
- Yuanwen Liang
- Beijing Key Laboratory for Bioengineering and Sensing Technology, School of Chemistry and Biological Engineering, University of Science and Technology Beijing, 30 Xueyuan Road, Haidian District, Beijing, 100083, China
| | - Desheng Chen
- Beijing Key Laboratory for Bioengineering and Sensing Technology, School of Chemistry and Biological Engineering, University of Science and Technology Beijing, 30 Xueyuan Road, Haidian District, Beijing, 100083, China
| | - Honghong Wang
- Beijing Key Laboratory for Bioengineering and Sensing Technology, School of Chemistry and Biological Engineering, University of Science and Technology Beijing, 30 Xueyuan Road, Haidian District, Beijing, 100083, China
| | - Hongru Pian
- Beijing Key Laboratory for Bioengineering and Sensing Technology, School of Chemistry and Biological Engineering, University of Science and Technology Beijing, 30 Xueyuan Road, Haidian District, Beijing, 100083, China
| | - Weiliang Liu
- Beijing Key Laboratory for Bioengineering and Sensing Technology, School of Chemistry and Biological Engineering, University of Science and Technology Beijing, 30 Xueyuan Road, Haidian District, Beijing, 100083, China
| | - Fangfang Wang
- Beijing Key Laboratory for Bioengineering and Sensing Technology, School of Chemistry and Biological Engineering, University of Science and Technology Beijing, 30 Xueyuan Road, Haidian District, Beijing, 100083, China
| | - Hui Wang
- Beijing Key Laboratory for Bioengineering and Sensing Technology, School of Chemistry and Biological Engineering, University of Science and Technology Beijing, 30 Xueyuan Road, Haidian District, Beijing, 100083, China.
| | - Zhengping Li
- Beijing Key Laboratory for Bioengineering and Sensing Technology, School of Chemistry and Biological Engineering, University of Science and Technology Beijing, 30 Xueyuan Road, Haidian District, Beijing, 100083, China.
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Quazi MZ, Hwang J, Song Y, Park N. Hydrogel-Based Biosensors for Effective Therapeutics. Gels 2023; 9:545. [PMID: 37504424 PMCID: PMC10378974 DOI: 10.3390/gels9070545] [Citation(s) in RCA: 1] [Impact Index Per Article: 0.5] [Reference Citation Analysis] [Abstract] [Key Words] [Grants] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 05/12/2023] [Revised: 06/22/2023] [Accepted: 06/26/2023] [Indexed: 07/29/2023] Open
Abstract
Nanotechnology and polymer engineering are navigating toward new developments to control and overcome complex problems. In the last few decades, polymer engineering has received researchers' attention and similarly, polymeric network-engineered structures have been vastly studied. Prior to therapeutic application, early and rapid detection analyses are critical. Therefore, developing hydrogel-based sensors to manage the acute expression of diseases and malignancies to devise therapeutic approaches demands advanced nanoengineering. However, nano-therapeutics have emerged as an alternative approach to tackling strenuous diseases. Similarly, sensing applications for multiple kinds of analytes in water-based environments and other media are gaining wide interest. It has also been observed that these functional roles can be used as alternative approaches to the detection of a wide range of biomolecules and pathogenic proteins. Moreover, hydrogels have emerged as a three-dimensional (3D) polymeric network that consists of hydrophilic natural or synthetic polymers with multidimensional dynamics. The resemblance of hydrogels to tissue structure makes them more unique to study inquisitively. Preceding studies have shown a vast spectrum of synthetic and natural polymer applications in the field of biotechnology and molecular diagnostics. This review explores recent studies on synthetic and natural polymers engineered hydrogel-based biosensors and their applications in multipurpose diagnostics and therapeutics. We review the latest studies on hydrogel-engineered biosensors, exclusively DNA-based and DNA hydrogel-fabricated biosensors.
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Affiliation(s)
- Mohzibudin Z Quazi
- Department of Chemistry and the Natural Science Research Institute, Myongji University, 116 Myongji-ro, Yongin-si 17058, Gyeonggi-do, Republic of Korea
| | - Jimin Hwang
- Department of Chemistry and the Natural Science Research Institute, Myongji University, 116 Myongji-ro, Yongin-si 17058, Gyeonggi-do, Republic of Korea
| | - Youngseo Song
- Department of Chemistry and the Natural Science Research Institute, Myongji University, 116 Myongji-ro, Yongin-si 17058, Gyeonggi-do, Republic of Korea
| | - Nokyoung Park
- Department of Chemistry and the Natural Science Research Institute, Myongji University, 116 Myongji-ro, Yongin-si 17058, Gyeonggi-do, Republic of Korea
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Yang LY, Xu XW, Lin Y, Ye CL, Liu WQ, Liu ZJ, Zhong GX, Xu YF, Lin XH, Chen JY. Nucleic Acid Amplification by Template-Dominated Click Chemistry for Ultrasensitive DNA/RNA Detection on an Electrochemical Readout Platform. Anal Chem 2023; 95:5331-5339. [PMID: 36926822 DOI: 10.1021/acs.analchem.2c05421] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 03/18/2023]
Abstract
As an enzyme-free exponential nucleic acid amplification method, the click chemistry-mediated ligation chain reaction (ccLCR) has shown great prospects in the molecular diagnosis. However, the current optics-based ccLCR is challenged by remarkable nonspecific amplification, severely hindering its future application. This study demonstrated that the severe nonspecific amplification was generated probably due to high random collision in the high DNA probe concentration (μM level). To solve this hurdle, a nucleic acid template-dominated ccLCR was constructed using nM-level DNA probes and read on an electrochemical platform (cc-eLCR). Under the optimal conditions, the proposed cc-eLCR detected a low-level nucleic acid target (1 fM) with a single-base resolution. Furthermore, this assay was applied to detect the target of interest in cell extracts with a satisfactory result. The proposed cc-eLCR offers huge possibility for click chemistry-mediated enzyme-free exponential nucleic acid amplification in the application of medical diagnosis and biomedical research.
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Affiliation(s)
- Liang-Yong Yang
- The Central Laboratory, Fujian Key Laboratory of Precision Medicine for Cancer, Key Laboratory of Radiation Biology of Fujian Higher Education Institutions, the First Affiliated Hospital, Fujian Medical University, Fuzhou 350005, China.,Department of Pharmaceutical Analysis, Higher Educational Key Laboratory for Nano Biomedical Technology of Fujian Province, Faculty of Pharmacy, Fujian Medical University, Fuzhou 350122, China
| | - Xiong-Wei Xu
- The Central Laboratory, Fujian Key Laboratory of Precision Medicine for Cancer, Key Laboratory of Radiation Biology of Fujian Higher Education Institutions, the First Affiliated Hospital, Fujian Medical University, Fuzhou 350005, China
| | - Yan Lin
- The Central Laboratory, Fujian Key Laboratory of Precision Medicine for Cancer, Key Laboratory of Radiation Biology of Fujian Higher Education Institutions, the First Affiliated Hospital, Fujian Medical University, Fuzhou 350005, China
| | - Chen-Liu Ye
- Department of Pharmacy, Longyan First Affiliated Hospital of Fujian Medical University, Longyan 364000, China
| | - Wei-Qiang Liu
- The Central Laboratory, Fujian Key Laboratory of Precision Medicine for Cancer, Key Laboratory of Radiation Biology of Fujian Higher Education Institutions, the First Affiliated Hospital, Fujian Medical University, Fuzhou 350005, China
| | - Zhou-Jie Liu
- The Central Laboratory, Fujian Key Laboratory of Precision Medicine for Cancer, Key Laboratory of Radiation Biology of Fujian Higher Education Institutions, the First Affiliated Hospital, Fujian Medical University, Fuzhou 350005, China
| | - Guang-Xian Zhong
- Department of Rehabilitation Medicine, School of Health, Fujian Medical University, Fuzhou 350122, China
| | - Yan-Fang Xu
- Department of Nephrology, the Central Laboratory, the First Affiliated Hospital, Fujian Medical University, Fuzhou 350005, China
| | - Xin-Hua Lin
- Department of Pharmaceutical Analysis, Higher Educational Key Laboratory for Nano Biomedical Technology of Fujian Province, Faculty of Pharmacy, Fujian Medical University, Fuzhou 350122, China
| | - Jin-Yuan Chen
- The Central Laboratory, Fujian Key Laboratory of Precision Medicine for Cancer, Key Laboratory of Radiation Biology of Fujian Higher Education Institutions, the First Affiliated Hospital, Fujian Medical University, Fuzhou 350005, China
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Shi H, Bi X, Zhang J, Duan S, Yan J, Jia H. Simple and sensitive detection of microRNA based on guanine-rich DNA-enhanced fluorescence of DNA-templated silver clusters. Talanta 2022. [DOI: 10.1016/j.talanta.2022.124065] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 11/12/2022]
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Yan X, Zhang J, Jiang Q, Jiao D, Cheng Y. Integration of the Ligase Chain Reaction with the CRISPR-Cas12a System for Homogeneous, Ultrasensitive, and Visual Detection of microRNA. Anal Chem 2022; 94:4119-4125. [PMID: 35195982 DOI: 10.1021/acs.analchem.2c00294] [Citation(s) in RCA: 26] [Impact Index Per Article: 8.7] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 12/26/2022]
Abstract
The ligase chain reaction (LCR), as a classic nucleic acid amplification technique, is popular in the detection of DNA and RNA due to its simplicity, powerfulness, and high specificity. However, homogeneous and ultrasensitive LCR detection is still quite challenging. Herein, we integrate the LCR with a CRISPR-Cas12a system to greatly promote the application of the LCR in a homogeneous fashion. By employing microRNA as the model target, we design LCR probes with specific protospacer adjacent motif sequences and the guide RNA. Then, the LCR is initiated by target microRNA, and the LCR products specifically bind to the guide RNA to activate the Cas12a system, triggering secondary signal amplification to achieve ultrasensitive detection of microRNA without separation steps. Moreover, by virtue of a cationic conjugated polymer, microRNA can not only be visually detected by naked eyes but also be accurately quantified based on RGB ratio analysis of images with no need of sophisticated instruments. The method can quantify microRNA up to 4 orders of magnitude, and the determination limit is 0.4 aM, which is better than those of other reported studies using CRISPR-Cas12a and can be compared with that of the reverse-transcription polymerase chain reaction. This study demonstrates that the CRISPR-Cas12a system can greatly expand the application of the LCR for the homogeneous, ultrasensitive, and visual detection of microRNA, showing great potential in efficient nucleic acid detection and in vitro diagnosis.
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Affiliation(s)
- Xinrong Yan
- Key Laboratory of Medicinal Chemistry and Molecular Diagnosis (Hebei University), Ministry of Education, Key Laboratory of Analytical Science and Technology of Hebei Province, College of Chemistry and Environmental Science, Hebei University, Baoding, 071002 Hebei, P. R. China
| | - Jiangyan Zhang
- Key Laboratory of Medicinal Chemistry and Molecular Diagnosis (Hebei University), Ministry of Education, Key Laboratory of Analytical Science and Technology of Hebei Province, College of Chemistry and Environmental Science, Hebei University, Baoding, 071002 Hebei, P. R. China
| | - Qi Jiang
- Key Laboratory of Medicinal Chemistry and Molecular Diagnosis (Hebei University), Ministry of Education, Key Laboratory of Analytical Science and Technology of Hebei Province, College of Chemistry and Environmental Science, Hebei University, Baoding, 071002 Hebei, P. R. China
| | - Dan Jiao
- Key Laboratory of Medicinal Chemistry and Molecular Diagnosis (Hebei University), Ministry of Education, Key Laboratory of Analytical Science and Technology of Hebei Province, College of Chemistry and Environmental Science, Hebei University, Baoding, 071002 Hebei, P. R. China
| | - Yongqiang Cheng
- Key Laboratory of Medicinal Chemistry and Molecular Diagnosis (Hebei University), Ministry of Education, Key Laboratory of Analytical Science and Technology of Hebei Province, College of Chemistry and Environmental Science, Hebei University, Baoding, 071002 Hebei, P. R. China
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11
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Li Y, Guo R, Deng C, Li D, Wu H. A Prussian blue nanoparticles-based fluorescent nanoprobe for monitoring microRNA-92a and microRNA-21. ANAL SCI 2022; 38:497-504. [PMID: 35359267 DOI: 10.2116/analsci.20p455] [Citation(s) in RCA: 2] [Impact Index Per Article: 0.7] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 12/25/2020] [Accepted: 02/05/2021] [Indexed: 11/23/2022]
Abstract
Since microRNA-92a (miR-92a) and microRNA-21 (miR-21) are crucial biomarkers for colorectal cancer (CRC), monitoring miR-92a and miR-21 in serum is very significant for the early diagnosis of CRC. In this work, we developed a simple and sensitive fluorescent biosensor for the detection of miR-92a and miR-21 based on the quenching ability of Prussian blue nanoparticles (PBNPs) to fluorophores. Carboxyl fluorescein (FAM)-modified ssDNA (P-92a) and Cyanine 5 (Cy5)-modified ssDNA (P-21) were completely complementary to miR-92a and miR-21 separately. They were adsorbed on PBNPs surface by the binding of PO43- in DNA and Fe3+ in PBNPs to fabricate the P-92a + P-21@PBNPs sensing system. The fluorescence responses from P-92a + P-21@PBNPs show good selection to miR-92a and a great linear process with the miR-92a concentration ranging from 1 to 30 nM (ΔF = 10.978 cmiR-92a + 71.457). Meanwhile, the fluorescence responses from P-92a + P-21@PBNPs is linearly relative to miR-21 from 3 to 30 nM; the linear equation is ΔF = 5.7560 cmiR-21 + 48.729. Furthermore, the detections of miR-92a and miR-21 added in serum samples were achieved. In summary, this method is sensitive, highly specific, time-saving, cost-effective and applicable for the detection of miR-92a and miR-21. Therefore, this present sensor was expected to be used in clinical applications, which lays a potential foundation for an early diagnosis of cancer.
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Affiliation(s)
- Yao Li
- Hunan Provincial Key Laboratory of Micro and Nano Materials Interface Science, College of Chemistry and Chemical Engineering, Central South University, Changsha, 410083, People's Republic of China
| | - Ren Guo
- Department of Pharmacy, The Third Xiangya Hospital, Central South University, Changsha, Hunan, People's Republic of China
| | - Chunyan Deng
- Hunan Provincial Key Laboratory of Micro and Nano Materials Interface Science, College of Chemistry and Chemical Engineering, Central South University, Changsha, 410083, People's Republic of China.
| | - Dai Li
- National Institution of Drug Clinical Trial, Xiangya Hospital, Central South University, Changsha, Hunan, People's Republic of China
| | - Huiyun Wu
- Academy of Military Medical Sciences, Academy of Military Sciences, Beijing, 100850, People's Republic of China
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12
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Zhao S, Yang S, Xu H, Tang X, Wang H, Yu L, Qiu X, Wang Y, Gao M, Chang K, Chen M. Enzyme-free and copper-free strategy based on cyclic click chemical-triggered hairpin stacking circuit for accurate detection of circulating microRNAs. Anal Chim Acta 2022; 1191:339282. [PMID: 35033257 DOI: 10.1016/j.aca.2021.339282] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 09/08/2021] [Revised: 11/10/2021] [Accepted: 11/11/2021] [Indexed: 11/01/2022]
Abstract
Accurate detection of circulating microRNAs (miRNAs) plays a vital role in the diagnosis of various diseases. However, enzyme-free amplification detection remains challenging. Here, we report an enzyme-free fluorescence resonance energy transfer assay termed "3C-TASK" (cyclic click chemical-triggered hairpin stacking kit) for the detection of circulating miRNA. In this strategy, the miRNA could initiate copper-free click chemical ligation reactions and the ligated products then trigger another hairpin stacking circuit. The first signal amplification was achieved through the recycling of the target miRNA in the click chemical ligation circuit, and the second signal amplification was realized through the recycling of ligated probes in a hairpin stacking circuit driven by thermodynamics. The two-step chain reaction event triggered by miRNAs was quantified by the fluorescence signal value so that accurate detection of target miRNA could be achieved. The 3C-TASK was easily controlled because no enzyme was involved in the entire procedure. Although simple, this strategy showed sensitivity with a detection limit of 8.63 pM and specificity for distinguishing miRNA sequences with single-base variations. In addition, the applicability of this method in complex biological samples was verified by detecting target miRNA in diluted plasma samples. Hence, our method achieved sensitive and specific detection of miRNA and may offer a new perspective for the broader application of enzyme-free chemical reaction and DNA circuits in biosensing.
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Affiliation(s)
- Shuang Zhao
- Department of Clinical Laboratory Medicine, Southwest Hospital, Army Medical University, 30 Gaotanyan, Shapingba District, Chongqing, 400038, China
| | - Sha Yang
- Department of Clinical Laboratory Medicine, Southwest Hospital, Army Medical University, 30 Gaotanyan, Shapingba District, Chongqing, 400038, China
| | - Hanqing Xu
- Department of Clinical Laboratory Medicine, Southwest Hospital, Army Medical University, 30 Gaotanyan, Shapingba District, Chongqing, 400038, China
| | - Xiaoqi Tang
- Department of Clinical Laboratory Medicine, Southwest Hospital, Army Medical University, 30 Gaotanyan, Shapingba District, Chongqing, 400038, China
| | - Hongwei Wang
- Department of Oncology, Southwest Hospital, Army Medical University, 30 Gaotanyan, Shapingba District, Chongqing, 400038, China
| | - Lianyu Yu
- Department of Clinical Laboratory Medicine, Southwest Hospital, Army Medical University, 30 Gaotanyan, Shapingba District, Chongqing, 400038, China
| | - Xiaopei Qiu
- Department of Clinical Laboratory Medicine, Southwest Hospital, Army Medical University, 30 Gaotanyan, Shapingba District, Chongqing, 400038, China
| | - Yunxia Wang
- Department of Clinical Laboratory Medicine, Southwest Hospital, Army Medical University, 30 Gaotanyan, Shapingba District, Chongqing, 400038, China
| | - Mingxuan Gao
- Department of Clinical Laboratory Medicine, Southwest Hospital, Army Medical University, 30 Gaotanyan, Shapingba District, Chongqing, 400038, China.
| | - Kai Chang
- Department of Clinical Laboratory Medicine, Southwest Hospital, Army Medical University, 30 Gaotanyan, Shapingba District, Chongqing, 400038, China.
| | - Ming Chen
- Department of Clinical Laboratory Medicine, Southwest Hospital, Army Medical University, 30 Gaotanyan, Shapingba District, Chongqing, 400038, China; College of Pharmacy and Laboratory Medicine, Army Medical University, 30 Gaotanyan, Shapingba District, Chongqing, 400038, China; State Key Laboratory of Trauma, Burn and Combined Injury, Army Medical University, 30 Gaotanyan, Shapingba District, Chongqing, 400038, China.
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13
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Lu X, Yao C, Sun L, Li Z. Plasmon-enhanced biosensors for microRNA analysis and cancer diagnosis. Biosens Bioelectron 2022; 203:114041. [DOI: 10.1016/j.bios.2022.114041] [Citation(s) in RCA: 5] [Impact Index Per Article: 1.7] [Reference Citation Analysis] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 01/02/2022] [Revised: 01/22/2022] [Accepted: 01/24/2022] [Indexed: 12/19/2022]
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14
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He Y, Long L, Yan W, Dong L, Xia W, Li C, Li F. Establishment and Application of Ligation Reaction-Based Method for Quantifying MicroR-156b. FRONTIERS IN PLANT SCIENCE 2021; 12:794752. [PMID: 34970292 PMCID: PMC8713971 DOI: 10.3389/fpls.2021.794752] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Figures] [Subscribe] [Scholar Register] [Received: 10/14/2021] [Accepted: 11/25/2021] [Indexed: 06/14/2023]
Abstract
Microribonucleic acids (miRNAs) play significant roles in the regulation of biological processes and in responses to biotic or abiotic environmental stresses. Therefore, it is necessary to quantitatively detect miRNAs to understand these complicated biological regulation mechanisms. This study established an ultrasensitive and highly specific method for the quantitative detection of miRNAs using simple operations on the ground of the ligation reaction of ribonucleotide-modified deoxyribonucleic acid (DNA) probes. This method avoids the complex design of conventional reverse transcription. In the developed assay, the target miRNA miR156b was able to directly hybridize the two ribonucleotide-modified DNA probes, and amplification with universal primers was achieved following the ligation reaction. As a result, the target miRNA could be sensitively measured even at a detection limit as low as 0.0001 amol, and differences of only a single base could be detected between miR156 family members. Moreover, the proposed quantitative method demonstrated satisfactory results for overexpression-based genetically modified (GM) soybean. Ligation-based quantitative polymerase chain reaction (PCR) therefore has potential in investigating the biological functions of miRNAs, as well as in supervising activities regarding GM products or organisms.
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15
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Ma GM, Huo LW, Tong YX, Wang YC, Li CP, Jia HX. Label-free and sensitive MiRNA detection based on turn-on fluorescence of DNA-templated silver nanoclusters coupled with duplex-specific nuclease-assisted signal amplification. Mikrochim Acta 2021; 188:355. [PMID: 34585278 DOI: 10.1007/s00604-021-05001-x] [Citation(s) in RCA: 9] [Impact Index Per Article: 2.3] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 04/06/2021] [Accepted: 08/24/2021] [Indexed: 11/30/2022]
Abstract
A novel strategy for microRNAs (miRNAs) detection has been developed utilizing duplex-specific nuclease-assisted signal amplification (DSNSA) and guanine-rich DNA-enhanced fluorescence of DNA-templated silver nanoclusters (AgNCs). The combination between target miRNA, DSNSA, and AgNCs is achieved by the unique design of DNA sequences. Target miRNA opens the hairpin structure of the Hairpin DNA probe (HP) by hybridizing with the HP and initiates the duplex-specific nuclease-assisted signal amplification (DSNSA) reaction. The DSNSA reaction generates the release of the guanine-rich DNA sequence, which can turn on the fluorescence of the dark AgNCs by hybridizing with the DNA template of the dark AgNCs. The fluorescence intensity of AgNCs corresponds to the dosage of the target miRNA. This is measured at 630 nm by exciting at 560 nm. The constructed method exhibits a low detection limit (~8.3 fmol), a great dynamic range of more than three orders of magnitude, and excellent selectivity. Moreover, it has a good performance for miR-21 detection in complex biological samples. A novel strategy for microRNAs (miRNAs) detection has been developed utilizing duplex-specific nuclease-assisted signal amplification (DSNSA) and guanine-rich DNA-enhanced fluorescence of DNA-templated silver nanoclusters (AgNCs).
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Affiliation(s)
- Gui-Min Ma
- Key Laboratory of Medicinal Chemistry and Molecular Diagnosis, Ministry of Education; Key Laboratory of Analytical Science and Technology of Hebei Province; Institute of Life Science and Green Development; College of Chemistry and Environmental Science, Hebei University, Baoding, 071002, Hebei Province, People's Republic of China
| | - Li-Wei Huo
- Key Laboratory of Medicinal Chemistry and Molecular Diagnosis, Ministry of Education; Key Laboratory of Analytical Science and Technology of Hebei Province; Institute of Life Science and Green Development; College of Chemistry and Environmental Science, Hebei University, Baoding, 071002, Hebei Province, People's Republic of China
| | - Yin-Xia Tong
- Key Laboratory of Medicinal Chemistry and Molecular Diagnosis, Ministry of Education; Key Laboratory of Analytical Science and Technology of Hebei Province; Institute of Life Science and Green Development; College of Chemistry and Environmental Science, Hebei University, Baoding, 071002, Hebei Province, People's Republic of China
| | - Yu-Cong Wang
- Key Laboratory of Medicinal Chemistry and Molecular Diagnosis, Ministry of Education; Key Laboratory of Analytical Science and Technology of Hebei Province; Institute of Life Science and Green Development; College of Chemistry and Environmental Science, Hebei University, Baoding, 071002, Hebei Province, People's Republic of China
| | - Cui-Ping Li
- Key Laboratory of Public Health Safety of Hebei Province; Key Laboratory of Medicinal Chemistry and Molecular Diagnosis, Ministry of Education; College of Public Health, Hebei University, Baoding, 071002, People's Republic of China
| | - Hong-Xia Jia
- Key Laboratory of Medicinal Chemistry and Molecular Diagnosis, Ministry of Education; Key Laboratory of Analytical Science and Technology of Hebei Province; Institute of Life Science and Green Development; College of Chemistry and Environmental Science, Hebei University, Baoding, 071002, Hebei Province, People's Republic of China.
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16
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Wang ZY, Li DL, Tian X, Zhang CY. A copper-free and enzyme-free click chemistry-mediated single quantum dot nanosensor for accurate detection of microRNAs in cancer cells and tissues. Chem Sci 2021; 12:10426-10435. [PMID: 34447534 PMCID: PMC8356811 DOI: 10.1039/d1sc01865e] [Citation(s) in RCA: 21] [Impact Index Per Article: 5.3] [Reference Citation Analysis] [Abstract] [Grants] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 04/03/2021] [Accepted: 07/06/2021] [Indexed: 12/31/2022] Open
Abstract
MicroRNAs (miRNAs) play key roles in the post-transcriptional regulation of genes, and their aberrant expression may disturb the normal gene regulation network to induce various diseases, and thus accurate detection of miRNAs is essential to early clinical diagnosis. Herein, we develop for the first time a single-quantum dot (QD)-based Förster resonance energy transfer (FRET) nanosensor to accurately detect miRNAs based on copper-free and enzyme-free cycling click chemistry-mediated tricyclic ligase chain reaction (LCR) amplification. We design four DNA probes namely DNA probes 1-4, with DNA probes 1 and 3 being modified with azide (N3) and DNA probes 2 and 4 being modified with dibenzocyclooctyne (DBCO). When target miRNA is present, DNA probes 1 and 2 can proceed via copper-free and enzyme-free click chemistry to generate the probes 1-2 ligation product. Subsequently, DNA probes 3 and 4 can hybridize with the probes 1-2 ligation product to generate the probes 3-4 ligation product. Both the probes 1-2 ligation product and probes 3-4 ligation product can act as the templates to initiate cycling click chemistry-mediated tricyclic LCR amplification whose products can be easily measured by the single-QD-based FRET nanosensor. This assay does not involve any enzymatic reverse transcription, copper catalyst, and ligase enzyme, and it exhibits excellent selectivity, high sensitivity, and the capability of differentiating even single-base mismatches. Moreover, this nanosensor can accurately quantify miRNA-155 even at the single-cell level, and it can distinguish the miRNA-155 expression in tissues of healthy persons and nonsmall cell lung cancer (NSCLC) patients.
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Affiliation(s)
- Zi-Yue Wang
- College of Chemistry, Chemical Engineering and Materials Science, Collaborative Innovation Center of Functionalized Probes for Chemical Imaging in Universities of Shandong, Key Laboratory of Molecular and Nano Probes, Ministry of Education, Shandong Provincial Key Laboratory of Clean Production of Fine Chemicals, Shandong Normal University Jinan 250014 China +86 0531-82615258 +86 0531-86186033
| | - Dong-Ling Li
- College of Chemistry, Chemical Engineering and Materials Science, Collaborative Innovation Center of Functionalized Probes for Chemical Imaging in Universities of Shandong, Key Laboratory of Molecular and Nano Probes, Ministry of Education, Shandong Provincial Key Laboratory of Clean Production of Fine Chemicals, Shandong Normal University Jinan 250014 China +86 0531-82615258 +86 0531-86186033
| | - Xiaorui Tian
- College of Chemistry, Chemical Engineering and Materials Science, Collaborative Innovation Center of Functionalized Probes for Chemical Imaging in Universities of Shandong, Key Laboratory of Molecular and Nano Probes, Ministry of Education, Shandong Provincial Key Laboratory of Clean Production of Fine Chemicals, Shandong Normal University Jinan 250014 China +86 0531-82615258 +86 0531-86186033
| | - Chun-Yang Zhang
- College of Chemistry, Chemical Engineering and Materials Science, Collaborative Innovation Center of Functionalized Probes for Chemical Imaging in Universities of Shandong, Key Laboratory of Molecular and Nano Probes, Ministry of Education, Shandong Provincial Key Laboratory of Clean Production of Fine Chemicals, Shandong Normal University Jinan 250014 China +86 0531-82615258 +86 0531-86186033
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17
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Wang L, He R, Lv B, Yu X, Liu Y, Yang J, Li W, Wang Y, Zhang H, Yan G, Mao W, Liu L, Wang F, Ma L. Pyrococcus furiosus Argonaute coupled with modified ligase chain reaction for detection of SARS-CoV-2 and HPV. Talanta 2021; 227:122154. [PMID: 33714462 PMCID: PMC7875706 DOI: 10.1016/j.talanta.2021.122154] [Citation(s) in RCA: 26] [Impact Index Per Article: 6.5] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 11/11/2020] [Revised: 01/17/2021] [Accepted: 01/24/2021] [Indexed: 12/16/2022]
Abstract
Infectious diseases caused by viruses such as SARS-CoV-2 and HPV have greatly endangered human health. The nucleic acid detection is essential for the early diagnosis of diseases. Here, we propose a method called PLCR (PfAgo coupled with modified Ligase Chain Reaction for nucleic acid detection) which utilizes PfAgo to only use DNA guides longer than 14-mer to specifically cleave DNA and LCR to precisely distinguish single-base mismatch. PLCR can detect DNA or RNA without PCR at attomolar sensitivities, distinguish single base mutation between the genome of wild type SARS-CoV-2 and its mutant spike D614G, effectively distinguish the novel coronavirus from other coronaviruses and finally achieve multiplexed detection in 70 min. Additionally, LCR products can be directly used as DNA guides without additional input guides to simplify primer design. With desirable sensitivity, specificity and simplicity, the method can be extended for detecting other pathogenic microorganisms.
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Affiliation(s)
- Longyu Wang
- State Key Laboratory of Biocatalysis and Enzyme Engineering, Hubei Collaborative Innovation Center for Green Transformation of Bio-resources, Hubei Key Laboratory of Industrial Biotechnology, School of Life Sciences, Hubei University, Wuhan, Hubei, PR China
| | - Ruyi He
- State Key Laboratory of Biocatalysis and Enzyme Engineering, Hubei Collaborative Innovation Center for Green Transformation of Bio-resources, Hubei Key Laboratory of Industrial Biotechnology, School of Life Sciences, Hubei University, Wuhan, Hubei, PR China
| | - Bin Lv
- State Key Laboratory of Biocatalysis and Enzyme Engineering, Hubei Collaborative Innovation Center for Green Transformation of Bio-resources, Hubei Key Laboratory of Industrial Biotechnology, School of Life Sciences, Hubei University, Wuhan, Hubei, PR China
| | - Xiao Yu
- Hubei Provincial Center for Disease Control and Prevention, Wuhan, Hubei, PR China
| | - Yang Liu
- State Key Laboratory of Biocatalysis and Enzyme Engineering, Hubei Collaborative Innovation Center for Green Transformation of Bio-resources, Hubei Key Laboratory of Industrial Biotechnology, School of Life Sciences, Hubei University, Wuhan, Hubei, PR China
| | - Jun Yang
- State Key Laboratory of Biocatalysis and Enzyme Engineering, Hubei Collaborative Innovation Center for Green Transformation of Bio-resources, Hubei Key Laboratory of Industrial Biotechnology, School of Life Sciences, Hubei University, Wuhan, Hubei, PR China
| | - Wenqiang Li
- State Key Laboratory of Biocatalysis and Enzyme Engineering, Hubei Collaborative Innovation Center for Green Transformation of Bio-resources, Hubei Key Laboratory of Industrial Biotechnology, School of Life Sciences, Hubei University, Wuhan, Hubei, PR China
| | - Yuan Wang
- Medical College of Hubei University of Arts and Sciences, Xiangyang, Hubei, PR China
| | - Hang Zhang
- State Key Laboratory of Biocatalysis and Enzyme Engineering, Hubei Collaborative Innovation Center for Green Transformation of Bio-resources, Hubei Key Laboratory of Industrial Biotechnology, School of Life Sciences, Hubei University, Wuhan, Hubei, PR China
| | - Guangbo Yan
- State Key Laboratory of Biocatalysis and Enzyme Engineering, Hubei Collaborative Innovation Center for Green Transformation of Bio-resources, Hubei Key Laboratory of Industrial Biotechnology, School of Life Sciences, Hubei University, Wuhan, Hubei, PR China
| | - Wuxiang Mao
- State Key Laboratory of Biocatalysis and Enzyme Engineering, Hubei Collaborative Innovation Center for Green Transformation of Bio-resources, Hubei Key Laboratory of Industrial Biotechnology, School of Life Sciences, Hubei University, Wuhan, Hubei, PR China
| | - Linlin Liu
- Hubei Provincial Center for Disease Control and Prevention, Wuhan, Hubei, PR China,Corresponding author
| | - Fei Wang
- State Key Laboratory of Biocatalysis and Enzyme Engineering, Hubei Collaborative Innovation Center for Green Transformation of Bio-resources, Hubei Key Laboratory of Industrial Biotechnology, School of Life Sciences, Hubei University, Wuhan, Hubei, PR China,Corresponding author
| | - Lixin Ma
- State Key Laboratory of Biocatalysis and Enzyme Engineering, Hubei Collaborative Innovation Center for Green Transformation of Bio-resources, Hubei Key Laboratory of Industrial Biotechnology, School of Life Sciences, Hubei University, Wuhan, Hubei, PR China,Corresponding author
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18
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Biocomputing label-free security system based on homogenous ligation chain reaction-induced dramatic change in melting temperature for screening single nucleotide polymorphisms. Talanta 2020; 218:121141. [PMID: 32797898 DOI: 10.1016/j.talanta.2020.121141] [Citation(s) in RCA: 3] [Impact Index Per Article: 0.6] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 02/13/2020] [Revised: 05/03/2020] [Accepted: 05/07/2020] [Indexed: 12/17/2022]
Abstract
The development of smart platform with accurate, inexpensive and reliable detection of single-nucleotide polymorphisms (SNPs) has long been concerned in the fields of medical diagnosis and basic research. Here, we present a ligation chain reaction (LCR)-based sensing system for the cost-effective screening of SNPs by simply conducting DNA melting analysis. No chemical modification is required and the signaling operation is accomplished in homogeneous solution, circumventing the complex modification process and possibly compromised enzymatic activity associated with heterogeneous materials, such as quantum dot (QD) and gold nanoparticle (GNP). Due to the enzymatic catalysis and high fidelity of ligase, the system is capable of executing signal amplification, providing a high sensitivity and selectivity. KRAS gene is easily recognized and the site-specific mutation of guanine (G) to adenine (A), thymine (T) or cytosine (C) is accurately screened. Moreover, the excellent reliability was demonstrated by blind test and recovery test. LCR-based signaling mechanism was further used to develop the biocomputing security system, and two logic gates consisting of four single-stranded DNAs (ssDNAs) offer a double insurance to protect the information against illegal invasion, guaranteeing the reliability of output information. Once in the absence of one essential factor, the security system was always locked regardless of target key, serving as a novel strategy to ensure the safety of output information at molecular level. As a proof-of-concept scheme, this contribution introduces new insight into the development of DNA security systems and the exploitation of powerful signal transduction strategy suitable for rapid and convenient disease diagnosis.
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19
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Wu A, Ji H, Li Y, Liu R, Hu Z, Ju S, Wang F. Establishment of a direct quantitative method for measurement of microRNA-224 in serum by UHPLC/MS/MS. J Chromatogr B Analyt Technol Biomed Life Sci 2020; 1152:122199. [DOI: 10.1016/j.jchromb.2020.122199] [Citation(s) in RCA: 5] [Impact Index Per Article: 1.0] [Reference Citation Analysis] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 04/03/2020] [Revised: 05/27/2020] [Accepted: 05/29/2020] [Indexed: 12/23/2022]
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20
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Guo Y, Yang H, Ren W, Gu H, Xu G, Xu H. A noise-free, ultrasensitive and accurate miRNAs detection using streptavidin coated magnetic microsphere based stem-loop ligation PCR. Talanta 2020; 213:120845. [DOI: 10.1016/j.talanta.2020.120845] [Citation(s) in RCA: 3] [Impact Index Per Article: 0.6] [Reference Citation Analysis] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 12/26/2019] [Revised: 02/09/2020] [Accepted: 02/13/2020] [Indexed: 02/08/2023]
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21
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Gao J, Zhang H, Wang Z. A DNA tetrahedron nanoprobe-based fluorescence resonance energy transfer sensing platform for intracellular tumor-related miRNA detection. Analyst 2020; 145:3535-3542. [PMID: 32314984 DOI: 10.1039/c9an02610j] [Citation(s) in RCA: 13] [Impact Index Per Article: 2.6] [Reference Citation Analysis] [Abstract] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 11/21/2022]
Abstract
Accurate and sensitive detection of disease-related microRNAs (miRNAs) is of great significance for early disease diagnosis. In this work, a DNA tetrahedron nanoprobe (DTNP)-based fluorescence resonance energy transfer (FRET) sensing platform (termed DTNP sensor) was constructed for sensitive detection of tumor-related miRNA (e.g., hsa-miR-146b-5p) with DNA assisted cyclic amplification. DTNP was synthesized by DNA self-assembly. In the absence of hsa-miR-146b-5p, the fluorescence DNA (HP) modified with FAM at the 5' terminal and TAMRA at the 3' terminal cannot form the hairpin structure because of the hybridization with the extended DNA strand of the DNA tetrahedron, resulting in a low FRET effect. In the presence of hsa-miR-146b-5p, it would complementarily hybridize with the extended DNA strand of the DNA tetrahedron, leading to the release of HP and occurrence of strong FRET. Thus, the concentration of hsa-miR-146b-5p can be revealed by the change in the fluorescence intensity. Moreover, an assistant DNA was employed to replace hsa-miR-146b-5p for cyclic signal amplification, which can further enhance the detection sensitivity. Under the optimal experimental conditions, the limit of detection for hsa-miR-146b-5p was as low as 6 pM (S/N = 3). Furthermore, the DTNP sensor was successfully applied to evaluate the hsa-miR-146b-5p expression levels in different cell lines. The inhibition of hsa-miR-146b-5p expression in different cells was also investigated and a satisfactory result was obtained.
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Affiliation(s)
- Jiaxue Gao
- State Key Laboratory of Electroanalytical Chemistry, Changchun Institute of Applied Chemistry, Chinese Academy of Sciences, Changchun, 130022, P. R. China.
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22
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Wang H, Wang H, Sun Y, Liu X, Liu Y, Wang C, Zhang P, Li Z. A general strategy for highly sensitive analysis of genetic biomarkers at single-base resolution with ligase-based isothermally exponential amplification. Talanta 2020; 212:120754. [DOI: 10.1016/j.talanta.2020.120754] [Citation(s) in RCA: 4] [Impact Index Per Article: 0.8] [Reference Citation Analysis] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 12/09/2019] [Revised: 01/12/2020] [Accepted: 01/14/2020] [Indexed: 01/17/2023]
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23
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Su F, Ji J, Zhang P, Wang F, Li Z. Real-time quantification of fusion transcripts with ligase chain reaction by direct ligation of adjacent DNA probes at fusion junction. Analyst 2020; 145:3977-3982. [PMID: 32319973 DOI: 10.1039/d0an00163e] [Citation(s) in RCA: 2] [Impact Index Per Article: 0.4] [Reference Citation Analysis] [Abstract] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 12/15/2022]
Abstract
Gene fusions, produced by aberrant juxtapositions of two or more genes even in different chromosomes, play important roles in the primary oncogenic mechanism and have been demonstrated to be typically associated with many cancers. So the fused genes or the transcripts can be specific predictive biomarkers for cancer diagnosis and therapy. Herein, we develop a direct ligation- and ligase chain reaction (LCR)-based method for a fusion transcript assay. In virtue of the high selectivity of ligase and the exponential amplification capacity of LCR, the proposed method can detect as low as 1 fM fusion transcripts with high specificity and has been successfully applied to real samples. With the real-time fluorescence measurements, the fusion transcripts can be assayed in a simple way. Therefore, the proposed method can provide a simple and cost-effective platform for fusion transcript detection in routine laboratories and clinical diagnosis.
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Affiliation(s)
- Fengxia Su
- School of Chemistry and Biological Engineering, University of Science and Technology Beijing, Beijing, 100083, P. R. China.
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24
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Wang G, Tian W, Liu X, Ren W, Liu C. New CRISPR-Derived microRNA Sensing Mechanism Based on Cas12a Self-Powered and Rolling Circle Transcription-Unleashed Real-Time crRNA Recruiting. Anal Chem 2020; 92:6702-6708. [DOI: 10.1021/acs.analchem.0c00680] [Citation(s) in RCA: 51] [Impact Index Per Article: 10.2] [Reference Citation Analysis] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 12/26/2022]
Affiliation(s)
- Gaoting Wang
- Key Laboratory of Applied Surface and Colloid Chemistry, Ministry of Education; Key Laboratory of Analytical Chemistry for Life Science of Shaanxi Province, School of Chemistry & Chemical Engineering, Shaanxi Normal University, Xi’an 710119, Shaanxi Province, P. R. China
| | - Weimin Tian
- Key Laboratory of Applied Surface and Colloid Chemistry, Ministry of Education; Key Laboratory of Analytical Chemistry for Life Science of Shaanxi Province, School of Chemistry & Chemical Engineering, Shaanxi Normal University, Xi’an 710119, Shaanxi Province, P. R. China
| | - Xiaoling Liu
- Key Laboratory of Applied Surface and Colloid Chemistry, Ministry of Education; Key Laboratory of Analytical Chemistry for Life Science of Shaanxi Province, School of Chemistry & Chemical Engineering, Shaanxi Normal University, Xi’an 710119, Shaanxi Province, P. R. China
| | - Wei Ren
- Key Laboratory of Applied Surface and Colloid Chemistry, Ministry of Education; Key Laboratory of Analytical Chemistry for Life Science of Shaanxi Province, School of Chemistry & Chemical Engineering, Shaanxi Normal University, Xi’an 710119, Shaanxi Province, P. R. China
| | - Chenghui Liu
- Key Laboratory of Applied Surface and Colloid Chemistry, Ministry of Education; Key Laboratory of Analytical Chemistry for Life Science of Shaanxi Province, School of Chemistry & Chemical Engineering, Shaanxi Normal University, Xi’an 710119, Shaanxi Province, P. R. China
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25
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Tang Y, He X, Yuan R, Liu X, Zhao Y, Wang T, Chen H, Feng X. Logic-signal-based multiplex detection of MiRNAs with high tension hybridization and multiple signal amplification. Analyst 2020; 145:4314-4320. [PMID: 32400825 DOI: 10.1039/d0an00550a] [Citation(s) in RCA: 2] [Impact Index Per Article: 0.4] [Reference Citation Analysis] [Abstract] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 11/21/2022]
Abstract
A multiplex miRNA detection scheme with simultaneous multiple signal output by single excitation has been reported.
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Affiliation(s)
- Yaqin Tang
- Department of Pharmaceutical Sciences
- Chongqing University
- Chongqing
- China
| | - Xiao He
- Department of Pharmaceutical Sciences
- Chongqing University
- Chongqing
- China
| | - Rui Yuan
- Chongqing University Cancer Hospital
- Chongqing Cancer Hospital
- Chongqing
- China
| | - Xingming Liu
- Chongqing University Cancer Hospital
- Chongqing Cancer Hospital
- Chongqing
- China
| | - Yi Zhao
- Chongqing University Cancer Hospital
- Chongqing Cancer Hospital
- Chongqing
- China
| | - Tingting Wang
- Biochemistry and Molecular Biology Laboratory
- Experimental Teaching and Management Center
- Chongqing Medical University
- Chongqing
- China
| | - Hui Chen
- Department of Pharmaceutical Sciences
- Chongqing University
- Chongqing
- China
| | - Xuli Feng
- Department of Pharmaceutical Sciences
- Chongqing University
- Chongqing
- China
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26
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Zhang P, Guo N, Gao K, Su F, Wang F, Li Z. Direct recognition and sensitive detection of circular RNA with ligation-based PCR. Org Biomol Chem 2020; 18:3269-3273. [DOI: 10.1039/d0ob00625d] [Citation(s) in RCA: 10] [Impact Index Per Article: 2.0] [Reference Citation Analysis] [Abstract] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 12/17/2022]
Abstract
A sensitive and specific method for circRNA detection is developed through the direct ligation of two ingeniously designed DNA probes.
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Affiliation(s)
- Pengbo Zhang
- School of Chemistry and Biology Engineering
- University of Science and Technology Beijing
- Beijing
- P. R. China
| | - Na Guo
- School of Chemistry and Biology Engineering
- University of Science and Technology Beijing
- Beijing
- P. R. China
| | - Kejian Gao
- School of Chemistry and Biology Engineering
- University of Science and Technology Beijing
- Beijing
- P. R. China
| | - Fengxia Su
- School of Chemistry and Biology Engineering
- University of Science and Technology Beijing
- Beijing
- P. R. China
| | - Fangfang Wang
- School of Chemistry and Biology Engineering
- University of Science and Technology Beijing
- Beijing
- P. R. China
| | - Zhengping Li
- School of Chemistry and Biology Engineering
- University of Science and Technology Beijing
- Beijing
- P. R. China
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27
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Biosensors for epigenetic biomarkers detection: A review. Biosens Bioelectron 2019; 144:111695. [PMID: 31526982 DOI: 10.1016/j.bios.2019.111695] [Citation(s) in RCA: 13] [Impact Index Per Article: 2.2] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 07/17/2019] [Revised: 08/24/2019] [Accepted: 09/06/2019] [Indexed: 12/11/2022]
Abstract
Epigenetic inheritance is a heritable change in gene function independent of alterations in nucleotide sequence. It regulates the normal cellular activities of the organisms by affecting gene expression and transcription, and its abnormal expression may lead to the developmental disorder, senile dementia, and carcinogenesis progression. Thus, epigenetic inheritance is recognized as an important biomarker, and the accurate quantification of epigenetic inheritance is crucial to clinical diagnosis, drug development and cancer treatment. Noncoding RNA, DNA methylation and histone modification are the most common epigenetic biomarkers. The conventional biosensors (e.g., northern blotting, radiometric, mass spectrometry and immunosorbent biosensors) for epigenetic biomarkers assay usually suffer from hazardous radiation, complicated manipulation, and time-consuming procedures. To facilitate the practical applications, some new biosensors including colorimetric, luminescent, Raman scattering spectroscopy, electrochemical and fluorescent biosensors have been developed for the detection of epigenetic biomarkers with simplicity, rapidity, high throughput and high sensitivity. In this review, we summarize the recent advances in epigenetic biomarkers assay. We classify the biosensors into the direct amplification-free and the nucleotide amplification-assisted ones, and describe the principles of various biosensors, and further compare their performance for epigenetic biomarkers detection. Moreover, we discuss the emerging trends and challenges in the future development of epigenetic biomarkers biosensors.
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Fluorometric determination of microRNA by using target-triggered cascade signal amplification and DNA-templated silver nanoclusters. Mikrochim Acta 2019; 186:669. [DOI: 10.1007/s00604-019-3789-7] [Citation(s) in RCA: 9] [Impact Index Per Article: 1.5] [Reference Citation Analysis] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 04/07/2019] [Accepted: 08/27/2019] [Indexed: 12/21/2022]
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29
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Qi Y, Lu X, Feng Q, Fan W, Liu C, Li Z. An Enzyme-Free MicroRNA Assay Based On Fluorescence Counting of Click Chemical Ligation-Illuminated Magnetic Nanoparticles with Total Internal Reflection Fluorescence Microscopy. ACS Sens 2018; 3:2667-2674. [PMID: 30456947 DOI: 10.1021/acssensors.8b01169] [Citation(s) in RCA: 23] [Impact Index Per Article: 3.3] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 12/18/2022]
Abstract
MicroRNAs (miRNAs) have been considered as promising cancer biomarkers. However, the simple but sensitive detection of low levels of miRNAs in biological samples still remains challenging. Herein, we wish to report an entirely enzyme-free, simple, and highly sensitive miRNA assay based on the counting of cycling click chemical ligation (3CL)-illuminated fluorescent magnetic nanoparticles (MNPs) with a total internal reflection fluorescence microscopy (TIRFM). In this strategy, each miRNA molecule can trigger many cycles of click chemical ligation reactions to produce plentiful ligated oligonucleotides (ODNs) with both 5'-biotin and 3'-fluorophore, resulting in efficient signal amplification. It is worth noting that only the ligated ODNs can bring fluorophores onto streptavidin-functionalized MNPs (STV-MNPs). Notably, merely 10 fluorescent molecules on each 50 nm MNP can make it bright enough to be clearly visualized by the TIRFM, which can significantly improve the detection sensitivity for miRNA. Through fluorescence counting of individual MNPs and integrating their fluorescence intensities, the amount of target miRNA can be quantitatively determined. This miRNA assay can be accomplished in a mix-and-read manner just by simply mixing the enzyme-free 3CL reaction system with the MNPs before TIRFM imaging, which avoids tedious immobilization, washing, and purification steps. Despite the extremely simple operation, this strategy exhibits high sensitivity with a quite low detection limit of 50 fM target miRNA as well as high specificity to well discriminate miRNA sequences with a single-base variation. Furthermore, the applicability of this method in real biological samples is also verified through the accurate detection of the miRNA target in cancer cells.
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Affiliation(s)
- Yan Qi
- Key laboratory of Analytical Chemistry for Life Science of Shaanxi Province, Key laboratory of Applied Surface and Colloid Chemistry, Ministry of Education, School of Chemistry and Chemical Engineering, Shaanxi Normal University, Xi’an, Shaanxi Province 710119, P. R. China
| | - Xiaohui Lu
- Key laboratory of Analytical Chemistry for Life Science of Shaanxi Province, Key laboratory of Applied Surface and Colloid Chemistry, Ministry of Education, School of Chemistry and Chemical Engineering, Shaanxi Normal University, Xi’an, Shaanxi Province 710119, P. R. China
| | - Qinya Feng
- Key laboratory of Analytical Chemistry for Life Science of Shaanxi Province, Key laboratory of Applied Surface and Colloid Chemistry, Ministry of Education, School of Chemistry and Chemical Engineering, Shaanxi Normal University, Xi’an, Shaanxi Province 710119, P. R. China
| | - Wenjiao Fan
- Key laboratory of Analytical Chemistry for Life Science of Shaanxi Province, Key laboratory of Applied Surface and Colloid Chemistry, Ministry of Education, School of Chemistry and Chemical Engineering, Shaanxi Normal University, Xi’an, Shaanxi Province 710119, P. R. China
| | - Chenghui Liu
- Key laboratory of Analytical Chemistry for Life Science of Shaanxi Province, Key laboratory of Applied Surface and Colloid Chemistry, Ministry of Education, School of Chemistry and Chemical Engineering, Shaanxi Normal University, Xi’an, Shaanxi Province 710119, P. R. China
| | - Zhengping Li
- Key laboratory of Analytical Chemistry for Life Science of Shaanxi Province, Key laboratory of Applied Surface and Colloid Chemistry, Ministry of Education, School of Chemistry and Chemical Engineering, Shaanxi Normal University, Xi’an, Shaanxi Province 710119, P. R. China
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30
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Wei K, Zhao J, Qin Y, Li S, Huang Y, Zhao S. A novel multiplex signal amplification strategy based on microchip electrophoresis platform for the improved separation and detection of microRNAs. Talanta 2018; 189:437-441. [DOI: 10.1016/j.talanta.2018.07.037] [Citation(s) in RCA: 14] [Impact Index Per Article: 2.0] [Reference Citation Analysis] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 04/11/2018] [Revised: 07/08/2018] [Accepted: 07/11/2018] [Indexed: 01/18/2023]
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31
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Wang RH, He LY, Zhou SH. The role of gene sculptor microRNAs in human precancerous lesions. Onco Targets Ther 2018; 11:5667-5675. [PMID: 30254459 PMCID: PMC6141127 DOI: 10.2147/ott.s171241] [Citation(s) in RCA: 9] [Impact Index Per Article: 1.3] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 12/12/2022] Open
Abstract
MicroRNAs (miRNAs) are small noncoding RNAs. These noncoding RNAs regulate the expression of target genes and inhibit the translation of target proteins at the post-transcriptional level. miRNAs also play an important role in human health, from the development and differentiation of cells to the occurrence and progression of disorders such as cancer, cardiovascular diseases, and neurodegenerative diseases. Precancerous lesions are lesions prior to invasive carcinomas, and carcinogenesis is a very complicated process, which is multistage and the result of multigene synergy. miRNAs exert effects as both oncogenes and tumor suppressor genes by regulating target genes involved in signaling pathways. Hence, precancerous lesions are accompanied by relevant miRNA changes. Based on the morphology of miRNAs in vivo and the specificity of miRNA, various novel miRNA analysis methods have been developed, including reverse transcription quantitative PCR, enzyme analysis, molecular beacons, and deep sequencing. For example, in the laryngeal epithelial precancerous lesions, the data demonstrate that the expression of miR-10a-5p is downregulated and miR-484 is the most abundant miRNA in hepatic precancerous lesions. In this review, we discuss the functional roles of miRNAs in human precancerous lesions.
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Affiliation(s)
- Ran-Hong Wang
- Department of Otolaryngology, The First Affiliated Hospital, College of Medicine, Zhejiang University, Hangzhou City, People's Republic of China, .,Department of Otolaryngology, The Third Hospital of Hangzhou City, Hangzhou City, People's Republic of China
| | - Lan-Ying He
- Department of Otolaryngology, The First Affiliated Hospital, College of Medicine, Zhejiang University, Hangzhou City, People's Republic of China,
| | - Shui-Hong Zhou
- Department of Otolaryngology, The First Affiliated Hospital, College of Medicine, Zhejiang University, Hangzhou City, People's Republic of China,
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32
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Tentori AM, Nagarajan MB, Kim JJ, Zhang WC, Slack FJ, Doyle PS. Quantitative and multiplex microRNA assays from unprocessed cells in isolated nanoliter well arrays. LAB ON A CHIP 2018; 18:2410-2424. [PMID: 29998262 PMCID: PMC6081239 DOI: 10.1039/c8lc00498f] [Citation(s) in RCA: 7] [Impact Index Per Article: 1.0] [Reference Citation Analysis] [Abstract] [MESH Headings] [Grants] [Track Full Text] [Subscribe] [Scholar Register] [Indexed: 05/03/2023]
Abstract
MicroRNAs (miRNAs) have recently emerged as promising biomarkers for the profiling of diseases. Translation of miRNA biomarkers to clinical practice, however, remains a challenge due to the lack of analysis platforms for sensitive, quantitative, and multiplex miRNA assays that have simple and robust workflows suitable for translation. The platform we present here utilizes functionalized hydrogel posts contained within isolated nanoliter well reactors for quantitative and multiplex assays directly from unprocessed cell samples without the need of prior nucleic acid extraction. Simultaneous reactor isolation and delivery of miRNA extraction reagents is achieved by sealing an array of wells containing the functionalized hydrogel posts and cells against another array of wells containing lysis and extraction reagents. The nanoliter well array platform features >100× better sensitivity compared to previous technology utilizing hydrogel particles without relying on signal amplification and enables >100 parallel assays in a single device. These advances provided by this platform lay the groundwork for translatable and robust analysis technologies for miRNA expression profiling in samples with small populations of cells and in precious, material-limited samples.
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Affiliation(s)
- Augusto M. Tentori
- Department of Chemical Engineering
, Massachusetts Institute of Technology
,
Cambridge
, USA
.
; Tel: +1 617 253 4534
| | - Maxwell B. Nagarajan
- Department of Chemical Engineering
, Massachusetts Institute of Technology
,
Cambridge
, USA
.
; Tel: +1 617 253 4534
| | - Jae Jung Kim
- Department of Chemical Engineering
, Massachusetts Institute of Technology
,
Cambridge
, USA
.
; Tel: +1 617 253 4534
| | - Wen Cai Zhang
- Department of Pathology
, Beth Israel Deaconess Medical Center/Harvard Medical School
,
Boston
, USA
| | - Frank J. Slack
- Department of Pathology
, Beth Israel Deaconess Medical Center/Harvard Medical School
,
Boston
, USA
| | - Patrick S. Doyle
- Department of Chemical Engineering
, Massachusetts Institute of Technology
,
Cambridge
, USA
.
; Tel: +1 617 253 4534
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33
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An innovative paradigm of methods in microRNAs detection: highlighting DNAzymes, the illuminators. Biosens Bioelectron 2018; 107:123-144. [DOI: 10.1016/j.bios.2018.02.020] [Citation(s) in RCA: 41] [Impact Index Per Article: 5.9] [Reference Citation Analysis] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 11/25/2017] [Revised: 01/22/2018] [Accepted: 02/07/2018] [Indexed: 12/15/2022]
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34
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Wang LJ, Ren M, Liang L, Zhang CY. Controllable fabrication of bio-bar codes for dendritically amplified sensing of human T-lymphotropic viruses. Chem Sci 2018; 9:4942-4949. [PMID: 29938021 PMCID: PMC5994793 DOI: 10.1039/c8sc01641k] [Citation(s) in RCA: 29] [Impact Index Per Article: 4.1] [Reference Citation Analysis] [Abstract] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 04/11/2018] [Accepted: 05/09/2018] [Indexed: 01/10/2023] Open
Abstract
We demonstrate for the first time the controllable fabrication of bio-bar codes for dendritically amplified sensing of low-abundant HTLV-II DNA.
Human T-lymphotropic virus type II (HTLV-II) is an important type-C retrovirus, closely related to a variety of human diseases. Here, we demonstrate for the first time the controllable fabrication of bio-bar codes for dendritically amplified sensing of low-abundant HTLV-II DNA by the integration of terminal deoxynucleotidyl transferase (TdT)-catalyzed template-free polymerization extension with bio-bar-code amplification (BCA). HTLV-II DNA hybridizes with magnetic microparticle (MMP)-modified capture probe 1, forming a stable DNA duplex with a protruding 3′-hydroxylated sequence which may function as a primer to initiate the TdT-catalyzed first-step polymerization extension for the generation of a poly-thymidine (T) sequence. The resultant poly-T products may hybridize with poly-adenine (A) capture probe 2, inducing the self-assembly of multiple capture probe 2-/reporter probe-functionalized Au nanoparticles (AuNPs) onto the MMP. Subsequently, the reporter probes may act as the primers to initiate the TdT-catalyzed second-step polymerization extension, producing large numbers of G-rich DNAzymes for the generation of an enhanced chemiluminescence signal. Taking advantage of the efficient polymerization extension reaction catalyzed by TdT, the high amplification efficiency of BCA, and the intrinsically high sensitivity of G-rich DNAzyme-driven chemiluminescence, this method exhibits ultrahigh sensitivity with a limit of detection of as low as 0.50 aM and a large dynamic range of 9 orders of magnitude from 1 aM to 1 nM. Moreover, this method can be applied for the discrimination of a single-base mismatch and the measurement of HTLV-II DNA in both human serum and human T-lymphocytic leukemia cells, holding great potential in biomedical research and clinical diagnosis.
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Affiliation(s)
- Li-Juan Wang
- College of Chemistry, Chemical Engineering and Materials Science , Collaborative Innovation Center of Functionalized Probes for Chemical Imaging in Universities of Shandong , Key Laboratory of Molecular and Nano Probes , Ministry of Education , Shandong Provincial Key Laboratory of Clean Production of Fine Chemicals , Shandong Normal University , Jinan 250014 , China . ; ; Tel: +86 0531 86186033
| | - Ming Ren
- College of Chemistry, Chemical Engineering and Materials Science , Collaborative Innovation Center of Functionalized Probes for Chemical Imaging in Universities of Shandong , Key Laboratory of Molecular and Nano Probes , Ministry of Education , Shandong Provincial Key Laboratory of Clean Production of Fine Chemicals , Shandong Normal University , Jinan 250014 , China . ; ; Tel: +86 0531 86186033
| | - Li Liang
- Department of Tumor Chemotherapy and Radiation Sickness , Peking University Third Hospital , Beijing 100191 , China
| | - Chun-Yang Zhang
- College of Chemistry, Chemical Engineering and Materials Science , Collaborative Innovation Center of Functionalized Probes for Chemical Imaging in Universities of Shandong , Key Laboratory of Molecular and Nano Probes , Ministry of Education , Shandong Provincial Key Laboratory of Clean Production of Fine Chemicals , Shandong Normal University , Jinan 250014 , China . ; ; Tel: +86 0531 86186033
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35
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Fan W, Qi Y, Qiu L, He P, Liu C, Li Z. Click Chemical Ligation-Initiated On-Bead DNA Polymerization for the Sensitive Flow Cytometric Detection of 3'-Terminal 2'-O-Methylated Plant MicroRNA. Anal Chem 2018; 90:5390-5397. [PMID: 29600844 DOI: 10.1021/acs.analchem.8b00589] [Citation(s) in RCA: 15] [Impact Index Per Article: 2.1] [Reference Citation Analysis] [Abstract] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 12/29/2022]
Abstract
A versatile flow cytometric strategy is developed for the sensitive detection of plant microRNA (miRNA) by coupling the target-templated click nucleic acid ligation (CNAL) with on-bead terminal enzymatic DNA polymerization (TEP). Unlike ligase-catalyzed ligation reaction, the plant miRNA-templated enzyme-free CNAL between two single-stranded DNA (ssDNA) probes, respectively modified with Aza-dibenzocyclooctyne (Aza-DBCO) and N3, can not only simplify the operation, but also achieve a much higher ligation efficiency. More importantly, the undesirable nonspecific ligation between the Aza-DBCO- and N3-modified ssDNA, can be effectively eliminated by adding Tween-20, which allows the use of cycling CNAL (CCNAL) in a background-free manner. So each plant miRNA can template many rounds of CNAL reaction to produce numerous ligation products, forming efficient signal amplification. The ligated ssDNA can be anchored on the magnetic beads (MBs) with the 3'-OH termini exposed outside. Then terminal deoxynucleotidyl transferase (TdT), a sequence-independent and template-free polymerase, would specifically catalyze the DNA polymerization along these 3'-OH termini on the MBs, forming poly(T) tails up to thousands of nucleotides long. Each poly(T) tail allows specific binding of numerous 6-carboxyfluorescein (FAM)-labeled poly(A)25 oligonucleotides to accumulate a lot of fluorophores on the MBs, leading to the second step of signal amplification. By integrating the advantages of CCNAL-TEP for highly efficient signal amplification and robust MBs signal readout with powerful flow cytometer, high sensitivity is achieved and the detection limit of plant miRNA has been pushed down to a low level of 5 fM with high specificity to well discriminate even single-base difference between miRNA targets.
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Affiliation(s)
- Wenjiao Fan
- Key Laboratory of Analytical Chemistry for Life Science of Shaanxi Province; Key Laboratory of Applied Surface and Colloid Chemistry, Ministry of Education, School of Chemistry and Chemical Engineering , Shaanxi Normal University , Xi'an 710119 , Shaanxi Province , People's Republic of China
| | - Yan Qi
- Key Laboratory of Analytical Chemistry for Life Science of Shaanxi Province; Key Laboratory of Applied Surface and Colloid Chemistry, Ministry of Education, School of Chemistry and Chemical Engineering , Shaanxi Normal University , Xi'an 710119 , Shaanxi Province , People's Republic of China
| | - Liying Qiu
- Key Laboratory of Analytical Chemistry for Life Science of Shaanxi Province; Key Laboratory of Applied Surface and Colloid Chemistry, Ministry of Education, School of Chemistry and Chemical Engineering , Shaanxi Normal University , Xi'an 710119 , Shaanxi Province , People's Republic of China
| | - Pan He
- Key Laboratory of Analytical Chemistry for Life Science of Shaanxi Province; Key Laboratory of Applied Surface and Colloid Chemistry, Ministry of Education, School of Chemistry and Chemical Engineering , Shaanxi Normal University , Xi'an 710119 , Shaanxi Province , People's Republic of China
| | - Chenghui Liu
- Key Laboratory of Analytical Chemistry for Life Science of Shaanxi Province; Key Laboratory of Applied Surface and Colloid Chemistry, Ministry of Education, School of Chemistry and Chemical Engineering , Shaanxi Normal University , Xi'an 710119 , Shaanxi Province , People's Republic of China
| | - Zhengping Li
- Key Laboratory of Analytical Chemistry for Life Science of Shaanxi Province; Key Laboratory of Applied Surface and Colloid Chemistry, Ministry of Education, School of Chemistry and Chemical Engineering , Shaanxi Normal University , Xi'an 710119 , Shaanxi Province , People's Republic of China
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36
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Dai W, Zhang J, Meng X, He J, Zhang K, Cao Y, Wang D, Dong H, Zhang X. Catalytic hairpin assembly gel assay for multiple and sensitive microRNA detection. Theranostics 2018; 8:2646-2656. [PMID: 29774065 PMCID: PMC5956999 DOI: 10.7150/thno.24480] [Citation(s) in RCA: 33] [Impact Index Per Article: 4.7] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 12/21/2017] [Accepted: 02/21/2018] [Indexed: 01/28/2023] Open
Abstract
As important modulators of gene expression, microRNAs (miRNAs) have been identified as promising biomarkers with powerful predictive value in diagnosis and prognosis for several diseases, especially for cancers. Here we report a facile, multiple and sensitive miRNA detection method that uses conventional gel electrophoresis and catalytic hairpin assembly (CHA) system without any complex nanomaterials or enzymatic amplification. Methods: In this study, three pairs of hairpin probes are rationally designed with thermodynamically and kinetically preferable feasibility for the CHA process. In the present of target miRNA, the stem of the corresponding hairpin detection probe (HDP) will be unfolded and expose the concealed domain. The corresponding hairpin assistant probe (HAP) then replaces the hybridized target miRNA to form specific HDP/HAP complexes and releases miRNA based on thermodynamically driven entropy gain process, and the released miRNA triggers the next recycle to produce tremendous corresponding HDP/HAP complexes. Results: The results showed that the CHA gel assay can detect miRNA at fM levels and shows good capability of discriminating miRNA family members and base-mismatched miRNAs. It is able to analyze miRNAs extracted from cell lysates, which are consistent with the results of conventional polymerase chain reaction (PCR) method. Depending on the length of the designed hairpin probes, the CHA gel assay consisting of different hairpin probes effectively discriminated and simultaneously detected multiple miRNAs in homogenous solution and miRNAs extracted from cell lysates. Conclusion: The work highlights the practical use of a conventional gel electrophoresis for sensitive interesting nucleic acid sequences detection.
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Affiliation(s)
- Wenhao Dai
- Research Center for Bioengineering and Sensing Technology, Beijing Key Lab for Bioengineering and Sensing Technology, School of Chemistry and bioengineering, University of Science & Technology Beijing, Beijing 100083, P.R. China
| | - Jing Zhang
- School of Petrochemical Engineering, School of Food Science and Technology, Changzhou University, Changzhou 213164, P.R. China
| | - Xiangdan Meng
- Research Center for Bioengineering and Sensing Technology, Beijing Key Lab for Bioengineering and Sensing Technology, School of Chemistry and bioengineering, University of Science & Technology Beijing, Beijing 100083, P.R. China
| | - Jie He
- School of Computer and Communication Engineering, University of Science & Technology Beijing, Beijing 100083, P.R. China
| | - Kai Zhang
- Research Center for Bioengineering and Sensing Technology, Beijing Key Lab for Bioengineering and Sensing Technology, School of Chemistry and bioengineering, University of Science & Technology Beijing, Beijing 100083, P.R. China
| | - Yu Cao
- Research Center for Bioengineering and Sensing Technology, Beijing Key Lab for Bioengineering and Sensing Technology, School of Chemistry and bioengineering, University of Science & Technology Beijing, Beijing 100083, P.R. China
| | - Dongdong Wang
- Research Center for Bioengineering and Sensing Technology, Beijing Key Lab for Bioengineering and Sensing Technology, School of Chemistry and bioengineering, University of Science & Technology Beijing, Beijing 100083, P.R. China
| | - Haifeng Dong
- Research Center for Bioengineering and Sensing Technology, Beijing Key Lab for Bioengineering and Sensing Technology, School of Chemistry and bioengineering, University of Science & Technology Beijing, Beijing 100083, P.R. China
| | - Xueji Zhang
- Research Center for Bioengineering and Sensing Technology, Beijing Key Lab for Bioengineering and Sensing Technology, School of Chemistry and bioengineering, University of Science & Technology Beijing, Beijing 100083, P.R. China
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37
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Wu W, Fan H, Lian X, Zhou J, Zhang T. Aligner-mediated cleavage-triggered exponential amplification for sensitive detection of nucleic acids. Talanta 2018; 185:141-145. [PMID: 29759180 DOI: 10.1016/j.talanta.2018.03.067] [Citation(s) in RCA: 5] [Impact Index Per Article: 0.7] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 01/29/2018] [Revised: 03/14/2018] [Accepted: 03/22/2018] [Indexed: 10/17/2022]
Abstract
Exponential amplification reaction (EXPAR), as a simple and high sensitive method, holds great promise in nucleic acids detection. One major challenge in EXPAR is the generation of trigger DNA with a definite 3'-end, which now relies on fingerprinting technology. However, the requirement of different endonucleases for varying target sequences and two head-to-head recognition sites in double stranded DNA, as well as the confinement of trigger DNA's 3'-end to be near/within the recognition site, usually subject EXPAR to compromised universality and/or repeated matching of reaction conditions. Herein, we report a simple and universal method for high sensitive detection of nucleic acids, termed aligner-mediated cleavage-triggered exponential amplification (AMCEA). The aligner-mediated cleavage (AMC) needs only one nicking endonuclease and can make a break at any site of choice in a programmable way. Thus, the 3'-end of target DNA can be easily redefined as required, a key step for initiating the amplification reaction. This capability endows the proposed AMCEA with excellent universality and simplicity. Moreover, it is sensitive and specific, with a detection limit at amol level, a broad dynamic range of 5～6 orders of magnitude and the ability to distinguish single nucleotide mutation. Experiments performed with human serum indicate that AMCEA is compatible with the complex biological sample, and thus has the potentials for practical applications.
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Affiliation(s)
- Wanghua Wu
- Research Center for Analytical Instrumentation, Institute of Cyber-Systems and Control, State Key Laboratory of Industrial Control Technology, Zhejiang University, Hangzhou 310027, PR China
| | - Hongliang Fan
- Department of Environmental Medicine, Institute of Hygiene, Zhejiang Academy of Medical Sciences, Hangzhou 310013, PR China
| | - Xiang Lian
- Research Center for Analytical Instrumentation, Institute of Cyber-Systems and Control, State Key Laboratory of Industrial Control Technology, Zhejiang University, Hangzhou 310027, PR China
| | - Jianguang Zhou
- Research Center for Analytical Instrumentation, Institute of Cyber-Systems and Control, State Key Laboratory of Industrial Control Technology, Zhejiang University, Hangzhou 310027, PR China
| | - Tao Zhang
- Research Center for Analytical Instrumentation, Institute of Cyber-Systems and Control, State Key Laboratory of Industrial Control Technology, Zhejiang University, Hangzhou 310027, PR China.
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38
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Luminescent quantum dots for miRNA detection. Talanta 2018; 179:456-465. [PMID: 29310260 DOI: 10.1016/j.talanta.2017.11.011] [Citation(s) in RCA: 36] [Impact Index Per Article: 5.1] [Reference Citation Analysis] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 09/03/2017] [Revised: 10/30/2017] [Accepted: 11/06/2017] [Indexed: 01/23/2023]
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39
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Abstract
Recent advances in miRNA detection methods and new applications.
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Affiliation(s)
- Yongqiang Cheng
- Key Laboratory of Medicinal Chemistry and Molecular Diagnosis
- Ministry of Education
- Key Laboratory of Analytical Science and Technology of Hebei Province
- College of Chemistry and Environmental Science
- Hebei University
| | - Lijuan Dong
- Key Laboratory of Medicinal Chemistry and Molecular Diagnosis
- Ministry of Education
- Key Laboratory of Analytical Science and Technology of Hebei Province
- College of Chemistry and Environmental Science
- Hebei University
| | - Jiangyan Zhang
- Key Laboratory of Medicinal Chemistry and Molecular Diagnosis
- Ministry of Education
- Key Laboratory of Analytical Science and Technology of Hebei Province
- College of Chemistry and Environmental Science
- Hebei University
| | - Yaqing Zhao
- Key Laboratory of Medicinal Chemistry and Molecular Diagnosis
- Ministry of Education
- Key Laboratory of Analytical Science and Technology of Hebei Province
- College of Chemistry and Environmental Science
- Hebei University
| | - Zhengping Li
- Key Laboratory of Medicinal Chemistry and Molecular Diagnosis
- Ministry of Education
- Key Laboratory of Analytical Science and Technology of Hebei Province
- College of Chemistry and Environmental Science
- Hebei University
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40
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Wei K, Zhao J, Luo X, Qiu S, He F, Li S, Zhao S. An ultrasensitive microchip electrophoresis assay based on separation-assisted double cycling signal amplification strategy for microRNA detection in cell lysate. Analyst 2018; 143:1468-1474. [DOI: 10.1039/c7an02082a] [Citation(s) in RCA: 11] [Impact Index Per Article: 1.6] [Reference Citation Analysis] [Abstract] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 01/03/2023]
Abstract
An ultrasensitive microchip electrophoresis assay was developed for microRNA detection.
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Affiliation(s)
- Kaiji Wei
- Key Laboratory for the Chemistry and Molecular Engineering of Medicinal Resources
- Guangxi Normal University
- Guilin, 541004
- China
| | - Jingjin Zhao
- Key Laboratory for the Chemistry and Molecular Engineering of Medicinal Resources
- Guangxi Normal University
- Guilin, 541004
- China
| | - Xiaoshu Luo
- College of Electronic Engineering
- Guangxi Normal University
- Guilin
- China
| | - Senghui Qiu
- College of Electronic Engineering
- Guangxi Normal University
- Guilin
- China
| | - Fuyun He
- College of Electronic Engineering
- Guangxi Normal University
- Guilin
- China
| | - Shuting Li
- Key Laboratory for the Chemistry and Molecular Engineering of Medicinal Resources
- Guangxi Normal University
- Guilin, 541004
- China
| | - Shulin Zhao
- Key Laboratory for the Chemistry and Molecular Engineering of Medicinal Resources
- Guangxi Normal University
- Guilin, 541004
- China
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41
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Wang H, Wang H, Duan X, Liu C, Li Z. Digital quantitative analysis of microRNA in single cell based on ligation-depended polymerase colony (Polony). Biosens Bioelectron 2017; 95:146-151. [DOI: 10.1016/j.bios.2017.04.001] [Citation(s) in RCA: 16] [Impact Index Per Article: 2.0] [Reference Citation Analysis] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 02/04/2017] [Revised: 04/03/2017] [Accepted: 04/06/2017] [Indexed: 12/16/2022]
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42
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Chen YX, Huang KJ, Niu KX. Recent advances in signal amplification strategy based on oligonucleotide and nanomaterials for microRNA detection-a review. Biosens Bioelectron 2017; 99:612-624. [PMID: 28837925 DOI: 10.1016/j.bios.2017.08.036] [Citation(s) in RCA: 167] [Impact Index Per Article: 20.9] [Reference Citation Analysis] [Abstract] [Key Words] [Journal Information] [Subscribe] [Scholar Register] [Received: 06/12/2017] [Revised: 08/13/2017] [Accepted: 08/14/2017] [Indexed: 01/01/2023]
Abstract
MicroRNAs (MiRNAs) play multiple crucial regulating roles in cell which can regulate one third of protein-coding genes. MiRNAs participate in the developmental and physiological processes of human body, while their aberrant adjustment will be more likely to trigger diseases such as cancers, kidney disease, central nervous system diseases, cardiovascular diseases, diabetes, viral infections and so on. What's worse, for the detection of miRNAs, their small size, high sequence similarity, low abundance and difficult extraction from cells impose great challenges in the analysis. Hence, it's necessary to fabricate accurate and sensitive biosensing platform for miRNAs detection. Up to now, researchers have developed many signal-amplification strategies for miRNAs detection, including hybridization chain reaction, nuclease amplification, rolling circle amplification, catalyzed hairpin assembly amplification and nanomaterials based amplification. These methods are typical, feasible and frequently used. In this review, we retrospect recent advances in signal amplification strategies for detecting miRNAs and point out the pros and cons of them. Furthermore, further prospects and promising developments of the signal-amplification strategies for detecting miRNAs are proposed.
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Affiliation(s)
- Ying-Xu Chen
- College of Chemistry and Chemical Engineering, Xinyang Normal University, Xinyang 464000, China; Henan Province Key Laboratory of Utilization of Non-metallic Mineral in the South of Henan, Xinyang Normal University, Xinyang 464000, China
| | - Ke-Jing Huang
- College of Chemistry and Chemical Engineering, Xinyang Normal University, Xinyang 464000, China; Henan Province Key Laboratory of Utilization of Non-metallic Mineral in the South of Henan, Xinyang Normal University, Xinyang 464000, China.
| | - Ke-Xin Niu
- College of Chemistry and Chemical Engineering, Xinyang Normal University, Xinyang 464000, China; Henan Province Key Laboratory of Utilization of Non-metallic Mineral in the South of Henan, Xinyang Normal University, Xinyang 464000, China
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43
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A Quasi-direct LC-MS/MS-based Targeted Proteomics Approach for miRNA Quantification via a Covalently Immobilized DNA-peptide Probe. Sci Rep 2017; 7:5669. [PMID: 28720752 PMCID: PMC5515972 DOI: 10.1038/s41598-017-05495-7] [Citation(s) in RCA: 16] [Impact Index Per Article: 2.0] [Reference Citation Analysis] [Abstract] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 01/30/2017] [Accepted: 05/30/2017] [Indexed: 12/21/2022] Open
Abstract
MicroRNAs (miRNAs) play a vital role in regulating gene expression and are associated with a variety of cancers, including breast cancer. Their distorted and unique expression is a potential marker in clinical diagnoses and prognoses. Thus, accurate determination of miRNA expression levels is a prerequisite for their applications. However, the assays currently available for miRNA detection typically require pre-enrichment, amplification and labeling steps, and most of the assays are only semi-quantitative. Therefore, we developed a quasi-direct liquid chromatography-tandem mass spectrometry (LC-MS/MS)-based targeted proteomics approach to quantify target miRNA by innovatively converting the miRNA signal into the mass response of a reporter peptide via a covalently immobilized DNA-peptide probe. Specifically, the probe containing the targeted proteomics-selected substrate/reporter peptide, GDRAVQLGVDPFR/AVQLGVDPFR, and the DNA sequence complementary to the target miRNA (i.e., miR-21) was first immobilized on APMTS modified silica nanoparticles using PDITC. After the immobilized probe was recognized and hybridized with the target miRNA, the excess probe was degraded using MBN and followed by a trypsin digestion of the hybrids. The reporter peptide was released and quantified using LC-MS/MS. The obtained LOQ was 5 pM. Finally, the developed assay was used for the quantitative analysis of miR-21 in breast cells and tissue samples.
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44
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Xu F, Zhou W, Cao J, Xu Q, Jiang D, Chen Y. A Combination of DNA-peptide Probes and Liquid Chromatography-Tandem Mass Spectrometry (LC-MS/MS): A Quasi-Targeted Proteomics Approach for Multiplexed MicroRNA Quantification. Theranostics 2017; 7:2849-2862. [PMID: 28824720 PMCID: PMC5562220 DOI: 10.7150/thno.19113] [Citation(s) in RCA: 16] [Impact Index Per Article: 2.0] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 01/08/2017] [Accepted: 05/08/2017] [Indexed: 01/11/2023] Open
Abstract
The distorted and unique expression of microRNAs (miRNAs) in cancer makes them an attractive source of biomarker. There is much evidence indicating that a panel of miRNAs, termed "miRNA fingerprints", is more specific and informative than an individual miRNA as biomarker. Thus, multiplex assays for simultaneous quantification of multiple miRNAs could be more potent in clinical practice. However, current available assays normally require pre-enrichment, amplification and labeling steps, and most of them are semi-quantitative or lack of multiplexing capability. In this study, we developed a quasi-targeted proteomics assay for multiplexed miRNA quantification by a combination of DNA-peptide probes and liquid chromatography-tandem mass spectrometry (LC-MS/MS). Specifically, the signal of target miRNAs (i.e., miR-21, miR-let7a, miR-200c, miR-125a and miR-15b) was converted into the mass response of reporter peptides by hybridization of miRNAs with DNA-peptide probes and subsequent tryptic digestion to release the peptides. After a careful optimization of conditions related to binding, conjugation, hybridization and multiple reaction monitoring (MRM) detection, the assay was validated for each miRNA and the limit of quantification (LOQ) for all the miRNAs can achieve 1 pM. Moreover, crosstalk between DNA-peptide probes in multiplex assay was sophisticatedly evaluated. Using this quasi-targeted proteomics assay, the level of target miRNAs was determined in 3 human breast cell lines and 36 matched pairs of breast tissue samples. Finally, simplex assay and qRT-PCR were also performed for a comparison. This approach grafts the strategy of targeted proteomics into miRNA quantification and may offer a new way for multiplexed miRNA profiling.
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Affiliation(s)
- Feifei Xu
- Nanjing Medical University, Nanjing, 211166, China
| | - Weixian Zhou
- Nanjing Medical University, Nanjing, 211166, China
| | | | - Qingqing Xu
- Nanjing Medical University, Nanjing, 211166, China
| | | | - Yun Chen
- Nanjing Medical University, Nanjing, 211166, China
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45
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Simultaneously electrochemical detection of microRNAs based on multifunctional magnetic nanoparticles probe coupling with hybridization chain reaction. Biosens Bioelectron 2017. [PMID: 28622643 DOI: 10.1016/j.bios.2017.06.022] [Citation(s) in RCA: 62] [Impact Index Per Article: 7.8] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 01/08/2023]
Abstract
We report a sensor combining two distinguishable magnetic nanoprobes (DNA1/Fe3O4 NPs/Thi and DNA2/Fe3O4 NPs/Fc) with target-triggered hybridization chain reaction (HCR) strategy for the simultaneous detection of microRNA-141 (miR-141) and microRNA-21 (miR-21). In the presence of targets, the thiol-modified hairpin capture probes (HCP1 and HCP2) specifically hybridize with miR-141 and miR-21 on a gold electrode, leading to the conformation change of HCP1 and HCP2, respectively. The conformation change subsequently triggers HCR to generate plentiful bonding sequences of magnetic nanoprobes. Thus, numerous thionine (Thi) modified DNA1/Fe3O4 NPs/Thi and ferrocene carboxaldehyde (Fc-CHO) modified DNA2/Fe3O4 NPs/Fc are captured by the well-designed HCR, via DNA hybridization respectively, giving rise to the dual magnified response of currents. The increase in the electrochemical currents at different potentials of the two magnetic nanoprobes enables us to simultaneously and quantitatively detect miR-141 and miR-21. Target-triggered HCR increases the amount of captured nanoprobes due to the increasing number of bonding sequences, greatly amplifying the currents of the two magnetic nanoprobes in the presence of targets, and ultimately realizing the dual signal amplification with increased sensitivity. The sensor can be applied for detecting miRNAs in cell lysates, thus, promising to be a clinic diagnosis of cancers by means of simultaneous detection of a variety of miRNA biomarkers.
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46
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Koo KM, Wee EJ, Trau M. High-speed biosensing strategy for non-invasive profiling of multiple cancer fusion genes in urine. Biosens Bioelectron 2017; 89:715-720. [DOI: 10.1016/j.bios.2016.11.024] [Citation(s) in RCA: 14] [Impact Index Per Article: 1.8] [Reference Citation Analysis] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 09/20/2016] [Revised: 11/03/2016] [Accepted: 11/08/2016] [Indexed: 11/29/2022]
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47
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Xue Q, Kong Y, Wang H, Jiang W. Liposome-encoded magnetic beads initiated by padlock exponential rolling circle amplification for portable and accurate quantification of microRNAs. Chem Commun (Camb) 2017; 53:10772-10775. [DOI: 10.1039/c7cc05686a] [Citation(s) in RCA: 37] [Impact Index Per Article: 4.6] [Reference Citation Analysis] [Abstract] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 12/20/2022]
Abstract
In this work, we propose a strategy for glucoamylase-encapsulated liposome-encoded magnetic beads initiated by padlock exponential rolling circle amplification (P-ERCA) for portable and accurate quantification of miRNA by using a glucometer (GM) for readout.
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Affiliation(s)
- Qingwang Xue
- Department of Chemistry
- Liaocheng University
- Liaocheng
- China
| | - Yancong Kong
- Department of Chemistry
- Liaocheng University
- Liaocheng
- China
| | - Huaisheng Wang
- Department of Chemistry
- Liaocheng University
- Liaocheng
- China
| | - Wei Jiang
- School of Chemistry and Chemical Engineering
- Shandong University
- Jinan 250100
- P. R. China
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48
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Qi Y, Qiu L, Fan W, Liu C, Li Z. An enzyme-free flow cytometric bead assay for the sensitive detection of microRNAs based on click nucleic acid ligation-mediated signal amplification. Analyst 2017; 142:2967-2973. [DOI: 10.1039/c7an00989e] [Citation(s) in RCA: 13] [Impact Index Per Article: 1.6] [Reference Citation Analysis] [Abstract] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 01/13/2023]
Abstract
An enzyme-free flow cytometric assay is developed for the sensitive detection of microRNAs based on click nucleic acid ligation-mediated signal amplification.
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Affiliation(s)
- Yan Qi
- Key Laboratory of Applied Surface and Colloid Chemistry
- Ministry of Education
- Key Laboratory of Analytical Chemistry for Life Science of Shaanxi Province
- School of Chemistry and Chemical Engineering
- Shaanxi Normal University
| | - Liying Qiu
- Key Laboratory of Applied Surface and Colloid Chemistry
- Ministry of Education
- Key Laboratory of Analytical Chemistry for Life Science of Shaanxi Province
- School of Chemistry and Chemical Engineering
- Shaanxi Normal University
| | - Wenjiao Fan
- Key Laboratory of Applied Surface and Colloid Chemistry
- Ministry of Education
- Key Laboratory of Analytical Chemistry for Life Science of Shaanxi Province
- School of Chemistry and Chemical Engineering
- Shaanxi Normal University
| | - Chenghui Liu
- Key Laboratory of Applied Surface and Colloid Chemistry
- Ministry of Education
- Key Laboratory of Analytical Chemistry for Life Science of Shaanxi Province
- School of Chemistry and Chemical Engineering
- Shaanxi Normal University
| | - Zhengping Li
- Key Laboratory of Applied Surface and Colloid Chemistry
- Ministry of Education
- Key Laboratory of Analytical Chemistry for Life Science of Shaanxi Province
- School of Chemistry and Chemical Engineering
- Shaanxi Normal University
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49
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Sun Y, Tian H, Liu C, Sun Y, Li Z. One-step detection of microRNA with high sensitivity and specificity via target-triggered loop-mediated isothermal amplification (TT-LAMP). Chem Commun (Camb) 2017; 53:11040-11043. [DOI: 10.1039/c7cc06140d] [Citation(s) in RCA: 52] [Impact Index Per Article: 6.5] [Reference Citation Analysis] [Abstract] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 12/16/2022]
Abstract
A target-triggered loop-mediated isothermal amplification (TT-LAMP) mechanism is developed for simple one-step but highly sensitive detection of microRNAs.
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Affiliation(s)
- Yuanyuan Sun
- Key Laboratory of Applied Surface and Colloid Chemistry
- Ministry of Education, Key Laboratory of Analytical Chemistry for Life Science of Shaanxi Province
- School of Chemistry and Chemical Engineering
- Shaanxi Normal University
- Xi’an 710062
| | - Hui Tian
- Key Laboratory of Applied Surface and Colloid Chemistry
- Ministry of Education, Key Laboratory of Analytical Chemistry for Life Science of Shaanxi Province
- School of Chemistry and Chemical Engineering
- Shaanxi Normal University
- Xi’an 710062
| | - Chenghui Liu
- Key Laboratory of Applied Surface and Colloid Chemistry
- Ministry of Education, Key Laboratory of Analytical Chemistry for Life Science of Shaanxi Province
- School of Chemistry and Chemical Engineering
- Shaanxi Normal University
- Xi’an 710062
| | - Yueying Sun
- Key Laboratory of Applied Surface and Colloid Chemistry
- Ministry of Education, Key Laboratory of Analytical Chemistry for Life Science of Shaanxi Province
- School of Chemistry and Chemical Engineering
- Shaanxi Normal University
- Xi’an 710062
| | - Zhengping Li
- Key Laboratory of Applied Surface and Colloid Chemistry
- Ministry of Education, Key Laboratory of Analytical Chemistry for Life Science of Shaanxi Province
- School of Chemistry and Chemical Engineering
- Shaanxi Normal University
- Xi’an 710062
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50
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Li X, Chen A, Chai Y, Yuan R. K-junction structure mediated exponential signal amplification strategy for microRNA detection in electrochemiluminescence biosensor. Analyst 2017; 142:2185-2190. [DOI: 10.1039/c7an00586e] [Citation(s) in RCA: 5] [Impact Index Per Article: 0.6] [Reference Citation Analysis] [Abstract] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 11/21/2022]
Abstract
Based on the novel designed K-junction structure, an economic and efficient exponential signal amplification strategy with simple protocol combining hemin/G-quadruplex, a mimetic peroxidase, as a catalyzer was proposed and utilized in an electrochemiluminescence biosensor for sensitive microRNA detection.
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Affiliation(s)
- Xue Li
- Key Laboratory of Luminescent and Real-Time Analytical Chemistry (Southwest University)
- Ministry of Education
- College of Chemistry and Chemical Engineering
- Southwest University
- Chongqing 400715
| | - Anyi Chen
- Key Laboratory of Luminescent and Real-Time Analytical Chemistry (Southwest University)
- Ministry of Education
- College of Chemistry and Chemical Engineering
- Southwest University
- Chongqing 400715
| | - Yaqin Chai
- Key Laboratory of Luminescent and Real-Time Analytical Chemistry (Southwest University)
- Ministry of Education
- College of Chemistry and Chemical Engineering
- Southwest University
- Chongqing 400715
| | - Ruo Yuan
- Key Laboratory of Luminescent and Real-Time Analytical Chemistry (Southwest University)
- Ministry of Education
- College of Chemistry and Chemical Engineering
- Southwest University
- Chongqing 400715
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