Nature Communications
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2022
·Vol. 13
·期 1
·1-14
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IF 17.694
Xu Zhichen,Chen Dongjuan,Li Tao,Yan Jiayu,Zhu Jiang,He Ting,Hu Rui,Li Ying,Yang Yunhuang,Liu Maili
Fast, inexpensive, and multiplexed detection of multiple nucleic acids is of great importance to human health, yet it still represents a significant challenge. Herein, we propose a nucleic acid testing platform, named MiCaR, which couples a mi crofluidic device with CRISPR- C as12a a nd multiplex r ecombinase polymerase amplification. With only one fluorescence probe, MiCaR can simultaneously test up to 30 nucleic acid targets through microfluidic space coding. The detection limit achieves 0.26 attomole, and the multiplexed assay takes only 40 min. We demonstrate the utility of MiCaR by efficiently detecting the nine HPV subtypes targeted by the 9-valent HPV vaccine, showing a sensitivity of 97.8% and specificity of 98.1% in the testing of 100 patient samples at risk for HPV infection. Additionally, we also show the generalizability of our approach by successfully testing eight of the most clinically relevant respiratory viruses. We anticipate this effective, undecorated and versatile platform to be widely used in multiplexed nucleic acid detection.
研究领域:
生物技术
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分子生物学
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医学诊断
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DOI
PubMed
高影响因子
ANALYTICA CHIMICA ACTA
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2022
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IF 6.911
Xiaofen Zhao,Xiaoxiao Tian,Yuwei Wang,Linbin Li,Yan Yu,Shiqiao Zhao,Juan Zhang
Accurate analysis of HIV DNA is valuable for the diagnosis of AIDS. Herein, an ultrasensitive and specific fluorescence method was developed for HIV-1 DNA detection based on CRISPR-Cas12a-activated palindrome-catalytic hairpin assembly (CRISPR-Cas12a-PCHA). The presence of HIV-1 DNA activated the trans-cleavage activity of CRISPR-Cas12a, which could continuously digest the DNA fragment of hairpins connected to magnetic beads to expose single-stranded RNA . After magnetic separation, the exposed RNA triggered multiple PCHA reactions, generating many Y-shaped DNA structures that were self-assembled into the DNA superstructures via the hybridization of palindromic sticky ends, leading to the release of amounts of fluorescence signal. Different from the reported recently biosensing strategies of nucleic acid amplification technologies-activated CRISPR-Cas12a, CRISPR-Cas12a-PCHA endowed the strategy with unique advantages of simple sample pretreatment , direct duplex target detection, and ultrahigh sensitivity. The strategy was able to resist the interference of the complex matrix in real sample and distinguish between HIV patients and healthy persons. Thus, the method is a promising tool for ultrasensitive and specific detection of HIV-1 DNA for AIDS diagnosis.
研究领域:
生物技术
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分子生物学
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医学诊断
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产品关联
DOI
PubMed
高影响因子
Device
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2024
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IF 0
Siyi Zou,Jingnan Li,Sheng Lu,Danyu Li,Yuxuan Liu,Weiguo Zhang,Xiaolin Cui,J. Justin Gooding,Kang Tian,Guozhen Liu
Inflammation quantification through the detection of cytokines and inflammatory factors in a point-of-care scenario is crucial for managing and preventing diseases. Achieving early-stage inflammation quantification in osteoarthritis (OA), before the radiographic findings become apparent, may provide an opportunity for accurate diagnosis and effective therapy, potentially preventing joint degeneration. Here, we developed a glass-fiber-based universal system with CRISPR-Cas12a-based immunosensors (GUSCI) for simultaneous detection of multiple inflammation biomarkers using a portable fluorescence detection device. GUSCI enables the detection of five inflammation biomarkers (interleukin-4 [IL-4], IL-6, tumor necrosis factor alpha [TNF-α], interferon γ [IFN-γ], and matrix metalloproteinase 3 [MMP-3]) with picomolar sensitivity in a 10-μL sample while having less than 75 min of assay time and a smartphone signal readout. A cohort study of 35 synovial samples from OA patients revealed differences in inflammation levels. This “sample in, answer out” platform not only aids in understanding OA progression but also offers a versatile strategy for point-of-care diagnosis of a wide range of diseases in resource-limited settings.
研究领域:
生物传感器
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分子诊断
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炎症疾病
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骨关节炎
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即时检测
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DOI
STAR Protocols
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2023
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IF 0
Xiaoqian Zeng,Shuliu Wang,Mindong Liang,Weishan Wang,Yue Jiang,Fei Xu,Leshi Liu,Hao Yan,Yaojun Tong,Lixin Zhang,Gao-Yi Tan
Summary Large biosynthetic gene cluster (BGC) cloning is important for discovering natural product-based drugs and remains challenging in high GC content microorganisms (e.g., Actinobacteria). Here, we present an in vitro CRISPR-Cas12a-mediated protocol for direct cloning of large DNA fragments. We describe steps for crRNA design and preparation, genomic DNA isolation, and CRISPR-Cas12a cleavage and capture plasmid construction and linearization. We then detail target BGC and plasmid DNA ligation and transformation and screening for positive clones. For complete details on the use and execution of this protocol, please refer to Liang et al. 1
研究领域:
分子生物学
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生物技术
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基因工程
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DOI
PubMed
MICROCHIMICA ACTA
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2025
·Vol. 192
·期 2
·1-11
·
IF 5.3
Zhang Xiaoyu,Sun Ruimeng,Zheng Haoran,Qi Yanfei
A spherical nucleic acid (SNA, AuNPs-aptamer) into CRISPR/Cas12a system combined with poly T-template copper nanoparticles as fluorescence reporter was fabricated to establish an amplification-free sensitive method for Staphylococcus aureus ( S. aureus ) detection. This method, named PTCas12a, utilizes the concept that the bifunction of SNA recognizes the S. aureus and triggers the Cas12a cleavage activity. Then, the Cas12a enzyme cleaves the Poly T40 to generate a signal change in Poly T-Cu fluorescence, indicating the presence or absence of the target bacteria. The PTCas12a platform demonstrated a detection limit as low as 3.0 CFU/mL (3 N/S) in a wide response range of 1.0 × 10 1 –1.0 × 10 6 CFU/mL for S. aureus detection, which holds significant potential in ensuring food safety and preventing the spread of diseases. Graphical abstract
研究领域:
生物技术
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医学诊断
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食品安全
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产品关联
DOI
PubMed
高影响因子
MICROCHEMICAL JOURNAL
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2025
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IF 5.1
Caihong Zhou,Jingjing Fan,Liangyuan Wang,Baoxue Gao,Chunhui Wei,Long Ma,Lijuan Yin
Norovirus (NoV) is a leading cause of nonbacterial acute gastroenteritis, primarily transmitted through contaminated food and water. Rapid, sensitive, and on-site detection is crucial for controlling NoV outbreaks and ensuring food safety. In this study, we developed a portable, one-tube CRISPR/Cas12a-driven colorimetric biosensor for the rapid and sensitive detection of NoV RNA without the need for pre-amplification. The biosensor integrates magnetic nanoprobes with gold nanoparticle (AuNP) bioprobes, connected via single-stranded DNA (ssDNA) linkers. Upon the specific recognition of NoV cDNA, the Cas12a protein’s trans -cleavage activity is activated, cleaving the ssDNA linkers and releasing AuNP bioprobes from the magnetic nanoprobes. This release results in a visually detectable red color change and enhanced UV–visible absorbance in the solution following magnetic separation. These changes can be easily detected using portable devices such as microplate readers or smartphones. This method achieves a detection limit of 10 copies/µL within 60 min, with no cross-reactivity from other viruses or bacteria. Validation using oyster samples showed perfect concordance with qPCR results. Designed for resource-limited settings, our biosensor can be adapted for detecting various pathogens by altering the target nucleic acids and crRNA sequences. This advancement offers significant potential for enhancing rapid diagnostics and outbreak management.
研究领域:
分子生物学
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食品科学
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诊断技术
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DOI
高影响因子
ANALYTICA CHIMICA ACTA
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2024
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IF 5.7
Tianyi Zhang,Xiaoqin Wang,Yingtao Jiang,Yunyun Zhang,Shuhao Zhao,Jian Hu,Fei Hu,Niancai Peng
Background Nucleic acid testing is the most effective detection method currently available for the diagnosis of respiratory infectious diseases. However, the conventional real-time fluorescent quantitative PCR technique, which is regarded as the gold standard method for nucleic acid detection, presents significant challenges for implementation in home self-testing and popularization in underdeveloped regions due to its rigorous experimental standards. It is therefore clear that an easy-to-use, miniaturized nucleic acid testing technology and products for nonprofessionals are of great necessity to define the pathogens and assist in controlling disease transmission. (87) Results In this study, we propose a strategy for self-testing of respiratory pathogen nucleic acid that is oriented towards the public and user-friendly. The proposed system integrates the processes of extraction-free nucleic acid release, RPA isothermal amplification, and CRISPR fluorescence detection into a compact configuration. A microfluidic testing chip actuated by air pouches and a battery/USB-powered reusable device has been developed to enable simultaneous detection of internal reference genes and viral targets in a fully enclosed condition. The system allows for sample-in, and result-out testing in less than 30 minutes with a detection limit of 2 copies/μL. Additionally, a straightforward signal-light-based result display method has been developed to make it easy and intuitive for users to access the results. Furthermore, freeze-drying reagent is introduced to guarantee the storage and transportation of testing chips in ambient conditions. (135) Significance This work presents a miniaturized, portable, and highly sensitive nucleic acid detection system, where simple operating procedures have been designed for unskilled users. It is our belief that the testing system developed in this work is well suited for home-based self-testing and infection diagnosis in resource-limited areas, due to the above-mentioned advantages. (52)
研究领域:
分子生物学
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医学检验
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生物工程
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DOI
PubMed
高影响因子
JOURNAL OF HAZARDOUS MATERIALS
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2024
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IF 12.2
Jingyi Yang,Yining Zhao,Lelin Qian,Ying Yu,Yuan Zhang,Juan Zhang
In this work, a dual recognized CRISPR/Cas12a system has been proposed, in which the activation chain is cleverly divided into two parts that can serve for precise dual target recognition, and hydrazone chemistry is introduced for the formation of a whole activation chain. It has been further explored to construct a new method for the specific and sensitive detection of Staphylococcus aureus (SA) as one of the most common pathogens in infectious diseases. In virtue of proximity effect contributed by complementary base pairing, hydrazone chemistry accelerates the formation of the whole activation strand and improves the specificity of the CRISPR/Cas12a system, serving for the accurate analysis of SA. Moreover, the temporary aggregation of CRISPR/Cas12a around SA enhances its catalytical efficiency so as to further amplify signal. With high sensitivity, stability, reproducibility and specificity, the established method has been successfully applied to detect SA in complex substrates. Meanwhile, our established method can well evaluate the inhibition effect of chlorogenic acid and congo red in comparison with flow cytometry. Environmental implication Bacterial pathogens exist widely in the environment and seriously threaten the safety of human health. Staphylococcus aureus (SA) is the most common pathogen of human suppurative infection, which can cause local suppurative infection, pneumonia, and even systemic infections such as sepsis. In this work, a dual recognized CRISPR/Cas12a system mediated by hydrazone chemistry has been proposed. With high sensitivity and low detection limit, the established method can specifically detect SA and effectively evaluate the antibacterial effect of inhibitors. This method is expected to be further developed into a detection method in different scenarios such as environmental monitoring and clinical diagnosis.
研究领域:
分子生物学
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病原微生物学
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分析化学
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产品关联
DOI
PubMed
高影响因子
FOOD CHEMISTRY
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2023
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IF 8.8
Xuening Shi,Hang Li,Shuo Yao,Yukun Ding,Xiuzhu Lin,Hui Xu,Yi Liu,Chao Zhao,Tong Zhang,Juan Wang
The total bacterial count is an important indicator of food contamination in food safety supervision and management. Recently, the CRISPR/Cas12a system integrated with nucleic acid amplification has increasingly shown tremendous potential in microorganism detection. However, a general quantification strategy for total bacteria count based on the CRISPR/Cas12a system has not yet been developed. Herein, we established a sensitive bacterial quantification strategy based on the CRISPR/Cas12a system combined with magnetic covalent organic frameworks (MCOFs) and hybridization chain reaction (HCR). MCOFs acted as a carrier, adsorbing the ssDNA as HCR trigger sequence through π-π stacking. Then, the HCR circuit produces DNA duplexes containing the PAM sequences that activate the trans -cleavage activity of Cas12a for further signal amplification. Under the optimal conditions, the proposed method can quantify total bacteria in 50 min with a minimum detection concentration of 10 CFU/mL. The successful applications in food samples confirmed the feasibility and broad application prospects.
研究领域:
生物技术
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食品科学
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分子生物学
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产品关联
DOI
PubMed
高影响因子
BIOSENSORS & BIOELECTRONICS
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2022
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IF 12.545
Feng Qiu,Xiaoyan Gan,Jianglong Yao,Bingying Jiang,Ruo Yuan,Yun Xiang
Transcription factors (TFs) are key substances in regulating the transcription, replication and expression of genes, and the detection of TFs can provide valuable information to diagnose a variety of diseases. By integrating hybridization chain reaction (HCR)-activated Cas12a enzyme with bio-responsive DNA hydrogels, we propose a dual amplification and label-free homogeneous electrochemical detection method to realize sensitive nuclear factor-kappa B p50 (NF-κB p50) detection. The presence of the target molecules protects the DNA duplex probes from digesting by exonuclease III and initiates HCR to generate long double stranded DNAs that can activate the activity of RNA-guided Cas12a enzymes. The single-stranded region of the DNA linkers that crosslink the DNA hydrogels can be cleaved by the activated Cas12a to release a large number of electroactive substances embedded in the gels, which exhibit highly enhanced electrochemical signals for detecting target molecules at the detection limit of 54.1 fM. In addition, the successful interrogation of NF-κB p50 spiked into lysate of HeLa cells by such method is also verified. The established method thus shows new opportunities for sensitive and convenient monitoring of other transcription factors and biomarkers.
研究领域:
生物化学
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分子生物学
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医学诊断
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产品关联
DOI
PubMed
高影响因子
ANALYTICAL CHEMISTRY
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2025
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IF 6.7
Junzhuo Shan,Yixiao Sheng,Lun Luo,Wenhai Wang,Xinjiang Liu,Yi Ma,Jufang Wang
Apurinic/apyrimidinic endonuclease 1 (APE1) is a key enzyme involved in DNA repair and cellular redox regulation, and is frequently overexpressed in tumor cells. This highlights the urgent need for a rapid and high-sensitive point-of-care testing (POCT) strategy for APE1 to facilitate early cancer diagnosis. Rolling circle amplification (RCA) is a widely used isothermal DNA amplification method; however, its application in APE1 detection remains rare. Here, we introduce a versatile RCA-Lock (Rlock) conversion platform that enables the transformation of RCA-based nucleic acid detection technologies into APE1-responsive assays. Building upon this platform, we further developed a novel POCT method for APE1 detection─Rlock-mediated, CRISPR/Cas12a-driven RCA cycle (RCRE)─which, for the first time, integrates CRISPR/Cas12a with RCA into a one-pot APE1 detection system. The RCRE assay achieves a limit of detection of 8.86 × 10–4 U/mL within 30 min, while requiring minimal equipment, low cost, and no complex handling procedures. This Rlock-based conversion strategy represents a transformative advance in the field of APE1 diagnostics and offers conceptual inspiration for the design of programmable nucleic acid–based biosensors. The resulting RCRE assay significantly broadens the technological landscape for early cancer detection and paves the way for future clinical translation.
研究领域:
分子诊断学
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生物化学
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生物技术
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癌症研究
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遗传学
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分析化学
✓ 已验证
产品关联
DOI
PubMed
高影响因子
ANALYTICAL CHEMISTRY
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2025
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IF 6.7
Tianyi Zhang,Yingying Zhao,Cong Zhu,Xi Zhu,Xiaohua Zhu,Ye Qiu,Zhou Nie,Chunyang Lei
Viral proteases are critical molecular targets in viral pathogenesis, representing pivotal biomarkers for understanding viral infection mechanisms and developing antiviral therapeutics. This study introduces a label-free electrochemical biosensor that enables sensitive viral protease detection by integrating protease-responsive CRISPR/Cas protein switches (CasPSs) with a hemin aptamer-functionalized electrochemical interface. The biosensor’s mechanism relies on viral protease-mediated proteolysis, which leads to the release of active Cas12a proteins from CasPSs and generates amplified electrochemical responses through continuous cleavage of immobilized redox-active hemin/aptamer complexes. This biosensor achieved specific hepatitis C virus NS3/4A protease sensing with femtomolar sensitivity and could be readily expanded to other viral proteases by replacing the CasPS module. The feasibility of this biosensor was demonstrated by monitoring enterovirus 71 3C protease activities in virus-infected cell samples with different viral loads and postinfection times. This study provides a promising strategy for integrating CRISPR biosensing with electrochemical platforms, offering a helpful analytical tool for viral infection monitoring and antiviral drug screening.
研究领域:
分子生物学
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病毒学
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生物传感技术
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医学诊断
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产品关联
DOI
PubMed
高影响因子
危险品化学品经营许可证（带存储）