SENSORS AND ACTUATORS B-CHEMICAL
·
2026
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IF 8.3
Zishu Lan,ZhongWei Yang,Xuexia Peng,Xinmei Li,Tangping Zhang,Tianjiao Chen,Shuqing Zhou,Yutong Shao,Junwen Li,Min Jin,Dong Yang
The rising global burden of foodborne illnesses necessitates the development of rapid, sensitive, and multiplex detection technologies for on-site screening. This study presents an innovative smartphone-assisted magnetic MOF-RPA-CRISPR platform for the simultaneous detection of four major foodborne pathogens: Salmonella Typhimurium , Staphylococcus aureus , Vibrio parahaemolyticus , and Listeria monocytogenes . The platform’s originality lies in the one-pot integrated workflow of three functional modules: a core-shell structured Fe₃O₄@UiO-66-NH₂ material for efficient bacterial enrichment with over 90% capture efficiency, a quadruplex RPA-CRISPR/Cas12a assay enabling specific amplification and detection with a sensitivity of 10 CFU/mL, and an intelligence visual system that provides quantitative, objective readouts. When validated with spiked food samples including milk, chicken, and shrimp, the entire assay was completed within 60 minutes without specialized equipment, demonstrating high accuracy and robustness against matrix interference. This integrated approach offers a powerful and user-friendly solution for rapid, on-site multiplex pathogen monitoring, significantly advancing food safety surveillance capabilities.
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高影响因子
DIAGNOSTIC MICROBIOLOGY AND INFECTIOUS DISEASE
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2026
·Vol. 116
·期 3
·117523
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IF 1.8
Huifeng Yang,Xiaoyi Li,Yimin Su,Jianxin Guo,Tengfei Wang,Zhigang Wang,Hai Fu,Zhi Chen,Zhongjian Xie,Defa Li
Purpose Acute respiratory infections caused by bacterial and viral pathogens pose a major global health burden. The recombinase polymerase amplification (RPA)-CRISPR-Cas12a system offers a promising point-of-care testing (POCT) platform, but its field deployment is limited by the instability of lyophilized reagents. This study aims to develop a composite lyoprotectant to enhance the stability of the RPA-CRISPR-Cas12a system for respiratory pathogen detection. Materials and methods A composite lyoprotectant composed of trehalose and carboxymethyl chitosan (Tre-CMC) was formulated at various mass ratios. The optimal ratio was identified by evaluating matrix microstructure, enzyme activity retention, and primer-dimer suppression. The lyophilized system was tested for sensitivity, specificity, and long-term stability against six respiratory pathogens (H1N1, IBV, Neisseria meningitidis , SARS-CoV-2, Streptococcus pneumoniae , and human adenovirus) using real-time fluorescence and gel electrophoresis. Results This study establishes a promising proof-of-concept framework for an integrated, lyophilized RPA-CRISPR–Cas12a diagnostic system. The optimal Tre: CMC ratio (2:1) produced a porous, non‑hygroscopic matrix that preserved reagent integrity. The lyophilized system achieved a detection limit of 10 copies/reaction within 30 min for all six pathogens, with 100% specificity. After six months of storage at 4°C, RPA enzyme activity remained above 89%, and Cas12a-crRNA complex functionality was fully retained. Tre-CMC significantly reduced primer-dimer formation and nonspecific background fluorescence. While clinical evaluation using 21 nasopharyngeal swab samples demonstrated 100% concordance with RT-qPCR—preliminarily supporting the system's potential diagnostic accuracy—we acknowledge that this initial methodological framework requires broader and more rigorous downstream validation with larger clinical cohorts to formally establish its robust diagnostic performance. Conclusion The Tre-CMC composite lyoprotectant effectively stabilizes the RPA-CRISPR-Cas12a system, enabling cold-chain-independent storage and reliable POCT for respiratory pathogens in resource-limited settings.
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高影响因子
ACS Sensors
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2026
·Vol. 11
·期 7
·5553-5565
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IF 10.9
Qiu Zhiqiang,Chen Jiabo,Wu Jinxian,Wang Jujing,Hu Yuting,Feng Wei,Zhao Jun,Minamoto Toshifumi,Chen Xiao,Zhou Lei,Liu Min,Wang Chen,Lin Mei,Hu Menglu,Zhou Xiaoming,Wang Junjie
Developing a rapid, sensitive, and field-deployable assay for on-site environmental DNA (eDNA) detection of endangered species is crucial, as current PCR-based assays are slow and expensive and rely on laboratory-based thermal cycling. Here, we adapted a previously reported optically controlled one-pot RPA-CRISPR-Cas12a (OORC) assay to detect eDNA of the critically endangered Bahaba taipingensis . The assay combines isothermal recombinase polymerase amplification (RPA) with the high specificity of a CRISPR-Cas12a trans -cleavage reaction in a single tube. The entire process is operated at a constant temperature, avoiding thermal cycling. Low-template replicate experiments conservatively redefined the OORC limit of detection as 6 copies/reaction. To support near-field application, we further integrated the assay with a portable handheld fluorescence detector capable of 365 nm photoactivation and fluorescence readout. This portable and highly sensitive workflow extends the potential of eDNA monitoring, offering a practical tool for the conservation of endangered species.
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高影响因子
JOURNAL OF AGRICULTURAL AND FOOD CHEMISTRY
·
2025
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IF 6.2
Wang Guo,Yiqing Guo,Hong Xu,Chen Li,Xinai Zhang,Xiaobo Zou,Zongbao Sun
Staphylococcus aureus (S. aureus) is a major pathogenic bacterium responsible for bacterial foodborne diseases, making its rapid, specific, and accurate detection crucial. In this study, we develop a ratiometric biosensor based on the recombinase polymerase amplification-clustered regularly interspaced short palindromic repeats/CRISPR associated protein 12a (RPA-CRISPR/Cas12a) system and Eu-metal–organic framework (Eu-MOF) fluorescent nanomaterials for the high-sensitivity detection of S. aureus, combining with RPA for efficient isothermal amplification, this sensor enhances specificity and sensitivity by utilizing the target activation of CRISPR/Cas12a. The Eu-MOF serves a dual function, providing stable red fluorescence as a reference signal and adsorbing FAM-labeled probes for fluorescence quenching, forming a dual-signal system that significantly reduces background interference. This ratiometric design enables accurate and quantitative detection over a wide range (7.9 × 100 to 7.9 × 108 CFU/mL) with a low detection limit of 3 CFU/mL. Overall, with these merits of simplicity, rapid response, high sensitivity, and specificity, this dual-signal biosensor offers a promising method for accurately evaluating S. aureus contamination in food under complex substrate conditions.
研究领域:
生物技术
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食品安全检测
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分子诊断
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高影响因子
Small Methods
·
2025
·
IF 9.1
Xiaoying Han,Juxin Yin,Yu Wang,Jianjian Zhuang,Kai Hu,Yehong Gui,Haohua Mei,Jizhi Tong,Ying Mu
The rapid, precise, and automated diagnosis of infectious diseases is crucial for effective disease management and control. Herein, the integrated portable and automatic digital detection system (IPADS), a novel diagnostic platform for nucleic acid detection is introduced. The device employs the hybrid magnetic system (HMS), which uses an electromagnet and a movable permanent magnet to modulate the magnetic field and control bead movement, increasing nucleic acid extraction efficiency to over 80%, while simplifying the traditional labor-intensive process and enabling quick, low-risk sample processing. Additionally, a disposable cartridge is designed for integrated HMS based preprocessing, with detection performed using digital RPA-Cas12a, enabling rapid, enclosed, and automation-friendly detection across a dynamic range spanning five orders of magnitude, with a sensitivity as low as 100 copies mL −1 in serum samples. An automated platform further optimizes workflow. As a proof of concept, IPADS is applied to detect hepatitis B virus (HBV) DNA in 20 clinical serum samples, demonstrating high concordance with gold-standard quantitative PCR (qPCR) methods. These results validate the potential of IPADS as a reliable point-of-care testing solution.
研究领域:
医学诊断
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分子生物学
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生物医学工程
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ANALYTICAL CHEMISTRY
·
2023
·
IF 7.4
Guobin Mao,Xing Luo,Silu Ye,Xun Wang,Jin He,Jilie Kong,Junbiao Dai,Wen Yin,Yingxin Ma
It is well-established that different detection modes are necessary for corresponding applications, which can effectively reduce matrix interference and improve the detection accuracy. Here, we reported a magnetic separation method based on recombinase polymerase amplification (RPA)-assisted clustered regularly interspaced short palindromic repeats (CRISPR)/Cas12a for dual-mode analysis of African swine fever virus (ASFV) genes, including colorimetry and fluorescence. The ASFV gene was selected as the initial RPA template to generate the amplicon. The RPA amplicon was then recognized by CRISPR-associated RNA (crRNA), activating the trans-cleavage activity of Cas12a and leading to the nonspecific cleavage of ssDNA as well as a significant release of alkaline phosphatase (ALP) in the ALP-ssDNA modified magnetic bead. The released ALP can catalyze para-nitrophenyl phosphate to generate para-nitrophenol, resulting in substantial changes in absorbance and fluorescence, both of which can be used for detection with the naked eye. This strategy allows the sensitive detection of ASFV DNA, with a 20 copies/mL detection limit; no cross-reactivity with other viruses was observed. A good linear relationship was obtained in serum. In addition, this sensor displayed 100% specificity and sensitivity for clinical sample analysis. This method integrates the high sensitivity of fluorescence with easy readout of colorimetry and enables a simple, low-cost, and highly sensitive dual-mode detection of viral nucleic acid, thereby providing a broad prospect for the practical application in the diagnosis of virus infection.
研究领域:
病毒学
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分子生物学
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生物技术
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分析化学
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高影响因子
ANALYTICAL AND BIOANALYTICAL CHEMISTRY
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2026
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IF 3.8
Sun Guofeng,Shi Kai
Meat adulteration poses significant social concerns, including the infringement of consumer rights, potential risks of food allergies, and conflicts with religious dietary practices. Therefore, a pressing need exists for the creation of swift and highly sensitive techniques to verify the authenticity of meat products. A portable and quantitative detection assay for pork identification in food was established by integrating a personal glucose meter (PGM) with a heparin-mediated one-pot RPA/Cas12a (called hRPA/SCas12a-PGM). In the hRPA/SCas12a-PGM assay, the target DNA sequence initiates the heparin-enhanced RPA/Cas12a reaction in a single tube, subsequently activating Cas12a’s DNase function. Once activated, Cas12a cleaves sucrase-labeled DNA probes immobilized on the electrode, which are labeled with sucrase. Sucrase-labeled DNA fragments are released into the solution through this cleavage process. Sucrase then catalyzes the conversion of sucrose into glucose, producing a quantifiable signal that is detected by the PGM. The integrated system exhibited a broad dynamic range (10 pg/μL to 100 ng/μL), a low detection limit (10 pg/μL), and strong specificity against non-target samples. The hRPA/SCas12a-PGM assay provides a powerful, field-deployable solution for meat authenticity testing, offering significant potential for application in food safety surveillance and regulatory enforcement. Graphical
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npj Science of Food
·
2026
·
IF 9.7
Wang Juan,Li Xiang,Zhang Jing,Zhao Chao,Shi Xuening
Microbial Contamination serves as an important issue in dairy safety. CRISPR/Cas12a-based methods possess tremendous potential, but are limited by nucleic acid pretreatment and specialized operations. Herein, a photothermal nanomaterial-assisted enhanced One-Tube CRISPR/Cas12a system was established to detect S. typhimurium in milk . S. typhimurium was lysed through the photothermal effect to obtain genomic DNA. A multi-crRNA strategy enables rapid signal accumulation, and the whole system was integrated into a single tube via trehalose-based aqueous biphasic systems. The proposed method requires only one step of sample addition to achieve sensitive detection within 40 min, with an LOD of 10 CFU/mL, showing potential in application.
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Frontiers in Chemistry
·
2025
·
IF 4.2
Ting Yuan,Jianbo Yuan,Jian Huang,Nana Li,Huan Cai,Yan Yang,Juan Li,Rui Chen,Xun Min
Introduction: The rapid and accurate identification of Mycobacterium tuberculosis (MTB) is essential for effective tuberculosis (TB) control. However, conventional diagnostic methods for MTB suffer from limitations such as low sensitivity, poor specificity, high cost, reliance on specialized instruments, and complex, time-consuming procedures. To address these challenges, there is an urgent need for a simple, rapid, and highly sensitive detection method that can be deployed in point-of-care settings.Methods: We developed a one-pot biosensing system combining recombinase polymerase amplification (RPA) and CRISPR/Cas12a-driven G4-hemin self-assembly for the colorimetric detection of MTB. Glycerol was employed as a phase-separation barrier to prevent interference between RPA amplification and CRISPR/Cas12a trans-cleavage. A single-stranded DNA (ssDNA) probe, designed to self-assemble with ssDNA-hemin into G4-hemin nanozymes upon CRISPR/Cas12a-mediated cleavage, served as the reaction substrate. The ssDNA-hemin further enhanced the catalytic activity of the generated G4-hemin DNAzyme. The entire assay was completed in a single step within 60 min without requiring complex instrumentation.Results and Discussion: Under optimized conditions, the biosensing system achieved ultrasensitive naked-eye detection of MTB with a limit of detection (LOD) of 10 copies/μL, comparable to traditional four-step fluorescent assays. Clinical validation using 104 patient samples demonstrated high concordance with standard diagnostic methods. This approach combines the advantages of recombinase polymerase amplification (RPA), CRISPR/Cas12a specificity, and G4-hemin DNAzyme-based colorimetric signal amplification, enabling simple, equipment-free visual detection. Given its speed, sensitivity, and ease of use, this biosensing system holds significant promise for point-of-care MTB nucleic acid testing in resource-limited settings.
研究领域:
生物传感器
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分子诊断
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CRISPR技术
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核酸扩增
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DNA酶技术
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结核病检测
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ANALYTICAL CHEMISTRY
·
2023
·
IF 7.4
Liping Xia,Juxin Yin,Jianjian Zhuang,Weihong Yin,Zheyu Zou,Ying Mu
Rapid and sensitive pathogen detection methods are critical for disease diagnosis and treatment. RPA-CRISPR/Cas12 systems have displayed remarkable potential in pathogen detection. A self-priming digital PCR chip is a powerful and attractive tool for nucleic detection. However, the application of the RPA-CRISPR/Cas12 system to the self-priming chip still has great challenges due to the problems of protein adsorption and two-step detection mode of RPA-CRISPR/Cas12. In this study, an adsorption-free self-priming digital chip was developed and a direct digital dual-crRNAs (3D) assay was established based on the chip for ultrasensitive detection of pathogens. This 3D assay combined the advantages of rapid amplification of RPA, specific cleavage of Cas12a, accurate quantification of digital PCR, and point-of-care testing (POCT) of microfluidics, enabling accurate and reliable digital absolute quantification of Salmonella in POCT. Our method can provide a good linear relationship of Salmonella detection in the range from 2.58 × 101 to 2.58 × 104 cells/mL with a limit of detection ∼0.2 cells/mL within 30 min in a digital chip by targeting the invA gene of Salmonella. Moreover, the assay could directly detect Salmonella in milk without nucleic acid extraction. Therefore, the 3D assay has the significant potential to provide accurate and rapid pathogen detection in POCT. This study provides a powerful nucleic detection platform and facilitates the application of CRISPR/Cas-assisted detection and microfluidic chips.
研究领域:
分子生物学
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生物技术
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微生物学
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分析化学
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即时诊断
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ANALYTICAL CHEMISTRY
·
2025
·
IF 6.7
Songkuan Zhuang,Xiaojun Huang,Muyan Diao,Ruiling Dong,Yueyu Dong,Jun Liu,Yizhen Liu
Current one-pot CRISPR diagnostics necessitate meticulous control of nucleic acid hybridization parameters or extensive modification of CRISPR components to achieve complete enzymatic silencing, a fundamental bottleneck limiting their robustness and generalizability. Here, we resolve this challenge through dynamic crRNA reconfiguration, a paradigm-shifting strategy that exploits the intrinsic structural duality of CRISPR RNA. We present CONVERT (CRISPR One-Pot Nucleic acid detection Via Engineered crRNA Reconfiguration Technology), a universal platform where nontarget intact crRNA acts as a universal suppressor, achieving complete Cas12a inhibition during RPA (recombinase polymerase amplification) by irreversible enzyme sequestration. Target detection is initiated through programmable conversion to split crRNA activators, bypassing sequence-specific design constraints entirely. This crRNA conformational switching mechanism is implemented via photocleavable linker cleavage and subsequent assembly with presupplied truncated crRNA (tcrRNA), activating trans-cleavage signal amplification. Three transformative advances emerge: First, the endogenous crRNA engineering eliminates dependency on exogenous nucleic acid blockers or chemical modifications, reducing optimization costs and time. Second, near-total enzymatic suppression ensures zero cross-talk between amplification and detection phases. Third, the unified workflow enables contamination-resistant operation through spatiotemporal control, requiring no physical partitioning. Validated for Enterocytozoon bieneusi detection, the system demonstrates attomolar sensitivity of 1 aM, 100% diagnostic concordance with qPCR, and absolute specificity against related pathogens. By effectively decoupling CRISPR regulation from sequence-context constraints through rational crRNA structural plasticity, this work establishes a generalized framework for next-generation molecular diagnostics.
研究领域:
分子生物学
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生物工程
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诊断技术
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合成生物学
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核酸扩增
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高影响因子
ANALYTICA CHIMICA ACTA
·
2022
·
IF 6.911
Mengyang Zhou,Haoyu Wang,Chao Li,Chao Yan,Panzhu Qin,Lin Huang
Single base mutations detection is crucial for the diagnosis and treatment of cancer. However, the current methods with poor selectivity and sensitivity required large instruments, which are difficult to meet clinical demands. Herein, we develop a CRISPR/Cas9 based visual colorimetric platform to specifically detect all single base mutations. In this strategy, the Recombinase Polymerase Amplification (RPA) was firstly used to amplify the target, and introduced the PAM site in the target DNA sequence by designing the point mutation primer, thus achieving detection for all single base mutations by the clustered regularly interspaced short palindromic repeats (CRISPR)/Cas9-mediated specific recognition. With the help of CRISPR/Cas9 system, those RPA products can release single strand DNA to hybridize with the padlock probe and trigger rolling circle amplification (RCA). Based on the magnetic separation, HRP-gold nanoparticles complex (hGNPs) and biotin modified probe (Bio-probe) were further used to achieve enhanced visual variations assay by hybridizing with RCA products. Benefiting from the RPA assisted triple signal amplification, this method not only showed enhanced sensitivity with a limit of detection (LOD) as low as 0.2 fM and 0.01% of KRAS-G12D mutation percentage, but the specificity against KRAS-G12D mutation also be synergistically enhanced by combining the CRISPR/Cas9-mediated specific recognition with the specific T4 ligation reaction of RCA system. Furthermore, this system has been successfully used to visually detect genome in serum, suggesting its great potential for point-of-care diagnosis in clinical.
研究领域:
分子生物学
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生物技术
·
医学诊断
·
癌症研究
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高影响因子
危险品化学品经营许可证（带存储）