Biosensors-Basel
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2025
·Vol. 15
·期 12
·821
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IF 5.6
Jie Chen,Shutao Liu,Sunyi Chen,Jingwen Mai,Maiwula Abudukadi,Yao Chen,Jie Lu,Guanglei Li,Chenchen Ge
Mycoplasma hominis (MH) is a prevalent opportunistic pathogen that is strongly associated with a wide range of urogenital tract infections and severe adverse pregnancy outcomes in clinical settings. Current MH detection methods, including microbial culture and qPCR, are time-consuming and rely on complex equipment, making them unsuitable for scenarios requiring rapid or simplified testing. In this study, we developed a visual readout biosensing platform by synergistically integrating recombinase polymerase amplification (RPA), CRISPR/Cas12a-mediated target nucleic acid recognition, and lateral flow biosensors for the rapid, sensitive, and specific identification of MH. The assay specifically targets the MH-specific 16S rRNA gene, achieving a limit of detection as low as 2 copies/reaction of recombinant plasmid containing the target gene with a total assay time of 60 min. Critical reaction parameters, including Cas12a-crRNA molar ratio, volume of RPA amplicon input, and Cas12a cleavage time, were systematically optimized to maximize the biosensor’s response efficiency and detection reliability. The platform exhibited exceptional specificity, with no cross-reactivity observed against common co-occurring urogenital pathogens, and effectively minimized aerosol contamination risks via a rigorous decontamination workflow. Furthermore, this work represents the first documented implementation of a contamination-control protocol for an MH-specific CRISPR-LFA assay. Notably, testing results from 18 clinical samples demonstrated the high specificity of this assay, highlighting its promising potential for clinical application.
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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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JOURNAL OF AGRICULTURAL AND FOOD CHEMISTRY
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2025
·
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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DIAGNOSTIC MICROBIOLOGY AND INFECTIOUS DISEASE
·
2026
·Vol. 116
·期 3
·117523
·
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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npj Science of Food
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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
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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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CHEMICAL ENGINEERING JOURNAL
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2024
·
IF 13.3
Haoyu Wang,Shiting Chen,Jiqing Liu,Wanyue Liu,Wei Chen,Lin Huang,Chao Yan
In recent years, RPA-CRISPR/Cas12a-based electrochemical biosensors have emerged as highly effective tools for nucleic acid detection. However, the random trans -cleavage characteristics of Cas12a on single-stranded nucleic acid sequences can result in the presence of uncertain nucleic acid residues on the electrode surface post-reaction. This situation complicates subsequent sensitization strategies. In this study, we devised a universal sensitization protocol mediated by terminal deoxynucleotidyl transferase (TdT) , a template-independent DNA polymerase and G-rich spherical nucleic acids (G-rich SNAs) with superior performance. Initially, Cas12a was activated by the specific RPA amplification product and cleaved the closed single stranded DNA (C-ssDNA) on the electrode surface. Then, the TdT enzyme extended residual single stranded DNA (R-ssDNA) on the electrode surface, forming G-Quadruplex structures. Subsequently, G-rich SNAs were immobilized on the electrode surface via π-π stacking interactions with the G-quadruplexes. Consequently, a substantial accumulation of hemin occurred within the G-quadruplex cluster structures, generating significant electrochemical signals. We applied this method to the trace detection of Staphylococcus aureus ( S. aureus ) and achieved a good linear calibration curve with concentrations ranging from 0 CFU/mL to 10 4 CFU/mL. Notably, the limit of detection (LOD) was as low as 1.29 CFU/mL, surpassing that of most similar sensors. These results demonstrate that our proposed universal sensitization method effectively addresses the limitations associated with the random trans -cleavage characteristics of Cas12a.
研究领域:
生物传感
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纳米技术
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分子生物学
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电化学
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