CHEMICAL ENGINEERING JOURNAL
·
2024
·
IF 13.3
Yongqiang He,Ruxiang Ma,Niu Feng,Yiping Chen
Due to the significant health risks posed by pathogenic bacteria, there is an urgent need to develop multiplex, sensitive detection methods for them. Here, we developed a highly sensitive fluorescent sensor for the detection of pathogens by leveraging the powerful programmability of Clostridium butyricum Argonaute ( Cb Ago)-assisted exponential isothermal amplification reaction (EXPAR). This system combined Cb Ago, which locally cleaved extracted genomic DNA to produce single-stranded DNA (ssDNA), with EXPAR, which induced the amplification of large DNAzyme fragments by the ssDNA. Each DNAzyme fragment cleaved a specific reporter to generate a fluorescent signal for quantification of pathogenic bacteria. The EXPAR templates were carefully designed to ensure sufficient specificity for multiplex detection. The assay demonstrated superior analytical performance, with a limit of detection of as low as 4 CFU mL −1 and a linear range of 10–10 7 CFU mL −1 . This method represents the integration of Cb Ago-mediated genome editing and EXPAR high-efficiency amplification, yielding a customizable nucleic acid detection tool with broad applications in pathogen detection
研究领域:
分子生物学
·
食品安全
·
生物传感技术
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高影响因子
Small
·
2024
·
IF 13
Jun Kou,Yaru Li,Zhiying Zhao,Jiali Qiao,Qiang Zhang,Xiao Han,Xinkuan Cheng,Shuli Man,Long Ma
The development of novel method for drug-resistant bacteria detection is imperative. A simultaneous dual-gene Test of methicillin-resistant Staphylococcus aureus (MRSA) is developed using an Argonaute-centered portable biosensor (STAR). This is the first report concerning Argonaute-based pathogenic bacteria detection. Simply, the species-specific mec A and nuc gene are isothermally amplified using loop-mediated isothermal amplification (LAMP) technique, followed by Argonaute-based detection enabled by its programmable, guided, sequence-specific recognition and cleavage. With the strategy, the targeted nucleic acid signals gene are dexterously converted into fluorescent signals. STAR is capable of detecting the nuc gene and mecA gene simultaneously in a single reaction. The limit of detection is 10 CFU/mL with a dynamic range from 10 to 10 7 CFU/mL. The sample-to-result time is <65 min. This method is successfully adapted to detect clinical samples, contaminated foods, and MRSA-infected animals. This work broadens the reach of Argonaute-based biosensing and presents a novel bacterial point-of-need (PON) detection platform.
研究领域:
微生物学
·
生物技术
·
分子生物学
·
病原体检测
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生物传感器
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高影响因子
JOURNAL OF HAZARDOUS MATERIALS
·
2025
·
IF 11.3
Xiaoqin Tang,Xinyue Li,Yuhan Tian,Qiang Zhang,Shuli Man,Weipan Peng,Long Ma
Foodborne illnesses and contamination are becoming increasingly severe global public health issues. In recent years, the resistance of pathogenic bacteria has increased significantly, which makes it particularly urgent to develop advanced detection technologies beyond traditional methods. Pf Ago ( Pyrocus furiosus Argonaute), a novel programmable nuclease with specific sequence targeting and cleavage capabilities, offers significant advantages over traditional CRISPR diagnostics (CRISPR-Dx) in molecular detection, particularly in flexible design and multiplexed detection. In this work, Pf Ago was innovatively integrated with the nucleic acid reverse-phase enhanced fluorescent lateral flow test strip (rLFTS) and smartphone for the first time, enabling ultrasensitive, portable and multiplexed molecular detection of pathogenic bacteria in a cross-validating manner. Pf Ago precisely recognized LAMP amplicons, triggering linker DNA cleavage that generated dual colorimetric/fluorescent signals on rLFTS. A 3D-printed visualizer equipped with a homemade App-enabled smartphone achieved single-cell sensitivity (1 CFU/mL) within 45 min, two orders of magnitude lower than traditional qPCR (∼100 CFU/mL). In summary, a conceptually novel cross-validating manner integrating Pf Ago with dual-mode rLFTS was constructed. It eliminated the reliance on large instruments, representing a significant step forward in developing an ultrasensitive, portable, accurate and multiplexed molecular detection technology for foodborne pathogens.
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CHEMICAL ENGINEERING JOURNAL
·
2024
·
IF 13.3
Nan Wang,Qiang Zhang,Yajie Liu,Baoyan Huang,Shuli Man,Shengying Ye,Long Ma
Foodborne pathogens endanger public health and rapid, sensitive and accurate detection of them is vital. Argonaute stands in the frontline as the next generation nucleic acid detection tool, bearing a few comparable advantages. Centrifugal microfluidic chips (CMCs) use centrifugal force to achieve liquid flowing, mixing and reaction, eliminating complicated designs of valves and pumps. In this study, for the first time, we devised a Thermus thermophilus A rgonaute ( Tt Ago)-powered centrifugal microfluidic chip reaction biosensor for S ample-in- A nswer-out detection of P athogen S. aureus (termed as ASAP) with ultrahigh rapidity and sensitivity in a one-pot fashion. The samples subjected to testing were mixed with a home-made nucleic acid fast extraction reagent and then the mixture was injected into the sample cell of CMC, which was centrifuged down into the reaction cell. The reaction cell was preloaded with both LAMP (Loop-mediated isothermal amplification) and Tt Ago systems. As such, the species-specific nuc genes were amplified by LAMP, while the Tt Ago performed site-specific cleavage to output fluorescent (FL) signals. The sample-to-answer time was 17 min, and the limit of detection (LOD) reached 1 CFU/mL. ASAP was able to perform simultaneous multiplexed detection and up to 16 samples can be detected at the same time. ASAP reduced reagent consumption and minimized the influence of carry-over and cross-contamination. ASAP was capable of detecting S. aureus in foods, but also detecting physiological samples from infect, holding great promise for practical applications. Overall, our work has enriched the Argonaute-powered biosensing and CMC biosensor technology by providing a conceptually novel bacterial detection platform.
研究领域:
生物技术
·
分子生物学
·
微生物学
·
食品安全
·
生物传感器
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高影响因子
BIOELECTROCHEMISTRY
·
2025
·
IF 4.5
Qiang Tang,Jiayi Zhang,Jialuo Pang,Yingying Huang,Ying Guan,Yuanxun Gong,Qianli Tang,Kai Zhang,Xianjiu Liao
This study introduces an innovative electrochemiluminescence (ECL) biosensor for the highly sensitive and specific detection of Argonaute 2 (Ago2) activity. Ago2, a key enzyme in the RNA interference (RNAi) pathway, plays a crucial role in gene regulation, and its dysregulation is associated with diseases such as cancer and viral infections. The biosensor integrates hybridization chain reaction (HCR) amplification and the CRISPR/Cas12a system, leveraging a multi-stage signal amplification strategy. The detection mechanism begins with Ago2-mediated cleavage of a designed hairpin RNA (HP-RNA), releasing single-stranded RNA (ssRNA) that triggers HCR. This amplification step generates long DNA polymers, which serve as activators for the CRISPR/Cas12a system. Cas12a's collateral cleavage activity amplifies the signal further by cleaving a DNA reporter labeled with a ruthenium-based luminophore, enhancing the ECL output. This dual amplification strategy achieves exceptional sensitivity, with a detection limit of 0.126 aM. The biosensor demonstrates excellent specificity, distinguishing Ago2 from other Argonaute proteins, and maintains high reproducibility and stability, retaining 94 % of its signal after two weeks of storage. Real-world applicability was confirmed by accurately detecting Ago2 in spiked cell lysates, with recovery rates exceeding 100 %. The combination of HCR, CRISPR/Cas12a, and ECL establishes a robust platform for biomarker detection, offering superior sensitivity and adaptability for clinical diagnostics, disease monitoring, and therapeutic evaluation. This biosensor represents a significant advancement in the development of next-generation diagnostic tools.
研究领域:
生物传感技术
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分子生物学
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临床诊断
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ANALYTICAL CHEMISTRY
·
2025
·
IF 6.7
Zhuoqun Su,Ziye Pei,Jianing Chen,Wenya Song,Di Wu,Ying Wang,Guoliang Li
Food samples are often simultaneously contaminated with multiple small-molecule hazardous substances. However, conventional sensing methods typically detect only a single contaminant due to the lack of accurate and multiplexed signal conversion and output mechanisms. In this work, we propose a versatile sensing platform, “programmable hairpin argonaute–nanopore detection” (PHAND), which integrates Thermus thermophilus Argonaute (TtAgo)-mediated cleavage and a modular library of DNA hairpin probes to enable multiplex and portable detection of analytes. In the presence of specific targets, guide DNAs are released to activate TtAgo proteins, which cleave the corresponding hairpin probes and generate distinct nanopore reporters. These reporters produce orthogonal, characteristic current blockade signals when blocking α-hemolysin nanopores. Using the PHAND system, we successfully achieved the simultaneous detection of three major mycotoxins─aflatoxin B1, ochratoxin A, and zearalenone with high sensitivity and specificity. Validation in spiked real samples demonstrated satisfactory recovery and confirmed the practical applicability of our method. The PHAND system developed here supports portable and multiplexed detection of small-molecule hazardous substances, offering potential for rapid food safety screening.
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ACS Nano
·
2024
·
IF 15.8
Zhipan Wang,Aimin Ma,Yiping Chen
Multiple and ultrasensitive detection of pathogenic bacteria is critical but remains a challenge. Here, we introduce a digital assay for multiplexed and target DNA amplification-free detection of pathogenic bacteria using botryoidal-like fluorescent polystyrene dots (PS-dots), which were first prepared through the hybridization reaction between primer exchange reaction chains and polystyrene nanospheres that encapsulated polymer dots for signal preamplification. The pathogenic bacteria’s DNA was cleavaged by the argonaute (Ago) protein-mediated multiple and precise cleavage reactions, where the obtained target sequences bridged the magnetic beads (MBs) and botryoidal-like PS-dots via a hybridization reaction, and the fluorescent MB-botryoidal PS-dot complexes were utilized as digital probes based on colors and sizes for digital encoding. An artificial-intelligence-fluorescent microsphere counting algorithm was applied to identify and count the fluorescent MBs for digital readout. This digital assay combined the ultrabright botryoidal-like PS-dots with Clostridium butyricum Ago’s precise enzyme cleavage properties, achieving simultaneous detection of three pathogenic bacteria with a linearity range from 102 to 106 CFU/mL without target DNA amplification within 1.5 h. This digital assay has also been applied to detect aquatic and clinical samples with accepted accuracy (98%), which offers an avenue for a next-generation multiplexed digital platform for pathogenic bacteria analysis.
研究领域:
生物技术
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医学检验
·
纳米技术
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高影响因子
JOURNAL OF AGRICULTURAL AND FOOD CHEMISTRY
·
2026
·Vol. 74
·期 28
·22227-22238
·
IF 6.7
Zhang Yumei,Li Letian,Feng Niu,Wang Mengjiao,Li Hui,Chen Yiping
Multiplexed and ultrasensitive identification of foodborne
pathogens
is crucial for food safety. However, conventional methods are limited
by complex matrices, insufficient signal conversion tools, and low
sensitivity. Herein, we constructed bionic nanorobots by optimizing
phenylboronic acid density on magnetic nanoparticles via polyethylene
glycol linkers, achieving 92.21% Gram-positive bacteria capture within
20 min with 25 day stability. Additionally, we developed an artificial
intelligence (AI)-powered microscopic imaging strategy based on polystyrene
microsphere size-encoding and Clostridium butyricum Argonaute (CbAgo) decoding. The rational-designed CbAgo activates
cyclic cleavage circuits for multiplexed decoding, with AI-mediated
counting as readout. Without DNA amplification, the entire detection
can be completed within 1.5 h, and the platform achieved dual-pathogen
detection across 5 orders of magnitude (10 2 –10 6 CFU/mL), with a detection limit of 10 2 CFU/mL.
Compared with quantitative polymerase chain reaction, the platform
demonstrates 10-fold sensitivity improvement. Overall, this platform
provides a robust tool for multiplexed pathogen detection in complex
environments.
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SENSORS AND ACTUATORS B-CHEMICAL
·
2024
·
IF 8
Jiali Qiao,Jingyu Jia,Weipan Peng,Minghui Lu,Yaru Li,Shuli Man,Shengying Ye,Long Ma
Early detection and control of foodborne diseases are crucial for ensuring food safety and consumer health. Therefore, there is a need for faster and more sensitive detection methods. We developed a rapid, portable and visible lateral flow biosensor (LFB) for foodborne pathogenic bacteria detection powered by a Pyrococcus furiosus Argonaute nuclease ( Pf Ago), named as Pf Ago-LFB. LAMP (Loop-mediated isothermal amplification) was used to amplify the target nucleic acid . By using Pf Ago as the “lighting up” technology, we can overcome the limitations of false-positive results and lack of effective endpoint monitoring in LAMP, greatly enhancing its applicability. It turned out that Pf Ago-LFB was able to provide yes-or-no (positive or negative) results with a limit of detection (LOD) as low as 1 CFU/mL for S. typhimurium and the detection range was between 10 0 to 10 8 CFU/mL. It was able to secure a sample-to-answer test for contaminated food samples in less than 45 min and demonstrated satisfactory selectivity. In summary, we designed a Pf Ago-LFB that rendered a novel, rapid, accurate, easy-to-deploy, on-site and yes or no detection of S. typhimurium , which could be generalized for building a “one-for-all” biosensing platform.
研究领域:
食品安全
·
生物技术
·
分子生物学
·
病原体检测
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高影响因子
CHEMICAL ENGINEERING JOURNAL
·
2026
·
IF 12.5
Long Ma,Yajie Liu,Yongpo Mao,Jiayi Wang,Yiping Chen,Fei Deng,Zhiwei Zhu,Lanxiang Ou,Myunghee Kim,Shengying Ye,Weipan Peng,Shuli Man
Helicobacter pylori ( H. pylori ) infection poses a significant threat to public health. In this work, we combined Thermus thermophilus A rgonaute ( Tt Ago) with l oop-mediated isothermal amplification (LAMP) to establish a t ri-color fluorescent biosensing for detection and genotyping of H . p ylori from human salivary samples, termed as ALT- Hp . Leveraging the programmability, site specific cleaving-activity of Tt Ago, ALT- Hp enabled the multiplexed detection of H. pylori -specific genes ureA , vacA , and cagA . Furthermore, a customized 3D-printed visualizer compatible with App-enabled smartphones was developed, highlighting its potential for on-site detection. ALT- Hp utilized the amplification capability of LAMP and the signal transduction and amplification “lighting-up” functions of Tt Ago, for building a highly sensitive, specific and robust platform for on-site detection. It demonstrated robust performance in complex matrices, achieving a LOD of 1 CFU/mL within 30 min. Clinical validation using 72 samples showed 100% sensitivity and specificity. ALT- Hp was not only able to detect H. pylori , but also capable of genotyping by classifying H. pylori as type I and type II. The classification is primarily based on whether the strains carry virulence factors, and the two types differed significantly in pathogenicity and clinical outcomes, serving as a critical reference for clinical diagnosis and treatment decision-making. Thus, this ALT- Hp biosensing represented a powerful molecular diagnostic tool for monitoring and management of H. pylori infections.
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BIOSENSORS & BIOELECTRONICS
·
2026
·
IF 10.5
Xuewen Peng,Sufen Yang,Zhengzhang Huang,Niu Feng,Hetong Zhang,Yiqi Li,Yating Han,Caiyang Zhang,Jimei Ma,Yiping Chen
Pathogens have long presented a significant threat to human lives and environmental security, and the rapid detection of infectious pathogens is therefore vital for mitigating such risks. However, the practical application of biosensors is often hampered by complex sample matrices and limited sensitivity. To address these challenges, we established magnetic nanoparticle (MNP) functionalized with phenylboronic acid (PBA) (MNP-PBA) that efficiently and non-destructively captures Staphylococcus aureus ( S. aureus ) from complex samples within 20 min, achieving 92.21% capture rate at 10 8 CFU/mL. In addition, an aggregation-induced emission luminogen, TTQ-PF 6 , was introduced as a detection probe. TTQ-PF 6 exhibits a large Stokes shift (265 nm), intense red fluorescence, and exceptional anti-photobleaching ability—representing a 5.67-fold and 9.40-fold enhancement compared to Rhodamine X, respectively. Moreover, TTQ-PF 6 allows multiple fluorophores to be conjugated to a single DNA strand without the need for chemical reagents. The combination of MNP-PBA and red-emitting TTQ-PF 6 provides strong resistance to matrix interference in bacterial detection, realizing a broad detection linear range for S. aureus from 10 2 to 10 7 CFU/mL, with a limit of detection as low as 13.74 CFU/mL. Overall, this work provides a robust and efficient platform for pathogen detection in complex environments.
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FOOD CHEMISTRY
·
2025
·
IF 9.8
Xiru Zhang,Manyan Qiu,Yiyi Zhang,Qianyu Zhao,Bo Qu,Wei Zhang,Xianlong Zhang,Yujun Jiang
Foodborne pathogens have posed a great threat on food safety and public health, and thus the development of accurate and sensitive methods for the detection of foodborne pathogens is of great significance. In this study, a novel Pyrococcus furiosus Argonaute ( Pf Ago) biosensing platform based on metal-organic framework (MOF) nanozyme (namely AgoZyme) was developed for the detection of Salmonella typhimurium . Inspired by the catalytic characteristics of horseradish peroxidase, we synthesized a Hemin@ZIF-90 nanozyme with peroxidase-like activity by encapsulating hemin into a zeolitic imidazolate framework-90 through a self-assembly strategy. By integrating the specific recognition and cleavage capabilities of Pf Ago with the highly catalytic performance of Hemin@ZIF-90, the AgoZyme biosensor exhibited excellent detection sensitivity and specificity. This biosensor had a limit of detection of 1 CFU/mL in a linear range from 10 1 to 10 8 CFU/mL. This work provided a new insight for designing and developing novel biosensors for food safety detection.
研究领域:
生物传感器
·
食品安全
·
纳米技术
·
分子诊断学
·
微生物学
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高影响因子
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