MICROCHEMICAL JOURNAL
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2024
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IF 4.9
Yuanxun Gong,Jiayi Zhang,Huiying Liang,Yanling Wang,Zichun Song,Jihua Wei,Qianli Tang,Kai Zhang,Xianjiu Liao
Matrix metalloproteinases (MMPs) are critical for the degradation of extracellular matrix components, which is essential for processes such as tissue remodeling, wound healing, and angiogenesis. MMP-2, in particular, degrades type IV collagen and is associated with diseases such as cancer and cardiovascular conditions, making it a significant biomarker. This study presents a novel electrochemical biosensor for the detection of MMP-2. The biosensor combines the specificity of a programmable RNA detection system with the amplification capabilities of T7 RNA polymerase and CRISPR/Cas12a. In the absence of MMP-2, Peptide Nucleic Acid (PNA) adsorbs on the DNA template, preventing RNA amplification and resulting in a low electrochemical signal. When MMP-2 is present, it cleaves a specific peptide, releasing the DNA template and enabling RNA synthesis. This activation leads to the cleavage of single-stranded DNA probes, increasing the electrochemical signal. The biosensor demonstrates high sensitivity with a detection limit of 0.496 fM for MMP-2 and excellent specificity, effectively distinguishing MMP-2 from other proteins. It successfully detected MMP-2 in biological samples, such as liver cancer cell supernatants, indicating its potential for clinical diagnostics. This innovative biosensor offers a powerful tool for the early detection and monitoring of diseases associated with MMP-2 activity.
研究领域:
生物医学工程
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分子诊断
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纳米技术
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DOI
高影响因子
Biosensors-Basel
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2023
·Vol. 13
·期 1
·111
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IF 5.4
Tianyi Yang,Yanju Chen,Jinsong He,Jian Wu,Meixia Wang,Xiaoping Zhong
A rapid and intuitive method for detecting Vibrio parahaemolyticus (VP) was established by a designed reaction vessel which coupled CRISPR/Cas12a with loop-mediated isothermal nucleic acid amplification (LAMP). There were two spaces in the vessel-holding LAMP reaction solution and CRISPR reaction solution, respectively, which were separated with a polyvinyl alcohol (PVA) membrane. The PVA membrane could be dissolved with a water solution. The thermolabile hemolysin (TLH) gene of VP was employed as the detection target. After the target sequence of the TLH gene was amplified with LAMP, the PVA membrane would be dissolved and the CRISPR reaction solution mixed with the LAMP reaction solution. In this way, amplicons could be detected with CRISPR/Cas12a in the reaction vessel. The fluorescent signals produced by the positive samples were clearly identified by the naked eye under a UV light, while the negative samples were dark. The whole detection procedure could be finished within 35 min with a detection limit of 100 copies/µL. The designed reaction vessel is easy to produce and can effectively prevent contamination due to the opening of the reaction vessel after the LAMP reaction. Thus, it will have the potential to provide a new solution for rapid detection in the field.Keywords:polyvinyl alcohol;dissolvable membrane;CRISPR/Cas12a;LAMP reaction;Vibrio parahaemolyticus
研究领域:
分子生物学
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病原体检测
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生物技术
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DOI
PubMed
高影响因子
ANALYTICAL CHEMISTRY
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2025
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IF 6.7
Jiaqi Xue,Kang Mao,Zihui Tang,Jiming Hu,Hua Zhang
Owing to the severe environmental and health issues posed by organophosphorus pesticides (OPs), a dual-enzyme cascade biosensing platform based on manganese dioxide (MnO2) and CRISPR/Cas12a was developed in this study. Smartphones were innovatively integrated with a stacked ensemble learning (SEL) model for ultrasensitive detection and dynamic monitoring of OPs in environmental water samples. A dual-enzyme cascade signal amplification strategy was used to construct this sensing platform. Acetylcholinesterase (AChE) catalyzes the generation of thiocholine (TCh), which, in turn, regulates the degradation of MnO2 nanosheets, releasing Cas12a activators and generating a fluorescence signal. Owing to the irreversible inhibition of AChE activity by OPs, dichlorvos (DDVP) was successfully detected, with a detection limit as low as 4.62 pg/mL. Additionally, the SEL model, integrated into the smartphone biosensing platform and incorporating random forest (RF), XGBoost, and ridge regression algorithms, exhibited strong performance in detecting OPs after optimization (R2 = 0.9985). In real water samples, the SEL model achieved a recovery rate of 93.1–103.1%, and the degradation kinetics of DDVP were successfully monitored over 24 h, revealing significant differences in DDVP degradation rates across various water matrices. This study is the first to report the integration of CRISPR/Cas12a biosensing technology with an SEL model-driven smartphone detection platform, providing a novel approach for sensitive, portable, and intelligent monitoring of OPs and offering new insights for water quality monitoring and early detection of environmental risks.
研究领域:
环境监测
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分析化学
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生物传感
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分子生物学
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环境毒理学
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水污染控制
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产品关联
DOI
PubMed
高影响因子
BIOELECTROCHEMISTRY
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2025
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IF 4.5
Yun Zhang,Liang Gao,Zhe Shi,Qiong Wu,Xiangmin Miao
Sensitive telomerase activity detection becomes particularly significance since the important value of it in early cancer diagnosis as a potential biomarker. Herein, we developed a paper-based analytical devices (PADs) for telomerase activity detection, using positively charged Au@luminol nanoparticles ((+)Au@luminol NPs) as electrochemiluminescence (ECL) signal probe coupling with hybridization chain reaction (HCR) and CRISPR/Cas12a dual signal amplification. Firstly, the initial strong ECL signal was obtained based on the electrostatic adsorption of (+)Au@luminol NPs onto the surface of HCR double-stranded hybrid aggregates. In the presence of telomerase, the primer was efficiently elongated with telomeric repeats of (TTAGGG) n to release activator DNA and trigger the CRISPR/Cas12a, which can prevent the happen of HCR and the adsorption of (+)Au@luminol NPs through cleaving the capture probe on the electrode surface, such results directly inducing the decrease of the ECL signal that was proportional to telomerase concentration, due to the efficient signal amplification of HCR and CRISPR/Cas12a, a low detection limit of 2.3 cells/mL for telomerase could be detected. Moreover, the sensor realized the effective application for telomerase extracts analysis in human serum samples, making it possess potential application value for telomerase activity assays in cancer diagnostics.
研究领域:
生物医学工程
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分子诊断学
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癌症早期诊断
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DOI
PubMed
高影响因子
JOURNAL OF AGRICULTURAL AND FOOD CHEMISTRY
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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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产品关联
DOI
PubMed
高影响因子
ACS Sensors
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2025
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IF 9.1
Shixian Liu,Tian Yu,Libing Song,Kourosh Kalantar-Zadeh,Guozhen Liu
Highly sensitive point-of-care early screening for high-risk human papillomavirus (HPV) infections is urgently needed, particularly in resource-limited settings. Nucleic acid amplification methods, especially CRISPR/Cas-based biosensors, have emerged as promising tools for sensitive HPV detection; however, current approaches typically rely on tedious tube-based formats coupled with lateral flow assays for signal readout in point-of-care testing (POCT). Here, we developed customized microfluidic paper-based analytical devices (μPADs) with valves that seamlessly integrated recombinase polymerase amplification (RPA) with CRISPR/Cas12a biosensing (RPA-CRISPR/Cas12a) on the filter paper substrate. This innovation achieved sensitive and cost-effective high-risk HPV detection in POCT. The RPA-CRISPR/Cas12a system with a linear reporter on μPADs, enabled fluorescence detection of the E7 gene, achieving a sensitivity of 1 pM at approximately 1 h. The sensitivity was further enhanced by introducing a circular reporter into the fluorescence-based RPA-CRISPR/Cas12a system on μPADs, enabling detection of the E7 gene with a detection limit of 1 fM and an assay time of 35 min. The system was validated using 50 cervical swab clinical samples, demonstrating 95% sensitivity and 100% specificity when compared to qPCR. This sample-to-answer detection platform holds significant promise for early screening of high-risk HPV infections in point-of-care scenarios.
研究领域:
分子生物学
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医学诊断
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生物传感技术
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DOI
PubMed
高影响因子
SENSORS AND ACTUATORS B-CHEMICAL
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2024
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IF 8
Honglian Wu,Ruolin Zhang,Huang Lai,Luogen Lai,Yi Liu,Yadi Fan,Yu Zhang,Jingyu Shi,Mo Yang
Endotoxins or lipopolysaccharides (LPS) are potent inflammatory agents that pose serious health risks even at low concentrations, thus requiring sensitive and reliable detection methods. Current methods for endotoxin detection, primarily the Limulus Amebocyte Lysate (LAL) assay and Enzyme-linked Immunosorbent Assays (ELISA) are limited by complex procedures, prolonged assay time, and susceptibility to interference. To address these challenges, we developed a single-reporter probe-based dual-mode biosensor integrating MXene-Au nanosheets with the CRISPR/Cas12a system for endotoxin detection. The sensing platform was constructed by conjugating ssDNA-Cy5 reporters onto MXene-Au nanosheets. MXene-Au nanosheets served dual functions, including efficient quenching of fluorescence and strong enhancement of SERS signals. In the presence of target endotoxins, CRISPR/Cas12a remains inactive, leaving the ssDNA-Cy5 reporters intact on the MXene-Au surface. The close proximity of Cy5 to the MXene-Au surface results in quenched fluorescence (“FL off”) but enhanced SERS signals (“SERS on”). Conversely, in the absence of endotoxins, activated CRISPR/Cas12a cleaves the ssDNA-Cy5, releasing Cy5 from the surface, thus leading to fluorescence recovery (“FL on”) but diminished SERS signals (“SERS off”). By measuring the changes in SERS and FL signals and analysing the ratiometric I SERS /I FL signals, the biosensor achieved a lower limit of detection (LOD) of 15.9 pg/mL within 30 minutes compared to single detection modes. Validation studies in complex matrices, including spiked water and milk samples, showed performance comparable to commercial endotoxin detection kits. Overall, our CRISPR-Cas12a-mediated fluorescent-SERS dual-mode biosensor not only improves detection sensitivity but also provides built-in quality control, improving the reliability of results through cross-validation.
研究领域:
生物医学工程
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分子诊断学
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产品关联
DOI
高影响因子
ANALYTICA CHIMICA ACTA
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2025
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IF 6
Xin Meng,Yuanyuan Yue,Mengyang Huang,Zhangqun Duan,Kang Liu,Lina Wu
Background Lead ion (Pb 2+ ) is a toxic heavy metal that poses severe threats to food safety. Traditional methods like inductively coupled plasma mass spectrometry (ICP-MS) and atomic absorption spectrometry (AAS) rely on bulky instrumentation, which are limited by high costs, complex sample preparation requirements, and inability to meet the demands for rapid on-site testing. Nanozyme-based biosensors have emerged as promising alternatives, yet single-mode sensors often suffer from matrix interference in complex food samples. Therefore, developing a rapid and reliable on-site method is critical for Pb 2+ detection. Results To address this challenge, a CRISPR/Cas12a-driven dual-mode biosensor integrating Pt/CeO 2 nanozyme-mediated peroxidase activity with GR-5 DNAzyme recognition was developed. The biosensor's design capitalizes on GR-5 DNAzyme for Pb 2+ -specific recognition, triggering CRISPR/Cas12a-mediated cleavage of electrochemical/colorimetric signal probe SH-ssDNA-Pt/CeO 2 to generate dual signals. This innovative platform synergizes electrochemical precision and colorimetric simplicity for Pb 2+ detection in complex food matrices. The biosensor achieved an ultra-sensitive electrochemical response (linear range: 0.002–200 nM; limit of detection: 0.14 pM) alongside a robust colorimetric readout (linear range: 0.5–2000 nM; limit of detection: 0.47 nM), representing significant sensitivity improvements over conventional single-mode sensors. Crucially, the intrinsic cross-verification mechanism between orthogonal signal modalities minimized false positives while ensuring >90.5 % recovery in spiked corn, edible oil, beef and red wine samples with RSD <5 %. These results highlight the potential of the dual-mode sensor as a practical, field deployable sensing platform for the detection of Pb 2+ . Significance This work innovatively integrates the specificity of CRISPR with the catalytic properties of nanozymes into a self-validating electrochemical/colorimetric dual-mode system. The detection method not only establishes a robust platform for highly sensitive, reliable and visualized detection of Pb 2+ in complex food matrices, but also pioneers a new paradigm for multi-modal biosensor design.
研究领域:
分析化学
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食品安全
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生物传感器
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环境毒理学
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分子诊断
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纳米生物技术
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产品关联
DOI
PubMed
高影响因子
Biosensors-Basel
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2025
·Vol. 15
·期 8
·479
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IF 5.6
Xiaotian Guan,Peizheng Wang,Yi Wang,Shuqing Sun
The CRISPR/Cas system has attracted increasing attention in accurate nucleic acid detection. Herein, we reported a CRISPR/Cas12a-chemiluminescence cascaded bioassay (CCCB) for the amplification-free and sensitive detection of human papillomavirus type 16 (HPV-16) and parvovirus B19 (PB-19). A magnetic bead (MB)-linking single-stranded DNA (LssDNA)-alkaline phosphatase (ALP) complex was constructed as the core component of the bioassay. During the detection process, the single-stranded target DNA was captured and enriched by LssDNA and then activated the trans-cleavage activity of Cas12a. Due to the Cas12a-mediated cleavage of LssDNA, ALP was released from the MB, subsequently catalyzing the substrate to generate a chemiluminescence (CL) signal. Given the cascade combination of CRISPR/Cas12a with the CL technique, the limits of detection for HPV-16 and PB-19 DNA were determined as 0.14 pM and 0.37 pM, respectively, and the whole detection could be completed within 60 min. The practicality and reliability of the platform were validated through target-spiked clinical specimens, and the recovery rate was 93.4–103.5%. This dual-amplification strategy—operating without target pre-amplification—featured high specificity, low contamination risk, facile preparation, and robust stability. It provides a novel approach for sensitive nucleic acid detection, with the potential for rapid extension to the diagnosis of various infectious diseases.
研究领域:
生物分析
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分子诊断
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CRISPR技术
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分析化学
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病毒学
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产品关联
DOI
PubMed
高影响因子
SENSORS AND ACTUATORS B-CHEMICAL
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2022
·
IF 9.221
Liu Wang,Fang He,Xueyun Chen,Kaiyu He,Linlin Bai,Qiang Wang,Fang Zhang,Xiahong Xu
CRISPR/Cas12a, as a powerful and programmable biosensing tool, has brought great convenience in visual detection of the target in a sequence-specific mode. Though fluorescent output is the predominant way of present visual methods, this strategy highly depends on cleaving fluorophore-/quencher-labeled oligonucleotides . Few research focuses on realizing fluorescent visual detection without DNA-labeling. Herein, we have proposed a CRISPR/Cas12a-based label-free fluorescent visual detection strategy. The basic principle of this strategy is that G-quadruplex can enhance the fluorescent emission of fluorescent ligands while dsDNA target-activated Cas12a make them impotent by disrupting the higher-ordered structure. After comparing four kinds of G-quadruplex-specific ligands, we selected Thioflavin T (ThT) to achieve the visual observation goal. The application feasibility was confirmed by combining with loop-mediated isothermal amplification (LAMP) for detection of Vibrio parahaemolyticus ( V. parahaemolyticus ), and it demonstrated a high sensitivity (1.36 × 10 2 copies) and specificity (among 12 kinds of bacteria). The method displayed similar performance as the real-time PCR for detection of real samples yet had lower requirement on the instrument. The signal output format could also distinguish Salmo salar from other seven kinds of sequence-similar Salmonidae. The findings and strategy proposed in this manuscript will expand the application of CRISPR/Cas12a-based label-free fluorescent analysis.
研究领域:
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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微生物学
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DOI
高影响因子
MICROCHIMICA ACTA
·
2025
·Vol. 192
·期 3
·1-10
·
IF 5.3
Liu Jianghua,Wang Yu,Zhang Xinyue,Huang Mingquan,Li Guoliang
A novel direct lysis method combined with amplification-free CRISPR/Cas12a-SERS genosensor was for the first time developed to rapidly and sensitively identify meat adulteration. Notably, polystyrene (PS) microspheres, with distinct shrinking and swelling properties, were dexterously employed to encapsulate biological-silent Raman reporter 4-mercaptobenzonitrile (4-MBN) and act as a controlled-release signal probe. Target DNA activated the trans -cleavage activity of CRISPR/Cas12a towards ssDNA linked with PS microsphere to liberate the signal probe, which was able to release numerous Raman reporters after treatment with THF solution, resulting in high signal amplification. Through this platform, trace target DNA was deftly transformed into a sensitive Raman signal and could be on-site determined through a portable Raman equipment. Under optimized conditions, this strategy displayed good linearity in the range 1–450 ng/μL ( R 2 = 0.9943) and favorable sensitivity with limit of detection as low as 0.23 ng/μL without any pre-amplification. Moreover, it exhibited good applicability to on-site identification of commercial meat samples in complicated food matrix. In addition, DNA extraction by direct lysis and amplification-free detection realized ultrafast meat adulteration determination within 35 min from sampling to result. This method possessed great potential in rapid and on-site accurate determination of meat authenticity. Graphical abstract
研究领域:
食品科学
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分子生物学
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分析化学
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产品关联
DOI
PubMed
高影响因子
BIOELECTROCHEMISTRY
·
2025
·
IF 4.5
Zhaofan Zhou,Ye Li,Dongya Wen,Xuyu Zhang,Ni Li,Jiangbing Shuai,Xiaofeng Zhang,Xiaoye Zheng,Zhaojun Ban,Farouk Amr,Xiaoping Yu,Jun Huang
Porcine epidemic diarrhea virus (PEDV) is a highly infectious pathogen responsible for porcine epidemic diarrhea, which causes severe diarrhea and high mortality rates in neonatal piglets, leading to substantial economic losses in the swine industry. Therefore, there is an urgent need for rapid, sensitive, and accurate detection methods for PEDV. In this study, we develop a rapid, ultrasensitive electrochemical CRISPR-based biosensor for detecting PEDV with high sensitivity and specificity. The sensor integrates reduced graphene oxide-polypyrrole‑gold nanoparticle (rGO-PPy-AuNP) nanocomposites to enhance sensitivity and CRISPR-Cas13a for target-specific recognition. This dual-signal amplification strategy achieves an excellent analytical detection limit of 1.01 fg/mL and high linearity (R 2 = 0.9979) across a broad dynamic range (0.005–100,000 pg/mL) within 45 min, eliminating the need for nucleic acid amplification. By synergizing Cas13a-assisted signal enhancement with nanoparticle-mediated electron transfer, the biosensor outperforms conventional methods in sensitivity while maintaining excellent stability, and specificity, demonstrating strong potential for PEDV detection in clinical diagnostics.
研究领域:
分子生物学
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生物传感技术
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兽医学
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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）