MICROCHEMICAL JOURNAL
·
2023
·
IF 4.8
Xianfeng Wang,Chengxiang Chu,Yuanyi Deng,Yi Ma,Mei Yang,Huibo Luo,Danqun Huo,Changjun Hou
Accurate identification of Staphylococcus aureus ( S. aureus ) is crucial for food safety and clinical diagnosis. Particularly, identifying drug-resistant strains guides effective treatment and prevention of infectious diseases. Herein, we presented a highly efficient programmable CRISPR/Cas12a system for identification of methicillin-resistant S. aureus (MRSA). Genus-specific gene (spec gene) and methicillin-resistant gene (mecA gene) of S. aureus were amplified by PCR. According to the sequences of spec gene and mecA gene, corresponding programmable crRNAs were designed and prepared to construct CRISPR/Cas12a system, which recognized and bound to the PCR products through complementary interactions to activate the trans -cleavage activity of Cas12a. This led to the indiscriminate digestion of surrounding FQ-reporter, releasing a distinct fluorescence signal for MRSA identification. In addition, we devised a pair of AuNPs-DNA probes for colorimetric recognition of S. aureus . Target-triggered trans -cleavage for linker probe led to an non-aggregation of AuNPs-DNA probes pair. This led to a noticeable appearance of red color, enabling naked-eye detection. Thus, MRSA identification was realized with remarkable sensitivity and exceptional selectivity. Successful identification of MRSA in clinical samples validated the practical applications of the proposed CRISPR/Cas12a assay, establishing it as a universal platform for diagnosing pathogenic microorganisms with versatility and compatibility.
研究领域:
分子生物学
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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
高影响因子
NUCLEIC ACIDS RESEARCH
·
2025
·Vol. 53
·期 7
·
IF 13.1
Liu Qiong,Jiang Zhou,Li Sheng,Li Yinfeng,Wan Yingfei,Hu Zhenyu,Ma Shimeng,Zou Zhen,Yang Ronghua
Accurate identification of single-nucleotide variants (SNVs) is critical in clinical diagnostics but remains challenging due to subtle free energy variations, particularly for hard-to-detect SNVs such as wobble base pairs and those in high guanine–cytosine (GC) regions. Here we report a high-energetic-penalty SNV detection (HEPSD) platform that redesigns the hybridization regions of CRISPR RNA (crRNA) in the CRISPR/Cas12a system. This system employs a binary crRNA architecture design that enables the activation of the cleavage activity of Cas12a while amplifying the energetic penalty for single-nucleotide mismatches through nonequilibrium hybridization-driven regulation. Consequently, the entire targeting region of CRISPR/Cas exhibits a marked preference for mutations in genomic DNA, while preventing false activation induced by sequences containing a single mismatched nucleotide. Moreover, HEPSD exhibits exceptional differentiation performance for hard-to-detect SNVs including wobble mutations at extreme GC contents. As proof of principle, profiling of BRAF V600E and EGFR L858R tumor mutations down to a 0.01% variant allele frequency was achieved, enabling accurate discrimination of 132 clinical sample pairs, which showed high consistency with quantitative polymerase chain reaction-based techniques and next-generation sequencing. The proven effectiveness of this platform showcases its potential for clinical molecular diagnostics and expands the fundamental scope of hybridization-based protocols.
研究领域:
分子生物学
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医学诊断
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基因组学
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产品关联
DOI
PubMed
高影响因子
SENSORS AND ACTUATORS B-CHEMICAL
·
2024
·
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
CHEMICAL ENGINEERING JOURNAL
·
2025
·
IF 13.2
Yuan Zhang,Kangzheng Lv,Yu Zhang,Qin Mi,Daixin Ye,Xi Chen,Juan Zhang
The emergence and robust advancement of the CRISPR/Cas system have presented novel opportunities for the development of paper-based microfluidics. In this study, the CRISPR/Cas system is firstly ingeniously integrated into a paper-based platform as an efficient signal amplification component. The Cas12a-crRNA system is integrated with nitrocellulose (NC) membrane to prepare an anchored CRISPR-Cas12a system-boosted paper-based microfluidic device, thereby increasing the local concentration of the CRISPR/Cas12a-crRNA system and enhancing catalytic efficiency. Compared with free CRISPR/Cas12a system, our explored split anchored CRISPR/Cas12a signal amplification component demonstrates a catalytic constant that is 22 times higher and a specificity constant that is 34 times greater. Further, an anchored split CRISPR/Cas12a boosted paper-based microfluidic array (ASCPMA) has been further constructed for the on-site detection of Streptococcus mutans ( S. mutans ) to assess the early risk of oral caries. ASCPMA exhibits a wide linear range from 10 CFU/mL to 10 8 CFU/mL, with a detection limit as low as 2.08 CFU/mL, and it has been successfully applied to test 140 clinical saliva samples. With rapidity, accuracy, and portability, ASCPMA will facilitate early diagnosis and prevention of dental caries.
研究领域:
生物技术
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微流控技术
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分子诊断
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口腔医学
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产品关联
DOI
高影响因子
TALANTA
·
2025
·
IF 6.1
Yafang Shen,Xingyue Tang,Jiangqi Wang,Huang Dai,Yanna Cui,Qiuyue Hu,Yuxin Wu,Fei Jia,Guijie Hao
Recent years have witnessed the flourishing of CRISPR/Cas-based biosensors in various fields. However, most of them were developed for nucleic acid detection because non-nucleic acid targets are unable to unleash the cleavage activity of the CRISPR/Cas system directly. To circumvent this problem, activator DNA and deoxyribonuclease I (DNase I) were introduced in this research to render the CRISPR/Cas12a system as a new powerful tool for the detection of enrofloxacin (ENR), a common veterinary drug. In this biosensor, target ENR competed with DNase I- and bovine serum albumin-ENR composite-modified gold nanoparticles (DNase I-AuNPs-BSA-ENR) for the binding sites on the surface of antibody-modified magnetic nanoparticles (immuno-MNPs). Then, the captured DNase I-AuNPs-BSA-ENR degraded the activator DNA in the solution, which inhibited the activation of the CRISPR/Cas12a system. Finally, the fluorescence released by the activated CRISPR/Cas12a system was measured for the quantitative detection of ENR. The ingenious use of activator DNA and DNase I helped transduce the target recognition event into the cleavage activity of the CRISPR/Cas12a system. Moreover, the dual enzymatic amplification from DNase I and the CRISPR/Cas12a system guaranteed the sensitivity of this method with a low detection limit of 0.04 ng/mL. The developed biosensor extended the application of the CRISPR/Cas12a system for the sensitive detection of non-nucleic acid targets, providing a powerful tool in various fields such as environmental monitoring, food safety and clinical diagnosis.
研究领域:
生物技术
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食品安全
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环境监测
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临床诊断
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产品关联
DOI
PubMed
高影响因子
SENSORS AND ACTUATORS B-CHEMICAL
·
2024
·
IF 8
Dayong Li,Yanheng Yao,Wenting Cheng,Feifan Yin,Miao He,Yang Xiang
In this work, we constructed a manganese ion enhanced CRISPR fluorescence analysis platform based on magnetic separation technology and CRISPR/Cas12a system for the amplification-free detection of SARS-CoV-2 RNA. Firstly, nucleic acid functionalized magnetic beads and dual-function signal probes were used to identify conserved sequences of target RNA at the same time, and then the target was captured and separated by magnetic enrichment. The dual-function signal probe is used to bridge the recognition of the target and the output of the fluorescence signal. And the CRISPR activation sequence on the signal probe is used to initiate the downstream CRISPR/Cas12a fluorescence system to generate fluorescent signals. In this method, we used manganese ions to replace the traditional magnesium ions to assist the CRISPR/Cas12a system, which significantly enhanced the analysis effect. Moreover, we designed a variety of signal probes for different regions of the target RNA to further improve the signal intensity. This method can accurately detect the target RNA within 45 minutes, and can effectively identify the positive cases of COVID-19 infection in clinical throat swab samples, which has great potential for clinical application.
研究领域:
分子生物学
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医学诊断
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生物化学
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产品关联
DOI
高影响因子
Exploration
·
2024
·
IF 0
Yiming Zhang,Zhi Chen,Songrui Wei,Yujun Zhang,Hai Fu,Han Zhang,Defa Li,Zhongjian Xie
Real-time polymerase chain reaction (RT-PCR) remains the most prevalent molecular detection technology for sewage analysis but is plagued with numerous disadvantages, such as time consumption, high manpower requirements, and susceptibility to false negatives. In this study, an automated robot-driven photoelectrochemical (PEC) biosensing platform is constructed, that utilizes the CRISPR/Cas12a system to achieve fast, ultrasensitive, high specificity detection of biological loads in sewage. The Shennong-1 robot integrates several functional modules, involving sewage sampling and pretreatment to streamline the sewage monitoring. A screen-printed electrode is employed with a vertical graphene-based working electrode and enhanced with surface-deposited Au nanoparticles (NPs). CdTe/ZnS quantum dots (QDs) are further fabricated through the double-stranded DNA (dsDNA) anchored on Au NPs. Using the cDNA template of Omicron BA.5 spike gene as a model, the PEC biosensor demonstrates excellent analytical performance, with a lower detection limit of 2.93 × 10 2 z m and an outstanding selectivity at the level of single-base mutation recognition. Furthermore, the rapid, accurate detection of BA.5 in sewage demonstrates the feasibility of the PEC platform for sewage monitoring. In conclusion, this platform allows early detection and tracking of infectious disease outbreaks, providing timely data support for public health institutions to take appropriate prevention and control measures.
研究领域:
环境科学
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生物传感技术
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分子生物学
·
公共卫生
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DOI
ANALYTICA CHIMICA ACTA
·
2024
·
IF 5.7
Haolin Sun,Xiaoyu Zhang,Hainan Ma,Lina Zhang,Yang Zhang,Ruimeng Sun,Haoran Zheng,Han Wang,Jiayu Guo,Yanqi Liu,Yurou wang,Yanfei Qi
Rapid and sensitive detection of pathogenic bacteria is crucial for disease prevention and control. The CRISPR/Cas12a system with the DNA cleavage capability holds promise in pathogenic bacteria diagnosis. However, the sensitivity of CRISPR-based assays remains a challenge. Herein, we report a versatile and sensitive pathogen sensing platform (HTCas12a) based on the CRISPR/Cas12a system, hybridization chain reaction (HCR) and Poly T-copper fluorescence nanoprobe . The sensitivity is improved by HCR and the Poly-T-Cu reporter probe reduces the overall experiment cost to less than one dollar per sample. Our results demonstrate the specific recognition of target nucleic acid fragments from other pathogens. Furthermore, a good linear correlation between fluorescence intensity and target quantities were achieved with detection limits of 23.36 fM for Target DNA and 4.17 CFU/mL for S.aureus , respectively. The HTCas12a system offers a universal platform for pathogen detection in various fields, including environmental monitoring, clinical diagnosis, and food safety.
研究领域:
生物技术
·
医学诊断
·
食品安全
✓ 已验证
产品关联
DOI
PubMed
高影响因子
BIOSENSORS & BIOELECTRONICS
·
2024
·
IF 10.7
Xueying Lei,Lihua Ding,Xiaonan Yang,Feng Xu,Yongjun Wu,Songcheng Yu
The CRISPR/Cas12a system is increasingly used in biosensor development. However, high background signal and low sensitivity for the non-nucleic acid targets detection is challenging. Here, a p adlock activator which could i nhibit the t rans -cleavage activity of CRISPR/Cas12a system in the intact form by steric hindrance effect (PAIT effect) was designed for non-nucleic acid targets detection. The PAIT effect disappeared when padlock activator was separated into two split activators. To verify the feasibility of padlock activator, a Ca 2+ sensor was developed based on PAIT effect with the assistance of DNAzyme, activity of which was Ca 2+ dependent. In the presence of Ca 2+ , DNAzyme was activated to cleave its substrate, a padlock activator modified with adenine ribonucleotide, into split padlock activators which would trigger the trans -cleavage activity of Cas12a to generate fluorescence. There was a mathematical relationship between the fluorescence intensity and the logarithm of Ca 2+ concentration ranging from 10 pM to 1 nM, with a limit of detection of 3.98 pM. The little interference of Mg 2+ , Mn 2+ , Cd 2+ , Cu 2+ , Na + , Al 3+ , K + , Fe 2+ , and Fe 3+ indicated high selectivity. Recovery ranged from 93.32% to 103.28% with RSD s from 1.87% to 12.74% showed a good accuracy and reliability. Furthermore, the proposed sensor could be applied to detect Ca 2+ in mineral water, milk powder and urine. The results were consistent with that of flame atomic absorption spectroscopy. Thus, PAIT effect is valuable for expanding the application boundary of CRISPR/Cas12a system.
研究领域:
生物技术
·
生物化学
·
分析化学
·
分子生物学
✓ 已验证
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）