MICROCHEMICAL JOURNAL
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2023
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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
LWT-FOOD SCIENCE AND TECHNOLOGY
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2023
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IF 6
Yingchun Li,Yaqin Zhang,Fange Kong,Chunxia Wang,Shanshan Chen,Jiasi Wang,Di Wang
Fusarium temperatum ( F. temperatum ) causes maize stalk rot disease, reducing grain yield and producing multiple harmful mycotoxins that threaten food safety and quality. However, current detection methods are limited by the requirement of well-trained personnel, complicated operations and expensive instruments. To address this challenge, gold nanoparticles (AuNPs)-based colorimetric assay using loop-mediated isothermal amplification (LAMP) coupled with CRISPR-Cas12a (Au-CRISPR) was tested for F. temperatum detection. The assay utilizes DNA-modified AuNPs as a colorimetric probe and the results can be readily observed by the naked eye. After optimization, the method can specifically detect as low as 100 copies/μL of target DNA or 10 −8 ng/μL of extracted DNA from F. temperatum . The detection platform allows high-throughput testing by integrating microtiter plates and microplate reader. Furthermore, a portable smartphone-based device was developed to realize point of care (POC) detection of F. temperatum in resource-limited settings. This simple, inexpensive and sensitive detection platform has great potential for the applications in field detection.
研究领域:
植物病理学
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分子生物学
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食品安全
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产品关联
DOI
高影响因子
ANALYST
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2025
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IF 3.3
Chenyan Li,Yilan Liang,Qincong Feng
Electrochemical biosensors are frequently employed to identify harmful microbes and disease indicators. However, their practical applicability is constrained by poor signal amplification efficiency and immobilization processes on the probe surface. To get over these restrictions, we here integrated roll-cycling amplification (RCA) and CRISPR/Cas12a gene editing tools with electrochemical biosensors. We constructed an electrochemical biosensor based on RCA to activate the cleavage activity of Cas12a. First, by double-stranded nucleic acid aptamer (ds Apt) specifically binding to E. coli-2571, Apt-b was competitively isolated to bind to T4 ligase and produce circular DNA. This in turn activates RCA, which in turn activates the accessory cleavage activity of CRISPR/Cas12a, which in turn cleaves DNA sequences loaded onto the electrode, changing electrochemical signals. With a linear range of 1 × 102–1 × 107 CFU mL−1, a detection limit of 5.28 CFU mL−1, and experimental recoveries of 93.01–101.53%, the measured electrochemical signals were positively connected with the concentration of E. coli-2571 under the optimal experimental conditions. Therefore, by combining two approaches—RCA and CRISPR/Cas12a—our electrochemical biosensor was able to detect E. coli-2571 specifically and sensitively, providing new research opportunities for the detection of other harmful bacteria.
研究领域:
分子生物学
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生物传感技术
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微生物检测
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产品关联
DOI
PubMed
高影响因子
ANALYTICAL CHEMISTRY
·
2024
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IF 6.7
Luyu Wei,Zhilong Wang,Yongzhen Dong,Deyang Yu,Yiping Chen
CRISPR/Cas12a fluorimetry has been extensively developed in the biosensing arena, on account of its high selectivity, simplicity, and rapidness. However, typical CRISPR/Cas12a fluorimetry suffers from low sensitivity due to the limited trans-cleavage efficiency of Cas12a, necessitating the integration of other preamplification techniques. Herein, we develop an enhanced CRISPR/Cas12a fluorimetry via a DNAzyme-embedded framework nucleic acid (FNAzyme) substrate, which was designed by embedding four CLICK-17 DNAzymes into a rigid tetrahedral scaffold. FNAzyme can not only enhance the trans-cleavage efficiency of CRISPR/Cas12a by facilitating the exposure of trans-substrate to Cas12a but also result in an exceptionally high signal-to-noise ratio by mediating enzymatic click reaction. Combined with a functional nucleic acid recognition module, this method can profile methicillin-resistant Staphylococcus aureus as low as 18 CFU/mL, whose sensitivity is approximately 54-fold higher than that of TaqMan probe-mediated CRISPR/Cas12a fluorimetry. Meanwhile, the method exhibited satisfactory recoveries in food matrices ranging from 80% to 101%. The DNA extraction- and preamplification-free detection format as well as the potent detection performance highlight its tremendous potential as a next-generation analysis tool.
研究领域:
生物传感
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分子诊断
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合成生物学
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产品关联
DOI
PubMed
高影响因子
FlatChem
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2024
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IF 5.9
Taojian Fan,Shaohui Zhang,Changle Meng,Lingfeng Gao,Li Yan,Hao Wang,Xin Shi,Yanqi Ge,Han Zhang,Junqing Hu
Ultra-sensitive nucleic acid detection is important for rapid prevention of infectious diseases. Recently, clustered regularly interspaced short palindromic repeat (CRISPR)/CRISPR-associated (Cas) systems with great target specificity and programmability have demonstrated amazing capabilities in the field of nucleic acid detection. However, most CRISPR/Cas systems-based strategies still rely on pre-amplification of nucleic acid, which limits their clinical application in point of care detection. Here, we designed a novel electrochemical CRISPR/Cas biosensor (called E-Sb CRISPR) that utilizes antimonene nanosheets (Sb NSs) modifications. Due to the strong interaction between Sb NSs and single stranded DNA (ssDNA), E-Sb CRISPR exhibited specific nucleic acid detection capabilities with a detection limit of 100 aM within 35 min. Notably, the sensor showed excellent selectivity to target DNA in serum in the presence of nucleus acid extracted from other viruses. The excellent stability (8 weeks) and reproducibility (50 cycles) of this sensor were observed. By developing microcircuits and software systems, rapid acquisition of detection results on mobile electronic devices is possible. Therefore, the E-Sb CRISPR showed great promise as a scalable nucleic acid detection platform for early diagnosis.
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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产品关联
DOI
高影响因子
BIOSENSORS & BIOELECTRONICS
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2024
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IF 10.7
Chuanyu Yang,Caiyi Du,Feiyu Yuan,Peitong Yu,Boxu Wang,Changshun Su,Ruiqi Zou,Junyang Wang,Xu Yan,Chunyan Sun,Hongxia Li
Benefiting from specific target recognition and trans-cleavage capabilities, the CRISPR/Cas12a system has great application prospects in the design of highly sensitive and rapid fluorescence biosensors. The CRISPR/Cas12a-based fluorophore-quencher molecular beacons exhibit single-color emission and are easily exposed to interference from environmental factors. Herein, we design a CRISPR/Cas12a-derived ratiometric fluorescence sensor for Pb 2+ detection based on embedded carbon dots@zeolitic imidazolate framework-8 (CDs@ZIF-8) composites and DNAzyme. The functions of ZIF-8 about encapsulating red emissive CDs in the inner cavity and adsorbing DNA on the outer surface are integrated to establish dual fluorescence signals, thereby reducing the possibility of interference and improving sensing accuracy. The presence of Pb 2+ is converted into the change of activator by the GR5 DNAzyme to activate the CRISPR/Cas12a system, which provides signal amplification through multiple turnovers of side branch cutting, achieving highly sensitive detection of Pb 2+ with a low detection limit of 18 pM. This method has the advantages of simplicity, universality, and excellent quantitative ability, and has broad prospects in sensing applications.
研究领域:
生物传感
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分子生物学
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环境科学
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产品关联
DOI
PubMed
高影响因子
MICROCHEMICAL JOURNAL
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2024
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IF 4.9
Yang Song,Yiwen Xu,Ruirui Wang,Jianping Wang,Xiaoping Yu,Xuping Shentu
Pathogenic bacterial contamination of food is a major challenge in food safety and public health management. To monitor pathogenic bacteria in food, a rapid, sensitive, and specific pathogen detection method is needed. CRISPR-Cas12a has been developed as a sensitive method for detecting pathogenic bacteria, but its output signal is fluorescent, which need equipment to detect. Here, the nanoparticle probe was designed to detect the activity of Cas12a and were able to visualize the assay results by observing the color change by naked eyes. The target sequences of pathogenic bacteria were amplified by recombinase polymerase, then recognized and bound by the crRNA and Cas12a complex. The activated Cas12a cleaved the linker-ssDNA connecting the nanoparticles, causing the nanoparticles to transition from aggregation to dispersion, and the color showed as purple. The sensitivity analysis showed that the limits of detection (LOD) reached 1 × 10°CFU/mL, 1 × 10 4 CFU/mL, 1 × 10 2 CFU/mL, 1 × 10 3 CFU/mL, 1 × 10 1 CFU/mL for Escherichia coli , Salmonella typhimurium , Vibrio parahaemolyticus , Staphylococcus aureus , and Listeria monocytogenes , which were all specific for five bacteria in the milk samples. Therefore, this method is a dependable, fast, sensitive and specific method, which make the CRIPSR-Cas12a detection method more useful in bacterial pathogenic detection.
研究领域:
食品科学
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分子生物学
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生物技术
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产品关联
DOI
高影响因子
SENSORS AND ACTUATORS B-CHEMICAL
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2022
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IF 9.221
Yu Tao,Ke Yi,Haixia Wang,Kai Li,Mingqiang Li
Since hepatitis B virus (HBV) infection can cause chronic viral hepatitis, increase the risk of liver fibrosis and cirrhosis, and ultimately result in hepatoma, it is of great significance to explore convenient, rapid, and sensitive diagnostic strategies for HBV DNA detection. In this study, we developed a novel and sensitive fluorescent biosensor for HBV detection based on the sensing system consisting of CRISPR-Cas12a enzymes and metal nanoclusters as the luminescent nanoprobes, involving gold nanoclusters (AuNCs), silver nanoclusters (AgNCs), and copper nanoclusters (CuNCs). The DNA probes could effectively be applied as the template for the preparation of fluorescent metal nanoclusters. In the presence of the HBV target, the trans-cleavage capability of Cas12a was able to be initiated to degrade DNA probes, which brought about the inhibition of metal nanoclusters formation, thereby giving minimal fluorescence signals. In comparison with AuNCs and AgNCs, the CuNCs-based nanosensor exhibited much higher sensitivity. In addition, the fluorescence response of CuNCs to HBV detection was significantly faster, which could be completed within 25 min without the requirements of sophisticated equipment or harsh reaction conditions. Moreover, the practical application of this analysis method was also demonstrated to detect HBV DNA target in human serum samples with satisfactory recovery, which was promising for simple, rapid, sensitive, and label-free HBV DNA bioanalysis .
研究领域:
分子生物学
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医学诊断
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纳米技术
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产品关联
DOI
高影响因子
CHEMICAL ENGINEERING JOURNAL
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2023
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IF 15.1
Yuqing Zhu,Xiaohe Zheng,Rui Zhu,Hongrui Zhao,Hongli Zhai,Feiyang Qian,Tenghua Zhang,Zihui Xie,Silan Liu,Bowen Jiang,Yan Sheng,Jiaming Hu
Detection of multiple biomarkers for accurate diagnosis of disease is highly desirable but remains challenging. Here, we developed a versatile diagnostic biosensor (SMARTLOCK) through the unique combination of DNA tetrahedron, nonlinear HCR and CRISPR-Cas12a system for simultaneous detection of multiple nucleic acids, small molecules, proteins and different types of combinations. Two DNA tetrahedrons are assembled into a functional dumbbell lock, which was unlocked by the target molecules, thereby triggering a two-stage signal amplification of nonlinear HCR and CRISPR-Cas12a to generate powerful fluorescence signals. The capability of multi-target simultaneous detection of SMARTLOCK is demonstrated in the detection of miR-141, miR-375, miR-21, ATP, cortisol, insulin and their combinations. SMARTLOCK could selectively (specificity = 93.3%) and sensitively (sensitivity = 90%) distinguish early-stage prostate cancer (PCa) patients (n = 10) from healthy donors (n = 30). Measurement of two PCa-related RNAs (miR-141 and miR-375) shows the potential of SMARTLOCK to be a highly accurate and low-cost tool for the screening, diagnosis and prognosis of various diseases.
研究领域:
生物技术
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医学诊断
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分子生物学
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产品关联
DOI
高影响因子
Nature Communications
·
2025
·Vol. 16
·期 1
·1-14
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IF 15.7
Hu Hao,Guo Songcheng,Li Yiyuan,Dong Kejun,Lu Yan,Ye Keyi,Li Longjie,Zhou Xiaoyu,Cheng Liming,Xiao Xianjin
Detecting small molecules is pivotal across fields like clinical diagnostics, environmental monitoring, and food safety. The CRISPR-Cas12a system, known for its simplicity and sensitivity, offers a promising basis for small molecule detection. However, current CRISPR-based detection methods face challenges, including complex design requirements, high background noise, and limited adaptability to different targets. In our study, we introduce the SBS-Cas system, leveraging a split crRNA mode to induce spatial hindrance on the scaffold strand through molecular binding. This approach prevents the assembly with Cas12a, effectively masking its trans-cleavage activity. By introducing small molecules that competitively bind to the macromolecule, we eliminate this spatial hindrance, activating Cas12a. Our results demonstrate high sensitivity, versatility, and adaptability in small molecule detection across multiple reactions, with successful intracellular imaging and responsive fluctuations in complex environments underscoring the system’s robustness. This innovative CRISPR-Cas12a-based approach establishes a low-background, highly sensitive platform for small molecule detection. SBS-Cas promises not only to enhance tools for clinical, environmental, and food safety applications but also to advance CRISPR research, providing insights and expanding possibilities in molecular detection science.
研究领域:
分子生物学
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临床诊断
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环境科学
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食品安全
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产品关联
DOI
PubMed
高影响因子
ANALYTICAL CHEMISTRY
·
2023
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IF 7.4
Yin Zhao,Dongjuan Chen,Zhichen Xu,Tao Li,Jiang Zhu,Rui Hu,Guoyong Xu,Ying Li,Yunhuang Yang,Maili Liu
Fast, simplified, and multiplexed detection of human papillomaviruses (HPVs) is of great importance for both clinical management and population screening. However, current HPV detection methods often require sophisticated instruments and laborious procedures to detect multiple targets. In this work, we developed a simple microfluidic dual-droplet device (M-D3) for the simultaneous detection of HPV16 and HPV18 by combining the CRISPR-Cas12a system and multiplexed recombinase polymerase amplification (RPA) assay. A new approach of combining pressure/vacuum was proposed for efficient droplet generation with minimal sample consumption. Two groups of droplets that separately encapsulate the relevant Cas12a/crRNA and the fluorescent green or red reporters are parallelly generated, followed by automatic imaging to discriminate the HPV subtypes based on the specific fluorescence of the droplets. The M-D3 platform performs with high sensitivity (∼0.02 nM for unamplified plasmids) and specificity in detecting HPV16 and HPV18 DNA. By combining the RPA and Cas12a assay, M-D3 allows on-chip detection of HPV16 and HPV18 DNA simultaneously within 30 min, reaching a detection limit of 10–18 M (∼1 copy/reaction). Moreover, the outstanding performance of M-D3 was validated in testing 20 clinical patient samples with HPV infection risk, showing a sensitivity of 92.3% and a specificity of 100%. By integrating the dual-droplet generator, CRISPR-Cas12a, and multiplexed RPA, the M-D3 platform provides an efficient way to discriminate the two most harmful HPV subtypes and holds great potential in the applications of multiplexed nucleic acid testing.
研究领域:
分子生物学
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医学检测
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生物技术
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产品关联
DOI
PubMed
高影响因子
ANALYTICAL CHEMISTRY
·
2021
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IF 6.986
Wei S. Zhang,Jianbin Pan,Feng Li,Min Zhu,Mengting Xu,Hongyan Zhu,Yanyan Yu,Gaoxing Su
The outbreak of the pandemic caused by the severe acute respiratory syndrome coronavirus-2 (SARS-CoV-2) calls for an urgent unmet need for developing a facial and cost-effective detection method. The requirement of well-trained personnel and sophisticated instrument of current primary mean (reverse transcription polymerase chain reaction, RT-PCR) may hinder the practical application worldwide. In this regard, a reverse transcription recombinase polymerase amplification (RT-RPA) coupled with CRISPR-Cas12a colorimetric assay is proposed for the SARS-CoV-2 detection. The methodology we have described herein utilizes DNA-modified gold nanoparticles (AuNPs) as a universal colorimetric readout and can specifically target ORF1ab and N regions of the SARS-CoV-2 genome. After the virus genome is amplified through RT-RPA, the resulting abundant dsDNA will bind and activate Cas12a. Under trans-cleavage degradation, the capped DNA substrate will be hydrolyzed gradually from AuNPs, demonstrating a change in the surface plasmon resonance (SPR), which can be facially monitored by UV–vis absorbance spectroscopy and naked eye observation. The high amplification efficiency from RT-RPA and Cas12a trans-cleavage process bring the sensitivity of our method to 1 copy of viral genome sequence per test. Notably, under the dual variations inspecting from the isothermal amplification and Cas12a activation process, the false positive events from other beta coronavirus members can be effectively avoided and thus significantly improve the specificity. Furthermore, the reliability of this colorimetric assay is validated by standard clinical samples from the hospital laboratory department. Through integration of the inherently high sensitivity and specificity from an RPA-coupled Cas12a system with the intrinsic simplicity of AuNP-based colorimetric assay, our method increases the practical testing availability of SARS-CoV-2.
研究领域:
分子生物学
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病毒学
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诊断学
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DOI
PubMed
高影响因子
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