Frontiers in Chemistry
·
2025
·
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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PubMed
高影响因子
TALANTA
·
2024
·
IF 5.6
Qiang Tang,Xiaoling Huang,Guangxing Liu,Hongran Huang,Jiayi Zhang,Jihua Wei,Yuanxun Gong,Qianli Tang,Kai Zhang,Xianjiu Liao
This study introduces a novel electrochemical biosensor for detecting Matrix Metalloproteinase-2 (MMP-2), a key biomarker in cancer diagnostics and tissue remodeling. The biosensor is based on a dual-amplification strategy utilizing T7 RNA polymerase isothermal amplification and CRISPR-Cas12a technology. The principle involves the release of a DNA template in the presence of MMP-2, leading to RNA synthesis by T7 RNA polymerase. This RNA activates CRISPR-Cas12a, which cleaves a DNA probe on the electrode surface, resulting in a measurable electrochemical signal.The biosensor demonstrated exceptional sensitivity, with a detection limit of 2.62 fM for MMP-2. This high sensitivity was achieved through the combination of transcriptional amplification and the collateral cleavage activity of CRISPR-Cas12a, which amplifies the signal. The sensor was able to detect MMP-2 across a wide dynamic range from 2 fM to 1 nM, showing a strong linear correlation between MMP-2 concentration and the electrochemical signal. In practical applications, the biosensor accurately detected elevated levels of MMP-2 in cell culture supernatants from HepG2 liver cancer cells, distinguishing them from normal LO2 liver cells. The use of an MMP-2 inhibitor confirmed the specificity of the detection. These results underscore the biosensor's potential for clinical diagnostics, particularly in early cancer detection and monitoring of tissue remodeling activities. The biosensor's design allows for rapid, point-of-care testing without the need for complex laboratory equipment, making it a promising tool for personalized healthcare and diagnostic applications.
研究领域:
生物传感器
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癌症生物学
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分子诊断
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电化学
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生物技术
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PubMed
高影响因子
Small
·
2025
·
IF 12.1
Chenggong Zhu,Qunfang Huang,Ruijie Fu,Zhen Xun,Qishui Ou,Yunlei Xianyu,Can Liu
Hepatitis B virus (HBV) infection remains a significant global public health issue, and rapid detection of HBV DNA is crucial for disease prevention and control. However, traditional methods for HBV DNA detection are limited by their reliance on precise instruments and single readout, which can hardly meet the requirements of on-site detection. In this study, the Mg 2+ -enhanced trans-cleavage activity of clustered regularly interspaced short palindromic repeats/associated protein 12a (CRISPR/Cas12a) is reported and coupled with loop-mediated isothermal amplification (LAMP) and Au@Pt nanoparticles as a signaling reporter for on-site detection of HBV DNA. This triple-modal biosensing strategy enables multiple signal readouts including UV–vis spectrum, RGB value, and temperature with high sensitivity and accuracy. The linear detection ranges using UV–vis spectroscopy, RGB color recognition, and photothermal modes are from 50 to 10,000 copies µL −1 , with the limitation of detection of 24.07, 39.65, and 23.33 copies µL −1 , respectively. This biosensing strategy is further employed for the qualitative detection of HBV DNA in 48 serum samples, achieving sensitivities of 100%, 100%, and 95.24% for triple modes. This work offers a promising tool of next-generation LAMP-CRISPR/Cas12a for the rapid and portable detection of nucleic acids.
研究领域:
生物传感器
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病毒学
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纳米技术
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分子诊断学
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CRISPR技术
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分析化学
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DOI
PubMed
高影响因子
ANALYTICA CHIMICA ACTA
·
2024
·
IF 5.7
Kai Shi,Yi Tian,Sujun Liu,Wenjie Luo,Keer Liu,Lin Zhang,Ying Zhang,Jiali Chang,Jiaheng Zhang,Shuo Wang
Background Clustered regularly interspaced short palindromic repeats (CRISPR)/Cas12a-powered biosensor with a G-quadruplex (G4) reporter offer the benefits of simplicity and sensitivity, making them extensively utilized in detection applications. However, these biosensors used for monitoring pollutants in environmental water samples may face the problem of high background signal and easy interference due to the “signal-off” output. It is obvious that a biosensor based on the CRISPR/Cas12a system and G4 with a “signal on” output mode needs to be designed for detecting environmental pollutants. Results By using phosphorothioate-modified G4 as a reporter and catalytic hairpin assembly (CHA) integrated with Cas12a as an amplification strategy, a “signal-on” colorimetric/photothermal biosensor (psG4-CHA/Cas) for portable detection of environmental pollutants was developed. With the help of functional nucleotides, the target pollutant (kanamycin or Pb 2+ ) triggers a CHA reaction to produce numerous double-strand DNA, which can activate Cas12a′s trans -cleavage activity. The active Cas12a cleaves locked DNA to release caged psG-rich sequences. Upon binding hemin, the psG-rich sequence forms a psG4/hemin complex, facilitating the oxidation of the colorless 3,3′,5,5′-tetramethylbenzidine (TMB) into the blue photothermal agent (oxTMB). The smartphone was employed for portable colorimetric detection of kanamycin and Pb 2+ . The detection limits were found to be 100 pM for kanamycin and 50 pM for Pb 2+ . Detection of kanamycin and Pb 2+ was also carried out using a portable thermometer with a detection limit of 10 pM for kanamycin and 8 pM for Pb 2+ . Significance Sensitive, selective, simple and robust detection of kanamycin and Pb 2+ in environmental water samples is achieved with the psG4-CHA/Cas system. This system not only provides a new perspective on the development of efficient CRISPR/Cas12a-based “signal-on” designs, but also has a promising application for safeguarding human health and environmental monitoring.
研究领域:
环境科学
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生物技术
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纳米技术
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分析化学
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生物化学
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DOI
PubMed
高影响因子
CHEMICAL ENGINEERING JOURNAL
·
2024
·
IF 13.3
Yuexin Wang,Bo Shen,Nini Luo,Cai Li,Haiping Wu,Yanshuang Wang,Shen Tian,Xuemiao Li,Rui Liu,Xinmin Li,Junman Chen,Wei Cheng,Shijia Ding,Rui Chen,Meifang Xiao,Qianfeng Xia
The simple, rapid, and accurate detection of highly lethal melioidosis is crucial for early clinical diagnosis and improving cure rates. Currently, due to time consumption, low sensitivity and detection rate the existing clinical detection methods cannot satisfy the needs of clinical diagnosis. Herein, a novel self-enhanced porous hydrogel material (Au@PEI-ABEI@Pt) for ultrasensitive ECL strategy of detection B. pseudomallei was report. The novel porous hydrogel material composed of PEI-ABEI porous hydrogel, gold nanoparticles (AuNP) and platinum nanoparticles (PtNP) have large specific surface area and porous structure, which not only fix more ABEI to realize self-enhanced ECL signal amplification, but also facilitate ion diffusion and efficient utilization of catalytic materials, realizing rapid electron transfer and zero-distance catalysis, significantly improving the initial signal of ECL sensor. Moreover, the most attractive aspect is that Au@PEI-ABEI@Pt hydrogels with good biocompatibility can achieve widespread application of CRISPR/Cas12a in solid-phase carriers without affecting the sensitivity, specificity and shearing activity of CRISPR/Cas12a. After coupling with the ECL system and CRISPR/Cas12a signal amplification strategy, the Au@PEI-ABEI@Pt can achieve an ultrasensitive ECL assay of B. pseudomallei with the LOD of 5 CFU mL −1 in complex samples, with high specificity and stability to effectively classify B. pseudomallei and other Gram-negative bacteria. This study shows that the developed porous hydrogel materials not only serve as an excellent ECL signal reporter to significantly improve the detection sensitivity of ECL biosensors, but also provide a new approach for the wide application of CRISPR/Cas systems in solid-phase carriers.
研究领域:
生物技术
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纳米技术
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分析化学
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生物传感器
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分子生物学
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医学诊断
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高影响因子
ANALYTICAL AND BIOANALYTICAL CHEMISTRY
·
2020
·Vol. 412
·期 3
·601-609
·
IF 3.637
Zhao Xianxian,Zhang Wenqing,Qiu Xiaopei,Mei Qiang,Luo Yang,Fu Weiling
Numerous studies have shown that exosomes are closely related to the pathogenesis of various diseases, especially cancers. Therefore, a rapid and sensitive method for exosome detection will be of great importance for the diagnosis and prognosis of diseases. We report here a method for exosome detection based on the CD63 aptamer and clustered regular interspaced short palindromic repeats (CRISPR)/Cas12a system. This method consists mainly of exosomal membrane protein recognition based on the CD63 aptamer and signal amplification based on CRISPR/Cas12a. The CD63 aptamer, as an easily adaptable nucleic acid strand, is responsible for the conversion of the amounts of exosomes into nucleic acid detection, whereas CRISPR/Cas12a is responsible for highly specific nucleic acid signal amplification. The detection range of the method was determined as 3 × 10 3 –6 × 10 7 particles per microliter. Additionally, we successfully applied this method to detect exosomes in clinical samples from both healthy individuals and patients with lung cancer, and the results were highly consistent with those obtained by nanoparticle tracking analysis. In general, this method provides a highly sensitive and specific method for the detection of exosomes and offers an avenue toward future exosome-based diagnosis of diseases.
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PubMed
高影响因子
New Biotechnology
·
2021
·
IF 5.079
Ke Liu,Yang Gao,Zhen-Hai Li,Min Liu,Feng-Qing Wang,Dong-Zhi Wei
Efficient and convenient genetic manipulation of mycobacteria, important microorganisms in human healthcare and the pharmaceutical industry, is limited. In this study, using a model strain Mycolicibacterium neoaurum ATCC 25795, the classical bacterium for the production of valuable steroidal pharmaceuticals, a genome editing system employing CRISPR-Cas12a to achieve efficient and precise genetic manipulation has been developed. Targeted genome mutations could be easily achieved by the CRISPR-Cas12a system without exogenous donor templates, assisted by innate non-homologous end-joining (NHEJ). CRISPR-Cas12a enabled rapid one-step genomic DNA fragment deletions of 1 kb, 5 kb, 10 kb, 15 kb, 20 kb and 24 kb with efficiencies of 70 %, 30 %, 30 %, 20 %, 20 % and 10 %, respectively. Combined with the pNIL/pGOAL system, CRISPR-Cas12a successfully integrated the gene of interest into the targeted genomic site by single crossover and double crossovers with efficiencies of 100 % and 9 %, respectively, using a two-plasmid system. The robust CRISPR systems developed demonstrated strong potential for precise genome editing in M. neoaurum , including targeted deletion of DNA sequences of various lengths and integration of targeted genes into desired sites in the genome.
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高影响因子
ANALYTICAL CHEMISTRY
·
2025
·
IF 6.7
Fei Hu,Kaihui Liu,Yunyun Zhang,Shuhao Zhao,Tianyi Zhang,Cuiping Yao,Xing Lv,Jing Wang,Xiaolong Liu,Niancai Peng
Infectious diseases, especially respiratory infections, have been significant threats to human health. Therefore, it is essential to develop rapid, portable, and highly sensitive diagnostic methods for their control. Herein, a short-time preamplified, one-pot clustered regularly interspaced short palindromic repeats (CRISPR) nucleic acid detection method (SPOC) is developed by combining the rapid recombinase polymerase amplification (RPA) with CRISPR-Cas12a to reduce the mutual interference and achieve facile and rapid molecular diagnosis. SPOC can reduce the detection time and stably detect up to 1 copy/μL of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) RNA without affecting the detection sensitivity. A highly sensitive one-pot assay integrated with reverse transcription RPA is achieved by wrapping paraffin with a specific melting point on the lyophilized CRISPR reagent surface. A self-heating pack is designed based on thermodynamic principles to melt the paraffin and release CRISPR reagents, enabling low-cost and time-saving detection. Notably, the designed system, coupled with RNA extraction-free technology, can achieve “sample-in-answer-out” detection of the SARS-CoV-2 Orf1ab gene within 22 min using smartphone imaging. The developed assay is validated on 12 clinical samples, and the results 100% correlate with real-time polymerase chain reaction. SPOC is time-saving, is easy to operate, and can eliminate centrifugal and complex hardware devices, satisfying the demand for point-of-care diagnostics in resource-constrained settings.
研究领域:
分子诊断学
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传染病学
·
生物技术
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DOI
PubMed
高影响因子
ANALYTICA CHIMICA ACTA
·
2025
·
IF 6
Fei Ding,Xiaomin Hang,Sen Tian,Wei Cao,Jingwen Wu,Li Wang
Primer exchange reaction (PER) is a novel and simple nucleic acid-templated extension technique that has recently attracted much attention in the field of biosensing. However, current PER reactions have shown relatively slow rates and low amplification performances, resulting in long assay times and limited detection sensitivities. Here we report a nicking endonuclease-mediated PER reaction (named NEPER) that rapidly releases amplified DNA products by adding a nicking endonuclease to hydrolyze the hybridized double-stranded DNA (dsDNA), and consequently has a maximum speed that is thirty orders of magnitude greater than the maximum for conventional PER. We further combined a CRISPR/Cas12a signal readout technique and developed a cascade NEPER-CRISPR/Cas12a method that can detect miRNA-155 with a limit of detection (LOD) down to 3.1 fM. We also show that the NEPER-CRISPR/Cas12a can be used to detect targets in serum samples.
研究领域:
分子生物学
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生物传感
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诊断学
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DOI
PubMed
高影响因子
Current Research in Food Science
·
2025
·
IF 7
Lijuan Yin,Lijing Sun,Yueqi Wang,Gailing Shi,Liangcai Lin,Cuiying Zhang
Baijiu is the product of the cooperation of a variety of core functional organisms in an open fermentation system. The differences in the structure of these core functional microorganisms are directly related to the quality of the base liquor. Acetilactobacillus jinshanensis is the dominant functional microorganism involved in the process of making Jiang-flavour Baijiu. To quantitatively evaluate the concentration of A. jinshanensis in the fermentation system, a CRISPR/Cas12a-coupled gold nanoparticle visual biosensor was developed. The limit of detection for A. jinshanensis was as low as 1 copy/μL, and its sensitivity exceeded that of the existing quantitative real-time PCR method. Furthermore, this system was employed to determine the concentration of A. jinshanensis during actual Baijiu fermentation. The average fluorescence value of A. jinshanensis in excellent-grade workshop was 4838, while that in ordinary workshops was 2145. The results revealed a linear relationship (R 2 =0.845) between the quality of the base liquor and the concentration of A. jinshanensis was obtained. Therefore, the concentration of A. jinshanensis might be used as a new parameter to predict the quality of base liquor and as a regulatory target in the fermentation process, thereby achieving a controllable fermentation process and high quality.
研究领域:
微生物学
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分子生物学
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食品科学
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DOI
PubMed
高影响因子
NUCLEIC ACIDS RESEARCH
·
2026
·Vol. 54
·期 5
·
IF 15
Wang Zhongzhong,Wu Yi,Wang Zixuan,Zhang Simeng,Liu Hao,Nie You,Chen Kexin,Huang Yanjun,Zhou Yu,Cao Yuanyuan,Sun Lin,Hao Rongzhang
CRISPR–Cas12a has transformative potential in molecular diagnostics owing to its robust signal amplification, but its sustained activity state severely limits temporal programmability and precise nuclease control in complex detection workflows. Here, we demonstrate that the conserved crRNA scaffold secondary structure itself can be repurposed as a reversible and programmable conformational switch to regulate Cas12a activity. By introducing short complementary DNA blockers of tunable length, we achieved length-dependent disruption and remodeling of scaffold secondary structure, shifting LbCas12a into an inactive conformation. Scaffold structure was subsequently reinstated through either single or cooperative strand displacement activation, enabling time-resolved and on-demand restoration of Cas12a activity. The conserved scaffold ensures intrinsic assay universality, while its programmable rewiring markedly improves SNVs discrimination and enables compatibility with one-pot isothermal amplification assays, delivering analytical sensitivity comparable to conventional two-step assays. This regulatory framework was further demonstrated in the detection of Klebsiella pneumoniae and Mycobacterium tuberculosis. By validating the crRNA scaffold as a practical and programmable switch for Cas12a activity control, this work establishes a universal and reversible framework for scaffold rewiring to modulate CRISPR nucleases and offers mechanistic insight to guide future assay engineering.
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高影响因子
Analytical Methods
·
2023
·Vol. 15
·期 17
·2105-2113
·
IF 3.1
Furong Zhao,Pei Wang,Haoxuan Wang,Sirui Liu,Muhammad Sohail,Xing Zhang,Bingzhi Li,He Huang
The spread of the monkeypox virus (MPXV) from Central and West Africa to previously non-endemic regions has caused a global panic. In this context, the rapid, specific, and ultrasensitive detection of MPXV is crucial to contain its spread, though such technology has seldom been reported. Herein, we proposed an MPXV assay combining recombinase-aided amplification (RAA) and CRISPR/Cas12a. This assay targeted the highly conserved MPXV F3L gene and demonstrates a low detection limit (LOD) of 101 copies per μL. By leveraging the high specificity nature of RAA and CRISPR/Cas12a, we rationally optimized probes and conditions to achieve high selectivity that differentiates MPXV from other orthopox viruses and current high-profile viruses. To facilitate on-site screening of potential MPXV carriers, a kit integrating lateral flow strips was developed, enabling naked-eye MPXV detection with a LOD of 104 copies per μL. Our RAA-Cas12a-MPXV assay was able to detect MPXV without the need for sophisticated operation and expensive equipment. We believe that this assay can be rapidly deployed in emerging viral outbreaks for on-site surveillance of MPXV.
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PubMed
高影响因子
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