ANALYTICAL CHEMISTRY
·
2023
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IF 7.4
Zhirun Ji,Bin Zhou,Zhaoyang Shang,Sirui Liu,Xue Li,Xing Zhang,Bingzhi Li
CRISPR-Cas12a is an accurate and responsive biosensing technique, but its limited stability has restricted its widespread applications. To address this, we propose a strategy using metal–organic frameworks (MOFs) to protect Cas12a from harsh environments. After screening multiple candidate MOFs, it was found that hydrophilic MAF-7 is highly compatible with Cas12a, and the as-formed Cas12a-on-MAF-7 (COM) not only retains high enzymatic activity but also possesses excellent tolerance to heat, salt, and organic solvents. Further investigation showed that COM can serve as an analytical component for nucleic acid detection, resulting in an ultrasensitive assay for SARS-CoV-2 RNA detection with a detection limit of 1 copy. This is the first successful attempt to create an active Cas12a nanobiocomposite that functions as a biosensor without the need for shell deconstruction or enzyme release.
研究领域:
生物传感
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纳米技术
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分子生物学
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分析化学
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PubMed
高影响因子
Synthetic and Systems Biotechnology
·
2025
·
IF 4.4
Wen Tian,Songcheng Yu,Kaiyang Zhang,Tao Liu,Lihua Ding,Peng Zhang
The CRISPR/Cas12a system holds significant promise for biomedical applications. Nevertheless, the commonly used reporter, fluorophore–quencher-labeled substrates, is hindered by labor-intensive synthesis procedures and high costs, while also relying on a single-photon method and being vulnerable to environmental interference. Herein, a label-free dual-fluorescent functional nucleic acid (DFFNA) was engineered, comprising an aptamer domain for auramine O (AO) recognition and a dSpacer-integrated DNA duplex region for 5,6,7-trimethyl-1,8-naphthyridin-2-amine (ATMND) binding. The fluorescence of AO and ATMND can be enhanced and quenched, respectively, when bound to DFFNAs. The fluorescence intensity ratio between ATMND and AO increased significantly following the cleavage of DFFNAs by activated Cas12a, thus offering a universal, label-free, ratiometric fluorescent reporter for the CRISPR/Cas12a system. To explore the application of the DFFNA-based CRISPR/Cas12a system, a novel biosensor was developed to detect site-specific DNA methylation. It employs a methylation-sensitive restriction enzyme to recognize methylation sites, Cas12a for site-specific DNA identification and signal amplification, and DFFNAs to produce ratiometric fluorescence. The assay demonstrated remarkable specificity and sensitivity, with a limit of detection of 152 pM, due to the high resolution and trans -cleavage activity of Cas12a. The rationally designed and label-free DFFNAs enhance stability, increase flexibility, and reduce cost. The observable color change and smartphone imaging capability facilitate portable, point-of-care testing. Specifically, the biosensor demonstrated excellent specificity by differentiating colorectal cancer patients from healthy individuals. Consequently, this work presents a superior label-free and ratiometric fluorescent reporter for the CRISPR/Cas12a system, which offers a promising strategy for DNA methylation detection in clinical settings.
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SPECTROCHIMICA ACTA PART A-MOLECULAR AND BIOMOLECULAR SPECTROSCOPY
·
2025
·
IF 4.6
Jiaxuan Chen,Kai Shi
Accurate detection of glyphosate (GLY) residues is essential for protecting public health, ensuring food safety, and preventing environmental pollution caused by excessive or improper herbicide application. Based on the asymmetric CRISPR/Cas12a system as a signal amplification technique, with phosphorothioate-modified hairpin G-quadruplex (psHG4) acting as a signal probe, a platform named ACC-GLY is developed for the sensitive and accurate detection of GLY. In the designed ACC-GLY platform, the target GLY specifically binds to the aptamer, and the cascading signal amplification strategy, driven by the DNase activity of a single Cas12a, is initiated by two competitive guide RNAs. Under the influence of Cas12a's DNase activity, the psHG4 probe is cleaved, releasing the psG4 sequence. The released psG4 sequence then binds to Thioflavin T (ThT), forming a complex that generates a fluorescence signal. Under optimal conditions, the detection platform can specifically detect GLY at a concentration as low as 0.3 pM. The detection platform demonstrates significant capability in detecting GLY in tap water and corn samples, highlighting its vast potential for applications in environmental monitoring and food safety. To the best of our knowledge, this is the first study to utilize the CRISPR/Cas12a system for GLY detection.
研究领域:
分析化学
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生物传感器
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环境监测
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食品安全
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分子生物学
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PubMed
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BIOSENSORS & BIOELECTRONICS
·
2021
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IF 10.618
Bin Qiao,Jiakun Xu,Wenhao Yin,Wanmeng Xin,Lixin Ma,Jie Qiao,Yi Liu
Herein, we report a method that combined “aptamer-locker” DNA with CRISPR/Cas12a-based biosensing for sensitive and rapid melamine analysis. Three strategies were harnessed for designing the DNA sensors that were well characterized by circular dichroism (CD) spectroscopy and isothermal titration calorimetry (ITC) in the absence and presence of melamine. The detection parameters were optimized to achieve good analytic performance. As a result, a limit of detection (LOD) as low as 38 nM was achieved, which is below the threshold (1.0 mg/kg) of allowable melamine in infant milk products. In addition, the sensors show high selectivity for melamine against other analogues such as cyanuric acid , ammeline and ammelide . Moreover, our method was effective for rapid melamine analysis in whole milk samples, with or without sample pretreatment , in less than 20 min. Adopting a commercially available portable fluorimeter, on-site analysis of melamine in milk was accomplished. The strategies demonstrated here can expand to detect other non-nucleic-acid targets by simply replacing the aptamers.
研究领域:
分析化学
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分子生物学
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生物技术
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食品安全
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PubMed
高影响因子
ANALYST
·
2024
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IF 3.6
Chengchen Tan,Xiaolong Yan,Xingchang Lu,Jianxiu Wang,Xinyao Yi
Breast cancer, as a malignant tumor with the highest incidence in the world, poses a great threat to human life and health. Early diagnosis of breast cancer may help enhancing the survival rate. In this work, a colorimetric and fluorescent dual-mode biosensor based on CRISPR-Cas12a system was constructed for the detection of breast cancer biomarker BRCA1. The intact G4 DNA could form DNAzyme in the assistance of K+ and hemin, catalyzed the oxidation of o-phenylenediamine (OPD) under the assistance of hydrogen peroxide (H2O2), and generate the oxidation product 2,3-diaminophenazine (DAP) with obvious absorption and fluorescence peaks. The presence of the target BRCA1 can activate the trans-cleavage activity of CRISPR-Cas12a, cleaving the G4 DNA and inhibiting the catalytic oxidation of OPD. The target BRCA1 was quantitatively determined by detecting both the absorbance and fluorescence intensity of DAP. Compared with single signal analysis, dual-signal output mode provided more reliable detection results. The detection limits were calculated to be 0.615 nM for colorimetric method and 0.289 nM for fluorescence method. The dual-mode biosensor showed good selectivity for BRCA1 and can resist the interference of complex substrates, which has great potential in biomedical detection.
研究领域:
生物医学工程
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分子生物学
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癌症研究
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生物技术
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高影响因子
MICROCHIMICA ACTA
·
2025
·Vol. 192
·期 6
·1-12
·
IF 5.3
Chen Yafei,Zhang Xuhui,Huang Wanjin,Jin Zhenhuan,Wei Xiaoping,Li Jianping
Atrazine (ATZ) is used extensively, resulting in residues in food and the environment, posing a serious threat to human health. Herein, Cd 0.5 Zn 0.5 S/Ti 3 C 2 photoelectric material was synthesized and immobilized on a FTO electrode as a photoanode. A photoelectrochemical (PEC) aptamer sensor was constructed for the highly sensitive and selective determination of ATZ based on signal amplification via cascade catalysis of CRISPR/Cas12a and G-quadruplex/hemin DNAzyme (G4/hemin DNAzyme). G4/hemin DNAzyme catalyses the oxidation reaction between H 2 O 2 and dopamine (DA) to form polydopamine (PDA) deposit. This process, in turn, inhibits the photocurrent at the photoanode, leading to a decrease in photocurrent. Concurrently, the depletion of DA as an electron donor for the PEC reaction at the photoelectrode further contributes to the decrease in photocurrent. ATZ can hybridize with ATZ aptamer (Apt) in Apt/cDNA to release activation strand (cDNA), which activates the activity of CRISPR/Cas12a and triggers cleavage of G4, causing the cleaving of G4/hemin DNAzyme immobilized on the electrode surface. This process leads to a decrease of G4/hemin DNAzymes amount on the electrode, consequently reducing both the PDA generation and the DA consumption. As a result, the photocurrent is restored. The cascade catalysis of CRISPR/Cas12a and G4/hemin DNAzyme has been demonstrated to result in photocurrent amplification. The photocurrent change was linear with the logarithmic value of ATZ concentration in the range 1.00 × 10 –12 to 1.00 × 10 –5 mol/L. The limit of detection was 3.47 × 10 –13 mol/L. The sensor has been successfully applied to the determination of trace ATZ in environmental and food samples. Graphical abstract
研究领域:
环境科学
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分析化学
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分子生物学
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PubMed
高影响因子
ANALYTICA CHIMICA ACTA
·
2023
·
IF 6.2
Yuanyuan Yue,Songtao Wang,Qiang Jin,Nan An,Lina Wu,He Huang
Lead ions (Pb 2+ ) are a well-known toxic heavy metal that poses a significant threat to human health. Therefore, the development of a simple and ultrasensitive technique for detecting Pb 2+ is essential. With their trans -cleavage properties, the newly discovered CRISPR-V effectors have become a potential high-precision biometric tool. In this regard, a CRISPR/Cas12a-based electrochemical biosensor (E-CRISPR) has been developed, which is combined with the GR-5 DNAzyme that can specifically recognize Pb 2+ . In this strategy, the GR-5 DNAzyme acts as a signal-mediated intermediary, which can convert Pb 2+ into nucleic acid signals, thereby becoming single-stranded DNA that triggers strand displacement amplification (SDA) reaction. This is coupled with following activated CRISPR/Cas12a cleavage of the electrochemical signal probe, enabling cooperative signal amplification for ultrasensitive Pb 2+ detection. The proposed method has a detection limit as low as 0.02 pM. Therefore, we have developed an E-CRISPR detection platform with GR-5 DNAzyme as a signal medium (called SM-E-CRISPR biosensor). This provides a method for the CRISPR system to specifically detect non-nucleic substances by converting the signal using a medium.
研究领域:
生物化学
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分子生物学
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分析化学
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环境科学
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高影响因子
Frontiers in Microbiology
·
2025
·
IF 4.5
Yayun Jiang,Zongyao Chen,Xiao Liu,Qi Xin,Dengchao Wang,Caixia Ji,Youwei Li,Gang Mai
Methicillin-resistant Staphylococcus aureus (MRSA), a widely drug-resistant bacterium, poses a significant threat to global health. Current culturing and nucleic acid detection methods are time-consuming and require complex instruments, which do not meet the detection needs. Herein, we developed a rapid and visual MRSA detection method (MCFHCR) using ssDNA-functionalized magnetic beads as a trigger chain combined with the trans-cleavage activity of the Cas12a protein and fluorescence signal amplification of the hybridization chain reaction (HCR). MCFHCR is a signal-off platform for the detection of MRSA. In the absence of DNA targets, the trans-cleavage activity of Cas12a is inactivated, allowing HCR to proceed and form long double-stranded DNA, resulting in an increased fluorescent signal. In the presence of the DNA targets, the trans-cleavage activity of CRISPR/Cas12a is activated to cleave the trigger strand, failing HCR and leading to a decrease in the fluorescence signal. Combined with RPA, MCFHCR was completed within 35 min, achieving a limit of detection (LOD) of five copies/μL for mecA DNA and 8 CFU/mL for MRSA. In detecting clinical strains, MCFHCR demonstrated comparable performance to qPCR and drug sensitivity testing. Therefore, with its simple, rapid operation and convenient signal acquisition, MCFHCR shows significant practical applicability in detecting MRSA.
研究领域:
微生物学
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分子诊断学
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CRISPR技术
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传染病检测
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生物传感器
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JOURNAL OF AGRICULTURAL AND FOOD CHEMISTRY
·
2023
·
IF 6.1
Xiaobo Zhang,Zhiru Li,Lili Yang,Bing Hu,Qiuyue Zheng,Jiang Man,Jijuan Cao
The sensitive and accurate detection of ochratoxin A (OTA) is crucial for public health due to its high toxicity. Herein, using Au nanoparticle (NP)-attached CdS/UiO-66-NH2 heterostructures as photoactive materials, a photoelectrochemical (PEC) aptasensor was presented for the ultrasensitive assay of OTA based on a competitive displacement reaction triggering the trans-cleavage ability of CRISPR/Cas12a. In this sensing strategy, methylene blue-labeled single-stranded DNA (MB-ssDNA) was immobilized on the Au NPs/CdS/UiO-66-NH2 electrode to accelerate the separation of the photogenerated carrier, thus producing a significantly increased PEC response. In the presence of OTA, it specifically bound with the aptamer (Apt) and resulted in the release of the activation chain, triggering the trans-cleavage characteristics of CRISPR/Cas12a. MB-ssDNA was cut randomly on the electrode surface to convert the PEC signal from the “on” to the “off” state, thereby achieving a quantitative and accurate detection of OTA. The CRISPR/Cas12a-derived PEC aptasensor exhibited excellent sensitivity and specificity, with a linear range from 100 to 50 ng/mL and a detection limit of 38 fg/mL. Overall, the proposed aptasensor could provide a rapid, accurate, and sensitive method for the determination of OTA in actual samples.
研究领域:
食品安全检测
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分子生物学
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分析化学
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PubMed
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MICROCHEMICAL JOURNAL
·
2024
·
IF 4.9
Xuan Wu,Ning-Ning Pi,Fei Deng,Shi-Yang Tang,Cheng-Chen Zhang,Lu Zhu,Fang-Fang Sun,Xiao-Yao He,Han-Qing Li,Shi-Long Zhao,Rong Xiang,Yi Li
Periodontitis not only leads to tooth loss but is also associated with various systemic symptoms. Porphyromonas gingivalis ( Pg ) is the microorganism most closely linked to periodontitis. A rapid and accurate Pg detection method holds significant potential health benefits for patients. While several nucleic acid amplification-based diagnostic methods have been developed, there is a current lack of rapid diagnostic tools for Pg , particularly at chair-side level. Here, two detection methods for the Pg 16S gene based on the CRISPR/Cas12a system have been developed. To achieve ultra-high sensitivity in the developed CRISPR/Cas12a assays without pre-amplification, we designed a circular DNA molecule (termed as Cir-amplifier) which amplifies the signal output of the Cas12a reactions in a cascade response manner. The Cir-amplifier enhanced Fluorescence-Cas12a reaction achieved a minimum detectable limit of 0.39 CFU of Pg within 80-minute under room-temperature. In addition, the Cir-amplifier enhanced Lateral Flow Detection (LFD)-Cas12a reaction has demonstrated the ability to detect a minimum of 3.9 CFU of Pg in 90 min, with results observable by the naked eye. The potential of the two methods for Pg detection in clinical samples has been demonstrated, showing 100 % sensitivity compared to the traditional real-time PCR method. In summary, we have established two efficient detection methods for Pg that eliminate the need for pre-amplification, hence no amplicon contaminations, making them promising candidates for chair-side Pg diagnosis. Furthermore, the Cir-amplifier has been demonstrated to be adaptable to CRISPR/Cas systems producing various signal formats. The Cir-amplifier is expected to become a universal CRISPR/Cas enhancer.
研究领域:
分子生物学
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微生物学
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诊断学
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生物技术
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DOI
高影响因子
ANALYTICAL CHEMISTRY
·
2023
·
IF 7.4
Guobin Mao,Xing Luo,Silu Ye,Xun Wang,Jin He,Jilie Kong,Junbiao Dai,Wen Yin,Yingxin Ma
It is well-established that different detection modes are necessary for corresponding applications, which can effectively reduce matrix interference and improve the detection accuracy. Here, we reported a magnetic separation method based on recombinase polymerase amplification (RPA)-assisted clustered regularly interspaced short palindromic repeats (CRISPR)/Cas12a for dual-mode analysis of African swine fever virus (ASFV) genes, including colorimetry and fluorescence. The ASFV gene was selected as the initial RPA template to generate the amplicon. The RPA amplicon was then recognized by CRISPR-associated RNA (crRNA), activating the trans-cleavage activity of Cas12a and leading to the nonspecific cleavage of ssDNA as well as a significant release of alkaline phosphatase (ALP) in the ALP-ssDNA modified magnetic bead. The released ALP can catalyze para-nitrophenyl phosphate to generate para-nitrophenol, resulting in substantial changes in absorbance and fluorescence, both of which can be used for detection with the naked eye. This strategy allows the sensitive detection of ASFV DNA, with a 20 copies/mL detection limit; no cross-reactivity with other viruses was observed. A good linear relationship was obtained in serum. In addition, this sensor displayed 100% specificity and sensitivity for clinical sample analysis. This method integrates the high sensitivity of fluorescence with easy readout of colorimetry and enables a simple, low-cost, and highly sensitive dual-mode detection of viral nucleic acid, thereby providing a broad prospect for the practical application in the diagnosis of virus infection.
研究领域:
病毒学
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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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DOI
PubMed
高影响因子
TALANTA
·
2022
·
IF 6.556
Biyun Fang,Zhenzhen Jia,Cui Liu,Kangsheng Tu,Mingzhen Zhang,Lei Zhang
Pathogen nucleic acid detection is of great significance to control the spread of diseases caused by the viruses. Nevertheless, traditional methods for nucleic acid detection such as polymerase chain reaction (PCR) and oligonucleotide microarrays require bulky instruments, which restrain their point-of-care (POC) testing application. Here, we proposed a POC method enabling sensitive detection of pathogen nucleic acids by combining the clustered regularly interspaced short palindromic repeat (CRISPR) Cas12a-based assay and personal glucometer readout (PGM). The quantification of target pathogen DNA by PGM was achieved based on pathogen DNA activates Cas12a ssDNase to cleave magnetic bead-DNA-invertase reporter probe, and separated free invertase to catalyze hydrolysis of sucrose to glucose. Without using nucleic acid amplification technology, we demonstrated here dual signal amplifications based on Cas12a and invertase-mediated catalytic reactions, making it possible to sensitively detect HIV-related DNA or SARS-CoV-2 pseudovirus with the limits of detection of 11.0 fM and 50 copies/μL, respectively. This strategy also showed excellent selectivity as well as potential applicability for detection of HIV in human serum samples or of SARS-CoV-2 in saliva samples. Therefore, our CRISPR-PGM-based dual signal amplifications detection platform might offer a great promise in POC diagnosis of pathogen nucleic acids.
研究领域:
分子生物学
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医学诊断
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生物技术
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DOI
PubMed
高影响因子
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