ANALYTICAL AND BIOANALYTICAL CHEMISTRY
·
2024
·
IF 3.8
Zhang Runze,Wang Yuzhu,Wang Xiaoxu,Ren Honglin,Du Junzheng,Yang Yongjie,Hu Xueyu,Shi Ruoran,Zhang Bo,Li Chengwei,Lu Shiying,Li Yansong,Liu Zengshan,Hu Pan
Listeria monocytogenes ( L. monocytogenes ) is a prevalent food-borne pathogen that can cause listeriosis, which manifests as meningitis and other symptoms, potentially leading to fatal outcomes in severe cases. In this study, we developed an aptasensor utilizing carboxylated magnetic beads and Cas12a to detect L. monocytogenes . In the absence of L. monocytogenes , the aptamer maintains its spatial configuration, keeping the double-stranded DNA attached and preventing the release of a startup template and activation of Cas12a’s trans-cleavage capability. Conversely, in the presence of L. monocytogenes , the aptamer undergoes a conformational change, releasing the double-stranded DNA to serve as a startup template, thereby activating the trans-cleavage capability of Cas12a. Consequently, as the concentration of L. monocytogenes increases, the observable brightness in a blue light gel cutter intensifies, leading to a rise in fluorescence intensity difference compared to the control. This Cas12a aptasensor demonstrates excellent sensitivity towards L. monocytogenes , with a lowest detection limit (LOD) of 57.15 CFU/mL and a linear range of 4×10 2 to 4×10 7 CFU/mL ( R 2 =0.9858). Notably, the proposed Cas12a aptasensor exhibited outstanding selectivity and recovery in beef samples, and could be employed for precise monitoring. This Cas12a aptasensor not only provides a novel fluorescent and visual rapid detection method for L. monocytogenes but also offers simplicity, speed, and stability compared to previous detection methods. Furthermore, it is suitable for on-site detection of beef samples. Graphical Abstract
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高影响因子
SENSORS AND ACTUATORS B-CHEMICAL
·
2020
·
IF 7.1
Lei Peng,Jin Zhou,Guozhen Liu,Lijuan Yin,Siyu Ren,Shuli Man,Long Ma
CRISPR-Cas based detection platform opens up a new avenue for biosensing owing to its programmability , sequence specificity and high base resolution upon nucleic acid targets. In current study, we utilised the target DNA-induced non-specific single-stranded DNA cutting activity ( trans -cleavage) of CRISPR-Cas12a to fabricate an aptamer mediated fluorescent biosensor for sensitive and selective detection of adenosine triphosphate (ATP). In this sensor, we designed an ATP-binding aptamer as the target ssDNA for Cas12a, thereby in the presence of ATP, the ATP-binding aptamer would be occupied by ATP, and less target ssDNA can be processed by Cas12a, which gave rise to an ATP concentration dependent change in fluorescence resulted from trans -cleavage of doubly labeled ssDNA reporter. In this way, we successfully “translated” ATP signals to nucleic acid signals that can be amplified by Cas12a process. The dynamic detection range was from 1 μM to 200 μM and the limit of detection was 400 nM. The entire sample-to-answer time for this biosensor was around 40 min. Overall, this novel biosensor balanced sensitivity, specificity, detection time and ease of use. Our proposed biosensor provided a principle-of-proof for detecting small molecule by using CRISPR-Cas12a system, which would unlock its potential and further its futuristic applications in the field of diagnostics.
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TALANTA
·
2022
·
IF 6.556
Zefeng Mao,Xiaojuan Wang,Ruipeng Chen,Zixuan Zhou,Shuyue Ren,Jun Liang,Zhixian Gao
The CRISPR-Cas system has achieved breakthrough applications in the field of molecular diagnostics. CRISPR/Cas12a can accurately identify subtle changes in a target nucleic acid sequence and has a wide range of applications such as in highly sensitive detection methods. In this study, an upconversion-magnetic probe-DNA-Fe 3 O 4 probe was designed to replace traditional fluorescent probes using nucleic acid aptamers to design a biosensing method powered by CRISPR/Cas12a. The CRISPR/Cas12a technology can be widely used for sensitive, rapid, and stable detection of ochratoxin A. In our sensing strategy, the ochratoxin A capture probe was used to capture ochratoxin A and release the Cas protein activation chain, further triggering the single-stranded DNA degradation activity of CRISPR/Cas12a and removal of the fluorescent probe to generate a fluorescent signal. Compared with traditional commercial kits, our method was more rapid and exhibited comparable detection capability.
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MICROCHEMICAL JOURNAL
·
2023
·
IF 4.8
Si Chen,Rong Fang,Yi Li,Fei Deng,Xing Liu,Danting Yang
Ochratoxin A (OTA) is classified as a Class 2B carcinogens and poses a significant risk bto the liver, kidney and other organs, potentially leading to carcinogenic and teratogenic effects. Accurate monitoring of OTA content in food is crucial for safeguarding human and animal health. Here, a novel approach called the C RISPR/Cas12a enzyme- l inked a ptamer- s orbent a ssay (CLASA) was developed, which integrates the high-performance trans -cleavage ability of CRISPR/Cas12a with the cost-effective and high-affinity properties of aptamer for ultra-sensitive OTA detection. In this strategy, we incubated aptamers on microwell plates, and used the fluorescence intensity changes caused by OTA and crRNA competitively bind OTA-specific aptamer to achieve the quantitative detection. Our sensitive CLASA method not only solves the problems of poor repeatability and relatively low sensitivity of fluorescence based-CRISPR/Cas12a reaction in solution, but also solves the defects of expensive antibody and false positive result in traditional ELISA method, achieving a limit of detection (LOD) as low as 171 fg·mL −1 with a broad linear range from 1 pg·mL −1 to 1000 pg·mL −1 . The presented CLASA approach was validated successfully by using commercial ELISA kit on rice and oat samples as well. Furthermore, by strategically modifying the sequences of aptamer and crRNA, our CLASA platform can be flexibly customized to detect a wide range of small molecular targets. This adaptability allows for the extension of our framework beyond nucleic acid substances, enabling the detection of non-nucleic acid compounds using the CRISPR-Cas12a system. As a result, our CLASA approach holds great promise as a versatile sensing kit with applications in various fields requiring sensitive and specific detection of diverse analytes.
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ANALYTICAL CHEMISTRY
·
2025
·
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.
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ACS Synthetic Biology
·
2023
·
IF 4.7
Yanqiang Liu,Ziwen Zhang,Ni Zuo,Weihong Jiang,Yang Gu
Protein lysine acetylation (PLA) is a crucial post-translational modification in organisms that regulates a variety of metabolic and physiological activities. Many advances have been made in PLA-related research; however, the quick and accurate identification of causal relationships between specific protein acetylation events and phenotypic outcomes at the proteome level remains challenging due to the lack of efficient targeted modification techniques. In this study, based on the characteristics of transcription-translation coupling in bacteria, we designed and constructed an in situ targeted protein acetylation (TPA) system integrating the dCas12a protein, guiding element crRNA, and bacterial acetylase At2. Rapid identification of multiple independent protein acetylation and cell phenotypic analyses in Gram-negative Escherichia coli and Gram-positive Clostridium ljungdahlii demonstrated that TPA is a specific and efficient targeting tool for protein modification studies and engineering.
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ANALYTICA CHIMICA ACTA
·
2026
·
IF 6.1
Songbai Tian,Xin Tang,Saijun Zhang,Yuexin Yao,Ran Bi,Haixia Li,Xiaoming Sun,Zhuo Chen
Digital immunoassay allows for the detection of proteins at fg/mL levels by leveraging the principles of Poisson distribution. Nevertheless, the inherent Poisson noise restricts digital sensitivity to targets exceeding 100 molecules, rendering standard digital assays incapable of detecting targets below this threshold. To overcome this constraint, we introduce an integrated CRISPR/Cas12a-digital immunoassay technology. In this system, the CRISPR/Cas12a machinery first acts as a molecular amplifier, converting each target protein into multiple enzymatic reporters. These enzyme molecules are then individually quantified via a digitized readout system, enabling ultrasensitive protein detection. By harnessing the catalytic amplification of CRISPR/Cas12a, the method achieves detection of proteins at copy numbers below 100, effectively surpassing the conventional sensitivity barrier of digital immunoassays. We validated this approach through highly selective and accurate detection of CD44 protein. A linear response was observed across a concentration range of 0.05 to 5 fg/mL, conforming to the calibration model: P(X > 0) = 0.1191c + 0.0036. The limit of detection was determined to be 0.018 fg/mL, equivalent to approximately 36 molecules of CD44. The method was further applied to quantify CD44 in plasma samples from colorectal cancer patients, demonstrating its strong potential for clinical use in early cancer diagnosis and treatment monitoring.
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TALANTA
·
2026
·
IF 6.7
Luqin Lv,Yizhuo Zhang,Yaxin Fan,Beining Guo,Yijian Chen
Therapeutic drug monitoring (TDM) of vancomycin (VAN) is critical for maximizing efficacy and minimizing toxicity, but conventional methods are constrained by high costs, slow turnaround times, and operational complexity. To address these limitations, we developed a novel Bivalent Aptamer-assisted CRISPR-Cas12a Sensor (termed BACS) for rapid and precise VAN detection. Central to this platform is a high-affinity bivalent aptamer (2AP33), engineered via molecular docking-guided truncation and rational linker design, which exhibits significantly enhanced binding avidity compared to its monovalent counterpart. This aptamer was integrated into a CRISPR-Cas12a system based on a competitive binding mechanism, where target binding modulates Cas12a trans-cleavage activity. The optimized BACS achieved a wide linear detection range (1-50 μM) with a low limit of detection (0.64 μM) in clinical serum, fully covering the clinical therapeutic window. Notably, the assay is rapid (within 10 min), cost-effective, and simple. Critically, the clinical practicality and reliability of BACS were rigorously validated with 175 clinical serum samples, showing exceptional concordance with both the gold standard method and a classical method. This work not only provides a reliable tool for VAN TDM but also offers an adaptable strategy for developing high-performance CRISPR-powered biosensors for diverse clinical analytes through a streamlined molecular engineering pipeline.
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NUCLEIC ACIDS RESEARCH
·
2025
·Vol. 53
·期 21
·
IF 13.1
Xiao Yao,Zhao Rujian,Bao Yin,Lu Baiyang,Jiang Yanfang,Tang Yidan,Li Bingling
The Cas12a–crRNA system possesses inherent sequence-specific recognition of double-stranded DNA/single-stranded DNA (dsDNA/ssDNA) coupled with trans-cleavage activity toward ssDNA, making it a powerful tool for nucleic acid diagnostics. However, its application beyond nucleic acid targets remains challenging, limiting its potential as a universal detection platform. In this study, we systematically explore the key parameters governing the activation of a Cas12a-split crRNA system and established a comprehensive set of design guidelines. Building on these findings, we developed CASCADE (Cas12a-Assisted Split crRNA Complex for Analysis and Detection of Diverse Entities), an adaptable detection platform that extends Cas12a’s application beyond nucleic acids. Using microRNA as a model, we validated the system’s sensitivity, specificity, and mismatch discrimination capability. Additionally, we successfully demonstrated its capability for non-nucleic acid target detection by detecting tobramycin, kanamycin, biotin, and tetracycline repressor protein, confirming its sensitivity and specificity. Finally, by integrating a lateral flow assay (LFA), we enhanced the portability of CASCADE, enabling user-friendly, on-site detection. This work expands the application scope of the Cas12a system and offers a promising strategy for point-of-care diagnostics or environmental monitoring.
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SENSORS AND ACTUATORS B-CHEMICAL
·
2022
·
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 .
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SENSORS AND ACTUATORS B-CHEMICAL
·
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.
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CHEMICAL ENGINEERING JOURNAL
·
2023
·
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.
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