ANALYTICA CHIMICA ACTA
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2025
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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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高影响因子
Current Research in Food Science
·
2025
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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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PubMed
高影响因子
TALANTA
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2022
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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
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2023
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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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ANALYTICA CHIMICA ACTA
·
2026
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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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SENSORS AND ACTUATORS B-CHEMICAL
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2022
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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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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 .
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CHEMICAL ENGINEERING JOURNAL
·
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.
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SENSORS AND ACTUATORS B-CHEMICAL
·
2025
·
IF 7.7
Decai Yuan,Cheng Qiu,Hui Chen,Feng Liu,Shanshan Feng,Peiyi Zhang,Ying Qin,Tingting Fan,Yan Chen,Yuyang Jiang
Colorectal cancer (CRC) represents a significant global health issue, necessitating innovative approaches for early screening and diagnosis. Recent advances in molecular diagnostics have highlighted the potential of aptamers for use as highly specific and sensitive probes for detecting cancer biomarkers. In this study, we focus on the identification of aptamers that selectively bind to Solobacterium moorei (S. moorei) , a bacterium associated with CRC. By integrating these aptamers into electrochemical sensor platforms, a reliable diagnostic tool is created for facile implementation in the clinical setting. More specifically, nucleic acid aptamers for S. moorei were obtained through whole-cell SELEX, and the affinity and specificity of these aptamers were validated. Subsequently, two types of electrochemical sensors were developed. Firstly, an electrochemical impedance spectroscopy sensor was developed to detect impedance changes on the electrode surface, which were caused by the binding of S. moorei to the aptamer. Secondly, a CRISPR/Cas12a-based electrochemical aptasensor was developed based on the ability of the Cas12a enzyme to be activated by specific single-stranded DNA, triggering its trans-cleavage activity. The limits of detection of these sensors for S. moorei were 30 and 6 CFU/mL, respectively. Clinical validation was performed using patient samples to assess the sensor efficacy in a real-world setting. The obtained results suggested that the abundance of S. moorei in the feces of CRC patients was significantly greater compared to that of healthy individuals. This integration of S. moorei aptamers into electrochemical sensors offers a non-invasive and cost-effective alternative to current screening methods available for CRC.
研究领域:
分子诊断学
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生物传感技术
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肿瘤学
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高影响因子
TALANTA
·
2025
·
IF 6.1
Tao Meng,Qi Kang,Jiashuo Xu,Shuang Zhao,Tianqi Liu,Dianming Zhou,Xiaoqun Gong,Jianyu Zhang
The CRISPR/Cas12a system has become a powerful tool in biosensing because of its specific target recognition ability and highly efficient trans -cleavage activity. However, a problem faced by the CRISPR/Cas12a system when directly used for trace detection is the linear amplification efficiency of single-cycle digestion. Here, we present a novel hairpin reporter-driven CRISPR/Cas12a (HR-CRISPR) amplification system that establishes a positive feedback loop within the CRISPR/Cas12a platform to finish an exponential and sensitive signal amplification in a one-step reaction. As proof of concept, we applied this strategy to the terminal deoxynucleotidyl transferase (TdT) activity assay without pre-amplification procedure. The polyT strand extended by TdT hybridizes with crRNA, activating Cas12a, which then cleaves the FQ-hairpin reporter. The cleavage products are further elongated by reverse transcriptase using crRNA as a template, reactivating Cas12a and producing exponentially amplified fluorescence signals. This assay offers a simple yet highly sensitive approach for quantifying TdT activity, achieving a low detection limit of 4.55 × 10 −6 U. Moreover, it is applicable for inhibitor screening and monitoring TdT activity in human serum samples.
研究领域:
分子生物学
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生物技术
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医学检测
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高影响因子
BIOSENSORS & BIOELECTRONICS
·
2026
·
IF 11.8
Mengqing Cheng,Xinyue Chen,Hongrui Cheng,Xianglin Gao,Haoyue Ao,Xinyang Bao,Xiaojun Song,Yulei Tai,Dazhi Jin,Lei Zhang
Clostridioides difficile ( C. difficile ) infection (CDI) represents a formidable global healthcare challenge, necessitating the development of rapid, accurate, and cost-effective diagnostic platforms to mitigate nosocomial transmission and improve patient outcomes. Compared with the conventional methods, CRISPR/Cas systems featured by specific target reorganization by a single chain RNA, coupled with electrochemical technology enables highly sensitive detection of various biomarkers. However, their application to CDI has remained unexplored due to the lack of tailored crRNAs. Herein, we present the integration of CRISPR/Cas12a with electrochemical transduction for the direct detection of C. difficile . A novel crRNA was engineered to specifically recognize the toxin B gene ( tcdB ), activating the trans-cleavage activity of Cas12a upon target binding. This cascade triggers the cleavage of immobilized ssDNA reporters on the electrode surface, generating measurable amperometric signal changes. The developed biosensor demonstrates exceptional performance, achieving a detection limit of pM level for tcdB DNA within 40 min, while exhibiting high specificity against non-target pathogens and robust stability over 7 days. This work establishes a rapid and reliable CRISPR-electrochemical diagnostic platform, offering significant potential for point-of-care CDI management.
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