TALANTA
·
2024
·
IF 5.6
Yu Tao,Haixia Wang,Enguo Ju,Yeh-Hsing Lao,Yu Zhang,Mingqiang Li
Diseases caused by viruses, such as monkeypox virus (MPXV) and human papillomavirus (HPV), pose serious threats to human health and safety. Although numerous strategies have been constructed for detecting MPXV and HPV DNA, most methods require either laborious procedures or complicated instruments involving skilled professionals. In this research, a CRISPR-Cas12a-mediated colorimetric detection platform for MPXV and HPV sensing was constructed for the first time by applying probe DNA to reprogram the catalytic properties of molybdenum disulfide quantum dots (MoS 2 QDs). In the presence of MPXV or HPV targets, the CRISPR-Cas12a trans -cleavage activity is effectively motivated to decompose the probe DNA, leading to the suppression of enzymatic activity DNA enhancer adsorbed on MoS 2 QDs, resulting in greatly decreased catalytic behaviors. The MoS 2 QDs-DNA nanohybrids displayed prominent specificity and sensitivity, with detection limits at subpicomolar levels, as well as excellent stability and accuracy for determining MPXV and HPV DNA in human sera biosamples. Furthermore, the proposed colorimetric biosensing approach can be ensembled with a smartphone platform, allowing visible analysis of DNA targets. Taken together, this colorimetric strategy offers a novel diagnosis method for MPXV and HPV DNA detection, particularly favorable for highly endemic developing countries with restricted medical and instrumental support.
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SENSORS AND ACTUATORS B-CHEMICAL
·
2025
·
IF 7.7
Kai Shi,Jiaxuan Chen,Ying Cheng,Jiuhua Song,Yang Li,Xin Cheng,Xiaofeng Bai,Jiali Chang,Tao Jiang
Microplastic (MP) pollution has attracted worldwide attention due to its harmful effects on human health and ecosystems. Herein, we develop a novel label-free electrochemical biosensor for sensitive and selective detection of MP based on split gRNA with CRISPR/Cas12a (SCas12a)-mediated cascade strand displacement and an aptamer (termed as CRISPR-MP). Within the CRISPR-MP system, the target polyvinyl chloride (PVC)/polystyrene (PS) triggers the SCas12a-mediated cascade strand displacement, and then activates the DNase activity of SCas12a. Upon cleaving the DNA probe of the G-quadruplex/hemin complex on the gold electrode, the active Cas12a triggers a decrease in the electrochemical signal.This electrochemical aptasensor shows a detection limit of 37 ng/mL of PVC and 45 ng/mL of PS, respectively. The developed CRISPR-MP system expands the utility of the CRISPR for MP detection and exhibits significant promise in protecting human health and monitoring the environment.
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TALANTA
·
2025
·
IF 6.1
Xiang Li,Yuting Zhang,Mengyuan He,Jiarui Sun,Bowen Xiong,Gongke Wang
In this work, we present an ultrasensitive, specific, and high-signal-to-background ratio fluorescence split-aptasensor for D-vasopressin (D-VP) detection. This sensor is based on target-induced self-propelled 3D DNA walkers in conjunction with CRISPR-Cas12a technology. Two split probes (SDA 1 and SDA 2) were designed to undergo structural recombination and function as a walking chain (SDA) under the induction of D-VP. Simultaneously, an intact Mg 2+ -dependent DNAzyme domain was formed at the tail of SDA and subsequently activated. The activated Mg 2+ -dependent DNAzyme continuously propelled the 3D DNA walker, enabling the generation of signal strand DNA (activator DNA). The activator DNA can subsequently trigger the activation of the Cas12a protein, enabling it to cleave the FAM-ssDNA-BHQ1 substrate. This process leads to signal amplification and the specific detection of D-VP. Under optimal conditions, the designed split-aptasensor exhibits excellent linearity across a concentration range of 5 ng/mL to 1215 ng/mL, with a detection limit (LOD) as low as 0.22 ng/mL. This split-aptasensor were employed to identify D-VP in human serum and urine samples, yielding highly satisfactory results. This unique design acts as a proof of concept and illustrates considerable promise for the detection of a wide range of analytes.
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INTERNATIONAL JOURNAL OF BIOLOGICAL MACROMOLECULES
·
2026
·
IF 8.5
Jingwen Zhang,Biru Han,Xiru Zhang,Xinna Xie,Feng Zhao,Wei Zhang,Yujun Jiang,Xianlong Zhang
The efficient and sensitive detection of mycotoxins is critical to ensure food safety and maintain public health worldwide. In this study, a CRISPR/Cas12a-mediated marker-free fluorescent biosensor based on an automated 3D DNA walker-enabled ‘one-to-many’ signal amplification was developed for sensitive detection of aflatoxin B1. Due to the efficient amplification effect of DNA walkers and the strong fluorescence properties of silver nanoclusters, a sensitive output of the amplified signal was produced by precisely regulating the activated trans-cleavage activity of the specific target DNA of Cas12a. In the established fluorescence biosensor, a small amount of aflatoxin B1 promoted the production of a large amount of activator by the established 3D DNA walker, which stimulated the trans-cleavage activity of Cas12a to degrade the DNA single strands for synthesis of silver nanoclusters, leading to a decreased fluorescent signal. The established biosensor was able to achieve the sensitive detection of aflatoxin B1 under optimal conditions, obtaining a detection limit of 45.38 pg/mL in a linear range of 0.05 to 10 ng/mL. In addition, the developed biosensor showed good recoveries in spiked food samples (peanut milk and drinking water) at different concentrations. This work provided new insights for the applications of DNA walker and the development of a marker-free fluorescent biosensing platform based on CRISPR/Cas for the detection of mycotoxins.
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CHEMICAL ENGINEERING JOURNAL
·
2026
·
IF 13.2
Lijuan Yin,Liangyuan Wang,Lu Yin,Lingzhi Yang,Baoxue Gao,Shuli Man,Fufeng Liu,Long Ma
Norovirus (NoV) represents a leading cause of foodborne illness worldwide and is the predominant agent of non-bacterial acute gastroenteritis. The development of rapid, highly sensitive, specific, and point-of-care detection methods is critical for the protection of public health and food safety. In this study, we developed a lyophilized paper-based microfluidic analytical device (μPAD) that integrates reverse transcription recombinase polymerase amplification (RT-RPA) with CRISPR/Cas12a technology. By incorporating a smartphone-assisted portable fluorescence detection device, the platform achieves a high level of integration, simplicity, and intelligence. It demonstrates a detection limit for NoV as low as 1 copy/μL within 45 min, with no cross-reactivity against other common microorganisms or the human genome. The fluorescence intensity on the chips exhibits a strong linear relationship with NoV RNA concentration, enabling accurate quantification. Moreover, the lyophilized RPA-CRISPR paper chips retain stability for up to 8 weeks at room temperature, underscoring their suitability for point-of-care applications. When applied to real food and environmental samples for NoV detection, the platform showed 100% concordance with RT-qPCR and maintained a detection sensitivity of 1 copy/μL. This work provides a robust approach for rapid, sensitive, and on-site detection of norovirus, and the platform architecture holds promise for broader application in detecting other microorganisms.
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MICROCHIMICA ACTA
·
2026
·Vol. 193
·期 8
·525
·
IF 5.4
Tang Yafang,Zhang Liwen,Wang Wenjing,Yang Xing
A novel biosensing platform is proposed that integrates catalytic hairpin assembly (CHA) and hybridization chain reaction (HCR) cascade isothermal amplification with the CRISPR/Cas12a system, enabling ultrasensitive detection of microRNAs (miRNAs) targets. Within this platform, two modules are integrated: a target recognition and signal amplification module constructed by the cascade of CHA and HCR, and a signal transduction module in which the CRISPR/Cas12a system acts in concert with DNA probes loaded onto gold nanoparticles (AuNPs). This design achieves cascaded amplification from target recognition to signal output, thereby conferring high signal gain. Experimental results demonstrate that the proposed biosensor had high sensitivity toward the target miRNA, with a detection limit as low as 37 fM. Moreover, it shows remarkable single-base discrimination capability, effectively distinguishing sequences with single-nucleotide mismatches. Notably, the sensor maintains stable and reliable performance in complex biological matrices, including serum samples and lysates from various tumor cells. This strategy effectively couples signal amplification with the CRISPR system, achieving both high sensitivity and specificity, making it a useful tool for miRNA detection and early cancer screening. Graphical
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SENSORS AND ACTUATORS B-CHEMICAL
·
2022
·
IF 9.221
Minghui Wang,Yuxin Lin,Shuai Wu,Ying Deng,Yuanyuan Zhang,Jie Yang,Genxi Li
Programmed death-ligand 1 protein positive (PD-L1 + ) exosome is one of the most prospective biomarkers for the diagnosis of cancer. However, the current methods for the detection of PD-L1 + exosomes are usually laborious, time-consuming and require numerous samples. Herein, we report in this paper an electrochemical biosensor for PD-L1 + exosomes detection based on ultra-thin two-dimensional covalent organic framework nanosheets (2D COF NSs) coupled with clustered regularly interspaced short palindromic repeats (CRISPR) Cas 12a for signal amplification . Firstly, COF-367 NSs functionalized with gold nanoparticles (AuNPs) have been synthesized for the modification of the working electrode , thus the proposed sensor can show highly improved electrochemical performance. Secondly, by utilizing the trans-cleavage activity of Cas12a, this sensor can achieve very high sensitivity for the detection of PD-L1 + exosomes. With such design, the biosensor can have a linear range from 1.2 × 10 2 to 1.2 × 10 7 particles/μL with a detection limit of 38 particles/μL. Moreover, this sensor can be employed to analyze non-small cell lung cancer (NSCLC) patient serum samples, and successfully identify NSCLC patients, demonstrating the great potential in clinical applications.
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TALANTA
·
2021
·
IF 6.057
Kai Zhang,Zhenqiang Fan,Yue Huang,Yuedi Ding,Minhao Xie
Early diagnosis and timely management of Severe Acute Respiratory Syndrome Coronavirus 2 (SARS-CoV-2) are the keys to preventing the spread of the epidemic and controlling new infection clues. Therefore, strengthening the surveillance of the epidemic and timely screening and confirming SARS-CoV-2 infection is the primary task. In this work, we first proposed the idea of activating CRISPR-Cas12a activity using double-stranded DNA amplified by a three-dimensional (3D) DNA walker. We applied it to the design of an electrochemiluminescent (ECL) biosensor to detect the SARS-CoV-2 RNA-dependent RNA polymerase (RdRp) gene. We first activated the cleavage activity of CRISPR-Cas12a by amplifying the target DNA into a segment of double-stranded DNA through the amplification effect of a 3D DNA walker. At the same time, we designed an MXene based ECL material: [email protected] 3 C 2 @AuNPs, and constructed an ECL biosensor to detect the RdRp gene based on this ECL material as a framework. Activated CRISPR-Cas12a cleaves the single-stranded DNA on the surface of this sensor and causes the ferrocene modified at one end of the DNA to move away from the electrode surface, increasing the ECL signal. The extent of the change in electrochemiluminescence reflects the concentration of the gene to be measured. Using this system, we detected the SARS-CoV-2 RdRp gene with a detection limit of 12.8 aM. This strategy contributes to the rapid and convenient detection of SARS-CoV-2-associated nucleic acids and promotes the clinical application of ECL biosensors based on CRISPR-Cas12a and novel composite materials.
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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.
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TALANTA
·
2024
·
IF 5.6
Jiajia Hu,Yang Yu,Xiaoyan Pan,Yue han,Xinyi She,Xiaojun Liu,Qingquan Zhang,Hongwei Gai,Chenghua Zong
Human papillomavirus (HPV) is a prevalent sexually transmitted pathogen associated with cervical cancer . Detecting high-risk HPV (hr-HPV) infections is crucial for cervical cancer prevention, particularly in resource-limited settings. Here, we present a highly sensitive and specific sensor for HPV-16 detection based on CRISPR/Cas12a coupled with enhanced single nanoparticle dark-field microscopy (DFM) imaging techniques . Ag–Au satellites were assembled through the hybridization of AgNPs-based spherical nucleic acid (Ag-SNA) and AuNPs-based spherical nucleic acid (Au-SNA), and their disassembly upon target-mediated cleavage by the Cas12a protein was monitored using DFM for HPV-16 quantification. To enhance the cleavage efficiency and detection sensitivity, the composition of the ssDNA sequences on Ag-SNA and Au-SNA was optimized. Additionally, we explored using the SynSed technique (synergistic sedimentation of Brownian motion suppression and dehydration transfer) as an alternative particle transfer method in DFM imaging to traditional electrostatic deposition. This addresses the issue of inconsistent deposition efficiency of Ag–Au satellites and their disassembly due to their size and charge differences. The sensor achieved a remarkable limit of detection (LOD) of 10 fM, lowered by 9-fold compared to traditional electrostatic deposition methods . Clinical testing in DNA extractions from 10 human cervical swabs demonstrated significant response differences between the positive and negative samples. Our sensor offers a promising solution for sensitive and specific HPV-16 detection, with implications for cancer screening and management.
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FOOD CHEMISTRY
·
2025
·
IF 9.8
Ruonan Wang,Baoshan He,Zhengyong Liang,Yao Liu,Jun Yang,Huali Jin,Min Wei,Wenjie Ren,Zhiguang Suo,Yiwei Xu
This study presents an integrated dual-mode sensing strategy, in which qualitative fluorescence screening is followed by quantitative electrochemical detection, improving detection efficiency and enabling rapid PCM analysis. It develops a novel fluorescence electrochemical aptasensor that combines in vitro “Dark-box” applications with CRISPR/Cas12a system electrode surface sensing technology. PCM activates the DNA walker, and the DNAzyme induces cyclic cleavage of DNA strands bearing the Carboxyfluorescein (FMA) group. After magnetic separation, the fluorescence reaction combined with the “Dark box” enables the preliminary qualitative analysis of procymidone (PCM). Following the preliminary qualitative detection, the solution is introduced to the electrochemical aptasensor platform integrated with the CRISPR/Cas12 system. The Cas12a system triggers cyclic cleavage, producing a signal change that enables the electrochemical quantitative detection of PCM. An fluorescence (FL) response occurs when the PCM concentration in spiked samples is at or above 1 pg·mL −1 , allowing for qualitative fluorescence detection. The EC platform's detection limit is 8.51 × 10 −6 ng·mL −1 , with a range of 1 × 10 −2 ng·mL −1 to 1 × 10 4 ng·mL −1 . The designed dual-mode sensor provides reliable monitoring of PCM in real samples.
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ANALYTICAL CHEMISTRY
·
2025
·
IF 6.7
Kai Shi,Wenjie Luo,Ying Cheng,Honglei Li,Liai Peng,Xiangrui Luo,Yu Hu,Jiaheng Zhang,Jiaxuan Chen
CRISPR/Cas12a-powered biosensors with guanine (G)-rich sequence reporters (e.g., G-quadruplex and G-triplex) are widely used in detection applications due to their simplicity and sensitivity. However, when these biosensors are employed for molecular detection in complex samples, they may encounter difficulties such as high background signal and susceptibility to interference because of the “turn-off” signal output. Herein, we explore, for the first time, a set of phosphorothioate (ps)-modified G-quadruplex (G4) and G-triplex (G3) sequences that can bind with thioflavin T (ThT) in an active split CRISPR/Cas12a system (SCas12a) to generate a “turn-on” fluorescent signal. To apply this new phenomenon, we develop a universal SCas12a-powered biosensor for “turn-on” fluorescent detection of nucleic acid (miRNA-21) and non-nucleic acid (kanamycin) targets by using ps-modified hairpin G3 as a reporter (SCas12a/psHG3). Target recognition activates SCas12a’s trans-cleavage activity, leading to cleavage at the loop region of the psHG3 reporter. The released prelocked psG3 DNA binds ThT to produce a strong fluorescence signal. Without preamplification, this strategy can detect miRNA-21 with a detection limit of 100 fM. Moreover, the SCas12a/psHG3 system was further utilized for detecting kanamycin by incorporating its aptamers, enabling the detection of kanamycin at concentrations as low as 100 pM. This work is the first to develop a “turn-on” SCas12a/psHG3 system, showcasing its improved performance and wide range of applications in synthetic biology-based sensing technology.
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