Analytical Methods
·
2024
·
IF 2.7
Qiong Liu,Mei Yang,He Zhang,Wenjie Ma,Xin Fu,Huiqing Li,Sainan Gao
Tuberculosis caused by Mycobacterium bovis poses a global infectious threat to humans and animals. Therefore, there is an urgent need to develop a sensitive, precise, and easy-to-readout strategy. Here, a novel tandem combination of CRISPR/Cas12a system with dual HCR (denoted as CRISPR/Cas12a-D-HCR) was constructed for detecting Mycobacterium bovis. Based on the efficient trans-cleavage activity of the active CRISPR/Cas12a system, tandem-dsDNA with PAM sites was established using two flexible hairpins, providing multiple binding sites with CRISPR/Cas12a for further amplification. Furthermore, the activation of Cas12a initiated the second hybridization chain reaction (HCR), which integrated complete G-quadruplex sequences to assemble the hemin/G-quadruplex DNAzyme. With the addition of H2O2 and ABTS, a colorimetric signal readout strategy was achieved. Consequently, the CRISPR/Cas12a-D-HCR achieved a satisfactory detection linear range from 20 aM to 50 fM, and the limit of detection was as low as 2.75 aM with single mismatched recognition capability, demonstrating good discrimination from different bacterial species. Notably, the practical application performance via the standard addition method was verified, with the recovery ranging from 96.0% to 105.2% and the relative standard deviations (RSD) ranging from 0.95% to 6.45%. The proposed CRISPR/Cas12a-D-HCR sensing system served as a promising application for accurate detection in food safety and agricultural fields.
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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.
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SENSORS AND ACTUATORS B-CHEMICAL
·
2025
·
IF 7.7
Yayun Jiang,E Jianfei,Youwei Li,Jian Chen,Jie Yang,Yang He,Gang Mai,Laibao Zheng
Group B Streptococcus (GBS) is a major cause of neonatal morbidity and mortality, underscoring the urgent need for rapid, accurate, and deployable diagnostics. Conventional culture and nucleic acid amplification tests are limited by long turnaround time, insufficient sensitivity, or dependence on sophisticated instrumentation, restricting their use in resource-limited settings. Here, we present a magnetically controlled dual-mode biosensing platform, termed c olorimetric/ p hotothermal–CRISPR/ Cas – Au @ Pt nanozyme (CP-CasAuPt), which integrates the sequence-specific trans-cleavage activity of CRISPR/Cas12a with the peroxidase-mimicking activity of Au@PtNPs. The catalytic oxidation of 3,3′,5,5′-Tetramethylbenzidine (TMB) generates oxidized TMB (oxTMB), which provides a visible colorimetric change and a near-infrared photothermal response, thereby enabling reliable dual-mode readouts with minimal instrumentation. Magnetic bead-assisted separation further reduces background signals, ensuring robust performance in complex matrices. Using GBS as a proof-of-concept, CP-CasAuPt achieved a detection limit of 10 3 CFU/mL after loop-mediated isothermal amplification, with results fully consistent with qPCR across 20 clinical samples. Beyond high sensitivity and specificity, this study establishes a generalizable strategy for multimodal molecular diagnostics, where orthogonal dual signals allow cross-validation, ensuring robust detection across diverse assay conditions. The CP-CasAuPt system thus offers a promising paradigm for rapid, instrument-free pathogen detection, particularly suited for resource-limited settings.
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BIOSENSORS & BIOELECTRONICS
·
2025
·
IF 10.5
Xiaobo Zhang,Xiuwen Wang,Longjiao Zhu,Jinyan Zhu,Qiuyue Zheng,Jingli Yuan,Wentao Xu,Jijuan Cao
Salmonella typhimurium ( S. typhimurium ), as common and highly pathogenic foodborne pathogen, poses a significant risk to public safety worldwide. The development of highly sensitive, rapid and on-site method for S. typhimurium analysis is urgently needed to prevent bacterial infections. Herein, we introduced a CRISPR/Cas12a-driven electrochemiluminescence (ECL) sensor based on luminol/O 2 binary systems for S. typhimurium detection, employing ultrafine Pt nanoclusters-anchored 2D delaminated-MXenes (Pt NCs/D-MXenes) as the co-reactant accelerator. The ultrathin D-MXenes support regulates the size and dispersibility of Pt NCs and facilitates the full exposure of active sites, and synergistic interactions between D-MXenes and Pt NCs improves electrocatalytic properties toward the reduction of O 2 , which promotes the generation of ROS for boosting ECL emission. Using target responsive-regulated CRISPR/Cas12a system, the ECL sensor for S. typhimurium showed a broad concentration range from 10 1 to 10 6 CFU/mL and limit of detection of 6 CFU/mL, with satisfactory recoveries in spiked-actual samples.
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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.
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MICROCHEMICAL JOURNAL
·
2025
·
IF 5.1
Caihong Zhou,Jingjing Fan,Liangyuan Wang,Baoxue Gao,Chunhui Wei,Long Ma,Lijuan Yin
Norovirus (NoV) is a leading cause of nonbacterial acute gastroenteritis, primarily transmitted through contaminated food and water. Rapid, sensitive, and on-site detection is crucial for controlling NoV outbreaks and ensuring food safety. In this study, we developed a portable, one-tube CRISPR/Cas12a-driven colorimetric biosensor for the rapid and sensitive detection of NoV RNA without the need for pre-amplification. The biosensor integrates magnetic nanoprobes with gold nanoparticle (AuNP) bioprobes, connected via single-stranded DNA (ssDNA) linkers. Upon the specific recognition of NoV cDNA, the Cas12a protein’s trans -cleavage activity is activated, cleaving the ssDNA linkers and releasing AuNP bioprobes from the magnetic nanoprobes. This release results in a visually detectable red color change and enhanced UV–visible absorbance in the solution following magnetic separation. These changes can be easily detected using portable devices such as microplate readers or smartphones. This method achieves a detection limit of 10 copies/µL within 60 min, with no cross-reactivity from other viruses or bacteria. Validation using oyster samples showed perfect concordance with qPCR results. Designed for resource-limited settings, our biosensor can be adapted for detecting various pathogens by altering the target nucleic acids and crRNA sequences. This advancement offers significant potential for enhancing rapid diagnostics and outbreak management.
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TALANTA
·
2025
·
IF 6.1
Jingwen Zhang,Xiru Zhang,Xinna Xie,Biru Han,Feng Zhao,Xinyan Yang,Wei Zhang,Yujun Jiang,Xianlong Zhang
The problem of mycotoxin contamination in foodstuffs has attracted widespread attention and posed a great threat to human health. Therefore, the sensitive and effective detection of mycotoxins is of great importance for preserving public health worldwide. In this study, a CRISPR/Cas12a-mediated marker-free fluorescent biosensing platform was constructed for highly sensitive and fast detection of aflatoxin B1. The copper nanoclusters were synthesized with marker-free DNA single strands within 5 min, showing the outstanding fluorescence properties. With the existence of aflatoxin B1, the released complementary DNA (cDNA) triggered multiple isothermal amplification reaction. Subsequently, the obtained amplification products triggered the trans -cleavage activity of CRISPR/Cas12a system, which degraded the DNA single strands for synthesis of copper nanoclusters, leading to a decreased fluorescent signal. Benefiting from good fluorescence properties of copper nanoclusters, the established biosensing platform for aflatoxin B1 detection had a high specificity and a limit of detection of 47.51 pg/mL in the linear range of 0.05–10 ng/mL. The proposed platform provided a new insight for the detection of non-nucleic acid targets.
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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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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.
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ANALYTICAL CHEMISTRY
·
2025
·
IF 6.7
Chen Huang,Ling-Jie Guo,Jia-Yi Li,Yue-Wen Chen,Bing Wu,Yi-Wei Liu,Chen-Zhi Zhang,Wei-Wei Lin,Ye Yang,Jin-Yuan Chen,Zhou-Jie Liu
The combination of CRISPR-Cas12a with aptamers can potentially improve the sensitivity, specificity, and speed of non-nucleic acid target detection. Nevertheless, current CRISPR-Cas12a aptasensors, solely dependent on aptamer affinity and overlooking the enzymatic regulation of CRISPR-Cas12a, may produce false-positive signals. We proposed a CRISPR-Cas12a aptasensor synergistically regulated by an aptamer and RNA, which incorporated aptamer-mediated drug recognition and CRISPR-powered signal amplification in a one-pot format. Herein, a regulatory RNA probe synergized with the conformational switching of a drug-induced aptamer, enabling precise regulation of Cas12a trans-cleavage activity via toehold-mediated strand displacement (TMSD). This dual regulatory mechanism transformed the aptamer-activated CRISPR-Cas12a sensing process into a TMSD-driven conditional reaction, avoiding false-positive signals and thus achieving better detection performance with a lower detection limit. With the vancomycin (VCM) aptamer as a model, the aptasensor can detect VCM within 30 min from 2% serum, 1% synovial fluid, and 1% cerebrospinal fluid, with a detection limit of 13.62, 7.56, and 6.75 nM, respectively. The proposed aptasensor was incorporated into a custom 3D-printed portable fluorometer and underwent clinical validation in 22 VCM serum samples, reporting no significant difference when compared with the enzyme-multiplied immunoassay technique, confirming the reliability for point-of-care quantification. It further received cross-validation with a quinine aptamer, suggesting universality [a linear range of 10–250 nM (R2 = 0.985) and a detection limit of 0.42 nM]. By integration of aptamer selectivity with CRISPR programmability, this work presents a novel robust biosensing paradigm for point-of-care drug concentration detection.
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SCIENCE OF THE TOTAL ENVIRONMENT
·
2023
·
IF 9.8
Junhua Chen,Gu Shi,Chong Yan
On-site and cost-effective monitoring of antibiotic residue in water samples using a ubiquitous device that is readily available to the general public is a big challenge. Herein, we developed a portable biosensor for kanamycin (KAN) detection based on a glucometer and CRISPR-Cas12a. The aptamer-KAN interactions liberate the trigger C strand, which can initiate the hairpin assembly to produce numerous double-stranded DNA. After recognition by CRISPR-Cas12a, Cas12a can cleave the magnetic bead and invertase-modified single-stranded DNA. After magnetic separation, the invertase can convert sucrose into glucose, which can be quantified by a glucometer. The linear range of the glucometer biosensor is from 1 pM to 100 nM and the detection limit is 1 pM. The biosensor also exhibited high selectivity and the nontarget antibiotics had no significant interference with KAN detection. The sensing system is robust and can work in complex samples with excellent accuracy and reliability. The recovery values were in the range of 89–107.2 % for water samples and 86–106.5 % for milk samples. The relative standard deviation (RSD) was below 5 %. With the advantages of simple operation, low cost, and easy accessibility to the public, this portable pocket-sized sensor can realize the on-site detection of antibiotic residue in resource-limited settings.
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Biosensors-Basel
·
2023
·Vol. 13
·期 1
·111
·
IF 5.4
Tianyi Yang,Yanju Chen,Jinsong He,Jian Wu,Meixia Wang,Xiaoping Zhong
A rapid and intuitive method for detecting Vibrio parahaemolyticus (VP) was established by a designed reaction vessel which coupled CRISPR/Cas12a with loop-mediated isothermal nucleic acid amplification (LAMP). There were two spaces in the vessel-holding LAMP reaction solution and CRISPR reaction solution, respectively, which were separated with a polyvinyl alcohol (PVA) membrane. The PVA membrane could be dissolved with a water solution. The thermolabile hemolysin (TLH) gene of VP was employed as the detection target. After the target sequence of the TLH gene was amplified with LAMP, the PVA membrane would be dissolved and the CRISPR reaction solution mixed with the LAMP reaction solution. In this way, amplicons could be detected with CRISPR/Cas12a in the reaction vessel. The fluorescent signals produced by the positive samples were clearly identified by the naked eye under a UV light, while the negative samples were dark. The whole detection procedure could be finished within 35 min with a detection limit of 100 copies/µL. The designed reaction vessel is easy to produce and can effectively prevent contamination due to the opening of the reaction vessel after the LAMP reaction. Thus, it will have the potential to provide a new solution for rapid detection in the field.Keywords:polyvinyl alcohol;dissolvable membrane;CRISPR/Cas12a;LAMP reaction;Vibrio parahaemolyticus
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