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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高影响因子
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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高影响因子
CHEMICAL ENGINEERING JOURNAL
·
2026
·
IF 12.5
Yongzhen Dong,Aoting Gu,Chunling Li,Huihui Wang,Yang Zhou,Zhixiong Pi,Chengjia Jiang,Yiping Chen
A CbAgo-CRISPR/Cas12a-powered cascade amplification sensing strategy was proposed. • It combined CbAgo's precise cleavage with the Cas12a's robust trans-cleavage activity. • An amplified fluorescent biosensor was developed for detecting Salmonella Typhimurium . • The proposed biosensor demonstrated comparable analytical performance to qPCR assay. Sensitive and accurate detection of foodborne pathogens constitutes a critical imperative for safeguarding food safety. Herein, we proposed a Clostridium butyricum argonaute (Cb A go)-CRISPR/ C as12a-powered synergistic amplification strategy and developed a DNA amplification-free f luorescent ( ACF ) biosensor for ultrasensitive detection of Salmonella Typhimurium . In this biosensor, magnetic beads-phage conjugates enabled the selective capture of viable Salmonella Typhimurium , followed by rapid DNA extraction within 5 min using commercial lysis buffers. A synergistic signal amplification strategy was designed for efficient biosensing by utilizing the primary cleavage product (tDNA) of the CbAgo system as a molecular bridge to integrate CbAgo and Cas12a systems. Specifically, CbAgo performed primary cleavage of target DNA to generate tDNA fragments. These tDNA subsequently activated the CRISPR/Cas12a system, initiating its trans-cleavage activity to degrade fluorescent reporter probes for quantitative analysis. This tDNA-mediated integration synergizes CbAgo's precision cleavage capability with Cas12a's efficient trans-cleavage activity, enabling exponential amplification. Under optimized conditions, the ACF biosensor exhibited a linear detection range of 10 2 –10 7 CFU/mL and a detection limit of 24.8 CFU/mL for Salmonella Typhimurium without DNA amplification steps. Moreover, the ACF biosensor demonstrated comparable analytical performance to the qPCR assay and achieved satisfactory results in the detection of Salmonella Typhimurium in 20 food samples, highlighting its potential as an ultrasensitive tool for foodborne pathogen monitoring. Download: Download high-res image (107KB) Download: Download full-size image
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SCIENCE OF THE TOTAL ENVIRONMENT
·
2023
·
IF 9.8
Fang Deng,Jiafeng Pan,Manjia Chen,Zhi Liu,Junhua Chen,Chengshuai Liu
Polychlorinated biphenyls (PCBs) are ubiquitous persistent organic pollutants that cause harmful effects on environmental safety and human health. There is an urgent need to develop an intelligent method for PCBs sensing. In this work, we proposed a logic gate biosensing platform for simultaneous detection of multiple PCBs. 2,3′,5,5′-tetrachlorobiphenyl (PCB72) and 3,3′,4,4′-tetrachlorobiphenyl (PCB77) were used as the two inputs to construct biocomputing logic gates. We used 0 and 1 to encode the inputs and outputs. The aptamer was used to recognize the inputs and release the trigger DNA. A catalytic hairpin assembly (CHA) module is designed to convert and amplify each trigger DNA into multiple programmable DNA duplexes, which initiate the trans -cleavage activity of CRISPR/Cas12a for the signal output. The activated Cas12 cleaves the BHQ-Cy5 modified single-stranded DNA (ssDNA) to yield the fluorescence reporting signals. In the YES logic gate, PCB72 was used as the only input to carry out the logic operation. In the OR, AND, and INHIBIT logic gates, PCB72 and PCB77 were used as the two inputs. The output signals can be visualized by the naked eye under UV light transilluminators or quantified by a microplate reader. Our constructed biosensing platform possesses the merits of multiple combinations of inputs, intuitive digital output, and high flexibility and scalability, which holds great promise for the intelligent detection of different PCBs.
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BIOSENSORS & BIOELECTRONICS
·
2023
·
IF 12.6
Hua Wang,Yang Sun,Yuan Zhou,Yujie Liu,Shuo Chen,Wenbo Sun,Zidong Zhang,Junqing Guo,Cheng Yang,Zhengping Li,Lei Chen
At present, the 100% case fatality and the cross-infection of virus strains make the ASFV 's harm to society continue to expand. The absence of an effective commercial vaccine poses early detection remains the most effective means of curbing ASFV infection. Here, we report a cascaded detection platform based on the CRISPR-Cas12a system combined with graphene field-effect transistor sensors. The cascade platform could detect ASFV as low as 0.5 aM within 30 min and achieve typing of wild and vaccine strains of ASFV in a single detection system. The evaluation of 16 clinical samples proved that, compared with the gold standard Real-time PCR method, this platform has outstanding advantages in sensitivity, specificity and typing. Combining CRISPR-Cas12a's high specificity with the bipolar electric field effect of graphene field-effect transistor, the cascade platform is expected to achieve clinical application in the field of DNA disease detection, and provides a new direction for multi-strain disease typing.
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Frontiers in Bioengineering and Biotechnology
·
2024
·
IF 4.3
Huang Hao,Li Shuang,Han Xianjing,Zhang Yule,Gao Lingfeng,Wang Xiangjiang,Wang Guiqing,Chen Zhi
Osteoarthritis (OA) has become a serious problem to the human society for years due to its high economic burden, disability, pain, and severe impact on the patient’s lifestyle. The importance of current clinical imaging modalities in the assessment of the onset and progression of OA is well recognized by clinicians, but these modalities can only detect OA in the II stage with significant structural deterioration and clinical symptoms. Blood vessel formation induced by vascular endothelial growth factor (VEGF) occurs in the early stage and throughout the entire course of OA, enables VEGF relating gene sequence to act as a biomarker in the field of early diagnosis and monitoring of the disease. Here in, a facile rapid detection of VEGF relating ssDNA sequence was developed, in which manganese-based zeolitic imidazolate framework nanoparticles (Mn-ZIF-NPs) were synthesized by a simple coprecipitation strategy, followed by the introduction and surficial absorption of probe ssDNAs and the CRISPR/Cas12a system components. Furthermore, fluorescence experiments demonstrated that the biosensor displayed a low detection limit of 2.49 nM, a good linear response to the target ssDNA ranging from 10 nM to 500 nM, and the ability of distinguishing single nucleotide polymorphism. This finding opens a new window for the feasible and rapid detection of ssDNA molecules for the early diagnose of OA.
研究领域:
生物医学工程
·
分子生物学
·
纳米技术
·
诊断学
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高影响因子
FOOD CHEMISTRY
·
2026
·
IF 10.4
Yipeng Zhang,Shijia Wu,Yisheng Chen
A portable biosensor for the peanut allergen Ara h 1 was developed by integrating a polydopamine-functionalized pipette tip, CRISPR/Cas12a signal amplification, and a personal glucose meter. Ara h 1 binding induced partial displacement of a cDNA activator, which activated Cas12a trans-cleavage and released glucose oxidase from GOx–DNA conjugates. The released enzyme catalyzed glucose oxidation, producing a quantitative decrease in glucose concentration. Under optimized conditions, the assay showed a linear range of 50–1000 ng/mL and a solution-based limit of detection of 22.19 ng/mL. Post-extraction matrix-spike tests in commercial peanut-biscuit and peanut-beverage extracts yielded recoveries of 101.58–104.68% with relative standard deviations below 3.44%. These results demonstrate the analytical feasibility and preliminary matrix compatibility of the platform, although validation using pre-extraction fortified, incurred, and naturally contaminated foods is still required.
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SENSORS AND ACTUATORS B-CHEMICAL
·
2025
·
IF 7.7
Zhengfang Liang,Shuwei Xiao,Junhong Li,Guoli Zuo,Jihua Wei,Yuanxun Gong,Qianli Tang,Kai Zhang,Xianjiu Liao
Apurinic/apyrimidinic endonuclease 1 (APE1), a key enzyme in the base excision repair pathway, is frequently overexpressed in multiple cancers and has emerged as an important biomarker for early diagnosis and therapeutic response evaluation. In this work, a highly sensitive electrochemiluminescence (ECL) biosensor was developed for APE1 detection by integrating a PAM-regulated CRISPR-Cas12a activation mechanism with a multifunctional nanocomposite-modified electrode. A Ti₃C₂/AuNPs/CsPbBr₃ ternary nanocomposite was fabricated through ultrasonic assembly to provide enhanced conductivity, water stability, and probe-loading capacity. Meanwhile, a DNA duplex containing an AP site within the PAM region was engineered to regulate Cas12a activation through APE1-mediated cleavage and polymerase-assisted repair, thereby minimizing background interference and enabling highly specific signal generation. Ferrocene-labeled DNA tetrahedrons (TDNs) were further introduced as a spatially ordered probe scaffold to improve hybridization efficiency and facilitate Cas12a trans-cleavage–mediated signal release. This work presents three key innovations: (1) Construction of a Ti₃C₂/AuNPs/CsPbBr₃ nanocomposite that simultaneously enhances ECL stability, conductivity, and electron-transfer efficiency, addressing the inherent water-instability of perovskite emitters. (2) Coupling of a PAM-regulated APE1–KF repair mechanism with Cas12a activation to achieve highly specific AP-site recognition and selective signal generation. (3) Introduction of a TDN-based probe architecture that provides rigid spatial orientation and high-density probe loading, markedly improving hybridization efficiency and Cas12a trans-cleavage performance. Benefiting from these synergistic innovations, the biosensor achieves a detection limit of 6.28 × 10⁻⁸ U μL⁻¹ and a wide linear range (1 × 10⁻⁷ to 1 × 10⁻ 1 U μL⁻¹). The platform also demonstrates excellent specificity, reproducibility, and reliable performance in diluted serum, highlighting its promise for clinical and biochemical applications.
研究领域:
生物传感器
·
纳米技术
·
癌症诊断
·
电分析化学
·
分子生物学
·
CRISPR技术
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Journal of Future Foods
·
2026
·
IF 7.2
Xiaobo Zhang,Zekun Long,Wei Wang,Yafeng Jiang,Qiuyue Zheng,Jijuan Cao
Aflatoxin B1 (AFB1), the most toxic mycotoxin among aflatoxin family, which presents a risk to food safety and human health at a trace level. Herein, the self-enhanced electrochemiluminescence (ECL) emitters of Au nanoclusters-anchored g-C 3 N 4 nanosheet (Au NCs@g-C 3 N 4 ) were synthesized, in which the Au NCs as a co-reaction accelerator enhanced the ECL stability and intensity. Integrating with CRISPR/Cas12a system, the Au NCs@g-C 3 N 4 behaved as sensing substrate to construct a “off-on” switch-type ECL sensor for AFB1 detection based on ECL resonance energy transfer (ECL-RET). The presence of targets AFB1 can specifically bind its aptamer to trigger the release of the activator. Using dual aptamer-regulated activation of CRISPR-Cas12a, AFB1 can be quantified via the ECL sensor, displaying a wide detection range from 0.2 pg/mL-100 ng/mL and a detection limit of 0.08 pg/mL. Furthermore, good practicability was verified in real samples assay, providing an efficient tool for the early warning of AFB1 pollution.
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MICROCHEMICAL JOURNAL
·
2026
·
IF 5.2
Chunlei Yu,Keyun Ren,Xuezhen Xu,Jinmiao Ma,Falan Li,Yanyan Zhang,Qingqing Yang
Most applications of CRISPR-Cas12a technology for mycotoxin detection rely on aptamers as recognition elements. However, although antibodies exhibit high affinity and specificity, they require converting immune signals into nucleic acid signals and cannot directly activate the CRISPR-Cas12a system, thereby limiting their utility for highly sensitive small-molecule target detection. In this work, we conjugated CRISPR-Cas12a activator DNA (aDNA) to antibodies via strain-promoted azide-alkyne cycloaddition (SPAAC) to achieve dual functionality: transducing small-molecule target signals into nucleic acid signals and partially immobilizing the Cas12a protein. Magnetic spherical nucleic acids (MSNAs) served as trans-cleavage substrates for the CRISPR-Cas12a system. Terminal deoxynucleotidyl transferase (TdT) was used to extend polyA sequences by adding dATP to the 3’-OH ends of numerous cleaved DNA fragments. This process, combined with high-concentration NaCl-induced aggregation and color change of gold nanoparticles (AuNPs), established a cascade signal amplification system. We developed a novel ultrasensitive immunosensor, termed Cas12a-TdT Cascade Amplification for Antigen-Toxin Immunoassay (CTCA-ATI), for mycotoxin quantification. The limits of detection for aflatoxin B1 and deoxynivalenol (DON) were 2.63 × 10 −5 ng/mL and 6.23 × 10 −4 ng/mL, respectively, with linear ranges of 1 × 10 −4 –10 ng/mL for aflatoxin B1 and 1 × 10 −3 –10 ng/mL for DON. This study provides new insights into the development of cascade-amplified biosensors using CRISPR-Cas12a and demonstrates substantial potential for integrating antibodies with CRISPR-Cas technology to detect other critical targets.
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BIOSENSORS & BIOELECTRONICS
·
2021
·
IF 10.618
Kai Zhang,Zhenqiang Fan,Bo Yao,Yuedi Ding,Jing Zhao,Minhao Xie,Jianbin Pan
Sialic acid-binding immunoglobulin (Ig)-like lectins (Siglecs) is a type I transmembrane receptor on the cell surface. Siglec-5, as one of the Siglecs family, play an important role as an inhibitory receptor for leukocytes in the human body. The development of novel siglec-5 assays can help to study the pathogenesis of related diseases as well as to develop novel therapeutic drugs. We use catalytic hairpin assembly (CHA) amplification strategy combined with CRISPR-Cas12a′s side-cutting feature to build a 2D ultra-thin Ti 3 C 2 T x (MXene) based electrochemiluminescence (ECL) biosensor for the detection of Siglec-5. By using this ECL biosensor, the cleavage of CRISPR-Cas12a is reasonably combined with CHA-mediated isothermal amplification, thereby realizing the sensitive amplification assay Siglec-5 with 20.22 fM sensitivity. By introducing pairs of sites that are not in the same double-stranded DNA into the DNA duplex, the hybridization sequence of CRISPR-Cas12a complements the targeting mechanism to enhance indirect Siglec-5 amplification assay. Also, the double-strand DNA (dsDNA) design based on CRISPR-Cas12a amplification allows the same CRISPR RNA (crRNA, also known as guide RNA (gRNA)) to detect the output of DNA duplexes from different intermediate DNAs, which provides a common way for biomarker detection based on the conversion of protein analytes to intermediate DNA strategy. This work extends the application scope of CRISPR-Cas12a to the construction of ECL biosensors, evaluates the role of lectins, which can be used for the biochemical research and clinical diagnosis of protein markers. This is the first investigative work exploring the Trans-Cleavage activity of CRISPR-Cas12a for Mxene-based ECL biosensor establishment to the best of our knowledge.
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SENSORS AND ACTUATORS B-CHEMICAL
·
2021
·
IF 7.46
Kai-Ren Zhao,Li Wang,Peng-Fei Liu,Xiao-Min Hang,Hui-Yi Wang,Shu-Ying Ye,Zhi-Jun Liu,Guo-Xi Liang
Herein, we report a “on-off” signal-switchable electrochemiluminescence (ECL) biosensor for simple, sensitive and quantitative detection of two kinds of virus genes, human immunodeficiency virus (HIV) and human papilloma virus (HPV-16) DNAs, by the integration of spherical nucleic acid (SNA) with CRISPR/Cas12a. The concept behind this approach is “on” signal based on a sandwich type assay after ECL luminophores of carbon dots (CDs) labeled SNA incubated with the biosensor in the presence of HIV DNA; and “off” signal upon the biosensor incubation with assistant DNA (HIV DNA sequence), CDs-labeled SNA and Cas12a/crRNA/HPV-16 DNA ternary complex relying on the collaterally cleavage of Cas12a. The cross-interference between HIV and HPV-16 DNAs can be avoided by adding Cas12a effector to the system or not. Using this strategy, HIV and HPV-16 DNAs are successfully quantitatively detection with detection limits of 30 fM and 0.32 pM (S/N = 3), respectively. Importantly, this system does not rely on multiplex signal reporting molecules, thus simplifying the detection system and avoiding the likelihood of cross interferences presented in the assay. Furthermore, multiplex detection of HIV/HPV-16 DNAs in human serum samples are successfully achieved within 2 h without additional nucleic acid amplification step. As demonstrated, the proposed biosensor has the potential for multiplex detection in clinical diagnosis.
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