JOURNAL OF HAZARDOUS MATERIALS
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2024
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IF 12.2
Manyan Qiu,Zhiyu Yuan,Nan Li,Xinyan Yang,Xianlong Zhang,Yujun Jiang,Qianyu Zhao,Chaoxin Man
Sensitive detection and point-of-care test of bacterial pathogens is of great significance in safeguarding the public health worldwide. Inspired by the characteristics of horseradish peroxidase (HRP), we synthesized a hybrid nanoflower with peroxidase-like activity via a three-component self-assembled strategy. Interestingly, the prepared nanozyme not only could act as an alternative to HRP for colorimetric biosensing, but also function as a unique signal probe that could be recognized by a pregnancy test strip. By combining the bifunctional properties of hybrid nanoflower, isothermal amplification of LAMP, and the specific recognition and non-specific cleavage properties of CRISPR/Cas12a system, the dual-readout CRISPR/Cas12a biosensor was developed for sensitive and rapid detection of Salmonella enterica . Moreover, this platform in the detection of Salmonella enterica had limits of detection of 1 cfu/mL (colorimetric assay) in the linear range of 10 1 –10 8 cfu/mL and 10 2 cfu/mL (lateral flow assay) in the linear range of 10 2 –10 8 cfu/mL, respectively. Furthermore, the developed biosensor exhibited good recoveries in the spiked samples (lake water and milk) with varying concentrations of Salmonella enterica . This work provides new insights for the design of multifunctional nanozyme and the development of innovative dual-readout CRISPR/Cas system-based biosensing platform for the detection of pathogens. Environmental implication The presence of bacterial pathogens is pervasive in the environment and poses a significant threat to public health safety. To prevent foodborne disease outbreaks, it is crucial to develop sensitive and reliable pathogen detection strategies for rapid and on-site detection of pathogens in both environmental and food samples. Herein, we developed a novel dual-readout CRISPR/Cas biosensing platform based on nanozyme with bifunctional properties and loop-mediated isothermal amplification (LAMP) for highly sensitive and on-site detection of Salmonella enterica in lake water and milk samples. The proposed method has good stability and high accuracy in the detection of Salmonella enterica .
研究领域:
生物技术
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纳米技术
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病原体检测
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生物传感器
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即时诊断
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DOI
PubMed
高影响因子
MOLECULAR AND CELLULAR BIOCHEMISTRY
·
2023
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IF 4.3
Lu Junnan,Tong Ying,Dong Rui,Yang Yijun,Hu Wen,Zhang Minghong,Liu Quan,Zhao Siting,Adams John H.,Qin Li,Chen Xiaoping
CRISPR/Cas9 technology applied to Plasmodium falciparum offers the potential to greatly improve gene editing, but such expectations including large DNA fragment knock-ins and sequential gene editing have remained unfulfilled. Here, we achieved a major advance in addressing this challenge, especially for creating large DNA fragment knock-ins and sequential editing, by modifying our suicide-rescue-based system that has already been demonstrated to be highly efficient for conventional gene editing. This improved approach was confirmed to mediate efficient knock-ins of DNA fragments up to 6.3 kb, to produce “marker-free” genetically engineered parasites and to show potential for sequential gene editing. This represents an important advancement in establishing platforms for large-scale genome editing, which might gain a better understanding of gene function for the most lethal cause of malaria and contribute to adjusting synthetic biology strategies to live parasite malaria vaccine development. Site-directed knock-in of large DNA fragments is highly efficient using suicide-rescue-based CRISPR/Cas9 system, and sequential gene insertion is feasible but further confirmation is still needed.
研究领域:
寄生虫学
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分子生物学
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基因组编辑
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高影响因子
SENSORS AND ACTUATORS B-CHEMICAL
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2023
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IF 8.4
Jia Wei,Wanjun Wang,Qiong Yu,Mingrui Zhang,Fengyu Xue,Beibei Fan,Tong Zhang,Yanpeng Gao,Juan Li,Xianying Meng,Bo Pang
Mpox (formerly referred as Monkeypox) outbreak has been declared a Public Health Emergency of International Concern. However, traditional polymerase chain reaction (PCR) diagnostic technology is not ideal for on-site applications. To conduct the sample-to-result Mpox viral particles detection outside the laboratories, we developed an easy-to-operate palm-size pouch, termed Mpox At-home Self-Test and point-of-caRe Pouch (MASTR Pouch). In this MASTR Pouch, the fast and accurate visualization was achieved by incorporating recombinase polymerase amplification (RPA) with clustered regularly interspaced short palindromic repeat (CRISPR)/Cas12a system. From viral particle lysis to naked eye readout, MASTR Pouch required only four simple steps to accomplish the analysis process within 35 min. Fifty-three Mpox pseudo-viral particles in exudate (10.6 particles/μL) were able to be detected. To verify the practicability, 104 mock Mpox clinical exudate specimens were tested. The clinical sensitivities were determined to be 91.7%− 95.8%. There was no false-positive result, validating the 100% clinical specificity. MASTR Pouch approaches the WHO's ASSURD criteria for point-of-care diagnostic, which will be beneficial for mitigating Mpox's global spread. The versatility potential of MASTR Pouch could further revolutionize infection diagnosis.
研究领域:
病毒学
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分子诊断
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公共卫生
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生物技术
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DOI
PubMed
高影响因子
ANALYST
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2024
·Vol. 149
·期 15
·3920-3927
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IF 3.6
Zhenhuan Jin,Wei Xiao,Lin Shen,Xiaoxue Shi,Jianping Li
Anti-cancer therapy is crucial in cancer prevention and anti-cancer, and thus, highly sensitive methods for detecting cancer biomarkers are essential for cancer early diagnosis. Herein, an electrochemical aptamer biosensor based on the CRISPR-Cas12a system was constructed for the detection of cancer tumor biomarker MUC1 mucin. The sensitivity was significantly prompted by enzyme-catalyzed signal amplification, and the selectivity was improved by the dual recognition of the aptamer to MUC1 and crRNA-Cas12a system to the aptamer. Glucose oxidase (GOD) was loaded on the surface of magnetic Fe3O4@Au (MGNP) via probe single-stranded DNA (pDNA) with the terminal modification of mercapto (–SH) to form GOD-pDNA/MGNP. The corresponding aptamer of MUC1 (MUC1 Apt) binds to its complementary ssDNA (cDNA) to form the activator Apt/cDNA, which is specifically recognized by crRNA-Cas12a and excites the trans-cleavage function of Cas12a, thus in turn trans-cleaves pDNA and detaches GOD from the magnetic particles. The magnetic beads were separated and transferred into a glucose solution, and the oxidation current of H2O2 produced by the catalytic reaction of GOD was measured on a Pt-modified magnetically-controlled glassy carbon electrode, resulting in an indirect determination of MUC1. The current change was linear with the logarithm of MUC1 concentration in the range from 1.0 × 10−17 g mL−1 to 1.0 × 10−10 g mL−1. The detection limit was as low as 7.01 × 10−18 g mL−1. The method was applied for the detection of MUC1 in medical samples.
研究领域:
生物化学
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分子生物学
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医学诊断
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DOI
PubMed
高影响因子
JOURNAL OF NANOBIOTECHNOLOGY
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2023
·Vol. 21
·期 1
·1-8
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IF 10.2
Zheng Laibao,Jiang Yayun,Huang Fuyuan,Wu Qiaoli,Lou Yongliang
A multimodal analytical strategy utilizing different modalities to cross-validate each other, can effectively minimize false positives or negatives and ensure the accuracy of detection results. Herein, we establish a colorimetric, photothermal, and fluorescent triple modal CRISPR/Cas12a detection platform (CPF-CRISPR). An MNPs-ssDNA-HRP signal probe is designed to act as a substrate to trigger three signal outputs. In the presence of the DNA target, MNPs-ssDNA-HRP is cleaved by the activated CRISPR/Cas12a, resulting in the release of HRP and generating short DNA strands with 3-terminal hydroxyl on magnetic beads. The released HRP subsequently catalyzed TMB-H2O2 reaction and oxidized TMB is used for colorimetric and photothermal signal detection. Under the catalysis of terminal deoxynucleotidyl transferase (TdT), the remaining short DNA strands are used as primers to form poly-T and function as scaffolds to form copper nanoclusters for fluorescent signal output. To verify the practical application of CPF-CRISPR, we employed MRSA as a model. The results demonstrate the platform’s high accuracy and sensitivity, with a limit of detection of 101 CFU/mL when combined with recombinase polymerase amplification. Therefore, by harnessing the programmability of CRISPR/Cas12a, the biosensor has the potential to detect various drug-resistant bacteria, demonstrating significant practical applicability.
研究领域:
生物技术
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分子生物学
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分析化学
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微生物学
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PubMed
高影响因子
BIOTECHNOLOGY LETTERS
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2023
·Vol. 45
·期 3
·401-410
·
IF 2.7
Tian Li,Yan Bingyu,Huo Dandan,Sun Wenhui,Cui Sufang,Li Xiaojing,Zhang Xiangmei,Dong Huijun
Objectives To develop a modified CRISPR/Cas9 system with the β-glucuronidase (GusA) reporter and a dual sgRNA cassette for Nonomuraea gerenzanensis ( N. gerenzanensis ). Results With the aid of a visual GusA reporter, the complicated and tedious process of cloning and gene identification could be abandoned entirely in the genetic editing of N. gerenzanensis . Moreover, introducing a dual sgRNA cassette into the CRISPR/Cas9 system significantly improved gene deletion efficiency compared to the single sgRNA element. Furthermore, the length of the homologous flanking sequences set to the lowest value of 500 bp in this system could still reach the relatively higher conjugation transfer frequency. Conclusions The enhanced CRISPR/Cas9 system could efficiently perform genetic manipulation on the rare actinomycete N. gerenzanensis .
研究领域:
微生物学
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分子生物学
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遗传工程
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高影响因子
ANALYTICAL CHEMISTRY
·
2021
·
IF 6.986
Chunyan Wang,Cuiyan Han,Xiaoxue Du,Weiwei Guo
In addition to their roles as revolutionary genome engineering tools, CRISPR-Cas systems are also highly promising candidates in the construction of biosensing systems and diagnostic devices, which have attracted significant attention recently. However, the CRISPR-Cas system cannot be directly applied in the sensing of non-nucleic acid targets, and the needs of synthesizing and storing different vulnerable guide RNA for different targets also increase the application and storage costs of relevant biosensing systems, and therefore restrict their widespread applications. To tackle these barriers, in this work, a versatile CRISPR-Cas12a-based biosensing platform was developed through the introduction of an enzyme-free and robust DNA reaction network, the entropy-driven dynamic DNA network. By programming the sequences of the system, the entropy-driven catalysis-based dynamic DNA network can respond to different types of targets, such as nucleic acids or proteins, and then activate the CRISPR-Cas12a to generate amplified signals. As a proof of concept, both nucleic acid targets (a DNA target with random sequence, T, and an RNA target, microRNA-21 (miR-21)) and a non-nucleic acid target (a protein target, thrombin) were chosen as model analytes to address the feasibility of the designed sensing platform, with detection limits at the pM level for the nucleic acid analytes (7.4 pM for the DNA target T and 25.5 pM for miR-21) and 0.4 nM for thrombin. In addition, the detection of miR-21 or thrombin in human serum samples further demonstrated the applicability of the proposed biosensing platform in real sample analysis.
研究领域:
生物技术
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分子生物学
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分析化学
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诊断设备
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DOI
PubMed
高影响因子
TALANTA
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2022
·
IF 6.556
Bin Zhou,Runlin Yang,Muhammad Sohail,Xiaoxue Kong,Xing Zhang,Ninghua Fu,Bingzhi Li
CRISPR is reshaping biosensing technology due to its programmability, sensitivity, and specificity. Most current CRISPR-based biosensors are developed based on Cas12 and Cas13, while the biosensing potentials of the newly discovered Cas14 have not been fully elucidated yet. Herein, a fluorometric biosensor named HARRY ( h ighly sensitive a ptamer- r egulated Cas14 R -loop for bioanal y sis) was developed. The diblock ssDNA is designed to contain the activator sequence of Cas14 and the aptamer sequence of specific targets. In the absence of targets, the ssDNA activates Cas14a, then the Cas14a trans -cleavages the fluorescent reporter, causing fluorescence enhancement. In the presence of the targets, ssDNA-target assembly is formed via aptamer interaction, resulting in the inhibition of Cas14a activation. HARRY can detect ATP , Cd 2+ , histamine, aflatoxin B1 , and thrombin with detection limits at the low-nanomolar level, which shows improvement compared with Cas12a-based aptasensors in sensitivity and versatility. We reasoned that the improvement is derived from the ssDNA specificity of Cas14a and found that the detection limit of HARRY is correlated to the binding affinities of aptamers. This study unlocks the potential of Cas14a in versatile aptasensing, which may inspire the development of CRISPR-based biosensors from the Cas14a branch.
研究领域:
生物技术
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分子生物学
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纳米技术
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分析化学
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DOI
PubMed
高影响因子
ACS Synthetic Biology
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2019
·
IF 5.571
Jing Fan,Yuchen Liu,Lisa Liu,Yikun Huang,Xuemei Li,Weiren Huang
The treatment of bladder cancer has recently shown minimal progress. Gene therapy mediated by CRISPR provides a new option for bladder cancer treatment. In this study, we developed a versatile liposome system to deliver the CRISPR-Cas13a gene circuits into bladder cancer cells. After in vitro studies and intravesical perfusion studies in mice, this system showed five advantages: (1) CRISPR-Cas13a, a transcriptional targeting and cleavage tool for gene expression editing, did not affect the stability of the cell genome; (2) the prepared liposome systems were targeted to hVEGFR2, which is always highly expressed in bladder cancer cells; (3) the CRISPR-Cas13a sequence was driven by an artificial tumor specific promoter to achieve further targeting; (4) a near-infrared photosensitizer released using near-infrared light was introduced to control the delivery system; and (5) the plasmids were constructed with three crRNA tandem sequences to achieve multiple targeting and wider therapeutic results. This tumor cell targeting lipid delivery system with near-infrared laser-controlled ability provided a versatile strategy for CRISPR-Cas13a based gene therapy of bladder cancer.
研究领域:
癌症生物学
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基因治疗
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生物技术
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药学
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DOI
PubMed
高影响因子
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.
研究领域:
生物传感技术
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食品安全检测
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分子生物学
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产品关联
DOI
PubMed
高影响因子
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.
研究领域:
分子生物学
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医学诊断
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纳米技术
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产品关联
DOI
PubMed
高影响因子
SENSORS AND ACTUATORS B-CHEMICAL
·
2021
·
IF 7.46
Yaqin Zhang,Minyan Chen,Chengrong Liu,Jiaqi Chen,Xinyi Luo,Yingying Xue,Qiming Liang,Li Zhou,Yu Tao,Mingqiang Li,Di Wang,Jianhua Zhou,Jiasi Wang
The outbreak of corona virus disease 2019 (COVID-19) caused by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) has led to a global pandemic. The high infectivity of SARS-CoV-2 highlights the need for sensitive, rapid and on-site diagnostic assays of SARS-CoV-2 with high-throughput testing capability for large-scale population screening. The current detection methods in clinical application need to operate in centralized labs. Though some on-site detection methods have been developed, few tests could be performed for high-throughput analysis. We here developed a gold nanoparticle-based visual assay that combines with CRISPR/Cas12a-assisted RT-LAMP, which is called C as12a-assisted RT- LA MP/AuN P (CLAP) assay for rapid and sensitive detection of SARS-CoV-2. In optimal condition, we could detect down to 4 copies/μL of SARS-CoV-2 RNA in 40 min. by naked eye. The sequence-specific recognition character of CRISPR/Cas12a enables CLAP a superior specificity. More importantly, the CLAP is easy for operation that can be extended to high-throughput test by using a common microplate reader. The CLAP assay holds a great potential to be applied in airports, railway stations, or low-resource settings for screening of suspected people. To the best of our knowledge, this is the first AuNP-based colorimetric assay coupled with Cas12 and RT-LAMP for on-site diagnosis of COVID-19. We expect CLAP assay will improve the current COVID-19 screening efforts, and make contribution for control and mitigation of the pandemic.
研究领域:
分子生物学
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病毒学
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诊断检测
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纳米技术
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公共卫生
✓ 已验证
产品关联
DOI
PubMed
高影响因子
危险品化学品经营许可证（带存储）