Autophagy
·
2026
·
IF 18.6
Hao Xu,Hailong Liu,Taozhen Lu,Yuken Chen,Yazhou Li,Yuchao Zhu,Liting Wei,Bolin Fan,Yonghui Zhang,Chuangshi Zhang,Rui Fang,Shengsong Xie,Bang Shen
Toxoplasma gondii is a globally prevalent foodborne zoonotic pathogen that threatens animal production and human health. Consumption of undercooked meat like pork and lamb is a major route of human infection. In this study, we performed a genome-wide CRISPR knockout screen in the porcine cell line PK15 to identify host factors that are critical for T. gondii replication. The results showed that disrupting the ELFN2 (extracellular leucine rich repeat and fibronectin type III domain containing 2) gene in PK15 did not affect host cell growth, but significantly reduced the proliferation of Toxoplasma parasites. Loss of ELFN2 decreased macroautophagy/autophagy in PK15 cells and impaired lipid metabolism, resulting in reduced lipid availability for the parasites and consequent suppression of T. gondii proliferation. Exogenous lipid supplementation or pharmacological activation of autophagy could fully restore the replication of parasites in ΔELFN2 cells. The requirement of host ELFN2 for optimal parasite proliferation in vivo was validated by constructing elfn2−/− mice, which showed increased resistance to T. gondii infection and reduced parasite burden, highlighting the value of ELFN2 in breeding Toxoplasma-resistant animals. Notably, naturally occurring loss-of-function mutations in ELFN2 could be found in certain pig breeds, further indicating the feasibility of breeding T. gondii-resistant animals like pigs, to reduce the transmission of Toxoplasma.
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ANALYTICAL CHEMISTRY
·
2020
·
IF 6.785
Xuan Sun,Yu Wang,Lu Zhang,Sha Liu,Man Zhang,Jiang Wang,Baoan Ning,Yuan Peng,Jing He,Yonggang Hu,Zhixian Gao
Escherichia coli O157:H7 has been reported as an important pathogenic bacteria causing serious infection and economic loss. However, detection of Escherichia coli O157:H7 needs improvement, given its current complexity and sensitivity. Herein, we attempt to build a fluorescence sensing method to detect Escherichia coli O157:H7 with easy operation and high efficiency. The target virulence gene sequences are recognized and cleaved by the CRISPR-Cas9 system, and trigger strand displacement amplification and rolling circle amplification. After amplification reactions, massive products can hybridize with the probes, the fluorescence of which are quenched based on a metal–organic framework platform, leading to the fluorescence recovery at typical excitation/emission wavelengths of 480/518 nm. This method exhibits high sensitivity with the detection limit at 4.0 × 101 CFU mL–1 and a wide range from 1.3 × 102 CFU mL–1 to 6.5 × 104 CFU mL–1. Meanwhile, this assay also shows significant specificity and applies to practical samples with high accuracy. Therefore, our method would have great potential application in bacterial detection, food safety monitoring, or clinical diagnostics.
研究领域:
微生物学
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生物技术
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食品科学
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分子生物学
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分析化学
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Advanced Healthcare Materials
·
2022
·Vol. 11
·期 16
·2201038
·
IF 11.092
Lin Huang,Mengyang Zhou,Ghulam Abbas,Chao Li,Mengmeng Cui,Xian-En Zhang,Dian-Bing Wang
Bimodal synergistic therapy produces superadditive effect for enhanced therapeutic efficacy. However, how to efficiently and simultaneously deliver several kinds of therapeutic agents is still challenging. A cancer cell membrane-derived nanocarrier (mCas9-sGNRs) is proposed for synergistic photothermal/gene therapy (PTT/GT) by efficient delivery of clustered regularly interspaced short palindromic repeat (CRISPR)/CRISPR-associated protein 9 (Cas9) and gold nanorods (GNRs). In this approach, Cas9 proteins can be efficiently loaded inside the cell membranes (mCas9) by electrostatic interactions. Similarly, single-guide RNAs, which target survivin, can be loaded onto GNRs (sGNRs) through electrostatic interactions and encapsulated by mCas9. As a result, the nanodelivery systems present advantages in biocompatibility, homologous targeting capacity and loading efficiency of cargoes. In addition, significant antitumor effects is achieved by gene editing of survivin which induces anticancer activity and reduces heat tolerance of cancer cells caused by GNRs mediated PTT due to the downregulation of HSP70. These results indicate the nanotherapeutic platform leads to enhanced PTT/GT efficacy. Therefore, this work not only provides a general strategy to construct a versatile nanoplatform for loading and target delivery of several therapeutic cargos but will also be valuable for PTT/GT and other bimodal synergistic therapy.
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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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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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SENSORS AND ACTUATORS B-CHEMICAL
·
2020
·
IF 7.1
Xianfeng Wang,Xiaolong Chen,Chengxiang Chu,Yuanyi Deng,Mei Yang,Zhong Ji,Faliang Xu,Danqun Huo,Yang Luo,Changjun Hou
High-performance detection of adenosine triphosphate (ATP) is of great importance due to its irreplaceable roles in various physiological processes. In this work, a four-stage signal amplification was established for ATP detection based on an allosteric probe-conjugated strand displacement amplification (SDA) integrated with CRISPR/Cpf1 system (ASD-Cpf1). An allosteric probe (AH) with a hairpin structure was designed to reduce background interference from self-extension and non-specific amplification during the SDA reaction. ATP bound with the aptamer segment at 5′-terminal of AH probe and subsequently opened the AH probe to activate the SDA reaction. The amplification products were recognized by CRISPR/Cpf1 system and triggered the ssDNase activity of Cpf1, resulting in robust cleavage of surrounding DNA FQ-reporter (fluorophore-quencher labeled short single-stranded DNA sequence). With the four-step amplification, ATP was measured with high sensitivity and low background interference. Under the optimal conditions, a linear relationship was obtained between the fluorescence response and ATP concentrations ranging from 2 pM to 10 μM with a limit of detection (LOD) as low as 1.8 pM. Successful detection of ATP in diluted human serum samples further indicated that ASD-Cpf1 system provided a potential platform for detection of small molecules.
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BIOSENSORS & BIOELECTRONICS
·
2024
·
IF 10.7
Han Jiang,Xiaofan Zhu,Jingran Jiao,Chao Yan,Kaiyong Liu,Wei Chen,Panzhu Qin
Accurate detection of multiple pathogens in the early stages of infection is critical for guiding treatment and saving patients' lives, but current methods are still challenged by low sensitivity, poor robustness and long turnaround times. Here, we report a CRISPR/dCas9-based hotspot self-assembling surface-enhanced Raman scattering (SERS) biosensor (called dCasSERS) and its integration with a smartphone to address these challenges. In this design, bacterial DNA was pre-amplified by loop-mediated isothermal amplification (LAMP), and the repeat sequences of the amplicons were recognized by CRISPR/dCas9, providing abundant sites for the assembly of gold nanoparticles (AuNPs) and forming numerous hotspots for SERS analysis. Using AuNPs labeled with different Raman molecules as reporters, the presence of Staphylococcus aureus ( S. aureus ), Pseudomonas aeruginosa ( P. aeruginosa ) and Escherichia coli O157:H7 ( E. coli O157:H7) could be dexterously converted into distinguishable SERS signals. CRISPR/dCas9-based amplicon-specific recognition and SERS hotspot self-assembly improved the specificity and sensitivity, enabling the biosensor to simultaneously detect three target pathogens down to 1 CFU/mL without any cross-reactivity. By introducing a rapid extraction procedure and a smartphone-integrated handheld Raman spectrometer, rapid on-site analysis of multiple pathogens could be achieved in less than 50 min. The accuracy and robustness of the biosensor were demonstrated by 500 real urine specimens. This study displays a new paradigm for CRISPR/Cas-based self-assembly of SERS hotspots and provides insight into the future development of pathogen screening tools.
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MICROCHEMICAL JOURNAL
·
2024
·
IF 4.9
Yuanxun Gong,Jiayi Zhang,Huiying Liang,Yanling Wang,Zichun Song,Jihua Wei,Qianli Tang,Kai Zhang,Xianjiu Liao
Matrix metalloproteinases (MMPs) are critical for the degradation of extracellular matrix components, which is essential for processes such as tissue remodeling, wound healing, and angiogenesis. MMP-2, in particular, degrades type IV collagen and is associated with diseases such as cancer and cardiovascular conditions, making it a significant biomarker. This study presents a novel electrochemical biosensor for the detection of MMP-2. The biosensor combines the specificity of a programmable RNA detection system with the amplification capabilities of T7 RNA polymerase and CRISPR/Cas12a. In the absence of MMP-2, Peptide Nucleic Acid (PNA) adsorbs on the DNA template, preventing RNA amplification and resulting in a low electrochemical signal. When MMP-2 is present, it cleaves a specific peptide, releasing the DNA template and enabling RNA synthesis. This activation leads to the cleavage of single-stranded DNA probes, increasing the electrochemical signal. The biosensor demonstrates high sensitivity with a detection limit of 0.496 fM for MMP-2 and excellent specificity, effectively distinguishing MMP-2 from other proteins. It successfully detected MMP-2 in biological samples, such as liver cancer cell supernatants, indicating its potential for clinical diagnostics. This innovative biosensor offers a powerful tool for the early detection and monitoring of diseases associated with MMP-2 activity.
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Theranostics
·
2025
·Vol. 15
·期 7
·2870
·
IF 13.3
Xuena Zhu,Shanshan Wang,Yuanyuan Xue,Xiaoyan Wang,Shaoqi Hu,Tingbo Liang,Wenjun Liu
Significant efforts and resources have been dedicated to developing CRISPR-Cas based point-of-care testing (POCT) and self-diagnosis methods for nucleic acid pathogens, aiming to complement the gold standard quantitative PCR tests, particularly in settings where centralized facilities, trained personnel, or resource-intensive equipment are unavailable. However, the reliance on stationary, high-cost readout machinery hinders their full deployment at the point of care. We aimed to develop a solid-phase invertase-labeled reporter (ILR) system that enables convenient readout of CRISPR-Cas reactions, facilitate HPV detection in a POCT-compatible manner. Methods: Through multiple chemical couplings, invertase is immobilized onto magnetic microbeads via a nucleic acid linker that responds to target nucleic acid-induced CRISPR-Cas activation. This activation releases active invertase, which then converts sucrose to glucose in proportion to the target's abundance. Enzymatic signal amplification by Cas12a/Cas13a and invertase compensates for the moderate sensitivity of personal glucose meters (PGMs). Results: When applied to human papillomavirus detection, the HPV18-targeted LAMP-Cas12a/ILR/PGM system can detect as few as 7 HPV18-positive HeLa cells out of 7,000, achieving 95.8% sensitivity and 100% specificity in cervical cell samples. Furthermore, minimal reagent adjustments allow for the rapid establishment of HPV16 and HPV52-targeted LAMP-Cas12a/ILR/PGM systems, offering satisfactory sensitivity, specificity, and cross-species detection. Conclusion: These findings demonstrate a highly efficient testing platform for a range of nucleic acid pathogens, suitable for both point-of-care and household use.
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MICROCHEMICAL JOURNAL
·
2024
·
IF 4.9
Jun Wang,Xueting Cui,Jing Zhang,Juan Li,Bo Pang,Jinhua Li
Microcystin-LR (MC-LR) is a significant environmental pollutant that presents a substantial risk to public health. The monitoring with simple approaches is essential for the prevention and management of MC-LR. In this study, we present an aptasensor platform utilizing recombinase polymerase amplification (RPA)-CRISPR/Cas12a technology to accurately identify MC-LR. The MC-LR aptamers were attached to magnetic beads (MBs) following combination with cDNA by hybridization, resulting in the formation of the MB aptasensor. Subsequently, glucose oxidase (GOx)-modified MB were synthesized using single-stranded DNA (MB-ssDNA-GOx) in order to induce alterations in color upon exposure to the TMB-glucose system. The colorimetric signal was visible to the naked eye and exhibited a positive correlation with MC-LR concentration. The sensing platform described in this study demonstrated great sensitivity and achieved a limit of detection of 0.1 μg/L. Furthermore, this methodology exhibited exceptional selectivity and favorable recovery rates, hence showcasing its strong suitability for the analysis of actual water samples. Hence, the assay exhibites significant potential for future environmental monitoring.
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CHEMICAL ENGINEERING JOURNAL
·
2024
·
IF 13.3
Yuexin Wang,Bo Shen,Nini Luo,Cai Li,Haiping Wu,Yanshuang Wang,Shen Tian,Xuemiao Li,Rui Liu,Xinmin Li,Junman Chen,Wei Cheng,Shijia Ding,Rui Chen,Meifang Xiao,Qianfeng Xia
The simple, rapid, and accurate detection of highly lethal melioidosis is crucial for early clinical diagnosis and improving cure rates. Currently, due to time consumption, low sensitivity and detection rate the existing clinical detection methods cannot satisfy the needs of clinical diagnosis. Herein, a novel self-enhanced porous hydrogel material (Au@PEI-ABEI@Pt) for ultrasensitive ECL strategy of detection B. pseudomallei was report. The novel porous hydrogel material composed of PEI-ABEI porous hydrogel, gold nanoparticles (AuNP) and platinum nanoparticles (PtNP) have large specific surface area and porous structure, which not only fix more ABEI to realize self-enhanced ECL signal amplification, but also facilitate ion diffusion and efficient utilization of catalytic materials, realizing rapid electron transfer and zero-distance catalysis, significantly improving the initial signal of ECL sensor. Moreover, the most attractive aspect is that Au@PEI-ABEI@Pt hydrogels with good biocompatibility can achieve widespread application of CRISPR/Cas12a in solid-phase carriers without affecting the sensitivity, specificity and shearing activity of CRISPR/Cas12a. After coupling with the ECL system and CRISPR/Cas12a signal amplification strategy, the Au@PEI-ABEI@Pt can achieve an ultrasensitive ECL assay of B. pseudomallei with the LOD of 5 CFU mL −1 in complex samples, with high specificity and stability to effectively classify B. pseudomallei and other Gram-negative bacteria. This study shows that the developed porous hydrogel materials not only serve as an excellent ECL signal reporter to significantly improve the detection sensitivity of ECL biosensors, but also provide a new approach for the wide application of CRISPR/Cas systems in solid-phase carriers.
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Analytical Methods
·
2025
·
IF 2.6
Zixun Wang,Dong Zhang,Yuxuan Wu,Xu Ye,Yayun Qian,Fengsong Chen
The low early diagnosis rate of hepatocellular carcinoma (HCC) severely impacts patient prognosis, making the development of highly sensitive and specific early diagnostic technologies crucial. MicroRNA-221 (miR-221), an aberrantly overexpressed biomarker in HCC, holds significant diagnostic potential. This paper constructed a capillary surface-enhanced Raman scattering (SERS) sensing platform utilizing CRISPR/Cas13a trans-cleavage and double-shell gold-silver nanorods (DS Au-AgNRs) to detect serum miR-221 in HCC patients. DS Au-AgNRs were synthesized and assembled onto aminated capillaries, followed by conjugation of Cy5-labeled single-stranded DNA (ssDNA) to the DS Au-AgNR surface via Au–S bonds. In the presence of miR-221, activated CRISPR/Cas13a trans-cleavage cleaves the ssDNA, releasing Cy5 from the sensor surface and diminishing the SERS signal, enabling miR-221 quantification. The synthesized DS Au-AgNRs exhibit uniform morphology and size, are uniformly distributed on the capillary, and form numerous “hotspots”, thereby significantly enhancing the SERS signal. According to the characteristic peak of Cy5 at 1074 cm−1, a linear relationship is established between the log concentration of miR-221 and the measured SERS intensity (y = −3527.97 × −35369.60, R2 = 0.97767), with a LOD as low as 4.17 × 10−17 M. The sensor demonstrated high specificity and high sensitivity, and its capacity to detect miR-221 expression aligned with qRT-PCR results when analyzing serum samples, confirming that hepatocellular carcinoma patients exhibited significantly higher miR-221 levels compared to healthy individuals. The capillary SERS sensor thus provides an accurate and convenient approach for early HCC diagnosis.
研究领域:
生物医学工程
·
分子诊断
·
纳米生物技术
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癌症生物标志物
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表面增强拉曼散射（SERS）
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CRISPR技术
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