JOURNAL OF CONTROLLED RELEASE
·
2025
·
IF 11.5
Tong Wang,Tianyi Chen,Dazhao Li,Xiaoxing Hang,Shuangshuang Zhang,Han Yi,Tianyue Jiang,Dawei Ding,Xuenong Zhang
Cancer stem cells (CSCs) play an important role in the development of triple-negative breast cancer (TNBC), including metastasis, invasion, tumorigenicity, and drug resistance. Moreover, non-CSCs can spontaneously transform into CSCs in special tumor microenvironments, thereby leading to poor prognosis or even failed treatments. Therefore, reversing CSCs into normal tumor cells in a sustained-acting manner is a promising strategy. It has been reported that down-regulation of FBXO44 protein expression inhibits tumor cell stemness. Moreover, CRISPR/Cas9 technology, a well-known precise gene editing tool, was adopted to permanently block FBXO44 within the genome upon its successful implementation. Given this, a core-shell nanoparticle (NP) consisting of amphiphilic polymer core and crosslinked-hyaluronic acid shell (nDOX-PL/pFBXO44 NPs) is developed in this work to concurrently deliver FBXO44-targeted CRISPR/Cas9 plasmids (pFBXO44) and doxorubicin (DOX) for combinational CSC reprogramming and chemotherapy of TNBC, which exhibits tumor cell targeting, endosomal escape, and reduction responsiveness to release DOX and plasmids in the cytoplasma. CRISPR/Cas9-mediated downregulation of FBXO44 expression could convert CSC into normal tumor cells, and effectively inhibit tumor growth without obvious side effects in vivo after combining with chemotherapy. In summary, we developed an intelligent system to co-deliver genetic and hydrophobic drugs, achieving effective cancer stemness reversal and synergistic suppression of contractable TNBC.
研究领域:
肿瘤学
·
基因编辑
·
纳米医学
·
药理学
✓ 已验证
产品关联
DOI
PubMed
高影响因子
Nature Communications
·
2022
·Vol. 13
·期 1
·1-14
·
IF 17.694
Xu Zhichen,Chen Dongjuan,Li Tao,Yan Jiayu,Zhu Jiang,He Ting,Hu Rui,Li Ying,Yang Yunhuang,Liu Maili
Fast, inexpensive, and multiplexed detection of multiple nucleic acids is of great importance to human health, yet it still represents a significant challenge. Herein, we propose a nucleic acid testing platform, named MiCaR, which couples a mi crofluidic device with CRISPR- C as12a a nd multiplex r ecombinase polymerase amplification. With only one fluorescence probe, MiCaR can simultaneously test up to 30 nucleic acid targets through microfluidic space coding. The detection limit achieves 0.26 attomole, and the multiplexed assay takes only 40 min. We demonstrate the utility of MiCaR by efficiently detecting the nine HPV subtypes targeted by the 9-valent HPV vaccine, showing a sensitivity of 97.8% and specificity of 98.1% in the testing of 100 patient samples at risk for HPV infection. Additionally, we also show the generalizability of our approach by successfully testing eight of the most clinically relevant respiratory viruses. We anticipate this effective, undecorated and versatile platform to be widely used in multiplexed nucleic acid detection.
研究领域:
生物技术
·
分子生物学
·
医学诊断
✓ 已验证
DOI
PubMed
高影响因子
ANALYTICA CHIMICA ACTA
·
2022
·
IF 6.911
Xiaofen Zhao,Xiaoxiao Tian,Yuwei Wang,Linbin Li,Yan Yu,Shiqiao Zhao,Juan Zhang
Accurate analysis of HIV DNA is valuable for the diagnosis of AIDS. Herein, an ultrasensitive and specific fluorescence method was developed for HIV-1 DNA detection based on CRISPR-Cas12a-activated palindrome-catalytic hairpin assembly (CRISPR-Cas12a-PCHA). The presence of HIV-1 DNA activated the trans-cleavage activity of CRISPR-Cas12a, which could continuously digest the DNA fragment of hairpins connected to magnetic beads to expose single-stranded RNA . After magnetic separation, the exposed RNA triggered multiple PCHA reactions, generating many Y-shaped DNA structures that were self-assembled into the DNA superstructures via the hybridization of palindromic sticky ends, leading to the release of amounts of fluorescence signal. Different from the reported recently biosensing strategies of nucleic acid amplification technologies-activated CRISPR-Cas12a, CRISPR-Cas12a-PCHA endowed the strategy with unique advantages of simple sample pretreatment , direct duplex target detection, and ultrahigh sensitivity. The strategy was able to resist the interference of the complex matrix in real sample and distinguish between HIV patients and healthy persons. Thus, the method is a promising tool for ultrasensitive and specific detection of HIV-1 DNA for AIDS diagnosis.
研究领域:
生物技术
·
分子生物学
·
医学诊断
✓ 已验证
产品关联
DOI
PubMed
高影响因子
ANALYTICAL AND BIOANALYTICAL CHEMISTRY
·
2024
·
IF 3.8
Qiao Yinuo,Wang Xiaoyan,Fan Zhenlin,Song Yuzhu,Zhang Jinyang,Han Qinqin
Tuberculosis is a highly infectious disease caused by the bacterium Mycobacterium tuberculosis , and the spread of this agent has caused serious health problems worldwide. The rapid and accurate detection of M. tuberculosis is essential for controlling the spread of infection and for preventing the emergence of multidrug-resistant strains. In this study, the powerful trans-cleavage ability of CRISPR-Cas12a for ssDNA was combined with a surface-enhanced Raman spectroscopy (SERS)–based strategy to establish a CRISPR-SERS sensor for the hypersensitive detection of M. tuberculosis DNA. We observed a linear relationship between the concentration of M. tuberculosis DNA and the output signal over the range of 5 to 100 pM. The equation describing the standard curve was y = 24.10 x + 1594, with R 2 = 0.9914. The limit of detection was as low as 4.42 pM for genomic DNA, and a plasmid containing an M. tuberculosis –specific sequence was detected at 5 copy/μL. A detection accuracy of 100% was achieved in the analysis of DNA isolated from the sputum of hospitalized patients with tuberculosis. The entire detection process is simple to deploy and only takes 50 min and results in the sensitive and specific detection of M. tuberculosis DNA. This study provides a new method for the detection of tuberculosis. The tool is stable and can be utilized on-site, and it thus broadens the diagnostic application of CRISPR-Cas12a-based sensor technology.
研究领域:
医学诊断
·
分子生物学
✓ 已验证
产品关联
DOI
PubMed
高影响因子
Device
·
2024
·
IF 0
Siyi Zou,Jingnan Li,Sheng Lu,Danyu Li,Yuxuan Liu,Weiguo Zhang,Xiaolin Cui,J. Justin Gooding,Kang Tian,Guozhen Liu
Inflammation quantification through the detection of cytokines and inflammatory factors in a point-of-care scenario is crucial for managing and preventing diseases. Achieving early-stage inflammation quantification in osteoarthritis (OA), before the radiographic findings become apparent, may provide an opportunity for accurate diagnosis and effective therapy, potentially preventing joint degeneration. Here, we developed a glass-fiber-based universal system with CRISPR-Cas12a-based immunosensors (GUSCI) for simultaneous detection of multiple inflammation biomarkers using a portable fluorescence detection device. GUSCI enables the detection of five inflammation biomarkers (interleukin-4 [IL-4], IL-6, tumor necrosis factor alpha [TNF-α], interferon γ [IFN-γ], and matrix metalloproteinase 3 [MMP-3]) with picomolar sensitivity in a 10-μL sample while having less than 75 min of assay time and a smartphone signal readout. A cohort study of 35 synovial samples from OA patients revealed differences in inflammation levels. This “sample in, answer out” platform not only aids in understanding OA progression but also offers a versatile strategy for point-of-care diagnosis of a wide range of diseases in resource-limited settings.
研究领域:
生物传感器
·
分子诊断
·
炎症疾病
·
骨关节炎
·
即时检测
✓ 已验证
DOI
STAR Protocols
·
2023
·
IF 0
Xiaoqian Zeng,Shuliu Wang,Mindong Liang,Weishan Wang,Yue Jiang,Fei Xu,Leshi Liu,Hao Yan,Yaojun Tong,Lixin Zhang,Gao-Yi Tan
Summary Large biosynthetic gene cluster (BGC) cloning is important for discovering natural product-based drugs and remains challenging in high GC content microorganisms (e.g., Actinobacteria). Here, we present an in vitro CRISPR-Cas12a-mediated protocol for direct cloning of large DNA fragments. We describe steps for crRNA design and preparation, genomic DNA isolation, and CRISPR-Cas12a cleavage and capture plasmid construction and linearization. We then detail target BGC and plasmid DNA ligation and transformation and screening for positive clones. For complete details on the use and execution of this protocol, please refer to Liang et al. 1
研究领域:
分子生物学
·
生物技术
·
基因工程
✓ 已验证
DOI
PubMed
POULTRY SCIENCE
·
2024
·
IF 3.8
Sha-Sha Chen,Yong-Lei Yang,Hong-Yun Wang,Tian-Kui Guo,Riaz-M Azeem,Chun-Wei Shi,Gui-Lian Yang,Hai-Bin Huang,Yan-Long Jiang,Jian-Zhong Wang,Xin Cao,Nan Wang,Yan Zeng,Wen-Tao Yang,Chun-Feng Wang
ABSTRACT Avian influenza virus (AIV) subtype H9N2 has significantly threatened the poultry business in recent years by having become the predominant subtype in flocks of chickens, ducks, and pigeons. In addition, the public health aspects of H9N2 AIV pose a significant threat to humans. Early and rapid diagnosis of H9N2 AIV is therefore of great importance. In this study, a new method for the detection of H9N2 AIV based on fluorescence intensity was successfully established using CRISPR/Cas13a technology. The Cas13a protein was first expressed in a prokaryotic system and purified using nickel ion affinity chromatography, resulting in a high-purity Cas13a protein. The best RPA(Recombinase polymerase amplification) primer pairs and crRNA were designed and screened, successfully constructing the detection of H9N2 AIV based on CRISPR/Cas13a technology. Optimal concentration of Cas13a and crRNA was determined to optimise the constructed assay. The sensitivity of the optimised detection system is excellent, with a minimum detection limit of 10° copies/μL and didn't react with other avian susceptible viruses, with excellent specificity. The detection method provides the basis for the field detection of the H9N2 AIV.
研究领域:
分子生物学
·
病毒学
·
生物技术
·
公共卫生
✓ 已验证
产品关联
DOI
PubMed
高影响因子
JOURNAL OF COLLOID AND INTERFACE SCIENCE
·
2025
·
IF 9.7
Shi-Cheng Tian,Xun-Huan Song,Ke-Ke Feng,Cheng-Lei Li,Yi-Fan Tu,Yong-Shan Hu,Jing-Wei Shao
Photodynamic therapy (PDT) has arisen as a promising method due to its spatiotemporal precision and minimal invasiveness. It encounters significant obstacles in solid tumors due to hypoxia-induced therapeutic resistance and the self-protective mechanisms of cancer cells facilitated by MutT homolog 1 (MTH1), an enzyme involved in oxidative damage repair. Herein, we fabricate a tumor-microenvironment responsive CRISPR nanoplatform based on hollow mesoporous manganese dioxide (H-MnO 2 ) for PDT. This platform utilizes H-MnO 2 to produce oxygen (O 2 ) through the decomposition of hydrogen peroxide (H 2 O 2 ) in TME, thereby mitigating hypoxia and enhancing reactive oxygen species (ROS) generation. The high concentration of glutathione (GSH) and hyaluronidase (HAase) in TME induces the release of CRISPR/Cas9 ribonucleoproteins (RNP) to target the MTH1 gene, thereby impairs oxidative damage repair pathways and amplifys ROS-mediated cytotoxicity. The released Mn 2+ ions function as immunomodulatory agents, activate innate immune responses via stimulating STING signal pathway. In vitro , IHMRH NPs markedly increased intracellular O 2 levels, ROS production, lipid peroxidation and DNA damage, leading to tumor cell death, immune activation, and effective gene editing. In vivo , the nanoplatform suppressed tumor growth, diminished MTH1 gene expression, stimulated dendritic cell (DC) maturation through immunogenic cell death (ICD). This multimodal nanosystem may amplifies oxidative stress, collaborates with innate and adaptive immune activation to surpass the constraints of traditional PDT. The research presents a novel framework for cancer combination therapy by systematically integrating nanotechnology with precision gene editing.
研究领域:
生物医学工程
·
肿瘤学
·
基因治疗
·
免疫学
✓ 已验证
产品关联
DOI
PubMed
高影响因子
MICROCHIMICA ACTA
·
2025
·Vol. 192
·期 2
·1-11
·
IF 5.3
Zhang Xiaoyu,Sun Ruimeng,Zheng Haoran,Qi Yanfei
A spherical nucleic acid (SNA, AuNPs-aptamer) into CRISPR/Cas12a system combined with poly T-template copper nanoparticles as fluorescence reporter was fabricated to establish an amplification-free sensitive method for Staphylococcus aureus ( S. aureus ) detection. This method, named PTCas12a, utilizes the concept that the bifunction of SNA recognizes the S. aureus and triggers the Cas12a cleavage activity. Then, the Cas12a enzyme cleaves the Poly T40 to generate a signal change in Poly T-Cu fluorescence, indicating the presence or absence of the target bacteria. The PTCas12a platform demonstrated a detection limit as low as 3.0 CFU/mL (3 N/S) in a wide response range of 1.0 × 10 1 –1.0 × 10 6 CFU/mL for S. aureus detection, which holds significant potential in ensuring food safety and preventing the spread of diseases. Graphical abstract
研究领域:
生物技术
·
医学诊断
·
食品安全
✓ 已验证
产品关联
DOI
PubMed
高影响因子
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.
研究领域:
分子生物学
·
食品科学
·
诊断技术
✓ 已验证
DOI
高影响因子
ANALYTICA CHIMICA ACTA
·
2024
·
IF 5.7
Tianyi Zhang,Xiaoqin Wang,Yingtao Jiang,Yunyun Zhang,Shuhao Zhao,Jian Hu,Fei Hu,Niancai Peng
Background Nucleic acid testing is the most effective detection method currently available for the diagnosis of respiratory infectious diseases. However, the conventional real-time fluorescent quantitative PCR technique, which is regarded as the gold standard method for nucleic acid detection, presents significant challenges for implementation in home self-testing and popularization in underdeveloped regions due to its rigorous experimental standards. It is therefore clear that an easy-to-use, miniaturized nucleic acid testing technology and products for nonprofessionals are of great necessity to define the pathogens and assist in controlling disease transmission. (87) Results In this study, we propose a strategy for self-testing of respiratory pathogen nucleic acid that is oriented towards the public and user-friendly. The proposed system integrates the processes of extraction-free nucleic acid release, RPA isothermal amplification, and CRISPR fluorescence detection into a compact configuration. A microfluidic testing chip actuated by air pouches and a battery/USB-powered reusable device has been developed to enable simultaneous detection of internal reference genes and viral targets in a fully enclosed condition. The system allows for sample-in, and result-out testing in less than 30 minutes with a detection limit of 2 copies/μL. Additionally, a straightforward signal-light-based result display method has been developed to make it easy and intuitive for users to access the results. Furthermore, freeze-drying reagent is introduced to guarantee the storage and transportation of testing chips in ambient conditions. (135) Significance This work presents a miniaturized, portable, and highly sensitive nucleic acid detection system, where simple operating procedures have been designed for unskilled users. It is our belief that the testing system developed in this work is well suited for home-based self-testing and infection diagnosis in resource-limited areas, due to the above-mentioned advantages. (52)
研究领域:
分子生物学
·
医学检验
·
生物工程
✓ 已验证
产品关联
DOI
PubMed
高影响因子
JOURNAL OF HAZARDOUS MATERIALS
·
2024
·
IF 12.2
Jingyi Yang,Yining Zhao,Lelin Qian,Ying Yu,Yuan Zhang,Juan Zhang
In this work, a dual recognized CRISPR/Cas12a system has been proposed, in which the activation chain is cleverly divided into two parts that can serve for precise dual target recognition, and hydrazone chemistry is introduced for the formation of a whole activation chain. It has been further explored to construct a new method for the specific and sensitive detection of Staphylococcus aureus (SA) as one of the most common pathogens in infectious diseases. In virtue of proximity effect contributed by complementary base pairing, hydrazone chemistry accelerates the formation of the whole activation strand and improves the specificity of the CRISPR/Cas12a system, serving for the accurate analysis of SA. Moreover, the temporary aggregation of CRISPR/Cas12a around SA enhances its catalytical efficiency so as to further amplify signal. With high sensitivity, stability, reproducibility and specificity, the established method has been successfully applied to detect SA in complex substrates. Meanwhile, our established method can well evaluate the inhibition effect of chlorogenic acid and congo red in comparison with flow cytometry. Environmental implication Bacterial pathogens exist widely in the environment and seriously threaten the safety of human health. Staphylococcus aureus (SA) is the most common pathogen of human suppurative infection, which can cause local suppurative infection, pneumonia, and even systemic infections such as sepsis. In this work, a dual recognized CRISPR/Cas12a system mediated by hydrazone chemistry has been proposed. With high sensitivity and low detection limit, the established method can specifically detect SA and effectively evaluate the antibacterial effect of inhibitors. This method is expected to be further developed into a detection method in different scenarios such as environmental monitoring and clinical diagnosis.
研究领域:
分子生物学
·
病原微生物学
·
分析化学
✓ 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）