Journal of Analytical Science and Technology
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2024
·Vol. 15
·期 1
·1-8
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IF 2.5
Li Yunxiao,Wang Qiuxia,Wang Yali
The abnormally expressed microRNAs (miRNAs) serve as crucial indicators for disease diagnosis and are strongly associated with the progression of various diseases. The quantification of miRNAs is highly significant for the clinical diagnosis and treatment of various types of cancers. This study utilizes the CRISPR/Cas13a system, which combines CRISPR RNA (crRNA) and CRISPR-associated Cas13a, to recognize the miRNA directly and specifically, thus activating the trans-cleavage activity of Cas13a. By integrating the CRISPR-Cas13a system with an exonuclease-iii (Exo-iii)-assisted chain cleavage of silver ions (Ag+)-aptamer and an Ag+ -based color reaction, a method for the detection of miRNA that is specific and sensitive is developed. This approach demonstrated exceptional sensitivity in detecting miRNA, with a low detection limit of 5.12 fM. Moreover, the suggested method was effectively utilized to precisely measure the amount of miRNA in intricate biological samples, demonstrating its practical applicability. Furthermore, due to its elevated sensitivity and simple probe design and fluorophore labeling, the suggested colorimetric technique holds great potential for use in clinical diagnostics.
研究领域:
分子生物学
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医学诊断
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生物技术
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产品关联
DOI
高影响因子
ANALYTICA CHIMICA ACTA
·
2024
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IF 5.7
Jihua Wei,Jiayi Zhang,Wei Wang,Haidong Zhou,Huade Ma,Yuanxun Gong,Qianli Tang,Kai Zhang,Xianjiu Liao
The article details a groundbreaking platform for detecting microRNAs (miRNAs), crucial biomolecules involved in gene regulation and linked to various diseases. This innovative platform combines the CRISPR-Cas13a system's precise ability to specifically target and cleave RNA molecules with the amplification capabilities of the hybridization chain reaction (HCR). HCR aids in signal enhancement by creating branched DNA structures. Additionally, the platform employs electrochemiluminescence (ECL) for detection, noted for its high sensitivity and low background noise, making it particularly effective. A key application of this technology is in the detection of miR-17, a biomarker associated with multiple cancer types. It exhibits remarkable detection capabilities, characterized by low detection limits (14.38 aM) and high specificity. Furthermore, the platform's ability to distinguish between similar miRNA sequences and accurately quantify miR-17 in cell lysates underscores its significant potential in clinical and biomedical fields. This combination of precise targeting, signal amplification, and sensitive detection positions the platform as a powerful tool for miRNA analysis in medical diagnostics and research.
研究领域:
生物技术
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分子生物学
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医学诊断
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癌症研究
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DOI
PubMed
高影响因子
JOURNAL OF EXPERIMENTAL & CLINICAL CANCER RESEARCH
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2024
·Vol. 43
·期 1
·1-19
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IF 11.4
Liang Mingkang,Wang Yongqiang,Liu Lisha,Deng Dashi,Yan Zeqin,Feng Lida,Kong Chenfan,Li Chenchen,Li Yuqing,Li Guangzhi
Background CRISPR-Cas13a is renowned for its precise and potent RNA editing capabilities in cancer therapy. While various material systems have demonstrated efficacy in supporting CRISPR-Cas13a to execute cellular functions in vitro efficiently and specifically, the development of CRISPR-Cas13a-based therapeutic agents for intravesical instillation in bladder cancer (BCa) remains unexplored.
Methods In this study, we introduce a CRISPR-Cas13a nanoplatform, which effectively inhibits PDL1 expression following intravesical instillation. This system utilizes a fusion protein CAST, created through the genetic fusion of CRISPR-Cas13 and the transmembrane peptide TAT. CAST acts as a potent transmembrane RNA editor and is assembled with the transepithelial delivery carrier fluorinated chitosan (FCS). Upon intravesical administration into the bladder, the CAST-crRNAa/FCS nanoparticles (NPs) exhibit remarkable transepithelial capabilities, significantly suppressing PDL1 expression in tumor tissues.To augment immune activation within the tumor microenvironment, we integrated a fenbendazole (FBZ) intravesical system (FBZ@BSA/FCS NPs). This system is formulated through BSA encapsulation followed by FCS coating, positioning FBZ as a powerful chemo-immunological agent. Results In an orthotropic BCa model, the FBZ@BSA/FCS NPs demonstrated pronounced tumor cell apoptosis, synergistically reduced PDL1 expression, and restructured the immune microenvironment. This culminated in an enhanced synergistic intravesical instillation approach for BCa. Consequently, our study unveils a novel RNA editor nanoagent formulation and proposes a potential synergistic therapeutic strategy. This approach significantly bolsters therapeutic efficacy, holding promise for the clinical translation of CRISPR-Cas13-based cancer perfusion treatments.
研究领域:
癌症治疗
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分子生物学
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纳米技术
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免疫学
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产品关联
DOI
PubMed
高影响因子
ANALYTICA CHIMICA ACTA
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2022
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IF 6.911
Xin Guo,Tian Tian,Xinchun Deng,Yang Song,Xiaoming Zhou,Erqun Song
Development of rapid and accurate detection of miRNAs in complex samples is of great significance for potential early diagnosis of disease. Herein, we report a magnetic relaxation switching (MRS)-based strategy for direct detection of miRNAs in complex samples via the assistance of signal amplification system of CRISPR/Cas13a which has the ability to specifically recognize target RNA. In the designed strategy, 30 nm-magnetic nanoparticles (MB 30 ) and 1000 nm-magnetic particles (MM 1000 ) linked by single-strand RNA 1 complexes (MB 30 -RNA 1 - MM 1000 ) were employed as signal probe. After the target miRNAs (taking miR-21 as model) recognition by CRISPR/Cas13a system, the resulted trans -cleavage degrades the MB 30 -RNA 1 -MM 1000 , releasing MB 30 which caused transverse relaxation time (T 2 ) signal change. The combination of CRISPR/Cas13a assisted signal amplification and the MRS assay achieved direct detection of miR-21 in the serum sample without extracting within 60min, with a detection limit of 0.22 pM. Moreover, the detection accuracy is confirmed by performing the detection of miR-21 using qRT-PCR. The CRISPR/Cas13a system assisted MRS assay successfully achieved accurate, simple, and rapid detection of miRNAs in complex samples, showing great potential for detection miRNAs in potential clinical applications.
研究领域:
分子生物学
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医学诊断
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生物技术
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产品关联
DOI
PubMed
高影响因子
ANALYST
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2025
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IF 3.3
Yang Li,Yikan Zhao,Zhihao Yi,Shitong Han
Timely and accurate diagnosis of RNA viruses, represented by the SARS-CoV-2, is the foundation for protecting people from health threats. Currently, direct detection of viral RNA genes remains the most accurate method. Herein, a rapid, ultrasensitive, and no heating equipment required CRISPR/Cas13a based evanescent wave fluorescence biosensing platform for quantitative detection of the SARS-CoV-2 gene is reported. The collateral effect of CRISPR/Cas13a for RNA is combined with a self-driven enzyme-free hybridization chain reaction (HCR) as a signal amplification step. When the initiator RNA strand is cleaved by Cas13a, the downstream signal amplification induced by HCR is blocked, and a multiple crRNA strategy is used to enhance the cleavage efficiency. The newly designed HCR assemblies are captured by the cDNA-modified optical fiber and generate a higher-intensity fluorescence signal induced by the evanescent field. The CRISPR/Cas13a-HCR evanescent wave fluorescence biosensing platform is capable of detection of SARS-CoV-2 with a LOD of 0.47 copies per μL and the detection time is within 35 min. The spike recovery tests in saliva and high specificity have demonstrated the potential of this method for point-of-care diagnosis. This method is also suitable for the detection of other RNA viruses, without the need to alter any design of the HCR component, and only the corresponding crRNA needs to be replaced.
研究领域:
生物技术
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医学诊断
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病毒学
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产品关联
DOI
PubMed
高影响因子
APPLIED BIOCHEMISTRY AND BIOTECHNOLOGY
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2024
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IF 3.1
Yan Na,Hu Zhiwei,Zhang Lingling
The development of a bio-sensing strategy based on CRISPR/Cas that is exceptionally sensitive is crucial for the identification of trace molecules. Colorimetric miRNA detection utilizing CRISPR/Cas13a-triggered DNAzyme signal amplification was described in this article. The developed strategy was implemented for miRNA-21 detection as a proof of concept. The cleavage activity of Cas13a was triggered when the target molecule bonded to the Cas13a-crRNA complex and cleaved uracil ribonucleotides (rU) in the substrate probe. As a consequence, the S chain was liberated from the T chain that had been modified on magnetic beads (MB). The G-rich sections were then exposed when the catalytic hairpin assembly between the H1 and H2 probes was activated by the released T@MB. G-rich section can fold into G-quadruplex. By catalyzing the formation of green ABTS 3 – via HRP-mimicking G-quadruplex/hemin complexes, colorimetric measurements of miRNA can be achieved visually through DNAzyme-mediated signal amplification. The method demonstrated a low limit of detection of 27 fM and a high selectivity towards target miRNA eventually. As a result, the developed strategy provides a clinical application platform for the detection of miRNAs that is both ultrasensitive and extremely specific.
研究领域:
生物技术
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分子生物学
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诊断方法
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DOI
PubMed
高影响因子
POULTRY SCIENCE
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2024
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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
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病毒学
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生物技术
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公共卫生
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产品关联
DOI
PubMed
高影响因子
BIOSENSORS & BIOELECTRONICS
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2024
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IF 10.7
Chengchen Tan,Guoyang Xie,Song Wu,Cailu Song,Jinhui Zhang,Xinyao Yi,Jianxiu Wang,Hailin Tang
Breast cancer is reported to be one of the most lethal cancers in women, and its multi-target detection can help improve the accuracy of diagnosis. In this work, a cluster regularly interspaced short palindromic repeats (CRISPR)-Cas13a/Cas12a-based system was established for the simultaneous fluorescence detection of breast cancer biomarkers circROBO1 and BRCA1. CRISPR-Cas13a and CRISPR-Cas12a were directly activated by their respective targets, resulting in the cleavage of short RNA and DNA reporters, respectively, thus the signals of 6-carboxyfluorescein (FAM) and 6-carboxy-xrhodamine (ROX) were restored. As the fluorescence intensities of FAM and ROX were dependent on the concentrations of circROBO1 and BRCA1, respectively, synchronous fluorescence scanning could achieve one-step detection of circROBO1 and BRCA1 with detection limits of 0.013 pM and 0.26 pM, respectively. The system was highly sensitive and specific, holding high diagnostic potential for the detection of clinical samples. Furthermore, the competing endogenous RNA mechanism between circROBO1 and BRCA1 was also explored, providing a reliable basis for the intrinsic regulatory mechanism of breast cancer.
研究领域:
分子生物学
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癌症研究
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生物技术
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医学诊断
✓ 已验证
产品关联
DOI
PubMed
高影响因子
BIOELECTROCHEMISTRY
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2025
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IF 4.5
Zhaofan Zhou,Ye Li,Dongya Wen,Xuyu Zhang,Ni Li,Jiangbing Shuai,Xiaofeng Zhang,Xiaoye Zheng,Zhaojun Ban,Farouk Amr,Xiaoping Yu,Jun Huang
Porcine epidemic diarrhea virus (PEDV) is a highly infectious pathogen responsible for porcine epidemic diarrhea, which causes severe diarrhea and high mortality rates in neonatal piglets, leading to substantial economic losses in the swine industry. Therefore, there is an urgent need for rapid, sensitive, and accurate detection methods for PEDV. In this study, we develop a rapid, ultrasensitive electrochemical CRISPR-based biosensor for detecting PEDV with high sensitivity and specificity. The sensor integrates reduced graphene oxide-polypyrrole‑gold nanoparticle (rGO-PPy-AuNP) nanocomposites to enhance sensitivity and CRISPR-Cas13a for target-specific recognition. This dual-signal amplification strategy achieves an excellent analytical detection limit of 1.01 fg/mL and high linearity (R 2 = 0.9979) across a broad dynamic range (0.005–100,000 pg/mL) within 45 min, eliminating the need for nucleic acid amplification. By synergizing Cas13a-assisted signal enhancement with nanoparticle-mediated electron transfer, the biosensor outperforms conventional methods in sensitivity while maintaining excellent stability, and specificity, demonstrating strong potential for PEDV detection in clinical diagnostics.
研究领域:
分子生物学
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生物传感技术
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兽医学
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产品关联
DOI
PubMed
高影响因子
SENSORS AND ACTUATORS B-CHEMICAL
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2024
·
IF 8
Xin Gao,Yun Yin,Junyu Xie,Shaohua Gong,Xuemei Li
CRISPR/Cas13a system has been combined with different isothermal amplification strategies for RNA analysis. Nevertheless, expensive enzymes are usually indispensable in these isothermal amplification methods, which increases the detection cost. In this study, a CRISPR/Cas13a-mediated triple signal amplification strategy has been designed for selective, sensitive and inexpensive detection of viral RNA. SARS-CoV-2 S RNA activated CRISPR/Cas13a to cut hairpin DNA, triggering catalytic hairpin assembly (CHA) and subsequent hybridization chain reaction (HCR) to form a long-stranded Y-type DNA. G-quadruplexes were distributed on the branch of the long-stranded DNA and bound with N-methyl mesoporphyrin IX (NMM) to enhance the fluorescence signal. SARS-CoV-2 S RNA was specifically detected with a limit of detection (LOD) of 285 fM. Notably, no additional expensive enzymes were required in the entire detection process. The CRISPR/Cas13a-mediated triple signal amplification strategy holds great potential for specific and sensitive viral RNA detection with low cost.
研究领域:
生物技术
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分子生物学
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病毒学
✓ 已验证
产品关联
DOI
高影响因子
SENSORS AND ACTUATORS B-CHEMICAL
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2022
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IF 9.221
Kai Zhang,Meina Yang,Helin Sun,Xueyin Wang,Hao Li,Jinxiang Han,Zhongwen Zhang
Using Peptide nucleic acid (PNA), this work provides a unique approach for controlling CRISPR/Cas13a activity by RNA Inhibitory PNA (RIP). This method reduces the action of CRISPR/Cas13a by inhibiting the binding of crRNA to CRISPR/Cas13a, which was subsequently utilized to detect metalloproteinase-2 (MMP-2) activity. We carefully constructed a PNA sequence that includes a peptide sequence (Gly-Pro-Leu-Gly-Val-Arg-Gly) that is particularly cleaved by MMP-2 in the middle. When MMP-2 is not present in the system, the PNA/crRNA complex reduces crRNA binding to CRISPR/Cas13a, hence inhibiting CRISPR/Cas13a cleavage activity. When MMP-2 is present in the system, it cleaves the peptide sequence (Gly-Pro-Leu-Gly-Val-Arg-Gly) selectively and so prevents PNA from binding to crRNA, enabling crRNA to reach the "free" form. Then, in the presence of an RNA activator, crRNA may bind to CRISPR/Cas13a and stimulate its activity. CRISPR/Cas13a triggered in this manner may precisely cleave the DNA sequence containing modified -UU- bases on the electrode surface . Thus, the ferrocene (Fc) is changed away from the electrode surface at one end of the DNA , enabling for the recovery of the Electrochemiluminescence (ECL) signal from the two-dimensional material transformed on the electrode surface. As a result, MMP-2 activity was detected utilizing this technique of inhibiting crRNA activity with a detection limit of 15.56 fM. Our suggested method of inhibiting CRISPR/Cas13a activity via PNA has a broad variety of potential therapeutic applications in gene editing and the development of CRISPR/Cas13a-based biosensors.
研究领域:
分子生物学
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基因编辑
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生物传感技术
✓ 已验证
产品关联
DOI
高影响因子
ANALYTICA CHIMICA ACTA
·
2020
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IF 5.977
Jin Zhou,Lijuan Yin,Yanan Dong,Lei Peng,Guozhen Liu,Shuli Man,Long Ma
The severity of foodborne diseases caused by foods contaminated by pathogens or their toxins creates an urgent need for the development of specific and sensitive method for detection of bacteria. In this study, taking advantages of CRISPR-Cas13a system, namely, the crRNA programmability and Cas13a “collateral effect” of promiscuous RNase activity upon target RNA recognition, we developed a bacterial sensing strategy with the name of CCB-Detection ( C RISPR- C as13a based B acterial Detection ). Staphylococcus aureus ( S. aureus ) was chosen as a model bacteria for validating the performance of CCB-Detection. Specifically, four steps were carried out: 1) simple extraction of genome DNA ; 2) specific gene amplification by PCR; 3) in vitro transcription; and 4) the “collateral effect” cleavage of reporter RNA to report the analyte signal. It was observed that CCB-Detection was capable to successfully detect the target genomic DNA (gDNA) as low as 10 0 aM. The limit of detection (LOD) was 1 CFU/mL with a dynamic detection range of S. aureus from 10 0 to 10 7 CFU/mL. The entire sample-to-answer time for this biosensor was less than 4 h. CCB-Detection demonstrated satisfactory selectivity for S. aureus without interference from other bacteria. Furthermore, CCB-Detection was successfully applied for sensing S. aureus in real food samples with both known and unknown amounts bacteria (spiked ones and non-spiked ones) and its performance is comparable to the conventional culture-based counting method but with short assay time and high sensitivity. With desirable reliability, sensitivity, specificity and simplicity, herein proposed CCB-Detection could be extended and generalised for other bacterial detection, and has great potential to be used in a wide range of applications such as food safety inspection, disease diagnosis, environment monitoring, etc .
研究领域:
食品科学
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微生物学
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分子生物学
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生物技术
✓ 已验证
DOI
PubMed
高影响因子
危险品化学品经营许可证（带存储）