ACS Synthetic Biology
·
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
·
基因治疗
·
生物技术
·
药学
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产品关联
DOI
PubMed
高影响因子
BIOSENSORS & BIOELECTRONICS
·
2022
·
IF 12.545
Yang Sun,Cheng Yang,Xiaolin Jiang,Pengbo Zhang,Shuo Chen,Fengxia Su,Hui Wang,Weiliang Liu,Xiaofei He,Lei Chen,Baoyuan Man,Zhengping Li
False detection of SARS-CoV-2 is detrimental to epidemic prevention and control. The scalar nature of the detected signal and the imperfect target recognition property of developed methods are the root causes of generating false signals. Here, we reported a collaborative system of CRISPR-Cas13a coupling with the stabilized graphene field-effect transistor, providing high-intensity vector signals for detecting SARS-CoV-2. In this collaborative system, SARS-CoV-2 RNA generates a “big subtraction” signal with a right-shifted feature, whereas any untargets cause the left-shifted characteristic signal. Thus, the false detection of SARS-CoV-2 is eliminated. High sensitivity with 0.15 copies/μL was obtained. In addition, the wide concerned instability of the graphene field-effect transistor for biosensing in solution environment was solved by the hydrophobic treatment to its substrate, which should be a milestone in advancing it's engineering application. This collaborative system characterized by the high-intensity vector signal and amazing stability significantly advances the accurate SARS-CoV-2 detection from the aspect of signal nature.
研究领域:
生物传感
·
纳米技术
·
分子生物学
·
电子学
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产品关联
DOI
PubMed
高影响因子
TALANTA
·
2025
·
IF 6.1
Haixiang Li,Shuo Lian,Zhiwei Zhang,Weiye Bi,Qingyou Meng,Qingwei Ding
Brain natriuretic peptide (BNP) is considered a reliable marker of heart failure disease, and its timely detection can provide important pathological information to prevent or treat heart failure. In this article, an electrochemiluminescence (ECL) sensing device based on a boron carbon nitride/gold nanoparticle (BCN/AuNPs) complex is developed to determine BNP. Prominently, the CRISPR/CAS 13a enzyme reverse cleavage mode, the entropy-driven and hammer hybridization chain reaction processes were involved in the entire detection scheme. Ultimately, with multiple reaction methods and amplification reactions of nucleic acids, this ECL sensing device is able to achieve a detection limit as low as of 0.03 pg/mL and linear range from 0.1 pg/mL to 30 ng/mL for BNP. In addition, the ECL sensing device based on BCN/AuNPs complex obtained satisfactory stability and specificity, and can also be extended to the detection of other pathological markers.
研究领域:
生物医学工程
·
分子诊断学
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产品关联
DOI
PubMed
高影响因子
ANALYTICA CHIMICA ACTA
·
2025
·
IF 6
Zhongheng Wei,Huini Luo,Danlu Huang,Peixin Wei,Jiayi Zhang,Jihua Wei,Qianli Tang,Longjian Huang,Kai Zhang,Xianjiu Liao
Flap endonuclease 1 (FEN1) is crucial for DNA replication, repair, and telomere maintenance. Its dysregulation is linked to various cancers and diseases. Accurate detection of FEN1 is essential for early diagnosis and therapeutic monitoring. Thus, a novel electrochemiluminescent (ECL) biosensor has been developed for the structure-specific and detection of FEN1. The strategy integrates dumbbell DNA probe-mediated transcription and CRISPR/Cas13a-induced trans-cleavage of RNA G-quadruplexes. In the presence of FEN1, the 5′-flap structure of the probe was selectively cleaved and subsequently ligated by T4 DNA ligase to form a closed circular template. This enabled T7 RNA polymerase to transcribe crRNA-encoded RNA strands, which activated Cas13a to cleave surface-tethered G-quadruplexes/hemin complexes on a Ru(II)/Ti 3 C 2 /AuNPs-modified electrode, thereby restoring the quenched ECL signal. The biosensor exhibited an ultralow detection limit of 4.82 × 10 -9 U μL -1 and a wide dynamic range (1 × 10 -8 to 1 × 10 -5 U μL -1 ), along with excellent specificity and stability. Successful application in human serum validated its reliability for complex biological samples. This work presents a powerful platform for sensitive FEN1 monitoring, holding potential for clinical diagnostics and enzymatic analysis.
研究领域:
生物分析化学
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分子诊断
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癌症研究
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酶检测
·
生物传感器与生物电子学
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产品关联
DOI
PubMed
高影响因子
SENSORS AND ACTUATORS B-CHEMICAL
·
2022
·
IF 9.221
Li Shuofeng,Wang Fangfang,Chun Wang,Zhi Wang,Qiuhua Wu
As vital biomarkers for diagnosis of diseases, the anomalously expressed microRNAs (miRNAs) are closely linked to the occurrence and development of many diseases. Quantitative analysis of miRNAs has remarkable significance to clinical diagnosis and therapy of different cancers. In this study, the clustered regularly interspaced short palindromic repeats (CRISPR) and CRISPR-associated (Cas) 13a (CRISPR/Cas13a) system was first exploited to directly and specifically recognize the miRNA at the guidance of CRISPR RNA (crRNA), which can activate the nonspecific trans-ribonuclease activity of Cas13a. Then, combining the CRISPR-Cas13a system with terminal deoxynucleotidyl transferase (TdT)-triggered template-free DNA extension to form the fluorescent copper nanoparticles (CuNCs), a simple but sensitive method (named as Cas13a-TFCN) was developed for highly specific detection of miRNA. Based on the CRISPR/Cas13a system coupled with TdT-triggered simple but efficient signal amplification strategy, the developed Cas13a-TFCN method showed high sensitivity for miRNA detection with the low detection limit of 0.7 fM. Furthermore, the proposed Cas13a-TFCN approach was successfully applied for accurate quantification of miRNA in complex biological samples, indicating its practical application ability. Moreover, featuring high sensitivity and simplicity without complicated probe design and fluorophore labeling, the proposed Cas13a-TFCN has a promising applications prospect in clinical diagnosis.
研究领域:
分子生物学
·
生物技术
·
纳米技术
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分析化学
·
临床诊断
✓ 已验证
产品关联
DOI
高影响因子
SENSORS AND ACTUATORS B-CHEMICAL
·
2023
·
IF 8.4
Qing Han,Xinlan Bian,Yue Chen,Bingzhi Li
Flap endonuclease I (FEN1) is a structure-specific nuclease that participates in DNA repair, and its overexpression is related to an increased risk of cancer. Currently, few FEN1 assays can meet the requirements for diagnostic purposes due to their high complexity, low specificity, and limited sensitivity. Herein, we developed a biosensor adopting synthetic biology strategy, called CLAIM (cleavage-ligation-activation-involved monitoring) for FEN1 quantification. CLAIMintegrates ligation-based T7 promoter repair, RNA generation, and CRISPR/Cas13a. FEN1 cleavages the DNA probe with a 5′-flap to generate a nick site that can be ligated to form a T7 promoter. Subsequent transcription produces abundant RNA that can activate Cas13a, generating a fluorescence signal due to the trans -cleavage of Cas13a to a fluorogenic reporter. CLAIM was able to detect FEN1 with low femtomolar sensitivity, and its accuracy was validated using samples of cancer and non-tumorigenic cell lines. Interestingly, we found that the utilization of thermostable T4 DNA ligase is critical to make the cascade of FEN1 cleavage, ligation, and transcription take place in one-pot. CLAIM was also repurposed for validating FEN1 inhibitors, indicating its potential in drug screening. This work provides a diagnostic tool for FEN1 analysis and opens up opportunities for FEN1 biosensing and related drug discovery.
研究领域:
分子生物学
·
合成生物学
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癌症诊断
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生物技术
✓ 已验证
DOI
高影响因子
MICROCHEMICAL JOURNAL
·
2025
·
IF 5.1
Zhengfang Liang,Yunzhan Xie,Hongbing Zhou,Zilu Zhang,Yuanxun Gong,Jiayi Zhang,Yue Huang,Qianli Tang,Kai Zhang,Xianjiu Liao
MicroRNA-21 (miR-21) is a critical biomarker involved in various pathological processes, yet its low abundance in biological fluids poses challenges for accurate detection. Herein, we present a highly sensitive electrochemiluminescence (ECL) biosensor for miR-21 detection. The system integrates a multi-stage amplification cascade that begins with toehold-mediated strand displacement (TMSD) and continues through T7 RNA polymerase transcription and CRISPR/Cas13a-mediated collateral cleavage. The sensing interface was constructed by immobilizing ferrocene-labeled DNA probes onto a conductive AuNPs/Ti₃C₂Tx/Ru(II)-PEI nanohybrid-modified electrode. In the absence of miR-21, ferrocene effectively quenched the ECL signal. Upon target recognition, TMSD activates a split T7 promoter/template system, triggering RNA transcription. The resulting single-stranded RNAs activate Cas13a, which cleaves DNA probes and restores the ECL emission. Under optimized conditions, the biosensor exhibited a wide dynamic range from 10 aM to 500 fM and a detection limit as low as 5.23 aM. It also demonstrated excellent specificity, repeatability (RSD = 4.3 %), and recovery (99.93–106.20 %) in spiked serum samples. This work provides a versatile and modular ECL sensing platform for ultrasensitive miRNA detection, offering promising applications in clinical diagnostics and precision medicine.
✓ 已验证
产品关联
DOI
高影响因子
BIOSENSORS & BIOELECTRONICS
·
2025
·
IF 10.5
Yingjing Fan,Weilun Pan,Qi Xiu,Yue Qiao,Peng Zhang,Tong Liao,Junjie Feng,Chengtao Nie,Shuai Chu,Ningcen Li,Xiuhua Wu,Lei Zheng,Zhen Cai,Bo Li
Tumor immunotherapy has significantly improved cancer treatment, yet immune checkpoint inhibitors only benefit a subset of patients. A critical challenge is non-invasive detection of tumor PD-L1 mRNA, a key biomarker that dynamically reflects PD-L1 expression and correlates with therapeutic outcomes. Current methods are hindered by the instability of circulating mRNA and the need for invasive tissue biopsies. Herein, we introduce the Cationic LIposome capsulated CRISPR-Cas13a Kit (CLICK) for in-situ detection of circulating PD-L1 mRNA in extracellular vesicles (EVs). CLICK achieves direct target identification in 10 μL plasma within 2 hours without EV purification or RNA extraction, demonstrating a detection limit of 10 3 EVs/mL for PD-L1 mRNA. By integrating membrane fusion-mediated CRISPR delivery with nanoflow cytometry, CLICK resolves PD-L1 mRNA (+) EV subpopulations at single-vesicle resolution, revealing heterogeneous distributions. Clinically validated in 53 immunotherapy patients, CLICK accurately stratifies progressive disease (PD) from stable disease (SD) patients (AUC = 0.8236; 95% CI: 0.7054–0.9417; p= 0.0006) based on PD-L1 mRNA (+) EV percentages. This work establishes a rapid, blood-based paradigm for therapy response monitoring, advancing liquid biopsy through precise EV RNA characterization.
研究领域:
肿瘤学
·
分子诊断
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转化医学
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免疫学
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RNA检测
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液体活检技术
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精准医学
✓ 已验证
DOI
PubMed
高影响因子
MICROCHIMICA ACTA
·
2024
·Vol. 191
·期 8
·1-12
·
IF 5.3
He Qian,Chen Qun,Lian Lijin,Qu Jiuxin,Yuan Xi,Wang Chuhui,Xu Lidan,Wei Jiazhang,Zeng Shaoling,Yu Dongmei,Dong Yuhan,Zhang Yongbing,Deng Lin,Du Ke,Zhang Canyang,Pandey Vijay,Gul Ijaz,Qin Peiwu
The CRISPR/Cas13 nucleases have been widely documented for nucleic acid detection. Understanding the intricacies of CRISPR/Cas13’s reaction components is pivotal for harnessing its full potential for biosensing applications. Herein, we report on the influence of CRISPR/Cas13a reaction components on its trans-cleavage activity and the development of an on-chip total internal reflection fluorescence microscopy (TIRFM)-powered RNA sensing system. We used SARS-CoV-2 synthetic RNA and pseudovirus as a model system. Our results show that optimizing Mg 2+ concentration, reporter length, and crRNA combination significantly improves the detection sensitivity. Under optimized conditions, we detected 100 fM unamplified SARS-CoV-2 synthetic RNA using a microtiter plate reader. To further improve sensitivity and provide a new amplification-free RNA sensing toolbox, we developed a TIRFM-based amplification-free RNA sensing system. We were able to detect RNA down to 100 aM. Furthermore, the TIRM-based detection system developed in this study is 1000-fold more sensitive than the off-coverslip assay. The possible clinical applicability of the system was demonstrated by detecting SARS-CoV-2 pseudovirus RNA. Our proposed sensing system has the potential to detect any target RNA with slight modifications to the existing setup, providing a universal RNA detection platform. Graphical Abstract
CAS / MDL:
研究领域:
生物传感
·
分子生物学
·
纳米技术
·
病毒学
✓ 已验证
产品关联
DOI
PubMed
高影响因子
ANALYTICA CHIMICA ACTA
·
2024
·
IF 5.7
Zaiyong Zhang,Jinglong Li,Chunlin Chen,Yuwei Tong,Dehui Liu,Cuizhi Li,Huan Lu,Li Huang,Wanling Feng,Xiaoting Sun
Brain natriuretic peptide (BNP) is considered to be an important biomarker of heart failure (HF) attracting attention. However, its low concentration and short half-life in blood lead to a low-sensitivity detection of BNP, which is a challenge that has to be overcome. In this work, we propose a highly specific, highly sensitive T7 RNA polymerase-assisted clustered regularly interspaced short palindromic repeats (CRISPR)/Cas13a system to detect BNP via an electrochemiluminescence (ECL) sensing platform and incorporate Exo III-hairpin and dumbbell-shaped hybridization chain reaction (HCR) technologies. In this detection scheme, the ECL sensing platform possesses low background signal and high sensitivity. Firstly, the T7 promoter-initiated T7 RNA polymerase acts as a signal amplification technique to generate large amounts of RNAs that can activate CRISPR/Cas13a activity. Secondly, CRISPR/Cas13a is able to trans -cleave the surrounding trigger strand to produce DNA1. Thirdly, DNA1 is involved in the co-amplification reaction of Exo III and hairpin DNA, which subsequently triggers a dumbbell-shaped HCR technology. Eventually, a large number of Ru (II) molecules are inserted into the interstitial space of the dumbbell-shaped HCR to generate a strong ECL signal. The CRISPR/Cas13a possesses outstanding specificity for a single base and increased sensitivity. The tightly conformed dumbbell-shaped HCR provides higher sensitivity than the traditional linear HCR amplification technique. Ultimately, the clever combination of several amplification reactions enables the limit of detection (LOD) as low as 3.2 fg/mL. It showed promise for clinical sample testing, with recovery rates ranging from 98.4% to 103% in 5% human serum samples. This detection method offered a valuable tool for early HF detection, emphasizing the synergy of amplification strategies and specificity conferred by CRISPR/Cas13a technology.
研究领域:
生物技术
·
分子生物学
·
分析化学
·
临床诊断
✓ 已验证
产品关联
DOI
PubMed
高影响因子
SENSORS AND ACTUATORS B-CHEMICAL
·
2023
·
IF 8.4
Pei Wang,Bohui Zhou,Ziye Liu,Yining Zhao,Robert Chunhua Zhao,Jinhui Peng,Juan Zhang
The accurate detection of immune checkpoint molecules on the surface of tumor cells is of great significance for immunotherapy. Herein, we propose a method based on cellular interface supported cascade signal amplification mediated by CRISPR/Cas13a system for in situ analysis of immune checkpoint molecules on bladder cancer cells. CD47 and EpCAM on bladder cancer cells can be sensitively monitored through T7 transcription amplification, CRISPR/Cas13a system and HCR triple signal amplification. Moreover, the established method can specifically differentiate EpCAM and CD47 positive bladder cancer cells and well serve for the evaluation of two kinds of membrane protein inhibitors. In a word, the method we proposed can successfully realize in situ analysis of immune checkpoint molecules on bladder cancer cells for guiding immunotherapy.
研究领域:
癌症生物学
·
分子生物学
·
免疫学
·
生物技术
✓ 已验证
产品关联
DOI
高影响因子
JOURNAL OF HAZARDOUS MATERIALS
·
2023
·
IF 13.6
Jihua Wei,Zichun Song,Jiuying Cui,Yuanxun Gong,Qianli Tang,Kai Zhang,Xinlei Song,Xianjiu Liao
In this study, we introduce an electrochemiluminescence (ECL) sensing platform based on the "Entropy-driven triggered T7 amplification-CRISPR/Cas13a system" (EDT-Cas). This platform combines a programmable entropy-driven cycling strategy, T7 RNA polymerase, and the CRISPR/Cas13a system to amplify the determination of the SARS-CoV-2 RdRp gene. The Ti 3 C 2 T x -compliant ECL signaling molecule offers unique benefits when used with the ECL sensing platform to increase the assay sensitivity and the electrode surface modifiability. To obtain the T7 promoter, the SARS-CoV-2 RdRp gene may first initiate an entropy-driven cyclic amplification response. Then, after recognizing the T7 promoter sequence on the newly created dsDNA, T7 RNA polymerase starts transcription, resulting in the production of many single-stranded RNAs (ssRNAs), which in turn trigger the action of CRISPR/Cas13a. Finally, Cas13a/crRNA identifies the transcribed ssRNA. When it cleaves the ssRNA, many DNA reporter probes carrying -U-U- are cleaved on the electrode surface, increasing the ECL signal and allowing for the rapid and highly sensitive detection of SARS-CoV-2. With a detection limit of 7.39 aM, our method enables us to locate the SARS-CoV-2 RdRp gene in clinical samples. The detection method also demonstrates excellent repeatability and stability. The SARS-CoV-2 RdRp gene was discovered using the "Entropy-driven triggered T7 amplification-CRISPR/Cas13a system" (EDT-Cas). The developed ECL test had excellent recoveries in pharyngeal swabs and environmental samples. It is anticipated to offer an early clinical diagnosis of SARS-CoV-2 and further control the spread of the pandemic.
研究领域:
生物技术
·
分子生物学
·
病毒学
·
诊断技术
·
纳米技术
✓ 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）