ANALYTICA CHIMICA ACTA
·
2015
·
IF 4.513
Yingfeng Qin,Yefei Ma,Xue Jin,Liangliang Zhang,Gaojie Ye,Shulin Zhao
As an important antitumor drug, bleomycin (BLM) is widely used in the treatment of a variety of cancers. In addition, nucleases play a crucial role in DNA replication, recombination and repair which are associated with cancer development. Thus, the development of BLM and nuclease detection methods is of great significance in cancer therapy and related biological mechanism research. Here, a WS 2 nanosheet-based turn-on fluorescent sensing platform for simple, fast and sensitive detection of BLM and nuclease was reported. WS 2 nanosheet exhibits different affinity toward ssDNA with different length and excellent fluorescence quenching ability. A fluorescein (FAM)-labeled long ssDNA could be adsorbed on the surface of WS 2 nanosheet and the fluorescence was therefore quenched. In the presence of BLM·Fe(II) or S1 nuclease (a ssDNA-specific nuclease which was used as a model enzyme), an irreversible scission of long ssDNA was underwent through the BLM-induced oxidation cleavage or S1 nuclease-induced enzymatic hydrolysis. Short FAM-linked oligonucleotide fragments which could not be adsorbed on the nanosheet surface were then produced, resulting in a weak fluorescence quenching after mixing WS 2 nanosheets. Thus, the fluorescence signal was restored. The proposed sensor displays a wide linear range and a high sensitivity with a detection limit of 0.3 nM for BLM and 0.01 U mL −1 for S1 nuclease. It also exhibits a good performance in complex biological samples. This method not only provides a strategy for BLM or S1 nuclease assay but also offers a potential application in biomedical and clinical study.
研究领域:
生物化学
·
分子生物学
·
分析化学
·
癌症研究
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DOI
PubMed
高影响因子
RSC Advances
·
2021
·Vol. 11
·期 52
·32898-32903
·
IF 3.361
Lancheng Wang,Huimin Zhou,Kun Yan,Peng Xu,Bin Di,Chi Hu,Mengxiang Su
The binding of small molecules to their DNA aptamers can modulate their susceptibility to digestion by exonucleases, however, absolute differentiation between digestion and inhibition has never been reported. Here, we show that the digestion of aptamers by T7 exonuclease can be completely inhibited upon binding of small-molecule targets and exploit this finding for the first time to achieve sensitive, label-free small-molecule detection. We use a quinine-binding aptamer to show that target binding entirely halts T7 exonuclease digestion, leaving behind an intact double-stranded product that retains strong target affinity. On the contrary, digestion of nontarget-bound aptamer produces a single-stranded product incapable of target binding. Exonuclease I efficiently eliminates these single-stranded products but is unable to digest the target-bound double-stranded product. The remaining products can be fluorescently quantified with SYBR Gold to determine target concentrations, giving a limit of detection of 100 nM with the linear range from 0 to 8 μM. We demonstrate the first example of a dual-exonuclease-mediated approach capable of producing a concentration-dependent response in terms of aptamer digestion modules, therefore improving performance of the current aptamer-based assay for small-molecule detection.
研究领域:
生物化学
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分子生物学
·
分析化学
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产品关联
DOI
PubMed
高影响因子
Science of Advanced Materials
·
2014
·Vol. 6
·期 9
·1936-1942
·
IF 2.908
Li Xiang,Ding Xuelian,Wang Gongke,Zhou Yixuan,Hou Ruipeng,Gao Shuyan,Lu Yan
In this work, we have developed a simple and highly sensitive fluorescent biosensor for the detection of HIV-1 RNase H activity and inhibition by using graphene oxide (GO) as sensing platform. In our approach, a DNA-RNA substrate was prepared which a carboxy fluorescein (FAM) was labeled
at the 3 termini of single strand DNA. The FAM labeled DNA-RNA substrate preserves most of the fluorescence when mixed with GO. However, in the present of RNase H, RNase H can cleavage the RNA into fragments, resulting in the dissociation of ssDNA and mononucleotides. The fluorescence intensity
was greatly quenched after the addition of GO. Because of the super quenching ability of GO, the as-proposed method provides a wide linear range from 1.0 to 26.3 units mL–1 and a low detection limit of 0.6 units mL–1 for HIV-1 RNase H activity analysis. Additionally,
this approach shows potential for high-throughput screening of HIV-1 RNase H inhibitors. The method not only provides a universal platform for monitoring activity and inhibition of RNase H but also shows great potential in biological process researches, drug discovery, and clinic diagnostics.
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
·
病毒学
·
药物发现
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DOI
高影响因子
ANALYTICAL CHEMISTRY
·
2017
·
IF 6.32
Xiangmin Miao,Zhiyuan Cheng,Haiyan Ma,Zongbing Li,Ning Xue,Po Wang
A novel strategy was developed for microRNA-155 (miRNA-155) detection based on the fluorescence quenching of positively charged gold nanoparticles [(+)AuNPs] to Ag nanoclusters (AgNCs). In the designed system, DNA-stabilized Ag nanoclusters (DNA/AgNCs) were introduced as fluorescent probes, and DNA–RNA heteroduplexes were formed upon the addition of target miRNA-155. Meanwhile, the (+)AuNPs could be electrostatically adsorbed on the negatively charged single-stranded DNA (ssDNA) or DNA–RNA heteroduplexes to quench the fluorescence signal. In the presence of duplex-specific nuclease (DSN), DNA–RNA heteroduplexes became a substrate for the enzymatic hydrolysis of the DNA strand to yield a fluorescence signal due to the diffusion of AgNCs away from (+)AuNPs. Under the optimal conditions, (+)AuNPs displayed very high quenching efficiency to AgNCs, which paved the way for ultrasensitive detection with a low detection limit of 33.4 fM. In particular, the present strategy demonstrated excellent specificity and selectivity toward the detection of target miRNA against control miRNAs, including mutated miRNA-155, miRNA-21, miRNA-141, let-7a, and miRNA-182. Moreover, the practical application value of the system was confirmed by the evaluation of the expression levels of miRNA-155 in clinical serum samples with satisfactory results, suggesting that the proposed sensing platform is promising for applications in disease diagnosis as well as the fundamental research of biochemistry.
研究领域:
生物化学
·
分子诊断
·
纳米生物学
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DOI
PubMed
高影响因子
Journal of Materials Chemistry B
·
2024
·
IF 6.1
Miao He,Zhiqiang Hou,Feifan Yin,Wenting Cheng,Yang Xiang,Zhongyun Wang
The detection of a single biomarker is prone to false negative or false positive results. Simultaneous analysis of two biomarkers can greatly improve the accuracy of diagnosis. In this work, we designed a new method for coinstantaneous detection of two breast cancer biomarkers miRNA-21 and HER2 using the properties of duplex-specific nuclease (DSN). Cy5-labeled DNA1 and FAM-labeled DNA2 are used as signal probes to distinguish the two signals. When the sample contains the targets HER2 and miRNA-21, HER2 binds to the HER2 aptamer on the double-stranded DNA2, while miRNA-21 binds to the complementary DNA1. Then, DSN enzyme is added to cut the DNA probes adsorbed on the HER2 aptamer and miRNA-21, releasing the fluorescent groups, which can be readsorbed to the empty sites, thus repeating the cutting of the probes and producing an exponential signal amplification with two distinct fluorescent signals. The detection limits of miRNA-21 and HER2 are 1.12 pM and 0.36 ng mL−1, respectively, with linear ranges of 5 pM to 50 pM and 1 ng mL−1 to 15 ng mL−1. The method was validated in real biological samples, providing a new approach for synchronous analysis of important markers in breast cancer.
研究领域:
生物化学
·
分子生物学
·
医学诊断
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产品关联
DOI
PubMed
高影响因子
NUCLEIC ACIDS RESEARCH
·
2025
·Vol. 53
·期 2
·
IF 13.1
Zhang Lihang,Qiu Xinyuan,Zhou Yuting,Luo Zhengyang,Zhu Lingyun,Shao Jiawei,Xie Mingqi,Wang Hui
The CRISPR-derived endoribonuclease Csy4 is a popular tool for controlling transgene expression in various therapeutically relevant settings, but adverse effects potentially arising from non-specific RNA cleavage remains largely unexplored. Here, we report a split-Csy4 architecture that was carefully optimized for in vivo usage. First, we separated Csy4 into two independent protein moieties whose full catalytic activity can be restored via various constitutive or conditional protein dimerization systems. Next, we show that introduction of split-Csy4 into human cells caused a substantially reduced extent in perturbation of the endogenous transcriptome when directly compared to full-length Csy4. Inspired by these results, we went on to use such split-Csy4 module to engineer inducible CRISPR- and translation-level gene switches regulated by the FDA-approved drug grazoprevir. This work provides valuable resource for Csy4-related biomedical research and discusses important issues for the development of clinically eligible regulation tools.
研究领域:
分子生物学
·
基因工程
·
治疗学
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产品关联
DOI
PubMed
高影响因子
BIOSENSORS & BIOELECTRONICS
·
2017
·
IF 7.78
Shuzhen Yue,Tingting Zhao,Sai Bi,Zhipeng Zhang
High throughput analysis of miRNAs is of great significance to clinical and biomedical applications . In this work, we have developed a highly sensitive and selective chemiluminescence imaging array (CLIA) for simultaneous detection of three miRNAs with high throughput, easy operation and low cost. In this assay, three kinds of hairpins that can specially recognize respective miRNAs (miR-155, miR-let-7a and miR-141) are parallel modified on the magnetic beads (MBs). The employment of MBs enables quick sorting of multiplex targets in high yield and purity with high throughput. Upon introduction of the target miRNAs, they hybridize to the corresponding hairpins and initiate the DNA machines with the assistance of Klenow fragment exo- DNA polymerase and Nb.BbvCI NEase , achieving exponential amplification of targets. After that, the toehold-mediated strand displacement (TMSD) reactions are performed through sequential addition of displacement probes under magnetic separation, resulting in the release of HRP-tagged DNA hybrids in solution for CL imaging and recovery of MBs for repetitive use. This CLIA method demonstrates ultrahigh sensitivity with detection limits down to fM level, wide linear range over 5 orders of magnitude, excellent selectivity to distinguish one-base mismatched target miRNA, and good performance in real sample analysis. Together with the advantages of high throughput, easy operation, acceptable accuracy, and good recyclability and reproducibility, the proposed multiplex CLIA method holds great potential in practical applications, such as profiling the pattern of miRNA expression, disease screening, biomedical research, and so on.
研究领域:
生物医学工程
·
分子诊断学
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DOI
PubMed
高影响因子
COLLOIDS AND SURFACES B-BIOINTERFACES
·
2019
·
IF 3.973
Weipan Peng,Yiwen Huang,Qian Zhao,Guowei Lian,Minghui Chen,Jiafang Piao,Xiaoqun Gong,Jin Chang
Considered as the next-generation biomarkers, microRNAs play an important role in the early diagnosis of cancers. Here, we designed a fluorescent signal “removal” sensor for one-step, sensitive and specific detection of multiple microRNAs by flow cytometry (FCM). In this work, single-stranded DNA (ssDNA), working as the interlinkage, immobilized the fluorescent nanosphere (FS) onto the SiO 2 microspheres surface to form the SiO 2 -ssDNA-FS probes. When target miRNAs integrated with SiO 2 -ssDNA-FS probes, the duplex-specific nuclease (DSN) could cleave the ssDNA selectively and release FS with numerous cycles to enhance the fluorescent signal attenuation of SiO 2 -ssDNA-FS, so as to remarkably improve the analysis sensitivity. It achieved a simple, accurate and quantitative microRNA-21 detection for clinical blood samples. Parallel multi-target detection of microRNA-21 and Let-7d was also realized by different color labeled FS. Moreover, our designed sensor was suitable for other targets’ detection with the corresponding probes.
研究领域:
分子生物学
·
医学诊断
·
生物技术
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产品关联
DOI
PubMed
高影响因子
ACS Applied Materials & Interfaces
·
2015
·
IF 6.723
Xuehong Min,Yuan Zhuang,Zhenyu Zhang,Yongmei Jia,Abdul Hakeem,Fuxin Zheng,Yong Cheng,Ben Zhong Tang,Xiaoding Lou,Fan Xia
We demonstrate an ultrasensitive microRNA detection method based on an extremely simple probe with only fluorogens but without quencher groups. It avoids complex and difficult steps to accurately design the relative distance between the fluorogens and quencher groups in the probes. Furthermore, the assay could accomplish various detection limits by tuning the reaction temperature due to the different activity of exonuclease III corresponding to the diverse temperature. Specifically, 1 pM miR-21 can be detected in 40 min at 37 °C, and 10 aM (about 300 molecules in 50 μL) miR-21 could be discriminated in 7 days at 4 °C. The great specificity of the assay guarantees that the real 21 urine samples from the bladder cancer patients are successfully detected by our method.
研究领域:
癌症研究
·
分子生物学
·
分析化学
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产品关联
DOI
PubMed
高影响因子
MICROCHEMICAL JOURNAL
·
2023
·
IF 4.8
Feng Xu,Xin Chen,Daofei Lv,Yong Liang
Here, we report a DNA hairpin strategy for electrochemical determination of Hg 2+ based on T-Hg 2+ -T pairs and exonuclease III (Exo III) assistance. The innovation of this work lies in the designed DNA structure and detection principles. Unlike the previously reported DNA hairpin structure, the ring portion of the hairpin DNA we designed consisting of T bases serves as a recognition probe, forming a T-Hg-T complex with Hg 2+ . The stem is composed of 5 pairs of complementary bases, which is conducive to the stability of the hairpin structure. After the formation of T-Hg-T in the ring, with the help of Exo III, Exo III can digest more DNA than the T bases designed in the stem. In this event, the shorter the length of the remaining DNA strand on the electrode, the weaker the repulsive force against [Fe(CN) 6 ] 3−/4− , and the stronger the differential pulse voltammetry (DPV) current signal, achieving “signal-on” transitions. Under the optimal conditions, the DPV current response increases with increasing Hg 2+ concentration and is linear with the logarithm of Hg 2+ concentration (1 nM–10 μM). The limit ofdetection is 0.16 nM. The electrochemical sensor was successfully applied to the determination of Hg 2+ in river water.
研究领域:
生物化学
·
环境科学
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DOI
高影响因子
APPLIED BIOCHEMISTRY AND BIOTECHNOLOGY
·
2021
·Vol. 193
·期 11
·3753-3764
·
IF 2.926
Chen Xiaochun,Huang Xiaoquan,Tang Yiwen,Zhang Lei
Nuclease P1 (NP1) can hydrolyze nucleic acids into four 5′-mononucleotides, which are widely used in the pharmaceutical and food industries. In this paper, an aqueous two-phase system (ATPS) was developed to purify NP1 from Penicillium citrinum . Polyethylene glycol (PEG) and nucleotides salts were studied to form ATPSs, among which PEG3000/disodium guanosine monophosphate (GMPNa 2 ) was researched, including the phase composition and pH. Using 14% (w/w) PEG3000 and 20% (w/w) GMPNa 2 ATPS at pH 5.0, the best recovery and purification factor, 82.4% and 3.59, were obtained. The recovery of NP1 was 98.3% by the separation of ultrafiltration from the PEG-rich phase. The recycling use of GMPNa 2 was also studied, and 95.1% of GMPNa 2 in the salt-rich phase was obtained with the addition of ethanol as the solvent. These results showed that the ATPS was effective for purification of NP1.
研究领域:
生物化学
·
工业微生物学
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产品关联
DOI
PubMed
高影响因子
BIOCHEMICAL ENGINEERING JOURNAL
·
2020
·
IF 3.475
Hong Zhang,Chuance Cheng,Nalin Dong,Xiaoming Ji,Jiandong Hu
S1 nuclease plays an important role in DNA transcription, recombination and repair. Here, we synthesized a positively charged [email protected] core-shell nanoparticles ((+) [email protected] CSNPs) and used as highly stable and enhanced fluorescence quenching platform for one-step S1 nuclease activity detection. In the sensor, fluorophore labeled DNA was used as signal probe and its fluorescence signal could be effectively quenched based on the electrostatic interaction of it with (+) [email protected] CSNPs. In the presence of S1 nuclease, DNA probe was hydrolyzed into DNA fragments and cannot interact with (+) [email protected] CSNPs, inducing an obvious fluorescence signal increase that was proportional with S1 nuclease concentration in the range of 2.5 × 10 −4 –3.0 × 10 −2 U/mL. The enhanced quenching efficiency of (+) [email protected] CSNPs paved the way for sensitive S1 detection with a detection limit of 6.0 × 10 −5 U/mL. Moreover, the high stability of such nanocomposite can achieve the one-step detection of S1 in complex solution containing salt, protein or other metal ions. In addition, the method also exhibited high selectivity for nuclease detection, making our sensing platform has a potential application value in bioanalysis and clinical diagnosis.
研究领域:
生物化学
·
分析化学
·
医学诊断
✓ 已验证
产品关联
DOI
高影响因子
危险品化学品经营许可证（带存储）