ANALYTICA CHIMICA ACTA
·
2018
·
IF 5.123
Fengzhou Xu,Lan Luo,Hui Shi,Xiaoxiao He,Yanli Lei,Jinlu Tang,Dinggeng He,Zhenzhen Qiao,Kemin Wang
Poly(thymine)-hosted copper nanoparticles (poly T-CuNPs) have emerged as a promising label-free fluorophore for bioanalysis, but its application in RNA-related studies is still rarely explored. Herein, by utilizing duplex-specific nuclease (DSN) as a convertor to integrate target recycling mechanism into terminal deoxynucleotidyl transferase (TdT)-mediated superlong poly T-CuNPs platform, a specific and sensitive method for microRNA detection has been developed. In this strategy, a 3′-phosphorylated DNA probe can hybridize with target RNA and then be cut by DSN to produce 3′-hydroxylated fragments, which can be further tailed by TdT with superlong poly T for fluorescent CuNPs synthesis. As proof of concept, an analysis of let-7d was achieved with a good linear correlation between 20 and 1000 pM (R 2 = 0.9965) and a detection limit of 20 pM. Moreover, both homologous and heterologous microRNAs were also effectively discriminated. This strategy might pave a brand-new way for designing label-free and sensitive microRNA assays.
研究领域:
生物化学
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分子生物学
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分析化学
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DOI
PubMed
高影响因子
STAR Protocols
·
2022
·
IF 0
Qianqian Fang,Zhengkang Hua,Zhonghui Lin
Summary The human MUS81-EME1&2 complexes are structure-selective endonucleases that play important roles in DNA damage repair. Here, we describe a protocol to determine the endonuclease activities of MUS81-EME1&2 complexes toward various DNA structures. We co-express MUS81 with EME1 or EME2 and purify the complexes with high purity, and determine their activities on the cleavages of 3ˊ flaps, 5ˊ flaps, nicked double-stranded DNAs, and Holliday junctions. This protocol can also be used for the determination of substrate preferences of other structure-selective endonucleases. For complete details on the use and execution of this protocol, please refer to Hua et al. (2022) .
研究领域:
分子生物学
·
细胞生物学
·
遗传学
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DOI
PubMed
ANALYST
·
2023
·Vol. 148
·期 7
·1492-1499
·
IF 4.2
Jiahai Wang,Lanfang Chen,Cenlin Gui,Jianji Zhu,Baian Zhu,Zhuobin Zhu,Yunhui Li,Daqi Chen
DNA methylation has been considered an essential epigenetic biomarker for diagnosing various diseases, such as cancer. A simple and sensitive way for DNA methylation level detection is necessary. Inspired by the label-free and ultra-high sensitivity of solid-state nanopores to double-stranded DNA (dsDNA), we proposed a nanopore counter for evaluating DNA methylation by integrating a dual-restriction endonuclease digestion strategy coupled with polymerase chain reaction (PCR) amplification. Simultaneous application of BstUI/HhaI endonucleases can ensure the full digestion of the unmethylated target DNA but shows no effect on the methylated ones. Therefore, only the methylated DNA remains intact and can trigger the subsequent PCR reaction, producing a large quantity of fixed-length PCR amplicons, which can be directly detected through glassy nanopores. By simply counting the event rate of the translocation signals, the concentration of methylated DNA can be determined to range from 1 aM to 0.1 nM, with the detection limit as low as 0.61 aM. Moreover, a 0.01% DNA methylation level was successfully distinguished. The strategy of using the nanopore counter for highly sensitive DNA methylation evaluation would be a low-cost but reliable alternative in the analysis of DNA methylation.
研究领域:
生物化学
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分子生物学
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医学诊断
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DOI
PubMed
高影响因子
ANALYTICA CHIMICA ACTA
·
2022
·
IF 6.911
Yunyun Ning,Cuiling Zhang,Chunyan Wang,Chuqing Zhou,Nana Gong,Qiang Wang,Ye Zhu
Accurate detection of microRNA (miRNA) is challenging but essential for disease diagnostics and therapeutics. To meet the high demands of miRNA analysis, we proposed a highly sensitive and specific electrochemical strategy based on duplex-specific nuclease (DSN)-assisted target recycling and DNA self-assembled FeN x C nanocatalytic network for precise quantitation of miRNA-21 as a model. FeN x C nanospherical catalyst with high peroxidase-mimicking activity was synthesized through high-temperature pyrolysis and modified with DNA. The identification of miRNA-21 by elaborately designed capture probe triggered DSN-assisted target recycling. Subsequently, self-assembled FeN x C nanocatalytic network was constructed on the sensor surface through DNA hybridization reaction, whose signal response was demonstrated to be 4.272-fold higher than the single layer of FeN x C. By virtue of such cascade signal amplification, ultrasensitive electrochemical detection of miRNA-21 was realized in a dynamic range of 0.5 fM - 1 pM with a detection limit of 0.2805 fM. Additionally, the proposed strategy exhibited high specificity for miRNA-21 and practical applicability in real sample analysis, demonstrating promising potential for the reliable miRNA analysis in disease diagnosis and therapy applications.
研究领域:
生物化学
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分子诊断
·
纳米医学
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分析化学
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产品关联
DOI
PubMed
高影响因子
BIOELECTROCHEMISTRY
·
2022
·
IF 5.76
Xianghui Li,Yichan Huang,Jiawen Chen,Shuangmu Zhuo,Zhenyu Lin,Jianxin Chen
A sensitive homogeneous electrochemiluminescence (ECL) biosensor for flap endonuclease 1 (FEN1) detection was developed by combining highly sensitive ECL detection, high efficiency of branched hybridization chain reaction (BHCR) amplification, a convenient homogeneous strategy, and simple ultrafiltration separation. Magnetic beads were first modified with well-designed double flap DNAs containing 5′-flaps. In the presence of FEN1, the 5′-flap can be cleaved, and a large amount of single-stranded DNA can be produced, which can be separated easily from the double-flap DNA-modified beads by a magnet. Then, the cleaved 5′-flap can be used to initiate BHCR amplification to produce a large amount of long-strand dsDNA. Ru(phen) 3 2+ can insert dsDNA to form Ru-dsDNAs, which can be easily separated from the main solution through ultrafiltration. The ECL signal from the separated Ru-dsDNAs has a good linear relationship with the logarithm of the FEN1 concentration ranging from 6.5 × 10 −2 ∼ 6.5 × 10 3 U/L with a detection limit of 2.2 × 10 −2 U/L. The proposed biosensor was used to evaluate FEN1 activity in real samples with satisfactory results.
研究领域:
生物传感
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分子诊断
·
分析化学
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DOI
高影响因子
SENSORS AND ACTUATORS B-CHEMICAL
·
2017
·
IF 5.401
Haiyan Wang,Pei Liu,Wenjing Jiang,Xue Li,Huanshun Yin,Shiyun Ai
DNA methylation is one of the important epigenetics events, and it is responsible for transcription, genomic imprinting and cellular differentiation. Aberrant DNA methylation is always contacted with various diseases. Herein, a simple and low-cost photoelectrochemical (PEC) biosensing method is proposed for detection of DNA methylation, assay of DNA methyltransferase (MTase) activity and screening of MTase inhibitor, which is based on M. SssI MTase- HpaII endonuclease system. The graphite-like C 3 N 4 (g-C 3 N 4 ) and CdS quantum dots (CdS QDs) are used as photoactive materials. After the double-stranded DNA (dsDNA) was treated with M. SssI MTase in the presence of S -adenosylmethionine, the methylated dsDNA cannot be digested by HpaII endonuclease and dithiol group at the terminal of dsDNA can be retained. As a result, CdS QDs could be coupled successfully with methylated dsDNA between the reaction of CdS QDs with dithiol of dsDNA, achieving the amplification of PEC response. Thus, the photocurrent of PEC biosensor is consistent with the M. SssI activity. The increased photocurrent was in proportion to M. SssI activity in the linear range from 1 to 80 U/mL with a detection limit of 0.316 U/mL. Furthermore, the effects of atrazine and azamethipos on M. SssI MTase activity were investigated, which might provide useful information on carcinogenic mechanism of pesticide. The PEC biosensor has potential to screen of DNA MTase inhibitor, providing valuable information for anti-cancer drug research and also for cancer therapy.
研究领域:
生物化学
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分子生物学
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分析化学
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DOI
高影响因子
ELECTROCHIMICA ACTA
·
2013
·
IF 3.777
Zhenning Xu,Mo Wang,Huanshun Yin,Shiyun Ai,Liang Wang,Junling Pang
A sensitive electrochemical method for detection of DNA methylation and assay of DNA methyltransferase (Dam) was investigated based on methylation-sensitive restriction endonuclease, DNA-Au bio bar code and enzymatic signal amplification. After the DNA probe was hybridized with complementary DNA sequence, and the obtained double-strand DNA (dsDNA) was further methylated by Dam MTase, it could be successively digested by Dpn I at the methylated sites. As a result, the bio bar code and streptavidin-horseradish peroxidase (S-HRP) could not be conjugated on the electrode surface through the specific interaction between biotin and S-HRP, where biotin was labeled at the DNA-Au bio bar code. Thus, only a low electrochemical reduction signal was obtained. However, the hybridized probe could hybrid with DNA-Au bio bar code, and then S-HRP could be conjugated on the electrode surface in the absence of Dam MTase and Dpn I, so a larger reduction current of enzymatic product was obtained. The change of the reduction current of enzymatic product was used to detect DNA methylation and Dam MTase activity. The detection limit of Dam MTase was 0.02 unit/mL (S/N = 3). Moreover, the inhibitions of mitomycin C, paclitaxel, chlorogenic acid and epicatechin were also investigated. Therefore, this method provided a sensitive platform for monitoring DNA methylation and DNA MTase activity.
研究领域:
生物化学
·
分子生物学
·
医学检测
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产品关联
DOI
高影响因子
BIOSENSORS & BIOELECTRONICS
·
2017
·
IF 7.78
Minqiang Hong,Mengyan Wang,Jing Wang,Xueqin Xu,Zhenyu Lin
In this paper, a novel and signal-on electrochemical biosensor based on Hg 2+ - triggered nicking endonuclease-assisted target recycling and hybridization chain reaction (HCR) amplification tactics was developed for sensitive and selective detection of Hg 2+ . The hairpin-shaped capture probe A (PA) contained a specific sequence which was recognized by nicking endonuclease (NEase). In the presence of Hg 2+ , probe B (PB) hybridized with PA to form stand-up duplex DNA strands via the Hg 2+ mediated thymine-Hg 2+ -thymine (T-Hg 2+ -T) structure, which automatically triggered NEase to selectively digest duplex region from the recognition sites, spontaneously dissociating PB and Hg 2+ and leaving the remnant initiators. The released PB and Hg 2+ could be reused to initiate the next cycle and more initiators were generated. The long nicked double helices were formed through HCR event, which was triggered by the initiators and two hairpin-shaped signal probes labeled with methylene blue , resulting in a significant signal increase. Under optimum conditions, the resultant biosensor showed the high sensitivity and selectivity for the detection of Hg 2+ in a linear range from 10 pM to 50 nM (R 2 =0.9990), and a detection limit as low as 1.6 pM (S/N=3). Moreover, the proposed biosensor was successfully applied in the detection of Hg 2+ in environment water samples with satisfactory results.
研究领域:
生物化学
·
环境科学
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分析化学
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DOI
PubMed
高影响因子
MICROCHEMICAL JOURNAL
·
2025
·
IF 5.1
Fulin Yang,Hongjie Liu,Hao Fu,Chaoxin Zhang,Changrui Ye,Shaopeng Wang,Man Zhang,Fang Shen,Liwei Wang
As one of the significant algal bloom species, Akashiwo sanguinea has long brought challenges to controlling red tide. Red tide outbreaks are often accompanied by the mass propagation of various algal bloom species, which brings problems to investigating red tides using traditional optical methods. Electrochemical biosensing technology has high sensitivity and specificity, yet its use in detecting A. sanguinea DNA in sea areas is insufficient. Here, the amplification system combining Nt.BbvCI endonuclease-assisted and hybridization chain reaction (HCR) has a detection limit of the same or even lower magnitude than other HCR systems. More importantly, the auxiliary short strands from the Nt.BbvCI system activates HCR, reducing the steric hindrance of long DNA molecules and enhancing HCR’s activity. Thus, the portable electrochemical biosensor based on this system successfully detected simulated A. sanguinea DNA samples, with a limit of detection (LOD) at 1.53 fg/μL and a limit of quantification (LOQ) at 4.27 fg/μL, far better than traditional optical-based methods. Also, the predicted cell density of A. sanguinea generally matches that of optical microscopy. This portable biosensor is expected to conduct an on-site investigation of A. sanguinea ’s cell density. It can provide data support for early warnings of red tide outbreaks and reduce the time needed for investigations.
研究领域:
海洋生物学
·
分子生物学
·
分析化学
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产品关联
DOI
高影响因子
Analytical Methods
·
2021
·Vol. 13
·期 35
·4001-4007
·
IF 2.896
Mengyang Zhou,Chao Li,Baolong Wang,Lin Huang
Exosomes as fluid biomarkers hold great promise for noninvasive cancer diagnosis. However, a method for the rapid and convenient detection of exosomes is still a challenge because current analysis processes involve multiple steps and yield low sensitivity. Here, we developed a wash-free fluorescent biosensor for the rapid and sensitive quantification of exosomes by combining aptamer and nicking endonuclease (Nb·BbvCI). In this system, an aptamer–trigger complex was used as the recognition element; the trigger probe could be released, and it hybridized with gold nanoparticles (GNPs)–DNA–FAM conjugates, thereby resulting in Nb·BbvCI-assisted target recycling. As a result, our method allowed the quantification of exosomes with lower analysis time by using a cocktail containing an aptamer–trigger complex, Nb·BbvCI, and GNPs–DNA–FAM. A high sensitivity with a limit of detection (LOD) of 1.0 × 104 particles per μL could be achieved. Besides, this biosensor exhibited potential application for the quantification of exosomes in human plasma, facilitating the development of exosome-based noninvasive cancer diagnosis.
研究领域:
分子生物学
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医学诊断
·
生物化学
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产品关联
DOI
PubMed
高影响因子
BIOSENSORS & BIOELECTRONICS
·
2015
·
IF 6.409
Linqun Zhang,Min Wei,Chunyan Gao,Wei Wei,Yuanjian Zhang,Songqin Liu
Detection of DNA methylation and methyltransferase (MTase) activity are important in determining human cancer because aberrant methylation was linked to cancer initiation and progression. In this work, we proposed an electrochemical method for sensitive detection of DNA methylation and MTase activity based on methylation sensitive restriction endonuclease HpaII and the deposition of polyaniline (PANI) catalyzed by HRP-mimicking DNAzyme. In the presence of methylated DNA, HRP-mimicking DNAzyme catalyzed the polymerization of aniline on the dsDNA template, producing huge DPV current. In the presence of non-methylated DNA, dsDNA are cleaved and digested by HpaII and exonuclease III , as a result, no PANI are deposited. This method can be used to determine DNA methylation at the site of CpG. It exhibits a wide linear response toward M.SssI MTase activity in the range of 0.5–0.6 U mL −1 with the detection limit of 0.12 U mL −1 . G-rich DNA forms HRP mimicking DNAzyme, which avoids complex labeling procedures and is robust. The method is simple, reliable, sensitive and specific, which has been successfully applied in human serum samples and been used to screen the inhibitors. Thus, the proposed method may be a potential and powerful tool for clinical diagnosis and drug development in the future.
研究领域:
生物化学
·
医学诊断
·
分子生物学
✓ 已验证
产品关联
DOI
PubMed
高影响因子
SENSORS AND ACTUATORS B-CHEMICAL
·
2022
·
IF 9.221
Yihan Wang,Jinfeng Quan,Jialei Zhang,Kejing Huang,Xuemei Wang,Hui Jiang
The fragments of molecular motors can be transported and operated autonomously along a track. Most molecular walker systems are assembled from DNA building modules, exhibiting great potential applications in biocomputing, drug transport, sensing, etc. Hereby, we report a DNA walker driven by endonuclease Nt.BbvC I that can autonomously induce “signal off” sensing platform for highly sensitive detection of tumor cells. DNA density and trajectory conformation can affect DNA walker properties. Molybdenum selenide@gold nanoparticles (MoSe 2 @AuNPs) are modified on the electrode surface as a scaffold to accommodate more hairpin probe (H1) and provide a good interface for DNA walking. Magnetic beads are used to enrich target tumor cells, release single-stranded DNA (S1) and trigger DNA walker-based amplification strategy. Upon addition of tumor cells, more DNA-methylene blue (DNA-MB) is cracked from the electrode surface. Benefiting from efficient electrode modification, magnetic enrichment, DNA walker and enzyme-assisted amplification, an ultra-high sensitivity electrochemical cell analysis is realized, with a detection limit of 2 cells mL −1 . More importantly, this DNA walker strategy is available for tumor cell analysis in clinical samples, suggesting the potential diagnostic applications.
研究领域:
生物传感
·
分子生物学
·
医学诊断
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产品关联
DOI
高影响因子
危险品化学品经营许可证（带存储）