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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DOI
高影响因子
ACS Omega
·
2019
·
IF 2.584
Yu Ding,Xianming Li,Xinfeng Zhang,Feng Li,Xiandeng Hou,Peng Wu
Exonuclease III (Exo III) is an important enzymatic tool that is being widely used in molecular biology, biotechnology, and bioassay development. Exo III prefers to cleave double-stranded DNA (dsDNA) with blunt and recessed 3′-termini rather than their protruding counterpart. While it has been accepted that a short 3′-overhang (e.g., >4 nt) is necessary to protect a dsDNA from Exo III cleavage, critical roles of the length and sequence of this 3′-overhang remain unexplored. Herein, we develop a novel light-induced colorimetric assay allowing the systematic probe of the sequence selectivity of Exo III in a rapid and high-throughput manner. Our finding that Exo III is highly specific to 3′-overhang in terms of both length and sequence will be valuable for guiding the design of bioassays and DNA manipulating tools mediated by Exo III.
研究领域:
分子生物学
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生物技术
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酶学研究
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DOI
PubMed
高影响因子
TALANTA
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2019
·
IF 4.916
Chen Liu,Jialun Han,Jingjing Zhang,Jie Du
A novel detection platform based on Exonuclease III dual-cycle signal amplification with graphene oxide as quenching agent and DNA labelling as recognition element has been developed for the fluorometric determination of insulin. Specific DNA sequences were cut by Exo III to control the fluorescence recovery of the system. Fluorescence intensity analysis was carried out by fluorescence spectrophotometer to obtain the fluorescence intensity corresponding to different concentrations of insulin. When insulin is added to the system, insulin binds to the adaptor and the fluorescence is quenched, whereas in the absence of insulin the fluorescence is restored. The sensor platform achieved multiple signal amplification affording rapid and sensitive detection of insulin with a detection range of 0.048–2.15 U/ml. The methodology is rapid, sensitive, of low cost and convenient to perform and has potential for routine screening of insulin in blood, and also has prospects for the detection of other proteins and biomolecules.
研究领域:
生物化学
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医学检测
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DOI
PubMed
高影响因子
TALANTA
·
2023
·
IF 6.1
Yaqi Hu,Fang Bao,Siyuan Fu,Shiyuan Feng,Jinfeng Miao,Peng Miao,Yuanyuan Xu
Coronaviruses are highly infectious and pose a serious threat to human and animal healths. In this work, a facile electrochemical method based on Exonuclease III (Exo III) catalyzed digestion and silver deposition is developed for coronavirus RNA analysis. A magnetic separation procedure is performed to specifically identify target sequence and release single-stranded DNA modified gold nanoparticles (AuNPs). The nanoparticles can thus be immobilized at a screen-printed electrode and catalyze silver deposition for signal readout. This method allows sensitive analysis of PEDV and SARS-CoV-2 RNAs in the concentration range from 1 to 1000 nM with the limits of detection as low as 0.47 nM and 0.17 nM, respectively. Good specificities are demonstrated. Thus, the proposed method may have great potential use in the applications of coronaviruses analysis.
研究领域:
生物医学工程
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分子生物学
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DOI
PubMed
高影响因子
Analytical Methods
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2014
·Vol. 6
·期 12
·4366-4372
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IF 1.938
Lina Ma,Lan Ma,Min Su,Zhenxin Wang
Herein, a gold nanoparticle (GNP)-based colorimetric aptasensor has been developed for detecting adenosine with exonuclease III (Exo III) assisted recycling amplification. In this aptasensor, two kinds of single-stranded oligonucleotide functionalized GNP probes (Oligo 1-GNPs and Oligo 2-GNPs) are firstly hybridized with one complementary single-stranded oligonucleotide (linker) with the motif of adenosine aptamer, resulting in the formation of GNP aggregates (cross-linked GNPs). In the presence of adenosine, the linker would form a double-stranded DNA (dsDNA) with a recessed 3′-terminus due to its structural switching property, which would lead to disassembly of the cross-linked GNPs. However, upon the addition of Exo III, the dsDNA is enzymatically digested from the 3′-hydroxyl termini, liberating the adenosine. The released adenosine can then interact with another linker and a new round of enzymatic digestion is started. Therefore, remarkable signal amplification is achieved because the disassembly of cross-linked GNPs is strongly dependent on the free adenosine in the reaction mixture. The experimental results demonstrate that the Exo III-assisted GNP-based colorimetric aptasensor is more sensitive (ca. 10 times) than that without Exo III amplification. The Exo III-assisted GNP-based colorimetric aptasensor detects adenosine downs to 5.6 nM, with a linear relationship from 10 nM to 1 mM. Moreover, the Exo III-assisted GNP-based colorimetric aptasensor has been successfully applied to detect Jinshuibao capsule (JSBC) containing adenosine in both a buffer solution and a diluted human serum, demonstrating that this assay has great potential to be employed for detecting adenosine in real samples.
研究领域:
生物化学
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分析化学
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纳米技术
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DOI
高影响因子
MICROCHEMICAL JOURNAL
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2026
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IF 5.2
Henan Wang,Baiang Chen,Xuran Zhao,Yue Huang
Ochratoxin A (OTA) as one of the most hazardous mycotoxins has drawn particular concern owing to its widespread occurrence in food and severe threat on human health. Herein, an exonuclease III (Exo III)-amplified electrochemical biosensor is developed for the sensitive detection of OTA by using bimetallic AuPt as signal enhancer. AuPt bimetallic nanoparticles loaded with massive electrochemical species thionine are employed as the excellent signal amplification probes (AuPt@Thi). The specific recognition of OTA triggers the conformational switching of DNA probes and induces the Exo III-assisted nucleic acid cycle amplification, generating a significant amount of DNA segments which are then introduced to the sensing interface to allow the attachment of signal probes AuPt@Thi. Bimetallic AuPt with outstanding electrical conductivity, exceptional loading capacity, and high catalytic activity enables the enhancement of Thi redox signal, resulting in a remarkable increase of signal response. Benefiting from the synergistic effect of Exo III-catalyzed cycle amplification and signal enhancement of electrochemically active AuPt@Thi, the proposed biosensor demonstrates superior analytical performance for OTA detection with the advantages of rapid response, fine robustness, cost-effectiveness and simple operation. Under the optimal conditions, a broad linear detection range of 0.1 pg mL −1 –100 ng mL −1 and the limit of detection down to 0.07 pg mL −1 are achieved. Furthermore, the as-prepared electrochemical biosensor shows good specificity, acceptable stability, satisfactory repeatability, and reliable capability for monitoring the OTA in actual food samples, implying promising practical applicability in the realm of food safety testing.
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高影响因子
MICROCHIMICA ACTA
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2018
·Vol. 185
·期 5
·1-6
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IF 5.705
Liu Renjie,Wu Hua,Lv Lei,Kang Xiaojiao,Cui Chengbi,Feng Jin,Guo Zhijun
This study describes an aptamer based assay for the mycotoxin ochratoxin A (OTA). The method is based on the use of an OTA-specific aptamer, exonuclease (Exo) III, SYBR Gold as a fluorescent probe, and a complementary strand that specifically combines with the aptamer. In the presence of OTA, the aptamer and OTA hybridize, thereby resulting in the formation of ssDNA, which is not digested by Exo III. Intense fluorescence is observed after addition of SYBR Gold (best measured at excitation/emission wavelengths of 495/540 nm). Fluorescence increases linearly with the log of the OTA concentration in the range from 8 to 1000 ng · mL −1 . The detection limit is 4.7 ng·mL −1 . The assay was applied to the determination of OTA in diluted [2%( v /v)] red wine, and recoveries and RSDs ranged between 93.5% and 113.8%, and between 3.2% and 5.7%, respectively.
In the presence of ochratoxin A (OTA), specific combinations of aptamer and OTA may occur and result in DNA double strands being untied, which avoids being digested by Exo III. Intense fluorescence is observed after SYBR Gold addition.
研究领域:
食品科学
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分子生物学
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DOI
PubMed
高影响因子
RSC Advances
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2017
·Vol. 7
·期 80
·50420-50424
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IF 3.108
Minhao Xie,Kai Zhang,Feifan Zhu,Hao Wu,Pei Zou
Herein, a simple, highly sensitive and selective exonuclease III (Exo III)-based sensor was developed for mercury ions Hg2+ detection without the need of complicated, high-cost and time-consuming labeling. In this strategy, two oligonucleotides were employed for the experiment design. DNA1 was prepared with one DNA strand which was designed as a quadruplex-forming oligomer, and which was constructed as 3′-protruding to ensure the probe cannot be digested by Exo III. When the target Hg2+ is introduced into the sensing system, it associates with DNA1 and DNA2 to form double strand DNAs (DNA1/DNA2 duplexes) which have a blunt 3′-terminus. Exo III then can degrade part of DNA1, releasing the DNA1 and Hg2+, and ultimately liberating the G-rich oligomer. This released G-rich oligomer folds into a G-quadruplex structure and thus allows the formation of DNAzyme in the presence of hemin. The formed DNAzyme can effectively catalyze the H2O2-mediated oxidation of ABTS, giving rise to a change in solution color. The released DNA1 and Hg2+ are free to bind to another 3′-protruding terminus of DNA1 to trigger a new digestion reaction, leading to significant amplification of the signal. The present new strategy shows a limit of detection as low as 66 pM and excellent selectivity toward Hg2+ over a wide range of metal ions.
研究领域:
分析化学
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环境科学
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生物化学
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DOI
高影响因子
SENSORS AND ACTUATORS B-CHEMICAL
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2019
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IF 6.393
Kai Wen,Xingdong Yang,Cuiling Zhang,Huiying Wei,Guizheng Zou,Ye Zhu
Diffusion-mediated homogeneous electrochemical assay has attracted substantial attention owing to its simplicity, rapid response, and high recognition efficiency, however, lack of inherent connection between signal reporter and electrode surface restricts the improvement of detection sensitivity and further limits its potential applications. Herein, a facile electrochemical strategy is proposed through integrating the advantages of traditional heterogeneous assay and homogenous assay. In this strategy, a small amount of target DNA can trigger the release of assist DNA from the electrode surface. Then, the assist DNA can induce the homogeneous amplification reaction, which in turn promotes the amplification on the electrode surface by generating numerous target DNA analogues, thereby realizing positive feedback signal amplification. As a result, ultrasensitive electrochemical detection of nucleic acids was achieved in the dynamic range from 5 aM to 5 fM with a detection limit down to 1.2 aM. There is about 2.5 orders of magnitude improvement in sensitivity compared with the control strategy without homogeneous amplification. Moreover, the proposed strategy was demonstrated with high selectivity, reproducibility, and capability of detecting nucleic acids in serum samples, displaying its promising potential in the development of ultrasensitive electrochemical biosensing platform for disease diagnosis applications.
研究领域:
生物化学
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分析化学
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DOI
高影响因子
ANALYST
·
2021
·Vol. 146
·期 8
·2664-2669
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IF 4.616
Liling Lu,Xiao Han,Jingwen Lin,Yingxin Zhang,Minghao Qiu,Ying Chen,Meijin Li,Dianping Tang
Herein a rapid and sensitive fluorometric bioanalysis platform for mercury(II) (Hg2+) detection was innovatively developed using ultrathin two-dimensional MXenes (Ti3C2) as fluorescence quencher and Hg2+-induced exonuclease III (Exo III)-assisted target recycling strategy for efficient signal amplification. Initially, fluorophore-labeled single-stranded DNA (FAM-labeled probe) can be easily adsorbed onto the surface of ultrathin Ti3C2 nanosheets by hydrogen bonding and metal chelating interaction, and the fluorescence signal emitted by the FAM-labeled probe is quenched strongly owing to the fluorescence resonance energy transfer between the FAM and ultrathin Ti3C2 nanosheets. Upon sensing the target Hg2+, the protruding DNA fragment at the 3′ end of hairpin will hybridize with primer (hairpin-Hg2+-primer), and then further digested by Exo III to produce a probe (nicker). The released target Hg2+ and primer continue to participate in the next recycling, resulting in more hairpin probes becoming nickers. The combination of a large number of nickers and FAM-probe resulted in a significant increase in the fluorescence signal of the system, which was attributed to the fact that the double helix DNA was more rigid and separated from the surface of the ultrathin Ti3C2 nanosheets. The obvious fluorescence signal change of the Ti3C2-based Exo III-assisted target recycling can be accurately monitored by fluorescence spectrometry, which is also proportional to the concentration of Hg2+. Under optimum operating conditions, the peak intensity (520 nm wavelength) of fluorescence increased with increasing Hg2+ within a wide dynamic working range from 0.05 nM to 50 nM (R2 = 0.9913) with a limit of detection down to 42.5 pM. The proposed strategy uses ultrathin MXenes as a platform for binding nucleic acids, which contributes to its potential in nucleic acid hybridization-based biosensing and/or nucleic acid signal amplification bio-applications.
研究领域:
分析化学
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环境科学
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纳米技术
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生物传感
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DOI
PubMed
高影响因子
RSC Advances
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2016
·Vol. 6
·期 49
·43501-43508
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IF 3.289
Hongzhi Wang,Yu Wang,Su Liu,Jinghua Yu,Yuna Guo,Ying Xu,Jiadong Huang
In this work, a novel electrochemical DNA sensor based on exonuclease III (Exo III)-assisted autocatalytic DNA biosensing platform for ultrasensitive detection of antibiotics has been reported. When the arched probe was challenged with target, the released primer caused by the specific recognition of the aptamer and target antibiotics hybridized with the 3′-protruding terminus in the HP1. Then, Exo III could catalyze the stepwise removal of mononucleotides from this flat terminus, resulting in the release of the primer and trigger. As a secondary target, trigger could replace Helper from the 5′-MB labeled HP2, and HP2 formed a “close” probe structure, confining MB close to the electrode surface for efficient electron transfer. To our best knowledge, such work is the first report about Exo III-assisted autocatalytic DNA biosensing platform combing with signal-on sensing strategy, which has been utilized for quantitative determination of antibiotics. It would be further used as a general strategy associated with more analytical techniques toward the detection of a wide spectrum of analytes. Thus, it holds great potential for the development of ultrasensitive biosensing platform for the applications in bioanalysis, disease diagnostics, and clinical biomedicine.
研究领域:
生物分析
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分子诊断
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临床生物医学
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DOI
高影响因子
SENSORS AND ACTUATORS B-CHEMICAL
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2019
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IF 6.393
Rufeng Zhang,Yu Wang,Xiaonan Qu,Shasha Li,Yihan Zhao,Su Liu,Jiadong Huang
Herein, an Exonuclease III (Exo III)-powered DNA walking machine has been integrated into electrochemical sensing for label-free and highly sensitive detection of antibiotics. The electrochemical sensing interface is prepared by co-assembly of hemin aptamer-containing DNA track (DT) and DNA walker (DW)-locking probe (LP) duplex on gold electrode surface. The specific target and aptamer interaction triggers Exo III-assisted cyclic amplification, releasing the unlocking probe (UP) that is used for the deblocking of DW. Then the autonomous moving of DW on electrode surface is propelled via Exo III digestion of hybridized DT. The consumption of DT induces the formation of G-quadruplex/hemin DNAzyme that can catalyze the reduction of H 2 O 2 , thus a significantly strong current signal is released. Using ampicillin as a model analyte, the proposed biosensor achieves a detection limit of 0.76 pM within a linear detection range from 1 pM to 10 nM. Additionally, this biosensing strategy can be readily extended for the detection of wide variety of analytes by using corresponding target recognition probes. Therefore, the proposed strategy indeed creates a highly sensitive and label-free electrochemical sensing platform for molecular diagnosis applications.
研究领域:
生物化学
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分析化学
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分子诊断
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DOI
高影响因子
危险品化学品经营许可证（带存储）