Biosensors-Basel
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2023
·Vol. 13
·期 9
·854
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IF 5.4
Yang Qing,Haobin Fang,Yuxing Yang,Yazhen Liao,Haiyu Li,Zhencui Wang,Jie Du
At present, a large number of studies have demonstrated that miRNAs can be used as biological indicators for the diagnosis and treatment of diseases such as tumours and cancer, so it is important to develop a new miRNA detection platform. In this work, miRNA-122 is used as the basis for targeting detection agents. We have designed an unlabelled DNA1 that undergoes partial hybridisation and has a 20 T base long strand. The fluorescent signal in this experiment is derived from copper nanoclusters (CuNCs) generated on the circular T-long strand of DNA1. At the same time, DNA1 is able to react with miRNA-122 and achieve hydrolysis of the part bound to miRNA-122 via the action of nucleic acid exonuclease III (Exo III), leaving a part of the DNA, called DNA3, while releasing miRNA-122 to participate in the next reaction, thus achieving circular amplification. DNA3 is able to react with DNA2, which is bound to streptavidin magnetic beads (SIBs) and separated from the reaction solution via the application of a magnetic field. Overall, this is a fluorescence signal reduction experiment, and the strength of the fluorescence signal from the copper nanoclusters can determine whether the target miRNA-122 is present or not. The degree of fluorescence reduction indicates how much DNA1, and thus the amount of target miRNA-122, has been hydrolysed. By evaluating the variations in the fluorescence signal under optimised conditions, we discovered that this method has good sensitivity, with a detection limit as low as 0.46 nM, better than many other previous works on fluorescence signal-based biosensors for miRNA detection. This technique offers high discrimination and selectivity and can serve as a persuasive reference for early diagnosis.Keywords:exonuclease III;miRNA-122;CuNCs;fluorescence reduction
研究领域:
分子生物学
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生物医学工程
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PubMed
高影响因子
SENSORS AND ACTUATORS B-CHEMICAL
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2020
·
IF 7.1
Yunyun Hu,Xin Guo,Peilin Gu,Qin Luo,Yang Song,Erqun Song
Since circulating tumor DNA (ctDNA) has been regarded as a promising noninvasive biomarker for early cancer screening, cancer condition monitoring, and prognosis evaluation, the accurate and simple detection of ctDNA has been extensive explored. Up to now, there is still a lot of space for following-up the assay of ctDNA, especially simply direct analysis ctDNA in complex biological samples such as blood sample. In this work, we develop a Mn 2+ -mediated magnetic relaxation switching (MRS) assay for direct and simple detection of ctDNA via exonuclease III-assisted signal amplification. In the presence of target ctDNA, exonuclease III selectively cleaves the hybridization complex of between ctDNA and its complementary DNA labeled with magnetic beads and alkaline phosphatase (MB 1000 -DNA 1 -ALP) to release ALP and ctDNA, the former further generating Mn 2+ and causing transverse relaxation time signal change while the later recycling to amplify the signal. The combination of efficient recognition of ctDNA, enzyme-assisted amplification, and the MRS assay could achieve direct and specific detection of ctDNA with a detection limit of 340 pM. In our opinion, this is the first successful direct detection of ctDNA in whole blood sample without ctDNA extraction from biospecimen, which make the assay for ctDNA much accurate and timely since a short half-life (2 h) of ctDNA in blood. The as-proposed method for ctDNA assay in this study shows potential tracking the evolution of tumor timely and serving as a liquid biopsy for diagnosis.
研究领域:
生物医学工程
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分子诊断学
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癌症研究
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DOI
高影响因子
BIOSENSORS & BIOELECTRONICS
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2013
·
IF 5.437
Wenjiao Zhou,Xue Gong,Yun Xiang,Ruo Yuan,Yaqin Chai
Visual detections have attracted great research attentions recently due to their convenient monitoring of the target analytes without using any advanced instruments. However, achieving visual detection of trace amounts of biomolecules with PCR-like sensitivity remains a major challenge. In current work, we describe a new quadratic signal amplification strategy for sensitive visual detection of HIV DNA biomarkers based on exonuclease III (Exo III)-assisted DNA recycling amplification and DNAzymes. The presence of the target HIV DNA leads to two independent and simultaneous DNA recycling processes to achieve quadratic signal amplification with the assistance of Exo III. This quadratic signal amplification results in catalytic cleavage of the G-quadruplex sequence-locked hairpin probes to release numerous active G-quadruplex sequences, which further associate with hemin to form DNAzymes and cause significantly intensified color change for sensitive and visual detection of HIV DNA down to 2.5 pM. The proposed visual detection method employs un-modified hairpin DNA as probes, avoids using any complex and expensive instruments for signal transduction and is essentially simple. This method also shows single-base mismatch discrimination capability as well. All these features make our developed DNA detection method holds great potential for visual monitoring of various DNA biomarkers at ultralow levels with careful and proper probe designs.
研究领域:
分子生物学
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分析化学
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PubMed
高影响因子
RSC Advances
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2016
·Vol. 6
·期 110
·108662-108667
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IF 3.289
Jin-Xiu Cao,Yong-Sheng Wang,Jin-Hua Xue,Yan-Qin Huang,Ming-Hui Li,Si-Han Chen,Bin Zhou,Xian Tang,Xiao-Feng Wang,Yu-Feng Zhu
We report on a novel and highly sensitive strategy for UO22+ detection. The designed single-labeled hairpin signal probe (SSP) was employed as a reporter, and a multipurpose RNA-cleaving DNAzyme as a target recognition element, catalytic DNAzyme and the primer of substrate fragment recycling amplification. The presence of UO22+ results in the substrate strand in DNAzyme being cleaved at the RNA site, releasing short substrate fragments. The fragment that is complementary partially with SSP could open the hairpin of SSP to form a double-stranded DNA with recessed 3′-termini, which could be selectively digested by exonuclease III. After the double-stranded DNA is fully consumed, the substrate fragment is released. This one could hybridize with another SSP to initiate the next round of digestion by exonuclease III. The recycling of substrate fragments can be repeated multiple times, resulting in the accumulation of the more 6-carboxyfluorescein in the solution along with an amplified fluorescence signal. By this strategy, we achieved hitherto an unreported UO22+ detection limit of 2.4 pM. Therefore, this novel exonuclease III-amplified strategy has great potential for highly sensitive and selective quantification of UO22+ in biomedical research and environmental science fields.
研究领域:
生物化学
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环境科学
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分析化学
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DOI
高影响因子
International Journal of Analytical Chemistry
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2024
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IF 1.5
Bingyuan Fan,Qian Wang,Shan Wang,Yahui Gao,Yan Liang,Jinru Pan,Xinrui Fu,Li Li,Wei Meng
MiR-378 is abnormally expressed in various cancers, such as hepatocellular carcinoma, renal cell carcinoma, and nonsmall cell lung cancer. Here, we developed a label- and immobilization-free ratiometric homogeneous electrochemical strategy based on exonuclease III (Exo III) for the facile and rapid determination of miR-378. Two 3′-protruding hairpin DNA probes (HPs) are designed in this strategy. Doxorubicin (DOX) and potassium ferrocyanide (Fe 2+ ) were used as label-free probes to produce a response signal (I DOX ) and a reference signal (I Fe 2+ ) in the solution phase. When no target was present in the solution, the HP was stable, most of the DOX was intercalated in the stem of the HP, and the diffusion rate of DOX was significantly reduced, resulting in reduced electrochemical signal response. When miR-378 was present, double-cycle signal amplification triggered by Exo III cleavage was initiated, ultimately disrupting the hairpin structures of HP1 and HP2 and releasing a large amount of DOX into the solution, yielding a stronger electrochemical signal, which was low to 50 pM. This detection possesses excellent selectivity, demonstrating high application potential in biological systems, and offers simple and low-cost electrochemical detection for miR-378.
研究领域:
生物化学
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分子生物学
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肿瘤学
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PubMed
高影响因子
SENSORS AND ACTUATORS B-CHEMICAL
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2019
·
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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高影响因子
BIOSENSORS & BIOELECTRONICS
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2015
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IF 6.409
Sai Bi,Xiaoqiang Jia,Jiayan Ye,Ying Dong
Gold nanomaterials promise a wide range of potential applications in chemical and biological sensing, imaging, and catalysis. In this paper, we demonstrate a facile method for room-temperature synthesis of gold nanostars (AuNSs) with a size of ~50 nm via seeded growth. Significantly, the AuNSs are found to have high light-scattering properties, which are successfully used as labels for sensitive and selective detection of nucleic acids and proteins by using exonuclease III (Exo III) as a biocatalyst . For DNA detection, the binding of targets to the functionalized AuNS probes leads to the Exo III-stimulated cascade recycling amplification. As a result, a large amount of AuNSs are released from magnetic nanoparticles (MNPs) into solution, providing a greatly enhanced light-scattering signal for amplified sensing process. Moreover, a binding-induced DNA three-way junction (DNA TWJ) is introduced to thrombin detection, in which the binding of two aptamers to thrombin triggers assembly of the DNA motifs and initiates the subsequent DNA strand displacement reaction (SDR) and Exo III-assisted cascade recycling amplification. The detection limits of 89 fM and 5.6 pM are achieved for DNA and thrombin, respectively, which are comparable to or even exceed that of the reported isothermal amplification methods. It is noteworthy that based on the DNA TWJ strategy the sequences are independent on target proteins. Additionally, the employment of MNPs in the assays can not only simplify the operations but also improve the detection sensitivity. Therefore, the proposed amplified light-scattering assay with high sensitivity and selectivity , acceptable accuracy, and satisfactory versatility of analytes provides various applications in bioanalysis .
研究领域:
生物传感
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分析化学
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纳米生物学
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PubMed
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
SPECTROCHIMICA ACTA PART A-MOLECULAR AND BIOMOLECULAR SPECTROSCOPY
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2023
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IF 4.4
Boxu Wang,Zheng Liu,Zhihong Li,Ningyi Xu,Xuejiao Zhang,Ruifang Su,Junyang Wang,Rui Jin,Chunyan Sun
Hg 2+ is highly toxic to human health and ecosystem. In this work, based on the unique fluorescent property of 2-Aminopurine (2-AP), the formation of T-Hg 2+ -T mismatch structure and the signal amplification of exonuclease III (Exo III) assisted target cycle, a fluorescent probe for facile and sensitive detection of Hg 2+ is constructed. The hairpin-looped DNA probe is rationally designed with 2-AP embedded in the stem and thymine-rich recognition overhangs extended at the termini. The cleavage of the double stranded DNA stem with stable T-Hg 2+ -T pairs catalyzed by Exo III is prompted to happen upon recognition of trace Hg 2+ . Under the optimal reaction conditions, there is an excellent linear relationship between Hg 2+ concentration and fluorescence intensity in the range of 7.5–200 nM with a detection limit of 0.38 nM. In addition, the detection results of Hg 2+ in Songhua River water and fish samples are satisfactory. The fluorescent probe avoids labeling additional quenchers or quenching materials and has strong anti-interference ability. Thus, the fluorescent probe has a broad prospect in practical application.
研究领域:
生物化学
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环境科学
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DOI
PubMed
高影响因子
TALANTA
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2021
·
IF 6.057
Cai-Xia Shi,Yan-Zi Li,Qing Liu,Zeng-Ping Chen,Sha-Sha Li,Ru-Qin Yu
Biosensors based on various spectroscopic techniques discriminate the target microRNA (miRNA) from non-target ones with single nucleotide polymorphisms (SNPs) according to the differences in signal intensities which can be caused by other factors besides SNPs. As a result, they are liable to produce false positive results. Herein, we report an attempt to develop a false-positive resistance, sensitive and reliable mass spectrometric platform for miRNA detection. In the proposed platform, the qualitative and quantitative information of the target miRNA was obtained through analyzing mass spectral responses of the multiply charged ions of the residual fragments of the probe DNA produced during exonuclease III assisted signal amplification reaction using an advanced data analysis method. The proposed platform could achieve sensitive and accurate quantitative results for the target miRNA (e.g., miRNA-141) in complex medium with a detection limit of about 1 pM, and unambiguously identify non-target miRNAs with SNPs based on the length distribution patterns of residual fragments of probe DNA. The findings obtained in this study might open an avenue for the design of new miRNA detection methods based on mass spectrometry in combination with various nuclease assisted signal amplification strategies.
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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PubMed
高影响因子
RSC Advances
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2014
·Vol. 5
·期 12
·9123-9129
·
IF 3.708
Fenglei Gao,Yan Du,Jingwen Yao,Yanzhuo Zhang,Jian Gao
We developed a novel dual-amplification strategy for ultrasensitive electrochemical detection of DNA based on exonuclease III-assisted target recycling and rolling circle amplification (RCA). In this assay, a gold electrode was used to immobilize a molecular beacon (MB) with a 3′ overhang as recognition probe and perform the dual signal amplification procedure. In the presence of target DNA, MB hybridized with the target DNA to form a duplex region and created a 3′-blunt end for Exo III to initiate the target DNA recycling amplification process to cleave numerous MB probes. Then, the remaining MB fragments hybridized as primers with the RCA template to initiate the RCA process. Subsequently, the detection probe's modified Ag NPs hybridized with the long, amplified DNA products, resulting in the multiplication of Ag NPs on the electrode surface, which were used for subsequent electrochemical strip analysis of silver. This novel signal amplification strategy could detect target DNA down to 6.4 amol L−1, with a dynamic range spanning five orders of magnitude. In addition, this method avoids the de-oxygenation procedure in standard electrochemical detection, thus presenting a promising application in clinical diagnosis.
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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高影响因子
Analytical Methods
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2016
·Vol. 8
·期 39
·7054-7060
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IF 1.915
Lili Yu,Hui Xu,Hou Chen,Liangjiu Bai,Wenxiang Wang
A label-free, exonuclease III (Exo III) assisted and cost-effective Hg2+ fluorometric assay based on a strand displacement amplification strategy was developed with high sensitivity and selectivity. Thymine–thymine (T–T) mismatches between the toehold domains in hairpin DNA and assistant DNA can serve as specific recognition elements for Hg2+ binding with the help of T–Hg2+–T base pairs to initiate a toehold-mediated strand displacement reaction. Exo III provides a reduced background signal, and the method is label-free using an intercalator, Sybr green I (SG). The number of T–T mismatches, the concentration of SG and Exo III, the incubation temperature and time were all optimized. Under the optimized condition, the limit of detection for Hg2+ can be as low as 9.42 pM with a linear range from 0.01 to 1 nM. Moreover, this proposed strategy shows excellent selectivity, and was successfully applied for the detection of Hg2+ in laboratory tap water and Yanjing lake water samples in our university.
研究领域:
生物化学
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环境科学
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DOI
高影响因子
危险品化学品经营许可证（带存储）