Analytical Methods
·
2016
·Vol. 8
·期 39
·7054-7060
·
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
·
环境科学
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DOI
高影响因子
RSC Advances
·
2016
·Vol. 6
·期 110
·108662-108667
·
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
·
分析化学
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DOI
高影响因子
BIOSENSORS & BIOELECTRONICS
·
2014
·
IF 6.451
Wang Ren,Zhong Feng Gao,Nian Bing Li,Hong Qun Luo
This work reported a novel, ultrasensitive, and selective platform for electrochemical detection of DNA , employing an integration of exonuclease III (Exo-III) assisted target recycling and hybridization chain reaction (HCR) for the dual signal amplification strategy. The hairpin capture probe DNA (C-DNA) with an Exo-III 3′ overhang end was self-assembled on a gold electrode. In the presence of target DNA (T-DNA), C-DNA hybridized with the T‐DNA to form a duplex region, exposing its 5′ complementary sequence (initiator). Exo-III was applied to selectively digest duplex region from its 3-hydroxyl termini until the duplex was fully consumed, leaving the remnant initiator. The intact T-DNA spontaneously dissociated from the structure and then initiated the next hybridization process as a result of catalysis of the Exo-III. HCR event was triggered by the initiator and two hairpin helper signal probes labeled with methylene blue , facilitating the polymerization of oligonucleotides into a long nicked dsDNA molecule. The numerous exposed remnant initiators can trigger more HCR events. Because of integration of dual signal amplification and the specific HCR process reaction, the resultant sensor showed a high sensitivity for the detection of the target DNA in a linear range from 1.0 fM to 1.0 nM, and a detection limit as low as 0.2 fM. The proposed dual signal amplification strategy provides a powerful tool for detecting different sequences of target DNA by changing the sequence of capture probe and signal probes, holding a great potential for early diagnosis in gene-related diseases.
研究领域:
生物化学
·
分析化学
·
纳米技术
·
分子生物学
·
生物医学工程
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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
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环境科学
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DOI
高影响因子
MICROCHIMICA ACTA
·
2019
·Vol. 186
·期 8
·1-7
·
IF 5.479
Li Mei,Xu Xiong,Cai QingYou,Luo XuJian,Zhou ZhongGao,Xu GuoHai,Xie YongRong
A graphene oxide-based method has been developed for ultrasensitive and selective determination of microRNA-141 by means of rolling circle amplification (RCA) and exonuclease III (Exo III)-assisted recycling amplification. The method uses (a) a padlock probe with a hybrid sequence that is complementary to the target microRNA-141 at both the 5'- and the 3'-end, and (b) a long binding region of a signalling reporter strand. On addition of microRNA-141, it acts as the primer for triggering the RCA reaction following ligation. This results in the formation of a repeatedly concatenated sequence of the padlock probe. Subsequently, the RCA product hybridizes with the fluorescein-labelled signal strand to form the duplex DNA containing the blunt 3'-termini of signal strand. Addition of Exo III causes signal strand digestion and leads to RCA product recycling and liberation of fluorescein. Added graphene oxide does not absorb the fluorescein liberated because of the poor mutual interaction. Therefore, microRNA-141 can be quantified by measurement of the green fluorescence under excitation/emission wavelengths of 470/520 nm. The method has a 100 aM detection limit towards microRNA-141 and works in the wide range from 1 fM to 1 nM. It can discriminate even single-mismatched microRNA and shows good selectivity and sensitivity when applied to spiked human serum.
Schematic representation of a graphene oxide (GO)-based method for fluorometric determination of microRNA by using rolling circle amplification and exonuclease III (Exo III)-aided recycling amplification. With microRNA, the fluorescein-labelled signal strand becomes digested, and this genererates a fluorescent signal.
研究领域:
分子生物学
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生物化学
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纳米医学
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DOI
PubMed
高影响因子
ANALYST
·
2014
·Vol. 139
·期 24
·6387-6392
·
IF 3.906
Chunyan Gao,Henan Li,Yuanjian Liu,Wei Wei,Yuanjian Zhang,Songqin Liu
Strategies to detect the methylation of site specific DNA and assay of M.SssI methyltransferase (M.SssI MTase) activity are important in determining human cancers due to aberrant methylation linked to cancer initiation and progression. Herein, we report a label-free fluorescence detection method for DNA methylation and MTase activity based on restriction endonuclease HpaII and exonuclease III (Exo III). A label-free probe DNA was designed, which hybridized with target DNA (one 32-mer DNA from the exon 8 promoter region of the Homo sapiens p53 gene) to form double stranded DNA (dsDNA). Upon the cleavage action of HpaII and degradation reaction of Exo III, dsDNA changed to single stranded DNA (ssDNA) and the fluorescence intensity of thiazole orange (TO) is weak. After the resulting dsDNA was methylated by M.SssI MTase, the action of HpaII and Exo III was prevented, then TO intercalates into the dsDNA and emits strong fluorescence. This method can determine DNA methylation at the site of CpG and distinguish a one-base mismatched target sequence. The fluorescence intensity has a linear relationship with M.SssI MTase activities in the range of 1–10 U mL−1 with a detection limit of 0.16 U mL−1 in terms of 3 times deviation of the blank sample. The methylation of DNA by a hydroxyl radical triggered by DMSO and CH3CHO was also measured. These results show that the proposed method can specifically and selectively detect DNA methylation and M.SssI MTase activity. Human serum has no obvious effects on the assay performance, indicating that the method has great potential for further application in complex samples.
研究领域:
生物化学
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分子生物学
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遗传学
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癌症研究
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DOI
PubMed
高影响因子
Biosensors-Basel
·
2026
·Vol. 16
·期 5
·279
·
IF 6.2
Lidan Niu,Siyu Huang,Jinmei Zhao,Wenjing Yang,Zhengnan Li,Siqi Niu,Jianchun Yang,Shiqi Chen,Qihui Wang
The safety of genetically modified crops, particularly the commercial cultivation of glyphosate-resistant genetically modified soybeans, has given rise to significant public concern. Consequently, there is an urgent need to develop efficient and precise methods for detecting genetically modified components. The present study constructed a novel electrochemical biosensor based on nucleic acid exonuclease III (Exo III)-assisted cyclic signal amplification and hairpin probe recognition for the highly sensitive and specific detection of the CP4-EPSPS gene in genetically modified soybeans. The sensor achieves exponential signal amplification by triggering Exo III to cyclically cleave the hairpin probe (H1) upon target DNA binding. Subsequent to this, the released DNA fragments hybridize with the methylene blue-labeled signal probe (HS-MB) that has been immobilized on the electrode surface. This process induces conformational changes and a decrease in the current signal, thereby enabling quantitative analysis of the target gene. The experimental phase of the study successfully validated the sensor’s mechanism and systematically optimized key parameters such as Exo III concentration and reaction time. In optimal conditions, the sensor demonstrated excellent linearity with target DNA concentrations ranging from 100 fM to 10 nM, achieving a detection limit as low as 0.1072 pM. Furthermore, it exhibited remarkable repeatability and stability. This study provides an analytical tool with broad application prospects for the rapid and precise detection of genetically modified crops.
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DOI
PubMed
高影响因子
MICROCHIMICA ACTA
·
2020
·Vol. 187
·期 9
·1-8
·
IF 6.232
Li Xiaoyun,Weng Chenyuan,Wang Jing,Yang Wei,Lu Qiaoyun,Yan Xiaoqiang,Sakran Marwan Ahmad,Hong Junli,Zhu Wanying,Zhou Xuemin
A novel label-free and exonuclease III (Exo III)–assisted signal amplification electrochemical aptasensor was constructed for the determination of carcinoembryonic antigen (CEA) via magnetic field–induced self-assembly of magnetic biocomposites (Fe 3 O 4 @Au NPs-S1-S2-S3). The magnetic biocomposites were acquired by modifying double-stranded DNA (S1-S2-S3) on the surface of Fe 3 O 4 @Au nanoparticles (Fe 3 O 4 @Au NPs). Among them, Fe 3 O 4 @Au NPs were used as carriers for magnetic separation, thiolated single-stranded DNA (S1) provided signal sequence, CEA aptamer (S2) worked as a recognition element, and complementary strand (S3) was used to form double strands. In the presence of CEA, S2 bonded with CEA competitively; the exposed S1 could not be cleaved since Exo III was inactive against ssDNA. The G-quadruplex/hemin complexes finally formed with the existence of K + , and the high electrochemical signal of G-quadruplex/hemin complexes was recorded by differential pulse voltammetry (DPV) at − 0.6 V. Conversely, in the absence of CEA, dsDNA was cleaved from the 3′ blunt end by Exo III; the disappearance of G-rich sequence blocked the generation of the signal. This method exhibited good selectivity and sensitivity for the determination of CEA; the linear range was from 0.1 to 200 ng mL −1 and the limit of detection was 0.4 pg mL −1 .
Graphical abstract
研究领域:
生物化学
·
分析化学
·
生物传感器
·
癌症诊断
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产品关联
DOI
PubMed
高影响因子
SENSORS AND ACTUATORS B-CHEMICAL
·
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
·
分子诊断学
·
癌症研究
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DOI
高影响因子
MICROCHEMICAL JOURNAL
·
2018
·
IF 2.746
Lili Yu,Hui Xu,Hou Chen,Shengxiao Zhang,Tingting Jiang,Liangjiu Bai,Wenxiang Wang,Shanmin Gao,Juan Jin
A label-free DNA Y junction sensor for amplified detection of Hg 2+ has been demonstrated by using thymine-Hg 2+ -thymine (T-Hg 2+ -T) recognition, cascade toehold-mediated DNA strand displacement and dye staining double-stranded DNA (dsDNA). In our work, assistant DNA could bind with the first hairpin DNA with toehold domains by the help of thymine-Hg 2+ -thymine (T–Hg 2+ –T) base pairs recognition. The second hairpin DNA with toehold domains will then hybridize with the complementary sequences in first hairpin DNA, which again initiates a new toehold domains to open the third hairpin DNA, triggering a cascade strand displacement reaction. Exonuclease III (Exo III) was added to digest the three hairpin DNA with recessed 3′ ends in the absence of Hg 2+ to reduce the background signal. As a comparison, Exo III is replaced by GO to reduce the background signal because single-strand DNA (ssDNA), three hairpin DNAs with intercalated dye could be adsorbed onto the surface of GO and lead to quenched fluorescence. The method, designed in a simple “mix, incubate, and detect” format, can detect concentrations of Hg 2+ as low as 0.031 nM, and exhibit good selectivity and practical application.
研究领域:
生物化学
·
环境科学
·
分析化学
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DOI
高影响因子
SENSORS AND ACTUATORS B-CHEMICAL
·
2018
·
IF 5.667
Xiao Fang Zhang,Na Li,Yu Ling,Nian Bing Li,Hong Qun Luo
Since uracil-DNA glycosylase (UDG) is closely related to some human diseases, monitoring and detection of UDG activity have great significance in its clinic diagnosis and functional study. Here we demonstrate a sensitive cascade multi-amplification fluorescence strategy for the active UDG assay with the help of exonuclease III (Exo III). Under the cleavage reaction of UDG, the double-stranded DNA containing peculiar uracil bases separated into two dissociative single-stranded DNA, which separately hybridized with two cyclic hairpin probes to launch the Exo III-assisted dual cycles. The hairpin DNA modified with fluorophore and quenching group was employed as the signal probe. After the dual cycles, countless same short DNA fragments were generated to hybridize with signal probes, initiating a new Exo III-assisted cyclic amplification and releasing numerous single-stranded DNA which carried only fluorophores. Thus, the fluorescence intensity of the detection system was enhanced. This study obtained the detection limit as low as 2.4 × 10 −4 U/mL for detecting the UDG activity. And it performed satisfactory selectivity and well practical applicability by analysis of the HeLa cell lysate, providing a potential method for clinic diagnosis and functional study of UDG activity.
研究领域:
生物化学
·
分子生物学
·
临床诊断
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DOI
高影响因子
BIOSENSORS & BIOELECTRONICS
·
2018
·
IF 8.173
Kangyao Zhang,Shuzhen Lv,Minghua Lu,Dianping Tang
In this work, a new “signal-on” split-type photoelectrochemical (PEC) sensing platform for prostate-specific antigen (PSA) detection was successfully constructed using p-type Cu-doped Zn 0.3 Cd 0.7 S as the photosensitive semiconductor material and target-triggered rolling circle amplification (RCA) for signal amplification . The signal derived from Cu-doped Zn 0.3 Cd 0.7 S was amplified by hemin/G-quadruplex. Upon target PSA introduction, the aptamer-primer probe (apt-pri) was captured by capture antibody-conjugated magnetic bead (MB-mAb) to form the sandwiched MB-mAb/PSA/apt-pri. The complex could initiate the RCA reaction to produce a long single-stranded DNA that provided binding sites for G-rich DNA and to form long single-stranded DNA/G-quadruplex/hemin. Upon the addition of exonuclease III (Exo III), the hemin/G-quadruplex immobilized on the RCA long product could be released by the digestion of Exo III. The hemin/G-quadruplex complexes in this study were used as efficient electron acceptors to neutralize the photoelectrons generated from the semiconductor and hindered the recombination of charges, thus enhancing the photocurrent . Under the optimum conditions, the developed sensing system displayed a good analytical performance with a linear range of 0.05–40 ng mL −1 PSA and a detection limit of 16.3 pg mL −1 . Furthermore, good selectivity , high anti-interference ability, satisfactory reproducibility, and good accuracy were also achieved. These prominent analytical properties revealed that our strategy might be a potential and reliable tool for the detection of PSA.
研究领域:
生物化学
·
分析化学
·
生物传感器
·
癌症诊断
✓ 已验证
DOI
PubMed
高影响因子
危险品化学品经营许可证（带存储）