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1.
Ecotoxicol Environ Saf ; 194: 110417, 2020 May.
Artigo em Inglês | MEDLINE | ID: mdl-32171958

RESUMO

A fluorescence aptasensor for the highly specific and sensitive determination of tetrodotoxin was established with tetrodotoxin-aptamer as the recognition unit, berberine as the signal reporter and exonuclease I as the elimination agent for the background. Berberine has a weak fluorescence emission at 540 nm, and it can form the tetrodotoxin-aptamer/berberine complex, resulted in an increased fluorescence. After introducing exonuclease I, it can degrade the single strand oligonucleotides of tetrodotoxin-aptamer into the single nucleotide in the absence of tetrodotoxin, which lead to dramatic fluorescence quenching, and reduce the background signal of sensing system. Once tetrodotoxin is in the presence, tetrodotoxin-aptamer is converted into the stable neck ring conformation, which resists the degradation of exonuclease I and provides a more rigid micro-environment for the excited state of berberine, and then the strong fluorescence is observed. Based on the above properties, an ultrasensitive label-free fluorescence aptasensor for tetrodotoxin is established. The fluorescence aptasensor shows good analytical performance with the linear increase of fluorescence intensity at the tetrodotoxin concentration from 0.030 nM to 6.0 × 103 nM. The detection limit of 11.0 pM is much lower than that of other reported sensor methods.


Assuntos
Aptâmeros de Nucleotídeos/química , Técnicas Biossensoriais/métodos , Exodesoxirribonucleases/química , Fluorometria/métodos , Tetrodotoxina/análise , Animais , Berberina/química , Humanos , Limite de Detecção , Músculos/química , Tetraodontiformes , Tetrodotoxina/sangue
2.
Nucleic Acids Res ; 48(5): 2457-2472, 2020 03 18.
Artigo em Inglês | MEDLINE | ID: mdl-31889185

RESUMO

DNA damage is the driving force for mutation and genomic instability, which can both lead to cell death or carcinogenesis. DNA double strand breaks are detected and processed in part by the Mre11-Rad50-Nbs1 protein complex. Although the Mre11-Rad50-Nbs1 complex is essential, several spontaneous mutations have been noted in various cancers. One of these mutations, within a conserved motif of Rad50, resulted in an outlier curative response in a clinical trial. We show through biochemical and biophysical characterization that this cancer-associated mutation and a second mutation to the adjacent residue, previously described in a breast cancer patient, both have gain-of-function Rad50 ATP hydrolysis activity that results not from faster association of the ATP-bound form but faster dissociation leading to less stable Rad50 dimer. This disruption impairs the regulatory functions of the protein complex leading to a loss of exonuclease activity from Mre11. Interestingly, these two mutations affect Rad50 structure and dynamics quite differently. These studies describe the relationship between function, structure, and molecular motions in improperly regulated Rad50, which reveal the underlying biophysical mechanism for how these two cancer-associated mutations affect the cell.


Assuntos
Trifosfato de Adenosina/metabolismo , Proteínas Arqueais/química , Proteínas Arqueais/metabolismo , Endodesoxirribonucleases/química , Endodesoxirribonucleases/metabolismo , Exodesoxirribonucleases/química , Exodesoxirribonucleases/metabolismo , Mutação/genética , Pyrococcus furiosus/genética , Regulação Alostérica , Sequência de Aminoácidos , Hidrólise , Modelos Biológicos , Modelos Moleculares , Complexos Multiproteicos/metabolismo , Estrutura Secundária de Proteína , Relação Estrutura-Atividade
3.
Talanta ; 209: 120550, 2020 Mar 01.
Artigo em Inglês | MEDLINE | ID: mdl-31891998

RESUMO

Developing the high selectivity and sensitivity strategy for nucleic acid detection is crucial for early diagnosis and therapy of diseases. In this work, a novel low back-ground fluorescent sensor platform for the detection of nucleic acid has been developed based on δ-FeOOH nanosheets integrating with exonuclease III-assisted target-recycling signal amplification. Because of the strong binding ability between the single-strand DNA (ssDNA) and the δ-FeOOH nanosheets, the dye-labeled ssDNA probe would be quenched by δ-FeOOH nanosheets through fluorescence resonance energy transfer (FRET). By using magnetic separate properties of δ-FeOOH, the background signal was separated from the sensor system, and the low background sensor system was obtained. After adding the target DNA, a double-strand DNA complex (dsDNA) would be formed between the target DNA and dye-labeled ssDNA probe. Then, the dye-labeled ssDNA probe in the dsDNA complex would be stepwise hydrolyzed into short fragments from 3'-terminus by Exonuclease III, and the fluorescence signal was recovered due to the weak bind affinity between the short fragments and δ-FeOOH nanosheets. By using the fluorescence quenching ability of δ-FeOOH nanosheets and enzyme-assisted target-recycling signal amplification, this strategy could show an excellent selectivity toward hepatitis C virus DNA with a low detection limit of 10 pM. By simply changing the dye-labeled ssDNA probe sequence, this sensing platform can be developed as a universal approach for the simple, sensitive, and selective detection of different target DNA.


Assuntos
DNA Viral/sangue , Exodesoxirribonucleases/química , Compostos Férricos/química , Hepacivirus/isolamento & purificação , Hepatite C/sangue , Técnicas Biossensoriais/métodos , Sondas de DNA/química , DNA de Cadeia Simples/química , DNA Viral/análise , Transferência Ressonante de Energia de Fluorescência/métodos , Corantes Fluorescentes/química , Hepatite C/virologia , Humanos , Limite de Detecção , Nanoestruturas/química
4.
Talanta ; 206: 120216, 2020 Jan 01.
Artigo em Inglês | MEDLINE | ID: mdl-31514829

RESUMO

In this work, an intriguing phenomenon was found and studied that the efficiency of exonuclease (Exo) III-assisted recycling amplification could be greatly promoted by the three-way junction structure. This was utilized as a visualizable detection strategy of DNA. Herein, highly bright DNA hairpin-templated silver nanoclusters (AgNCs) with quantum yield of 69.2% was firstly employed as the label-free signal output. The self-designed DNA hairpin was used as a molecular beacon by the relationship between the hairpin structure and the fluorescence signal of AgNCs. By recycling amplification of Exo-III, a significant change in fluorescence signal generated. Eventually, a label-free, visual, universal DNA detection platform based on Exo-III assisted amplification and DNA hairpin-AgNCs with high quantum yield was proposed.


Assuntos
DNA/análise , Corantes Fluorescentes/química , Nanopartículas Metálicas/química , Prata/química , DNA/química , DNA/genética , Sondas de DNA/química , Sondas de DNA/genética , Exodesoxirribonucleases/química , Fluorescência , Sequências Repetidas Invertidas , Limite de Detecção , Técnicas de Amplificação de Ácido Nucleico/métodos , Hibridização de Ácido Nucleico , Espectrometria de Fluorescência/métodos
5.
Biosens Bioelectron ; 149: 111847, 2020 Feb 01.
Artigo em Inglês | MEDLINE | ID: mdl-31733487

RESUMO

A promising electrochemical system was explored for DNA methylation detection according to the construction of a signal-on biosensor. Based on the ingenious design of probe DNA and auxiliary DNA, methylated target DNA triggered the exonuclease III (Exo III) digestion of auxiliary DNA from 3'-terminus, resulting in the conformational change of probe DNA with an electroactive methylene blue (MB) tag at 5'-terminus. Consequently, the MB tag in the probe DNA was close to the electrode surface for electron transfer, generating an increased current signal. Because of the target recycling of methylated DNA, significant signal amplification was obtained. Moreover, bisulfite conversion conferred an efficient approach for the universal analysis of any CpG sites without the restriction of specific DNA sequence. As a result, the target DNA with different methylation statuses were clearly recognized, and the fully methylated DNA was quantified in a wide range from 10 fM to 100 pM, with a detection limit of 4 fM. The present work realized the assay of methylated target DNA in serum samples with satisfactory results, illustrating the application performance of the system in complex sample matrix.


Assuntos
Aptâmeros de Nucleotídeos/química , Técnicas Biossensoriais , Metilação de DNA/genética , Técnicas Eletroquímicas , Sondas de DNA/química , Exodesoxirribonucleases/química , Ouro/química , Limite de Detecção , Conformação de Ácido Nucleico
6.
Biosens Bioelectron ; 147: 111769, 2020 Jan 01.
Artigo em Inglês | MEDLINE | ID: mdl-31630030

RESUMO

Rapid and accurate detection of nucleic acids plays a major role in biological research and clinical diagnostics. Here, a 3D multiple self-cleaning electrochemical ratiometric microfluidic paper-based analytical device (SER-µPAD) has been constructed for manganese super oxide dismutase (MnSOD) gene detection on the basis of the inner reference probe and exonuclease Ш (Exo Ш)-assisted analytes recycling amplification method. To simplify manual operations, a multipath self-cleaning tab that could manipulate fluid transport was introduced into the paper-based device, realizing time-programmable self-cleaning of the electrode. For achieving sensitive detection of MnSOD gene, the methylene blue (MB)-modified capture probe (CP) as the inner reference element was first self-assembled on triangular Au nanosheets modified paper working electrode to provide a built-in correction and improve the detection accuracy. When MnSOD gene existed, it hybridized with the hairpin-structured signal probe, triggering the cyclic amplification with the assistance of Exo Ш selective digestion to engender numerous residual DNA labeled with ferrocene (Fc) that could be captured on electrode surface by CPs. Hence, the Fc tags were close to the electrode surface, resulting in the oxidation peak current of Fc (IFc) increase, while that of MB (IMB) was constant on account of the unchanged distance between the MB tags and the electrode. The value of IFc/IMB was linear with MnSOD gene concentration from 10 nM to 1200 nM, and the detection limit was 3.91 nM. This strategy provides an accurate, robust, and sensitive method for nucleic acids detection and shows great potential in the construction of portable devices.


Assuntos
Técnicas Biossensoriais , DNA/isolamento & purificação , Técnicas Eletroquímicas , Exodesoxirribonucleases/química , DNA/química , Eletrodos , Ouro/química , Limite de Detecção , Azul de Metileno/química , Hibridização de Ácido Nucleico , Oxirredução
7.
Mikrochim Acta ; 186(12): 760, 2019 11 11.
Artigo em Inglês | MEDLINE | ID: mdl-31712919

RESUMO

A fluorometric method is described for nucleic acid signal amplification through target-induced catalytic hairpin assembly with DNA-templated copper nanoparticles (Cu NPs). The toehold-mediated self-assembly of three metastable hairpins is triggered in presence of target DNA. This leads to the formation of a three-way junction structure with protruding mononucleotides at the 3' terminus. The target DNA is released from the formed branched structure and triggers another assembly cycle. As a result, plenty of branched DNA becomes available for the synthesis of Cu NPs which have fluorescence excitation/emission maxima at 340/590 nm. At the same time, the branched structure protects the Cu NPs from digestion by exonuclease III. The unreacted hairpins are digested by exonuclease III, and this warrants a lower background signal. The method can detect ssDNA (24 nt) at low concentration (44 pM) and is selective over single-nucleotide polymorphism. On addition of an aptamer, the strategy can also be applied to the quantitation of thrombin at levels as low as 0.9 nM. Graphical abstractSchematic representation of target-induced catalytic hairpin assembly to form branched DNA template for the in situ synthesis of fluorescent Cu nanoparticles.


Assuntos
DNA/sangue , Corantes Fluorescentes/química , Nanopartículas Metálicas/química , Espectrometria de Fluorescência/métodos , Trombina/análise , Aptâmeros de Nucleotídeos/química , Aptâmeros de Nucleotídeos/genética , Cobre/química , DNA/química , DNA/genética , Sondas de DNA/química , Sondas de DNA/genética , Exodesoxirribonucleases/química , Corantes Fluorescentes/síntese química , Humanos , Sequências Repetidas Invertidas , Limite de Detecção , Hibridização de Ácido Nucleico
8.
Mol Biotechnol ; 61(12): 938-944, 2019 Dec.
Artigo em Inglês | MEDLINE | ID: mdl-31641996

RESUMO

The Hot Start polymerase chain reaction (Hot Start PCR) is designed to reduce off-target amplification by blocking DNA polymerase extension at room temperature until the desired temperature is reached. In this study, we investigated a new method of Hot Start PCR that uses a modified Escherichia coli Exonuclease III (EcoExoIIIM) by substituting residues in the DNA-binding pocket and catalytic center. The results showed that PCR amplification yield and specificity were significantly promoted by the addition of EcoExoIIIM. We hypothesize that non-specific binding of primers at room temperature is prevented by binding of the primed template by EcoExoIIIM, which is then released from the DNA by heat denaturation before the first PCR cycle. Through this mechanism, PCR would be enhanced by reducing off-target extension at room temperature.


Assuntos
Escherichia coli/enzimologia , Escherichia coli/genética , Exodesoxirribonucleases/genética , Reação em Cadeia da Polimerase/métodos , Primers do DNA/química , Proteínas de Ligação a DNA/genética , Proteínas de Ligação a DNA/metabolismo , DNA Polimerase Dirigida por DNA/metabolismo , Exodesoxirribonucleases/química , Exodesoxirribonucleases/metabolismo , Temperatura Alta , Mutação , Temperatura
9.
Analyst ; 144(22): 6689-6697, 2019 Nov 04.
Artigo em Inglês | MEDLINE | ID: mdl-31598619

RESUMO

A sensitive and label-free fluorometric method has been developed for the determination of polynucleotide kinase (PNK) activity, by employing exonuclease III (Exo III)-assisted cyclic signal amplification and poly(thymine)-templated copper nanoparticles (polyT-CuNPs). In the presence of PNK, cDNA with 5'-hydroxyl termini was phosphorylated and then hybridized with tDNA to form the cDNA/tDNA duplex, which subsequently triggered the λ exonuclease cleavage reaction, eventually resulting in the release of tDNA. The released tDNA could unfold the hairpin structure of HP DNA to generate partially complementary duplex (tDNA/HP DNA), wherein the HP DNA possessed T-rich sequences (T30) and tDNA recognition sequence. With the help of Exo III digestion, the tDNA was able to initiate the cycle for the generation of T-rich sequences, the template for the formation of fluorescent CuNPs. Conversely, the cDNA could not be cleaved by λ exonuclease without PNK and individual HP DNA could not be hydrolyzed by Exo III. The T-rich sequence was caged in HP DNA, resulting in a weak fluorescence signal. Under optimized conditions, the fluorescence intensity was linearly correlated to a concentration range of 0.001 to 1 U mL-1 with a low detection limit of 2 × 10-4 U mL-1. Considering the intriguing analytical performance, this approach could be explored to screen T4 PNK inhibitors and hold promising applications in drug discovery and disease therapy.


Assuntos
Ensaios Enzimáticos/métodos , Exodesoxirribonucleases/química , Nanopartículas Metálicas/química , Poli T/química , Polinucleotídeo 5'-Hidroxiquinase/análise , Espectrometria de Fluorescência/métodos , Bacteriófago T4/enzimologia , Sequência de Bases , Técnicas Biossensoriais/métodos , Cobre/química , DNA/química , DNA/genética , Sondas de DNA/química , Sondas de DNA/genética , Células HeLa , Humanos , Sequências Repetidas Invertidas , Limite de Detecção , Hibridização de Ácido Nucleico , Reprodutibilidade dos Testes
10.
Mikrochim Acta ; 186(11): 716, 2019 10 25.
Artigo em Inglês | MEDLINE | ID: mdl-31654133

RESUMO

A fluorometric method is described for the determination of DNA. It involves the use of graphene oxide (GO), exonuclease III (Exo III), and two specially designed fluorophore-labeled hairpin probes (HP1 and HP2). Different from other GO-based DNA assays, the method takes advantage of the distinct binding abilities of GO with hairpin DNA probes and single nucleotides. GO serves as a strong quencher for fluorescent labels to ensure a very low background signal. Two reaction cycles mediated by Exo III are employed to enhance the signals. The combination of GO-induced quenching and Exo III-mediated dual regeneration of analytes leads to a detection limit as low as 1 pM for the model analyte human hemochromatosis protein (HFE) gene. The method is also applicable for the determination of HFE gene spiked into fetal bovine serum. Graphical abstract Schematic representation of a GO-based, Exo III-assisted method for dual-signal amplified detection of DNA, for which human haemochromatosis protein (HFE) gene is designed as the model target. The assay involves graphene oxide (GO), exonuclease (Exo III), and two specially designed, fluorophore-labelled hairpin probes (HP1 and HP2).


Assuntos
DNA/sangue , Exodesoxirribonucleases/química , Grafite/química , Espectrometria de Fluorescência/métodos , Animais , Bovinos , DNA/química , DNA/genética , Sondas de DNA/química , Sondas de DNA/genética , Fluoresceínas/química , Corantes Fluorescentes/química , Sequências Repetidas Invertidas , Limite de Detecção , Hibridização de Ácido Nucleico
11.
Mikrochim Acta ; 186(11): 692, 2019 10 12.
Artigo em Inglês | MEDLINE | ID: mdl-31605242

RESUMO

A photocathode is described for the determination of microRNA-21 by using CuInS2 as an active photocathode material. Exonuclease III assisted target recycling amplification was employed to enhance the detection sensitivity. The TATA-binding protein (TBP) was applied to enhance steric hindrance which decreases the photoelectrochemical intensity. This strategy is designed by combining the anti-interference photocathode material, enzyme assisted target recycling amplification and TBP induced signal off, showing remarkable amplification efficiency. Under the optimized conditions, the detection limit for microRNA-21 is as low as 0.47 fM, and a linear range was got from 1.0 × 10-15 M to 1.0 × 10-6 M. Graphical abstract Schematic representation of sensitive photoelectrochemical detection of microRNA-21.CuInS2 is used as an active photocathode material. Combined Exonuclease III assisted target recycling amplification and TATA-binding protein decreased of photoelectrochemical intensity, the detection limit was 0.47 fM with good selectivity. (miR-21: microRNA-21; CS: chitosan).


Assuntos
DNA/química , Técnicas Eletroquímicas/métodos , Exodesoxirribonucleases/química , MicroRNAs/sangue , Fotoquímica/métodos , Sulfetos/química , Aptâmeros de Nucleotídeos/química , Aptâmeros de Nucleotídeos/genética , Sequência de Bases , Biomarcadores Tumorais/sangue , Biomarcadores Tumorais/química , Biomarcadores Tumorais/genética , Cobre/química , Cobre/efeitos da radiação , DNA/genética , Técnicas Eletroquímicas/instrumentação , Eletrodos , Humanos , Índio/química , Índio/efeitos da radiação , Sequências Repetidas Invertidas , Luz , Limite de Detecção , MicroRNAs/química , MicroRNAs/genética , Hibridização de Ácido Nucleico , Sulfetos/efeitos da radiação , Compostos de Estanho/química
12.
Analyst ; 144(21): 6231-6239, 2019 Oct 22.
Artigo em Inglês | MEDLINE | ID: mdl-31552930

RESUMO

The 3'-5' exonuclease enzyme plays a dominant role in multiple pivotal physiological activities, such as DNA replication and repair processes. In this study, we designed a sensitive graphene oxide (GO)-based probe for the detection of exonuclease enzymatic activity. In the absence of Exo III, the strong π-π interaction between the fluorophore-tagged DNA and GO causes the efficient fluorescence quenching via a fluorescence resonance energy transfer (FRET). In contrast, in the presence of Exo III, the fluorophore-tagged 3'-hydroxyl termini of the DNA probe was digested by Exo III to set the fluorophore free from adsorption when GO was introduced, causing an inefficient fluorescence quenching. As a result, the fluorescence intensity of the sensor was found to be proportional to the concentration of Exo III; towards the detection of Exo III, this simple GO-based probe demonstrated a highly sensitive and selective linear response in the low detection range from 0.01 U mL-1 to 0.5 U mL-1 and with the limit of detection (LOD) of 0.001 U mL-1. Compared with other fluorescent probes, this assay exhibited superior sensitivity and selectivity in both buffer and fetal bovine serum samples, in addition to being cost effective and having a simple setup.


Assuntos
Ensaios Enzimáticos/métodos , Exodesoxirribonucleases/sangue , Grafite/química , Animais , Sequência de Bases , Bovinos , DNA/química , Sondas de DNA/química , Sondas de DNA/genética , Exodesoxirribonucleases/química , Fluoresceínas/química , Fluorescência , Transferência Ressonante de Energia de Fluorescência , Corantes Fluorescentes/química , Sequências Repetidas Invertidas , Cinética , Limite de Detecção
13.
Biosens Bioelectron ; 143: 111609, 2019 Oct 15.
Artigo em Inglês | MEDLINE | ID: mdl-31476597

RESUMO

In this research, a sensitive and specific electrochemical biosensor for DNA detection was constructed. The highly sensitivity of this biosensor is due to the exploitation of exonuclease III-assisted double recycling and toehold-mediated strand displacement recycling to achieve the target triple recycling amplification, thus generating a large amount of Y-shaped DNA structures. Combination with a terminal deoxynucleotidyl transferase (TDT)-mediated cascaded signal amplification strategy can catalyze the repetitive incorporation of biotin-dUTP to the 3'-OH of the Y-shaped DNA. Via biotin-streptavidin interaction, multiple streptavidin-alkaline phosphatases were conjugated to the surface of an Au electrode and generated a sharply increasing electrochemical signal in a 1-naphthyl phosphate (1-NP) solution. In this method, an impressive detection limit of 0.05 fM was obtained, presenting outstanding selectivity with a dynamic response scope between 0.1 fM and 1 nΜ. Thus, the designed biosensor opens an avenue for DNA detection in clinical molecular diagnostics, pathogen detection, gene therapy, food safety and environmental monitoring.


Assuntos
Técnicas Biossensoriais , DNA Nucleotidilexotransferase/química , DNA/isolamento & purificação , Técnicas Eletroquímicas , Fosfatase Alcalina/química , Biotina/química , Catálise , DNA/química , Eletrodos , Exodesoxirribonucleases/química , Limite de Detecção , Técnicas de Amplificação de Ácido Nucleico , Estreptavidina/química
14.
Biosens Bioelectron ; 143: 111613, 2019 Oct 15.
Artigo em Inglês | MEDLINE | ID: mdl-31450095

RESUMO

Incorporating elements of triple-helix aptamer probes (TAP), catalyzed hairpin assembly (CHA) signal amplification and host-guest recognition, a novel "signal-on" sensing strategy for sensitive electrochemical quantification of tetracycline (TC) was reported unprecedentedly. TAP was formed involving an aptamer loop, two-segment stems and a triplex oligonucleotide serving as trigger probe. Then, the trigger probe would be released from TAP once the target presented due to the conformational variation of TAP induced by aptamer binding event, sparking off the upcoming CHA amplification reaction, in which two coexisting DNA hairpins (H1 and H2 both modified with the electroactive molecules) would hybridize into plentiful H1-H2 double helices. Afterwards, the Exonuclease III was added, demolishing double helices and simultaneously releasing plentiful electroactive molecules which were capable of diffusing onto the electrode surface under the assistance of ß-cyclodextrin due to host-guest recognition, where appreciable signals were enriched and generated. As thus, considerably slight amounts of targets though, emitted trigger probes, yet efficiently engining spectacular CHA cycles of reactions through which amplified signals were yielded, and in turn progressively enabling the sensitive target detection done. Under optimal conditions, the growing signal stayed a linear relation along with the logarithm of the target concentrations ranging from 0.2 nM to 100 nM, the detection limit reaching as low as 0.13 nM. This approach was desirable regarding to sensitivity, detection limit and range, prospectively rendering a service for diverse targets detection by easily replacing the matched aptamer loop of TAP.


Assuntos
Antibacterianos/isolamento & purificação , Técnicas Biossensoriais , Técnicas Eletroquímicas , Tetraciclina/isolamento & purificação , Antibacterianos/química , Aptâmeros de Nucleotídeos/química , DNA/química , Eletrodos , Exodesoxirribonucleases/química , Técnicas de Amplificação de Ácido Nucleico , Tetraciclina/química
15.
Chem Commun (Camb) ; 55(71): 10603-10606, 2019 Aug 29.
Artigo em Inglês | MEDLINE | ID: mdl-31424058

RESUMO

A truly ratiometric homogeneous electrochemical biosensor has been developed for sensitive miRNA detection based on the unique diffusion/intercalation properties of electroactive dyes without the need for electrode modification or materials preparation.


Assuntos
Técnicas Biossensoriais/métodos , Corantes/química , Técnicas Eletroquímicas/métodos , Substâncias Intercalantes/química , MicroRNAs/análise , DNA/química , Eletrodos , Exodesoxirribonucleases/química , Compostos Ferrosos/química , Limite de Detecção , Metalocenos/química , Azul de Metileno/química , Oxirredução
16.
Biosens Bioelectron ; 142: 111532, 2019 Oct 01.
Artigo em Inglês | MEDLINE | ID: mdl-31377576

RESUMO

In this paper, a novel label-free electrochemical impedance aptasensor for highly sensitive detection of IFN-γ based on target-induced exonuclease inhibition was constructed. For this purpose, we designed a DNA hairpin modified on the gold electrode whose loop was the aptamer of the IFN-γ, and the stem was 5'-thiol-modified. In the absence of IFN-γ, Exonuclease III (Exo III) and Exonuclease I (Exo I) digested the double-stranded and single-stranded strands of the hairpin DNA, respectively, causing smaller impedance value on the surface of the electrode. In the presence of IFN-γ, the function of Exo III was greatly inhibited by the binding of the aptamer with the target, and it stopped after cutting three bases of the hairpin DNA. Forming a major target-bound aptamer digestion product, it could not be digested by Exo I, so there was larger impedance on the electrode surface. The calibration curve for IFN-γ was linear in the range of 1 pM-50 nM with the detection limit (LOD) of 0.7 pM. The proposed aptasensor proved good selectivity and reproducibility, and low cost. In addition, the biosensor was able to detect IFN-γ in serum samples successfully, which is expected to provide an efficient method for TB diagnosis at early stages.


Assuntos
Aptâmeros de Nucleotídeos/química , Técnicas Biossensoriais/métodos , Interferon gama/sangue , Técnicas Biossensoriais/instrumentação , Impedância Elétrica , Técnicas Eletroquímicas/instrumentação , Técnicas Eletroquímicas/métodos , Eletrodos , Desenho de Equipamento , Exodesoxirribonucleases/química , Humanos , Interferon gama/análise , Limite de Detecção
17.
Biosens Bioelectron ; 142: 111574, 2019 Oct 01.
Artigo em Inglês | MEDLINE | ID: mdl-31408824

RESUMO

The monitoring of transcription factors (TFs) is critical for understanding the regulation of gene transcriptions. Here, by programming nucleic acid sequence-based and cascaded recycling amplifications, we developed a sensitive and non-label electrochemical biosensor for detecting TFs from tumor cell extracts. The binding of the target nuclear factor-kappa B p50 (NF-κB p50) with the dsDNA probes protects them from being digested by exonuclease III for subsequent initiation of three cascaded recycling cycles, which causes the generation of tremendous free G-quadruplex special sequences on the sensing electrode. Such G-quadruplexes can specifically bind and confine hemin within the vicinity of the sensor, generating substantially enhanced reduction current to achieve determination of NF-κB p50 within the range from 0.5 pM to 5 nM with the detection limit down to 0.13 pM. The proposed sensing system also has high selectivity and it can be used to interrogate the presence of NF-κB p50 in tumor cell extracts, demonstrating its potential for disease diagnosis and gene transcription-related studies.


Assuntos
Técnicas Biossensoriais/métodos , Subunidade p50 de NF-kappa B/análise , Sondas de DNA/química , DNA Catalítico/química , Técnicas Eletroquímicas/métodos , Exodesoxirribonucleases/química , Quadruplex G , Células HeLa , Hemina/química , Humanos , Neoplasias/patologia , Técnicas de Amplificação de Ácido Nucleico/métodos
18.
Bioelectrochemistry ; 130: 107341, 2019 Dec.
Artigo em Inglês | MEDLINE | ID: mdl-31400568

RESUMO

Telomerase is considered a pivotal biomarker for early cancer diagnosis and a valuable therapeutic target. However, the current methods to detect telomerase activity have some limitations. Herein, we propose a homogeneous electrochemical strategy to develop a simple, rapid, and highly sensitive assay to detect human telomerase activity from crude cancer cell extracts. Our strategy is based on magnetic bead separation and exonuclease III-aided target recycling amplification. The complementary probes can hybridize with the extended telomeric repeats, which allows exonuclease III to recognize and digest the latter once the hybrid product is separated with magnetic beads. The released complementary probes can hybridize with and open multiple methylene blue (MB)-labeled hairpin (HP) DNA probes, allowing exonuclease III to digest the duplex. Then, the opened hairpin couples with the captured mononucleotides on the surface of the gold electrode. By taking advantage of the exonuclease III-aided target recycling strategy, the present assay enables the detection of telomerase activity at a single-cell level. Furthermore, the assay is carried out in a homogeneous solution achieved by magnetic purification, which removes the interferents present in crude lysates and avoids false negatives, thus, providing a powerful platform to detect telomerase activity in samples of early-stage cancer.


Assuntos
Técnicas Biossensoriais/métodos , Exodesoxirribonucleases/química , Telomerase/análise , Sondas de DNA/química , Técnicas Eletroquímicas/métodos , Ensaios Enzimáticos/métodos , Células HeLa , Humanos , Imãs/química , Neoplasias/enzimologia , Hibridização de Ácido Nucleico
19.
Biosens Bioelectron ; 142: 111537, 2019 Oct 01.
Artigo em Inglês | MEDLINE | ID: mdl-31376709

RESUMO

Electrochemical detection of specific nucleic acid sequence remains a hot topic in current bioanalytical research. Here, a novel ratiometric electrochemical biosensor based on Exo III-assisted recycling amplification and graphene-modified electrode was fabricated for quantitative detection of trinucleotide repeat sequence d(CAG)n. The double-signals used are the hairpin DNAs labeled with ferrocene and methylene blue respectively as report DNAs, which can hybridize to target DNA. The hybridized DNA was digested by Exo III, resulting in the release of target and report fragments. The graphene-modified electrode can selectively adsorb the released report fragments to generate double electrochemical signals. The signal ratio (F/M) of ferrocene and methylene blue was used to determine the repeat length accurately: a linear relationship was found between F/M and numbers of repeats (n), F/M = 0.061 n + 1.97, with a correlation coefficient of 0.992. Moreover, any electrochemical signal can be used to test repeat concentration with detection limit of 0.22 pM. Therefore, this novel ratiometric electrochemical biosensor provided a reliable and efficient method for the analysis of d(CAG)n trinucleotide repeat and a potential simplified clinical tool for neurodegenerative diseases.


Assuntos
Técnicas Biossensoriais/métodos , Sondas de DNA/química , Exodesoxirribonucleases/química , Técnicas de Amplificação de Ácido Nucleico/métodos , Repetições de Trinucleotídeos , Técnicas Eletroquímicas/métodos , Compostos Ferrosos/química , Grafite/química , Humanos , Limite de Detecção , Metalocenos/química , Azul de Metileno/química , Hibridização de Ácido Nucleico/métodos
20.
Mol Cell Probes ; 46: 101423, 2019 08.
Artigo em Inglês | MEDLINE | ID: mdl-31323319

RESUMO

Polydeoxyadenosine (poly (dA)) has been extensively applied for detecting many drug molecules. Herein, we developed a sensitive method for detecting coralyne and heparin using a modified DNA probe with poly (dA) at one end. In the absence of coralyne, the DNA probe was digested by the Exonuclease I (Exo I), and therefore the SYBR Green I (SG I) emitted an extremely low fluorescent signal. While coralyne specifically binding to poly (dA) with strong propensity could remarkably restrain the disintegration of the DNA probe, through which as a template the second strand of DNA sequence was formed with the introduction of DNA polymerase. Therefore, the fluorescent signal of SG I was intensified to quantify coralyne. Based on this method, heparin can be determined due to its strong affinity towards coralyne. This method showed a linear range from 2 to 500 nM for coralyne with a low detection limit of 0.98 nM, and the linear range of heparin was from 1 to 100 nM when 1.25 nm was the detection limit. The proposed method was also implemented successfully in biological samples and showed a potential application for screening potential therapeutic molecules.


Assuntos
Alcaloides de Berberina/isolamento & purificação , Técnicas Biossensoriais , Exodesoxirribonucleases/genética , Heparina/isolamento & purificação , Alcaloides de Berberina/química , DNA/química , Sondas de DNA/química , Sondas de DNA/genética , Desoxiadenosinas/química , Desoxiadenosinas/genética , Exodesoxirribonucleases/química , Heparina/química , Heparina/genética , Humanos , Limite de Detecção , Compostos Orgânicos/química
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