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1.
Mikrochim Acta ; 186(8): 594, 2019 08 01.
Artigo em Inglês | MEDLINE | ID: mdl-31372831

RESUMO

A method is described for the colorimetric determination of the activity of CpG methyltransferase (M.SssI). It is based on (a) the crosslinking effect between dsDNA-modified gold nanoparticles (AuNPs) and graphene oxide (GO), and (b) an amplification reaction with the aid of a nicking enzyme. To avoid the aggregation of AuNPs (which would produce false signals), a hairpin DNA was connected to the AuNPs. Thus, the red color of the solution (measured at 530 nm) increases linearly with the activity of M.SssI from 0.2 to 60 U·mL-1, and the limit of detection is 67 U·mL-1. This is superior to some reported strategies. The method was successfully applied to analyze spiked serum samples. Conceivably, it represents a powerful tool for use in drug development and diagnosis. Graphical abstracts A method based on the conjugated cross-linking effect between dsDNA modified Au NPs and GO coupled with an amplification reaction of nicking enzyme has been developed for colorimetric detection of the activity of CpG methyltransferase (M.SssI).

2.
Mikrochim Acta ; 186(4): 241, 2019 03 13.
Artigo em Inglês | MEDLINE | ID: mdl-30868262

RESUMO

The activity of terminal deoxynucleotidyl transferase (TdTase) is a biomarker for routine diagnosis of acute leukemia. A method has been developed for the determination of TdTase activity. It is based on the use of silver nanoclusters (AgNCs) whose yellow fluorescence is enhanced by an in-situ grown DNA tail of TdTase-polymerized and guanine-rich DNA at the 3' end of a hairpin DNA. The fluorescence, best measured at excitation/emission peaks of 530/585 nm, increases linearly in the 1 to 35 mU mL-1 TdTase activity range. The detection limit is 0.8 mU mL-1. The method is cost-efficient, selective and convenient. It integrates enhancement of the fluorescence of AgNCs and target recognition into a single process. Graphical abstract Schematic presentation of a method for determination of TdTase activity. It is based on AgNCs fluorescence enhanced by in-situ grown TdTase-polymerized G-rich DNA tail. The method integrates AgNCs fluorescence enhancement and the target recognition into a single process.


Assuntos
DNA Nucleotidilexotransferase/sangue , DNA/química , Ensaios Enzimáticos/métodos , Nanopartículas Metálicas/química , Sequência de Bases , Biomarcadores/sangue , Técnicas Biossensoriais/métodos , DNA/genética , Fluorescência , Humanos , Sequências Repetidas Invertidas , Leucemia/diagnóstico , Limite de Detecção , Prata/química , Espectrometria de Fluorescência/métodos
3.
Mikrochim Acta ; 185(5): 280, 2018 05 03.
Artigo em Inglês | MEDLINE | ID: mdl-29725866

RESUMO

A method is reported for the fluorometric quantitation of microRNA. It is making use of a luminescent probe deribed from terbium(III) ion whose fluorescence is sensitized with a guanine-rich (G-rich) nucleotide. The probe has a large Stokes' shift and strong and sharp emission bands. The assay relies on the wide substrate specificity of terminal deoxynucleotidyl transferase (TdTase), which catalyzes the formation of long G-rich nucleotides when using microRNA primer as a trigger to start the polymerization. The addition of Tb(III) induces the formation of a G-quadruplex from the G-rich nucleotide, and this strongly enhances the green fluorescence of Tb(III) (peaking at 545 nm upon photoexcitation at 290 nm). Specifically, microRNA-21 was chosen as the analyte. The fluorescence intensity of Tb(III) increases linearly in the 1 pM to 1 nM microRNA concentration range, and the detection limit is as low as 0.11 pM. The method can distinguish between family members of microRNA and performs excellently even when applied to extracts of cancer cells. Graphical abstract A fluorometric technique is reported for the determination of microRNA. It is based on signal enhancement based on the sensitization of terbium(III) via a guanine-rich nucleotide sequence. Klenow Fragment exo- (KFexo-) generates DNA sequence at the 3'-OH of microRNA, and terminal deoxynucleotidyl transferase (TdTase) catalyzes the formation of long G-rich nucleotides.


Assuntos
Técnicas Biossensoriais/métodos , DNA Nucleotidilexotransferase/metabolismo , Nucleotídeos de Guanina/química , Nucleotídeos de Guanina/metabolismo , Medições Luminescentes/métodos , MicroRNAs/análise , Térbio/química , Células A549 , Humanos , Células MCF-7
4.
Biosens Bioelectron ; 102: 211-216, 2018 Apr 15.
Artigo em Inglês | MEDLINE | ID: mdl-29145074

RESUMO

We propose a ratiometric electrochemical assay for detecting microRNA (miRNA) on the basis of dual-amplification mechanism by using distinguishable electrochemical signals from thionine (Thi) and ferrocene (Fc). The thiol-modified and ferrocene-labeled hairpin capture probes (CP) are first immobilized on an Au electrode via Au-S reaction. The target miRNA hybridizes with CP and unfolding the hairpin structure of CP to form miRNA-DNA duplexes. Then, kamchatka crab duplex specific nuclease (DSN) specifically cleaves the DNA in miRNA-DNA duplexes, leading to the release of miRNA and another cleaves cycle, meanwhile, numerous Fc leaves away from the electrode surface and leads to the signal-off of Fc. The residual fragment on electrode surface acts as a HCR primer to form dsDNA polymers through in situ HCR with the presence of the primer and two probes (HDNA and HDNA'), resulting in the capture of numerous DNA/Au NPs/Thi and the signal-on of Thi. The dual-amplification mechanism significantly amplifies the decrease of Fc signal and the increase of Thi signal for ratiometric readout (IThi/IFc), thus providing a sensitive method for the selective detection of miR-141 with a detection limit down to 11aM. The dual-signal ratiometric outputs have an intrinsic self-calibration to the effects from system, which is promising to be applied in biosensing and clinical diagnosis.


Assuntos
Técnicas Biossensoriais/métodos , Técnicas Eletroquímicas/métodos , Nanopartículas Metálicas/química , MicroRNAs/isolamento & purificação , Compostos Ferrosos/química , Ouro/química , Humanos , Limite de Detecção , Metalocenos/química , MicroRNAs/química , Hibridização de Ácido Nucleico , Fenotiazinas/química
5.
Biosens Bioelectron ; 97: 325-331, 2017 Nov 15.
Artigo em Inglês | MEDLINE | ID: mdl-28622643

RESUMO

We report a sensor combining two distinguishable magnetic nanoprobes (DNA1/Fe3O4 NPs/Thi and DNA2/Fe3O4 NPs/Fc) with target-triggered hybridization chain reaction (HCR) strategy for the simultaneous detection of microRNA-141 (miR-141) and microRNA-21 (miR-21). In the presence of targets, the thiol-modified hairpin capture probes (HCP1 and HCP2) specifically hybridize with miR-141 and miR-21 on a gold electrode, leading to the conformation change of HCP1 and HCP2, respectively. The conformation change subsequently triggers HCR to generate plentiful bonding sequences of magnetic nanoprobes. Thus, numerous thionine (Thi) modified DNA1/Fe3O4 NPs/Thi and ferrocene carboxaldehyde (Fc-CHO) modified DNA2/Fe3O4 NPs/Fc are captured by the well-designed HCR, via DNA hybridization respectively, giving rise to the dual magnified response of currents. The increase in the electrochemical currents at different potentials of the two magnetic nanoprobes enables us to simultaneously and quantitatively detect miR-141 and miR-21. Target-triggered HCR increases the amount of captured nanoprobes due to the increasing number of bonding sequences, greatly amplifying the currents of the two magnetic nanoprobes in the presence of targets, and ultimately realizing the dual signal amplification with increased sensitivity. The sensor can be applied for detecting miRNAs in cell lysates, thus, promising to be a clinic diagnosis of cancers by means of simultaneous detection of a variety of miRNA biomarkers.


Assuntos
Técnicas Eletroquímicas/métodos , Nanopartículas de Magnetita/química , MicroRNAs/análise , Hibridização de Ácido Nucleico/métodos , Técnicas Biossensoriais/métodos , Eletrodos , Compostos Ferrosos/química , Humanos , Células MCF-7 , Nanopartículas de Magnetita/ultraestrutura , Metalocenos/química
6.
Biosens Bioelectron ; 87: 216-221, 2017 Jan 15.
Artigo em Inglês | MEDLINE | ID: mdl-27566394

RESUMO

A new strategy based on enzymatically engineered primer extension poly-thymine (EPEPT) and nanomaterials in situ generation technology is reported for direct detection of microRNA (miRNA) in a fluorescence turn-on format using the sequential and complementary reactions catalyzed by Klenow Fragment exo- (KFexo-) and terminal deoxynucleotidyl transferase (TdTase). The short miRNA can be efficiently converted into long poly-thymine (polyT) sequences, which function as template for in situ formation of fluorescence copper nanoparticles (CuNPs) as nano-dye for detecting miRNA. The polyT-CuNPs can effectively form and emit intense red fluorescence under the 340nm excitation. For the proof of concept, microRNA-21 (miR-21) was selected as the model target to testify this strategy as a versatile assay platform. By directly using miR-21 as the primer, the simple, rapid and sensitive miRNA detection was successfully achieved with a good linearity between 1pM and 1nM and a detection limit of 100fM. Thus, the EPEPT strategy holds great potential in biochemical sensing research as an efficient and universal platform.


Assuntos
Técnicas Biossensoriais/métodos , Cobre/química , Corantes Fluorescentes/química , Nanopartículas Metálicas/química , MicroRNAs/análise , Poli T/química , Células A549 , DNA Nucleotidilexotransferase/química , DNA Polimerase I/química , Fluorescência , Humanos , Limite de Detecção , Células MCF-7 , Espectrometria de Fluorescência/métodos
7.
Chem Commun (Camb) ; 51(52): 10543-6, 2015 Jul 04.
Artigo em Inglês | MEDLINE | ID: mdl-26040236

RESUMO

This assay, termed branched cascade enzymatic amplification (BCEA), can be a novel and straightforward method for sensitive and specific microRNA detection in crude cellular extracts of cancer cells at physiological temperature, by coupling two ordinary polymerases, Klenow fragment exo(-) and terminal deoxynucleotidyl transferase.


Assuntos
DNA Nucleotidilexotransferase/metabolismo , DNA Polimerase I/metabolismo , MicroRNAs/análise , MicroRNAs/biossíntese , Técnicas de Amplificação de Ácido Nucleico/métodos , Humanos , Células MCF-7
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