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1.
Chem Commun (Camb) ; 55(69): 10300-10303, 2019 Aug 22.
Artigo em Inglês | MEDLINE | ID: mdl-31397452

RESUMO

Shorter DNA probes provide better specificity for hybridization, but they may not form stable duplexes at room temperature. In this study, we used thiazole orange to follow DNA hybridization upon freezing and achieved stable 5-mer duplex DNA. Using multiple short probes in tandem, long DNA could also be studied. This study provides insights into DNA hybridization in the frozen state and expands the application of freezing for nucleic acid chemistry.


Assuntos
DNA/química , Hibridização de Ácido Nucleico , Oligonucleotídeos/química , Pareamento Incorreto de Bases , Sequência de Bases , Benzotiazóis/análise , DNA/genética , Sondas de DNA/química , Sondas de DNA/genética , Corantes Fluorescentes/análise , Congelamento , Oligonucleotídeos/genética , Quinolinas/análise , Espectrometria de Fluorescência
3.
Chem Commun (Camb) ; 55(61): 8963-8966, 2019 Aug 07.
Artigo em Inglês | MEDLINE | ID: mdl-31290488

RESUMO

We develop a simple method for sensitive detection of alkaline phosphatase (ALP) based on the ligase amplification reaction-catalyzed assembly of a single quantum dot (QD)-based nanosensor. This nanosensor requires only a single ligase enzyme to achieve ultrahigh sensitivity with a detection limit of 5.63 × 10-7 U mL-1, and can be applied for kinetic analysis, inhibitor screening, and ALP measurement in cell extracts.


Assuntos
Fosfatase Alcalina/análise , Técnicas Biossensoriais/métodos , DNA Ligases/química , Ensaios Enzimáticos/métodos , Transferência Ressonante de Energia de Fluorescência/métodos , Pontos Quânticos/química , Biotina/química , Carbocianinas/química , DNA/química , DNA/genética , Sondas de DNA/química , Sondas de DNA/genética , Fluorescência , Células HEK293 , Humanos , Limite de Detecção , Células MCF-7 , Hibridização de Ácido Nucleico , Imagem Individual de Molécula , Estreptavidina/química
4.
Chem Commun (Camb) ; 55(58): 8466-8469, 2019 Jul 25.
Artigo em Inglês | MEDLINE | ID: mdl-31265022

RESUMO

We presented a branch migration based PCR in which a branch migration blocker was introduced to selectively reduce the amplification efficiency of the wild-type target and enrich the mutant-type target. The low-abundance mutations could be enriched and then detected by high resolution melting, Sanger sequencing or fluorescent DNA probe.


Assuntos
Análise Mutacional de DNA/métodos , DNA/análise , DNA/genética , Reação em Cadeia da Polimerase/métodos , Sequência de Bases , Sondas de DNA/química , Sondas de DNA/genética , DNA Polimerase Dirigida por DNA/química , Fluorescência , Corantes Fluorescentes/química , Genes , Humanos , Limite de Detecção , Mutação , Hibridização de Ácido Nucleico
5.
Anal Chim Acta ; 1078: 24-31, 2019 Oct 31.
Artigo em Inglês | MEDLINE | ID: mdl-31358225

RESUMO

A novel electrochemical DNA biosensor was developed and MON89788 of soybean transgenic gene sequence was detected based on a strategy of rolling circle amplification (RCA) and gold nanoparticle cube (AuNPC)-labeled multiple probes. First, the mercapto-modified capture DNA was immobilized on the surface of the Fe3O4@Au magnetic nanoparticles via an Au-S bond, and the capture DNA was opened and complementarily hybridized with the target DNA to form a double-stranded DNA. In the 10 × reaction buffer, Exonuclease III (ExoIII) specifically recognized and sheared the double-stranded DNA to release the target DNA, which led to the next round of reaction. Afterward, AuNP cube-loaded ssDNA (AuNPC/DNA) was added with the rolling circle reaction with the help of Phi29 DNA polymerase and T4 ligase. Finally, [Ru(NH3)6]3+ was attracted directly by the anionic phosphate of ssDNA via electrostatic interaction. The determination was carried out by using chronocoulometry (CC), and the CC signal was recorded. The mass amount of DNA strands extended infinitely on the AuNPs cube and numerous [Ru(NH3)6]3+ were absorbed, thus the detected signal was highly amplified. The corresponding CC signal showed a good linear relationship with the logarithm of the target DNA concentration in the range of 1 × 10-16 to 1 × 10-7 mol L-1, with a detection limit of 4.5 × 10-17 mol L-1. Specific gene sequence of MON89788 in soybean samples was determined, and the recoveries ranged from 97.3% to 102.0%. This sensor is one of the most sensitive sensors for genetic sequence assessment at present. Moreover, it demonstrates good selectivity, stability, and reproducibility.


Assuntos
Técnicas Biossensoriais/métodos , DNA de Plantas/análise , Técnicas Eletroquímicas/métodos , Plantas Geneticamente Modificadas/genética , Soja/genética , Sequência de Bases , Calibragem , Sondas de DNA/química , Sondas de DNA/genética , DNA de Plantas/química , DNA de Plantas/genética , Exodesoxirribonucleases/química , Ouro/química , Limite de Detecção , Nanopartículas de Magnetita/química , Técnicas de Amplificação de Ácido Nucleico/métodos , Hibridização de Ácido Nucleico , Oligodesoxirribonucleotídeos/química , Oligodesoxirribonucleotídeos/genética , Reprodutibilidade dos Testes , Compostos de Rutênio/química
6.
Anal Chim Acta ; 1075: 137-143, 2019 Oct 10.
Artigo em Inglês | MEDLINE | ID: mdl-31196419

RESUMO

Nucleic acid probes are very useful tools in biological and medical science. However, the essential sensing mechanism of nucleic acid probes was prone to the interference of surrounding sequences. Especially when the target sequences formed secondary structures such as hairpin or quadruplex, the nucleic acid probes were hindered from hybridizing with target strands, greatly disabled the function of probes. Herein, we have established an Open strand based strategy for eliminating the influence of secondary structures on the performance of nucleic acid probes. The strategy was general toward different lengths, secondary structures and sequences of the targeting strand, and we found that the improvement was higher when the secondary structure of the targeting strand was more complicated. Experiments on synthetic single stranded DNA and real clinical genomic DNA samples were conducted for low abundance mutation detection, and the limit of detection for TERT-C228T and BRCA2 rs80359065 mutations could be 0.02% and 0.05% respectively, demonstrating the clinical practicability of our proposed strategy in low abundance mutation detection.


Assuntos
Sondas de DNA/química , DNA de Cadeia Simples/análise , Proteína BRCA2/genética , Sondas de DNA/genética , DNA de Cadeia Simples/genética , Desoxirribonuclease IV (Fago T4-Induzido)/química , Feminino , Corantes Fluorescentes/química , Humanos , Limite de Detecção , Medições Luminescentes/métodos , Conformação de Ácido Nucleico , Hibridização de Ácido Nucleico , Neoplasias Ovarianas/genética , Mutação Puntual , Telomerase/genética
7.
Anal Chim Acta ; 1076: 110-117, 2019 Oct 17.
Artigo em Inglês | MEDLINE | ID: mdl-31203954

RESUMO

Encoded hydrogel microparticles, synthesized by Stop Flow Lithography (SFL), have shown great potential for microRNA assays for their capability to provide high multiplexing capacity and solution-like hybridization kinetics. However, due to the low conversion of copolymerization during particle synthesis, current hydrogel microparticles can only utilize ∼10% of the input probes that functionalize the particles for miRNA assay. Here, we present a novel method of functionalizing hydrogel microparticles after particle synthesis by utilizing unconverted double bonds remaining inside the hydrogel particles to maximize functional probe incorporation and increase the performance of miRNA assay. This allows covalent bonding of functional probes to the hydrogel network after particle synthesis. Because of the abundance of the unconverted double bonds and accessibility of all probes, the probe density increases about 8.2 times compared to that of particles functionalized during the synthesis. This results lead to an enhanced miRNA assay performance that improves the limit of detection from 4.9 amol to 1.5 amol. In addition, higher specificity and shorter assay time are achieved compared to the previous method. We also demonstrate a potential application of our particles by performing multiplexed miRNA detections in human plasma samples.


Assuntos
Hidrogéis/química , MicroRNAs/sangue , Biomarcadores/sangue , DNA/química , DNA/genética , Sondas de DNA/química , Sondas de DNA/genética , Humanos , Hidrogéis/síntese química , Dispositivos Lab-On-A-Chip , MicroRNAs/genética , Técnicas Analíticas Microfluídicas/métodos , Hibridização de Ácido Nucleico , Polietilenoglicóis/química , Porosidade
8.
Anal Chim Acta ; 1076: 138-143, 2019 Oct 17.
Artigo em Inglês | MEDLINE | ID: mdl-31203958

RESUMO

The detection and quantification of microRNA (miRNA) plays essential roles in clinical and biomedical research. Yet, it is of major challenge to sense miRNA with high degree of selectivity and sensitivity due to its unique characteristics of short length, similarity of sequence among family members and low abundance. Here, with the design of a new hairpin/DNA ring ternary probe, we describe the development of a rolling circle amplification (RCA) method for sensitively and selectively sensing miRNA from cancer cells. The target miRNA binds the hairpin/DNA ring probes through toehold-mediated strand displacement (TSD) to form the ternary structures, in which the bound miRNA and DNA ring are respectively used as the primer and template to realize RCA, leading to the generation of many repeated metal ion-dependent DNAzyme sequences. The fluorescently quenched hairpin signal probes can be cyclically cleaved by these DNAzyme sequences with co-existence of the corresponding metal ions in buffer to show drastically enhanced fluorescence recovery for highly sensitive sensing of miRNA in the range between 10 fM and 10 nM with a detection limit of 1.51 fM. Besides, owing to the high base variation discrimination ability of TSD, selective detection of the target miRNA among the corresponding family members can be achieved by this method. Moreover, such a method can also be employed to differentiate miRNA expression variations in cancer cells for screening potential therapeutic drugs.


Assuntos
Sondas de DNA/química , DNA/química , MicroRNAs/análise , Sequência de Bases , Linhagem Celular Tumoral , DNA/genética , Sondas de DNA/genética , DNA Catalítico/química , Fluorescência , Corantes Fluorescentes/química , Humanos , Sequências Repetidas Invertidas , Limite de Detecção , MicroRNAs/genética , Técnicas de Amplificação de Ácido Nucleico/métodos , Hibridização de Ácido Nucleico , Espectrometria de Fluorescência/métodos
9.
Anal Chim Acta ; 1076: 55-63, 2019 Oct 17.
Artigo em Inglês | MEDLINE | ID: mdl-31203964

RESUMO

In this work, an implantable and minimally invasive micro-aptasensor for adenosine monitoring in vivo, based on flexible integrated electrodes, was developed. Firstly the sensor was made by the modification of a needle-type electrode with reduced graphene oxide and gold nanoclusters (rGO-AuNCs) using two-step electrodeposition. Secondly Sulfhydryl-terminated capture probe (ssDNA1) was immobilized on rGO-AuNCs modified electrode surface by self-assembly, and then it was hybridized with adenosine aptamer (ssDNA2). Lastly methylene blue (MB) as an electrochemical indicator was adsorbed on the aptamer through specific interaction of MB with guanine base. The peak current of MB decreased linearly with increasing adenosine concentration due to the formation of aptamer-adenosine complex and displacement of the aptamer from the modified electrode surface. The sensor showed a low detection limit of 0.1 nM with signal-to-noise ratio equal to 3 as well as a wide linear response range (0.1 nM-1 mM) in vitro. Also, a high selectivity was demonstrated for adenosine in relation to uridine, guanosine, and cytidine. Experiments in vivo demonstrated fast responses for a range of adenosine concentrations. This work demonstrates a promising path for implantable devices for the determination of biomolecules in vivo, thus allowing for health tests, detection of infectious diseases, and other medical conditions.


Assuntos
Adenosina/análise , Aptâmeros de Nucleotídeos/química , DNA/química , Animais , Aptâmeros de Nucleotídeos/genética , Técnicas Biossensoriais/instrumentação , Técnicas Biossensoriais/métodos , DNA/genética , Sondas de DNA/química , Sondas de DNA/genética , Técnicas Eletroquímicas/instrumentação , Técnicas Eletroquímicas/métodos , Eletrodos , Ouro/química , Grafite/química , Indicadores e Reagentes/química , Limite de Detecção , Masculino , Nanopartículas Metálicas/química , Azul de Metileno/química , Hibridização de Ácido Nucleico , Ratos Wistar
10.
Analyst ; 144(11): 3649-3658, 2019 Jun 07.
Artigo em Inglês | MEDLINE | ID: mdl-31074470

RESUMO

Serious healthcare concerns have been raised on the issue of antibiotic residues after overuse, especially by accumulation in the human body through food webs. Here, we report a methodological development for sensitive detection of antibiotics with aptamer conformation cooperated enzyme-assisted SERS (ACCESS) technology. We design and integrate a set of nucleic acid oligos, realizing specific recognition of chloramphenicol (CAP) and efficient exonuclease III-assisted DNA amplification. It features a "signal-on" analysis of CAP with the limit of detection (15 fM), the lowest concentration detectable in the literature. Our method exhibits a high selectivity on the target analyte, free of interference of other potential antibiotic contaminants. The ACCESS assay promises an ultrasensitive and specific detection tool for trace amounts of antibiotic residues in samples of our daily life.


Assuntos
Antibacterianos/análise , Aptâmeros de Nucleotídeos/química , Cloranfenicol/análise , Sondas de DNA/química , DNA/química , Animais , Aptâmeros de Nucleotídeos/genética , Sequência de Bases , Técnicas Biossensoriais/métodos , DNA/genética , Sondas de DNA/genética , Exodesoxirribonucleases/química , Contaminação de Alimentos/análise , Ouro/química , Limite de Detecção , Nanopartículas Metálicas/química , Leite/química , Técnicas de Amplificação de Ácido Nucleico/métodos , Conformação de Ácido Nucleico , Hibridização de Ácido Nucleico , Reprodutibilidade dos Testes , Silício/química , Análise Espectral Raman/métodos , Poluentes Químicos da Água/análise
11.
Anal Chim Acta ; 1071: 78-85, 2019 Sep 13.
Artigo em Inglês | MEDLINE | ID: mdl-31128758

RESUMO

The development of a low-cost and disposable biosensing technologies has received a great interest of healthcare for the sensitive and reliable detection of single nucleotide mutation related to single nucleotide polymorphisms (SNPs). In the present study, an impedimetric biosensing platform based on zip nucleic acids (ZNA) was developed for the sensitive detection of Factor V Leiden (FV Leiden) mutation. After optimization of experimental parameters, the sequence selective hybridization between ZNA probe and target related to FV Leiden mutation was evaluated via electrochemical impedance spectroscopy technique (EIS) by measuring changes at the charge transfer resistance, Rct. Sensitive and selective impedimetric analysis was performed using carbon nanofiber (CNF) modified screen printed electrodes (SPE) and multi-channel screen printed array of electrodes (MULTIx8 CNF-SPE) resulting in a relatively shorter time in comparison to conventional methods. The selectivity of ZNA probe to mutation-free DNA sequences was also investigated. The applicability of single-use ZNA biosensor was also tested in synthetic PCR samples containing a single base mutation.


Assuntos
Sondas de DNA/química , DNA/análise , Fator V/genética , Técnicas Biossensoriais/métodos , Carbono/química , DNA/genética , Sondas de DNA/genética , Espectroscopia Dielétrica/métodos , Técnicas Eletroquímicas/instrumentação , Técnicas Eletroquímicas/métodos , Eletrodos , Limite de Detecção , Nanofibras/química , Hibridização de Ácido Nucleico , Mutação Puntual
12.
Analyst ; 144(13): 3972-3979, 2019 Jul 07.
Artigo em Inglês | MEDLINE | ID: mdl-31140473

RESUMO

Hepatitis C virus (HCV) is a major cause of chronic liver disease, which affects 2-3% of the world population. Until now, the early detection of HCV has been a great challenge, especially for those who live in developing countries. In this study, we developed a novel and ultrasensitive assay for the detection of HCV RNA based on the reduced graphene oxide nanosheets (rGONS) and hybridization chain reaction (HCR) amplification technique. This detection system contains a pair of single fluorophore-labeled hairpin probes that can freely exist in the solution in the absence of target RNA. The introduction of target RNA can robustly trigger a HCR with the two probes and produce long nanowires containing a double-stranded structure. The weak adsorption to rGONS makes the long nanowires emit a strong fluorescence. Using this enzyme-free amplification strategy, we developed a new method for the HCV RNA assay with a detection limit of 10 fM, which is far more sensitive than the common GO-based fluorescence method. Furthermore, the new method exhibits high selectivity for the discrimination of perfectly complementary and mismatched sequences. Finally, the new method was successfully used as a HCV RNA assay in biological samples with a strong anti-interference capability in complicated environments. In summary, these remarkable characteristics of the new method highlight its potential use in a clinical sample primary screening.


Assuntos
Bioensaio/métodos , Técnicas Biossensoriais/métodos , Grafite/química , Hepacivirus/isolamento & purificação , RNA Viral/análise , Linhagem Celular Tumoral , DNA/química , DNA/genética , Sondas de DNA/química , Sondas de DNA/genética , Fluoresceínas/química , Fluorescência , Corantes Fluorescentes/química , Grafite/síntese química , Células HEK293 , Humanos , Limite de Detecção , Técnicas de Amplificação de Ácido Nucleico/métodos , Hibridização de Ácido Nucleico , Oxirredução , Estudo de Prova de Conceito , RNA Viral/genética , Espectrometria de Fluorescência/métodos
13.
Analyst ; 144(13): 4033-4044, 2019 Jul 07.
Artigo em Inglês | MEDLINE | ID: mdl-31143920

RESUMO

Epigenetic biomarkers are powerful tools for early disease detection and are particularly useful for elusive conditions like preeclampsia. Predicting preeclampsia at an early stage is one of the most important goals of maternal-fetal medicine. To this end, recent studies have identified microRNAs-such as microRNA-17-as early biomarkers for preeclampsia. Yet clinical applications are lagging, owing in part to the sensing challenges presented by the biomarkers' small size and complex environment. Surface enhanced Raman spectroscopy (SERS) is an emergent optical technique that is recognized for its potential to overcome these challenges. In this study, DNA functionalized nanoparticles were designed as probes to capture and quantify miRNA-17 in solution. SERS was used to determine the presence and concentration of miRNA-17 based on the formation of plasmonic nanoparticle aggregates. The miRNA-17 assay was tested at concentrations of 1 pM to 1 nM in both PBS and a representative complex biological sample. In both situations the assay was unaffected by non-complementary microRNA samples. These results demonstrate SERS's specificity and sensitivity for a new biomarker (miRNA-17) that may ultimately be used in a detection platform for early diagnosis of preeclampsia.


Assuntos
Sondas de DNA/química , DNA/química , Nanopartículas Metálicas/química , MicroRNAs/sangue , Animais , Biomarcadores/sangue , Bovinos , DNA/genética , Sondas de DNA/genética , Feminino , Limite de Detecção , MicroRNAs/genética , Hibridização de Ácido Nucleico , Pré-Eclâmpsia/diagnóstico , Gravidez , Prata/química , Análise Espectral Raman/métodos
14.
Analyst ; 144(12): 3817-3825, 2019 Jun 21.
Artigo em Inglês | MEDLINE | ID: mdl-31086898

RESUMO

Herein, an ultrasensitive and label-free electrochemical biosensor was developed for microRNA (miRNA) based on rolling circle amplification (RCA)-mediated palladium nanoparticles (PdNPs). The sensor was fabricated by immobilizing dual-functionalized hairpin probes onto an electrode. The specific recognition of target miRNA-21 by the hairpin probes could trigger the RCA reaction, which produced numerous guanine (G)-rich long single-stranded DNAs (ssDNAs). Based on the interaction of the PdII species with the nitrogen atoms of the G bases, these G-rich long ssDNAs served as specific templates in the in situ synthesis of massive PdNPs as electrochemical indicators. The formation of PdNPs was demonstrated to be exactly along the RCA products by high-resolution transmission electron microscopy. Using this cascade signal amplification strategy, the developed biosensor achieved a linear range of 50 aM-100 fM with an ultralow detection limit of 8.6 aM miRNA-21. Furthermore, the developed biosensor exhibited good selectivity, reproducibility, stability and satisfactory feasibility for miRNA-21 detection in human serum samples; this ensured significant potential of this biosensor in disease diagnosis and prognosis applications.


Assuntos
Técnicas Biossensoriais/métodos , Sondas de DNA/química , Ácidos Nucleicos Imobilizados/química , Nanopartículas Metálicas/química , MicroRNAs/sangue , Platina/química , Calibragem , Sondas de DNA/genética , Técnicas Eletroquímicas/métodos , Humanos , Ácidos Nucleicos Imobilizados/genética , Sequências Repetidas Invertidas , Limite de Detecção , MicroRNAs/genética , Técnicas de Amplificação de Ácido Nucleico/métodos , Hibridização de Ácido Nucleico , Reprodutibilidade dos Testes
15.
Analyst ; 144(12): 3836-3842, 2019 Jun 21.
Artigo em Inglês | MEDLINE | ID: mdl-31095133

RESUMO

A rapid and label-free fluorescence biosensing strategy for highly sensitive detection of microRNA-122 (miR-122) has been developed by the combination of DNA three-way junction (TWJ)-actuated strand displacement and a fluorescence light-up Ag nanocluster (AgNC) probe. In the presence of target miR-122, the attachment of miR-122 to its complementary DNA results in the unblocking of the toehold and branch migration domains in the TWJ, activating the strand displacement reaction (SDR) accompanied by the proximity between the G-rich DNA probe and DNA-AgNC probe; thus a remarkably enhanced fluorescence signal of AgNCs can be obtained owing to the G-rich fluorescence enhancement mechanism. The results reveal that this biosensor exhibits superb specificity and high sensitivity toward miR-122 with a detection limit of 0.030 nM. In addition, the practicality of the biosensor is demonstrated by analyzing miR-122 in three cell lines with satisfactory results. Furthermore, by the utilization of the toehold-mediated SDR and DNA-AgNC conjugates, this proposed strategy offers the advantages of rapidness, convenience, low cost, and simplified operation without the need for biological labeling and the addition of enzymes. Thus, the constructed biosensor might provide a valuable and practical tool for detecting miRNA and the related clinical diagnosis and fundamental biomedicine research.


Assuntos
Técnicas Biossensoriais/métodos , DNA/química , Nanopartículas Metálicas/química , MicroRNAs/análise , Prata/química , Sequência de Bases , Linhagem Celular Tumoral , DNA/genética , Sondas de DNA/química , Sondas de DNA/genética , Fluorescência , Humanos , Limite de Detecção , MicroRNAs/genética , Hibridização de Ácido Nucleico , Espectrometria de Fluorescência/métodos
16.
Analyst ; 144(12): 3886-3891, 2019 Jun 21.
Artigo em Inglês | MEDLINE | ID: mdl-31115404

RESUMO

Food allergy is an abnormal immune response of the immune system to some foods, which has caused great harm to people's health. Therefore, it is particularly important to detect allergens in food. In this article, a hybridization chain reaction (HCR) was coupled with gold nanoparticles (AuNPs) to detect the allergen genes of peanut, soybean and sesame DNAs. Two hairpin probes (H1 and H2) were designed for the allergen target genes of peanut, soybean and sesame DNAs. In the presence of target DNA, the hybridization chain reaction was triggered by it producing long double-stranded DNA (dsDNA) products. In the gold nanoparticle system, long dsDNA couldn't be adsorbed on the surface of AuNPs. When the concentration of salt ions in the solution increased, gold nanoparticles accumulated and led to a decrease of ultraviolet absorption. In the absence of target DNA, no hybridization chain reaction occurred. The hairpin probes could be adsorbed on the surface of AuNPs and no accumulation happened for gold nanoparticles even if the concentration of salt ions in the solution was increased. This method required no enzymes and had a strong specificity, so it was very easy to distinguish target DNA from non-target DNA. The detection limit of three allergens detected by this method was as low as 0.5 nM. The feasibility of this method for the detection of commercial commodities had been demonstrated by the successful detection of the DNAs extracted from commercial commodities, which were treated with extreme thermostable single-stranded binding protein (ET SSB).


Assuntos
Alérgenos/genética , DNA/análise , Ouro/química , Nanopartículas Metálicas/química , Adsorção , Arachis/genética , Sequência de Bases , Colorimetria/métodos , DNA/genética , Sondas de DNA/química , Sondas de DNA/genética , Sequências Repetidas Invertidas , Limite de Detecção , Técnicas de Amplificação de Ácido Nucleico/métodos , Hibridização de Ácido Nucleico , Sesamum/genética , Soja/genética
17.
Analyst ; 144(12): 3800-3806, 2019 Jun 21.
Artigo em Inglês | MEDLINE | ID: mdl-31116196

RESUMO

In typical photoelectrochemical (PEC) biosensing assays, electrodes are generally modified with photoactive probes and/or target recognition probes, which makes the processes complicated, time-consuming, and difficult to achieve excellent reproducibility. Hence, to overcome such shortcomings, we propose here an immobilization-free and label-free PEC aptasensor using solution-phase methylene blue (MB) as the PEC signal probe. Based on the unique T-Hg2+-T base pairs, and the diffusivity difference between free MB molecules and the MB/G-quadruplex composite towards the ITO electrode surface with negative charge, the "signal-off" approach for Hg2+ detection is developed. In the presence of target Hg2+, via the T-Hg2+-T bond formation, the two sticky ends of the hairpin DNA probe form a rigid duplex stem, which triggers the exonuclease III-facilitated target cycling amplification, and the formation of multiple G-quadruplexes. Upon the intercalation of MB in G-quadruplexes, significantly decreased photocurrent is obtained owing to the increased electrostatic repulsion between the MB/G-quadruplex composite and the ITO electrode. Therefore, highly sensitive and ultrasensitive Hg2+ determination is achieved, with a low detection limit of 1.2 pM, well below the maximum allowable Hg2+ level in drinking water defined by the WHO, China's Ministry of Health, and the US EPA. Due to the avoidance of sophisticated electrode modification and recognition probe immobilization processes, as well as an expensive labeling procedure, the PEC aptasensor proposed here demonstrates the advantages of simplicity, good reproducibility, rapidness and low cost.


Assuntos
Aptâmeros de Nucleotídeos/química , Técnicas Biossensoriais/métodos , Sondas de DNA/química , Exodesoxirribonucleases/química , Mercúrio/análise , Azul de Metileno/química , Aptâmeros de Nucleotídeos/genética , Pareamento de Bases , DNA/química , DNA/genética , Sondas de DNA/genética , Água Potável/análise , Técnicas Eletroquímicas/instrumentação , Técnicas Eletroquímicas/métodos , Eletrodos , Quadruplex G , Substâncias Intercalantes/química , Substâncias Intercalantes/efeitos da radiação , Sequências Repetidas Invertidas , Lagos/análise , Limite de Detecção , Mercúrio/química , Azul de Metileno/efeitos da radiação , Técnicas de Amplificação de Ácido Nucleico/métodos , Reprodutibilidade dos Testes , Timina/química , Compostos de Estanho/química , Poluentes Químicos da Água/análise , Poluentes Químicos da Água/química
18.
Vet Parasitol ; 269: 2-6, 2019 May.
Artigo em Inglês | MEDLINE | ID: mdl-31079823

RESUMO

Neospora caninum is an apicomplexan protozoan parasite that is a leading cause of abortion in cattle. Detection of parasite-specific DNA by PCR is a highly sensitive method for identifying the presence of N. caninum in a variety of tissues. We developed and validated a probe-based real-time PCR assay targeting the conserved Nc5 gene of N. caninum. Using N. caninum strain Nc-1 genomic DNA and a synthetic gene fragment as amplification standards, we determined the PCR amplification efficiency and the limit of detection to be 95.60% and 3 copies, respectively. Five pathogens frequently associated with bovine abortions, namely bovine viral diarrhea virus types I and II, bovine alphaherpesvirus-1, Chlamydia, and Leptospira, were tested to ensure analytical exclusivity. A total of 103 clinical samples from aborted fetuses were tested concurrently with a standard conventional PCR and the new probe-based real-time PCR assay. All tested samples showed 100% agreement between these two assays. In conclusion, the probe-based real-time PCR assay facilitates accurate and rapid detection of N. caninum from abortions in cattle.


Assuntos
Aborto Animal/diagnóstico , Doenças dos Bovinos/diagnóstico , Coccidiose/veterinária , Neospora/isolamento & purificação , Complicações Parasitárias na Gravidez/veterinária , Reação em Cadeia da Polimerase em Tempo Real/veterinária , Feto Abortado/parasitologia , Aborto Animal/parasitologia , Animais , Encéfalo/parasitologia , Bovinos , Doenças dos Bovinos/parasitologia , Coccidiose/diagnóstico , Coccidiose/parasitologia , Primers do DNA/genética , Sondas de DNA/genética , Feminino , Coração/parasitologia , Neospora/genética , Gravidez , Complicações Parasitárias na Gravidez/diagnóstico , Complicações Parasitárias na Gravidez/parasitologia
19.
Talanta ; 201: 358-363, 2019 Aug 15.
Artigo em Inglês | MEDLINE | ID: mdl-31122435

RESUMO

Single base mismatch can always connect with various gene-related diseases, whose determination has aroused widespread interest. So far, various methods have been developed to determine the common base mismatch. However most of them are complex, time-consuming. Herein, we report a novel method, which only need one conventional endonuclease (NEase) and achieve site-specific cleavage in a programmable way, to detect single base mismatch, termed aligner-mediated cleavage-based single base mismatch discrimination (AMCMD). The DNA aligner (DA) is in a stem-loop structure, consistent with an incomplete recognition site of NEase on its stem and a 5'-side arm complementary to the target sequence (TS). Once TS contains matched base and hybridizes with DA, the complete recognition site of NEase is formed, and the TS will be cleavaged with fast speed, while converse is not. Based on it, the method can clearly distinguish mismatched and complementary bases. Without sample pre-processing, we were able to obtain and verify all the test result in about 30 min through the polyacrylamide gel electrophoresis analysis. This endows the proposed method with a simpler advantage. Then we combined AMCMD and EXPAR to create a new method for single base mismatch discrimination, the short sequence obtained by AMCMD as a target to trigger EXPAR, with a detection limit at 1pM level. Another process with human serum underlines that AMCMD is compatible with the complex biological sample, thus it has the potentials for practical applications.


Assuntos
Pareamento Incorreto de Bases , Técnicas Biossensoriais/métodos , Citidina Monofosfato/sangue , Sondas de DNA/química , DNA/química , Sequência de Bases , Citidina Monofosfato/genética , DNA/genética , Sondas de DNA/genética , Desoxirribonucleases de Sítio Específico do Tipo II/química , Humanos , Sequências Repetidas Invertidas , Limite de Detecção , Hibridização de Ácido Nucleico
20.
Genome ; 62(5): 329-339, 2019 May.
Artigo em Inglês | MEDLINE | ID: mdl-30933665

RESUMO

Fluorescence in situ hybridization (FISH) using oligonucleotides is a simple and convenient method for chromosome research. In this study, 34 of 46 previously developed oligonucleotides produced signals in barley. Together with two plasmid clones and one PCR-amplified cereal centromere repeat (CCS1) probe, 37 repetitive sequences were chromosomally located produced three types of signals covering different positions on the chromosomes. The centromeric and pericentric regions had a more complex genomic organization and sequence composition probably indicative of higher contents of heterochromatin. An efficient multi-plex probe containing eight oligonucleotides and a plasmid clone of 45S rDNA was developed. Thirty-three barley karyotypes were developed and compared. Among them, 11 irradiation-induced mutants of cultivar 08-49 showed no chromosomal variation, whereas 22 cultivar and landrace accessions contained 28 chromosomal polymorphisms. Chromosome 4H was the most variable and 6H was the least variable based on chromosome polymorphic information content (CPIC). Five polymorphic chromosomes (1H-2, 2H-1, 3H-3, 5H-2, and 6H-2) were dominant types, each occurring in more than 50% of accessions. The multi-plex probe should facilitate identification of further chromosomal polymorphisms in barley.


Assuntos
Cromossomos de Plantas/genética , Hordeum/genética , Polimorfismo Genético/genética , Sequências Repetitivas de Ácido Nucleico/genética , Centrômero/genética , Sondas de DNA/genética , Hibridização in Situ Fluorescente , Cariótipo , Cariotipagem , Oligonucleotídeos/genética , RNA de Plantas/genética , RNA Ribossômico/genética
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