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1.
Nat Commun ; 11(1): 5473, 2020 10 29.
Artigo em Inglês | MEDLINE | ID: mdl-33122648

RESUMO

Combining experimental and simulation strategies to facilitate the design and operation of nucleic acid hybridization probes are highly important to both fundamental DNA nanotechnology and diverse biological/biomedical applications. Herein, we introduce a DNA equalizer gate (DEG) approach, a class of simulation-guided nucleic acid hybridization probes that drastically expand detection windows for discriminating single nucleotide variants in double-stranded DNA (dsDNA) via the user-definable transformation of the quantitative relationship between the detection signal and target concentrations. A thermodynamic-driven theoretical model was also developed, which quantitatively simulates and predicts the performance of DEG. The effectiveness of DEG for expanding detection windows and improving sequence selectivity was demonstrated both in silico and experimentally. As DEG acts directly on dsDNA, it is readily adaptable to nucleic acid amplification techniques, such as polymerase chain reaction (PCR). The practical usefulness of DEG was demonstrated through the simultaneous detection of infections and the screening of drug-resistance in clinical parasitic worm samples collected from rural areas of Honduras.


Assuntos
Sondas de DNA/química , Corantes Fluorescentes/química , Animais , DNA/química , Helmintos/genética , Helmintos/isolamento & purificação , Modelos Teóricos , Hibridização de Ácido Nucleico/métodos , Nucleotídeos , Reação em Cadeia da Polimerase/métodos , Polimorfismo de Nucleotídeo Único , Termodinâmica
2.
Clin Chem ; 66(8): 1047-1054, 2020 08 01.
Artigo em Inglês | MEDLINE | ID: mdl-32384153

RESUMO

BACKGROUND: The current outbreak of SARS-CoV-2 has spread to almost every country with more than 5 million confirmed cases and over 300,000 deaths as of May 26, 2020. Rapid first-line testing protocols are needed for outbreak control and surveillance. METHODS: We used computational and manual designs to generate a suitable set of reverse transcription recombinase polymerase amplification (RT-RPA) primer and exonuclease probe, internally quenched (exo-IQ), sequences targeting the SARS-CoV-2 N gene. RT-RPA sensitivity was determined by amplification of in vitro transcribed RNA standards. Assay selectivity was demonstrated with a selectivity panel of 32 nucleic acid samples derived from common respiratory viruses. To validate the assay against full-length SARS-CoV-2 RNA, total viral RNA derived from cell culture supernatant and 19 nasopharyngeal swab samples (8 positive and 11 negative for SARS-CoV-2) were screened. All results were compared to established RT-qPCR assays. RESULTS: The 95% detection probability of the RT-RPA assay was determined to be 7.74 (95% CI: 2.87-27.39) RNA copies per reaction. The assay showed no cross-reactivity to any other screened coronaviruses or respiratory viruses of clinical significance. The developed RT-RPA assay produced 100% diagnostic sensitivity and specificity when compared to RT-qPCR (n = 20). CONCLUSIONS: With a run time of 15 to 20 minutes and first results being available in under 7 minutes for high RNA concentrations, the reported assay constitutes one of the fastest nucleic acid based detection methods for SARS-CoV-2 to date and may provide a simple-to-use alternative to RT-qPCR for first-line screening at the point of need.


Assuntos
Betacoronavirus/genética , Infecções por Coronavirus/diagnóstico , Técnicas de Amplificação de Ácido Nucleico/métodos , Pneumonia Viral/diagnóstico , RNA Viral/metabolismo , Betacoronavirus/isolamento & purificação , Infecções por Coronavirus/virologia , Sondas de DNA/química , Sondas de DNA/metabolismo , Exonucleases/metabolismo , Humanos , Pandemias , Pneumonia Viral/virologia , Testes Imediatos , Reação em Cadeia da Polimerase em Tempo Real , Sensibilidade e Especificidade
3.
Chem Commun (Camb) ; 56(40): 5409-5412, 2020 May 19.
Artigo em Inglês | MEDLINE | ID: mdl-32286590

RESUMO

A simple multiplexed digital microRNA detection strategy with fluorescence flow cytometry was proposed. By isothermal ligation-rolling circle amplification, multiplexed microRNAs could be simultaneously converted to a series of nanoflower balls (NFBs) and counted by flow cytometry directly.


Assuntos
Citometria de Fluxo/métodos , MicroRNAs/análise , Sondas de DNA/química , Sondas de DNA/genética , Corantes Fluorescentes/química , Humanos , Limite de Detecção , MicroRNAs/química , MicroRNAs/genética , Nanopartículas/química , Técnicas de Amplificação de Ácido Nucleico/métodos , Hibridização de Ácido Nucleico , Estudo de Prova de Conceito
4.
J Infect Chemother ; 26(5): 523-526, 2020 May.
Artigo em Inglês | MEDLINE | ID: mdl-32147375

RESUMO

Transmissible gastroenteritis virus (TGEV) and porcine epidemic diarrhea virus (PEDV) are the main pathogens causing viral diarrhea in pig, mixed infections of these two viruses are very common in intensive pig rearing. However, there is a lack of a method to simultaneously detect and distinguish PEDV and TGEV in preclinical levels. In this study, we aimed to establish a dual ultrasensitive nanoparticle DNA probe-based PCR assay (dual UNDP-PCR) based on functionalized magnetic bead enrichment and specific nano-technology amplification for simultaneous detection and distinguish diagnosis of PEDV and TGEV. The detection limit of dual UNDP-PCR for single or multiple infections of PEDV and TGEV is 25 copies/g, which is 400 times more sensitive than the currently known duplex RT-PCR, showing better specificity and sensitivity without cross-reaction with other viruses. For pre-clinical fecal samples, the dual UNDP-PCR showed a markedly higher positive detection rate (52.08%) than conventional duplex RT-PCR (13.21%), can rapidly and accurately identify targeted pathogens whenever simple virus infection or co-infection. In summary, this study provides a technique for detecting and distinguishing PEDV and TGEV in preclinical levels, which is high sensitivity, specificity, repeatability, low cost and broad application prospect.


Assuntos
Sondas de DNA/química , Gastroenterite Suína Transmissível/diagnóstico , Nanopartículas/química , Vírus da Diarreia Epidêmica Suína/isolamento & purificação , Reação em Cadeia da Polimerase Via Transcriptase Reversa/veterinária , Vírus da Gastroenterite Transmissível/isolamento & purificação , Animais , Sondas de DNA/genética , Diarreia/veterinária , Diarreia/virologia , Fezes/virologia , Gastroenterite Suína Transmissível/virologia , Limite de Detecção , Imãs , Vírus da Diarreia Epidêmica Suína/genética , RNA Viral/genética , RNA Viral/isolamento & purificação , Reprodutibilidade dos Testes , Reação em Cadeia da Polimerase Via Transcriptase Reversa/métodos , Sensibilidade e Especificidade , Suínos , Doenças dos Suínos/diagnóstico , Doenças dos Suínos/virologia , Vírus da Gastroenterite Transmissível/genética
5.
Sci Rep ; 10(1): 4018, 2020 03 04.
Artigo em Inglês | MEDLINE | ID: mdl-32132554

RESUMO

The characteristic features of stem-loop structured probes make them robust tools to detect targets with high sensitivity and selectivity. The basis of the hairpin based sensors operation is a conformational change that occurs upon hybridization of target with stem-loop probe. The design of the stem-loop probe has an important role in target recognition. Therefore, we designed a label-free stem loop probe for targeting miR-21 as a cancer biomarker investigated by web-based tools; its thermodynamic parameters obtained by thermal UV spectroscopy. The efficiency of stem-loop structure opening in the presence of target and non-target sequences was evaluated by fluorescence spectroscopy and circular dichroism spectro-polarimetry. The results showed that the target sequence opens the structure of hairpin efficiently in comparison to non-target sequences. To optimize the stem-loop hybridization to its target, the buffer ionic strength was changed by adding different concentrations of NaCl, KCl and MgCl2. It was shown that buffering conditions have a significant role in loop structure opening and its optimization, led to an increase in sensitivity detection and have improved LOD from 60 pM to 45 pM.


Assuntos
Técnicas Biossensoriais , Sondas de DNA , MicroRNAs , Dicroísmo Circular , Sondas de DNA/química , Sondas de DNA/genética , Humanos , MicroRNAs/análise , MicroRNAs/química , MicroRNAs/genética , Hibridização de Ácido Nucleico , Espectrometria de Fluorescência
6.
Nat Commun ; 11(1): 1543, 2020 03 24.
Artigo em Inglês | MEDLINE | ID: mdl-32210235

RESUMO

Field-effect transistor (FET)-based biosensors allow label-free detection of biomolecules by measuring their intrinsic charges. The detection limit of these sensors is determined by the Debye screening of the charges from counter ions in solutions. Here, we use FETs with a deformed monolayer graphene channel for the detection of nucleic acids. These devices with even millimeter scale channels show an ultra-high sensitivity detection in buffer and human serum sample down to 600 zM and 20 aM, respectively, which are ∼18 and ∼600 nucleic acid molecules. Computational simulations reveal that the nanoscale deformations can form 'electrical hot spots' in the sensing channel which reduce the charge screening at the concave regions. Moreover, the deformed graphene could exhibit a band-gap, allowing an exponential change in the source-drain current from small numbers of charges. Collectively, these phenomena allow for ultrasensitive electronic biomolecular detection in millimeter scale structures.


Assuntos
Técnicas Biossensoriais/instrumentação , Sondas de DNA/análise , DNA de Cadeia Simples/análise , Grafite/química , MicroRNAs/análise , Sondas de DNA/química , DNA de Cadeia Simples/química , Estudos de Viabilidade , Humanos , Íons , Limite de Detecção , MicroRNAs/química , Simulação de Dinâmica Molecular , Sensibilidade e Especificidade , Transistores Eletrônicos
7.
Mikrochim Acta ; 187(3): 194, 2020 03 02.
Artigo em Inglês | MEDLINE | ID: mdl-32124079

RESUMO

A controllable approach for preparing a portable colloidal photonic crystal (CPC) array chip is presented. The approach was inspired by the confinement effect of nanoparticle self-assembly on patterned surface. Hydrophobic polydimethylsiloxane substrate with reproducible micro-region array was fabricated by soft-lithography. The substrate was employed as the patterned template for self-assembly of monodisperse polystyrene nanoparticles. The CPC units can be prepared in several minutes, and exhibit consistent reflection wavelength. By adjusting the size of polystyrene nanoparticles and the shape of micro-regions, CPC units with multiple structure, colors and geometries were obtained. The CPC array chip features fluorescence enhancement owing to the optical modulation capability of the periodic nanostructure of the self-assembled CPC. With the reflection wavelength (523 nm) of green CPC units overlapping the emission wavelength (520 nm, with excitation wavelength of 490 nm) of 6-carboxyfluorescein-labeled DNA probe, the fluorescence intensity increased more than 10-fold. For signal-amplified assay of adenosine, the concentration range of linear response was 5.0 × 10-5 mol L-1 to 1.0 × 10-3 mol L-1, and the limit of detection was 1.3 × 10-6 mol L-1. Because of the enhancement effect of photonic crystal, the fluorescence images were more readable from the CPC array chip, compared with those from the planar substrate. The chip has potential applications in multiplex determination with high-throughput via encoding strategy based on the tunable structure, color or geometric shape. Graphical abstractSchematic diagram of signal-enhanced fluorescent detection of adenosine based on the colloidal photonic crystal array chip (PDMS, polydimethylsiloxane; PS NPs, polystyrene nanoparticles; CPC, colloidal photonic crystal; GO, graphene oxide; FAM, 6-carboxyfluorescein).


Assuntos
Adenosina/análise , Técnicas Biossensoriais/métodos , Fluoresceínas/química , Corantes Fluorescentes/química , Dispositivos Lab-On-A-Chip , Coloides , Cristalização , Sondas de DNA/química , Dimetilpolisiloxanos/química , Interações Hidrofóbicas e Hidrofílicas , Limite de Detecção , Fótons , Espectrometria de Fluorescência , Propriedades de Superfície
8.
Talanta ; 212: 120735, 2020 May 15.
Artigo em Inglês | MEDLINE | ID: mdl-32113527

RESUMO

Changes in expression of Klotho gene are associated with chronic kidney disease and its potential as early biomarker is being studied. We report, for the first time, the detection of Klotho gene by a biosensor platform. Self-assembled mixed monolayers (SAMs) as DNA immobilization method in screen-printed gold electrodes and a sandwich format detection were used in the development of an electrochemical genosensor for the detection of a 100-mer DNA fragment, copy of the partial region of the mRNA Klotho gene. The use of different binary and ternary SAMs based on aliphatic (mercaptohexanol, MCH, and hexanedithiol, HDT) and aromatic (mercaptophenylacetic acid, MPAA) thiol diluents and capture probe (CP) as sensing phases was evaluated by cyclic voltammetry and electrochemical impedance spectroscopy. Multiple configurations were studied, changing the order of component addition and comparing co-immobilization and two-step immobilization processes. The procedure for binary SAM preparation consisting of sequential addition of a thiol diluent followed by CP was found to have the least detrimental impact on electrochemical performance. The signal-to-blank ratios increased considerably in the case of thioaromatic binary DNA monolayers, MPPA/CP, compared to the values obtained for aliphatic SAMs. Ternary monolayers formed by MCH and HDT rendered good fractional coverage levels and generated more reversible redox reactions at the surface, mostly when CP was firstly immobilized, CP/HDT/MCH. A significant reduction of the blank and non-specific (non-complementary sequence) signals was obtained with this ternary SAM, compared to binary SAMs and an increase of 2.42-fold of the S/B ratio (10 nM of target) compared with MPAA/CP SAMs. A linear response in the range of 5·10-10 to 5·10-8 M was obtained with CP/HDT/MCH monolayer, with a detection limit of 0.5 nM and RSD of 8.10%.


Assuntos
Técnicas Biossensoriais/métodos , DNA/análise , Técnicas Eletroquímicas/métodos , Glucuronidase/genética , Compostos de Sulfidrila/química , Fosfatase Alcalina/química , DNA/química , DNA/genética , Sondas de DNA/química , Sondas de DNA/genética , Fluoresceínas/química , Corantes Fluorescentes/química , Humanos , Ácidos Nucleicos Imobilizados/química , Ácidos Nucleicos Imobilizados/genética , Limite de Detecção , Naftalenos/química , Hibridização de Ácido Nucleico , Compostos Organofosforados/química
9.
Talanta ; 212: 120754, 2020 May 15.
Artigo em Inglês | MEDLINE | ID: mdl-32113533

RESUMO

Robust, reliable, and sensitively quantitative detection of genetic biomarkers at single-base resolution has the potential to revolutionize medical diagnostics, especially for precision medicine. Here, taking the advantages of the high specificity of ligase reaction and the powerful amplification features of the isothermally exponential amplification, we have demonstrated a novel methodology to sensitively quantify genetic biomarkers at one-base resolution. The methodology is based on the ligase reaction of two stem-loop DNA probes templated by the nucleic acid targets to form a double stem-loop DNA, which subsequently initiates the isothermally exponential amplification reaction with high amplification efficiency. With the proposed method, high sensitivity to determine as low as 0.01 fM DNA or 0.1 fM RNA targets and high specificity to detect single-base changes can be achieved. The new methodology is robust to be performed by using a pair of universal primers under isothermal conditions, which should be employed to quantitatively detect any genetic biomarkers because all DNA/RNA targets can be directly used as the templates to ligate the stem-loop DNA probes with single-base resolution.


Assuntos
DNA/análise , MicroRNAs/análise , Técnicas de Amplificação de Ácido Nucleico/métodos , Bacteriófago T4/enzimologia , Biomarcadores/análise , DNA/química , DNA/genética , DNA Ligases/química , Metilação de DNA , Sondas de DNA/química , Sondas de DNA/genética , Humanos , Sequências Repetidas Invertidas , Limite de Detecção , Células MCF-7 , MicroRNAs/química , MicroRNAs/genética , Hibridização de Ácido Nucleico , Polimorfismo de Nucleotídeo Único , RNA Ligase (ATP)/química , Proteínas Virais/química
10.
Talanta ; 212: 120764, 2020 May 15.
Artigo em Inglês | MEDLINE | ID: mdl-32113537

RESUMO

A magnetic-separation-dual-targets fluorescent biosensor was fabricated to detect terminator nopaline synthase (TNOS) and promoter of cauliflower mosaic virus 35s (P35S) in transgenic soybean based on incorporation of bicolor CdTe quantum dots carried by silica nanospheres. In this protocol, the fixed probes for TNOS or P35S were magnetized firstly with Fe3O4@Au magnetic nanosphere by Au-S covalent bonding to achieve magnetized probes. Meanwhile, the capture probes for TNOS or P35S were functionalized with green or red fluorescent microspheres respectively to obtain fluorescently-labeled probes, which could emit relative strong green or red fluorescent signal. Two terminals of TNOS or P35S were recognized by magnetized probes and fluorescently-labeled probes respectively to form the sandwiched structures in the process of biosensor development subsequently, and it was separated by a magnet instantly. The fluorescence intensities of remnant supernatant were measured and analyzed accordingly to achieve simultaneous detection of TNOS and P35S. This biosensor exhibited a good dynamic range, low limit of detection and excellent selectivity in detecting transgenic soybean.


Assuntos
Aminoácido Oxirredutases/genética , Técnicas Biossensoriais/métodos , DNA Viral/análise , Corantes Fluorescentes/química , Nanosferas/química , Proteínas Virais/genética , Compostos de Cádmio/química , Caulimovirus/química , Caulimovirus/enzimologia , Sondas de DNA/química , Sondas de DNA/genética , DNA Viral/genética , Óxido Ferroso-Férrico/química , Ouro/química , Limite de Detecção , Hibridização de Ácido Nucleico , Plantas Geneticamente Modificadas , Regiões Promotoras Genéticas , Pontos Quânticos/química , Reprodutibilidade dos Testes , Soja , Telúrio/química
11.
Talanta ; 213: 120816, 2020 Jun 01.
Artigo em Inglês | MEDLINE | ID: mdl-32200921

RESUMO

Nucleic acid-based biosensors have become powerful tools in biomedical applications. But the stability issue seriously limits their wide applications. Fortunately, the emergence of carbon nanoparticles (CNPs), which can effectively protect DNA probes from enzymatic digestion and unspecific protein binding, provides a good solution. In this work, a DNase I-aided cyclic enzymatic amplification method (CEAM) for microRNA analysis has been developed based on the coupling use of nucleic acid probes with specific molecular recognition ability as well as CNPs with excellent biostability. The method is simple and sensitive, with a detection limit down to 3.2 pM. Furthermore, satisfactory results are achieved for miRNA analysis in breast cancer cell lysate, demonstrating the applicability in disease diagnosis. The ingenious combination of CNPs and nucleic acid probes can open a new chapter in the development of versatile analytical strategies that holds great potentials for clinical diagnosis, food safety, and environmental monitoring.


Assuntos
Técnicas Biossensoriais/métodos , Carbono/química , Desoxirribonuclease I/química , MicroRNAs/análise , Nanopartículas/química , Neoplasias da Mama/genética , Linhagem Celular Tumoral , Sondas de DNA/química , Sondas de DNA/genética , Feminino , Humanos , Limite de Detecção , MicroRNAs/genética , Técnicas de Amplificação de Ácido Nucleico/métodos , Espectrometria de Fluorescência/métodos
12.
J Colloid Interface Sci ; 566: 369-374, 2020 Apr 15.
Artigo em Inglês | MEDLINE | ID: mdl-32018176

RESUMO

Single-molecule Förster Resonance Energy Transfer was used to observe the adsorption of fluorescently-labeled "target" DNA oligonucleotides and their association and hybridization with complementary DNA "probes" tethered to the surface as a function of surface grafting density. Ionic strength was varied systematically to disentangle the potentially competing effects of probe accessibility and electrostatic repulsion. At high ionic strength, when the Debye length was ~1 nm, the adsorption of target DNA was not significantly inhibited by the presence of tethered probe DNA, even at high grafting density, and the fraction of adsorbed target strands undergoing hybridization increased systematically with grafting density, leading to a dramatic increase in the net hybridization rate at high grafting density. However, at lower ionic strength, when the Debye length was ≥3 nm, the adsorption rate of target DNA decreased and the fraction of adsorbed target strands undergoing hybridization saturated at high probe grafting density (≥7,000 strands/µm2), presumably due to electrostatic repulsion. As a result, the net rate of hybridization exhibited a maximum as a function of grafting density. This has important consequences for the design of systems that optimize surface-mediated DNA hybridization under low-salt high-stringency conditions.


Assuntos
DNA/química , Hibridização de Ácido Nucleico , Adsorção , Sondas de DNA/química , Concentração Osmolar , Tamanho da Partícula , Eletricidade Estática , Propriedades de Superfície
13.
Talanta ; 211: 120726, 2020 May 01.
Artigo em Inglês | MEDLINE | ID: mdl-32070590

RESUMO

MiRNAs are known to be involved in a series of diseases, including breast cancer, and they have the potential to serve as diagnostic/prognostic markers and therapeutic targets. A prerequisite for miRNAs to be applied in clinical practice is the quantitative profiling of their expression. However, the majority of current assays used in miRNA detection are highly enzyme-dependent. In this study, a novel enzyme-free assay was developed that relies on stacking hybridization and a photocleavable DNA-PL-peptide probe, which contains a reporter peptide (AVLGVDPFR), a photocleavable o-nitrobenzyl derivative linker and a detection DNA sequence that is complementary to a part of the target miRNA (e.g., miR-21, miR-125a or miR-200c). Stacking hybridization enabled the DNA-PL-peptide probe to capture DNA in a contiguous tandem arrangement to generate a long DNA single strand complementary to the target miRNA. Then, photolysis was initiated to rapidly release the reporter peptide, and the reporter peptide was ultimately monitored by liquid chromatography-tandem mass spectrometry (LC-MS/MS). In this experiment, the parameters linked with photorelease, binding, conjugation and hybridization were characterized. The results showed that the assay time was significantly shortened, and the detection specificity was improved. After validation of the assay, the target miRNA level was determined in human breast cells and tissue samples. The results demonstrated that photocleavable materials coupled with mass spectrometric detection have great potential in clinical practice.


Assuntos
Biomarcadores Tumorais/análise , Neoplasias da Mama/genética , Sondas de DNA/química , MicroRNAs/análise , Fragmentos de Peptídeos/química , Espectrometria de Massas em Tandem/métodos , Biomarcadores Tumorais/genética , Neoplasias da Mama/patologia , Cromatografia Líquida , Feminino , Humanos , MicroRNAs/genética , Células Tumorais Cultivadas
14.
Clin Chem ; 66(3): 463-473, 2020 03 01.
Artigo em Inglês | MEDLINE | ID: mdl-32068788

RESUMO

BACKGROUND: The specific characteristics of copy number variations (CNVs) require specific methods of detection and characterization. We developed the Easy One-Step Amplification and Labeling procedure for CNV detection (EOSAL-CNV), a new method based on proportional amplification and labeling of amplicons in 1 PCR. METHODS: We used tailed primers for specific amplification and a pair of labeling probes (only 1 labeled) for amplification and labeling of all amplicons in just 1 reaction. Products were loaded directly onto a capillary DNA sequencer for fragment sizing and quantification. Data obtained could be analyzed by Microsoft Excel spreadsheet or EOSAL-CNV analysis software. We developed the protocol using the LDLR (low density lipoprotein receptor) gene including 23 samples with 8 different CNVs. After optimizing the protocol, it was used for genes in the following multiplexes: BRCA1 (BRCA1 DNA repair associated), BRCA2 (BRCA2 DNA repair associated), CHEK2 (checkpoint kinase 2), MLH1 (mutL homolog 1) plus MSH6 (mutS homolog 6), MSH2 (mutS homolog 2) plus EPCAM (epithelial cell adhesion molecule) and chromosome 17 (especially the TP53 [tumor protein 53] gene). We compared our procedure with multiplex ligation-dependent probe amplification (MLPA). RESULTS: The simple procedure for CNV detection required 150 min, with <10 min of handwork. After analyzing >240 samples, EOSAL-CNV excluded the presence of CNVs in all controls, and in all cases, results were identical using MLPA and EOSAL-CNV. Analysis of the 17p region in tumor samples showed 100% similarity between fluorescent in situ hybridization and EOSAL-CNV. CONCLUSIONS: EOSAL-CNV allowed reliable, fast, easy detection and characterization of CNVs. It provides an alternative to targeted analysis methods such as MLPA.


Assuntos
Variações do Número de Cópias de DNA , Reação em Cadeia da Polimerase/métodos , Receptores de LDL/genética , Sondas de DNA/química , Sondas de DNA/metabolismo , Corantes Fluorescentes/química , Humanos , Hibridização in Situ Fluorescente , Reação em Cadeia da Polimerase Multiplex , Análise de Sequência de DNA
15.
Analyst ; 145(4): 1174-1178, 2020 Feb 17.
Artigo em Inglês | MEDLINE | ID: mdl-32016264

RESUMO

We present a novel ratiometric fluorescent biosensor for ctDNA analysis based on the construction of a DNA four-way junction (FWJ). Three fuel strands for the FWJ are firstly designed and prepared. Another essential strand for the formation of the structure is the DNA product generated from target ctDNA initiated strand displacement amplification. With the transformation of the DNA structure, the FRET states of two fluorophores change and the ratiometric fluorescence response can be recorded to indicate the level of the initial ctDNA. The proposed method also has excellent capability to discriminate mismatches and shows potential practical utility for clinical samples.


Assuntos
Técnicas Biossensoriais/métodos , DNA Tumoral Circulante/sangue , Espectrometria de Fluorescência/métodos , Sondas de DNA/química , Fluorescência , Humanos , Limite de Detecção , Técnicas de Amplificação de Ácido Nucleico/métodos
16.
Chem Commun (Camb) ; 56(17): 2658-2661, 2020 Feb 27.
Artigo em Inglês | MEDLINE | ID: mdl-32022034

RESUMO

A DNAzyme-powered micromachine with anti-interfering properties and displaying resistance to being inhibited by biological matrices was built. This micromachine was able to respond to a specific target in high-concentration serum or whole blood.


Assuntos
Técnicas Biossensoriais/instrumentação , DNA Catalítico/metabolismo , Sondas de DNA/química , DNA Catalítico/antagonistas & inibidores
17.
Chem Commun (Camb) ; 56(19): 2901-2904, 2020 Mar 05.
Artigo em Inglês | MEDLINE | ID: mdl-32037435

RESUMO

The enzymatic-assisted signal amplification of DNA sensors is rarely applied in living cells due to the difficulties in protein delivery. In this study, we have proposed a biomineralization-based DNA nanoprobe to transport nucleases and DNA sensors for enzyme-assisted imaging of microRNA in living cells.


Assuntos
Biomineralização , Sondas de DNA/química , DNA/metabolismo , Exodesoxirribonucleases/metabolismo , Nanopartículas/química , Humanos , Estruturas Metalorgânicas/química , MicroRNAs/metabolismo
18.
Mikrochim Acta ; 187(3): 176, 2020 02 19.
Artigo em Inglês | MEDLINE | ID: mdl-32076892

RESUMO

The authors describe a microfluidic chip-based aptasensor platform combined with magnetic tripartite DNA structure-functionalized nanocomposites to achieve simultaneous determination of kanamycin (KANA), aflatoxin M1 (AFM1), and 17ß-estradiol (E2) in milk. The two-duplex tripartite DNA nanostructure was first assembled on the surface of magnetic beads. When the aptamer on the probes recognized the specific target, the aptamer-target would be released into the supernatant. The pre-primer@circular DNA template structure initiates rolling circle amplification (RCA) by phi29 polymerase. After magnetic separation, the magnetic nanocomposites were added into a solution containing three different lengths of complementary strands to the RCA products. The number of complementary strands significantly decrease, and this can be quantitated by the microfluidic chip. Further, the employment of magnetic nanocomposites and microfluidic chip not only resolve the complex matrix interference, but also dramatically enhances the determination selectivity and sensitivity. This aptasensor allows for determination of KANA, AFM1, and E2 with limits of detection as low as 0.32 pg mL-1, 0.95 pg mL-1, and 6.8 pg mL-1, respectively. This novel method exhibits the advantages of excellent stability and fast response time (< 3 min on microfluidic chip platform) for simultaneous determination of KANA, AFM1, and E2 in milk samples and ensures food safety. Graphical abstract.


Assuntos
Aflatoxina M1/química , Sondas de DNA/química , Estradiol/química , Canamicina/química , Microfluídica/métodos , Nanoestruturas/química , Técnicas Biossensoriais/métodos , Humanos , Fenômenos Magnéticos
19.
Mikrochim Acta ; 187(2): 119, 2020 01 11.
Artigo em Inglês | MEDLINE | ID: mdl-31927667

RESUMO

A colorimetric microplate assay for determination of Staphylococcus aureus DNA is described. Linear padlock probes were designed to recognize target sequences. After DNA binding, the linear padlock probes were circularized by ligation and then hybridize with biotin-labeled capture probes. Biotin-labeled capture probes act as primers to initiate the RCA. The biotin-labeled RCA products hybridize with digoxin-labeled signal probes fixed on streptavidin-functionalized wells of a 96-well plate. To enhance sensitivity, an AuNP-anti-digoxigenin-POx-HRP conjugate was added to the wells and then bound to digoxin-labeled signalling probes. The oxidation of tetramethylbenzidine (TMB) by H2O2 produces a color change from colorless to blue via HRP catalysis. After the reaction was terminated, absorbance is measured at 450 nm. For target sequences of Staphylococcus aureus, the detection limit is 1.2 pM. For genomic DNA, the detection limit is 7.4 pg.µL-1. The potential application of the method was verified by analyzing spiked food samples. Graphical abstractSchematic representation of rolling circle amplification and functionalized AuNP-based colorimetric determination of Staphylococcus aureus. The method uses streptavidin-functionalized 96-well plates and RCA as a molecular tool and AuNP-anti-digoxigenin-POx-HRP as signal transduction markers to increase sensitivity.


Assuntos
Colorimetria/métodos , DNA Bacteriano/análise , Staphylococcus aureus/isolamento & purificação , Animais , Armoracia/enzimologia , Benzidinas/química , Galinhas , Corantes/química , Sondas de DNA/química , Sondas de DNA/genética , DNA Bacteriano/genética , Contaminação de Alimentos/análise , Microbiologia de Alimentos , Limite de Detecção , Leite/microbiologia , Técnicas de Amplificação de Ácido Nucleico/métodos , Hibridização de Ácido Nucleico , Aves Domésticas/microbiologia , Staphylococcus aureus/química
20.
Biosens Bioelectron ; 150: 111926, 2020 Feb 15.
Artigo em Inglês | MEDLINE | ID: mdl-31929081

RESUMO

DNA-templated silver nanoclusters (DNA-AgNCs) have demonstrated pervasive applications in analytical chemistry recently. As a way of signal output in DNA-based detection methods, DNA-AgNCs have prominent advantages: first, the recognition and synthesizing sequences are naturally integrated in one DNA probe without any chemical modification or connection; second, the emissive wavelength of DNA-AgNCs can be adjusted in a wide range by employing different sequences; third, DNA-AgNCs can be utilized for producing not only fluorescence, also electrochemiluminescence and electrochemical signals. Besides, they also show potential applications for cell imaging, and are considered to be one of the most ideal nanomaterials for in-vivo imaging due to their ultra-small particle size. In this review, a brief and comprehensive introduction of DNA-AgNCs is firstly given, then label-free probes using DNA-AgNCs are classified and summarized, lastly concluding perspectives are provided on the defects and application potentials.


Assuntos
Técnicas Biossensoriais/métodos , Ácidos Nucleicos Imobilizados/química , Nanopartículas Metálicas/química , Prata/química , Animais , Técnicas Biossensoriais/instrumentação , Sondas de DNA/química , Técnicas Eletroquímicas/instrumentação , Técnicas Eletroquímicas/métodos , Humanos , Medições Luminescentes/instrumentação , Medições Luminescentes/métodos , Imagem Óptica/instrumentação , Imagem Óptica/métodos
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