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1.
Phys Chem Chem Phys ; 21(45): 24884-24890, 2019 Dec 07.
Artigo em Inglês | MEDLINE | ID: mdl-31584588

RESUMO

The controlled synthesis of hybrid two-dimensional (2D) materials and the development of atomically precise nanopore fabrication techniques have opened up entirely new possibilities for sensing applications via nanoelectronics. Here, we investigate the electronic transport properties of an in-plane hybrid graphene/h-BN device, containing a graphene nanopore, to assess its feasibility to act as a molecular sensor. The results from our calculations based on density functional theory and the non-equilibrium Green's function formalism reveal the capability to confine the electric current pathways to the two carbon wires lining either edge of the nanopore, thereby creating conditions in which the conductance is highly sensitive to any changes in the electrical potential inside the nanopore. We apply this setup to assess whether it is possible to electrically determine the base sequence in a DNA molecule. Indeed, the modulation of the device conductance reveals a characteristic fingerprint of each nucleotide, which manifests itself in a pronounced difference in the sensitivity of the four different nucleotides, thereby allowing electrical discrimination. These findings lead us to propose this device architecture as a promising nanobiosensor. While fabrication in the lab may represent a profound experimental challenge, it should nevertheless in principle be feasible with existing contemporary techniques of hybrid 2D material synthesis, in conjunction with approaches for highly controlled nanopore creation.


Assuntos
DNA/análise , Nanoporos , Nanotecnologia , Compostos de Boro/química , Teoria da Densidade Funcional , Eletricidade , Transporte de Elétrons , Grafite/química
2.
Exp Parasitol ; 207: 107770, 2019 Dec.
Artigo em Inglês | MEDLINE | ID: mdl-31586454

RESUMO

Neutrophils respond differently to violations of the body's physiological barriers during infections. Extracellular traps comprise one of the mechanisms used by these cells to reduce the spread of pathogens to neighboring tissues, as well as ensure a high concentration of antimicrobial agents at the site of infection. To date, this innate defense mechanism has not been previously demonstrated in neutrophils of cats exposed to Toxoplasma gondii. The aim of this study was to characterize the in vitro release of neutrophil extracellular traps (NETs) when neutrophils isolated from cats were exposed to T. gondii. First, cellular viability was tested at different time points after parasite exposure. The production of reactive oxygen species (ROS) and lactate dehydrogenase and the amount of extracellular DNA were quantified. In addition, the number of parasites associated with neutrophils was determined, and the observed NETs formed were microscopically characterized. Results showed that (i) in culture, neutrophils isolated from cats presented diminished cellular viability after 4 h of incubation, and when neutrophils were incubated with T. gondii, they displayed cytotoxic effects after 3 h of interaction; (ii) neutrophils were able to release structures composed of DNA and histones, characterized as NETs under optical, immunofluorescence, and electron scanning microscopy, when stimulated with T. gondii; (iii) only 11.4% of neutrophils were able to discharge NETs during 3 h of incubation; however, it was observed through extracellular quantification of DNA that this small number of cells were able to display different behavior compared to a negative control (no parasite) group; (iv) significant differences in ROS production were observed in neutrophils exposed to T. gondii. In conclusion, our results showed that neutrophils isolated from cats exposed to T. gondii release structures composed of DNA and histones, similar to what has already been described in other neutrophil species infected with the parasite.


Assuntos
Armadilhas Extracelulares/metabolismo , Neutrófilos/parasitologia , Toxoplasma/imunologia , Animais , Gatos , Sobrevivência Celular , Cercopithecus aethiops , DNA/análise , Formazans/metabolismo , L-Lactato Desidrogenase/metabolismo , Microscopia Eletrônica de Varredura , Microscopia de Fluorescência , Neutrófilos/imunologia , Neutrófilos/ultraestrutura , Espécies Reativas de Oxigênio/metabolismo , Superóxidos/análise , Sais de Tetrazólio/metabolismo , Células Vero
3.
Chem Commun (Camb) ; 55(88): 13219-13222, 2019 Oct 31.
Artigo em Inglês | MEDLINE | ID: mdl-31589231

RESUMO

We demonstrate that short single-stranded DNA generated by Pyrococcus furiosus Argonaute (PfAgo) can initiate a second round of cleavage. Based on this principle, we established a molecular diagnostic method, termed PfAgo-mediated Nucleic acid Detection (PAND). This method could detect DNA at attomolar sensitivities, distinguish single-nucleotide mutants and accomplish multiplexed detection.


Assuntos
DNA/análise , Pyrococcus furiosus/química , Humanos
4.
Gene ; 720: 144078, 2019 Dec 15.
Artigo em Inglês | MEDLINE | ID: mdl-31473321

RESUMO

Short tandem repeats (STRs) are a widely utilized tool in forensic applications, the latter of which range from human identification and paternity testing to population analysis. The GlobalFiler STR loci, which includes 21 autosomal STRS, were analyzed in the Chechen subpopulation of Jordan. Whole blood samples were withdrawn from 159 Jordanian Chechen individuals, and genomic DNA was extracted from each sample. The GlobalFiler™ kit PCR Amplification Kit amplified and analyzed the STR loci on the 3130xl Genetic Analyzer using GeneMapper ID-X software. The combined match probability for the 21 autosomal STR loci was calculated to be 1.06 × 10-24, a number that is highly discriminatory and informative. The SE33 (0.983) and TPOX (0.806) loci exhibited the highest and lowest powers of discrimination, respectively. Conclusively, the current study indicates that the GlobalFiler loci have a high utility in the Jordanian Chechen population, possibly paving the way for the future establishment of a reference population database in Jordan.


Assuntos
DNA/análise , DNA/genética , Grupos Étnicos/genética , Genética Forense/estatística & dados numéricos , Genética Populacional , Repetições de Microssatélites , Polimorfismo Genético , Impressões Digitais de DNA , Feminino , Frequência do Gene , Loci Gênicos , Humanos , Masculino
5.
Anal Bioanal Chem ; 411(23): 6119-6128, 2019 Sep.
Artigo em Inglês | MEDLINE | ID: mdl-31388714

RESUMO

A new approach employing a microchip in combination with photothermal lens microscopy has been described for a DNA hybridization assay using gold nanoparticles. The difference in adsorption propensities of single- and double-stranded DNAs on gold nanoparticles was used for a highly sensitive DNA hybridization assay through a photothermal lens effect in a femtoliter scale of detection volume. Under the optimized conditions, the results showed that the variation of photothermal lens signal in the focal volume of 105 fL (10-15 L) was linearly proportional to the target DNA concentration over the range of 50-500 nM with detection limits of 30.7 nM and 27.3 nM for target DNA I and II, respectively. The lowest amount of target DNA that was measured using gold nanoparticles was 2.6 zepto mole. The assay was completed within 5 min and the relative standard deviations (n = 8) for both target DNAs were about 2.34%. The hybridization process was proved by two different common methods including gel electrophoresis and in situ fluorescence monitoring of DNA hybridization. The performance of this detection method was investigated in diluted human serum sample as a complex sample. The recovery values were between 98 and 104.9%. Graphical abstract.


Assuntos
DNA/análise , Ouro/química , Dispositivos Lab-On-A-Chip , Nanopartículas Metálicas/química , Hibridização de Ácido Nucleico , Técnicas Biossensoriais/instrumentação , DNA/genética , Desenho de Equipamento , Limite de Detecção , Nanopartículas Metálicas/ultraestrutura , Microscopia/instrumentação
6.
Nat Biotechnol ; 37(9): 1080-1090, 2019 09.
Artigo em Inglês | MEDLINE | ID: mdl-31427819

RESUMO

Spatial mapping of proteins in tissues is hindered by limitations in multiplexing, sensitivity and throughput. Here we report immunostaining with signal amplification by exchange reaction (Immuno-SABER), which achieves highly multiplexed signal amplification via DNA-barcoded antibodies and orthogonal DNA concatemers generated by primer exchange reaction (PER). SABER offers independently programmable signal amplification without in situ enzymatic reactions, and intrinsic scalability to rapidly amplify and visualize a large number of targets when combined with fast exchange cycles of fluorescent imager strands. We demonstrate 5- to 180-fold signal amplification in diverse samples (cultured cells, cryosections, formalin-fixed paraffin-embedded sections and whole-mount tissues), as well as simultaneous signal amplification for ten different proteins using standard equipment and workflows. We also combined SABER with expansion microscopy to enable rapid, multiplexed super-resolution tissue imaging. Immuno-SABER presents an effective and accessible platform for multiplexed and amplified imaging of proteins with high sensitivity and throughput.


Assuntos
Anticorpos/imunologia , Anticorpos/metabolismo , Imuno-Histoquímica/métodos , Proteínas/metabolismo , Coloração e Rotulagem , Animais , Linhagem Celular , DNA/análise , Código de Barras de DNA Taxonômico , Corantes Fluorescentes , Humanos , Hibridização in Situ Fluorescente/métodos , Camundongos , Microscopia de Fluorescência/métodos , Retina/citologia
7.
Anal Bioanal Chem ; 411(26): 6867-6875, 2019 Oct.
Artigo em Inglês | MEDLINE | ID: mdl-31401669

RESUMO

A novel label-free photoelectrochemical biosensing method for highly sensitive and specific detection of DNA hybridization using a CdS quantum dot (QD)-dendrimer nanocomposite is presented. A molecular beacon (MB) was assembled on a gold-nanoparticle-modified indium tin oxide electrode surface. Hybridization to a complementary target DNA disrupts the stem-loop structure of the MB, which was afterward labeled with the QD-dendrimer nanocomposite. The modified indium tin oxide electrode showed a stable anodic photocurrent response at 300 mV (vs Ag/AgCl) to light excitation at 410 nm in the presence of 0.1 M ascorbic acid as an electron donor. The protocol developed integrates the specificity of an MB for molecular recognition and the advantages of gold nanoparticles for increasing the loading capacity of the MB on the electrode surface and accelerating the electron transfer. Moreover, the photocurrent was greatly enhanced because of the high loading of QDs by the dendrimer, which eliminated the surface defects of CdS QDs and prevented recombination of their photogenerated electron-hole pairs. Under the optimal conditions, a linear relationship between the increase of photocurrent and target DNA concentration was obtained in the range from 1 fM to 0.1 nM, with a detection limit of 0.5 fM. The sequence-specificity experiment showed that one or three mismatches of DNA bases could be discriminated. This photoelectrochemical method is a prospective technique for DNA hybridization detection because of its great advantages: label-free, high sensitivity and specificity, low cost, and easy fabrication. This could create a new platform for the application of CdS QD-dendrimer nanocomposites in photoelectrochemical bioanalysis. Graphical abstract.


Assuntos
Técnicas Biossensoriais/métodos , DNA/análise , Dendrímeros/química , Nanocompostos/química , Pontos Quânticos/química , Compostos de Cádmio/química , Técnicas Eletroquímicas/métodos , Ouro/química , Limite de Detecção , Nanopartículas Metálicas/química , Hibridização de Ácido Nucleico , Processos Fotoquímicos , Sulfetos/química
9.
Nat Methods ; 16(8): 675, 2019 08.
Artigo em Inglês | MEDLINE | ID: mdl-31363210
10.
Anal Chim Acta ; 1077: 30-66, 2019 Oct 24.
Artigo em Inglês | MEDLINE | ID: mdl-31307723

RESUMO

This review summarizes progress in electroanalysis of organic compounds and biomacromolecules by means of bare BDD-based electrodes for the period of 2009-2018. New trends, which have emerged in the reported decade and which have improved their performance in batch voltammetric and amperometric methods and electrochemical detection in liquid flow techniques are commented. Importance of BDD surface termination, effect of boron doping level, and utilization of adsorption of analytes on BDD surfaces enabling development of adsorptive voltammetric techniques are addressed. Further, possibilities of simultaneous determination of analytes by means of voltammetric techniques utilizing computational approaches and multiple-pulse amperometric detection are discussed. Strategies leading to enhancement of sensitivity such as nanostructuring of the BDD surface, fabrication of BDD-based composite materials or new approaches in construction of microelectrodes and microelectrode arrays for biosensing represent another area of interest. Attention is paid to possibilities in detection of amino acids, peptides and proteins, nucleobases, nucleos(t)ides and DNA/RNA.


Assuntos
Boro/química , Diamante/química , Microeletrodos , Compostos Orgânicos/análise , DNA/análise , Técnicas Eletroquímicas/instrumentação , Técnicas Eletroquímicas/métodos , Peptídeos/análise , Proteínas/análise , RNA/análise
11.
Cell Mol Life Sci ; 76(23): 4689-4704, 2019 Dec.
Artigo em Inglês | MEDLINE | ID: mdl-31342119

RESUMO

The review includes information on the current state of knowledge of immunometric methods with emphasis on the possibility of deoxyribonucleic acid (DNA) damage detection. Beginning with basic immunoassay enzyme-linked immunosorbent assay (ELISA), this review describes methods such as tyramide signal amplification (TSA), enhanced polymer one-step staining (EPOS), and time resolved amplified cryptate emission (TRACE) as improvements of ELISA's developed over time to obtain more accurate results. In the second part of the review, surface plasmon resonance (SPR) and quantum dots (QDs) are presented as the newest outlooks in the context of immunoanalysis of biological material and molecular studies. The aim of this review is to briefly present immunoassays with emphasis on DNA damage detection; therefore, the types of methods are listed and described, types of signal indicators, basic definitions such as antigen and antibody are given. Every method is considered with an exemplary application focusing on DNA studies, DNA damage and instability detection.


Assuntos
Dano ao DNA , DNA/análise , Imunoensaio/métodos , DNA/química , DNA/metabolismo , Ensaio de Imunoadsorção Enzimática/métodos , Compostos Organometálicos/química , Pontos Quânticos/química , Ressonância de Plasmônio de Superfície , Tiramina/química
12.
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
13.
Nature ; 572(7767): 136-140, 2019 08.
Artigo em Inglês | MEDLINE | ID: mdl-31316204

RESUMO

Many genome-processing reactions, including transcription, replication and repair, generate DNA rotation. Methods that directly measure DNA rotation, such as rotor bead tracking1-3, angular optical trapping4 and magnetic tweezers5, have helped to unravel the action mechanisms of a range of genome-processing enzymes that includes RNA polymerase (RNAP)6, gyrase2, a viral DNA packaging motor7 and DNA recombination enzymes8. Despite the potential of rotation measurements to transform our understanding of genome-processing reactions, measuring DNA rotation remains a difficult task. The time resolution of existing methods is insufficient for tracking the rotation induced by many enzymes under physiological conditions, and the measurement throughput is typically low. Here we introduce origami-rotor-based imaging and tracking (ORBIT), a method that uses fluorescently labelled DNA origami rotors to track DNA rotation at the single-molecule level with a time resolution of milliseconds. We used ORBIT to track the DNA rotations that result from unwinding by the RecBCD complex, a helicase that is involved in DNA repair9, as well as from transcription by RNAP. We characterized a series of events that occur during RecBCD-induced DNA unwinding-including initiation, processive translocation, pausing and backtracking-and revealed an initiation mechanism that involves reversible ATP-independent DNA unwinding and engagement of the RecB motor. During transcription by RNAP, we directly observed rotational steps that correspond to the unwinding of single base pairs. We envisage that ORBIT will enable studies of a wide range of interactions between proteins and DNA.


Assuntos
DNA/análise , DNA/metabolismo , Exodesoxirribonuclease V/metabolismo , Genoma/genética , Conformação de Ácido Nucleico , Rotação , Pareamento de Bases , DNA/química , Quebras de DNA de Cadeia Dupla , DNA Helicases/metabolismo , RNA Polimerases Dirigidas por DNA/metabolismo , Transcrição Genética
14.
Anal Bioanal Chem ; 411(23): 6091-6100, 2019 Sep.
Artigo em Inglês | MEDLINE | ID: mdl-31289897

RESUMO

Two 600-bp DNA solutions (DNA600-G and DNA600-T) were developed as certified reference material, NMIJ CRM 6205-a, for the validation of DNA quantification methods. Both DNA600-G and DNA600-T are ideal as "spike-in control" because these materials have artificial nucleic acid sequences. The certified values were determined as the mass concentration of total DNA (whole DNA materials in sample solution regardless of sequence) at 25 °C by formic acid hydrolysis/liquid chromatography-isotope dilution mass spectrometry (LC-IDMS) and inductively coupled plasma-mass spectrometry (ICP-MS) based on the amount of phosphorus. DNAs were synthesized, and plasmids including the synthesized DNAs were cloned into Escherichia coli DH5α. The amplified plasmids were digested with a restriction enzyme and highly purified. Then, the purified DNAs were diluted with water to approximately 1 ng/µL. By using the CRM-validated methods in fields where DNA quantification is required, the reliability of DNA quantification could be improved. Graphical abstract.


Assuntos
DNA/análise , Espectrometria de Massas/métodos , Sequência de Bases , Cromatografia Líquida de Alta Pressão/métodos , Cromatografia Líquida de Alta Pressão/normas , Cromatografia Líquida/métodos , Cromatografia Líquida/normas , DNA/genética , Formiatos/química , Hidrólise , Espectrometria de Massas/normas , Plasmídeos/análise , Plasmídeos/genética , Reação em Cadeia da Polimerase/métodos , Reação em Cadeia da Polimerase/normas , Padrões de Referência
15.
Anal Bioanal Chem ; 411(23): 6021-6029, 2019 Sep.
Artigo em Inglês | MEDLINE | ID: mdl-31289898

RESUMO

Cytobiological methods for cell nucleus-related studies start with the extraction processes of intranuclear components with many cell lysis buffers, following with the structural characterizations and quantitative analysis of the extracted components. In this study, we tried to evaluate the availability and reliability of these extraction-based analytical methods from their spectral features. We implemented an in situ surface-enhanced Raman scattering spectroscopy (SERS) strategy with the help of the nucleus-targeting nanoprobes to investigate the molecular information of nucleus, in comparison with these ex situ methods. This study provides valuable references for choosing an appropriate detection method according to different detection purposes, and also points out the risks of many developing cell-related analytical methods that combine the traditional cytobiological techniques from exogenous interferences during sample preprocesses. Graphical abstract.


Assuntos
Núcleo Celular/química , DNA/análise , Nanopartículas Metálicas/química , Proteínas Nucleares/análise , Análise Espectral Raman/métodos , Fluoresceína-5-Isotiocianato/química , Corantes Fluorescentes/química , Ouro/química , Células Hep G2 , Humanos , Nanopartículas Metálicas/ultraestrutura , Peptídeos/química , Polietilenoglicóis/química
16.
Genome Biol ; 20(1): 132, 2019 07 01.
Artigo em Inglês | MEDLINE | ID: mdl-31262344

RESUMO

CRISPR-based nucleic acid detection methods are reported to facilitate rapid and sensitive DNA detection. However, precise DNA detection at the single-base resolution and its wide applications including high-fidelity SNP genotyping remain to be explored. Here we develop a Cas12b-mediated DNA detection (CDetection) strategy, which shows higher sensitivity on examined targets compared with the previously reported Cas12a-based detection platform. Moreover, we show that CDetection can distinguish differences at the single-base level upon combining the optimized tuned guide RNA (tgRNA). Therefore, our findings highlight the high sensitivity and accuracy of CDetection, which provides an efficient and highly practical platform for DNA detection.


Assuntos
Sistemas CRISPR-Cas , DNA/análise , Técnicas Genéticas , Testes Genéticos/métodos , Escherichia coli , Humanos , Sensibilidade e Especificidade
17.
Chem Commun (Camb) ; 55(62): 9084-9087, 2019 Jul 30.
Artigo em Inglês | MEDLINE | ID: mdl-31287464

RESUMO

An isothermal amplification circuit for specific DNA molecules was implemented in giant unilamellar vesicles. Using this circuit, over 5000-fold amplification of output DNAs was achieved, and the amplification behaviour depended on the concentration of input signal DNAs in a cell-sized compartment. Moreover, initiation of the amplification by photo-stimulation was demonstrated.


Assuntos
DNA/análise , Lipossomas Unilamelares/química , DNA/síntese química , Técnicas de Amplificação de Ácido Nucleico , Tamanho da Partícula , Propriedades de Superfície
18.
Analyst ; 144(14): 4175-4179, 2019 Jul 08.
Artigo em Inglês | MEDLINE | ID: mdl-31237576

RESUMO

In this work, we report a facile, sensitive, selective, and reproducible DNA impedimetric sensor device. We demonstrate that, combined with exonuclease III, the easily prepared electrochemically reduced graphene oxide (rGO) could be a desirable platform to amplify signals in electrochemical impedance spectroscopy for ultrasensitive DNA detection. Guided by enzyme assisted target recycling, efficient interfacial tuning can be obtained, from the situation with high impedance caused by single-stranded DNA probes directly adsorbed onto rGO to the one with low impedance due to the continuous desorption of target-probe DNA hybrids and the consequent digestion of DNA probes. Just a few DNA targets can specifically trigger the enzymatic digestion of a large number of DNA probes. It is the excellent electrical conductivity of rGO that further enlarges the changes of electron transfer resistance after the removal of DNA probes. As a result of synergistically combining both enzymatic and electrical amplification, the enlarged changes of impedimetric signals can be measured to sensitively report DNA targets. The specificity has been guaranteed by the intrinsic recognition of hybrids through both rGO and exonuclease III. A limit of detection as low as 10 aM target DNA in the matrix of cell culture medium, as well as a wide linear range and good discrimination of mismatched sequences even at the one-base level, suggests its great application prospect in biosensing and biomedical analysis. It also has other advantages including easy operation, low cost, and convenient regeneration, with more competitive performance in developing impedimetric biosensors.


Assuntos
DNA/análise , Técnicas Biossensoriais/métodos , DNA/química , DNA/genética , Sondas de DNA/genética , Espectroscopia Dielétrica/métodos , Exodesoxirribonucleases/química , Grafite/química , Limite de Detecção , Técnicas de Amplificação de Ácido Nucleico/métodos , Hibridização de Ácido Nucleico
19.
Talanta ; 202: 207-213, 2019 Sep 01.
Artigo em Inglês | MEDLINE | ID: mdl-31171171

RESUMO

In this work, a simple, sensitive and specific assay for DNA was proposed by combining inductively coupled plasma mass spectrometry (ICP-MS) detection with gold nanoparticle (AuNPs) amplification and isothermal circular strand-displacement polymerization reaction (ICSDPR). First, AuNPs were decorated with hairpin-structured DNA (HP-DNA) through Au-S bond to form the AuNPs probe. The ICSDPR was conducted on AuNPs probe in a homogeneous phase to realize the dual amplification and simplify the analytical process at the same time. By using a biotin modified primer, AuNPs were connected with biotins after the ICSDPR, then captured by the streptavidin modified magnetic beads (SA-MBs), and finally detected by ICP-MS. Many key factors including probe volume, hybridization time, polymerase amount, primer concentration, enzyme reaction time, SA-MBs capture time and desorption time were optimized. Under the optimized condition, the proposed method could detect target DNA as low as 45 zmol (8.9 fM in 5 µL) in a relative short time (about 4.5 h) with good specificity, and the linear range of this method is 0.1-10000 pM, the relative standard deviations are in the range of 3.6-6.4%. The proposed method was applied for determination of target DNA in human serum samples, the recovery for the spiked human serum samples is in the range of 84-120%. It demonstrates a good application potential of the developed method for biological studies and clinical diagnosis of human pathogenic diseases.


Assuntos
DNA/análise , Ouro/química , Nanopartículas Metálicas/química , Técnicas de Amplificação de Ácido Nucleico , Espectrometria de Massas , Polimerização
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