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1.
Forensic Sci Int Genet ; 57: 102657, 2022 03.
Artigo em Inglês | MEDLINE | ID: mdl-34973558

RESUMO

In recent years, extraordinary progress has been made in genome sequencing technologies, which has led to a decrease in cost and an increase in the diversity of sequenced genomes. Nanopore sequencing is one of the latest genome sequencing technologies. It aims to sequence longer contiguous pieces of DNA, which are essential for resolving structurally complex regions, and provides a new approach for forensic genetics to detect longer markers in real time. To date, multiple studies have been conducted to sequence forensic markers using MinION from Oxford Nanopore Technologies (ONT), and the results indicate that nanopore sequencing holds promise for forensic applications. Qitan Technology (QitanTech) recently launched its first commercial nanopore genome sequencer, QNome. It could achieve a read length of more than 150 kbp, and could generate approximately 500 Mb of data in 8 h. In this pilot study, we explored and validated this alternative nanopore sequencing device for microhaplotype (MH) profiling using a custom set of 15 MH loci. Seventy single-contributor samples were divided into 7 batches, each of which included 10 samples and control DNA 9947A and was sequenced by QNome. MH genotypes generated from QNome were compared to those from Ion Torrent sequencing (Ion S5XL system) to evaluate the accuracy and stability. Twelve samples randomly selected from the last three batches and Control DNA 9947A were also subjected to ONT MinION sequencing (with R9.4 flow cell) for parallel comparison. Based on MHtyper, a bioinformatics workflow developed for automated MH designation, all MH loci can be genotyped and reliably phased using the QNome data, with an overall accuracy of 99.83% (4 errors among 2310 genotypes). Three occurred near or in the region of homopolymer sequences, and one existed within 50 bp of the start of the sequencing reaction. In the last 15 samples (12 individual samples and 3 replicates of control DNA 9947A), two SNPs located at 4-mer homopolymers failed to obtain reliable genotypes on the MinION data. This study shows the potential of state-of-the-art nanopore sequencing methods to analyze forensic MH markers. Given the rapid pace of change, sporadic and nonrepetitive errors presented in this study are expected to be resolved by further developments of nanopore technologies and analysis tools.


Assuntos
Sequenciamento por Nanoporos , Nanoporos , Sequenciamento de Nucleotídeos em Larga Escala , Humanos , Projetos Piloto , Análise de Sequência de DNA/métodos
2.
Nano Lett ; 22(15): 6350-6358, 2022 Aug 10.
Artigo em Inglês | MEDLINE | ID: mdl-35912616

RESUMO

First-aid hemostatic agents for acute bleeding can save lives in emergency situations. However, rapid hemostasis remains challenging when uncontrolled hemorrhage occurs on lethal noncompressible and irregular wounds. Herein, cellulose-based cryogel microspheres with deliberately customized micromorphologies for ultrafast water transportation and diffusion, including the shark skin riblet-inspired wrinkled surface with low fluid drag and the hydrophilic nanoporous 3D networks, are developed to deal with the acute noncompressible bleeding within seconds. These cryogel microspheres can rapidly absorb a large amount of blood over 6 times their own weight in 10 s and form a robust barrier to seal a bleeding wound without applying pressure. Remarkably, massive bleeding from a cardiac penetrating hole is effectively stopped using the microspheres within 20 s and no blood leakage is observed after 30 min. Additionally, these microspheres could be readily removed without rebleeding and capillary thrombus, which is highly favorable to rapid hemostasis in emergency rescue.


Assuntos
Criogéis , Nanoporos , Celulose , Hemorragia/terapia , Hemostasia , Humanos , Microesferas
3.
Toxins (Basel) ; 14(7)2022 Jul 09.
Artigo em Inglês | MEDLINE | ID: mdl-35878208

RESUMO

Ricin is a toxin which enters cells and depurinates an adenine base in the sarcin-ricin loop in the large ribosomal subunit, leading to the inhibition of protein translation and cell death. We postulated that this depurination event could be detected using Oxford Nanopore Technologies (ONT) direct RNA sequencing, detecting a change in charge in the ricin loop. In this study, A549 cells were exposed to ricin for 2-24 h in order to induce depurination. In addition, a novel software tool was developed termed RIPpore that could quantify the adenine modification of ribosomal RNA induced by ricin upon respiratory epithelial cells. We provided demonstrable evidence for the first time that this base change detected is specific to RIP activity using a neutralising antibody against ricin. We believe this represents the first detection of depurination in RNA achieved using ONT sequencers. Collectively, this work highlights the potential for ONT and direct RNA sequencing to detect and quantify depurination events caused by ribosome-inactivating proteins such as ricin. RIPpore could have utility in the evaluation of new treatments and/or in the diagnosis of exposure to ricin.


Assuntos
Nanoporos , Ricina , Adenina/metabolismo , RNA/metabolismo , Ribossomos/metabolismo , Ricina/metabolismo , Ricina/toxicidade , Análise de Sequência de RNA
4.
ACS Appl Mater Interfaces ; 14(28): 32618-32624, 2022 Jul 20.
Artigo em Inglês | MEDLINE | ID: mdl-35798544

RESUMO

Nanopores in two-dimensional (2D) materials have emerged to offer in principle necessary spatial resolution for high-throughput DNA sequencing. However, their fidelity is severely limited by the fast DNA translocation. A recent experiment indicates that introducing ionic liquids could slow down DNA translocation in a MoS2 nanopore. However, the corresponding in-depth molecular mechanism underlying the experimental findings is not fully understood, which is crucial for the future improvement of rational DNA translocation control. Here, we computationally investigate and then experimentally identify the effect of BmimCl ionic liquid on the retardation of ssDNA translocation through a single-layer MoS2 nanopore. Our all-atom molecular dynamics simulations demonstrate that the strong interaction between Bmim+ and ssDNA offers a considerable dragging force to decelerate the electrophoretic motion of ssDNA in the BmimCl solution. Moreover, we show that Bmim+ ions exhibit preferential binding on the sulfur edges of the nanopore. These Bmim+ in the pore region can not only act as a steric blockage but also form π-π stackings with nucleobases, which provide a further restriction on the ssDNA motion. Therefore, our molecular dynamics simulation investigations deepen the understanding of the critical role of ionic liquid in DNA translocation through a nanopore from a molecular landscape, which may benefit practical implementations of ionic liquids in nanopore sequencing.


Assuntos
Líquidos Iônicos , Nanoporos , DNA/química , DNA de Cadeia Simples , Dissulfetos , Simulação de Dinâmica Molecular , Molibdênio/química , Análise de Sequência de DNA/métodos
5.
Genome Biol ; 23(1): 153, 2022 07 08.
Artigo em Inglês | MEDLINE | ID: mdl-35804393

RESUMO

Nanopore sequencing enables the efficient and unbiased measurement of transcriptomes. Current methods for transcript identification and quantification rely on mapping reads to a reference genome, which precludes the study of species with a partial or missing reference or the identification of disease-specific transcripts not readily identifiable from a reference. We present RATTLE, a tool to perform reference-free reconstruction and quantification of transcripts using only Nanopore reads. Using simulated data and experimental data from isoform spike-ins, human tissues, and cell lines, we show that RATTLE accurately determines transcript sequences and their abundances, and shows good scalability with the number of transcripts.


Assuntos
Sequenciamento por Nanoporos , Nanoporos , Sequenciamento de Nucleotídeos em Larga Escala/métodos , Humanos , Isoformas de Proteínas/genética , Transcriptoma
6.
Nat Methods ; 19(7): 823-826, 2022 07.
Artigo em Inglês | MEDLINE | ID: mdl-35789207

RESUMO

Long-read Oxford Nanopore sequencing has democratized microbial genome sequencing and enables the recovery of highly contiguous microbial genomes from isolates or metagenomes. However, to obtain near-finished genomes it has been necessary to include short-read polishing to correct insertions and deletions derived from homopolymer regions. Here, we show that Oxford Nanopore R10.4 can be used to generate near-finished microbial genomes from isolates or metagenomes without short-read or reference polishing.


Assuntos
Metagenoma , Nanoporos , Genoma Bacteriano , Sequenciamento de Nucleotídeos em Larga Escala , Análise de Sequência de DNA
7.
J Am Chem Soc ; 144(30): 13717-13728, 2022 Aug 03.
Artigo em Inglês | MEDLINE | ID: mdl-35867993

RESUMO

Alditols, which have a sweet taste but produce much lower calories than natural sugars, are widely used as artificial sweeteners. Alditols are the reduced forms of monosaccharide aldoses, and different alditols are diastereomers or epimers of each other and direct and rapid identification by conventional methods is difficult. Nanopores, which are emerging single-molecule sensors with exceptional resolution when engineered appropriately, are useful for the recognition of diastereomers and epimers. In this work, direct distinguishing of alditols corresponding to all 15 monosaccharide aldoses was achieved by a boronic acid-appended hetero-octameric Mycobacterium smegmatis porin A (MspA) nanopore (MspA-PBA). Thirteen alditols including glycerol, erythritol, threitol, adonitol, arabitol, xylitol, mannitol, sorbitol, allitol, dulcitol, iditol, talitol, and gulitol (l-sorbitol) could be fully distinguished, and their sensing features constitute a complete nanopore alditol database. To automate event classification, a custom machine-learning algorithm was developed and delivered a 99.9% validation accuracy. This strategy was also used to identify alditol components in commercially available "zero-sugar" drinks and healthcare products, suggesting their use in rapid and sensitive quality control for the food and medical industry.


Assuntos
Nanoporos , Atenção à Saúde , Monossacarídeos , Mycobacterium smegmatis , Porinas , Sorbitol , Álcoois Açúcares
8.
Viruses ; 14(7)2022 06 29.
Artigo em Inglês | MEDLINE | ID: mdl-35891411

RESUMO

The Eurasian otter (Lutra lutra) is a piscivorous apex predator in aquatic habitats, and a flagship species of conservation biology throughout Europe. Despite the wide distribution and ecological relevance of the species, there is a considerable lack of knowledge regarding its virological and veterinary health context, especially in Central Europe. Canine morbillivirus (Canine distemper virus (CDV)) is a highly contagious viral agent of the family Paramyxoviridae with high epizootic potential and veterinary health impact. CDV is present worldwide among a wide range of animals; wild carnivores are at particular risk. As part of a retrospective study, lung-tissue samples (n = 339) from Eurasian otters were collected between 2000 and 2021 throughout Hungary. The samples were screened for CDV using a real-time RT-PCR method. Two specimens proved positive for CDV RNA. In one sample, the complete viral genome was sequenced using a novel, pan-genotype CDV-specific amplicon-based sequencing method with Oxford Nanopore sequencing technology. Both viral sequences were grouped to a European lineage based on the hemagglutinin-gene phylogenetic classification. In this article, we present the feasibility of road-killed animal samples for understanding the long-term dynamics of CDV among wildlife and provide novel virological sequence data to better understand CDV circulation and evolution.


Assuntos
Vírus da Cinomose Canina , Cinomose , Nanoporos , Lontras , Animais , Vírus da Cinomose Canina/genética , Cães , Genômica , Lontras/genética , Filogenia , Estudos Retrospectivos , Tecnologia
9.
Anal Chem ; 94(28): 10027-10034, 2022 07 19.
Artigo em Inglês | MEDLINE | ID: mdl-35786863

RESUMO

Holliday junctions (HJs) are an important class of nucleic acid structure utilized in DNA break repair processes. As such, these structures have great importance as therapeutic targets and for understanding the onset and development of various diseases. Single-molecule fluorescence resonance energy transfer (smFRET) has been used to study HJ structure-fluctuation kinetics, but given the rapid time scales associated with these kinetics (approximately sub-milliseconds) and the limited bandwidth of smFRET, these studies typically require one to slow down the structure fluctuations using divalent ions (e.g., Mg2+). This modification limits the ability to understand and model the underlying kinetics associated with HJ fluctuations. We address this here by utilizing nanopore sensing in a gating configuration to monitor DNA structure fluctuations without divalent ions. A nanopore analysis shows that HJ fluctuations occur on the order of 0.1-10 ms and that the HJ remains locked in a single conformation with short-lived transitions to a second conformation. It is not clear what role the nanopore plays in affecting these kinetics, but the time scales observed indicate that HJs are capable of undergoing rapid transitions that are not detectable with lower bandwidth measurement techniques. In addition to monitoring rapid HJ fluctuations, we also report on the use of nanopore sensing to develop a highly selective sensor capable of clear and rapid detection of short oligo DNA strands that bind to various HJ targets.


Assuntos
DNA Cruciforme , Nanoporos , Sequência de Bases , DNA/metabolismo , Transferência Ressonante de Energia de Fluorescência
10.
Proc Natl Acad Sci U S A ; 119(30): e2202527119, 2022 Jul 26.
Artigo em Inglês | MEDLINE | ID: mdl-35858428

RESUMO

Despite an extensive theoretical and numerical background, the translocation ratchet mechanism, which is fundamental for the transmembrane transport of biomolecules, has never been experimentally reproduced at the nanoscale. Only the Sec61 and bacterial type IV pilus pores were experimentally shown to exhibit a translocation ratchet mechanism. Here we designed a synthetic translocation ratchet and quantified its efficiency as a nanopump. We measured the translocation frequency of DNA molecules through nanoporous membranes and showed that polycations at the trans side accelerated the translocation in a ratchet-like fashion. We investigated the ratchet efficiency according to geometrical and kinetic parameters and observed the ratchet to be only dependent on the size of the DNA molecule with a power law [Formula: see text]. A threshold length of 3 kbp was observed, below which the ratchet did not operate. We interpreted this threshold in a DNA looping model, which quantitatively explained our results.


Assuntos
DNA , Nanoporos , Transporte Biológico , DNA/metabolismo , Fímbrias Bacterianas/metabolismo , Cinética
11.
Biochem Biophys Res Commun ; 621: 67-73, 2022 09 17.
Artigo em Inglês | MEDLINE | ID: mdl-35810593

RESUMO

Nonsense-mediated mRNA decay (NMD) and its regulation play an important role in eliminating faulty transcripts and controlling gene expression. However, measuring NMD activity and characterizing its targets remain challenging. In this study, we set out to establish Nanopore direct RNA sequencing in combination with quantitative real-time PCR (qPCR) as a method for analyzing NMD activity and its targets in cultured cell lines and clinical tissue samples. Nanopore RNA sequencing could detect more isoforms than short-read sequencing, especially in identifying novel isoforms and predicting isoforms annotated with premature termination codon (PTC). Changes in transcriptional isoforms of five genes (PRS, RPL12, SRSF2, PPIA, and TMEM208) could faithfully reflect NMD activity in the three cell lines and prostate cancer (PCA) samples. NMD activity in PCA samples varied, but some patients showed an increased trend. Together, Nanopore sequencing was superior in identifying NMD targets and evaluating NMD activity compared with short-read sequencing, and the NMD markers we screened may be used for measuring NMD activity in clinical patients.


Assuntos
Sequenciamento por Nanoporos , Nanoporos , Humanos , Masculino , Proteínas de Membrana/metabolismo , Degradação do RNAm Mediada por Códon sem Sentido , Isoformas de Proteínas/metabolismo , RNA/metabolismo , Estabilidade de RNA/genética , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , Análise de Sequência de RNA
12.
Appl Environ Microbiol ; 88(15): e0078522, 2022 Aug 09.
Artigo em Inglês | MEDLINE | ID: mdl-35867567

RESUMO

Whole-genome sequencing (WGS) for public health surveillance and epidemiological investigation of foodborne pathogens predominantly relies on sequencing platforms that generate short reads. Continuous improvement of long-read nanopore sequencing, such as Oxford nanopore technologies (ONT), presents a potential for leveraging multiple advantages of the technology in public health and food industry settings, including rapid turnaround and onsite applicability in addition to superior read length. Using an established cohort of Salmonella Enteritidis isolates for subtyping evaluation, we assessed the technical readiness of nanopore long read sequencing for single nucleotide polymorphism (SNP) analysis and core-genome multilocus sequence typing (cgMLST) of a major foodborne pathogen. By multiplexing three isolates per flow cell, we generated sufficient sequencing depths in <7 h of sequencing for robust subtyping. SNP calls by ONT and Illumina reads were highly concordant despite homopolymer errors in ONT reads (R9.4.1 chemistry). In silico correction of such errors allowed accurate allelic calling for cgMLST and allelic difference measurements to facilitate heuristic detection of outbreak isolates. IMPORTANCE Evaluation, standardization, and implementation of the ONT approach to WGS-based, strain-level subtyping is challenging, in part due to its relatively high base-calling error rates and frequent iterations of sequencing chemistry and bioinformatic analytics. Our study established a baseline for the continuously evolving nanopore technology as a viable solution to high-quality subtyping of Salmonella, delivering comparable subtyping performance when used standalone or together with short-read platforms. This study paves the way for evaluating and optimizing the logistics of implementing the ONT approach for foodborne pathogen surveillance in specific settings.


Assuntos
Nanoporos , Salmonella enteritidis , Genoma Bacteriano , Sequenciamento de Nucleotídeos em Larga Escala , Humanos , Tipagem de Sequências Multilocus , Polimorfismo de Nucleotídeo Único , Salmonella enteritidis/genética , Sequenciamento Completo do Genoma
13.
J Phys Chem B ; 126(30): 5689-5694, 2022 Aug 04.
Artigo em Inglês | MEDLINE | ID: mdl-35867912

RESUMO

Ion current rectification is highly reported in aqueous electrochemical systems and sensors but lacks exploration in organic systems due to the additional complexity introduced by non-aqueous solvents. Herein, a detailed study on ion current rectification with highly polar and mildly polar aprotic organic solvents as a function of tetraethylammonium tetrafluoroborate supporting electrolyte concentration is presented. To explain our experimental results, we introduce a previously unreported phenomenon: the formation of a double-junction diode within the nanopore that arises due to a complex interplay between ion and solvent enrichment effects. Finite element simulations are used to explore this phenomenon and the subsequent effect on the rectifying behavior of conical quartz nanopores.


Assuntos
Nanoporos , Eletrólitos , Transporte de Íons , Solventes
14.
Nano Lett ; 22(14): 5982-5989, 2022 07 27.
Artigo em Inglês | MEDLINE | ID: mdl-35816451

RESUMO

One main challenge of realizing high-energy-density lithium-sulfur batteries is low active materials utilization, excessive use of inert components, high electrolyte intake, and mechanical instability of high-mass-loading sulfur cathodes. Herein, chunky sulfur/graphene particle electrodes were designed, where active sulfur was confined in vertically aligned nanochannels (width ∼12 nm) of chunky graphene-based particles (∼70 µm) with N, O-containing groups. The short charge transport distance and low tortuosity enabled high utilization of active materials for high-mass-loading chunky sulfur/graphene particle electrodes. The intermediate polysulfide trapping effect by capillary effect and heteroatoms-containing groups, and a mechanically robust graphene framework, helped to realize stable electrode cycling. The as-designed electrode showed high areal capacity (10.9 mAh cm-2) and high sulfur utilization (72.4%) under the rigorous conditions of low electrolyte/active material ratio (∼2.5 µL mg-1) and high sulfur loading (9.0 mg cm-2), realizing high energy densities (520 Wh kg-1, 1635 Wh L-1).


Assuntos
Grafite , Nanoporos , Eletrodos , Lítio , Enxofre
15.
Nucleic Acids Res ; 50(13): 7479-7492, 2022 07 22.
Artigo em Inglês | MEDLINE | ID: mdl-35819189

RESUMO

Genome assembly has been benefited from long-read sequencing technologies with higher accuracy and higher continuity. However, most human genome assembly require large amount of DNAs from homogeneous cell lines without keeping cell heterogeneities, since cell heterogeneity could profoundly affect haplotype assembly results. Herein, using single-cell genome long-read sequencing technology (SMOOTH-seq), we have sequenced K562 and HG002 cells on PacBio HiFi and Oxford Nanopore Technologies (ONT) platforms and conducted de novo genome assembly. For the first time, we have completed the human genome assembly with high continuity (with NG50 of ∼2 Mb using 95 individual K562 cells) at single-cell levels, and explored the impact of different assemblers and sequencing strategies on genome assembly. With sequencing data from 30 diploid individual HG002 cells of relatively high genome coverage (average coverage ∼41.7%) on ONT platform, the NG50 can reach over 1.3 Mb. Furthermore, with the assembled genome from K562 single-cell dataset, more complete and accurate set of insertion events and complex structural variations could be identified. This study opened a new chapter on the practice of single-cell genome de novo assembly.


Assuntos
Genoma Humano , Nanoporos , Mapeamento Cromossômico , Sequenciamento de Nucleotídeos em Larga Escala/métodos , Humanos , Análise de Sequência de DNA/métodos
16.
Phys Chem Chem Phys ; 24(30): 18340-18346, 2022 Aug 03.
Artigo em Inglês | MEDLINE | ID: mdl-35880670

RESUMO

Predicting the precipitation of solids is important in both natural systems and subsurface energy applications. The factors controlling reaction mechanisms, phase selection and conversion between phases are particularly important. In this contribution the precipitation and growth of an amorphous calcium carbonate species from flowing aqueous solution in a nanoporous controlled pore glass is followed in situ with differential X-ray pair distribution function analysis. It is discovered that the local atomic structure of this phase indicates monohydrocalcite-like pair-pair correlations, yet is functionally amorphous because it lacks long-range structure. The unexpected occurrence of synthetic proto-monohydrocalcite amorphous calcium carbonate, precipitated from a solution undersaturated with respect to published solubilities, suggests that nanopore confinement facilitates formation of an amorphous phase at the expense of more favorable crystalline ones. This result illustrates that confinement and interface effects are physical factors exerting control on mineral nucleation behavior in natural and geological systems.


Assuntos
Carbonato de Cálcio , Nanoporos , Carbonato de Cálcio/química , Minerais/química
17.
PLoS One ; 17(7): e0270897, 2022.
Artigo em Inglês | MEDLINE | ID: mdl-35877652

RESUMO

The unintentional movement of agronomic pests and pathogens is steadily increasing due to the intensification of global trade. Being able to identify accurately and rapidly early stages of an invasion is critical for developing successful eradication or management strategies. For most invasive organisms, molecular diagnostics is today the method of choice for species identification. However, the currently implemented tools are often developed for certain taxa and need to be adapted for new species, making them ill-suited to cope with the current constant increase in new invasive species. To alleviate this impediment, we developed a fast and accurate sequencing tool allowing to modularly obtain genetic information at different taxonomical levels. Using whole genome amplification (WGA) followed by Oxford nanopore MinION sequencing, our workflow does not require any a priori knowledge on the investigated species and its classification. While mainly focusing on harmful plant pathogenic insects, we also demonstrate the suitability of our workflow for the molecular identification of bacteria (Erwinia amylovora and Escherichia coli), fungi (Cladosporium herbarum, Colletotrichum salicis, Neofabraea alba) and nematodes (Globodera rostochiensis). On average, the pairwise identity between the generated consensus sequences and best GenBank BLAST matches was 99.6 ± 0.6%. Additionally, assessing the generated insect genomic dataset, the potential power of the workflow to detect pesticide resistance genes, as well as arthropod-infecting viruses and endosymbiotic bacteria is demonstrated.


Assuntos
Ascomicetos , Sequenciamento por Nanoporos , Nanoporos , Ascomicetos/genética , Bactérias/genética , Biosseguridade , Sequenciamento de Nucleotídeos em Larga Escala , Análise de Sequência de DNA , Sequenciamento Completo do Genoma
18.
Sci Rep ; 12(1): 11305, 2022 Jul 04.
Artigo em Inglês | MEDLINE | ID: mdl-35787637

RESUMO

We report Brownian dynamics simulation results with the specific goal to identify key parameters controlling the experimentally measurable characteristics of protein tags on a dsDNA construct translocating through a double nanopore setup. First, we validate the simulation scheme in silico by reproducing and explaining the physical origin of the asymmetric experimental dwell time distributions of the oligonucleotide flap markers on a 48 kbp long dsDNA at the left and the right pore. We study the effect of the electric field inside and beyond the pores, critical to discriminate the protein tags based on their effective charges and masses revealed through a generic power-law dependence of the average dwell time at each pore. The simulation protocols monitor piecewise dynamics at a sub-nanometer length scale and explain the disparate velocity using the concepts of nonequilibrium tension propagation theory. We further justify the model and the chosen simulation parameters by calculating the Péclet number which is in close agreement with the experiment. We demonstrate that our carefully chosen simulation strategies can serve as a powerful tool to discriminate different types of neutral and charged tags of different origins on a dsDNA construct in terms of their physical characteristics and can provide insights to increase both the efficiency and accuracy of an experimental dual-nanopore setup.


Assuntos
Nanoporos , DNA , Eletricidade , Simulação de Dinâmica Molecular , Peso Molecular
19.
J Chem Phys ; 156(24): 244902, 2022 Jun 28.
Artigo em Inglês | MEDLINE | ID: mdl-35778106

RESUMO

DNA capture with high fidelity is an essential part of nanopore translocation. We report several important aspects of the capture process and subsequent translocation of a model DNA polymer through a solid-state nanopore in the presence of an extended electric field using the Brownian dynamics simulation that enables us to record statistics of the conformations at every stage of the translocation process. By releasing the equilibrated DNAs from different equipotentials, we observe that the capture time distribution depends on the initial starting point and follows a Poisson process. The field gradient elongates the DNA on its way toward the nanopore and favors a successful translocation even after multiple failed threading attempts. Even in the limit of an extremely narrow pore, a fully flexible chain has a finite probability of hairpin-loop capture, while this probability decreases for a stiffer chain and promotes single file translocation. Our in silico studies identify and differentiate characteristic distributions of the mean first passage time due to single file translocation from those due to translocation of different types of folds and provide direct evidence of the interpretation of the experimentally observed folds [M. Gershow and J. A. Golovchenko, Nat. Nanotechnol. 2, 775 (2007) and Mihovilovic et al., Phys. Rev. Lett. 110, 028102 (2013)] in a solitary nanopore. Finally, we show a new finding-that a charged tag attached at the 5' end of the DNA enhances both the multi-scan rate and the uni-directional translocation (5' → 3') probability that would benefit the genomic barcoding and sequencing experiments.


Assuntos
Nanoporos , DNA , Eletricidade , Simulação de Dinâmica Molecular , Polímeros
20.
Molecules ; 27(13)2022 Jun 30.
Artigo em Inglês | MEDLINE | ID: mdl-35807482

RESUMO

Matrix-assisted laser desorption/ionization mass spectrometry (MALDI-MS) on cluster-assembled super-hydrophilic nanoporous titania films deposited on hydrophobic conductive-polymer substrates feature a unique combination of surface properties that significantly improve the possibilities of capturing and processing biological samples before and during the MALDI-MS analysis without changing the selected sample target (multi-dimensional MALDI-MS). In contrast to pure hydrophobic surfaces, such films promote a remarkable biologically active film porosity at the nanoscale due to the soft assembling of ultrafine atomic clusters. This unique combination of nanoscale porosity and super-hydrophilicity provides room for effective sample capturing, while the hydrophilic-hydrophobic discontinuity at the border of the dot-patterned film acts as a wettability-driven containment for sample/reagent droplets. In the present work, we evaluate the performance of such advanced surface engineered reactive containments for their benefit in protein sample processing and characterization. We shortly discuss the advantages resulting from the introduction of the described chips in the MALDI-MS workflow in the healthcare/clinical context and in MALDI-MS bioimaging (MALDI-MSI).


Assuntos
Nanoporos , Interações Hidrofóbicas e Hidrofílicas , Polímeros , Espectrometria de Massas por Ionização e Dessorção a Laser Assistida por Matriz/métodos , Propriedades de Superfície
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