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1.
J Phys Chem Lett ; 15(14): 3900-3906, 2024 Apr 11.
Artigo em Inglês | MEDLINE | ID: mdl-38564363

RESUMO

Nanopores with two-dimensional materials have various advantages in sensing, but the fast translocation of molecules hinders their scale-up applications. In this work, we investigate the influence of -F, -O, and -OH surface terminations on the translocation of peptides through MXene nanopores. We find that the longest dwell time always occurs when peptides pass through the Ti3C2O2 nanopores. This elongated dwell time is induced by the strongest interaction between peptides and the Ti3C2O2 membrane, in which the van der Waals interactions dominate. Compared to the other two MXene nanopores, the braking effect is indicated during the whole translocation process, which evidence the advantage of Ti3C2O2 in nanopore sensing. Our work demonstrates that membrane surface chemistry has a great influence on the translocation of peptides, which can be introduced in the design of nanopores for a better performance.


Assuntos
Nanoporos , Nitritos , Elementos de Transição , Peptídeos
2.
Nat Commun ; 15(1): 2964, 2024 Apr 05.
Artigo em Inglês | MEDLINE | ID: mdl-38580638

RESUMO

The high sequencing error rate has impeded the application of long noisy reads for diploid genome assembly. Most existing assemblers failed to generate high-quality phased assemblies using long noisy reads. Here, we present PECAT, a Phased Error Correction and Assembly Tool, for reconstructing diploid genomes from long noisy reads. We design a haplotype-aware error correction method that can retain heterozygote alleles while correcting sequencing errors. We combine a corrected read SNP caller and a raw read SNP caller to further improve the identification of inconsistent overlaps in the string graph. We use a grouping method to assign reads to different haplotype groups. PECAT efficiently assembles diploid genomes using Nanopore R9, PacBio CLR or Nanopore R10 reads only. PECAT generates more contiguous haplotype-specific contigs compared to other assemblers. Especially, PECAT achieves nearly haplotype-resolved assembly on B. taurus (Bison×Simmental) using Nanopore R9 reads and phase block NG50 with 59.4/58.0 Mb for HG002 using Nanopore R10 reads.


Assuntos
Diploide , Nanoporos , Alelos , Haplótipos , Heterozigoto , Análise de Sequência de DNA/métodos , Sequenciamento de Nucleotídeos em Larga Escala/métodos
3.
Int J Mol Sci ; 25(7)2024 Mar 22.
Artigo em Inglês | MEDLINE | ID: mdl-38612388

RESUMO

Styryl dyes are generally poor fluorescent molecules inherited from their flexible molecular structures. However, their emissive properties can be boosted by restricting their molecular motions. A tight confinement into inorganic molecular sieves is a good strategy to yield highly fluorescent hybrid systems. In this work, we compare the confinement effect of two Mg-aluminophosphate zeotypes with distinct pore systems (the AEL framework, a one-dimensional channeled structure with elliptical pores of 6.5 Å × 4.0 Å, and the CHA framework, composed of large cavities of 6.7 Å × 10.0 Å connected by eight-ring narrower windows) for the encapsulation of 4-DASPI styryl dye (trans-4-[4-(Dimethylamino)styryl]-1-methylpyridinium iodide). The resultant hybrid systems display significantly improved photophysical features compared to 4-DASPI in solution as a result of tight confinement in both host inorganic frameworks. Molecular simulations reveal a tighter confinement of 4-DASPI in the elliptical channels of AEL, explaining its excellent photophysical properties. On the other hand, a singular arrangement of 4-DASPI dye is found when confined within the cavity-based CHA framework, where the 4-DASPI molecule spans along two adjacent cavities, with each aromatic ring sitting on these adjacent cavities and the polymethine chain residing within the narrower eight-ring window. However, despite the singularity of this host-guest arrangement, it provides less tight confinement for 4-DASPI than AEL, resulting in a slightly lower quantum yield.


Assuntos
Nanoporos , Corantes , Movimento (Física) , Extremidade Superior
4.
Proc Natl Acad Sci U S A ; 121(16): e2400203121, 2024 Apr 16.
Artigo em Inglês | MEDLINE | ID: mdl-38598338

RESUMO

Viral outbreaks can cause widespread disruption, creating the need for diagnostic tools that provide high performance and sample versatility at the point of use with moderate complexity. Current gold standards such as PCR and rapid antigen tests fall short in one or more of these aspects. Here, we report a label-free and amplification-free nanopore sensor platform that overcomes these challenges via direct detection and quantification of viral RNA in clinical samples from a variety of biological fluids. The assay uses an optofluidic chip that combines optical waveguides with a fluidic channel and integrates a solid-state nanopore for sensing of individual biomolecules upon translocation through the pore. High specificity and low limit of detection are ensured by capturing RNA targets on microbeads and collecting them by optical trapping at the nanopore location where targets are released and rapidly detected. We use this device for longitudinal studies of the viral load progression for Zika and Severe Acute Respiratory Syndrome Coronavirus-2 (SARS-CoV-2) infections in marmoset and baboon animal models, respectively. The up to million-fold trapping-based target concentration enhancement enables amplification-free RNA quantification across the clinically relevant concentration range down to the assay limit of RT-qPCR as well as cases in which PCR failed. The assay operates across all relevant biofluids, including semen, urine, and whole blood for Zika and nasopharyngeal and throat swab, rectal swab, and bronchoalveolar lavage for SARS-CoV-2. The versatility, performance, simplicity, and potential for full microfluidic integration of the amplification-free nanopore assay points toward a unique approach to molecular diagnostics for nucleic acids, proteins, and other targets.


Assuntos
Nanoporos , Infecção por Zika virus , Zika virus , Animais , RNA Viral/genética , RNA Viral/metabolismo , SARS-CoV-2/genética , SARS-CoV-2/metabolismo , Primatas/genética , Zika virus/genética , Sensibilidade e Especificidade , Técnicas de Amplificação de Ácido Nucleico
5.
Shanghai Kou Qiang Yi Xue ; 33(1): 6-12, 2024 Feb.
Artigo em Chinês | MEDLINE | ID: mdl-38583018

RESUMO

PURPOSE: Bioactive magnesium ions were successfully incorporated into the nanoporous titanium base coating by micro-arc oxidation(MAO), and its physical properties and osteogenic effects were explored. METHODS: Non-magnesium-containing and magnesium-containing titanium porous titanium coatings(MAO, MAO-mg) were prepared by changing the composition of MAO electrolyte and controlling the doping of magnesium in porous titanium coatings. The samples were characterized by scanning electron microscope (SEM), roughness, contact angle and energy dispersive X-ray spectrometer (EDS). Mg2+ release ability of magnesium-doped nanoporous titanium coatings was determined by inductively coupled plasma/optical emission spectrometer(ICP-OES). The structure of the cytoskeleton was determined by live/dead double staining, CCK-8 detection of material proliferation-toxicity, and staining of ß-actin using FITC-phalloidin. The effects of the coating on osteogenic differentiation in vitro were determined by alizarin red (ARS), alkaline phosphatase (ALP) staining and real-time polymerase chain reaction (qRT-PCR). SPSS 25.0 software package was used for statistical analysis. RESULTS: The MAO electrolyte with magnesium ions did not change the surface characteristics of the porous titanium coating. Each group prepared by MAO had similar microporous structure(P>0.05). There was no significant difference in surface roughness and contact angle between MAO treatment group (MAO, MAO-mg)(P>0.05), but significantly higher than that of Ti group (P<0.05). With the passage of cell culture time, MAO-mg group promoted cell proliferation (P<0.05). MAO-mg group was significantly higher than other groups in ALP and ARS staining. The expression of Runx2 mRNA (P<0.05), ALP(P<0.05) and osteocalcin OCN(P<0.05) in MAO-mg group was significantly higher than that in Ti and MAO groups. CONCLUSIONS: MAO successfully prepared magnesium-containing nanoporous titanium coating, and showed a significant role in promoting osteogenic differentiation.


Assuntos
Nanoporos , Titânio , Titânio/farmacologia , Magnésio/química , Magnésio/farmacologia , Osteogênese/genética , Eletrólitos/farmacologia , Íons/farmacologia , Propriedades de Superfície , Materiais Revestidos Biocompatíveis/farmacologia , Materiais Revestidos Biocompatíveis/química
6.
Gigascience ; 132024 Jan 02.
Artigo em Inglês | MEDLINE | ID: mdl-38608279

RESUMO

BACKGROUND: As adoption of nanopore sequencing technology continues to advance, the need to maintain large volumes of raw current signal data for reanalysis with updated algorithms is a growing challenge. Here we introduce slow5curl, a software package designed to streamline nanopore data sharing, accessibility, and reanalysis. RESULTS: Slow5curl allows a user to fetch a specified read or group of reads from a raw nanopore dataset stored on a remote server, such as a public data repository, without downloading the entire file. Slow5curl uses an index to quickly fetch specific reads from a large dataset in SLOW5/BLOW5 format and highly parallelized data access requests to maximize download speeds. Using all public nanopore data from the Human Pangenome Reference Consortium (>22 TB), we demonstrate how slow5curl can be used to quickly fetch and reanalyze raw signal reads corresponding to a set of target genes from each individual in large cohort dataset (n = 91), minimizing the time, egress costs, and local storage requirements for their reanalysis. CONCLUSIONS: We provide slow5curl as a free, open-source package that will reduce frictions in data sharing for the nanopore community: https://github.com/BonsonW/slow5curl.


Assuntos
Sequenciamento por Nanoporos , Nanoporos , Humanos , Algoritmos , Disseminação de Informação , Registros
7.
Int J Mol Sci ; 25(7)2024 Mar 26.
Artigo em Inglês | MEDLINE | ID: mdl-38612506

RESUMO

Positronium (Ps) is a valuable probe to investigate nanometric or sub-nanometric cavities in non-metallic materials, where Ps can be confined. Accessible experimental measurements concern the lifetime of trapped Ps, which is largely influenced by pick-off processes, depending on the size of the cavity as well as on the density of the electrons belonging to the surface of the host trap. Another relevant physical quantity is the contact density, that is the electron density at the positron position, which is usually found to be well below the vacuum value. Here, we review the principal models that have been formulated to account and explain for these physical properties of confined Ps. Starting with models, treating Ps as a single particle formulated essentially to study pick-off, we go on to describe more refined two-particle models because a two-body model is the simplest approach able to describe any change in the contact density, observed in many materials. Finally, we consider a theory of Ps annihilation in nanometric voids in which the exchange correlations between the electron of Ps and the outer electrons play a fundamental role. This theory is not usually taken into account in the literature, but it has to be considered for a correct theory of pick-off annihilation processes.


Assuntos
Nanoporos , Ligante de CD40 , Elétrons , Vácuo
8.
Talanta ; 273: 125873, 2024 Jun 01.
Artigo em Inglês | MEDLINE | ID: mdl-38460425

RESUMO

Heparin, being a widely employed anticoagulant in numerus clinical complications, requires strict quantification and qualitative screening to ensure the safety of patients from potential threat of thrombocytopenia. However, the intricacy of heparin's chemical structures and low abundance hinders the precise monitoring of its level and quality in clinical settings. Conventional laboratory assays have limitations in sensitivity and specificity, necessitating the development of innovative approaches. In this context, nanosensors emerged as a promising solution due to enhanced sensitivity, selectivity, and ability to detect heparin even at low concentrations. This review delves into a range of sensing approaches including colorimetric, fluorometric, surface-enhanced Raman spectroscopy, and electrochemical techniques using different types of nanomaterials, thus providing insights of its principles, capabilities, and limitations. Moreover, integration of smart-phone with nanosensors for point of care diagnostics has also been explored. Additionally, recent advances in nanopore technologies, artificial intelligence (AI) and machine learning (ML) have been discussed offering specificity against contaminants present in heparin to ensure its quality. By consolidating current knowledge and highlighting the potential of nanosensors, this review aims to contribute to the advancement of efficient, reliable, and economical heparin detection methods providing improved patient care.


Assuntos
Heparina , Nanoporos , Humanos , Inteligência Artificial , Anticoagulantes , Smartphone
9.
Talanta ; 273: 125971, 2024 Jun 01.
Artigo em Inglês | MEDLINE | ID: mdl-38521020

RESUMO

T-2 is one of the most potent cytotoxic food-borne mycotoxins. In this work, we have developed and characterized an electrochemical microfluidic immunosensor for T-2 toxin quantification in wheat germ samples. T-2 toxin detection was carried out using a competitive immunoassay method based on monoclonal anti-T-2 antibodies immobilized on the poly(methyl methacrylate) (PMMA) microfluidic central channel. The platinum wire working electrode at the end of the channel was in situ modified by a single-step electrodeposition procedure with reduced graphene oxide (rGO)-nanoporous gold (NPG). T-2 toxin in the sample was allowed to compete with T-2-horseradish peroxidase (HRP) conjugated for the specific recognizing sites of immobilized anti-T-2 monoclonal antibodies. The HRP, in the presence of hydrogen peroxide (H2O2), catalyzes the oxidation of 4-tert-butylcatechol (4-TBC), whose back electrochemical reduction was detected on the nanostructured electrode at -0.15 V. Thus, at low T-2 concentrations in the sample, more enzymatically conjugated T-2 will bind to the capture antibodies, and, therefore, a higher current is expected. The detection limits found for electrochemical immunosensor, and commercial ELISA procedure were 0.10 µg kg-1 and 10 µg kg-1, and the intra- and inter-assay coefficients of variation were below 5.35% and 6.87%, respectively. Finally, our microfluidic immunosensor to T-2 toxin will significantly contribute to faster, direct, and secure in situ analysis in agricultural samples.


Assuntos
Técnicas Biossensoriais , Grafite , Nanopartículas Metálicas , Micotoxinas , Nanoporos , Toxina T-2 , Grafite/química , Imunoensaio/métodos , Microfluídica , Ouro/química , Técnicas Biossensoriais/métodos , Peróxido de Hidrogênio/química , Técnicas Eletroquímicas/métodos , Limite de Detecção , Nanopartículas Metálicas/química
10.
ACS Sens ; 9(3): 1602-1610, 2024 Mar 22.
Artigo em Inglês | MEDLINE | ID: mdl-38451864

RESUMO

Solid-state (SS-) nanopore sensing has gained tremendous attention in recent years, but it has been constrained by its intrinsic lack of selectivity. To address this, we previously established a novel SS-nanopore assay that produces translocation signals only when a target biotinylated nucleic acid fragment binds to monovalent streptavidin (MS), a protein variant with a single high-affinity biotin-binding domain. While this approach has enabled selective quantification of diverse nucleic acid biomarkers, sensitivity enhancements are needed to improve the detection of low-abundance translational targets. Because the translocation dynamics that determine assay efficacy are largely governed by constituent charge characteristics, we here incorporate a polyhistidine-tagged MS (hMS) to alter the component detectability. We investigate the effects of buffer pH, salt concentration, and SS-nanopore diameter on the performance with the alternate reagent, achieve significant improvements in measurement sensitivity and selectivity, and expand the range of device dimensions viable for the assay. We used this improvement to detect as little as 1 nM miRNA spiked into human plasma. Overall, our findings improve the potential for broader applications of SS-nanopores in the quantitative analyses of molecular biomarkers.


Assuntos
Histidina , Nanoporos , Ácidos Nucleicos , Humanos , Estreptavidina/química , Biomarcadores
11.
Phys Rev E ; 109(2-1): 024404, 2024 Feb.
Artigo em Inglês | MEDLINE | ID: mdl-38491642

RESUMO

Free energies (FEs) in molecular sciences can be used to quantify the stability of folded molecules. In this article, we introduce nanopores for measuring FEs. We pull DNA hairpin-forming molecules through a nanopore, measure work, and estimate the FE change in the slow limit, and with the Jarzynski fluctuation theorem (FT) at fast pulling times. We also pull our molecule with optical tweezers, compare it to nanopores, and explore how sampling single molecules from equilibrium or a folded ensemble affects the FE estimate via the FT. The nanopore experiment helps us address and overcome the conceptual problem of equilibrium sampling in single-molecule pulling experiments. Only when molecules are sampled from an equilibrium ensemble do nanopore and tweezer FE estimates mutually agree. We demonstrate that nanopores are very useful tools for comparing FEs of two molecules at finite times and we propose future applications.


Assuntos
Nanoporos , DNA
12.
Langmuir ; 40(11): 5799-5808, 2024 Mar 19.
Artigo em Inglês | MEDLINE | ID: mdl-38501264

RESUMO

Nanopores are powerful single-molecule sensors for analyzing biomolecules such as DNA and proteins. Understanding the dynamics of DNA capture and translocation through nanopores is essential for optimizing their performance. In this study, we examine the effects of applied voltage and pore diameter on current blockage, translocation time, collision, and capture location by translocating λ-DNA through 5.7 and 16 nm solid-state nanopores. Ionic current changes are used to infer DNA conformations during translocation. We find that translocation time increases with pore diameter, which can be attributed to the decrease of the stall force. Linear and exponential decreases of collision frequency with voltage are observed in the 16 and 5.7 nm pores, respectively, indicating a free energy barrier in the small pore. Moreover, the results reveal a voltage-dependent bias in the capture location toward the DNA ends, which is explained by a "pulley effect" deforming the DNA as it approaches the pore. This study provides insights into the physics governing DNA capture and translocation, which can be useful for promoting single-file translocation to enhance nanopore sensing.


Assuntos
Nanoporos , DNA , Nanotecnologia/métodos , Transporte de Íons , Conformação de Ácido Nucleico
13.
Nat Chem ; 16(3): 314-334, 2024 Mar.
Artigo em Inglês | MEDLINE | ID: mdl-38448507

RESUMO

Sequencing of nucleic acids with nanopores has emerged as a powerful tool offering rapid readout, high accuracy, low cost and portability. This label-free method for sequencing at the single-molecule level is an achievement on its own. However, nanopores also show promise for the technologically even more challenging sequencing of polypeptides, something that could considerably benefit biological discovery, clinical diagnostics and homeland security, as current techniques lack portability and speed. Here we survey the biochemical innovations underpinning commercial and academic nanopore DNA/RNA sequencing techniques, and explore how these advances can fuel developments in future protein sequencing with nanopores.


Assuntos
Nanoporos , Proteômica , Tecnologia , Análise de Sequência de DNA , DNA
14.
Nat Commun ; 15(1): 1970, 2024 Mar 05.
Artigo em Inglês | MEDLINE | ID: mdl-38443335

RESUMO

Natural herbs, which contain pharmacologically active compounds, have been used historically as medicines. Conventionally, the analysis of chemical components in herbal medicines requires time-consuming sample separation and state-of-the-art analytical instruments. Nanopore, a versatile single molecule sensor, might be suitable to identify bioactive compounds in natural herbs. Here, a phenylboronic acid appended Mycobacterium smegmatis porin A (MspA) nanopore is used as a sensor for herbal medicines. A variety of bioactive compounds based on salvianolic acids, including caffeic acid, protocatechuic acid, protocatechualdehyde, salvianic acid A, rosmarinic acid, lithospermic acid, salvianolic acid A and salvianolic acid B are identified. Using a custom machine learning algorithm, analyte identification is performed with an accuracy of 99.0%. This sensing principle is further used with natural herbs such as Salvia miltiorrhiza, Rosemary and Prunella vulgaris. No complex sample separation or purification is required and the sensing device is highly portable.


Assuntos
Alcenos , Nanoporos , Plantas Medicinais , Polifenóis , Algoritmos , Extratos Vegetais
15.
Nat Commun ; 15(1): 1969, 2024 Mar 05.
Artigo em Inglês | MEDLINE | ID: mdl-38443434

RESUMO

Natural fruits contain a large variety of cis-diols. However, due to the lack of a high-resolution sensor that can simultaneously identify all cis-diols without a need of complex sample pretreatment, direct and rapid analysis of fruits in a hand-held device has never been previously reported. Nanopore, a versatile single molecule sensor, can be specially engineered to perform this task. A hetero-octameric Mycobacterium smegmatis porin A (MspA) nanopore modified with a sole phenylboronic acid (PBA) adapter is prepared. This engineered MspA accurately recognizes 1,2-diphenols, alditols, α-hydroxy acids and saccharides in prune, grape, lemon, different varieties of kiwifruits and commercial juice products. Assisted with a custom machine learning program, an accuracy of 99.3% is reported and the sample pretreatment is significantly simplified. Enantiomers such as DL-malic acids can also be directly identified, enabling sensing of synthetic food additives. Though demonstrated with fruits, these results suggest wide applications of nanopore in food and drug administration uses.


Assuntos
Citrus , Nanoporos , Estados Unidos , Frutas , Álcoois Açúcares , Ácidos Carboxílicos , Mycobacterium smegmatis , Porinas
16.
Methods Mol Biol ; 2778: 345-366, 2024.
Artigo em Inglês | MEDLINE | ID: mdl-38478288

RESUMO

Biological nanopores incorporated into synthetic membranes are widely used for single-molecule analytical applications such as DNA sequencing. The ability to engineer custom membrane proteins with a pore would allow the generation of a multitude of nanopores for the sensing/sequencing of small molecules and (bio)polymers. The de novo design of transmembrane ß-barrel pores has recently enabled the generation of nanopores with custom size, shape, and properties. In this chapter, I describe the rationale of transmembrane ß-barrel design and computational methods to assemble the backbones, design sequences, and select the designs for experimental validation.


Assuntos
Proteínas de Membrana , Nanoporos
17.
J Phys Chem B ; 128(11): 2792-2798, 2024 Mar 21.
Artigo em Inglês | MEDLINE | ID: mdl-38471969

RESUMO

The transport behavior of biomolecules at the confined nanoscale is very different from that of the bulk state. Numerous disease diagnostics and targeted drug treatments are performed based on nanochannels in cells. The specific structure and shape of nanochannels play an important role in the behavior and efficiency of substance transport. In this paper, we fabricated nanopores with different tilt angles and the same diameters using focused ion beam. The capture frequency and the blocking current amplitude of λ-DNA within large-angle nanopores decrease obviously, suggesting an increase in the energy barrier of large-angle nanopores and the fact that they stretch biomolecules to thinness. Most importantly, large-angle nanopores slow down λ-DNA transport by 2-4 times. MD simulations find that the sloped electroosmotic flow inside the tilted nanopores is the main factor contributing to the transport phenomena. The increase in the capture time of biomolecules by nanopores assists in obtaining more biological information from the current trajectories. Our study provides a new understanding of substance transport in specially shaped nanopores, which can be instrumental in providing fresh inspiration and approaches to the biomedical field.


Assuntos
Nanoporos , DNA/química , Transporte Biológico , Eletro-Osmose
18.
IEEE Trans Nanobioscience ; 23(2): 310-318, 2024 Apr.
Artigo em Inglês | MEDLINE | ID: mdl-38546987

RESUMO

In nanopore sequencers, single-stranded DNA molecules (or k-mers) enter a small opening in a membrane called a nanopore and modulate the ionic current through the pore, producing a channel output in the form of a noisy piecewise constant signal. An important problem in DNA-based data storage is finding a set of k-mers, i.e. a DNA code, that is robust against noisy sample duplication introduced by nanopore sequencers. Good DNA codes should contain as many k-mers as possible that produce distinguishable current signals (squiggles) as measured by the sequencer. The dissimilarity between squiggles can be estimated using a bound on their pairwise error probability, which is used as a metric for code design. Unfortunately, code construction using the union bound is limited to small k's due to the difficulty of finding maximum cliques in large graphs. In this paper, we construct large codes by concatenating codewords from a base code, thereby packing more information in a single strand while retaining the storage efficiency of the base code. To facilitate decoding, we include a circumfix in the base code to reduce the effect of the nanopore channel memory. We show that the decoding complexity scales as [Formula: see text], where m is the number of concatenated k-mers. Simulations show that the base code error rate is stable as m increases.


Assuntos
DNA Concatenado , Nanoporos , DNA/genética , Análise de Sequência de DNA , Armazenamento e Recuperação da Informação
19.
Bioinformatics ; 40(4)2024 Mar 29.
Artigo em Inglês | MEDLINE | ID: mdl-38478392

RESUMO

MOTIVATION: Oxford Nanopore Technologies (ONT) sequencers enable real-time generation of sequence data, which allows for concurrent analysis during a run. Adaptive sampling leverages this real-time capability in extremis, rejecting or accepting reads for sequencing based on assessment of the sequence from the start of each read. This functionality is provided by ONT's software, MinKNOW (Oxford Nanopore Technologies). Designing and developing software to take advantage of adaptive sampling can be costly in terms of sequencing consumables, using precious samples and preparing sequencing libraries. MinKNOW addresses this in part by allowing the replay of previously sequenced runs for testing. However, as we show, the sequencing output only partially changes in response to adaptive sampling instructions. Here we present Icarust, a tool enabling more accurate approximations of sequencing runs. Icarust recreates all the required endpoints of MinKNOW to perform adaptive sampling and writes output compatible with current base-callers and analysis pipelines. Icarust serves nanopore signal simulating a MinION or PromethION flow cell experiment from any reference genome using either R9 or R10 pore models. We show that simulating sequencing runs with Icarust provides a realistic testing and development environment for software exploiting the real-time nature of Nanopore sequencing. AVAILABILITY AND IMPLEMENTATION: All code is open source and freely available here-https://github.com/LooseLab/Icarust. Icarust is implemented in Rust, with a docker container also available. The data underlying this article will be shared on reasonable request to the corresponding author.


Assuntos
Sequenciamento por Nanoporos , Nanoporos , Análise de Sequência de DNA , Software , Sequenciamento de Nucleotídeos em Larga Escala
20.
Langmuir ; 40(14): 7300-7309, 2024 Apr 09.
Artigo em Inglês | MEDLINE | ID: mdl-38536237

RESUMO

DNA sequencing and sensing using nanopore technology delves critically into the alterations in the measurable electrical signal as single-stranded DNA is drawn through a tiny passage. To make such precise measurements, however, slowing down the DNA in the tightly confined passage is a key requirement, which may be achieved by grafting the nanopore walls with a polyelectrolyte layer (PEL). This soft functional layer at the wall, under an off-design condition, however, may block the DNA passage completely, leading to the complete loss of output signal from the nanobio sensor. Whereas theoretical postulates have previously been put forward to explain the essential physics of DNA translocation in nanopores, these have turned out to be somewhat inadequate when confronted with the experimental findings on functionalized nanopores, including the prediction of the events of complete signal losses. Circumventing these constraints, herein we bring out a possible decisive role of the interplay between the inevitable variabilities in the ionic distribution along the nanopore axis due to its finite length as opposed to its idealized "infinite" limit as well as the differential permittivity of PEL and bulk solution that cannot be captured by the commonly used one-dimensional variant of the electrical double layer theory. Our analysis, for the first time, captures variations in the ionic concentration distribution across multidimensional physical space and delineates its impact on the DNA translocation characteristics that have hitherto remained unaddressed. Our results reveal possible complete blockages of DNA translocation as influenced by less-than-threshold permittivity values or greater-than-threshold grafting densities of the PEL. In addition, electrohydrodynamic blocking is witnessed due to the ion-selective nature of the nanopore at low ionic concentrations. Hence, our study establishes a functionally active regime over which the PEL layer in a finite-length nanopore facilitates controllable DNA translocation, enabling successful sequencing and sensing through the explicit modulation of translocation speed.


Assuntos
Nanoporos , Polieletrólitos , DNA , DNA de Cadeia Simples , Íons
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