Nanopore Device Research Articles

Resolving the Size and Charge of Small Particles: A Predictive Model of Nanopore Mechanics.

The movement of small particles and molecules through nanopore membranes is widespread and has far-reaching implications. Consequently, the development of mathematical models is essential for understanding these processes on a micro level, leading to deeper insights. In this endeavor, we suggested a model based on a set of empirical equations to predict the transport of substances through a solid-state nanopore and the associated signals generated during their translocation. This model establishes analytical relationships between the ionic current and electrical double-layer potential observed during analyte translocation and their size, charge, and mobility in an electrolyte solution. This framework allows for rapid interpretation and prediction of the nanopore system's behavior and provides a means for quantitatively determining the physical properties of molecular analytes. To illustrate the analytical capability of this model, ceria nanoparticles were investigated while undergoing oxidation or reduction within an original nanopore device. The results obtained were found to be in good agreement with predictions from physicochemical methods. This developed approach and model possess transferable utility to various porous materials, thereby expediting research efforts in membrane characterization and the advancement of nano- and ultrafiltration or electrodialysis technologies.

Sequence-Dependent Single-Molecule DNA Sensing Using Covalent Organic Framework Nanopores.

Enzyme-free single-molecule sequencing has the potential to significantly expand the application of nanopore technology to DNA, proteins, and saccharides. Despite their advantages over biological nanopores and natural suitability for enzyme-free single-molecule sequencing, conventional solid-state nanopores have not yet achieved single-molecule DNA sequencing. The biggest challenge for the accuracy of single-molecule sequencing using solid-state nanopores lies in the precise control of the pore size and conformity. In this study, we fabricated nanopore devices by covering the tip of a quartz nanopipette with ultrathin two-dimensional (2D) covalent organic framework (COF) nanosheets (pore size ≈ 1.1 nm). The size of the periodically arranged nanopores in COF is comparable to that of protein nanopores, and the structure of each COF nanopore is consistent at the atomic scale. The COF nanopore device could roughly distinguish dAMP, dCMP, dGMP, and dTMP. Furthermore, a certain percentage of the current blockades originating from 150 nucleotides model DNA molecules (13.5% for dA50dC50dA50 and 11.1% for dC50dA50dC50) show distinct DNA sequence-specific concave and convex resistive current patterns. The finite element simulation confirmed that the current blockade pattern of a DNA molecule passing through a COF nanopore is dependent on the relative location of the nanopore with respect to the wall of the nanopipette. Our study is a significant step toward single-molecule DNA sequencing by solid-state nanopores.

High Sensitivity and Specificity Platform to Validate MicroRNA Biomarkers in Cancer and Human Diseases.

We developed a technology for detecting and quantifying trace nucleic acids using a bracketing protocol designed to yield a copy number with approximately ± 20% accuracy across all concentrations. The microRNAs (miRNAs) let-7b, miR-15b, miR-21, miR-375 and miR-141 were measured in serum and urine samples from healthy subjects and patients with breast, prostate or pancreatic cancer. Detection and quantification were amplification-free and enabled using osmium-tagged probes and MinION, a nanopore array detection device. Combined serum from healthy men (Sigma-Aldrich, St. Louis, MO, USA #H6914) was used as a reference. Total RNA isolated from biospecimens using commercial kits was used as the miRNA source. The unprecedented ± 20% accuracy led to the conclusion that miRNA copy numbers must be normalized to the same RNA content, which in turn illustrates (i) independence from age, sex and ethnicity, as well as (ii) equivalence between serum and urine. miR-21, miR-375 and miR-141 copies in cancers were 1.8-fold overexpressed, exhibited zero overlap with healthy samples and had a p-value of 1.6 × 10-22, tentatively validating each miRNA as a multi-cancer biomarker. miR-15b was confirmed to be cancer-independent, whereas let-7b appeared to be a cancer biomarker for prostate and breast cancer, but not for pancreatic cancer.

In-tube micro-pyramidal silicon nanopore for inertial-kinetic sensing of single molecules

Electrokinetic force has been the major choice for driving the translocation of molecules through a nanopore. However, the use of this approach is limited by an uncontrollable translocation speed, resulting in non-uniform conductance signals with low conformational sensitivity, which hinders the accurate discrimination of the molecules. Here, we show the use of inertial-kinetic translocation induced by spinning an in-tube micro-pyramidal silicon nanopore fabricated using photovoltaic electrochemical etch-stop technique for biomolecular sensing. By adjusting the kinetic properties of a funnel-shaped centrifugal force field while maintaining a counter-balanced state of electrophoretic and electroosmotic effect in the nanopore, we achieved regulated translocation of proteins and obtained stable signals of long and adjustable dwell times and high conformational sensitivity. Moreover, we demonstrated instantaneous sensing and discrimination of molecular conformations and longitudinal monitoring of molecular reactions and conformation changes by wirelessly measuring characteristic features in current blockade readouts using the in-tube nanopore device.

Single-Molecule Discrimination of Saccharides Using Carbon Nitride Nanopores.

Structural complexity brings a huge challenge to the analysis of sugar chains. As a single-molecule sensor, nanopores have the potential to provide fingerprint information on saccharides. Traditionally, direct single-molecule saccharide detection with nanopores is hampered by their small size and weak affinity. Here, a carbon nitride nanopore device is developed to discern two types of trisaccharide molecules (LeApN and SLeCpN) with minor structural differences. The resolution of LeApN and SLeCpN in the mixture reaches 0.98, which has never been achieved in solid-state nanopores so far. Monosaccharide (GlcNAcpN) and disaccharide (LacNAcpN) can also be discriminated using this system, indicating that the versatile carbon nitride nanopores possess a monosaccharide-level resolution. This study demonstrates that the carbon nitride nanopores have the potential for conducting structure analysis on single-molecule saccharides.

A Universal Approximation for Conductance Blockade in Thin Nanopore Membranes.

Nanopore-based sensing platforms have transformed single-molecule detection and analysis. The foundation of nanopore translocation experiments lies in conductance measurements, yet existing models, which are largely phenomenological, are inaccurate in critical experimental conditions such as thin and tightly fitting pores. Of the two components of the conductance blockade, channel and access resistance, the access resistance is poorly modeled. We present a comprehensive investigation of the access resistance and associated conductance blockade in thin nanopore membranes. By combining a first-principles approach, multiscale modeling, and experimental validation, we propose a unified theoretical modeling framework. The analytical model derived as a result surpasses current approaches across a broad parameter range. Beyond advancing our theoretical understanding, our framework's versatility enables analyte size inference and predictive insights into conductance blockade behavior. Our results will facilitate the design and optimization of nanopore devices for diverse applications, including nanopore base calling and data storage.

Free-energy measuring nanopore device.

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.

DNA Barcodes Using a Dual Nanopore Device.

We report a novel method based on the current blockade (CB) characteristics obtained from a dual nanopore device that can determine DNA barcodes with near-perfect accuracy using a Brownian dynamics simulation strategy. The method supersedes our previously reported velocity correction algorithm (S. Seth and A. Bhattacharya, RSC Advances, 11:20781-20787, 2021), taking advantage of the better measurement of the time-of-flight (TOF) protocol offered by the dual nanopore setup. We demonstrate the efficacy of the method by comparing our simulation data from a coarse-grained model of a polymer chain consisting of 2048 excluded volume beads of diameter 𝜎 = 24bp using with those obtained from experimental CB data from a 48,500bp λ-phage DNA, providing a base pair resolution in simulation. The simulation time scale is compared to the experimental time scale by matching the simulated time-of-flight (TOF) velocity distributions with those obtained experimentally (Rand et al., ACS Nano, 16:5258-5273, 2022). We then use the evolving coordinates of the dsDNA and the molecular features to reconstruct the current blockade characteristics on the fly using a volumetric model based on the effective van der Waal radii of the species inside and in the immediate vicinity of the pore. Our BD simulation mimics the control-zoom-in-logic to understand the origin of the TOF distributions due to the relaxation of the out-of-equilibrium conformations followed by a reversal of the electric fields. The simulation algorithm is quite general and can be applied to differentiate DNA barcodes from different species.

Nanopore Electroporation Device for DNA Transfection into Various Spreading and Nonadherent Cell Types.

Cell transfection plays a crucial role in the study of gene function and regulation of gene expression. The existing gene transfection methods, such as chemical carriers, viruses, electroporation, and microinjection, suffer from limitations, including cell type dependence, reliance on cellular endocytosis, low efficiency, safety concerns, and technical complexity. Nanopore-coupled electroporation offers a promising approach to localizing electric fields for efficient cell membrane perforation and nucleic acid transfection. However, the applicability of nanopore electroporation technology across different cell types lacks a systematic investigation. In this study, we explore the potential of nanopore electroporation for transfecting DNA plasmids into various cell types. Our nanopore electroporation device employs track-etched membranes as the core component. We find that nanopore electroporation efficiently transfects adherent cells, including well-spreading epithelial-like HeLa cells, cardiomyocyte-like HL-1 cells, and dendritic-cell-like DC2.4 cells. However, it shows a limited transfection efficiency in weakly spreading macrophages (RAW264.7) and suspension cells (Jurkat). To gain insights into these observations, we develop a COMSOL model, revealing that nanopore electroporation better localizes the electric field on adherent and well-spreading cells, promoting favorable membrane poration conditions. Our findings provide valuable references for advancing nanopore electroporation as a high-throughput, safe, and efficient gene transfection platform.

Quantitation of Circulating Mycobacterium tuberculosis Antigens by Nanopore Biosensing in Children Evaluated for Pulmonary Tuberculosis in South Africa.

Nanopore sensing of proteomic biomarkers lacks accuracy due to the ultralow abundance of targets, a wide variety of interferents in clinical samples, and the mismatch between pore and analyte sizes. By converting antigens to DNA probes via click chemistry and quantifying their characteristic signals, we show a nanopore assay with several amplification mechanisms to achieve an attomolar level limit of detection that enables quantitation of the circulating Mycobacterium tuberculosis (Mtb) antigen ESAT-6/CFP-10 complex in human serum. The assay's nonsputum-based feature and low-volume sample requirements make it particularly well-suited for detecting pediatric tuberculosis (TB) disease, where establishing an accurate diagnosis is greatly complicated by the paucibacillary nature of respiratory secretions, nonspecific symptoms, and challenges with sample collection. In the clinical assessment, the assay was applied to analyze ESAT-6/CFP-10 levels in serum samples collected during baseline investigation for TB in 75 children, aged 0-12 years, enrolled in a diagnostic study conducted in Cape Town, South Africa. This nanopore assay showed superior sensitivity in children with confirmed TB (94.4%) compared to clinical "gold standard" diagnostic technologies (Xpert MTB/RIF 44.4% and Mtb culture 72.2%) and filled the diagnostic gap for children with unconfirmed TB, where these traditional technologies fell short. We envision that, in combination with automated sample processing and portable nanopore devices, this methodology will offer a powerful tool to support the diagnosis of pulmonary TB in children.

Single-molecule recognition of Nucleolin and the interactions with DNA/RNA G-quadruplexes via nanopore decoding

DNA/RNA quadruplexes (G4) are significant secondary structures that exist in human genes, playing essential part in transcription and replication. Nucleolin (NCL) is a ubiquitous protein mainly found in the human nucleus. It is frequently overexpressed in cancer cells and displays a remarkable preference for DNA/RNA G4 binding, playing a crucial role in controlling nucleic acid metabolism and chromatin structure. Additionally, NCL is involved in microRNA (miRNA) biogenesis.This work presents the discrimination of DNA/RNA G4 topologies harboring close sequence length with size-matched nanopore devices. The single-molecule identification of NCL under various parameters was implemented, and a low detection limit of 0.5 nM was achieved, NCL could specifically bind with DNA G4 AS1411 and RNA G4 (rG4) from pre-miRNA 92b and pre-miRNA 149, forming stable complexes that demonstrate distinct nanopore translocation properties. The augmentation of the molar ratio of G4 to NCL induces elevated complex formation. Recognition of both NCL and NCL-G4 complex in human serum is realized. Our work established a precise platform for the differentiation of nucleic acid secondary structure with similar conformation, we also proved the binding behaviors between NCL and DNA/RNA G4, as well as the serum interference test, which is valuable for the investigation of the interactions between proteins and nucleic acids as well as for further understanding the mechanism of the NCL-related diseases processes.

Estimated Nucleotide Reconstruction Quality Symbols of Basecalling Tools for Oxford Nanopore Sequencing.

Currently, one of the fastest-growing DNA sequencing technologies is nanopore sequencing. One of the key stages involved in processing sequencer data is the basecalling process, where the input sequence of currents measured on the nanopores of the sequencer reproduces the DNA sequences, called DNA reads. Many of the applications dedicated to basecalling, together with the DNA sequence, provide the estimated quality of the reconstruction of a given nucleotide (quality symbols are contained on every fourth line of the FASTQ file; each nucleotide in the FASTQ file corresponds to exactly one estimated nucleotide reconstruction quality symbol). Herein, we compare the estimated nucleotide reconstruction quality symbols (signs from every fourth line of the FASTQ file) reported by other basecallers. The conducted experiments consisted of basecalling the same raw datasets from the nanopore device by other basecallers and comparing the provided quality symbols, denoting the estimated quality of the nucleotide reconstruction. The results show that the estimated quality reported by different basecallers may vary, depending on the tool used, particularly in terms of range and distribution. Moreover, we mapped basecalled DNA reads to reference genomes and calculated matched and mismatched rates for groups of nucleotides with the same quality symbol. Finally, the presented paper shows that the estimated nucleotide reconstruction quality reported in the basecalling process is not used in any investigated tool for processing nanopore DNA reads.

High durability and stability of 2D nanofluidic devices for long-term single-molecule sensing

Nanopores in two-dimensional (2D) membranes hold immense potential in single-molecule sensing, osmotic power generation, and information storage. Recent advances in 2D nanopores, especially on single-layer MoS2, focus on the scalable growth and manufacturing of nanopore devices. However, there still remains a bottleneck in controlling the nanopore stability in atomically thin membranes. Here, we evaluate the major factors responsible for the instability of the monolayer MoS2 nanopores. We identify chemical oxidation and delamination of monolayers from their underlying substrates as the major reasons for the instability of MoS2 nanopores. Surface modification of the substrate and reducing the oxygen from the measurement solution improves nanopore stability and dramatically increases their shelf-life. Understanding nanopore growth and stability can provide insights into controlling the pore size, shape and can enable long-term measurements with a high signal-to-noise ratio and engineering durable nanopore devices.

Characterization of Avidin and Case9 single protein molecules by a solid-state nanopore device.

Solid-state nanopores made of silicon nitride have emerged as novel platforms for biosensing such as real-time single-molecule characterization of protein molecules. Solid-state nanopore detection is based on measuring the variations in ionic conductance as charged biomolecules translocate through nanometer-sized channels driven by an external voltage applied across the membrane. In this study, we characterize Avidin (MW: ∼67 kDa) and Case9 (MW: ∼160 kDa) proteins with nanopore current signal. We study how voltage and glycerol, impact on the time durations and current blockage amplitudes produced by the proteins passing through a nanopore.

Toward protein fingerprinting with a solid-state nanopore.

Solid-state nanopores have shown great potential for single-molecule protein identification in recent years. As a first step toward single-molecule protein fingerprinting using a solid-state nanopore device, thorough characterization of the passage of proteins under the different conformational states is required. For this purpose, we use green fluorescent protein (GFP), antibiotin immunoglobulin G (IgG), and cytochrome c (cyt c) as model proteins, and show results from field-driven translocations through nanopores with diameters ranging from 2 to 10 nm in SiN membranes of thicknesses from 10 to 20 nm. As expected, the distinguishable current blockades observed during individual translocations allow the identification of different protein conformations adopted throughout its passage, ranging from partially unfolded to completely unfolded conformations. Here we show how different temperatures and voltages facilitate protein unfolding and alter their folding kinetics. This is achieved by varying voltages from 0.1 V to 1 V and by varying the temperature from room temperature to 60 oC while maintaining low-noise ionic current recording at 1 MHz bandwidth using a custom instrument with thermal control offering ±0.2 oC precision and ±1 oC repeatability. These results will serve to better understand the accessible states in the protein energy landscape and to benchmark future studies furthering protein fingerprinting.

Exploring the dynamics of DNA nucleotides in graphene/h-BN nanopores: insights from ab initio molecular dynamics.

Nanopore devices based on graphene and h-BN heterostructures show outstanding electrical and physical characteristics for high throughput label-free DNA sequencing. On top of their suitability for DNA sequencing with the ionic current method, G/h-BN nanostructures are promising for DNA sequencing by employing the in-plane electronic current. The influence of the nucleotide/device interaction on the in-plane current has been widely explored for static-optimized geometries. Therefore, it is essential to investigate the dynamics of the nucleotides within the G/h-BN nanopores to gain a comprehensive view of their interaction with the nanopores. In this study, we investigated the dynamic interaction between nucleotides and nanopores in horizontal graphene/h-BN/graphene heterostructures. The insulating h-BN layer, where the nanopores are implemented, changes the in-plane charge transport mechanism into the quantum mechanical tunneling regime. We employed the Car-Parrinello molecular dynamics (CPMD) formalism to investigate the interaction of the nucleotides with nanopores in a vacuum as well as in an aqueous environment. The simulation was performed in the NVE canonical ensemble with an initial temperature of 300 K. The results indicate that the interaction between the electronegative ends of the nucleotides and the atoms at the nanopore edge is essential for the dynamic behavior of the nucleotides. Moreover, water molecules have a substantial effect on the dynamics and interactions of the nucleotides with nanopores.

Use of a solid-state nanopore for profiling the transferrin receptor protein and distinguishing between transferrin receptor and its ligand protein.

A nanopore device is capable of providing single-molecule level information of an analyte as they translocate through the sensing aperture-a nanometer-sized through-hole-under the influence of an applied electric field. In this study, a silicon nitride (Six Ny )-based nanopore was used to characterize the human serum transferrin receptor protein (TfR) under various applied voltages. The presence of dimeric forms of TfR was found to decrease exponentially as the applied electric field increased. Further analysis of monomeric TfR also revealed that its unfolding behaviors were positively dependent on the applied voltage. Furthermore, a comparison between the data of monomeric TfR and its ligand protein, human serum transferrin (hSTf), showed that these two protein populations, despite their nearly identical molecular weights, could be distinguished from each other by means of a solid-state nanopore (SSN). Lastly, the excluded volumes of TfR were experimentally determined at each voltage and were found to be within error of their theoretical values. The results herein demonstrate the successful application of an SSN for accurately classifying monomeric and dimeric molecules while the two populations coexist in a heterogeneous mixture.

Direct RNA sequencing of respiratory syncytial virus infected human cells generates a detailed overview of RSV polycistronic mRNA and transcript abundance.

To characterize species of viral mRNA transcripts generated during respiratory syncytial virus (RSV) infection, human fibroblast-like MRC-5 lung cells were infected with subgroup A RSV for 6, 16 and 24 hours. In addition, we characterised the viral transcriptome in infected Calu-3 lung epithelial cells at 48 hours post infection. Total RNA was harvested and polyadenylated mRNA was enriched and sequenced by direct RNA sequencing using an Oxford nanopore device. This platform yielded over 450,000 direct mRNA transcript reads which were mapped to the viral genome and analysed to determine the relative mRNA levels of viral genes using our in-house ORF-centric pipeline. We examined the frequency of polycistronic readthrough mRNAs were generated and assessed the length of the polyadenylated tails for each group of transcripts. We show a general but non-linear decline in gene transcript abundance across the viral genome, as predicted by the model of RSV gene transcription. However, the decline in transcript abundance is not uniform. The polyadenylate tails generated by the viral polymerase are similar in length to those generated by the host polyadenylation machinery and broadly declined in length for most transcripts as the infection progressed. Finally, we observed that the steady state abundance of transcripts with very short polyadenylate tails less than 20 nucleotides is less for N, SH and G transcripts in both cell lines compared to NS1, NS2, P, M, F and M2 which may reflect differences in mRNA stability and/or translation rates within and between the cell lines.

Environmental surveillance and clinical assessment of SARS-CoV-2 epidemiology and variant prevalence in an urban area in Spain

BACKGROUND AND AIM: Wastewater-based epidemiology (WBE) provides the means to monitor pathogens prevalence in the sewage system of a community. SARS-CoV-2, the virus responsible for the COVID-19 global pandemic, can be detected in the feces of infected individuals. Therefore, viral concentrations in wastewater can be taken as an indication of SARS-CoV-2 prevalence in the entire population. This work was aimed at the detection and sequence analysis of SARS-CoV-2 RNA in wastewater and its correlation with clinical data. METHODS: Samples collected over a nine-month period (2020-21) at a wastewater treatment plant in Elche (Spain) were concentrated by ultrafiltration and analyzed for the presence of SARS-CoV-2 RNA by reverse transcriptase quantitative polymerase chain reaction (RT-qPCR). Sequences generated from cDNA samples with a MinION Nanopore device were compared to SARS-CoV-2 sequence databases. RESULTS: The appearance of the first qPCR positive signals correlated with the increasing number of covid-19 cases in Elche at the onset of the second wave of COVID-19 (july 2020). Measurements of viral genomes in water samples in October-November 2020 reflected the higher number of clinical cases within that period. Nanopore sequencing assay allowed the detection of spike (S) protein mutations associated with circulating SARS-CoV-2 variants both in environmental as well as in clinical samples, reflecting the emergence of the B.1.1.7 variant in Elche in late 2020. CONCLUSIONS: This study highligted the potential of wastewater-based epidemiology (WBE) in revealing the presence of pathogenic viruses in urban populations. By the use of the nanopore sequencing technology we were able to assess the changes in circulating SARS-CoV-2 variants in a whole population. KEYWORDS: environmental epidemiology, SARS-CoV-2 variants, wastewater, nanopore sequencing

DNA Origami in the Quest for Membrane Piercing

The tool kit for label-free single-molecule sensing, nucleic acid sequencing (DNA, RNA and protein) and other biotechnological applications has been significantly broadened due to the wide range of available nanopore-based technologies. Currently, various sources of nanopores, including biological, fabricated solid-state, hybrid and recently de novo chemically synthesized ion-like channels have put in use for rapid single-molecule sensing of biomolecules and other diagnostic applications. At length scales of hundreds of nanometers, DNA nanotechnology, particularly DNA origami-based devices, enables the assembly of complex and dynamic 3-dimensional nanostructures, including nanopores with precise control over the size/shape. DNA origami technology has enabled to construct nanopores by DNA alone or hybrid architects with solid-state nanopore devices or nanocapillaries. In this review, we briefly discuss the nanopore technique that uses DNA nanotechnology to construct such individual pores in lipid-based systems or coupled with other solid-state devices, nanocapillaries for enhanced biosensing function. We summarize various DNA-based design nanopores and explore the sensing properties of such DNA channels. Apart from DNA origami channels we also pointed the design principles of RNA nanopores for peptide sensing applications.