Nanopore-based Devices Research Articles

Pore-Size-Dependent Role of Functional Elements at the Outer Surface and Inner Wall in Single-Nanochannel Biosensors.

Biological nanopores feature functional elements on the outer surfaces (FEOS) and inner walls (FEIW), enabling precise control over ions and molecules with exceptional sensitivity and specificity. This provides valuable inspiration to scientists for the development of intelligent artificial nanochannel-based platforms, with a wide range of potential applications, including biosensors. Much effort has been dedicated to investigating the distinct contribution of FEOS and FEIW of multichannel membrane biosensors. However, the intricate interactions among neighboring pores in multichannel biosensors have presented challenges. This underscores the untapped potential of single nanochannels as ideal candidates in this field. Here, we employed single nanochannel membranes with different pore sizes to investigate the distinct contributions of FEIW and FEOS to single-nanochannel biosensors, combined with numerical simulations. Our findings revealed that alterations in the negative charges of FEIW and FEOS, induced by target binding, have differential effects on ion transport, contingent upon the degree of nanoconfinement. In the case of smaller pores, such as 20 nm, the ion concentration polarization driven by FEIW can independently control ion transport through the surface's electric double layer. However, as the pore size increases to 40-60 nm, both FEIW and FEOS become essential for effective ion concentration polarization. When the pore size reaches 100 nm, both FEIW and FEOS are ineffective and thus unsuitable for biosensors. Simulations demonstrate that the observed phenomena can be attributed to the interactions between the charges of FEIW and FEOS within the overlapping electric double layer under confinement. These results underscore the critical role of pore size as a key parameter in governing the functionality of probes within or on nanopore-based biosensors as well as in the design of nanopore-based devices.

A high density nanopore 3-triangulene kagome lattice.

Nanopore-containing two-dimensional materials have been explored for a wide range of applications including filtration, sensing, catalysis, energy storage and conversion. Triangulenes have recently been experimentally synthesized in a variety of sizes. In this regard, using these systems as building blocks, we theoretically examined 3-triangulene kagome crystals with inherent holes of ∼12 Å diameter and a greater density array of nanopores (≥1013 cm-2) compared to conventional 2D systems. The energetic, electronic, and transport properties of pristine and B/N-doped 3-triangulene kagome crystals were evaluated through a combination of density functional theory and non-equilibrium Green's function method. The simulated scanning tunneling microscopy images clearly capture electronic perturbation around the doped sites, which can be used to distinguish the pristine system from the doped systems. The viability of precisely controlling the band structure and transport properties by changing the type and concentration of doping atoms is demonstrated. The findings presented herein can potentially widen the applicability of these systems that combine unique electronic properties and intrinsically high-density pores, which can pave the way for the next generation of nanopore-based devices.

Electroosmotic flow, ionic current rectification, and selectivity of a conical nanopore modified with a pH-regulated polyelectrolyte layer: Influence of functional groups profile

Nanopores functionalized with polyelectrolyte (PE) brushes on their surface are widely used to improve the performance of solid-state nanopore-based devices. Since the profile of the functional groups, and therefore, fixed charge density, inside PE layer can be influenced by surrounding conditions and the way a nanopore is modified, it is usually nonuniformly distributed. However, a theoretical model taking account of the presence of a PE layer on the nanopore surface assumes that its functional groups are uniformly distributed. In this study we consider a conical nanopore surface modified with a pH-regulated PE layer. Two types of PE layer are considered: the functional groups are clustered near the PE-liquid interface (type 1), and near the nanopore surface (type 2). We theoretically study the influence of pH, types of PE layer, and bulk salt concentration on the ion transport behavior of the nanopore, and the associated mechanisms are discussed. The dependence of the average electroosmotic flow on relevant driving force and resistive force, including shear stress and drag, is analyzed. The influence of the types of PE layer on the I−V, rectification, and selectivity behaviors of the nanopore depends highly on the levels of pH and applied potential. The larger diffusion flux of type2 polyelectrolyte layer is the most important factor affecting current performance. Knowing the rectification behavior of a nanopore is helpful for estimating the solution pH and understanding the profile of the functional groups in PE. A reversal in selectivity is observed when both pH and bulk salt concentration are low, which can be attributed to that the ionic flux is dominated by H+.

Single-molecule DNA sequencing using two-dimensional Ti2C(OH)2 MXene nanopores: A first-principles investigation

Nanopore-based devices have provided exciting opportunities to develop affordable label-free DNA sequencing platforms. Over a decade ago, graphene has been proposed as a two-dimensional (2D) nanopore membrane in order to achieve single-base resolution. However, it was experimentally revealed that clogging of the graphene nanopore can occur due to the hydrophobic nature of graphene, thus hindering the translocation of DNA. To overcome this problem, the exploration of alternative 2D materials has gained considerable interest over the last decade. Here we show that a Ti2C-based MXene nanopore functionalized by hydroxyl groups (−OH) exhibits transverse conductance properties that allow for the distinction between all four naturally occurring DNA bases. We have used a combination of density functional theory and non-equilibrium Green’s function method to sample over multiple orientations of the nucleotides in the nanopore, as generated from molecular dynamics simulations. The conductance variation resulting from sweeping an applied gate voltage demonstrates that the Ti2C-based MXene nanopore possesses high potential to rapidly and reliably sequence DNA. Our findings open the door to further theoretical and experimental explorations of MXene nanopores as a promising 2D material for nanopore-based DNA sensing.

Species- and strain-level assessment using rrn long-amplicons suggests donor’s influence on gut microbial transference via fecal transplants in metabolic syndrome subjects

ABSTRACT Fecal microbiota transplantation (FMT) is currently used for treating Clostridium difficile infection and explored for other clinical applications in experimental trials. However, the effectiveness of this therapy could vary, and partly depend on the donor’s bacterial species engraftment, whose evaluation is challenging because there are no cost-effective strategies for accurately tracking the microbe transference. In this regard, the precise identification of bacterial species inhabiting the human gut is essential to define their role in human health unambiguously. We used Nanopore-based device to sequence bacterial rrn operons (16S-ITS-23S) and to reveal species-level abundance changes in the human gut microbiota of a FMT trial. By assessing the donor and recipient microbiota before and after FMT, we further evaluated whether this molecular approach reveals strain-level genetic variation to demonstrate microbe transfer and engraftment. Strict control over sequencing data quality and major microbiota covariates was critical for accurately estimating the changes in gut microbial species abundance in the recipients after FMT. We detected strain-level variation via single-nucleotide variants (SNVs) at rrn regions in a species-specific manner. We showed that it was possible to explore successfully the donor-bacterial strain (e.g., Parabacteroides merdae) engraftment in recipients of the FMT by assessing the nucleotide frequencies at rrn-associated SNVs. Our findings indicate that the engraftment of donors’ microbiota is to some extent correlated with the improvement of metabolic health in recipients and that parameters such as the baseline gut microbiota configuration, sex, and age of donors should be considered to ensure the success of FMT in humans. The study was prospectively registered at the Dutch Trial registry – NTR4488 (https://www.trialregister.nl/trial/4488).

Applications of Photo-Responsive Molecules in Nanopore-based Devices

Abstract In biochemistry, it is crucial to explore the conformation, response to external environment and interaction rules of biomacromolecules and small molecules for understanding the correlation of the structures and properties among organic matters. However, it is a challenge for the traditional techniques to observe the conformation heterogeneity and interpret the interaction mechanism of the biomolecules in living cells. As the emerged the third generation single-molecule gene sequencing technology, nanopore is used to monitor the conformation change of the molecules in situ and therefore demonstrate good applications in single-molecule sensing and nucleic acid and protein sequencing. To further improve the detection resolution and precision, the combined photoelectric detection that can achieve higher accuracy via introducing optical-responsive molecules is adopted. In view of the extensive applications and development status of nanopore technique in single-molecule conformation resolution, this paper summarizes the research progress of the nanopore devices and the design and applications of some typical photo-responsive molecules in nanopore sensing, including azobenzenes and their derivatives (Azos), spiropyrane (SP) and diarylethene (DAE). Light regulation is a simple and effective approach for the monitoring of the molecular structures, and the further integration with nanopore devices exhibits great potential in single-molecule detection meanwhile sheds light on the design and applications of multi-responsive nano-devices.

Advanced DNA Nanopore Technologies.

Diverse nanopore-based technologies have substantially expanded the toolbox for label-free single-molecule sensing and sequencing applications. Biological protein pores, lithographically fabricated solid-state and graphene nanopores, and hybrid pores are in widespread use and have proven to be feasible devices for detecting amino acids, polynucleotides, and their specific conformations. However, despite the indisputable and remarkable advantages in technological exploration and commercialization of such equipment, the commonly used methods may lack modularity and specificity in characterization of particular phenomena or in development of nanopore-based devices. In this review, we discuss DNA nanopore techniques that harness the extreme addressability, precision, and modularity of DNA nanostructures that can be incorporated as customized gates or plugs into for example lipid membranes, solid-state pores, and nanocapillaries, thus forming advanced hybrid instruments. In addition to these, there exist a number of diverse DNA-assisted nanopore-based detection and analysis methods. Here, we introduce different types of DNA nanostructure-based pore designs and their intriguing properties as well as summarize the extensive collection of current and future technologies and applications that can be realized through combining DNA nanotechnology with common nanopore approaches.

Current and Emerging Trends in Point-of-Care Technology and Strategies for Clinical Validation and Implementation.

Point-of-care technology (POCT) provides actionable information at the site of care to allow rapid clinical decision-making. With healthcare emphasis shifting toward precision medicine, population health, and chronic disease management, the potential impact of POCT continues to grow, and several prominent POCT trends have emerged or strengthened in the last decade. This review summarizes current and emerging trends in POCT, including technologies approved or cleared by the Food and Drug Administration or in development. Technologies included have either impacted existing clinical diagnostics applications (e.g., continuous monitoring and targeted nucleic acid testing) or are likely to impact diagnostics delivery in the near future. The focus is limited to in vitro diagnostics applications, although in some sections, technologies beyond in vitro diagnostics are also included given the commonalities (e.g., ultrasound plug-ins for smart phones). For technologies in development (e.g., wearables, noninvasive testing, mass spectrometry and nuclear magnetic resonance, paper-based diagnostics, nanopore-based devices, and digital microfluidics), we also discuss their potential clinical applications and provide perspectives on strategies beyond technological and analytical proof of concept, with the end goal of clinical implementation and impact. The field of POCT has witnessed strong growth over the past decade, as evidenced by new clinical or consumer products or research and development directions. Combined with the appropriate strategies for clinical needs assessment, validation, and implementation, these and future POCTs may significantly impact care delivery and associated outcomes and costs.

Zero-mode waveguide detection of DNA translocation through FIB-organised arrays of engineered nanopores

Zero-mode waveguides (ZMWs) are optical nanostructures fabricated in a thin metallic film capable of confining the excitation volume to the zeptoliter range.In this work we describe the batch fabrication of a nanopore-based device, based upon high-resolution arrays of nanopores (of various sizes), which is used to directly measure the passage of DNA. In our method, nanopores are fabricated in ultrathin dielectric films with a deposited gold layer. The gold layer on the device induces a zero-mode waveguide illumination at the cis end of the nanopores.The method presented allows for optical detection, in real time, at the level of a single molecule and a single pore. The detection of fluorescently labelled single molecules passing through the pores, measured using an electron multiplying charge coupled device camera, is described. Molecules inside the nanopore were invisible until they reached the volume illuminated by the evanescent field.This fabrication methodology appears to be very promising for the development and batch fabrication of a new generation of nanopore-based sensor devices.

Sequence-Specific Detection of MicroRNAs Related to Clear Cell Renal Cell Carcinoma at fM Concentration by an Electroosmotically Driven Nanopore-Based Device.

MicroRNAs (miRs) are small noncoding RNAs that play a critical role in gene regulation. Recently, traces of cancer-related miRs have been identified in body fluids, which make them remarkable noninvasive biomarkers. In this study, a new nanopore-based detection scheme utilizing a borosilicate micropipette and an assay of complementary γ-peptide nucleic acid (γ-PNA) probes conjugated to polystyrene beads have been reported for the detection of miR-204 and miR-210 related to the clear cell Renal Cell Carcinoma (ccRCC). Electroosmotic flow (EOF) is induced as the driving force to transport PNA-beads harboring target miRs to the tip of the pore (sensing zone), which results in pore blockades with unique and easily distinguishable serrated shape electrical signals. The concentration detection limit is investigated to be 1 and 10 fM for miR-204 and miR-210, respectively. The EOF transport mechanism enables highly sensitive detection of molecules with low surface charge density with 97.6% detection accuracy compared to the conventional electrophoretically driven methods. Furthermore, resistive-pulse experiments are conducted to study the correlation of the particles' surface charge density with their translocation time and verify the detection principle.

Three-dimensional structures of magnesium nanopores

The optimization of nanopore-based devices is closely related to the nanopore three-dimensional (3D) structures. In this paper, faceted nanopores were fabricated in magnesium (Mg) by aligning the electron beam (e-beam) along the [0001] direction. Detailed structural characterization by transmission electron microscopy reveals the existence of two 3D structures: hexagonal prism-shaped and hourglass-shaped 3D morphologies. Moreover, the 3D structures of nanopores are also found to depend on the widest nanopore diameter-to-thickness ratio (D/t). A plausible formation mechanism for different 3D structures is discussed. Our results incorporate a critical piece of information regarding the nanopore 3D structures in Mg and may serve as an important design guidance for the size- and shape-controllable fabrication of solid-state nanopores applying the e-beam sculpting technique.

MD Study of Solution Concentrations on Ion Distribution in a Nanopore-Based Device Inspired from Red Blood Cells.

A molecular dynamics model of a nanopore-based device, which is similar to the nanopores in a cell membrane, was used to determine the influence of solution concentration on radial ion distribution, screening effects, and the radial potential profile in the nanopore. Results from these simulations indicate that as the solution concentration increases, the density peaks for both the counterion and coion near the charged wall increase at different speeds as screening effects appeared. Consequently, the potential near the charged wall of the nanopore changed from negative to positive during the simulation. The detailed understanding of ion distribution in nanopores is important for controlling the ion permeability and improving the cell transfection and also the design and application of nanofluidic devices.

Atomic layer deposition modified track-etched conical nanochannels for protein sensing.

Nanopore-based devices have recently become popular tools to detect biomolecules at the single-molecule level. Unlike the long-chain nucleic acids, protein molecules are still quite challenging to detect, since the protein molecules are much smaller in size and usually travel too fast through the nanopore with poor signal-to-noise ratio of the induced transport signals. In this work, we demonstrate a new type of nanopore device based on atomic layer deposition (ALD) Al2O3 modified track-etched conical nanochannels for protein sensing. These devices show very promising properties of high protein (bovine serum albumin) capture rate with well time-resolved transport signals and excellent signal-to-noise ratio for the transport events. Also, a special mechanism involving transient process of ion redistribution inside the nanochannel is proposed to explain the unusual biphasic waveshapes of the current change induced by the protein transport.

Capture, Unfolding, and Detection of Individual tRNA Molecules Using a Nanopore Device.

Transfer RNAs (tRNA) are the most common RNA molecules in cells and have critical roles as both translators of the genetic code and regulators of protein synthesis. As such, numerous methods have focused on studying tRNA abundance and regulation, with the most widely used methods being RNA-seq and microarrays. Though revolutionary to transcriptomics, these assays are limited by an inability to encode tRNA modifications in the requisite cDNA. These modifications are abundant in tRNA and critical to their function. Here, we describe proof-of-concept experiments where individual tRNA molecules are examined as linear strands using a biological nanopore. This method utilizes an enzymatically ligated synthetic DNA adapter to concentrate tRNA at the lipid bilayer of the nanopore device and efficiently denature individual tRNA molecules, as they are pulled through the α-hemolysin (α-HL) nanopore. Additionally, the DNA adapter provides a loading site for ϕ29 DNA polymerase (ϕ29 DNAP), which acts as a brake on the translocating tRNA. This increases the dwell time of adapted tRNA in the nanopore, allowing us to identify the region of the nanopore signal that is produced by the translocating tRNA itself. Using adapter-modified Escherichia coli tRNAfMet and tRNALys, we show that the nanopore signal during controlled translocation is dependent on the identity of the tRNA. This confirms that adapter-modified tRNA can translocate end-to-end through nanopores and provide the foundation for future work in direct sequencing of individual transfer RNA with a nanopore-based device.

PH-regulated ionic conductance in a nanopore

Analytical expressions for the pH-regulated ionic current/conductance in a cylindrical nanopore are derived for taking into account practical effects of multiple ionic species, surface chemistry reactions, and electroosmotic flow. Our model is verified by the experimental data of the conductance in the silica nanopore available in the literature. The validated analytical models are applicable to investigate electrokinetic ion transport, and to design the next-generation nanopore-based devices.

Electrochemistry at the Edge of a Single Graphene Layer in a Nanopore

We study the electrochemistry of single layer graphene edges using a nanopore-based structure consisting of stacked graphene and Al(2)O(3) dielectric layers. Nanopores, with diameters ranging from 5 to 20 nm, are formed by an electron beam sculpting process on the stacked layers. This leads to a unique edge structure which, along with the atomically thin nature of the embedded graphene electrode, demonstrates electrochemical current densities as high as 1.2 × 10(4) A/cm(2). The graphene edge embedded structure offers a unique capability to study the electrochemical exchange at an individual graphene edge, isolated from the basal plane electrochemical activity. We also report ionic current modulation in the nanopore by biasing the embedded graphene terminal with respect to the electrodes in the fluid. The high electrochemical specific current density for a graphene nanopore-based device can have many applications in sensitive chemical and biological sensing, and energy storage devices.

Controlling pH-Regulated Bionanoparticles Translocation through Nanopores with Polyelectrolyte Brushes

A novel polyelectrolyte (PE)-modified nanopore, comprising a solid-state nanopore functionalized by a nonregulated PE brush layer connecting two large reservoirs, is proposed to regulate the electrokinetic translocation of a soft nanoparticle (NP), comprising a rigid core covered by a pH-regulated, zwitterionic, soft layer, through it. The type of NP considered mimics bionanoparticles such as proteins and biomolecules. We find that a significant enrichment of H(+) occurs near the inlet of a charged solid-state nanopore, appreciably reducing the charge density of the NP as it approaches there, thereby lowering the NP translocation velocity and making it harder to thread the nanopore. This difficulty can be resolved by the proposed PE-modified nanopore, which raises effectively both the capture rate and the capture velocity of the soft NP and simultaneously reduces its translocation velocity through the nanopore so that both the sensing efficiency and the resolution are enhanced. The results gathered provide a conceptual framework for the interpretation of relevant experimental data and for the design of nanopore-based devices used in single biomolecules sensing and DNA sequencing.

Modeling and simulation of nanoparticle separation through a solid‐state nanopore

Recent experimental studies show that electrokinetic phenomena such as electroosmosis and electrophoresis can be used to separate nanoparticles on the basis of their size and charge using nanopore-based devices. However, the efficient separation through a nanopore depends on a number of factors such as externally applied voltage, size and charge density of particle, size and charge density of membrane pore, and the concentration of bulk electrolyte. To design an efficient nanopore-based separation platform, a continuum-based mathematical model is used for fluid. The model is based on Poisson-Nernst-Planck equations along with Navier-Stokes equations for fluid flow and on the Langevin equation for particle translocation. Our numerical study reveals that membrane pore surface charge density is a vital parameter in the separation through a nanopore. In this study, we have simulated high-density lipoprotein (HDL) and low-density lipoprotein (LDL) as the sample nanoparticles to demonstrate the capability of such a platform. Numerical results suggest that efficient separation of HDL from LDL in a 0.2 M KCL solution (resembling blood buffer) through a 150 nm pore is possible if the pore surface charge density is ∼ -4.0 mC/m(2). Moreover, we observe that pore length and diameter are relatively less important in the nanoparticle separation process considered here.

Nanopore-Based Devices for Bioanalytical Applications

With over a decade passed since the first reported use of a Staphylococcal α-hemolysin pore to study single molecules of single-stranded DNA, research in the field of nanopores has advanced rapidly. We discuss the technological progression of nanopore-based devices from the initial use of α-hemolysin pores to the advent of solid-state nanopores to the burgeoning of organic-inorganic hybrid pores driven by the desire to achieve fast and inexpensive DNA sequencing. Additional nanopore-based efforts are also discussed that study other classes of molecules, such as proteins. We discuss the use of nanopores for protein folding and binding analysis. In addition to single-molecule analysis, we report on the introduction of nanopore arrays on thin film membranes for ultrafiltration. Owing to their reduced spatial dimensionality, such membranes offer greater control over how the pores interact with analytes thus leading to very efficient separation. With several technical hindrances yet to be overcome, the devices we report are still works in progress. The realization of these devices will enhance laboratory processes by permitting superior spatial and temporal analytical resolution at the single-molecule level resulting in laboratory capacities of great impact.

Distinguishing Single- and Double-Stranded Nucleic Acid Molecules Using Solid-State Nanopores

Solid-state nanopores offer a promising method for rapidly probing the structural properties of biopolymers such as DNA and RNA. We have for the first time translocated RNA molecules through solid-state nanopores, comparing the signatures of translocating double-stranded RNA molecules and of single-stranded homopolymers poly(A), poly(U), poly(C). On the basis of their differential blockade currents, we can rapidly discriminate between both single- and double-stranded nucleic-acid molecules, as well as separate purine-based homopolymers from pyrimidine-based homopolymers. Molecule identification is facilitated through the application of high voltages ( approximately 600 mV), which contribute to the entropic stretching of these highly flexible molecules. This striking sensitivity to relatively small differences in the underlying polymer structure greatly improves the prospects for using nanopore-based devices for DNA or RNA mapping.