Solid-state Nanopores Research Articles

Solid-State Nanopores for Spatially Resolved Chemical Neuromodulation.

Most neural prosthetic devices are based on electrical stimulation, although the modulation of neuronal activity by a localized chemical delivery would better mimic physiological synaptic machinery. In the past decade, various drug delivery approaches attempted to emulate synaptic transmission, although they were hampered by poor retention of their cargo while reaching the target destination, low spatial resolution, and poor biocompatibility and stability of the materials involved. Here, we propose a planar solid-state device for multisite neurotransmitter translocation at the nanoscale consisting of a nanopatterned ceramic membrane connected to a reservoir designed to store neurotransmitters. We achieved diffusion-mediated glutamate stimulation of primary neurons, while we showed the feasibility to translocate other molecules through the pores by either pressure or diffusion, proving the versatility of the proposed technology. Finally, the system proved to be a promising neuronal stimulation interface in mice and nonhuman primates ex vivo, paving the way toward a biomimetic chemical stimulation in neural prosthetics and brain machine interfaces.

Detecting and Controlling DNA Translocation through a Nanopore using a van der Waals Heterojunction Diode.

A long-unrealized goal in solid-state nanopore sensing is to achieve out-of-plane electrical sensing and control of DNA during translocation, which is a prerequisite for base-by-base ratcheting that enables DNA sequencing in biological nanopores. Two-dimensional (2D) heterostructures, with their capability to construct out-of-plane electronics with atomic layer precision, are ideal yet unexplored candidates for use as electrical sensing membranes. Here we demonstrate a nanopore architecture using a vertical 2D heterojunction diode consisting of p-type WSe2 on n-type MoS2. This diode exhibits rectified interlayer tunneling currents modulated by ionic potential, while the heterojunction potential reciprocally rectifies ionic transport through the nanopore. We achieve concurrent detection of DNA translocation using both ionic and diode currents and demonstrate a 2.3-fold electrostatic slowing of translocation speed. Encapsulation layers enable robust operation while preserving the spatial resolution of atomically sharp 2D heterointerface for sensing. These results establish a paradigm for out-of-plane electrical sensing and control of single biomolecules.

Graphite-Based Bio-Mimetic Nanopores for Protein Sequencing and Beyond.

Protein sequencing using nanopores represents the next frontier in bio-analytics. However, linearizing unfolded proteins and controlling their translocation speed through solid-state nanopores pose significant challenges in protein sequencing. In order to address these issues, this work proposes a biomimetic graphite-based nanopore construction. These nanopores feature a nanometer-sized pore with a constriction zone, mimicking the structure of the α-hemolysin protein pore. Ourall-atom Molecular Dynamics simulations demonstrate the high practical potential of these nanopores by revealing how their charge state renders them complete ion-selective and generates an electro-osmotic flow. This study shows that this nanopore construction can detect peptides at the single amino acid level by analyzing the ionic current traces generated as peptides traverse the nanopore. The novelty of the proposed nanopore lies in its ability to modulate the hydrodynamic drag induced by electro-osmotic flow, relative to the electro-phoretic force. This investigation reveals that tuning these forces helps to linearize translocating peptides and extend the residence time of individual amino acids at the constriction zone of the pore. This significantly enhances the detection and sequencing efficiency of the pore. Furthermore, the high relevance of the proposed nanopores is underscored for seawater desalination through electrodialysis and extends to ion separation under salinitygradients.

Automated-Screening Oriented Electric Sensing of Vitamin B1 Using a Machine Learning Aided Solid-State Nanopore.

Micronutrient detection and identification at the single-molecule level are paramount for both clinical and home diagnostics. Analytical tools such as high-performance liquid chromatography and liquid chromatography-tandem mass spectrometry have been widely used but include a high instrument cost and prolonged analysis time. Here, as a model system, by merging nanopore signatures with machine learning algorithms, we propose an automated electric sensing strategy to identify vitamin B1 and its phosphorylated derivatives with good accuracy. Further, the relationship between vitamin B1 dynamics and nanopore signatures is examined. To understand the machine-decision-making process, Shapley additive explanations are made. Using a machine learning aided solid-state nanopore, we pave the way for next-generation micronutrient detection.

Hydrogel interfaced glass nanopore for high-resolution sizing of short DNA fragments

Solid-state nanopores, known for their label-free detection and operational simplicity, face challenges in accurately sizing the short nucleic acids due to fast translocation and a lack of enzyme-based control mechanisms as compared to their biological counterparts with sizing resolutions still limited to ≥100 bp. Here, we present a facile polyethylene glycol-dimethacrylate (PEG-DMA) hydrogel interfaced glass nanopore (HIGN) system by inserting glass nanopore into the hydrogel to achieve sub-100 base pair (bp) resolution in short DNA sizing analysis. We systematically investigated the effects of hydrogel mesh size, spatial configurations of glass nanopores about the hydrogel, applied bias voltage, and analyte concentration on the transport dynamics of 200 bp double-stranded DNA (dsDNA). A 7.5 w/v% PEG-DMA hydrogel induced ∼11x increase in the mean dwell times compared with bare solution nanopore system. The insertion locations and depths of the glass nanopore into the hydrogel resulted in 7.16% and 5.28% coefficients of variation (CV) for mean normalized event frequencies. This enhancement of dwell times and invariability in translocation characteristics enables precise sizing of dsDNA fragments under 400 bp using HIGN, with an achieved size resolution of 50 bp with observable mean normalized peak amplitude (ΔI/Io) of ∼0.005. Furthermore, we have demonstrated the capability of HIGN to perform multiplex detection of influenza A virus (IAV) and severe acute respiratory syndrome-coronavirus-2 (SARS-CoV-2) through reverse transcriptase-polymerase chain reaction (RT-PCR). These results demonstrated the potential of HIGN as a versatile tool in nucleic acid analysis and multiplexed label-free molecular diagnostics.

DNA Origami Incorporated into Solid-State Nanopores Enables Enhanced Sensitivity for Precise Analysis of Protein Translocations.

The rapidly advancing field of nanotechnology is driving the development of precise sensing methods at the nanoscale, with solid-state nanopores emerging as promising tools for biomolecular sensing. This study investigates the increased sensitivity of solid-state nanopores achieved by integrating DNA origami structures, leading to the improved analysis of protein translocations. Using holo human serum transferrin (holo-hSTf) as a model protein, we compared hybrid nanopores incorporating DNA origami with open solid-state nanopores. Results show a significant enhancement in holo-hSTf detection sensitivity with DNA origami integration, suggesting a unique role of DNA interactions beyond confinement. This approach holds potential for ultrasensitive protein detection in biosensing applications, offering advancements in biomedical research and diagnostic tool development for diseases with low-abundance protein biomarkers. Further exploration of origami designs and nanopore configurations promises even greater sensitivity and versatility in the detection of a wider range of proteins, paving the way for advanced biosensing technologies.

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.

Genomic Analysis of Aeromonas salmonicida ssp. salmonicida Isolates Collected During Multiple Clinical Outbreaks Supports Association with a Single Epidemiological Unit.

Outbreaks of furunculosis cause significant losses in salmonid aquaculture worldwide. With a recent rise in antimicrobial resistance, regulatory measures to minimize the use of antibiotics in animal husbandry, including aquaculture, have increased scrutiny and availability of veterinary medical products to control this disease in production facilities. In such a regulatory environment, the utility of autogenous vaccines to assist with disease prevention and control as a veterinary-guided prophylactic measure is of high interest to the producers and veterinary services alike. However, evolving concepts of epidemiological units and epidemiological links need to be considered during approval and acceptance procedures for the application of autogenous vaccines in multiple aquaculture facilities. Here, we present the results of solid-state nanopore sequencing (Oxford Nanopore Technologies, ONT) performed on 54 isolates of Aeromonas salmonicida ssp. salmonicida sampled during clinical outbreaks of furunculosis in different aquaculture facilities from Bavaria, Germany, from 2017 to 2020. All of the performed analyses (phylogeny, single nucleotide polymorphism and 3D protein modeling for major immunogenic proteins) support a high probability that all studied isolates belong to the same epidemiological unit. Simultaneously, we describe a cost/effective method of whole genome analysis with the usage of ONT as a viable strategy to study outbreaks of other pathogens in the field of aquatic veterinary medicine for the purpose of developing the best autogenous vaccine candidates applicable to multiple aquaculture establishments.

Solid-State Nanopore-Based Nanosystem for Registration of Enzymatic Activity of a Single Molecule of Cytochrome P450 BM3.

Experimental methods of single-molecule enzymology allow scientists to determine physicochemical properties of distinct single molecules of various enzymes and to perform direct monitoring of functioning of enzymes at different steps of their catalytic cycle. The approach based on the use of solid-state nanopores is a promising tool for studying the functioning of single-enzyme molecules. Herein, this approach is employed for monitoring the functioning of cytochrome P450 BM3, which represents a very convenient model of cytochrome P450-containing monooxygenase systems. A nanopore of ~5 nm in diameter has been formed in a 40 nm-thick silicon nitride chip by electron beam drilling (EBD), and a single molecule of the BM3 enzyme has been entrapped in the pore. The functioning of the enzyme molecule has been monitored by recording the time dependence of the ion current through the nanopore during the reaction of laurate hydroxylation. In our experiments, the enzyme molecule has been found to be active for 1500 s. The results of our research can be further used in the development of highly sensitive detectors for single-molecule studies in enzymology.

Investigating the expansion behavior of silicon nitride nanopores

Nanopores are a single-molecule detection platform that has attracted more and more researchers’ attention, but so far there is still a lack of systematic research on their stability. In this study, we investigated the mechanism behind the expansion behavior of solid-state nanopores and its effect on the electrical properties of nanopores. Through the analysis of electron energy loss spectroscopy, it was found that the expansion process of nanopores is accompanied by the loss of silicon elements. Three nanopore expansion models can well explain the differential impact of silicon element loss in different regions on the electrical properties of nanopores. Molecular dynamics simulations analyzed the differences in nanopore conductivity and expansion rate corresponding to different expansion models from the perspective of single-atom loss. Finally, we demonstrated that chemical modification can isolate direct contact between the nanopore wall and the solution, thereby greatly delaying the expansion behavior of solid-state nanopores.

Detection of nanopores with the scanning ion conductance microscopy: A simulation study

During the dielectric breakdown process of thin solid-state nanopores, the application of high voltages may cause the formation of multi-nanopores on one chip, which number and sizes are important for their applications. Here, simulations were conducted to mimic the investigation of in situ nanopore detection with scanning ion conductance microscopy (SICM). Results show that SICM can provide accurate nanopore location and relative pore size. Detection resolution is influenced by the dimensions of the applied probe and separation between the probe and membranes, which can be enhanced under large voltages or a concentration gradient.

Computational and experimental study of AC measurements performed by a double-nanohole plasmonic nanopore sensor on 20 nm silica nanoparticles

A novel method of AC sensing is presented that uses a double nanohole (DNH) nanoaperture atop a solid-state nanopore (ssNP) to trap analytes and measure their optical and electrical properties. In this method analytes are propelled by an external applied voltage towards the sensor until they are trapped at the DNH-ssNP interface via a self-induced back action (SIBA) plasmonic force. We have previously named this method SIBA Actuated Nanopore Electrophoresis (SANE) sensing and have shown its ability to perform concurrent optical and DC electrical measurements. Here, we extend this method to AC sensing of 20 nm SiO2 (silica) nanoparticles, using voltage modulation over a wide range of frequencies applied on top of a baseline DC bias. The sensor was constructed using two-beam GFIS Focused Ion Beam (FIB) lithography, incorporating Ne FIB to mill the DNH and He FIB to drill a central 30 nm ssNP. We utilized COMSOL Multiphysics simulations to explore the multi-frequency AC current conductance properties of the silica nanoparticles trapped at the SANE sensor. These simulations computed conductance changes and phase shifts induced by the presence of the nanoparticle over an AC frequency range of 20 Hz to 100 kHz. Experimental measurements confirmed the trends seen in the computational data. Additional computational studies were then performed to dissect the underlying mechanisms driving the observed AC measurements. Looking forward, we aim to adapt this technology for probing therapeutic nanoparticles non-invasively, offering a promising tool for enhancing quality control of nanoparticle-mediated drug and gene delivery systems.

Bidirectional Peptide Translocation through Ultrasmall Solid-State Nanopores.

It is important to obtain the configuration of polypeptides and the sequence information on amino acids for understanding various life processes and many biological applications. Nanopores, as a newly developed single-molecule detection technology, exhibit unique advantages in real-time dynamics detection. Here, we designed a special peptide chain with 10 arginine in the head and achieved successful single-molecule detection by ultrasmall solid-state nanopores (2-3 nm). Unique bidirectional translocation signals were observed and explained under the framework of charge distribution of the peptide and interaction with the nanopore wall. Two natural peptide chains, histatin-5 and angiopep-2, were also explored by nanopore experiments to confirm our conjecture. Our designed peptide chain could realize multiple detections of the same peptide chain, offering possibilities for high-resolution peptide detection and fingerprinting by solid-state nanopores in the future.

Nucleosomal DNA unwinding pathway through canonical and non-canonical histone disassembly

The nucleosome including H2A.B, a mammalian-specific H2A variant, plays pivotal roles in spermatogenesis, embryogenesis, and oncogenesis, indicating unique involvement in transcriptional regulation distinct from canonical H2A nucleosomes. Despite its significance, the exact regulatory mechanism remains elusive. This study utilized solid-state nanopores to investigate DNA unwinding dynamics, applying local force between DNA and histones. Comparative analysis of canonical H2A and H2A.B nucleosomes demonstrated that the H2A.B variant required a lower voltage for complete DNA unwinding. Furthermore, synchronization analysis and molecular dynamics simulations indicate that the H2A.B variant rapidly unwinds DNA, causing the H2A-H2B dimer to dissociate from DNA immediately upon disassembly of the histone octamer. In contrast, canonical H2A nucleosomes unwind DNA at a slower rate, suggesting that the H2A-H2B dimer undergoes a state of stacking at the pore. These findings suggest that nucleosomal DNA in the H2A.B nucleosomes undergoes a DNA unwinding process involving histone octamer disassembly distinct from that of canonical H2A nucleosomes, enabling smoother unwinding. The integrated approach of MD simulations and nanopore measurements is expected to evolve into a versatile tool for studying molecular interactions, not only within nucleosomes but also through the forced dissociation of DNA-protein complexes.

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.

Registration of activity of a single molecule of horseradish peroxidase using a detector based on a solid-state nanopore.

This work demonstrates the use of a solid-state nanopore detector to monitor the activity of a single molecule of a model enzyme, horseradish peroxidase (HRP). This detector includes a measuring cell, which is divided into cis- and trans- chambers by a silicon nitride chip (SiN structure) with a nanopore of 5 nm in diameter. To entrap a single HRP molecule into the nanopore, an electrode had been placed into the cis-chamber; HRP solution was added into this chamber after application of a negative voltage. The reaction of the HRP substrate, 2,2-azino-bis(3-ethylbenzothiazoline-6-sulfonate) (ABTS), oxidation by the enzyme molecule was performed in the presence of hydrogen peroxide. During this reaction, the functioning of a single HRP molecule, entrapped in the nanopore, was monitored by recording the time dependence of the ion current flowing through the nanopore. The approach proposed in our work is applicable for further studies of functioning of various enzymes at the level of single molecules, and this is an important step in the development of single-molecule enzymology.

De-wetted gold nanostructures for SERS-based sensing of static and dynamic targets

De-wetting of metal thin film is one of the simplest fabrication techniques to produce metallic nanostructures for Surface-Enhanced Raman Spectroscopy (SERS)-based sensing. In this manuscript, we explore the sensitivity of a de-wetted metal SERS substrate by testing it with the organic dye Rhodamine 6G (R6G) as well as with the single amino acids (Glycine, Serine, and Histidine) and a binary mixture of Glycine and Serine mixture. We observe that repeated de-wetting of the metal thin film provides a more than ten times higher SERS enhancement factor compared to a single de-wetting. This is attributed to the presence of more closely spaced metal nanoclusters, enabling a higher electromagnetic field enhancement at the hotspots. We characterized the sensing capability of our SERS substrates by detecting R6G dye drop-casted onto the substrates at sub-femtomolar concentrations. We were also able to detect R6G in a dynamic state while it was transported across a 500 nm diameter pore with a nanomolar detection limit. Finally, we were also able to detect amino acids with a micromolar detection limit using our SERS substrate. These results indicate the promise in the integration of our SERS substrate with solid-state nanopores for SERS-based signal readout where concentrations higher than nanomolar exist in the pore due to high confinement.

Research Progress on Saccharide Molecule Detection Based on Nanopores.

Saccharides, being one of the fundamental molecules of life, play essential roles in the physiological and pathological functions of cells. However, their intricate structures pose challenges for detection. Nanopore technology, with its high sensitivity and capability for single-molecule-level analysis, has revolutionized the identification and structural analysis of saccharide molecules. This review focuses on recent advancements in nanopore technology for carbohydrate detection, presenting an array of methods that leverage the molecular complexity of saccharides. Biological nanopore techniques utilize specific protein binding or pore modifications to trigger typical resistive pulses, enabling the high-sensitivity detection of monosaccharides and oligosaccharides. In solid-state nanopore sensing, boronic acid modification and pH gating mechanisms are employed for the specific recognition and quantitative analysis of polysaccharides. The integration of artificial intelligence algorithms can further enhance the accuracy and reliability of analyses. Serving as a crucial tool in carbohydrate detection, we foresee significant potential in the application of nanopore technology for the detection of carbohydrate molecules in disease diagnosis, drug screening, and biosensing, fostering innovative progress in related research domains.

PH and potential-controlled multi-modal mass transport in block copolymer nanochannel membranes

Inspired by the hydrophobic gating for achieving fast and selective ion/molecular transport in cell membranes, wetting/dewetting transition in solid-state nanopores controlled by external stimuli such as voltage, pH, electrostatics, and light have attracted increasing attention. For an accurate and better understanding, a single nanopore or low-density array of nanopores was preferred to investigate the wetting and dewetting transitions owing to their well-defined chemical functions and physical structures. However, high-density nanochannel membranes capable of processing high-throughput and multi-modal mass transport are more beneficial with the aim of practical use. In this regard, pH- and potential-responsive nanochannel membranes consisting of a polystyrene-b-poly(4-vinylpyridine) (PS-b-P4VP) block copolymer (BCP) are prepared to demonstrate a multi-modal transport system with high-throughput capability. At pH < pKa(P4VP) (pKa ∼ 4.8), the cylindrical P4VP nanodomains are hydrophilic and positively charged, acting as an anion-exchange membrane. In contrast, at pH > pKa(P4VP), the P4VP domains switch to be charge-neutral and hydrophobic, naturally blocking the mass transport through the nanochannels. Applying a sufficiently positive potential to a BCP membrane-coated electrode may induce oxidative wetting in the hydrophobic nanochannels to facilitate mass transport across the membrane with no charge-selectivity. Releasing the bias makes the hydrophobic nanochannel membranes retrieve the original dewetted state, blocking the transport again. In addition, direct observation of the wetting-dewetting transition dynamics in the hydrophobic nanochannels is investigated by monitoring potential-correlated electrochemiluminescence (ECL) signals arising from Ru(bpy)32+ and co-reactant tripropylamine (TPA) under potential modulations. ECL signals tend to decrease with increasing membrane thickness ranging from 0 nm to 820 nm because it requires higher potentials to induce wetting in the nanochannels due to elongated hydrophobic nanochannels. The multi-modal transport system developed in the present work will be useful for applications such as water treatment, biosensors, and smart valve systems like controlled drug release/delivery.

Ten Thousand Recaptures of a Single DNA Molecule in a Nanopore and Variance Reduction of Translocation Characteristics.

Nanopores have emerged as highly sensitive biosensors operating at the single-molecule level. However, the majority of nanopore experiments still rely on averaging signals from multiple molecules, introducing systematic errors. To overcome this limitation and obtain accurate information from a single molecule, the molecular ping-pong methodology provides a precise approach involving repeated captures of a single molecule. In this study, we have enhanced the molecular ping-pong technique by incorporating a customized electronic system and control algorithm, resulting in a recapture number exceeding 10,000. During the ping-pong process, we observed a significant reduction in the variance of translocation characteristics, providing fresh insights into single-molecule translocation dynamics. An inhomogeneous translocation velocity of folded DNA has been revealed, illustrating a strong interaction between the molecule and the solid-state nanopore. The results not only promise heightened experimental efficiency with reduced sample volume but also increase the precision in statistical analysis of translocation events, marking a significant stride toward authentic single-molecule nanopore sensing.