Solid-state Nanopores Research Articles

Ultrahigh Spatial Resolution Cross-Disjoint Mortise-Confined Solid-State Nanopores with an Ultrathin Middle Layer

Ultrahigh Spatial Resolution Cross-Disjoint Mortise-Confined Solid-State Nanopores with an Ultrathin Middle Layer

In-Plane, In-Series Nanopores with Circular Cross Sections for Resistive-Pulse Sensing.

Resistive-pulse sensing with solid-state nanopores is a sensitive, label-free technique for analyzing single molecules in solution. To add functionality to resistive-pulse measurements, direct coupling of the nanopores to other pores and nanoscale fluidic elements, e.g., reactors, separators, and filters, in the same device is an important next step. One approach is monolithic fabrication of the fluidic elements in the plane of the substrate, but methods to generate pores with circular cross sections are needed to improve sensing performance with in-plane devices. Here, we report a fabrication method that directly patterns nanopores with circular cross sections in series and in plane with the substrate. A focused ion beam instrument is used to mill a lamella in a nanochannel and, subsequently, bore a nanopore through the lamella. The diameter and geometry of the nanopore are controlled by the current and dose of the ion beam and by the tilt angle and thickness of the lamella. We fabricated devices with vertical and tilted lamellae and nanopores with diameters from 40 to 90 nm in cylindrical and conical geometries. To test device performance, we conducted resistive-pulse measurements of hepatitis B virus capsids. Current pulses from T = 3 capsids (∼31 nm diameter) and T = 4 capsids (∼35 nm diameter) were well resolved and exhibited relative pulse amplitudes (Δi/i) up to 5 times higher than data obtained on nanopores with rectangular cross sections. For smaller pore diameters (<45 nm), which approach the diameters of the capsids, a dramatic increase in the pulse amplitude was observed for both T = 3 and T = 4 capsids. Two and three pores fabricated in series further improved the resolution between the relative pulse amplitude distributions for the T = 3 and T = 4 capsids by up to 2-fold.

Single-molecule analysis of DNA structures using nanopore sensors

Nanopore has been studied for a lot of applications in DNA sequencing, multifarious single-molecule detection, detection of molecular conformational change, and biomolecular interaction. There are many categories of nanopores, such as biological nanopores, solid-state nanopores and newly developed hybrid nanopores. All of them play an important role in the detection of DNA structures because of their unique properties, such as the nano-scale pore size, dynamic detection in solution, and low sample loading. This review elaborates on the detection of several DNA structures by nanopore, including the dynamic analysis of conformational changes, and the factors that promote conformational changes. In addition, the detection of DNA origami, a type of artificial structured DNA, is also discussed. In these tests, current signals were produced by the interaction of analyte and nanopore. The signals were often studied by the statistical features of the currents’ amplitude, duration time, or both of them. Finally, taking advantage of the nanopore's ability to detect the conformational changes of these folds, the application of DNA structures detection in nanopore is introduced.

Light-Controlled Ionic/Molecular Transport through Solid-State Nanopores and Nanochannels.

Biological nanochannels perfectly operate in organisms and exquisitely control mass transmembrane transport for complex life process. Inspired by biological nanochannels, plenty of intelligent artificial solid-state nanopores and nanochannels are constructed based on various materials and methods with the development of nanotechnology. Specially, the light-controlled nanopores/nanochannels have attracted much attention due to the unique advantages in terms of that ion and molecular transport can be regulated remotely, spatially and temporally. According to the structure and function of biological ion channels, light-controlled solid-state nanopores/nanochannels can be divided into light-regulated ion channels with ion gating and ion rectification functions, and light-driven ion pumps with active ion transport property. In this review, we present a systematic overview of light-controlled ion channels and ion pumps according to the photo-responsive components in the system. Then, the related applications of solid-state nanopores/nanochannels for molecular sensing, water purification and energy conversion are discussed. Finally, a brief conclusion and short outlook are offered for future development of the nanopore/nanochannel field.

Fine-tuning-based Transfer Learning for Characterization of Adeno-Associated Virus.

Accurate and precise identification of adeno-associated virus (AAV) vectors play an important role in dose-dependent gene therapy. Although solid-state nanopore techniques can potentially be used to characterize AAV vectors by capturing ionic current, the existing data analysis techniques fall short of identifying them from their ionic current profiles. Recently introduced machine learning methods such as deep convolutional neural network (CNN), developed for image identification tasks, can be applied for such classification. However, with smaller data set for the problem in hand, it is not possible to train a deep neural network from scratch for accurate classification of AAV vectors. To circumvent this, we applied a pre-trained deep CNN (GoogleNet) model to capture the basic features from ionic current signals and subsequently used fine-tuning-based transfer learning to classify AAV vectors. The proposed method is very generic as it requires minimal preprocessing and does not require any handcrafted features. Our results indicate that fine-tuning-based transfer learning can achieve an average classification accuracy between 90 and 99% in three realizations with a very small standard deviation. Results also indicate that the classification accuracy depends on the applied electric field (across nanopore) and the time frame used for data segmentation. We also found that the fine-tuning of the deep network outperforms feature extraction-based classification for the resistive pulse dataset. To expand the usefulness of the fine-tuning-based transfer learning, we have tested two other pre-trained deep networks (ResNet50 and InceptionV3) for the classification of AAVs. Overall, the fine-tuning-based transfer learning from pre-trained deep networks is very effective for classification, though deep networks such as ResNet50 and InceptionV3 take significantly longer training time than GoogleNet.

Lifetime of glass nanopores in a PDMS chip for single-molecule sensing

SummaryNanopore sensing is an emerging technology that has many biosensing applications ranging from DNA sequencing using biological pores to biomolecular analysis using solid-state pores. Solid-state nanopores that are more stable are an attractive choice for biosensing applications. Still, biomolecule interactions with the nanopore surface reduce nanopore stability and increase usage costs. In this study, we investigated the biosensing capability for 102 quartz glass nanopores with a diameter of 11–18 nm that were fabricated using laser-assisted capillary pulling. Nanopores were assembled into multiple microfluidic chips that were repeatedly used for up to 19 weeks. We find that using vacuum storage combined with minimal washing steps improved the number of use cycles for nanopores. The single-molecule biosensing capability over repeated use cycles was demonstrated by quantitative analysis of a DNA carrier designed for detection of short single-stranded DNA oligonucleotides.

Electronic Mapping of a Bacterial Genome with Dual Solid-State Nanopores and Active Single-Molecule Control.

We present an electronic mapping of a bacterial genome using solid-state nanopore technology. A dual-nanopore architecture and active control logic are used to produce single-molecule data that enables estimation of distances between physical tags installed at sequence motifs within double-stranded DNA. Previously developed "DNA flossing" control logic generates multiple scans of each captured DNA. We extended this logic in two ways: first, to automate "zooming out" on each molecule to progressively increase the number of tags scanned during flossing, and second, to automate recapture of a molecule that exited flossing to enable interrogation of the same and/or different regions of the molecule. Custom analysis methods were developed to produce consensus alignments from each multiscan event. The combined multiscanning and multicapture method was applied to the challenge of mapping from a heterogeneous mixture of single-molecule fragments that make up the Escherichia coli (E. coli) chromosome. Coverage of 3.1× across 2355 resolvable sites of the E. coli genome was achieved after 5.6 h of recording time. The recapture method showed a 38% increase in the merged-event alignment length compared to single-scan alignments. The observed intertag resolution was 150 bp in engineered DNA molecules and 166 bp natively within fragments of E. coli DNA, with detection of 133 intersite intervals shorter than 200 bp in the E. coli reference map. We present results on estimating distances in repetitive regions of the E. coli genome. With an appropriately designed array, higher throughput implementations could enable human-sized genome and epigenome mapping applications.

Optimizing the sensitivity and resolution of hyaluronan analysis with solid-state nanopores

Hyaluronan (HA) is an essential carbohydrate in vertebrates that is a potentially robust bioindicator due to its critical roles in diverse physiological functions in health and disease. The intricate size-dependent function that exists for HA and its low abundance in most biological fluids have highlighted the need for sensitive technologies to provide accurate and quantitative assessments of polysaccharide molecular weight and concentration. We have demonstrated that solid state (SS-) nanopore technology can be exploited for this purpose, given its molecular sensitivity and analytical capacity, but there remains a need to further understand the impacts of experimental variables on the SS-nanopore signal for optimal interpretation of results. Here, we use model quasi-monodisperse HA polymers to determine the dependence of HA signal characteristics on a range of SS-nanopore measurement conditions, including applied voltage, pore diameter, and ionic buffer asymmetry. Our results identify important factors for improving the signal-to-noise ratio, resolution, and sensitivity of HA analysis with SS-nanopores.

Single-Molecule Identification of the Conformations of Human C-Reactive Protein and Its Aptamer Complex with Solid-State Nanopores.

Human C-reactive protein (CRP) is an established inflammatory biomarker and was proved to be potentially relevant to disease pathology and cancer progression. A large body of methodologies have been reported for CRP analysis, including electrochemical/optical biosensors, aptamer, or antibody-based detection. Although the detection limit is rather low until pg/uL, most of which are time-consuming and relatively expensive, and few of them provided CRP single-molecule information. This work demonstrated the nanopore-based approach for the characterization of CRP conformation under versatile conditions. With an optimized pore of 14 nm in diameter, we achieved the detection limit as low as 0.3 ng/μL, voltage polarity significantly influences the electro-osmotic force and CRP translocation behavior, and the pentameric conformation of CRP may dissociate into pro-inflammatory CRP isoforms and monomeric CRP at bias potential above 300 mV. CRP tends to translocate through nanopores faster along with the increase in pH values, due to more surface charge on both CRP and pore inner wall and stronger electro-osmotic force. The CRP could specifically bind with its aptamer of different concentrations to form complexes, and the complexes exhibited distinguishable nanopore translocation behavior compared with CRP alone. The variation of the molar ratio of aptamer significantly influences the orientation of CRP translocation. The plasma test under physiological conditions displayed the ability of the nanopore system on the CRP identification with a concentration of 3 ng/μL.

Back Cover: Protein‐enabled detection of ibuprofen and sulfamethoxazole using solid‐state nanopores

The cover image is based on the Research Article Protein-enabled detection of ibuprofen and sulfamethoxazole using solid-state nanopores by Zehui Xia, Chih-Yuan Lin, and Marija Drndić., https://doi.org/10.1002/pmic.202100071.

Low-Area Four-Channel Controlled Dielectric Breakdown System Design for Point-of-Care Applications.

In this study, we propose a low-area multi-channel controlled dielectric breakdown (CDB) system that simultaneously produces several nanopore sensors. Conventionally, solid-state nanopores are prepared by etching or drilling openings in a silicon nitride (SiNx) substrate, which is expensive and requires a long processing time. To address these challenges, a CDB technique was introduced and used to fabricate nanopore channels in SiNx membranes. However, the nanopore sensors produced by the CDB result in a severe pore-to-pore diameter variation as a result of different fabrication conditions and processing times. Accordingly, it is indispensable to simultaneously fabricate nanopore sensors in the same environment to reduce the deleterious effects of pore-to-pore variation. In this study, we propose a four-channel CDB system that comprises an amplifier that boosts the command voltage, a 1-to-4 multiplexer, a level shifter, a low-noise transimpedance amplifier and a data acquisition device. To prove our design concept, we used the CDB system to fabricate four nanopore sensors with diameters of <10 nm, and its in vitro performance was verified using λ-DNA samples.

Label-Free Optical Analysis of Biomolecules in Solid-State Nanopores: Toward Single-Molecule Protein Sequencing.

Sequence identification of peptides and proteins is central to proteomics. Protein sequencing is mainly conducted by insensitive mass spectroscopy because proteins cannot be amplified, which hampers applications such as single-cell proteomics and precision medicine. The commercial success of portable nanopore sequencers for single DNA molecules has inspired extensive research and development of single-molecule techniques for protein sequencing. Among them, three challenges remain: (1) discrimination of the 20 amino acids as building blocks of proteins; (2) unfolding proteins; and (3) controlling the motion of proteins with nonuniformly charged sequences. In this context, the emergence of label-free optical analysis techniques for single amino acids and peptides by solid-state nanopores shows promise for addressing the first challenge. In this Perspective, we first discuss the current challenges of single-molecule fluorescence detection and nanopore resistive pulse sensing in a protein sequencing. Then, label-free optical methods are described to show how they address the single-amino-acid identification within single peptides. They include localized surface plasmon resonance detection and surface-enhanced Raman spectroscopy on plasmonic nanopores. Notably, we report new data to show the ability of plasmon-enhanced Raman scattering to record and discriminate the 20 amino acids at a single-molecule level. In addition, we discuss briefly the manipulation of molecule translocation and liquid flow in plasmonic nanopores for controlling molecule movement to allow high-resolution reading of protein sequences. We envision that a combination of Raman spectroscopy with plasmonic nanopores can succeed in single-molecule protein sequencing in a label-free way.

Ionic heat dissipation in solid-state pores.

Energy dissipation in solid-state nanopores is an important issue for their use as a sensor for detecting and analyzing individual objects in electrolyte solution by ionic current measurements. Here, we report on evaluations of heating via diffusive ion transport in the nanoscale conduits using thermocouple-embedded SiNx pores. We found a linear rise in the nanopore temperature with the input electrical power suggestive of steady-state ionic heat dissipation in the confined nanospace. Meanwhile, the heating efficiency was elucidated to become higher in a smaller pore due to a rapid decrease in the through-water thermal conduction for cooling the fluidic channel. The scaling law suggested nonnegligible influence of the heating to raise the temperature of single-nanometer two-dimensional nanopores by a few kelvins under the standard cross-membrane voltage and ionic strength conditions. The present findings may be useful in advancing our understanding of ion and mass transport phenomena in nanopores.

Nanopore Current Enhancements Lack Protein Charge Dependence and Elucidate Maximum Unfolding at Protein's Isoelectric Point.

Protein sequencing, as well as protein fingerprinting, has gained tremendous attention in the electrical sensing realm of solid-state nanopores and is challenging due to fast translocations and the use of high molar electrolytes. Despite providing an appreciable signal-to-noise ratio, high electrolyte concentrations can have adverse effects on the native protein structure. Herein, we present a thorough investigation of low electrolyte sensing conditions across a broad pH and voltage range generating conductive pulses (CPs) irrespective of protein net charge. We used Cas9 as the model protein and demonstrated that unfolding is noncooperative, represented by the gradual elongation or stretching of the protein, and sensitive to both the applied voltage and pH (i.e., charge state). The magnitude of unfolding and the isoelectric point (pI) of Cas9 was found to be correlated and a critical factor in our experiments. Electroosmotic flow (EOF) was always aligned with the transit direction, whereas electrophoretic force (EPF) was either reinforcing (pH < pI) or opposing (pH > pI) the protein's movement, which led to slower translocations at higher pH values. Further exploration of higher pH values led to slowing down of protein with > 30% of the population being slower than 0.5 ms. Our results would be critical for protein sensing at very low electrolytes and to retard their translocation speed without resorting to high-bandwidth equipment.

Molecular insights into geometric and electrophoretic effects on DNA translocation speed through graphene nanoslit sensor

Slowing down DNA translocation speeds through solid-state nanopores is of vital importance to detect temporal and electrical signals with single-base resolution. To control DNA translocation speed and maintain sequencing sensitivity, the graphene nanoslit sensor is proposed and molecular dynamics simulations are performed to investigate geometric and electrophoretic effects. Results show that the translocation speed is slowed down and the detection range of nanoslit sensors maintains when reducing nanoslit width. Energy barriers of translocation increase with a narrower width, while the ions flow remains because of invariant nanoslit length. Adjusting applied voltage can further control the translocation speed but in a non-linear way. Generally, the speed of a single-base translocation is expected to be well adjusted by integrating geometric and electrophoretic factors, which can separately result in a 66-fold and 25-fold de-speeding. Meanwhile, the distinguishability of the ionic current signal maintains. This study provides molecular insights in controlling translocation speed and sequencing DNA bases by nanoslit sensor. The unique geometry of nanoslit with two adjustable dimensions makes it a promising pore candidate for solid-state nanopore sequencing.

Identifying Single-Stranded DNA by Tuning the Graphene Nanogap Size: An Ionic Current Approach.

Solid nanopore-based deoxyribonucleic acid (DNA) sequencing has led to low-cost, fast, reliable, controlled, and amplified or label-free and high-resolution recognition and identification of DNA nucleotides. Solid-state materials and biological nanopores have a low signal-to-noise ratio (SNR) and generally are too thick to read at single-nucleotide resolution. The issue with solid-state nanopores is that the DNA strands stick to the nanopore sides and on the surface during the translocation process. The coexistence of DNA nucleotides on the surface and the nanopore sides will complicate the ionic current signals, making nucleotide detection difficult. Therefore, different sized nanogaps can be promising to overcome some of these issues. Using all-atom molecular dynamics (MD) simulations, we have studied the translocation of single-stranded (ss) DNA through solid-state nanogaps embedded in a graphene membrane device. A nucleotide-specific DNA sequencing technique is proposed based on unique differences in the ionic current responses for all the four ssDNA nucleotides (dAMP16, dGMP16, dTMP16, and dCMP16). As the individual homogeneous ssDNA translocate through the nanogaps, characteristic changes are observed in the ionic current. Our results show that ssDNA nucleotides can translocate through the proposed graphene nanogap devices by applying an external electric field. In addition, the sticking issue can be resolved using graphene nanogaps during the ssDNA translocation processes. Therefore, the significant difference in ionic current sensitivity and the translocation event/time yielded by the graphene nanogap-based devices reveal possibilities for utilizing it for ultrafast nanogap-based DNA sequencing.

Development of single-molecule assays with solid-state nanopores using computational tools and DNA nanostructures

Solid-state nanopores are versatile nanoscale sensors to electrically characterize a range of biomolecules at the single-molecule level. When combined with DNA nanotechnology (nanostructures self-assembled via specific base pairing of DNA), solid-state nanopores can enable the specific and sensitive detection of disease biomarkers, opening exciting opportunities in clinical diagnostics. Yet challenges remain to improve the performance of such single-molecule assays. To that end, tools and methods through which to design ultra-sensitive, target-specific assays are critical to advancing these digital schemes.

Electrokinetic transport phenomena in solid-state nanopores: dynamics of protein and DNA translocations

Electrokinetic transport mechanism through solid-state nanopores (SSNs) results from a complex interplay of electrophoresis, electroosmosis, and/or diffusion. These processes are influenced by a host of factors, viz., applied voltage, electrolyte type, concentration and pH, nanopore surface chemistry, analyte charge, etc. In this study, we investigated the transport phenomena associated with the translocation of protein and DNA. Unlike DNA, which has a uniform linear charge density, protein charge is heterogeneous and highly pH-dependent.

Controlled laser-induced fabrication of multichannel solid-state nanopore sensors for studying single-molecule protein dynamics

Nanopores have emerged in recent years as a sensitive and informative sensor for probing biomolecules at a single-molecule level. Nanopore sensing is achieved by recording the ion flux through a small pore submerged in an aqueous electrolyte by applying a voltage bias across it. The essential parameters of an analyte are then deduced through resistive pulses in the ionic current as it is electrokinetically captured and driven through the pore. Solid-state nanopores have recently gained traction as high-resolution sensors for protein characterization because unlike their lipid-embedded biological pore analogs, the solid-state membrane is stable at higher voltages and the pore size can be tailored to be compatible with the studied molecule of choice.

Multi-layered heterostructure nanopore sensor for single biomolecule detection

Nanopores have shown to be a promising tool for fast and real-time sequencing of DNA. Due to the chemical and mechanical robustness and design flexibility, two-dimensional (2D) solid-state nanopore (SSNP) sensors are a contesting candidate for next-generation sequencing devices. However, the major hindrance in detecting DNA nucleotides via 2D SSNPs lies in the low signal-to-noise ratio (SNR) due to thermal noise and the lack of statistically significant current signals. In this regard, we propose a stacked 2D SSNP heterostructure to naturally quench the conformational fluctuations of the translocating biomolecule while obtaining multiple independent electronic in-plane current signals.