Solid-state Nanopores Research Articles

Rapid detection of novel coronavirus SARS-CoV-2 by RT-LAMP coupled solid-state nanopores

The current pandemic of COVID-19 caused by SARS-CoV-2 (severe acute respiratory syndrome coronavirus-2) has raised significant public health concerns. Rapid and accurate testing of SARS-CoV-2 is urgently needed for early detection and control of the disease spread. Here, we present an RT-LAMP coupled glass nanopore digital counting method for rapid detection of SARS-CoV-2. We validated and compared two one-pot RT-LAMP assays targeting nucleocapsid (N) and envelop (E) genes. The nucleocapsid assay was adopted due to its quick time to positive and better copy number sensitivity. For qualitative positive/negative classification of a testing sample, we used the glass nanopore to digitally count the RT-LAMP amplicons and benchmarked the event rate with a threshold. Due to its intrinsic single molecule sensitivity, nanopore sensors could capture the amplification dynamics more rapidly (quick time to positive). We validated our RT-LAMP coupled glass nanopore digital counting method for SARS-CoV-2 detection by using both spiked saliva samples and COVID-19 clinical nasopharyngeal swab samples. The results obtained showed excellent agreement with the gold standard RT-PCR assay. With its integration capability, the electronic nanopore digital counting platform has significant potential to provide a rapid, sensitive, and specific point-of-care assay for SARS-CoV-2.

Front Cover: Investigation of entrance effects on particle electrophoretic behavior near a nanopore for resistive pulse sensing

DOI: 10.1002/elps.202100162 The cover picture illustrates entrance effects of a solid-state nanopore immersed in an electrolyte solution on the electrophoretic behavior of a charged biomolecule. The intense electric field in the high electrical resistance nanopore generated by an external electric potential bias across the membrane gives rise to a significant electroosmotic flow opposing the molecule migration. Whether or not the molecule can translocate through the nanopore for resistive pulse sensing is subject to both the zeta potential difference between the molecule and nanopore and the variation of the local electric field strength from the nanopore-reservoir junction into the nanopore.

Shaped DNA origami carrier nanopore translocation influenced by aptamer based surface modification

DNA origami is widely used as a translocation carrier to assist solid-state nanopore analysis, e.g., soft linear origami carrier and special-shaped origami structures. In the linear origami carriers based nanopore sensing, molecular modifications induced tiny structural and charge changes, can result in significant variations on translocation signals to facilitating single-molecule sensing. However, an understanding on the influences of surface modifications on special-shaped DNA origami structures during solid-state (SS) nanopores translocation is still far elusive. Herein, we reported a surface modification strategy using aptamer/target-binding to influence the translocation of the shaped origami ribbon carrier through SS-nanopore. Our measurements indicate that the translocation signal variations can respond to ATP/aptamer binding on the carrier surface, even to the surface modifications induced by spatial distributions and enzyme catalysis. Meanwhile, the results also suggest a possibility to identify small spatial and electronic changes on DNA origami by using SS-nanopore. We envision that the surface aptamer-binding influenced origami translocation strategy could find more applications in origami carrier assisted SS-nanopore sensing and detection.

Current Fluctuations in Nanopores Reveal the Polymer-Wall Adsorption Potential.

Modification of surface properties by polymer adsorption is a widely used technique to tune interactions in molecular experiments such as nanopore sensing. Here, we investigate how the ionic current noise through solid-state nanopores reflects the adsorption of short, neutral polymers to the pore surface. The power spectral density of the noise shows a characteristic change upon adsorption of polymer, the magnitude of which is strongly dependent on both polymer length and salt concentration. In particular, for short polymers at low salt concentrations no change is observed, despite the verification of comparable adsorption in these systems using quartz crystal microbalance measurements. We propose that the characteristic noise is generated by the movement of polymers on and off the surface and perform simulations to assess the feasibility of this model. Excellent agreement with experimental data is obtained using physically motivated simulation parameters, providing deep insight into the shape of the adsorption potential and underlying processes. This paves the way toward using noise spectral analysis for insitu characterization of functionalized nanopores.

Simple Fabrication of Solid-State Nanopores on a Carbon Film.

Solid-state nanopores are widely used as a platform for stochastic nanopore sensing because they can provide better robustness, controllable pore size, and higher integrability than biological nanopores. However, the fabrication procedures, including thin film preparation and nanopore formation, require advanced micro-and nano-fabrication techniques. Here, we describe the simple fabrication of solid-state nanopores in a commercially available material: a flat thin carbon film-coated micro-grid for a transmission electron microscope (TEM). We attempted two general methods for nanopore fabrication in the carbon film. The first method was a scanning TEM (STEM) electron beam method. Nanopores were fabricated by irradiating a focused electron beam on the carbon membrane on micro-grids, resulting in the production of nanopores with pore diameters ranging from 2 to 135 nm. The second attempt was a dielectric breakdown method. In this method, nanopores were fabricated by applying a transmembrane voltage of 10 or 30 V through the carbon film on micro-grids. As a result, nanopores with pore diameters ranging from 3.7 to 1345 nm were obtained. Since these nanopores were successfully fabricated in the commercially available carbon thin film using readily available devices, we believe that these solid-state nanopores offer great utility in the field of nanopore research.

Fast Fabrication of Solid-State Nanopores for DNA Molecule Analysis.

Solid-state nanopores have been developed as a prominent tool for single molecule analysis in versatile applications. Although controlled dielectric breakdown (CDB) is the most accessible method for a single nanopore fabrication, it is still necessary to improve the fabrication efficiency and avoid the generation of multiple nanopores. In this work, we treated the SiNx membranes in the air–plasma before the CDB process, which shortened the time-to-pore-formation by orders of magnitude. λ-DNA translocation experiments validated the functionality of the pore and substantiated the presence of only a single pore on the membrane. Our fabricated pore could also be successfully used to detect short single-stranded DNA (ssDNA) fragments. Using to ionic current signals, ssDNA fragments with different lengths could be clearly distinguished. These results will provide a valuable reference for the nanopore fabrication and DNA analysis.

Investigation of entrance effects on particle electrophoretic behavior near a nanopore for resistive pulse sensing.

Resistive pulse sensing using solid-state nanopores provides a unique platform for detecting the structure and concentration of molecules of different types of analytes in an electrolyte solution. The capture of an entity into a nanopore is subject not only to the electrostatic force but also the effect of electroosmotic flow originating from the charged nanopore surface. In this study, we theoretically analyze spherical particle electrophoretic behavior near the entrance of a charged nanopore. By investigating the effects of pore size, particle-pore distance, and salt concentration on particle velocity, we summarize dominant mechanisms governing particle behavior for a range of conditions. In the literature, the Helmholtz-Smoluchowski equation is often adopted to evaluate particle translocation by considering the zeta potential difference between the particle and nanopore surfaces. We point out that, due to the difference of the electric field inside and outside the nanopore and the influence from the existence of the particle itself, the zeta potential of the particle, however, needs to be at least 30% higher than that of the nanopore to allow the particle to enter into the nanopore when its velocity is close to zero. Accordingly, we summarize the effective salt concentrations that enable successful particle capture and detection for different pore sizes, offering direct guidance for nanopore applications.

Digital immunoassay for biomarker concentration quantification using solid-state nanopores

ABSTRACTSingle-molecule counting is the most accurate and precise method for determining the concentration of a biomarker in solution and is leading to the emergence of digital diagnostic platforms enabling precision medicine. In principle, solid-state nanopores—fully electronic sensors with single-molecule sensitivity—are well suited to the task. Here we present a digital immunoassay scheme capable of reliably quantifying the concentration of a target protein in complex biofluids that overcomes specificity, sensitivity, and consistency challenges associated with the use of solid-state nanopores for protein sensing. This is achieved by employing easily-identifiable DNA nanostructures as proxies for the presence (“1”) or absence (“0”) of the target protein captured via a magnetic bead-based sandwich immunoassay. As a proof-of-concept, we demonstrate quantification of the concentration of thyroid-stimulating hormone from human serum samples down to the high femtomolar range. Further optimization to the method will push sensitivity and dynamic range, allowing for development of precision diagnostic tools compatible with point-of-care format.

Investigation on the competition of duplex/ G-quadruplex/ i-motif in telomere sequences and c-MYC gene with a solid-state nanopore sensor

Apart from duplex helix, the coexisted noncanonical DNA may be involved in inhibiting DNA replication, and controlling gene expression. The competition of duplex/ G4/ i-motif depends on many factors, including sequence information, external conditions and binding effects. This work looked into the human telomere sequences of different length (hTelo-1, 2) and intramolecular G-rich and C-rich strands (hTelo-3, 4, 5). Nanopore translocation properties suggested that in the presence of their complementary sequences in the mixed K+/Cs+ buffer, G4 was the major species, and G4 from hTelo-2 that holds multiple G-quartets formed slower than shorter hTelo-1. The intramolecular G/C-rich strands hTelo-3, 4, 5 predominantly formed duplex rather than quadruplex under K+ or acidic conditions, indicating that intramolecular quadruplex is less stable than duplex due to the strand strain. Translocation behaviors of mixed proto-oncogene cMYC/c-cMYC illustrated that G4 was formed in both K+ and Cs+, and Cs+ inhibited the duplex formation in K+/Cs+ buffer. The increase of the ratio of Cs+ will decrease the duplexes species. Our work demonstrated the relative stability of duplex/quadruplex under various conditions with distinct strands, which is useful for the investigation of DNA secondary structural dynamics and transition mechanism in biological function regions, and paved the way for the potential of disease-targeted diagnosis.

Nanopore electro-osmotic trap for the label-free study of single proteins and their conformations.

Many strategies have been pursued to trap and monitor single proteins over time to detect the molecular mechanisms of these essential nanomachines. Single-protein sensing with nanopores is particularly attractive because it allows label-free high-bandwidth detection on the basis of ion currents. Here we present the nanopore electro-osmotic trap (NEOtrap) that allows trapping and observing single proteins for hours with submillisecond time resolution. The NEOtrap is formed by docking a DNA-origami sphere onto a passivated solid-state nanopore, which seals off a nanocavity of a user-defined size and creates an electro-osmotic flow that traps nearby particles irrespective of their charge. We demonstrate the NEOtrap's ability to sensitively distinguish proteins on the basis of size and shape, and discriminate between nucleotide-dependent protein conformations, as exemplified by the chaperone protein Hsp90. Given the experimental simplicity and capacity for label-free single-protein detection over the broad bio-relevant time range, the NEOtrap opens new avenues to study the molecular kinetics underlying protein function.

Hyaluronic acid synthesis, degradation, and crosslinking in equine osteoarthritis: TNF-\u03b1-TSG-6-mediated HC-HA formation

BackgroundTNF-α-stimulated gene 6 (TSG-6) protein, a TNF-α-responsive hyaladherin, possesses enzymatic activity that can catalyze covalent crosslinks of the polysaccharide hyaluronic acid (HA) to another protein to form heavy chain-hyaluronic acid (HC-HA) complexes in pathological conditions such as osteoarthritis (OA). Here, we examined HA synthase and inflammatory gene expression; synovial fluid HA, TNF-α, and viscosity; and TSG-6-mediated HC-HA complex formation in an equine OA model. The objectives of this study were to (1) evaluate the TNF-α-TSG-6-HC-HA signaling pathway across multiple joint tissues, including synovial membrane, cartilage, and synovial fluid, and (2) determine the impact of OA on synovial fluid composition and biophysical properties.MethodsHA and inflammatory cytokine concentrations (TNF-α, IL-1β, CCL2, 3, 5, and 11) were analyzed in synovial fluid from 63 OA and 25 control joints, and HA synthase (HAS1-3), TSG-6, and hyaluronan-degrading enzyme (HYAL2, HEXA) gene expression was measured in synovial membrane and cartilage. HA molecular weight (MW) distributions were determined using agarose gel electrophoresis and solid-state nanopore measurements, and HC-HA complex formation was detected via immunoblotting and immunofluorescence. SEC-MALS was used to evaluate TSG-6-mediated HA crosslinking, and synovial fluid and HA solution viscosities were analyzed using multiple particle-tracking microrheology and microfluidic measurements, respectively.ResultsTNF-α concentrations were greater in OA synovial fluid, and TSG6 expression was upregulated in OA synovial membrane and cartilage. TSG-6-mediated HC-HA complex formation was greater in OA synovial fluid and tissues than controls, and HC-HA was localized to both synovial membrane and superficial zone chondrocytes in OA joints. SEC-MALS demonstrated macromolecular aggregation of low MW HA in the presence of TSG-6 and inter-α-inhibitor with concurrent increases in viscosity.ConclusionsSynovial fluid TNF-α concentrations, synovial membrane and cartilage TSG6 gene expression, and HC-HA complex formation were increased in equine OA. Despite the ability of TSG-6 to induce macromolecular aggregation of low MW HA with resultant increases in the viscosity of low MW HA solutions in vitro, HA concentration was the primary determinant of synovial fluid viscosity rather than HA MW or HC-HA crosslinking. The TNF-α-TSG-6-HC-HA pathway may represent a potential therapeutic target in OA.

Electronic Conductance and Current Modulation through Graphdiyne Nanopores for DNA Sequencing

Solid-state nanopore devices have emerged as one of the most promising technologies that may decode the sequence of DNA nucleobases by reading electronic conductance and electric current signals. Herein we study the potential of atomically thick graphdiyne (GDY) to act as an all-electronic nanopore (NP)-based DNA sequencing device. The first-principles density functional theory method is used to study the structural properties, interaction energy (Ei) values, translocation time (τ), charge transfer [Q(e)] values, charge density difference [Δρ(r)], molecular orbital (MO) positions, and electronic density of states (DOS) properties of the GDYNP and GDYNP + nucleobase (nucleobases: adenine (A), guanine (G), thymine (T), and cytosine (C)) systems. Our results suggest that the GDYNP system could be a suitable device for the detection of nucleobases due to their specific translocation times inside the GDYNP device. The Δρ(r) results reveal that the charge fluctuates around the GDYNP edges close to the target nucleobase. This charge reorganization causes the modulation of charge transport in the GDYNP device. Furthermore, the GDYNP device is used to read-out the conductance and current signals of each DNA nucleobase while located inside the GDYNP by using nonequilibrium Green’s functions formalism. Our findings show a change in conductance with respect to the reference bare “GDYNP” device for energy values below/above the Fermi energy. The conductance as well as current sensitivity (%) values (C > G > T > A) indicates that the nucleobases could possibly be sequenced through the GDYNP device. Further, each nucleobase transmits unique current signals when transported through the GDYNP device. A distinction between purine- and pyrimidine-type nucleobases seems possible due to their different current signals. Thus, our study highlights the potentials that would motivate research toward the development of the GDYNP device, which may be even a better DNA nucleobase detector compared to graphene-based devices.

Storage and Cleaning Extend Lifetime of Glass Nanopores for Biosensing

Nanopore 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 can withstand non-physiological conditions are an attractive choice for biosensing applications. Still, biomolecule interactions with the nanopore surface reduces nanopore reusability and increases usage cost. In this study, we present a protocol for storage and repeated use of glass nanopores lasting up to a few months. We investigated the biosensing capability for 90 quartz glass nanopores with a diameter of 11-18 nm that were fabricated using laser-assisted pipette pulling. Nanopores were assembled into multiple microfluidic chips. The storage protocol allows for repeated use of our nanopore chips for up to 19 weeks. The single-molecule biosensing capability over repeated use cycles was demonstrated by quantitative analysis of a DNA nanostructure designed for detection of short single-stranded DNA oligos.

Detection of Small-Sized DNA Fragment in a Glassy Nanopore by Utilization of CRISPR-Cas12a as Converter System

The fabrication of nanopore with matched pore size, and the existence of multi interferents make the reproducible detection of small-sized molecules by means of solid-state nanopore still challenging. A useful method to solve these problems is based on the detection of large DNA nanostructure related to the existence of small-sized target. In particular, DNA tetrahedron with well-defined 3D nanostructure is the ideal candidate to be used as signal transducer. Here, we demonstrate the detection of L1-encoding gene of HPV18 as test DNA target sequence in a reaction buffering solution, where long single-stranded DNA linking DNA tetrahedrons onto the surface of magnetic beads are cleaved by target DNA-activated CRISPR-cas12 system. The DNA tetrahedrons are subsequently released and can be detected by the current pulse in a glassy nanopore. This approach has several advantages: (1) One signal transducer can be used to detect different targets; (2) Glassy nanopore with pore size much larger than the target DNA fragment can boost the tolerance of contaminants and interferents which often degrade the performance of nanopore sensor.

Nanopore Data Analysis: Baseline Construction and Abrupt Change-Based Multilevel Fitting.

Solid-state nanopore technology delivers single-molecule resolution information, and the quality of the deliverables hinges on the capability of the analysis platform to extract maximum possible events and fit them appropriately. In this work, we present an analysis platform with four baseline fitting methods adaptive to a wide range of nanopore traces (including those with a step or abrupt changes where pre-existing platforms fail) to maximize extractable events (2× improvement in some cases) and multilevel event fitting capability. The baseline fitting methods, in the increasing order of robustness and computational cost, include arithmetic mean, linear fit, Gaussian smoothing, and Gaussian smoothing and regressed mixing. The performance was tested with ultra-stable to vigorously fluctuating current profiles, and the event count increased with increasing fitting robustness prominently for vigorously fluctuating profiles. Turning points of events were clustered using the dbscan method, followed by segmentation into preliminary levels based on abrupt changes in the signal level, which were then iteratively refined to deduce the final levels of the event. Finally, we show the utility of clustering for multilevel DNA data analysis, followed by the assessment of protein translocation profiles.

Understanding Electrical Conduction and Nanopore Formation During Controlled Breakdown.

Controlled breakdown has recently emerged as a highly appealing technique to fabricate solid-state nanopores for a wide range of biosensing applications. This technique relies on applying an electric field of approximately 0.4-1 V nm-1 across the membrane to induce a current, and eventually, breakdown of the dielectric. Although previous studies have performed controlled breakdown under a range of different conditions, the mechanism of conduction and breakdown has not been fully explored. Here, electrical conduction and nanopore formation in SiNx membranes during controlled breakdown is studied. It is demonstrated that for Si-rich SiNx , oxidation reactions that occur at the membrane-electrolyte interface limit conduction across the dielectric. However, for stoichiometric Si3 N4 the effect of oxidation reactions becomes relatively small and conduction is predominately limited by charge transport across the dielectric. Several important implications resulting from understanding this process are provided which will aid in further developing controlled breakdown in the coming years, particularly for extending this technique to integrate nanopores with on-chip nanostructures.

Diameter distribution and formation mechanism of solid-state nanopores prepared by dielectric breakdown

The diameter distribution and dielectric breakdown mechanism of solid-state nanopores in a liquid environment are discussed based on the experimental results. We discussed the nanopore formation mechanism from the viewpoint of the statistical correlation between the diameter distribution, the charge to breakdown, and the dissipated energy across the nanopore by changing the field stress. We found that high field stress increases the instability of the uniformity of the nanopore diameter. Similar to the mechanism in the solid situation, the diameter of the nanopore can be uniquely correlated to the dissipated energy in the pore formation process.

Central Limit Theorem-Based Analysis Method for MicroRNA Detection with Solid-State Nanopores

Although nanopore as a single-molecule sensing platform has proven its potential in various applications, data analysis of nanopores remains challenging. Herein, we introduce a method with increased accuracy in nanopore analysis based on the central limit theorem (CLT). An optimal voltage used in detection is determined from the standard deviations of blockage currents and time constants at various voltage biases. Compared with the conventional data analysis method, blockage signals processed with the CLT result in more concentrated distributions of blockage currents and durations. It allows fitting a Gaussian to the duration histogram and avoids the influence of bin sizes on time constants in duration analysis. The proposed method is further validated by applying it to detect isolated microRNAs with solid-state nanopores. Under the optimal voltage, different nucleic acids present in the isolation process are translocated through the nanopore. By processing the event signals with the CLT, all the nucleic acids including the microRNA are well differentiated. The method proposed here should also be applicable for sensing other biomolecules with the solid-state nanopores.

Detection and Separation of Single-Stranded DNA Fragments Using Solid-State Nanopores.

Many biological assays require effectively and sensitively sorting DNA fragments. Here, we demonstrate a solid-state nanopore platform for label-free detection and separation of short single-stranded DNA (ssDNA) fragments (<100 nt), based on their length-dependent translocation behaviors. Our experimental data show that each sized pore has a passable length threshold. The negative charged ssDNA fragments with length smaller than the threshold can be electrically facilitated driven through the correspondingly sized nanopore along the direction of electric field. In addition, the passable length threshold increases with the pore size enlarging. As a result, this phenomenon is able to be applicable for the controllable selectivity of ssDNA by tuning nanopore size, and the selectivity limitation is up to 30nt. Numerical simulation results indicate the translocation direction of ssDNA is governed by the competition of electroosmosis and electrophoresis effects on the ssDNA and offer the relationship between passable length threshold and pore size.

Devices for Nanoscale Guiding of DNA through a 2D Nanopore.

We fabricate on-chip solid-state nanofluidic-2D nanopore systems that can limit the range of motion for DNA in the sensing region of a nanopore. We do so by creating devices containing one or more silicon nitride pores and silicon nitride pillars supporting a 2D pore that orient DNA within a nanopore device to a restricted geometry, yet allow the free motion of ions to maintain a high signal-to-noise ratio. We discuss two concepts with two and three independent electrical connections and corresponding nanopore chip device architectures to achieve this goal in practice. Here, we describe device fabrication and transmission electron microscope (TEM) images, and provide simulated translocations based on the finite element analysis in 3D to demonstrate its merit. In both methods, there is a main 2D nanopore which we refer to as a "sensing" nanopore (monolayer MoS2 in this paper). A secondary layer is either an array of guiding pores sharing the same electrode pair as the sensing pore (Method 1) or a single, independently contacted, guiding pore (Method 2). These pores are constructed parallel to the "sensing" pore and serve as "guiding" elements to stretch and feed DNA into the atomically thin sensing pore. We discuss the practical implementation of these concepts with nanofluidic and Si-based technology, including detailed fabrication steps and challenges involved for DNA applications in solution.