Nanopore Translocation Reveals Electrophoretic Force on Noncanonical RNA:DNA Double Helix.

Electrophoretic transport plays a pivotal role in advancing sensing technologies. So far, systematic studies have focused on the translocation of canonical B-form or A-form nucleic acids, while direct RNA analysis is emerging as the new frontier for nanopore sensing and sequencing. Here, we compare the less-explored dynamics of noncanonical RNA:DNA hybrids in electrophoretic transport to the well-researched transport of B-form DNA. Using DNA/RNA nanotechnology and solid-state nanopores, the translocation of RNA:DNA (RD) and DNA:DNA (DD) duplexes was examined. Notably, RD duplexes were found to translocate through nanopores faster than DD duplexes, despite containing the same number of base pairs. Our experiments reveal that RD duplexes present a noncanonical helix, with distinct transport properties from B-form DD molecules. We find that RD and DD molecules, with the same contour length, move with comparable velocity through nanopores. We examined the physical characteristics of both duplex forms using atomic force microscopy, atomistic molecular dynamics simulations, agarose gel electrophoresis, and dynamic light scattering measurements. With the help of coarse-grained and molecular dynamics simulations, we find the effective force per unit length applied by the electric field to a fragment of RD or DD duplex in nanopores with various geometries or shapes to be approximately the same. Our results shed light on the significance of helical form in nucleic acid translocation, with implications for RNA sensing, sequencing, and the molecular understanding of electrophoretic transport.

Electroosmotic Sensing of Uncharged Peptides and Differentiating Their Phosphorylated States Using Nanopores.

The correct characterization and identification of different kinds of proteins is crucial for the survival and development of living organisms, and proteomics research promotes the analysis and understanding of future genome functions. Nanopore technique has been proved to accurately identify individual nucleotides. However, accurate and rapid protein sequencing is difficult due to the variability of protein structures that contains more than 20 amino acids, and it remains very challenging especially for uncharged peptides as they can not be electrophoretically driven through the nanopore. Graphene nanopores have the advantages of high accuracy, sensitivity and low cost in identifying protein phosphorylation modifications. Here, by using all-atom molecular dynamics simulations, charged graphene nanopores are employed to electroosmotically capture and sense uncharged peptides. By further mimicking AFM manipulation of single molecules, it is also found that the uncharged peptides and their phosphorylated states could also be differentiated by both the ionic current and pulling force signals during their pulling processes through the nanopore with a slow and constant velocity. The results shows ability of using nanopores to detect and discriminate single amino acid and its phosphorylation, which is essential for the future low-cost and high-throughput sequencing of protein residues and their post-translational modifications.

Emerging Data Processing Methods for Single-Entity Electrochemistry.

Single-entity electrochemistry is a powerful tool that enables the study of electrochemical processes at interfaces and provides insights into the intrinsic chemical and structural heterogeneities of individual entities. Signal processing is a critical aspect of single-entity electrochemical measurements and can be used for data recognition, classification, and interpretation. In this review, we summarize the recent five-year advances in signal processing techniques for single-entity electrochemistry and highlight their importance in obtaining high-quality data and extracting effective features from electrochemical signals, which are generally applicable in single-entity electrochemistry. Moreover, we shed light on electrochemical noise analysis to obtain single-molecule frequency fingerprint spectra that can provide rich information about the ion networks at the interface. By incorporating advanced data analysis tools and artificial intelligence algorithms, single-entity electrochemical measurements would revolutionize the field of single-entity analysis, leading to new fundamental discoveries.

Counter-Intuitive Features of Particle Dynamics in Nanopores.

Using the framework of a continuous diffusion model based on the Smoluchowski equation, we analyze particle dynamics in the confinement of a transmembrane nanopore. We briefly review existing analytical results to highlight consequences of interactions between the channel nanopore and the translocating particles. These interactions are described within a minimalistic approach by lumping together multiple physical forces acting on the particle in the pore into a one-dimensional potential of mean force. Such radical simplification allows us to obtain transparent analytical results, often in a simple algebraic form. While most of our findings are quite intuitive, some of them may seem unexpected and even surprising at first glance. The focus is on five examples: (i) attractive interactions between the particles and the nanopore create a potential well and thus cause the particles to spend more time in the pore but, nevertheless, increase their net flux; (ii) if the potential well-describing particle-pore interaction occupies only a part of the pore length, the mean translocation time is a non-monotonic function of the well length, first increasing and then decreasing with the length; (iii) when a rectangular potential well occupies the entire nanopore, the mean particle residence time in the pore is independent of the particle diffusivity inside the pore and depends only on its diffusivity in the bulk; (iv) although in the presence of a potential bias applied to the nanopore the "downhill" particle flux is higher than the "uphill" one, the mean translocation times and their distributions are identical, i.e., independent of the translocation direction; and (v) fast spontaneous gating affects nanopore selectivity when its characteristic time is comparable to that of the particle transport through the pore.

Nanopore translocation reveals electrophoretic force on A- and B-form nucleic acids.

Electrophoretic transport plays a pivotal role in advancing sensing technologies, with A-form nucleic acids, predominantly RNA-containing, emerging as the new frontier for nanopore sensing and sequencing. Here, we compare the less-explored dynamics of A-form electrophoretic transport with the well-researched transport of B-form DNA. Using DNA/RNA nanotechnology and solid-state nanopores, the translocation of RNA:DNA (RD) and DNA:DNA (DD) duplexes was examined. Notably, RD duplexes were found to translocate through nanopores up to 1.8 times faster than DD duplexes, despite containing the same number of base pairs. Our experiments reveal that A- and B-form duplex molecules with the same contour length move with comparable velocity through nanopores. We examined the physical characteristics of both duplex forms using atomic force microscopy, agarose gel electrophoresis, and dynamic light scattering measurements. With the help of coarse-grained and atomistic molecular dynamics simulations, we find the effective force applied by the electric field to a fragment of A-form or B-form duplex in a nanopore to be approximately the same. Our results shed light on the significance of helical form in nucleic acid translocation, with implications for RNA sensing, sequencing, and molecular understanding of electrophoretic transport.

DNA Volume, Topology, and Flexibility Dictate Nanopore Current Signals.

Nanopores have developed into powerful single-molecule sensors capable of identifying and characterizing small polymers, such as DNA, by electrophoretically driving them through a nanoscale pore and monitoring temporary blockades in the ionic pore current. However, the relationship between nanopore signals and the physical properties of DNA remains only partly understood. Herein, we introduce a programmable DNA carrier platform to capture carefully designed DNA nanostructures. Controlled translocation experiments through our glass nanopores allowed us to disentangle this relationship. We vary DNA topology by changing the length, strand duplications, sequence, unpaired nucleotides, and rigidity of the analyte DNA and find that the ionic current drop is mainly determined by the volume and flexibility of the DNA nanostructure in the nanopore. Finally, we use our understanding of the role of DNA topology to discriminate circular single-stranded DNA molecules from linear ones with the same number of nucleotides using the nanopore signal.

Improving Single-Molecule Antibody Detection Selectivity through Optimization of Peptide Epitope Presentation in OmpG Nanopore.

Outer membrane protein G (OmpG) is a monomeric porin found in Escherichia coli, which possesses seven flexible loops. OmpG has been engineered as a nanopore sensor, where its loops can host affinity epitopes for selective detection of biological molecules. In this study, we investigated various loop positions to incorporate a FLAG peptide antigen epitope in the most flexible loop 6 and tested the efficacy and sensitivity of these nanopore constructs in antibody detection. We observed an OmpG construct containing inserted FLAG sequence, which exhibited strong interaction with anti-FLAG antibodies in flow cytometry; however, it could not translate molecule interactions into a readable signal in current recordings. Further optimization of the peptide presentation strategy by replacing specific sections of loop 6 sequences with the FLAG tag created a construct capable of generating unique and distinct signals when interacting with various monoclonal or polyclonal anti-FLAG clones IgG antibodies in the mixture. The peptide display scheme demonstrated in this study can be generalized for the engineering of OmpG sensors, which can be used for screening and validating positive clones during antibody development, as well as for real-time quality control of cell cultures in monoclonal antibody production.

Multi-Stimuli-Responsive and Mechano-Actuated Biomimetic Membrane Nanopores Self-Assembled from DNA.

In bioinspired design, biological templates are mimicked in structure and function by highly controllable synthetic means. Of interest are static barrel-like nanopores that enable molecular transport across membranes for use in biosensing, sequencing, and biotechnology. However, biological ion channels offer additional functions such as dynamic changes of the entire pore shape between open and closed states, and triggering of dynamic processes with biochemical and physical stimuli. To better capture this complexity, this report presents multi-stimuli and mechano-responsive biomimetic nanopores which are created with DNA nanotechnology. The nanopores switch between open and closed states, whereby specific binding of DNA and protein molecules as stimuli locks the pores in the open state. Furthermore, the physical stimulus of high transmembrane voltage switches the pores into a closed state. In addition, the pore diameters are larger and more tunable than those of natural templates. These multi-stimuli-responsive and mechanically actuated nanopores mimic several aspects of complex biological channels yet offer easier control over pore size, shape and stimulus response. The designer pores are expected to be applied in biosensing and synthetic biology.

Two Detection Modes of Nanoslit Sensing Based on Planar Heterostructure of Graphene/Hexagonal Boron Nitride.

Solid-state nanopore sequencing is now confronted with problems of stochastic pore clogging and too fast speed during the DNA permeation through a nanopore, although this technique is revolutionary with long readability and high efficiency. These two problems are related to controlling molecular transportation during sequencing. To control the DNA motion and identify the four bases, we propose nanoslit sensing based on the planar heterostructure of two-dimensional graphene and hexagonal boron nitride. Molecular dynamics simulations are performed on investigating the motion of DNA molecules on the heterostructure with a nanoslit sensor. Results show that the DNA molecules are confined within the hexagonal boron nitride (HBN) domain of the heterostructure. And the confinement effects of the heterostructure can be optimized by tailoring the stripe length. Besides, there are two ways of DNA permeation through nanoslits: the DNA can cross or translocate the nanoslit under applied voltages along the y and z directions. The two detection modes are named cross-slit and trans-slit, respectively. In both modes, the ionic current drops can be observed when the nanoslit is occupied by the DNA. And the ionic currents and dwell times can be simultaneously detected to identify the four different DNA bases. This study can shed light on the sensing mechanism based on the nanoslit sensor of a planar heterostructure and provide theoretical guidance on designing devices controlling molecular transportation during nanopore sequencing.

Glass Capillary-Based Nanopores for Single Molecule/Single Cell Detection.

A glass capillary-based nanopore (G-nanopore), due to its tapered tip, easy tunability in orifice size, and especially its flexible surface modifications that can be tailored to effectively capture and enhance the ionic current signal of single entities (single molecules, single cells, and single particles), offers a powerful and nanoconfined sensing platform for diverse biological measurements of single cells and single molecules. Compared with other artificial two-dimensional solid-state nanopores, its conical tip and high spatial and temporal resolution characteristics facilitate noninvasive single molecule and selected area (subcellular) single cell detections (e.g., DNA mutations, highly expressed proteins, and small molecule markers that reflect the change characteristics of the tumor), as a small G-nanopore (≤100 nm) does negligible damage to cell functions and cell membrane integrity when inserted through the cell membrane. In this brief review, we summarize the preparation of G-nanopores and discuss the advantages of them as solid-state sensing platforms for single molecule and single cell detection applications as well as for cancer diagnosis and treatment applications. We also describe the current bottlenecks that limit the widespread use of G-nanopores in clinical applications and provide an outlook on future developments. The brief review will provide the reader with a quick survey of this field and facilitate the rapid development of a G-nanopore sensing platform for future tumor diagnosis and personalized medicine based on single-molecule/single-cell bioassay.

Scaled-Up Synthesis of Freestanding Molybdenum Disulfide Membranes for Nanopore Sensing.

2D materials are ideal for nanopores with optimal detection sensitivity and resolution. Among these, molybdenum disulfide (MoS2 ) has gained traction as a less hydrophobic material than graphene. However, experiments using 2D nanopores remain challenging due to the lack of scalable methods for high-quality freestanding membranes. Herein, a site-directed, scaled-up synthesis of MoS2 membranes on predrilled nanoapertures on 4-inch wafer substrates with 75% yields is reported. Chemical vapor deposition (CVD), which introduces sulfur and molybdenum dioxide vapors across the sub-100 nm nanoapertures results in exclusive formation of freestanding membranes that seal the apertures. Nucleation and growth near the nanoaperture edges is followed by nanoaperture decoration with MoS2 , which proceeds until a critical flake curvature is achieved, after which fully spanning freestanding membranes form. Intentional blocking of reagent flow through the apertures inhibits MoS2 nucleation around the nanoapertures, promoting the formation of large-crystal monolayer MoS2 membranes. The in situ grown membranes along with facile membrane wetting and nanopore formation using dielectric breakdown enables the recording of dsDNA translocation events at an unprecedentedly high 1 MHz bandwidth. The methods presented here are important steps toward the development of scalable single-layer membrane manufacture for 2D nanofluidics and nanopore applications.

Nipple fluid for breast cancer diagnosis using the nanopore of Phi29 DNA-packaging motor.

Detection of cancer in its early stage is a challenging task for oncologists. Inflammatory breast cancer has symptoms that are similar to mastitis and can be mistaken for microbial infection. Currently, the differential diagnosis between mastitis and Inflammatory breast cancer via nipple aspirate fluid (NAF) is difficult. Here, we report a label-free and amplification-free detection platform using an engineered nanopore of the phi29 DNA-packaging motor with biomarker Galectin3 (GAL3), Thomsen-Friedenreich (TF) binding peptide as the probe fused at its C-terminus. The binding of the biomarker in NAF samples from breast cancer patients to the probe results in the connector's conformational change with a current blockage of 32 %. Utilization of dwell time, blockage ratio, and peak signature enable us to detect basal levels of biomarkers from patient NAF samples at the single-molecule level. This platform will allow for breast cancers to be resolved at an early stage with accuracy and thoroughness.

Dynamics of DNA Through Solid-state Nanopores Fabricated by Controlled Dielectric Breakdown.

Controlled dielectric breakdown (CDB) is gaining popularity for fabricating solid-state nanopores in situ with size control in a simple, low-cost, and scalable way. This technique could be used for a broad type of applications in the field of nucleic acid analysis and even for protein studies. In this work, we studied the entry and transport of double-stranded DNAs using a solid-state nanopore fabricated by CDB as a function of applied voltage for two different DNA lengths. We showed that the blockade rate increases exponentially with voltage up to 120 mV. The energy barrier depends on the chain length, and the dwell times decrease with applied voltage up to 120 mV. Moreover, no matter the chain length, it is possible to differentiate two families of blockade amplitudes, high and low ones, due to DNA folding.

Nanopore Electrochemistry for Pathogen Detection.

Pathogen infections seriously threaten human health, and there is an urgent demand for rapid and efficient pathogen identification to provide instructions in clinical diagnosis and therapeutic intervention. Recently, nanopore technology, a rapidly maturing technology which delivers ultrasensitive sensing and high throughput in real-time and at low cost, has achieved success in pathogen detection. Furthermore, the remarkable development of nanopore sequencing, for example, the MinION sequencer from Oxford Nanopore Technologies (ONT) as a competitive sequencing technology, has facilitated the rapid analysis of disease-related microbiomes at the whole-genome level and on a large scale. Here, we highlighted recent advances in nanopore approaches for pathogen detection at the single-molecule level. We also overviewed the applications of nanopore sequencing in pathogenic bacteria identification and diagnosis. In the end, we discussed the challenges and future developments of nanopore technology as promising tools for the management of infections, which may be helpful to aid understanding as well as decision-making.

A Generalized Transformer-Based Pulse Detection Algorithm.

Pulse-like signals are ubiquitous in the field of single molecule analysis, e.g., electrical or optical pulses caused by analyte translocations in nanopores. The primary challenge in processing pulse-like signals is to capture the pulses in noisy backgrounds, but current methods are subjectively based on a user-defined threshold for pulse recognition. Here, we propose a generalized machine-learning based method, named pulse detection transformer (PETR), for pulse detection. PETR determines the start and end time points of individual pulses, thereby singling out pulse segments in a time-sequential trace. It is objective without needing to specify any threshold. It provides a generalized interface for downstream algorithms for specific application scenarios. PETR is validated using both simulated and experimental nanopore translocation data. It returns a competitive performance in detecting pulses through assessing them with several standard metrics. Finally, the generalization nature of the PETR output is demonstrated using two representative algorithms for feature extraction.

Localized Nanopore Fabrication via Controlled Breakdown.

Nanopore sensors provide a unique platform to detect individual nucleic acids, proteins, and other biomolecules without the need for fluorescent labeling or chemical modifications. Solid-state nanopores offer the potential to integrate nanopore sensing with other technologies such as field-effect transistors (FETs), optics, plasmonics, and microfluidics, thereby attracting attention to the development of commercial instruments for diagnostics and healthcare applications. Stable nanopores with ideal dimensions are particularly critical for nanopore sensors to be integrated into other sensing devices and provide a high signal-to-noise ratio. Nanopore fabrication, although having benefited largely from the development of sophisticated nanofabrication techniques, remains a challenge in terms of cost, time consumption and accessibility. One of the latest developed methods-controlled breakdown (CBD)-has made the nanopore technique broadly accessible, boosting the use of nanopore sensing in both fundamental research and biomedical applications. Many works have been developed to improve the efficiency and robustness of pore formation by CBD. However, nanopores formed by traditional CBD are randomly positioned in the membrane. To expand nanopore sensing to a wider biomedical application, controlling the localization of nanopores formed by CBD is essential. This article reviews the recent strategies to control the location of nanopores formed by CBD. We discuss the fundamental mechanism and the efforts of different approaches to confine the region of nanopore formation.

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.

DNA Coil Dynamics and Hydrodynamic Gating of Pressure-Biased Nanopores.

Nanopores are ideally suited for the analysis of long DNA fragments including chromosomal DNA and synthetic DNA with applications in genome sequencing and DNA data storage, respectively. Hydrodynamic fluid flow has been shown to slow down DNA transit time within the pore, however other influences of hydrodynamic forces have yet to be explored. In this report, a broad analysis of pressure-biased nanopores and the impact of hydrodynamics on DNA transit time, capture rate, current blockade depth, and DNA folding are conducted. Using a 10 nm pore, it is shown that hydrodynamic flow inhibits the early stages of linearization of DNA and produces predominately folded events which are initiated by folded DNA (2-strands) entering the pore. Furthermore, utilizing larger pores (30 nm) leads to unique DNA gating behavior in which DNA events can be switched on and off with the application of pressure. A computational model, based on combining electrophoretic drift velocities with fluid velocities, accurately predicts the pore size required to observe DNA gating. Hydrodynamic fluid flow generated by a pressure bias, or potentially more generally by other mechanisms like electroosmotic flow, is shown to have significant effects on DNA sensing and can be useful for DNA sensing technologies.

A Nanopore Sensing Assay Resolves Cascade Reactions in a Multienzyme System.

Enzymatic cascade reactions are widely used to synthesize complex molecules from simple precursors. The major underlying mechanism of cascade reactions is substrate channelling, where intermediates of different enzymatic steps are not in equilibrium with the bulk solution. Here, we report a nanopore sensing assay that allows accurate quantification of all the reaction intermediates and the product of an artificial three-enzyme system. A DNA-peptide complex is used as the initial substrate which undergoes sequential enzymatic cleavages in solution. All the temporal changes of the intermediates and product can be obtained through nanopore translocation recordings. Furthermore, we find that in a confined environment such as liposome, substrate channelling occurs between two sets of the three enzymes. Our results demonstrate a novel and powerful approach to determine and quantify substrate channelling effects, which is potentially useful for designing and evaluating multienzyme systems.

Current pulse signature of native kanamycin aptamer and its implication for molecular interactions on a single protein nanopore sensing interface.

Due to the pore size limitation of single α-hemolysin (α-HL) nanopore sensing interface, ssDNA with secondary conformations can only pass through the nanopore after unzipping as linear ssDNA. For hairpin DNA, a tail with 15-50 bases was usually added to the stem terminal (5' or 3') to facilitate the capture rate and unzipping process, and the typical translocation signal behaves as a square wave with a short dip at the end of the pulse. In this work, the pulse signal of native kanamycin aptamer, a hairpin DNA without the added long tail, was investigated with the single nanopore sensing interface, and different current pulse pattern was observed. The pulse signal exhibited two precise current levels with significantly extended duration of the second, and both duration of the two levels correlate to the interaction of the aptamer to kanamycin. Moreover, the pulse signal not only reveals the selectivity of the aptamer to its target, but also sensitive to the loop sequence change of the aptamer. This work shows that a single nanopore sensing interface could be used as a unique alternative means for interaction investigation of hairpin DNA aptamer without labeling or adding the extra-long tail.