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
(
) chromosome. Coverage of 3.1× across 2355 resolvable sites of the
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
DNA, with detection of 133 intersite intervals shorter than 200 bp in the
reference map. We present results on estimating distances in repetitive regions of the
genome. With an appropriately designed array, higher throughput implementations could enable human-sized genome and epigenome mapping applications.
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.
This Minireview summarizes the latest advances in data processing techniques for single‐entity electrochemistry towards achieving automation, real‐time monitoring, increased sensitivity, as well as improved temporal and current resolution.
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.
By using molecular dynamics simulations, charged graphene nanopores are employed to electroosmotically capture and sense uncharged peptides, and their phosphorylated states are differentiated by both the ionic current and pulling force signals. The results show ability of using nanopores to discriminate single amino acid and its modification, which is essential for the future low‐cost and high‐throughput protein sequencing.
Protein post-translational modification (PTM) is crucial to modulate protein interactions and activity in various biological processes. Emerging evidence has revealed PTM patterns participate in the ...pathology onset and progression of various diseases. Current PTM identification relies mainly on mass spectrometry-based approaches that limit the assessment to the entire protein population in question. Here we report a label-free method for the detection of the single peptide with only one amino acid modification via electronic fingerprinting using reengineered durable channel of phi29 DNA packaging motor, which bears the deletion of 25-amino acids (AA) at the C-terminus or 17-AA at the internal loop of the channel. The mutant channels were used to detect propionylation modification via single-molecule fingerprinting in either the traditional patch-clamp or the portable MinION™ platform of Oxford Nanopore Technologies. Up to 2000 channels are available in the MinION™ Flow Cells. The current signatures and dwell time of individual channels were identified. Peptides with only one propionylation were differentiated. Excitingly, identification of single or multiple modifications on the MinION™ system was achieved. The successful application of PTM differentiation on the MinION™ system represents a significant advance towards developing a label-free and high-throughput detection platform utilizing nanopores for clinical diagnosis based on PTM.
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.
Biomimetic nanopores of complex function are built with DNA nanotechnology. The pores undergo dynamic changes in shape and can be locked in an open state by specific binding of DNA and protein stimuli, and close at high transmembrane potentials. The pores may be used in biosensing and synthetic biology.
The ability to measure biomarkers, both specifically and selectively at the single‐molecule level in biological fluids, has the potential to transform the diagnosis, monitoring, and therapeutic ...intervention of diseases. The use of nanopores has been gaining prominence in this area, not only for sequencing but more recently in screening applications. The selectivity of nanopore sensing can be substantially improved with the use of labels, but substantial challenges remain, especially when trying to differentiate between bound from unbound targets. Here highly sensitive and selective molecular probes made from nanoparticles (NPs) that self‐assemble and dimerize upon binding to a biological target are designed. It is shown that both single and paired NPs can be successfully resolved and detected at the single‐molecule nanopore sensing and can be used for applications such as antigen/antibody detection and microRNA (miRNA) sequence analysis. It is expected that such technology will contribute significantly to developing highly sensitive and selective strategies for the diagnosis and screening of diseases without the need for sample processing or amplification while requiring minimal sample volume.
A single‐molecule nanopore‐based sensing strategy is developed to screen for biomarkers. In the presence of the target biomolecules, AuNPs dimerize and are detected electrically using a nanopore. This approach is highly sensitive and selective and can potentially be used for diagnosis and screening of diseases without the need for sample processing or amplification.
Accurate detection and identification of conformational evolutions are essential to describe the micro-structural mechanisms of protein allostery. Here we establish an approach by integrating ...nanopore sensing technique with atomic force microscopy, based on molecular dynamics simulations. A theoretical method is further developed to real-time estimate the low-resolution structures of those evolved conformations. Tests for the forced allostery of αXβ2 integrin, a typical protein with multiple functional states, demonstrate that the spheroidal shape identifiers of those intermediate conformers present sufficient sensitivity to resolve two allosteric patterns of αXβ2 from bent-down to stand-up states under the nanopore confinement. The strong steric confinement from the nanopore restricts the geometric displacements of key domains in conformational extension dynamics, thereby increasing the intramolecular interaction energy barrier to be overcome for standing. Prolongation of the neck-shoulder region of αXβ2 and opening at the thigh-calf joint form a competitive compensation for the stretched height, resulting in two tendentious allosteric patterns under various confined diameters and steered speeds. Thus, this study proposes a new method to real-time visualize the conformational dynamics during protein allostery, favoring to depict structural characteristics for the conformations of those transient states and evaluate the impacts of the nanopore confinement on protein allostery.
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•A MD approach of combining nanopore and AFM to probe protein conformational dynamics.•Shape signatures of evolutive conformations resolved by SMD-spheroid approximation.•Nanopore confinement enhanced the energy barrier of structural extension of αXβ2.•Intramolecular domains were competitive in compensating for the stretched height.•Two allosteric patterns under varied confinements and steered speeds were formed.
Nucleic acid nanoparticles (NANPs) are an emerging class of programmable structures with tunable shape and function. Their promise as tools for fundamental biophysics studies, molecular sensing, and ...therapeutic applications necessitates methods for their detection and characterization at the single-particle level. In this work, we study electrophoretic transport of individual ring-shaped and cube-shaped NANPs through solid-state nanopores. In the optimal nanopore size range, the particles must deform to pass through, which considerably increases their residence time within the pore. Such anomalously long residence times permit detection of picomolar amounts of NANPs when nanopore measurements are carried out at a high transmembrane bias. In the case of a NANP mixture, the type of individual particle passing through nanopores can be efficiently determined from analysis of a single electrical pulse. Molecular dynamics simulations provide insight into the mechanical barrier to transport of the NANPs and corroborate the difference in the signal amplitudes observed for the two types of particles. Our study serves as a basis for label-free analysis of soft programmable-shape nanoparticles.
We present a novel method to trap nanoparticles in double nanohole (DNH) nanoapertures integrated on top of solid-state nanopores (ssNP). The nanoparticles were propelled by an electrophoretic force ...from the cis towards the trans side of the nanopore but were trapped in the process when they reached the vicinity of the DNH-ssNP interface. The self-induced back action (SIBA) plasmonic force existing between the tips of the DNH opposed the electrophoretic force and enabled simultaneous optical and electrical sensing of a single nanoparticle for seconds. The novel SIBA actuated nanopore electrophoresis (SANE) sensor was fabricated using two-beam GFIS FIB. Firstly, Ne FIB milling was used to create the DNH features and was combined with end pointing to stop milling at the metal-dielectric interface. Subsequently, He FIB was used to drill a 25 nm nanopore through the center of the DNH. Proof of principle experiments to demonstrate the potential utility of the SANE sensor were performed with 20 nm silica and Au nanoparticles. The addition of optical trapping to electrical sensing extended translocation times by four orders of magnitude. The extended electrical measurement times revealed newly observed high frequency charge transients that were attributed to bobbing of the nanoparticle driven by the competing optical and electrical forces. Frequency analysis of this bobbing behavior hinted at the possibility of distinguishing single from multi-particle trapping events. We also discuss how SANE sensor measurement characteristics differ between silica and Au nanoparticles due to differences in their physical properties and how to estimate the charge around a nanoparticle. These measurements show promise for the SANE sensor as an enabling tool for selective detection of biomolecules and quantification of their interactions.
Nanopores play essential roles in biological processes, such as ion channels and pumps in cellular membranes, and in technological applications such as DNA sequencing. Advancements in nanofabrication ...techniques have enabled the routine integration of nanopores into solid-state devices, resulting in a plethora of analytical applications. This review explores recent developments in nanopore-enabled electrochemical systems, which have transcended traditional resistive pulse sensing to offer novel capabilities in single-entity studies, stimulus-responsive gating, and point-of-care diagnostics. We highlight recent studies on the design and utility of nanopore electrode arrays, which serve as nanocontainers capable of isolating and analyzing single entities, and extend the discussion to hierarchically organized, stimulus-responsive systems that regulate species transport across nanopores, enriching analytes for ultrasensitive detection. In addition, we review the utilization of probe-assisted nanopore sensing, demonstrating its efficacy in selectively binding and detecting target molecules and ions. Finally, we outline future directions for nanopore-based systems to enhance robustness, achieve high-throughput analysis, and incorporate artificial intelligence for materials design and data analysis, promising transformative impacts on diagnostics and biological research.
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