A Robust Parallel Computing Data Extraction Framework for Nanopore Experiments.

The success of a nanopore experiment relies not only on the quality of the experimental design but also on the performance of the analysis program utilized to decipher the ionic perturbations necessary for understanding the fundamental molecular intricacies. An event extraction framework is developed that leverages parallel computing, efficient memory management, and vectorization, yielding significant performance enhancement. The newly developed abf-ultra-simple function extracts key parameters from the header critical for the operation of open-seek-read-close data loading architecture running on multiple cores. This underpins the swift analysis of large files where an ≈ × 18 improvement is found for a 100 min-long file (≈4.5 GB) compared to the more traditional single (cell) array data loading method. The application is benchmarked against five other analysis platforms showcasing significant performance enhancement (>2 ×-1120 ×). The integrated provisions for batch analysis enable concurrently analyzing multiple files (vital for high-bandwidth experiments). Furthermore, the application is equipped with multi-level data fitting based on abrupt changes in the event waveform. The application condenses the extracted events to a single binary file improving data portability (e.g., 16 GB file with 28 182 events reduces to 47.9 MB-343 × size reduction) and enables a multitude of post-analysis extractions to be done efficiently.

Electromigrated Gold Nanogap Tunnel Junction Arrays: Fabrication and Electrical Behavior in Liquid and Gaseous Media.

Tunnel junctions have been suggested as high-throughput electronic single molecule sensors in liquids with several seminal experiments conducted using break junctions with reconfigurable gaps. For practical single molecule sensing applications, arrays of on-chip integrated fixed-gap tunnel junctions that can be built into compact systems are preferable. Fabricating nanogaps by electromigration is one of the most promising approaches to realize on-chip integrated tunnel junction sensors. However, the electrical behavior of fixed-gap tunnel junctions immersed in liquid media has not been systematically studied to date, and the formation of electromigrated nanogap tunnel junctions in liquid media has not yet been demonstrated. In this work, we perform a comparative study of the formation and electrical behavior of arrays of gold nanogap tunnel junctions made by feedback-controlled electromigration immersed in various liquid and gaseous media (deionized water, mesitylene, ethanol, nitrogen, and air). We demonstrate that tunnel junctions can be obtained from microfabricated gold nanoconstrictions inside liquid media. Electromigration of junctions in air produces the highest yield (61-67%), electromigration in deionized water and mesitylene results in a lower yield than in air (44-48%), whereas electromigration in ethanol fails to produce viable tunnel junctions due to interfering electrochemical processes. We map out the stability of the conductance characteristics of the resulting tunnel junctions and identify medium-specific operational conditions that have an impact on the yield of forming stable junctions. Furthermore, we highlight the unique challenges associated with working with arrays of large numbers of tunnel junctions in batches. Our findings will inform future efforts to build single molecule sensors using on-chip integrated tunnel junctions.

Nanopore Blockade Sensors for Quantitative Analysis Using an Optical Nanopore Assay.

Nanopore sensing is a popular biosensing strategy that is being explored for the quantitative analysis of biomarkers. With low concentrations of analytes, nanopore sensors face challenges related to slow response times and selectivity. Here, we demonstrate an approach to rapidly detect species at ultralow concentrations using an optical nanopore blockade sensor for quantitative detection of the protein vascular endothelial growth factor (VEGF). This sensor relies on monitoring fluorescent polystyrene nanoparticles blocking nanopores in a nanopore array of 676 nanopores. The fluorescent signal is read out using a wide-field fluorescence microscope. Nonspecific blockade events are then distinguished from specific blockade events based on the ability to pull the particles out of the pore using an applied electric field. This allows the detection of VEGF at sub-picomolar concentration in less than 15 min.

Aptamer-Functionalized Interface Nanopores Enable Amino Acid-Specific Peptide Detection.

Single-molecule proteomics based on nanopore technology has made significant advances in recent years. However, to achieve nanopore sensing with single amino acid resolution, several bottlenecks must be tackled: controlling nanopore sizes with nanoscale precision and slowing molecular translocation events. Herein, we address these challenges by integrating amino acid-specific DNA aptamers into interface nanopores with dynamically tunable pore sizes. A phenylalanine aptamer was used as a proof-of-concept: aptamer recognition of phenylalanine moieties led to the retention of specific peptides, slowing translocation speeds. Importantly, while phenylalanine aptamers were isolated against the free amino acid, the aptamers were determined to recognize the combination of the benzyl or phenyl and the carbonyl group in the peptide backbone, enabling binding to specific phenylalanine-containing peptides. We decoupled specific binding between aptamers and phenylalanine-containing peptides from nonspecific interactions (e.g., electrostatics and hydrophobic interactions) using optical waveguide lightmode spectroscopy. Aptamer-modified interface nanopores differentiated peptides containing phenylalanine vs. control peptides with structurally similar amino acids (i.e., tyrosine and tryptophan). When the duration of aptamer-target interactions inside the nanopore were prolonged by lowering the applied voltage, discrete ionic current levels with repetitive motifs were observed. Such reoccurring signatures in the measured signal suggest that the proposed method has the possibility to resolve amino acid-specific aptamer recognition, a step toward single-molecule proteomics.

"Turbo-Charged" DNA Motors with Optimized Sequence Enable Single-Molecule Nucleic Acid Sensing.

DNA motors that consume chemical energy to generate processive mechanical motion mimic natural motor proteins and have garnered interest due to their potential applications in dynamic nanotechnology, biosensing, and drug delivery. Such motors translocate by a catalytic cycle of binding, cleavage, and rebinding between DNA "legs" on the motor body and RNA "footholds" on a track. Herein, we address the well-documented trade-off between motor speed and processivity and investigate how these parameters are controlled by the affinity between DNA legs and their complementary footholds. Specifically, we explore the role of DNA leg length and GC content in tuning motor performance by dictating the rate of leg-foothold dissociation. Our investigations reveal that motors with 0 % GC content exhibit increased instantaneous velocities of up to 150 nm/sec, three-fold greater than previously reported DNA motors and comparable to the speeds of biological motor proteins. We also demonstrate that the faster speed and weaker forces generated by 0 % GC motors can be leveraged for enhanced capabilities in sensing. We observe single-molecule sensitivity when programming the motors to stall in response to the binding of nucleic acid targets. These findings offer insights for the design of high-performance DNA motors with promising real-world biosensing applications.

Deep Learning-Assisted Single-Molecule Detection of Protein Post-translational Modifications with a Biological Nanopore.

Protein post-translational modifications (PTMs) play a crucial role in countless biological processes, profoundly modulating protein properties on both spatial and temporal scales. Protein PTMs have also emerged as reliable biomarkers for several diseases. However, only a handful of techniques are available to accurately measure their levels, capture their complexity at a single molecule level, and characterize their multifaceted roles in health and disease. Nanopore sensing provides high sensitivity for the detection of low-abundance proteins, holding the potential to impact single-molecule proteomics and PTM detection, in particular. Here, we demonstrate the ability of a biological nanopore, the pore-forming toxin aerolysin, to detect and distinguish α-synuclein-derived peptides bearing single or multiple PTMs, namely, phosphorylation, nitration, and oxidation occurring at different positions and in various combinations. The characteristic current signatures of the α-synuclein peptide and its PTM variants could be confidently identified by using a deep learning model for signal processing. We further demonstrate that this framework can quantify α-synuclein peptides at picomolar concentrations and detect the C-terminal peptides generated by digestion of full-length α-synuclein. Collectively, our work highlights the advantage of using nanopores as a tool for simultaneous detection of multiple PTMs and facilitates their use in biomarker discovery and diagnostics.

Selective Single-Molecule Nanopore Detection of mpox A29 Protein Directly in Biofluids.

Single-molecule antigen detection using nanopores offers a promising alternative for accurate virus testing to contain their transmission. However, the selective and efficient identification of small viral proteins directly in human biofluids remains a challenge. Here, we report a nanopore sensing strategy based on a customized DNA molecular probe that combines an aptamer and an antibody to enhance the single-molecule detection of mpox virus (MPXV) A29 protein, a small protein with an M.W. of ca. 14 kDa. The formation of the aptamer-target-antibody sandwich structures enables efficient identification of targets when translocating through the nanopore. This technique can accurately detect A29 protein with a limit of detection of ∼11 fM and can distinguish the MPXV A29 from vaccinia virus A27 protein (a difference of only four amino acids) and Varicella Zoster Virus (VZV) protein directly in biofluids. The simplicity, high selectivity, and sensitivity of this approach have the potential to contribute to the diagnosis of viruses in point-of-care settings.

Nanopore Signatures of Nucleoside Drugs.

Nucleoside drugs, which are analogues of natural nucleosides, have been widely applied in the clinical treatment of viral infections and cancers. The development of nucleoside drugs, repurposing of existing drugs, and combined use of multiple drug types have made the rapid sensing of nucleoside drugs urgently needed. Nanopores are emerging single-molecule sensors that have high resolution to resolve even minor structural differences between chemical compounds. Here, an engineered Mycobacterium smegmatis porin A hetero-octamer was used to perform general nucleoside drug analysis. Ten nucleoside drugs were simultaneously detected and fully discriminated. An accuracy of >99.9% was consequently reported. This sensing capacity was further demonstrated in direct nanopore analysis of ribavirin buccal tablets, confirming its sensing reliability against complex samples and environments. No sample separation is needed, however, significantly minimizing the complexity of the measurement. This technique may inspire nanopore applications in pharmaceutical production and pharmacokinetics measurements.

Metasurface Biosensors Enabling Single-Molecule Sensing of Cell-Free DNA.

In this study, we have revealed that highly fluorescence (FL)-enhancing all-dielectric metasurface biosensors are capable of detecting single-target DNA, which is cell-free DNA (cfDNA) specific to the human practice effect. The ultimately high-precision detection was achieved in a scheme combining the metasurface biosensors with a short-time nucleic acid amplification technique, that is, a reduced-cycle polymerase chain reaction (PCR). In this combined scheme, we obtained a series of FL signals at a single-molecule concentration, reflecting the Poisson distribution, and moreover elucidated that the FL signals exhibit the single-molecule cfDNA detection with more than 84% statistical confidence in an automated FL detection system and with 99.9% statistical confidence in confocal FL microscopy. As a result, we have found a simple and practical test to discriminate the target of 1 copy/test from zero using metasurface biosensors, which has not been realized by other elaborate techniques such as digital PCR.

Observation of Enantiomeric Switching of Individual Plasmonic Metamolecules.

Active plasmonic metamolecules under microscopic observation are promising for optical reporters in single molecule sensing applications. While self-assembled reconfigurable chiral plasmonic metamolecules can be conveniently engineered with sensing functionalities, their observation is usually based on ensemble measurements, where the chiroptical response of enantiomers tend to cancel each other in ensemble circular dichroism. Herein, we demonstrate microscopic observation of enantiomeric switching of individual active DNA origami-assembled plasmonic metamolecules. The metamolecules are immobilized on a glass substrate in a microfluidic chamber, in which the plasmonic metamolecule can maintain their activities upon certain local stimuli as in solution. In circular differential scattering, two enantiomeric states controlled by the strand-displacement reaction display opposite spectral signals to each other, representing successful enantiomeric switching of the chirality. Moreover, in a close-to-racemic mixture of chiral metamolecules controlled by pH-sensitive strands, the coexistence of enantiomeric individuals, which is concealed in ensemble measurements, is clearly identified.

Over 30-Fold Enhancement in DNA Translocation Dynamics through Nanoscale Pores Coated with an Anionic Surfactant.

Solid-state nanopores (ssNPs) are single-molecule sensors capable of label-free quantification of different biomolecules, which have become highly versatile with the introduction of different surface treatments. By modulating the surface charges of the ssNP, the electro-osmotic flow (EOF) can be controlled in turn affecting the in-pore hydrodynamic forces. Herein, we demonstrate that negative charge surfactant coating to ssNPs generates EOF that slows-down DNA translocation speed by >30-fold, without deterioration of the NP noise, hence significantly improving its performances. Consequently, surfactant-coated ssNPs can be used to reliably sense short DNA fragments at high voltage bias. To shed light on the EOF phenomena inside planar ssNPs, we introduce visualization of the electrically neutral fluorescent molecule's flow, hence decoupling the electrophoretic from EOF forces. Finite elements simulations are then used to show that EOF is likely responsible for in-pore drag and size-selective capture rate. This study broadens ssNPs use for multianalyte sensing in a single device.

Polarity Nano-Mapping of Polymer Film Using Spectrally Resolved Super-Resolution Imaging.

With the rapid development of the nanofabrication of polymer materials, the local measurement of the chemical properties of polymer nanostructures has become crucial because they can be highly heterogeneous at the nanoscale. We developed a spectroscopic imaging approach to characterize the nanoscale local polarity of polymer films via spectrally resolved super-resolution microscopy. We demonstrate the capability of the recently developed single-molecule sensing and imaging method to probe the polarity of polymers either inside a polymer matrix or on the external surface of a polymer. The nanoscale polarity sensing capability of our method facilitates the differentiation of various polymer surfaces based on chemical polarities, and it can further differentiate the polarity of functional side chain groups. Moreover, we demonstrate that a two-component polymer mixture can be locally distinguished based on the contrasting polarities of the lateral phase separation, further allowing for the investigation of nanoscale phase separation depending on the composition of the polymer blend film. This approach is anticipated to open the door to further characterizations of various nanocomposite materials.

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.

Single-File Translocation Dynamics of SDS-Denatured, Whole Proteins through Sub-5 nm Solid-State Nanopores.

The ability to routinely identify and quantify the complete proteome from single cells will greatly advance medicine and basic biology research. To meet this challenge of single-cell proteomics, single-molecule technologies are being developed and improved. Most approaches, to date, rely on the analysis of polypeptides, resulting from digested proteins, either in solution or immobilized on a surface. Nanopore biosensing is an emerging single-molecule technique that circumvents surface immobilization and is optimally suited for the analysis of long biopolymers, as has already been shown for DNA sequencing. However, proteins, unlike DNA molecules, are not uniformly charged and harbor complex tertiary structures. Consequently, the ability of nanopores to analyze unfolded full-length proteins has remained elusive. Here, we evaluate the use of heat denaturation and the anionic surfactant sodium dodecyl sulfate (SDS) to facilitate electrokinetic nanopore sensing of unfolded proteins. Specifically, we characterize the voltage dependence translocation dynamics of a wide molecular weight range of proteins (from 14 to 130 kDa) through sub-5 nm solid-state nanopores, using a SDS concentration below the critical micelle concentration. Our results suggest that proteins' translocation dynamics are significantly slower than expected, presumably due to the smaller nanopore diameters used in our study and the role of the electroosmotic force opposing the translocation direction. This allows us to distinguish among the proteins of different molecular weights based on their dwell time and electrical charge deficit. Given the simplicity of the protein denaturation assay and circumvention of the tailor-made necessities for sensing protein of different folded sizes, shapes, and charges, this approach can facilitate the development of a whole proteome identification technique.

Protein-enabled detection of ibuprofen and sulfamethoxazole using solid-state nanopores.

Enabled by proteins, we present an all-electrical method for rapid detection of small pharmaceuticals (ibuprofen and sulfamethoxazole [SMZ]) in aqueous media using silicon nitride pores. Specifically, we use carrier proteins, bovine serum albumin (BSA), and take advantage of their interactions with two small drug molecules to form BSA-drug complexes which can be detected by nm-diameter pores, thereby confirming the presence of small pharmaceuticals. We demonstrate detection of ibuprofen and SMZ at concentrations down to 100 nM (∼21 µg/L) and 48.5 nM (12 µg/L), respectively. We observe changes in electrical signal characteristics (reflected in event durations, rates, current magnitudes, and estimated particle diameters) of BSA-drug complexes compared to BSA-only, and differences between these two small pharmaceuticals, possibly paving a path toward developing selective sensors by identifying "electrical fingerprints" of these molecules in the future. These distinct electrical signals are likely a combined result of diffusion, electrophoretic and electroosmotic effects, interactions between the pore and particles, which depend on pore diameters, pH, and the resulting surface charges. The use of single-molecule-counting nanopores allows sensing of small pharmaceuticals, studies of protein conformational changes, and may aid in efforts to evaluate the impact of small drug molecules on aquatic and human life.

Optical Nanopore Sensors for Quantitative Analysis.

Nanopore sensors have received significant interest for the detection of clinically important biomarkers with single-molecule resolution. These sensors typically operate by detecting changes in the ionic current through a nanopore due to the translocation of an analyte. Recently, there has been interest in developing optical readout strategies for nanopore sensors for quantitative analysis. This is because they can utilize wide-field microscopy to independently monitor many nanopores within a high-density array. This significantly increases the amount of statistics that can be obtained, thus enabling the analysis of analytes present at ultralow concentrations. Here, we review the use of optical nanopore sensing strategies for quantitative analysis. We discuss optical nanopore sensing assays that have been developed to detect clinically relevant biomarkers, the potential for multiplexing such measurements, and techniques to fabricate high density arrays of nanopores with a view toward the use of these devices for clinical applications.

A Multiplexable Plasmonic Hairpin-DNA Sensor Based On Target-specific Tether Dynamics.

The need for measurements of multiple biomarkers simultaneously at subnanomolar concentrations asks for the development of new sensors with high sensitivity, specificity, precision, and accuracy. Currently, multiplexed sensing in single molecule sensors increases the complexity of the system in terms of reagents and sample read-out. In this letter, we propose a novel approach to multiplex hairpin-based single-DNA molecule sensors, which overcomes the limitations of the present approaches for multiplexing. By target-dependent ssDNA hairpin design, we can create DNA tethers that have distinct tether dynamics upon target binding. Our numerical model shows that by changing the stem length of the ssDNA hairpin, significantly different dynamic tether behavior will be observed. By exploiting the distance-dependent coupling of AuNPs to gold films, we can probe this dynamic behavior along the z-axis using a simple laser equipped microscope.

Nanopores in Graphene and Other 2D Materials: A Decade's Journey toward Sequencing.

Nanopore techniques offer a low-cost, label-free, and high-throughput platform that could be used in single-molecule biosensing and in particular DNA sequencing. Since 2010, graphene and other two-dimensional (2D) materials have attracted considerable attention as membranes for producing nanopore devices, owing to their subnanometer thickness that can in theory provide the highest possible spatial resolution of detection. Moreover, 2D materials can be electrically conductive, which potentially enables alternative measurement schemes relying on the transverse current across the membrane material itself and thereby extends the technical capability of traditional ionic current-based nanopore devices. In this review, we discuss key advances in experimental and computational research into DNA sensing with nanopores built from 2D materials, focusing on both the ionic current and transverse current measurement schemes. Challenges associated with the development of 2D material nanopores toward DNA sequencing are further analyzed, concentrating on lowering the noise levels, slowing down DNA translocation, and inhibiting DNA fluctuations inside the pores. Finally, we overview future directions of research that may expedite the emergence of proof-of-concept DNA sequencing with 2D material nanopores.

Amplification-free smartphone-based attomolar HBV detection.

Classical gold standard HBV detection relies on expensive devices and complicated procedures, thus is always restricted in large-scale hospitals and centers for disease control and prevention. To extend HBV detection to primary clinics especially in underdeveloped areas, we design amplification-free smartphone-based attomolar HBV detecting technique based on single molecule sensing. Verified by synthesized HBV target DNA, this technique reaches a detection limit at attomolar concentration (100 aM); and verified by 110 clinical samples, it also reaches a rather high sensitivity of 104 copy/mL (≈2000 IU/mL) with a high accuracy of 93.64% certificated by gold standard HBV detecting devices. Besides, this technique can quantify HBV viral load in 70 min only using portable and inexpensive devices as well as simple operations. Because of its cost-effective, field-portable and operable design, highly sensitive and selective detecting capability and wireless data connectivity, this technique can be potentially used in mobile HBV diagnoses and share HBV epidemic information especially in resource limited situations.

Influence of Electrolyte Concentration on Single-Molecule Sensing of Perfluorocarboxylic Acids.

Perfluorocarboxylic acids (PFCAs) are an emerging class of persistent organic pollutants. During the fabrication process, it is unavoidable to form PFCA homologs or isomers which exhibit distinct occurrence, bioaccumulation, and toxicity. The precision measurement of PFCAs is therefore of significant importance. However, the existing characterization techniques, such as LC-MS/MS, cannot fully meet the requirement of isomer-specific analysis, largely due to the lack of authentic standards. Single-molecule sensors (SMSs) based on nanopore electrochemistry may be a feasible solution for PFCAs determination, thanks to their ultra-high spatiotemporal resolutions. Hence, as a first step, this work was to elucidate the influence of electrolyte concentration on the four most critical indicators of nanopore measurements, and furthermore, performance of nanopore SMSs. More specifically, three of the most representative short-chain PFCAs, perfluoropentanoic acid (PFPeA), perfluorohexanoic acid (PFHxA) and perfluoroheptanoic acid (PFHpA), were adopted as the target analytes, aerolysin nanopore was employed as the sensing interface, and 2, 3 and 4 M KCl solutions were used as electrolytes. It was found that, when the concentration of KCl solution increased from 2 to 4 M, the conductance of aerolysin nanopore increased almost linearly at a rate of 0.5 nS per molar KCl within the whole voltage range, the current blockade of PFPeA at -50 mV increased from 61.74 to 66.57% owing to the enhanced steric exclusion effect, the maximum dwell time was more than doubled from 14.5 to 31.5 ms, and the barrier limited capture rate increased by 8.3 times from 0.46 to 3.85 Hz. As a result, when using 4 M KCl as the electrolyte, over 90% of the PFPeA, PFHxA and PFHpA were accurately identified from a mixed sample, and the calculated limit of detection of PFPeA reached 320 nM, more than 24 times lower than in 2 M KCl. It was thus clear that tuning the electrolyte concentration was a simple but very effective approach to improve the performance of nanopore SMSs for PFCAs determination.