While a common workflow for data analysis exists in the nanopore research field, limitations remain. Notably, the fragmentation of methods and tools hinders interoperability and limits broader collaboration. Poriscope addresses these challenges by providing a unifying, modular framework for the analysis of single-molecule time series data acquired with nanopore sensors. It implements a plugin-based architecture that enables extensibility and interoperability while allowing users to customize each analytical stage independently from the overall framework. Any existing analysis methodology can be implemented as a self-contained plugin that integrates seamlessly into the overall pipeline via the Poriscope application programming interface (API). Poriscope is available both as a graphical desktop application and as a scripting module that can be imported into custom analysis scripts as needed. For more information, visit: https://tcossalab.github.io/poriscope/.

Digital immunoassays enable highly sensitive detection of biomolecules, offering absolute quantification rather than relying on bulk signal intensity. We adapt a digital immunoassay scheme for a nanopore sensor, a versatile platform for single-molecule counting. Current nanopore sensors have demonstrated great progress when counting nucleic acids but struggle with proteins due to variability in translocation behavior and limited recognition strategies. While recent advancements have highlighted the promise of nanopore platforms for protein studies, precise quantification remains a challenge. Here, building on previous work, we present a nanopore-based digital immunoassay that employs gold nanoparticle-mediated molecular amplification with a single-molecule readout. This approach translates protein recognition into quantifiable DNA, enabling a precise digital assay. This assay employs a DNA NanoLock probe combined with a paramagnetic bead-based immunocapture, where the target proteins trigger a structural transformation of the NanoLock, converting their presence into a binary DNA-based signal. By incorporating AuNPs carrying hundreds of DNA proxy reporters, we effectively amplify the detectable signal by 2 orders of magnitude, significantly improving sensitivity. We validate the performance of this system by detecting the glial fibrillary acidic protein, a biomarker for traumatic brain injury and neurodegenerative diseases, in plasma samples and demonstrate high femtomolar-level sensitivity (∼40 pg/mL). Using the NanoLock probe, we further mitigate previous challenges, with reduced assay times (hours) and extended dynamic range (3-log). The self-calibrating nature of this digital approach offers robust, reproducible measurements across different nanopores, eliminating interdevice variability.

Carbohydrates are one of the many biomolecular building blocks of life, with many practical applications in medicine. However, current methods for analysing these biomolecules struggle to operate at the single-molecule level. Carbohydrate sensing with solid-state nanopores has recently been explored but has proved difficult due to their structural complexity and the rapid translocation through the pores in the case of short polysaccharide chains. Among the numerous forms they can take, the best-known example is heparin, a widely used anticoagulant that has a closely related but toxic contaminant. In this work, we study the kinetics of capture and translocation of heparin molecules passing through a solid-state nanopore based on experimental results and signal simulations, seeking to understand the dynamics of passage. We examine the molecular interaction with the pore as a function of pH to elucidate the conformational dynamics of translocation. Despite using state-of-the-art electronics, we find that the durations of translocation through bare silicon nitride pores are too short to provide the resolution needed to reliably distinguish heparin from its contaminants. En route, we shed light on the physical mechanisms that govern translocation and suggest practical means by which future work might overcome these limitations.

Solid-state nanopores, nm-sized holes in thin, freestanding membranes, are powerful single-molecule sensors capable of interrogating a wide range of target analytes, from small molecules to large polymers. Interestingly, due to their high spatial resolution, nanopores can also identify tags on long polymers, making them an attractive option as the reading element for molecular information storage strategies. To fully leverage the compact and robust nature of solid-state nanopores, however, they will need to be packaged in a highly parallelized manner with on-chip electronic signal processing capabilities to rapidly and accurately handle the data generated. Additionally, the membrane itself must have specific physical, chemical, and electrical properties to ensure sufficient signal-to-noise ratios are achieved, with the traditional membrane material being SiNX . Unfortunately, the typical method of deposition, low-pressure vapour deposition, requires temperatures beyond the thermal budget of CMOS back-end-of-line integration processes, limiting the potential to generate an on-chip solution. To this end, we explore various lower-temperature deposition techniques that are BEOL-compatible to generate SiNx membranes for solid-state nanopore use, and successfully demonstrate the ability for these alternative methods to generate low-noise nanopores that are capable of performing single-molecule experiments.

As the complexity of solid-state nanopore experiments increases, analysis of the resulting electrical signals to determine biomolecular details becomes a challenge. State of the art techniques for this task perform poorly when transient signal characteristics approach the bandwidth limitations of the measurement electronics. In this work, we address this challenge through an algorithm, called Nano Trees, for fitting piecewise constant functions. Nano Trees leverages machine learning algorithms to provide accurate fits to the noisy piecewise constant data that is characteristic of nanopore ionic current signals, producing accurate fits on transients as short as twice the rise time of the measurement system. We demonstrate the performance of our algorithm on several real and synthetic datasets. These findings underscore the generalizability and accuracy of this approach in the regime of fast molecular translocations.

Nanopores are versatile single-molecule sensors thatare beingused to sense increasingly complex mixtures of structured moleculeswith applications in molecular data storage and disease biomarkerdetection. However, increased molecular complexity presents additionalchallenges to the analysis of nanopore data, including more translocationevents being rejected for not matching an expected signal structureand a greater risk of selection bias entering this event curationprocess. To highlight these challenges, here, we present the analysisof a model molecular system consisting of a nanostructured DNA moleculeattached to a linear DNA carrier. We make use of recent advances inthe event segmentation capabilities of Nanolyzer, a graphical analysistool provided for nanopore event fitting, and describe approachesto the event substructure analysis. In the process, we identify anddiscuss important sources of selection bias that emerge in the analysisof this molecular system and consider the complicating effects ofmolecular conformation and variable experimental conditions (e.g.,pore diameter). We then present additional refinements to existinganalysis techniques, allowing for improved separation of multiplexedsamples, fewer translocation events rejected as false negatives, anda wider range of experimental conditions for which accurate molecularinformation can be extracted. Increasing the coverage of analyzedevents within nanopore data is not only important for characterizingcomplex molecular samples with high fidelity but is also becomingessential to the generation of accurate, unbiased training data asmachine-learning approaches to data analysis and event identificationcontinue to increase in prevalence.

Due to their programmability via specific base pairing, self-assembled DNA origami structures have proven to be useful for a wide variety of applications, including diagnostics, molecular computation, drug delivery, and therapeutics. Measuring and characterizing these structures is therefore of great interest and an important part of quality control. Here, we show the extent to which DNA nanostructures can be characterized by a solid-state nanopore; a non-destructive, label-free, single-molecule sensor capable of electrically detecting and characterizing charged biomolecules. We demonstrate that in addition to geometrical dimensions, nanopore sensing can provide information on the mechanical properties, assembly yield, and stability of DNA nanostructures. For this work, we use a model structure consisting of a 3 helix-bundle (3HB), i.e. three interconnected DNA double helices using a M13 scaffold folded twice on itself by short DNA staple strands, and translocate it through solid-state nanopores fabricated by controlled breakdown. We present detailed analysis of the passage characteristics of 3HB structures through nanopores under different experimental conditions which suggest that segments of locally higher flexibility are present along the nanostructure contour that allow for the otherwise rigid 3HB to fold inside nanopores. By characterizing partially melted 3HB structures, we find that locally flexible segments are likely due to short staple oligomers missing from the fully assembled structure. The 3HB used herein is a prototypical example to establish nanopores as a sensitive, non-destructive, and label-free alternative to conventional techniques such as gel electrophoresis with which to characterize DNA nanostructures.

Solid-state nanopores are a technology poised to disrupt multiple critical fields, including but not limited to nucleic acid sequencing, proteomics, in vitro diagnostics, drug discovery, and next-generation information storage. While several proof-of-concept studies have been demonstrated the feasibility of many of these promising applications, work involving solid-state nanopores has been limited to research labs with specific nanofabrication expertise due to challenges in fabrication methods that have made them difficult to use outside of highly specialized academic settings. Over the past two decades, numerous methods of nanopore fabrication have arisen to tackle the problem of producing individual nanopores in the 1–30 nm range with angstrom precision in thin solid-state membranes. In this Chapter, we review the main methods of solid-state nanopore fabrication that have been developed so far to drive research in the field. We review nanopore formation by drilling with a transmission electron microscopeTransmission electron microscope, by ion beam milling, by pipette pulling, and provide a special emphasis on nanopore fabrication by controlled breakdownControlled breakdown, which shows promise to expand the user base and accelerate progress in the field. We compare and contrast each method in terms of the accessible range of pore sizes, the precision of size control, the shape of the resulting nanopores, and present an outlook for each method in terms of its potential to address the overarching scalability challenges on the way toward solid-state nanopore technologies.

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

Single molecule detection methods are becoming increasingly important for diagnostic applications. Practical Early detection of disease requires sensitivity down to the level of single copies of the targeted biomarkers. Of the candidate technologies that can address this need, solid-state nanopores show great promise as digital sensors for single-molecule detection. Here, we present work detailing the use of solid-state nanopores as downstream sensors for a PCR-based assay targeting group A streptococcus (strep A) which can be readily extended to detect any pathogen that can be identified with a short nucleic acid sequence. We demonstrate that with some simple modifications to the standard PCR reaction mixture, nanopores can be used to reliably identify strep A in clinical samples. We also discuss methodological best practices both for adapting PCR-based assays to solid-state nanopore readout as well as analytical approaches by which to decide on sample status.

The ability to digitally count single molecules enables accurate and precise determination of the concentration of a disease biomarker. Owing to their intrinsic single-molecule sensitivity and fully electronic detection capability, solid-state nanopores show great promise for this task toward point-of-care diagnostic applications. Here, we describe the protocols for implementing a magnetic bead-based immunoassay strategy coupled with digital detection and downstream solid-state nanopore electrical readout. The digital scheme employs DNA nanostructures, as proxy labels for the presence (“1”) or absence (“0”) of the target protein. We provide step-by-step protocols for assembling and purifying DNA nanostructures; Preparing magnetic beads decorated with capture antibodies; Conjugating secondary detection antibodies to bind the DNA label; Functionalizing gold nanoparticles; and Running the full assay where thyroid stimulating hormone (TSH) from human serum samples is quantified down to the femtomolar range. The protocols and assay scheme presented herein are easily generalized to the quantification of a wide range of target proteins, by selecting the appropriate antibody pair.

Single-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. The concentration of a biomarker in solution can be determined by counting single molecules. Here the authors report a digital immunoassay scheme with solid-state nanopore readout to quantify a target protein and use this to measure thyroid-stimulating hormone from human serum.

Traditional ELISA, long the workhorse for specific target protein detection using microplate wells, is nearing its fundamen-tal limit of sensitivity. New opportunities in healthcare call for in vitro diagnostic tests with ultra-high sensitivity. Magnetic bead-based ELISA formats have been developed that can reach unprecedented sensitivities order of magnitude better than are allowed for by the rate constants for a single ligand-receptor interaction. However, these ultra-high sensitivity assays are highly vulnerable to a host of confounding factors, including nonspecific binding from background molecules and loss of low-abundance target to tube walls and during wash steps. Moreover, optimization of workflow is often time-consuming and expensive. In this work, we present a simulation tool that allows users to graphically define arbitrary binding assays, includ-ing fully reversible first-order binding kinetics, timed addition of extra components, and timed wash steps. The tool is freely available as a user-friendly webapp. The framework is lightweight and fast, allowing for inexpensive simulation and visuali-zation of arbitrarily complex assay schemes, including but not limited to digital immunoassays, DNA hybridization, and enzyme kinetics, for validation and optimization of assay designs without requiring any programming knowledge from the user. We demonstrate some of these capabilities and provide practical guidance on assay simulation design.

We present a thorough exploration of nanopore growth under electrical stress in electrolyte solution, and demonstrate that despite their superficial similarities, nanopore formation by controlled breakdown (CBD) and nanopore growth under moderate voltage stress are fundamentally different processes. In particular, we demonstrate that unlike the CBD process, nanopore growth is primarily driven by the level of ionic current passing through the nanopore, rather than the strength of the electric field generating the current, and that enlargement has a much weaker pH dependence than does CBD pore formation. In combination with other works in the field, our results suggest that despite clear current-dependence, Joule heating is unlikely to be the main driver of pore growth during electrical stress, pointing instead toward electrochemical dissolution of membrane material along the pore walls. While the chemistry underlying the growth process remains unclear, the dependence of growth rate on current allows decoupling of the pore enlargement mechanism from the possibility of forming additional nanopores during the growth process, providing a practical method by which to rapidly enlarge a nanopore without risking opening a second nanopore.

The capture rate of linear dsDNA through solid-state nanopores is subject to significant variations, the nature of which remains unknown. To better understand the underlying physical processes responsible for these variations, we present nanopore capture rate data of dsDNA fragments of varying sizes for a wide range of experimental conditions. We use CBD-fabricated (controlled breakdown) nanopores to study the capture kinetics of DNA in pores of different sizes, solutions of different salt concentrations and pH. This is achieved by studying the dependencies of nanopore-capture on the applied voltage and DNA length, through which the two known capture regimes emerge. Furthermore, we investigate the translocation and capture properties under a salt asymmetry across the membrane and show that the conditions for the two capture regimes to emerge change compared to the symmetric case. These results are helping to develop a better fundamental understanding of the nanopore capture process, and may find applications in quantification of biological molecule concentrations using solid-state nanopores.

We use solid-state nanopores (fabricated by controlled breakdown, ranging from 3 to 30 nm in size in thin 10 nm thick SiN membranes) to study the electrophoretic capture and translocation characteristics of various DNA nanostructures from two-dimensional multi-branched molecules to three-dimensional ribbon-like origami structures. We discuss the capture regime, folding characteristics, and molecular configuration during passage for different, applied voltages and LiCl concentrations. We find that 12-arm short DNA stars with a relaxed core (carbon spacer) can be assembled with high yield and produce high signal-to-noise ratio (6x that of dsDNA). A 200 bp tail can be added to these DNA stars to produce shooting star-like nanostructures, alternatively the two stars can be linked together to form a dumbbell-like structure, thus further enhancing their multiplexing potential. Furthermore, 3 helix bundle (HB) and 6-HB show unique translocation and folding characteristics related to their flexibility. Finally, we show that the capture kinetics of these nanostructures can be enhanced by over 100-fold using salt concentration gradients while maintaining high quality nanopore temporal and ionic signals. The rapid capture and unique electrical blockages and signatures produced by these DNA nanostructures make them promising candidates for the development of multiplexed nanopore-based assays.

Solid-state nanopores are now well established as single-biomolecule sensors that hold great promise as sensing elements in diagnostic and sequencing applications. However, until recently this promise has been limited by the expensive, labor-intensive, and low-yield methods used to fabricate low-noise and precisely sized pores. To address this problem, we pioneered a low-cost and scalable solid-state nanopore fabrication method, termed controlled breakdown (CBD), which is rapidly becoming the method of choice for fabricating solid-state nanopores. Since its initial development, nanopore research groups around the world have applied and adapted the CBD method in a variety of ways, with varying levels of success. In this work, we present our accumulated knowledge of nanopore fabrication by CBD, including a detailed description of the instrumentation, software, and procedures required to reliably fabricate low-noise and precisely sized solid-state nanopores with a yield of >85% in less than 1 h. The assembly instructions for the various custom instruments can be found in the Supplementary Manual, and take approximately a day to complete, depending on the unit that the user is building and their level of skill with mechanical and electrical assembly. Unlike traditional beam-based nanopore fabrication technologies, the methods presented here are accessible to non-experts, lowering the cost of, and technical barriers to, fabricating nanoscale pores in thin solid-state membranes.

Elucidating the kinetics of DNA passage through a solid-state nanopore is a fertile field of research, and mechanisms for controlling capture, passage, and trapping of biopolymers are likely to find numerous technological applications. Here we present a nanofiltered nanopore device which forms an entropic cage for DNA following first passage through the nanopore, trapping the translocated DNA, and permitting recapture for subsequent reanalysis and investigation of kinetics of passage under confinement. We characterize the trapping properties of this nanodevice by driving individual DNA polymers into the nanoscale gap separating the nanofilter and the pore, forming an entropic cage similar to a "two pores in series" device, leaving polymers to diffuse in the cage for various time lengths, and attempting to recapture the same molecule. We show that the cage results in effectively permanent trapping when the radius of gyration of the target polymer is significantly larger than the radii of the pores in the nanofilter. We also compare translocation dynamics as a function of translocation direction to study the effects of confinement on DNA just prior to translocation, providing further insight into the nanopore translocation process. This nanofiltered nanopore device realizes simple fabrication of a femtoliter nanoreactor in which to study fundamental biophysics and biomolecular reactions on the single-molecule level. The device provides an electrically permeable single-molecule trap with a higher entropic barrier to escape than previous attempts to fabricate similar structures.

Using a solid-state nanopore to measure the concentration of clinically relevant target analytes, such as proteins or specific DNA sequences, is a major goal of nanopore research. This is usually achieved by measuring the capture rate of the target analyte through the pore. However, progress is hindered by sources of systematic error that are beyond the level of control currently achievable with state-of-the-art nanofabrication techniques. In this work, we show that the capture rate process of solid-state nanopores is subject to significant sources of variability, both within individual nanopores over time and between different nanopores of nominally identical size, which are absent from theoretical electrophoretic capture models. We experimentally reveal that these fluctuations are inherent to the nanopore itself and make nanopore-based molecular concentration determination insufficiently precise to meet the standards of most applications. In this work, we present a simple method by which to reduce this variability, increasing the reliability, accuracy, and precision of single-molecule nanopore-based concentration measurements. We demonstrate controlled counting, a concentration measurement technique, which involves measuring the simultaneous capture rates of a mixture of both the target molecule and an internal calibrator of precisely known concentration. Using this method on linear DNA fragments, we show empirically that the requirements for precisely controlling the nanopore properties, including its size, height, geometry, and surface charge density or distribution, are removed while allowing for higher-precision measurements. The quantitative tools presented herein will greatly improve the utility of solid-state nanopores as sensors of target biomolecule concentration.

Nanoscale preconfinement of DNA has been shown to reduce the variation of passage times through solid-state nanopores. Preconfinement has been previously achieved by forming a femtoliter-sized cavity capped with a highly porous layer of nanoporous silicon nitride (NPN). This cavity was formed by sealing a NPN nanofilter membrane against a substrate chip using water vapor delamination. Ultimately, this method of fabrication cannot keep a consistent spacing between the filter and solid-state nanopore due to thermal fluctuations and wrinkles in the membrane, nor can it be fabricated on thousands of individual devices reliably. To overcome these issues, we present a method to fabricate the femtoliter cavity monolithically, using a selective XeF2 etch to hollow out a polysilicon spacer sandwiched between silicon nitride layers. These monolithically fabricated cavities behave identically to their counterparts formed by vapor delamination, exhibiting similar translocation passage time variation reduction and folding suppression of DNA without requiring extensive manual assembly. The ability to form nanocavity sensors with nanometer-scale precision and to reliably manufacture them at scale using batch wafer processing techniques will find numerous applications, including motion control of polymers for single-molecule detection applications, filtering of dirty samples prior to nanopore detection, and simple fabrication of single-molecule nanobioreactors.