Lithium Chloride Effects Field-Induced Protein Unfolding and the Transport Energetics Inside a Nanopipette.

The tapered geometry of nanopipettes offers a unique perspective on protein transport through nanopores since both a gradual and fast confinement are possible depending on the translocation direction. The protein capture rate, unfolding, speed of translocation, and clogging probability are studied by toggling the LiCl concentration between 2 and 4 M. Interestingly, the proteins in this study could be transported with or against electrophoresis and offer vastly different attributes of sensing. Herein, a ruleset for studying proteins is developed that prevents irreversible pore clogging and yields upward of >100,000 events/nanopore. The extended duration of experiments further revealed that the capture rate takes ∼2 h to reach a steady state, emphasizing the importance of reaching equilibrated transport for studying the energetics and kinetics of protein transport (i.e., diffusion vs barrier-limited). Even in the equilibrated transport state, improper lowpass filtering was shown to distort the classification of diffusion-limited vs barrier-limited transport. Finally, electric-field-induced protein unfolding was found to be most prominent in electroosmotic-dominant transport, whereas electrophoretic-dominant events show no evidence of unfolding. Thus, our findings showcase the optimal conditions for protein translocations and the impact on studying protein unfolding, transporting energetics, and acquiring high bandwidth data.

Enhanced Signal to Noise Ratio Enables High Bandwidth Nanopore Recordings and Molecular Weight Profiling of Proteins.

Fast protein translocations often lead to bandwidth-limited amplitude-attenuated event signatures. In this study, we developed a protein- and electrolyte chemistry-centric pathway to construct a readily executable decision tree for the detection of non-attenuated protein translocations using conventional electronics. Each optimization encompasses increasing capture rate (CR), signal-to-noise ratio (SNR), and minimizing irreversible analyte clogging to collect >104 events/pipette spanning a host of electric fields. This was demonstrated using 11 proteins ranging from ∼12 kDa to ∼720 kDa. Moreover, both symmetric and asymmetric electrolyte conditions (cis and trans chamber electrolyte concentration ratios <> 1) were explored. As a result, asymmetric electrolyte conditions were favorable on the extreme ends of the size spectrum (i.e., larger, and smaller proteins) and while the remainder of proteins were best sensed under symmetric electrolyte conditions. Under these optimal conditions, only ≲10% of events were attenuated at 500 mV (≲ 5% for most proteins at 500 mV with only ≲1-5% of the population faster than ∼7 µs, which is the theoretical attenuation threshold for 100 kHz bandwidth). Finally, applied voltage (Vapp), peak current drop (ΔIp), electrolyte conductivity (K), and open-pore conductance (G0) were used to generate a linear relationship to evaluate the molecular weight of the protein (Mw) using plots of (dΔIp)/(dVapp) vs Mw/(G0/K).

Protein and DNA Yield Current Enhancements, Slow Translocations, and an Enhanced Signal-to-Noise Ratio under a Salt Imbalance.

Nanopores are a promising single-molecule sensing device class that captures molecular-level information through resistive or conductive pulse sensing (RPS and CPS). The latter has not been routinely utilized in the nanopore field despite the benefits it could provide, specifically in detecting subpopulations of a molecule. A systematic study was conducted here to study the CPS-based molecular discrimination and its voltage-dependent characteristics. CPS was observed when the cation movement along both electrical and chemical gradients was favored, which led to an ∼3× improvement in SNR (i.e., signal-to-noise ratio) and an ∼8× increase in translocation time. Interestingly, a reversal of the salt gradient reinstates the more conventional resistive pulses and may help elucidate RPS-CPS transitions. The asymmetric salt conditions greatly enhanced the discrimination of DNA configurations including linear, partially folded, and completely folded DNA states, which could help detect subpopulations in other molecular systems. These findings were then utilized for the detection of a Cas9 mutant, Cas9d10a─a protein with broad utilities in genetic engineering and immunology─bound to DNA target strands and the unbound Cas9d10a + sgRNA complexes, also showing significantly longer event durations (>1 ms) than typically observed for proteins.

On the origins of conductive pulse sensing inside a nanopore.

Nanopore sensing is nearly synonymous with resistive pulse sensing due to the characteristic occlusion of ions during pore occupancy, particularly at high salt concentrations. Contrarily, conductive pulses are observed under low salt conditions wherein electroosmotic flow is significant. Most literature reports counterions as the dominant mechanism of conductive events (a molecule-centric theory). However, the counterion theory does not fit well with conductive events occurring via net neutral-charged protein translocation, prompting further investigation into translocation mechanics. Herein, we demonstrate theory and experiments underpinning the translocation mechanism (i.e., electroosmosis or electrophoresis), pulse direction (i.e., conductive or resistive) and shape (e.g., monophasic or biphasic) through fine control of chemical, physical, and electronic parameters. Results from these studies predict strong electroosmosis plays a role in driving DNA events and generating conductive events due to polarization effects (i.e., a pore-centric theory).

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.

Nanopore Current Enhancements Lack Protein Charge Dependence and Elucidate Maximum Unfolding at Protein's Isoelectric Point.

Protein sequencing, as well as protein fingerprinting, has gained tremendous attention in the electrical sensing realm of solid-state nanopores and is challenging due to fast translocations and the use of high molar electrolytes. Despite providing an appreciable signal-to-noise ratio, high electrolyte concentrations can have adverse effects on the native protein structure. Herein, we present a thorough investigation of low electrolyte sensing conditions across a broad pH and voltage range generating conductive pulses (CPs) irrespective of protein net charge. We used Cas9 as the model protein and demonstrated that unfolding is noncooperative, represented by the gradual elongation or stretching of the protein, and sensitive to both the applied voltage and pH (i.e., charge state). The magnitude of unfolding and the isoelectric point (pI) of Cas9 was found to be correlated and a critical factor in our experiments. Electroosmotic flow (EOF) was always aligned with the transit direction, whereas electrophoretic force (EPF) was either reinforcing (pH < pI) or opposing (pH > pI) the protein's movement, which led to slower translocations at higher pH values. Further exploration of higher pH values led to slowing down of protein with > 30% of the population being slower than 0.5 ms. Our results would be critical for protein sensing at very low electrolytes and to retard their translocation speed without resorting to high-bandwidth equipment.

Pressure-Biased Nanopores for Excluded Volume Metrology, Lipid Biomechanics, and Cell-Adhesion Rupturing.

Nanopore sensing has been widely used in applications ranging from DNA sequencing to disease diagnosis. To improve these capabilities, pressure-biased nanopores have been explored in the past to-primarily-increase the residence time of the analyte inside the pore. Here, we studied the effect of pressure on the ability to accurately quantify the excluded volume which depends on the current drop magnitude produced by a single entity. Using the calibration standard, the inverse current drop (1/ΔI) decreases linearly with increasing pressure, while the dwell drop reduces exponentially. We therefore had to derive a pressure-corrected excluded volume equation to accurately assess the volume of translocating species under applied pressure. Moreover, a method to probe deformation in nanoliposomes and a single cell is developed as a result. We show that the soft nanoliposomes and even cells deform significantly under applied pressure which can be probed in terms of the shape factor which was introduced in the excluded volume equation. The proposed work has practical applications in mechanobiology, namely, assessing the stiffness and mechanical rigidity of liposomal drug carriers. Pressure-biased pores also enabled multiple observations of cell-cell aggregates as well as their subsequent rupture, potentially allowing for the study of microbial symbioses or pathogen recognition by the human immune system.

Nanodiagnostics: A review of the medical capabilities of nanopores.

Modern diagnostics strive to be accurate, fast, and inexpensive in addition to properly identifying the presence of a disease, infection, or illness. Early diagnosis is key; catching a disease in its early stages can be the difference between fatality and treatment. The challenge with many diseases is that detectability of the disease scales with disease progression. Since single molecule sensors, e.g., nanopores, can sense biomolecules at low concentrations, they have the potential to become clinically relevant in many of today's medical settings. With nanopore-based sensing, lower volumes and concentrations are required for detection, enabling it to be clinically beneficial. Other advantages to using nanopores include that they are tunable to an enormous variety of molecules and boast low costs, and fabrication is scalable for manufacturing. We discuss previous reports and the potential for incorporating nanopores into the medical field for early diagnostics, therapeutic monitoring, and identifying relapse/recurrence.

Constricted Apertures for Dynamic Trapping and Micro-/Nanoscale Discrimination Based on Recapture Kinetics.

Sensing via analyte passage through a constricted aperture is a powerful and robust technology which is being utilized broadly, from DNA sequencing to single virus and cell characterization. Micro- and nanoscale structures typically translocate a constricted aperture, or pore, using electrophoretic force. In the present work, we explore the advances in metrology which can be achieved through rapid directional switching of hydrodynamic forces. Interestingly, multipass measurements of microscale and nanoscale structures achieve cell discrimination. We explore this cell-discrimination phenomenon as well as other features of hydrodynamic focusing such as dynamic trapping and discrete interval sensing.

Measuring trapped DNA at the liquid-air interface for enhanced single molecule sensing.

Nanopore sensing is a promising tool with widespread application in single-molecule detection. Borosilicate glass nanopores are a viable alternative to other solid-state nanopores due to low noise and cost-efficient fabrication. For dielectric materials, including borosilicate glass, the capacitive noise is one of the major contributors to noise, which depends on the wall thickness and the surface area submerged in an ionic solution. Here, we investigated the root mean square (IRMS) noise and ionic conductance for borosilicate nanopores in different depths (i.e., tip submersion depth) ranging from the solution surface (assumed to be zero) to 5000 µm. Our findings demonstrate a decrease in IRMS noise as the pipette moves toward the surface. We further demonstrate that borosilicate nanopores can detect single lambda DNA (λ-DNA) molecules with a high signal-to noise ratio close to the liquid-air interface. Specifically, our results indicate a higher signal to noise ratio as the submersion depth is reduced owing to the reduced surface area and thus capacitive noise. Further, our experimental results show higher DNA capture frequency at the air-water interface due to a combined effect of evaporation and an evaporation-induced thermal gradient at the surface. Therefore, our findings demonstrate that borosilicate glass nanopores are suitable for studying interfacial concentration gradients of molecules, specifically DNA, with a higher signal to noise.

Substrate Dependent Ad-Atom Migration on Graphene and the Impact on Electron-Beam Sculpting Functional Nanopores.

The use of atomically thin graphene for molecular sensing has attracted tremendous attention over the years and, in some instances, could displace the use of classical thin films. For nanopore sensing, graphene must be suspended over an aperture so that a single pore can be formed in the free-standing region. Nanopores are typically drilled using an electron beam (e-beam) which is tightly focused until a desired pore size is obtained. E-beam sculpting of graphene however is not just dependent on the ability to displace atoms but also the ability to hinder the migration of ad-atoms on the surface of graphene. Using relatively lower e-beam fluxes from a thermionic electron source, the C-atom knockout rate seems to be comparable to the rate of carbon ad-atom attraction and accumulation at the e-beam/graphene interface (i.e., Rknockout ≈ Raccumulation). Working at this unique regime has allowed the study of carbon ad-atom migration as well as the influence of various substrate materials on e-beam sculpting of graphene. We also show that this information was pivotal to fabricating functional graphene nanopores for studying DNA with increased spatial resolution which is attributed to atomically thin membranes.

Nanopore sensing at ultra-low concentrations using single-molecule dielectrophoretic trapping.

Single-molecule techniques are being developed with the exciting prospect of revolutionizing the healthcare industry by generating vast amounts of genetic and proteomic data. One exceptionally promising route is in the use of nanopore sensors. However, a well-known complexity is that detection and capture is predominantly diffusion limited. This problem is compounded when taking into account the capture volume of a nanopore, typically 10(8)-10(10) times smaller than the sample volume. To rectify this disproportionate ratio, we demonstrate a simple, yet powerful, method based on coupling single-molecule dielectrophoretic trapping to nanopore sensing. We show that DNA can be captured from a controllable, but typically much larger, volume and concentrated at the tip of a metallic nanopore. This enables the detection of single molecules at concentrations as low as 5 fM, which is approximately a 10(3) reduction in the limit of detection compared with existing methods, while still maintaining efficient throughput.

Nonequilibrium capture rates induce protein accumulation and enhanced adsorption to solid-state nanopores.

Single molecule capturing of analytes using an electrically biased nanopore is the fundamental mechanism in which nearly all nanopore experiments are conducted. With pore dimensions being on the order of a single molecule, the spatial zone of sensing only contains approximately a zeptoliter of volume. As a result, nanopores offer high precision sensing within the pore but provide little to no information about the analytes outside the pore. In this study, we use capture frequency and rate balance theory to predict and study the accumulation of proteins at the entrance to the pore. Protein accumulation is found to have positive attributes such as capture rate enhancement over time but can additionally lead to negative effects such as long-term blockages typically attributed to protein adsorption on the surface of the pore. Working with the folded and unfolded states of the protein domain PDZ2 from SAP97, we show that applying short (e.g., 3-25 s in duration) positive voltage pulses, rather than a constant voltage, can prevent long-term current blockades (i.e., adsorption events). By showing that the concentration of proteins around the pore can be controlled in real time using modified voltage protocols, new experiments can be explored which study the role of concentration on single molecular kinetics including protein aggregation, folding, and protein binding.

High precision fabrication and positioning of nanoelectrodes in a nanopore.

A simple and versatile method for the direct fabrication of tunneling electrodes with controllable gap distance by using electron-beam-induced deposition (EBID) is presented. We show that tunneling nanogaps smaller than the minimum feature size realizable by conventional EBID can be achieved with a standard scanning electron microscope. These gaps can easily be embedded in nanopores with high accuracy. The controllability of this fabrication method and the nanogap geometry was verified by SEM and TEM imaging. Furthermore, tunneling spectroscopy in a group of solvents with different barrier heights was used to determine the nanogap functionality. Ultimately, the presented fabrication method can be further applied for the fabrication of arrays of nanogap/nanopores or nanogap electrodes with tunable electrode materials. Additionally, this method can also offer direct fabrication of nanoscale electrode systems with tunable spacing for redox cycling and plasmonic applications, which represents an important step in the development of tunneling nanopore structures and in enhancing the capabilities of nanopore sensors.

Gold nanoparticle translocation dynamics and electrical detection of single particle diffusion using solid-state nanopores.

This paper describes the use of gold nanoparticles to study particle translocation dynamics through silicon nitride solid-state nanopores. Gold nanoparticles were dispersed in 20 mM KCl solution containing nonionic surfactant Triton X-100 and their translocation was studied at different applied voltages. The use of low electrolyte concentration resulted in current enhancement upon particle translocation. The counterion cloud around the nanoparticles is proposed to be the reason for current enhancement phenomena because associated counterion cloud is believed to increase the ion density inside the pore during particle translocation. Further, single particle diffusion events were also recorded at 0 mV voltage bias and 0 pA background ionic current with high signal-to-noise ratio as the particles moved down their concentration gradient. The ability of nanopore sensors to detect single particle diffusion can be extended to field-free analysis of biomolecules in their native state and at or near physiological salt concentrations.

Detection of long and short DNA using nanopores with graphitic polyhedral edges.

Graphene is a unique material with a thickness as low as a single atom, high in-plane conductivity and a robust lattice that is self-supporting over large length scales. Schematically, graphene is an ideal solid-state material for tuning the properties of a nanopore because self-supported sheets, ranging from single to multiple atomic layers, can create pores with near-arbitrary dimensions which can provide exquisite control of the electric field drop within the pore. In this study, we characterize the drilling kinetics of nanopores using a thermionic electron source and various electron beam fluxes to minimize secondary hole formation. Once established, we investigated the use of multilayer graphene to create highly tailored nanostructures including nanopores with graphite polyhedral crystals formed around the nanopore edge. Finally, we report on the translocation of double stranded and single stranded DNA through such graphene pores and show that the single stranded DNA translocates much slower allowing detection of extremely short fragments (25 nucleotides in length). Our findings suggest that the kinetic and controllable properties of graphene nanopores under sculpting conditions can be used to further enhance the detection of DNA analytes.

Single molecule unfolding and stretching of protein domains inside a solid-state nanopore by electric field.

Single molecule methods have provided a significantly new look at the behavior of biomolecules in both equilibrium and non-equilibrium conditions. Most notable are the stretching experiments performed by atomic force microscopes and laser tweezers. Here we present an alternative single molecule method that can unfold a protein domain, observed at electric fields greater than 10(6) V/m, and is fully controllable by the application of increasing voltages across the membrane of the pore. Furthermore this unfolding mechanism is characterized by measuring both the residence time of the protein within the nanopore and the current blockade. The unfolding data supports a gradual unfolding mechanism rather than the cooperative transition observed by classical urea denaturation experiments. Lastly it is shown that the voltage-mediated unfolding is a function of the stability of the protein by comparing two mutationally destabilized variants of the protein.

Single-molecule studies of intrinsically disordered proteins using solid-state nanopores.

Partially or fully disordered proteins are instrumental for signal-transduction pathways; however, many mechanistic aspects of these proteins are not well-understood. For example, the number and nature of intermediate states along the binding pathway is still a topic of intense debate. To shed light on the conformational heterogeneity of disordered protein domains and their complexes, we performed single-molecule experiments by translocating disordered proteins through a nanopore embedded within a thin dielectric membrane. This platform allows for single-molecule statistics to be generated without the need of fluorescent labels or other modification groups. These studies were performed on two different intrinsically disordered protein domains, a binding domain from activator of thyroid hormone and retinoid receptors (ACTR) and the nuclear coactivator binding domain of CREB-binding protein (NCBD), along with their bimolecular complex. Our results demonstrate that both ACTR and NCBD populate distinct conformations upon translocation through the nanopore. The folded complex of the two disordered domains, on the other hand, translocated as one conformation. Somewhat surprisingly, we found that NCBD undergoes a charge reversal under high salt concentrations. This was verified by both translocation statistics as well as by measuring the ζ-potential. Electrostatic interactions have been previously suggested to play a key role in the association of intrinsically disordered proteins, and the observed behavior adds further complexity to their binding reactions.

Solid-state nanopore detection of protein complexes: applications in healthcare and protein kinetics.

Protein conjugation provides a unique look into many biological phenomena and has been used for decades for molecular recognition purposes. In this study, the use of solid-state nanopores for the detection of gp120-associated complexes are investigated. They exhibit monovalent and multivalent binding to anti-gp120 antibody monomer and dimers. In order to investigate the feasibility of many practical applications related to nanopores, detection of specific protein complexes is attempted within a heterogeneous protein sample, and the role of voltage on complexed proteins is researched. It is found that the electric field within the pore can result in unbinding of a freely translocating protein complex within the transient event durations measured experimentally. The strong dependence of the unbinding time with voltage can be used to improve the detection capability of the nanopore system by adding an additional level of specificity that can be probed. These data provide a strong framework for future protein-specific detection schemes, which are shown to be feasible in the realm of a 'real-world' sample and an automated multidimensional method of detecting events.

Fabrication of tunable silica-mineralized nanotubes using flagella as bio-templates.

Bacterial flagella are particularly attractive bio-templates for nanotubes due to their tubular structures and small inner and outer diameters. In this work, flagella isolated from Salmonella typhimurium were used as templates for silica-mineralized nanotubes. The process involved pretreatment of flagella with aminopropyltriethoxysilane (APTES), followed by the addition of tetraethoxysilane (TEOS). By controlling the concentration of TEOS and the reaction time, we developed a simple and precise method for creating silica-mineralized flagella nanotubes (SMFNs) with various thicknesses of the silica layer. It is demonstrated that flagella can be utilized for the fabrication of SMFNs with tunable thickness. A thicker silica layer was obtained as the concentration ratio of TEOS and reaction time was increased. The present experimental evidence has shown the feasibility of using such fabrication techniques to manufacture nanotubes without genetic modification of flagella which retain the original morphology.