Nanopore Conductance Research Articles

Development of Solid-State Nanopore Life Detection Technology

Biomarkers collected from past and present life on Earth are a central guide in the development of flight-ready technologies designed to search for life on other planets and moons. Data from numerous laboratory and field studies have revealed the importance of incorporating capabilities that minimize the risk of false negatives or false positives during life detection missions. Biomolecules are the most unambiguous and information-rich of all known biosignatures. Indeed, the identification in a sample of biopolymers akin to DNA, RNA or proteins, would be difficult to refute as a successful life detection experiment. In the case of Mars, the search for biomolecules is inescapable, but few technological solutions exist for in situ identification. Thus, our work has focused on the development of technologies designed to detect biochemical polyelectrolyte molecules, which have been argued to be a universal signature for life due to their ability to store information while reducing their tendency to adopt complex tertiary structures that would prohibit their replication. To that end we have developed a solid-state nanopore device that has the ability to detect electrically charged organic polymers, such as those found in deoxyribonucleic acid. Our nanopore technology, when used as a biosensor, consists of two or three ion-filled chambers separated by voltage-biased thin layer (Figure 1). Nanopores are milled into the thin layer, allowing the chambers to be connected electrically via an ionic solution. Polyelectrolytes are introduced into one of the chambers, and a patch clamp amplifier is connected to the chip via electrodes that serve as a voltage source and an ammeter. When a voltage is applied across the membrane, the ionic current going only through this pore is measured. The polyelectrolytes are captured by the electric field in the pore, then and electrophoretically drawn through the nanopore (translocation) causing the nanopore conductance to decrease. This conductance decrease, or transient current blockage, is evaluated for: 1) mean blockage current (which is caused by the physical passage of the polyelectrolyte through the nanopore, and is measured by a sensitive ammeter in pA); 2) translocation duration (physical time of the translocation, for DNA this is typically 30 bases per second when translocation is controlled by a molecular motor [1]); and 3) integrated area of a blockage, revealing key features of the particle. Solid-state nanopore technology is being aggressively advanced for use as an electrochemistry sensor for a wide variety of biological molecules. Testing of the solid-state nanopore membranes includes the establishment of criteria used to distinguish informational from non-informational output signals. Our work highlights past, current, and future efforts in the development of solid-state nanopore technology for life detection.

Positivity preserving finite difference methods for Poisson–Nernst–Planck equations with steric interactions: Application to slit-shaped nanopore conductance

To study ion transport in electrolyte solutions, we propose numerical methods for a modified Poisson–Nernst–Planck model with ionic steric effects (SPNP). Positivity preserving schemes based on harmonic-mean approximations are proposed on a nonuniform mesh for the spatial discretization of the SPNP equations. Both explicit and semi-implicit discretization are considered in time. Numerical analysis shows that explicit forward Euler and semi-implicit trapezoidal discretization lead to schemes that maintain fully discrete solution positivity under a constraint on a mesh ratio, while the semi-implicit backward Euler discretization maintain fully discrete solution positivity unconditionally. We further study the condition numbers of the matrix associated with the semi-implicit backward Euler discretization, and establish an upper bound on condition numbers, indicating that the developed discretization based on harmonic-mean approximations effectively solves the issue of the presence of large condition numbers when using the Slotboom-type variables. Further numerical simulations confirm the analysis results on accuracy, positivity, and bounded condition numbers. The proposed schemes are also applied to study practical applications, such as the impact of self and cross steric interactions on ion distribution and rectifying behavior in a slit-shaped nanopore with surface charges. Possible extensions of the numerical methods to other modified PNP models with ion correlations and steric effects are also discussed.

Rapid and Accurate Determination of Nanopore Ionic Current Using a Steric Exclusion Model.

Nanopore sensing has emerged as a versatile approach to detection and identification of biomolecules. Presently, researchers rely on experience and intuition for choosing or modifying the nanopores to detect a target analyte. The field would greatly benefit from a computational method that could relate the atomic-scale geometry of the nanopores and analytes to the blockade nanopore currents they produce. Existing computational methods are either computationally too expensive to be used routinely in experimental laboratories or not sensitive enough to account for the atomic structure of the pore and the analytes. Here, we demonstrate a robust and inexpensive computational approach-the steric exclusion model (SEM) of nanopore conductance-that is orders of magnitude more efficient than all-atom MD and yet is sensitive enough to account for the atomic structure of the nanopore and the analyte. The method combines the computational efficiency of a finite element solver with the atomic precision of a nanopore conductance map to yield unprecedented speed and accuracy of ionic current prediction. We validate our SEM approach through comparison with the current blockades computed using the all-atom molecular dynamics method for a range of proteins confined to a solid-state nanopore, biological channels embedded in a lipid bilayer membranes, and blockade currents produced by DNA homopolymers in MspA. We illustrate potential applications of SEM by computing blockade currents produced by nucleosome proteins in a solid-state nanopore, individual amino acids in MspA, and by testing the effect of point mutations on amino acid distinguishability. We expect our SEM approach to become an integral part of future development of the nanopore sensing field.

Atomic-Scale Fluidic Diodes Based on Triangular Nanopores in Bilayer Hexagonal Boron Nitride.

Nanofluidic diodes based on nanochannels have been studied theoretically and experimentally for applications such as biosensors and logic gates. However, when analyzing attoliter-scale samples or enabling high-density integration of lab-on-a-chip devices, it is beneficial to miniaturize the size of a nanofluidic channel. Using molecular dynamics simulations, we investigate conductance of nanopores in bilayer hexagonal boron nitride (h-BN). Remarkably, we found that triangular nanopores possess excellent rectifications of ionic currents while hexagonal ones do not. It is worth highlighting that the pore length is only about 0.7 nm, which is about the atomic limit for a bipolar diode. We determined scaling relations between ionic currents I and pore sizes L for small nanopores, that are I ∼ L1 in a forward biasing voltage and I ∼ L2 in a reverse biasing voltage. Simulation results qualitatively agree with analytical ones derived from the one-dimensional Poisson-Nerst-Planck equations.

High fidelity moving Z-score based controlled breakdown fabrication of solid-state nanopore

We investigate the current transport characteristics in the electrolyte-dielectric-electrolyte structure commonly used in the in situ controlled breakdown (CBD) fabrication of solid-state nanopores. It is found that the stochastic breakdown process could lead to fidelity issues of false positives (an incorrect indication of a true nanopore formation) and false negatives (inability to detect initial nanopore formation). Robust and deterministic detection of initial physical breakdown to alleviate false positives and false negatives is critical for precise nanopore size control. To this end, we report a high fidelity moving Z-score method based CBD fabrication of solid-state nanopore. We demonstrate 100% success rate of realizing the initial nanopore conductance of 3 ± 1 nS (corresponds to size of 1.7 ± 0.6 nm) regardless of the dielectric membrane characteristics. Our study also elucidates the Joule heating is the dominant mechanism for electric field-based nanopore enlargement. Single DNA molecule sensing using nanopores fabricated by this method was successfully demonstrated. We anticipate the moving Z-score based CBD method could enable broader access to the solid state nanopore-based single molecule analysis.

Controlling ionic current through a nanopore by tuning pH: a local equilibrium Monte Carlo study

ABSTRACTThe purpose of this work is to create a model of a nanofluidic transistor which is able to mimic the effects of pH on nanopore conductance. The pH of the electrolyte is an experimentally controllable parameter through which the charge pattern can be tuned: pH affects the ratio of the protonated/deprotonated forms of the functional groups anchored to the surface of the nanopore (for example, amino and carboxyl groups). Thus, the behaviour of the bipolar transistor changes as it becomes ion selective in acidic/basic environments. We relate the surface charge to pH and perform particle simulations (Local Equilibrium Monte Carlo) with different nanopore geometries (cylindrical and double conical). The simulations form a self consistent system with the Nernst–Planck equation with which we compute ionic flux. We discuss the mechanism behind pH-control of ionic current: formation of depletion zones.

Wavelet Denoising of High-Bandwidth Nanopore and Ion-Channel Signals.

Recent work has pushed the noise-limited bandwidths of solid-state nanopore conductance recordings to more than 5 MHz and of ion channel conductance recordings to more than 500 kHz through the use of integrated complementary metal-oxide-semiconductor (CMOS) integrated circuits. Despite the spectral spread of the pulse-like signals that characterize these recordings when a sinusoidal basis is employed, Bessel filters are commonly used to denoise these signals to acceptable signal-to-noise ratios (SNRs) at the cost of losing many of the faster temporal features. Here, we report improvements to the SNR that can be achieved using wavelet denoising instead of Bessel filtering. When combined with state-of-the-art high-bandwidth CMOS recording instrumentation, we can reduce baseline noise levels by over a factor of 4 compared to a 2.5 MHz Bessel filter while retaining transient properties in the signal comparable to this filter bandwidth. Similarly, for ion-channel recordings, we achieve a temporal response better than a 100 kHz Bessel filter with a noise level comparable to that achievable with a 25 kHz Bessel filter. Improvements in SNR can be used to achieve robust statistical analyses of these recordings, which may provide important insights into nanopore translocation dynamics and mechanisms of ion-channel function.

Formation of Single Nanopores with Diameters of 20-50 nm in Silicon Nitride Membranes Using Laser-Assisted Controlled Breakdown.

Nanopores with diameters from 20 to 50 nm in silicon nitride (SiN x) windows are useful for single-molecule studies of globular macromolecules. While controlled breakdown (CBD) is gaining popularity as a method for fabricating nanopores with reproducible size control and broad accessibility, attempts to fabricate large nanopores with diameters exceeding ∼20 nm via breakdown often result in undesirable formation of multiple nanopores in SiN x membranes. To reduce the probability of producing multiple pores, we combined two strategies: laser-assisted breakdown and controlled pore enlargement by limiting the applied voltage. Based on laser power-dependent increases in nanopore conductance upon illumination and on the absence of an effect of ionic strength on the ratio between the nanopore conductance before and after laser illumination, we suggest that the increased rate of controlled breakdown results from laser-induced heating. Moreover, we demonstrate that conductance values before and after coating the nanopores with a fluid lipid bilayer can indicate fabrication of a single nanopore versus multiple nanopores. Complementary flux measurements of Ca2+ through the nanopore typically confirmed assessments of single or multiple nanopores that we obtained using the coating method. Finally, we show that thermal annealing of CBD pores significantly increased the success rate of coating and reduced the current noise before and after lipid coating. We characterize the geometry of these nanopores by analyzing individual resistive pulses produced by translocations of spherical proteins and demonstrate the usefulness of these nanopores for estimating the approximate molecular shape of IgG proteins.

Femtosecond photonic viral inactivation probed using solid-state nanopores

We report on detection of virus inactivation using femtosecond laser radiation by measuring the conductance of a solid state nanopore designed for detecting single particles. Conventional methods of assaying for viral inactivation based on plaque forming assays require 24–48 h for bacterial growth. Nanopore conductance measurements provide information on morphological changes at a single virion level. We show that analysis of a time series of nanopore conductance can quantify the detection of inactivation, requiring only a few minutes from collection to analysis. Morphological changes were verified by dynamic light scattering. Statistical analysis maximizing the information entropy provides a measure of the log reduction value. This work provides a rapid method for assaying viral inactivation with femtosecond lasers using solid-state nanopores.

Role of charge regulation and flow slip in the ionic conductance of nanopores: An analytical approach.

The number of precise conductance measurements in nanopores is quickly growing. To clarify the dominant mechanisms at play and facilitate the characterization of such systems for which there is still no clear consensus, we propose an analytical approach to the ionic conductance in nanopores that takes into account (i) electro-osmotic effects, (ii) flow slip at the pore surface for hydrophobic nanopores, (iii) a component of the surface charge density that is modulated by the reservoir pH and salt concentration c_{s} using a simple charge regulation model, and (iv) a fixed surface charge density that is unaffected by pH and c_{s}. Limiting cases are explored for various ranges of salt concentration and our formula is used to fit conductance experiments found in the literature for carbon nanotubes. This approach permits us to catalog the different possible transport regimes and propose an explanation for the wide variety of currently known experimental behavior for the conductance versus c_{s}.

Investigation of Ionic Transport Through Track-Etched Conical Nanopores of PET Membrane

The control of ionic transportation inside the multi asymmetric conical nanopores in polyethylene terephthalate (PET) membrane was investigated. The conical nanopores were prepared by chemical etching in irradiated PET foil using etchant (9 M NaOH) and stopping solution (1 M NaCl [Formula: see text] 1 M HCOOH). The behavior of ionic current was recorded under stepping voltage [Formula: see text]2[Formula: see text]V to [Formula: see text]2[Formula: see text]V at different molar concentrations of potassium halides (KCl, KBr and KI) under symmetric bathing condition in electrochemical cell. It is found that the presence of multiple ionic species and the occurrence of counterion condensation of charge regulated polyelectrolyte play an effective role in ionic current rectification (ICR). The electrical conductance of conical nanopores may be estimated by measuring the ionic current rectification properties of track-etched nanopores. The charge transport properties vary with molar concentration and pH of electrolyte. Moreover, ICR may be used as a voltage gating phenomena with wide technological applications.

Conductance-based profiling of nanopores: Accommodating fabrication irregularities.

Solid-state nanopores are nanoscale channels through otherwise impermeable membranes. Single molecules or particles can be passed through electrolyte-filled nanopores by, e.g. electrophoresis, and then detected through the resulting physical displacement of ions within the nanopore. Nanopore size, shape, and surface chemistry must be carefully controlled, and on extremely challenging <10nm-length scales. We previously developed a framework to characterize nanopores from the time-dependent changes in their conductance as they are being formed through solution-phase nanofabrication processes with the appeal of ease and accessibility. We revisited this simulation work, confirmed the suitability of the basic conductance equation using the results of time-dependent experimental conductance measurements during nanopore fabrication by Yanagi etal., and then deliberately relaxed the model constraints to allow for (i) the presence of defects; and (ii) the formation of two small pores instead of one larger one. Our simulations demonstrated that the time-dependent conductance formalism supports the detection and characterization of defects, as well as the determination of pore number, but with implementation performance depending on the measurement context and results. In some cases, the ability to discriminate numerically between the correct and incorrect nanopore profiles was slight, but with accompanying differences in candidate nanopore dimensions that could yield to post-fabrication conductance profiling, or be used as convenient uncertainty bounds. Time-dependent nanopore conductance thus offers insight into nanopore structure and function, even in the presence of fabrication defects.

Aberrantly Large Single-Channel Conductance of Polyhistidine Arm-Containing Protein Nanopores.

There have been only a few studies reporting on the impact of polyhistidine affinity tags on the structure, function, and dynamics of proteins. Because of the relatively short size of the tags, they are often thought to have little or no effect on the conformation or activity of a protein. Here, using membrane protein design and single-molecule electrophysiology, we determined that the presence of a hexahistidine arm at the N-terminus of a truncated FhuA-based protein nanopore, leaving the C-terminus untagged, produces an unusual increase in the unitary conductance to ∼8 nS in 1 M KCl. To the best of our knowledge, this is the largest single-channel conductance ever recorded with a monomeric β-barrel outer membrane protein. The hexahistidine arm was captured by an anti-polyhistidine tag monoclonal antibody added to the side of the channel-forming protein addition, but not to the opposite side, documenting that this truncated FhuA-based protein nanopore inserts into a planar lipid bilayer with a preferred orientation. This finding is in agreement with the protein insertion in vivo, in which the large loops face the extracellular side of the membrane. The aberrantly large single-channel conductance, likely induced by a greater cross-sectional area of the pore lumen, along with the vectorial insertion into a lipid membrane, will have profound implications for further developments of engineered protein nanopores.

TRNA Modification Detection Using Graphene Nanopores: A Simulation Study.

There are over 100 enzyme-catalyzed modifications on transfer RNA (tRNA) molecules. The levels and identity of wobble uridine (U) modifications are affected by environmental conditions and diseased states, making wobble U detection a potential biomarker for exposures and pathological conditions. The current detection of RNA modifications requires working with nucleosides in bulk samples. Nanopore detection technology uses a single-molecule approach that has the potential to detect tRNA modifications. To evaluate the feasibility of this approach, we have performed all-atom molecular dynamics (MD) simulation studies of a five-layered graphene nanopore by localizing canonical and modified uridine nucleosides. We found that in a 1 M KCl solution with applied positive and negative biases not exceeding 2 V, nanopores can distinguish U from 5-carbonylmethyluridine (cm5U), 5-methoxycarbonylmethyluridine (mcm5U), 5-methoxycarbonylmethyl-2-thiouridine (mcm5s2U), and 5-methoxycarbonylmethyl-2′-O-methyluridine (mcm5Um) based on changes in the resistance of the nanopore. Specifically, we observed that in nanopores with dimensions less than 3 nm diameter, a localized mcm5Um and mcm5U modifications could be clearly distinguished from the canonical uridine, while the other modifications showed a modest yet detectable decrease in their respective nanopore conductance. We have compared the results between nanopores of various sizes to aid in the design, optimization, and fabrication of graphene nanopores devices for tRNA modification detection.

Solid State Nanopore Kit for Real-Time Analysis of DNA and Other Analytes

Recent improvements in nanofabrication techniques have greatly increased the reproducibility and stability of nanopores fabricated in ultrathin dielectrics such as silicon nitride or silicon oxide. These nanopores are now suitable for a wide range of accurate and reliable molecular analyses. Here we present a compact, portable and easy to use nanopore kit, intended for laboratory experiments and development of prototype devices. Experiments range from simple nanopore conductance and stability determination, to the more complex analysis of translocations of DNA and other analytes (resistive pulse sensing/nanopore spectroscopy). The kit consists of: -A Teflon flow-cell with two 1.0 ml chambers, hosting a central cassette containing the silicon chip with the nanopore. -A miniaturized low-noise and high bandwidth amplifier. The entire kit is contained within a 16x10x5 cm Faraday cage and connects to a laptop via a USB cable. The real-time analyses are performed using the amplifier control software, enabling a fast characterization of nanopore conductance, production of I/V curves and production of histograms for blockade sizes and dwell times. The kit was tested under different conditions, using nanopores in the range from 5 to 50nm. A 3,000bp fragment of DNA was successfully translocated on a 6 nm nanopore. A 48,500bp phage lambda DNA was successfully translocated on a 11 nm nanopore. Translocations were performed between potentials of 200mV and 380mV in 1M KCl buffer, 10mM HEPES pH 8.0. The versatility of the nanopore kit combined with the real-time analysis feature allows for quick and easy characterizations of nanopores and their interaction with analytes, for both educational and research purposes.

Transport of Water and Ions through a Graphene Nanopore in an Electric Field

Graphene nanopore has been widely used in a nanofluidic device for sensing or analyzing biomolecules, as its single atom thickness provides high spatial resolution. The ionic conductance of nanopore is of primary importance for analyzing biomolecules transported through the pore. The continuum theory holds that conductance comprise of three contributions: bulk, surface (for a charged nanopore) and access ones. However, when the pore diameter approaches the size of the molecule, ion and water dynamics may deviate from the bulk values and continuum analysis cannot be used to account for experimental data. Here we perform molecular dynamics simulations to systematically investigate the relationship between conductance and the coupling transport of water and ions through a graphene nanopore in electric fields. The results show that the ionic conductance of nanopore strongly depend on the ionic conditions, including the salt concentration, ion mobility in the pore. More surprisingly, with the increase of the electric field strength, ion concentration in the nanopore sharply drops from the bulk concentration, while ion mobility in the nanopore increases. Ion mobility in the nanopore will decrease as the salt concentration in the nanopore is increased. These unordinary ion behaviors should be due to be a sum of three contributions: collision of ions, Wien effect, desolvation. The results presented here will be helpful not only in understanding the behavior of ions and water transport but also in the design of novel nanofluidic devices.

Solid-State Nanopore Detection of Hydrophobic Proteins

We report on a modified solid-state nanopore measurement scheme to probe hydrophobic molecules that cannot be studied in conventional systems. Taking advantage of the intrinsic alcohol solubility of LiCl as an electrolyte, we show that the devices can be operated in alcohol-based azeotropic mixtures. We first characterize nanopore conductivity as a function of ethanol content, nanopore diameter, and salt concentration, showing predictable ionic response. Then, as a demonstration of resistive-pulse sensing, we measure and interpret electrical translocations of zeins, a class of water-insoluble maize protein.

DNA Origami-Graphene Hybrid Nanopore for DNA Detection.

DNA origami nanostructures can be used to functionalize solid-state nanopores for single molecule studies. In this study, we characterized a nanopore in a DNA origami-graphene heterostructure for DNA detection. The DNA origami nanopore is functionalized with a specific nucleotide type at the edge of the pore. Using extensive molecular dynamics (MD) simulations, we computed and analyzed the ionic conductivity of nanopores in heterostructures carpeted with one or two layers of DNA origami on graphene. We demonstrate that a nanopore in DNA origami-graphene gives rise to distinguishable dwell times for the four DNA base types, whereas for a nanopore in bare graphene, the dwell time is almost the same for all types of bases. The specific interactions (hydrogen bonds) between DNA origami and the translocating DNA strand yield different residence times and ionic currents. We also conclude that the speed of DNA translocation decreases due to the friction between the dangling bases at the pore mouth and the sequencing DNA strands.

Real-Time Profiling of Solid-State Nanopores During Solution-Phase Nanofabrication.

We describe a method for simply characterizing the size and shape of a nanopore during solution-based fabrication and surface modification, using only low-overhead approaches native to conventional nanopore measurements. Solution-based nanopore fabrication methods are democratizing nanopore science by supplanting the traditional use of charged-particle microscopes for fabrication, but nanopore profiling has customarily depended on microscopic examination. Our approach exploits the dependence of nanopore conductance in solution on nanopore size, shape, and surface chemistry in order to characterize nanopores. Measurements of the changing nanopore conductance during formation by etching or deposition can be analyzed using our method to characterize the nascent nanopore size and shape, beyond the typical cylindrical approximation, in real-time. Our approach thus accords with ongoing efforts to broaden the accessibility of nanopore science from fabrication through use: it is compatible with conventional instrumentation and offers straightforward nanoscale characterization of the core tool of the field.

Fabrication of Low Noise Borosilicate Glass Nanopores for Single Molecule Sensing.

We show low-cost fabrication and characterization of borosilicate glass nanopores for single molecule sensing. Nanopores with diameters of ~100 nm were fabricated in borosilicate glass capillaries using laser assisted glass puller. We further achieve controlled reduction and nanometer-size control in pore diameter by sculpting them under constant electron beam exposure. We successfully fabricate pore diameters down to 6 nm. We next show electrical characterization and low-noise behavior of these borosilicate nanopores and compare their taper geometries. We show, for the first time, a comprehensive characterization of glass nanopore conductance across six-orders of magnitude (1M-1μM) of salt conditions, highlighting the role of buffer conditions. Finally, we demonstrate single molecule sensing capabilities of these devices with real-time translocation experiments of individual λ-DNA molecules. We observe distinct current blockage signatures of linear as well as folded DNA molecules as they undergo voltage-driven translocation through the glass nanopores. We find increased signal to noise for single molecule detection for higher trans-nanopore driving voltages. We propose these nanopores will expand the realm of applications for nanopore platform.