Voltage gated inter-cation selective ion channels from graphene nanopores

With the ability to selectively control ionic flux, biological protein ion channels perform a fundamental role in many physiological processes. For practical applications that require the functionality of a biological ion channel, graphene provides a promising solid-state alternative, due to its atomic thinness and mechanical strength. Here, we demonstrate that nanopores introduced into graphene membranes, as large as 50 nm in diameter, exhibit inter-cation selectivity with a ~20x preference for K+ over divalent cations and can be modulated by an applied gate voltage. Liquid atomic force microscopy of the graphene devices reveals surface nanobubbles near the pore to be responsible for the observed selective behavior. Molecular dynamics simulations indicate that translocation of ions across the pore likely occurs via a thin water layer at the edge of the pore and the nanobubble. Our results demonstrate a significant improvement in the inter-cation selectivity displayed by a solid-state nanopore device and by utilizing the pores in a de-wetted state, offers an approach to fabricating selective graphene membranes that does not rely on the fabrication of sub-nm pores

PEG Equilibrium Partitioning in the α-Hemolysin Channel: Neutral Polymer Interaction with Channel Charges

We study the interaction of neutral polyethylene glycol (PEG) molecules of different molecular weights (MWs) with the charged residues of the α-hemolysin channel secreted by Staphylococcus aureus. Previously reported experiments of PEG equilibrium partitioning into this nanopore show that the charge state of the channel changes the ability of PEG entry in an MW-dependent manner. We explain such an effect by parameter-free calculations of the PEG self-energy from the channel 3D atomic structure that include repulsive dielectrophoretic and hydrostatic forces on the polymer. We found that the pH-induced shift in the measured free energy of partitioning ΔΔGexp from single-channel conductance measurements agrees with calculated energy changes ΔΔEcalc. Our results show that the PEG-sizing technique may need corrections in the case of charged biological pores

Rationally Designed DNA Origami Carriers for Quantitative Single Molecule Detection with Nanopipettes

The ability to detect small concentrations of biomarkers in patient samples is one of the cornerstones of modern healthcare. In general, biosensing approaches employed to address this need are based on measuring signals resulting from the interaction of a large ensemble of molecules with the sensor. Here, a biosensor platform using DNA origami, featuring a central cavity with a target–specific DNA aptamer, as carriers for translocation through nanopores which enables individual biomarkers to be identified and counted to compile a sensing signal is reported. It is shown that the modulation of the ion current through the nanopore upon the DNA origami translocation strongly depends on the presence and in fact the size of a central cavity. While DNA origami without a central cavity cause a single peak in the ion current, DNA origami of the same dimensions but featuring a central cavity lead to double peaks in the ion current. This is also true for DNA origami (with and without central cavities) made of similar sized DNA but of different dimensions. It is also observed that the peak characteristics, peak amplitude and the dwell time, are different depending on the presence or absence of a central cavity. This work exploits these parameters to generate a biosensing platform capable of detecting human C–reactive protein (CRP) in clinically relevant fluids. DNA origami frames with cavities large enough to lead to clear ion current double peaks were designed and a CRP–specific DNA aptamer was introduced into the cavity. Also, upon binding of CRP, the ion current peak changes to a single peak and the peak characteristics change. Using this three–parameter classification, CRP–occupied and unoccupied carriers can be distinguished when they translocate through the nanopore. Thus CRP biosensing by computing the ratio of occupied vs total number of frames with a limit of detection of 3 nM is successfully demonstrated

Access resistance in protein nanopores. A structure-based computational approach

Single-channel conductance measurements in biological pores have demonstrated the importance of interfacial effects in nanopores, particularly in protein channels with low aspect ratio (length over aperture radius). Access resistance (AR), the contribution to the total measured resistance arising from the electrodiffusive limitation that ions experience in passing from bulk solution to confinement within the pore, becomes essential in the description of ionic transport across these biological channels. Common analytical estimates of AR are based on idealized nanopore models, cylindrical in shape, electrically neutral and embedded in a neutral substrate. Here we calculate the AR of five protein channels by using their atomic structure and a mean-field approach based on solving 3D Poisson and Nernst-Planck equations. Our approach accounts for the influence of the protein charged ionizable residues, the geometry of the pore mouth and the ion concentration gradients near the pore. We compare numerical calculations with the few available AR measurements and show for several protein channels that analytical predictions tend to overestimate AR for physiological concentrations and below. We also discuss the relationship between AR and the size of the channel aperture in single-pore channels and three-pore channels and demonstrate that in the latter case, there is an enhancement of AR

Resistive Pulse Sensing of Protein Unfolding and Transport in Solid-State Nanopores

Solid-state nanopore sensors have attracted considerable attraction as a tool for solution-based single-molecule studies and have been successfully utilized for characterization of biomolecules such as nucleic acids, proteins, glycans, viruses, etc. Among these, characterization of proteins has been more challenging due to their charge heterogeneity and the complex energy landscape associated with different protein conformations. Presented in this thesis is the fabrication of solid-state nanopores and their application for characterizing proteins and understanding their transport through nanopores. Fabrication of nanometer-sized pores in SixNy membranes was achieved using the conventional controlled dielectric breakdown method as well as a simple modified version of it that resulted in ultra-stable nanopores devoid of legacy issues associated with the conventional nanopores. The noise characteristics of the fabricated nanopores were studied as a function of solution pH, electrolyte type and concentration, applied voltage and pore diameter. SixNy-based solid-state nanopores were used for studying the voltage and pH-induced conformational changes of the human serum transferrin protein and for distinguishing between its two forms – apo (iron-free) and holo (iron-rich). Finally, the transport of protein through nanopores was studied, first in symmetric salt conditions and then in asymmetric salt conditions. Investigating protein transport phenomena in different electrolyte types and concentrations as well as different electrolyte concentration gradients provided valuable insights into the electrokinetic phenomena such as electrophoresis and electroosmosis that govern analyte capture and transport through solid-state nanopores

Molecular Sensing with Protein and Solid-State Nanopores

In the past 15 years nanopore sensing has proven to be a successful method for probing a variety of molecules of biological interest, such as DNA, RNA and proteins. Of particular appeal is this technique\u27s ability to probe these molecules without the need for chemical modification or labeling, to do so at physiological conditions, and to probe single molecules at a time, allowing the possibility for results masked in bulk measurements to come to light. In this thesis these advantageous properties will be used in work on both a synthetic (solid-state) nanopore system and an engineered biological nanopore. I will describe the techniques for producing solid-state nanopores in thin membranes of silicon nitride and how these nanopores can be integrated into a fully functioning nanopore sensor system. I will then explore two applications of this system. First, a study of adsorption of bovine serum albumin (BSA), a protein found in blood serum, to the inorganic surface of nitride at the single molecule level. A simple physical model describing the behavior of this protein in the nanopore will be shown. Second, a study of the binding of the nucleocapsid protein of HIV-1 (NCp7) to three aptamers of different affinity, specifically three sequence 20mer mimics of the stem-loop 3 (SL3) RNA--the packaging domain of genomic RNA. Additionally, N-ethylmaleimide, which is known to inhibit the binding of NCp7 to a high-affinity SL3 RNA aptamer, will be used to demonstrate that the inhibition of the binding can be monitored in real time. Following these applications of the solid-state nanopore system, I will explore the geometry of a newly engineered biological nanopore, FhuA [Delta]C/[Delta]4L, by using inert polymers to probe the nanopore interio

Nanopores with Fluid Walls for Characterizing Proteins and Peptides.

Nanopore-based, resistive-pulse sensing is a simple single-molecule technique, is label free, and employs basic electronic recording equipment. This technique shows promise for rapid, multi-parameter characterization of single proteins; however, it is limited by transit times of proteins through a nanopore that are too fast to be resolved, non-specific interactions of proteins with the nanopore walls, and poor specificity of nanopores for particular proteins. This dissertation introduces the concept of nanopores with fluid walls and their applications in sensing and characterization of proteins, disease-relevant aggregates of amyloid-beta peptides, and activity of membrane-active enzymes. Inspired by lipid-coated nanostructures found in the olfactory sensilla of insect antennae, this work demonstrates that coating nanopores with a fluid lipid bilayer confers unprecedented capabilities to a nanopore such as precise control and dynamic actuation of nanopore diameters with sub-nanometer precision, well-defined control of protein transit times, simultaneous multi-parameter characterization of proteins, and an ability to monitor phospholipase D. Using these bilayer-coated nanopores with lipids presenting a ligand, proteins binding to the ligand were captured, concentrated on the surface, and selectively transported to the nanopore, thereby, conferring specificity to a nanopore. These assays enabled the first combined determination of a protein’s volume, shape, charge, and affinity for the ligand using a single molecule technique. For non-spherical proteins, the dipole moment and rotational diffusion coefficient could be determined from a single protein. Additionally, the fluid, biomimetic surface of a bilayer-coated nanopore was non-fouling and enabled characterization of Alzheimer’s disease-related amyloid-beta aggregates. The presented method and analysis fulfills a previously unmet need in the amyloid research field: a method capable of determining the size distributions and concentrations of amyloid-beta aggregates in solution. The experiments presented here demonstrate that the concept of a nanopore with fluid walls enables new nanopore-based assays. In particular, it demonstrates the benefits of this concept for simultaneous, multi-parameter characterization of proteins with a single-molecule method; this technique may, therefore, be well-suited for identification of proteins directly in complex biological fluids. Based on these findings, the addition of fluid walls to nanopores holds great promise as a tool for simple, portable single-molecule assays and protein characterization.PHDBiomedical EngineeringUniversity of Michigan, Horace H. Rackham School of Graduate Studieshttp://deepblue.lib.umich.edu/bitstream/2027.42/96049/1/ecyus_1.pd

Expanding the Functionality and Applications of Nanopore Sensors

Nanopore sensors have developed into powerful tools for single-molecule studies since their inception two decades ago. Nanopore sensors function as nanoscale Coulter counters, by monitoring ionic current modulations as particles pass through a nanopore. While nanopore sensors can be used to study any nanoscale particle, their most notable application is as a low cost, fast alternative to current DNA sequencing technologies. In recent years, signifcant progress has been made toward the goal of nanopore-based DNA sequencing, which requires an ambitious combination of a low-noise and high-bandwidth nanopore measurement system and spatial resolution. In this dissertation, nanopore sensors in thin membranes are developed to improve dimensional resolution, and these membranes are used in parallel with a high-bandwidth amplfier. Using this nanopore sensor system, the signals of three DNA homopolymers are differentiated for the first time in solid-state nanopores. The nanopore noise is also reduced through the addition of a layer of SU8, a spin-on polymer, to the supporting chip structure. By increasing the temporal and spatial resolution of nanopore sensors, studies of shorter molecules are now possible. Nanopore sensors are beginning to be used for the study and characterization of nanoparticles. Nanoparticles have found many uses from biomedical imaging to next-generation solar cells. However, further insights into the formation and characterization of nanoparticles would aid in developing improved synthesis methods leading to more effective and customizable nanoparticles. This dissertation presents two methods of employing nanopore sensors to benet nanoparticle characterization and fabrication. Nanopores were used to study the formation of individual nanoparticles and serve as nanoparticle growth templates that could be exploited to create custom nanoparticle arrays. Additionally, nanopore sensors were used to characterize the surface charge density of anisotropic nanopores, which previously could not be reliably measured. Current nanopore sensor resolution levels have facilitated innovative research on nanoscale systems, including studies of DNA and nanoparticle characterization. Further nanopore system improvements will enable vastly improved DNA sequencing capabilities and open the door to additional nanopore sensing applications

Metallic Nanopores for Single Molecule Biosensing

This thesis describes a novel approach to the fabrication and characterisation of metallic nanopores and their application for the detection of single DNA molecules. Metallic nanopores with apparent diameters below 20 nm are produced using electrochemical deposition and real-time ionic current feedback. Beginning with large nanopores (diameter 100-200 nm) milled into gold silicon nitride membranes using a focused ion beam, platinum metal is electrodeposited onto the gold surface, thus reducing the effective pore diameter. By simultaneously observing the ion current feedback, the shrinking of the nanopore can be monitored and terminated at any pre-defined value of the pore conductance in a precisely controlled and reproducible way. The ion transport properties of the metallic nanopore system are investigated by characterising the pore conductance at varying potentials across the nanopore and concentrations of electrolyte. The results are compared to conventional bare silicon nitride nanopore systems. Chemical modification at the nanopore surface is also studied using thiolisation to reduce the capacitive charging effects observed with metallic nanopores. Further to this, impedance measurements are carried out to study the resistive behaviour exhibited in these systems. An equivalent circuit model is proposed to validate the results obtained from the experimental studies. To evaluate the suitability of these nanopores for applications in single-molecule biosensing, translocation experiments using λ-DNA are performed. DNA molecules are electrokinetically driven through the nanopore under an applied electric field, hence as the DNA translocates through the pore, current blockade events are detected. Each event is the result of a single molecular interaction of DNA with the nanopore and is characterised by its dwell time and amplitude. Characterisation studies and noise analysis towards the applicability of metallic nanopores as single molecule detectors are also studied and compared to current bare silicon nitride pore systems

Doctor of Philosophy

dissertationThis dissertation presents experimental and computational investigations of electrolyte negative differential resistance, nanoparticle dynamics in nanopores, and nanobubble formation at nanoelectrodes. Chapter 1 provides an introduction to negative differential resistance and other nonlinear electrical responses in nanopores, an overview of resistive pulse analysis of nanoparticles using nanopores, and current nanobubble research. Chapter 2 describes the first example of electrolyte negative differential resistance (NDR) discovered in nanopores, where the current decreases as the voltage is increased. The NDR turn-on voltage was found to be tunable over a ~1 V window by adjusting the applied external pressure. Finite-element simulations yielded predictions of the NDR behavior that are in qualitative agreement with the experimental observations. Chapter 3 presents the extension of NDR to an aqueous system and demonstrates the potential for chemical sensing based on NDR behavior. Solution pH and Ca2+ in the solution were separately employed as the stimulus to investigate the surface charge density dependence of the NDR behavior. The NDR turn-on voltage was found to be exceedingly sensitive to the nanopore surface charge density, suggesting possible analytical applications in detecting as few as several hundred of molecules. Chapter 4 discusses the technique of controlling the dynamics of single 8 nm diameter gold nanoparticles in nanopores, which is extended from traditional resistive pulse analysis of nanoparticles. A pressure was applied to balance electrokinetic forces acting on the charged Au nanoparticles as they translocate through a ~10 nm diameter orifice at an electric field. This force balance provides a means to vary the velocity of nanoparticles by three orders of magnitude. Finite-element simulations yielded predictions in semiquantitative agreement with the experimental results. Chapter 5 reports the electrochemical generation of individual H2 nanobubbles at Pt nanodisk electrodes immersed in a H2SO4 solution. A sudden drop in current associated with the transport-limited reduction of protons was observed in the i-V response at Pt nanodisk electrodes of radii less than 50 nm. Finite element simulation based on Fick's first law, combined with the Young-Laplace equation and Henry's Law, were employed to investigate the bubble formation and its stabilization mechanism