Investigation of entrance effects on particle electrophoretic behavior near a nanopore for resistive pulse sensing

Resistive pulse sensing using solid-state nanopores provides a unique platform for detecting the structure and concentration of molecules of different types of analytes in an electrolyte solution. The capture of an entity into a nanopore is subject not only to the electrostatic force but also the effect of electroosmotic flow originating from the charged nanopore surface. In this study, we theoretically analyze spherical particle electrophoretic behavior near the entrance of a charged nanopore. By investigating the effects of pore size, particle–pore distance, and salt concentration on particle velocity, we summarize dominant mechanisms governing particle behavior for a range of conditions. In the literature, the Helmholtz–Smoluchowski equation is often adopted to evaluate particle translocation by considering the zeta potential difference between the particle and nanopore surfaces. We point out that, due to the difference of the electric field inside and outside the nanopore and the influence from the existence of the particle itself, the zeta potential of the particle, however, needs to be at least 30% higher than that of the nanopore to allow the particle to enter into the nanopore when its velocity is close to zero. Accordingly, we summarize the effective salt concentrations that enable successful particle capture and detection for different pore sizes, offering direct guidance for nanopore applications.     

Electric field-controlled water permeation coupled to ion transport through a nanopore

We report molecular dynamics simulations of a generic hydrophobic nanopore connecting two reservoirs which are initially at different Na(+) concentrations, as in a biological cell. The nanopore is impermeable to water under equilibrium conditions, but the strong electric field caused by the ionic concentration gradient drives water molecules in. The density and structure of water in the pore are highly field dependent. In a typical simulation run, we observe a succession of cation passages through the pore, characterized by approximately bulk mobility. These ion passages reduce the electric field, until the pore empties of water and closes to further ion transport, thus providing a possible mechanism for biological ion channel gating.     

Resistive-pulse detection of multilamellar liposomes

The resistive-pulse method was used to monitor the pressure-driven translocation of multilamellar liposomes with radii between 190 and 450 nm through a single conical nanopore embedded in a glass membrane. Liposomes (0% and 5% 1,2-dioleoyl-sn-glycero-3-phospho-l-serine (sodium salt) in 1,2-dilauroyl-sn-glycero-3-phosphocholine or 0%, 5%, and 9% 1,2-dipalmitoyl-sn-glycero-3-phospho(1'-rac-glycerol) (sodium salt) in 1,2-dipalmitoyl-sn-glycero-3-phosphocholine) were prepared by extrusion through a polycarbonate membrane. Liposome translocation through a glass nanopore was studied as a function of nanopore size and the temperature relative to the lipid bilayer transition temperature, T(c). All translocation events through pores larger than the liposome, regardless of temperature, show translocation times between 30 and 300 μs and current pulse heights between 0.2% and 15% from the open pore baseline. However, liposomes at temperatures below the T(c) were captured at the pore orifice when translocation was attempted through pores of smaller dimensions, but squeezed through the same pores when the temperature was raised above T(c). The results provide insights into the deformation and translocation of individual liposomes through a porous material.     

Mean force potential of interaction between Na<Superscript>+</Superscript> and Cl<Superscript>−</Superscript> ions in planar nanopores in contact with water under pressure

The mean force potential (MFP) of interaction between counterions Na⁺ and Cl^? in a planar nanopore with structureless hydrophobic walls is calculated via computer simulation under the condition that the nanopore is in contact with water at an external pressure that exceeds the saturation pressure but remains insufficient to fill the nanopore with water. For a nanopore with a liquid phase, the MFP dependence on the interionic distance indicates the dissociation of an ion pair into two hydrated ions in a nanopore that is not completely filled with water. Fluctuations in the number of water molecules drawn into the interionic space decisively influence the dissociation. The attraction between counterions, averaged over thermal fluctuations, depends largely on the pore width and grows as the shielding of the ions’ electric field by water molecules in a narrow pore diminishes. The contributions from energy and entropy to the free energy of hydration are analyzed.     

Modeling the induction of lipid membrane electropermeabilization

Experiments show significant effects of an electric field on lipid membrane, leading to a pore formation when a high intensity field is applied. The phenomenon of electroporation is preceded by the induction and expansion of defects, responsible for the pre-pore excitation. We examine the mechanism of the induction of the field-driven defects by Monte Carlo simulations. The study is based on the improved Pink's model, which includes explicit interactions between the polar heads and energy of interactions between the heads and the field. No anomalous deformation of the molecules is considered. The study, provided for bilayer dipalmitoyl-phosphatidylcholine (DPPC) membrane in the gel (300 K) and fluid (330 K) phases, shows dependence of the membrane conformational and energetical state on the value of the electric field. We observe that the electric field affects the number of molecules in the gel and in the fluid states. In the layer at the negative potential, when the transmembrane voltage is above U(c) approximately 280 mV, lipid heads abruptly reorient and the number of local spots with fluid conformation increases. The other layer slightly tends to tighten its structure, producing additional mechanical stress between layers. Lipids showed complete insensitivity to the electric field within physiological limits, U<70 mV.     

Structure of a Na<Superscript>+</Superscript> cation hydration shell on heating in a planar nanopore

Resistance to heating above the boiling point of water of the molecular structure of a single-charged sodium cation hydration shell growing under the conditions of a model planar nanopore with a width of 5 Å is studied by computer simulation. Monte Carlo calculations of spatial correlation functions are performed in a detailed model with regard to many-body interactions between the ion and water molecules. The system demonstrates an increased resistance to thermal fluctuations along the pore plane and a decreased one in the transverse direction. The heating is accompanied by an enhanced coating effect of molecules around the ion and a diminished effect of extruding the ion out of its own hydration shell. The orientational molecular order due to strong spatial anisotropy inside the nanopore is much more stable than the hydrogen bonds between the molecules.     

Bioactive Structure of Membrane Lipids and Natural Products Elucidated by a Chemistry‐Based Approach

Determining the bioactive structure of membrane lipids is a new concept, which aims to examine the functions of lipids with respect to their three‐dimensional structures. As lipids are dynamic by nature, their “structure” does not refer solely to a static picture but also to the local and global motions of the lipid molecules. We consider that interactions with lipids, which are completely defined by their structures, are controlled by the chemical, functional, and conformational matching between lipids and between lipid and protein. In this review, we describe recent advances in understanding the bioactive structures of membrane lipids bound to proteins and related molecules, including some of our recent results. By examining recent works on lipid‐raft‐related molecules, lipid–protein interactions, and membrane‐active natural products, we discuss current perspectives on membrane structural biology.     

Gating of single synthetic nanopores by proton-driven DNA molecular motors

Switchable ion channels that are made of membrane proteins play different roles in cellular circuits. Since gating nanopore channels made of proteins can only work in the environment of lipid membrane, they are not fully compatible to the application requirement as a component of those nanodevice systems in which lipid membranes are hard to establish. Here we report a synthetic nanopore-DNA system where single solid-state conical nanopores can be reversibly gated by switching DNA motors immobilized inside the nanopores. High- (on-state) and low- (off-state) conductance states were found within this nanopore-DNA system corresponding to the single-stranded and i-motif structures of the attached DNA motors. The highest gating efficiency indicated as current ratio of on-state versus off-state was found when the length of the attached DNA molecule matched the tip diameter of the nanopore well. This novel nanopore-DNA system, which was gated by collective folding of structured DNA molecules responding to the external stimulus, provided an artificial counterpart of switchable protein-made nanopore channels. The concept of this DNA motor-driven nanopore switch can be used to build novel, biologically inspired nanopore machines with more precisely controlled functions in the near future by replacing the DNA molecules with other functional biomolecules, such as polypeptides or protein enzymes.     

Atomistic simulations of an antimicrobial molecule interacting with a model bacterial membrane

The dynamics of an antimicrobial molecule (end-only oligo(p-phenylene ethynylene) or EO-OPE-1 (C3)) interacting with a model bacterial membrane is simulated using all-atom molecular dynamics. It is found that the molecule spontaneously adheres to the membrane at the membrane?Cwater interface, but no insertion into the bilayer was observed within the nanosecond simulation time. However, when the simulations start from an inserted configuration, this molecule aligns with the lipid molecules in the membrane and interacts strongly through electrostatic interactions with the anionic phosphoryl groups of the lipid molecules. Due to the hydrophobic mismatch between the molecule and lipids, the inserted molecule induces the deformation of the membrane in the form of local thinning. When more than one molecule were inserted, self-assembling was observed on a nanosecond scale. However, no transmembrane pore formation was observed, due presumably to the hydrophobic backbone of the molecule. Implications in the biocidal action of this molecule are discussed.     

Fluorescence microscopy of the pressure-dependent structure of lipid bilayers suspended across conical nanopores

Glass and fused-quartz nanopore membranes containing a single conically shaped pore are promising solid supports for lipid bilayer ion-channel recordings due to the high inherent stability of lipid bilayers suspended across the nanopore orifice, as well as the favorable electrical properties of glass and fused quartz. Fluorescence microscopy is used here to investigate the structure of the suspended lipid bilayer as a function of the pressure applied across a fused-quartz nanopore membrane. When a positive pressure is applied across the bilayer, from the nanopore interior relative to the exterior bulk solution, insertion or reconstitution of operative ion channels (e.g., α-hemolysin (α-HL) and gramicidin) in the bilayer is observed; conversely, reversing the direction of the applied pressure results in loss of all channel activity, although the bilayer remains intact. The dependence of the bilayer structure on pressure was explored by imaging the fluorescence intensity from Nile red dye doped into suspended 1,2-diphytanoyl-sn-glycero-3-phosphocholine bilayers, while simultaneously recording the activity of an α-HL channel. The fluorescence images suggest that a positive pressure results in compression of the bilayer leaflets and an increase in the bilayer curvature, making it suitable for ion-channel formation and activity. At negative pressure, the fluorescence images are consistent with separation of the lipid leaflets, resulting in the observed loss of the ion-channel activity. The fluorescence data indicate that the changes in the pressure-induced bilayer structure are reversible, consistent with the ability to repeatedly switch the ion-channel activity on and off by applying positive and negative pressures, respectively.     

Investigating the translocation of λ-DNA molecules through PDMS nanopores

We investigate the translocation of λ-DNA molecules through resistive-pulse polydimethylsiloxane (PDMS) nanopore sensors. Single molecules of λ-DNA were detected as a transient current increase due to the effect of DNA charge on ionic current through the pore. DNA translocation was found to deviate from a Poisson process when the interval between translocations was comparable to the duration of translocation events, suggesting that translocation was impeded during the presence of another translocating molecule in the nanopore. Characterization of translocation at different voltage biases revealed that a critical voltage was necessary to drive DNA molecules through the nanopore. Above this critical voltage, frequency of translocation events was directly proportional to DNA concentration and voltage bias, suggesting that transport of DNA from the solution to the nanopore was the rate limiting step. These observations are consistent with experimental results on transport of DNA through nanopores and nanoslits and the theory of hydrodynamically driven polymer flow in pores.     

Ionic conductivity of the aqueous layer separating a lipid bilayer membrane and a glass support

The in-plane ionic conductivity of the approximately 1-nm-thick aqueous layer separating a 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine (POPC) bilayer membrane and a glass support was investigated. The aqueous layer conductivity was measured by tip-dip deposition of a POPC bilayer onto the surface of a 20- to 75-microm-thick glass membrane containing a single conical-shaped nanopore and recording the current-voltage (i-V) behavior of the glass membrane nanopore/POPC bilayer structure. The steady-state current across the glass membrane passes through the nanopore (45-480 nm radius) and spreads radially outward within the aqueous layer between the glass support and bilayer. This aqueous layer corresponds to the dominant resistance of the glass membrane nanopore/POPC bilayer structure. Fluorescence recovery after photobleaching measurements using dye-labeled lipids verified that the POPC bilayer maintains a significant degree of fluidity on the glass membrane. The slopes of ohmic i-V curves yield an aqueous layer conductivity of (3 +/- 1) x 10(-3) Omega(-1) cm(-1) assuming a layer thickness of 1.0 nm. This conductivity is essentially independent of the concentration of KCl in the bulk solution (10-4 to 1 M) in contact with the membrane. The results indicate that the concentration and mobility of charge carriers in the aqueous layer between the glass support and bilayer are largely determined by the local structure of the glass/water/bilayer interface.     

Free Energy of Bare and Capped Gold Nanoparticles Permeating through a Lipid Bilayer

Herein, we study the permeation free energy of bare and octane‐thiol‐capped gold nanoparticles (AuNPs) translocating through a lipid membrane. To investigate this, we have pulled the bare and capped AuNPs from bulk water to the membrane interior and estimated the free energy cost. The adsorption of the bare AuNP on the bilayer surface is energetically favorable but further loading inside it requires energy. However, the estimated free‐energy barrier for loading the capped AuNP into the lipid membrane is much higher compared to bare AuNP. We also demonstrate the details of the permeation process of bare and capped AuNPs. Bare AuNP induces the curvature in the lipid membrane whereas capped AuNP creates an opening in the interacting monolayer and get inserted into the membrane. The insertion of capped AuNP induces a partial unzipping of the lipid bilayer, which results in the ordering of the local lipids interacting with the nanoparticle. However, bare AuNP disrupts the lipid membrane by pushing the lipid molecules inside the membrane. We also analyze pore formation due to the insertion of capped AuNP into the membrane, which results in water molecules penetrating the hydrophobic region.     

Ion pairs in aqueous electrolyte microdrops under conditions of a flat nanopore

The molecular mechanisms of aqueous solvent penetration into a flat nanopore with hydrophobic structureless walls containing a Na⁺Cl^? ion pair with nonfixed distance between ions is studied by computer simulations. A detailed many-body polycenter model of intermolecular interactions calibrated with respect to experimental data for the free energy of attachment of water vapor molecules and quantum-chemical calculations in clusters is used. The ion pair hydration results in its decomposition. Drawing the molecules into the gap between ions makes easier penetration of solvent and filling of the nanopore with electrolyte. The ion-pair dissociation is accompanied by dramatic changes in the chemical potential of molecules and electric properties of the whole system. The thermodynamic characteristics of decomposition are stable as regards variations in the pore width. The post-decomposition electric polarizability demonstrates strong anisotropy associated with the nanopore flatness.     

Effect of crown ether on ion currents through synthetic membranes containing a single conically shaped nanopore

Ion-current measurements were made on synthetic polymer membranes that contained a single conically shaped nanopore. This entailed placing an electrolyte solution on either side of the membrane, using an electrode placed in each solution to control the transmembrane potential, and measuring the resulting transmembrane ion current. The effect of the crown ether commonly called 18-crown-6 (18C6) on the measured ion current was investigated. Adding 18C6 to the electrolyte solution on one side of a conical nanopore membrane provides a way to rectify the ion current flowing through the nanopore. This chemical rectification is observed only when the cation of the electrolyte is complexed by 18C6 (e.g., K+), and when the mouth diameter of the conical nanopore is of molecular dimensions, in this case approximately 1.5 nm. This chemical rectification can either augment or diminish the inherent electrostatic rectification observed with these small mouth-diameter nanopores. We have interpreted these results using a model based on the formation of a junction potential at the membrane-solution interface. This junction potential arises because the transference number for the K+-18C6 complex in bulk solution is larger than its transference number in the mouth of the conical nanopore.     

Spot Moiré Fringes: Determination of Domain Boundaries and Structural Parameters in Ordered Nanoporous Structures

Spot moiré fringes are generated by the superposition between a nanoporous structure and a digital three‐way grating. The spot moiré fringes are useful for the characterization of the domain boundaries and structural parameters in ordered nanoporous materials. The pitches and the orientations of the nanopore arrays in three directions can be simultaneously determined in a large view field.     

Modeling and simulation of nanoparticle separation through a solid-state nanopore

Recent experimental studies show that electrokinetic phenomena such as electroosmosis and electrophoresis can be used to separate nanoparticles on the basis of their size and charge using nanopore‐based devices. However, the efficient separation through a nanopore depends on a number of factors such as externally applied voltage, size and charge density of particle, size and charge density of membrane pore, and the concentration of bulk electrolyte. To design an efficient nanopore‐based separation platform, a continuum‐based mathematical model is used for fluid. The model is based on Poisson–Nernst–Planck equations along with Navier–Stokes equations for fluid flow and on the Langevin equation for particle translocation. Our numerical study reveals that membrane pore surface charge density is a vital parameter in the separation through a nanopore. In this study, we have simulated high‐density lipoprotein (HDL) and low‐density lipoprotein (LDL) as the sample nanoparticles to demonstrate the capability of such a platform. Numerical results suggest that efficient separation of HDL from LDL in a 0.2 M KCL solution (resembling blood buffer) through a 150 nm pore is possible if the pore surface charge density is ～ ?4.0 mC/m². Moreover, we observe that pore length and diameter are relatively less important in the nanoparticle separation process considered here.     

Modeling the selective partitioning of cations into negatively charged nanopores in water

Partitioning and transport of water and small solutes into and through nanopores are important to a variety of chemical and biological processes and applications. Here we study water structure in negatively charged model cylindrical [carbon nanotube (CNT)-like] nanopores, as well as the partitioning of positive ions of increasing size (Na+, K+, and Cs+) into the pore interior using extensive molecular dynamics simulations. Despite the simplicity of the simulation system-containing a short CNT-like nanopore in water carrying a uniformly distributed charge of qpore=-ne surrounded by n (=0,...,8) cations, making the overall system charge neutral-the results provide new and useful insights on both the pore hydration and ion partitioning. For n=0, that is, for a neutral nanopore, water molecules partition into the pore and form single-file hydrogen-bonded wire spanning the pore length. With increasing n, water molecules enter the pore from both ends with preferred orientations, resulting in a mutual repulsion between oriented water molecules at the pore center and creating a cavity-like low density region at the center. For low negative charge densities on the pore, the driving force for partitioning of positive ions into the pore is weak, and no partitioning is observed. Increasing the pore charge gradually leads to partitioning of positive ions into the pore. Interestingly, over a range of intermediate negative charge densities, nanopores display both thermodynamic as well as kinetic selectivity toward partitioning of the larger K+ and Cs+ ions into their interior over the smaller Na+ ions. Specifically, the driving force is in the order K+>Cs+>Na+, and K+ and Cs+ ions enter the pore much more rapidly than Na+ ions. At higher charge densities, the driving force for partitioning increases for all cations-it is highest for K+ ions-and becomes similar for Na+ and Cs+ ions. The variation of thermodynamic driving force and the average partitioning time with the pore charge density together suggest the presence of free energy barriers in the partitioning process. We discuss the role of ion hydration in the bulk and in the pore interior as well as of the pore hydration in determining the barrier heights for ion partitioning and the observed thermodynamic and kinetic selectivities.     

Single molecule measurements of DNA transport through a nanopore

We examined the voltage-driven movement of single-stranded DNA molecules in a membrane channel or "nanopore". Using single channel recording methods and a statistical analysis of many single molecule events, we determined how voltage influences capture and translocation in the nanopore. We verified that the mean time between capture events follows a simple exponential distribution, whereas the translocation times follow a unique distribution that is partly Gaussian and partly exponential. Measurements of polymer sequence effects demonstrated that translocation duration is heavily influenced by specific or nonspecific purine-channel interactions. The single molecule approach we used revealed molecular interactions that can influence both capture rates and translocation velocities in a manner that enriches naive barrier crossing models.     

Hydrogen bond-assisted macrocyclic oligocholate transporters in lipid membranes

Three macrocyclic oligocholates containing a carboxyl group, a guanidinium ion, and a Cbz-protected amine, respectively, were studied as membrane transporters for hydrophilic molecules. To permeate glucose across lipid bilayers, the macrocycles stacked over one another to form a transmembrane nanopore, driven by a strong tendency of the water molecules in the internal cavities of the amphiphilic macrocycles to aggregate in a nonpolar environment. To transport larger guests such as carboxyfluorescein (CF), the macrocycles acted as carriers to shuttle the guest across the membrane. Hydrogen-bonds between the side chains of the macrocycles strongly affected the transport properties. Surprisingly, the carboxyl group turned out to be far more effective at assisting the aggregation of the oligocholate macrocycles in the membrane than the much stronger carboxylate-guanidinium salt bridge, likely due to competition from the phosphate groups of the lipids for the guanidinium.