Table of contents

Biological and solid-state nanopores are an exciting field of research, which has seen a rapid development over the last 10 to 20 years. Activities in this area range from theoretical and experimental work on the underlying fundamental (bio)physics to applications in single-molecule biosensing. And while the prospect of DNA sequencing continues to be a major driving force, other applications with potentially similar impact begin to emerge, for example the detection of small molecules, proteins, protein/protein and protein/DNA complexes, and RNA to name just a few.

It has also become apparent that both classes of nanopore devices have intrinsic advantages and disadvantages; hybrid structures combining the better of the two worlds would be a logical consequence and are beginning to appear in the literature. Many other highly innovative ideas and concepts continue to emerge and the number of nanopore-related publications has increased drastically over recent years. We found that more than 100 research groups worldwide are active in this area; several commercial settings are in the process of translating fundamental research into real-life applications.

We therefore felt that now is the right time to showcase these new developments in a special issue: to inspire researchers active in the field, to liberate inherent synergies, and not least, to demonstrate to the outside world the current state-of-the-art and future opportunities. The title 'New developments in nanopore research—from fundamentals to applications' in some way reflects these ambitions and, even though not everyone invited was able to contribute, we were able to assemble 34 high-quality research papers from all over the world.

We would like to acknowledge and thank all the contributors for their submissions, which made this special issue possible in the first place. Moreover, we would like to thank the staff at IOP Publishing for helping us with the administrative aspects and for coordinating the refereeing process, and Ms Natalia Goehring for the beautiful cover artwork.

Finally, to the readers, we hope you find this special issue a valuable source of information and insight into the field of nanopores.

New developments in nanopore research—from fundamentals to applications contents

Mathematical modeling and simulation of nanopore blocking by precipitationM-T Wolfram, M Burger and Z S Siwy

Protein conducting nanoporesAnke Harsman, Vivien Krüger, Philipp Bartsch, Alf Honigmann, Oliver Schmidt, Sanjana Rao, Christof Meisinger and Richard Wagner

Electrically sensing protease activity with nanoporesMikiembo Kukwikila and Stefan Howorka

Electrical characterization of DNA-functionalized solid state nanopores for bio-sensingV Mussi, P Fanzio, L Repetto, G Firpo, P Scaruffi, S Stigliani, M Menotta, M Magnani, G P Tonini and U Valbusa

Automatable lipid bilayer formation and ion channel measurement using sessile dropletsJ L Poulos, S A Portonovo, H Bang and J J Schmidt

Critical assessment of OmpF channel selectivity: merging information from different experimental protocolsM L López, E García-Giménez, V M Aguilella and A Alcaraz

Chemically modified solid state nanopores for high throughput nanoparticle separationAnmiv S Prabhu, Talukder Zaki N Jubery, Kevin J Freedman, Rafael Mulero, Prashanta Dutta and Min Jun Kim

Changes in ion channel geometry resolved to sub-ångström precision via single molecule mass spectrometryJoseph W F Robertson, John J Kasianowicz and Joseph E Reiner

Entropic transport of finite size particlesW Riefler, G Schmid, P S Burada and P Hänggi

Osmotic stress regulates the strength and kinetics of sugar binding to the maltoporin channelPhilip A Gurnev, Daniel Harries, V Adrian Parsegian and Sergey M Bezrukov

Detection of urea-induced internal denaturation of dsDNA using solid-state nanoporesnAlon Singer, Heiko Kuhn, Maxim Frank-Kamenetskii and Amit Meller

Translocation events in a single-walled carbon nanotubeJin He, Hao Liu, Pei Pang, Di Cao and Stuart Lindsay

Probing DNA with micro- and nanocapillaries and optical tweezersL J Steinbock, O Otto, D R Skarstam, S Jahn, C Chimerel, J L Gornall and U F Keyser

Fabrication of nanopores with embedded annular electrodes and transverse carbon nanotube electrodesZhijun Jiang, Mirna Mihovilovic, Jason Chan and Derek Stein

Fabrication and electrical characterization of a pore–cavity–pore deviceD Pedone, M Langecker, A M Münzer, R Wei, R D Nagel and U Rant

Use of tunable nanopore blockade rates to investigate colloidal dispersionsG R Willmott, R Vogel, S S C Yu, L G Groenewegen, G S Roberts, D Kozak, W Anderson and M Trau

Facilitated translocation of polypeptides through a single nanoporeRobert Bikwemu, Aaron J Wolfe, Xiangjun Xing and Liviu Movileanu

Mechanistic insight into gramicidin-based detection of protein–ligand interactions via sensitized photoinactivationTatyana I Rokitskaya, Michael X Macrae, Steven Blake, Natalya S Egorova, Elena A Kotova, Jerry Yang and Yuri N Antonenko

Sequence-dependent unfolding kinetics of DNA hairpins studied by nanopore force spectroscopyStephan Renner, Andrey Bessonov, Ulrich Gerland and Friedrich C Simmel

Hydration properties of mechanosensitive channel pores define the energetics of gatingA Anishkin, B Akitake, K Kamaraju, C-S Chiang and S Sukharev

Dynamic translocation of ligand-complexed DNA through solid-state nanopores with optical tweezersAndy Sischka, Andre Spiering, Maryam Khaksar, Miriam Laxa, Janine König, Karl-Josef Dietz and Dario Anselmetti

Force fluctuations assist nanopore unzipping of DNAV Viasnoff, N Chiaruttini, J Muzard and U Bockelmann

Control and reversal of the electrophoretic force on DNA in a charged nanoporeBinquan Luan and Aleksei Aksimentiev

The properties of the outer membrane localized Lipid A transporter LptDRaimund Haarmann, Mohamed Ibrahim, Mara Stevanovic, Rolf Bredemeier and Enrico Schleiff

Structural and dynamical properties of the porins OmpF and OmpC: insights from molecular simulationsAmit Kumar, Eric Hajjar, Paolo Ruggerone and Matteo Ceccarelli

Dehydration and ionic conductance quantization in nanoporesMichael Zwolak, James Wilson and Massimiliano Di Ventra

Current oscillations generated by precipitate formation in the mixing zone between two solutions inside a nanoporeErik C Yusko, Yazan N Billeh and Michael Mayer

Precise electrochemical fabrication of sub-20 nm solid-state nanopores for single-molecule biosensingMariam Ayub, Aleksandar Ivanov, Jongin Hong, Phillip Kuhn, Emanuele Instuli, Joshua B Edel and Tim Albrecht

The distribution of DNA translocation times in solid-state nanoporesJiali Li and David S Talaga

Crowding effects in non-equilibrium transport through nano-channelsA Zilman and G Bel

Permeation through nanochannels: revealing fast kineticsKozhinjampara R Mahendran, Pratik Raj Singh, Jürgen Arning, Stefan Stolte, Ulrich Kleinekathöfer and Mathias Winterhalter

LILBID-mass spectrometry of the mitochondrial preprotein translocase TOMFrauke Mager, Lucie Sokolova, Julia Lintzel, Bernhard Brutschy and Stephan Nussberger

Evidence that small proteins translocate through silicon nitride pores in a folded conformationRadu I Stefureac, Dhruti Trivedi, Andre Marziali and Jeremy S Lee

Methods for controlling the pore properties of ultra-thin nanocrystalline silicon membranesD Z Fang, C C Striemer, T R Gaborski, J L McGrath and P M Fauchet

High surface charges of polymer pore walls and applied electric fields can lead to the formation and subsequent dissolution of precipitates in nanopores. These precipitates block the pore, leading to current fluctuations.

We present an extended Poisson–Nernst–Planck system which includes chemical reactions of precipitation and dissolution. We discuss the mathematical modeling and present 2D numerical simulations.

About 50% of the cellular proteins have to be transported into or across cellular membranes. This transport is an essential step in the protein biosynthesis. In eukaryotic cells secretory proteins are transported into the endoplasmic reticulum before they are transported in vesicles to the plasma membrane. Almost all proteins of the endosymbiotic organelles chloroplasts and mitochondria are synthesized on cytosolic ribosomes and posttranslationally imported. Genetic, biochemical and biophysical approaches led to rather detailed knowledge on the composition of the translocon-complexes which catalyze the membrane transport of the preproteins. Comprehensive concepts on the targeting and membrane transport of polypeptides emerged, however little detail on the molecular nature and mechanisms of the protein translocation channels comprising nanopores has been achieved. In this paper we will highlight recent developments of the diverse protein translocation systems and focus particularly on the common biophysical properties and functions of the protein conducting nanopores. We also provide a first analysis of the interaction between the genuine protein conducting nanopore Tom40_SC as well as a mutant Tom40_SC () containing an additional negative charge at the channel vestibule and one of its native substrates, CoxIV, a mitochondrial targeting peptide. The polypeptide induced a voltage-dependent increase in the frequency of channel closure of Tom40_SC corresponding to a voltage-dependent association rate, which was even more pronounced for the Tom40_SC S54E mutant. The corresponding dwelltime reflecting association/transport of the peptide could be determined with  ms for the wildtype, whereas the mutant Tom40_SC S54E displayed a biphasic dwelltime distribution ( ms;  ms).

The enzymatic activity of a protease was electrically detected using nanopore recordings. A peptide substrate was tethered to microscale beads, and cleavage by the enzyme trypsin released a soluble fragment that was electrophoretically driven through the α-hemolysin protein pore, leading to detectable blockades in the ionic current. Owing to its simplicity, this approach to sense enzymatic activity may be applied to other proteases.

We present data concerning the electrical properties of a class of biosensor devices based on bio-functionalized solid state nanopores able to detect different kinds of interactions between probe molecules, chemically attached to the pore surface, and target molecules present in solution and electrophoretically drawn through the nanometric channel. The great potentiality of this approach resides in the fact that the functionalization of a quite large pore (up to 50–60 nm) allows a sufficient diameter reduction for the attainment of a single molecule sensing dimension and selective activation, without the need for further material deposition, such as metal or oxides, or localized surface modification. The results indicate that it will be possible, in the near future, to conceive and design devices for parallel analysis of biological samples made of arrays of nanopores differently functionalized, fabricated by standard lithographic techniques, with important applications in the field of molecular diagnosis.

Artificial lipid bilayer membranes have been used to reconstitute ion channels for scientific and technological applications. Membrane formation has traditionally involved slow, labor intensive processes best suited to small scale laboratory experimentation. We have recently demonstrated a high throughput method of membrane formation using automated liquid-handling robotics. We describe here the integration of membrane formation and measurement with two methods compatible with automation and high throughput liquid-handling robotics. Both of these methods create artificial lipid bilayers by joining lipid monolayers self-assembled at the interface of aqueous and organic phases using sessile aqueous droplets in contact with a measurement electrode; one using a pin tool, commonly employed in high throughput fluid handling assays, and the other using a positive displacement pipette. Membranes formed with both methods were high quality and supported measurement of ion channels at the single molecule level. Full automation of bilayer production and measurement with the positive displacement pipette was demonstrated by integrating it with a motion control platform.

The ion selectivity of a channel can be quantified in several ways by using different experimental protocols. A wide, mesoscopic channel, the OmpF porin of the outer membrane of E. coli, serves as a case study for comparing and analysing several measures of the channel cation–anion permeability in chlorides of alkali metals (LiCl, NaCl, KCl, CsCl). We show how different insights can be gained and integrated to rationalize the global image of channel selectivity. To this end, reversal potential, channel conductance and bi-ionic potential (two different salts with a common anion on each side of the channel but with the same concentration) experiments are discussed in light of an electrodiffusion model based on the Poisson–Nernst–Planck formalism. Measurements and calculations based on the atomic crystal structure of the channel show that each protocol displays a particular balance between the different sources of selectivity.

The separation of biomolecules and other nanoparticles is a vital step in several analytical and diagnostic techniques. Towards this end we present a solid state nanopore-based set-up as an efficient separation platform. The translocation of charged particles through a nanopore was first modeled mathematically using the multi-ion model and the surface charge density of the nanopore membrane was identified as a critical parameter that determines the selectivity of the membrane and the throughput of the separation process. Drawing from these simulations a single 150 nm pore was fabricated in a 50 nm thick free-standing silicon nitride membrane by focused-ion-beam milling and was chemically modified with (3-aminopropyl)triethoxysilane to change its surface charge density. This chemically modified membrane was then used to separate 22 and 58 nm polystyrene nanoparticles in solution. Once optimized, this approach can readily be scaled up to nanopore arrays which would function as a key component of next-generation nanosieving systems.

The ion channel formed by Staphylococcus aureus alpha-hemolysin switches between multiple open conducting states. We describe a method for precisely estimating the changes in the ion channel geometry that correspond to these different states. Experimentally, we observed that the permeability of a single channel to differently sized poly(ethylene glycol) molecules depends on the magnitude of the open state conductance. A simple theory is proposed for determining changes in channel length of 4.2% and in cross-sectional area of − 0.4%.

Transport of spherical Brownian particles of finite size possessing radii R ≤ R_max through narrow channels with varying cross-section width is considered. Applying the so-called Fick–Jacobs approximation, i.e. assuming fast equilibration in the direction orthogonal to the channel axis, the 2D problem can be described in terms of a 1D effective dynamics in which bottlenecks cause entropic barriers. Geometrical confinements result in entropic barriers which the particles have to overcome in order to proceed in the transport direction. The analytic findings for the nonlinear mobility for the transport are compared with precise numerical simulation results. The dependence of the nonlinear mobility on the particle size exhibits a striking resonance-like behavior as a function of the relative particle size ρ = R/R_max; this latter feature renders possible new effective particle separation scenarios.

We study the effect of osmotic stress, exerted by salts, on carbohydrate binding to the sugar-specific bacterial channel maltoporin. When the channel is reconstituted into planar lipid bilayers, single events of its occlusion by sugar are seen as transient interruptions in the flow of small ions. We find that, for most salts, changes in the free energy of maltoporin–sugar binding vary linearly with solution osmotic pressure. Such a change in binding with solution osmolarity indicates that for each salt a constant number of salt-excluding water molecules is released upon sugar–maltoporin association at all salt concentrations. We find that larger numbers of water molecules are released upon binding of the cyclic carbohydrate β-cyclodextrin (CD) than upon binding of the corresponding linear homologue maltoheptaose (m7). Remarkably, the extent to which salts affect the binding constants and rates depends sensitively on the type of salt; dehydration in solutions of different anions corresponds to the Hofmeister series. In sodium sulfate solutions, CD and m7 respectively release about 120 and 35 salt-excluding water molecules; in sodium chloride solutions, 35 and 15 waters. No water release is observed with sodium bromide. Finally, by adding adamantane, known to form an inclusion complex with CD, we can infer that CD not only dehydrates but also undergoes a conformational change upon binding to the channel. As a practical outcome, our results also demonstrate how osmotic stress can improve single-molecule detection of different solutes using protein-based nanopores.

The ability to detect and measure dsDNA thermal fluctuations is of immense importance in understanding the underlying mechanisms responsible for transcription and replication regulation. We describe here the ability of solid-state nanopores to detect sub-nanometer changes in DNA structure as a result of chemically enhanced thermal fluctuations. In this study, we investigate the subtle changes in the mean effective diameter of a dsDNA molecule with 3–5 nm solid-state nanopores as a function of urea concentration and the DNA's AT content. Our studies reveal an increase in the mean effective diameter of a DNA molecule of approximately 0.6 nm at 8.7 M urea. In agreement with the mechanism of DNA local denaturation, we observe a sigmoid dependence of these effects on urea concentration. We find that the translocation times in urea are markedly slower than would be expected if the dynamics were governed primarily by viscous effects. Furthermore, we find that the sensitivity of the nanopore is sufficient to statistically differentiate between DNA molecules of nearly identical lengths differing only in sequence and AT content when placed in 3.5 M urea. Our results demonstrate that nanopores can detect subtle structural changes and are thus a valuable tool for detecting differences in biomolecules' environment.

Translocation of DNA oligomers through a single-walled carbon nanotube was demonstrated recently. Translocation events are accompanied by giant current pulses, the origin of which remains obscure. Here, we show that the introduction of a nucleotide, guanosine triphosphate, alone into the input reservoir of a carbon nanotube nanofluidic device also gives giant current pulses. Taken together with data on oligomer translocation, these new results suggest that the pulse width has a nonlinear, power-law dependence on the number of nucleotides in a DNA molecule. We have also measured the time for the onset of DNA translocation pulses after bias reversal, finding that the time for the onset of translocation is directly proportional to the period of the bias reversal.

We combine for the first time optical tweezer experiments with the resistive pulse technique based on capillaries. Quartz glass capillaries are pulled into a conical shape with tip diameters as small as 27 nm. Here, we discuss the translocation of λ-phage DNA which is driven by an electrophoretic force through the nanocapillary. The resulting change in ionic current indicates the folding state of single λ-phage DNA molecules. Our flow cell design allows for the straightforward incorporation of optical tweezers. We show that a DNA molecule attached to an optically trapped colloid is pulled into a capillary by electrophoretic forces. The detected electrophoretic force is in good agreement with measurements in solid-state nanopores.

Nanopores with one or two embedded nanoelectrodes can be fabricated by high resolution, milling-based methods. We first demonstrate how a focused ion beam, whose sputtering mechanism is well understood, can create a nanopore containing an annular electrode of an arbitrary metal, and with a regular perimeter. The inner surface of the nanopore can be insulated, and its diameter can be reduced with nanometer precision, by conformally coating a dielectric material by atomic layer deposition. We then investigate the mechanism of pore formation using a transmission electron microscope (TEM) through studies of the milling rate, and its dependence on the flux of electrons and on the atomic number of different target metals. Sputtering from the surface is identified as the dominant mechanism. Accordingly, light element conductors should be chosen to enhance the rate and resolution of TEM milling, which we demonstrate by articulating a nanopore with transverse carbon nanotube electrodes. Finally, we electrochemically verify that TEM milling preserves the quality of an annular gold electrode through cyclic voltammetry measurements performed at various stages of the fabrication.

We present a solid state nanopore device structure comprising two nanopores which are stacked above each other and connected via a pyramidal cavity of 10 fl volume. The process of fabrication of the pore–cavity–pore device (PCP) relies on the formation of one pore in a Si₃N₄ membrane by electron beam lithography, while the other pore is chemically etched into the Si carrier by a feedback controlled process. The dimensions of the two nanopores as well as the cavity can be adjusted independently, which is confirmed by transmission electron microscopy. The PCP device is characterized with respect to its electrical properties, including noise analysis and impedance spectroscopy. An equivalent circuit model is identified and resistance, capacitance, and dielectric loss factors are obtained. Potential and electric field distributions inside the electrically biased device are simulated by finite element methods. The low noise characteristics of the PCP device (comparable to a single solid state nanopore) make it suitable for the stochastic sensing of single molecules; moreover, the pore–cavity–pore architecture allows for novel kinds of experiments including the trapping of single nano-objects and single molecule time-of-flight measurements.

Tunable nanopores fabricated in elastomeric membranes have been used to study the dependence of ionic current blockade rate on the concentration and electrophoretic mobility of particles in aqueous suspensions. A range of nanoparticle sizes, materials and surface functionalities has been tested. Using pressure-driven flow through a pore, the blockade rate for 100 nm carboxylated polystyrene particles was found to be linearly proportional to both transmembrane pressure (between 0 and 1.8 kPa) and particle concentration (between 7 × 10⁸ and 4.5 × 10¹⁰ ml ^{− 1}). This result can be accurately modelled using Nernst–Planck transport theory, enabling measurement of particle concentrations. Using only an applied potential across a pore, the blockade rates for carboxylic acid and amine coated 500 and 200 nm silica particles were found to correspond to changes in their mobility as a function of the solution pH. Scanning electron microscopy and confocal microscopy have been used to visualize changes in the tunable nanopore geometry in three dimensions as a function of applied mechanical strain. The pores were conical in shape, and changes in pore size were consistent with ionic current measurements. A zone of inelastic deformation adjacent to the pore has been identified as important in the tuning process.

The transport of polypeptides through nanopores is a key process in biology and medical biotechnology. Despite its critical importance, the underlying kinetics of polypeptide translocation through protein nanopores is not yet comprehensively understood. Here, we present a simple two-barrier, one-well kinetic model for the translocation of short positively charged polypeptides through a single transmembrane protein nanopore that is equipped with negatively charged rings, simply called traps. We demonstrate that the presence of these traps within the interior of the nanopore dramatically alters the free energy landscape for the partitioning of the polypeptide into the nanopore interior, as revealed by significant modifications in the activation free energies required for the transitions of the polypeptide from one state to the other. Our kinetic model permits the calculation of the relative and absolute exit frequencies of the short cationic polypeptides through either opening of the nanopore. Moreover, this approach enabled quantitative assessment of the kinetics of translocation of the polypeptides through a protein nanopore, which is strongly dependent on several factors, including the nature of the translocating polypeptide, the position of the traps, the strength of the polypeptide–attractive trap interactions and the applied transmembrane voltage.

Among the many challenges for the development of ion channel-based sensors is the poor understanding of how to engineer modified transmembrane pores with tailored functionality that can respond to external stimuli. Here, we use the method of sensitized photoinactivation of gramicidin A (gA) channels in planar bilayer lipid membranes to help elucidate the underlying mechanistic details for changes in macroscopic transmembrane ionic current observed upon interaction of C-terminally attached gA ligands with specific proteins in solution. Three different systems were studied: (i) carbonic anhydrase (CA) and gA-sulfonamide, (ii) PSD-95 protein (belonging to the 'PDZ domain-containing protein') and a gA analog carrying the KGGHRRSARYLESSV peptide sequence at the C-terminus, and (iii) an anti-biotin antibody and gA-biotin. The results challenge a previously proposed mechanistic hypothesis suggesting that protein-induced current suppression is due to steric blockage of the ion passage through gA channels, while they reveal new insight for consideration in alternative mechanistic models. Additionally, we demonstrate that the length of a linker between the ligand and the gA channel may be less important for gramicidin-based detection of monovalent compared to multivalent protein–ligand interactions. These studies collectively shed new light on the mechanism of protein-induced current alterations in bilayer recordings of gA derivatives, which may be important in the design of new gramicidin-based sensors.

Nanopore force spectroscopy is used to study the unzipping kinetics of two DNA hairpin molecules with a 12 base pair long stem containing two contiguous stretches of six GC and six AT base pairs in interchanged order. Even though the thermodynamic stabilities of the two structures are nearly the same, they differ greatly in their unzipping kinetics. When the GC segment has to be broken before the AT segment, the unfolding rate is orders of magnitude smaller than in the opposite case. We also investigated hairpins with stem regions consisting only of AT or GC base pairs. The pure AT hairpins translocate much faster than the other hairpins, whereas the pure GC hairpins translocate on similar timescales to the hairpins with only an initial GC segment. For each hairpin, nanopore force spectroscopy is performed for different loading rates and the resulting unzipping distributions are mathematically transformed to a master curve that yields the unfolding rate as a function of applied voltage. This is compared with a stochastic model of the unfolding process for the two sequences for different voltages. The results can be rationalized in terms of the different natures of the free energy landscapes for the unfolding process.

Opening of ion channels directly by tension in the surrounding membrane appears to be the most ancient and simple mechanism of gating. Bacterial mechanosensitive channels MscL and MscS are the best-studied tension-gated nanopores, yet the key physical factors that define their gating are still hotly debated. Here we present estimations, simulations and experimental results showing that hydration of the pore might be one of the major parameters defining the thermodynamics and kinetics of mechanosensitive channel gating. We associate closing of channel pores with complete dehydration of the hydrophobic gate (occlusion by 'vapor lock') and formation of two water–vapor interfaces above and below the constriction. The opening path is the expansion of these interfaces, ultimately leading to wetting of the hydrophobic pore, which does not appear to be the exact reverse of the closing path, thus producing hysteresis. We discuss specifically the role of polar groups (glycines) buried in narrow closed conformations but exposed in the open states that change the wetting characteristics of the pore lining and stabilize conductive states of the channels.

We investigated the threading and controlled translocation of individual lambda-DNA (λ-DNA) molecules through solid-state nanopores with piconewton force sensitivity, millisecond time resolution and picoampere ionic current sensitivity with a set-up combining quantitative 3D optical tweezers (OT) with electrophysiology. With our virtually interference-free OT set-up the binding of RecA and single peroxiredoxin protein molecules to λ-DNA was quantitatively investigated during dynamic translocation experiments where effective forces and respective ionic currents of the threaded DNA molecule through the nanopore were measured during inward and outward sliding. Membrane voltage-dependent experiments of reversible single protein/DNA translocation scans yield hysteresis-free, asymmetric single-molecule fingerprints in the measured force and conductance signals that can be attributed to the interplay of optical trap and electrostatic nanopore potentials. These experiments allow an exact localization of the bound protein along the DNA strand and open fascinating applications for label-free detection of DNA-binding ligands, where structural and positional binding phenomena can be investigated at a single-molecule level.

We experimentally study the statistical distributions and the voltage dependence of the unzipping time of 45 base-pair-long double-stranded DNA through a nanopore. We then propose a quantitative theoretical description considering the nanopore unzipping process as a random walk of the opening fork through the DNA sequence energy landscape biased by a time-fluctuating force. To achieve quantitative agreement fluctuations need to be correlated over the millisecond range and have an amplitude of order k_BT/bp. Significantly slower or faster fluctuations are not appropriate, suggesting that the unzipping process is efficiently enhanced by noise in the kHz range. We further show that the unzipping time of short 15 base-pair hairpins does not always increase with the global stability of the double helix and we theoretically study the role of DNA elasticity on the conversion of the electrical bias into a mechanical unzipping force.

Electric field driven transport of DNA through solid-state nanopores is the key process in nanopore-based DNA sequencing that promises dramatic reduction of genome sequencing costs. A major hurdle in the development of this sequencing method is that DNA transport through the nanopores occurs too quickly for the DNA sequence to be detected. By means of all-atom molecular dynamics simulations, we demonstrate that the velocity of DNA transport through a nanopore can be controlled by the charge state of the nanopore surface. In particular, we show that the charge density of the nanopore surface controls the magnitude and/or direction of the electro-osmotic flow through the nanopore and thereby can significantly reduce or even reverse the effective electrophoretic force on DNA. Our work suggests a physical mechanism to control DNA transport in a nanopore by chemical, electrical or electrochemical modification of the nanopore surface.

Gram-negative bacteria are surrounded by a cell wall including the outer membrane. The outer membrane is composed of two distinct monolayers where the outer layer contains lipopolysaccharides (LPS) with the non-phospholipid Lipid A as the core. The synthesis of Lipid A is initiated in the cytosol and thereby the molecule has to be transported across the inner and outer membranes. The β-barrel lipopolysaccharide-assembly protein D (LptD) was discovered to be involved in the transfer of Lipid A into the outer membrane of Gram-negative bacteria. At present the molecular procedure of lipid transfer across the outer membrane remains unknown. Here we approached the functionality of the transfer system by an electrophysiological analysis of the outer membrane protein from Escherichia coli named ecLptD. In vitro the protein shows cation selectivity and has an estimated pore diameter of about 1.8 nm. Addition of Lipid A induces a transition of the open state to a sub-conductance state with two independent off-rates, which might suggest that LptD is able to bind and transport the molecule in vitro. To generalize our findings with respect to the Lipid A transport system of other Gram-negative bacteria we have explored the existence of the proteins involved in this pathway by bioinformatic means. We were able to identify the membrane-inserted components of the Lipid A transport system in all Gram-negative bacteria, whereas the periplasmic components appear to be species-specific. The LptD proteins of different bacteria are characterized by their periplasmic N-terminal domain and a C-terminal barrel region. The latter shows distinct sequence properties, particularly in LptD proteins of cyanobacteria, and this specific domain can be found in plant proteins as well. By electrophysiological experiments on LptD from Anabaena sp. PCC 7120 we are able to confirm the functional relation of anaLptD to Lipid A transport.

In this paper we investigate the structural and dynamical properties of the two major porins (OmpF and OmpC) in Escherichia coli, using molecular dynamics (MD) simulations. In particular we characterized the atomic fluctuations, correlated motions, temperature dependence, solvent-accessible cross-sectional area and water dynamics in the key regions of the two channels. Our in-depth analysis allows us to highlight the importance of both the key conserved and substituted residues between OmpF and OmpC. The latter is characterized by a narrower and longer constriction region with respect to OmpF. OmpC also showed a higher stability upon increasing temperature. We then present the results of transport properties by using accelerated MD simulations to probe the diffusion of norfloxacin (a fluoroquinolone antibiotic) through the two porins OmpF/OmpC. Our study constitutes a step forward towards understanding the structure–function relationship of the two porins' channels. This will benefit the research of antibacterials with improved permeation properties and nanopores that aim to use these porins as sensing systems.

There has been tremendous experimental progress in the last decade in identifying the structure and function of biological pores (ion channels) and fabricating synthetic pores. Despite this progress, many questions still remain about the mechanisms and universal features of ionic transport in these systems. In this paper, we examine the use of nanopores to probe ion transport and to construct functional nanoscale devices. Specifically, we focus on the newly predicted phenomenon of quantized ionic conductance in nanopores as a function of the effective pore radius—a prediction that yields a particularly transparent way to probe the contribution of dehydration to ionic transport. We study the role of ionic species in the formation of hydration layers inside and outside of pores. We find that the ion type plays only a minor role in the radial positions of the predicted steps in the ion conductance. However, ions with higher valency form stronger hydration shells, and thus, provide even more pronounced, and therefore, more easily detected, drops in the ionic current. Measuring this phenomenon directly, or from the resulting noise, with synthetic nanopores would provide evidence of the deviation from macroscopic (continuum) dielectric behavior due to microscopic features at the nanoscale and may shed light on the behavior of ions in more complex biological channels.

Unlike biological protein pores in lipid membranes, nanopores fabricated in synthetic materials can withstand a wide range of environmental conditions including the presence of organic solvents. This capability expands the potential of synthetic nanopores to monitor chemical reactions occurring at the interface between solutions of organic and aqueous character. In this work, nanopores fabricated in borosilicate glass or silicon nitride connected a predominantly organic solvent to an aqueous solvent, thereby generating a mixing zone between these solutions inside the pore. This configuration was exploited to precipitate small organic molecules with low aqueous solubility inside the nanopores, and concomitantly, to monitor this precipitation by the decrease of the ionic conductance through the nanopores over time. Hence, this method provides a means to induce and investigate the formation of nanoprecipitates or nanoparticles. Interestingly, precipitates with a slight electric charge were cleared from the pore, causing the conductance of the pore to return to its original value. This process repeated, resulting in stable oscillations of the ionic current. Although such oscillations might be useful in fluidic logic circuits, few conditions capable of generating oscillations in ionic currents have been reported. The frequency and amplitude of oscillations could be tuned by changing the concentration of the precipitating molecule, the pH of the electrolyte, and the applied potential bias. In addition to generating oscillations, nanopores that separate two different solutions may be useful for monitoring and mediating chemical reactions in the mixing zone between two solutions.

It has recently been shown that solid-state nanometer-scale pores ('nanopores') can be used as highly sensitive single-molecule sensors. For example, electrophoretic translocation of DNA, RNA and proteins through such nanopores has enabled both detection and structural analysis of these complex biomolecules. Control over the nanopore size is critical as the pore must be comparable in size to the analyte molecule in question. The most widely used fabrication methods are based on focused electron or ion beams and thus require (scanning) transmission electron microscopy and focused ion beam (FIB) instrumentation. Even though very small pores have been made using these approaches, several issues remain. These include the requirement of being restricted to rather thin, mechanically less stable membranes, particularly for pore diameters in the single-digit nanometer range, lack of control of the surface properties at and inside the nanopore, and finally, the fabrication cost. In the proof-of-concept study, we report on a novel and simple route for fabricating metal nanopores with apparent diameters below 20 nm using electrodeposition and real-time ionic current feedback in solution. This fabrication approach inserts considerable flexibility into the kinds of platforms that can be used and the nanopore membrane material. Starting from much larger pores, which are straightforward to make using FIB or other semiconductor fabrication methods, we electrodeposit Pt at the nanopore interface while monitoring its ionic conductance at the same time in a bi-potentiostatic setup. Due to the deposition of Pt, the nanopore decreases in size, resulting in a decrease of the pore conductance. Once a desired pore conductance has been reached, the electrodeposition process is stopped by switching the potential of the membrane electrode and the fabrication process is complete. Furthermore, we demonstrate that these pores can be used for single-biomolecule analysis, such as that of λ-DNA, which we use in a proof-of-concept study. Importantly, our approach is applicable to single nanopores as well as nanopore arrays, and can easily be extended to deposits of metal other than Pt.

This paper systematically investigates the effects of solution viscosity, applied voltage and DNA chain length on the distribution of DNA translocation times through 8 ± 2 nm diameter silicon nitride nanopores. Linear dsDNA translocation events were selected based on the magnitude of current blockage and accumulated into scatter plots of current blockage and event duration (translocation time). The translocation time distribution was fitted to the solution of a Smoluchowski-type equation for 1D biased diffusion to a sink. The DNA drifting speed under bias and diffusion constant were extracted from the fits as functions of solution viscosity, applied voltage and DNA chain length. Combined with the Einstein–Smoluchowski relation, this model allowed evaluation of the viscous drag force on DNA molecules. This model also allowed estimation of the uncertainty in determining the DNA chain length due to the influence of friction on the spread of translocation times in a nanopore measurement. The data analysis suggests that the simple 1D biased diffusion model fits the experimental data well for a wide range of conditions. Some deviations from predicted behavior were observed and show where additional phenomena are likely to contribute to the distribution of DNA translocation times.

Transport through nano-channels plays an important role in many biological processes and industrial applications. Gaining insights into the functioning of biological transport processes and the design of man-made nano-devices requires an understanding of the basic physics of such transport. A simple exclusion process has proven to be very useful in explaining the properties of several artificial and biological nano-channels. It is particularly useful for modeling the influence of inter-particle interactions on transport characteristics. In this paper, we explore several models of the exclusion process using a mean field approach and computer simulations. We examine the effects of crowding inside the channel and in its immediate vicinity on the mean flux and the transport times of single molecules. Finally, we discuss the robustness of the theory's predictions with respect to the crucial characteristics of the hindered diffusion in nano-channels that need to be included in the model.

The permeation of water soluble molecules across cell membranes is controlled by channel-forming proteins and, in particular, the channel surface determines the selectivity. An adequate method to study the properties of these channels is electrophysiology and, in particular, analyzing the ion current fluctuation in the presence of permeating solutes. Ion current fluctuation analysis provides information on possible interactions of solutes with the channel surface. Due to the limited time resolution, fast permeation events are not visible using standard techniques. Here, we demonstrate that miniaturization of the lipid bilayer; varying the temperature or changing the solvent may enhance the resolution. Although electrophysiology is considered as a single molecule technique, it does not provide atomic resolution. Molecular details of solute permeation can be revealed by combining electrophysiology and all-atom computer modeling; these methods include ion conductance, selectivity, ion pair formation, and rate limiting interactions of the solute with the channel walls during permeation.

In the present work we applied a novel mass spectrometry method termed laser-induced liquid bead ion desorption mass spectrometry (LILBID-MS) to the outer mitochondrial membrane protein translocon TOM to analyze its subunit composition and stoichiometry. With TOM core complex, purified at high pH, we demonstrate that a TOM core complex of Neurospora crassa is composed of at least two Tom40 and Tom22 molecules, respectively, and more than five small Tom subunits between 5.5 and 6.4 kDa. We show that the multiprotein complex has a total molecular mass higher than 170 depending on the number of Tom5, Tom6 and Tom7 molecules bound.

The interaction of three proteins (histidine-containing phosphocarrier protein, HPr, calmodulin, CaM, and maltose binding protein, MBP) with synthetic silicon nitride (SiN_x) membranes has been studied. The proteins which have a net negative charge were electrophoretically driven into pores of 7 and 5 nm diameter with a nominal length of 15 nm. The % blockade current and event duration were measured at three different voltages. For a translocation event it was expected that the % block would be constant with voltage whilst the event duration would decrease with increasing voltage. On the basis of these criteria, we deduce that MBP whose largest dimension is 6.5 nm does not translocate whereas up to 40% of CaM molecules can translocate the 7 nm pore as can a majority of HPr molecules, with some translocations being observed for the 5 nm pore. For translocation events the magnitude of the % blockade current is consistent with a folded conformation of the proteins surrounded by a hydration shell of 0.5–1.0 nm.

Porous nanocrystalline silicon (pnc-Si) membranes are a new class of solid-state ultra-thin membranes with promising applications ranging from biological separations to use as a platform for electron imaging and spectroscopy. Because the thickness of the membrane is only 15–30 nm, on the order of that of the molecules to be separated, mass transport through the membrane is greatly enhanced. For applications involving molecular separations, it is crucial that the membrane is highly permeable to some species while being nearly impermeable to others. An important approach to adjusting the permeability of a membrane is by changing the size and density of the pores. With pnc-Si, a rapid thermal treatment is used to induce nanopore formation in a thin film of nanocrystalline silicon, which is then released over a silicon scaffold using an anisotropic etchant. In this study, we examine the influence of thin film deposition and thermal treatment parameters on pore size and density.

Biopolymers are of dynamic nature and undergo functional motions spanning a large spectrum of timescales. To study the internal dynamics of nano-sized molecular complexes that exceed hundred thousands of atoms with atomic detail is computationally inefficient. Therefore, to achieve both the spatial and temporal scales of biological interest coarse-grained models of macromolecules are often used. By uniting groups of atoms into single interacting centers one decreases the resolution of the system and gets rid of the irrelevant degrees of freedom. This simplification, even though it requires parameterization, makes the studies of biomolecular dynamics computationally tractable and allows us to reach beyond the microsecond time frame. Here, I review the coarse-grained models of macromolecules composed of proteins and nucleic acids. I give examples of one-bead models that were developed to investigate the internal dynamics and focus on their applications to the ribosome—the nanoscale protein synthesis machine.

The mass dependence of the mutual diffusion coefficient, in a binary equimolar mixture of Lennard-Jones fluids, is studied within Mori's memory function formalism. A phenomenological form of the memory function is used to study the time evolution of the self- and relative velocity correlation functions. The diffusion coefficients are calculated from the relevant velocity correlation functions using the Green–Kubo integral formula. Like the self-diffusion coefficient, the mutual diffusion coefficient is also found to be weakly dependent on the mass ratio. The present study shows that the minimum value that the mutual diffusion coefficient in an equimolar mixture of isotopic fluids can have is times the self-diffusion coefficient of any of the species when in isolation. Further, the contribution of the dynamic/distinct cross correlations to the mutual diffusion coefficient is found to be small and positive for the whole range of the mass ratio which is consistent with earlier molecular dynamics results.

We present a quantitative confocal-microscopy study of the transient and final microstructure of particle-stabilized emulsions formed via demixing in a binary liquid. To this end, we have developed an image-analysis method that relies on structure factors obtained from discrete Fourier transforms of individual frames in confocal image sequences. Radially averaging the squared modulus of these Fourier transforms before peak fitting allows extraction of dominant length scales over the entire temperature range of the quench. Our procedure even yields information just after droplet nucleation, when the (fluorescence) contrast between the two separating phases is scarcely discernible in the images. We find that our emulsions are stabilized on experimental timescales by interfacial particles and that they are likely to have bimodal droplet-size distributions. We attribute the latter to coalescence together with creaming being the main coarsening mechanism during the late stages of emulsification and we support this claim with (direct) confocal-microscopy observations. In addition, our results imply that the observed droplets emerge from particle-promoted nucleation, possibly followed by a free-growth regime. Finally, we argue that creaming strongly affects droplet growth during the early stages of emulsification. Future investigations could clarify the link between quench conditions and resulting microstructure, paving the way for tailor-made particle-stabilized emulsions from binary liquids.

X-ray scattering measurements of an expanded liquid Se₈₀Te₂₀ mixture in the temperature range between 300 and 1000 °C and at pressures of 10, 200, and 1600 bar have been carried out. The reverse Monte Carlo simulation and Voronoi–Delaunay void analyses have been applied to clarify the relationship between the voids and chain geometries in the intermediate scale near the semiconductor to metal (SC–M) transition at 800 °C under 200 bar accompanied by a shrinkage of molar volume. The structure of the liquid mixture can be envisaged in terms of a packing of covalently bonded chains and interstitial voids. The thermal expansion leads to a decrease in the number of chains around a chain and is compensated for by empty spaces (voids). The packing density of helical chains decreases and so voids supported by chains increase with rising temperature. At high temperature the shortening of chains and frequent transfer of lone pair electrons on Se (or Te) atoms cause modification of the helical chain to ring and zigzag chain conformations. The stacking of zigzag chains, joined to layers above 800 °C, leads to the formation of metallic domains separated by voids. The shrinkage of molar volume near the SC–M transition arises through the progressive filling of the voids around chains with (Se, Te)_{5, 6, 7, 8} rings.

The reported relaxation time for several typical glass-forming liquids was analyzed by using a kinetic model for liquids which invoked a new kind of atomic cooperativity—thermodynamic cooperativity. The broadly studied 'cooperative length' was recognized as the kinetic cooperativity. Both cooperativities were conveniently quantified from the measured relaxation data. A single-exponential activation behavior was uncovered behind the super-Arrhenius relaxations for the liquids investigated. Hence the mesostructure of these liquids and the atomic mechanism of the glass transition became clearer.

We study the equilibrium structure of large but finite aggregates of magnetic dipoles, representing a colloidal suspension of magnetite particles in a ferrofluid. With increasing system size, the structural motif evolves from chains and rings to multi-chain and multi-ring assemblies. Very large systems form single- and multi-wall coils, tubes and scrolls. These structural changes result from a competition between various energy terms, which can be approximated analytically within a continuum model. We also study the effect of external parameters such as magnetic field on the relative stability of these structures. Our results may give insight into experimental data obtained during solidification of ferrofluid aggregates at temperatures where thermal fluctuations become negligible in comparison to inter-particle interactions. These data may also help to experimentally control the aggregation of magnetic particles.

We present a study about the crystallization behavior of a two-dimensional particle system via molecular dynamics simulation. The interaction potential used here is a modified version of the Lennard-Jones potential which has the square lattice as the stable crystal structure at zero-pressure. The growth rate and the thermodynamic melting point are determined using a system with a free surface in the x direction. We also performed simulations of a supercooled liquid system with periodic boundary conditions to estimate the nucleation rate, the critical nucleus size, and the time lag of nucleation. These properties are important tools for understanding the crystallization phenomenon in a variety of materials.