Nanopore translocation reveals electrophoretic force on A- and B-form nucleic acids.

Electrophoretic transport plays a pivotal role in advancing sensing technologies, with A-form nucleic acids, predominantly RNA-containing, emerging as the new frontier for nanopore sensing and sequencing. Here, we compare the less-explored dynamics of A-form electrophoretic transport with the well-researched transport of B-form DNA. Using DNA/RNA nanotechnology and solid-state nanopores, the translocation of RNA:DNA (RD) and DNA:DNA (DD) duplexes was examined. Notably, RD duplexes were found to translocate through nanopores up to 1.8 times faster than DD duplexes, despite containing the same number of base pairs. Our experiments reveal that A- and B-form duplex molecules with the same contour length move with comparable velocity through nanopores. We examined the physical characteristics of both duplex forms using atomic force microscopy, agarose gel electrophoresis, and dynamic light scattering measurements. With the help of coarse-grained and atomistic molecular dynamics simulations, we find the effective force applied by the electric field to a fragment of A-form or B-form duplex in a nanopore to be approximately the same. Our results shed light on the significance of helical form in nucleic acid translocation, with implications for RNA sensing, sequencing, and molecular understanding of electrophoretic transport.

Spatially multiplexed single-molecule translocations through a nanopore at controlled speeds.

In current nanopore-based label-free single-molecule sensing technologies, stochastic processes influence the selection of translocating molecule, translocation rate and translocation velocity. As a result, single-molecule translocations are challenging to control both spatially and temporally. Here we present a method using a glass nanopore mounted on a three-dimensional nanopositioner to spatially select molecules, deterministically tethered on a glass surface, for controlled translocations. By controlling the distance between the nanopore and glass surface, we can actively select the region of interest on the molecule and scan it a controlled number of times and at a controlled velocity. Decreasing the velocity and averaging thousands of consecutive readings of the same molecule increases the signal-to-noise ratio by two orders of magnitude compared with free translocations. We demonstrate the method's versatility by assessing DNA-protein complexes, DNA rulers and DNA gaps, achieving down to single-nucleotide gap detection.

Noise properties of rectifying and non-rectifying nanopores.

Achieving a full understanding of the noise in resistive pulse sensing experiments is central to the development of this important single molecule technique. Here, we present a comprehensive study of the noise properties of conical glass nanopores as components in an ionic circuit by studying the power spectral density of the system in salt solutions at a range of concentrations. We begin by investigating the ionic current rectification of the pores, showing that it is only observed above a critical Dukhin number in agreement with theoretical predictions. We then investigate the noise properties of the pores and demonstrate that the fluctuations in the ionic current at no applied potential difference can be well modelled over four decades of frequency as thermal fluctuations over a complex impedance. Finally, we show that-when an ionic current flows-1/f noise dominates the power spectrum below ∼100 Hz. Fluctuations in the surface current govern the low-frequency 1/f noise, with the asymmetric shape of the pore leading the magnitude to scale with [Formula: see text], faster than predicted by Hooge's empirical relation.

A microfluidic platform for the characterisation of membrane active antimicrobials.

The spread of bacterial resistance against conventional antibiotics generates a great need for the discovery of novel antimicrobials. Polypeptide antibiotics constitute a promising class of antimicrobial agents that favour attack on bacterial membranes. However, efficient measurement platforms for evaluating their mechanisms of action in a systematic manner are lacking. Here we report an integrated lab-on-a-chip multilayer microfluidic platform to quantify the membranolytic efficacy of such antibiotics. The platform is a biomimetic vesicle-based screening assay, which generates giant unilamellar vesicles (GUVs) in physiologically relevant buffers on demand. Hundreds of these GUVs are individually immobilised downstream in physical traps connected to separate perfusion inlets that facilitate controlled antibiotic delivery. Antibiotic efficacy is expressed as a function of the time needed for an encapsulated dye to leak out of the GUVs as a result of antibiotic treatment. This proof-of-principle study probes the dose response of an archetypal polypeptide antibiotic cecropin B on GUVs mimicking bacterial membranes. The results of the study provide a foundation for engineering quantitative, high-throughput microfluidics devices for screening antibiotics.

Experimental evidence of symmetry breaking of transition-path times.

While thermal rates of state transitions in classical systems have been studied for almost a century, associated transition-path times have only recently received attention. Uphill and downhill transition paths between states at different free energies should be statistically indistinguishable. Here, we systematically investigate transition-path-time symmetry and report evidence of its breakdown on the molecular- and meso-scale out of equilibrium. In automated Brownian dynamics experiments, we establish first-passage-time symmetries of colloids driven by femtoNewton forces in holographically-created optical landscapes confined within microchannels. Conversely, we show that transitions which couple in a path-dependent manner to fluctuating forces exhibit asymmetry. We reproduce this asymmetry in folding transitions of DNA-hairpins driven out of equilibrium and suggest a topological mechanism of symmetry breakdown. Our results are relevant to measurements that capture a single coordinate in a multidimensional free energy landscape, as encountered in electrophysiology and single-molecule fluorescence experiments.

Nondeterministic self-assembly with asymmetric interactions.

We investigate general properties of nondeterministic self-assembly with asymmetric interactions, using a computational model and DNA tile assembly experiments. By contrasting symmetric and asymmetric interactions we show that the latter can lead to self-limiting cluster growth. Furthermore, by adjusting the relative abundance of self-assembly particles in a two-particle mixture, we are able to tune the final sizes of these clusters. We show that this is a fundamental property of asymmetric interactions, which has potential applications in bioengineering, and provides insights into the study of diseases caused by protein aggregation.

A label-free microfluidic assay to quantitatively study antibiotic diffusion through lipid membranes.

With the rise in antibiotic resistance amongst pathogenic bacteria, the study of antibiotic activity and transport across cell membranes is gaining widespread importance. We present a novel, label-free microfluidic assay that quantifies the permeability coefficient of a broad spectrum fluoroquinolone antibiotic, norfloxacin, across lipid membranes using the UV autofluorescence of the drug. We use giant lipid vesicles as highly controlled model systems to study the diffusion through lipid membranes. Our technique directly determines the permeability coefficient without requiring the measurement of the partition coefficient of the antibiotic.

Note: Direct force and ionic-current measurements on DNA in a nanocapillary.

We have developed optical tweezers, with force measurements based on fast video tracking, for analysis and control of DNA translocation through nanocapillaries. Nanocapillaries are single-molecule biosensors with very similar characteristics to solid-state nanopores. Our novel experimental setup allows for ionic-current measurements in which the nanocapillary is oriented perpendicular to the trapping laser. Using video-based particle tracking, we are able to measure the position of DNA coated colloids at sub-millisecond resolution and in real-time. We present the first electrophoretic force and simultaneous ionic-current measurements of a single DNA molecule inside the orifice of a nanocapillary.

Micro-rheology on (polymer-grafted) colloids using optical tweezers.

Optical tweezers are experimental tools with extraordinary resolution in positioning (± 1 nm) a micron-sized colloid and in the measurement of forces (± 50 fN) acting on it-without any mechanical contact. This enables one to carry out a multitude of novel experiments in nano- and microfluidics, of which the following will be presented in this review: (i) forces within single pairs of colloids in media of varying concentration and valency of the surrounding ionic solution, (ii) measurements of the electrophoretic mobility of single colloids in different solvents (concentration, valency of the ionic solution and pH), (iii) similar experiments as in (i) with DNA-grafted colloids, (iv) the nonlinear response of single DNA-grafted colloids in shear flow and (v) the drag force on single colloids pulled through a polymer solution. The experiments will be described in detail and their analysis discussed.

Indole transport across Escherichia coli membranes.

Indole has many, diverse roles in bacterial signaling. It regulates the transition from exponential to stationary phase, it is involved in the control of plasmid stability, and it influences biofilm formation, virulence, and stress responses (including antibiotic resistance). Its role is not restricted to bacteria, and recently it has been shown to include mutually beneficial signaling between enteric bacteria and their mammalian hosts. In many respects indole behaves like the signaling component of a quorum-sensing system. Indole synthesized within the producer bacterium is exported into the surroundings where its accumulation is detected by sensitive cells. A view often repeated in the literature is that in Escherichia coli the AcrEF-TolC and Mtr transporter proteins are involved in the export and import, respectively, of indole. However, the evidence for their involvement is indirect, and it has been known for a long time that indole can pass directly through a lipid bilayer. We have combined in vivo and in vitro approaches to examine the relative importance of protein-mediated transport and direct passage across the E. coli membrane. We conclude that the movement of indole across the E. coli membrane under normal physiological conditions is independent of AcrEF-TolC and Mtr. Furthermore, direct observation of individual liposomes shows that indole can rapidly cross an E. coli lipid membrane without the aid of any proteinaceous transporter. These observations not only enhance our understanding of indole signaling in bacteria but also provide a simple explanation for the ability of indole to signal between biological kingdoms.

Probing DNA with micro- and nanocapillaries and optical tweezers.

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.

Inserting and manipulating DNA in a nanopore with optical tweezers.

The translocation of small molecules and polymers is an integral process for the functioning of living cells. Many of the basic physical, chemical, and biological interactions have not yet been studied because they are not directly experimentally accessible. We have shown that a combination of optical tweezers, single solid-state nanopores, and electrophysiological ionic current detection enable deeper insight into the behavior of polymers in confinement. Here we describe the experimental procedures that are necessary to manipulate single biopolymers in a single nanopore, not only by electrical fields, but also through mechanical forces using optical tweezers.

Phase-state dependent current fluctuations in pure lipid membranes.

Current fluctuations in pure lipid membranes have been shown to occur under the influence of transmembrane electric fields (electroporation) as well as a result from structural rearrangements of the lipid bilayer during phase transition (soft perforation). We demonstrate that the ion permeability during lipid phase transition exhibits the same qualitative temperature dependence as the macroscopic heat capacity of a D15PC/DOPC vesicle suspension. Microscopic current fluctuations show distinct characteristics for each individual phase state. Although current fluctuations in the fluid phase show spikelike behavior of short timescales (approximately 2 ms) with a narrow amplitude distribution, the current fluctuations during lipid phase transition appear in distinct steps with timescales of approximately 20 ms. We propose a theoretical explanation for the origin of timescales and permeability based on a linear relationship between lipid membrane susceptibilities and relaxation times near the phase transition.

Single colloid electrophoresis.

Optical tweezers enable one to trap a single particle without any mechanical contact and to measure its position and the forces acting on it with high resolution (+/-4 nm, +/-160 fN). Taking advantage of a specially designed microfluidic cell the electrophoretic response of the colloid under study and the electroosmotic effect on the surrounding medium are determined using the identical colloid. The former is found to be by more than one order of magnitude larger than the electroosmotic effect. It is shifted in phase with respect to the external field, hence giving rise to a complex electrophoretic mobility which can be theoretically described by a strongly damped driven harmonic oscillator model. By exchanging the medium surrounding the colloid it is possible to deduce the (KCl) concentration dependence of the single colloid electrophoretic response. The results are compared with conventional Zetasizer measurements.

Sensing DNA-coatings of microparticles using micropipettes.

The resistive pulse technique is widely used to detect the size of small particles in aqueous solutions. This work demonstrates that a few tens of DNA molecules and thus the charges on a particle can be simply detected by pressure-driven translocation through a microcapillary based Coulter counter. The typical opening of the capillaries ranges from 2 to 6 microm. The custom-built system gives optical access using a high numerical aperture objective allowing to observe colloids passing the sensing volume by optical means. We show the feasibility of our setup by distinguishing colloids with one and two micron diameters. Our measurements prove that a few ten strands of DNA bound to the colloids can be detected. This can be achieved by simple comparison of current amplitudes for blank and coated colloids at low salt concentrations (2-40 mmol [NaCl]). Our results clearly demonstrate that the Coulter counter can be used to detect the surface charges on colloids. Moreover, the results are in good agreement with a dynamical computer model taking into account the full geometry of the capillary.

Noise in solid-state nanopores.

We study ionic current fluctuations in solid-state nanopores over a wide frequency range and present a complete description of the noise characteristics. At low frequencies (f approximately < 100 Hz) we observe 1/f-type of noise. We analyze this low-frequency noise at different salt concentrations and find that the noise power remarkably scales linearly with the inverse number of charge carriers, in agreement with Hooge's relation. We find a Hooge parameter alpha = (1.1 +/- 0.1) x 10(-4). In the high-frequency regime (f approximately > 1 kHz), we can model the increase in current power spectral density with frequency through a calculation of the Johnson noise. Finally, we use these results to compute the signal-to-noise ratio for DNA translocation for different salt concentrations and nanopore diameters, yielding the parameters for optimal detection efficiency.

Forces between single pairs of charged colloids in aqueous salt solutions.

Forces between single pairs of negatively charged micrometer-sized colloids in aqueous solutions of monovalent, divalent, or trivalent counter-ions at varying concentrations have been measured by employing optical tweezers. The experimental data have been analyzed by using the Derjaguin-Landau-Verwey-Overbeek (DLVO) theory and a numerical solution of the Poisson-Boltzmann (PB) equation. With monovalent counterions, the data are well described by the DLVO and PB theories, suggesting that the DLVO theory is adequate to describe the colloidal forces at these conditions. At higher counter-ion valence, the approximations within the two theories become evident.

Nanobubbles in solid-state nanopores.

From conductance and noise studies, we infer that nanometer-sized gaseous bubbles (nanobubbles) are the dominant noise source in solid-state nanopores. We study the ionic conductance through solid-state nanopores as they are moved through the focus of an infrared laser beam. The resulting conductance profiles show strong variations in both the magnitude of the conductance and in the low-frequency noise when a single nanopore is measured multiple times. Differences up to 5 orders of magnitude are found in the current power spectral density. In addition, we measure an unexpected double-peak ionic conductance profile. A simple model of a cylindrical nanopore that contains a nanobubble explains the measured profile and accounts for the observed variations in the magnitude of the conductance.

Kondo effect in a few-electron quantum ring.

A small quantum ring with less than ten electrons was studied by transport spectroscopy. For strong coupling to the leads a Kondo effect is observed and used to characterize the spin structure of the system in a wide range of magnetic fields. At small magnetic fields Aharonov-Bohm oscillations influenced by Coulomb interaction appear. They exhibit phase jumps by pi at the Coulomb-blockade resonances. Inside Coulomb-blockade valleys the Aharonov-Bohm oscillations can also be studied due to the finite conductance caused by the Kondo effect. Astonishingly, the maxima of the oscillations show linear shifts with increasing magnetic field and gate voltage.