Cell-inspired, massive electromodulation of friction via transmembrane fields across lipid bilayers.

Transient electric fields across cell bilayer membranes can lead to electroporation and cell fusion, effects crucial to cell viability whose biological implications have been extensively studied. However, little is known about these behaviours in a materials context. Here we find that transmembrane electric fields can lead to a massive, reversible modulation of the sliding friction between surfaces coated with lipid-bilayer membranes-a 200-fold variation, up to two orders of magnitude greater than that achieved to date. Atomistic simulations reveal that the transverse fields, resembling those at cell membranes, lead to fully reversible electroporation of the confined bilayers and the formation of inter-bilayer bridges analogous to the stalks preceding intermembrane fusion. These increase the interfacial dissipation through reduced hydration at the slip plane, forcing it to revert in part from the low-dissipation, hydrated lipid-headgroup plane to the intra-bilayer, high-dissipation acyl tail interface. Our results demonstrate that lipid bilayers under transmembrane electric fields can have striking materials modification properties.

Nanopore Translocation Reveals Electrophoretic Force on Noncanonical RNA:DNA Double Helix.

Electrophoretic transport plays a pivotal role in advancing sensing technologies. So far, systematic studies have focused on the translocation of canonical B-form or A-form nucleic acids, while direct RNA analysis is emerging as the new frontier for nanopore sensing and sequencing. Here, we compare the less-explored dynamics of noncanonical RNA:DNA hybrids in electrophoretic transport to 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 faster than DD duplexes, despite containing the same number of base pairs. Our experiments reveal that RD duplexes present a noncanonical helix, with distinct transport properties from B-form DD molecules. We find that RD and DD 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, atomistic molecular dynamics simulations, agarose gel electrophoresis, and dynamic light scattering measurements. With the help of coarse-grained and molecular dynamics simulations, we find the effective force per unit length applied by the electric field to a fragment of RD or DD duplex in nanopores with various geometries or shapes 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 the molecular understanding of electrophoretic transport.

Multiplexed Nanopore-Based Nucleic Acid Sensing and Bacterial Identification Using DNA Dumbbell Nanoswitches.

Multiplexed nucleic acid sensing methods with high specificity are vital for clinical diagnostics and infectious disease control, especially in the postpandemic era. Nanopore sensing techniques have developed in the past two decades, offering versatile tools for biosensing while enabling highly sensitive analyte measurements at the single-molecule level. Here, we establish a nanopore sensor based on DNA dumbbell nanoswitches for multiplexed nucleic acid detection and bacterial identification. The DNA nanotechnology-based sensor switches from an "open" into a "closed" state when a target strand hybridizes to two sequence-specific sensing overhangs. The loop in the DNA pulls two groups of dumbbells together. The change in topology results in an easily recognized peak in the current trace. Simultaneous detection of four different sequences was achieved by assembling four DNA dumbbell nanoswitches on one carrier. The high specificity of the dumbbell nanoswitch was verified by distinguishing single base variants in DNA and RNA targets using four barcoded carriers in multiplexed measurements. By combining multiple dumbbell nanoswitches with barcoded DNA carriers, we identified different bacterial species even with high sequence similarity by detecting strain specific 16S ribosomal RNA (rRNA) fragments.

Simultaneous identification of viruses and viral variants with programmable DNA nanobait.

Respiratory infections are the major cause of death from infectious disease worldwide. Multiplexed diagnostic approaches are essential as many respiratory viruses have indistinguishable symptoms. We created self-assembled DNA nanobait that can simultaneously identify multiple short RNA targets. The nanobait approach relies on specific target selection via toehold-mediated strand displacement and rapid readout via nanopore sensing. Here we show that this platform can concurrently identify several common respiratory viruses, detecting a panel of short targets of viral nucleic acids from multiple viruses. Our nanobait can be easily reprogrammed to discriminate viral variants with single-nucleotide resolution, as we demonstrated for several key SARS-CoV-2 variants. Last, we show that the nanobait discriminates between samples extracted from oropharyngeal swabs from negative- and positive-SARS-CoV-2 patients without preamplification. Our system allows for the multiplexed identification of native RNA molecules, providing a new scalable approach for the diagnostics of multiple respiratory viruses in a single assay.

DNA-Based Optical Quantification of Ion Transport across Giant Vesicles.

Accurate measurements of ion permeability through cellular membranes remains challenging due to the lack of suitable ion-selective probes. Here we use giant unilamellar vesicles (GUVs) as membrane models for the direct visualization of mass translocation at the single-vesicle level. Ion transport is indicated with a fluorescently adjustable DNA-based sensor that accurately detects sub-millimolar variations in K+ concentration. In combination with microfluidics, we employed our DNA-based K+ sensor for extraction of the permeation coefficient of potassium ions. We measured K+ permeability coefficients at least 1 order of magnitude larger than previously reported values from bulk experiments and show that permeation rates across the lipid bilayer increase in the presence of octanol. In addition, an analysis of the K+ flux in different concentration gradients allows us to estimate the complementary H+ flux that dissipates the charge imbalance across the GUV membrane. Subsequently, we show that our sensor can quantify the K+ transport across prototypical cation-selective ion channels, gramicidin A and OmpF, revealing their relative H+/K+ selectivity. Our results show that gramicidin A is much more selective to protons than OmpF with a H+/K+ permeability ratio of ∼104.

Quantifying proton-induced membrane polarization in single biomimetic giant vesicles.

Proton gradients are utilized by cells to power the transport activity of many membrane proteins. Synthetic cells, such as proteo-giant unilamellar vesicles, offer an advanced approach for studying the functionality of membrane proteins in isolation. However, understanding of protein-based transport in vitro requires accurate measurements of proton flux and its accompanying electrochemical gradient across the lipid bilayer. We present an approach to directly quantify the flux of protons across single cell-sized lipid vesicles under modulated electrochemical gradients. Our measurements reveal the corresponding association between proton permeation and transmembrane potential development and its relation to the chemical nature of the conjugated anion (base). In the case of formic acid, we showed that, out of the total amount of permeated protons, a fraction of ≈0.2 traverse the lipid bilayer as H+, with the rest (≈0.8) in the form of a neutral acid. For strong acids (HCl or HNO3), proton permeation was governed by translocation of H+. Accordingly, a larger proton motive force (pmf) was generated for strong acids (pmf=14.2 mV) relative to formic acid (pmf=1.3 mV). We anticipate that our approach will guide the development of protein-based transport driven by proton gradient in artificial cell models and enable a deeper understanding of how vital acids, such as fatty acids, amino acids, bile acids, and carboxylic acid-containing drugs, traverse the lipid bilayer.

A Microfluidic Platform for Sequential Assembly and Separation of Synthetic Cell Models.

Cell-sized vesicles like giant unilamellar vesicles (GUVs) are established as a promising biomimetic model for studying cellular phenomena in isolation. However, the presence of residual components and byproducts, generated during vesicles preparation and manipulation, severely limits the utility of GUVs in applications like synthetic cells. Therefore, with the rapidly growing field of synthetic biology, there is an emergent demand for techniques that can continuously purify cell-like vesicles from diverse residues, while GUVs are being simultaneously synthesized and manipulated. We have developed a microfluidic platform capable of purifying GUVs through stream bifurcation, where a vesicles suspension is partitioned into three fractions: purified GUVs, residual components, and a washing solution. Using our purification approach, we show that giant vesicles can be separated from various residues─which range in size and chemical composition─with a very high efficiency (e = 0.99), based on size and deformability of the filtered objects. In addition, by incorporating the purification module with a microfluidic-based GUV-formation method, octanol-assisted liposome assembly (OLA), we established an integrated production-purification microfluidic unit that sequentially produces, manipulates, and purifies GUVs. We demonstrate the applicability of the integrated device to synthetic biology through sequentially fusing SUVs with freshly prepared GUVs and separating the fused GUVs from extraneous SUVs and oil droplets at the same time.

Modulating Interfacial Energy Dissipation via Potential-Controlled Ion Trapping.

As a metal (gold) surface at a given, but variable potential slides past a dielectric (mica) surface at a fixed charge, across aqueous salt solutions, two distinct dissipation regimes may be identified. In regime I, when the gold potential is such that counterions are expelled from between the surfaces, which then come to adhesive contact, the frictional dissipation is high, with coefficient of friction µ ≈ 0.8-0.9. In regime II, when hydrated counterions are trapped between the compressed surfaces, hydration lubrication is active and friction is much lower, µ = 0.05 ± 0.03. Moreover, the dissipation regime as the surfaces contact is largely retained even when the metal potential changes to the other regime, attributed to the slow kinetics of counterion expulsion from or penetration into the subnanometer intersurface gap. Our results indicate how frictional dissipation between such a conducting/nonconducting couple may be modulated by the potential applied to the metal.

Multiplexed DNA Identification Using Site Specific dCas9 Barcodes and Nanopore Sensing.

Decorating double-stranded DNA with dCas9 barcodes to identify characteristic short sequences provides an alternative to fully sequencing DNA samples for rapid and highly specific analysis of a DNA sample. Solid state nanopore sensors are especially promising for this type of single-molecule sensing because of the ability to analyze patterns in the ionic current signatures of DNA molecules. Here, we systematically demonstrate the use of highly specific dCas9 probes to create unique barcodes on the DNA that can be read out using nanopore sensors. Single dCas9 probes are targeted to various positions on DNA strands up to 48 kbp long and are effectively measured in high salt conditions typical of nanopore sensing. Multiple probes bound to the same DNA strand at characteristic target sequences create distinct barcodes of double and triple peaks. Finally, double and triple barcodes are used to simultaneously identify two different DNA targets in a background mixture of bacterial DNA. Our method forms the basis of a fast and versatile assay for multiplexed DNA sensing applications in complex samples.

Publisher Correction: Charging dynamics of an individual nanopore.

The original version of this Article contained an error throughout in which an incorrect symbol was used for the diffusion coefficient: it should be cambria math, italicized, and not bold. These have been corrected in both the PDF and HTML versions of the Article.

Charging dynamics of an individual nanopore.

Meso-porous electrodes (pore width « 1 µm) are a central component in electrochemical energy storage devices and related technologies, based on the capacitive nature of electric double-layers at their surfaces. This requires that such charging, limited by ion transport within the pores, is attained over the device operation time. Here we measure directly electric double layer charging within individual nano-slits, formed between gold and mica surfaces in a surface force balance, by monitoring transient surface forces in response to an applied electric potential. We find that the nano-slit charging time is of order 1 s (far slower than the time of order 3 × 10-2 s characteristic of charging an unconfined surface in our configuration), increasing at smaller slit thickness, and decreasing with solution ion concentration. The results enable us to examine critically the nanopore charging dynamics, and indicate how to probe such charging in different conditions and aqueous environments.

Modifying surface forces through control of surface potentials.

Combining direct surface force measurements with in situ regulation of surface potential provides an exceptional opportunity for investigating and manipulating interfacial phenomena. Recently, we studied the interaction between gold and mica surfaces in water with no added salt, while controlling the metal potential, and found that the surface charge at the metal may vary, and possibly even change its sign, as it progressively approaches the (constant-charge) mica surface [Langmuir, 2015, 31(47), 12845-12849]. Such a variation was found to directly affect the nature of the contact and adhesion between them due to exclusion of all mobile counterions from the intersurface gap. In this work, we extend this to examine the potential-dependent response of the adhesion and interaction between gold and mica to externally applied voltages and in electrolyte solution. Using a surface force balance (SFB) combined with a three-electrode electrochemical cell, we measured the normal interaction between gold and mica under surface potential regulation, revealing three interaction regimes - pure attraction, non-monotonic interaction from electrostatic repulsion to attraction (owing to charge inversion) and pure repulsion. Accordingly, the adhesion energy between the surfaces was found to vary both in no added salt water and, more strongly, in electrolyte solution. We justify this potential-dependent variation of adhesion energy in terms of the interplay between electrostatic energy and van der Waals (vdW) interaction at contact, and attribute the difference between the two cases to the weaker vdW interaction in electrolyte solution. Finally, we showed that through abruptly altering the gold surface potential from negative to positive and vice versa, the adhesion between gold and mica can be reversibly switched on and off. We surmise that the process of bringing the surface into contact is associated with the formation of a strong electric field O (108 V m-1) in the intersurface gap.

Probing the Surface Properties of Gold at Low Electrolyte Concentration.

Using the surface force balance (SFB), we studied the surface properties of gold in aqueous solution with low electrolyte concentration (∼10(-5) M and pH = 5.8), i.e., water with no added salt, by directly measuring the interaction between an ultrasmooth gold surface (ca. 0.2 nm rms roughness) and a mica surface. Under these conditions, specific adsorption of ions is minimized and its influence on the surface charge and surface potential of gold is markedly reduced. At open circuit potential, the electrostatic interaction between gold and mica was purely attractive and gold was found to be positively charged. This was further confirmed by force measurements against a positively charged surface, poly-l-lysine coated mica. Successive force measurements unambiguously showed that once gold and mica reach contact all counterions are expelled from the gap, confirming that at contact the surface charge of gold is equal and opposite in charge to that of mica. Further analysis of adhesion energy between the surfaces indicated that adhesion is mostly governed by vdW dispersion force and to a lesser extent by electrostatic interaction. Force measurements under external applied potentials showed that the gold-mica interaction can be regulated as a function of applied potential even at low electrolyte concentration. The gold-mica interaction was described very precisely by the nonlinearized Poisson-Boltzmann (PB) equation, where one of the surfaces is at constant charge, i.e., mica, and the other, i.e., gold, is at constant potential. Consequently, the gold surface potential could be determined accurately both at open circuit potential (OCP) and under different applied potentials. Using the obtained surface potentials, we were able to derive fundamental characteristics of the gold surface, e.g., its surface charge density and potential of zero charge (PZC), at very low electrolyte concentration.

Direct Observation of Confinement-Induced Charge Inversion at a Metal Surface.

Surface interactions across water are central to areas from nanomedicine to colloidal stability. They are predominantly a combination of attractive but short-ranged dispersive (van der Waals) forces, and long-ranged electrostatic forces between the charged surfaces. Here we show, using a surface force balance, that electrostatic forces between two surfaces across water, one at constant charge while the other (a molecularly smooth metal surface) is at constant potential of the same sign, may revert smoothly from repulsion to attraction on progressive confinement of the aqueous intersurface gap. This remarkable effect, long predicted theoretically in the classic Gouy-Chapman (Poisson-Boltzmann) model but never previously experimentally observed, unambiguously demonstrates surface charge reversal at the metal-water surface. This experimental confirmation emphasizes the implications for interactions of dielectrics with metal surfaces in aqueous media.

Biomedical imaging of colorectal cancer by near infrared fluorescent nanoparticles.

In this paper we describe the preparation of novel Near Infrared (NIR) fluorescent nanoparticles for application in medical imaging of colorectal tumors. The nanoparticles are prepared by using only non-covalent binding processes of molecules which are approved for clinical use. The preparation process is based on the precipitation of a polycation, Eudragit-RS, followed by sequential adsorption of a blocking protein, sodium caseinate, NIR fluorescent dye, Indocyanine Green (ICG) and optionally, a targeting molecule, anti-CEA antibody. Fluorescence measurements have shown that these nanoparticles have higher resistance to photobleaching and higher quantum yield relatively to free ICG. Imaging experiments in orthotopic colorectal cancer mice models have shown that these fluorescent nanoparticles are capable of binding to LS174T human colon tumors in vivo with high specificity, even without the targeting molecule. These nanoparticles, composed of all FDA approved materials, open the way to clinical bioimaging and diagnostics of colon cancer.

Penetration and biological effects of topically applied cyclosporin A nanoparticles in a human skin organ culture inflammatory model.

Systemic antipsoriatic therapies have potentially life-threatening, long-term side effects. The efficacy of topical drugs is poor, but may be improved by the use of delivery systems based on drug nanoparticles. To produce nanoparticles (NP) composed of cyclosporin A, a classical antipsoriatic drug, and to investigate their penetration and biological effects in human skin affected by psoriatic symptoms, poly-ε-caprolactone (PCL) and cyclosporin A (CsA) NP were prepared by the solvent evaporation method. Skin penetration was followed using fluorescently labeled NP in human skin organ cultures (hSOC). Psoriatic symptoms were mimicked in hSOC by the treatment with epidermal growth factor (EGF) and bacterial lipopolysaccharide (LPS). Cell viability in hSOC was evaluated by the resazurin test, and cytokine secretion into the growth medium was measured by immunodetection. We showed that topically applied NP diffused throughout the epidermis within two hours and through the dermis within the following day. They significantly reduced the secretion of inflammatory cytokines IL-1ß, IL-6, IL-8, IL-20 and IL-23. At active doses, no cytotoxicity was detected. This type of NP display relevant properties for the use as topical anti-inflammatory agents and may help to resorb psoriatic lesions.