Silvia Hernández-Ainsa

Single Protein Molecule Detection by Glass Nanopores

Nanopores can be used to detect and analyze single molecules in solution. We have used glass nanopores made by laser-assisted capillary-pulling, as a high-throughput and low cost method, to detect a range of label-free proteins: lysozyme, avidin, IgG, β-lactoglobulin, ovalbumin, bovine serum albumin (BSA), and β-galactosidase in solution. Furthermore, we show for the first time solid state nanopore measurements of mammalian prion protein, which in its abnormal form is associated with transmissible spongiform encephalopathies. Our approach provides a basis for protein characterization and the study of protein conformational diseases by nanopore detection.

Multiplexed ionic current sensing with glass nanopores

We report a method for simultaneous ionic current measurements of single molecules across up to 16 solid state nanopore channels. Each device, costing less than

Ionic Conductivity, Structural Deformation, and Programmable Anisotropy of DNA Origami in Electric Field

20, contains 16 glass nanopores made by laser assisted capillary pulling. We demonstrate simultaneous multichannel detection of double stranded DNA and trapping of DNA origami nanostructures to form hybrid nanopores.

DNA-Tile Structures Induce Ionic Currents through Lipid Membranes

The DNA origami technique can enable functionalization of inorganic structures for single-molecule electric current recordings. Experiments have shown that several layers of DNA molecules, a DNA origami plate, placed on top of a solid-state nanopore is permeable to ions. Here, we report a comprehensive characterization of the ionic conductivity of DNA origami plates by means of all-atom molecular dynamics (MD) simulations and nanocapillary electric current recordings. Using the MD method, we characterize the ionic conductivity of several origami constructs, revealing the local distribution of ions, the distribution of the electrostatic potential and contribution of different molecular species to the current. The simulations determine the dependence of the ionic conductivity on the applied voltage, the number of DNA layers, the nucleotide content and the lattice type of the plates. We demonstrate that increasing the concentration of Mg(2+) ions makes the origami plates more compact, reducing their conductivity. The conductance of a DNA origami plate on top of a solid-state nanopore is determined by the two competing effects: bending of the DNA origami plate that reduces the current and separation of the DNA origami layers that increases the current. The latter is produced by the electro-osmotic flow and is reversible at the time scale of a hundred nanoseconds. The conductance of a DNA origami object is found to depend on its orientation, reaching maximum when the electric field aligns with the direction of the DNA helices. Our work demonstrates feasibility of programming the electrical properties of a self-assembled nanoscale object using DNA.

Studying DNA translocation in nanocapillaries using single molecule fluorescence

Self-assembled DNA nanostructures have been used to create man-made transmembrane channels in lipid bilayers. Here, we present a DNA-tile structure with a nominal subnanometer channel and cholesterol-tags for membrane anchoring. With an outer diameter of 5 nm and a molecular weight of 45 kDa, the dimensions of our synthetic nanostructure are comparable to biological ion channels. Because of its simple design, the structure self-assembles within a minute, making its creation scalable for applications in biology. Ionic current recordings demonstrate that the tile structures enable ion conduction through lipid bilayers and show gating and voltage-switching behavior. By demonstrating the design of DNA-based membrane channels with openings much smaller than that of the archetypical six-helix bundle, our work showcases their versatility inspired by the rich diversity of natural membrane components.

Voltage-dependent properties of DNA origami nanopores.

We demonstrate simultaneous measurements of DNA translocation into glass nanopores using ionic current detection and fluorescent imaging. We verify the correspondence between the passage of a single DNA molecule through the nanopore and the accompanying characteristic ionic current blockage. By tracking the motion of individual DNA molecules in the nanocapillary perpendicular to the optical axis and using a model, we can extract an effective mobility constant for DNA in our geometry under high electric fields.

Philic and Phobic Segregation in Liquid‐Crystal Ionic Dendrimers: An Enthalpy–Entropy Competition

We show DNA origami nanopores that respond to high voltages by a change in conformation on glass nanocapillaries. Our DNA origami nanopores are voltage sensitive as two distinct states are found as a function of the applied voltage. We suggest that the origin of these states is a mechanical distortion of the DNA origami. A simple model predicts the voltage dependence of the structural change. We show that our responsive DNA origami nanopores can be used to lower the frequency of DNA translocation by 1 order of magnitude.

The Indole Pulse: A New Perspective on Indole Signalling in Escherichia coli

This work was supported by CICYT-FEDER Spanish project CTQ2006-15611-CO2-01, UE project (7th FP—THE PEOPLE PROGRAMME. The Marie Curie Actions—ITN, No. 215884-2), and by the Gobierno de Aragon. S.H.-A. thanks the MICINN (Spain) for a grant.

Nanoobjects coming from mesomorphic ionic PAMAM dendrimers

Indole has diverse signalling roles, including modulation of biofilm formation, virulence and stress responses. Changes are induced by indole concentrations of 0.5–1.0 mM, similar to those found in the supernatant of Escherichia coli stationary phase culture. Here we describe an alternative mode of indole signalling that promotes the survival of E. coli cells during long-term stationary phase. A mutant that has lost the ability to produce indole demonstrates reduced survival under these conditions. Significantly, the addition of 1 mM indole to the culture supernatant is insufficient to restore long-term survival to the mutant. We provide evidence that the pertinent signal in this case is not 1 mM indole in the culture supernatant but a transient pulse of intra-cellular indole at the transition from exponential growth to stationary phase. During this pulse the cell-associated indole reaches a maximum of approximately 60 mM. We argue that this is sufficient to inhibit growth and division by an ionophore-based mechanism and causes the cells to enter stationary phase before resources are exhausted. The unused resources are used to repair and maintain cells during the extended period of starvation.

Controlling the Reversible Assembly of Liposomes through a Multistimuli Responsive Anchored DNA.

A series of ionic amphiphilic dendrimers constituted by the grafting of poly(amidoamine) (PAMAM) of different generations (G = 0–4) with linear carboxylic acids bearing hydrophobic chains has been prepared. Their self-assembly tendency both in bulk and in water has been investigated. Almost all of the compounds present liquid crystalline behaviour as shown by differential scanning calorimetry (DSC), polarizing optical microscopy (POM), and X-ray diffractometry (XRD) studies. Smectic A mesomorphism has been found for all of the compounds and rectangular columnar mesophase is displayed for the highest generation compound at low temperature. Interestingly these amphiphilic dendrimers are also capable to self-assemble in water depending on their hydrophobic/hydrophilic balance forming some nanoobjects. In most of the cases these nanoobjects resemble nanospheres whose morphology has been studied by means of transmission electronic microscopy (TEM). The stability of these nanospheres is disrupted in acid or basic media and their amphiphilic nature makes it possible for them to host both hydrophobic (β-carotene) and hydrophilic (Rhodamine B) molecules.