Mapping shifts in nanopore signal to changes in protein and protein-DNA conformation.

Solid-state nanopores have been used extensively in biomolecular studies involving DNA and proteins. However, the interpretation of signals generated by the translocation of proteins or protein-DNA complexes remains challenging. Here, we investigate the behavior of monovalent streptavidin and the complex it forms with short biotinylated DNA over a range of nanopore sizes, salts, and voltages. We describe a simple geometric model that is broadly applicable and employ it to explain observed variations in conductance blockage and dwell time with experimental conditions. The general approach developed here underscores the value of nanopore-based protein analysis and represents progress toward the interpretation of complex translocation signals.

Instrumentation for low noise nanopore-based ionic current recording under laser illumination.

We describe a nanopore-based optofluidic instrument capable of performing low-noise ionic current recordings of individual biomolecules under laser illumination. In such systems, simultaneous optical measurements generally introduce significant parasitic noise in the electrical signal, which can severely reduce the instrument sensitivity, critically hindering the monitoring of single-molecule events in the ionic current traces. Here, we present design rules and describe simple adjustments to the experimental setup to mitigate the different noise sources encountered when integrating optical components to an electrical nanopore system. In particular, we address the contributions to the electrical noise spectra from illuminating the nanopore during ionic current recording and mitigate those effects through control of the illumination source and the use of a PDMS layer on the SiNx membrane. We demonstrate the effectiveness of our noise minimization strategies by showing the detection of DNA translocation events during membrane illumination with a signal-to-noise ratio of ∼10 at 10 kHz bandwidth. The instrumental guidelines for noise minimization that we report are applicable to a wide range of nanopore-based optofluidic systems and offer the possibility of enhancing the quality of synchronous optical and electrical signals obtained during single-molecule nanopore-based analysis.

Solid-state nanopore localization by controlled breakdown of selectively thinned membranes.

We demonstrate precise positioning of nanopores fabricated by controlled breakdown (CBD) on solid-state membranes by spatially varying the electric field strength with localized membrane thinning. We show 100 × 100 nm2 precision in standard SiN x membranes (30-100 nm thick) after selective thinning by as little as 25% with a helium ion beam. Control over nanopore position is achieved through the strong dependence of the electric field-driven CBD mechanism on membrane thickness. Confinement of pore formation to the thinned region of the membrane is confirmed by TEM imaging and by analysis of DNA translocations. These results enhance the functionality of CBD as a fabrication approach and enable the production of advanced nanopore devices for single-molecule sensing applications.

Interfacing solid-state nanopores with gel media to slow DNA translocations.

We demonstrate the ability to slow DNA translocations through solid-state nanopores by interfacing the trans side of the membrane with gel media. In this work, we focus on two reptation regimes: when the DNA molecule is flexible on the length scale of a gel pore, and when the DNA behaves as persistent segments in tight gel pores. The first regime is investigated using agarose gels, which produce a very wide distribution of translocation times for 5 kbp dsDNA fragments, spanning over three orders of magnitude. The second regime is attained with polyacrylamide gels, which can maintain a tight spread and produce a shift in the distribution of the translocation times by an order of magnitude for 100 bp dsDNA fragments, if intermolecular crowding on the trans side is avoided. While previous approaches have proven successful at slowing DNA passage, they have generally been detrimental to the S/N, capture rate, or experimental simplicity. These results establish that by controlling the regime of DNA movement exiting a nanopore interfaced with a gel medium, it is possible to address the issue of rapid biomolecule translocations through nanopores-presently one of the largest hurdles facing nanopore-based analysis-without affecting the signal quality or capture efficiency.

Membrane thickness dependence of nanopore formation with a focused helium ion beam.

Solid-state nanopores are emerging as a valuable tool for the detection and characterization of individual biomolecules. Central to their success is the realization of fabrication strategies that are both rapid and flexible in their ability to achieve diverse device dimensions. In this paper, we demonstrate the membrane thickness dependence of solid-state nanopore formation with a focused helium ion beam. We vary membrane thickness in situ and show that the rate of pore expansion follows a reproducible trend under all investigated membrane conditions. We show that this trend shifts to lower ion dose for thin membranes in a manner that can be described quantitatively, allowing devices of arbitrary dimension to be realized. Finally, we demonstrate that thin, small-diameter nanopores formed with our approach can be utilized for high signal-to-noise ratio resistive pulse sensing of DNA.

Selective detection and quantification of modified DNA with solid-state nanopores.

We demonstrate a solid-state nanopore assay for the unambiguous discrimination and quantification of modified DNA. Individual streptavidin proteins are employed as high-affinity tags for DNA containing a single biotin moiety. We establish that the rate of translocation events corresponds directly to relative concentration of protein-DNA complexes and use the selectivity of our approach to quantify modified oligonucleotides from among a background of unmodified DNA in solution.

Solid-state nanopores and nanopore arrays optimized for optical detection.

While conventional solid-state nanopore measurements utilize ionic current, there is a growing interest in alternative sensing paradigms, including optical detection. However, a limiting factor in the application of optical schemes in particular is the inherent background fluorescence created by the solid-state membrane itself, which can interfere with the desired signal and place restrictions on the fluorophores that can be employed. An ideal device would incorporate a localized reduction in membrane fluorescence using a method that can be integrated easily with the nanopore fabrication process. Here, we demonstrate that in addition to forming nanopores and nanopore arrays, a focused helium ion beam can be used to reduce the fluorescence of a conventional silicon nitride membrane controllably. The reduction in background produces low-fluorescence devices that can be used for optical detection of double-strand DNA, as well as for conventional resistive pulse sensing. This approach is used to identify the translocation of short single-strand DNA through individual nanopores within an array, creating potential for a massively-parallel detection scheme.

Interpreting the conductance blockades of DNA translocations through solid-state nanopores.

Solid-state nanopore electrical signatures can be convoluted and are thus challenging to interpret. In order to better understand the origin of these conductance changes, we investigate the translocation of DNA through small, thin pores over a range of voltage. We observe multiple, discrete populations of conductance blockades that vary with applied voltage. To describe our observations, we develop a simple model that is applicable to solid-state nanopores generally. These results represent an important step toward understanding the dynamics of the electrokinetic translocation process.

Metallization of a genetically engineered polypeptide.

Recently, well-ordered biological materials have been exploited to pattern inorganic nanoparticles into linear arrays that are of particular interest for nanoelectronic applications. In this work, a de novo designed E. coli-expressed polypeptide (previously shown to form highly rectilinear, ß-sheet-containing structures) operates as a template for divalent metal cations. EDX and TEM analysis verify the attachment of platinum ions to the histidine-rich fibril surface, which was designed specifically to facilitate attachment of chemical moieties. Following chemical reduction, TEM further confirms the formation of localized zero-valent metal aggregates with sub-nanometer interparticle spacing.

Block copolymer vesicle permeability measured by osmotic swelling and shrinking.

Vesicle response to osmotic shock provides insight into membrane permeability, a highly relevant value for applications ranging from nanoreactor experimentation to drug delivery. The osmotic shock approach has been employed extensively to elucidate the properties of phospholipid vesicles (liposomes) and of varieties of polymer vesicles (polymersomes). This study seeks to compare the membrane response for two varieties of polymersomes, a comb-type siloxane surfactant, poly(dimethylsiloxane)-g-poly(ethylene oxide) (PDMS-g-PEO), and a diblock copolymer, polybutadiene-b-poly(ethylene oxide) (PBut-b-PEO). Despite similar molecular weights and the same hydrophilic block (PEO), the two copolymers possess different hydrophobic blocks (PBut and PDMS) and corresponding glass transition temperatures (-31 and -123 °C, respectively). Dramatic variations in membrane response are observed during exposure to osmotic pressure differences, and values for polymer membrane permeability to water are extracted. We propose an explanation for the observed phenomena based on the respective properties of the PBut-b-PEO and PDMS-g-PEO membranes in terms of cohesion, thickness, and fluidity.

Self-assembly of 1-D organic semiconductor nanostructures.

This review focuses on the molecular design and self-assembly of a new class of crowded aromatics that form 1-D nanostructures via hydrogen bonding and pi-pi interactions. These molecules have a permanent dipole moment that sums as the subunits self assemble into molecular stacks. The assembly of these molecular stacks can be directed with electric fields. Depending on the nature of the side-chains, molecules can obtain the face-on or edge-on orientation upon the deposition onto a surface via spin cast technique. Site-selective steady state fluorescence, time-resolved fluorescence, and various types of scanning probe microscopy measurements detail the intermolecular interactions that drive the aromatic molecules to self-assemble in solution to form well-ordered columnar stacks. These nanostructures, formed in solution, vary in their number, size, and structure depending on the functional groups, solvent, and concentration used. Thus, the substituents/side-groups and the proper choice of the solvent can be used to tune the intermolecular interactions. The 1-D stacks and their aggregates can be easily transferred by solution casting, thus allowing a simple preparation of molecular nanostructures on different surfaces.

Electrically excited, localized infrared emission from single carbon nanotubes.

Carbon nanotube field-effect transistors (CNTFETs) produce band gap derived infrared emission under both ambipolar and unipolar transport conditions. We demonstrate here that heterogeneities/defects in the local environment of a CNTFET perturb the local potentials and, as a result, the characteristic bias dependent motion of the ambipolar light emission. Such defects can also introduce localized infrared emission due to impact excitation by carriers accelerated by a voltage drop at the defect. The correlation of the change in the motion of the ambipolarlight emission and of the stationary electroluminescence with the electrical characteristics of the CNTFETs shows that stationaryelectroluminescence can identify "environmental defects" in carbon nanotubes and help evaluate their influence on electrical transport and device operation. A number of different defects are studied involving local dielectric environment changes (partially polymer-covered nanotubes), nanotube-nanotube contacts in looped nanotubes, and nanotube segments close to the electronic contacts. Random defects due to local charging are also observed.

Bilayer fibril formation by genetically engineered polypeptides: preparation and characterization.

A de novo, genetically engineered 687 residue polypeptide expressed in E. coli has been found to form highly rectilinear, beta-sheet containing fibrillar structures. Tapping-mode atomic force microscopy, deep-UV Raman spectroscopy, and transmission electron microscopy definitively established the tendency of the fibrils to predominantly display an apparently planar bilayer or ribbon assemblage. The ordered self-assembly of designed, extremely repetitive, high molecular weight peptides is a harbinger of the utility of similar materials in nanoscience and engineering applications.