Assembly of DNA curtains using hydrogen silsesquioxane as a barrier to lipid diffusion.

We have established a single-molecule imaging experimental platform called "DNA curtains" in which DNA molecules tethered to a lipid bilayer are organized into patterns at nanofabricated metallic barriers on the surface of a microfluidic sample chamber. This technology has wide applications for real-time single-molecule imaging of protein-nucleic acid interactions. Here, we demonstrate that DNA curtains can also be made from hydrogen silsesquioxane (HSQ). HSQ offers important advantages over metallic barriers because it can be lithographically patterned directly onto fused silica slides without any requirement for further processing steps, thereby offering the potential for rapid prototype development and/or scale up for manufacturing.

Quantum dot nanoarrays: self-assembly with single-particle control and resolution.

The develpoment of a highly selective immobilization strategy for the self-assembly of quantum dots (QDs) from solution on lithographically defined, biochemically functionalized metal nanopatterns is presented. Nanosale control is achieved for the formation of predominantly single-particle structures consisting of a QD coupled to a metal nanoparticle, and assembled into an ordered nanoarray.

High-resolution imaging of the immunological synapse and T-cell receptor microclustering through microfabricated substrates.

T-cell activation via antigen presentation is associated with the formation of a macromolecular membrane assembly termed the immunological synapse (IS). The genesis of the IS and the onset of juxtacrine signalling is characterized by the formation of cell membrane microclusters and the organization of such into segregated microdomains. A central zone rich in T-cell receptor (TCR)-major histocompatibility complex microclusters termed the central supramolecular activation cluster (cSMAC) forms the bullseye of this structure, while the cellular interface surrounding the cSMAC is characterized by regions enriched in adhesion and co-stimulatory molecules. In vitro, the study of dynamic TCR microcluster coalescence and IS genesis in T-cell populations is hampered by cell migration within the culture system and resolution constraints resulting from lateral cell-cell contact. Here, we detail a novel system describing the fabrication of micropit arrays designed to sequester single T-cell-antigen presenting cell (APC) conjugates and promote IS formation in the horizontal imaging plane for high-resolution studies of microcluster dynamics. We subsequently use this system to describe the formation of the cSMAC in T-cell populations and to investigate the morphology of the interfacial APC membrane.

Fabrication of Nanoscale Bioarrays for the Study of Cytoskeletal Protein Binding Interactions Using Nanoimprint Lithography.

We describe a high throughput patterning process used to create arrays of molecular-scale features for the study of cytoskeletal protein binding interactions. The process uses a shadow-evaporated metal mask to facilitate lift-off of features defined by nanoimprint lithography. This simple and robust approach alleviates difficulties in pattern transfer of ultra-small features and results in arrays of highly ordered sub-10 nm features which are then functionalized with extracellular matrix proteins. Application of these arrays is demonstrated in cell spreading assays.

Plasma fluorination of diamond-like carbon surfaces: mechanism and application to nanoimprint lithography.

Diamond-like carbon (DLC) films, used as molds for nanoimprint lithography, were treated with a fluorocarbon-based plasma in order to enhance their anti-adhesion properties. While ellipsometry and atomic force microscope measurements showed negligible changes in thickness and surface roughness after plasma processing, contact angle measurement found fluorine plasma-treated DLC surfaces to be highly hydrophobic, with surface energy values reduced from approximately 45 mJ m(-2) for untreated films to approximately 20-30 mJ m(-2) after fluorination. X-ray photoelectron spectroscopy revealed a thin (from approximately 0.5 to approximately 3 nm) fluorocarbon layer on the DLC surface. Proposed mechanisms for the formation of this layer include two competing processes: etching of DLC and deposition of fluorocarbon material, with one or the other mechanism dominant, depending on the plasma conditions. Fluorocarbon plasma-treated DLC molds for nanoimprint lithography were used to pattern sub-20 nm size features with a high degree of repeatability, demonstrating an extended lifetime of the anti-adhesion coating.

Gold-Tipped Elastomeric Pillars for Cellular Mechanotransduction.

We describe a technique for the fabrication of arrays of elastomeric pillars whose top surfaces are treated with selective chemical functionalization to promote cellular adhesion in cellular force transduction experiments. The technique involves the creation of a rigid mold consisting of arrays of circular holes into which a thin layer of Au is deposited while the top surface of the mold and the sidewalls of the holes are protected by a sacrificial layer of Cr. When an elastomer is formed in the mold, the Au adheres to the tops of the molded pillars. This can then be selectively functionalized with a protein that induces cell adhesion, while the rest of the surface is treated with a repellent substance. An additional benefit is that the tops of the pillars can be fluorescently labeled for improved accuracy in force transduction measurements.

Fabrication of Nanoscale "Curtain Rods" for DNA Curtains Using Nanoimprint Lithography.

We have developed a new lithographically-based patterning process which significantly increases the throughput of experiments which probe how repair proteins scan DNA molecules for errors. In this process, nanoscale barriers are formed to interrupt the flow of a lipid bilayer in which DNA is tethered to proteins in the bilayer. The barriers trap the DNA, which is then stretched out by hydrodynamic flow, resulting in the formation of "DNA curtains." Nanoimprint lithography is used to facilitate massively parallel data collection for protein diffusion experiments on DNA.

Plasma fluorination of carbon-based materials for imprint and molding lithographic applications.

Diamondlike carbon nanoimprint templates are modified by exposure to a fluorocarbon-based plasma, yielding an ultrathin layer of a fluorocarbon material on the surface which has a very low surface energy with excellent antiwear properties. We demonstrate the use of these plasma fluorinated templates to pattern features with dimensions approximately 20 nm and below. Furthermore, we show that this process is extendable to other carbon-based materials. Plasma fluorination can be applied directly to nanoimprint resists as well as to molds used to form elastomer stamps for microcontact printing and other applications requiring easy mold release.

Fabrication of elastomer Pillar Arrays with Modulated Stiffness for Cellular Force Measurements.

The mechanical properties of a cell's environment can alter behavior such as migration and spreading, and control the differentiation path of stem cells. Here we describe a technique for fabricating substrates whose rigidity can be controlled locally without altering the contact area for cell spreading. The substrates consist of elastomeric pillar arrays in which the top surface is uniform but the pillar height is changed across a sharp step. Preliminary results demonstrate the effects on cell migration and morphology at the step boundary.

Lateral scaling in carbon-nanotube field-effect transistors.

We have fabricated carbon-nanotube (CN) field-effect transistors with multiple, individually addressable gate segments. The devices exhibit markedly different transistor characteristics when switched using gate segments controlling the device interior versus those near the source and drain. We ascribe this difference to a change from Schottky-barrier modulation at the contacts to bulk switching. We also find that the current through the bulk portion is independent of gate length for any gate voltage, offering direct evidence for ballistic transport in semiconducting carbon nanotubes over at least a few hundred nanometers, even for relatively small carrier velocities.