Silicon Nitride Nanopores Formed by Simple Chemical Etching: DNA Translocations and TEM Imaging.

We demonstrate DNA translocations through silicon nitride pores formed by simple chemical etching on glass substrates using microscopic amounts of hydrofluoric acid. DNA translocations and transmission electron microscopy (TEM) prove the fabrication of nanopores and allow their characterization. From ionic measurements on 318 chips, we report the effective pore diameters ranging from zero (pristine membranes) and sub-nm to over 100 nm, within 50 µm diameter membranes. The combination of ionic conductance, DNA current blockades, TEM imaging, and electron energy loss spectroscopy (EELS) provides comprehensive information about the pore area and number, from single to few pores, and pore structure. We also show the formation of thinned membrane regions as precursors of pores. The average pore density, about 5 × 10-4 pores/µm2, allows pore number adjustment statistically (0, 1, or more). This simple and affordable chemical method for making solid-state nanopores accelerates their adoption for DNA sensing and characterization applications.

Devices for Nanoscale Guiding of DNA through a 2D Nanopore.

We fabricate on-chip solid-state nanofluidic-2D nanopore systems that can limit the range of motion for DNA in the sensing region of a nanopore. We do so by creating devices containing one or more silicon nitride pores and silicon nitride pillars supporting a 2D pore that orient DNA within a nanopore device to a restricted geometry, yet allow the free motion of ions to maintain a high signal-to-noise ratio. We discuss two concepts with two and three independent electrical connections and corresponding nanopore chip device architectures to achieve this goal in practice. Here, we describe device fabrication and transmission electron microscope (TEM) images, and provide simulated translocations based on the finite element analysis in 3D to demonstrate its merit. In both methods, there is a main 2D nanopore which we refer to as a "sensing" nanopore (monolayer MoS2 in this paper). A secondary layer is either an array of guiding pores sharing the same electrode pair as the sensing pore (Method 1) or a single, independently contacted, guiding pore (Method 2). These pores are constructed parallel to the "sensing" pore and serve as "guiding" elements to stretch and feed DNA into the atomically thin sensing pore. We discuss the practical implementation of these concepts with nanofluidic and Si-based technology, including detailed fabrication steps and challenges involved for DNA applications in solution.

Surface characterization of nanoparticles using near-field light scattering.

The effect of nanoparticle surface coating characteristics on colloidal stability in solution is a critical parameter in understanding the potential applications of nanoparticles, especially in biomedicine. Here we explored the modification of the surface of poly(ethylene glycol)-coated superparamagnetic iron oxide nanoparticles (PEG-SPIOs) with the synthetic pseudotannin polygallol via interpolymer complexation (IPC). Changes in particle size and zeta potential were indirectly assessed via differences between PEG-SPIOs and IPC-SPIOs in particle velocity and scattering intensity using near-field light scattering. The local scattering intensity is correlated with the distance between the particle and waveguide, which is affected by the size of the particle (coating thickness) as well as the interactions between the particle and waveguide (related to the zeta potential of the coating). Therefore, we report here the use of near-field light scattering using nanophotonic force microscopy (using a NanoTweezerTM instrument, Halo Labs) to determine the changes that occurred in hydrated particle characteristics, which is accompanied by an analytical model. Furthermore, we found that altering the salt concentration of the suspension solution affected the velocity of particles due to the change of dielectric constant and viscosity of the solution. These findings suggest that this technique is suitable for studying particle surface changes and perhaps can be used to dynamically study reaction kinetics at the particle surface.

Using single nanoparticle tracking obtained by nanophotonic force microscopy to simultaneously characterize nanoparticle size distribution and nanoparticle-surface interactions.

Comprehensive characterization of nanomaterials for medical applications is a challenging and complex task due to the multitude of parameters which need to be taken into consideration in a broad range of conditions. Routine methods such as dynamic light scattering or nanoparticle tracking analysis provide some insight into the physicochemical properties of particle dispersions. For nanomedicine applications the information they supply can be of limited use. For this reason, there is a need for new methodologies and instruments that can provide additional data on nanoparticle properties such as their interactions with surfaces. Nanophotonic force microscopy has been shown as a viable method for measuring the force between surfaces and individual particles in the nano-size range. Here we outline a further application of this technique to measure the size of single particles and based on these measurement build the distribution of a sample. We demonstrate its efficacy by comparing the size distribution obtained with nanophotonic force microscopy to established instruments, such as dynamic light scattering and differential centrifugal sedimentation. Our results were in good agreement to those observed with all other instruments. Furthermore, we demonstrate that the methodology developed in this work can be used to study complex particle mixtures and the surface alteration of materials. For all cases studied, we were able to obtain both the size and the interaction potential of the particles with a surface in a single measurement.

Near-field Light Scattering Techniques for Measuring Nanoparticle-Surface Interaction Energies and Forces.

Nanoparticles are quickly becoming commonplace in many commercial and industrial products, ranging from cosmetics to pharmaceuticals to medical diagnostics. Predicting the stability of the engineered nanoparticles within these products a priori remains an important and difficult challenge. Here we describe our techniques for measuring the mechanical interactions between nanoparticles and surfaces using near-field light scattering. Particle-surface interfacial forces are measured by optically "pushing" a particle against a reference surface and observing its motion using scattered near-field light. Unlike atomic force microscopy, this technique is not limited by thermal noise, but instead takes advantage of it. The integrated waveguide and microfluidic architecture allow for high-throughput measurements of about 1000 particles per hour. We characterize the reproducibility of and experimental uncertainty in the measurements made using the NanoTweezer surface instrument. We report surface interaction studies on gold nanoparticles with 50 nm diameters, smaller than previously reported in the literature using similar techniques.

Rac1 pathway mediates stretch response in pulmonary alveolar epithelial cells.

Alveolar epithelial cells (AECs) maintain the pulmonary blood-gas barrier integrity with gasketlike intercellular tight junctions (TJ) that are anchored internally to the actin cytoskeleton. We have previously shown that AEC monolayers stretched cyclically and equibiaxially undergo rapid magnitude- and frequency-dependent actin cytoskeletal remodeling to form perijunctional actin rings (PJARs). In this work, we show that even 10 min of stretch induced increases in the phosphorylation of Akt and LIM kinase (LIMK) and decreases in cofilin phosphorylation, suggesting that the Rac1/Akt pathway is involved in these stretch-mediated changes. We confirmed that Rac1 inhibitors wortmannin or EHT-1864 decrease stretch-stimulated Akt and LIMK phosphorylation and that Rac1 agonists PIP3 or PDGF increase phosphorylation of these proteins in unstretched cells. We also confirmed that Rac1 pathway inhibition during stretch modulated stretch-induced changes in occludin content and monolayer permeability, actin remodeling and PJAR formation, and cell death. As further validation, overexpression of Rac GTPase-activating protein ß2-chimerin also preserved monolayer barrier properties in stretched monolayers. In summary, our data suggest that constitutive activity of Rac1, which is necessary for stretch-induced activation of the Rac1 downstream proteins, mediates stretch-induced increases in permeability and PJAR formation.

Cyclic stretch-induced oxidative stress increases pulmonary alveolar epithelial permeability.

Mechanical ventilation with high tidal volumes has been associated with pulmonary alveolar flooding. Understanding the mechanisms underlying cyclic stretch-induced increases in alveolar epithelial permeability may be important in designing preventive measures for acute lung injury. In this work, we assessed whether cyclic stretch leads to the generation of reactive oxygen species in type I-like alveolar epithelial cells, which increase monolayer permeability via activation of NF-κB and extracellular signal-regulated kinase (ERK). We cyclically stretched type I-like rat primary alveolar epithelial cells at magnitudes of 12, 25, and 37% change in surface area (ΔSA) for 10 to 120 minutes. High levels of reactive oxygen species and of superoxide and NO specifically were detected in cells stretched at 37% ΔSA for 10 to 120 minutes. Exogenous superoxide and NO stimulation increased epithelial permeability in unstretched cells, which was preventable by the NF-κB inhibitor MG132. The cyclic stretch-induced increase in permeability was decreased by the superoxide scavenger tiron and by MG132. Furthermore, tiron had a dramatic protective effect on in vivo lung permeability under mechanical ventilation conditions. Cyclic stretch increased the activation of the NF-κB signaling pathway, which was significantly decreased with the ERK inhibitor U0126. Altogether, our in vitro and in vivo data demonstrate the sensitivity of permeability to stretch- and ventilation-induced superoxide production, suggesting that using antioxidants may be helpful in the prevention and treatment of ventilator-induced lung injury.

Sepsis enhances epithelial permeability with stretch in an actin dependent manner.

Ventilation of septic patients often leads to the development of edema and impaired gas exchange. We hypothesized that septic alveolar epithelial monolayers would experience stretch-induced barrier dysfunction at a lower magnitude of stretch than healthy alveolar epithelial monolayers. Alveolar epithelial cells were isolated from rats 24 hours after cecal ligation and double puncture (2CLP) or sham surgery. Following a 5-day culture period, monolayers were cyclically stretched for 0, 10, or 60 minutes to a magnitude of 12% or 25% change in surface area (ΔSA). Barrier function, MAPk and myosin light chain (MLC) phosphorylation, tight junction (TJ) protein expression and actin cytoskeletal organization were examined after stretch. Significant increases in epithelial permeability were observed only in 2CLP monolayers at the 12% ΔSA stretch level, and in both 2CLP and sham monolayers at the 25% ΔSA stretch level. Increased permeability in 2CLP monolayers was not associated with MAPk signaling or alterations in expression of TJ proteins. 2CLP monolayers had fewer actin stress fibers before stretch, a more robust stretch-induced actin redistribution, and reduced phosphorylated MLCK than sham monolayers. Jasplakinolide stabilization of the actin cytoskeleton in 2CLP monolayers prevented significant increases in permeability following 60 minutes of stretch to 12% ΔSA. We concluded that septic alveolar epithelial monolayers are more susceptible to stretch-induced barrier dysfunction than healthy monolayers due to actin reorganization.

Rho kinase signaling pathways during stretch in primary alveolar epithelia.

Alveolar epithelial cells (AECs) maintain integrity of the blood-gas barrier with actin-anchored intercellular tight junctions. Stretched type I-like AECs undergo magnitude- and frequency-dependent actin cytoskeletal remodeling into perijunctional actin rings. On the basis of published studies in human pulmonary artery endothelial cells (HPAECs), we hypothesize that RhoA activity, Rho kinase (ROCK) activity, and phosphorylation of myosin light chain II (MLC2) increase in stretched type I-like AECs in a manner that is dependent on stretch magnitude, and that RhoA, ROCK, or MLC2 activity inhibition will attenuate stretch-induced actin remodeling and preserve barrier properties. Primary type I-like AEC monolayers were stretched biaxially to create a change in surface area (ΔSA) of 12%, 25%, or 37% in a cyclic manner at 0.25 Hz for up to 60 min or left unstretched. Type I-like AECs were also treated with Rho pathway inhibitors (ML-7, Y-27632, or blebbistatin) and stained for F-actin or treated with the myosin phosphatase inhibitor calyculin-A and quantified for monolayer permeability. Counter to our hypothesis, ROCK activity and MLC2 phosphorylation decreased in type I-like AECs stretched to 25% and 37% ΔSA and did not change in monolayers stretched to 12% ΔSA. Furthermore, RhoA activity decreased in type I-like AECs stretched to 37% ΔSA. In contrast, MLC2 phosphorylation in HPAECs increased when HPAECs were stretched to 12% ΔSA but then decreased when they were stretched to 37% ΔSA, similar to type I-like AECs. Perijunctional actin rings were observed in unstretched type I-like AECs treated with the Rho pathway inhibitor blebbistatin. Myosin phosphatase inhibition increased MLC2 phosphorylation in stretched type I-like AECs but had no effect on monolayer permeability. In summary, stretch alters RhoA activity, ROCK activity, and MLC2 phosphorylation in a manner dependent on stretch magnitude and cell type.

Stretch magnitude and frequency-dependent actin cytoskeleton remodeling in alveolar epithelia.

Alveolar epithelial cells (AEC) maintain integrity of the blood-gas barrier with gasket-like intercellular tight junctions (TJ) that are anchored internally to the actin cytoskeleton. We hypothesize that stretch rapidly reorganizes actin (<10 min) into a perijunctional actin ring (PJAR) in a manner that is dependent on magnitude and frequency of the stretch, accompanied by spontaneous movement of actin-anchored receptors at the plasma membrane. Primary AEC monolayers were stretched biaxially to create a change in surface area (DeltaSA) of 12%, 25%, or 37% in a cyclic manner at 0.25 Hz for up to 60 min, or held tonic at 25% DeltaSA for up to 60 min, or left unstretched. By 10 min of stretch PJARs were evident in 25% and 37% DeltaSA at 0.25 Hz, but not for 12% DeltaSA at 0.25 Hz, or at tonic 25% DeltaSA, or with no stretch. Treatment with 1 muM jasplakinolide abolished stretch-induced PJAR formation, however. As a rough index of remodeling rate, we measured spontaneous motions of 5-mum microbeads bound to actin focal adhesion complexes on the apical membrane surfaces; within 1 min of exposure to DeltaSA of 25% and 37%, these motions increased substantially, increased with increasing stretch frequency, and were consistent with our mechanistic hypothesis. With a tonic stretch, however, the spontaneous motion of microbeads attenuated back to unstretched levels, whereas PJAR remained unchanged. Stretch did not increase spontaneous microbead motion in human alveolar epithelial adenocarcinoma A549 monolayers, confirming that this actin remodeling response to stretch was a cell-type specific response. In summary, stretch of primary rat AEC monolayers forms PJARs and rapidly reorganized actin binding sites at the plasma membrane in a manner dependent on stretch magnitude and frequency.

7-Oxo-4,7-dihydrothieno[3,2-b]pyridine-6-carboxamides: synthesis and biological activity of a new class of highly potent inhibitors of human cytomegalovirus DNA polymerase.

We report a new class of non-nucleoside antivirals, the 7-oxo-4,7-dihydrothieno[3,2-b]pyridine-6-carboxamides, some of which possess remarkable potency versus a broad spectrum of herpesvirus DNA polymerases and excellent selectivity compared to human DNA polymerases. A critical factor in the level of activity is hypothesized to be conformational restriction of the key 2-aryl-2-hydroxyethylamine sidechain by an adjacent methyl group.