Nano-stenciled RGD-gold patterns that inhibit focal contact maturation induce lamellipodia formation in fibroblasts

Cultured fibroblasts adhere to extracellular substrates by means of cell-matrix adhesions that are assembled in a hierarchical way, thereby gaining in protein complexity and size. Here we asked how restricting the size of cell-matrix adhesions affects cell morphology and behavior. Using a nanostencil technique, culture substrates were patterned with gold squares of a width and spacing between 250 nm and 2 µm. The gold was functionalized with RGD peptide as ligand for cellular integrins, and mouse embryo fibroblasts were plated. Limiting the length of cell-matrix adhesions to 500 nm or less disturbed the maturation of vinculin-positive focal complexes into focal contacts and fibrillar adhesions, as indicated by poor recruitment of ?5-integrin. We found that on sub-micrometer patterns, fibroblasts spread extensively, but did not polarize. Instead, they formed excessive numbers of lamellipodia and a fine actin meshwork without stress fibers. Moreover, these cells showed aberrant fibronectin fibrillogenesis, and their speed of directed migration was reduced significantly compared to fibroblasts on 2 µm square patterns. Interference with RhoA/ROCK signaling eliminated the pattern-dependent differences in cell morphology. Our results indicate that manipulating the maturation of cell-matrix adhesions by nanopatterned surfaces allows to influence morphology, actin dynamics, migration and ECM assembly of adhering fibroblasts.

Vapor bubble generation around gold nano-particles and its application to damaging of cells

We investigated vapor bubbles generated upon irradiation of gold nanoparticles with nanosecond laser pulses. Bubble formation was studied both with optical and acoustic means on supported single gold nanoparticles and single nanoparticles in suspension. Formation thresholds determined at different wavelengths indicate a bubble formation efficiency increasing with the irradiation wavelength. Vapor bubble generation in Bac-1 cells containing accumulations of the same particles was also investigated at different wavelengths. Similarly, they showed an increasing cell damage efficiency for longer wavelengths. Vapor bubbles generated by single laser pulses were about half the cell size when inducing acute damage.

An integrated nano-scale approach to profile miRNAs in limited clinical samples

Profiling miRNA expression in cells that directly contribute to human disease pathogenesis is likely to aid the discovery of novel drug targets and biomarkers. However, tissue heterogeneity and the limited amount of human diseased tissue available for research purposes present fundamental difficulties that often constrain the scope and potential of such studies. We established a flow cytometry-based method for isolating pure populations of pathogenic T cells from bronchial biopsy samples of asthma patients, and optimized a high-throughput nano-scale qRT-PCR method capable of accurately measuring 96 miRNAs in as little as 100 cells. Comparison of circulating and airway T cells from healthy and asthmatic subjects revealed asthma-associated and tissue-specific miRNA expression patterns. These results establish the feasibility and utility of investigating miRNA expression in small populations of cells involved in asthma pathogenesis, and set a precedent for application of our nano-scale approach in other human diseases. The microarray data from this study (Figure 7) has been submitted to the NCBI Gene Expression Omnibus (GEO; http://ncbi.nlm.nih.gov/geo) under accession no. GSE31030.

Proteome analysis of human plasma and amniotic fluid by Off-Gel isoelectric focusing followed by nano-LC-MS/MS

This paper presents a comparative proteomic analysis of human maternal plasma and amniotic fluid (AF) samples from the same patient at term of pregnancy in order to find specific AF proteins as markers of premature rupture of membranes, a complication frequently observed during pregnancy. Maternal plasma and the corresponding AF were immunodepleted in order to remove the six most abundant proteins before the systematic analysis of their protein composition. The protein samples were then fractionated by IEF Off-Gel electrophoresis (OGE), digested and analyzed with nano-LC-MS/MS separation, revealing a total of 73 and 69 proteins identified in maternal plasma and AF samples, respectively. The proteins identified in AF have been compared to those identified in the mother plasma as well as to the reference human plasma protein list reported by Anderson et al. (Mol. Cell. Proteomics 2004, 3, 311-326). This comparison showed that 26 proteins were exclusively present in AF and not in plasma among which 10 have already been described to be placenta or pregnancy specific. As a further validation of the method, plasma proteins fractionated by OGE and analysed by nano-LC-MS/MS have been compared to the Swiss 2-D PAGE reference map by reconstructing a map that matches 2-D gel and OGE experimental data. This representation shows that 36 of 49 reference proteins could be identified in both data sets, and that isoform shifts in pI are well conserved in the OGE data sets.

Structure and properties of graphene nano disks (GND) with and without edge-dopants

Graphene is one of the most important materials. In this research, the structures and properties of graphene nano disks (GND) with a concentric shape were investigated by Density Functional Theory (DFT) calculations, in which the most effective DFT methods - B3lyp and Pw91pw91 were employed. It was found that there are two types of edges - Zigzag and Armchair in concentric graphene nano disks (GND). The bond length between armchair-edge carbons is much shorter than that between zigzag-edge carbons. For C24 GND that consists of 24 carbon atoms, only armchair edge with 12 atoms is formed. For a GND larger than the C24 GND, both armchair and zigzag edges co-exist. Furthermore, when the number of carbon atoms in armchair-edge are always 12, the number of zigzag-edge atoms increases with increasing the size of a GND. In addition, the stability of a GND is enhanced with increasing its size, because the ratio of edge-atoms to non-edge-atoms decreases. The size effect of a graphene nano disk on its HOMO-LUMO energy gap was evaluated. C6 and C24 GNDs possess HOMO-LUMO gaps of 1.7 and 2.1eV, respectively, indicating that they are semi-conductors. In contrast, C54 and C96 GNDs are organic metals, because their HOMO-LUMO gaps are as low as 0.3 eV. The effect of doping foreign atoms to the edges of GNDs on their structures, stabilities, and HOMO-LUMO energy gaps were also examined. When foreign atoms are attached to the edge of a GND, the original unsaturated carbon atoms become saturated. As a result, both of the C-C bonds lengths and the stability of a GND increase. Furthermore, the doping effect on the HOMO-LUMO energy gap is dependent on the type of doped atoms. The doping H, F, or OH into the edge of a GND increases its HOMO-LUMO energy gap. In contrast, a Li-doped GND has a lower HOMO-LUMO energy gap than that without doping. Therefore, Li-doping can increase the electrical conductance of a GND, whereas H, F, or OH-doping decreases its conductance.

Regional comparisons of nano-mechanical properties of the human meniscus; structure and function

Osteoarthritis (OA) is a debilitating disease that is becoming more prevalent in today’s society. OA affects approximately 28 million adults in the United States alone and when present in the knee joint, usually leads to a total knee replacement. Numerous studies have been conducted to determine possible methods to halt the initiation of OA, but the structural integrity of the menisci has been shown have a direct effect on the progression of OA. Menisci are two C-shaped structures that are attached to the tibial plateau and aid in facilitating proper load transmission within the knee. The meniscal cross-section is wedge-like to fit the contour of the femoral condyles and help attenuate stresses on the tibial plateau. While meniscal tears are common, only the outer 1/3 of the meniscus is vascularized and has the capacity to heal, hence tears of the inner 2/3rds are generally treated via meniscectomy, leading to OA. To help combat this OA epidemic, an effective biomimetric meniscal replacement is needed. Numerous mechanical and biochemical studies have been conducted on the human meniscus, but very little is known about the mechanical properties on the nano-scale and how meniscal constituents are distributed in the meniscal cross-section. The regional (anterior, central and posterior) nano-mechanical properties of the meniscal superficial layers (both tibial and femoral contacting) and meniscal deep zone were investigated via nanoindentation to examine the regional inhomogeneity of both the lateral and medial menisci. Additionally, these results were compared to quantitative histological values to better formulate a structure-function relationship on the nano-scale. These data will prove imperative for further advancements of a tissue engineered meniscal replacement.

Quantum dot / optical protein bio-nano hybrid system biosensing

The integration of novel nanomaterials with highly-functional biological molecules has advanced multiple fields including electronics, sensing, imaging, and energy harvesting. This work focuses on the creation of a new type of bio-nano hybrid substrate for military biosensing applications. Specifically it is shown that the nano-scale interactions of the optical protein bacteriorhodopsin and colloidal semiconductor quantum dots can be utilized as a generic sensing substrate. This work spans from the basic creation of the protein to its application in a novel biosensing system. The functionality of this sensor design originates from the unique interactions between the quantum dot and bacteriorhodopsin molecule when in nanoscale proximity. A direct energy transfer relationship has been established between coreshell quantum dots and the optical protein bacteriorhodopsin that substantially enhances the protein’s native photovoltaic capabilities. This energy transfer phenomena is largely distance dependent, in the sub-10nm realm, and is characterized experimentally at multiple separation distances. Experimental results on the energy transfer efficiency in this hybrid system correlate closely to theoretical predictions. Deposition of the hybrid system with nano-scale control has allowed for the utilization of this energy transfer phenomena as a modulation point for a functional biosensor prototype. This work reveals that quantum dots have the ability to activate the bacteriorhodopsin photocycle through both photonic and non-photonic energy transfer mechanisms. By altering the energy transferred to the bacteriorhodopsin molecule from the quantum dot, the electrical output of the protein can be modulated. A biosensing prototype was created in which the energy transfer relationship is altered upon target binding, demonstrating the applicability of a quantum dot/bacteriorhodopsin hybrid system for sensor applications. The electrical nature of this sensing substrate will allow for its efficient integration into a nanoelectronics array form, potentially leading to a small-low power sensing platform for remote toxin detection applications.

Nano-engineering of composite material via reactive mechanical alloying/milling (RMA/M)

Attempts to strengthen a chromium-modified titanium trialuminide by a combination of grain size refinement and dispersoid strengthening led to a new means to synthesize such materials. This Reactive Mechanical Alloying/Milling process uses in situ reactions between the metallic powders and elements from a process control agent and/or a gaseous environment to assemble a dispersed small hard particle phase within the matrix by a bottom-up approach. In the current research milled powders of the trialuminide alloy along with titanium carbide were produced. The amount of the carbide can be varied widely with simple processing changes and in this case the milling process created trialuminide grain sizes and carbide particles that are the smallest known from such a process. Characterization of these materials required the development of x-ray diffraction means to determine particle sizes by deconvoluting and synthesizing components of the complex multiphase diffraction patterns and to carry out whole pattern analysis to analyze the diffuse scattering that developed from larger than usual highly defective grain boundary regions. These identified regions provide an important mass transport capability in the processing and not only facilitate the alloy development, but add to the understanding of the mechanical alloying process. Consolidation of the milled powder that consisted of small crystallites of the alloy and dispersed carbide particles two nanometers in size formed a unique, somewhat coarsened, microstructure producing an ultra-high strength solid material composed of the chromium-modified titanium trialuminide alloy matrix with small platelets of the complex carbides Ti2AlC and Ti3AlC2. This synthesis process provides the unique ability to nano-engineer a wide variety of composite materials, or special alloys, and has shown the ability to be extended to a wide variety of metallic materials.

Colloidal Nano-apatite Particles with Active Luminescent and Magentic Properties for Biotechnology Applications

Colloidal Nano-apatite Particles with Active Luminescent and Magentic Properties for Biotechnology Applications. The synthesis of functional nano-materials is a burgeoning field that has produced remarkable and consistent breakthroughs over the last two decades. Individual particles have become smaller and shown potential for well defined functionality. However, there are still unresolved problems, a primary one being the loss of functionality and novelty due to uncontrolled aggregation driven by surface energy considerations. As such the first design criteria to harness the true potential of nanoparticles is to prevent unwanted agglomeration by: (1) improving, and, if possible, (2) controlling aggregation behavior. This requires specific knowledge of the chemistry of the immediate locale of the intended application; especially for biologically relevant applications. The latter criterion is also application driven but should be considered, generally, to diversify the range of functional properties that can be achieved. We have now reason to believe that such a novel system with multifunctional capabilities can be synthesized rather conveniently and have far reaching impact in biotechnology and other applications in the near future. We are presently experimenting with the syntheses of spheroidal, metal-doped, colloidal apatite nano-particles (~10 nm) for several potential biomedical applications.