ordering-single-cells-single-embryos-3d-confinement-new-device-for

Ordering Single Cells and Single Embryos in 3D Confinement: A New Device for High Content Screening

Biological cells are usually observed on flat (2D) surfaces. This condition is not physiological, and phenotypes and shapes are highly variable. Screening ...

Institut de Science et d'Ingénierie Supramoléculaires (ISIS), CNRS and Université de Strasbourg

Institut de Génétique et de Biologie Moléculaire et Cellulaire (IGBMC), CNRS and Université de Strasbourg

In this work we report the fabrication and characterization of a label-free impedimetric immunosensor based on a silicon nitride (Si(3)N(4)) surface for the specific detection of human serum albumin (HSA) proteins. Silicon nitride provides several advantages compared with other materials commonly used, such as gold, and in particular in solid-state physics for electronic-based biosensors. However, few Si(3)N(4)-based biosensors have been developed; the lack of an efficient and direct protocol for the integration of biological elements with silicon-based substrates is still one of its the main drawbacks. Here, we use a direct functionalization method for the direct covalent binding of monoclonal anti-HSA antibodies on an aldehyde-functionalized Si-p/SiO(2)/Si(3)N(4) structure. This methodology, in contrast with most of the protocols reported in literature, requires less chemical reagents, it is less time-consuming and it does not need any chemical activation. The detection capability of the immunosensor was tested by performing non-faradaic electrochemical impedance spectroscopy (EIS) measurements for the specific detection of HSA proteins. Protein concentrations within the linear range of 10(-13)-10(-7) M were detected, showing a sensitivity of 0.128 Ω μM(-1) and a limit of detection of 10(-14) M. The specificity of the sensor was also addressed by studying the interferences with a similar protein, bovine serum albumin. The results obtained show that the antibodies were efficiently immobilized and the proteins detected specifically, thus, establishing the basis and the potential applicability of the developed silicon nitride-based immunosensor for the detection of proteins in real and more complex samples.

Impedimetric immunosensor for human serum albumin detection on a direct aldehyde-functionalized silicon nitride surface.

The shape and dimensions of an atomic force microscope tip are crucial factors to obtain high resolution images at the nanoscale. When measuring samples with narrow trenches, inclined sidewalls near 90 degrees or nanoscaled structures, standard silicon atomic force microscopy (AFM) tips do not provide satisfactory results. We have combined deep reactive ion etching (DRIE) and focused ion beam (FIB) lithography techniques in order to produce probes with sharp rocket-shaped silicon AFM tips for high resolution imaging. The cantilevers were shaped and the bulk micromachining was performed using the same DRIE equipment. To improve the tip aspect ratio we used FIB nanolithography technique. The tips were tested on narrow silicon trenches and over biological samples showing a better resolution when compared with standard AFM tips, which enables nanocharacterization and nanometrology of high-aspect-ratio structures and nanoscaled biological elements to be completed, and provides an alternative to commercial high aspect ratio AFM tips.

Sharp high-aspect-ratio AFM tips fabricated by a combination of deep reactive ion etching and focused ion beam techniques.

We report high-precision calculations that correctly include the rearrangement channels by solving the modified Faddeev equations for energies between the Ps(n = 2) and H(n = 3) thresholds, which involve six and eight open channels. We find that 99% of the antihydrogen is formed in H(n = 2). Just above the Ps(n = 2) threshold the S, P, and D partial waves contribute nearly 1400pi(a(2)0) near the maximum. We find evidence that the induced long-range dipole potential is responsible for such a large H formation cross section. The possibility of utilizing this resonance to synthesize low-energy H is discussed.

Induced long-range dipole-field-enhanced antihydrogen formation in the p + Ps(n = 2) --> e(-) + H(n < or = 2) reaction.

We have performed thermal (differential scanning calorimetry) and rheological studies in the preformulation phase of ethosuximide suppositories to determine the influence of the active ingredient and adjuvants on the melting characteristics and rheological performance of the suppositories. Both types of studies were performed on the fatty bases witepsol H-19 and suppocire AP, each mixed with 5% w/w polysorbate 80, 0.1% w/w docusate sodium and 3% w/w tetranyl AT-1/DP and on the suppository formulations obtained by the addition of 150 mg of ethosuximide to these excipients. Pure lipophilic excipients have two peaks in the DSC curve. The first one occurs at lower temperatures (27.35 degrees C for the suppocire AP and 34.3 degrees C for the witepsol H19) and the second peak at higher temperatures (36.27 and 35.95 degrees C for the suppocire AP and witepsol H19, respectively). Polysorbate 80, docusate sodium and tetranyl AT-1/DP cause the first melting peak to disappear and the second peak to occur at a lower temperature. The thermograms of the formulas show two melting peaks and the endothermic peak does not appear due to the melting of ethosuximide (47.8 degrees C). We attribute this behavior to the solubility of the drug in the melted excipient, thus originating a decrease of the melting temperature of the second peak in the DSC scan. The excipients and formulations behave as Newtonian fluids and the influence of ethosuximide and adjuvants can be noted in an increase in the viscosity of the suppository mass.

Thermal and rheological study of lipophilic ethosuximide suppositories.

There is substantial interest in engineering solid supports to achieve functional immobilization of membrane receptors both for investigation of their biological function and for the development of novel biosensors. Three simple and practical strategies for immobilization of a human olfactory receptor carried by nanosomes are presented. The basis of the functionalization of solid gold surfaces is a self-assembled monolayer (SAM) containing biotinyl groups. Biotinyl groups are subsequently used to attach neutravidin and then biotinylated monoclonal antibody directed against the receptor to allow its specific grafting. Surface plasmon resonance technique is implemented for real-time monitoring of step-by-step surface functionalization and, in addition, for testing the functional response of immobilized olfactory receptors. We show that OR1740 is functional when immobilized via a tag attached to its C-terminus, but not via its N-terminus. Finally, we demonstrate that gold surfaces can be patterned by the SAMs tested using microcontact printing. AFM images of immobilized nanosomes onto a patterned surface suggest that small nanosomes flatten and fuse into larger vesicles but do not merge into a continuous layer. The whole study emphasizes the outstanding performances of the BAT/PEGAT SAM, which could be useful for developing on-a-chip sensor formats for membrane receptor investigations and use.

Gold surface functionalization and patterning for specific immobilization of olfactory receptors carried by nanosomes.

Direct patterning of anti-human serum albumin antibodies on aldehyde-terminated silicon nitride surfaces for HSA protein detection.

Biological patterned surfaces having sub-micron scale resolution are of great importance in many fields of life science and biomedicine. Different techniques have been proposed for surface patterning at the nanoscale. However, most of them present some limitations regarding the patterned area size or are time-consuming. Micro/nanocontact printing is the most representative soft lithography-based technique for surface patterning at the nanoscale. Unfortunately, conventional micro/nanocontact printing also suffers from problems such as diffusion and stamp collapsing that limit pattern resolution. To overcome these problems, a simple way of patterning thiols under liquid media using submerged nanocontact printing (SnCP) over large areas (approximately cm2) achieving nanosize resolution is presented. The technique is also low cost and any special equipment neither laboratory conditions are required. Nanostructured poly(dimethyl siloxane) stamps are replicated from commercially available digital video disks. SnCP is used to stamp patterns of 200 nm 1-octadecanethiol lines in liquid media, avoiding ink diffusion and stamp collapsing, over large areas on gold substrates compared with conventional procedures. Atomic force microscopy measurements reveal that the patterns have been successfully transferred with high fidelity. This is an easy, direct, effective and low cost methodology for molecule patterning immobilization which is of interest in those areas that require nanoscale structures over large areas, such as tissue engineering or biosensor applications.

Submerged nanocontact printing (SnCP) of thiols.

Adhesion between cells and the extracellular matrix is mediated by different types of transmembraneous proteins. Their associations to specific partners lead to the assembly of contacts such as focal adhesions and hemidesmosomes. The spatial overlap between both contacts within cells has however limited the study of each type of contact. Here we show that with "stampcils" focal contacts and hemidesmosomes can be spatially separated: cells are plated within the cavities of a stencil and the grids of the stencil serve as stamps for grafting an extracellular matrix protein-fibronectin. Cells engage new contacts on stamped zones leading to the segregation of adhesions and their associated cytoskeletons, i.e., actin and intermediate filaments of keratins. This new method should provide new insights into cell contacts compositions and dynamics.

Separation of distinct adhesion complexes and associated cytoskeleton by a micro-stencil-printing method.

Cellular protrusions involved in motile processes are driven by site-directed assembly of actin filaments in response to Rho-GTPase signalling. So far, only chemical compounds depolymerizing actin or stabilizing filaments, inhibiting N-WASP, Arp2/3 or formins, have been used to eliminate the formation of protrusions, while Rho-GTPase-dominant positive strategies have been designed to stimulate protrusions. Here we describe the design of four polyamines (macrocyclic and branched acyclic), and show that they enter the cell and induce specific growth of actin-enriched lamellipodia within minutes. The largest increase in cell area is obtained with micromolar amounts of a branched polyamine harbouring an 8-carbon chain. These polyamines specifically target actin both in vitro and in vivo. Analysis of their effects on filament assembly dynamics and its regulation indicates that the polyamines act by slowing down filament dynamics and by enhancing actin nucleation. These compounds provide new opportunities to study the actin cytoskeleton in motile and morphogenetic processes.

Synthetic polyamines promote rapid lamellipodial growth by regulating actin dynamics.

Many physiological phenomena involve directional cell migration. It is usually attributed to chemical gradients in vivo. Recently, other cues have been shown to guide cells in vitro, including stiffness/adhesion gradients or micropatterned adhesive motifs. However, the cellular mechanism leading to these biased migrations remains unknown, and, often, even the direction of motion is unpredictable. In this study, we show the key role of fluctuating protrusions on ratchet-like structures in driving NIH3T3 cell migration. We identified the concept of efficient protrusion and an associated direction index. Our analysis of the protrusion statistics facilitated the quantitative prediction of cell trajectories in all investigated conditions. We varied the external cues by changing the adhesive patterns. We also modified the internal cues using drug treatments, which modified the protrusion activity. Stochasticity affects the short- and long-term steps. We developed a theoretical model showing that an asymmetry in the protrusion fluctuations is sufficient for predicting all measures associated with the long-term motion, which can be described as a biased persistent random walk.

Protrusion fluctuations direct cell motion.

Cell migration is a crucial event during development and in disease. Mechanical constraints and chemical gradients can contribute to the establishment of cell direction, but their respective roles remain poorly understood. Using a microfabricated topographical ratchet, we show that the nucleus dictates the direction of cell movement through mechanical guidance by its environment. We demonstrate that this direction can be tuned by combining the topographical ratchet with a biochemical gradient of fibronectin adhesion. We report competition and cooperation between the two external cues. We also quantitatively compare the measurements associated with the trajectory of a model that treats cells as fluctuating particles trapped in a periodic asymmetric potential. We show that the cell nucleus contributes to the strength of the trap, whereas cell protrusions guided by the adhesive gradients add a constant tunable bias to the direction of cell motion.

Cells as active particles in asymmetric potentials: motility under external gradients.

New silicon nitride coated optical gratings were tested by means of Optical Waveguide Lightmode Spectroscopy (OWLS). A thin layer of 10 nm of transparent silicon nitride was deposited on commercial optical gratings by means of sputtering. The quality of the layer was tested by x-ray photoelectron spectroscopy and atomic force microscopy. As a proof of concept, the sensors were successfully tested with OWLS by monitoring the concentration dependence on the detection of an antibody-protein pair. The potential of the Si3N4 as functional layer in a real-time biosensor opens new ways for the integration of optical waveguides with microelectronics.

Optical Gratings Coated with Thin Si3N4 Layer for Efficient Immunosensing by Optical Waveguide Lightmode Spectroscopy.

Foreword: physics of cell migration.

Many physiological and pathological processes involve directed cell motion. In general, migrating cells are represented with a polarized morphology with extending and retracting protrusions at the leading edge. However, cell motion is a more complex phenomenon. Cells show heterogeneous morphologies and high protrusive dynamics is not always related to cell shape. This prevents the quantitative prediction of cell motion and the identification of cellular mechanisms setting directionality. Here we discuss the importance of protrusion fluctuations in directed cell motion. We show how their spatiotemporal distribution and dynamics determine the fluctuations and directions of cell motion for NIH3T3 fibroblasts plated on micro-patterned adhesive ratchets. (1) We introduce efficient protrusions and direction index which capture short-term cell motility over hours: these new read-outs allow the prediction of parameters characteristic for the long-term motion of cells over days. The results may have important implications for the study of biological phenomena where directed cell migration is involved, in morphogenesis and in cancer.

The cell ratchet: interplay between efficient protrusions and adhesion determines cell motion.

Directed cell migration is usually thought to depend on the presence of long-range gradients of either chemoattractants or physical properties such as stiffness or adhesion. However, in vivo, chemical or mechanical gradients have not systematically been observed. Here we review recent in vitro experiments, which show that other types of spatial guidance cues can bias cell motility. Introducing local geometrical or mechanical anisotropy in the cell environment, such as adhesive/topographical microratchets or tilted micropillars, show that local and periodic external cues can direct cell motion. Together with modeling, these experiments suggest that cell motility can be viewed as a stochastic phenomenon, which can be biased by various types of local cues, leading to directional migration.

David Caballero

Laboratory of Cell Physics

Institut de Science et d'Ingénierie Supramoléculaires (ISIS), CNRS and Université de Strasbourg

Ordering Single Cells and Single Embryos in 3D Confinement: A New Device for High Content Screening

David Caballero

.css-o250i3{font-size:18px;font-family:Open Sans,sans-serif;line-height:21.6px;}Laboratory of Cell Physics

Institut de Science et d'Ingénierie Supramoléculaires (ISIS), CNRS and Université de Strasbourg

Ordering Single Cells and Single Embryos in 3D Confinement: A New Device for High Content Screening

Laboratory of Cell Physics