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International audience; In nature, cells exist in three-dimensional (3D) microenvironments with topography, stiffness, surface chemistry, and biological factors that strongly dictate their phenotype and behavior. The cellular microenvironment is an organized structure or scaffold that, together with the cells that live within it, make up living tissue. To mimic these systems and understand how the different properties of a scaffold, such as adhesion, proliferation, or function, influence cell behavior, we need to be able to fabricate cellular microenvironments with tunable properties. In this work, the nanotopography and functionality of scaffolds for cell culture were modified by coating 3D printed materials (DS3000 and poly(ethylene glycol)diacrylate, PEG-DA) with cellulose nanocrystals (CNCs). This general approach was demonstrated on a variety of structures designed to incorporate macro- and microscale features fabricated using photopolymerization and 3D printing techniques. Atomic force microscopy was used to characterize the CNC coatings and showed the ability to tune their density and in turn the surface nanoroughness from isolated nanoparticles to dense surface coverage. The ability to tune the density of CNCs on 3D printed structures could be leveraged to control the attachment and morphology of prostate cancer cells. It was also possible to introduce functionalization onto the surface of these scaffolds, either by directly coating them with CNCs grafted with the functionality of interest or with a more general approach of functionalizing the CNCs after coating using biotin–streptavidin coupling. The ability to carefully tune the nanostructure and functionalization of different 3D-printable materials is a step forward to creating in vitro scaffolds that mimic the nanoscale features of natural microenvironments, which are key to understanding their impact on cells and developing artificial tissues.

International audience; The development of advanced techniques of fabrication of three-dimensional (3D) microenvironments for the study of cell growth and proliferation has become one of the major motivations of material scientists and bioengineers in the past decade. Here, we present a novel residueless 3D structuration technique of poly(dimethylsiloxane) (PDMS) by water-in-PDMS emulsion casting and subsequent curing process in temperature-/pressure-controlled environment. Scanning electron microscopy and X-ray microcomputed tomography allowed us to investigate the impact of those parameters on the microarchitecture of the porous structure. We demonstrated that the optimized emulsion casting process gives rise to large-scale and highly interconnected network with pore size ranging from 500μm to 1.5 mm that turned out to be nicely adapted to 3D cell culture. Experimental cell culture validations were performed using SaOS-2 (osteosarcoma) cell lines. Epifluorescence and deep penetration imaging techniques as two-photon confocal microscopy unveiled information about cell morphology and confirmed a homogeneous cell proliferation and spatial distribution in the 3D porous structure within an available volume larger than 1 cm3. These results open alternative scenarios for the fabrication and integration of porous scaffolds for the development21of 3D cell culture platforms.

The realization of biomimetic microenvironments for cell biology applications such as organ-on-chip, in vitro drug screening, and tissue engineering is one of the most fascinating research areas in the field of bioengineering. The continuous evolution of additive manufacturing techniques provides the tools to engineer these architectures at different scales. Moreover, it is now possible to tailor their biomechanical and topological properties while taking inspiration from the characteristics of the extracellular matrix, the three-dimensional scaffold in which cells proliferate, migrate, and differentiate. In such context, there is therefore a continuous quest for synthetic and nature-derived composite materials that must hold biocompatible, biodegradable, bioactive features and also be compatible with the envisioned fabrication strategy. The structure of the current review is intended to provide to both micro-engineers and cell biologists a comparative overview of the characteristics, advantages, and drawbacks of the major 3D printing techniques, the most promising biomaterials candidates, and the trade-offs that must be considered in order to replicate the properties of natural microenvironments.

Droplets with colloidal biological suspensions evaporating on substrates with defined wetting properties generate confined environments for initiating aggregation and self-assembly processes. We describe smart micro- and nanostructured surfaces, optimized for probing single droplets and residues by synchrotron radiation micro- and nanobeam diffraction techniques. Applications are presented for Ac-IVD and β-amyloid (1–42) peptides capable of forming cross-β sheet structures. Complementary synchrotron radiation FTIR microspectroscopy addresses secondary structure formation. The high synchrotron radiation source brilliance enables fast raster-scan experiments.

International audience; The realization of 2D and 3D meso-scale architectures is an area of research involving a wide range of disciplines ranging from materials science, microelectronics, phononics, microfluidics to biomedicine requiring millimeter to centimeter-sized objects embedding micrometric features. In the recent years, several technologies have been employed to provide optimal features in terms of object size flexibility, printing resolution, large materials library and fabrication speed. In this work, we report a fully customizable single-photon absorption 3D fabrication methodology based on direct laser fabrication. To validate this approach and highlight the versatility of the setup, we have fabricated a comprehensive ensemble of 2D and 3D designs with potential applications in biomimetics, 3D scaffolding and microfluidics. The high degree of tunability of the reported fabrication system allows tailoring the laser power, slicing and fabrication speed for each single area of the design. These unique features enable a rapid prototyping of millimeter to centimeter-sized objects involving 3D architectures with true freestanding subunits and micrometric feature reproducibility. The presented strategy fills indeed the current technological gap related to the development of meso-scale architectures required in multidisciplinary fields of research.

International audience; The realization of 3D architectures for the study of cell growth, proliferation and differentiation is a task of fundamental importance for both technological and biological communities involved in the development of biomimetic cell culture environments. Here we report the combination of 3D freestanding scaffolds realized by multi-photon direct laser writing (DLW), seeded with neuroblastoma cells, and their multi-technique characterization using advanced 3D fluorescence imaging techniques. The high accuracy of the fabrication process (≈ 200 nm) provides a much finer control of the meso-, micro-and nano-scale features compared to other 3D printing technologies based on fused deposition modeling, inkjet printing, selective laser sintering or polyjet technology. Scanning electron microscopy (SEM) provided detailed insights about the morphology of both cells and cellular Complete Manuscript interconnections around the 3D architecture. On the other hand, the nature of the seeding in the inner core of the 3D scaffold, inaccessible by conventional SEM imaging, was unveiled by light sheet fluorescence microscopy and multi-photon confocal imaging which highlighted an optimal cell colonization both around and within the 3D scaffold as well as the formation of long neuritic extensions. The results open appealing scenarios for the use of the developed 3D fabrication/3D imaging protocols in several neuroscientific contexts.

A comprehensive review about the use of micro- and nanostructured superhydrophobic surfaces as a tool for in situ X-ray scattering investigations of soft matter and biological materials., Droplets on artificially structured superhydrophobic surfaces represent quasi contact-free sample environments which can be probed by X-ray microbeams and nanobeams in the absence of obstructing walls. This review will discuss basic surface wettability concepts and introduce the technology of structuring surfaces. Quasi contact-free droplets are compared with contact-free droplets; processes related to deposition and evaporation on solid surfaces are discussed. Droplet coalescence based on the electrowetting effect allows the probing of short-time mixing and reaction processes. The review will show for several materials of biological interest that structural processes related to conformational changes, nucleation and assembly during droplet evaporation can be spatially and temporally resolved by raster-scan diffraction techniques. Orientational ordering of anisotropic materials deposited during solidification at pinning sites facilitates the interpretation of structural data.

International audience; Here, the authors report the fabrication of lotus-leaf-like tailored SU8 micropillars and their application in the context of a multitechnique characterization protocol for the investigation of the structural properties of the two estrogen receptors (ERα66/ERα46). ER (α) expression is undoubtedly the most important biomarker in breast cancer, as it provides the index for sensitivity to endocrine treatment. Beside the well-characterized ERα66 isoform, a shorter one (ERα46) is also expressed in ERα positive breast cancers and breast cancer cell lines. The superhydrophobic supports were developed by using a two-step approach including an optical lithography process and a plasma reactive ion roughening one. Upon drying on the micropillars, the biological samples resulted in stretched fibers of different diameters which were then characterized by synchrotron x-ray diffraction (XRD), Raman and Fourier-transform infrared spectroscopy. The evidence of both different spectroscopic vibrational responses and XRD signatures in the two estrogen receptors suggests the presence of conformational changes between the two biomarkers. The SU8 micropillar platform therefore represents a valid tool to enhance the discrimination sensitivity of structural features of this class of biomarkers by exploiting a multitechnique in situ characterization approach.

Super-hydrophobic (SH) surfaces are bio-inspired, nanotechnology artifacts which feature a reduced friction coefficient whereby they can be used for a number of very practical applications including, on the medical side, the manipulation of biological solutions. These surfaces can be combined with bio-photonic devices to obtain an integrated lab-on-a-chip system where, on a first stage, the SH surface would vehicle or transport the analytes of interest into a small area and, on a second stage, the bio-sensors would permit, in that area, the detection of the solute with the resolution of a single molecule. This novel diagnostic modality offers realistic possibilities for the early detection of cancers. Nevertheless, as it stands, the device still suffers from the severe disadvantage that the exact final position of the solute, upon evaporation, is unpredictable, and thus the localization and recognition of few molecules would be impractical. Conventional SH surfaces typically comprise micro pillars combined to form a regular hexagonal motif. Here, the periodicity of those pillars was broken introducing artificial gradients of wettability over the surface. In doing so, some regions are rendered more hydrophilic than others and, on account of this, a solute would preferentially target these hydrophilic regions upon evaporation. In this work, such non regular geometries were realized and used to condense diluted Rhodamine solutions in a small area. Randomly distributed silver nano aggregates, conveniently positioned upon the micropillars, permitted the identification of few molecules using enhanced Fourier transform infrared spectroscopy (FTIR) spectroscopy.

‘Metal structures as advanced materials in nanotechnology’ is a collection of fabrication and characterization techniques which involve metallic materials for the realization of advanced micro- and nanostructured devices.

Superhydrophobic surfaces were fabricated comprising cylindrical micro-pillars arranged in a regular hexagonal lattice. These structures are hierarchical in that the pillars incorporate, on the top, nanoporous silicon films. In sight of the superhydrophobicity of the system, the devices retain the ability of manipulating and concentrating diluted biological solutions, while, on account of the nanoporous films, they would efficiently filter the biological content of such solutions. The major advance, here, is the simultaneous use of the properties above, and thus important applications are envisioned where extremely small quantities of bio-markers are efficiently extracted from serum and analyzed employing conventional and well assessed spectroscopy techniques. Ultra low concentrated small Rhodamine molecules were here efficiently analyzed using FTIR spectroscopy techniques, thus demonstrating that these devices are effective.

The detection of a few molecules in a highly diluted solution is of paramount interest in fields including biomedicine, safety and eco-pollution in relation to rare and dangerous chemicals. Nanosensors based on plasmonics are promising devices in this regard, in that they combine the features of high sensitivity, label-free detection and miniaturization. However, plasmonic-based nanosensors, in common with general sensors with sensitive areas on the scale of nanometres, cannot be used directly to detect molecules dissolved in femto- or attomolar solutions. In other words, they are diffusion-limited and their detection times become impractical at such concentrations. In this Article, we demonstrate, by combining super-hydrophobic artificial surfaces and nanoplasmonic structures, that few molecules can be localized and detected even at attomolar (10−18 mol l−1) concentration. Moreover, the detection can be combined with fluorescence and Raman spectroscopy, such that the chemical signature of the molecules can be clearly determined.

International audience; We developed a new generation of superhydrophobic chips optimized for probing ultrasmall sample quantities by X-ray scattering and fluorescence techniques. The chips are based on thin Si3N4 membranes with a tailored pattern of SU-8 photoresist pillars. Indeed, aqueous solution droplets can be evaporated and concentrated at predefined positions using a non-periodic pillar pattern. We demonstrated quantitatively the deposition and aggregation of gold glyconanoparticles from the evaporation of a nanomolar droplet in a small spot by raster X-ray nanofluorescence. Further, raster nanocrystallography of biological objects such as rod-like tobacco mosaic virus nanoparticles reveals crystalline macro-domain formation composed of highly oriented nanorods

Recent advances in the single-molecule detection and manipulation provided unexpected solutions for the understanding of the physio-pathological behavior of individual biological macromolecules. Modern techniques of patterning at the micro- and nanometer scale combined with chemical treatments are being used to create surfaces that stretch the hydrophobic behavior to the limit. The ability to create surfaces with high static water contact angles (usually greater than 150°) is essential for a variety of applications, ranging from the development of biosensors to the implementation of sensitive and reliable single-molecule collection and sample preparation methods. Thus, superhydrophobic devices could be considered as nano-biotechnological single-molecule detection tools that can be applied to a wide range of high-resolution studies. To outline the paper, single-molecule detection topics and theoretical principles of superhydrophobicity are first introduced. A comprehensive overview is then given, describing how different types of devices with superhydrophobic surfaces are realized. Finally, the usefulness of the presented devices for a wide range of applications and the concluding comments are proposed.

Drops of exosome dispersions from healthy epithelial colon cell line and colorectal cancer cells were dried on a superhydrophobic PMMA substrate. The residues were studied by small- and wide-angle X-ray scattering using both a synchrotron radiation micrometric beam and a high-flux table-top X-ray source. Structural differences between healthy and cancerous cells were detected in the lamellar lattices of the exosome macro-aggregates.

Modification and functionalization of substrates can lead to the presence at materials surfaces of molecular nanostructures. Their 2D packing and chemical functionalities may impart to surfaces particular properties that can be very different from the pristine ones. Modification is a very general term concerning every kind of treatment leading to change the physical morphology or surface chemistry of a given material in order to obtain tailored properties with dependence on the material application field. Functionalization is mainly related to the introduction at a surface of chemical groups with a variable surface density which are requested to subsequently react with other species. When a process or a group of processes are applied to the surface of an inorganic or organicmaterial, affecting both topography/morphology and surface chemistry at the micro- and/or nanoscale level, the material surface results physically and chemically patterned. In the following the main modification, functionalization, and patterning surface techniques will be described according to process methods, substrate chemistry, and nanotechnological application fields

Exosomes are very small vesicles that are shed from a variety of cell types. They are present in body fluids comprising blood, urine and ascites, and they hold the potential to serve as indicators in the diagnosis, prognosis and surveillance of a variety of health conditions. In consideration of this, there is broad interest worldwide in deriving and understanding their biochemical composition. Here, small drops of solutions containing exosomes, isolated from either healthy (CCD841-CoN) or tumor (HCT116) colon cells, were manipulated using super-hydrophobic surfaces (SHSs) decorated with nano-geometry based photonics structures. SHSs are surfaces with superior properties, including a reduced friction coefficient, on account of which they can be used to control diluted solutions of biological or medical interest. The combination of this technology with optical resonance phenomena, open up terrific opportunities in the detection, analysis or characterization of biological compounds. Therefore, exosomes were conveniently concentrated and conveyed onto the Surface Enhanced Raman Spectroscopy (SERS) active areas of the substrate, whereby they were analyzed and characterized with extreme precision. Results indicate a difference in the concentration of some biological compounds, and namely: exosomes derived from healthy cells exhibit higher intensities of the peaks corresponding to lipids vibrations, while exosomes extracted from tumor cell lines exhibit a richer RNA content.

Graphical abstractDisplay Omitted Highlights? Si micro-structured superhydrophobic surfaces by plasma etch and optical lithography. ? High water contact angles up to 168?. ? Facile Electrowetting On Dielectric platform working at low DC voltages. ? Potential coupling of the setup with analytical in situ techniques (X-Rays, Raman). A simple and ready to use approach for combining silicon superhydrophobic surfaces with ElectroWetting On Dielectrics (EWOD) phenomenon is presented. The substrate is fabricated using a two-phases process, where a first optical lithography step is used to define the position of the micro-pillars and a second one exploits the characteristics of reactive ion etch Bosch technique. The fabricated substrate has been then coupled with a micro-manipulator tip to show the local changes mechanism of contact angle by applying very low DC voltages in the range from 5 to 30V. The device can be of interest for a wide variety of microfluidics applications related to the biomedical field.

Superhydrophobic poly(methyl methacrylate) surfaces with contact angles of ∼170° and high optical and X-ray transparencies have been fabricated through the use of optical lithography and plasma etching. The surfaces contain either a microscale pattern of micropillars or a random nanofibrillar pattern. Nanoscale asperities on top of the micropillars closely resemble Nelumbo nucifera lotus leaves. The evolution of the contact angle of water and lysozyme solution droplets during evaporation was studied on the micro- and nanopatterned surfaces, showing in particular contact-line pinning for the protein solution droplet on the nanopatterned surface. The microstructural evolution of lysozyme solution droplets was studied on both types of surfaces in situ under nearly contact-free conditions by synchrotron radiation microbeam wide-angle and small-angle X-ray scattering revealing the increasing protein concentration and the onset of precipitation. The solid residuals show hollow sphere morphologies. Rastermicrodiffraction of the detached residuals suggests about a 1/3 volume fraction of ≥17 nm lysozyme nanocrystalline domains and about a 2/3 short-range-order volume fraction. About 5-fold larger nanocrystalline domains were observed at the attachment points of the sphere to the substrates, which is attributed to particle growth in a shear flow. Such surfaces represent nearly contact-free sample supports for studies of inorganic and organic solution droplets, which find applications in biochips.

Micro and nano-patterned devices were fabricated which disclose the possibility of concentrating, localizing, detecting and thus analyzing with unprecedented accuracy few molecules (at the limit a single molecule) of biological and medical interest. Important applications are envisioned where proteins poorly concentrated in blood are detected thus (early) revealing the appearance of diseases. The major advance of the paper is that it combines micro/nano-fabrication and spectroscopical nano-optics based techniques to study and individualize the biomolecules at study. Super hydrophobic surface would concentrate the diluted solution into a small area whereas spectroscopical nano-optics techniques would gather the molecular/chemical information of the residual solute. Fluorescence and SERS techniques were used to analyze Rhodamine molecules initially highly diluted (10^-^1^8M).