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Here we report an investigation on conformational changes of Alpha-synuclein (α-syn) and one of its mutations (E46K), whose fibrillar assemblies in neural cells are the molecular hallmark of Parkinsons' disease. By varying the environmental conditions of the two peptides in terms of temperature (21-60°C), the presence of lipid systems (in homogeneous fluid phase or presenting raft-like domains) and nanostructured surfaces, we observed significant secondary structure alterations in both proteins. In order to probe such transitions, we exploited synchrotron µFTIR directly on solid ring-like residues formed immediately upon evaporation. We concluded that high temperatures and the presence of a lipid fluid phase induce a transition from α-helical materials to β-sheet ones (the characteristic conformation of α-syn fibrils) on pristine flat substrates. The presence of lipid rafts hindered this transition. On the other hand, the inclination to form β-sheet phases in the presence of high temperatures or lipid systems was sensitively lowered by drying the proteins on top of nanostructured highly hydrophilic supports which allow a more homogeneous distribution of the analyte throughout the residue. These results and the presented protocol can indeed pave the basis for the structural characterization of this controversially debated protein and of other neurodegenerative peptides. Display Omitted µFTIR investigation of two α-synuclein proteins involved in Parkinson's diseaseHigh temperature and fluid phase of lipid systems favor β-sheet configuration.Hydrophilic nanostructured surfaces hinder the formation of β-sheet material.

Here we are presenting a comparative analysis of conformational changes of two amyloid β peptides, Aβ(25-35) and Aβ(1-42), in the presence and absence of a phospholipid system, namely, POPC/POPS (1-palmitoyl-2-oleoylphospatidylcholine/palmitoyl-2-oleoylphospatidylserine), through Raman spectroscopy, synchrotron radiation micro Fourier-transform infrared spectroscopy, and micro X-ray diffraction. Ringlike samples were obtained from the evaporation of pure and mixed solutions of the proteins together with the POPC/POPS system on highly hydrophilic substrates. The results confirm the presence of a α-helical to β-sheet transition from the internal rim of the ringlike samples to the external one in the pure Aβ(25-35) residual, probably due to the convective flow inside the droplets sitting on highly hydrophilic substrates enhancing the local concentration of the peptide at the external edge of the dried drop. In contrast, the presence of POPC/POPS lipids in the peptide does not result in α-helical structures and introduces the presence of antiparallel β-sheet material together with parallel β-sheet structures and possible β-turns. As a control, Aβ(1-42) peptide was also tested and shows β-sheet conformations independently from the presence of the lipid system. The μXRD analysis further confirmed these conclusions, showing how the absence of the phospholipid system induces in the Aβ(25-35) a probable composite α/β material while its coexistence with the peptide leads to a not oriented β-sheet conformation. These results open interesting scenarios on the study of conformational changes of Aβ peptides and could help, with further investigations, to better clarify the role of enzymes and alternative lipid systems involved in the amyloidosis process of Aβ fragments.

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.

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.

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.

The liquid/air interface of calcium bicarbonate solution drops was probed by synchrotron radiation microbeam scattering. The drops were deposited on a nanopatterned superhydrophobic poly(methyl methacrylate) surface and raster-scanned during evaporation by small-angle and wide-angle X-ray scattering. The appearance of about 200-nm-size calcite crystallites at the interface could be spatially resolved at the onset of crystallization. Diffuse scattering from the interface is attributed to a dense nanoscale amorphous calcium carbonate phase. Calcite was found to be the major phase in the solid residue with vaterite as minor phase.

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).

The evaporation of a lysozyme solution drop containing Ca2+ ions on a superhydrophobic poly(methyl methacrylate) surface was probed by X-ray microbeam raster-diffraction. We observe the rapid deposition of fibrillar lysozyme with amyloidic cross-β structure at the interface of the drop with the substrate under conditions of weak acidity, close to physiological.