Hyperspectral Chemical Imaging of Single Bacterial Cell Structure by Raman Spectroscopy and Machine Learning

6openopenBarzan, Giulia; Sacco, Alessio; Mandrile, Luisa; Giovannozzi, Andrea Mario; Portesi, Chiara; Rossi, Andrea MarioBarzan, Giulia; Sacco, Alessio; Mandrile, Luisa; Giovannozzi, Andrea Mario; Portesi, Chiara; Rossi, Andrea Mari

Advanced characterization of albumin adsorption on a chemically treated surface for osseointegration: An innovative experimental approach

Surface chemistry, charge, wettability, and roughness affect the adsorbed protein layer, influencing biocompatibility and functionality of implants. Material engineering seeks innovative, sensitive, and reliable characterization techniques for study the adsorbed proteins. These techniques must be suitable to be directly used on the surfaces of clinical interest. In this paper, the characterization of surfaces with topography and chemistry developed for osseointegration is performed by innovative surface analysis techniques to investigate the properties of adsorbed bovine serum albumin. Ti6Al4V alloy chemically treated with an oxidative process to obtain peculiar surface features (roughness and surface hydroxylation) was tested and compared with mirror-polished titanium. Albumin forms a continuous layer on both Ti surfaces when adsorbed from near physiological concentrations, as proved by Kelvin force probe microscopy. It was observed that the hydroxylation degree plays a pivotal role in determining the conformation of proteins after adsorption, where it strongly drives protein unfolding, as confirmed by Surface Enhanced Raman scattering, and in influencing the mechanism and chemical stability of protein-surface interactions, which was highlighted by zeta potential titration curves.(c) 2022 The Authors. Published by Elsevier Ltd. This is an open access article under the CC BY-NC-ND license (http://creativecommons.org/licenses/by-nc-nd/4.0/)

Development of innovative antioxidant food packaging systems based on natural extracts from food industry waste and Moringa oleifera leaves

Active packaging that prolongs food shelf life, maintaining its quality and safety, is an increasing industrial demand, especially if integrated in a circular economy model. In this study, the fabrication and characterization of sustainable cellulose-based active packaging using food-industry waste and natural extracts as antioxidant agents was assessed. Grape marc, olive pomace and moringa leaf extracts obtained by supercritical fluid, antisolvent and maceration extraction in different solvents were compared for their antioxidant power and phenolic content. Grape and moringa macerates in acetone and methanol, as the most efficient and cost-effective extracts, were incorporated in the packaging as coatings or in-between layers. Both systems showed significant free-radical protection in vitro (antioxidant power 50%) and more than 50% prevention of ground beef lipid peroxidation over 16 days by indirect TBARS and direct in situ Raman microspectroscopy measurements. Therefore, these systems are promising for industrial applications and more sustainable farm-to-fork food production systems

Effect of silver ion incorporation into a bioactive glass surface on the adsorption of albumin

Introduction Nowadays, bacterial infection is one of the major causes for orthopedic implants failure. While it is well known how to manufacture materials that are able to stimulate osseointegration and to firmly bond with the bone, the fight against pathogenic microorganisms is carried on mainly by antibiotics, with consequent problems of poorly localized actions and antibiotic-resistance. Therefore, novel antibacterial strategies have been deeply researched. Among those, incorporation of silver in biomaterials, such as bioactive glasses, is acknowledged as an effective way to reduce bacteria proliferation. Osseointegration of biomaterials is dependent on the surface properties of the implants and on the interactions with the biological environment. In particular, a protein layer is formed on the surface within minutes after the contact between the surface and the biological fluids and it will dictate how the cells will respond to the implanted foreign body. As consequence, it is important to understand how antibacterial modifications of bioactive materials affect their interactions with proteins. In this work, the adsorption of albumin was investigated onto a silica-based bioactive glass where silver ions were incorporated through ionic exchange (Ag-SBA2) in order to understand eventual differences with the untreated surface (SBA2)[1]. Experimental Methods SBA2 bioactive glass (mol %: 48% SiO2, 18% Na2O, 30% CaO, 3% P2O5, 0.43% B2O3, 0.57% Al2O3) was prepared via precursors melting and casting, cut into disks and grinded (up to 1000 grit). Ag-SBA2 was prepared by soaking glass slices for 1h in 0.03M AgNO3 solution. Protein adsorption was obtained by soaking the samples for 2h at 37°C in albumin solution in PBS, in near physiological conditions (20 mg/ml, pH 7.4). The glass substrates were characterized in terms of topography and roughness (SEM, AFM and confocal microscopy), chemical composition (EDS and XPS), surface charge and potential (solid surface zeta potential, Kelvin Probe Force Microscopy (KPFM)) and surface energy (contact angle, Owens-Wendt method). The adsorbed proteins were quantified by using different methods (BCA assay, fluorescent proteins and XPS) and the BSA layer was also imaged (fluorescent microscopy and KPFM). Substrate-protein interactions and albumin conformation were investigated, too (solid surface zeta potential and ATR-FTIR). Results and Discussion After silver incorporation, confirmed by chemical analysis, the surface properties of Ag-SBA2 were mostly similar to the undoped glass. In particular, topography and roughness were unchanged during soaking in the silver solution, as expected. Wettability and surface free energy, both the dispersive and polar components, were also similar between the two substrates. Instead, zeta potential titration curve showed that the incorporation of Ag3+ ions increased the surface potential, in particular around physiological pH (7.4). Quantification of adsorbed BSA showed that both surfaces adsorb a similar amount of albumin, with a little higher amount on Ag-SBA2. This can be possibly related to a couple of different factors: the high affinity of silver for proteins and the presence of a more positive charge on the surface, which is able to attract the negatively charged albumin. On both surfaces, albumin forms a complete and homogeneous layer, as detected by imaging techniques. Adsorption of proteins was confirmed also by zeta potential measurement on the surfaces, with a shift of the IEP of both glassestowards the IEP of albumin. Thanks to ATR-FTIR measurement, it was found that albumin retains more its native conformation on the undoped glass with respect to the silver containing glass, where a more disordered structure was found. This fact can be ascribed to a greater interaction between the proteins and the doped surface, due to the presence of metal ions and more positive charges. Conclusion In conclusion, even though the incorporation of silver ions in a bioactive glass surface does not affect surface properties that are usually addressed as pivotal in protein adsorption, such as roughness and surface energy, the presence of a more positive charge on the surface of the glass and affinity of proteins towards metallic ions seems to be enough to increase adsorption of albumin and strength of the protein-biomaterial interaction. The increased interaction with proteins may be beneficial for the cells response to antibacterial silver containing materials

Fabrication of flexible silicon nanowires by self-assembled metal assisted chemical etching for surface enhanced Raman spectroscopy

A homogenous array of flexible gold coated silicon nanowires was fabricated by the combination of nano spheres lithography and metal assisted chemical etching to obtain highly effective Surface Enhanced Raman Spectroscopy (SERS) substrates. 3D nanostructures with different aspect ratios and well-defined geometries were produced by adjusting the fabrication parameters in order to select the best configuration for SERS analysis. The optimum flexible nanowires with an aspect ratio of 1 : 10 can self-close driven by the microcapillary force under exposure to liquid and trap the molecules at their metallic coated ``fingertips'', thus generating hot spots with ultrahigh field enhancement. The performance of these SERS substrates was evaluated using melamine as the analyte probe with various concentrations from the millimolar to the picomolar range. Flexible gold coated SiNWs demonstrated high uniformity of the Raman signal over large area with a variability of only 10% and high sensitivity with a limit of detection of 3.20 x 10(-7) mg L-1 (picomolar) which promotes its application in several fields such food safety, diagnostic and pharmaceutical. Such an approach represents a low-cost alternative to the traditional nanofabrication processes to obtain well ordered silicon nanostructures, offering multiple degrees of freedom in the design of different geometries such as inter-wire distance, density of the wires on the surface as well as their length, thus showing a great potential for the fabrication of SERS substrates

Influence of the long-range ordering of gold-coated Si nanowires on SERS

Controlling the location and the distribution of hot spots is a crucial aspect in the fabrication of surface-enhanced Raman spectroscopy (SERS) substrates for bio-analytical applications. The choice of a suitable method to tailor the dimensions and the position of plasmonic nanostructures becomes fundamental to provide SERS substrates with significant signal enhancement, homogeneity and reproducibility. In the present work, we studied the influence of the long-range ordering of different flexible gold-coated Si nanowires arrays on the SERS activity. The substrates are made by nanosphere lithography and metal-assisted chemical etching. The degree of order is quantitatively evaluated through the correlation length (ξ) as a function of the nanosphere spin-coating speed. Our findings showed a linear increase of the SERS signal for increasing values of ξ, coherently with a more ordered and dense distribution of hot spots on the surface. The substrate with the largest ξ of 1100 nm showed an enhancement factor of 2.6 · 103 and remarkable homogeneity over square-millimetres area. The variability of the signal across the substrate was also investigated by means of a 2D chemical imaging approach and a standard methodology for its practical calculation is proposed for a coherent comparison among the data reported in literature

Indicators of sustainable forest management: application and assessment

Report of the international project Life ManForCBD

Nanometrology

In the present paper an overview of metrology at the nanometric scale is given. After an introductory part on terminology and definitions concerning generic objects at the nanoscale, nanoparticles are taken as example to describe the overall features of these systems and typical characterization issues occurring from a metrological point of view. The case of nanoparticles is then further examined in depth with reference to gold nanoparticles, whose peculiar properties deriving from nano-scaled effects, like surface plasmon resonance, are discussed in detail, along with the state of the art on the main fabrication methods and applications in different sectors. Finally, a case study on the detection, by gold nanoparticles, of a contaminant used for food adulteration, namely melamine, is presented and analyzed