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The influence of the physical, rheological, and process parameters on the cellulose nanocrystal (CNC) drops before and after external gelation in a CaCl2 solution was investigated. The dominant role of the CNC’s colloidal suspension properties, such as the viscous force, inertial, and surface tension forces in the fluid dynamics was quantitatively evaluated in the formation of drops and jellified beads. The similarity and difference between the behavior of carbohydrate polymers and rod-like crystallites such as CNC were enlightened. Pump-driven and centrifugally-driven external gelation approaches were followed to obtain CNC hydrogel beads with tunable size and regular shape. A superior morphological control—that is, a more regular shape and smaller dimension of the beads—were obtained by centrifugal force-driven gelation. These results suggest that even by using a simple set-up and a low-speed centrifuge device, the extrusion of a colloidal solution through a small nozzle under a centrifugal field is an efficient approach for the production of CNC hydrogel beads with good reproducibility, control over the bead morphology and size monodispersion.

Porous silicon micro-particles (micro-pSi) with size in the range of 1–10 μm are obtained by etching of silicon wafers followed by sonication. The derivatization of the micro-pSi surface by wet chemistry (silylation and coupling with a diamine) yields an interface, which exposes negative (carboxylic) or positive (amine) groups at pH 7.4. The surface modification, beyond the introduction of groups for the drug loading by covalent or electrostatic interactions, stabilizes the intense orange luminescence characteristic of the silicon nano-crystallites. Derivatization by amines introduces also a second emission in the blue region, which follows a different excitation pathway and can be attributed to the interface defects. The micro-pSi are efficiently internalized by human dendritic cells and do not show any toxic effect even at a concentration of 1 mg mL−1. The intrinsic luminescence of the differently functionalized micro-pSi is preserved inside the cells and permits the selective and efficient tracking of the microparticles without using molecular tags and thus leaving the organic coating available for the interaction with the drug. The results obtained suggest that the functionalized micro-pSi are an efficient platform for simultaneous imaging and delivery of therapeutic agents to the disease site.

Porous silicon (pSi) microparticles obtained by porosification of crystalline silicon wafers have unique optical properties that, together with biodegradability, biocompatibility and absence of immunogenicity, are fundamental characteristics to candidate them as tracers in optical imaging techniques and as drug carriers. In this work, we focus on the possibility to track down the pSi microparticles also by MRI (magnetic resonance imaging), thus realizing a comprehensive tool for theranostic applications, i.e., the combination of therapy and diagnostics. We have developed and tested an easy, quick and low-cost protocol to infiltrate the COOH-functionalized pSi microparticles pores (tens of nanometers about) with magnetic nanospheres (SPIONs&mdash, Super Paramagnetic Iron Oxide Nanoparticles, about 5&ndash, 7 nm) and allow an electrostatic interaction. The structural properties and the elemental composition were investigated by electron microscopy techniques coupled to elemental analysis to demonstrate the effective attachment of the SPIONs along the pores&rsquo, surface of the pSi microparticles. The magnetic properties were investigated under an external magnetic field to determine the relaxivity properties of the material and resulting in an alteration of the relaxivity of water due to the SPIONs presence, clearly demonstrating the effectiveness of the easy functionalization protocol proposed.

The European food sector generates about 250 million tons/yr of by-products and waste, of which around 10% from fruit and vegetable processing, with a heavy environmental burden. The agri-food residues (AFR) contain a significant fraction of cellulose and bioactive compounds (mainly antioxidants), which, if recovered, are high added-value material components. The reduction of cellulose down to nano-sized crystalline structures (nanocellulose, NC) provides versatile building blocks, which self-assemble into new materials with superior performances. Despite wood-derived NC is generally considered a green material, its production process is environmentally unfriendly and its large scale utilization would contribute to deforestation. Therefore, more sustainable sources, such as AFR, are desired. The PANACEA project, within the frame of PRIN 2017 call supported by the Italian Ministry of University and Research, proposes an approach based on the recovery of cellulose and bioactive compounds from AFR, with high yield, at various degrees of hierarchical organization, by cascading different physical and chemical processes of increasing complexity. More specifically, physical processes and microbial digestion are exploited to obtain micro-sized cellulose structures while preserving their bioactivity. Chemical and enzymatic processes are used to isolate, purify and functionalize NC at different levels of hierarchical organization, and to design advanced functional materials such as food ingredients, edible coatings, functional colloids, biocides and flame retardants. The sustainable integrated valorization of AFR is addressed through (i) ad hoc pre-treatment of different AFR (Tomato peels, Wheat straw, Coffee residues, Rice bran, Grape marcs, Orange peels), available all year round and with different composition, (ii) application of proprietary high-pressure homogenization (HPH) techniques to micronize the residues in water and completely disintegrate the vegetable cells, with integrated physical fractionation to recover insoluble fibers, (iii) combination with chemical fractionation or high P/T autohydrolysis, or enzymatic treatments. The fabrication of NC structures with tunable size, crystallinity, and surface properties is important to bridge the gap between the production of cellulose and cellulose hybrids and the final applications and is pursued through (i) the size-reduction of NC, or enzymatic lysis, (ii) functionalization via amination or phosphorylation, or (iii) via physical immobilization or covalent bonding with limonene. The characterization of NC in terms of (i) yields, purity, physicochemical, structural, and functional properties, using a multi-technique approach, is associated to the evaluation of (ii) bio-accessibility (through the simulated digestive process) of bioactive compounds and fibers, (iii) film-forming capacity, (iv) gas-barrier properties, (v) rheological behavior, as well as (vi) energy, water, and reagents consumptions. Finally, the PANACEA project also addresses the exploitation of NC-based colloids for novel materials and applications, such as (i) edible coatings, antimicrobial varnishes, and oil structuring materials, (ii) packaging films with gas-barrier properties, and (iii) fabrics and foams with flame-retardant properties (iv) advanced nanocomposite films, (v) Pickering emulsions, and (vi) antimicrobial films and coatings. The main contribution of the PANACEA project to the advancement of the knowledge is expected in (i) the deployment of greener and sustainable, cascading processes exploiting raw AFR functionalities, (ii) the tailored valorization of different AFR by developing physical, chemical, and biological procedures for sculpting the nanostructures, and (iii) the development of sustainable, high performing advanced materials such as edible coatings and gas barrier packages for the food industry, foams, and textiles with flame retardant properties, biocides for organic pest control in agriculture.

In recent decades, biocompatible and light emitting porous silicon (pSi) showed the possibility for use in biomedical applications. Unfortunately, until now the biological applications of light-emitting pSi have been severely limited due the immediate surface oxidation of this nanostructured material, followed by loss of optical properties and degradation in aqueous solutions. In view of drug delivery, the red-ox activity of pSi damages the carried drug. Herein, we have investigated the stabilization of pSi microparticles in aqueous solution by covalent attachment of polymers to a short-chain organic coating grafted onto inorganic silicon. The morphological, chemical and optical stability obtained using polyethylene glycol confirm this biocompatible polymer as a superior shell matrix nanostructure for use in biological environments.

Sol-gel transition of carboxylated cellulose nanocrystals has been investigated using rheology, SAXS, NMR and optical spectroscopies to unveil the distinctive roles of ultrasound treatments and addition of various cations. Besides cellulose fiber fragmentation, sonication treatment induces fast gelling of the solution. The gelation is independent of the addition of cations, while the final rheological properties are highly influenced by the type, concentration and sequence of the operations since the cations must be added prior to sonication to produce stiff gels. The gel elastic modulus was found to increase proportionally to the ionic charge rather than the cationic size. In cases where ions were added after sonication, SAXS analysis of the Na+ hydrogel and Ca2+ hydrogel indicated the presence of structurally ordered domains in which water is confined, and 1H-NMR investigation showed the dynamics of water exchange within the hydrogels. Conversely, separated phases containing essentially free water were characteristic of the hydrogels obtained by sonication after Ca2+ addition, confirming that this ion induces irreversible fiber aggregation. The rheological properties of the hydrogels depend on the duration of the ultrasound treatments, enabling the design of programmed materials with tailored energy dissipation response.

The structures of copper amine oxidases from various sources show good similarity, suggesting similar catalytic mechanisms for all members of this enzyme family. However, the optimal substrates for each member differ, depending on the source of the enzyme and its location. The structural factors underlying substrate selectivity still remain to be discovered. With this in view, we examined the kinetic behaviour of pea seedling amine oxidase with cadaverine and hexylamine, the first bearing two, and the second only one, positively charged amino group. The dependence of Km and catalytic constant (kc) values on pH, ionic strength and temperature indicates that binding of the monoamine is driven by hydrophobic interactions. Instead, binding of the diamine is strongly facilitated by electrostatic factors, controlled by polar side-chains and two titratable residues present in the active site. The position of the docked substrate is also essential for the participation of titratable amino acid residues in the following catalytic steps. A new mechanistic model explaining the substrate-dependent kinetics of the reaction is discussed.

Porous silicon solid supports with pore diameter 0.5–1 μm, infiltrated with Ag nanostructures for surface enhanced Raman scattering (SERS) were prepared according to two procedures: spontaneous Ag+ reduction on the surface of freshly etched porous silicon immersed in Ag+ aqueous solutions, or anchoring colloidal Ag nanoparticles on the surface previously functionalized by aminosilane. Using Rhodamine 6G (RH6G) as analyte the detection limits were of the order of 20 μM and 20 nM with porous silicon metalized by the first and second procedure, respectively. This large increase of sensitivity notwithstanding a reduced surface density of Rhodamine 6G obtained on porous silicon metalized by the second procedure is discussed in terms of better hot spot efficiency and reduced aspecific binding out of the hot regions obtained depositing the colloids on the aminosilane functionalized surface.

Silver nanocrystallites are obtained through immersion of porous silicon samples in AgNO 3 solutions and a successive thermal annealing. The efficiency of nanostructures as surface enhanced Raman scattering (SERS) substrates is checked on cyanine-based dyes and horseradish peroxidase, evidencing detectable concentrations as low as 10 −7 to 10 −8 M. The substrate efficiency is strictly related to the Ag particle morphology, which could yield to either local surface plasmons (LSP) coupled to individual particles or to inter-particle short-range interaction.

TPP+ (tetraphenylphosphonium ion) and its analogues were found to act as powerful competitive inhibitors of BSAO (bovine serum amine oxidase). The binding of this new class of inhibitors to BSAO was characterized by kinetic measurements. TPP+ can bind to the BSAO active site by hydrophobic and by coulombian interactions. The binding probably occurs in the region of the ‘cation-binding site’[Di Paolo, Scarpa, Corazza, Stevanato and Rigo (2002) Biophys. J. 83, 2231–2239]. Under physiological conditions, the association constant of TPP+ for this site is higher than 106 M−1, the change of enthalpy being the main free-energy term controlling binding. Analysis of the relationships between substrate structure and extent of inhibition by TPP+ reveals some new molecular features of the BSAO active site.

A steady-state kinetic study of bovine serum amine oxidase activity was performed, over a wide range of pH values (5.4-10.2) and ionic strength (10-200 mM), using various (physiological and analogue) substrates as specific probes of the active-site binding region. Relatively small changes in k (cat) values (approx. one order of magnitude) accompanied by marked changes in K(m) and k(cat)/K(m) values (approx. six orders of magnitude) were observed. This behaviour was correlated with the presence of positively charged groups or apolar chains in the substrates. In particular, it was found that the docking of the physiological polyamines, i.e. spermidine and spermine, appears to be modulated by three amino acid residues of the active site, which we have named L(-)H(+), G(-)H(+) and IH(+), characterized by p K (a) values of 6.2+/-0.2 [Di Paolo, Scarpa, Corazza, Stevanato and Rigo (2002) Biophys. J. 83, 2231-2239], 8.2+/-0.3 and 7.8+/-0.4 respectively. The electrostatic interaction between the protonated substrates and the enzyme containing the residues L(-)H(+), G(-)H(+) and IH(+) in the deprotonated form, the on/off role of the IH(+) residue and the role of hydrophobic interactions with substrates characterized by apolar chains are discussed.

Nanoporous materials are ideal candidates to fabricate sensors with high sensitivities. Using nanoporous materials both the strength and the type of interactions between analytes and host matrix can be tuned at will, and their large surface area permit to achieve ultralow detection limit and to keep high loading capacity and, thus, large dynamic range. Despite these intriguing possibilities their use in sensing technology is still limited because several fundamental aspects are not fully understood. By comparing supported and free standing Porous Silicon membranes, we demonstrate that sensor’s sensitivity is clearly overestimated if the sample is not properly washed. Moreover we underline the fact that, despite the reduced Q-factor of the resonant cavities, the strong interaction between the analyte and the optical modes in porous materials produces a large net sensitivity of the sensor. Finally, we show that the non-specific signal may have a linear response with the analyte concentration and it thus extremely difficult to separate it from the specific one. The work presented here is of importance in the design of nanoporous materials for sensing application. In fact it demonstrates that the final sensitivity of the device is not defined by its optical quality alone and that several other effects have to be considered to fabricate a reliable sensor.

In this paper, we report on the presence of cation binding areas on bovine serum amine oxidase, where metal ions of the groups IA and IIA, such as Na(+), K(+), Cs(+), Mg(2+), and Ca(2+), bind with various affinities. We found a cation-binding area that influences the enzyme activity if occupied, so that the catalytic reaction may be altered by some physiologically relevant cations, such as Ca(2+) and K(+). This binding area appears to be localized inside the enzyme active site, because some of these cations act as competitive inhibitors when highly charged amines, such as spermine and spermidine, are used as substrates. In particular, dissociation constant values (K(d)) of 23 and 27 mM were measured for Cs(+) and Ca(2+), respectively, using, as substrate, spermine, a polyamine of plasma. An additional cation-binding area, where metal ions such as Cs(+) (K(d) congruent with 0.1 mM) and Na(+) (K(d) congruent with 54 mM) bind without affecting the enzyme activity, was found by NMR.

A patient with a severe amnesic syndrome following a glioma of the splenium of the corpus callosum is reported. The long-term memory deficit involved anterograde as well as retrograde events dating back to 40 years and causing topographical disorientation. Short-term memory test performance was in the normal range, with the exception of tactile memory which was severely impaired. The patient also showed disconnection symptoms, due to severing of occipito-parietal and parieto-temporal connections, while parieto-parietal connections were undamaged.

The increased control over nanoscale materials structuring is reshaping a number of applications and technologies at a fast pace. Semiconducting materials are playing an increasingly important role and Silicon is so far the most investigated one thanks to its broad availability and its fundamental role in microelectronics. Despite all its favorable properties, a major drawback of silicon is the lack of an efficient light emission mechanism that prevents its use in active photonics. Quantum size effects has been demonstrated a viable strategy to overcome this limit and to achieve light emission from silicon nanostructures. Si nanostructures are well controlled and can be tuned to achieve the desired properties, often exploiting the mature CMOS technological platform. Simultaneously several synthetic strategies have been developed in order to fabricate silicon quantum dots from chemical synthesis. Silicon chemistry is (rather) well known and permits to synthesize nanoparticles and to functionalize their surface using a broad number of reactions. Such flexibility allows for the grafting of both organic and inorganic species onto the Si QDs surface, to add functionalities and to protect the nanoparticle from their natural oxidations that, in turn, quenches the photoluminescence properties. Thus, despite the initial research was focused on the possibility to fabricate inorganic light sources that might rival with III-V or II-VI semiconductors. More recently a large research effort has been devoted to the use of Silicon nanostructures in biological-related applications. In such research topics Silicon has several properties take renders it an ideal candidate: it is considered a biocompatible material; it is readily dissolved in biological environment without releasing toxic byproducts; its red and tunable luminescence make it an interesting fluorescent tag able to exploit the tissue transparency window. During the presentation I will make a brief review of the fabrications methods proposed so far and I will discuss in details some of the recent research activities performed in our group in ICT- as well as in bio-related topics, all sharing the common denominator of Si QDs as active optical element. About the ICT-related topics I will discuss two applications of Si QDS. The first is about the use of Si QDs in quantum technologies. Data encryption and cryptography are core technologies hidden in our daily life and their importance is constantly growing. We have developed a robust physical random number generator that exploits Si QDs as a random bit source. The preliminary results are promising and the random sequences pass the standard NIST test suite. Furthermore the system is fully compatible with CMOS technology and can be scaled up to achieve large bit-rate generation. The second case in point is about the large optical nonlinearities possessed by Si QDs and their exploitation to develop integrated photonic devices. As a bio-related application I will discuss how nanostructured silicon microparticles can be used as fluorescent tags and are successfully up-taken by human dendritic cells without stimulating apoptosis up to a very large concentration. Porous silicon micro-particles (micro-pSi) with size in the range of 1-10 um are functionalized obtaining an interface that exposes either positive or negative charges at neutral pH. Their porous structure enables their use as drug carriers. Moreover, the surface modification stabilizes their intense photoluminescence and suggests their use for simultaneous imaging and drug delivery. The few cases discussed underline the great potentialities of Silicon nanostructures in different technologies and suggest its possible implementation as a functional interface between organic and inorganic systems.

Clinical Audit is a term which has acquired different meanings over time in relation to health care quality. Ten years ago the National Institute for Clinical Excellence (NICE, 2002) published the first manual of Clinical Audit, with the classical definition “Clinical audit is a quality improvement process that seeks to improve patient care and outcomes through systematic review of care against explicit criteria and the implementation of change”.

The ionic-strength-dependence of steady-state kinetic parameters (kc and Km') for non-biogenic (benzylamine, butylamine) and biogenic (spermine, spermidine) amines has been measured in the bovine serum amine oxidase reaction. The catalytic rate constant (kc) values are similar (0.9-2.5 s-1) for all the substrates studied and are almost constant over the experimental ionic strength range (24-155 mM). In contrast, Km' values are in the range 6-2300 microM and undergo a 4-12-fold increase with increasing ionic strength, parallelled by a decrease in catalytic efficiency. From an analysis of the kc and Km' values and their dependence on ionic strength, we conclude that more than one negative site is involved in the binding of these amines and that the relative dielectric constant of the binding site is lower than that of aqueous solutions.

Silicon nanocrystals were made hydrophilic by 10-undecenoic acid grafting and were then coated with sodium deoxycholate, a detergent-like compound belonging to the bile acid class which is crucial for absorption of lipids in the small intestine. The resulting silicon nanocrystals have an average diameter of 3-5 nm, can be dispersed in aqueous solutions and show stable photoluminescence. Coating with non-biological surfactants, which are dangerous for cell safety, was investigated for comparison. Results indicate that deoxycholate is a stabilizer of luminescent silicon nanocrystals. Deoxycholate coated nanocrystals appear suitable for applications as multifunctional probes in biomedicine.

N.m.r. and e.p.r. were used to measure the oxidation state of copper in Cu, Zn superoxide dismutase treated with reducing agents such as NaBH4, K4Fe(CN)6, Na2S2O4 and H2O2. The activity and the electrophoretic pattern of the treated enzyme were also studied. On the basis of the reducing ability and of the absence of inactivating effects, NaBH4 was the most suitable reducer of those tested. Some characteristics of the reduction of superoxide dismutase by NaBH4 were further investigated. The results obtained indicate that NaBH4 can be used to prepare, in a few minutes, solutions of completely reduced enzyme without any apparent change of the activity and of the structure.

The amount of Mn superoxide dismutase (MnSOD) and the activity of Cu,Zn-superoxide dismutase (CuZnSOD) have been studied in human fibroblasts of five subjects with trisomy 21 and five subjects with normal karyotype, using nuclear magnetic relaxation and polarographic methods. In the trisomic fibroblasts we have found a mean molar amount of MnSOD 25.4% lower than in the control, and an amount of CuZnSOD 54.7% higher. A positive significant correlation between the activities of both enzymes has been observed indicating that the two enzymes dismute the O2- cooperatively. However, the increase of MnSOD per unit of CuZnSOD appears significantly lower in the trisomic fibroblasts, an effect that is not due to a diminished inducibility of MnSOD. These findings suggest that the MnSOD and CuZnSOD genes interact to preserve the normal level of total SOD activity.

The oxidation of ascorbic acid by molecular oxygen was investigated by optical and magnetic resonance methods in the presence and in the absence of copper and iron complexes. The results show that one superoxide ion is generated for each oxidized molecule of ascorbate. The effect of addition of Cu,Zn superoxide dismutase, which at concentrations higher than 10(-7) M halves the oxidation rate, is discussed and a reaction mechanism is proposed. An upper limit for the kinetic rate constant of the reaction between the ascorbate radical and the superoxide ion is reported.

The rate of oxidation of reduced bovine Cu,Zn superoxide dismutase [ECu(I)] by molecular O2 was studied by magnetic-resonance techniques and was found to be low under physiological conditions. The analysis of the kinetic data and of the experiments carried out in the presence of tetranitromethane confirms that O2.- is a product of the oxidation process. At [ECu(I)]/([ECu(I)] + [ECu(II)]) greater than 0.5 the O2.- produced reacts mainly with ECu(I), increasing the oxidation rate of the enzyme, whereas at [ECu(I)]/([ECu(I)] + [ECu(II)]) less than 0.5 it reacts mainly with ECu(II), decreasing the oxidation rate, the kinetics, at constant O2 concentration, being an apparent second-order process. The oxidation rate increased linearly with both O2 and OH- concentration, indicating that only a deprotonated form of the ECu(I) reacts with O2.-.

We review here our work on the photonic and sensor applications of nanostructured silicon. As we change the dimensionality of silicon very fascinating and new optical properties of the material appear. Light sources, modulators, waveguides, logical gates are a few examples of the various photonic devices which have been developed based on silicon nanocrystals. Needless to say, all these devices rely on quantum confinement and on the interplay between bulk and surface properties. Size effects and surface reconstructions are two critical issues which one has to master to employ silicon nanocrystals. Finally the use of silicon nanocrystals to develop innovative sensing mechanisms to reveal biomolecules or poisoning gasses will be discussed.

The generation of superoxide ion in human red blood cell lysates was investigated by an experimental method employing Cu,Zn superoxide dismutase as O2- scavenger and EPR to probe the oxidation state of the enzyme. The average value of the O2- flux in the erythrocytes of 8 normal individuals was (2.02 +/- 0.97) X 10(-8) M S-1. A progressive saturation of the rate of O2- production was found increasing PO2, KM = 1.04 X 10(-4) M, while the autoxidation of oxyhemoglobin did not contribute significantly to the measured O2- production.