Pierluigi Reschiglian - Academia.edu (original) (raw)

Papers by Pierluigi Reschiglian

Multi-analyte Detection 13.1.8 Protein Switches 13.2 Genetic and Chemical Engineering of Lucifera... more Multi-analyte Detection 13.1.8 Protein Switches 13.2 Genetic and Chemical Engineering of Luciferases for Bioanalytical Applications 13.2.1

Journal of Chromatography A

Analytica Chimica Acta

Protein misfolding and aggregation are the common mechanisms in a variety of aggregation-dependen... more Protein misfolding and aggregation are the common mechanisms in a variety of aggregation-dependent diseases. The compromised proteins often assemble into toxic, accumulating amyloid-like structures of various lengths and their toxicity can also be transferred both in vivo and in vitro a prion-like behavior. The characterization of protein interactions, degradation and conformational dynamics in biological systems still represents an analytical challenge in the prion-like protein comprehension. In our work, we investigated the nature of a transferable cytotoxic agent, presumably a misfolded protein, through the coupling of a multi-detector, non-destructive separation platform based on hollow-fiber flow field-flow fractionation with imaging and downstream in vitro tests. After purification with ion exchange chromatography, the transferable cytotoxic agentwas analyzed with Atomic Force Microscopy and statistical analysis, showing that the concentration of protein dimers and low n-oligomer forms was higher in the cytotoxic sample than in the control preparation. To assess whether the presence of these species was the actual toxic and/or self-propagating factor, we employed HF5 fractionation, with UV and Multi-Angle Light Scattering detection, to define proteins molar mass distribution and abundance, and fractionate the sample into size-homogeneous fractions. These fractions were then tested individually in vitro to investigate the direct correlation with cytotoxicity. Only the later-eluted fraction, which contains high-molar mass aggregates, proved to be toxic onto cell cultures. Moreover, it was observed that the selective transfer of toxicity also occurs for one lower-mass fraction, suggesting that two different mechanisms, acute and later induced toxicity, are in place. These results strongly encourage the efficacy of this platform to enable the identification of protein toxicants.

Analytical and bioanalytical chemistry, 2018

Asymmetric flow field-flow fractionation (AF4) coupled with UV-Vis spectroscopy, multi-angle ligh... more Asymmetric flow field-flow fractionation (AF4) coupled with UV-Vis spectroscopy, multi-angle light scattering (MALS) and refractive index (RI) detection has been applied for the characterization of MIL-100(Fe) nanoMOFs (metal-organic frameworks) loaded with nucleoside reverse transcriptase inhibitor (NRTI) drugs for the first time. Empty nanoMOFs and nanoMOFs loaded with azidothymidine derivatives with three different degrees of phosphorylation were examined: azidothymidine (AZT, native drug), azidothymidine monophosphate (AZT-MP), and azidothymidine triphosphate (AZT-TP). The particle size distribution and the stability of the nanoparticles when interacting with drugs have been determined in a time frame of 24 h. Main achievements include detection of aggregate formation in an early stage and monitoring nanoMOF morphological changes as indicators of their interaction with guest molecules. AF4-MALS proved to be a useful methodology to analyze nanoparticles engineered for drug delive...

Molecular pharmaceutics, Jan 4, 2018

Clofazimine (CLZ) is an antibiotic with a promising behavior against Gram-positive bacteria; howe... more Clofazimine (CLZ) is an antibiotic with a promising behavior against Gram-positive bacteria; however, the drug is completely insoluble in water and accumulates in fat tissues. We explored nanocarriers, labeled and not labeled with rhodamine, consisting of negatively charged sulfobutylether-β-cyclodextrins for CLZ loading. A new oligomeric carrier was obtained cross-linking βCyD with epichlorohydrin followed by sulfonation in a strongly alkaline aqueous medium. The oligomeric carrier has a MW of 53 kDa and forms small nanoparticles of a few tens of nm. With aqueous solutions containing a 25 mg/mL oligomeric carrier, we loaded up to 0.5 mg/mL of drug. The oligomers exhibited a 10-fold better loading capacity compared to monomers and formed nanoparticles with a size in the 20-60 nm range after drug loading. Circular dichroism confirmed encapsulation of the CLZ in the nanocarriers. All carriers with or without CLZ are not cytotoxic up to 1 μM, while CLZ alone is highly cytotoxic at the ...

Aesthetic surgery journal, Jan 14, 2018

New microfat preparations provide material suitable for use as a regenerative filler for differen... more New microfat preparations provide material suitable for use as a regenerative filler for different facial areas. To support the development of new robust techniques for regenerative purposes, the cellular content of the sample should be considered. To evaluate the stromal vascular fraction (SVF) cell components of micro-superficial enhanced fluid fat injection (SEFFI) samples via a technique to harvest re-injectable tissue with minimum manipulation. The results were compared to those obtained from SEFFI samples. Microscopy analysis was performed to visualize the tissue structure. Micro-SEFFI samples were also fractionated using Celector ®, an innovative non-invasive separation technique, to provide an initial evaluation of sample fluidity and composition. SVFs obtained from SEFFI and micro-SEFFI were studied. Adipose stromal cells (ASCs) were isolated and characterized by proliferation and differentiation capacity assays. Microscopic and quality analyses of micro-SEFFI samples by Ce...

Royal Society open science, 2018

Silver nanoparticle-based antimicrobials can promote a long lasting bactericidal effect without d... more Silver nanoparticle-based antimicrobials can promote a long lasting bactericidal effect without detrimental toxic side effects. However, there is not a clear and complete protocol to define and relate the properties of the particles (size, shape, surface charge, ionic content) with their specific activity. In this paper, we propose an effective multi-step approach for the identification of a 'purpose-specific active applicability window' to maximize the antimicrobial activity of medical devices containing silver nanoparticles (Ag NPs) (such as surface coaters), minimizing any consequent risk for human health (safety by design strategy). The antimicrobial activity and the cellular toxicity of four types of Ag NPs, differing in their coating composition and concentration have been quantified. Through the implementation of flow-field flow fractionation, Ag NPs have been characterized in terms of metal release, size and shape. The particles are fractionated in the process while ...

Microchemical Journal

In previous papers it has been demonstrated that by using a special atomiser and power supply it ... more In previous papers it has been demonstrated that by using a special atomiser and power supply it was possible with elements of high volatility like Hg, Cd, Pb and Ag to obtain absorbance vs. time curves of a form consistent with the assumption that all atoms present in a condensed state at the beginning of the atomisation step are simultaneously present in the vapour phase inside the atomiser. In fact, these absorbance vs. time curves closely fit a model, which assumes a very rapid atomisation and homogenisation of the atoms at the centre of the atomiser followed by diffusion out of the graphite tube. The absorbance in these curves is, theoretically, an asymptotic maximum, which is related, if the experimental conditions are well chosen, to the total number of atoms injected, through the Lambert᎐Beer law. In this case the absorbance data can be used, if the proportionality constant is known, for standardless analysis. The increase of the atomisation temperature, as a consequence of a new design of the atomisation system, has made it possible to extend the method to elements of medium volatility like Co, Cr, Cu, Fe, Mn and Ni. The elements cited are those so far analysed but it is easy to predict that the method can be applied to the majority of the elements, thus greatly increasing its usefulness in a large number of real samples.

Analytical and Bioanalytical Chemistry, 2016

An integrated sensing system is presented for the first time, where a metal oxide semiconductor s... more An integrated sensing system is presented for the first time, where a metal oxide semiconductor sensor-based electronic olfactory system (MOS array), employed for pathogen bacteria identification based on their volatile organic compound (VOC) characterisation, is assisted by a preliminary separative technique based on gravitational field-flow fractionation (GrFFF). In the integrated system, a preliminary step using GrFFF fractionation of a complex sample provided bacteria-enriched fractions readily available for subsequent MOS array analysis. The MOS array signals were then analysed employing a chemometric approach using principal components analysis (PCA) for a first-data exploration, followed by linear discriminant analysis (LDA) as a classification tool, using the PCA scores as input variables. The ability of the GrFFF-MOS system to distinguish between viable and non-viable cells of the same strain was demonstrated for the first time, yielding 100 % ability of correct prediction. The integrated system was also applied as a proof of concept for multianalyte purposes, for the detection of two bacterial strains (Escherichia coli O157:H7 and Yersinia enterocolitica) simultaneously present in artificially contaminated milk samples, obtaining a 100 % ability of correct prediction. Acquired results show that GrFFF band slicing before MOS array analysis can significantly increase reliability and reproducibility of pathogen bacteria identification based on their VOC production, simplifying the analytical procedure and largely eliminating sample matrix effects. The developed GrFFF-MOS integrated system can be considered a simple straightforward approach for pathogen bacteria identification directly from their food matrix. Graphical abstract An integrated sensing system is presented for pathogen bacteria identification in food, in which field-flow fractionation is exploited to prepare enriched cell fractions prior to their analysis by electronic olfactory system analysis.

AIP Conference Proceedings, 2005

ABSTRACT Water-soluble, functionalized multi-walled carbon nanotubes (MWNTs) are length-separated... more ABSTRACT Water-soluble, functionalized multi-walled carbon nanotubes (MWNTs) are length-separated and purified from amorphous material through direct flow field-flow fractionation. MWNTs subpopulations of relatively homogeneous, different length are obtained from collecting fractions of the raw, highly polydispersed (200-5000 nm) MWNT sample.

Fresenius' Journal of Analytical Chemistry, 1998

A new method for standardless analysis in electrothermal atomic absorption spectroscopy has been ... more A new method for standardless analysis in electrothermal atomic absorption spectroscopy has been recently proposed whose implementation requires the use of special atomizers and power supplies not available in the market. Up to now, with the proposed method, only volatile elements have been determined with good results because it can be applied only if all atoms injected are simultaneously present in the atomizer for a time interval sufficiently long that a good value of absorbance can be measured. This can be obtained if the heating rate is sufficiently high and the removal of atoms from the atomizer slow. From a theoretical model based only on diffusion, it can be deduced that the possibility of standardless analysis in the mode described above can be enhanced if the length of the atomizer is increased and the diffusion coefficient of the analyte is decreased. From the results obtained by comparing data from atomizers of 36 and 50 mm in length and at different pressures, it can be inferred that the removal mechanism is basically controlled by diffusion but that other mechanisms, like convection, are also present. Experimental All instruments and procedures for measuring A 0 have been previously described [1-3].

Scientific Reports, 2016

Protein aggregation is a common biological phenomenon, observed in different physiological and pa... more Protein aggregation is a common biological phenomenon, observed in different physiological and pathological conditions. Decreased protein solubility and a tendency to aggregate is also observed during physiological aging but the causes are currently unknown. Herein we performed a biophysical separation of aging-related high molecular weight aggregates, isolated from the bone marrow and splenic cells of aging mice and followed by biochemical and mass spectrometric analysis. The analysis indicated that compared to younger mice an increase in protein post-translational carbonylation was observed. The causative role of these modifications in inducing protein misfolding and aggregation was determined by inducing carbonyl stress in young mice, which recapitulated the increased protein aggregation observed in old mice. Altogether our analysis indicates that oxidative stress-related posttranslational modifications accumulate in the aging proteome and are responsible for increased protein aggregation and altered cell proteostasis. Protein aggregation is a general terminology used to describe the association of proteins into larger assemblies following loss of secondary, tertiary or quaternary structure and often loss of biological activity 1. Protein aggregation is a common biological phenomenon associated with the inability of the cell to maintain homeostasis of the proteome (proteostasis) 1. Under physiological conditions, the tendency of de novo synthesized unfolded proteins to aggregate is regulated by several chaperones that aid in their folding 2,3. Soluble aggregation is also commonly observed in ubiquitinated unfolded proteins before proteasome degradation or in damaged proteins before translocation into lysosomes by chaperone-mediated autophagy 4-6. Additionally, temporal changes to cellular homeostasis (temperature, pH, water content and salt/ions concentration) can induce transitory protein unfolding and soluble aggregation 1. During pathological conditions, protein aggregation is a common occurrence giving rise to the group of diseases collectively known as protein conformational diseases. In many degenerative diseases of the CNS, such as Alzheimer's, Parkinson's and Huntington's disease protein aggregation is a common pathological hallmark due to amino acid mutation and changes in the primary structure of the proteins 7-11. Size-wise, aggregates can range considerably, from protein oligomers up to visible cytosolic inclusions, known as the aggresome 12. The sub cellular location of these aggregates can also vary, from perinuclear to peri-endolasmic reticulum (ER) or intra-endosomal. Perinuclear aggregates (aggresomes) co-localize with the microtubule organizing center and mostly contain terminally aggregated proteins 12 , whereas ER-associated aggregates are mostly formed by soluble aggregates of ubiquitinated misfolded proteins 3 and endosomal aggregates are inclusions that co-localize with autophagic markers 13-15. The functional consequences of aggregation are also variable, from up-regulation of autophagy 16 , to cellular apoptosis due to aggregate-related cytotoxicity. Recently, it has been reported that during physiological aging proteostasis gradually becomes compromised and several hundred proteins tend to become more insoluble and aggregate 17,18. A few of these proteins have been shown to have common biochemical and biological properties, such as a primary structure with amino acids stretches often found in proteins associated with neurodegenerative diseases and a secondary structure with

The Journal of Physical Chemistry B, Nov 1, 2010

Very monodisperse water-soluble silica core-surfactant shell nanoparticles (SCSS NPs) doped with ... more Very monodisperse water-soluble silica core-surfactant shell nanoparticles (SCSS NPs) doped with a rhodamine B derivative were prepared using micelles of F127 as nanoreactors for the hydrolysis and condensation of the silica precursor tetraethoxysilane (TEOS). The functionalization of the rhodamines with a triethoxysilane group allowed the covalent binding of the fluorophores to the silica core: no leaking of the dye was observed when the NPs were purified either by ultrafiltration (UF) or dialysis. The diameter of the core (d(c) = 10 ± 1 nm) was determined by TEM and subtracted from the hydrodynamic diameter, measured by DLS, (d(H) = 24 nm, PdI = 0.1) to calculate the shell thickness (∼7 nm). The presence of a single population of NPs with a radius compatible with the one measured by DLS after UF was confirmed by AF4-MALS-RI measurements. The concentration of the NPs was measured by MALS-RI. This allowed us to determine the average number of rhodamine molecules per NP (10). The ability of the NPs to host hydrophobic species as cyanines in the SS was confirmed by fluorescence anisotropy measurements. Steady-state and time-resolved fluorescence measurements allowed us to observe the occurrence of a very efficient Förster resonance energy transfer process from the covalently linked rhodamines to the hosted cyanines. In particular, the analysis of the TCSPC data and steady-state measurements revealed that the adsorption of a single cyanine molecule causes an almost complete quenching of the fluorescence of the NP. Thanks to these observations, it was possible to easily determine the concentration of the NPs by fluorescence titration experiments. Results are in good agreement with the concentration values obtained by MALS-RI. Finally, the hosted cyanine molecule could be extracted with (±)-2-octanol, demonstrating the reversibility of the adsorption process.

I contenuti della presentazione potranno subire variazioni durante il corso. Gli studenti sono pr... more I contenuti della presentazione potranno subire variazioni durante il corso. Gli studenti sono pregati di prenderne nota. In caso di modifiche, al termine del corso il materiale verra aggiornato in rete.

Annali di Chimica

Cereal starch occurs as two types of micrometer-sized granules, large and small. Large starch gra... more Cereal starch occurs as two types of micrometer-sized granules, large and small. Large starch granules are more susceptible to enzymatic hydrolysis. When cereal starch is used for fermentation processes, as in brewing of barley malt, the barley strains with the highest content of large starch granules should be preferred. Gravitational field-flow fractionation (GFFF) is a separation method able to fractionate starch samples at low cost and short analysis time. In this work, the search for the best GFFF conditions for the analytical separation of barley starch within an inter-laboratory approach is presented. For different barley strains cultivated under monitored conditions the size distributions of starch granules is here quickly monitored and characterized by GFFF. As a consequence, dimensional characterization of barley starch can allow for the selection of the most suitable strains with the lowest content of non-degradable starch.

Encyclopedia of Chromatography, Second Edition, 2005

Review of Scientific Instruments, 1997

A simple high current power supply is described. It can be assembled from readily available and r... more A simple high current power supply is described. It can be assembled from readily available and relatively cheap components which can be itemized as follows: (1) commercial batteries; (2) fast high current switch; (3) feedback sensor; (4) PC with peripherals for controlling the switch. The apparatus was assembled for heating at very high heating rate with a low resistant load such as a graphite or vitreous carbon tube used as an atomizer in electrothermal atomization-atomic absorption spectroscopy measurements. The high heating rate must, however, be combined with a fairly good control of the temperature of the tube at various levels. This was obtained with a PC and relative peripherals which, directly or through a feedback circuit, control the switch. The system can be easily modified and adapted for other kinds of measurements as long as the high noise of the switching does not interfere with signal recovery. With the atomic spectrometer used (Perkin–Elmer Model 1100B), however, no worsening of the signal noise was observed. The level of current that can be used is mainly determined by the switch since, within a certain range, the voltage and the internal resistance of the power supply can be changed by the type of batteries and the way they are connected.

Annali di chimica, 2002

Cereal starch occurs as two types of micrometer-sized granules, large and small. Large starch gra... more Cereal starch occurs as two types of micrometer-sized granules, large and small. Large starch granules are more susceptible to enzymatic hydrolysis. When cereal starch is used for fermentation processes, as in brewing of barley malt, the barley strains with the highest content of large starch granules should be preferred. Gravitational field-flow fractionation (GFFF) is a separation method able to fractionate starch samples at low cost and short analysis time. In this work, the search for the best GFFF conditions for the analytical separation of barley starch within an inter-laboratory approach is presented. For different barley strains cultivated under monitored conditions the size distributions of starch granules is here quickly monitored and characterized by GFFF. As a consequence, dimensional characterization of barley starch can allow for the selection of the most suitable strains with the lowest content of non-degradable starch.

Journal of environmental pathology, toxicology and oncology : official organ of the International Society for Environmental Toxicology and Cancer, 1997

We describe a new method and the relevant instrumentation necessary for its implementation in the... more We describe a new method and the relevant instrumentation necessary for its implementation in the analysis of metals associated with particulate matter in air. The procedure can be divided into two steps: in the first step the sample is accumulated in a device through electrostatic precipitation whose center is a graphite tube; in the second step the graphite tube itself is used as an atomization device for the determination of the metals present in the sample through the electrothermal atomic absorption technique. The method is simple, fast, accurate, and inexpensive. Moreover, if the experimental conditions are well chosen, there is no need for calibration, which is very convenient in the case of samples such as particulate matter in the air. The elements that can be determined with the present apparatus are Hg, Cd, Tl, Ag, Mg, and Mn. These are highly or medium volatile because the materials used cannot reach very high temperatures for long periods. The experiments are confined t...

Multi-analyte Detection 13.1.8 Protein Switches 13.2 Genetic and Chemical Engineering of Lucifera... more Multi-analyte Detection 13.1.8 Protein Switches 13.2 Genetic and Chemical Engineering of Luciferases for Bioanalytical Applications 13.2.1

Journal of Chromatography A

Analytica Chimica Acta

Protein misfolding and aggregation are the common mechanisms in a variety of aggregation-dependen... more Protein misfolding and aggregation are the common mechanisms in a variety of aggregation-dependent diseases. The compromised proteins often assemble into toxic, accumulating amyloid-like structures of various lengths and their toxicity can also be transferred both in vivo and in vitro a prion-like behavior. The characterization of protein interactions, degradation and conformational dynamics in biological systems still represents an analytical challenge in the prion-like protein comprehension. In our work, we investigated the nature of a transferable cytotoxic agent, presumably a misfolded protein, through the coupling of a multi-detector, non-destructive separation platform based on hollow-fiber flow field-flow fractionation with imaging and downstream in vitro tests. After purification with ion exchange chromatography, the transferable cytotoxic agentwas analyzed with Atomic Force Microscopy and statistical analysis, showing that the concentration of protein dimers and low n-oligomer forms was higher in the cytotoxic sample than in the control preparation. To assess whether the presence of these species was the actual toxic and/or self-propagating factor, we employed HF5 fractionation, with UV and Multi-Angle Light Scattering detection, to define proteins molar mass distribution and abundance, and fractionate the sample into size-homogeneous fractions. These fractions were then tested individually in vitro to investigate the direct correlation with cytotoxicity. Only the later-eluted fraction, which contains high-molar mass aggregates, proved to be toxic onto cell cultures. Moreover, it was observed that the selective transfer of toxicity also occurs for one lower-mass fraction, suggesting that two different mechanisms, acute and later induced toxicity, are in place. These results strongly encourage the efficacy of this platform to enable the identification of protein toxicants.

Analytical and bioanalytical chemistry, 2018

Asymmetric flow field-flow fractionation (AF4) coupled with UV-Vis spectroscopy, multi-angle ligh... more Asymmetric flow field-flow fractionation (AF4) coupled with UV-Vis spectroscopy, multi-angle light scattering (MALS) and refractive index (RI) detection has been applied for the characterization of MIL-100(Fe) nanoMOFs (metal-organic frameworks) loaded with nucleoside reverse transcriptase inhibitor (NRTI) drugs for the first time. Empty nanoMOFs and nanoMOFs loaded with azidothymidine derivatives with three different degrees of phosphorylation were examined: azidothymidine (AZT, native drug), azidothymidine monophosphate (AZT-MP), and azidothymidine triphosphate (AZT-TP). The particle size distribution and the stability of the nanoparticles when interacting with drugs have been determined in a time frame of 24 h. Main achievements include detection of aggregate formation in an early stage and monitoring nanoMOF morphological changes as indicators of their interaction with guest molecules. AF4-MALS proved to be a useful methodology to analyze nanoparticles engineered for drug delive...

Molecular pharmaceutics, Jan 4, 2018

Clofazimine (CLZ) is an antibiotic with a promising behavior against Gram-positive bacteria; howe... more Clofazimine (CLZ) is an antibiotic with a promising behavior against Gram-positive bacteria; however, the drug is completely insoluble in water and accumulates in fat tissues. We explored nanocarriers, labeled and not labeled with rhodamine, consisting of negatively charged sulfobutylether-β-cyclodextrins for CLZ loading. A new oligomeric carrier was obtained cross-linking βCyD with epichlorohydrin followed by sulfonation in a strongly alkaline aqueous medium. The oligomeric carrier has a MW of 53 kDa and forms small nanoparticles of a few tens of nm. With aqueous solutions containing a 25 mg/mL oligomeric carrier, we loaded up to 0.5 mg/mL of drug. The oligomers exhibited a 10-fold better loading capacity compared to monomers and formed nanoparticles with a size in the 20-60 nm range after drug loading. Circular dichroism confirmed encapsulation of the CLZ in the nanocarriers. All carriers with or without CLZ are not cytotoxic up to 1 μM, while CLZ alone is highly cytotoxic at the ...

Aesthetic surgery journal, Jan 14, 2018

New microfat preparations provide material suitable for use as a regenerative filler for differen... more New microfat preparations provide material suitable for use as a regenerative filler for different facial areas. To support the development of new robust techniques for regenerative purposes, the cellular content of the sample should be considered. To evaluate the stromal vascular fraction (SVF) cell components of micro-superficial enhanced fluid fat injection (SEFFI) samples via a technique to harvest re-injectable tissue with minimum manipulation. The results were compared to those obtained from SEFFI samples. Microscopy analysis was performed to visualize the tissue structure. Micro-SEFFI samples were also fractionated using Celector ®, an innovative non-invasive separation technique, to provide an initial evaluation of sample fluidity and composition. SVFs obtained from SEFFI and micro-SEFFI were studied. Adipose stromal cells (ASCs) were isolated and characterized by proliferation and differentiation capacity assays. Microscopic and quality analyses of micro-SEFFI samples by Ce...

Royal Society open science, 2018

Silver nanoparticle-based antimicrobials can promote a long lasting bactericidal effect without d... more Silver nanoparticle-based antimicrobials can promote a long lasting bactericidal effect without detrimental toxic side effects. However, there is not a clear and complete protocol to define and relate the properties of the particles (size, shape, surface charge, ionic content) with their specific activity. In this paper, we propose an effective multi-step approach for the identification of a 'purpose-specific active applicability window' to maximize the antimicrobial activity of medical devices containing silver nanoparticles (Ag NPs) (such as surface coaters), minimizing any consequent risk for human health (safety by design strategy). The antimicrobial activity and the cellular toxicity of four types of Ag NPs, differing in their coating composition and concentration have been quantified. Through the implementation of flow-field flow fractionation, Ag NPs have been characterized in terms of metal release, size and shape. The particles are fractionated in the process while ...

Microchemical Journal

In previous papers it has been demonstrated that by using a special atomiser and power supply it ... more In previous papers it has been demonstrated that by using a special atomiser and power supply it was possible with elements of high volatility like Hg, Cd, Pb and Ag to obtain absorbance vs. time curves of a form consistent with the assumption that all atoms present in a condensed state at the beginning of the atomisation step are simultaneously present in the vapour phase inside the atomiser. In fact, these absorbance vs. time curves closely fit a model, which assumes a very rapid atomisation and homogenisation of the atoms at the centre of the atomiser followed by diffusion out of the graphite tube. The absorbance in these curves is, theoretically, an asymptotic maximum, which is related, if the experimental conditions are well chosen, to the total number of atoms injected, through the Lambert᎐Beer law. In this case the absorbance data can be used, if the proportionality constant is known, for standardless analysis. The increase of the atomisation temperature, as a consequence of a new design of the atomisation system, has made it possible to extend the method to elements of medium volatility like Co, Cr, Cu, Fe, Mn and Ni. The elements cited are those so far analysed but it is easy to predict that the method can be applied to the majority of the elements, thus greatly increasing its usefulness in a large number of real samples.

Analytical and Bioanalytical Chemistry, 2016

An integrated sensing system is presented for the first time, where a metal oxide semiconductor s... more An integrated sensing system is presented for the first time, where a metal oxide semiconductor sensor-based electronic olfactory system (MOS array), employed for pathogen bacteria identification based on their volatile organic compound (VOC) characterisation, is assisted by a preliminary separative technique based on gravitational field-flow fractionation (GrFFF). In the integrated system, a preliminary step using GrFFF fractionation of a complex sample provided bacteria-enriched fractions readily available for subsequent MOS array analysis. The MOS array signals were then analysed employing a chemometric approach using principal components analysis (PCA) for a first-data exploration, followed by linear discriminant analysis (LDA) as a classification tool, using the PCA scores as input variables. The ability of the GrFFF-MOS system to distinguish between viable and non-viable cells of the same strain was demonstrated for the first time, yielding 100 % ability of correct prediction. The integrated system was also applied as a proof of concept for multianalyte purposes, for the detection of two bacterial strains (Escherichia coli O157:H7 and Yersinia enterocolitica) simultaneously present in artificially contaminated milk samples, obtaining a 100 % ability of correct prediction. Acquired results show that GrFFF band slicing before MOS array analysis can significantly increase reliability and reproducibility of pathogen bacteria identification based on their VOC production, simplifying the analytical procedure and largely eliminating sample matrix effects. The developed GrFFF-MOS integrated system can be considered a simple straightforward approach for pathogen bacteria identification directly from their food matrix. Graphical abstract An integrated sensing system is presented for pathogen bacteria identification in food, in which field-flow fractionation is exploited to prepare enriched cell fractions prior to their analysis by electronic olfactory system analysis.

AIP Conference Proceedings, 2005

ABSTRACT Water-soluble, functionalized multi-walled carbon nanotubes (MWNTs) are length-separated... more ABSTRACT Water-soluble, functionalized multi-walled carbon nanotubes (MWNTs) are length-separated and purified from amorphous material through direct flow field-flow fractionation. MWNTs subpopulations of relatively homogeneous, different length are obtained from collecting fractions of the raw, highly polydispersed (200-5000 nm) MWNT sample.

Fresenius' Journal of Analytical Chemistry, 1998

A new method for standardless analysis in electrothermal atomic absorption spectroscopy has been ... more A new method for standardless analysis in electrothermal atomic absorption spectroscopy has been recently proposed whose implementation requires the use of special atomizers and power supplies not available in the market. Up to now, with the proposed method, only volatile elements have been determined with good results because it can be applied only if all atoms injected are simultaneously present in the atomizer for a time interval sufficiently long that a good value of absorbance can be measured. This can be obtained if the heating rate is sufficiently high and the removal of atoms from the atomizer slow. From a theoretical model based only on diffusion, it can be deduced that the possibility of standardless analysis in the mode described above can be enhanced if the length of the atomizer is increased and the diffusion coefficient of the analyte is decreased. From the results obtained by comparing data from atomizers of 36 and 50 mm in length and at different pressures, it can be inferred that the removal mechanism is basically controlled by diffusion but that other mechanisms, like convection, are also present. Experimental All instruments and procedures for measuring A 0 have been previously described [1-3].

Scientific Reports, 2016

Protein aggregation is a common biological phenomenon, observed in different physiological and pa... more Protein aggregation is a common biological phenomenon, observed in different physiological and pathological conditions. Decreased protein solubility and a tendency to aggregate is also observed during physiological aging but the causes are currently unknown. Herein we performed a biophysical separation of aging-related high molecular weight aggregates, isolated from the bone marrow and splenic cells of aging mice and followed by biochemical and mass spectrometric analysis. The analysis indicated that compared to younger mice an increase in protein post-translational carbonylation was observed. The causative role of these modifications in inducing protein misfolding and aggregation was determined by inducing carbonyl stress in young mice, which recapitulated the increased protein aggregation observed in old mice. Altogether our analysis indicates that oxidative stress-related posttranslational modifications accumulate in the aging proteome and are responsible for increased protein aggregation and altered cell proteostasis. Protein aggregation is a general terminology used to describe the association of proteins into larger assemblies following loss of secondary, tertiary or quaternary structure and often loss of biological activity 1. Protein aggregation is a common biological phenomenon associated with the inability of the cell to maintain homeostasis of the proteome (proteostasis) 1. Under physiological conditions, the tendency of de novo synthesized unfolded proteins to aggregate is regulated by several chaperones that aid in their folding 2,3. Soluble aggregation is also commonly observed in ubiquitinated unfolded proteins before proteasome degradation or in damaged proteins before translocation into lysosomes by chaperone-mediated autophagy 4-6. Additionally, temporal changes to cellular homeostasis (temperature, pH, water content and salt/ions concentration) can induce transitory protein unfolding and soluble aggregation 1. During pathological conditions, protein aggregation is a common occurrence giving rise to the group of diseases collectively known as protein conformational diseases. In many degenerative diseases of the CNS, such as Alzheimer's, Parkinson's and Huntington's disease protein aggregation is a common pathological hallmark due to amino acid mutation and changes in the primary structure of the proteins 7-11. Size-wise, aggregates can range considerably, from protein oligomers up to visible cytosolic inclusions, known as the aggresome 12. The sub cellular location of these aggregates can also vary, from perinuclear to peri-endolasmic reticulum (ER) or intra-endosomal. Perinuclear aggregates (aggresomes) co-localize with the microtubule organizing center and mostly contain terminally aggregated proteins 12 , whereas ER-associated aggregates are mostly formed by soluble aggregates of ubiquitinated misfolded proteins 3 and endosomal aggregates are inclusions that co-localize with autophagic markers 13-15. The functional consequences of aggregation are also variable, from up-regulation of autophagy 16 , to cellular apoptosis due to aggregate-related cytotoxicity. Recently, it has been reported that during physiological aging proteostasis gradually becomes compromised and several hundred proteins tend to become more insoluble and aggregate 17,18. A few of these proteins have been shown to have common biochemical and biological properties, such as a primary structure with amino acids stretches often found in proteins associated with neurodegenerative diseases and a secondary structure with

The Journal of Physical Chemistry B, Nov 1, 2010

Very monodisperse water-soluble silica core-surfactant shell nanoparticles (SCSS NPs) doped with ... more Very monodisperse water-soluble silica core-surfactant shell nanoparticles (SCSS NPs) doped with a rhodamine B derivative were prepared using micelles of F127 as nanoreactors for the hydrolysis and condensation of the silica precursor tetraethoxysilane (TEOS). The functionalization of the rhodamines with a triethoxysilane group allowed the covalent binding of the fluorophores to the silica core: no leaking of the dye was observed when the NPs were purified either by ultrafiltration (UF) or dialysis. The diameter of the core (d(c) = 10 ± 1 nm) was determined by TEM and subtracted from the hydrodynamic diameter, measured by DLS, (d(H) = 24 nm, PdI = 0.1) to calculate the shell thickness (∼7 nm). The presence of a single population of NPs with a radius compatible with the one measured by DLS after UF was confirmed by AF4-MALS-RI measurements. The concentration of the NPs was measured by MALS-RI. This allowed us to determine the average number of rhodamine molecules per NP (10). The ability of the NPs to host hydrophobic species as cyanines in the SS was confirmed by fluorescence anisotropy measurements. Steady-state and time-resolved fluorescence measurements allowed us to observe the occurrence of a very efficient Förster resonance energy transfer process from the covalently linked rhodamines to the hosted cyanines. In particular, the analysis of the TCSPC data and steady-state measurements revealed that the adsorption of a single cyanine molecule causes an almost complete quenching of the fluorescence of the NP. Thanks to these observations, it was possible to easily determine the concentration of the NPs by fluorescence titration experiments. Results are in good agreement with the concentration values obtained by MALS-RI. Finally, the hosted cyanine molecule could be extracted with (±)-2-octanol, demonstrating the reversibility of the adsorption process.

I contenuti della presentazione potranno subire variazioni durante il corso. Gli studenti sono pr... more I contenuti della presentazione potranno subire variazioni durante il corso. Gli studenti sono pregati di prenderne nota. In caso di modifiche, al termine del corso il materiale verra aggiornato in rete.

Annali di Chimica

Cereal starch occurs as two types of micrometer-sized granules, large and small. Large starch gra... more Cereal starch occurs as two types of micrometer-sized granules, large and small. Large starch granules are more susceptible to enzymatic hydrolysis. When cereal starch is used for fermentation processes, as in brewing of barley malt, the barley strains with the highest content of large starch granules should be preferred. Gravitational field-flow fractionation (GFFF) is a separation method able to fractionate starch samples at low cost and short analysis time. In this work, the search for the best GFFF conditions for the analytical separation of barley starch within an inter-laboratory approach is presented. For different barley strains cultivated under monitored conditions the size distributions of starch granules is here quickly monitored and characterized by GFFF. As a consequence, dimensional characterization of barley starch can allow for the selection of the most suitable strains with the lowest content of non-degradable starch.

Encyclopedia of Chromatography, Second Edition, 2005

Review of Scientific Instruments, 1997

A simple high current power supply is described. It can be assembled from readily available and r... more A simple high current power supply is described. It can be assembled from readily available and relatively cheap components which can be itemized as follows: (1) commercial batteries; (2) fast high current switch; (3) feedback sensor; (4) PC with peripherals for controlling the switch. The apparatus was assembled for heating at very high heating rate with a low resistant load such as a graphite or vitreous carbon tube used as an atomizer in electrothermal atomization-atomic absorption spectroscopy measurements. The high heating rate must, however, be combined with a fairly good control of the temperature of the tube at various levels. This was obtained with a PC and relative peripherals which, directly or through a feedback circuit, control the switch. The system can be easily modified and adapted for other kinds of measurements as long as the high noise of the switching does not interfere with signal recovery. With the atomic spectrometer used (Perkin–Elmer Model 1100B), however, no worsening of the signal noise was observed. The level of current that can be used is mainly determined by the switch since, within a certain range, the voltage and the internal resistance of the power supply can be changed by the type of batteries and the way they are connected.

Annali di chimica, 2002

Cereal starch occurs as two types of micrometer-sized granules, large and small. Large starch gra... more Cereal starch occurs as two types of micrometer-sized granules, large and small. Large starch granules are more susceptible to enzymatic hydrolysis. When cereal starch is used for fermentation processes, as in brewing of barley malt, the barley strains with the highest content of large starch granules should be preferred. Gravitational field-flow fractionation (GFFF) is a separation method able to fractionate starch samples at low cost and short analysis time. In this work, the search for the best GFFF conditions for the analytical separation of barley starch within an inter-laboratory approach is presented. For different barley strains cultivated under monitored conditions the size distributions of starch granules is here quickly monitored and characterized by GFFF. As a consequence, dimensional characterization of barley starch can allow for the selection of the most suitable strains with the lowest content of non-degradable starch.

Journal of environmental pathology, toxicology and oncology : official organ of the International Society for Environmental Toxicology and Cancer, 1997

We describe a new method and the relevant instrumentation necessary for its implementation in the... more We describe a new method and the relevant instrumentation necessary for its implementation in the analysis of metals associated with particulate matter in air. The procedure can be divided into two steps: in the first step the sample is accumulated in a device through electrostatic precipitation whose center is a graphite tube; in the second step the graphite tube itself is used as an atomization device for the determination of the metals present in the sample through the electrothermal atomic absorption technique. The method is simple, fast, accurate, and inexpensive. Moreover, if the experimental conditions are well chosen, there is no need for calibration, which is very convenient in the case of samples such as particulate matter in the air. The elements that can be determined with the present apparatus are Hg, Cd, Tl, Ag, Mg, and Mn. These are highly or medium volatile because the materials used cannot reach very high temperatures for long periods. The experiments are confined t...