Davide Baratella

A glucose biosensor based on surface active maghemite nanoparticles.

A simple carbon paste (CP) electrode, modified with novel maghemite (γ-Fe2O3) nanoparticles, called SAMNs (surface active maghemite nanoparticles) and characterized by a mean diameter of about 10nm, has been developed. The electrode catalyzes the electro-reduction of hydrogen peroxide at low applied potentials (-0.1 V vs SCE). In order to improve the electrocatalytic properties of the modified electrode an ionic liquid, namely 1-butyl-3-methylimidazolium hexafluorophosphate (BMIM-PF6), was introduced. At -0.1 V, the sensitivity of the SAMN-BMIM-PF6-CP electrode was 206.51 nA μM(-1)cm(-2), with a detection limit (S/N=3) of 0.8 μM, in the 0-1.5mM H2O2 concentration range. Furthermore, glucose oxidase was immobilized on the surface of maghemite nanoparticles as a monomolecular layer, by a bridge constituted of rhodamine B isothiocyanate, leading to a fluorescent, magnetic drivable nanocatalyst, containing 10 ± 2 enzyme molecules per nanoparticle. The resulting enzyme electrode presents a linear calibration curve toward glucose in solution in the concentration range of 0-1.5mM glucose, characterized by a sensitivity of 45.85 nA μM(-1)cm(-2) and a detection limit (S/N=3) of 0.9 μM. The storage stability of the system was evaluated and a half-life of 2 months was calculated, if the electrode is stored at 4°C in buffer. The present work demonstrates the feasibility of these surface active maghemite nanoparticles as efficient hydrogen peroxide electro-catalyst, which can be easily coupled to hydrogen peroxide producing enzymes in order to develop oxidase based reagentless biosensor devices.

Avidin functionalized maghemite nanoparticles and their application for recombinant human biotinyl-SERCA purification.

We report on the surface characterization, functionalization, and application of stable water suspensions of novel surface active maghemite nanoparticles (SAMNs), characterized by a diameter of 11 ± 2 nm and possessing peculiar colloidal properties and surface interactions. These features permitted the acquisition of titration curves and aqueous UV-vis spectra and suggested a role played by surface under-coordinated iron atoms. This new class of nanoparticles was obtained through an easy, inexpensive, one-step, green procedure and functionalized with ligands of high biotechnological interest, such as biotin and avidin, by simple incubation in aqueous solution. Bound avidin was determined by measuring the disappearance of free avidin absorbance at 280 nm, as a function of increasing nanoparticle concentration, showing the presence of 10 ± 3 avidin molecules per nanoparticle. The biological activity of the SAMN@avidin complex was evaluated and the number of available biotin binding sites was determined, using biotinyl-fluorescein as a probe, showing that each bound avidin molecule is able to bind 2.8 ± 0.8 biotin molecules, confirming the maintenance of biological activity and excellent binding capacity of the SAMN@avidin complex. Furthermore a Langmuir isotherm model was used to describe the biomolecule specific monolayer adsorption onto the particle surface, and in the case of avidin, the maximum adsorption capacity was 100 ± 27 μg avidin/mg SAMN, whereas the binding constant is 45.18 μL μg(-1). The SAMN@avidin complex was characterized by UV-vis spectroscopy, quartz crystal microbalance, FTIR spectroscopy, and transmission electron microscopy. Finally, SAMN@avidin was applied for the large scale purification of recombinant biotinylated human sarco/endoplasmic reticulum Ca(2+)-ATPase (hSERCA-2a), expressed by Saccharomyces cerevisiae. The protein was magnetically purified, and about 500 μg of a 70% pure hSERCA-2a were recovered from 4 L of yeast culture, with a purification yield of 64%.

Core-shell hybrid nanomaterial based on prussian blue and surface active maghemite nanoparticles as stable electrocatalyst.

A novel core-shell nanomaterial based on prussian blue (PB) coating on peculiar surface active maghemite nanoparticles (SAMNs), was developed. The synthetic process involves the direct crystallization of Fe(II)(CN)6(4-) onto the surface of SAMNs by simple incubation in water at controlled pH, demonstrating the presence of under-coordinated Fe(III) on nanoparticle surface. The coating reaction occurs in a narrow pH range and the synthetic procedure was optimized. The resulting SAMN@PB hybrid nanostructures were characterized by transmission and scanning electron microscopy, Mössbauer, UV-vis and FTIR spectroscopy and X-ray powder diffraction. The nanomaterial, characterized by high stability in alkaline media, behave as excellent electro-catalyst for hydrogen peroxide reduction. The stability of SAMN@PB hybrid has been investigated as a function of pH, showing excellent stability up to pH 9.0 and demonstrating the feasibility of SAMNs, superficially derivatized with prussian blue, to produce an efficient and extremely stable nanostructured material. This maghemite supported nanostructured prussian blue was applied to develop a sensor, based on a simple carbon paste electrode, which was able to catalyze the electro-reduction of hydrogen peroxide, in aqueous solutions, buffered at pH 7.0, at low applied potentials (0.0 V vs. SCE).

A Magnetically Drivable Nanovehicle for Curcumin with Antioxidant Capacity and MRI Relaxation Properties

Curcumin possesses wide-ranging anti-inflammatory and anti-cancer properties and its biological activity can be linked to its potent antioxidant capacity. Superparamagnetic maghemite (γ-Fe2 O3 ), called surface-active maghemite nanoparticles (SAMNs) were surface-modified with curcumin molecules, due to the presence of under-coordinated Fe(III) atoms on the nanoparticle surface. The so-obtained curcumin-modified SAMNs (SAMN@curcumin) had a mean size of 13±4 nm. SAMN@curcumin was characterized by transmission and scanning electron microscopy, UV/Vis, FTIR, and Mössbauer spectroscopy, X-ray powder diffraction, bulk susceptibility (SQUID), and relaxometry measurements (MRI imaging). The high negative contrast proclivity of SAMN@curcumin to act as potential contrast agent in MRI screenings was also tested. Moreover, the redox properties of bound curcumin were probed by electrochemistry. SAMN@curcumin was studied in the presence of different electroactive molecules, namely hydroquinone, NADH and ferrocyanide, to assess its redox behavior. Finally, SAMN@curcumin was electrochemically probed in the presence of hydrogen peroxide, demonstrating the stability and reactivity of bound curcumin.

Protein corona as a proteome fingerprint: The example of hidden biomarkers for cow mastitis.

Proteome modifications in a biological fluid can potentially indicate the occurrence of pathologies, even if the identification of a proteome fingerprint correlated to a specific disease represents a very difficult task. When a nanomaterial is introduced into a biological fluid, macromolecules compete to form a protein corona on the nanoparticle surface, and depending on the specific proteome, different patterns of proteins will form the final protein corona shell depending on their affinity for the nanoparticle surface. Novel surface active maghemite nanoparticles (SAMNs) display a remarkable selectivity toward protein corona formation, and they are able to concentrate proteins and peptides presenting high affinities for their surface even if they are present in very low amounts. Thus, SAMNs may confer visibility to hidden biomarkers correlated to the occurrence of a pathology. In the present report, SAMNs were introduced into milk samples from healthy cows and from animals affected by mastitis, and the selectively bound protein corona shell was easily analyzed and quantified by gel electrophoresis and characterized by mass spectrometry. Upon incubation in mastitic milk, SAMNs were able to selectively bind αs2-casein fragments containing the FALPQYLK sequence, as part of the larger casocidin-1 peptide with strong antibacterial activity, which were not present in healthy samples. Thus, SAMNs can be used as a future candidate for the rapid diagnosis of mastitis in bovine milk. The present report proposes protein competition for SAMN protein corona formation as a means of mirroring proteome modifications. Thus, the selected protein shell on the nanoparticles results in a fingerprint of the specific pathology.

Ternary Hybrid γ‐Fe2O3/CrVI/Amine Oxidase Nanostructure for Electrochemical Sensing: Application for Polyamine Detection in Tumor Tissue

Dichromate binds to surface-active maghemite nanoparticles (SAMNs) to form a stable core-shell nanostructures (SAMN@Cr(VI) ). The hybrid was characterized by Mössbauer spectroscopy, high-angle annular dark-field imaging, electron energy-loss spectroscopy, and electrochemical techniques, which revealed a strong interaction of dichromate with the nanoparticle surface. Electrochemical characterization showed lower charge-transfer resistance, better electrochemical performance, and more reversible electrochemical behavior with respect to naked SAMNs. Moreover, SAMN@Cr(VI) is an excellent electrocatalyst for hydrogen peroxide reduction. Furthermore, an enzyme, namely, bovine serum amine oxidase (BSAO: EC 1.4.3.6), was immobilized on SAMN@Cr(VI) by self-assembly to give a ternary hybrid nanostructured catalyst for polyamine oxidation (SAMN@Cr(VI) -BSAO). SAMN@Cr(VI) -BSAO was applied for the development of a reagentless, fast, inexpensive, and interference-free polyamine biosensor, which was successfully exploited for the discrimination of tumorous tissue from healthy tissue in human crude liver extracts.

Colloidal Surface Active Maghemite Nanoparticles for Biologically Safe CrVI Remediation: from Core‐Shell Nanostructures to Pilot Plant Development

The present study is aimed at the exploration of achievable improvements for Cr(VI) ex situ and in situ water remediation by using novel naked colloidal maghemite (γ-Fe2 O3 ) nanoparticles (surface active maghemite nanoparticles, SAMNs). The reliability of SAMNs for Cr(VI) binding and removal was demonstrated, and SAMN@Cr(VI) complex was characterized, as well as the covalent nature of the absorption was unequivocally proved. SAMNs were structurally and magnetically well conserved after Cr(VI) binding. Thus, in consideration of their affinity for Cr(VI) , SAMNs were exploited in a biological model system, mimicking a real in situ application. The assay evidenced a progressive reduction of revertant colonies of Salmonella typhimurium TA100 strain, as maghemite nanoparticles concentration increased, till the complete suppression of Cr(VI) mutagen effect. Finally, an automatic modular pilot system for continuous magnetic removal and recovery of Cr(VI) from water is proposed. SAMNs, thanks to their colloidal, binding, and catalytic properties, represent a promising tool as a reliable nanomaterial for water remediation by Cr(VI) .

Electrocatalytic Nanostructured Ferric Tannates: Characterization and Application of a Polyphenol Nanosensor

A novel core-shell hybrid nanomaterial composed of peculiar maghemite nanoparticles (surface-active maghemite nanoparticles (SAMNs)) as the core and tannic acid (TA) as the shell was developed by self-assembly of ferric tannates onto the surface of SAMNs by simple incubation in water. The hybrid nanomaterial (SAMN@TA) was characterized by using UV/Vis, FTIR, and Mössbauer spectroscopies, magnetization measurements, and X-ray powder diffraction, which provide evidence of a drastic reorganization of the iron oxide surface upon reaction with TA and the formation of an outer shell that consists of a cross-linked network of ferric tannates. According to a Langmuir isotherm analysis, SAMN@TA offers one of most stable iron complexes of TA reported in the literature to date. Moreover, SAMN@TA was characterized by using electrical impedance spectroscopy, voltammetry, and chronoamperometry. The nanostructured ferric tannate interface showed improved conductivity and selective electrocatalytic activity toward the oxidation of polyphenols. Finally, a carbon-paste electrode modified with SAMN@TA was used for the determination of polyphenols in blueberry extracts by square-wave voltammetry.

Magnetic Purification of Curcumin from Curcuma longa Rhizome by Novel Naked Maghemite Nanoparticles

Naked maghemite nanoparticles, namely, surface active maghemite nanoparticles (SAMNs), characterized by a diameter of about 10 nm, possessing peculiar colloidal stability, surface chemistry, and superparamagnetism, present fundamental requisites for the development of effective magnetic purification processes for biomolecules in complex matrices. Polyphenolic molecules presenting functionalities with different proclivities toward iron chelation were studied as probes for testing SAMN suitability for magnetic purification. Thus, the binding efficiency and reversibility on SAMNs of phenolic compounds of interest in the pharmaceutical and food industries, namely, catechin, tyrosine, hydroxytyrosine, ferulic acid, coumaric acid, rosmarinic acid, naringenin, curcumin, and cyanidin-3-glucoside, were evaluated. Curcumin emerged as an elective compound, suitable for magnetic purification by SAMNs from complex matrices. A combination of curcumin, demethoxycurcumin, and bis-demethoxycurcumin was recovered by a single magnetic purification step from extracts of Curcuma longa rhizomes, with a purity >98% and a purification yield of 45%, curcumin being >80% of the total purified curcuminoids.

Enlightening mineral iron sensing in Pseudomonas fluorescens by surface active maghemite nanoparticles: Involvement of the OprF porin

BACKGROUND Mineral iron(III) recognition by bacteria is considered a matter of debate. The peculiar surface chemistry of novel naked magnetic nanoparticles, called SAMNs (surface active maghemite nanoparticles) characterized by solvent exposed Fe(3+) sites on their surface, was exploited for studying mineral iron sensing in Pseudomonas fluorescens. METHODS SAMNs were applied for mimicking Fe(3+) ions in solution, acting as magnetically drivable probes to evaluate putative Fe(3+) recognition sites on the microorganism surface. Culture broths and nano-bio-conjugates were characterized by UV-Vis spectroscopy and mass spectrometry. RESULTS The whole heritage of a membrane porin (OprF) of P. fluorescens Ps_22 cells was recognized and firmly bound by SAMNs. The binding of nanoparticles to OprF porin was correlated to a drastic inhibition of a siderophore (pyoverdine) biosynthesis and to the stimulation of the production and rate of formation of a secondary siderophore. The analysis of metabolic pathways, based on P. fluorescens Ps_22 genomic information, evidenced that this putative secondary siderophore does not belong to a selection of the most common siderophores. CONCLUSIONS In the scenario of an adhesion mechanism, it is plausible to consider OprF as the biological component deputed to the mineral iron sensing in P. fluorescens Ps_22, as well as one key of siderophore regulation. GENERAL SIGNIFICANCE The present work sheds light on mineral iron sensing in microorganisms. Peculiar colloidal naked iron oxide nanoparticles offer a useful approach for probing the adhesion of bacterial surface on mineral iron for the identification of the specific recognition site for this iron uptake regulation in microorganisms.