Livia Giotta

Photosynthetic Machineries in Nano-Systems

Photosynthetic reaction centres are membrane-spanning proteins, found in several classes of autotroph organisms, where a photoinduced charge separation and stabilization takes place with a quantum efficiency close to unity. The protein remains stable and fully functional also when extracted and purified in detergents thereby biotechnological applications are possible, for example, assembling it in nano-structures or in optoelectronic systems. Several types of bionanocomposite materials have been assembled by using reaction centres and different carrier matrices for different purposes in the field of light energy conversion (e.g., photovoltaics) or biosensing (e.g., for specific detection of pesticides). In this review we will summarize the current status of knowledge, the kinds of applications available and the difficulties to be overcome in the different applications. We will also show possible research directions for the close future in this specific field.

Reversible Binding of Metal Ions onto Bacterial Layers Revealed by Protonation-Induced ATR-FTIR Difference Spectroscopy

The ability of microorganisms to adhere to abiotic surfaces and the potentialities of attenuated total reflection Fourier transform infrared (ATR-FTIR) spectroscopy have been exploited to study protonation and heavy metal binding events onto bacterial surfaces. This work represents the first attempt to apply on bacteria the recently developed method known as perfusion-induced ATR-FTIR difference spectroscopy. Such a technique allows measurement of even slight changes in the infrared spectrum of the sample, deposited as a thin layer on an ATR crystal, while an aqueous solution is perfused over its surface. Solutions at different pH have been used for inducing protonation/deprotonation of functional groups lying on the surface of Rhodobacter sphaeroides cells, chosen as a model system. The interaction of Ni(2+) with surface protonable groups of this microorganism has been investigated with a double-difference approach exploiting competition between nickel cations and protons. Protonation-induced difference spectra of simple model compounds have been acquired to guide band assignment in bacterial spectra, thus allowing identification of major components involved in proton uptake and metal binding. The data collected reveal that carboxylate moieties on the bacterial surface of R. sphaeroides play a role in extracellular biosorption of Ni(2+), establishing with this ion relatively weak coordinative bonds.

Evaluation of Possible Contamination Sources in the 14C Analysis of Bone Samples by FTIR Spectroscopy

In the sample preparation laboratory of CEDAD (CEnter for DAting and Diagnostics) of the University of Lecce, a protocol for the quality control of bone samples based on infrared spectroscopy has been set up. The protocol has been recently developed as a check-in test with the aim to identify the presence of collagen in the samples, assess its preservation status, and determine whether the submitted bone samples are suitable for accelerator mass spectrometry (AMS) radiocarbon measurements or not. We discuss in this paper the use of infrared-based techniques to identify the presence of contaminants such as restoration and consolidation materials, humic acids, and soil carbonates, which, if not removed by the sample processing chemistry, can be sources of exogenous carbon and can thus influence the accuracy of the 14C determinations. Bone samples recovered in well-defined and previously dated archaeological contexts were intentionally contaminated, submitted to the standard method for collagen extraction and purification, and then characterized by means of Fourier transform infrared (FTIR) spectroscopy analyses performed in attenuated total reflection (ATR) mode before being combusted, converted to graphite, and measured by AMS. The study shows that the ATR-FTIR technique is an extremely powerful method for the identification of both the collagen and its contaminants and can supply important information during the selection and processing of samples to be submitted for 14C dating.

Development and characterization of a novel bioactive polymer with antibacterial and lysozyme-like activity

The development and characterization of a novel bioactive polymer based on the immobilization of glucose oxidase enzyme (GOx) in a polyvinyl alcohol (PVA) film showing antibacterial activity is presented. The PVA‐GOx composite material was extensively characterized by UV‐vis, X‐ray Photoelectron (XPS) spectroscopy and by Fourier Transform Infrared (FTIR) spectroscopy to verify the preservation of enzyme structural integrity and activity. The antimicrobial activity of this composite material against Escherichia coli and Vibrio alginolyticus was assessed. Furthermore the lysozyme‐like activity of PVA‐GOx was highlighted by a standard assay on Petri dishes employing Micrococcus lysodeikticus cell walls. The findings from this study have implications for future investigations related to the employment of PVA‐GOx system as a composite material of pharmaceutical and technological interest.

Phenol chemisorption onto phthalocyanine thin layers probed by ATR-FTIR difference spectroscopy

Attenuated total reflection Fourier transform infrared (ATR-FTIR) difference spectroscopy has been employed as a powerful method for the comprehension of active layer-analyte interactions, revealing interesting mechanistic aspects concerning the binding of halogen-substituted phenols onto phthalocyanine Langmuir-Schaefer films.

Electrochemical and Spectroscopic Behavior of Iron(III) Porphyrazines in Langmuir−Schäfer Films

Thin films of a newly synthesized iron(III) porphyrazine, LFeOESPz ( L = ClEtO, OESPz = ethylsulfanylporphyrazine), have been deposited by the Langmuir-Schafer (LS) technique (horizontal lifting) on ITO or gold substrates. Before deposition, the floating films have been investigated at the air-water interface by pressure/area per molecule (pi/ A) experiments, Brewster angle microscopy (BAM) and UV-vis reflection spectroscopy (RefSpec). The complex reacts with water subphase (pH 6.2) forming the mu-oxo dimer, which becomes the predominant component of the LS films ( LS-Fe) as indicated by optical, IR, XPS, and electrochemical data. LS-Fe multilayers exhibit, between open circuit potential (OCP) and +0.90 V (vs SCE), two independent peak pairs with formal potentials, E surf (I) and E surf(II) of +0.56 V and +0.78 V, respectively. According to dynamic voltammetric and coulometric experiments the peak pair at +0.56 V is attributed to one-electron process at the iron(III) centers on the monomer, while the peak pair at +0.78 V is associated to a four-electron process involving mu-oxo-dimer oligomers. LS-Fe films prove to be quite stable electrochemically between OCP and +0.90 V. The electrochemical stability decreases, however, when the potential range is extended both anodically and cathodically outside these limits, due to formation of new species. Upon incubation with TCA solutions, LS-Fe films show remarkable changes in the UV-vis spectra, which are consistent with a significant mu-oxo dimer --> monomer conversion. Addition of TCA to the electrochemical cell using a LS-Fe film as working electrode, results in a linear increase of a cathodic current peak near -0.40 V as the TCA concentration varies in the 0.1-2.0 mM range. This behavior is interpreted in terms of TCA inducing a progressive change in the composition of the LS-Fe films in favor of the monomeric iron(III) porphyrazine, which is responsible for the observed increase in the cathodic current near -0.40 V.

Lipid/detergent mixed micelles as a tool for transferring antioxidant power from hydrophobic natural extracts into bio-deliverable liposome carriers: the case of lycopene rich oleoresins

This work demonstrates that lipid-detergent mixed micelles can be employed successfully in order to achieve and modulate the transfer of bio-active hydrophobic compounds into lipid carriers by means of a simple and bio-safe procedure. In our specific investigation, liposome preparations incorporating mixtures of natural carotenoids with high lycopene content were developed and characterized, aiming to obtain formulations of potential nutraceutical and pharmaceutical interest. The starting material was a solvent-free high-quality lycopene rich oleoresin (LRO) obtained by extracting a freeze-dried tomato matrix with supercritical carbon dioxide (SC-CO2). Mixed micelles containing 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine (POPC) and cholate were loaded with LRO antioxidants by means of two slightly different procedures, which surprisingly resulted in significant differences in both quality and quantity of incorporated carotenoids. In particular, the selective incorporation of (all-E)-lycopene was achieved by extracting the oleoresin with a pre-formed cholate/POPC micelle suspension whilst (Z)-isomers were preferentially integrated when treating a POPC/LRO mixed film with cholate. The micelle to vesicle transition (MVT) method was employed in order to produce vesicles of well-defined lamellarity and size. Visible and infrared (IR) spectroscopy as well as Dynamic Light Scattering (DLS) and Transmission Electron Microscopy (TEM) measurements allowed the extensive characterization of LRO-loaded micelles and liposomes. The antioxidant potential of preparations was assessed by measuring the radical scavenging activity towards the coloured radical cation of 2,2′-azino-bis(3-ethylbenzothiazoline-6-sulphonate) (ABTS). Important information about the reliability of different approaches for antioxidant capacity evaluation of micelle and liposome preparations was gained and the successful incorporation of LRO antioxidant power in a bio-deliverable water-dispersed form was demonstrated.

Efficient stabilization of natural curcuminoids mediated by oil body encapsulation

Curcumin is a natural hydrophobic polyphenol found in the powdered rhizomes of Curcuma longa. Due to its capacity to interfere with many signalling pathways, it has been shown that curcumin has potential beneficial pharmacological effects including antioxidant, anti-inflammatory, anticarcinogenic properties. However, the use of curcumin is fairly restricted because of its poor water solubility, low bioavailability, inadequate tissue absorption and degradation at alkaline pH. In the present contribution, we first verified the anti-proliferative effects of natural curcuminoids towards two different cell lines derived from an ovarian and a breast adenocarcinoma cancer. Later, curcuminoids were successfully encapsulated into reconstituted oil bodies. Once encapsulated into the triacylglycerol cores of the reconstituted oil bodies, curcumin, the most hydrophobic and active of the three curcuminoids, was better stabilized in comparison with albumin stabilization. Oil body encapsulated curcuminoids showed the same effects on cancer cell viability as the free drug, confirming the great potential of natural oil bodies as micro/nano-capsules in drug delivery applications.

Light induced transmembrane proton gradient in artificial lipid vesicles reconstituted with photosynthetic reaction centers

Photosynthetic reaction center (RC) is the minimal nanoscopic photoconverter in the photosynthetic membrane that catalyzes the conversion of solar light to energy readily usable for the metabolism of the living organisms. After electronic excitation the energy of light is converted into chemical potential by the generation of a charge separated state accompanied by intraprotein and ultimately transmembrane proton movements. We designed a system which fulfills the minimum structural and functional requirements to investigate the physico/chemical conditions of the processes: RCs were reconstituted in closed lipid vesicles made of selected lipids entrapping a pH sensitive indicator, and electron donors (cytochrome c2 and K4[Fe(CN)6]) and acceptors (decylubiquinone) were added to sustain the photocycle. Thanks to the low proton permeability of our preparations, we could show the formation of a transmembrane proton gradient under illumination and low buffering conditions directly by measuring proton-related signals simultaneously inside and outside the vesicles. The effect of selected ionophores such as gramicidin, nigericin and valinomycin was used to gain more information on the transmembrane proton gradient driven by the RC photochemistry.

Effect of Cobalt Ions on the Soluble Proteome of a Rhodobacter sphaeroides Carotenoidless Mutant

Rhodobacter sphaeroides strain R26.1 showed tolerance to Co2+ ions, up to 10 mM concentration. Interestingly the bacteriochlorophyll biosynthesis was found to decrease upon addition of such metal in the growth medium. Analysis of R. sphaeroides proteome from cells grown in control and in Co2+ enriched media was performed by two dimensional electrophoresis (2DE) followed by mass spectrometry. Proteome and functional analyses of differentially expressed proteins in cobalt response clearly highlighted the involvement of several metabolic pathways, and in particular of some enzymes involved in tetrapyrrole biosynthesis pathway.