Nanostructured materials based on the integration of ferrocenyl-tethered dendrimer and redox proteins on self-assembled monolayers: an efficient biosensor interface.

In this paper we report the use of ferrocenyl-tethered dendrimer (Fc-D) as an electrode modifier supported by a self-assembled monolayer coated gold surface. The pretreatment of electrodes with Fc-D allows the covalent immobilization of glucose oxidase. The resulting integrated hybrid system provides electrical contact between the redox center of the enzyme and the electrode, and improves the overall bioelectrocatalyzed oxidation of glucose. Cyclic voltammetry combined with surface plasmon resonance (SPR) is used to investigate the redox-induced orientation changes of ferrocene-tethered dendrimers and the optimal electrical wiring of the enzyme, depending on the length of the alkyl chain of the ferrocene-tethered groups. The amount of substrate controls the steady-state concentration ratio of Fc/Fc(+) in the film composition. Therefore, the SPR spectrum of the film is controlled by the reversible change in the refractive index of the enzyme-integrated redox film. The proposed method demonstrates a new procedure for developing a stable amperometric redox enzyme-based sensor by designing a new nanostructured material that control the biosensing performance.

Morphological and physiological responses of barley plants grown on soils characterised by metal toxicity and metal deficiency.

Aim of this work was to investigate which are the effects on barley crops grown on two different soils: a soil lacking in Cu, an essential micronutrient (A) and a naturally polluted soil rich in lead, zinc, copper (B). In particular we investigated the relationship between some ecophysiological parameters such as biomass, chlorophyll concentration and guaiacolo peroxidase activity and the chemical-physical properties of the soils like pH, organic matter and heavy metal content. Because metals uptake by plants is strongly correlated with the bioavailable fraction rather then their total amount in a soil, we have measured also metal exchangeable forms, using a single extraction method (MgCl2 as extractant). Plants grown on soil B showed a metal content higher than background limits, whereas plants grown on soil A were characterised by a background Fe and Zn concentrations and by a tolerant Pb concentration. Conversely, Cu content in tissues of plants grown in soil A is found to be under the background limits. Copper-deficiency plants present chlorotic leaves followed by a reduced clorophyll content, while plants grown on metals contaminated soil showed an increase of peroxidase activity.

Determination of the minimal functional ligand-binding domain of the GABAB1b receptor.

In the mammalian central nervous system, slow inhibitory neurotransmission is largely mediated by metabotropic GABA(B) receptors (where GABA stands for gamma-aminobutyric acid), which belong to the G-protein-coupled receptor gene family. Functional GABA(B) receptors are assembled from two subunits GABA(B1) (GABA(B) receptor subtype 1) and GABA(B2). For the GABA(B1) subunit, which binds the neurotransmitter GABA, two variants GABA(B1a) (GABA(B) receptor subtype 1 variant a) and GABA(B1b) have been identified. They differ at the very N-terminus of their large glycosylated ECD (extracellular domain). To simplify the structural characterization, we designed truncated GABA(B1) receptors to identify the minimal functional domain which still binds a competitive radioligand and leads to a functional, GABA-responding receptor when co-expressed with GABA(B2). We show that it is necessary to include all the portion of the ECD encoded by exon 6 to exon 14. Furthermore, we studied mutant GABA(B1b) receptors, in which single or all potential N-glycosylation sites are removed. The absence of oligosaccharides does not impair receptor function, suggesting that the unglycosylated ECD of GABA(B1) can be used for further functional or structural investigations.

Laccase-based biosensor for the determination of polyphenol index in wine.

In this work we have developed and characterized the use of Laccases from Trametes versicolor (TvL) and Trametes hirsuta (ThL) as biocatalytic components of electrochemical biosensors for the determination of polyphenol index in wines. Polyazetidine prepolimer (PAP) was used as immobilizing agent, multi-walled and single-walled carbon nanotubes screen-printed electrodes as sensors (MWCNTs-SPE and SWCNTs-SPE) and gallic acid as standard substrate. The amperometric measurements were carried out by using a flow system at a fixed potential of -100 mV vs. silver/silver chloride electrode in Britton-Robinson buffer 0.1 mol L(-1), pH 5. The results were compared with those obtained with the Folin-Ciocalteau reference method. The results obtained in the analysis of twelve Italian wines put in evidence the better suitability of ThL-MWCNTs-based biosensor in the determination of the polyphenol index in wines. This biosensor shows fast and reliable amperometric responses to gallic acid with a linear range 0.1-18.0 mg L(-1) (r(2)=0.999). The influence of the interferences on both spectrophotometric and electrochemical measurements have been carefully evaluated.

Spectroscopic and electrochemical characterization of cytochrome c encapsulated in a bio sol-gel matrix.

Sol-gel technique represents a remarkably versatile method for protein encapsulation. To enhance sol-gel biocompatibility, systems envisaging the presence of calcium and phosphates in the sol-gel composition were recently prepared and investigated. Unfortunately, the low pH at which solutions were prepared (pH < 2.5) dramatically limited their application to proteins, because the acidic environment induces protein denaturation. In this paper we apply a new protocol based on the introduction of calcium nitrate to the inorganic phase, with formation of a binary bioactive system. In this case protein encapsulation results versatile and secure, being achieved at a pH close to neutrality (pH 6.0); also, the presence of calcium is expected to enhance system biocompatibility. To determine the properties of the salt-doped sol-gel and the influence exerted on entrapped biosystems, the structural and functional properties of embedded cytochrome c have been investigated. Data obtained indicate that the salt-doped sol-gel induces no significant change in the structure and the redox properties of the embedded protein; also, the matrix increases protein stability. Interestingly, the presence of calcium nitrate appears determinant for refolding of the acid-denatured protein. This is of interest in the perspective of future applications in biosensoristic area.

Structure and oligomeric state of the mammalian tumour-associated antigen UK114.

The tumour-associated antigen UK114, isolated from goat liver, belongs to the YER057c/YIL051c/YjgF protein family, which has members in both the prokaryotes and eukaryotes. The crystal structure of a mammalian representative, goat UK114, was determined, revealing a trimeric arrangement in the crystal. It was confirmed by ultracentrifugation that UK114 is a trimer in solution. These results are in agreement with the published structures of homologues from unicellular organisms, but contrast with those reported for the rat homologue of UK114, for which a dimeric quaternary structure was proposed.