Biologically friendly room temperature ionic liquids and nanomaterials for the development of innovative enzymatic biosensors.

Main purpose of the work is assembling, testing and optimizing new disposable amperometric biosensors to analyze substances in different application fields as agribusiness, clinical chemistry and environment protection. Many kinds of modified electrodes have been prepared and tested to build portable devices to analyze quickly many analytes, in a simple and cost-effective manner. Bare electrodes of the screen-printed type, with silver as reference, have been used for modification. The glassy carbon electrodes with multi-walled carbon nanotubes or graphene or gold nanoparticles depositions were modified with generation IV ionic liquids. Choline as cation and amino acids, such as glycine, serine, phenylalanine and histidine, as anions have been employed for these ionic liquids. The presence of nanostructured materials on the electrode brings an increased contact surface between analytes and receptor and, consequently, an amplification of the amperometric signal and a better sensibility. Moreover the use of new ionic liquids of generation IV, biologically friendly and water soluble, improves the electronic transfer, facilitating and strengthening the redox reaction nearby the electrode. By immobilizing the proper enzymes onto the modified electrode surface, different compounds of analytical interest can be determined by means of sensitive, properly designed amperometric biosensors. Analytes such as antioxidant components in extra-virgin olive oils, alcohols in beverages and glucose in food matrices have been tested, using a suitable enzyme: microbial lipase, alcohol dehydrogenase and glucose oxidase, respectively.

Nanostructured enzymatic biosensor based on fullerene and gold nanoparticles: preparation, characterization and analytical applications.

In this work a novel electrochemical biosensing platform based on the coupling of two different nanostructured materials (gold nanoparticles and fullerenols) displaying interesting electrochemical features, has been developed and characterized. Gold nanoparticles (AuNPs) exhibit attractive electrocatalytic behavior stimulating in the last years, several sensing applications; on the other hand, fullerene and its derivatives are a very promising family of electroactive compounds although they have not yet been fully employed in biosensing. The methodology proposed in this work was finalized to the setup of a laccase biosensor based on a multilayer material consisting in AuNPs, fullerenols and Trametes versicolor Laccase (TvL) assembled layer by layer onto a gold (Au) electrode surface. The influence of different modification step procedures on the electroanalytical performance of biosensors has been evaluated. Cyclic voltammetry, chronoamperometry, surface plasmon resonance (SPR) and scanning tunneling microscopy (STM) were used to characterize the modification of surface and to investigate the bioelectrocatalytic biosensor response. This biosensor showed fast amperometric response to gallic acid, which is usually considered a standard for polyphenols analysis of wines, with a linear range 0.03-0.30 mmol L(-1) (r(2)=0.9998), with a LOD of 0.006 mmol L(-1) or expressed as polyphenol index 5.0-50 mg L(-1) and LOD 1.1 mg L(-1). A tentative application of the developed nanostructured enzyme-based biosensor was performed evaluating the detection of polyphenols either in buffer solution or in real wine samples.

Construction, assembling and application of a trehalase-GOD enzyme electrode system.

Trehalose is a disaccharide important in foods, serving as a glucose source in many and also as an additive in the food preparation. Because of its peculiar physico-chemical properties it plays an important role as preservative in drying and deep-freezing treatments. A new biosensor for trehalose determination has been realized by means of a flow system, based on a reactor in which the trehalase enzyme catalyses its hydrolysis into two alpha,d-glucose molecules, and a GOD (glucose oxidase) amperometric biosensor is employed for the glucose determination. The optimum operative conditions have been laid out and a particular attention has been paid to the immobilization procedure of the two enzymes. The electrode used is of the SPE (screen-printed electrode) type and has been activated with the Prussian Blue (PB) and then assembled using GOD immobilized with Nafion. The reactor has been prepared with the trehalase enzyme chemically immobilized on an Immunodyne ABC membrane. As demonstration of its utility, the biosensor has been tested on a real sample of Boletus edulis mushroom.

Determination of free fatty acids and lipase activity in milk: quality and storage markers.

The free fatty acid (FFA) content together with the lipase activity control can be considered as useful indexes of good quality and correct storage of food, especially for milk. The quantitative analysis of FFA in different kinds of milk has been performed by a potentiometric method, using a new extractive methodology outlined herein. The lipase activity has been controlled by a sensitive calorimetric method, previously validated by us, based on the direct measure of the heat quantity involved in the enzymatic reaction. In order to verify the milk quality after the healing treatments and/or during the shelf life, the behaviours of FFA content and of lipase activity have been outlined in function of storage time and pH variations on different typologies of milk. The FFA content in sample of fresh pasteurised milk was found to be quite high after the 5th/6th day of storage at +4 degrees C, meanwhile the pH values were always constant and only after the 9th day begun to decrease. At the same time the lipase activity, directly measured, was found to be appreciable after the 6th day of storage, giving an exothermic answer at the calorimeter, similar to that of a milk sample where only three international units of standard lipase were added.

Food analyses: a new calorimetric method for ascorbic acid (vitamin C) determination.

The analysis of the ascorbic acid (vitamin C; A.A.) contained in some foodstuff and pharmaceutical samples was performed by a new microcalorimetric method. It uses the oxidation of the vitamin C catalysed by the enzyme ascorbate oxidase (A.O.), which gets the specificity of the reaction. The calibration curve was built under the following operative conditions: 25.00 degrees C, pH 5.6, [A.O.]=11 IU ml(-1), the linearity range is: 3

Orotic acid: a milk constituent Enzymatic determination by means of a new microcalorimetric method.

The presence of orotic acid (a precursor of nucleic acid) in milk is very important in order to ensure its nutritional value and good conservation. In the literature, chromatographic, spectrophotometric and polarographic methods are reviewed. The reported values have a very wide interval range (19-664 mg l(-1)) and a low precision. The new method proposed in this article employs an enzymatic reaction. It has been improved on standards and then tested on milk samples. The same samples were also tested by means of a known spectrophotometric method. The new analytical method for orotic acid determination is reliable; the results are more accurate and more precise if compared with the usual methods, and it shows the same sensibility. The calorimetric analysis is faster and easier, owing to the fact that no sample treatments are required.

Studies about the adsorption on lichen Evernia prunastri by enthalpimetric measurements.

Lichens are bioaccumulators of divalent metal ions and the interaction between the lichens and five heavy metals was studied. In order to have a better understanding of the metal-uptake process by the lichens, data from atomic absorption spectroscopy were used to calculate the apparent coordination constants of the lichen-metal ion interactions by means of the Langmuir elaboration and microcalorimetric measurements to obtain enthalpimetric information. The results showed a correlation between the Langmuir constants and enthalpimetric measurements.

Noncovalent interactions in ternary complexes of spermine and copper(II) with adenosine 5'-triphosphate and tripolyphosphate.

Thermodynamic characteristics pertinent to the formation equilibria of two ternary systems: 1) Copper(II), 4,9-diazadodecane-1,12-diamine (spermine, Spe), and adenosine 5'-triphosphate (ATP) and 2) Copper(II), Spe, and tripolyphosphate (TPP) have been determined by means of potentiometric and calorimetric techniques, together with the parent binary complex characteristics. Ternary complexes involving ATP can give information useful in the interpretation of bioenergetic reactions and of biological interactions between nucleic acids and polyamines. As a model system, the TPP-containing ternary complexes have been studied, together with the parent binary complexes. The thermodynamic study of these systems is very important because it can give information about the structural environment of the complexes; moreover, it can help in outlining different noncovalent interactions such as coulombic forces and hydrogen bonds.

Thermodynamics of simple and mixed complexes of copper(II) with adenosine 5'-monophosphate and spermine.

Thermodynamic parameters pertinent to the equilibria of ternary system copper(II), adenosine 5'-monophosphate (AMP), and 4,9-diazadodecane-1,12-diamine (Spermine, Spe) were determined by means of potentiometric and calorimetric techniques, together with the corresponding parent binary complex parameters. This system could be considered as a model in the study of more complicated biological systems. In particular, it could give useful evidence in the investigation of the interaction between tetramine and DNA. The experimental results seem to suggest that the major contribution in DNA stabilization by means of tetramines is due to the electrostatic interactions occurring between the protonated nitrogen atoms of the amine molecules and the phosphate groups of nucleotides in the nucleic chains.