Marta Letizia Antonelli

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.

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</=A.A.</=270 mg l(-1). The method has been applied to many samples to determine the vitamin C quantity. Each analysis takes about 20 min (the assay time is instead about 5 min, the slow step is due to the time necessary for the calorimeter to return to the operative temperature after washing). No pre-treatments of the samples are requested, unless their solubilisation in buffer. The variations of vitamin C concentration were studied in function of storage time and light exposition. Using a known HPLC assay to compare its reliability on a drug analysis has validated the proposed method.

Fast determination of biogenic amines in beverages by a core–shell particle column

A fast and reliable HPLC method for the determination of 11 biogenic amines in beverages has been performed. After pre-column derivatization with dansyl-chloride a Kinetex C18 core-shell particle column (100 mm × 4.6 mm, 2.6 μm particle size) has been employed and the biogenic amines were identified and quantified in a total run time of 13 min with ultraviolet (UV) or fluorescence detection (FLD). Chromatographic conditions such as column temperature (kept at 50 °C), gradient elution and flow rate have been optimized and the method has been tested on red wine and fruit nectar. The proposed method is enhanced in terms of reduced analysis time and eluent consumption with respect of classical HPLC method as to be comparable to UHPLC methods. Green and cost-effective, this method can be used as a quality-control tool for routine quantitative analysis of biogenic amines in beverages for the average laboratory.

Solution properties of octyl-β-D-glucoside. Part 2: Thermodynamics of micelle formation

Solution properties of octyl-β-D-glucoside have been examined in a wide temperature and concentration range, by means of freezing point depression, volumetric and calorimetric methods. Partial molal quantities and the variations consequent to micellization have been evaluated and discussed. Strong support to the hypothesis of a growth of the micellar aggregates was inferred from heat capacity findings.

Simultaneous quantitation of free and conjugated phytoestrogens in leguminosae by liquid chromatography-tandem mass spectrometry

Phytoestrogens are diphenolic compounds that are present in several edible plants and are particularly abundant in soybeans. Because of their estrogenical, antriestrogenical, anticarcinogenic and antioxidant activities in animal and humans, they became of great interest. Dietary factors are considered important in determination of risks, in fact, studies have revealed beneficial or protective effects of the consumption of vegetables, in particular soy and soybean products. So that in the present paper the simultaneous determination of eight isoflavones and coumestrol in vegetables is reported. The quantitative analysis has been made by means of LC separation combined with tandem mass spectrometry. In particular, a new simple and fast extraction methodology and a clean-up, based on cold aided de-fatting, is proposed. Method performance was evaluated by comparison with a reference procedure. The developed procedure was then used for a survey of phytoestrogens concentration in some selected vegetables.

Microcalorimetric study of the enzymatic hydrolysis of starch : an α-amylase catalyzed reaction

Abstract Microcalorimetric studies of enzyme activities have been made on the hydrolysis of starch catalyzed by α-amylase. The best conditions for the hydrolysis of starch in the presence of α-amylase have been pointed out and the α-amylase activity was determined by isothermal batch microcalorimetry. The heat changes involved during the hydrolytic reaction can be related to the starch concentrations in a linear way and the heights of the calorimetric curves can be directly related to the amylase activities. Calibration curves were obtained. The new method for starch assay and α-amylase activity determination is direct, simple, accurate and it needs no pretreatment of the sample or auxiliary reactions. The α-amylase catalytic activity has also been investigated by microcalorimetry in the presence of some metal ions.

A microcalorimetric sensor for food and cosmetic analyses: l-Malic acid determination.

Enzymatic microcalorimetry has been successfully employed in the reliable determination of the l-malic acid concentration in some foods and cosmetic products. The l-malic acid concentration during the wine-making process is particularly useful in order to control the progress of the malo-lactic fermentation. Total acidity, taste and flavour characteristics of wine depend on the l-malic acid quantity still present. To point out the analytical methodology the dehydration process of l-malic acid, in the presence of Fumarase enzyme, has been used. The new method has been compared with a common spectrophotometric one. By the proposed calorimetric method the l-malic acid concentration in different types of food (white and red wines, fruits and soft beverages) has been determined. In some cosmetic products too the l-malic acid was quantified. The method outlined resulted simple, direct and reliable (good accuracy and precision), in particular it does not require any pre-treatment or clean up of the samples, save the dilution in buffer.

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.

Rapid determination of polycyclic aromatic hydrocarbons in rainwater by liquid–liquid microextraction and LC with core‐shell particles column and fluorescence detection

Liquid-liquid microextraction coupled to LC with fluorescence detection for the determination of Environmental Protection Agencys 16 priority pollutant polycyclic aromatic hydrocarbons in rainwater has been developed. The optimization of the extraction method has involved several parameters, including the comparison between an ultrasonic bath and a magnetic stirrer as extractant apparatus, the choice of the extractant solvent, and the optimization of the extraction time. Liquid-liquid microextraction gave good results in terms of recoveries (from 73.6 to 102.8% in rainwater) and repeatability, with a very simple procedure and low solvent consumption. The reported chromatographic method uses a Core-Shell technology column, with particle size <3 μm instead of classical 5-μm particles column. The resulting backpressure was below 300 bar, allowing the use of a conventional HPLC system rather than the more expensive ultrahigh performance LC (UHPLC). An average decrease of 59% in run time and 75% in eluent consumption has been obtained, compared to classical HPLC methods, keeping good separation, sensitivity, and repeatability. The proposed conditions were successfully applied to the determinations of polycyclic aromatic hydrocarbons in genuine rainwater samples.

COMPLEXING CAPACITY OF DIFFERENT MOLECULAR WEIGHT FRACTIONS OF SEDIMENTARY HUMIC SUBSTANCES

In this paper we studied the complexing capacity of different molecular weight fractions of humic substances, subdivided into humic (HA) and fulvic acids (FA), extracted from Arno river sediment. Humic acids are characterised by a high degree of aromaticity and a low nitrogen content whereas fulvic acids display aliphatic features and are characterised by a number of oxygen and nitrogenous functional groups such as carboxylic, alcoholic and/or phenolic and peptide groups. The higher degree of condensation of humic acids than fulvic acids is confirmed by their different molecular weight distribution; HA molecules are characterised by a narrow range of molecular weights while fulvic acids are distributed over a wider range of molecular weights. The complexation capacity for HA and FA increases with increasing pH value and at the same pH value the CC for HA and FA is equal even if structural features and molecular weight distribution are different. Moreover, the calorimetric measurements relative to fulvic acids at different pH values show that by increasing the pH, the quantity of heat involved in the metal ion-fulvate interactions changes from an exothermic response to an endothermic one. This shows that pH value influences the different reactions involved in the binding process, such as coordination reactions, electrostatic interactions, deprotonation due to exchange of copper with proton, interactions between hydrolysis products of copper and fulvic acids, in different ways.