Avanzate metodologie biosensoristiche per la sicurezza alimentare applicate nel rilevamento di ocratossina A

Ochratoxins are a group of toxic fungal secondary metabolites produced by several fungi of the genera Aspergillus and Penicillium. They can be frequently found in a variety of foodstuffs and feeds, including cereals, coffee, cocoa, spices, beer, wine, blood derived meat products (in particular pork ), etc. The family of ochratoxins consists of several members in which the ochratoxin A (OTA) is the most toxic ones. Indeed, several studies in animals have shown that OTA is a nephrotoxin, hapatotoxic, carcinogen and teratogen and it enters into the alimentary chain by different sources: cultivation practices, production processes, food transportation and storing processes. The identification of the effect of mycotoxins on human health has increased the attention on the detection of these compounds in human and animal food. Although it is difficult to remove mycotoxins from human and animal diets, it is possible to decrease the risk of exposure through a rigorous program of monitoring these mycotoxins in foods and feeds. Monitoring of mycotoxins in animal feeds is especially important because it provides not only a healthier diet for animals, but it also may indirectly prevent any mycotoxin residue carryover in animals for human consumption. Conventional methods used for detection and identification of OTA comprise thin-layer chromatography (TLC), gas chromatography, high-pressure liquid chromatography (HPLC) and mass spectroscopy (MS). Recently, some ELISA assays have been developed too. While these methods can be sensitive and give both qualitative and quantitative information about presence of mycotoxins, they are greatly restricted by assay time and their high cost. In addition they do not allow analysis of mycotoxins in real time.As a consequence, the need for a more rapid, reliable, at low cost, specific and sensitive method for detecting these analytes, is the focus of a great deal of research, especially for applications outside the laboratory environments. In this perspective, the aim of my thesis has been the development of two easy immunosensors based on two advanced optical methodologies for the detection of traces of ochratoxin A. We used Fluorescence Correlation Spectroscopy (FCS) and Surface Plasmone Resonance (SPR) as optical techniques for toxin detection. The FCS immunoassay is based on the measurement of the fluctuations of a fluorescein-labeled compound by a focused laser beam in the absence and in the presence of specific antibodies anti-compound. In this respect, a competitive assay based on the utilization of unlabeled analytes was developed. The obtained results indicated that the combination of high-avidity IgG antibodies together with an innovative fluorescence immunoassay strategy resulted in the detection limit of 0.0078 ng for ochratoxin A, suggesting the application of this experimental strategy for analyses in which a high sensitivity detection is required. As regard the immunoassay-based on SPR technique, we monitored the presence of free ochratoxin A by using an indirect sensing method. In particular, we measured the changes in refractive index of the complex by BIAcore. OTA conjugated to OVA was covalently immobilized onto the surface of a sensor gold chip, a mix of antibodies anti-OTA and free mycotoxins, at different concentrations, were flow continuously over the surface of the chip.We observed that the binding of antibody to the surface of the chip was inversely proportional to the amount of free ochratoxin A in solution.The results presented showed a limit of quantification of OTA in solution was 25 ng/mL, this value is under the low limits for this mycotoxin

FCS-besed sensing for the detection of ochratoxin and neomycin in food

In this work, we present an advanced fluorescence assay for the detection of traces of ocratoxin A and neomycin in food. The described assay is based on measurement of the fluctuations of the fluorescein-labeled analytes by a focused laser beam in the absence and in the presence of the specific antibodies anti-analytes. A competitive assay based on the utilization of unlabeled analytes was developed. The obtained results indicated that the combination of high-avidity IgG antibodies together with an innovative fluorescence immunoassay strategy resulted in the detection limit of 0.0078 ng and 0.0156 ng for ochratoxin A and neomycin, respectively

Molecular strategies for protein stabilization: the case of a trehalose/maltose-binding protein from Thermus thermophilus.

The trehalose/maltose-binding protein (MalE1) is one component of trehalose and maltose uptake system in the thermophilic organism Thermus thermophilus. MalE1 is a monomeric 48 kDa protein predominantly organized in alpha-helix conformation with a minor content of beta-structure. In this work, we used Fourier-infrared spectroscopy and in silico methodologies for investigating the structural stability properties of MalE1. The protein was studied in the absence and in the presence of maltose as well as in the absence and in the presence of SDS at different p(2)H values (neutral p(2)H and at p(2)H 9.8). In the absence of SDS, the results pointed out a high thermostability of the MalE1 alpha-helices, maintained also at basic p(2)H values. However, the obtained data also showed that at high temperatures the MalE1 beta-sheets underwent to structural rearrangements that were totally reversible when the temperature was lowered. At room temperature, the addition of SDS to the protein solution slightly modified the MalE1 secondary structure content by decreasing the protein thermostability. The infrared data, corroborated by molecular dynamics simulation experiments performed on the structure of MalE1, indicated that the protein hydrophobic interactions have an important role in the MalE1 high thermostability. Finally, the results obtained on MalE1 are also discussed in comparison with the data on similar thermostable proteins already studied in our laboratories

Nanobeads-based assays. the case of gluten detection

In order to verify if the use of nanobeads of poly[phenylacetylene-(co- acrylic acid)] (PPA/AA) in the ELISA test would affect the immune-activity of the antibodies (Ab) and/or the activity of the enzymes used to label the Ab anti-rabbit IGg, in this work we immobilized the horse liver peroxidase labelled Ab anti-rabbit IGg onto PPA/AA nanobeads. The gluten test was chosen as the model to demonstrate the usefulness of these nanobeads in immunoassays. The synthesis of PPA/AA nanobeads was performed by a modified emulsion polymerization. Self-assembly of nanospheres with mean diameter equal to 200nm was achieved by casting aqueous suspensions. The materials were characterized by traditional spectroscopic techniques, while the size and dispersion of the particles were analysed by scanning electron microscopy (SEM) measurements. The obtained results show that the immobilization process of the Abs onto PPA/AA did not affect either the immune-response of the Abs or the functional activity of the peroxidase suggesting the usefulness of PPA/AA for the design of advanced nanobeads-based assays for the simultaneous screening of several analytes in complex media. © 2008 IOP Publishing Ltd