Avanzate metodologie biosensoristiche per la sicurezza alimentare applicate nel rilevamento di ocratossina A
Iozzino, Luisa (2010) Avanzate metodologie biosensoristiche per la sicurezza alimentare applicate nel rilevamento di ocratossina A. [Tesi di dottorato] (Inedito)
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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.
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