Biosonde a cellule per l'identificazione dei pericoli chimico-tossicologici negli alimenti

Contamination of the food production chain can be both natural (eg. mycotoxins) and anthropogenic (eg. pesticides, veterinary antibiotics, monomers released from plastic materials). In this PhD project, the use of cell-based bioprobes has been studied and tested for the identification of chemical-toxicological hazards in food, by employing three cell-based bioprobes: 1) yeast cells-based amperometric bioprobe; 2) bacterial cells-based optical bioprobe; 3) mammalian origin-cells- based potentiometric bioprobe; 1. Wild-type (wt) and modified Saccharomyces cerevisiae cells amperometric bioprobe: The interference on cellular aerobic respiration was studied for different doses and exposure time to bisphenol A and B, monomers of plastic materials for foods, agro-herbicide diuron and wood preservatives (based on heavy metals) used for example vineyard trellis: • Bisphenol A and B (from 5 to 100 ppm; 24 h exposure); the obtained results with wt exposed cells show two types of interference: (a) hyperstimulation-type (respiratory capacity beyond the maximum); (b) inhibitory-type (reduced respiration capacity). The obtained trend of respiratory interference leads to hypothesise, with the support of the literature, the involvement of two mechanisms of: i) uncoupling of oxidative phosphorylation; ii) mitochondrial oxidative damage. • Diuron (10-10 - 10-6 M; 6 and 24 h exposure); the obtained results with wt cells showed a 6 h exposure to 10-10 - 10-7 M a partial inhibition of cellular respiration (~ 11-13%) and no effect at 10-6 M, while at 24 h exposure the results show a partial recovery of respiration activity of up to 10-7 M (~ 7-12%), while an inhibitory effect appears (~ 6%) for 10-6 M. These results, with the support of the literature, lead us to assume the involvement of three mechanisms: i) toxicity, ii) detoxification and iii) cellular repair; • Wood preservatives based on heavy metals; the obtained results with modified cells (spheroplasts), which effects seem to be related to the onset of a mitochondrial oxidative stress, show an increased sensitivity of the cells. 2. Wt and transgenic Escherichia coli-based optical bioprobe for the detection of ciprofloxacin (a quinolone veterinary antibiotic) in bovine milk. Two different strains of E. coli (ATCC 11303 and DH5α) were used as well as different methodologies. With the wt E. coli ATCC 11303 strain, cell proliferation tests were performed with the following methods: • Spectrophotometric monitoring of β-galactosidase enzyme activity induction; • Fluorimetric monitoring of the fluorophore mCherry which gene expression is modulated by the lactose inducible lac promoter. With the first method best results were obtained in terms of speed of response (60 min) in the presence of ciprofloxacin 1 x MRL (maximum residual limit). Using engineered E. coli DH5α strain (inducible bioreporter) fluorimetric tests were performed monitoring fluorophore mCherry (modulated by the quinolone inducible yorB promoter). The development of the experimental protocol allowed to reduce observed interferences and obtain meaningful answers after 120 min for cultures exposed to ciprofloxacin 20 x MRL and after 180 min for cultures exposed to ciprofloxacin 1x MRL; the use of the Green Fluorescent Protein as fluorophore showed no improvement in the method. 3. Modified (by insertion of antibodies) Vero cells-based potentiometric bioprobe for the detection of aflatoxin B1 (AFB1) This probe was developed at the Agricultural University of Athens (BERA System). In particular Vero cells were modified (insertion of anti-AFB1 antibodies) and they were employed for potentiometric measurements in the presence of AFB1. Based on the obtained results, the bioprobe seems to be able to detect the presence of AFB1 in concentration range 2-20 ppb.