MYCODIAG

Ochratoxin A (OTA) is the most common mycotoxin found in our temperate regions that contaminates food commodities prior to harvest or more commonly during storage. OTA inhibits protein synthesis and lipid peroxidation by oxidative processes. These mechanisms may generate nephrotoxic, neurotoxic and immunotoxic effects. After its ingestion or its inhalation, OTA reaches blood streams and is transported to kidney that ensured its biotransformation in metabolites that are responsible of its toxicity and that are present in biological fluids. Therefore, there is an important request for fast, reliable and low-cost analytical methods for the monitoring of OTA in food. Moreover, inhalation in the workplace could be considered as a route of exposure additional to the consumption. The conventional method used for the identification of OTA is based on the use of liquid chromatography and fluorescence detection (LC/Fluo), the native fluorescence of OTA favoring the development of a very sensitive method. An immunoaffinity column (IAC) for the sample treatment is currently associated to LC/Fluo to remove matrix components. The study of the impact of OTA in inhalation studies requires a very sensitive method. It also requires a method of sampling adapted to small-size samples preventing the use of IACs. Moreover, underestimation of OTA contamination in food has been already reported. At last, antibodies involved in IAC are not adapted to the selective extraction of the OTA metabolites and of OTA DNA adducts that have to be studied in biological fluids and tissue to understand the action mode of OTA and then explain its toxicity. So, the lack of powerful methods for these OTA analogues constitutes a real limitation for toxicological studies. The aim of MYCODIAG is to propose more powerful and less expensive extraction devices as an alternative to IAC by developing molecularly imprinted polymers (MIP) and aptamer based sorbents for the selective extraction of OTA and its structural analogs from complex samples. Both approaches will be developed in conventional format to be evaluated and compared with IAC and also in miniaturized formats (pipet tips, strips, microsystems) to circumvent the drawbacks of IAC for small-size samples and to develop analytical devices that can be directly applied on field. MIPs are obtained by the polymerization of monomers in the presence of a template molecule, i.e OTA or an analogue, in the presence of cross-linking agent. The non-covalent bonds taking place between the monomers and the template allows the removal of the template after the polymerization and thus to obtain a polymer that possesses cavities complementary in term of shape and functionalities to the template. Different templates will be envisaged for obtaining a MIP with the properties to trap OTA but also its structural analogues. The aptamers are composed by a well defined nucleotides sequence that is able to bind selectively the target analyte but also structural analogues. Moreover, the highest capacity already demonstrated for MIPs (developed for other molecules and compared to IAC) and expected for oligosorbents (because of the smaller size of the oligonucleotides compared to the size of antibodies thus allowing higher binding ratio) makes them very attractive for the development of miniaturized devices. Moreover, the retention process on these chemical and biological tools should be different thus rendering them complementary for the trapping of target compounds. These tools will be evaluated for the analysis of OTA in well-defined airborne particles and be used for estimating workers exposure and in the future to inhalation studies. They will be also evaluated for toxicological studies in biological fluids (blood, urine) from animal submitted to OTA ingestion to analyze OTA and also metabolites and DNA adducts. This evaluation in real biological matrices also constitutes an important point before to start inhalation studies.