Nanosonden-basierte Mykotoxin-Detektion in Agrarprodukten
Pietschmann, Jan; Commandeur, Ulrich Heinrich (Thesis advisor); Schillberg, Stefan (Thesis advisor)
Aachen : RWTH Aachen University (2022)
Dissertation / PhD Thesis
Dissertation, RWTH Aachen University, 2022
The natural infestation of feed and food with ubiquitously occurring molds causes high economic damage and, due to mold toxins (mycotoxins), severe health problems. Various factors, such as late harvest and incorrect storage, favor the growth of the fungi and the appearance of the toxins. Since up to 25 % of all foods are contaminated with at least one mycotoxin, routine and reliable food testing is required by law from international organizations and is essential. Current analytical methods rely predominantly on laboratory-bound liquid chromatography (LC) coupled with tandem mass spectrometers (LC MS/MS). This extremely expensive and sensitive equipment allows a precise detection of the toxins, however, due to the required laboratory equipment and the need for highly trained personnel, a fast on-site analysis of the food by non-professional is not possible. Therefore, other methods based on immunological principles have been developed, such as ELISA or lateral flow assays (LFA). Although more user-friendly test systems could be developed, they are only suitable for rapid control due to the still necessary laboratory equipment (ELISA) or the missing precise quantification possibility (LFA). Due to the low reliability, food processing companies and farmers still rely on cost-intensive analyses in certified analytical laboratories. In order to provide a possibility for highly sensitive as well as quantitative on-site detection of the toxins aflatoxin B1, zearalenone, ochratoxin A and deoxynivalenol in wheat, which are particularly relevant for the industry, a novel competitive magnetic immunodetection (cMID) assay was conceptualized and established in this work. By generating aflatoxin B1- as well as ochratoxin A-specific monoclonal antibodies (mAb) by hybridoma technology and using commercial mAb against zearalenone and deoxynivalenol, optimized reference assays based on the cELISA were established. With this, indications for the potential sensitivity could be obtained. Subsequently, suitable parameters for coating concentration, respective antibody amount, and nanoparticle-type or amount were identified in order to establish the novel cMID assay. By this, a competitive binding reaction of the functionalized mAb could be achieved, whereby the mAb can be enriched in the immunofiltration column (IFC) coated with mycotoxin protein conjugate depending on the amount of mycotoxin previously bound in the sample. Subsequent labeling of retained mAb with functionalized nanoparticles, which are flushed through the IFC by gravity flow, allows the magnetic signal-based detection as a function of the retained mAb using a portable magnetic reader. This allows a highly sensitive and quantitative detection of the toxin concentration within the sample. By developing an extraction buffer using LC MS/MS and statistical design of experiments (DoE), all four toxins could be simultaneously extracted from the sample wheat with high precision and reliability and quantitatively detected by cMID with over 90 % accuracy, confirmed by spiking experiments. By developing a separation demonstrator, a possible increase in sensitivity by magnetic enrichment of the nanoparticles was shown as a proof of concept, resulting in a separation efficiency of over 95 %. Finally, the possibility of increasing user-friendliness by analyzing the storage stability of coated IFCs as well as a reduction of assay time was investigated, demonstrating the potential of cMID.
- Department of Biology 
- Chair of Molecular Biotechnology