Detection and removal approaches for food mycotoxins

Mycotoxins, secondary metabolites from diverse fungal strains, are relevant compounds whose occurrence affects not only low-income countries, but high-income nations through imported products. Their threatening effects to human health, along with the negative impact on economic, social, and agricultural levels, have established an area of study for the development of better agricultural practices, more sensitive detection techniques and effective detoxification methods. Despite their great sensitivity and selectivity, conventional methods for mycotoxin quantification require long assay times, expensive equipment and instrumentation, specialized operators, and multiple steps of sample pre-treatment. Hence, less complex yet effective techniques are required for such targets. In addition, decontamination is sometimes the last alternative for some agricultural and food products in which the use of waste-based adsorbents results in a cheap, ecological, and affordable solution to this problem, when compared to other utilized materials. In this regard, natural polymers have been explored with promising results over the las years.
This thesis was focused on the development and improvement of bulk and paper-based biosensing techniques for the quantification of mycotoxins, and the exploration of natural polymeric materials as potential adsorbents for food decontamination and biosensing platforms. In order to examine the application of paper as a sensing matrix, five model samples (aqueous ink) were applied on 3MM chromatographic paper for the analysis of their flow-related phenomena by mathematical fitting to diffusion and imbibition equations. The characterized paper matrix was then applied on the colorimetric detection of aflatoxin B1 (AFB1), by exploiting its inhibitory effect towards acetylcholinesterase (AChE) in Ellman’s reaction, where the immobilization of AChE was assessed in two types of chitosan (high and low degree of acetylation (DA)/ molecular weight (MW). The application of a high MW/high DA cross-linked chitosan resulted in the linear quantification of AFB1. In addition, an aptamer (ssDNA)-based detection of fumonisin B1 (FB1) was developed with two aptamers (96 and 40 nt) incubated with FB1, followed by another incubation step with gold nanoparticles (AuNPs) and the addition of NaCl. From all the tested conditions, only the 96 nt aptamer was specific to FB1, whose biosensing properties were regulated by the formation of an aptamer-FB1-AuNP conjugate in MgCl2. These conjugates were stable to NaCl-induced aggregation at increasing concentrations of FB1 and characterized by Asymmetric Flow Field-Flow Fractionation (AF4), a technique showing low LODs in the fg/mL level. Finally, preliminary results for the mycotoxin adsorption of β-chitin scaffolds and N-isobutyryl chitosan (NIBC) hydrogels were obtained for FB1-spiked beer and milk. In this regard, although more exploration is necessary, the preliminary results indicated a slightly better performance from NIBC gels, specially at lower pH values. Furthermore, the potential application of NIBC gels as support materials in molecular imprinting, were explores through the use of L-phenylalanine as an analogue of ochratoxin A, in which no interference with the gel formation process and a complete template removal were observed in the selected material. Nevertheless, further optimization is required for the successful completion of both applications in the selected polymeric materials. In general, through this thesis more understanding on the diffusive and capillary-driven phenomena in a paper matrix was reported with a subsequent improvement of its performance in mycotoxin biosensing, which led to a space for improving other sensing and biosensing techniques through such immobilization method. Besides, the sole application of an aptamer in the sensitive detection of mycotoxins was achieved by denoting the effect of the incubation conditions and the selected sequence, which outlines another route for tackling aptasensing methods depending on the selected supports and sequences, while leaving an open door for its future implementation as a paper-based detection for mycotoxins. Lastly, the exploratory results on the application of chitin and chitosan-based structured materials, left a promising scope for more research and projects dedicated to the exploitation of these materials in similar and multiple applications.