Development of biosensors for mycotoxins detection in food and beverages

Mycotoxins are secondary metabolites of mould, which are ubiquitous in a large variety of food and feed commodities. Thousands of mycotoxins exist, but only a few present significant damages and poisonous properties. Among them, the aflatoxins and ochratoxins are considered to be the most toxic and widely spread in the world and therefore, represent a real threat for human/animal life. Depending on a number of factors like the intake levels, duration of exposure, mechanisms of action, metabolism and defense mechanisms, mycotoxins elicit a wide spectrum of toxicological effects leading to both acute and chronic disease, liver and kidney damage, skin irritation, cancer, immune suppression, birth defects or even death. To address the adverse effects of mycotoxin contaminants in food and feed, health authorities in many countries all over the world have become active in establishing regulations to protect their citizens and livestock from the potential damages caused by those compounds. The European Commission, the US Food and Drug Administration (FDA), the World Health Organization and the Food and Agriculture Organization of the United Nations have set up regulations and maximum levels for major mycotoxins in foods and feeds. To fulfill expectations of these regulatory limits, there is an increasing need for the development and validation of new, simple, fast and precise methods for toxins detection. Therefore, this thesis reveals different strategies for rapid, cost-effective and ultrasensitive bioanalysis of two major mycotoxins: aflatoxin M1 and ochratoxin A. Inhibition competitive assays with surface plasmon resonance spectroscopy (SPR, optical technique), quartz crystal microbalance (QCM, acoustic device) and electrochemical based readout were developed and compared. Presented biosensors were challenged in a red wine and milk samples with no need for pre-treatment or pre-concentration of the sample extract. In order to prevent fouling on the sensor surface by the constituents present in milk samples, the gold surface of the sensor chip was modified and different surface architecture and compared (antifouling polymer brushes and self-assembled monolayer - SAM). Complete resistance to the non-specific interactions was observed for coating with p(HEMA) brushes resulting in two times lower LOD compared to that on thiol SAM. The SPR biosensor for AFM1 allowed for highly sensitive detection in milk with an excellent precision (the average calculated CV was below 4%), limit of detection of 18 pg mL−1 for p(HEMA) brushes and 38 pg mL-1 for thiol SAM and with the analysis time of 55 min. It is worth highlighting that it is the first time that an SPR chip modified with such polymer brushes was used for real time detection of a small target antigen opening a new avenue for highly precise analysis. In the case of wine samples tested for OTA detection, a simple but very effective pre-treatment procedure was successfully applied. It was proved that the addition of the 3% of the binding agent poly(vinylpyrrolidone) (PVP) to red wine completely reduces non-specific interactions by binding polyphenolic compounds (which may be responsible for inactivation of antibody and blocking the sensor surface) through hydrogen bonding, making their elimination easier. Moreover, in this study, the authors evaluated the influence of gold nanoparticles (AuNPs) on signal enhancement and thereby biosensor sensitivity. For this purpose two assays were performed: with and without implementation of NPs. Obtained results allowed for OTA detection at concentrations as low as 0.75 ng mL−1 however, its limit of detection was improved by more than one order of magnitude to 0.068 ng mL−1 by applying AuNPs as a signal enhancer. The combination of indirect competitive assay and AuNPs with QCM-D gave a straightforward tool, which can simultaneously measure frequency and dissipation changes resulting in information about the sensitivity but also about the mass attached to the sensor surface as well as viscoelastic properties and the hydration state of the film. A linear detection range of 0.2–40 ng mL-1 has been achieved with LOD of 0.16 ng mL-1. The same assay format was also tested in voltammetric detection of mycotoxins using modified gold screen printed electrodes (AuSPE). An excellent LOD of 15 ng mL-1 for OTA and 37 pg mL-1 for AFM1 were obtained. Additionally, AuSPE modified with SAMs based on different types of alkanethiols (long and short chains) were tested and compared in terms of electron transfer resistance. Proposed biosensors offer vast range of advantages such as high sensitivity (at pg or ng levels), short analysis time (55 min) in comparison to for example, ELISA which require multiple steps that translates to prolonged analysis time, possibility for online monitoring, characterization of binding kinetics, low consumption of primary antibody (cost reduction), excellent antifouling surface and simple pre-treatment procedure. Combining all most desirable aspects of a good biosensor such as high sensitivity, low costs, short analysis time and simple but effective cleaning-up technique make proposed approaches an important and very promising tools for widespread biosensing applications.