Fast and accurate determination of minute Ochratoxin A levels in flours: towards the application at the field

Ochratoxins are a group of mycotoxins produced as secondary metabolites by several fungi of Aspergillus and Penicillium species. Ochratoxin A (OTA) is the most toxic member of the group and can be found in a large variety of highly consumed foods, such as coffee, cocoa, wine, and flour. Reliable determination of ΟΤΑ levels in food samples is therefore indispensable to assure compliance with national/European regulations concerning MRLs and consequently minimize health risks posed for living beings. In the current study, a label-free biosensor based on White Light Reflectance Spectroscopy (WLRS) for rapid and accurate determination of OTA in flour specimens is demonstrated. The transducer employed is a Si chip with a 1-μm thick thermal SiO₂ on top, and is transformed to biosensing element through immobilization of a suitable OTA-protein conjugate on the SiO₂ surface, while the determination is based on a dedicated competitive immunoassay format. For the assay performance, a mixture of an in-house developed anti-OTA antibody with the calibrators or the samples is injected over the chip surface followed by reaction with secondary biotinylated antibody and streptavidin for signal amplification. The WLRS biosensing platform allows for the label-free, real-time monitoring of biomolecular interactions carried out onto the SiO₂/Si chip by transforming the shift in the reflected interference spectrum caused by the immunoreaction to effective biomolecular adlayer thickness. After optimization, the sensor was capable of real-time detection of OTA in flour samples with LoD of 60 pg/mL, within 25 min total analysis time. The intra- and inter-assay CVs were ≤5.9% and ≤9.0%, respectively. The excellent analytical characteristics and short analysis time in combination with its small size render the proposed WLRS system ideal for the quantitative determination of minute OTA levels at the Point-of-Need for food-, environment-, and health-related purposes.