Optical biosensors for mycotoxin detection in milk

Optical biosensors, and in particular label-free optical biosensors have become one of the most active and attractive fields within the biosensing devices. The portability and the possibility to set free from the laboratory settings gave a new hint for integrated photonic biosensors development and use in numerous applications. Integrated photonic sensors have shown very promising results, and in particular, devices like WGM resonators and interferometers are showing high sensitivities and miniaturization abilities, which allow the realization of an integrated complete lab-on-chip device. The main goal of this thesis is the development of an optical biosensor for the fast and comprehensive detection of carcinogenic Aflatoxin M1 (AFM1) mycotoxin. The acceptable maximum level of AFM1 in milk according to European Union regulations is 50 ng/L equivalent to 152 pM for the adults and 25 ng/L equivalent to 76 pM for the infants, respectively. Within a European Project named SYMPHONY, we develop an integrated silicon-photonic biosensor based on the optical microring resonators (MRR) and the asymmetric Mach-Zehnder Interferometers (aMZI). The sensing is performed by measuring the resonance wavelength shift in the MRR transmission or the phase shift of aMZI caused by the binding of the analyte to the ligand immobilized on the sensor surface. The experimental characterization of the bulk refractometric sensing of the devices is performed in a continuous flow. This characterization assesses the high resolution of both device types, which are able to resolve variations in the refractive index of the liquids with a limit of detection down to 10E-6 refractive index units (RIU). Furthermore, the SYMPHONY sensor optimization based on the Fab' and DNA-aptamer functionalization strategies is realized. It is therefore demonstrated, that the Fab' functionalization strategy provides more reproducible results with respect to the DNA-aptamer one. However, for both strategies, the specificity of the sensor functionalization to detect AFM1 molecules is achieved with respect to non-specific Ochratoxin molecules at high concentrations. In the final stage of the SYMPHONY project, the Fab'-based functionalized aMZI sensor is tested with real milk samples (eluates) prepared in the SYMPHONY system that consists of the three main modules: the defatting module, the concentrator module and the sensor module. The system calibration yields the minimum concentration of AFM1 at 40 pM to be detectable. The detection of the ligand-analyte binding in real-time enabled the study of the kinetics of the binding reaction, and we measured for the first time the kinetic rate constants of the Fab'-AFM1 interaction with our sensors. Finally, a MRR based affinity biosensor is developed dedicated to the biotinylated BSA - anti-biotin binding study. An affinity constant of 10E6 1/M is measured. The sensor is successfully regenerated up to eight times by applying a longer incubation period