Development of Novel Optical Planar Waveguide-based Biosensors using a Nanotechnology Approach

This work aims at the development of novel biosensor based on optical planar waveguide (OPW) for detection of mycotoxins, which are common contaminants in agriculture products (grains, beans, nuts, fruits) and associated food and feed. These low molecular weight toxins produced by various fungi species possess a substantial danger to human and animals, and thus are under strict legislated limits in sub-ppm (part per million) level. The detection of mycotoxins in such low concentrations is of great interest nowadays. A novel detection principle of polarization interferometry (PI) exploited in this system (which can be considered as a logical continuation of ellipsometry) in based on tracking changes in the polarization state of a laser beam passing through the waveguide and affected by immobilized in the waveguide sensing window. The key element of this sensor is a planar optical waveguide consisting of 190 nm thick silicon nitride core layer sandwiched between two thick layers of silicon dioxide; a sensing window was etched in the top silicon oxide layer to allow monitoring molecular adsorption. A 630 nm polarized light from a laser diode coupled through the slant edge of the waveguide experiences a large number of reflections (about 500 per mm) when propagating through the waveguide. The p- component of polarized light is affected by changes in refractive index in the sensing window, while s- component is less affected and thus serves as a reference. Therefore, the changes in either the medium refractive index or molecular adsorption cause the phase shift between p- and s- components. The observation of the light polarization state is enabled by a polarizer converting the changes in polarization to variations of light intensity which is then recoded with CCD linear array interfaced to PC. The refractive index sensitivity of the OPW PI sensor of about 1600 rad/RIU/mm (the highest value known for optical detection) was found by both the theoretical modelling and experimental testing. The developed experimental set-up was used for detection of mycotoxins, i.e. aflatoxin B1 (AFT B1), ochratoxin A (OTA), and zearalenone (ZEN), in direct assay with two types of bio-receptors immobilized within the sensing window: (i) antibodies electrostatically bound onto silicon nitride surface via layers of poly-allylamine hydrochloride and protein A, or (ii) aptamers covalently bound via SH groups on aminated surface of silicon nitride. The outcome of such biosensing tests was successful; all three mycotoxins were detected in a wide concentration range from10 pg/ml up to 1 g/ml in direct immunoassays with their respective antibodies. The use of specific aptamers as bioreceptors in the latest upgrade of the OPW PI set-up has resulted in much lower detected concentrations of AFT B1 and OTA down to 1pg/ml, with LDL estimated as 0.6 -0.7 pg/ml. The obtained sensitivity in sub-ppt (part per trillion) level is the highest known for optical biosensors, and it is particularly remarkable for a label-free detection of low molecular weight analyte molecules in direct assay format. The developed OPW PI biosensor is universal and can be easily adapted for detection of different analyte molecules by choosing suitable bio-receptors. It can be used equally for detection of small and large molecules, and in different assay formats, e.g. direct, sandwich, and competitive assays, and therefore can be considered as a platform biosensing technology for a wide range of applications, i.e. environmental monitoring, security, agriculture and food industry, and biomedical.