Development of Optical Biosensors Based on Metal Nanostructures for Pollution (Mycotoxins) Detection

This work aims to develop optical biosensors for detection of the bio-toxins, particularly mycotoxins. The main detection technology chosen is a combination of a localized surface plasmon resonance (LSPR) transducer with direct immunoassay with specific bio-receptors antibodies or aptamers immobilized on the gold surface. The LSPR platform is based on gold nano-islands produced by thermal annealing of thin gold films deposited on glass. Thermal annealing was substituted with much quicker and more efficient microwave annealing in later stages of the project. The gold nano-structures produced were analyzed with scanning electron microscopy (SEM), atomic force microscopy (AFM), X-ray diffraction (XRD), optical absorption spectroscopy, and spectroscopic ellipsometry. The method of total internal reflection ellipsometry (TIRE) was chosen for LSPR bio-sensing because of its superior sensitivity as compared to conventional UV-vis absorption spectroscopy. The TIRE spectroscopic measurements were performed using the experimental set-up developed on the basis of J.A. Woollam M2000 spectroscopic instrument. The sensitivity of LSPR measurements is however limited by a finite evanescent field decay length in gold nano-islands which is typically in the range of tens of nanometres. In order to achieve the best results, the bio-receptors must be small and placed as close to the gold surface as possible. In our case the problem was solved using either halved antibodies or aptamers immobilized covalently on the surface of gold. A series of bio-sensing experiments to detect mycotoxins, i. e. aflatoxin B1, M1, zearalenone, and ochratoxin A, went successfully and resulted in the detection of the above mycotoxins in concentrations down to 0.01ng/mL. In this work we attempted, for the first time, the TIRE detection of Aflatoxin B1, M1 and ochratoxin A (OTA) in an assay with a specific aptamer. We compared the results obtained for the detection of mycotoxins using bio-receptors of different dimensions: large-size whole antibodies electrostatically immobilized on the surface of gold nano-islands, and small-size split antibodies or specific aptamers immobilised via thiol (SH) groups. For small-size receptors, the low detection limit (LDL) was 0.01ng/ml which is one order of magnitude lower than for whole antibodies. The results obtained demonstrate the advantages of using small bio-receptors in LSPR bio-sensing. The minimal detected concentration OTA was 0.01ng/ml, which is a remarkable result for direct aptamer assay format. The mycotoxin/aptamer binding kinetics were analysed using dynamic TIRE measurements and yeilded an association constant KA in the range of 107 Mol-1 which confirmed the high specificity of aptamer. An attempt to make gold nanostructures for SERS biosensing using nanosphere lithography was successful