Development of immobilization methods for detection of biomolecules in immunobiosensors

Biosensors are devices that consist of a combination of a transducer that generates a signal from the change caused by the interaction between a biological sensor and the target analyte. Performance parameters of biosensors are fast response time, dynamic response, reproducibility, selectivity, quantitative measurement limit, detection limit, wide measurement range, linearity and precision. The immobilization activities, which affect sensitivity of the biosensors, are achieved by different methods on the biosensor surface. In this thesis, analyte immobilization was performed by forming single layers (self assembled monolayer (SAM)) on the surface of the electrode. Fumonisin (FUM) biomolecule was used as a model to determine immobilization activity in developed quartz crystal microbalance (QCM) immunosensor in thesis study. FUM is a mycotoxin species produced by the fungus microorganisms named Fusarium Moniliforme and Fusarium Proliferatum and it is followed by the US Food and Drug Administration (FDA), European Commission (E.C.), Turkish Food Codex(T.F.C) regulations as other mycotoxins. In addition to laboratory-focused instrumental analytical systems in FUM contamination, biosensors and identification methods have also been developed depending on the availability of on-site detection facilities. In the thesis, for the first time, QCM immunosensor capable of FUM detection was developed by using Monoclonal Antibodies (Mab) produced against FUM. In this study FUM immunosensors were developed that can be identified at intervals determined by the regulation because of strategies that demonstrate the importance of immobilization effectiveness. Surface immobilization was performed by using 11-mercaptoundecanamide (MUD) and 11-mercaptoundecanol (MUO) from linear agents as well as 11-mercaptoundecanoic acid, which is the most commonly used SAM agent in surface immobilization of FUM. Dendrons were used as an alternative to linear agents for enhancing immobilization efficiency due to spontaneous SAM formation, the ability to present / capture analytes with different topographic features, high degree of wettability and conductivity. Three different generations of dendrons were used to determine the effect of the immobilized analyte on the sensitivities of the electrodes and their distances to each other. For this purpose, FUM immobilization was performed with dendrons of bis-MPA dendron-8-hydroxyl-1 thiol (G3 DND), bis-MPA dendron-16 hydroxyl-1 thiol (G4 DND) and bis-MPA dendron-32 hydroxyl-1 thiol (G5 DND). High sensor responses were obtained by using the dendrons on the surface but due to the confidence constraint in the data, two layer sensor surfaces were created and dendrons were used as a second layer on the surface after MUD and SAM were created. All developed electrode surfaces were investigated in terms of perception, precision, accuracy and repeatability parameters in measurements. Through the studies the effect of immobilization on biosensors, QCM immunosensor has been developed for FUM detection, which have 0.008 ppm detection limit, reliable, reproducible, capable of high precision measurement.