A novel biosensor system based on gold nanoparticles-bilayer thiol modified diamine oxidase-peroxidase coupled enzymes system

In this thesis, a biosensor system based on a gold nanoparticles(AuNP)-bilayer thiol modified diamine oxidase-peroxidase coupled enzyme system was intended to be developed to determine serotonin which brings a new and original perspective to biosensor technology. Primarily, in this developed biosensor system, binding of 1,6-hexanedithiol, having functional thiol group, were performed to the gold electrode (Au) surface and self assembled monolayers (SAM) formation was carried out. After the binding of dithiol to the electrode surface, the gold nanoparticles were allowed to bind to the other reagent (-SH) terminal. After that, the gold nanoparticles (AuNP) were reacted with 3- mercaptopropionic acid. After the addition of diamine oxidase-peroxidase on to the modified electrode surface, covalent immobilization was provided by dropping of glutaraldehyde on to the electrode surface. The serotonin measurements were made using 0,2 V amperometric method with the designed biosensor system. The determination was made on the basis of increasing current values in direct proportion with serotonin concentration. In the optimization studies of the biosensor firstly bioactive layer components such as SAM formation time, diamine oxidase-peroxidase activity and glutaraldehyde concentration were carried out. In the optimization of working conditions; optimum pH was also investigated. Afterwards, in the characterization studies of the biosensor some paramaters such as linearity, reproducibility, storage stability were determined. From the result of the optimization studies optimum 1,6-hexanedithiol amount , optimum diamine oxidase – peroxidase activitiy and optimum glutaraldehyde concentration were determined to be 20 hour, 0.11 Units/ml- 113 Units/ml and 2.5 % respectively. From the experiments the phosphate buffer (50 mM, pH 7.0) and 30 °C were chosen to be optimum working conditions for the biosensor. It was determined from the characterization studies of the biosensor that linear curves were obtained serotonin concentration between the ranges of 1 µM to 10 µM. At the repeatability experiments (n=6), the average value ( ), standard deviation, (SD) and coefficent of variation (CV %) were calculated to be 4.97 µM mM, ± 0.241 and 4.84 % for 5 µM serotonin concentration, respectively. Keywords : 1,6- hexanedithiol, gold nanoparticles, diamine oxidase, peroxidase, serotonin, biosensor