Determination of the effect of some nanotechnological immobilization methods on the activity of catalase enzyme by a biosensor method

The aim of this thesis is to determine the effect of nanotechnological immobilization methods on catalase enzyme which has attracted the attention of many researchers recently by biosensor method. The enzyme-based biosensors designed with two different immobilization methods to investigate the effect of these nanotechnological materials on the biosensor are: I. Au/3-MPA/EDC-NHS/Cys/AuNP/CAT/GA enzyme biosensor II. GCE/MWCNT/MWCNT-CAT enzyme biosensor For the development of the Au / 3-MPA / EDC-NHS / Cys / AuNP / CAT / Glutaraldehyde enzyme biosensor, self assambled monolayer formation on the gold electrode surface was provided with 3-MPA. EDC-NHS was used to activate the carboxyl groups of 3-MPA. The electrode was then immersed in cysteamine solution and incubated. After addition of AuNP and catalase onto the surface and forming the bioactive layer, cross-linking wal achieved between catalase enzyme molecules by glutaraldehyde. A multi-walled carbon nanotube (MWCNT) suspended in DMF was used to design the GCE / MWCNT / MWCNT-CAT enzyme biosensor. The first step in biosensor preparation was to drop the MWCNT susponded in DMF onto the electrode to increase the concluctivity of the electrode. In the second step the biosensor design was completed by the dropping the solution formed by mixing the MWCNT with 1/3 ratio (v/v) of the catalase after sonication, onto the electrode surface. Hydrogen peroxide measurements with the biosensors developed using two different immobilization methods were performed by differential pulse voltammetry (DPV) between -0.2 V & +0.5 V potentials. For both biosensors measurements were taken from the linear relationship between the observed changes in the oxidation peaks of K3[Fe (CN)6] and the hydrogen peroxide concentration between -0.2 V & +0.5 V potentials, In order to optimize the working conditions of designed enzyme-based biosensors, temperature, pH, ion intensity and different buffers were studied. For the characterization of the designed enzyme-based biosensors, same parameters such as determination of the linear range, repeatability and determination of hydrogen peroxide in milk were carried out. When the results obtanied from the experiments were examined, it was detected that the best working conditions for the enzyme biosensors developed by both immobilization methods were 50 mM, pH: 7.0 potassium phosphate buffer and 30ºC. The linear range for hydrogen peroxide was obtained the same in both biosensors designed as to be 150 µM -1000 µM. In the reproducibility studies performed for the enzyme biosensor designed using AuNP, the average value (n = 6) for 600 µM hydrogen peroxide concentration was found to be 591.4 µM, standard deviation (S.S) = ± 6.54 µM and coefficient of variation % (CV %) = 1.105 respectively. In the repeatability tests for the enzyme-based biosensor developed using MWCNT, the average value (n = 6) for 600 µM hydrogen peroxide concentration was found to be 597.3 µM, standard deviation (S.S) = ± 26.8 µM and coefficient of variation % (CV %) = 4.5 respectivity. In addition, hydrogen peroxide analysis was performed with enzyme biosensors in milk samples. Keywords: Gold nanoparticle, multi-walled carbon nanotube, hydrogen peroxide, 3-Mercaptopropionic acid, cysteamine, enzyme-based biosensor.