| Abstract [eng] |
In this doctoral thesis the development of surface plasmon resonance (SPR) and electrochemical (EL) immunosensors has been examined and used for the detection of human growth hormone (HGH) and antibodies against human growth hormone (anti-HGH). The designed immunosensors were applied for the real sample analysis. In this research work random and oriented techniques of anti-HGH immobilization on the SPR sensor chip were compared and the interaction between immobilized anti-HGH and HGH was evaluated. Fragments of anti-HGH immobilization via their native thiol-groups directly coupled to the SPR chip is the most suitable technique for the development of an SPR immunosensor for the HGH detection. Fragments of antibody were obtained by chemical reduction of 2-mercaptoethylamine (2-MEA). In our study atomic force microscopy has been successfully applied for the imaging of the the differently modified surfaces. The achieved results indicate how much adequately the fragments of anti-HGH molecules bound onto the gold substrate and how immune complexes were formed. SPR HGH immunosensor is suitable for HGH detection with a limit of detection (3.4 nmol/L), good stability and selectivity. Furthermore, active SPR-chip surface is fully regenerated that allows multiply usage of designed immunosensor. Direct and indirect anti-HGH immunosensor was studied by combining two analytical methods – SPR and EL (pulsedamperometry (PA) and cyclic voltammetry (CV)). The limit of detection values of anti-HGH immunosensors is low (0.056, 0.051 and 0.027 nmol/L for CV-, SPR- and PA-based immunosensors, respectively). Electrochemical immunosensors are single-use only. The developed PA and SPR immunosensors were successfully applied for the anti-HGH and HGH detection in real samples. |
