Development of biosensors for the determination of apoptosis marker

Apoptosis is an essential physiological process that plays a critical role in development and tissue homeostasis. This process is regulated by a series of signals under certain conditions. The caspase cascade system plays a vital role in the induction, transduction, and amplification of intracellular apoptotic signals. Caspase-3, a cysteine-aspartic acid protease, has been associated with poor prognosis in non-small cell lung cancer, hepatocellular carcinoma, intracerebral hemorrhages, lung adenocarcinoma, severe endometriosis cases, and bladder carcinoma. Therefore, detection of caspase-3 is of great importance for apoptosis imaging, early tumor therapy, and evaluation of the surveillance effect of other diseases. Electrochemical immunosensors are a type of affinity biosensor based on interactions between antibodies and specific antigens immobilized on the active layer surface. In this thesis, it is aimed to compare the results obtained by performing the electrochemical immunosensor design for the quantitative measurement of caspase-3 and the characterization and optimization studies of this biosensor with the ELISA method. In the first stage of the thesis work, the gold electrode surface was coated with anticaspase-3, and in the next stage, the current difference created by interacting with caspase-3 in the reaction medium was used. In the optimization of the bioactive layer, immobilization gels were prepared using different combinations of chitosan, alginate, gelatin, and BSA. In SEM micrographs and FTIR spectra, the optimal immobilization gel combination of Alginate-Gelatin was obtained. The surface images obtained from the AFM analysis support the SEM and FTIR results. The optimal crosslinker is EDC/NHS (0,3M/0,1M), and optimal results were obtained in a 0.1 M acetic acid/acetate buffer with a working pH of 6.5. The linear detection range of the caspase-3 biosensor we designed is 1.5 100 ng/mL, the mean value (x̄)=10.02 obtained in the intraday repeatability study, the standard deviation value (SD)=0.18, the coefficient of variation (CV)= 1.8%. The standard deviation value obtained from the interday repeatability study is 2.52. The shelf life of the electrode was determined to be usable until the 30th day, when 40% activity loss was observed. The correlation coefficient obtained as a result of the comparison of the ELISA method and the designed biosensor data is 0.9927, which shows that the designed caspase-3 biosensor is suitable for practical use.