Biological sensing elements in electrochemical biosensors for detection of effects of cholinesterase inhibitors with cytotoxic actionEghbalroknabadi, Minaalsadat (Avtor) Drobne, Damjana (Mentor) cell-based biosensorenzyme-based biosensorimpedance spectroscopymicroscopyEllman methodIn this thesis, enzyme-based and cell-based biosensing elements were investigated to study the biological effects of selected substances with neurotoxic potential, with a particular focus on acetylcholinesterase (AChE) inhibitors. The enzyme-based sensing element used AChE which was immobilized on gold surfaces via self-assembling monolayer (SAM). The immobilization process was optimized on different metal surfaces. The enzyme activity was measured after immobilization, and the stability of the immobilized surfaces was monitored over time. The activity of the immobilized enzymes was then tested upon exposure to inhibitors. Eserine caused complete inhibition above 25 µM. Rivastigmine, a reversible inhibitor, caused a concentration-dependent decrease in AChE activity from 57% ± 10 at 10 µM to 17% ± 10 at 50 µM. Diazinon, an irreversible inhibitor, led to inhibition at 10 µM (82% ± 6). The activity decreased to 52% ± 6 at 50 µM. The performance of the sensing element was confirmed as a proof of concept by electrochemical impedance spectroscopy (EIS) in the presence of eserine. In the cell-based biosensor approach, A549 human lung adenocarcinoma cells were cultured for 48 hours on stainless-steel surfaces and exposed to substances with neurotoxic potential. The cell responses to these substances were monitored by microscopy and confirmed as a proof of concept by electrochemical cell-based impedance spectroscopy (ECIS). Microscopic images showed that diazinon induced pronounced cytotoxic effects with morphological changes, while rivastigmine produced milder effects and reduced metabolic activity by 16% ± 7 at the highest concentrations tested. ZnCl₂, a non-cholinesterase substance with neurotoxic potential, was also tested due to its known effects on A549 cells. For all three compounds, surface coverage and the number of attached cells decreased as the concentration of the substance with neurotoxic potential increased. The study of the enzyme-based and cell-based systems revealed that systems responded differently to substances with neurotoxic potential. The enzyme system assessed how chemicals specifically affected enzymatic activity, while the cell-based system reflected the broader effects on living cells. Taken together, these complementary approaches provided a better understanding of the biological effects of substances with neurotoxic potential in toxicology, as well as their potential application in biosensor systems.20262026-06-10 07:15:10Doktorsko delo/naloga183270sl