20.500.12556/RUL-183270 Biological sensing elements in electrochemical biosensors for detection of effects of cholinesterase inhibitors with cytotoxic action Biološki senzorski elementi v elektrokemijskih biosenzorjih za odkrivanje učinkov inhibitorjev holinesteraze s citotoksičnim delovanjem In 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. Disertacija obravnava encimske in celične biološke senzorske elemente za preučevanje bioloških učinkov izbranih substanc z nevrotoksičnim potencialom, s posebnim poudarkom na inhibitorjih acetilholinesteraze (AChE). Encimski senzorski element je temeljil na AChE, ki je bil imobiliziran na zlatih površinah preko enosloja samoorganiziranih molekul. Postopek imobilizacije je bil optimiziran na različnih kovinskih površinah. Encimska aktivnost je bila izmerjena po imobilizaciji, stabilnost imobiliziranih površin pa je bila spremljana skozi čas. Aktivnost imobiliziranega encima je bila nato preizkušena po izpostavitvi inhibitorjem. Eserin je povzročil skoraj popolno inhibicijo nad 25 µM. Rivastigmin, reverzibilni inhibitor, je povzročil koncentracijsko odvisno zmanjšanje aktivnosti AChE, s 57 % ± 10 pri 10 µM na 17 % ± 10 pri 50 µM. Diazinon, ireverzibilni inhibitor, je povzročil inhibicijo pri 10 µM (82 % ± 6). Aktivnost se je zmanjšala na 52 % ± 6 pri 50 µM. Delovanje senzorskega elementa je bilo kot dokaz koncepta potrjeno z elektrokemijsko impedančno spektroskopijo (EIS) v prisotnosti eserina. Pri pristopu s celičnim biosensorskim elementom so bile celice A549 človeškega adenokarcinoma pljuč za 48 ur gojene na površinah iz nerjavečega jekla in izpostavljene substancam z nevrotoksičnim potencialom. Odziv celic, ki so bile izpostavljene substancam, so se spremljali z mikroskopijo in kot dokaz koncepta potrdili z elektrokemijsko spektroskopijo impedance na celični osnovi (ECIS). Mikroskopske slike so pokazale, da je diazinon povzročil izrazite citotoksične učinke z morfološkimi spremembami, medtem ko je rivastigmin povzročil blažje učinke in pri najvišjih testiranih koncentracijah zmanjšal presnovno aktivnost za 16 % ± 7. ZnCl₂, substanca brez holinesteraze z nevrotoksičnim potencialom, je bila prav tako testirana zaradi svojih znanih učinkov na celice A549. Pri vseh treh spojinah sta se površinska pokritost in število pritrjenih celic zmanjšala s povečanjem koncentracije snovi z nevrotoksičnim potencialom. Študija encimskih in celičnih sistemov je pokazala, da sta se sistema odzivala različno na substance z nevrotoksičnim potencialom. Encimski sistem je ocenil, kako snovi specifično vplivajo na encimsko aktivnost, medtem ko je celični sistem odražal širši vpliv na žive celice. Ti dopolnjujoči se pristopi skupaj omogočajo boljše razumevanje bioloških učinkov snovi z nevrotoksičnim potencialom v toksikologiji ter njihove možne uporabe v biosenzorskih sistemih. cell-based biosensor enzyme-based biosensor impedance spectroscopy microscopy Ellman method celično osnovan biosenzor encimsko osnovan biosenzor impedančna spektroskopija mikroskopija Ellmanova metoda true false false Angleški jezik Slovenski jezik Doktorsko delo/naloga 2026-06-10 07:15:10 2026-06-10 07:15:24 2026-06-16 07:43:56 0000-00-00 00:00:00 2026 0 0 0000-00-00 Zaloznikova Objavljeno NiDoloceno 0000-00-00 0000-00-00 0000-00-00 280066 281712131 57801.pdf 57801.pdf 1 5FB094BF59A6F1AB77F39E25B6E8649D 7783de2baed284d46c0fd2e66fe330bb57b2243ff2e23f72b36cb82225c8d53d 60ee0fbd-648b-11f1-9b0d-0050569b8976 https://repozitorij.uni-lj.si/Dokument.php?lang=slv&id=235234 Biotehniška fakulteta 0 0 0