Ugotavljanje reaktivnosti 2D materialov s testiranjem inhibicije in adsorpcije izoliranega encima AChE

Saje, Špela (author), Kononenko, Veno (mentor)

Razvoj nanotehnologije je privedel do postopnega izboljšanja lastnosti nanomaterialov (NM), ki se že uporabljajo za številne aplikacije v elektrotehniki, medicini, gradbeništvu in kmetijstvu. Zato je pomembno, da poznamo in razumemo biološko reaktivnost in morebitno strupenost ter potencialna tveganja NM. V okviru magistrskega dela smo testirali biološko reaktivnost 12 različnih izbranih grafenovih oksidov (GO), ki jih uvrščamo med 2D nanomateriale. Biološko reaktivnost izbranih GO smo ugotavljali s testiranjem inhibicije in adsorpcije na izoliranem encimu acetilholinesterazi (AChE) po Ellmanovi metodi, prilagojenim na mikrotitrske plošče. Dva GO, ki sta se najbolj razlikovala v reaktivnosti, izmerjeni s testiranjem aktivnosti AChE, smo testirali še s testi citotoksičnosti na celičnih linijah A549 in Beas-2B, in sicer s testom privzema barvila nevtralno rdeče in testom z resazurinom. Ugotovili smo, da so GO zelo reaktivni materiali, saj dobimo visok delež inhibicije in adsorpcije že pri zelo nizkih koncentracijah materiala. Reaktivnost je bila odvisna od koncentracije GO, pri višjih koncentracijah GO smo izmerili manjšo aktivnost encima AChE. Iz podatkov lahko sklepamo, da je velik delež inhibicije encima AChE posledica adsorpcije GO na encim. Rezultati testov citotoksičnosti in testov inhibicije dveh izbranih GO so se ujemali, biološko bolj reaktiven GO je bil bolj citotoksičen. Rezultati korelacijske analize so pokazali, da je reaktivnost GO odvisna od razmerja med kisikom in ogljikom, ki gradita GO. Naši rezultati so pokazali, da so GO biološko reaktivni materiali, stopnja njihove reaktivnosti pa je odvisna od njihovih fizikalno-kemijskih lastnosti.

• Language: Slovenian
• Keywords: acetilholinesteraza, 2D nanomateriali, grafenov oksid, inhibicija, adsorpcija, citotoksičnost
• Work type: Master's thesis/paper
• Typology: 2.09 - Master's Thesis
• Organization: BF - Biotechnical Faculty
• Publisher: [Š. Saje]
• Year: 2024
• PID: 20.500.12556/RUL-160134
• UDC: 577.151:620.3(043.2)
• COBISS.SI-ID: 205046531
• Publication date in RUL: 22.08.2024

SAJE, Špela, 2024, Ugotavljanje reaktivnosti 2D materialov s testiranjem inhibicije in adsorpcije izoliranega encima AChE [online]. Master’s thesis. Š. Saje. [Accessed 5 April 2025]. Retrieved from: https://repozitorij.uni-lj.si/IzpisGradiva.php?lang=eng&id=160134

Secondary language

• Language: English
• Title: Determining the reactivity of 2D materials by testing the inhibition and adsorption of the isolated enzyme AChE

Abstract

The development of nanotechnology has led to a gradual improvement in the properties of nanomaterials (NMs), which are already used for a wide range of applications in electrical engineering, medicine, construction and agriculture. Therefore, it is important to know and understand the biological reactivity and potential toxicity and risks of NMs. In this MSc thesis, the biological reactivity of 12 different selected graphene oxides (GOs), classified as 2D nanomaterials, was tested. The biological reactivity of the selected GOs was determined by testing inhibition and adsorption on the isolated acetylcholinesterase (AChE) enzyme using the Ellman method adapted to microtitre plates. The two GOs that differed most in reactivity as measured by AChE activity assays were further tested by cytotoxicity assays on the A549 and Beas-2B cell lines, namely the neutral red dye uptake assay and the resazurin assay. We found that GOs are highly reactive materials, with high inhibition and adsorption rates even at very low material concentrations. The reactivity was dependent on the concentration of GO, with lower AChE activity measured at higher GO concentrations. From the data it can be concluded that much of the inhibition of the AChE is due to adsorption of GO onto the enzyme. The cytotoxicity and reactivity results of the two GOs tested in the AChE assay and cytotoxicity assays were in agreement, the more biologically reactive GO being more cytotoxic. The results of the correlation analysis showed that the reactivity of GO depends on the ratio of oxygen to carbon. Our results showed that GOs are biologically reactive materials, and that the degree of their reactivity depends on their physicochemical properties.

• Keywords: acetylcholinesterase, 2D nanomaterials, graphene oxide, enzyme inhibition, enzyme adsorption, cytotoxicity