prikaz prve stranice dokumenta Functionalisation of mica with (3-Aminopropyl)triethoxysilane: influence on imaging of extracellular vesicles from human cerebrospinal fluid by atomic force microscopy
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master's thesis
Functionalisation of mica with (3-Aminopropyl)triethoxysilane: influence on imaging of extracellular vesicles from human cerebrospinal fluid by atomic force microscopy
2022. urn:nbn:hr:193:235297
Svetić, Ana
University of Rijeka
Faculty of Biotechnology and Drug Development

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Svetić, A. (2022). Functionalisation of mica with (3-Aminopropyl)triethoxysilane: influence on imaging of extracellular vesicles from human cerebrospinal fluid by atomic force microscopy (Master's thesis). Rijeka: University of Rijeka. Retrieved from https://urn.nsk.hr/urn:nbn:hr:193:235297

Svetić, Ana. "Functionalisation of mica with (3-Aminopropyl)triethoxysilane: influence on imaging of extracellular vesicles from human cerebrospinal fluid by atomic force microscopy." Master's thesis, University of Rijeka, 2022. https://urn.nsk.hr/urn:nbn:hr:193:235297

Svetić, Ana. "Functionalisation of mica with (3-Aminopropyl)triethoxysilane: influence on imaging of extracellular vesicles from human cerebrospinal fluid by atomic force microscopy." Master's thesis, University of Rijeka, 2022. https://urn.nsk.hr/urn:nbn:hr:193:235297

Svetić, A. (2022). 'Functionalisation of mica with (3-Aminopropyl)triethoxysilane: influence on imaging of extracellular vesicles from human cerebrospinal fluid by atomic force microscopy', Master's thesis, University of Rijeka, accessed 16 November 2024, https://urn.nsk.hr/urn:nbn:hr:193:235297

Svetić A. Functionalisation of mica with (3-Aminopropyl)triethoxysilane: influence on imaging of extracellular vesicles from human cerebrospinal fluid by atomic force microscopy [Master's thesis]. Rijeka: University of Rijeka; 2022 [cited 2024 November 16] Available at: https://urn.nsk.hr/urn:nbn:hr:193:235297

A. Svetić, "Functionalisation of mica with (3-Aminopropyl)triethoxysilane: influence on imaging of extracellular vesicles from human cerebrospinal fluid by atomic force microscopy", Master's thesis, University of Rijeka, Rijeka, 2022. Available at: https://urn.nsk.hr/urn:nbn:hr:193:235297

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Metadata
TitleFunctionalisation of mica with (3-Aminopropyl)triethoxysilane: influence on imaging of extracellular vesicles from human cerebrospinal fluid by atomic force microscopy
Title (croatian)Funkcionalizacija tinjca 3-aminopropiltrietoksisilanom: utjecaj na vizualizaciju izvanstaničnih vezikula iz cerebrospinalne tekućine ljudi mikroskopijom atomskih sila
AuthorAna Svetić
MentorMladenka Malenica (mentor)
Committee memberIvan Gudelj (predsjednik povjerenstva)
Committee memberMarin Tota (član povjerenstva)
Committee memberMarko Perčić (član povjerenstva)
Committee memberMladenka Malenica (član povjerenstva)
GranterUniversity of Rijeka
(Faculty of Biotechnology and Drug Development)
Rijeka
Defense date and country2022, Croatia
Scientific / art field,
discipline and subdiscipline		BIOTECHNICAL SCIENCES
Biotechnology
Molecular Biotechnology
Abstract
Extracellular vesicles (EV) are nanoparticles that are present in biofluids, including human cerebrospinal fluid (CSF). Their visualisation is performed by Electronic Microscopy (EM) and atomic force microscopy (AFM). AFM is advanced nanotechnology that enables the determination of morphology in nanometer resolution. The most common substrate used for visualisation is mica, however, its functionalisation is needed with the aim of stronger binding of EVs, especially in the AFM air mode. (3-Aminopropyl)triethoxysilane (APTES) is an amino silane linker that is often used in the functionalisation of surfaces, whose adsorption on the surface increases the number of positively charged sites for EV binding. Functionalisation improves the electrostatic interaction between the negatively charged EV cell membrane and the positively charged surface of the mica. The aim of the work is to establish a protocol for the functionalisation of mica with APTES and to investigate the effect of binding on visualisation in the air mode of AFM by applying different methods for dehydration and drying of the sample. Treatment of EVs before their visualisation included: fixation with paraformaldehyde (PFA, 3 %) and glutaraldehyde (GA, 1.5 %), dehydration with ethanol or 2,2-dimethoxypropane (2,2-DMP) and drying at either critical point of CO2 (CPD) or with hexamethyldisilazane (HMDS). The most optimal sample preparation protocol for visualisation is the one that involves functionalisation of mica by APTES in vapour, dehydration with 2,2-DMP and HMDS drying. This protocol showed the best overlap in total detected EVs according to diameter and height, highest diameter median and mean and lowest number of outliers out of all other protocols. The detected shapes of EVs were round, “cup-shaped” and multilobed. Future research should focus on methods that can differentiate EVs from other particles of the same outward appearance, like immuno-based interaction of EV antigens with antibodies.
Abstract (croatian)
Izvanstanične vezikule (IV) su nanočestice koje su prisutne u tjelesnim tekućinama uključujući i cerebrospinalnu tekućinu ljudi. Njihova vizualizacija se provodi elektronskom mikroskopijom i mikroskopijom atomskih sila, engl. Atomic Force Microscopy (AFM). AFM je napredna nano-tehnologija koja omogućava vizualizaciju morfologije u nanometarskoj rezoluciji. Najčešći supstrat koji se koristi za vizualizaciju je tinjac međutim potrebna je njegova funkcionalizacija s ciljem jačeg vezivanje IV-a, pogotovo u zračnom modu AFM-a. 3-aminopropiltrietoksisilan (APTES) je amino-silanski linker koji se često koristi u funkcionalizaciji površina, a čijom adsorpcijom na površinu se povećava broj pozitivno nabijenih mjesta za vezanje IV-a. Time se poboljšava elektrostatska interakcija između negativno nabijene stanične membrane IV-a i pozitivno nabijene površine tinjca. Cilj rada je uspostaviti protokol funkcionalizacije tinjca s APTES-om i istražiti učinak vezivanja na vizualizaciju u zračnom modu AFM-a primjenjujući različite metode sušenja uzorka. Prije vizualizacije IV-e su fiksirane otopinom paraformaldehida (PFA, 3 %) i glutaraldehida (GA, 1.5 %), dehidrirane etanolom ili 2,2-dimetoksipropanom (2,2-DMP) i osušene pri kritičnoj točki CO2 ili heksametildisilazanom (HMDS). Optimalni protokol pripreme uzorka za vizualizaciju uključuje: fukcionalizaciju tinjca isparavanjem APTES-a, dehidraciju sa 2,2-DMP-om i sušenje HMDS-om. Ovaj protokol je pokazao najbolje preklapanje promjera i visine po ukupnom broju IV-a, najveći medijan promjera i srednju vrijednost te najmanji broj čestica koje odstupaju u usporedbi s ostalim protokolima. Detektirane čestice bile su okruglog, višerežnjastog i konkavnog oblika. Budući da su ove metode usredotočene na otkrivanje IV-a prema njihovom obliku, promjeru i visini, naredna istraživanja trebala bi se usredotočiti na metode koje mogu razlikovati IV-e od drugih čestica istog ili sličnog vanjskog izgleda, primjerice imunointerakcija antigena IV-a s protutijelima.
Keywords
Keywords (croatian)
Languageenglish
URN:NBNurn:nbn:hr:193:235297
Study programmeTitle: Biotechnology in medicine
Study programme type: university
Study level: graduate
Academic / professional title: magistar/magistra biotehnologije u medicini (magistar/magistra biotehnologije u medicini)
Type of resourceText
File originBorn digital
Access conditionsOpen access
Terms of useLicence In copyright icon
Created on2022-09-29 09:28:51