Razvoj au nanodelcev z metodo ultrazvočne razpršilne pirolize za detekcijo protiteles proti SARS-CoV-2 : doktorska disertacija

Jelen, Žiga

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Title:	Razvoj au nanodelcev z metodo ultrazvočne razpršilne pirolize za detekcijo protiteles proti SARS-CoV-2 : doktorska disertacija
Authors:	ID Jelen, Žiga (Author) ID Rudolf, Rebeka (Mentor) More about this mentor... ID Rakuša, Martin (Comentor)
Files:	DOK_Jelen_Ziga_2024.pdf (5,61 MB) MD5: D624F45D75EE4D4285161A90054A72D8
Language:	Slovenian
Work type:	Doctoral dissertation
Typology:	2.08 - Doctoral Dissertation
Organization:	FS - Faculty of Mechanical Engineering
Abstract:	V doktorskem delu so predstavljene študije, eksperimentalno delo, tehnike karakterizacije, rezultati in analize, s pomočjo katerih smo potrdili hipotezo, da je možno Au nanodelce (AuND) pripravljene z metodo ultrazvočne razpršilne pirolize (USP), konjugirati s ciljnimi antigeni SARS-CoV-2 in rekombinantnim IgG, da bodo delovali v imunoserološkem hitrem testu na lateralni tok (LFIA), za potrditev potencialne prisotnosti protiteles proti virusu SARS-CoV-2. S ciljno izbranimi parametri (0,5 g/l Au, 5 l/min N2, 2,5 l/min H2) smo z USP sintetizirali AuND iz prekurzorja na osnovi zlatovega (III) klorida. AuND smo zbirali v steklenicah z demineralizirano vodo. Določili smo optimalne stabilizatorje in njihove koncentracije: 2,5 g/L PVP 40K ((C6H9NO)n) ter naknadno dodani 1 g/L trinatrijevega citrata (Na3C6H5O7) in saharoze (C12H22O11), da bi preprečili AuND aglomeracijo. Presevna elektronska mikroskopija AuND je pokazala, da so okrogli, s povprečno velikostjo okoli 50 nm in ozko velikostno porazdelitvijo, brez defektov in nizkim ZETA potencialom, 2,4 mV, pri pH 7,5. Za odstranitev vode iz suspenzije z AuND in stabilizatorji je bila uporabljena liofiliazcija, ki je bila izvedena pri naslednjih pogojih: zamrzovanje pri –40 °C, 4 h, atmosferski tlak; primarno sušenje pri +20 °C, 12 h, 1–4 Pa; sekundarno sušenje pri +30 °C, 33 h, 1–4 Pa. Nastali posušeni AuND so ohranili vse lastnosti, ki so potrebne za funkcijo oznak v hitrih LFIA testih: visoka površinska energija, površinska plazmonska resonanca in proste karboksilne skupine za konjugacijo. V nadaljevanju izvajanja eksperimentov smo, da bi potrdili doktorske hipoteze, izvedli raziskavo in optimizacijo konjugacije AuND z antigenom SARS-CoV-2, ki je bodičasti protein S1 (ak 1-681), in s protitelesi na zajčji IgG (RMG03). Za določitev poteka konjugacije AuND smo uporabili posredne (gelske elektroforeze, UV-Vis, preizkus delovanja) in direktne (XPS, SIMS) metode. Rezultati so pokazali, da se med našim postopkom konjugacije, v fazah čiščenja odstranimo večinski delež PVP in saharoze, ter s tem sprostimo proste karboksilne funkcionalne skupine citratnih ionov, ki so vezani na povšini AuND. Na teh prostih mestih lahko poteče konjugacija prek prostih -NH2 skupin na proteinih. Njihovo funkcionalnost smo ovrednotili z eksperimentalnimi testiranji lastno razvitega LFIA testa. Na osnovi tega smo postavili model in mehanizem konjugacije AuND, ki predpostavlja, da poteka konjugacija z antigeni oziroma protitelesi prek tvorbe peptidnih vezi med N-koncem proteina in prostimi karboksilnimi skupinami citratnega iona, ki je vezan na površino AuND prek svojih karboksilnih skupin. Za dodatno potrditev funkcionalizacije AuND z izbranimi biomolekulami smo v sklepnem delu doktorskega dela izvedli klinično študijo na pacientih UKC MB, ki je bila odobrena s sklepom KME RS št. 0120-148/2021/3. Rezultati študije so pokazali, da testi dosegajo visoko občutljivost (83 %) tako na vzorcih nosnega sluza kot na vzorcih seruma, slabšo specifičnost (66 % sluz, 74 % serum), visoko negativno napovedno vrednost (94 %) in slabšo pozitivno napovedno vrednost (38 % sluz, 45 % serum). S študijo smo potrdili delovanje AuND v funkciji oznak, saj so se testne in kontrolne linije v lastno razvitem LFIA hitrem testu obarvale.
Keywords:	Nanodelci zlata, SARS-CoV-2 virus, stabilizacija, konjugacija, LFIA testi, karakterizacija
Place of publishing:	Maribor
Place of performance:	Maribor
Publisher:	[Ž. Jelen]
Year of publishing:	2023
Number of pages:	VI, 96 str.
PID:	20.500.12556/DKUM-84331
UDC:	[620.3:669.21]:616-07(043.3)
COBISS.SI-ID:	190864899
Publication date in DKUM:	28.03.2024
Views:	522
Downloads:	86
Metadata:
Categories:	KTFMB - FS
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Licences

License:	CC BY-NC-ND 4.0, Creative Commons Attribution-NonCommercial-NoDerivatives 4.0 International

Link:	http://creativecommons.org/licenses/by-nc-nd/4.0/
Description:	The most restrictive Creative Commons license. This only allows people to download and share the work for no commercial gain and for no other purposes.
Licensing start date:	23.05.2023

Secondary language

Language:	English
Title:	Development of au nanoparticles by the method of ultrasonic spray pyrolysis for the detection of antibodies against SARS-CoV-2 : doctoral disertation
Abstract:	This doctoral thesis presents the studies, experimental work, characterisation techniques, results and analyses with the help of which we confirmed the hypothesis that it is possible to conjugate Au nanoparticles (AuNPs) prepared by the ultrasonic spray pyrolysis (USP) method with the target antigens of SARS-CoV-2 and recombinant IgG to function in a rapid lateral flow immunoserological assay (LFIA) to confirm the potential presence of antibodies against the SARS-CoV-2 virus. AuNPs were synthesised with the chosen parameters (0.5 g/l Au, 5 l/min N2, 2.5 l/min H2) from a precursor based on Au-chloride, using USP. AuNPs were collected in gas wash bottles filled with demineralized water. We determined the optimal stabilisers and their concentrations with the goal of preventing AuNPs agglomeration: 2.5 g/L PVP 40K ((C6H9NO)n), and subsequently added 1 g/L of trisodium citrate (Na3C6H5O7) and sucrose (C12H22O11). Microstructural analysis of the AuNPs showed them to be round, with sizes of around 50 nm and a narrow size distribution, without defects and with a low ZETA potential of 2.4 mV at a pH of 7.5. To remove water from the suspension with AuNPs and stabilisers, freeze-drying was used, which was carried out under the following conditions: freezing at -40 °C, 4 h, atmospheric pressure; primary drying at +20 °C, 12 h, 1-4 Pa, secondary drying at +30 °C, 33 h, 1-4 Pa. The resulting dried AuNPs retained all the properties required for their use as labels in LFIA assays: high surface energy, surface plasmon resonance, and free carboxyl groups for conjugation. To confirm the doctoral hypothesis, we continued with the experiments and the optimisation of the conjugation process of AuNPs with the SARS-CoV-2 S1 antigen, with the S1 (ak 1-681) subunit, and with anti-rabbit IgG antibodies (RMG03). Indirect methods such as gel electrophoresis, UV-Vis and LFIA experiments, and direct methods such as XPS and SIMS, were used to determine the mechanism of AuNPs conjugation. The results showed that during our conjugation process, the majority of PVP and sucrose is removed in the purification stages, thereby releasing free carboxyl functional groups of citrate ions, which are bound on the AuNPs surface. Conjugation happens via the free -NH2 groups of proteins and the vacant carboxyl sites on the surface of AuNPs. We evaluated their functionality by testing through our own LFIA test. Based on this, we set up a model and mechanism of AuNPs conjugation, which assumes that conjugation with antigens or antibodies takes place through the formation of peptide bonds between the N terminus of the protein and the free carboxyl groups of the citrate ion, which is bound to the AuNPs surface via its carboxyl groups. To further confirm the functionalisation of AuNPs with the selected biomolecules, in the concluding part of the doctoral thesis, we conducted a clinical study on patients of the University Medical Centre in Maribor, which was approved by The Commission of the Republic of Slovenia for Medical Ethics, with decision no. 0120-148/2021/3. The results of the study showed that the tests achieve a high sensitivity of 83% on both nasal mucus and serum samples and lower specificities of 66% (mucus) and 74% (serum), as well as a high negative predictive value of 94% and lower positive predictive values of 38% (mucus) and 45% (serum). Through the study, we confirmed the function of AuNPs as labels, with a successful staining of the test and control lines in our self-developed LFIA test.
Keywords:	Gold nanoparticles, SARS-CoV-2 virus, stabilization, conjugation, LFIA assays, characterization

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