Naslov
Optimization of microchip-based capillary electrophoresis for studying oncolytic adenovirus-peptide binding ratio

Autori
Batelić, Laura

Vrsta, podvrsta i kategorija rada
Ocjenski radovi, diplomski rad, diplomski

Fakultet
Farmaceutsko-biokemijski fakultet

Mjesto
Zagreb

Datum
16.09

Godina
2019

Stranica
39

Mentor
Nigović, Biljana

Neposredni voditelj
Sikanen, Tiina

Ključne riječi
Mikročip, elektroforeza, tiol-en, adsorpcija peptida, prevlake mikrokapilara, PEG
(Microchip, electrophoresis, thiol-ene, peptide adsorption, microchannel coatings, PEG)

Sažetak
One of the limitations in protein analysis by microchip capillary electrophoresis (MCE) is the adsorption of the analytes onto the walls of the separation channel because of the electrostatic or hydrophobic interactions of proteins with the channel. Capillary coatings are required to prevent protein adsorption phenomena onto the capillary wall and to increase the repeatability of the separations. In addition of that, capillary coatings should thus ensure high separation efficiency and good repeatability. In this work, thiol-ene based microchips were used for analysis of PolyK-SIINFEKL peptide. MCE- LIF(laser-induced fluorescence) method was developed in this work for future studying of binding ratios between oncolytic adenovirus and MHC-I epitope peptide (PolyK-SIINFEKL peptide). Since the peptides used in research have been fluorescently labeled with fluorescein isothiocyanate (FITC- peptides) optimization of the MCE-LIF method involved measurements of the fluorescein to adjust settings such as a detection slit size, injection time and photomultiplier tube settings. Validation characteristics such as detection limit, quantitation limit and linearity were demonstrated only on fluorescein samples due to the protein adsorption onto the microchannels (SIINFEKL peptide -non interactive with virus and PolyK-SIINFEKL peptide-interactive with virus). Successful microchannel coatings should have been done prior to method validation by FITC-peptides. Conventional PEGylation on allyl-rich thiol-ene microchannels was a viable approach to reduce both the surface charge and hydrophobicity of the native surfaces, as expected, but was not enough to eliminate nonspecific adsorption of the peptide on allyl-rich thiol-ene surfaces. Using the peptide non fluorescent analogue as the dynamic coating on thiol-rich thiol-ene microchannels showed more promising results. The dynamic peptide coating facilitated MCE analysis of the fluorescent polyK-SIINFEKL peptide on thiol-rich thiolene surfaces.

Izvorni jezik
Engleski

Znanstvena područja
Farmacija

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