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

master's thesis

• Author: Laura Batelić
• Mentor: Biljana Nigović (mentor)
• Committee member: Biljana Nigović (predsjednik povjerenstva)
• Committee member: Miranda Sertić (član povjerenstva)
• Committee member: Ivana Perković (član povjerenstva)
• Granter: University of Zagreb; Faculty of Pharmacy and Biochemistry; (Department of pharmaceutical analysis); Zagreb
• Defense date and country: 2019-09-16, Croatia
• Scientific / art field, discipline and subdiscipline: BIOMEDICINE AND HEALTHCARE; Pharmacy; Pharmacy

Abstract

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 thiol-ene surfaces.

Abstract (croatian)

Jedno od najvećih ograničenja u analizi proteina kapilarnom elektroforezom na mikročipu je njihova adsoprcija na stijenku kapilare kao posljedica elektrostatskih i hidrofobnih interakcija. Prevlačenje kapilarnih stijenki nužno je kako bi se spriječila adsorpcija proteina i time povećala reproducibilnost separacije.
U ovom radu, korištene su tiol-en mikrokapilare za analizu PolyK-SIINFEKL peptida. Kapilarna elektroforeza na mikročipu s laser-induciranom fluorescencijom kao načinom detekcije (MCE-LIF), pokušala se razviti u svrhu proučavanja vezanja onkolitičkog CpG adenovirusa i MHC-I epitopa peptida (PolyK-SIINFEKL peptid) kako bi se našla točka zasićenja u kojoj bi virus bio kompletno prekriven PolyK-SIINFEKL peptidom. Budući da su peptidi korišteni u istraživanju obilježeni s fluorescein izotiocijanatom (FITC), fluorescein je korišten u svrhu optimizacije MCE-LIF metode odnosno podešavanja veličine detekcijskog prozora, vremena injektiranja i postavki fotomultiplikatora. Fluorescein je također korišten u svrhu validacije metode za određivanje linearnosti, granice detekcije i kvanitifikacije. Budući da su se FITC-peptidi korišteni u istraživanju adsorbirali na stijenku nemodificiranih kapilara, metoda se nije mogla validirati za prvobitnu primjenu te se istraživanje usredotočilo na prevlake kapilara putem kojih bi PolyK-SIINFEKL peptid i SIINFEKL peptid neometano stizali do detekcijskog prozora na kapilari. Unatoč tome što je kovalentna imobilizacija polietilen glikola na kapilarnim stijenkama očekivano snizila hidrofobnost stijenki kapilara, to nije bilo dovoljno da se eliminiraju nespecifične adsoprcije peptida na stijenci kapilare. Fizikalna adsoprcija peptidnog analoga PolyK-SIINFEKL peptida (nefluorescentnog) na kapilarnim stijenkama pokazala je obećavajuće rezultate. Ubrzala je MCE-LIF analizu PolyK-SIINFEKL peptida na tiol-en mikrokapilarama.

• Keywords: microchip; electrophoresis; thiol-ene; peptide adsorption; microchannel coatings; PEG
• Keywords (croatian): mikročip; elektroforeza; tiol-en; adsorpcija peptida; prevlake mikrokapilara; PEG
• Language: english
• URN:NBN: urn:nbn:hr:163:198939
• Study programme: Title: Pharmacy; Study programme type: university; Study level: integrated undergraduate and graduate; Academic / professional title: magistar/magistra farmacije (magistar/magistra farmacije)
• Type of resource: Text
• File origin: Born digital
• Access conditions: Open access
• Created on: 2020-02-21 12:41:37