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Author:Vaittinen, Maija
Title:Oriented antibody biointerfaces on nanofibrillated cellulose materials
Orientoidut vasta-aine -biointerfaasit nanofibrilloiduilla selluloosamateriaaleilla
Publication type:Master's thesis
Publication year:2013
Pages:viii + 95 s. + liitt. 2      Language:   eng
Department/School:Puunjalostustekniikan laitos
Main subject:Puunjalostuksen kemia   (Puu-19)
Supervisor:Laine, Janne
Instructor:Orelma, Hannes
OEVS:
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Location:P1 Ark Aalto  2257   | Archive
Keywords:NFC film
paper
NFC model surface
oriented immobilization
antibodies
protein A of Staphylococcus aureus
avidin - biotin interaction
NFC-kalvo
paperi
NFC-mallipinta
orientoitu kiinnittäminen
vasta-aineet
proteiini A
avidiini - biotiini -vuorovaikutus
Abstract (eng): Utilization of cellulose based materials in immunodiagnostic applications have recently gained increased interest.
The literature part of this work reviews the basic structure of cellulosic fibers and some of the preparation methods, properties and functionalization techniques for nanofibrillated cellulose (NFC) and NFC films as well as some recent developments in diagnostic applications of cellulose based materials.

In the experimental part a potential cellulose based support material for immunodiagnostic applications was developed by combining paper and an NFC film to a layer-structured material.
The paper part of the layered material wicked water and hydrophobic channels were prepared on it as the NFC layer provided stiffness for the material.
For analysing the interactions of proteins with cellulosic materials, the adsorption of proteins on NFC was analysed by the Surface Plasmon Resonance technique with NFC-model cellulose surfaces.

BSA and human IgG were adsorbed on NFC model surfaces and the adsorption was determined to be influenced by electrostatic interaction at least at pH below the isoelectric point (IEP) of the respective protein.
At pH above their IEP non-electrostatic interactions seemed to dominate the protein adsorption on NFC.
Increased ionic strength of solution seemed to increase BSA adsorption on NFC at pH above its IEP due to reduced electrostatic repulsion between BSA and NFC.
Additionally two different approaches were utilized to immobilize antibodies with specific orientation on NFC.

The first approach utilized the specific interaction of protein A of Staphylococcus aureus with most IgG molecules.
Protein A was pre-adsorbed on unmodified and CMC-modified NFC-surfaces and anti-human IgG was then adsorbed on top of the protein A treated NFC.
These prepared bio interfaces were found to increase substantially the hIgG recognition sensitivity compared to a system where anti-hIgG was directly adsorbed on NFC.

The other approach utilized the specific interaction of avidin with biotin.
Chitosan-modified NFC surface was biotinylated after which avidin was allowed to adsorb on the surface.
Biotinylated anti-hIgG was then adsorbed on the avidin treated surface.
The avidin based bio interface also increased the recognition sensitivity of hIgG compared to the system with directly adsorbed anti-hIgG on NFC.
The best bio interface for hIgG recognition in this work was the protein A based bio interface on CMC-modified NFC when protein A was adsorbed at pH 5.
Abstract (fin): Kiinnostus selluloosapohjaisten materiaalien hyödyntämiseen immuno-diagnostisissa sovelluksissa on viime aikoina lisääntynyt.
Tämän työn kirjallisuusosa käsittelee lyhyesti selluloosakuitujen rakennetta sekä joitain nanofibrilloidun selluloosan (NFC) ja NFC-kalvojen valmistusmenetelmiä, ominaisuuksia ja funktionalisointitapoja.
Myös joitain uusia kehityssuuntia selluloosapohjaisten materiaalien hyödyntämisessä diagnostisissa sovelluksissa käsitellään.

Kokeellisessa osassa kehitettiin mahdollinen kerrosrakenteinen tukimateriaali immunodiagnostisille testeille yhdistämällä paperiarkki ja NFC-kalvo.
Kerrosrakenteisen materiaalin paperiosa imi vettä ja hydrofobisia kanavia pystyttiin valmistamaan sen päälle NFC-kalvon tarjotessa kaivattua jäykkyyttä materiaalille.
Myös proteiinien adsorptiota NFC-mallipinnoille analysoitiin pintaplasmoniresonanssimenetelmän (SPR) avulla.

Sähköstaattisten vuorovaikutusten havaittiin ohjaavan naudan seerumin albumiin (BSA) ja ihmisen IgG:n (hIgG) adsorptioita NFC:hen proteiinien isoelektrisen pisteen alapuolisessa pH:ssa.
Proteiinien isoelektrisen pisteen yllä olevassa pH:ssa adsorptioon vaikutti myös muutkin kuin sähköstaattiset vuorovaikutukset sekä liuoksen suurempi ionivahvuus lisäsi BSA:n adsorptiota NFC:hen pienentyneen sähköstaattisen repulsion vuoksi.
Lisäksi kahta eri lähestymistapaa hyödynnettiin vasta-aineiden orientoituun kiinnittämiseen NFC-pinnoille.

Ensimmäisessä tavassa hyödynnettiin Staphylocccus aureksen proteiini A:n spesifistä vuorovaikutusta IgG molekyylien kanssa.
Proteiini A:ta adsorboitiin ensin muokkaamattomalle ja CMC-muokatulle NFC-pinnalle, jonka jälkeen anti-hIgG:tä adsorboitiin sen päälle.
Nämä biointerfaasit lisäsivät huomattavasti hIgG:n tunnistusta verrattuna systeemiin, jossa anti-hIgG oli suoraan adsorboitu NFC:hen.

Toinen lähestymistapa hyödynsi avidiinin spesifistä vuorovaikutusta biotiinin kanssa.
Kitosaanimuokattu NFC-pinta biotinyloitiin, jonka jälkeen sille adsorboitiin avidiinia.
Biotinyloitua anti-hIgG:ta adsorboitiin sitten avidiinikäsitellylle NFC-pinnalle.
Avidiini-pohjainen biointerfaasi lisäsi myös huomattavasti hIgG:n tunnistuskykyä sellaiseen systeemiin verrattuna, jossa anti-hIgG oli suoraan adsorboitu NFC-pinnalle.
Paras biointerfaasi hIgG:n tunnistukseen tässä työssä oli proteiini A -pohjainen biointerfaasi CMC-muokatulla NFC-pinnalla, johon proteiini A oli adsorboitu pH 5:ssä.
ED:2013-04-02
INSSI record number: 46023
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