Date of Graduation

12-2016

Document Type

Dissertation

Degree Name

Doctor of Philosophy in Chemistry (PhD)

Degree Level

Graduate

Department

Chemistry & Biochemistry

Advisor

Julie A. Stenken

Committee Member

David Paul

Second Committee Member

Shannon Servoss

Third Committee Member

Wesley Stites

Fourth Committee Member

Suresh Thallapuranam

Keywords

Pure sciences; 4-Hydroxybenzoic acid; Cytokine; Heparin; Microdialysis; Nonspecific adsorption; Relative recovery

Abstract

Microdialysis (MD) sampling is a diffusion-based separation method which has the ability to sample any analyte that can diffuse across the semi-permeable membrane. However one challenge for MD is that for soluble proteins greater than 10 kDa, the relative recovery (RR) using a 100 kDa MD probe is between 1-5%.1 There are two major barriers that lead to these low recovery values - nonspecific adsorption (NSA) and poor solute mass transport. To overcome these two barriers, the modification of PES-based MD membranes has been initiated by laccase. Previous researchers have used laccase to modify PES flat sheet and hollow fiber membranes using 4HBA to create a hydrophilic polymer chain network.2 Furthermore by functionalizing the MD membranes with carboxylic acid functional groups from 4HBA, one can easily modify the surface.3 This study focuses on characterization of the PES membrane surface before and after attachment of 4HBA polymers and heparin. First the attachment of 4HBA and heparin has been confirmed using XPS and ATR-FTIR. Next protein adsorption measurements were performed for 4HBA modified flat sheets which showed an initial increase in BSA adsorption followed by a decrease in BSA adsorption after 24 hours of modification. However, for positively charged lysozyme the protein adsorption increased upon modification. RR experiments were performed using FITC-labeled dextrans, lysozyme, CCL2, VEGF, TNF-a, KC/GRO and aFGF. After modification with 4HBA for 2 hours, RR of CCL2, KC/GRO, and VEGF increased 2 to 3 times compared to the control relative recovery however, this increase in RR was not observed for aFGF and TNF-a. This difference could be due to the isoelectric points (pI) of these proteins indicating an electrostatic interaction between the surface and the protein. For 24 hour 4HBA-heparin modified membranes CCL2 RR increased twofold for hours 3 and 4 and for 2 hour 4HBA-heparin modified membranes aFGF RR increase threefold.

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