Protein transport through mesoporous materials
Creators: name: Chan, Sze-Lun
Publisher: name: Monash University
Year: 2017
Subjects: Uncategorized

description: Liquid chromatography is a widely used technique for biomolecule separation and analysis. Conventional chromatographic stationary supports mostly have specific limitations in relation to mass transfer and kinetics of adsorption when larger size biomolecules are applied. This results in poor target binding and high levels of non-specific binding in the intra-particle volume. Monolithic supports represent a relatively new class of stationary phases that address the issues of poor binding and slow mass transfer kinetics for large biomolecule applications. A monolithic microfluidic system was designed and applied for on-chip biomolecular separation. Another potential stationary phase material based on lyotropic liquid crystalline system was studied and characterized experimentally. Miniaturization of chromatographic systems is a major research endeavor; however, there are drawbacks relating to the fabrication of these systems. These include manufacturing cost of microdevices and effective introduction of stationary phase materials into microchannels. This thesis presents a detailed design for the fabrication of low-cost microchips that can be used for chromatographic analysis. Self-assembled materials based on lyotropic liquid crystalline systems have a unique bicontinuous nano scale porosity which has led to their use in controlled release and bioseparation applications. This study investigated the potential for these materials to enhance the efficiency of electrophoretic separation. Specifically, this work demonstrated the potential molecular separation capacity of a lyotropic liquid crystalline material and studied its behavior under natural diffusion and electrophoretic conditions. Solute transport through a membrane filled with the mesophase material was monitored by a fluorescence detection technique. A dye mixture of fluorescein and Rhodamine B, and a biomolecule mixture of dextran and Green Fluorescence Protein were separated under natural diffusion condition. The separation mechanism was found to be dependent on the molecular size and other physicochemical properties of the molecules such as polarity. The effect of applied electrical field on solute transport was investigated using fluorescein as the target molecule. The estimated diffusion coefficient and electrophoretic mobility of fluorescien transport through the mesophase material was 2 x 10-13 m2/s and 4.4 x 10-9 m2/SV, respectively. The observed relationship between migration velocity and electrical field was linear, consistent with the current theory of electrophoresis. The design and fabrication of a disposable and inexpensive pressure-driven microfluidic chip, that incorporates a mini stationary phase column for chromatographic separation, is presented. A poly(dimethylsiloxane) (PDMS) microfluidic chip was fabricated using a standard photolithography technique, and the performance for on-chip biomolecular separation using mesoporous silica as reversed-phase chromatography and functionalized monolithic polymer as weak anionic exchange chromatography was investigated. The chromatographic column incorporated with mesoporous silica beads of Ia3d space group was used to verify the chip design and study the performance as a reversed-phase liquid chromatographic column. Separation of dyes and proteins were performed to verify the performance of the chip. A mixture of dyes (fluorescein and Rhodamine B) and a biomolecule mixture (dextran, 10kDa and bovine serum albumin (BSA), 66kDa) were separated. The fluorescence technique was employed to detect the movement of the molecules. Fluorescein molecule was not retained whilst the Rhodamine B was bound onto the silica surface when the dye mixture in deionized water was injected into the microchannel. In the case of the biomolecule mixture, both dextran and BSA molecules were bound onto the silica. The retention times for dextran and BSA molecules were 45s and 120s, respectively. The retention factor was estimated to be 3.3 for dextran and 10.4 for BSA. The selectivity was 3.2 and the resolution was 10.7. The separation of dyes and biomolecules was effectively achieved with the chip. A microfabricated PDMS chip with channel filled with polymer monolith was developed for on-chip biomolecular separation. A methacrylate monolithic polymer was prepared by photoinitiated polymerization within the channel to serve as a continuous stationary phase. The monolithic polymer was functionalized with a weak anion-exchange ligand, and different factors affecting the binding characteristics were investigated. The total binding capacity remained constant regardless of the mobile phase flow rate while changes in the ionic strength and pH significantly affected the binding characteristics. The binding capacity decreased with increasing buffer ionic strength due to limited available binding sites for protein adsorption. Similarly, the binding capacity decreased with decreasing buffer pH towards the isoelectric point of the protein. Also, a protein mixture of BSA and ovalbumin was successfully used to illustrate the capacity of the methacrylate-based microfluidic chip for rapid biomolecule separation.