This dissertation focuses on the synthesis and characterization of glyconanomaterials, as well as their applications in studying carbohydrate-protein interactions. A new and versatile method for coupling underivatized carbohydrates to nanomaterials including gold and silica nanoparticles was developed via the photochemically induced coupling reaction of perfluorophenylazide (PFPA). A wide range of carbohydrates including mono-, oligo- and poly-saccharides were conjugated to the nanoparticles with high yields and efficiency. New analytical methods were developed to determine the binding affinities of glyconanoparticles (GNPs) with lectins; these include fluorescence-based competition assay, dynamic light scattering (DLS) and isothermal titration calorimetry (ITC). Results showed that the multivalent presentation of carbohydrate ligands significantly enhanced the binding affinity of GNPs by several orders of magnitude compared to the free ligands. Systematic studies were carried out to investigate the impact of ligand presentation, i.e., the type and length of spacer linkage, the ligand density and the nanoparticle size on the binding affinity of the resulting glyconanoparticles. We used gold GNPs to study interactions with anti-HIV lectin cyanovirin-N (CV-N), and dye-doped silica nanoparticles for labeling glyans and developing high-throughput screening technique.

Biofouling is an ongoing problem in the development and usage of biomaterials for biomedical implants, microfluidic devices, and water-based sensors. Antifouling coatings involving surface modification of biomaterials is widely utilized to reduce unwanted protein adsorption and cell adhesion. Surface modification strategies, however, are reliant on the working material’s chemical properties. Thus, published procedures are often not applicable to a wide range of material classes. This constitutes a serious limitation in using surface modification on assembled multi-material devices, i.e on whole device modification. The objective of this research is to develop an antifouling coating with non-aggressive reaction conditions that can universally modify polymers and other material classes. Two strategies using polydopamine (PDA) as an anchor for polyethylene glycol (PEG) surface attachment were investigated: (1) PDA-PEG backfilled with bovine serum albumin (BSA), and (2) PDA-PEG with light activated perfluorophenyl azide (PFPA) conjugated to the PEG. Three materials varying in surface wettability were studied to evaluate the coatings for multi-material applications: porous polycarbonate membrane (PC), polydimethyl siloxane (PDMS), and soda lime glass cover slips. Atomic force microscopy (AFM) and ellipsometry studies revealed substantial structural differences of PDA. Differences in PDA surface roughness affected PEG grafting in solution (the first method), with higher PEG coverage achieved on PC with intermediate surface roughness to PDMS and glass. Radiolabeled Fg adsorption and E. coli adhesion experiments showed reduced fouling on all PDA-PEG modified materials when backfilled with BSA. The ability for BSA to penetrate the PEG layer indicated that low PEG grafting densities were achieved using this grafting-to approach. The use of a photoactive labeling agent, PFPA, to tether PEG was proposed to improve PEG grafting on PDA. The PFPA-PEG modification protocol was optimized by quantifying Fg adsorption. Two treatments of PFPA-PEG were required to fully block PDA active sites. Fg adsorption was not significantly improved on PFPA-PEG modified PC and glass when backfilled with BSA, indicating sufficient PEG coverage of PDA. High Fg adsorption on PFPA-PEG surfaces indicate that high density PEG brushes were still not achieved with this method. PDMS surfaces were damaged with this procedure due to increased surface handling in the protocol. This is the first, to our knowledge, successful demonstration of PFPA modification on PDA surfaces. Photopatterning of polymer-based materials can be achieved, providing opportunities for utilising new materials in cell patterned platforms. Due to low PEG coverage on PDA surfaces from solution and using PFPA, ultra-low protein adsorption cannot be achieved using these aqueous-based methods. Antifouling modifications using PDA and PEG should be applied for short-term cell studies. / Thesis / Master of Applied Science (MASc)

This thesis focuses on developing a new photocoupling surface, base on polyallyamine (PAAm), to increase the efficiency of the photocoupling agent perfluorophenyl azide (PFPA) in the immobilization of nanoparticles, carbohydrates and graphene. Extensive studies have been carried out in our lab on the covalent immobilization of polymers and graphene using PFPA-functionalized surfaces. Here we show that PAAm-based PFPA surface can be used to efficiently immobilize not only graphene and polymers but also nanomaterials and small molecules. This was accomplished by first silanizing silicon wafers with PFPA-silane followed by attaching a thin film of PAAm by UV radiation. Treating the PAAm surface with N-hydroxysuccinimide-derivatized PFPA (PFPA-NHS) yielded the PAAm-PFPA surface. The functionalized surfaces were characterized by ellipsometry (layer thickness), contact angle (surface tension), and ATR-FTIR. The PAAm surface was further characterized by determining the density of amino groups on the surface. The PAAm-PFPA surfaces were subsequently used to covalently immobilize polymers, nanomaterials, carbohydrates and graphene by a simple procedure of coating the molecules or materials on the PAAm-PFPA surface followed by UV irradiation. The resulting surfaces were characterized using ellipsometry, AFM, optical microscopy. The attached carbohydrates were further evaluated using lectins, i.e., carbohydrate-binding proteins.

This thesis focuses on the development of carbohydrate coupling chemistry on nanomaterials and their biological activity studies. It is divided into two parts: In part one, two carbohydrate immobilization approaches, based on perfluorophenyl azide (PFPA)-functionalized silica nanoparticles (SNPs), are presented, where the binding affinity of the glyconanoparticles was evaluated through carbohydrate-lectin interaction. In the first approach, PFPAfunctionalized SNPs were treated with propargylated glycosides and functionalized under copper-catalyzed azide-alkyne cycloaddition (CuAAC) conditions to give glyconanoparticles. For the second approach, a metal-free coupling chemistry based on perfluorophenyl azide-aldehyde-amine cycloaddition (AAAC) was developed for carbohydrate immobilization on PFPA-functionalized SNPs using glycosyl amine and phenylacetaldehyde. Subsequently, a quantitative fluorine nuclear magnetic resonance (19F qNMR) technique was developed to determine the carbohydrate density on the glyconanoparticles. The addition of an internal standard allowed the accurate determination of carbohydrate density, which was then used to calculate the apparent dissociation constant (Kd ) of the glyconanoparticles with lectin by a ligand competition assay. The developed approaches proved general and versatile, and the carbohydrate-presenting nanoplatforms showed high binding specificity in lectin binding. In part two, microwave irradiation was used to functionalize carbon nanomaterials with PFPA followed by carbohydrate conjugation. The microwave-assisted method proved efficient for a number of carbon nanomaterials including carbon nanotubes (CNTs), graphene and fullerene. The carbohydrates on the glyconanomaterials retained their binding patterns towards cognate lectins. / <p>QC 20150907</p>