Synthesis and Characterization of Functionalized Bio-Molecular Surfaces with Self-Assembled Monolayers and Bioreactive Ligands for Nano/Biotechnological Applications

Abstract

In this work, the synthesis and characterization of functionalized biosurfaces that can be used for bioseparations and bio-nanotechnology are reported. A novel protein purification technique that incorporates chelating ligands and polymers onto the same chromatographic matrix is explored. A polysaccharide based gel, agarose, was modified systematically with hybrid ligands of the chelator iminodiacetic acid (IDA) and the polymer polyethylene glycol (PEG). The PEG molecule acts as a blocking polymer that can allow only small proteins to permeate onto the matrix surfaces and form conventional immobilized metal ion affinity chromatographic (IMAC) interactions with the chelators. Kinetic studies of chelator and polymer attachment were performed in order to effectively control the chelator and polymer densities on the matrix. Studies with different PEG surface densities and their effects on the adsorption of several proteins (e.g. myoglobin, lysozyme and bovine serum albumin (BSA)) were evaluated to characterize these new hybrid size exclusion IMAC (SEIMAC) matrices. An exclusion effect was observed while adsorption as observed in IMAC systems took place.Functionalization schemes and procedures were extended in the activation and incorporation of affinity ligands on inorganic surfaces such as gold surfaces. Functional gold platforms were explored for development of nano-interconnects via functionalized self assembled monolayers (FSAMs) on gold to attach specific affinity ligands as linkers to immobilize biomolecules, such as microtubules (MTs). MTs eventually could be utilized as self assembling structures and templates for fabrication of nano-scale bio-interconnect arrays and networks. In this work, different organothiols were used to form FSAMs and anti-glutathione S-transferase was attached as a linker to utilize the attachment of MT cap proteins, gamma-tubulin. The gamma-tubulin could recognize specifically a heterodimer of the MTs and can provide a nucleation center for MT growth. Several methodologies were employed including photolithographic methods and the use of photoreactive compounds for proper micro/nano scale dual protein functionalization of surfaces with homogeneous affinity ligands and with heterogeneous ligands as well.