The Detection of Biomolecules and Their Secondary Structures in Aqueous Media via Overlayer Enhanced Attenuated Total Reflectance Infrared Spectroscopy

The detection of proteins on substrates in aqueous solution is of great importance for the development of biosensors. It has been shown that it is possible to tether proteins near the surface via self-assembled monolayers (SAMs) without the protein losing its structure and function. Once the protein is detected, it is important to understand the structure it takes on. The secondary structure of a protein is generally determined via X-ray diffraction or nuclear magnetic resonance spectroscopy but in the past thirty years infrared spectroscopy has seen a boom in use for determination of secondary structure determination. Still there is some discrepancy on whether to use curve fitting or second derivative integration to determine the secondary structure of a protein using infrared spectroscopy.;Avidin, a protein commonly used in association with biotin, is adsorbed onto various SAMs to determine the ideal surface for maximum adsorption. Of the SAMs tested, 11-mercaproundecanoic acid (11-MUA) adsorbed the most avidin. The secondary structure of avidin adsorbed on each SAM is determined using both second derivative integration and curve fitting to figure out which technique gives more accurate results. The results show that curve fitting and second derivative integration can be used interchangeably as long as there is no negative band due to desorbed water molecules leaving the surface as protein molecules are adsorbing onto the surface.;An antifreeze protein extracted from the beetle Dendroides canadensis is then adsorbed onto a SAM of 11-MUA to test the adsorption of this protein with unknown structure. Antifreeze proteins are known to suppress the freezing point of the solution but there is not sufficient evidence on how the protein does this. Determining the secondary structure of this protein would be a start to figure out the mechanism for the freezing point depression. The 11-MUA surface is a suitable surface to tether this protein and based on the results the mechanism for the depression of the solution may be from increases in turn structures which trap ice crystals and inhibit the aggregation of ice.;Multi-layer systems, especially those of avidin-biotin, are important because of their high affinity to one another. Biotin is routinely introduced to avidin using another protein molecule, such as bovine serum albumin (BSA), and the protein is called a biotinylated protein. Even though the avidin-biotin system has been studied extensively, there are not very many studies involving avidin with the biotinylated proteins. The investigation of avidin and BSA in multi-layer formations is studied to determine whether avidin and BSA interact with each other. According to the results, the protein-protein interactions cannot be ignored. Therefore, caution must be used when using a biotinylated protein with avidin since the protein-avidin interaction can have an effect on the system.