ATR-IR Spectroscopic Studies of Chlorhexidine, Lysozyme, Micrococcus Luteus Films and their Interactions at Solid-Liquid Interfaces

Abstract

Understanding the interactions of biologically important materials at interfaces is becoming increasingly important especially for the advancement of medicine, dentistry and industrial engineering. As bacteria cells are difficult to remove once adhered to a surface, strategies to prevent or limit adhesion have become a requirement. Adsorption of enzymes and adhesion of bacteria have been extensively reported, however, there is limited information on the actual mechanisms involved. Even more limited are reports of the activity of an adsorbed enzyme against bacterial cells. Important questions include physical factors which influence enzyme adsorption, the activity of adsorbed enzymes and means to monitor enzymatic activity in situ. In this thesis, attenuated total reflection infrared (ATR-IR) spectroscopy was used to probe the solid-liquid interfacial phenomena involving 2 antimicrobials, chlorhexidine (CHX) and lysozyme (LYS), and the effectiveness of free and adsorbed LYS against the bacterium Micrococcus luteus (ML). Phosphate had previously been shown to form bidentate linkages to titanium dioxide surfaces (Connor and McQuillan, 1999). In the current study, the addition of phosphate to a CHX dihydrochloride (DHC) solution specifically and dramatically enhanced its adsorption on to a TiO2-coated ZnSe IR prism. The more soluble digluconate (DG) salts of CHX showed a lesser increase in adsorption than that of the DHC salt. Also, the enhanced CHX adsorption due to the contribution of adsorbed gluconate was inferior to that obtained by the addition of phosphate. By varying the bulk pH and ionic strength (IS) of the LYS solution flowing across a ZnSe IR prism, the influence of electrostatic attractions on LYS adsorption was highlighted. Further, LYS adsorbed more to the hydrophilic TiO2-coated ZnSe IR prism than to an uncoated ZnSe IR prism. Discrete adsorption rates of LYS with increasing LYS concentration suggested that LYS was adsorbing as a multi-layer assemblage on to the ZnSe prism. LYS adsorbed and desorbed at faster rates on an uncoated compared to a TiO2-coated ZnSe IR prism. A novel phosphate-modified TiO2 (pmTiO2) coating material for ZnSe IR prism was prepared by mixing phosphate buffer with a TiO2 suspension. This approach increased the negative charge on the TiO2 surface via coordinated linkages with the phosphate, and increased the initial rate of LYS adsorption. However, the rate of LYS adsorption onto the pmTiO2-coated ZnSe prism was still lower than that on to an uncoated ZnSe prism. Enhanced adsorption of LYS was achieved by increasing the solution pH (from pH 4 to pH 11 and at IS 0.01 mol L-1) and IS (from IS 0.001 to 1.0 mol L-1 and pH 6). Maximum LYS adsorption occurred at pH 11 which corresponded to the isoelectric pH (pI) of LYS. The adsorption of LYS to ZnSe could be explained in terms of electrostatic interactions between the protein and the surface. ML adhesion on to a ZnSe prism, from an ML suspension flowing over a ZnSe prism surface was generally rapid but a surface saturation state did not generally occur. The effects of pH and IS variations on ML adhesion over a low to medium IS could be explained in terms of attractive electrostatic interactions between the ML cells and the ZnSe surface. Exposure of adhered ML cells to a ML-free 0.1 mol L-1 NaCl solution rinse resulted in an increase in the absorbance of all characteristic IR peaks suggesting the involvement of a non-electrostatic surface-bacteria interaction where the DLVO theory could not be applied. This increase in the ML absorption spectra was temporary, thereby indicating that a disaggregation of ML cell clusters while adhering to the surface was occurring. Physical compression of the outer ML cell polymers when the adhered cells were exposed to a 0.1 mol L-1 NaCl solution resulted in the cells moving closer to the surface, occupying more adhesion sites and enhancing all ML IR peaks. The positively-charged LYS molecules were found to secondarily adsorb onto the negatively-charged pre-adhered ML cells. A flow of a LYS solution across ML cells already adhered to ZnSe caused alterations in the carboxylate-related (1400 cm-1) and carbohydrate, phospholipids and carboxylic acid components (1200-800 cm-1). In parallel studies where LYS was pre-adsorbed, ML cells from a suspension were electrostatically attracted to the positively-charged LYS layer. However, LYS under these conditions was not hydrolytic, presumably due to the enzyme being compressed as it moved physically closer to the surface. There was a discernible reduction in the concentration of viable ML cells attached to a surface after they were exposed to LYS in solution. In conclusion, ATR-IR spectroscopy has proved to be a useful method to study adsorption behaviour of antibacterial agents such as CHX and LYS as single entities and to explore the biologically important interfacial interactions between bacteria cells and enzymes. Such information will be useful in the development of robust strategies to control bacteria-fouled surfaces.