No abstract

A new biosensor platform that provides simultaneous fluorescence detection and electrochemical control of biospecific binding has been developed and investigated using antibody-antigen and streptavidin-biotin interactions. Specifically, biotin was covalently bound to a transparent indium-tin oxide (ITO) working electrode, which also served as an integral part of a total internal reflection fluorescence (TIRF) flow cell. TIRF was used to monitor biospecific interactions, while electrochemical polarization was employed to control interactions between biotin and streptavidin or between biotin and anti-biotin antibodies. Both streptavidin and polyclonal anti-biotin antibodies bound kinetically irreversibly to the biotinylated surface. In the absence of electrochemical control, the assay exhibited an extremely slow release of the bound analytes, causing poor regeneration ability of the biosensor surface. However, electrochemical polarization was found to stimulate dissociation of kinetically irreversibly bound biotin-streptavidin and antibody-antigen complexes. A "square wave" polarization function stimulated dissociation more effectively than a "saw tooth" function over the same voltage range. Application of the square wave polarization resulted in regeneration of an active biotinylated surface. Electrochemical polarization also modified affinity and kinetics of protein adsorption, which could likely be used to promote biospecific interactions and/or to suppress nonspecific adsorption.

Nonspecific interactions related to physicochemical properties of bacterial cell surfaces, such as hydrophobicity and electrostatic charge, are known to have important roles in bacterium-host cell encounters. Streptococcus pneumoniae (pneumococcus) expresses multiple, surface-exposed, choline-binding proteins (CBPs) which have been associated with adhesion and virulence. The purpose of this study was to determine the contribution of CBPs to the surface characteristics of pneumococci and, consequently, to learn how CBPs may affect nonspecific interactions with host cells. Pneumococcal strains lacking CBPs were derived by adapting bacteria to a defined medium that substituted ethanolamine for choline. Such strains do not anchor CBPs to their surface. Cell surface hydrophobicity was tested for the wild-type and adapted strains by using a biphasic hydrocarbon adherence assay, and electrostatic charge was determined by zeta potential measurement. Adherence of pneumococci to human-derived cells was assessed by fluorescence-activated cell sorter analysis. Strains lacking both capsule and CBPs were significantly more hydrophobic than nonencapsulated strains with a normal complement of CBPs. The effect of CBPs on hydrophobicity was attenuated in the presence of capsule. Removal of CBPs conferred a greater electronegative net surface charge than that which cells with CBPs possessed, regardless of the presence of capsule. Strains that lack CBPs were poorly adherent to human monocytelike cells when compared with wild-type bacteria with a full complement of CBPs. These results suggest that CBPs contribute significantly to the hydrophobic and electrostatic surface characteristics of pneumococci and may facilitate adherence to host cells partially through nonspecific, physicochemical interactions.

The Haas -Drenth -Wilson (HDW) (Haas et al., 1999) theoretical model was used to correlate osmotic second virial coefficient (B) values with solubility (S) values for equine serum albumin (ESA) and ovalbumin for corresponding solution conditions. The best fit from the theoretical model was compared to experimental S versus B data. B values were experimentally measured using static light scattering. Solubilities of ESA were estimated using a sitting drop method. When the experimental data for S versus B were plotted, an excellent fit for ESA was obtained according to the HDW model. The results showed that the coordination number (z) in the crystal lattice was 6, and the adjustable parameter (A) was 0.072. For ovalbumin, previously reported solubility data in aqueous ammonium sulfate solutions were utilized. The solubility data for ovalbumin were correlated with the measured B values obtained in our laboratory. The resulting best fit from the HDW model showed that z = 6 and A = 0.084.

A biomass pretreatment process called wet oxidation that utilized water, oxygen (240-480 psi), and temperatures above 120 °C was applied to loblolly pine, black oak, and a mixture of low-grade hardwoods. The process was found to be effective for fractionating the hemicellulose, lignin, and cellulose components of wood. Acid hydrolysis studies showed that the wet oxidation also enhanced the rate at which cellulose was hydrolyzed by acids to glucose.