Development of a RP-HPLC method for determination of glucose in Shewanella oneidensis cultures utilizing 1-phenyl-3-methyl-5-pyrazolone derivatization.

A method was developed and validated for low-level detection of glucose. The method involves quantitation of glucose though derivitization with 1-phenyl-3-methyl-5-pyrazolone (PMP) and HPLC-DAD analysis. The developed method was found to be accurate and robust achieving detection limits as low as 0.09 nM. The applicability of the method was tested against microbial samples with glucose acting as a carbon fuel source. The method was shown to be able to accurately discriminate and quantify PMP-glucose derivatives within Shewanella oneidensis MR-1 samples. The method proved capable at examining glucose usage during the early hours of microbial growth, with detectable usage occurring as early as two hours. S. oneidensis cultures were found to grow more effectively in the presence of oxygen which coincided with more efficient glucose usage. Glucose usage further increased in the presence of competing electron acceptors. The rate at which S. oneidensis reached exponential growth was affected by the presence of ferric iron under microaerobic conditions. Such samples reached exponential growth approximately two hours sooner than aerobic samples.

Electroactive planar waveguide studies of Ru(bpy)3(2+) intercalated in a thin clay film: I. Transport and electrochemical phenomena.

Electroactive planar waveguide (EAPW) instrumentation was used to perform potential modulated absorbance (PMA) experiments at indium tin oxide (ITO) electrodes coated with 0-, 300-, 800-, and 1200-nm-thick SWy-1 montmorillonite clay. PMA experiments performed at low potential modulation monitor mass transport events within 100 nm of the ITO surface and, thus, when used in conjunction with cyclic voltammetry (CV), can elucidate charge transport mechanisms. The data show that at very thin films electron transfer is controlled by electron hopping (sensitive to the anion species in the electrolyte) in an adsorbed Ru(bpy)(3)(2+) layer. As the thickness of the clay film grows, electron transfer may become controlled by mass transfer of Ru(bpy)(3)(2+) within the clay film to and from the electrode surface, a mechanism that is affected by the swelling of the film. Film swelling is controlled by the cation of the electrolyte. Films loaded with Ru(bpy)(3)(2+) while being subjected to evanescent wave stimulation demonstrate a large hydrophobic layer. The growth of the hydrophobic layer is attributed to the formation of Ru(bpy)(3)(2+*), which has negative charge located at the periphery of the molecule enhancing clay/complex repulsion. The results suggest that the structure of the film and the mechanism of charge transport can be rationally controlled. Simultaneous measurements of the ingress of Ru(bpy)(3)(2+) into the clay film by CV and PMA provide a means to determine the diffusion coefficient of the complex.

Spectroelectrochemical study of hemoglobin A, alpha- and beta-fumarate crosslinked hemoglobins; implications to autoxidation reaction.

The thermodynamics and kinetics of the reaction DeoxyHb-Fe(2+)<-->MetHb-Fe(3+) for human hemoglobin A (HbA), alpha- and beta-fumarate crosslinked hemoglobins were investigated by spectroelectrochemistry. Information from this study is used to determine what structural features and experimental conditions stabilize ferrous vs. ferric form of hemoglobin, and what implications this stabilization may have on the autoxidation reaction. Alpha- and beta-fumarate crosslinked hemoglobins, alphaXL-HbA and betaXL-HbA, were obtained by crosslinking deoxyhemoglobin and oxyhemoglobin, respectively, with bis(3,5-dibromosalicyl) fumarate (DBSF). Formal redox potentials, E(0), and reduction/oxidation rates were measured in the presence of mediator, hexammineruthenium(III) chloride. It was found that E(0) shifted positive for the alpha-, and negative for the beta-fumarate crosslinked hemoglobin compared to HbA for all experimental conditions investigated. This shift was consistent with stabilization of the tense (positive shift) or relaxed conformation (negative shift) conferred by crosslinking. Formal redox potentials shifted positive with addition of nitrate and chloride ions for alphaXL-HbA, indicating additional stabilization of the T quaternary. The slopes of the Nernst plots showed evidence of cooperativity as expressed by n(max). The data points (E(0), n(max)) were fitted by the MWC model which states that the electron transfer and the addition/removal of water are concerted. The set of K(R) and c values, where the parameter c is the ratio K(R)/K(T) and K(R) and K(T) are the ligand (water molecule and an electron-hole) dissociation constants for the R and T states, for the beta-crosslinked hemoglobin compared to that of HbA and alpha-crosslinked hemoglobin indicated that crosslinking of oxyhemoglobin affected differently the inner-coordination sphere at the heme site. By modulating the electrolyte concentration the reduction rates were measured as a function of DeltaE(0), the difference in E(0) between hemoglobin molecules and mediator. Linearization of the Marcus cross-relationship (based on the concerted water and electron transfer) was good for HbA, and poor for alphaXL-HbA and betaXL-HbA, consistent with results obtained by the MWC analysis. This may imply that the reduction of HbA is controlled by the driving force, DeltaE(0), whereas the reduction of alphaXL-HbA and betaXL-HbA occurs by a non-concerted mechanism controlled by structural features brought about by crosslinking. The autoxidation reaction, conversion of oxygen-bound ferrous hemoglobin to ferric hemoglobin, was found independent of E(0). Alpha-fumarate crosslinked hemoglobin showed the highest autoxidation rate despite its positive shift in formal redox potential as compared to HbA, followed by beta-fumarate crosslinked hemoglobin, and by native hemoglobin. These data suggest that the chemical mechanism of oxygen dissociation and accessibility of water and oxygen radicals to heme site control autoxidation.

Use of ferrocenyl surfactants of varying chain lengths to study electron transfer reactions in native montmorillonite clay.

A series of ferrocenyl surfactants was tested as model compounds to study electron transfer reactions involving structural Fe(III) in clay minerals. The surfactants contain trimethylammonium headgroups, ferrocene tail groups, and intervening hydrocarbon chain lengths of one, six, or 11 carbons. Two factors considered to be decisive for electron transfer were addressed: (1) physical access of the surfactant ferrocene to the reactive sites through hexagonal holes in the clay lattice by X-ray diffraction (XRD) and small-angle X-ray scattering (SAXS) and (2) thermodynamic favorability of the overall oxidation/reduction reaction based on experimentally determined oxidation/reduction potentials. In suspensions of clay with the longer chain surfactants, (ferrocenylhexyl)trimethylammonim (FHTMA+) and (ferrocenylundecyl)trimethylammonium (FUTMA+), where electron transfer may be expected to be favored by both factors, physical accessibility, and thermodynamic favorability, ferroecene oxidation was observed by diffuse reflectance infrared spectroscopy (DRIFT), ultraviolet-visible spectroscopy (UV-vis), and visual color changes. In contrast, the shorter chain length surfactant, (ferrocenylmethyl)trimethylammonium (FMTMA+), did not participate in electron transfer with the clay, as substantiated by UV-vis and no visible color changes. Rigid conformation and/or higher oxidation/reduction potential than clay Fe can accountforthe lack of reaction. The utility and limitations of using these surfactants as model compounds is discussed.