A Versatile Strategy for Characterization and Imaging of Drip Flow Microbial Biofilms.

The inherent architectural and chemical complexities of microbial biofilms mask our understanding of how these communities form, survive, propagate, and influence their surrounding environment. Here we describe a simple and versatile workflow for the cultivation and characterization of model flow-cell-based microbial ecosystems. A customized low-shear drip flow reactor was designed and employed to cultivate single and coculture flow-cell biofilms at the air-liquid interface of several metal surfaces. Pseudomonas putida F1 and Shewanella oneidensis MR-1 were selected as model organisms for this study. The utility and versatility of this platform was demonstrated via the application of several chemical and morphological imaging techniques-including matrix-assisted laser desorption/ionization mass spectrometry imaging, secondary ion mass spectrometry imaging, and scanning electron microscopy-and through the examination of model systems grown on iron substrates of varying compositions. Implementation of these techniques in combination with tandem mass spectrometry and a two-step imaging principal component analysis strategy resulted in the identification and characterization of 23 lipids and 3 oligosaccharides in P. putida F1 biofilms, the discovery of interaction-specific analytes, and the observation of several variations in cell and substrate morphology present during microbially influenced corrosion. The presented workflow is well-suited for examination of both single and multispecies drip flow biofilms and offers a platform for fundamental inquiries into biofilm formation, microbe-microbe interactions, and microbially influenced corrosion.

Single nanopore transport of synthetic and biological polyelectrolytes in three-dimensional hybrid microfluidicnanofluidic devices.

This paper presents a study of electrokinetic transport in single nanopores integrated into vertically stacked three-dimensional hybrid microfluidicnanofluidic structures. In these devices, single nanopores, created by focused ion beam (FIB) milling in thin polymer films, provide fluidic connection between two vertically separated, perpendicular microfluidic channels. Experiments address both systems in which the nanoporous membrane is composed of the same (homojunction) or different (heterojunction) polymer as the microfluidic channels. These devices are then used to study the electrokinetic transport properties of synthetic (i.e., polystyrene sulfonate and polyallylamine) and biological (i.e., DNA) polyelectrolytes across these nanopores using both electrical current measurements and confocal microscopy. Both optical and electrical measurements indicate that electro-osmotic transport is predominant over electrophoresis in single nanopores with d>180 nm, consistent with results obtained under similar conditions for nanocapillary array membranes.

pH-Dependent cis --> trans isomerization rates for azobenzene dyes in aqueous solution.

Azobenzenes can function as molecular switches driven by their unusual cis <--> trans photoisomerization properties. The stability of an azobenzene-based switch depends on its rate of thermal relaxation, which is known to depend on the solvent environment, but few kinetic studies in aqueous media have been reported. We use nanosecond UV laser flash photolysis-transient absorption spectroscopy to measure thermal cis --> trans isomerization rates for mono- and disubstituted p-aminoazobenzenes and p-hydroxyazobenzenes in water at 23 degrees C over the pH range of 4 to 11. Observed absorption transients are fit to first-order relaxation rate constants between 10(5) and 10(1) s(-1), which is generally much faster than in nonpolar solvents, and the relaxation rates vary systematically and predictably with pH as the equilibrium shifts to ionized forms of the dyes that isomerize much more rapidly. Acid ionization constants for these dyes determined from our kinetic mechanism are compared with the pH dependence of their equilibrium UV-vis spectra. New kinetics results may enable pH control of azobenzene-based molecular switching times.

Enzymatic activity of surface-immobilized horseradish peroxidase confined to micrometer- to nanometer-scale structures in nanocapillary array membranes.

Horseradish peroxidase (HRP) was immobilized on the planar surfaces and inside the cylindrical nanopores of nanocapillary array membranes (NCAMs) to study how the enzyme-catalyzed oxidation of a fluorigenic substrate, Amplex Red (AR), to fluorescent resorufin by hydrogen peroxide is influenced by confinement. Because AR was also found to be converted to resorufin photolytically at high laser fluences, a modified laser-induced fluorescence protocol was developed to characterize the enzyme-catalyzed reaction in the absence of interference from the photolytic reaction. Surface-immobilized HRP was studied in two environments: bound to the surface of a microfluidic channel, and bound to the interior of cylindrical nanopores in NCAMs connecting crossed microfluidic channels. HRP was immobilized through reaction of solvent-accessible primary amines with the epoxy group of the methyl methacrylate-glycidyl methacrylate copolymer synthesized in either planar or annular geometries to construct the test structures for enzymatic activity. HRP immobilized on planar surfaces shows high activity (approximately 10 microM min(-1)) meaning that the copolymer membrane exhibits good potential for immobilizing the enzyme, especially since active structures are obtained in a one-step reaction. HRP was also immobilized inside nanopores via physisorption. Enzymatic reactions inside the nanopores were characterized and compared to finite element simulations of a modified Eley-Rideal mechanism to bracket the value of the overall rate constant for the confined enzyme. Reaction velocities were estimated to be approximately 10-fold higher in the nanopores than for the same enzyme bound to a planar microfluidic surface.