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The behavior of pH-responsive polymers poly(N-methacryloyl-l-valine) (P1), poly(N-methacryloyl-l-phenylalanine) (P2), and poly(N-methacryloylglycyne-l-leucine) (P3) has been studied in the presence of the nonionic surfactant Brij98. The pure polymers phase-separate in an acidic medium with critical pHtr values of 3.7, 5.5, and 3.4, respectively. The addition of the surfactant prevents phase separation and promotes reorganization of polymer molecules. The nature of the interaction between polymer and surfactant depends on the amino acid structure in the side chain of the polymer. This effect was investigated by dynamic light scattering, isothermal titration calorimetry, electrophoretic measurements, small-angle neutron scattering, and infrared spectroscopy. Thermodynamic analysis revealed an endothermic association reaction in P1/Brij98 mixture, whereas a strong exothermic effect was observed for P2/Brij98 and P3/Brij98. Application of regular solution theory for the analysis of experimental enthalpograms indicated dominant hydrophobic interactions between P1 and Brij98 and specific interactions for the P2/Brij98 system. Electrophoretic and dynamic light scattering measurements support the applicability of the theory to these cases. The specific interactions can be ascribed to hydrogen bonds formed between the carboxylic groups of the polymer and the oligo(ethylene oxide) head groups of the surfactant. Thus, differences in polymer-surfactant interactions between P1 and P2 polymers result in different structures of polymer-surfactant complexes. Specifically, small-angle neutron scattering revealed pearl-necklace complexes and "core-shell" structures for P1/Brij98 and P2/Brij98 systems, respectively. These results may help in the design of new pH-responsive site-specific micellar drug delivery systems or pH-responsive membrane-disrupting agents.

Cellular response of wild-type Saccharomyces cerevisiae and the Delta yfh1 mutant to oxidative stress (OS) was examined by stressing cells through the addition of H(2)O(2) to the growth medium. The Delta yfh1 mutant is unusual in that it accumulates iron in it is mitochondria. Wild-type growth was immediately arrested and recovered in 2 h following H(2)O(2) treatment. No change in viability was observed. Growth of the mutant, on the other hand, was similar to wild-type yeast for 4 h but then rapidly declined, eventually reaching zero. Levels of carbonyl groups and reactive oxygen species (ROS) reached their maximum at 3 h following exposure. The impact of OS on protein function was also evaluated by proteomic techniques targeting protein carbonylation. Oxidized proteins were selected by affinity chromatography, and following trypsin digestion, peptide fragments were identified by RPLC-MS/MS. A total of 53 proteins were identified in both wild-type and mutant cells, respectively.

Comprehensive analysis of intracellular metabolites is a critical component of elucidating cellular processes. Although the resolution and flexibility of reversed-phase liquid chromatography-mass spectrometry (RPLC-MS) makes it one of the most powerful analytical tools for metabolite analysis, the structural diversity of even the simplest metabolome provides a formidable analytical challenge. Here we describe a robust RPLC-MS method for identification and quantification of a diverse group of metabolites ranging from sugars, phosphosugars, and carboxylic acids to phosphocarboxylics acids, nucleotides, and coenzymes. This method is based on in vitro derivatization with a (13)C-labeled tag that allows internal standard based quantification and enables separation of structural isomer pairs like glucose 6-phosphate and fructose 6-phosphate in a single chromatographic run. Calibration curves for individual metabolites showed linearity ranging over more than 2 orders of magnitude with correlation coefficients of R(2) > 0.9975. The detection limits at a signal-to-noise ratio of 3 were below 1.0 microM (20 pmol) for most compounds. Thirty common metabolites involved in glycolysis, the pentose phosphate pathway, and tricarboxylic acid cycle were identified and quantified from yeast lysate with a relative standard deviation of less than 10%.

Channel geometry combined with surface chemistry enables a stable liquid boundary flow to be attained along the surfaces of a 12 microm diameter hydrophilic glass fiber in a closed semi-elliptical channel. Surface free energies and triangular corners formed by PDMS/glass fiber or OTS/glass fiber surfaces are shown to be responsible for the experimentally observed wetting phenomena and formation of liquid boundary layers that are 20-50 microm wide and 12 microm high. Viewing this stream through a 20 microm slit results in a virtual optical window with a 5 pL liquid volume suitable for cell counting and pathogen detection. The geometry that leads to the boundary layer is a closed channel that forms triangular corners where glass fiber and the OTS coated glass slide or PDMS touch. The contact angles and surfaces direct positioning of the fluid next to the fiber. Preferential wetting of corner regions initiates the boundary flow, while the elliptical cross-section of the channel stabilizes the microfluidic flow. The Young-Laplace equation, solved using fluid dynamic simulation software, shows contact angles that exceed 105 degrees will direct the aqueous fluid to a boundary layer next to a hydrophilic fiber with a contact angle of 5 degrees. We believe this is the first time that an explanation has been offered for the case of a boundary layer formation in a closed channel directed by a triangular geometry with two hydrophobic wetting edges adjacent to a hydrophilic surface.

We report a rapid microfluidic device construction technique which does not employ lithography or stamping methods. Device assembly physically combines a silicon wafer, an elastomer (poly(dimethylsiloxane) (PDMS)), and microfibers to form patterns of hydrophobic channels, wells, elbows, or orifices that direct fluid flow into controlled boundary layers. Tweezers are used to place glass microfibers in a defined pattern onto an elastomeric (PDMS) hydrophobic film. The film is then manually pressed onto a hydrophobic silicon wafer, causing it to adhere to the silicon wafer and form a liquid-tight seal around the fibers. Completed in 15 min, the technique results in an operable microdevice with micrometer-scale features of nanoliter volume. Microfiber-directed boundary flow is achieved by use of the surface wetting properties of the hydrophilic glass fiber and the hydrophobicity of surrounding surfaces. The simplicity of this technique allows quick prototyping of microfluidic components, as well as complete biosensor systems, such as we describe for the detection of pathogenic bacteria.