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Single Substrate (single + substrate)
Selected AbstractsElectrokinetic-driven microfluidic system in poly(dimethylsiloxane) for mass spectrometry detection integrating sample injection, capillary electrophoresis, and electrospray emitter on-chipELECTROPHORESIS, Issue 24 2005Sara Thorslund Abstract A novel microsystem device in poly(dimethylsiloxane) (PDMS) for MS detection is presented. The microchip integrates sample injection, capillary electrophoretic separation, and electrospray emitter in a single substrate, and all modules are fabricated in the PDMS bulk material. The injection and separation flow is driven electrokinetically and the total amount of external equipment needed consists of a three-channel high-voltage power supply. The instant switching between sample injection and separation is performed through a series of low-cost relays, limiting the separation field strength to a maximum of 270,V/cm. We show that this set-up is sufficient to accomplish electrospray MS analysis and, to a moderate extent, microchip separation of standard peptides. A new method of instant in-channel oxidation makes it possible to overcome the problem of irreversibly bonded PDMS channels that have recovered their hydrophobic properties over time. The fast method turns the channel surfaces hydrophilic and less prone to nonspecific analyte adsorption, yielding better separation efficiencies and higher apparent peptide mobilities. [source] Microfluidic device for capillary electrochromatography-mass spectrometryELECTROPHORESIS, Issue 21 2003Iulia M. Lazar Abstract A novel microfabricated device that integrates a monolithic polymeric separation channel, an injector, and an interface for electrospray ionization-mass spectrometry detection (ESI-MS) was devised. Microfluidic propulsion was accomplished using electrically driven fluid flows. The methacrylate-based monolithic separation medium was prepared by photopolymerization and had a positively derivatized surface to ensure electroosmotic flow (EOF) generation for separation of analytes in a capillary electrochromatography (CEC) format. The injector operation was optimized to perform under conditions of nonuniform EOF within the microfluidic channels. The ESI interface allowed hours of stable operation at the flow rates generated by the monolithic column. The dimensions of one processing line were sufficiently small to enable the integration of 4,8 channel multiplexed structures on a single substrate. Standard protein digests were utilized to evaluate the performance of this microfluidic chip. Low- or sub-fmol amounts were injected and detected with this arrangement. [source] Influence of Transient Flow on Contaminant BiodegradationGROUND WATER, Issue 2 2001Mario Schirmer The rate of biodegradation in contaminated aquifers depends to a large extent on dispersive mixing processes that are now generally accepted to result from spatial variations in the velocity field. It has been shown, however, that transient flow fields can also contribute to dispersive mixing. The influence of transient flow on biodegrading contaminants is particularly important since it can enhance mixing with electron acceptors, further promoting the reactive process. Using numerical simulations, the effect of transient flow on the behavior of a biodegradable contaminant is evaluated here both with respect to the development of apparently large horizontal transverse dispersion and also with respect to enhanced mixing between the substrate (electron donor) and electron acceptor. The numerical model BIO3D, which solves for advective-dispersive transport coupled with Monod-type biodegradation of substrates in the presence of an electron acceptor, was used for the simulations. The model was applied in a two-dimensional plan view mode considering a single substrate. Transient flow fields were found to yield larger apparent transverse dispersion because the longitudinal dispersivity also acts transverse to the mean flow direction. In the reactive case, the transient flow field increases substrate-oxygen mixing, which in turn enhances the overall rate of biodegradation. The results suggest that in the case of moderate changes of flow directions, a steady-state flow field can be justified, thereby avoiding the higher computational costs of a fully transient simulation. The use of a higher transverse horizontal dispersivity in a steady flow field can, under these conditions, adequately forecast plume development. [source] Reduction of a set of elementary modes using yield analysisBIOTECHNOLOGY & BIOENGINEERING, Issue 2 2009Hyun-Seob Song Abstract This article proposes a new concept termed "yield analysis" (YA) as a method of extracting a subset of elementary modes (EMs) essential for describing metabolic behaviors. YA can be defined as the analysis of metabolic pathways in yield space where the solution space is a bounded convex hull. Two important issues arising in the analysis and modeling of a metabolic network are handled. First, from a practical sense, the minimal generating set spanning the yield space is recalculated. This refined generating set excludes all the trivial modes with negligible contribution to convex hull in yield space. Second, we revisit the problem of decomposing the measured fluxes among the EMs. A consistent way of choosing the unique, minimal active modes among a number of possible candidates is discussed and compared with two other existing methods, that is, those of Schwartz and Kanehisa (Schwartz and Kanehisa, 2005. Bioinformatics 21: 204,205) and of Provost et al. (Provost et al., 2007. Proceedings of the 10th IFAC Symposium on Computer Application in Biotechnology, 321,326). The proposed idea is tested in a case study of a metabolic network of recombinant yeasts fermenting both glucose and xylose. Due to the nature of the network with multiple substrates, the flux space is split into three independent yield spaces to each of which the two-staged reduction procedure is applied. Through a priori reduction without any experimental input, the 369 EMs in total was reduced to 35 modes, which correspond to about 91% reduction. Then, three and four modes were finally chosen among the reduced set as the smallest active sets for the cases with a single substrate of glucose and xylose, respectively. It should be noted that the refined minimal generating set obtained from a priori reduction still provides a practically complete description of all possible states in the subspace of yields, while the active set covers only a specific set of experimental data. Biotechnol. Bioeng. 2009;102: 554,568. © 2008 Wiley Periodicals, Inc. [source] Kinetic studies on aminopeptidase M-mediated degradation of human hemorphin LVV-H7 and its N -terminally truncated productsJOURNAL OF PEPTIDE SCIENCE, Issue 7 2008Harald John Abstract The human hemorphin LVV-H7 belongs to the class of µ-opiod receptor-binding peptides, which also exhibits significant affinity to insulin-regulated aminopeptidase (IRAP) thereby affecting IRAP inhibition. The inhibitory potency towards IRAP is of pharmaceutical interest for the treatment of Alzheimer's disease. Consecutive N -terminal cleavage of the first two amino acid residues of LVV-H7 affects a drastic increase of the binding affinity (V-H7) but ultimately leads to its complete abolition after cleavage of the next amino acid residue (H7). Therefore, we investigated LVV-H7 truncation by aminopeptidase M (AP-M) identified as a LVV-H7 degrading enzyme potentially regulating hemorphin activity towards IRAP in vivo. Using a selective quantitative multi-component capillary zone electrophoretic method (CZE-UV), we analyzed the AP-M-mediated subsequent proteolysis of the hemorphins LVV-H7 (L32 -F41), VV-H7 (V33 -F41), and V-H7 (V34 -F41) in vitro. Incubations were carried out with synthetic hemorphins applied as single substrates or in combination. Maximum velocities (Vmax), catalytic constants (turnover numbers, kcat), and specific enzyme activities (EA) were calculated. L32 cleavage from LVV-H7 happens more than two-times faster (kcat: 140 min,1 ± 9%, EA: 1.0 U/mg ± 9%) than V33 cleavage from VV-H7 (kcat: 61 min,1 ± 10%, EA: 0.43 U/mg ± 10%) or V32 deletion from V-H7 (kcat: 62 min,1 ± 8%, EA: 0.46 U/mg ± 8%). In contrast, we showed that H7 (Y35 -F41) was neither degraded by porcine AP-M nor did it act as an inhibitor for this enzyme. Determined turnover numbers were in the same dimension as those reported for dynorphin degradation. This is the first time that AP-M-mediated truncation of natural underivatized LVV-H7 and its physiological metabolites was analyzed to determine kinetic parameters useful for understanding hemorphin processing and designing hemorphin-derived drug candidates. Copyright © 2008 European Peptide Society and John Wiley & Sons, Ltd. [source] Active Transport of Amino Acids by a Guanidiniocarbonyl,Pyrrole ReceptorCHEMISTRY - A EUROPEAN JOURNAL, Issue 31 2010Christian Urban Dr. Abstract Herein we report the synthesis and characterization of a transporter 9 for N-acetylated amino acids. Transporter 9 is a conjugate of a guanidiniocarbonyl pyrrole cation, one of the most efficient carboxylate binding motifs reported so far, and a hydrophobic tris(dodecylbenzyl) group, which ensures solubility in organic solvents. In its protonated form, 9 binds N-acetylated amino acid carboxylates in wet organic solvents with association constants in the range of 104,M,1 as estimated by extraction experiments. Aromatic amino acids are preferred due to additional cation-,-interactions of the amino acid side chain with the guanidiniocarbonyl pyrrole moiety. U-tube experiments established efficient transport across a bulk liquid chloroform phase with fluxes approaching 10,6,mol,m,2,s,1. In experiments with single substrates, the release rate of the amino acid from the receptor,substrate complex at the interface with the receiving phase is rate determining. In contrast to this, in competition experiments with several substrates, the thermodynamic affinity to 9 becomes decisive. As 9 can only transport anions in its protonated form and has a pKa value of approximately 7, pH-driven active transport of amino acids is also possible. Transport occurs as a symport of the amino acid carboxylate and a proton. [source] | |||||||||||||