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Coulometry has been demonstrated to be effective for determining the analyte in a liquid plug on the nanoliter-scale confined in a flow channel. A plug prepared in a rhombus structure of an auxiliary flow channel was placed on a thin-film three-electrode system, and hydrogen peroxide was detected as a model analyte. Under a fixed potential, the current decayed rapidly, particularly in shallow flow channels, thus making reproducible amperometric detection difficult. On the other hand, the increasing charge during coulometry facilitated the measurements. A constricted flow channel structure with an array of platinum strips for the working electrode was effective at efficiently consuming the analyte to improve the sensitivity and lower the detection limit. Compared to the case of a single short working electrode with the same area, a 4-fold increase in sensitivity was observed for the best combination of flow channel height and interstrip distance. With an increase in the generated current while maintaining the background at a low level, the detection limit was lowered from 1.3 [micro]M to 410 nM using working electrodes with the same area. Furthermore, the processing of solutions containing L-glutamate or L-glutamate oxidase and the detection of L-glutamate were demonstrated. 10.1021/ac102289a

Autonomous control of microfluidic transport was realized through the use of chemically actuated diaphragm micro-pumps connected to a network of controlling flow channels. A hydrogen peroxide ([H.sub.2][O.sub.2]) solution was transported in the controlling flow channel by capillary action. Upon the solution's arrival at the lower compartment of a micropump filled with manganese dioxide (Mn[O.sub.2]) powder, a volume change that accompanied the production of oxygen caused by the catalytic decomposition of [H.sup.2][O.sup.2] induced inflation of the diaphragm. This in turn caused the movement of a solution in another network of flow channels formed in the upper layer. Micropumps that only exert pressure were also fabricated. By positioning the micropumps at appropriate locations in conjunction with additional flow-delaying components, the ejection of solutions from the reservoir of each micropump could be initiated at coordinated times. Furthermore, the solutions could be transported by the application of pressure from other micropumps. In other words, the information for switching from one micropump to another could be described on the chip in the form of a network of flow channels. This autonomous processing of solutions was demonstrated for enzymatic analyses of [H.sub.2][O.sub.2], glucose, and lactate. 10.1021/ac1009657

We present a microarray system that enables simultaneous monitoring of multiple ionic currents through transmembrane a-hemolysin nanopores arrayed at bilayer lipid membranes. We applied the self-assembling ability of lipid molecules interfaced between an aqueous solution and organic solvent to induce bilayer membrane formation at a microfluidic device; the device consists of a hydrophobic polymer film that serves to suspend the lipid-containing solvent at micrometer-sized apertures as well as to separate the aqueous solution into two chambers. In this study, we confirmed that expeditious and reproducible bilayer formation is realized by control of the composition of the solvent, a mixture of n-decane and 1-hexanol, which permits simultaneous incorporation of the a-hemolysin nanopores to the membrane array. Monitoring the eight wells on the array at once, we obtained a maximum of four relevant, synchronous signals of translocating ionic current through the nanopores. The system was also able to detect translocation events of nucleic acid molecules through the pore via the profile of a blocked current, promising its potential for high-throughput applications. 10.1021/ac901732z

Mechanisms of fragmentation of giycosyl bond linkages in various saccharides were investigated by using computational calculations to find general rules of fragmentation of sodiated oligosaccharides in mass spectrometry. The calculations revealed that [alpha]-Glc, [alpha]-Gal, [beta]-Man, [alpha]-Fuc, [beta]-GlcNAc, and [beta]-GalNAc linkages were cleaved more easily than [beta]-Glc, [beta]-Gal, and roman linkages because the transition states of the former were stabilized by the anomeric effect. The 1-6 linkage was more stable than the others, since saccharides with flexible 1-6 linkages were more stabilized in energy than the other linkages by the sodium cation. The sialyl linkage was the most labile of all the linkages investigated. Comparison of activation energies and binding affinities to the sodium cation revealed an increase in activation energy in proportion to the increment in binding affinity. The calculated stabilities of glycosyl bonds were: [alpha]-Man (Man[alpha] 1-3 Man, Man[alpha] 1-4Man, Man[alpha]l-6Man) > [beta]-Gal (Gal[beta]1-4Gal) > [alpha]-GalNAc (GalNAc[alpha]1-4GalNAc) > t-Man (Man[beta]ll-4GlcNAc) > [alpha]-Gal (Galc[alpha]1-3Gal, Gal[alpha]1-4Gal, Galc[alpha]l-6Gal) > [beta]-Man(Man[beta]l-4Man) > [beta]-GalNAc (GalNAc[beta]l-4GalNAc) > [alpha]-Fuc (Fuc[alpha]l-6GlcNAc) > [alpha]-Fuc (Fuc[alpha]1-4GlcNAc) > [beta]-GlcNAc (GlcNAc[beta]l-4GlcNAc) > [alpha]-Fuc (Fuc[alpha]1-3GlcNAc) > [alpha]-NeuNAc (NeuNAc[alpha]2-3Gal, NeuNAc[alpha]2-6Gal); this result was close to the experimentally deduced trend. These theoretically and experimentally derived general rules for fragmentation should be useful for analyzing the experimentally obtained mass spectra of oligosaccharides.

A microfluidic device and operation to handle liquid plugs for biochemical analyses were developed for efficient handling of plugs of many solutions. A major part of the device was a T-junction consisting of a main flow channel and a handling flow channel. Unit operations including attachment of plugs, division of a plug, sorting of plugs, and formation of plugs of various lengths enabled controlled sequential reactions in a microflow channel. Rapid mixing could easily be achieved by moving a plug formed by merging two plugs back and forth. The device could be used for efficient characterization of performance in bio/chemical sensing. In experiments using L-glutamate oxidase, plugs containing an enzyme or a substrate were formed, mixed sequentially, and the intensity of fluorescence from plugs of different concentrations of L-glutamate or pHs could be measured simultaneously. Cross-contamination of plugs by neighboring plugs poses a problem in using the same flow channel repeatedly. However, the influence could be minimized by using a cleansing plug placed between them in a sufficiently hydrophobic flow channel and by processing the plugs at a low velocity. The device can be a critical component for microprocessing in various bio/chemical analyses.

A microelectrochemical pH-stat with an automatic feedback function was fabricated. The operation of the device is based on the nonstandard use of an electrochemical three-electrode system with a pH-sensitive reference electrode, a Ag/AgCl working electrode, and an iridium auxiliary electrode that functions as an actuator to adjust the solution pH. The combination of the electrodes caused a negative feedback in response to a pH change. The shift of the potential of the pH-sensitive reference electrode caused an overpotential on the Ag/AgCl working electrode, which then caused a significant current increase. This led to the electrolysis of water on the auxiliary electrode and the rapid recovery of the pH. The negative feedback function to stabilize the initial state could be confirmed for changes to both the acidic and basic directions. The performance of the pH-stat was characterized in the titration of acetic acid or ammonia. The charge generated in the feedback process changed linearly with respect to the concentration. The pH-stat was also used in the determination of urea by urease and that of the activities of trypsin and pepsin while maintaining the optimum pH for the enzymes. The pH to be fixed could be changed by changing the working electrode potential. Moreover, the two pH-stats could be used to form a pH gradient in a microflow channel by firing the pH values at two positions.

This paper describes a multiwell biochip for simultaneous parallel recording of ion current through transmembrane pores reconstituted in planar lipid bilayer arrays. Use of a thin poly(p-xylylene) (parylene) film having micrometersized apertures ([theta] = 15-50 [micro]m, t = 20 [micro]m) led to formation of highly stable bilayer lipid membranes (BLMs) for incorporation of transmembrane pores; thus, a large number of BLMs could be arrayed without any skillful technique. We optically confirmed the simultaneous formation of BLMs in a 5 x 5 matrix, and in our durability test, the BLM lasted more than 15 h. Simultaneous parallel recording of alamethicin and gramicidin transmembrane pores in multiple contiguous recording sites demonstrated the feasibility of high-throughput screening of transmembrane ion currents in artificial lipid bilayers.

Insulin resistance is often associated with obesity and can precipitate type 2 diabetes. To date, most known approaches that improve insulin resistance must be preceded by the amelioration of obesity and hepatosteatosis. Here, we show that this provision is not mandatory; insulin resistance and hyperglycemia are improved by the modification of hepatic fatty acid composition, even in the presence of persistent obesity and hepatosteatosis. Mice deficient for Elovl6, the gene encoding the elongase that catalyzes the conversion of palmitate to stearate, were generated and shown to become obese and develop hepatosteatosis when fed a high-fat diet or mated to leptin-deficient ob/ob mice. However, they showed marked protection from hyperinsulinemia, hyperglycemia and hyperleptinemia. Amelioration of insulin resistance was associated with restoration of hepatic insulin receptor substrate-2 and suppression of hepatic protein kinase C [epsilon] activity resulting in restoration of Akt phosphorylation. Collectively, these data show that hepatic fatty acid composition is a new determinant for insulin sensitivity that acts independently of cellular energy balance and stress. Inhibition of this elongase could be a new therapeutic approach for ameliorating insulin resistance, diabetes and cardiovascular risks, even in the presence of a continuing state of obesity., Author(s): Takashi Matsuzaka [1, 2]; Hitoshi Shimano (corresponding author) [1, 2]; Naoya Yahagi [2, 3]; Toyonori Kato [1]; Ayaka Atsumi [1]; Takashi Yamamoto [1]; Noriyuki Inoue [1]; Mayumi Ishikawa [1]; [...]

Multidimensional NMR spectroscopy is used to determine the solution structure of the Rhodobacter sphaeroides PufX membrane protein expressed in Escherichia coli. It is found that the overall structure of the PufX is similar to light-harvesting complexes (LH1) [alpha]- and [beta]-polypeptides from Rhodospidrillum rubrum and LH2 polypeptides.

As the clock frequency and physical address space of 64-bit microprocessors continue to grow, one major critical path is the access to the on-die cache memory that includes a tag comparator, a tag SRAM and a data SRAM. To improve the delay of the tag comparator, a diode-partitioned (DP) domino circuit is proposed. DP domino reduces the parasitic capacitance and enables a smaller keeper in high fan-in gates. The diode circuit is also improved by an enhanced diode that boosts up the gate voltage of the nMOS diode. Delay of a 40-bit tag comparator using the proposed scheme is 33% faster than an optimized complex domino circuit in 1.8-V 180-nm CMOS technology. Index Terms--High-speed cache memory, high-speed domino circuit, keeper design, tag comparator.