Highly sensitive detection of monosaccharides on microchip electrophoresis using pH discontinuous solution system.

We improved the detection sensitivity of neutral and amino monosaccharides labeled with 2-aminoacridone by a factor of 19.6-48.7 and 44.4-65.9, respectively, with no deterioration in separation resolution, using a pH discontinuous solution system instead of the conventional solution system. The pH discontinuous solution system is simple, using only a borate solution at pH 6.0 as a sample solution and a Tris-borate solution at pH 9.3 as a separation solution, enabling an enhancement of the sensitivity by monosaccharide stacking. This technique is fully compatible with conventional microchip electrophoresis, thereby allowing us to develop efficient tools for highly sensitive and resolved detection of monosaccharides.

Light-triggered thermoelectric conversion based on a carbon nanotube-polymer hybrid gel.

Lights? Nanotubes? Action! A hydrogel comprising lysozymes, poly(ethylene glycol), phospholipids, and functionalized single-walled carbon nanotubes is employed for light-driven thermoelectric conversion. A photoinduced thermoelectric conversion module based on the hydrogel functions as a novel electric power generator (see image). This concept may find application in various industries, such as robotics and aerospace engineering.

Laser-triggered carbon nanotube microdevice for remote control of biocatalytic reactions.

We have developed a near-infrared laser-driven carbon nanotube (CNT) microdevice. Powerful photo-exothermy of CNT was coupled with a microdevice for remote control of temperature-dependent biocatalytic transformations. We succeeded in ultrafast temperature change (<0.03 s), wide range of controlled temperature (25-55 degrees C) and high-precision thermal cycle in a microspace owing to the following physical factors: (1) high efficiency of photothermal conversion of the CNTs; (2) high thermal conductivity of the CNTs; and (3) low heat capacity of the microspaces. Furthermore, this is the first report, supported by direct observations, of the optical control of biocatalytic reactions, such as DNA extension, DNA amplification and enzymatic cyclodextrin production, by employing a laser-triggered CNT microdevice. Our present work constitutes important progress for various lab-on-a-chip applications.

Use of a heterogeneous buffer combination in microchip electrophoresis for high-resolution separation by on-line concentration of DNA samples.

We have developed a novel high-resolution separation technique of DNA fragments in a heterogeneous combination of a sample buffer and a separation buffer. The use of a heterogeneous buffer combination is a simple method for on-line concentration of DNA fragments, in which a sample buffer is simply exchanged with one including taurine anions. The mobility of taurine anions, co-ions for DNA, is lower than the that of acetate anions in a separation buffer. The difference in the mobility invokes transient isotachophoresis. The current technique allows DNA fragments to be effectively concentrated and the separation length of microchips to be shorter than that of conventional ones by a factor of three without deterioration in separation resolution and any modification of a chip design. Fragments of 100-bp DNA ladders (100-1000 bp) were separated with high resolution (0.72-10.7) within 60 s with a 10 mm separation length on a polymethyl methacrylate chip. Furthermore, fragments of 10-bp DNA ladders (10-330 bp) were separated with high resolution (0.69-2.00) with a 10 mm separation length within 50 s without band broadening. The current achievements will make it possible to fabricate compact devices for microchip electrophoresis.

BMP2-induced gene profiling in dental epithelial cell line.

Tooth development is regulated by epithelial-mesenchymal interactions and their reciprocal molecular signaling. Bone morphogenetic protein 2 (BMP2) is known as one of the inducers for tooth development. To analyze the molecular mechanisms of BMP2 on ameloblast differentiation (amelogenesis), we performed microarray analyses using rat dental epithelial cell line, HAT-7. After confirming that BMP2 could activate the canonical BMP-Smads signaling in HAT-7 cells, we analyzed the effects of BMP2 on 14,815 gene expressions and profiled them. Seventy-three genes were up-regulated and 28 genes were down-regulated by BMP2 treatment for 24 hours in HAT-7 cells. Functional classification revealed that 18% of up-regulated genes were ECM/adhesion molecules present in the enamel organ. Furthermore, we examined the expression of several differentiation markers in dental epithelial four cell-lineages including inner enamel epithelium (ameloblasts), stratum intermedium, stratum reticulum, and outer enamel epithelium. The results indicated that BMP2 might induce at least two different cell-lineage markers including a BMP antagonist expressed in HAT-7 cells, suggesting that BMP2 could accelerate amelogenesis via BMP signaling.

Sizing of single globular DNA molecules by using a circular acceleration technique with laser trapping.

We describe a method for in situ sizing individual huge DNA molecules by laser trapping. Single DNA molecules are reversibly transformed, without mechanical fragmentation of fragile huge-sized DNA, from their random coil state into their globular state induced by condensing agents poly(ethylene glycol) and Mg(2+). With the use of a globular DNA molecule folded by condensation, the critical velocity of the circularly accelerated single globular DNA molecule by laser trapping was found to be proportional to the size of the DNA. Yeast, Saccharomyces cerevisiae, chromosome III (285 kbp) was successfully sized (281 +/- 40 kbp) from a calibration curve scaled using lambda, T4, and yeast chromosome VI (48.5, 166, and 385 kbp, respectively). The use of critical velocity as a sizing parameter makes it possible to size single DNA molecules without prior conformational information, i.e., the radius of a single globular huge DNA molecule as a nanoparticle. A sized single globular DNA molecule could be trapped again for subsequent manipulation, such as transportation of it anywhere. We also investigated a possibility of reusing the globular DNA molecules condensed by PEG and Mg(2+) for PCR and found that PCR efficiency was not deteriorated in the presence of the condensation agents.

Photoinduced antiviral carbon nanohorns.

Nanocarbons, such as carbon nanohorns (CNH) and carbon nanotubes, are materials of interest in many fields of science and technology because of their remarkable physical properties. We report here a novel approach for using NIR laser-driven CNH as an antiviral agent. NIR laser-driven functional CNH complexes could open the way to a new range of antiviral materials.

Genome wide expression analysis of white blood cells and liver of pre-diabetic Otsuka Long-Evans Tokushima Fatty (OLETF) rats using a cDNA microarray.

In a prior study, we reported on a significant decrease in calpain10 gene expression in white blood cells (WBC) as well as the major insulin-target tissues including liver and adipose tissue, before the onset of diabetes in Otsuka Long-Evans Tokushima Fatty (OLETF) rats. In this study, we extended our hypothesis that some type 2 diabetes mellitus (NIDDM) susceptible genes are up/down-regulated before the onset in WBC of OLETF rats, reflecting their up/down-regulation in major insulin-target tissues, such as the liver. We tested this hypothesis using rat cDNA microarrays. The findings show that 1080 genes are up/down-regulated by more than 2-fold compared to the controls, Long-Evans Tokushima Otsuka rats, before the onset in WBC and liver under fasted or insulin administered condition. Fifty-seven of the 1080 genes were up/down-regulated in both WBC and the liver. More than half have been reported to NIDDM susceptible genes and the remainder have not been reported to be related to NIDDM. These results indicate that there some NIDDM related genes are up/down-regulated in WBC before the onset of diabetes.

Conformational separation of monosaccharides of glycoproteins labeled with 2-aminoacrydone using microchip electrophoresis.

The conformational separation of monosaccharides labeled with fluorescent 2-aminoacrydone (AMAC) was performed by electrophoresis on a plastic microchip with light-emitting diode confocal fluorescence detection. The AMAC-labeled five neutral monosaccharide mixture (D-glucose (Glc), D-mannose, D-galactose, L-fucose, and D-xylose) or two amino monosaccharide mixture (N-acetyl-D-glucosamine and N-acetyl-D-galactosamine) were well separated at pH 8.5 and 0.5% w/v methylcellulose of 200 mM borate buffer conditions using microchip electrophoresis. The separation was successfully performed considering the difference in stability of the complex between the hydroxyl residue of the monosaccharide and borate ions, and we found that 200 mM and pH 8.5 of borate buffer conditions were critical. High-speed separation for the neutral monosaccharides (50 s) and for amino monosaccharides (70 s) was attained at a 400 V/cm of electric field condition, showing all peak resolutions were greater than 0.9% and RSD of mobility were less than 1.9%. The detection limits of 0.86 microM for Glc and <1 microM for all other monosaccharides were enhanced with the addition of 0.5% w/v methylcellulose to the buffer. These attainments are fully compatible with conventional CE. The analysis of the subtle differences in the conformational stability and the value of the hydroxyl residue of the borate complex allowed the development of an efficient prospective tool for attaining high-resolution separation of monosaccharide mixtures having complicated and analogous conformations.

On-line microdevice for stress proteomics.

The handling of the cells or tissues is essential for proteomics research or drug screening, where labor is not avoidable. The steps of cell wash, protein extraction, protein denaturing are complicated procedures in conventional method using centrifugation and pipetting in the laboratory. This is the bottle-neck for proteome research. To solve these problems, we propose to utilize the nanotechnology, which will improve the proteomics methodology. Utilizing the nanotechnology, we developed a novel microseparation system, where centrifugation and pipetting are needless. This system has a nanostructured microdevice, by which the cell handling, protein extraction, and antibody assay can be performed. Since cell transfer is needless, all cells are corrected without any loss during the cell-pretreatment procedures, which allowed high reproducibility and enabled the detection of low amount of protein expression. Utilizing the microdevice, we analyzed the stress induced proteins. We further succeeded the screening of food that was useful for immunity and found that an extraction from seaweed promoted the apoptosis of T-lymphoblastic cells. Here, we present an on-line microdevice for stress proteomics.

High-speed separation of proteins by microchip electrophoresis using a polyethylene glycol-coated plastic chip with a sodium dodecyl sulfate-linear polyacrylamide solution.

In this paper, we describe a method for size-based electrophoretic separation of sodium dodecyl sulfate (SDS)-protein complexes on a polymethyl methacrylate (PMMA) microchip, using a separation buffer solution containing SDS and linear polyacrylamide as a sieving matrix. We developed optimum conditions under which protein separations can be performed, using polyethylene glycol (PEG)-coated polymer microchips and electrokinetic sample injection. We studied the performance of protein separations on the PEG-coated PMMA microchip. The electrophoretic separation of proteins (21.5-116.0 kDa) was completed with separation lengths of 3 mm, achieved within 8 s on the PEG-coated microchip. This high-speed method may be applied to protein separations over a large range of molecular weight, making the PEG-coated microchip approach applicable to high-speed proteome analysis systems.

Microchip electrophoretic protein separation using electroosmotic flow induced by dynamic sodium dodecyl sulfate-coating of uncoated plastic chips.

Separation of sodium dodecyl sulfate (SDS)-protein complexes is difficult on plastic microchips due to protein adsorption onto the wall. In this paper, we elucidated the reasons for the difficulties in separating SDS-protein complexes on plastic microchips, and we then demonstrated an effective method for separating proteins using polymethyl methacrylate (PMMA) microchips. Separation difficulties were found to be dependent on adsorption of SDS onto the hydrophobic surface of the channel, by which cathodic electroosmotic flow (EOF; reversed flow) was generated. Our developed method effectively utilized the reversed flow from this cathodic EOF as a driving force for sample proteins using permanently uncoated but dynamic SDS-coated PMMA microchips. High-speed (6 s) separation of proteins and peptides up to 116 kDa was successfully achieved using this system.

A design of nanosized PEGylated-latex mixed polymer solution for microchip electrophoresis.

We report here advanced microchip electrophoresis using a nanoparticle doped polymer solution that enables greater separation of DNA. The proposed system is simple and effective without any new apparatus or complicated procedures. Various amounts and sizes (80 nm, 110 nm, and 193 nm) of polymer nanoparticle solutions (PEGylated-latex) were mixed with a conventional polymer solution for microchip electrophoresis. When a 0.49 wt% hydroxyl propyl methyl cellulose (HPMC) buffer solution was mixed with a 2.25 wt% 80 nm-PEGylated-latex a higher separation efficiency and a higher mobility of a wider molecular range of dsDNA (10 bp to 2 kbp) was achieved under low viscosity conditions (<5.5 cP) than in conventional 0.7% HPMC. The separation performance was dependent on the particle size and concentration. Furthermore, the effectiveness of the larger PEGylated-latex (193 nm) was not as high as the smaller one (80 to 110 nm). The observed separation improvement by polymer solution with latex-nanoparticles seems to derive from the balance between wider polymer mesh size and the structural obstacles of particles in the buffer.

Influence of the pH on separating DNA by high-speed microchip electrophoresis.

Various factors are critical in resolving DNA molecules at high speed, including the separation medium, concentration, composition, and pH of the buffer, as well as the electric field strength. To this study, considered the composition of a buffer and the difference in the pH, while paying attention to whether the separation ability changes in the microchip electrophoresis of DNA. DNA separation was particularly affected by both the buffer composition and the pH. Under the optimal microchip electrophoresis conditions that were determined in this study, an improved resolution of a wider range of DNA fragment sizes was achieved. Moreover, the total separation time decreased from 240 s to 100 s. Thus, by simplifying and improving the DNA electrophoresis in the microchip, this technique is now widely applicable to several different scientific fields.