Immunoassay on a power-free microchip with laminar flow-assisted dendritic amplification

We demonstrate a rapid (<30 min) and ultrasensitive (sub-picomolar) immunoassay on a microchip which needs no external power sources for fluid transport. We previously reported a rapid immunoassay of human C-reactive protein (CRP) on the power-free microchip with moderate sensitivity, i.e., a limit of detection (LOD) in sub-nanomolar range, due to the lack of signal amplification. In the current work, we have improved the LOD by 3 orders of magnitude by employing dendritic amplification (DA) methods. Specifically, a sandwich immunocomplex with a biotinylated secondary antibody was constructed on the inner surface of the microchannel as described in the previous report. Onto the immunocomplex, solutions of FITC-labeled streptavidin (F-SA) and biotinylated anti-streptavidin (B-anti-SA) were supplied to grow a dendritic structure. First, we alternately supplied the two solutions for layer-by-layer growth up to three layers. As a result, we obtained an LOD of 0.21 pM with a CRP sample volume of 1.0 microL and assay time of approximately 30 min under an ordinary fluorescence microscope. Second, to reduce the number of incubation steps, we have devised a new DA method: laminar flow-assisted dendritic amplification (LFDA). In this method, F-SA and B-anti-SA were simultaneously and continuously supplied from two laminar streams formed by a Y-shaped microchannel. The immunoassay with the LFDA for 10 min (total assay time of approximately 23 min) with a CRP sample volume of 0.5 microL yielded an LOD of 0.15 pM, which is equivalent to 75 zmol. The combination of the power-free microchip and the LFDA will provide a new opportunity for ultrasensitive point-of-care testing.     

Effect of medium-chain triacylglycerols on anti-obesity effect of fucoxanthin

Dietary effects of medium-chain triacylglycerols (MCT) and fucoxanthin (Fc) on abdominal fat weight were determined using KK-Ay obese mouse. Experimental diet contained MCT(0.9%), Fc (0.1%), or MCT (0.9%) +Fc (0.1%). The abdominal fat weight of mice fed with Fc was significantly lower than that of mice fed with MCT. Uncoupling protein 1 (UCP1), a key molecule for metabolic thermogenesis, was clearly expressed in the white adipose tissue (WAT) of mice fed Fc, but little expression in that of the mice fed MCT. The anti-obesity effect of Fc was increased by mixing Fc with MCT. This increase would be due to the increase in the absorption rate of Fc by MCT.     

Therapy of myeloma in vivo using marine phospholipid in combination with Agaricus blazei Murill as an immune respond activator

Mushroom (Agaricus blazei Murill) extract has been reported to possess antitumor effects through immune activation. Here, we investigated the beneficial effects of combining A. blazei extract with marine phospholipids in comparison to A. blazei extract alone on myeloma sp2 tumor suppression when orally administrated. The experimental groups designed for sp2 tumor bearing BALB/c nu/nu mice were drinks of: (1)control; (2)1.0 mg/mL squid phospholipid liposome alone; (3)0.5 mg/mL A. blazei Murill water extract alone; (4)1.0 mg/mL squid phospholipid liposome with 0.5 mg/mL A. blazei Murill water extract in the form of those simple mixture; and (5)1.0 mg/mL squid phospholipid liposome with 0.5 mg/mL A. blazei Murill water extract partially encapsulated. Orally administrated volumes amounted to approximately 5 mL per day per mouse for all groups. A. blazei Murill water extract alone and squid phospholipid alone served groups show moderate tumor suppression with total administrations of approximately 105 mg/mouse for squid phospholipid through out the experimental term. When both A. blazei Murill water extract and squid phospholipid were administrated simultaneously in a simple mixture form, promotional effect on cancer tumor suppression was observed. And when A. blazei Murill water extract was partially encapsulated in the squid phospholipid liposomes with total administrations being 105 mg/mouse for squid phospholipid, effect on cancer tumor suppression was more pronounced. Though there was no statistically significant difference in tumor sizes between the simple mixture form administrated group i.e. group (4) and the partially encapsulated form administrated group i.e. group (5), the tumor vanished mouse was seen in the partially encapsulated form administrated group. Thus it was concluded that combinational administration of the A. blazei Murill water extract and the marine phospholipid may be useful in myeloma sp2 therapy.     

Selective labeling of proteins by using protein farnesyltransferase

The challenging task of identifying and studying protein function has been greatly aided by labeling proteins with reporter groups. Here, we present a strategy that utilizes an enzyme that labels a four-residue sequence appended onto the C terminus of a protein, with an alkyne-containing substrate. By using a bio-orthogonal cycloaddition reaction, a fluorophore that carried an azide moiety was then covalently coupled to the alkyne appended on the protein. FRET was used to calculate a F?rster (R) distance of 40 A between the eGFP chromophore and the newly appended Texas Red fluorophore. This experimental value is in good agreement with the predicted R value determined by using molecular modeling. The small recognition tag, the high specificity of the enzyme, and the orthogonal nature of the derivatization reaction will make this approach highly useful in protein chemistry.     

Material performance of age-hardened beryllium–copper alloy,CDA-C17200, in a high-pressure,gaseous hydrogen environment

In order to develop safer and more energy-efficient, hydrogen pre-cooling systems for use in hydrogen refueling stations, it is necessary to identify a high-strength metallic material with greater thermal conductivity and lower susceptibility to hydrogen embrittlement, as compared with ordinary, stable austenitic stainless steels. To accomplish this task, the hydrogen compatibility of a precipitation-hardened, high-strength, copper-based alloy was investigated by slow-strain-rate tensile (SSRT), fatigue-life, fatigue-crack-growth (FCG) and fracture toughness tests in 115-MPa hydrogen gas at room temperature. The hydrogen solubility and diffusivity of the alloy were also determined. The hydrogen solubility of the alloy was two or three orders of magnitude lower than that of austenitic stainless steels. The alloy also demonstrated absolutely no hydrogen-induced degradation of its strength properties, a factor which could contribute to the reduction of costs related to the construction and maintenance of hydrogen refueling stations, owing to the downsizing and improved cooling performance of the pre-cooling systems.     

Fatigue limit of carbon and CrMo steels as a small fatigue crack threshold in high-pressure hydrogen gas

The fatigue limit properties of a carbon steel and a low-alloy CrMo steel were investigated via fully-reversed tension-compression tests, using smooth specimens in air and in 115-MPa hydrogen gas. With respect to the CrMo steel, specimens with sharp notches were also tested in order to investigate the threshold behavior of small cracks. The obtained SN data inferred that the fatigue limit was not negatively affected by hydrogen in either of the steels. Observation of fatigue cracks in the unbroken specimens revealed that non-propagating cracks can exist even in 115-MPa hydrogen gas, and that the crack growth threshold is not degraded by hydrogen. The experimental results provide justification for the fatigue limit design of components that are to be exposed to high-pressure hydrogen gas.     

Synthesis of nanocrystalline rare earth oxides by glycothermal method

The reaction of yttrium acetate hydrate in 1,2-propanediol at 300 °C yielded a product containing acetate groups and glycol moieties. From this product, Y₂O₃ was directly crystallized at 400 °C without the formation of a carbonate oxide phase. The thus-obtained Y₂O₃ samples had a small crystallite size (2.2 nm) and significantly large surface area (280 m²/g). Other nanocrystalline rare earth (Gd-Yb) oxides were also obtained by this method.     

Characterization of polycyclic aromatic hydrocarbons (PAHs) in different size fractions in deposited road particles (DRPs) from Lake Biwa area, Japan

Deposited road particles (DRPs) collected from 13 heavily traveled roadways in an urban area of Southern Lake Biwa, Japan, were analyzed for polycyclic aromatic hydrocarbons (PAHs) in seven different particle size fractions (< or = 20 to 1000-2000 microm) and evaluated for the aryl hydrocarbon receptor (AhR) ligand activity by using a yeast bioassay. The mean compositions of individual PAHs to total PAH concentrations in different particle size fractions were 19-21% for pyrene, which was the most dominant component, 14-16% for fluoranthene, and 7-13% for benzo[g,h,i]perylene, which were the next dominant components. The total PAH distribution pattern in different particle size fractions of DRPs was different from the organic matter distribution pattern which increased with decreasing particle size of DRPs, and could be explained by the differences in their sources. Moreover, AhR ligand activities were observed in the DRP extracts of all size fractions. The activity of the DRP extracts from the smallest size fraction was approximately 5 times more potent than that of the largest size fraction in a yeast AhR ligand activity assay. The mean contribution (%) of benzo[k]-fluoranthene, chrysene, benzo[b]fluoranthene, and benzo[a]pyrene to the AhR activity of DRP extracts in all size fractions was 1.43-4.11%, 1.63-3.53%, 0.63-1.69%, and 0.31-1.42%, respectively. Although the contribution of PAHs to AhR ligand activity presented in the DRP extracts was relatively low (below 10%), it may be best to remove these DRPs before discharge to receiving water environments.