Real-time PCR microfluidic devices with concurrent electrochemical detection

Electrochemistry-based detection methods hold great potential towards development of hand-held nucleic-acid analyses instruments. In this work, we demonstrate the implementation of in situ electrochemical (EC) detection method in a microfluidic flow-through EC-qPCR (FTEC-qPCR) device, where both the amplification of the target nucleic-acid sequence and subsequent EC detection of the PCR amplicon are realized simultaneously at selected PCR cycles in the same device. The FTEC-qPCR device utilizes methylene blue (MB), an electroactive DNA intercalator, for electrochemical signal measurements in the presence of PCR reagent components. Our EC detection method is advantageous, when compared to other existing EC methods for PCR amplicon analysis, since FTEC-qPCR does not require probe-modified electrodes, or asymmetric PCR, or solid-phase PCR. Key technical issues related to surface passivation, electrochemical measurement, PCR inhibition by metal electrode, bubble-free PCR, were investigated. By controlling the concentration of MB and the exposure of PCR mixture to the bare metal electrode, we successfully demonstrated electrochemical measurement of MB in solution-phase, symmetric PCR by amplifying a fragment of lambda phage DNA.

Preparation and chracaterization of novel graft copolymers of LLDPE

A series of novel fluorine surfactants, a, b, c, d, e and their acrylates, A, 13, C, D and E, were synthesized via poly( ethylene oxide) ( PEG) ( 200, 600, 1000, 2000, 5000) and perfluorooctane poly (ethylene oxide) ether as the main starting materials. Their chemical structures were characterized by means of FTIR and H-1 NMR. The surface activity and surface tension( y) of surfactants a, b, c, d and e were evaluated by maximum bubble pressure method. Surfactants A, 13, C, D and E were adopted as the grafting monomers of linear low density polyethere( LLDPE), and grafting reaction was carried out by melt reactive extrusion procedure. Their surface properties were characterized with measuring contact angle and XPS. It was found that the hydrophilic property of the graft copolymers was better than the palin LLDPE. Thermal properties of graft copolymers were studied by DSC. It was found that their crystalline temperatures of graft copolymers were faster than that of the plain LLDPE.

Morphology of electrodeposited poly(3,4-ethylenedioxythiophene)/poly(4-styrene sulfonate) films

Poly(4-styrene sulfonate)-doped poly(3,4-ethylenedioxythiophene) (PEDOT/PSS) films with ring-, arrow-, and bubble-like microstructures have been electrochemically generated simply by a one-step cyclic voltammetry in an aqueous media. Influences of applied potentials and surfactant/dopant-PSS on morphology of the resulting film were investigated, and a gas bubble template mechanism has been proposed. The result confirmed a well-doping of PSS in the PEDOT film. Electrochemical property and conductivity of the micro-structured PEDOT/PSS film were investigated further. Similar preparation with potential applications in fabrication of microdevices and micro-sensors can be extended to other micro-structured conducting polymers.

Direct electrochemical generation of conducting polymer micro-rings

Self-doped polyaniline (PANI) micro-rings have been successfully generated electrochemically. The polymer forming rings were about 100 nm wide, and the ring diameter is tunable from several to dozens of micrometres depending on deferent current densities. The morphology of such nanostructured polyaniline rings was investigated and further confirmed with field-emission scanning electron microscopy (FE-SEM). Furthermore, the film was characterized using UV/visible spectroscopy and cyclic voltammetry. The bubble template formation mechanism of the micro-rings was also proposed. Such nanostructured materials synthesized electrochemically open up a new approach to surface morphology control.

Geometries and electronic properties of AunPdm (n=1-4, m =-1, 0, 1) clusters

Possible conformers for AunPdm (n = 1-4, m = -1, 0, 1) clusters have been presented and studied by use of density functional theory. The results indicate that for n = 2, linear conformer with C-infinityv symmetry is the most stable for anion species, while for cation and neutral species, conformer with C-2v symmetry is the most stable. For n = 3, 4, conformers with C-2v symmetry (kite-shape) are energetically favored. The calculated electron affinities (EAs) and vertical detachment energies (VDEs) are in good agreement with experiments for n = 1-4. It is also interesting to note that for even n (n = 2, 4), the most stable conformers do not give the best agreement between calculated and experimental EA and VDE values, while for odd n (n = 3), the lowest energy conformer also gives the best agreement. The ionization potentials (IPs) of AunPd clusters are calculated as well.

Comparison of photobioreactors and optimal light regime for rapid vegetative propagation of transgenic Undaria pinnatifida gametophytes

BACKGROUND: Previously, tachyplesin gene (tac) has been successfully transferred into Undaria pinnatifida gametophytes using the method of microprojectile bombardment transformation. The objectives of this study were to compare and evaluate the performance of bubble-column and airlift bioreactors to determine a preferred configuration of bioreactor for vegetative propagation of transgenic U. pinnatifida gametophytes, and to then investigate the influence of light on vegetative propagation of these gametophytes, including incident light intensity, photoperiod and light quality to resolve the problems of rapid vegetative propagation within the selected bioreactor. RESULTS: Experimental results showed that final dry cell density in the airlift bioreactor was 12.7% higher than that in the bubble-column bioreactor under the optimal aeration rate of 1.2 L air min(-1) L-1 culture. And a maximum final dry cell density of 2830 mg L-1 was obtained within the airlift bioreactor using blue light at 40 mu mol m(-2) s(-1) with a light/dark cycle of 14/10 (h). Polymerase chain reaction (PCR) analysis indicated that genes (bar and tac) were not lost during rapid vegetative propagation within the airlift bioreactor. CONCLUSION: The airlift bioreactor was shown to be much more suitable for rapid vegetative propagation of transgenic U. pinnatifida gametophytes than the bubble-column bioreactor in the laboratory. The use of blue light allows improvement of vegetative propagation of transgenic U. pinnatifida gametophytes. (C) 2009 Society of Chemical Industry

Suspension culture of gametophytes of transgenic kelp in a photobioreactor

Transgenic Laminaria japonica gametophytes producing a recombinant tissue-type plasminogen activator (rtPA) protein, which is an effective third-generation thrombolytic agent for acute myocardial infarction (AMI), were cultured in an illuminated bubble column bioreactor. A maximum final dry cell weight of 1120 mg l(-1) was obtained in batch culture with an initial dry cell weight of 126 mg l(-1) and with aeration rate of 1.2 l air min(-1) l(-1) culture, nitrate at 1.5 mM and phosphate at 0.17 mM. The yield of rtPA was 56 mu g g(-1) dry cell wt. This is the first report regarding cultivation of a transgenic macroalga in a bioreactor.

Comparison of photobioreactors for cultivation of Undaria pinnatifida gametophytes

Undaria pinnatifida gametophytes were grown in 2.5 l bubble column and airlift reactor at 25 degreesC and light intensity of 40 mumol m(-2) s(-1) for 6 days. With aeration at 1 l min(-1), the airlift reactor yielded higher growth rate (0.12 mg DW ml(-1) d(-1)) than a bubble column (0.08 mg DW ml(-1) d(-1)). The advantages were related to the more homogeneous fluid dynamic characteristics of the airlift reactor.

Cultivation of the brown alga Hizikia fusiformis (Harvey) Okamura: stress resistance of artificially raised young seedlings revealed by chlorophyll fluorescence measurement

Commercial farming of the intertidal brown alga Hizikia fusiformis (Harvey) Okamura in China and South Korea in the sea depends on three sources of seedlings: holdfast-derived regenerated seedlings, young plants from wild population and zygote-derived seedlings. Like many successfully farmed seaweed species, the sustainable development of Hizikia farming will rely on a stable supply of artificial seedlings via sexual reproduction under controlled conditions. However, the high rate of detachment of seedlings after transfer to open sea is one of the main obstacles, and has limited large-scale application of zygote-derived seedlings. To seek the optimal condition for growing seedlings on substratum in land-based tanks for avoidance of detachment in this investigation, young seedlings were grown in both outdoor tanks exposed directly to sunlight and in indoor raceway tanks in reduced, filtered sunlight. Results showed that young seedlings, immediately after fertilization, could withstand a daily fluctuation of direct solar irradiance up to a level of 1800 mu mol photons m(-1)s(-1), and maintained a faster growth rate than seedlings grown in indoor tanks. Detailed experiments by use of chlorophyll fluorescence measurements further demonstrated that the overnight (12 h) recovery of optimal fluorescence quantum yield (F-v/F-m) of seedlings after 1 h treatment at 40 degrees C was 98%, and the 48 h recovery of F-v/F-m of seedlings after 1 h exposure to 1800 mu mol m(-2)s(-1) was 92%. Forty-one-day-old seedlings showed no significant decrease of optimal fluorescence quantum yield at salinity ranging from 30 to 5 ppt for a treatment up to 17 h. Six-hour desiccation treatment did not have any influence on the optimal fluorescence quantum yield. Exposure to 18 mmol L-1 sodium hypochlorite for 10 min did not damage the PSII efficiency, and thus could be used to remove epiphytic algae. The strong tolerance of young seedlings to high temperature, high irradiance, low salinity and desiccation found in this investigation supports the view that mass production of Hizikia seedlings should be performed in ambient light and temperature instead of in shaded greenhouse tanks.

Recombinant expression of rt-PA gene (encoding Reteplase) in gametophytes of the seaweed Laminaria japonica (Laminariales, Phaeophyta)

The life cycle of seaweed Laminaria japonica involves a generation alternation between diploid sporophyte and haploid gametophte. The expression of foreign genes in sporophte has been proved. In this research, the recombinant expression in gametophyte was investigated by particle bombardment with the rt-PA gene encoding the recombinant human tissue-type plasminogen activator (Reteplase), which is a thrombolytic agent for acute myocardial infarction (AMI). Transgenic gametophytes were selected by their resistance to herbicide phosphiothricin (PPT), and proliferated in an established bubble column photo-bioreactor. According to the results from quantitative ELISA, Southern blotting, and fibrin agarose plate assay (FAPA) for bioactivity, it was showed that the rt-PA gene had been integrated into the genome of gametophytes of L. japonica, and the expression product showed the expected bioactivity, implying the proper post-transcript modification in haploid gametophyte.

Cultivation of the brown alga Hizikia fusiformis (Harvey) Okamura: controlled fertilization and early development of seedlings in raceway tanks in ambient light and temperature

Commercial cultivation of the dioecious brown macroalga Hizikia fusiformis (Harvey) Okamura in East Asia depends on the supply of young seedlings from regenerated holdfasts or from wild population. Recent development of synchronized release of male and female gametes in tumble culture provides a possibility of mass production of young seedlings via sexual reproduction. In this paper, we demonstrate that controlled fertilization can be efficiently realized in ambient light and temperature in a specially designed raceway tank in which the sperm-containing water has been recirculated. The effective fertilization time of eggs by sperm was found to be within six hours. Fast growth and development of the young seedlings relied on the presence of water currents. Velocity tests demonstrated that young seedlings of 2-3 mm in length could withstand a water current of 190 cm s(-1) stop without detachment. Culture experiments at 24 h postfertilization showed that elongation of both the seedlings and their rhizoids were not hampered by high irradiance up to 600 mu mol photons m(-2) stop s(-1) stop. However, growth was slightly retarded if cultured at a temperature of 16 degrees C compared to other culture temperatures of 22, 25 and 29 degrees C. No seedling detachment was observed after transfer of the young seedlings to raft cultivation in the sea after one and 1.5 months post-fertilization, indicating the feasibility of obtaining large quantity of seedlings in such a system.

Effect of Stokes drift on upper ocean mixing

Stokes drift is the main source of vertical vorticity in the ocean mixed layer. In the ways of Coriolis - Stokes forcing and Langmuir circulations, Stokes drift can substantially affect the whole mixed layer. A modified Mellor-Yamada 2.5 level turbulence closure model is used to parameterize its effect on upper ocean mixing conventionally. Results show that comparing surface heating with wave breaking, Stokes drift plays the most important role in the entire ocean mixed layer, especially in the subsurface layer. As expected, Stokes drift elevates both the dissipation rate and the turbulence energy in the upper ocean mixing. Also, influence of the surface heating, wave breaking and wind speed on Stokes drift is investigated respectively. Research shows that it is significant and important to assessing the Stokes drift into ocean mixed layer studying. The laboratory observations are supporting numerical experiments quantitatively.

Application of effective medium approximation theory to ocean remote sensing under wave breaking

Based on the effective medium approximation theory of composites, the empirical model proposed by Pandey and Kakar is remedied to investigate the microwave emissivity of sea surface under wave breaking driven by strong wind. In the improved model, the effects of seawater bubbles, droplets and difference in temperature of air and sea interface (DTAS) on the emissivity of sea surface covered by whitecaps are discussed. The model results indicate that the effective emissivity of sea surface increases with DTAS increasing, and the impacts of bubble structures and thickness of whitecaps layer on the emissivity are included in the model by introducing the effective dielectric constant of whitecaps layer. Moreover, a good agreement is obtained by comparing the model results with the Rose's experimental data.

渤海湾地区前第三系构造特征及其演化研究

Based on fine structural interpretation on seismic profiles of buried-hills in Huanghua depression, structural interpretation and balanced cross-section restoration of regional seismic profiles, drawing structural maps of main seismic interfaces, residual strata distribution of different ages in the Bohai Bay region and structural survey in the western Shandong uplifted area and the intracontinental orogeny of Yanshan mountain, the paper has studied pre-tertiary structural styles and tectonic evolution of the Bohai Bay region. There mainly develop 5 types of pre-tertiary structural style that are extension structure, compression structure, strike-slip structure, negative inversion structure and sliding structure in the Bohai Bay region. Among these 5 types of structural style, extension structure develops detachment fault and its controlling fault terrain structure and fault break slop; compression structure develops reverted fold, fault propagation fold, fault bent fold, imbricate thrust structure and triangle zone; strike-slip structure develops positive flower structure, negative flower structure, en-echelon structure and brush structure; negative reversion structure develops Indosinian compression and Yanshanian extension negative reversion structure, late Yanshanian compression and Cenozoic extension negative reversion structure; sliding structure develops interlayer sliding structure and detachment structure. According to Cangdong fault of SN direction, Zhangjiakou – Penglai fault and Qihe – Guangrao fault of NWW direction, the Bohai Bay region can be divided into 6 sub-regions in which structural direction and style is different from each other. Structural maps of bottom boundary of Cenozoic and upper Paleozoic manifest that main NNE structural direction is formed from late Yanshanian to Himalayan movement and minor NWW structural direction and a string of area more than 8000m are mainly suggest that Indosinian tectonic pattern strongly influence on Yanshanian and Himalayan movement. Residual strata distribution characteristics of middle to upper Neoproterozoic in the Bohai Bay region manifest that middle- to neo- aulacogen position may be corresponding to late Mesozoic uplifted zone. Residual Paleozoic distribution characteristics of main ENN suggest that structural alteration should be resulted from late Yanshanian to Himalayan movement while which of minor NWW structures suggest that deeper structure should restrict shallower structure. Structural patterns of main EW fold direction in the Bohai Bay region and thrust structure in eastern part are formed late Triassic in studied area. Granite magma intrusion of early to middle Jurassic mainly develops Yanshan mountain zone. Late Mesozoic rifting basins of NEE direction are widely distributed in the Bohai Bay region and granite magma intrusions are mainly distributed in Tancheng – Rongcheng zone. Mesozoic structural evolution in the Bohai Bay region is related to scissor convergent from east to west between North China plate and Yangtze plate and gradually reinforcing of the west circum-pacific tectonic tract while basin and range province of late Jurassic and early Cretaceous may be mainly related to lithospheric thinning of North China craton in late Mesozoic.

拉萨地块中新生代火山作用-从大洋到大陆俯冲转换的构造演化纪录

Late Mesozoic－Cenozoic volcanic rocks are well exposed in Lhasa Terrane, southern Tibet. This research attempts to apply 40Ar/39Ar geochronology, major, trace element and Sr-Nd-O isotopic geochemistry data to constrain the spatio-temporal variations, the composition of source, geodynamic setting. The results indicate that Lhasa Terrane mainly went through three tectonic-magmatic cycle: (1) Phase of Oceanic subduction (140-80Ma). Along with the subducting beneath the Eurasian Plate of Neo-Tethys slab, the oceanic sediment and/or the subducting slab released fluids/melts to metasomatize the subcontinental lithospheric mantle, and induced the mantle wedge partially melt and produced the calc-alkaline continental arc volcanic rocks; (2) Phase of continental-continental collision. Following the subducting of the Neo-Tethys slab, the Indian Plate collided with the Eurasian Plate dragged by the dense Neo-Tethys oceanic lithosphere. The oceanic lithosphere detached from continental lithosphere during roll-back and break-off and the asthenosphere upwelled. The resulting conducted thermal perturbation leads to the melting of the overriding mantle lithosphere and produced the syn-collisional magmatism: the Linzizong Formation and dykes; (3) Following by the detachment of the Tethys oceanic lithosphere, the Indian Lithosphere subducted northward by the drive from the expanding of Indian Ocean. The dense Indian continental lithospheric mantle (±the thickened lower crust) break off, disturb the asthenosphere, and lead to the melting of the overriding mantle lithosphere, which has been metasomatized by the melts/fluids from the subducting oceanic/continental lithosphere and the asthenosphere, and produced the rift-related ultrapotassic rocks.