Scientific Program, Oral Presentations
WEDNESDAY, 25 January
SESSION 9B		BIOMARKER DISCOVERY:
	O30	Replaced by:
12:10 – 12:30		Introduction of a technology platform for human serum/plasma biomarker research based on the automated isolation of formerly N- linked glycopeptides and reproducible LC-MS analysis R. Ossola, L.N. Mueller, H. Lee, B. Domon , M. Mueller, H. Zhang, J. Watts and R. Aebersold, Institute for Molecular Systems Biology, ETH Hönggerberg, Wolfgang Pauli-Str. 16 CH 8093  Zürich (Switzerland)

Scientific Program: Poster Presentations

PA09, PD10 and PK14 have been withdrawn

PD36	Micro Flow Injection Analysis System for Environmental Pollutants K. Sakamoto^1, Y. Matsuoka^1, T. Kawakami^1, M. Kitaoka^1 and T. Kitamori^2 , ^1The Research Association of Micro Chemical Process Technology, Kanagawa (JAPAN), ^2The University of Tokyo, Tokyo (JAPAN)
PE10	Improved and easy subtyping of Campylobacter using microfluidic automated electrophoresis of repetitive-PCR (rep-PCR) fragments C.-H. Brogren, M. Malakauskas, O. Medvedeva, J. Elmerdahl-Olsen, H. Christensen  Affiliation University of Copenhagen, Copenhagen (DENMARK); Lithuanian Veterinary Academy, Kaunas (LITHUANIA); The Royal Veterinary and Agricultural University, Frederiksberg (DENMARK)
PJ37	Microchip-based Ammonia Gas Analysis System H.Hachiya^1,2, M.Kitaoka^1, T.Kitamori^3, ^1The Research Association of Micro Chemical Technology, Kawasaki (JAPAN), ^2DKK-TOA Corporation, Tokyo (JAPAN), ^3University of Tokyo, Tokyo (JAPAN)
PJ38	Sliding Micro Valve Device for Quantitative Analysis in Microchip M.Kuwata^1, Y.Murakami^1, M.Kitaoka^1and T.Kitamori^2, ^1The Research Association of Micro Chemical Process Technology, Kanagawa, (JAPAN), ^2The University of Tokyo, Tokyo (JAPAN)
PM10	Analysis of nucleotides by capillary zone electrophoresis C. Drăghici1, H. Billiet2, J. van Dam2, G van Dedem2, Gh Coman1, M. Badea1, S Gocan3, 1Transilvania University of Brasov, (ROMANIA), 2Delft University of Technology, Delft (THE NETHERLANDS), 3Babas-Bolyai University of Cluj-Napoca, (ROMANIA)

Scientific Program: Late Poster Abstracts

PD36

MICRO FLOW INJECTION ANALYSIS SYSTEM FOR ENVIRONMENTAL POLLUTANTS

Katsumasa Sakamoto 1, Yshinori Matsuoka1, Tomohiko Kawakami 1, Mitsuo Kitaoka1 andTakehiko Kitamori2

1The Research Association of Micro Chemical Process Technology, kanagawa, Japan 2The University of Tokyo, Tokyo, Japan

Recently, micro total analysis systems have been gaining much attention because of　wide applications in the fields of analysis and chemical synthesis. Especially, an analysis on a microchip features small size and high throughput screening (HTS). In this study, we report a compact microchip system for fast analysis of environmental pollutants. Our system has two main features; a microinjector and a miniaturized thermal lens microscope (TLM) detection system.　 With the microinｊector, just 2 μL of a sample can be automatically injected to the microchip and mixed in laminar flow. Sequential analyses of multiple components become possible, because exchange of reagents is possible with the injector. TLM is of our originally developed detection technology, and it is very sensitive and widely applicable for non-fluorescent molecules in a microchip. We have succeeded in miniaturizing TLM by using a SELFOC micro lens on a microchip. The miniaturized TLM was incorporated into the compact microchip system. As a demonstration of applications, we performed flow analyses of environmental pollutants, such as hexavalent chromium, copper and phosphoric acid in water. A sample having concentration close to environmental standards was injected into the flow of a coloring reagent. The analysis time of hexavalent chromium was as short as 1 minute by utilizing our system. This paper describes development of a compact microchip system with a microinjector and TLM. The system proposed here worked well and is expected to be useful in a number of multiple analyses in micro-scale. ACKNNNOWLEDGEMENTS A part of the study presented in this paper was supported by New Energy and Industrial Technology Development Organization (NEDO) in Japan

PE10

Improved and easy subtyping of Campylobacter using microfluidic automated electrophoresis of repetitive-PCR (rep-PCR) fragments.

C.-H. Brogren, M. Malakauskas, O. Medvedeva, J. Elmerdahl-Olsen, H. Christensen

University of Copenhagen, Copenhagen, Denmark; Lithuanian Veterinary Academy, Kaunas, Lithuania; The Royal Veterinary and Agricultural University, Frederiksberg, Denmark

Genetic PCR-based molecular sub-typing of pathogens is increasingly utilized to characterize agents for epidemiological analyses during disease outbreaks. The so far most reliably utilized nucleic acid based techniques such as pulsed-field gel electrophoresis (PFGE) and multi-locus sequence typing (MLST) requires many person-hours and is costly. Consequently, we have initiated investigations of a semi-automated microfluidic electrophoretic screening method (Agilent 2110 BioAnalyser) of repetitive extragenic palindromic-PCR (rep-PCR) as a viable method for sub-type analysis of Campylobacter spp. The rep-PCR subtyping technique was optimized for Campylobacter jejuni using the commonly used GTGGTGGTGGTGGTG rep-PCR primer and used for comparison with the classical established genotyping methods of PFGE. Classical subtyping of Campylobacter jejuni strains was done using macrorestriction enzyme SmaI as described by Ribot et. al (2001). Rep-PCR amplification reaction was performed in 50l of a solution with the following temperature profiles: 1 cycle at 94oC for 2 min., 31 cycles at 94oC for 30 s, at 40oC for 60 s. at 72oC for 110 s, and 1 cycle at 72oC for 16 min. Rep-PCR amplicons were directly separated by electrophoresis on a 1.3% agarose gel. The DNA-7500 Kit for the Agilent 2100 Bioanalyser was used for semiautomated microfluidic analysis. Highly reproducible fingerprints were generated from a library of 72 Campylobacter jejuni isolates recovered from a variety of epidemiologic backgrounds and sources. For data treatment of the electropherogram images, we used phylogenetic tree dendrogram construction using the un-weighted pair group method with arithmetic mean (UPGMA; 1% position tolerance) of the generated fingerprints placed isolates into epidemiologic relevant groups (Gelcompare, BioNumerics). The comparison of phylogenetic relationships obtained by PFGE with those obtained by rep-PCR was essentially equivalent, but the rep-PCR methods were the most discriminatory. Seventy-two tested strains were clustered into 55 clusters by microfluidic rep-PCR, 61 clusters by conventional gel rep-PCR and 53 clusters by PFGE respectively. Discriminatory power (D) was slightly higher of microfluidic rep-PCR (D=0,990) in comparison to PFGE (D=0,988) but lower then conventional gel rep-PCR (D=0,994). The Rep-PCR analyses of the Campylobacter isolates were highly reproducible following both gel-based and chip-based rep-PCR analysis. However, the variation in band intensity within one fingerprint could sometime cause difficulties, which largely was overcome by applying approximately the same amount of amplicon from each samples in the microfluidic chip. These studies strongly highlight improved and reproducible rep-PCR subtyping of Campylobacter isolates compared to PFGE, which rapidly and easily could be analysed using the semiautomatic microfluidic electrophoresis provided by the Agilent 2100 Bioanalyzer combined with the Bionumeric phylogeny software. Recently, we have further improved the discriminatory power of the rep-PCR subtyping by multiplexing various rep-PCR primers, which directly gave fragments over a large range of 100 bp to 3000 bp, and an even higher discriminatory power.

PJ36

Micro Distillation System on Chip Utilizing Gas/Liquid Two Phase Flow and Capillary Condensation in Nanopillars

K.Mawatari1, K.Toshin2, A.Hibara2, M.Kitaoka1, T.Kitamori12

1 The Research Association of Micro Chemical Process Technology, Kanagawa, Japan, 2 The University of Tokyo, Tokyo, Japan

Microchip systems are recognized as one of the key technologies for future progress both in analytical and chemical synthesis fields. So far, we have realized various chemical processes on microchip by developing micro-unit operations (MUOs) utilizing multiphase flow and continuous flow chemical processing (CFCP). The applications widely range from environmental analysis (heavy metal ions, dioxin, microparticles etc.), clinical diagnosis (blood, urine etc.) and cell biology to chemical synthesis fields[1]. However, gas-liquid two-phase flow system is still one of the challenges in spite of large requirement in analytical fields. The difficulties come from the phase transition from liquid to gas or from gas to liquid accompanied by a very large volume change. In addition, gas phase flow is compressive flow in contrast to incompressible liquid flow which leads to instability in gas/liquid flow. Recently, we have reported capillarity restricted modification method (CARM) for hydrophobic-hydrophilic patterning of a microchannel[2]. By utilizing the patterning, stable and several gas-liquid flow processes such as bubble purge, and degassing have been successfully demonstrated. In this report, the gas/liquid flow processes are extended to a novel distillation system on microchip by realizing gas/liquid separation utilizing CARM and capillary condensation in nanopillars. [1] T. Kitamori, M. Tokeshi, K. Sato and H. Akihide, Anal.Chem. 76 (2004) 52A-60A. [2] A. Hibara, S. Iwayama, S. Matsuoka, M. Ueno, Y. Kikutani, M. Tokeshi and T. Kitamori, Anal.Chem.76 (2005) 943-947.

PJ37

Microchip-based Ammonia Gas Analysis System

H.Hachiya 1,2, M.Kitaoka 1, T.Kitamori 3

1 The Research Association of Micro Chemical Technology, Kawasaki, Japan 2 DKK-TOA Corporation, Tokyo, Japan 3 University of Tokyo, Tokyo, Japan

A microchip-based ammonia gas analysis system using a gas-liquid two phase flow was developed. Although a gas sensing microchip has been desired for many applications because of its possibility of excellent performances, it had some serious problems to be solved, e.g. the difficulty of gas-liquid reaction in micro space. Therefore we have proposed a novel gas analysis microchip based on the direct gas-liquid extraction at a gas-liquid two phase flow in a microchannel instead of the gas permeation through a gas-liquid separator. Since this gas extraction mechanism did not cause the loss of the gas permeability, which was observed in case of using a gas-liquid separator, some superior performances on gas sensing were expected, such as high sensitivity, quick response and long-term stability. In this study, we developed a microchip for two-step gas-liquid reactions through gas-liquid interfaces inside microchannels. This microchip with asymmetric channels was made by Pyrex substrates. The dimensions were 190 um (W) and 80 um (D) for a deep channel, and were 50 um (W) and 20 um (D) for a shallow channel, respectively. The interfering gases like acidic gases were removed at primary reaction, which used 0.001 M NaOH solution as an absorbent. At secondary reaction, phenolphthalein solution (PP) was used as a reactant. The pH of the reactant was changed by ammonia absorption, and PP became colored. The color-change was detected by using a thermal lens microscopic detector. Since all of conventional gas pumps were impossible to use because of its small expiring pressure, we also developed a novel gas sampling and controlling device for a micro analysis system. A mass-flow controller combined with a compressor was built in this device. The size of this device was 260 mm (W) x 230 mm (D) x 99 mm (H). The flow rate and pressure of the gas-phase were able to be controlled 0.15 to 3 mL min-1 and 0.03 to 0.2 MPa. The stable flow of gas-phase was achieved inside a microchannel. By this analysis system, the regression line and the correlation factor for 0 to 106 ppm ammonia gases were Y = 3.89X + 1.59 and R2 = 1.00, respectively. The repeatability for 106 ppm ammonia gas was less than 3 %. The similar degree of sensitivities was observed for ammonia gas in 500 ppm carbon dioxide gas. The removal of carbon dioxide gas at primary reaction was confirmed. This microchip-based system was successfully applied for ammonia gas analysis. Acknowledgements : This work was supported by “Project of Micro-Chemical Technology for Production, Analysis and Measurement Systems” of New Energy and Industrial Technology Development Organization (NEDO), Japan

PJ38

Sliding Micro Valve Device for Quantitative Analysis in Microchip

M.Kuwata1, Y.Murakami1, M.Kitaoka1 and T.Kitamori2
1The Research Association of Micro Chemical Process Technology, Kanagawa, Japan and 2The University of Tokyo, Tokyo, Japan

A novel sliding micro valve device is proposed. A part of microchip was cut into pieces parallel to each other across microchannels. The central part is able to slide in parallel to right-and-left parts. Thus the connections of channels in a microchip can be changed and quantitative 1-10nL injecting, switching, multi-dispensing, and so on are capable in microchip. Before performing the cut, these channels were originally a single channel. Thus, when these are reconnected, the shape, depth and width are the same. Therefore there is no dead volume left in between these channel connections. So it has a very good repeatability in terms of quantity. The coefficient of variation of injected sample peaks was 0.9% (n=50). Surface treatment of the slide structure is very important for pressure tightness. The contact surfaces of each part are coated with fluorocarbon to prevent the leakage. All the valves could withstand a pressure of at least 5 MPa. Since the device is made of Pyrex glass and fluorocarbon coating, it has high chemical tolerance. This device can be applied to almost all liquids (e.g., organic solvent, acid, alkali, and so on). The channel design is easily changed in accordance with the intended use. We can set any micro fluid control components – such as quantitative injection valve, switching valve, quantitative multi dispensing valve, and so on – in any place in microchip. As a demonstration to justify the applicability of this device, we performed flow injection analysis (FIA) of environmental pollutant, such as hexavalent chromium, nitrite ion, and so on. Quantitative 10nL sample having concentration of near environmental quality standards was injected into the flow of a coloring reagent. The injected sample was mixed and reacted with reagent in the reagent flow. The degree of color change was measured with thermal lens microscope (TLM). The thermal lens signal was linearly increased with the environmental pollutant concentration. This result shows that the slide micro valve device works well for the quantitative analysis. This paper describes the development of a novel micro valve device. This device is capable of controlling liquids with sufficient quantitative accuracy in nanoliter range. This proposed device is expected to serve a number of multiple analyses in micro-scale. ACKNOWLEDGEMENTS A part of the study presented in this paper was supported by New Energy and Industrial Technology Development Organization (NEDO), Japan.

PM10

ANALYSIS OF NUCLEOTIDES BY CAPILLARY ZONE ELECTROPHORESIS

Drăghici, C.1, Billiet, H.2, van Dam, J.2, van Dedem, G.2, Coman, Gh.1, Badea, M.1, Gocan, S.3

1 Transilvania University of Brasov, Romania 2 Delft University of Technology, The Netherlands 3 Babas-Bolyai University of Cluj-Napoca, Romania

Capillary zone electrophoresis was used to develop a method for nucleotides detection. The method is developed for standard solutions of three adeninic nucleotides (AMP, ADP, ATP) and four nicotinamidic nucleotides (NAD, NADH, NADP, NADPH). The influence of the electrolyte composition (buffer type and concentration), pH, voltage, and organic modifiers (acetonitrile, iso-propanol, EDTA, and β-cyclodextrine) was studied with CZE technique. Several electrolytes were tested to perform the separation in a concentration range of 10-100 mM and a pH range of 7 to 11. Among phosphate, borate, CHES, CAPS and TRICINE, the last one has been found as the most suitable for these compounds. Separation was resolved in less than 30 minutes with good resolution and reproducibility.