Vol.3 No.1 2010

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Research paper : Biomarker analysis on microchips (M. Kataoka et al.)−47−Synthesiology - English edition Vol.3 No.1 (2010) micro flow channel on the microchip to reduce the sample quantity and to reduce the antigen-antibody reaction time by molecular scattering using the microspace. We investigated the sandwich enzyme-linked immuno-sorbent assay (ELISA) method (Fig. 5A) that is widely used for biomarker detection and has excellent quantitative quality.The blood procollagen I carboxyterminal propeptide (PICP), which is a biomarker for osteoporosis and cancer metastasis in which highly specific antibodies are commercially available, was selected as the measurement model. In the sandwich ELISA method, the primary antibody is fixed on to the solid phase[13]. In the conventional test, the 96-well plate (Fig. 5B) was mainly used as the solid phase, but here, the microchip (Fig. 5C) was used. After fixing the antibody and conducting blockingTerm 1, the plasma sample or the purified PICIP with known concentration and the peroxidase-labeled secondary antibodies were added. Fixing on to the solid phase was done via the bonding of PICP bonded to the labeled secondary antigen to the PICIP primary antibody. After washing away the labeled secondary antibody that did not bond to the antigen, peroxidase substrate was added, and the chemiluminescence was detected by a CCD camera. When using this as the POCT device, the user carries out the procedure after the blocking procedure. In the conventional 96-well plate, three hours was needed for the antigen-antibody reaction using 20 µl plasma. The cyclic olefin copolymer (COC) (Sumitomo Bakelite Co., Ltd.) substrate, in which the surface is treated to fix the protein and has three micro flow channels on one microchip, was used as the microchip substrate. The introduction of each solution at µl level into the micro flow channel was done using the Pipetman. Blocking was done after introducing the primary antibody from the sample well (1) in the direction of (2) and fixing it, and the antigen and the peroxidase-labeled secondary antibody were introduced in the direction from (3) to (2). Washing was done after the antigen-antibody reaction, the enzyme substrate was added in the direction from (1) to (2), and chemical luminescence was detected. The amount of plasma necessary per micro flow channel was 1 µl or less, and the antigen-antibody reaction time was 30 min. Dramatic reduction of the detection time and the sample volume was realized compared to the conventional method.For the antigen-antibody reaction in microspace, the method in which the micro-beads with diameter of several µm were fixed on the antibody, and then introducing and fixing the beads in the micro flow channel had been reported. In the beads method, there are issues such as a necessity of designing a micro flow channel with a complex shape to contain the beads in the channel. Therefore, we selected the method of fixing the antibody directly to the micro flow channel surface. As shown in Fig. 5C, the increased chemiluminescence intensity was observed in accordance to the concentration of the purified PICP, but the uneven luminescence in the same flow channel was observed, and it could be seen that quantitative property was not maintained. The reasons were thought to be: 1) uneven fixing of the primary antibody in the flow channel surface, and 2) insufficient washing or partial residue of labeled secondary antibody in each steps of blocking. In the Y-shaped flow channel used (Fig. 5C), the increased chemical luminescence was observed particularly at the fork point of the flow channel. It was necessary to design a flow channel that could be washed easily. Therefore, we attempted improvement of the quantitative property by fixing the primary antibody to the specific part of the micro flow channel using the inkjet for 1), and by increasing the washing efficiency by changing the flow channel design for 2).Fig. 5 Antigen-antibody reaction by sandwich ELISA method.Principle of the sandwich ELISA method and its experimental procedure (A), 96-well plate (B), and PICP detection in the micro flow channel (C).Measurement of chemiluminescenceEnzyme substrateWashingPlasma sample+Peroxidase-labeled secondary antibodyBlockingFixing of first antibodyCBALength 480 mmDepth 50 µmWidth 300 µm321321321PICP conc.(ng/ml)64032080160400Labeled secondaryantibodyPlate surfaceAntigenFirst antibody

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