Vol.3 No.1 2010

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Research paper : Biomarker analysis on microchips (M. Kataoka et al.)−48−Synthesiology - English edition Vol.3 No.1 (2010) 3.2.2 Fixing the antigen to the micro flow channel surface using the miniaturized inkjetTo fix a certain amount of antigen on to an arbitrary part on the micro flow channel, miniaturized inkjet that can be programmed to discharge ultralow volume of solution at pl level was used. The miniaturized inkjet used was the piezo-driven Pulse Injector (Cluster Technology Co., Ltd.) (Fig. 6). This inkjet device could discharge 65 pl of diluted anti-PICIP primary antigen in one droplet. When 100 droplets of primary antibody was discharged and fixed using this device, the droplet diameter reached approximately the width of the flow channel, and the anti-PICIP primary antibody was fixed (Fig. 6). As mentioned earlier, in the design of the micro flow channel with a fork, nonspecific chemiluminescenceTerm 2 was seen, where a strong chemiluminescence was observed in the fork part due to the problem of washing, and it was difficult to maintain the quantitative property. Therefore, four linear micro flow channels that allowed easy washing was formed on one COC microchip (Fig. 7A) to construct the quantitative detection system. After discharging and fixing the primary antigen to the micro flow channel surface using the inkjet, blocking and washing were done in the direction of (1) to (2) to prevent the nonspecific adsorption and residue antibody, and after 30 min of antigen-antibody reaction, the chemiluminescence was detected using a CCD camera (Fig. 7B). In this reaction system, the amount of plasma needed per micro flow channel was 1.8 µl and the antigen-antibody reaction time was 30 min. These were 1/10 or less and 1/6, respectively, compared to the conventional 96-well plate method, and a low sample, high-sensitivity detection system was constructed. As a negative control, the antibody for the cardiac infarction marker, heart type fatty acid binding protein (H-FABP), that did not recognize the PICIP was discharged and fixed on to the micro flow channel surface using the inkjet. No nonspecific luminescence was observed for H-FABP, and good quantitative property was observed in the concentration range of 0~600 ng/ml (Fig. 7B, C). In the case where the plasma sample was used, rapid, low-volume, and accurate detection system that allowed measurement accuracy equivalent to the current sandwich ELISA method using the 96-well plate was possible. By conducting the antigen-antibody reaction in the micro flow channel, we were able to construct a blood protein detection method that could be applied to the POCT technology.In the method where the antibody is discharged and fixed in the micro flow channel using the miniaturized inkjet, the discharge and fixing of an arbitrary amount of antibody on to an arbitrary part becomes possible (Fig. 7D). The principle of the blood protein detection by sandwich ELISA method is basically similar regardless of the type of biomarker. Multiple types of antibody solution can be discharged by changing the head of the inkjet including the antibody solution, and multiple biomarker detection is possible from only 1.8 µl plasma sample in one micro flow channel. Currently, we are investigating the concentrations of the various primary and secondary antibodies as optimal conditions of the antigen- antibody reaction system to quantitatively detect multiple types of blood biomarkers on one micro flow channel. We are aiming to fabricate the multi-marker detection microchip, particularly the diagnostic chip for diabetes and osteoporosis that are gathering attention as life-style related diseases. For the diabetes diagnosis, in addition to blood glucose, measurements of insulin and high-sensitivity CRP that could be detected by antigen-antibody reaction could be mounted on the chip for accurate diagnosis with very small amount of blood. In osteoporosis, the detailed state of the disease can be observed by measuring both the osteogenesis marker PICP and the absorption marker NTx. Considering the cost advantages, the current insulin test is Fig. 6 Schematic diagram of the antibody fixing on the micro flow channel using the miniaturized inkjet, and the antibody fixed on the flow channel surface.Drive circuitCOC substrateAntibody fixed on theflow channel surfaceMiniaturized inkjet300 µmFig. 7 Schematic diagram of the microchip substrate with micro flow channel (A) and the PICP detection image using this chip (B), calibration curve (C) and the image of chemiluminescence when antibody of arbitrary amount is fixed on each flow channel (D). D.C.B.A.Luminescence intensityPICP ng/ml0R2 = 0.9958y = 2167.5x + 808326003001500PICP(ng/ml)H-FABP antibodyPICP antibody30060012

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