Vol.3 No.1 2010

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Research paper : Biomarker analysis on microchips (M. Kataoka et al.)−46−Synthesiology - English edition Vol.3 No.1 (2010) into maltohexaose (G6) and maltotriose (G3) that are oligosaccharides, we focused on the high separation capacity of the fluorescence-labeled glucose using the microchip electrophoresis that became apparent in the blood glucose measurement. We used the APTS-G6 that was fluorescence-labeled by 8-aminopyrene-1,3,6-trisulfonic acid (APTS) as a substrate, separated the breakdown product APTS-G3 by microchip electrophoresis, and then quantitatively measured the amylase activity (Fig. 4B)[10]. Here, the boric acid buffer solution was used as the driving force of migration, as in the blood glucose separation. In this method, the quantitative detection of blood amylase activity was possible in the range of 5~500 U/L at detection limit of 4.38 U/L. There are two isozymes of blood amylase originating from the pancreas or the salivary gland. By conducting the plasma pretreatment using the anti-amylase antibody of salivary gland origin for the differential diagnosis of pancreatic disease, the specific measurement of pancreatic-origin amylase activity became possible. When the plasma sample was used, it was shown that the amylase activity measurement was accurate as the current clinical testing method, and this indicated the possibility for practical application of the microchip electrophoresis in amylase activity measurement.In the glucose and amylase measurements described above, about one hour is necessary for blood glucose labeling and enzyme treatment of APTS-G6, and the treatment time must be reduced for application as POCT. Therefore, it is necessary to shorten the detection time by achieving high sensitivity by changing the fluorescence material or the detection system. However, the blood glucose and amylase activity are measured using the supplemented DNA analysis software, after the fluorescence-labeling and electrophoresis of the plasma using the commercially-available microchip electrophoresis device and the supplemented electrophoresis chip, or by mixing the fluorescence-labeled oligosaccharide with the plasma and then conducting electrophoresis. The major advantages are that the quantitative detection can be done extremely easily, and that it has accuracy and reproducibility equivalent to the current clinical test method. Also, only µl level of plasma sample is required, the device is compact, and the plastic substrate can be sterilized in an autoclave. Combining all these factors, the high potential of the microchip electrophoresis in biomarker analysis such as blood glucose and amylase is indicated. However, the glucose and amylase measurements are relatively inexpensive at 110 yen per test as covered by the National Health Insurance system, and the economic feasibility is low when only one item is tested with the microchip electrophoresis. However, sufficient economic feasibility can be maintained by measuring a combination of multiple test items, such as the detection of various blood proteins that will be mentioned later, on one chip for a particular disease.3.2 Construction of the antigen-antibody reaction system on the microchip substrate3.2.1 Construction of the sandwich ELISA on the micro flow channelMany of the biomarkers in the blood are various metabolites and proteins, and specific detection is possible even in blood in which many foreign substances are present. In the current clinical test methods, the antigen-antibody reaction system that does not require molecule sorting by electrophoresis is widely used. In the current clinical test methods, the antigen-antibody reaction using 96-well plate is generally used, but over one hour is needed for the reaction time, and several tens of µl of samples are necessary. Therefore, we attempted construction of the antigen-antibody reaction system in the Fig. 4 Measurements of blood glucose (A) and amylase activity (B) using the microchip electrophoresis.(A) A peak for plasma glucose was observed in a similar migration time as the glucose preparation. The blood glucose concentration was measured by calculating the calibration curve from the fluorescence intensity of the glucose preparation with known concentration.(B) A single peak was observed in the solo electrophoresis of APTS-G6. Treatment of APTS-G6 with purified amylase, the single peaks of APTS-G6 and APTS-G3, its breakdown product, were observed. By reacting the APTS-G6 with plasma, it was broken down into APTS-G3 by the action of blood amylase. The blood amylase activity was measured by calculating the calibration curve from the fluorescence intensity corresponding to APTS-G3 by treatment of APTS-G6 with amylase with known concentration. B.A.APTS-G6 + amylaseAPTS-G6 + plasmaAPTS-G6G6G3907050PlasmaFluorescence intensityFluorescence intensityMigration time (sec)Migration time (sec)Purified Glucose160800160800

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