Vol.3 No.1 2010

48/110

Research paper : Biomarker analysis on microchips (M. Kataoka et al.)−45−Synthesiology - English edition Vol.3 No.1 (2010) the analysis software, by optimizing the electrophoresis conditions by considering the gel and buffer solution compositions. We found that in the various experimental methods conducted daily in the biology and biochemistry laboratories, the presence of the proteins such as enzymes and ions such as Mg2+ ions necessary for restriction enzymatic activities did not affect the electrophoresis, by conducting the electrophoresis analysis after the on-chip restriction enzymatic treatment using the sample reservoir as a reaction field. Based on this result, we designed the on-chip restriction enzyme treatment method, and conducted quick restriction fragment length polymorphism (RFLP) analysis. We also reported that a biology researcher could conduct the mitochondrial membrane potential measurement, as well as application to synthetic RNA analysis and DNA ligation reaction analysis, simply by changing the electrophoresis condition, without changing the electrophoresis device or the analysis software. We also reported the high applicability of the microchip electrophoresis to biological and biochemical analyses utilizing the advantages, as it allows not only for nucleic acid separation analysis but also rapid, small sample, and high sensitivity analysis and various enzyme treatments[4]-[8]. These results indicate that the microchip electrophoresis can be applied to various experimental procedures, and cost reduction can be expected.3.1.2 Application of the microchip electrophoresis to blood glucose analysisBased on these findings and considering the application to POCT, we applied the blood biomarker analysis using the commercially-available microchip electrophoresis device and microchip. The Hitachi SV1100 was used as the microchip electrophoresis device, because it allowed easy handling of the solution by Pipetman due to its 10 µl reservoir capacity, and the gel and buffer solution could be changed easily. The supplementary chip was used as the microchip. Figure 3 shows the i-chip made of polymethylmethacrylate (PMMA) to be used with the SV1100. The i-chip has three micro flow channels with width of 100 µm and depth of 30 µm, and simultaneous analysis of three samples was possible (Fig. 3A). The electrophoresis procedure is simple. After adding the gel from the gel reservoir (GR), total 10 µl sample solutions including the internal control DNA were placed in the sample reservoir (SR). Electrophoresis and separation were conducted, and the separation and analysis of the DNA were done by fluorescence detection (Fig. 3B). In this microchip electrophoresis, the detection sensitivity was about 10 times higher than that of conventional agarose gel electrophoresis with fewer samples. The analysis result was obtained in a few minutes after the start of the electrophoresis, and the DNA could be separated with error of only a few bases. We focused on the high DNA separation capacity including the glucose structure of the microchip electrophoresis, and used the supplied DNA analysis software for the analyses for blood glucose and amylase that have glucose structure or use glucose as enzyme substrates[9][10].In the blood glucose measurement, we reported that the blood glucose could be specifically detected, after directly fluorescence-labeling the glucose by adding the fluorescent pigment 2-aminoacridone (AMAC) to the blood plasma, and then charging the glucose negatively using the boric acid buffer solution as the driving force of electrophoresis (Fig. 4A)[8]. It was found that the separation and analysis by electrophoresis of fluorescence-labeled glucose could be done in the blood plasma sample in which diverse proteins and other substances were present. This method had detection limit of 0.92 µM, allowed quantitative detection in the range of 1~300 µM, and enabled detection of blood glucose as accurate as the blood glucose level obtained by the conventional clinical test. Moreover, it showed high reproducibility both in within-a-day and between-days reproducibility, and indicated the possibility of practical application to blood glucose measurement by microchip electrophoresis. In the hexokinase-G-6-P-dehydrogenase method used in current clinical tests, there is a major problem where the value higher than the actual glucose measurement is obtained due to the presence of the disaccharide maltose in the infusion. However, by using the microchip electrophoresis, the monosaccharide glucose and the disaccharide maltose can be easily identified due to the difference in migration time[11]. As a result, the risk of hypoglycemia due to the false high-value reading of the glucose measurements in patients receiving infusion containing maltose can be prevented.3.1.3 Measurement of blood amylase activity by microchip electrophoresisBlood amylase is a biomarker used in the diagnosis of pancreatitis and sialadenitis. Amylase hydrolyses the glycoside bond and converts starch into glucose, maltose, and oligosaccharides. In the current clinical test, the oligosaccharide is used as the enzyme substrate and a qualitative measurement is done by the colimetric method[12]. Since it is already known that amylase is hydrolyzed Fig. 3 Schematic diagram of Hitachi i-chip (A) and sample separation in the cross flow channel (B).The “+” indicates the anode, “G” indicates the ground, and arrows show the migration direction of the sample DNA.85 mm30 mm50 mmGRGRGRSRA.B.Fluorescence detectionGGG+GGG+G+++

※このページを正しく表示するにはFlashPlayer9以上が必要です