Vol.3 No.1 2010

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Research paper : Biomarker analysis on microchips (M. Kataoka et al.)−51−Synthesiology - English edition Vol.3 No.1 (2010) Discussions with Reviewers1 Practical useQuestion (Kazunori Nakamura, Evaluation Department, AIST)The authors mention, “There are many issues that remain before this can be actually used as a POCT device in clinical practice,” and I think the technology is still quite far from practical use. You should add this point to the title and the introduction of the paper to let the readers know.Please provide an analysis of the many remaining issues, organize their solutions and the actual efforts, and summarize the future direction. Also, before the device and the method can be used in actual clinical tests, there are issues of cost and approval by the Ministry of Health, Labour and Welfare, as well as its quantitative property and reliability. Since getting the MHLW approval involves setting the National Health Insurance points, it is important to establish the cost efficiency compared to the existing method. What is your thought on this?Answer (Masatoshi Kataoka)The future issues were described in chapter 4. The technologies include (1) mounting the blood cell separation system on to the chip, (2) construction of pump system using the micropump, and (3) micro flow channel design. The prototype of the device will be fabricated, and its efficacy as a clinical test device must be demonstrated. Also, we described the necessity of the approval as a medical test device according to the Pharmaceutical Affairs Law. On the issue of cost, we explained that there is sufficient economic feasibility when compared to the current test costs in terms of the NHI points.For the title, we changed it to emphasize the development of the core technology.2 Comparison with current technologyComment (Kazunori Nakamura)This technology is applied to the measurement of blood glucose and amylase activity. Particularly for blood glucose, the glucose sensors that the patient can use daily are already widely in use. Therefore, please state the issues in the currently used methods, and clarify the things you did to solve those problems. You also mention that the method in this study has equivalent performance to the current clinical test method, and therefore, I think you should explain the future developments such as whether this method will replace the current clinical test, whether there is possibility of diffusion as a POCT device, and the path to practical use including the cost aspects.Answer (Masatoshi Kataoka)As you mentioned, there are blood glucose sensors that are commercially available as POCT device. However, in the glucose measurement using the hexokinase-G-6-P-dehydrogenase method, higher-than-actual glucose value may be indicated because the disaccharide maltose is identified as monosaccharide glucose. This is a major issue when the patient is receiving infusion that contains maltose (there are actual fatal cases due to hypoglycemia). In such cases, the identification of monosaccharide and disaccharide by electrophoresis based on the Leader of Biodevice Group, Health Research Institute, AIST from 2010. Specialty is microfabrication using lasers. Develops the plastic disposable chip, the units to be embedded in integrated chip, and others to enable multi-item diagnosis using blood, to realize a device that can numerically express the human health condition. In this paper, constructed the discharge and fixing of antibody using the miniaturized inkjet for multi-marker analysis.migration time provides great clinical advantage.For amylase measurement, we described that the conditions needed as a POCT device are cleared, such as quantitative property, simple operation, smaller amount of samples, compactness of the device, and chip that can be autoclaved. In terms of cost, we described that the cost of the electrophoresis device will be reduced due to the high universal application of the microchip electrophoresis to various experimental procedures. Also, we explained that in clinical tests, because the costs of blood glucose and amylase measurements are already low in the NHI, the cost of a new device will not be feasible for single tests, but the cost competitiveness will be sufficient if it is used as a multiple analysis chip where several test items are combined.3 Performance of individual technologiesComment (Kazunori Nakamura)You indicated the problem of sandwich ELISA currently used. However, it is not really clear how effective the reduction of measurement time is in the diagnosis of the long-term disease like osteoporosis. You write that the reaction time was reduced from conventional three hours to 30 minutes, but the measurement principles are the same as the antigen-antibody reaction and enzyme reaction, and you don’t sufficiently explain why it is reduced even if they are basically the same principle.Comment (Motoyuki Akamatsu, Human Technology Research Institute, AIST)Please explain, why the microchip electrophoresis can use small amounts of samples and have high detection sensitivity compared to the conventional agarose electrophoresis.Answer (Masatoshi Kataoka)When constructing the antigen-antibody reaction system, the antigen identification capacity of the antibody becomes the issue. We selected the PICP because: it is a marker for osteoporosis, which is a social issue being a life-style related disease with high number of patients (fracture due to osteoporosis may cause geriatric patients to become bedridden); the highly specific antibody is readily available (the antibody is commercially available independently); and the PICP blood concentration is measurable in healthy individuals (data is always obtainable from the blood sample; markers such as inflammatory cytokine will fall below measurement limit in healthy individuals, and data analysis is difficult difficult to establish an experiment system). We added these points to the paper.Since the principle of antigen-antibody reaction is basically the same regardless of the marker type, the basics are the same whether it is PICP or any other marker under investigation. Therefore, we selected PICP for investigation because it is an osteoporosis marker for which highly specific antibody is commercially available. The reduction of time from three hours to 30 minutes will allow use in a doctor’s office, which is a requirement of POCT. In antigen-antibody reaction, specific bonding starts with the collision of the antibody and antigen in space. In the microspace, the dispersal time is reduced due to the molecular dispersal effect, and this, as a result, is thought to lead to the reduction of antigen-antibody reaction time. We also added this explanation.For electrophoresis, we stated: “Compared to the conventional electrophoresis method for the separation analysis of nucleic acid and proteins, the microchip electrophoresis has high separation capacity by application of high voltage, because the sample volume can be reduced by using the micro flow channel, and because the efficiency of heat release during electrophoresis is increased due to the increased surface volume against the sample volume in the flow channel. Moreover, higher sensitivity can be achieved by using the LED-excited fluorescence detection system.”

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