| Co-Investigator(Kenkyū-buntansha) |
KATSURA Shinji Gunma University, Department of Engineering, Professor, 工学部, 教授 (10260598)
HATSUKADE Yoshimi Toyohashi University of Technology, Department of Engineering, Research Associate, 工学部, 助教 (90339713)
大西 徳幸 チッソ(株), 横浜研究所, 主任研究員(研究職)
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| Budget Amount *help |
¥11,200,000 (Direct Cost: ¥11,200,000)
Fiscal Year 2006: ¥2,900,000 (Direct Cost: ¥2,900,000)
Fiscal Year 2005: ¥3,400,000 (Direct Cost: ¥3,400,000)
Fiscal Year 2004: ¥4,900,000 (Direct Cost: ¥4,900,000)
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| Research Abstract |
Immunoassay using ultra sensitive SQUID magnetic sensors and chemically-stable magnetic nano-particle markers has been proposed. In this method, so far, a single marker has been connected with one antibody. If it is possible to connect much more magnetic particles with one antibody, the sensitivity of this method must be notably improved. Use of thermo responsive magnetic nano-particles (Therma-Max : TM) was proposed by our research group. TM can aggregate and disperse by themselves associated with temperature of solution around them. However, there was a problem that reproducibility and quantization of the results were hard to achieve. Therefore, in this work, the detail of the characteristics of aggregation and dispersion of the TMs was researched, and the number of aggregated TMs was quantified. Besides, confirmation of all reaction from antigen to antibody to TM was studied by using enzymes. Finally, the detection of the antigen-antibody reaction through magnetism from the labeled
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and aggregated TMs by a SQUID was examined respectively. At first, by means of optical method, the size of aggregated TMs in solution was researched. TMs have Fe_3O_4 core surrounded by thermo-responsive polymer. It was found that the averaged diameter of aggregated TMs increased with temperature above 29 degrees, and became 400 nm at 30.5 degrees. Above 31 degrees, aggregated particles became so large that most particles went down. Next, magnetic detection of antigen-antibody reaction labeled by TMs was examined using a SQUID. Myosin was used as antigen, while connecting anti-Myosin (first antibody), biotin-antibody (second antibody) and TMs in turn with Myosin. Furthermore, TMs were labeled by third anti-avidin antibody and fourth antibody with enzyme. By detecting the enzyme, all reactions from Myosin to TMs were confirmed. By means of SQUID measurements using AC magnetization method, magnetic signals of about 2.4 mφ_0 from aggregated TMs in the reaction plates were detected. However, the signal amplitude was not approximately proportional to the amount of antigen. It was found that this disproportion was caused by remaining surplus aggregated TMs on the bottom and wall of the reaction plates. A possible solution to remove the remaining surplus TMs was discussed. We believe that immunoassay using TMs and SQUID will realize higher sensitivity than ever by establish this technique. Less
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