成果報告書詳細
管理番号20110000001155
タイトル*平成22年度中間年報 ナノテク・先端部材実用化研究開発/高機能性蛍光磁性ビーズによる高速・高感度疾患診断システムの開発(3)
公開日2011/9/9
報告書年度2010 - 2010
委託先名凸版印刷株式会社
プロジェクト番号P05023
部署名電子・材料・ナノテクノロジー部
和文要約和文要約等以下本編抜粋:1. 研究開発の内容及び成果等 (1) 時間分解測定装置(顕微鏡)の開発 時間分解蛍光顕微鏡は完成し、チップ上の蛍光磁性ビーズの定量的な検出に利用した。コスト低減のために検討を開始したイムノクロマトグラフィーも、顕微鏡でのイメージングによってメンブレン上での蛍光ビーズの展開挙動を観察することができた。顕微鏡による時間分解測定とモジュールによる測定を比較するために、ユーロピウムドープガラスと低反射ND(neutral density)フィルターを組み合わせた校正板(波長620nmの微弱蛍光サンプル)を用いて検討した。
英文要約1. Development of time-resolved fluorescence microscopy: We have developed time-resolved fluorescence microscopy. It was possible to observe the movement of the fluorescent magnetic beads on a lateral flow chip, which we selected as an alternative chip for microfluidics type, and also to measure the fluorescence of the beads quantitatively. 2. Module assembly: In order to increase the sensitivity of detection, we tried to adopt a brighter objective lens, reduce the size of spots energized, introduce the photon-counting type to PMT (photomultiplier tube), and incorporate the UV-semiconductor laser and its detector. We tried to find a low-cost excitation and detection device, and constitution of optical system for the lateral flow chip. 3. Construction of immunoassay: We evaluated the fluorescent magnetic beads labeled with antibodies by immunoassay on 96 well plates. The fluorescence signal depended on the lot of the preparation of beads. It is important to measure the amount of beads and the fluorescence in the steps of preparation. We tried to fix antibodies on the surface of the beads using a tag fused ProteinG but we could not confirm the effect of the antibody's orientation because of the low amount of fixation of antibodies compared to the random fixation with EDC (1-ethyl-3-(3-dimethylaminopropyl) carbodiimide hydrochloride). After we confirmed that we can use the fluorescent magnetic beads on the lateral flow system, we have searched for the best composition of solution to spread the beads using a half-strip membrane in the lateral flow. PSA (prostate specific antigen) was detected at 1ng/mL using colloidal gold, otherwise, it was detected more sensitively using fluorescent magnetic bead. As this data is not stable, it will necessary to analyze the reasons. 4. Utilization of magnetic effects: The beads were migrated in the nitrocellulose membrane in the lateral flow by magnetic field. When the beads were concentrated by ferrite magnet in the test line where the antigen binds, PSA was detected at 0.01ng/mL in response to the concentration of PSA. Otherwise, it was detected at 0ng/ml nonspecifically. 5. Development of microfluidics: To the surface of PDMS (polydimethylsiloxane) in the microfluidics, we screened out the condition for protein binding because the elastomeric microvalves need the PDMS's thin membrane. Using the valves, we constructed microfluidics capable of making a series of both diluted samples and diluted standards. In the microfluidics, the sample and buffer were mixed by circulation driven by the consecutive peristalsis. However, bubbles appeared at times and the complexity made it difficult to produce with low cost. 6. Survey of technological direction: There are some reports of lateral flow using time-resolved fluorescence and other kinds of fluorescence but it is necessary to accumulate technologies for the quantitative and multiplexed assay.
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