| 研究実績の概要 |
We have integrated our previously proposed motor-protein based tau protein detection method with an on-chip format. The method was consisted of kinesin motor protein motion along microtubules that were incubated with tau protein for attachment. As healthy tau protein attaches on microtubules unlike the degenerated tau protein with reduced microtubule affinity, the average velocity of kinesin molecules provided the existence of tau protein on the microtubules. During the first year of this project, the proposed idea was implemented on an on-chip format. PDMS microfluidic device with multiple channels were fabricated for the experiments. At first, the surface coating was tested. Poly-L-lysine-coated surfaces showed good microtubule attachment but suffer from efficient kinesin motion. Protein-coated surfaces, on the other hand, provided better kinesin motion. However, this time, microtubule attachment on the surface was not very strong. Therefore, the flow in the channels were optimized to minimize the microtubule detachment. Finally, the system was tested for detecting different tau concentrations. Four channels were used to test different tau concentrations concurrently using a motorized stage that cycled through observation areas successively. The experimental results showed that different channels resulted in different kinesin velocity in parallel with the amount of tau flushed into the channels. The results indicate that the proposed device permits multiplex parallel measurements of tau protein content in different samples.
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| 現在までの達成度 (区分) |
現在までの達成度 (区分)
3: やや遅れている
理由
Although Task 1 (Assay development and on-chip integration) and Task 2 (Examining the detection sensitivity) were successfully demonstrated as FY2014 goals, the detection sensitivity limits have not been pushed to the limits. Further optimization was required but this was delayed due to delivery issues of tau protein from the USA as optimization often require certain amount of proteins to be used in the experiments.
As a result, the final sensitivity check of Task 2 will be performed in FY2015.
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| 今後の研究の推進方策 |
As proposed, Task 3 (Characterizing the effect of tau isoforms and mutants) and Task 4 (Device demonstration) will be demonstrated after the final optimization experiments on Task 2.
First, further optimization of the device will be performed to push the detection limits for achieving the final goal of Task 2. Then, the device will be tested on different tau isoforms and mutants as the goal of Task 3. 6 different isoforms (2N3R, 2N4R, 1N3R, 1N4R, 0N3R, 0N4R) and 4 commercially available Tau 2N4R-mutants that were tested earlier on flow cells (Tarhan et al, Lab Chip, 2013) will be demonstrated. As the final step, the device will be used to detect the amount of tau in a sample solution in parallel with the calibration solutions for Task 4. Concequently, the device will be demonstrated as a tau protein detection system.
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