| 研究課題/領域番号 |
18K04905
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| 研究機関 | 北陸先端科学技術大学院大学 |
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研究代表者 |
ビヤニ マニッシュ 北陸先端科学技術大学院大学, 先端科学技術研究科, 客員教授 (00599780)
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| 研究分担者 |
高村 禅 北陸先端科学技術大学院大学, 先端科学技術研究科, 教授 (20290877)
山下 啓子 北海道大学, 大学病院, 教授 (70332947)
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| 研究期間 (年度) |
2018-04-01 – 2021-03-31
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| キーワード | Peptide aptamer / Voltammetric sensor / Brest Cancer / Cathepsin E |
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| 研究実績の概要 |
Two research themes were followed:
(Research 1) Immobilization of functional peptide aptamer on carrier magnetic microbeads: First, a biotinylated alpha-helix inducing ligand is utilized to immobilize the CatE-aptamer peptide onto a carrier bead, i.e., streptavidin coated magnetic beads. Second, a pentapeptide ligand coupled with alpha-helix inducing ligand is utilized to immobilize the CatE-substrate peptide onto gold nanoparticles. The terminal Cysteine of pentapeptide is coupled with BSA protein via NHS-ester of BSA protein and pyridyldithiol reactive group of SPDP crosslinker. The complex is purifed using gel filtration column and confirmd by SDS-PAGE analysis. Next, the CatE-substrate peptide coupled BSA is physically adsorbed onto the gold nanoparticles. The binding efficiency of peptide on nanoparticles is confirmed by lateral flow assay.
(Research 2) Binding, activity assay and sensitivity evaluation of CatE in a known sample solution: The binding of CatE-aptamer peptide with CatE enzyme which is bounded on magnetic beads are performed. The protease activity assay of CatE enzyme to cleave CatE-substrate peptide is performed at neutral pH. The protease activity assay is evaluated by electrochemical measurement. The peak height which represents the cleaved part of CatE-substrate peptide increased with the concentration of CatE. In next, the activity assay of CatE is evaluated in the presence of CatE-aptamer peptide. A 0.01 nmol concentration of CatE-aptamer peptide is optimized with higher current signals.
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| 現在までの達成度 (区分) |
現在までの達成度 (区分)
2: おおむね順調に進展している
理由
The designed research themes were successfully performed. However, the detection range of desired concentration of CatE in the clinical sample is in nanoM range. And the present detection limit of designed CatE-substrate peptide is near 5 microM. In order to increase the substrate specificity, a new sequence of CatE-substrate peptide is designed which is distinctly cleaved at neutral pH with special preference for Arg-Arg bond. Presently the peptide is under synthesis process and is expected to obtain after recovering from the current state of COVID-19.
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| 今後の研究の推進方策 |
Improved sensitivity will be evaluated by using re-designed CatE-substrate Peptide. In case, if sensitivity is not much improved, an alternative electrochemical approach of OCP (Open Circuit Potential) will be challenged.
After this, we will enter in the final phase of this project and sensor evaluation using clinical sample will be performed at Hokkaido University Hospital.
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| 次年度使用額が生じた理由 |
The unused budget in FY2019 was mainly due to the slow progress in peptide synthesis that delayed the experiments related to the development of sensor optimization. Since, we already evaluated the peptide now so the allocated budget to develop sensor will be utilized in FY2020.
1) Cost for development of software and hardware for OCP-based sensor; 2) Fee to hire a researcher to perform voltammetric experiments; 3) Cost for reagents and performing evaluation experiments at Hokkaido University Hospital; 4) Travel cost to make a presentation of project work in one international conference
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