2019 Fiscal Year Research-status Report
乳がん予後の高感度簡易検出のためのペプチドFETセンサーの開発
| Project/Area Number |
18K04905
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| Research Institution | Japan Advanced Institute of Science and Technology |
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Principal Investigator |
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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| Co-Investigator(Kenkyū-buntansha) |
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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| Project Period (FY) |
2018-04-01 – 2021-03-31
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| Keywords | Peptide aptamer / Voltammetric sensor / Brest Cancer / Cathepsin E |
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| Outline of Annual Research Achievements |
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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| Current Status of Research Progress |
Current Status of Research Progress
2: Research has progressed on the whole more than it was originally planned.
Reason
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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| Strategy for Future Research Activity |
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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| Causes of Carryover |
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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Research Products
(12 results)
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[Journal Article] Inhibition of HIV-1 Reverse Transcriptase Activity by the Extractsof Indian Plants2020
Author(s)
K. Ishizuka, Y. Tsutsumi, M. Baba, R. Biyani, C.W. Meng, M. Biyani, M. Takagi, J. Jaiswa, B. Sharma, K. Kojima, T. Takita, K. Yasukawa
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Journal Title
J. Biol. Macromol
Volume: 20
Pages: 17-22
Peer Reviewed
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