2021 Fiscal Year Research-status Report
Development of a generalizable toolkit for rapidly improving the sensitivity of fluorescent immunosensor proteins
| Project/Area Number |
21K14468
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| Research Institution | Tokyo Institute of Technology |
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Principal Investigator |
朱 博 東京工業大学, 科学技術創成研究院, 助教 (70886605)
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| Project Period (FY) |
2021-04-01 – 2024-03-31
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| Keywords | Immunosensor / mRNA display / Quenchbody / SARS-CoV-2 |
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| Outline of Annual Research Achievements |
The research project is about making the toolkit for improving the performance of quenchbody-based immunosensor (quenchbody) in a short period. It will increase the feasibility of the quenchbody while responding to the needs of society in a fast manner, such as developing the homogeneous immunoassay for the viruses.
In the first year of the project, the applicant optimized the cleavable linker regions of the anti-Bone Gla protein (BGP) and anti-cortisol quenchbodies to find a better design of the cleaver cleavable version of quenchbody. The cleavage site closer to the dye-labeling site for the anti-BGP quenchbody was found to be more effective. The amino acid sequence after the cleavage site was also optimized to give a better recognition by the protease Factor Xa. The optimized linker sequence will be the starting point of the next directed evolution-based linker optimization.
The mRNA display workflow for the quenchbody has been started in the first year as planned. The large amount of the mRNA of the anti-BGP quenchbody has been prepared successfully as the material for the next mRNA and quenchbody linkering process.
As a new part of the toolkit, the additives for the quenchbody-based immunoassay have been optimized for an anti-SARS-CoV-2 nucleocapsid protein quenchbody. A crowding agent was found to be able to increase both the response speed and the limit of detection of the immunosensor. And the crowding agent was tested for other TAMRA double-labeled qunechbodies with similar effect, therefore could be another important component in the developing toolkit.
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| Current Status of Research Progress |
Current Status of Research Progress
3: Progress in research has been slightly delayed.
Reason
The reseach progress was slightly affected by the COVID-19 pandemic due to the limited reseach time for reducing the on campus working time. The linker regions for two quenchbodies were optimized accoroding to the research plan, but the mathmatical model for the linker-cleavable quenchbody is not well modified due to the moderate performance of the current best linkers.
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| Strategy for Future Research Activity |
The research will mainly focus on the establishment of the mRNA display-based quenchbody optimization method as an important part of the toolkit. The anti-thyroxine antibody expression level and the response of the related quenchbody are not ideal as a model quenchbody during the toolkit development. Therefore, the following mRNA-display work will focus on the anti-BGP and anti-cortisol quenchbodies. The new component of the toolkit, the crowding agent in quenchbody immunoassay, will be further tested on more quenchbodies to find the most suitable application scenario.
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| Causes of Carryover |
The usage of the mRNA crosslinking reagent, fluorescent dye-labeled peptides and capturing antibody/bead is reduced due to the slightly delayed progress. And this part of the budget will be mainly used for pruchasing the same consumables mentioned above as planned. The conferences I attended last year were mainly hosted on-line, so the bussiness trip expense was not used last year. This part of the budget will be used for bussiness trip for conference attendence in the next fiscal year.
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