2019 Fiscal Year Research-status Report
Nanometer scale resolution imaging of pH and reactive oxygen species on the outer membrane surface for fast and effective cell discrimination in cancer tissues.
| Project/Area Number |
19K05228
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| Research Institution | Kanazawa University |
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Principal Investigator |
プップリン レオナルド 金沢大学, ナノ生命科学研究所, 特任助教 (40771580)
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| Co-Investigator(Kenkyū-buntansha) |
山田 忠明 京都府立医科大学, 医学(系)研究科(研究院), 講師 (00507048)
細木 誠之 京都薬科大学, 薬学部, 准教授 (30433254)
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| Project Period (FY) |
2019-04-01 – 2022-03-31
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| Keywords | hydrogen peroxides / cell surface / Raman spectroscopy / nano-sensor |
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| Outline of Annual Research Achievements |
In the first year of the project, we developed a nano-sensor for quantification of extracellular hydrogen peroxide (H2O2) on the cell surface using surface enhanced Raman spectroscopy. We tested the nano-sensor on lung cancer cells (A59 cell line). The sensor is selective for hydrogen peroxide. We were invited as speaker at the Symposium "Frontiers of biophotonics in physiology: Ion environments and molecule behaviors in the nano-space detected by Raman spectrum" at The 97th Annual Meeting of the Physiological Society of Japan. Based on the results, we published a proceeding in the Journal of Physiological Science (Volume 70 supplement 1, 2020) titled: "Surface enhanced Raman spectroscopy probe for nanometer-scale measurement of pH and hydrogen peroxides on the outer membrane of cells".
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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
We successfully functionalized the surface of gold nanoparticles with H2O2-sensitive 4-mercaptophenylboronic ester (4-MPBE). We optimized the conjugation of 4-MPBE on the gold surface to obtain the best Raman spectrum possible. Raman intensity of selected 4-MPBE bands are dependent on H2O2 concentration. Raman spectra were collected from solution at different H2O2 concentrations to calibrate the sensor. Built upon an experimental procedure established in our previous publication (Puppulin et al., Nature communication, 9, 5278 (2018)), we anchored the sensor to the surface of adenocarcinomic human alveolar basal epithelial cells (A59 cell line) for highly localized quantitative measurements of H2O2. We correlated the cell surface H2O2 to kinetic parameters of the cell H2O2 consumption.
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| Strategy for Future Research Activity |
We are writing the manuscript of the first scientific article based on the results obtained during the first year of this project. We will continue the experiments using the nano-sensor on different types of cells. We will acquire data not only on hydrogen peroxide, but also cell surface pH. We will compare the results to discriminate the type of cell depending on the data obtained by our Raman analysis. We should improve the acquisition time of Raman spectra to speed up the analysis on cell tissues. In order to achieve this goal, we are planning to test gold nanostructures different from the commercially available 90 nm gold nano-urchins that we used so far. New type of nano-structures may increase the intensity of the Raman signal of the 4MPBE molecules assembled on the gold surface.
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| Causes of Carryover |
We will buy enzymes and reagents that we will use for experiments of redox biology on the cancer cells under investigation. The final goal of these experiments is to calculate the kinetic constants of hydrogen peroxide consumption by the cells under investigation and the hydrogen peroxide reduction by enzymes inside the cells. These experiments are necessary to validate the results obtained using our new nano-sensor and Raman spectroscopy. The paper that we are preparing requires these results to increase the quality and the impact of our research.
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