2020 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 / redox biology |
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| Outline of Annual Research Achievements |
We completed the development of the biosensor designed for estimation of endogenous extracellular hydrogen peroxide (H2O2) on the surface of cells using surface enhanced Raman spectroscopy (SERS). The SERS analysis was combined with analytical methods of redox biology to estimate also the associated intracellular H2O2. Based on these results, we prepared the manuscript titled "Plasma membrane anchored nanosensor for quantifying endogenous production of H2O2 in living cells", which was accepted for publication in Biosensors and Bioelectronics (DOI:10.1016/j.bios.2021.113077).
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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 completed the production, the calibration and the testing of the hydrogen peroxide (H2O2) nano-sensor. Unfortunately, we verified that the previously developed pH-nanosensor cannot be used in combination with the newly developed H2O2 nano-sensor for simultaneous cell surface analysis. In fact, the Raman intensities bands of the two compounds sensitive to pH and H2O2 are overlapping. The two Raman signals cannot be separated with reliable accuracy. We are carrying on the analyses of different types of cells using the two nano-sensors separately.
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| Strategy for Future Research Activity |
Following the results obtained in the first two years, we will continue to exploit and develop the imaging technique of pH and hydrogen peroxides (H2O2) in many types of cells, such as HepG2 human liver cancer cells, MKN45 gastric cancer cells, A549 lung cancer cells, M30966 melanoma cells, human epidermal keratinocyte and human bronchial ciliary cells. In particular, we would like to optimize the nano-sensor anchoring to the cells in order to reduce the acquisition time of the Raman spectra. This may enable us to obtain pH or H2O2 images from cell tissues rather than from a single cell. We will try to collect statistically meaningful sets of data to test the possibility of creating a machine learning-based cell classifier.
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| Causes of Carryover |
We need to purchase enzymes and reagents for experiments of redox biology on the cells under investigation. We will purchase the software for statistical analysis of the data acquired from different types of cells.
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