光学的方法を用いた組織間質液イオン環境の非侵襲的測定法開発

2017 Fiscal Year Research-status Report

• Project/Area Number: 16K18990
• Research Institution: Kyoto Prefectural University of Medicine
• Principal Investigator: プップリン レオナルド 京都府立医科大学, 医学部, 研究員 (40771580)
• Project Period (FY): 2016-04-01 – 2019-03-31
• Keywords: 表面強調ラマン分光法 / interstitial fluid / pH / gold nanoparticles

Outline of Annual Research Achievements

Conventional Raman Spectroscopy was not sufficiently sensitive in detecting variation of ion concentration in the micro- and nano-molar range. As stated in the last year activity report, in order to effectively measure proton concentration in the interstitial fluid,it was necessary to exploit a more sophisticated experimental approach.The newly developed spectroscopic method of analysis is based on the use of gold nanoparticles functionalized with a pH sensitive molecule (4-mercaptobenzoic acid). In fact, the nonometer size gold nanoparticles enhance the Raman scattering of the molecules attached to their surface, which can be detected even at concentration less than nanomole. This advanced spectroscopic approach named Surface Enhanced Raman Spectroscopy enabled us to measure variation of pH with high spatial resolution (nanometer scale). In addition, the pH nano-sensor was designed to target the outer membrane surface of cells. We used a combination of biochemical compounds to provide strong attachment between the gold nanoparticles and the cell surface proteins. We succeeded in testing our new method on mkn28 gastric cancer cells. Highly localized acidic pH was measured at the surface of the gastric cancer cells. The results have been presented at the annual meeting of the Physiological Society of Japan.

Current Status of Research Progress

3: Progress in research has been slightly delayed.

Reason

We are currently performing further experiments on MKN28, gastric cancer cells, to provide full validation of the new protocol for pH assessment by Surface Enhanced Raman Spectroscopy. Based on these results we are preparing a manuscript that will be soon submitted for publication on a scientific journal. In addition we are testing the variation of interstitial pH in the basolateral and apical outer membrane of airway ciliary cells.

Strategy for Future Research Activity

The new method was tested in-vitro on MKN28, gastric cancer cells, but not yet implemented for measurement of pH in the interstitial fluid of the brain.Further studies will be aimed at optimizing the conjugation procedure for attachment of gold nanoparticles to the membrane surface of different kind of cellsin vivo.We are also planning to patent this new method for interstitial pH assessment at the cell surface without invasive harmful method.

Causes of Carryover

ラマン分光解析装置に置いてレーザー出力の不具合により修理期間、使用不可期間が１２月より２月まであり、実験が遅延したため次年度に繰り越すこととなった。現在primary culture cell や種々のがん細胞において表面pHの測定を行い、また、今後ガン細胞移植したマウスにおいてのガン細胞表面のpH測定を遂行して行く予定である。