Ratiometric Fluorescence Mesoporous Silica Nanoparticles for Intracellular pH Sensing

本研究的目標在於設計一具有偵測pH值的中孔洞奈米矽材(mesoporous silica nanoparticle, MSN)，並將其應用在偵測細胞不同部位的pH值研究。使用CTAB當作模板，TEOS為矽源，在鹼性條件下與兩種不同的螢光染劑FITC和TRITC進行共縮合(co-condensation)。接著用後修飾法(post-modication)修飾上帶有負電荷的PP或是帶有正電荷的TA。最後用HCl進行酸萃取，合成出粒徑約50奈米且具有ratiometric pH sensing 功能的MSN。 細胞內不同的胞器有不同的pH值，其pH值大約在pH5~7的範圍，FITC (pH-sensitive dye)的pKa為6.4，在pH5.4~7.4其螢光強度會隨著pH由酸至鹼而劇烈增強，因此非常適合用於偵測細胞內的pH值。TRITC(pH-insensitive dye)螢光強度則不隨著pH值變化，因此TRITC可當成是referencing fluorophore增加pH值偵測的準確度。 未進行後修飾的MSN，其isoelectric point落在pH~5，為了避免MSN在pH5~7之間會聚集(aggregation)，使用後修飾法修飾上帶有負電荷的PP或帶有正電荷TA使isoelectric point能不在pH4~8使用範圍之間。 所得的材料經由XRD、N2 adsorption-desorption isotherm、TEM鑑定，可得知MSN具有很好的六角結構、大表面積和規則孔道。由In-vitro的pH-fluorescence檢測可得一pH-fluorescence ratio校正曲線。由fluorescence microscopy、FLIM和Confocal fluorescence microscopy可知細胞不同部位的pH值。 關鍵字:中孔洞，奈米材料，比例計數螢光，細胞內pH值，後修飾，共縮合Mesoporous silica Nanoparticles (MSNs) exhibits many advantages such as large pore volume, high surface area, rigid structure, great capability of surface modification and good biocompatibility. In this research we synthesis and characterization MSNs, with pH-sensing capability and their application for intracellular pH measurements in HeLa cell. The MSNs are labeled with FITC (a pH-sensitive dye) and RITC (a pH-insensitive dye) covalently attached to the particle surface, which serves as a ratiometric Fluorescence for increased accuracy of the pH measurements. The pH dynamic range of the sensing particles is between 5.5 and 7.5 which is suitable be used to imaging intracellular pH localization. Key word: mesoporous, nanoparticles, ratiometric fluorescence, intracellular pH, post modification, co-condensatio