奈米生物感測粒子用於細胞內氧化壓力即時偵測之發展及研究

Abstract

要維持生物細胞功能正常運作，細胞中的氧化劑及抗氧化劑的平衡作用是不可或缺的。一般來說，在自然的情況下，細胞會有相對應的防禦機制將氧化劑還原並維持體內氧化鈦平衡。當細胞因為自然因素或是外在因素致使細胞內產生額外的氧化劑，且防禦機制無法平衡時，過多的氧化劑與細胞中的各物質發生交互作用並造成氧化傷害，即氧化壓力(oxidative stress)；關於氧化壓力及其所造城的傷害在近年來已經被證實與老化與多種疾病有關。在本文中，提出以光學比率測量原理為基礎的奈米生物感測粒子[PEBBLE (probes encapsulated by biologically localized embedding) nanosensor]。粒子中含有過氧化還原酵素－catalase (EC 1.11.1.6)以及兩種螢光染劑－Ru(II)-tris(4,7-diphenyl-1,10-phenanthroline) chloride ([Ru(dpp)3]2+)和Oregon Green 488-dextran。並使用sol-gel技術將上述物質包覆於例子中以用於偵測體內過氧化氫濃度。此光學比率測量奈米生物感測粒子粒子為圓球體，平均尺寸約為200±50nm，並具有約8U/mg的catalase活性。Catalase可以將細胞中造成氧化壓力的過氧化氫還原生成氧氣。同時，粒子中的[Ru(dpp)3]2+的螢光訊號會因為氧氣的存在而產生quenching，造成螢光強度的改變；而相反的Oregon Green 488-dextran之螢光訊號則不受氧氣濃度影響，可作為測量的基準值，與[Ru(dpp)3]2+訊號相較以提供細胞內過氧化氫偵測資訊。

Balance between oxidants and antioxidants is important to maintain normal cellular functions. Although the most oxidative events are carefully monitored and controlled by the natural defense systems of cells, sustained perturbation of this balance may result in oxidative stress. Oxidative stress-induced damages are usually key factors for aging and many disorders in human cell. In this study, a ratiometric optical PEBBLE (probes encapsulated by biologically localized embedding) nanosensor, which encapsulates catalase and two fluorescent dyes, Ru(II)-tris(4,7-diphenyl-1,10- phenanthroline) chloride ([Ru(dpp)3]2+) and Oregon Green 488-dextran was developed by sol-gel technology for the detection oxidative stress in vivo. The developed ratiometric optical PEBBLE nanosensor is spherical with an average size of 200±50 nm. The catalase encapsulated in the nanosensor is around 8U/mg nanoparticle. Catalase in nanosensor converts H2O2 to oxygen that quenches the fluorescence of [Ru(dpp)3]2+; whereas, the fluorescence of Oregon Green 488-dextran is unaffected, acting as a reference for ratiometric intensity measurement. The developed optical nanosensor exhibits potential for the real-time detection of oxidative stress in living cells.