即時追蹤單粒螢光奈米鑽石於細胞膜通道與神經突中

Abstract

螢光奈米鑽石(fluorescent nanodiamond)是一種碳系相關的新穎奈米材料，有著獨特的光學特性與優良的生物相容性，且奈米鑽石表面容易進行化學修飾。這邊我們使用高能量氦離子束轟炸100nm Type Ib的鑽石，再經過高溫焠火後，會創造出大量且帶有負電的氮-空缺(NV-)缺陷中心，缺陷中心便可以在黃綠光的激發下放出遠紅外波段的螢光。優異的光學性質，像是沒有光漂白(no photobleaching)、沒有光閃爍(no photoblinking)與無毒性，使得螢光奈米鑽石不同於一般的螢光探針。因此，螢光奈米鑽石非常適合應用於標記細胞與長時間的追蹤。

我們使用螢光奈米鑽石作為單粒子在細胞膜通道(tunneling nanotubes)與神經突(neurites)中追蹤。細胞經由細胞膜通道與神經突的傳遞溝通行為，被認為與許多疾病有關，像是愛滋病毒的傳遞、普利昂蛋白的感染、阿茲罕默症與帕金森氏症…等。為了研究傳遞的過程，我們使用表面包覆牛血清蛋白(bovine serum albumin)的螢光奈米鑽石去做標記與追蹤。細胞膜通道上的研究用的是HEK293T細胞，神經突則是用N18細胞，我們使用共軛式聚焦顯微鏡拍攝螢光奈米鑽石的傳遞，追蹤與分析影片，並計算出移動速率介於0.05 - 1μm/s。我們成功建立了一個可以追蹤非專一性奈米鑽石標記的技術，同時也為未來追蹤專一性奈米鑽石標記，這種更深入研究細胞傳遞的方式打開了大門。

Fluorescent nanodiamond (FND), a relatively new nanocarbon material, has recently emerged as a novel fluorescent probe for biological applications. The material exhibits unique optical properties and is highly biocompatible with very low toxicity. Also, the surface of FND is easy to be functionalized for specific targeting. In this work, high energy helium ion beam was used to irradiate 100-nm type Ib nanodiamonds, followed by annealing, to create a high density of negatively charged nitrogen-vacancy (NV−) defect centers. The center emits far red fluorescence under excitation by green yellow light. Excellent optical properties such as no photobleaching, no photoblinking and nontoxicity make FND distinct from conventional fluorescent probes for cell labeling and long-term tracking applications.

This work applies FNDs as a single particle tracker in tunneling nanotubes (TNTs) and neurites. The intercellular vesicles transportation through TNTs formed between cells and organelle trafficking in neurites is related to many diseases, such as HIV infection, prion protein infection, Alzheimer's disease and Parkinson's disease. In order to study the transportation, we first coated FNDs with bovine serum albumin (BSA) and then performed single particle tracking of these nanoparticle bioconjugates inside TNTs (HEK293T cells) and neurites (N18 cells) by confocal fluorescence microscopy. We analyzed the transportation of the BSA-coated FNDs individually and obtained an average speed of 0.05 to 1 μm/s in both TNTs and neurites. The success of these experiments opens new ways to explore cellular transports in detail by using specifically labeled FNDs in future experiments.