新穎奈米鑽石之開發與性質探討及其生物應用

Advanced nanodiamonds as a versatile probe for biological applications

方嘉謚(Chia-Yi Fang)

指導教授 ： 張煥正

共同指導教授 ： 牟中原

國立臺灣大學/理學院/化學研究所/博士(2014年)

https://doi.org/10.6342/NTU.2014.10237

摘要

近年來，奈米鑽石在許多不同研究領域上逐漸嶄露頭角，因為此材料具有眾多優越的性質，例如低細胞毒性，高生物相容性，極佳的熱傳導性，此外經離子束照射並高溫焠火後，鑽石內部將生成氮-缺陷中心，此缺陷中心會放出極穩定的紅色螢光，並且還具有獨特的自旋性質。這些特性讓奈米鑽石在眾多的研究上大放異彩。於此，將針對鑽石的螢光性質與作為生物上長時間追蹤的應用做進一步的探討。
螢光奈米鑽石由於內部存在著氮-缺陷中心，所以可發出近紅外波段的螢光，然而有許多因素都會影響到該物質的螢光強度，因此在本研究中，在可以控制範圍內，針對其製造方式，鑽石粒徑大小，粒子內部含氮濃度與晶體的結構做詳細的探討。首先對各式樣品進行氮濃度的量測並以球磨研磨方式將微米級鑽石顆粒減小至奈米等級，再以不同能量之離子源對奈米鑽石進行照射，最終製備成螢光奈米鑽石。接著量測各式樣品之螢光強度與螢光生命週期，從結果顯示，於一定氮濃度範圍內，螢光強度會與該物質內部氮含量有正相關之趨勢，然而若鑽石中有部分結構缺陷，此趨勢將不復存在。根據實驗結果與對發光中心濃度的探討，相信未來可以發展出更小粒徑與亮度更高的螢光奈米鑽石，將有助於提高該物質於科學研究與生物應用上的價值。
為了證實螢光奈米鑽石於生物應用上的優勢，我們特地將該材料作為一種追蹤器，並針對癌細胞與幹細胞進行長時間追蹤與其胞吐作用的研究。首先以100 nm的螢光奈米鑽石對細胞做標記，接著觀察細胞於短時間內細胞周期分裂之情形與長時間細胞分裂成長狀況，結果顯示鑽石對細胞分裂與其增長現象不會產生實質影響，另一方面，鑽石經胞吞作用進入細胞後，隨即以流式細胞儀對細胞進行胞吐作用的觀察，配合細胞倍增時間量測，可以發現奈米鑽石於常見HeLa癌細胞與489_2.1幹細胞中經過六天觀察後約最多只有15%的鑽石會經胞吐作用而排出細胞外，而3T3-L1細胞則約為30%。與其他奈米粒子比較後，該數值相對小了許多，表示此物質更適合用於長時間追蹤的研究。此外，再與生物上常的染料分子CFSE比較後，發現鑽石的效果並不亞於染料分子，而且在某些細胞類型上鑽石的效果更是優於CFSE。根據本實驗研究結果，螢光奈米鑽石可以發展成適合用於細胞長時間標記與長時間追蹤的一種平台。有了這項技術，未來可望該物質能適用於活體內幹細胞的研究。
除了上述螢光奈米鑽石以外，我們也致力開發具有其他特性的奈米鑽石。近來以高劑量的氦離子束照射奈米鑽石製作出一種新材料，稱為輻照鑽石(INDs)，此新型鑽石具有良好的光聲性質，是一種能夠作為光聲對比試劑的材料。以粒徑為40 nm的INDs與常用於光聲研究上的金奈米粒子(10 nm x 67 nm)相比較，前者在莫耳吸收係數上雖然遠小於後者，但在同樣波長為1064 nm光源照射後，INDs卻可以產生比金奈米粒子更強的光聲訊號，造成此現象的因素可能是鑽石具有較高的熱穩定性、與水分子有良好的作用力及奈米微泡的生成。即使以高能量(>100mJ/cm2)的光源長時間照射，亦不會造成INDs的損害。此外也利用INDs進行了細胞活性的測試，結果顯示即使在100 μg/mL的濃度下，細胞的成長與活性並 沒有受到影響，代表INDs是不具毒性，而且適合運用在生物光聲影像的研究上，相信未來將具有很好發展的潛力。

關鍵字

螢光奈米鑽石 ； 含氮量 ； 光穩定性 ； 長時間追蹤 ； 光聲影像

並列摘要

Most researches show that Nanodiamonds (NDs) possess several significant advantages, such as low cytotoxic, high biocompatibility and good thermal conductivity. Fluorescent nanodiamonds (FNDs), produced by ion beam irradiation and subsequent annealing, emit bright and stable fluorescence. NDs are promising for applications in many fields owing to their outstanding properties.
In our study, we made a further discussion on how production method, particle size, nitrogen content and the crystallographic structure of diamond affect the fluorescence property of FNDs. First we measured nitrogen content for all kinds of samples and reduced the particle size from micrometer to submicrometer level by ball milling. We used different ion sources to irradiate NDs to form FNDs. A systematic investigation of fluorescence performance and lifetime of as-prepared nanodiamonds was done. We found that fluorescence intensity increased with the nitrogen density ranging from 100 to 200 ppm. This trend, however, failed to continue for NDs with higher nitrogen density (up to 390 ppm) but poorer crystallinity. Our results indicate that it is possible to produce smaller and brighter FNDs in the future.
In order to demonstrate FNDs as a fluorescent probe for biological applications, we use FNDs as a cell tracker for cell long-term tracking and cell exocytosis studies. FNDs (~100 nm) were introduced into cell by endocytosis through incubation. No significant alteration in growth or proliferation of FND-labeled cells is observed for up to 8 day. Flow cytometric analysis, in combination with parallel cell doubling-time measurements, indicates that there are about 15% excretion of the endocytosed FNDs after 6 days of labeling for both HeLa and 489_2.1 cell, and about 30 % for 3T3-L1 cell. A comparative experiment with FND and the widely used dye, CFSE (Carboxy-fluorescein diacetate succinimidyl ester), demonstrates that the nanoparticle platform is a promising alternate probe for long-term cell labeling and tracking applications.
In addition to fluorescent nanodiamonds, we make efforts in developing other features of nanodiamonds. Irradiated nanodiamonds (INDs) are a new type of nanomaterial served as a potential photoacoustic (PA) contrast agent. This work characterized in detail the photophysical properties of these materials prepared by ion irradiation of natural diamond powders using various spectroscopic methods. Compared with gold nanorods of similar dimensions, the INDs have a substantially smaller molar extinction coefficient and can generate stronger photoacoustic signal. This phenomenon may be attributed by INDs having much higher thermal stability, stronger hydrophilic interaction with water, and a lower nanobubble formation threshold. Besides, no sign of photodamage was detected after high-energy illumination of the INDs for hours. Cell viability assays with 100 μg/mL INDs concentration showed that the nanomaterial is non-cytotoxic and potentially useful for long-term PA bioimaging application.

並列關鍵字

Fluorescent nanodiamonds ； nitrogen dendity ； photostability ； long-term tracking ； photoacoustic image

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