本研究目的在於發展批次化製造可應用於標定癌細胞後投藥，並觀測細胞內分子層級變化之表面增強拉曼散射螢光奈米粒子。此奈米粒子系統建構於一容易取得、奈米至微米尺寸齊全、及包埋之螢光波段可選擇的聚苯乙烯奈米球上。此包埋螢光的奈米粒子，藉由聚苯乙烯本身的化學特性，在電漿環境下進行物理轟擊與化學反應處理，則可形成上半球面粗糙化及全球面羧基官能基分佈的結果。接著以電子束垂直沈積金膜於粒子上半球面，即可形成具備高增強係數之表面增強拉曼散射奈米螢光粒子。由於沈積金屬後，粒子上半粗糙球面與下半球面分別可與硫醇分子(-SH)及蛋白質分子的胺基 (-NH2)，形成選擇性的金硫鍵(Au-S)與肽鍵(peptide)自組裝分子修飾反應。
實驗中利用聚苯乙烯奈米粒子陣列排球技術，將250 μL含1-10%直徑220奈米至920奈米的粒子溶液滴在4吋玻璃晶片上，再以三段式旋轉參數(400rpm/10s、800rpm/120s、3000rpm/2-5s)，將粒子以單層最密排列的方式旋佈於4吋晶片上。接著的電漿處理程序，為了探討聚苯乙烯粒子表面粗糙形成的原因，實驗中選用了化學惰性的氬電漿及強化學活性的氧電漿，對含有不同表面羧基密度(0-14.7 carboxyl groups/nm2)的聚苯乙烯奈米粒子作處理。同時採用X射線光電子能譜(XPS)分析表面官能基的變化，另外以原子力顯微鏡(AFM)分析表面羧基密度與粗糙度的關係。因而推論粗糙表面源自於電漿環境下的選擇性蝕刻，即氧電漿改質純聚苯乙烯形成的羧基(或氬電漿環境下處理本身聚苯乙烯球修飾有的羧基)，相對於苯乙烯本體具有較高鍵結能，而形成奈米遮罩分部於球體表面。在垂直的電漿轟擊下，而造成粒子上半球面粗糙結構的形成。除此之外，為了最佳化表面增強拉曼散射的能力，實驗中比較了100 μM若丹明6G(Rhodamine 6G)在632奈米的雷射光激發下的拉曼散射強度，於電漿處理並鍍上金膜後的奈米球陣列上。經由分析二次電子顯微鏡(SEM)影像、原子力顯微鏡影像、局部表面等離子共振(LSPR)光譜圖，各種試片的拉曼增強效應可整理為受以下三個因子影響：(1)粒子與臨近粒子間的電漿耦合效應，(2)奈米次結構於粒子表面的粗糙度，以及(3)奈米次結構間距離。而其中奈米次結構間距離主要決定了拉曼增強散射的能力，且增強效益為待測溶液不在奈米粒子上的10E6。
為了使此拉曼散射螢光粒子具備更多的應用性，實驗中選用了一個抗體(anti-CD44)，選擇性修飾在粒子下表面羧基官能基上，使粒子得以辨識大部分癌細胞表面(例如子宮頸癌HeLa與乳癌MCF-7細胞) 過量表現的CD44表面跨膜糖蛋白，而使該粒子具備辨認癌細胞的能力。另外選用可修飾在金膜與胺基上的磺酸基丁二醯聯胺生物素化試劑(Sulfo-NHS-SS-biotin)，由於該試劑的中的雙硫鍵(-SS-)可在細胞膜內環境被切斷，因而可作為釋放藥物的鍵結分子(linker)。實驗中以卵白素量子點 (streptavidin-QDs)接在該鏈結分子的生物素(biotin)上作多重修飾的相容性測試。並針對各種修飾順序比較此粒子辨認癌細胞的能力，及在細胞外測試雙硫鍵斷鍵的能力與時間。此經過修飾的表面增強拉曼散射螢光奈米粒子，會經由超音波振盪的方式從4吋基材上取下，均勻散佈在去離子水(DI water) 中保存，每片4吋基材可取得約10E10個粒子於2mL溶液中，且其純度可高達99%以上。該奈米粒子辨識癌細胞(HeLa與MCF-7)能力，呈現高達12倍的標記數量相對於在正常細胞(軟骨細胞chondrocyte) 上。在論文最末，初步驗證了本技術應用在奈米粒子胞吞動態追蹤及癌細胞治療診斷研究的潛力。成功展示200奈米的表面增強拉曼散射螢光奈米粒子，在長時間的共軛焦螢光顯微鏡三維追蹤實驗中，配合拉曼掃瞄成像(Raman mapping)感測生物分子的能力。

The purpose of this study is to develop a batch manufacturing approach to massively produce surface-enhanced Raman scattering (SERS) fluorescent nanoparticles, mushroom-like Au semishell fluorescent nanoparticle (AuFNMs), for a potential application of targeting cancer cells, delivering drug, and subsequently observing cell behavior via SERS detection of intracellular biomolecule. This nanoparticle system was established on commercial available polystyrene beads with full range of sizes (from tens of nanometer to micrometers in diameter) and many fluorescent specifications. Based on the chemical property of polystyrene, the fluorescent nanoparticle can be treated with surface roughness on the upper hemisphere and carboxyl groups on the entire surface through plasma ion bombardment and chemical oxidation. Followed by the electron-beam deposition of gold film on the upper hemisphere, the nanoparticle can perform as a SERS-active fluorescent bead with high enhancement factor. After the deposition of metal, thiol (-SH) molecules and the amine (-NH2) of protein molecules could be modified simultaneously and selectively onto the top gold surfaces and bottom carboxyl groups through Au-S and peptide bonds, respectively.
In the experiments, the technique of densely packed nanoparticle array was employed to obtain a monolayer distribution of polystyrene beads on a 4-inch glass wafer. A 250-μL droplet containing 1-10% polystyrene bead, ranging from 220 nm to 920 nm in diameter, was sufficient to cover the entire surface of 4-inch wafer by a three-step spin-coating, 400rpm/10s, 800rpm/120s, and 3000rpm/2-5s. The following plasma treatment process was processed under inert argon plasma and/or vigorous oxygen plasma for the investigation and comparison of surface roughness on polystyrene nanoparticles with different intrinsic carboxyl density (0-14.7 carboxyl groups/nm2). In the meanwhile X-ray photoelectron spectroscopy (XPS) and atomic force microscopy (AFM) were utilized to analyze the change of surface chemistry and the correlation coefficient between carboxyl group density and surface roughness, respectively. The results suggest that bare polystyrene bead surface can be oxidized with carboxyl groups under oxygen plasma treatment, and the higher bonding energy of the carboxyl groups play the role as nanomasks decorating on the surface of polystyrene. As a result, the upper hemisphere of the polystyrene beads were treated with corrugated surface due to the selective etching under vertical plasma ion bombardment. Besides, in order to optimize the SERS enhancement factor on the nanoparticles, various gold-coated and plasma-treated polystyrene bead arrays with 100 μM Rhodamine 6G solution were observed their Raman scattering intensities under 632 nm laser excitation. Raman intensity enhancement on a 20-nm gold coated nanocorrugated polystyrene bead array is summarized by three factors: (1) the effect of plasmonic coupling among neighboring particles, (2) the nanocorrugation-contributed roughness, and (3) the pitch size of nanocorrugations, through the analysis of SEM images, AFM height images, and LSPR signals. Among these factors, the pitch size of nanocorrugations (ranging from ~6 nm to ~12 nm on the surface of polystyrene beads) dominates the SERS enhancement, and the average enhancement factor can reach up to 10E6.
To equip the SERS fluorescent nanoparticles (AuFNMs) with more applications, an anti-CD44 antibody was selectively modified onto the carboxyl hemisphere for the recognition of overexpressive CD44 transmembrane glycoprotein on most cancer cells, such as HeLa and MCF-7 cells. A cleavable disulfide linker of Sulfo-NHS-SS-with biotin was chosen for the modification either on the gold film or on the primary amine for releasing drug in the cell intracellular environment, via the reduction of a disulfide bond (-SS-) to two thiols. In the experiments, streptavidin-linked QDots was modified onto the disulfide linker of Sulfo-NHS-SS-biotin for the compatibility testing of multiple modifications. Moreover, the cancer cell targeting ability was compared among AuFNMs with a variety of modification order, and the cleavage of disulfide bond was examined in the extracellular environment. The AuFNMs suspension, which was verified with >99% purity and uniformed particle size in a concentration of ~10E10 numbers/mL in 2-mL DI water, can be employed to target cell-surface overexpressive glycoproteins (CD44) on cancer cells and release the loads via cleaving the disulfide bonds in cytoplasm after endocytosis of 30 minutes. A 12-fold cancer targeting ability of our AuFNMs was achieved on HeLa cells when compared to a normal cell of chondrocyte. For the applications of 3D confocal particle tracking and Raman mapping, the ~200 nm AuFNMs demonstrate excellent long-lasting single-particle fluorescence and superior biomolecule sensing ability. This technique provides a potential platform for the research of cell endocytosis pathway and the cancer cell theranostics.

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