高分子奈米結構在生物分子上之應用

Polymer Base Nanostructures for Biological Applications

徐昭業(Jau-Ye Shiu)

指導教授 ： 黃華宗

國立交通大學/工學院/材料科學與工程系所/博士(2009年)

https://doi.org/10.6842/NCTU.2009.01043

摘要

具有奈米形貌的材料表面已被廣泛的應用於生物材料上，特別是因表面形貌所造成的超疏水表面(Super Hydrophobic Surface)，由於大部分生物材料需要附著於物體上才能夠表現其應有的行為，例如：osteoclast cell吸附於骨骼上之行為，因此了解材料表面與細胞之間的交互關係為重要的議題。本論文分為兩部分，其一主要是利用高分子材料加上奈米結構製程技術，來製備具有不同形貌之奈米基材，並用來了解生物分子在此表面上的吸附行為；更進一步利用材料表面的特性來達到操控細胞的貼附行為，如蛋白質陣(Protein arrays)列及細胞陣列(Cell arrays)；其二是利用奈米球微影術(Nanosphere Lithogrophy)及微流體系統(Microfludic system)製備三維多孔性結構於微流道中，並用來偵測單一DNA分子在其中的行為。

本論文研究第一部分主要探討如何在高分子材料表面製造出奈米尺度的形貌，其主要可分為兩種方法，一為將含氟的高分子旋轉塗佈在基材表面再利用氧電漿漿做表面處理，藉由改變氧電漿的處理時間可得到不同粗糙度的表面，其對應的水滴接觸角(Water Contact Angle)可從120o改變成169o。第二個製程是利用奈米壓印技術將高分子材料轉為具有週期性的奈米陣列形貌，其主要是先利用奈米球微影術將奈米球緊密排列於矽基板上，再利用氧電漿將奈米球縮小到適當的大小，最後再經由濺鍍金屬薄膜、舉離及蝕刻的步驟可得到具有奈米結構的矽基板；稱之為母模。將母模放置於高分子薄膜表面並加熱、加壓，等待溫度回到室溫將其分離便可得與母模相反的高分子奈米結構，其對應的水滴接觸角可達167度。

本論文研究第二部分是將細胞培養在這些不同粗糙度的含氟高分子表面，並進一步了解其吸附行為，本研究選擇三種不同的細胞，其中包含NIH 3T3小鼠纖維母細胞、CHO (Chinese Hamster Ovary)中國倉鼠卵巢細胞及HeLa (Human cervical epithelioid carcinoma)子宮頸癌細胞。其結果顯示NIH 3T3及CHO 細胞較容易吸附在越粗糙的表面上。因此當把材料表面製備成具有圖案化的(Square 200*200μm)週期陣列，只有圖案內為粗糙的表面，其餘表面皆保持平整。細胞吸附之行為也會隨之改變而形成細胞陣列，最後結果也發現可利此材料表面來增加細胞基因轉染效率(Transfection Efficiency)。

本論文研究第三部分是利用電濕潤效應(Electrowetting Effect)結合超疏水表面作為蛋白質陣列的表面材料，其原理是將含氟的高分子塗佈在ITO電極表面，再將含有塩離子的溶液放置於材料表面並施加電壓於電極與溶液上使其產生電場，此電場會使溶液更濕潤於表面，換句話說，當施加電壓後會使材料從疏水狀態轉為親水狀態。實驗結果發現當材料經過氧電漿處理後而成超疏水表面再施加相同的的電壓下，會從超疏水(接觸角為163度)變成超親水(接觸角為10度)。當把材料表面設計成與前一部分細胞培養表面的相同圖案後，將蛋白質分子放置於其表面並施加150V之電壓後，發現大部份蛋白質分子僅吸附於處理過的表面，也就是說蛋白質分子可被吸附在原本是超疏水表面藉由電濕潤效應而轉為超親水的表面上。經由不同電極設計可將特殊的細胞吸附分子(Fibronectin)吸附在特定的位置，再將細胞培養於其表面上，藉此亦可得到可操控的細胞陣列，此應用可與細胞吸附於粗糙表面特性結合，最後可將兩種不同的細胞共培養(Co-culture)在同一個表面上。

本論文研究最後一部分是利用具有三維奈米多孔性結構之微流道來分析單一DNA分子之行為，利用奈米膠體球(Colloidal Particle)自組裝的特性將其堆疊於微流道中，等溶液揮發後並形成六角最密堆積(Hexagonal close-pack)的光子晶體於微流道內，再將其填入凝膠(So-gel)或光阻(Photoresist)充滿其餘空間，最後將膠體球溶解便可得一三維多孔性的反向結構(Inver Opal structure)，此結構由兩種不同大小所組成，一個來自於膠體球本身所佔據之空間(330nm&570nm)，另一來自於球與球連接處(40~62nm)。DNA分子可被放入此結構中並施加電場，藉由電場的誘導帶負墊的DNA分子會向正極靠近，當DNA分子經過這些微小的奈米孔洞時會被拉伸而產成形變，最後利用螢光顯微鏡可觀測到單一DNA分子在此結構中的行為。

關鍵字

超疏水表面 ； 細胞陣列 ； 微流體 ； 奈米結構

並列摘要

Nanotecnology has been wildly used for biological applications. One of the most interesting examples is the so-called superhydrophobic surface. This type of structure is influenced by material property (hydrophbicity) and surface mophorogy (nanostructures). Since most cells can’t express celluar behavior without adhere on surfaces, it is very important to investiget the cellular adhesion on surface. For example osteoclast cells have to attach on the bone to behave normally. To understand the cell-substrate interactions, it is very important to investigate how cells adhere to the substrates and how the substrates respond to forces exerted by cells. There are two parts in this thesis; one is using low toxcisity polymeric nanostructure with different morphology to study the celluar adhesion behavior by it. Further more, the cell can be controlled to pattern on seleted area, as cell arrays. In the second part, the three dimentional periodic nano-porous structure in the integrated microfludic channel was used to study single DNA behavior.

In the first part of the dissertation, there were two simple techniques to impart superhydrophobic properties to the surfaces of microdevices. In the first approach, thin films of a fluoropolymer were spin-coated on the device surfaces followed by an oxygen plasma treatment. By varying the oxygen plasma treatment time, the water contact angles
on device surface could be tuned from 120° to 169°. In the second approach, a nanoimprint process was used to create nanostructures on the devices. To fabricate the nanoimprint stamps with various feature sizes, nanosphere lithography was employed to produce a monolayer of well-ordered close-packed nanoparticle array on the silicon surfaces. After oxygen plasma trimming, metal deposition and dry etching process, silicon stamps with different nanostructures were obtained. These stamps were used to imprint nanostructures on hydrophobic coatings, such as Teflon, over the device surfaces. The water contact angle as high as 167° was obtained by the second approach.

In the second part of this dissertation, the patterned nanostructure fluropolymer surfaces were used for the study of the cell adhesion. By a combination of photolithography and oxygen plasma treatment, patterned fluropolymer surfaces with various roughnesses have been obtain. The water contact angles measured on the surface were range from 120° to 163°, and surface roughness was measured from 2 nm to 65 nm. When these pattern surfaces were used as the substrates for the cell cultures of HeLa, NIH3T3, and CHO cells, it was found that those cell lines did not adhere to the flat fluropolymer surfaces. However, the number of NIH3T3 and CHO cells adhered on the surfaces increase with the surface roughness. Such nanostructure materials could be used as scaffold for selected cell growth.

In the third part of this dissertation, I will describe an approach to fabricate addressable cell microarrays, which are based on the patterned switchable superhydrophobic surfaces. The switchable superhydrophobic surfaces were prepared by roughening the surface of fluoropolymers on the electrodes. Upon the application of 150 V, the water contact angle on the roughened fluoropolymer surface could be changed from 1630 to less than 100 allowing the deposition of fibronectin, which could guide the growth of the cell. To patten the cells on such device,the HeLa cell was first seeded on pre-patterned fibbronectin area for incubatoring. After 3 hours incubation and removing suspension cell, the NIH 3T3 cell was incubated on same chip. Two different cell lines can be patterned on the same chip using the technique.

In the last part of this dissertation, I will describe a simple approach to fabricate robust three-dimensional periodic porous nanostructures inside the microchannels. In this approach, the colloidal crystals were first grown inside the microchannel using an evaporation-assisted self-assembly process. Then the void spaces among the colloidal crystals were filled with epoxy-based negative tone photoresist. After subsequent development and nanoparticle removal, thewell-ordered nanoporous structures inside the microchannel could be fabricated. Depending on the size of the colloidal nanoparticles, periodic porous nanostructures inside the microchannels with cavity size of 330 and 570 nm have been obtained. The dimensions of interconnecting pores for these cavities were around 40 and 64 nm, respectively. The behavior of single λ-phage DNA molecules in these nanoporous structures was studied using fluorescence microscopy. It was found that the length of DNA molecules oscillated in the nanoporous structures. The measured length for λ-phage DNA was larger in the 330 nm cavity than those measured in the 570 nm cavity.

並列關鍵字

Superhydrophobic surface ； Cell arrays ； Microfluidic channel ； Nanostructure

參考文獻

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