利用自組雷射光鉗系統量測膠原蛋白熱降解之黏性變化

A study of the effect on collagen Type I viscosity caused by thermal denaturation using self-assembled optical tweezers system

莊迺民(Juang, Benjamin)

指導教授 ： 吳見明

國立清華大學/原子科學院/生醫工程與環境科學系/碩士(2013年)

摘要

將近40年前，Athur Ashkin等人第一次使用雷射光壓將微小粒子固定在焦點上。至今，單一雷射光組成的雷射光鉗已成為生物及物理領域中經常被應用的技術。在老化的社會中，保健及保養商品成為迅速成長的產業。組織工程與生醫材料的主要建材為膠原蛋白，是人體內佔有最大比例的單白質。然而，膠原蛋白與大多數的蛋白質擁有一個相同的特性，在較高溫的環境中(約 40 °C)，膠原蛋白的結構將開始降解。本研究利用自組光鉗系統，結合擁有高位移解析度的壓電平台，應用於測量膠原蛋白熱降解所造成的黏性變化。光鉗系統首先以兩種不同的方式以校正電壓¬¬-位移常數。本研究利用不同的緩衝溶液與添加物，將大鼠尾膠原蛋白一號(Rat tail collagen Type I)稀釋10倍並且配置不同pH值之膠原蛋白樣品。實驗首先將樣品加熱至目標溫度停留10分鐘，再取出放置室溫冷卻，並測量黏度。利用光鉗系統抓取聚乙烯微球(d = 1.78 μm)，測量微球在樣品溶液中的熱擾動，以計算黏度。測量黏度方式並非及時量測，因此加熱速度及目標溫度停留時間，對於本實驗並無顯著影響。本研究測量結果紀錄膠原蛋白pH接近中性時(pH=6)，擁有較好的耐熱能力，熱降解溫度此時延後約1 °C。當膠原蛋白pH接近中性時，會因纖維化造成黏度大幅上升，pH= 7時已經無法以本實驗方式測量黏度。pH= 7時加入1M葡萄糖(glucose)可大幅降低膠原蛋白之纖維化現象，此時測得熱降解溫度較pH= 6時，延後約2 °C。在酸性環境的蛋白質溶液中添加甘油(glycerol)，將測得熱降解溫度相較無添加時，延後約3 °C。本研究將不同pH環境與添加物，對於膠原蛋白一號的耐熱保護能力進行比較與討論。

關鍵字

雷射光鉗 ； 膠原蛋白 ； 黏彈度 ； 流變儀

並列摘要

Athur Ashkin first performed the trapping of particles by optical pressure nearly 40 years ago. Since then, single gradient optical tweezers have been developed and broadly applied in fields of biology and physics. In an aging society, health care and cosmetics are now a rapidly growing industry. Collagen is one of the most abundant proteins in the human body, and is a main building material in tissue engineering and for other biomaterials. Similar to most proteins, collagen is sensitive to high thermal conditions, denaturing at about 40 °C. This research applies a self-assembled optical trapping system, modified with a piezo-stage for nanometer resolutions, to measure the thermal denaturing effect on the viscosity of collagen Type I. Calibration of the voltage-displacement coefficient was completed by two methods and compared. Rat tail collagen Type I was diluted with various buffers and additives, collagen samples are then heated to target temperatures before cooling down to room temperature (27 °C) for measurement. Viscosities of collagen samples were then determined by measuring the thermal motion of polystyrene microspheres (d = 1.78 μm) immersed within the samples. The measurement of viscosity is not real time, therefore different heating rates and incubation times were applied, but both results had minor effect on the denaturation temperature. Our results indicate that collagen Type I has best thermal resistance at neutral pH 6, a slight delay of denaturation temperature of ≈ 1 °C is discovered. As the pH of collagen samples approach neutral, the aggregation of collagen fibrils causes the viscosity to increase significantly, restricting the thermal motion of the microsphere. This result makes it impossible to measure viscosity at pH 7, but the addition of glucose inhibits collagen aggregation, and the denaturation temperature is measured to have a ≈ 2 °C increase when compared to pH 6. The addition of glycerol in acidic environments can also raise the denaturation temperature up to 3 °C. In this research, pH environments and additives are compared to conclude the best conditions for the protection of collagen Type I against thermal denaturation.

並列關鍵字

Optical tweezer ； collagen ； viscosity ； viscometer

參考文獻

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