利用雷射光鉗系統量測pH值與添加物對膠原蛋白熱降解之黏度變化影響

Using optical tweezers to study the effect on collagen type I viscosity caused by thermal denaturation with varying pH and additives

王傑(Wang, Jie)

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(2014年)

摘要

雷射光鉗量測膠原蛋白熱變性過程的黏度變化，比起傳統黏度計和流變儀的測量方法，使用雷射光鉗與微流變學的方法可以量到高頻的部分而且可以大幅減少消耗樣本的量。這些黏度和微流變的量測數據將來可以應用在生醫材料和生物科技的研究上。
膠原蛋白是一種天然生醫材料，現今常運用在組織工程和生醫材料的研究當中。膠原蛋白會在大約40°C熱變性，造成膠原蛋白的三股螺旋結構變成隨機線圈結構。
本研究利用雷射光鉗系統量測膠原蛋白在不同的pH值和加入其他的添加物，包括甘油、尿素、葡萄醣、鹽之後，加熱熱變性過程的黏度和黏彈模數。本研究利用PBS緩衝液和醋酸將大鼠尾巴第一型膠原蛋白(Rat tail collagen type I )稀釋10倍到不同的pH值和加入其他不同的添加物，接著使用雷射光鉗捕捉聚苯乙烯微球(d=2.07μm)測量微球在膠原蛋白溶液中的熱擾動，並計算膠原蛋白溶液在加熱過程中的黏度變化和微流變特性。
本研究首先量測純水溶液計算求得系統的電壓-位移轉換因子 ，和雷射光鉗彈性係數(trap stiffness)。得知系統參數後，藉由量測無彈性的純水溶液樣本，利用Kramers-Kornig關係式求得純水的彈性模數G'和黏性模數G''。接著量測膠原蛋白在不同的pH環境下和加入不同添加物在熱變性過程中黏度和黏彈模數的變化，並探討在這過程中膠原蛋白的變性溫度、結構變化等。膠原蛋白在中性pH值時，會聚集形成膠原蛋白纖維造成黏度的大幅增加。葡萄醣會抑制膠原蛋白纖維的形成，造成黏度的下降，變性溫度比起在pH6.8時增加了1°C。甘油會保護膠原蛋白纖維，變性溫度增加大約2°C。尿素會打斷膠原蛋白的分子間氫鍵，造成黏度的下降，變性溫度下降大約4°C。鹽會降解膠原蛋白，造成黏度的下降，變性溫度下降大約4°C。
膠原蛋白在不同的pH值下和加入不同的添加物會導致膠原蛋白的交聯程度、分子間作用力、分子內作用力、氫鍵、靜電作用力、疏水作用力發生改變。而這將會影響膠原蛋白的變性溫度、摺疊的平衡狀態、分子結構、熱穩定度、黏度和微流變性質。

關鍵字

黏度 ； 黏彈模數 ； 膠原蛋白 ； 雷射光鉗 ； 微流變

並列摘要

We combined optical tweezers and microrheological technique to measure the viscosity and viscoelasticity of collagen solutions in the denature process. Using optical tweezers to measure the collagen viscosity and viscoelasticity enabled us to extend the range of frequency and reduced the amount of samples used compared to traditional viscometers and rheometers. This study could be used in biomaterials and biotechnology research in the future.
Collagen is a natural biomaterial and is one of the most abundant proteins in the human body. It has often been applied in the fields of tissue engineering and biomaterials. Collagen denatures at about 40°C and causes the collagen’s triple helix structure becoming random coils.
In this study, we utilized optical tweezers system to measure the effects of thermal denaturation with varying pH and additives, (i.e. glycerol, urea, glucose, and NaCl) on viscosity of collagen Type I. Rat tail collagen Type I was diluted with PBS buffer and acetic acid to varying pH, with different additives. Collagen samples are then heated to target temperature and cooled down to room temperature (25°C) for measurement. Viscosity of collagen samples were determined by measuring the thermal motion of polystyrene microspheres (d=2.07μm) by optical tweezers.
We used distilled water to calibrate the voltage-displacement coefficient and trap stiffness. After obtaining the system parameters, we used Kramers-Kornig relation to calculate water’s elastic modulus G' and viscous modulus G''. And then, we measured the collagen’s viscosity with varying pH and additives under thermal denaturation conditions.
Our results indicate that as the pH of collagen approaches neutral, the aggregation of collagen fibrils causes the viscosity to increase significantly. The addition of glucose would inhibit collagen aggregation, causes the viscosity to decrease, and the denaturation temperature was measured to be 1°C higher when compared to pH 6.8. The addition of glycerol would protect collagen fibrils, and increase the denaturation temperature about 2°C. The addition of urea would break the collagen’s intermolecular hydrogen bonds, causes the viscosity to decrease, and lower the denaturation temperature about 4°C. The addition of NaCl would degrade collagen, causes the viscosity to decrease, and lower the denaturation temperature about 4°C.
Different additives and pH would alter the collagen’s degree of cross-linking, intermolecular interactions, intramolecular interactions, hydrogen bonds, hydrophobic interactions, and electrostatic interactions. This would influence the collagen’s denature temperature, folding equilibrium, molecular structure, thermal stability, viscosity, and viscoelasticity.

並列關鍵字

viscosity ； viscoelasticity ； collagen ； optical tweezers ； microrheology

參考文獻

29. 張世熙“利用雷射光鉗探討膠原蛋白黏彈度受紫外光照射的影響”NTHU, 2012年.

1. Ashkin, A., Acceleration and Trapping of Particles by Radiation Pressure. Physical Review Letters, 1970. 24(4): p. 156-159.

2. James, R.S. and Veigel, C., Direct observation of the myosin-Va power stroke and its reversal. Nature Structural & Molecular Biology, 2010. 17(5): p. 590-595.

4. Bormuth, V., Varga, V., Howard, J. and Schaffer., Protein friction limits diffusive and directed movements of kinesin motors on microtubules. Science, 2009. 325(5942): p. 870-873.

5. Lyubin, E.V., Khokhlova, M.D., Skryabina, M.N. and Fedyanin, A.A., Cellular viscoelasticity probed by active rheology in optical tweezers. Journal of Biomedical Optics, 2012. 17(10): p. 1-4.