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。

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.

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.
3. Wang, M.D., Yin, H., Landick, R., Gelles, J. and Block, S.M., Stretching DNA with optical tweezers. Biophysical Journal, 1997. 72(3): p. 1335-1346.
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.
6. Gittes, F., Schnurr, B., Olmsted, P.D., MacKintosh, F.C. and Schmidt, C.F., Microscopic viscoelasticity: Shear moduli of soft materials determined from thermal fluctuations. Physical Review Letters, 1997. 79(17): p. 3286-3289.
7. Pesce, G., Sasso, A., Fusco, S., Borzacchiello, A. and Netti, P., Optical Tweezers as a tool for microrheology of simplex and complex fluids. SPIE Proceedings, 2004. 5514: p. 487-493.
8. Mason, T.G. and Weitz, D.A., Optical measurements of frequency-dependent linear viscoelastic moduli of complex fluids. Physical Review Letters, 1995. 74(7): p. 1250-1253.
9. Yao, A., Tassieri, M., Padgett, M. and Cooper, J., Microrheology with optical tweezers. Lab on a Chip, 2009. 9(17): p. 2568-2575.
10. Chvapil, M., Collagen sponge: theory and practice of medical applications. Journal of Biomedical Materials Research, 1977. 11(5): p. 721-741.
11. Leikina, E., Mertts, M.V., Kuznetsova, N. and Leikin, S., Type I collagen is thermally unstable at body temperature. Proceedings of the National Academy of sciences of the United States of America, 2002. 99(3): p. 1314–1318.
12. Olde Damink, L.H.H., Dijkstra, P.J., Van Luyn, M.J.A., Van Wachem, P.B., Nieuwenhuis, P. and Feijen, J., Influence of ethylene oxide gas treatment on the in vitro degradation behavior of dermal sheep collagen. Journal of Biomedical Materials Ressearch, 1995. 29(2): p.149-155.
13. Kuo, S.M., Tsai, S.W., Huang, L.H. and Wang, Y.J., Plasma-modified nylon meshes as supports for cell culture. Artificial cells, Blood substitutes and Biotechnology, 1997. 25(6): p.551-562.
14. Hayashi, T. and Nagai, Y., Effect of pH on the stability of collagen molecule in solution. The Journal of Biochemistry, 1973. 73(5): p. 999-1006.
15. Li, J.and Li, G., The thermal behavior of collagen in solution: effect of glycerol and 2-propanol. International Journal of Biological Macromolecules, 2011. 48(2): p.364-368.
16. http://www.anton-paar.com/ca-en/products/details/mcr-rheometer-series/
17. 溫偉源、陸駿逸“微流變學”物理雙月刊2005年(廿七卷三期) p. 479-482.
18. Brau, R.R., Ferrer, J.M., Lee, H., Castro, C.E., Tam, B.K., Tarsa, P.B., Matsudaira, P., Boyce, M.C., Kamm, R.D. and Lang, M.J., Passive and active microrheology with optical tweezers. Journal of Optics A: Pure and Applied Optics, 2007. 9(8): p. 103-112.
19. Latinovic, O., Hough, L.A. and Ou-Yang, D., Structural and micromechanical characterization of type I collagen gels. Journal of Biomechanics, 2010. 43(3): p. 500-505.
20. Pesce, G., Rusciano, G. and Sasso, A., Blinking Optical Tweezers for microrheology measurements of weak elasticity complex fluids. Optics Express, 2010. 18(3): p. 2116-2126.
21. Pesce, G., Luca, A.C.De., Rusciano, G., Netti, P.A., Fusco, S. and Sasso, A., Microrheology of complex fluids using optical tweezers: a comparison with macrorheological measurements. Journal of Optics A: Pure and Applied Optics, 2009. 11(3): p. 1-11.
22. Shayegan, M. and Forde, N.R., Microrheological Characterization of Collagen System: From Molecular Solutions to Fibrillar Gels. PLOS ONE, 2013. 8(8): p. 1-12.
23. Li, Y., Li, Y., Du, Z. and Li, G., Comparison of dynamic denaturation temperature of collagen with its static denaturation temperature and the configuration characteristics in collagen denaturation processes. Thermochimica Acta, 2008. 469(1-2): p. 71-76.
24. Lai, G., Li, Y. and Li, G., Effect of concentration and temperature on the rheological behavior of collagen solution. International Journal of Biological Macromolecules, 2008. 42(3): p. 285-291.
25. Shoulders, M.D. and Raines, R.T., Collagen Structure and Stability. Annual Review of Biochemistry, 2009. 78: p. 929-958.
26. Shayegan, M., Rezaei, N., Lam, N.H., Altindal, T., Wieczorek, A. and Forde, N.R., Probing multiscale mechanics of collagen with optical tweezers. SPIE Proceedings, 2013. 8810: p. 1-10.
27. Mayne, J. and Robinson, J.J., Comparative Analysis of the Structure and Thermal Stability of Sea Urchin Peristome and Rat Tail Tendon Collagen. Journal of Cellular Biochemistry, 2002. 84(3): p. 567-574.
28. Ashkin, A., Dziedzic, J.M., Bjorkholm, J.E. and Chu, S., Observation of a single-beam gradient force optical trap for dielectric particles. Optics Letters, 1986. 11(5): p. 288-290.
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.
30. Grier, D.G., A revolution in optical manipulation. Nature, 2003. 424: p. 810–816.
31. Ashkin, A., Force of a Single-Beam Gradient Laser Trap on a Dielectric Sphere in the Ray Optics Regime. Biophysical Journal, 1992. 61(2): p. 569-582.
32. Berg-Sorensen, K. and Flyvbjerg, H., Power spectrum analysis for optical tweezers. Review of Scientific Instruments, 2004. 75(3): p. 594-612.
33. Neuman, K.C. and Block, S.M., Optical trapping. Review of Scientific Instruments, 2004. 75(9): p. 2787-2809.
34. Kubo, R., The fluctuation-dissipation theorem. Reports on Progress in Physics, 1966. 25: p. 255-284.
35. 莊迺民“利用自組裝雷射光鉗系統量測膠原蛋白熱降解之黏性變化”NTHU, 2013年.
36. Vermeulen, K. C., Wuite, G.J.L., Stienen, G.J.M.and Schmidt, C.F., Optical trap stiffness in the presence and absence of spherical aberrations. Applied Optics, 2006. 45(8): p. 1812-1819.
37. Korson, L., Drost-Hansen, W. and Millero, F. J., Viscosity of Water at Various Temperatures. The Journal of Physical Chemistry, 1969. 73(1): p. 34-39.
38. Mu, C., Li, D., Lin, W., Ding, Y. and Zhang, G., Temperature induced denaturation of collagen in acidic solution. Bipolymers, 2007. 86(4): p.282-287.
39. Jiang, F., Horber, H., Howerd, J. and Muller, D.J., Assembly of collagen into microribbons: effects of pH and electrolytes. Journal of Structural Biology, 2004. 148(3): p. 268-278.
40. Usha, R. and Ramasami, T., Effect of pH on Dimensional Stability of Rat Tail Tendon Collagen Fiber. Journal of Applied Polymer Science, 2000. 75(13): p. 1577-1584.
41. Li, Y., Asadi, A., Monroe, M.R. and Douglas, E.P., pH effects on collagen fibrillogenesis in vitro: Electrostatic interactions and phosphate binding. Materials Science and Engineering, 2009. 29(5): p. 1643-1649.
42. Jamilah, B. and Harvinder, K.G., Properties of gelatins from skins of fish-black tilapia (Oreochromis mossambicus) and red tilapia (Oreochromis nilotica). Food Chemistry, 2002. 77(1): p. 81-84.
43. Hayashi, T. and Nagai, Y., Factors Affecting the Interactions of Collagen Molecules as Observed by in Vitro Fibril Formation. Journal of Biochemistry, 1972. 72(3): p. 749-758.
44. Kuznetsova, N., Chi, S.L. and Leikin, S., Sugars and Polyols Inhibit Fibrillogenesis of Type I Collagen by Disrupting Hydrogen-Bonded Water Bridges between the Helices. Biochemistry, 1998. 37(34): p. 11888-11895.
45. Lien, Y.H., Stern, R., Fu, J.C. and Siegel, R.C., Inhibition of collagen fibril formation in vitro and subsequent cross-linking by glucose. Science, 1984. 25(4669): p. 1489-1491.
46. Rathi, A.N., Padmavathi, P. and Chandrakasan, G., Influence of Monosaccharides on the Fibrillogenesis of Type I Collagen. Biochemical Medicine and Metabolic Biology, 1989. 42(3): p. 209-215.
47. Schnider, S.L. and Kohn, R.R., Glucosylation of Human Collagen in Aging and Diabetes Mellitus. The Journal of Clinical Investigation, 1980. 66(5): p. 1179-1181.
48. Penkova, R., Goshev, I., Gorinstein, S. and Nedkov, P., Stability of Collagen During Denaturation. Journal of Protein Chemistry, 1999. 18(4): p. 397–401.
49. Usha, R. and Ramasami, T., Effect of Hydrogen-Bond-Breaking Reagent (Urea) on the Dimensional Stability of Rat Tail Tendon (RTT) Collagen Fiber. Journal of Applied Polymer Science, 2002. 84(5): p. 975–982.
50. Usha, R. and Ramasami, T., The effects of urea and n-propanol on collagen denaturation: using DSC, circular dicroism and viscosity. Thermochimica Acta, 2004. 409(2): p. 201–206.
51. Duan, L., Li, J., Li, C. and Li, G., Effects of NaCl on the rheological behavior of collagen solution. Korea-Australia Rheology Journal, 2013. 25(3): p. 137-144.
52. Komsa-Penkova, R., Koynova, R., Kostov, G. and Tenchov, B.G., Thermal stability of calf skin collagen type I in salt solutions. Biochimica et Biophysica Acta, 1996. 1297(2): p. 171-181.
53. Paul Attfield, J., Sleight, A.W. and Cheetham, A.K., Structure determination of α-CrPO4 from powder synchrotron X-ray data. Nature, 1986. 322(14): p. 620-622.
54. Antonov, Y.A. and Wolf, B.A., Calorimetric and Structural Investigation of the Interaction between Bovine Serum Albumin and High Molecular Weight Dextran in Water. Biomacromolecules, 2005. 6(6): p. 2980-2989.
55. Yamada, K., Sato, J., Oku, H. and Katakai, R., Conformation of the transmembrane domains in peripheral myelin protein 22. Part 1. Solution-phase synthesis and circular dichroism study of protected 17-residue partial peptides in the first putative transmembrane domain. The Journal of Peptide Research, 2003. 62(2): p. 78-87.
56. Mackenzie, R.C., Nomenclature in thermal analysis, part IV. Thermochimica Acta, 1979. 28(1): p. 1-6.
57. 曾楷涵“利用外差式光學偏光儀研究三氟乙醇與甘油對膠原蛋白熱變性之影響”NTHU, 2013年.
58. Lee, C.H., Singla, A. and Lee, Y., Biomedical applications of collagen. International Journal of Pharmaceutics, 2001. 221(1-2): p. 1-22.
59. Friess, W., Collagen-biomaterial for drug delivery. European Journal of Pharmaceutics and Biopharmaceutics, 1998. 45(2): p. 113-136.