本研究探討葡萄糖氧化酵素(GOx)由水溶液中吸附至氣/液界面的吸附行為，且分析GOx的吸附行為對固定酵素薄膜感測特性的影響。藉由GOx的表面壓-吸附時間曲線(π-t)、表面壓-每分子佔據面積等溫線(π-A)、遲滯曲線及布魯斯特角顯微鏡(BAM)，了解GOx的吸附行為及其與Langmuir單分子層的交互作用。以原子力顯微鏡(AFM)觀察GOx LB膜表面形態。GOx二級結構及GOx含量分別由圓二色光譜儀(CD)及紅外線光譜儀(FTIR)量測。葡萄糖感測器特性分析則採用安培法進行討論。
GOx由溶液吸附至氣/液界面，根據其表面壓變化可以分成二個階段。於第一吸附階段，GOx以單分子膜形式存在於氣/液界面上，由於界面-分子間作用力，GOx構形以β-sheet結構為主。持續吸附至界面的GOx促使第二吸附階段的發生，原先吸附於界面的GOx被推擠出氣/液界面，形成多層GOx分子膜，引起GOx構形轉換成α-helix結構。
由葡萄糖感測實驗結果得知，α-helix構形的GOx具有較高的電化學活性，藉由硬酯胺(ODA)單分子膜為模板吸附GOx，除了可以增加GOx的吸附量，利用分子之間的靜電吸引力，GO分子能分布均勻地併入ODA單分子膜，避免GOx聚集堆疊的行為而降低酵素催化能力。具有緊密均相的固定化酵素薄膜也能藉由阻隔棒的壓縮控制來製備。利用逐層沉積於白金電極基板上修飾緊密規則排列的金奈米粒子分子膜，及GOx/金奈米粒子分子膜，可製備一高靈敏度(0.521 μA•cm-2•mM-1)及短應答時間(12 s)之生物感測器。

The adsorption behavior of glucose oxidase (GOx) from aqueous solution to the air/liquid interface was studied. The GOx monolayer at the air/liquid was transferred on the surstrate for application to glucose sensor. With the variation of surface pressure in the adsorption stage (π-t), pressure-area (π-A) isotherms, hysteresis curves, and the Brewster angle microscopy (BAM), the GOx adsorption and the interaction between GOx and Langmuir monolayer were elucidated. The morphology of GOx LB films was also examined by the observation of atomic force microscopy (AFM). The GOx secondary structure and the GOx amount were examined by a circular dichroism (CD) spectroscopy and Fourier transform infrared spectroscopy (FTIR). Amperometric measurements were made for determining the performance of assembled glucose sensors.
The adsorption of GOx from aqueous solution to an air/liquid interface can be classified into two stages according to the surface pressure variation. In the first adsorption stage, GOx adsorb as a single-molecular layer, extended by the interface-molecule interaction, and organize as a mainly β-sheet conformation. The second adsorption stage is initiated by further adsorption of GOx into the interface, pushing up the pre-adsorbed proteins away from the interface, forming GOx multilayer, and inducing a conformational transition into α-helix.
The glucose sensing experiments demonstrate that GOx with α-helix conformation has a much higher electroacivity than the β-sheet. The electrostatic attractive interaction between octadecylamine (ODA) and GOx not only enchance the amount of adsorbed GOx, but also promote the uniformity of ODA and GOx to avoid the reduction in GOx catalytic ability. It is possible to prepare an immobilized enzyme film with condensed and homogeneous phase by surface-film compression. Overall, the {AuNPs-1/GOxAuNPs-2} sensor prepared by layer-by-layer deposition with a close-packed AuNP monolayer and the GOx/AuNPs monolayer exhibited the highest current sensitivity (0.521 μA•cm-2•mM-1) and the fastest response time (within 12 s).

1. Ellis, R. J.; Pinheiro, T. J. T. Nature 2002, 416, 483.
2. Dobson, C. M. Nature 2002, 418, 729.
3. Dobson, C. M. Nature 2003, 426, 884.
4. Kim, P. S.; Baldwin, R. L. Annu. Rev. Biochem. 1990, 59, 631.
5. Radford, S. E.; Dobson, C. M. Cell 1999, 97, 291.
6. Sawaya, M. R.; Sambashivan, S.; Nelson, R.; Ivanova, M. I.; Sievers, S. A.; Apostol, M. I.; Thompson, M. J.; Balbirnie, M.; Wiltzius, J. J. W.; McFarlane, H. T.; Madsen, A. O.; Riekel, C.; Eisenberg, D. Nature 2007, 447, 453.
7. Botello, E.; Harris, N. C.; Sargent, J.; Chen, W.-H.; Lin, K.-J.; Kiang, C.-H. J. Phys. Chem. B 2009, 113, 10845.
8. Maekawa, H.; Toniolo, C.; Broxterman, Q. B.; Ge, N.-H. J. Phys. Chem. B 2007, 111, 3222.
9. Polsky, R.; Harper, J. C.; Dirk, S. M.; Arango, D. C.; Wheeler, D. R.; Brozik, S. M. Langmuir 2007, 23, 364.
10. Puleo, D. A.; Nanci, A. Biomaterials 1999, 20, 2311.
11. Chaki, N. K.; Vijayamohanan, K. Biosens. Bioelectron. 2002, 17, 1.
12. Agnihotri, A.; Siedlecki, C. A. Langmuir 2004, 20, 8846.
13. Loladze,V.V.; Ibarra-Molero, B.; Sanchez-Ruiz, J.M.;Makhatadze, G. I. Biochemistry 1999, 38, 16419.
14. Spector, S.; Wang, M.; Carp, S. A.; Robblee, J.; Hendsch, Z. S.; Fairman, R.; Tidor, B.; Raleigh, D. P. Biochemistry 2000, 39, 872.
15. Ahmad, A.; Akhtar, M. S.; Bhakuni, V. Biochemistry 2001, 40, 1945.
16. Guiseppi-Elie, A.; Choi, S. H.; Geckeler, K. E. J. Mol. Catal. B-Enzym. 2009, 58, 118.
17. Zhao, H. Z.; Sun, J. J.; Song, J.; Yang, Q. Z. Carbon 2010, 48, 1508.
18. Wang, Q.; Xu, W.; Wu, P.; Zhang, H.; Cai, C.; Zhao, B. J. Phys. Chem. B 2010, 114, 12754.
19. Liu, Y.; Wang, M. K.; Zhao, F.; Xu, Z. A.; Dong, S. J. Biosens. Bioelectron. 2005, 21, 984.
20. Fears, K. P.; Sivaraman, B.; Powell, G. L.; Wu, Y.; Latour, R. A. Langmuir 2009, 25, 9319.
21. Shao, Q.; Wu, P.; Gu, P. A.; Xu, X. Q.; Zhang, H.; Cai, C. X. J. Phys. Chem. B 2011, 115, 8627.
22. Lechevalier, V.; Croguennec, T.; Pezennec, S.; Guerin-Dubiard, C.; Pasco, M.; Nau, F. J. Agr. Food Chem. 2003, 51, 6354.
23. Lad, M. D.; Birembaut, F.; Matthew, J. M.; Frazier, R. A.; Green, R. J. Phys. Chem. Chem. Phys. 2006, 8, 2179.
24. Gaines, G. L. Jr., “Insoluble Mnonlayers at Liquid-Gas Interface,” Wiley Press, Chapter 6, 1966.
25. Gaines, G.L. Thin Solid Films 1983, 99, R9-R13.
26. Myers, D. “Surface, Interfaces, and Colloids: Priciples and Applications,” VCH, Chapter 8, 1999.
27. Roberts, G., “Langmuir-Blodgett Films,” Plenum, 1990.
28. Neuman, R.D. J. Colloid Interface Sci. 1975, 53, 161.
29. Xing, W.; Shan, Y.; Guo, D.; Lu, T.; Xi, S. Corrosion 1995, 51, 45.
30. Leonard, M.; Morelis, R. M.; Coulet, P. R. Thin Solid Films 1995, 260, 227.
31. Angelova, A.; Penacorada, F.; Stiller, B.; Zetzsche, T.; Ionov, R. Kamusewitz, H.; Brehmer, L. J. Phys. Chem. 1994, 98, 6790.
32. Georgiou, G.; Bernardez-Clerk, E. D. American Chemical Society 1991, 1.
33. Lehninger, A. L.; Nelson, D. L.; Cox, M. M. “Principles of Biochemistry,” Second Edition, 1993.
34. Nyström, M.; Aimar, P.; Luque, S.; Kulovaara, M. Colloids and Surfaces A: Physicochem. Eng. Aspects 1998, 138, 185.
35. Rothwarf, D. M.; Scheraga, H. A. Biochemistry 1996, 35, 13797.
36. Hecht, H. J.; Schomburg, D. Biosensors and Bioelectronics 1993, 197
37. Graham, D. E.; Phillips, M. C.; Smith A. L “Theory and Practice of Emulsion Technology,” Academic Press, 75-98, 1974.
38. Graham, D. E.; Phillips, M. C. J. Colloid Interface Sci. 1979, 70, 403.
39. Rosilio, V.; Boissonnade, M. M.; Zhang, J. Y.; Jiang, L.; Baszkin, A. Langmuir 1997, 13, 4669.
40. Hunter, J. R.; Kilpatrick, P. K.; Carbonell, R. G. Journal of Colloid and Interface Science 1990, 137, 462.
41. Douillard, R.; Lefebvre, J. Journal of Colloid and Interface Science 1990, 139, 488.
42. Macritchie, F. Colloid Surf. A-Physicochem. Eng. Asp. 1993, 76, 159.
43. Tornberg, J. M, J. Colloid Interface Sci. 1978, 64, 391-398.
44. Xu, S.; Damodaran, S. Langmuir 1994, 10, 472.
45. Ward, A. F. M.; Tordaї, L. J. Chem. Phys. 1946, 14, 453.
46. Gazová, Z.; Antalík, M.; Bágel’ová, J.; Tomori, Z. Biochimica et Biophysica Acta 1999, 1432, 82.
47. Suttiprasit, P.; Krisdhasima, V.; McGuire, J. J. Colloid Interface Sci. 1992, 154, 316.
48. Graham, D. E.; Phillips, M. C. J. Colloid Interface Sci. 1979, 70, 415.
49. Graham, D. E.; Phillips, M. C. J. Colloid Interface Sci. 1979, 70, 427.
50. Macritchie, F.; Alexander, A. E. J. Colloid Interface Sci. 1963, 18, 464.
51. Xu, S.; Damodaran, S. Langmuir 1992, 8, 2021.
52. Xu, H.;Schlenoff, J. B. Langmuir 1994, 10, 241.
53. Li, J.; Rosilio, V.; Boissonnade, M. M.; Baszkin, A. Colloids Surf B Biointerfaces 2003, 29, 13.
54. Chovelon, J. M.; Wan, K.; Jaffrezic-Renault, N. Langmuir 2000, 16, 6223.
55. Yin, F.; Shin, H. K.; Kwon, Y. S. Thin Solid Films 2006, 499, 1.
56. Zaitsev, S. Y. Colloid Surf. A-Physicochem. Eng. Asp. 1993, 75, 211.
57. Lee, Y. L.; Lin, J. Y.; Lee, S. Langmuir 2007, 23, 2042.
58. Zhang, J.; Rosilio, V.; Goldmann, M.; Boissonnade, M. M.; Baszkin, A. Langmuir 2000, 16, 1226.
59. Kamilya, T.; Pal, P.; Talapatra, G. B. Journal of Colloid and Interface Science 2007, 315, 464.
60. Rosilio, V.; Boissonnade, M. M.; Zhang, J. Y.; Jiang, L.; Baszkin, A. Langmuir 1997, 13, 4669.
61. Li, J. R.; Rosilio, V.; Boissonnade, M. M.; Baszkin, A. Colloids and Surfaces B-Biointerfaces 2003, 29, 13.
62. Dong, S. J.; Wang, B. X.; Liu, B. F. Biosens. Bioelectron 1992, 7, 215.
63. Ghica, M. E.; Brett, C. M. A. Anal. Lett. 2005, 38, 907.
64. Nien, P. C.; Tung, T. S.; Ho, K. C. Electroanalysis, 18, 1408.
65. Tumer, A. P.; Chen, B.; Piletsky, S. Clin. Chem. 1999, 45, 1596.
66. Yang, M.; Yang, Y.; Yang, H.; Shen, G.; Yu,R. Biomaterials 2006, 27, 246.
67. Zhang, S.; Wang, N.; Yu, H.; Niu, Y.; Sun, C. Bioelectrochemistry 2005, 67, 15.
68. Wang, C.; Chen, S.; Xiang, Y.; Li, W.; Zhong, X.; Che, X.; Li, J. Journal of Molecular Catalysis B: Enzymatic 2011, 69, 1.
69. Pandey, P.; Singh, S. P.; Arya, S. K.; Gupta, V.; Datta, M.; Singh, S.; Malhotra, B. D. Langmuir 2007, 23, 3333.
70. Matharu, Z.; Pandey, P.; Pandey, M. K.; Gupta, V.; Malhotra, B. D. Electroanalysis 2009, 21, 1587.
71. Lee, J. M. “Biochemical Engineering”, Prentice Hall, 1992.
72. Shuler, M.L.; Kargi, F. “Bioprocess Engineering : Basic Concepts”, Prentice Hall, 2nd ed., 2002.
73. Li, J. R.; Cai, M.; Chen, T. F.; Jiang, L. Thin Solid Films 1989,180, 205.
74. Moriizumi, T. Thin Solid Films 1988,160, 413
75. Arisawa, S.; Arise, T.; Yamamoto, R. Thin Solid Films 1992, 209, 259.
76. Pal, P.; Nandi, D.; Misra T. N. Thin Solid Films 1994, 239, 138.
77. Zaitsev, S. Y. Sens Actuators B 1995, 24, 177.
78. Choi, J. W.; Min, J.; Jung, J.W.; Rhee, H. W.; Lee, W. H. Thin Solid Films 1998, 327, 329.
79. Yasuzawa, M.; Hashimoto, M.; Fujii, S.; Kunugi, A.; Nakaya, T. Sens Actuators B 2000, 65, 241.
80. Sriyudthsak, M.; Yamagishi, H.; Moriizumi, T. Thin Solid Films 1988, 160, 463.
81. Miyauchi, S.; Arisawa, S.; Arise, T.; Yamamoto, R. Thin Solid Films 1989, 80, 293.
82. Fiol, C.; Valleton, J. M.; Delpire, N.; Barbey, G.; Barraud, A.; Ruaudel -Texier A. Thin Solid Films 1992, 210, 489.
83. Fiol, C.; Alexandre, S.; Delpire, N.; Valleton, J. M.; Paris, E. Thin Solid Films 1992, 215, 88.
84. Zhang, A.; Hou, Y.; Jaffrezic-Renault, N.; Wan, J.; Soldatkin, A.; Chovelon, J. M. Bioelectrochemistry 2002, 56,157.
85. Heckl, W. M.; Zaba, B. N.; Möhwald, H. Biochim Biophys Acta 1987, 903, 166.
86. Caseli, L.; Furriel, R. P. M.; de Andrade, J. F.; Leone, F. A.; Zaniquelli M. E. D. J. Colloid Interface Sci 2004, 275, 123.
87. Singhal, R.; Gambhir, A.; Pandey, M. K.; Annapoorni, S.; Malhotra, B. D. Biosens Bioelectron 2002,17, 697.
88. Singhal, R.; Takashima, W.; Kaneto, K.; Samanta, S. B.; Annapoorni, S.; Malhotra, B. D. Sens Actuators B 2002, 86, 42.
89. Sharma, S. K.; Singhal, R.; Malhotra B. D.; Sehgal, N.; Kumar, A. Electrochim Acta 2004, 49, 2479.
90. Ohnuki, H.; Saiki, T.; Kusakari, A.; Endo, H.; Ichihara, M.; Izumi, M. Langmuir 2007, 23, 4675.
91. Ohnuki, H.; Saiki, T.; Kusakari, A.; Ichihara, M.; Izumi, M. Thin Solid Films 2008, 516, 8860.