Please use this identifier to cite or link to this item: http://hdl.handle.net/11455/3044
標題: 以固定化金屬親和吸附薄膜固定開關蛋白作為開發生物感測器之研究
Immobilization of Protein Switches on Affinity Membrane for the Development of Biosensors
作者: 劉育銘
Liu, Yu-Ming
關鍵字: 固定化金屬親合吸附薄膜;immobilized metal ion affinity membrane;開關蛋白;再生纖維素膜;生物感測器;protein switches;regenerated cellulose membrane;biosensors
出版社: 化學工程學系所
引用: 1. Lei, Y., W. Chen, and A. Mulchandani, Microbial biosensors. Anal Chim Acta, 2006. 568(1-2): p. 200-10. 2. Jose’ I. Reyes De Corcuera, R.P.C., Biosensors, in Encyclopedia of Agricultural, Food, and Biological Engineering, 2003, Marcel Dekker, Inc: New York. 3. Schubert, F.W., U.; Scheller, F.W.; Mu‥ller, H.G., Artificially Coupled Reactions with Immobilized Enzymes: Biological Analogs and Technical Consequences. Bioinstrumentation and Biosensors1991, New York: Marcel Dekker, Inc. 19. 4. Clark, L.C. and C. Lyons, ELECTRODE SYSTEMS FOR CONTINUOUS MONITORING IN CARDIOVASCULAR SURGERY. Annals of the New York Academy of Sciences, 1962. 102(1): p. 29-45. 5. 蘇宏基, 化學生物感測器, 2011, 國立東華大學: 花蓮. 6. Cao, L., Immobilised enzymes: science or art? Current Opinion in Chemical Biology, 2005. 9(2): p. 217-226. 7. Tischer, W. and F. Wedekind, Immobilized Enzymes: Methods and Applications Biocatalysis - From Discovery to Application, W.-D. Fessner, et al., Editors. 1999, Springer Berlin / Heidelberg. p. 95-126. 8. Buchholz, K., Reaction engineering parameters for immobilized biocatalysts Reaction Engineering, 1982, Springer Berlin / Heidelberg. p. 39-71. 9. Sheldon, R.A., Enzyme Immobilization: The Quest for Optimum Performance. Advanced Synthesis & Catalysis, 2007. 349(8-9): p. 1289-1307. 10. Martinek, K., et al., The principles of enzyme stabilization I. Increase in thermostability of enzymes covalently bound to a complementary surface of a polymer support in a multipoint fashion. Biochimica et Biophysica Acta(BBA) - Enzymology, 1977. 485(1): p. 1-12. 11. Cao, L., L.v. Langen, and R.A. Sheldon, Immobilised enzymes: carrier-bound or carrier-free? Current Opinion in Biotechnology, 2003. 14(4): p. 387-394. 12. Tischer, W. andV. Kasche, Immobilized enzymes: crystals or carriers? Trends in Biotechnology, 1999. 17(8): p. 326-335. 13. Avnir, D., et al., Enzymes and Other Proteins Entrapped in Sol-Gel Materials. Chemistry of Materials, 1994. 6(10): p. 1605-1614. 14. Hartmann, M. and D. Jung, Biocatalysis with enzymes immobilized on mesoporous hosts: the status quo and future trends. Journal of Materials Chemistry, 2010. 20(5): p. 844-857. 15. Chaga, G.S., Twenty-five years of immobilized metal ion affinity chromatography: past, present and future. Journal of Biochemical and Biophysical Methods, 2001. 49(1–3): p. 313-334. 16. Sulkowski, E., Purification of proteins by IMAC. Trends in Biotechnology, 1985. 3(1): p. 1-7. 17. Ueda, E.K.M., P.W. Gout, and L. Morganti, Current and prospective applications of metal ion–protein binding. Journal of Chromatography A, 2003. 988(1): p. 1-23. 18. Gaberc-Porekar,V. andV. Menart, Perspectives of immobilized-metal affinity chromatography. Journal of Biochemical and Biophysical Methods, 2001. 49(1–3): p. 335-360. 19. Suen, S.-Y., Y.-C. Liu, and C.-S. Chang, Exploiting immobilized metal affinity membranes for the isolation or purification of therapeutically relevant species. Journal of Chromatography B, 2003. 797(1–2): p. 305-319. 20. Zhou, F.L., et al., Coated silica supports for high-performance affinity chromatography of proteins. Journal of Chromatography A, 1989. 476(0): p. 195-203. 21. Xi, F. and J. Wu, Macroporous chitosan layer coated on non-porous silica gel as a support for metal chelate affinity chromatographic adsorbent. Journal of Chromatography A, 2004. 1057(1–2): p. 41-47. 22. Zou, H., Q. Luo, and D. Zhou, Affinity membrane chromatography for the analysis and purification of proteins. Journal of Biochemical and Biophysical Methods, 2001. 49(1–3): p. 199-240. 23. Gutierrez, R., E.M. Martin delValle, and M.A. Galan, Immobilized Metal‐Ion Affinity Chromatography: Status and Trends. Separation & Purification Reviews, 2007. 36(1): p. 71-111. 24. Arıca, M.Y., G. Bayramoǧlu, and N. Bıcak, Characterisation of tyrosinase immobilised onto spacer-arm attached glycidyl methacrylate-based reactive microbeads. Process Biochemistry, 2004. 39(12): p. 2007-2017. 25. Denizli, A., S. Şenel, and M.Y. Arıca, Cibacron Blue F3GA and Cu(II) derived poly(2-hydroxyethylmethacrylate) membranes for lysozyme adsorption. Colloids and Surfaces B: Biointerfaces, 1998. 11(3): p. 113-122. 26. Pearson, R.G., Hard and soft acids and bases. Benchmark papers in inorganic chemistry1973, Stroudsburg, Pa.,: Dowden. xiii, 480 p. 27. Smith, M.C., T.C. Furman, and C. Pidgeon, Immobilized iminodiacetic acid metal peptide complexes. Identification of chelating peptide purification handles for recombinant proteins. Inorganic Chemistry, 1987. 26(12): p. 1965-1969. 28. Sharma, S. and G.P. Agarwal, Interactions of Proteins with Immobilized Metal Ions: A Comparative Analysis UsingVarious Isotherm Models. Analytical Biochemistry, 2001. 288(2): p. 126-140. 29. Langmuir., I., The adsorption of gases on plane surfaces of glass, mica and platinum. Journal of the American Chemical Society, 1918. 40(9): p. 1361-1403. 30. Gunn, D.J., Process analysis by statistical methods, D. M. Himmelblau, Wiley, New York, 1968. No. of pages: 460. Price: £8‧75; $19.95. International Journal for Numerical Methods in Engineering, 1971. 3(1): p. 151-151. 31. Anderson-Cook, C.M., C.M. Borror, and D.C. Montgomery, Response surface design evaluation and comparison. Journal of Statistical Planning and Inference, 2009. 139(2): p. 629-641. 32. Gunaraj,V. and N. Murugan, Application of response surface methodology for predicting weld bead quality in submerged arc welding of pipes. Journal of Materials Processing Technology, 1999. 88(1–3): p. 266-275. 33. Box, G.E.P., Hunter, W. and Hunter, J.S., Statistics for experimenters1978, New York: John Wiley and Sons. 34. Thompson, D., RESPONSE SURFACE EXPERIMENTATION1. Journal of Food Processing and Preservation, 1982. 6(3): p. 155-188. 35. Bezerra, M.A., et al., Response surface methodology(RSM) as a tool for optimization in analytical chemistry. Talanta, 2008. 76(5): p. 965-977. 36. Guntas, G., S.F. Mitchell, and M. Ostermeier, A molecular switch created by inVitro recombination of nonhomologous genes. Chem Biol, 2004. 11(11): p. 1483-7. 37. Liu, Y.-C., et al., Effects of spacer arm on penicillin G acylase purification using immobilized metal affinity membranes. Journal of Membrane Science, 2005. 251(1–2): p. 201-207. 38. Bayramoğlu, G., B. Kaya, and M. Yakup Arıca, Immobilization of Candida rugosa lipase onto spacer-arm attached poly(GMA-HEMA-EGDMA) microspheres. Food Chemistry, 2005. 92(2): p. 261-268. 39. Liang, J., et al., Ligand binding and allostery can emerge simultaneously. Protein Science, 2007. 16(5): p. 929-937. 40. Wu, C.-Y., et al., Analysis of protein adsorption on regenerated cellulose-based immobilized copper ion affinity membranes. Journal of Chromatography A, 2003. 996(1–2): p. 53-70.
摘要: 
開關蛋白(protein switches)具有辨認特定物質,同時即時隨著特定物質濃度不同而產生不同酵素活性的特質;因此開關蛋白適合做為生物感測器中生物感測元件的部分。為了應用在生物感測器上,本研究中將探討以固定化金屬親和方式將開關蛋白(RG13)固定在再生纖維素膜上的特性,此種開關蛋白會因為環境中麥芽醣濃度的不同產生不同的酵素活性。
先以反應曲面法(response surface methodology,RSM)找出改質再生纖維素膜所使用活化劑的最適化條件,再選用亞胺乙二酸(iminodiacetic acid,IDA)做為螯合劑,最後鍵結上金屬離子。接著以Langmuir等溫吸附方程式分析RG13固定於再生纖維素膜上的吸附情形,並與固定於含次氨基三乙酸(nitrilotriacetic acid,NTA) QIAGEN商業膠體上RG13的特性做比較。發現固定後RG13的比活性均下降,且固定於膜上的RG13對麥芽醣濃度敏感性和比活性的增幅程度比自由態RG13降低許多。經實驗證實,銅離子與鎳離子對自由態RG13比活性會有抑制的效果,因此推測是有少許的金屬離子從螯合劑上脫附造成固定RG13比活性下降。而固定於膜上的RG13對麥芽醣濃度敏感性和比活性的增幅程度下降的情形則可能是因為基材形狀所產生的擴散限制而造成。以IDA-Ni2+膜固定的RG13雖會受到金屬離子影響但仍保有對麥芽醣的專一性;而在重複使用8次循環後,雖然相對比活性會隨著使用次數而逐漸下降,但仍保有對不同麥芽醣濃度產生不同大小的比活性的特性。因此在選用以固定化金屬親和方式固定開關蛋白做為未來開發生物感測器時,選擇適當的螯合劑及基材種類以降低金屬離子脫附和擴散限制的影響是很重要的。

Protein switches can identify specific chemical substrates, and concurrently express different activities according to the concentration of substrates. Therefore, it is suitable for the biological sensing element in the application of biosensors. In this study, we immobilized RG13, whose activity depends on the concentration of maltose, on the immobilized metal ion affinity regenerated cellulose membrane(RC membrane) for applications in biosensors.
First, active agent’s condition for membrane modification is optimized by RSM(response surface methodology, RSM). Then, iminodiacetic acid(IDA) is selected as the chelating ligand to capture to chelate metal ions. Finally, the adsorption phenomenon is analyzed by Langmuir isotherm, and then the performances of RG13 on membrane and on QIAGEN nitrilotriacetic acid(NTA) agarose were compared. The result shows that the specific activity of immobilized RG13 is lower than free RG13. And, the sensitivity and response of specific activity to various maltose concentration are also lower than the free RG13. According to the result, the specific activity of free RG13 might be inhibited by Cu(II) and Ni(II), therefore, in which was probably due to the leakage of metal ion. And, the lower sensitivity and response of specific activity of immobilized RG13 on membrane possibly comes from the diffusion limitation caused the form of matrix. Although the specific activity of immobilized RG13 on IDA-NI2+ membrane is affected by the metal ion, it still shows selectivity for maltose. In the period of repetitive 8 operations, although its relative specific activity declines as increasing used times, it still showed different specific activity depend on maltose concentration. To immobilize protein switches on immobilized metal affinity adsorbents for the development of biosensors, it is important to choose appropriate chelating ligand and matrix materials to reduce metal ion leakage and diffusion limitation.
URI: http://hdl.handle.net/11455/3044
其他識別: U0005-0211201215342200
Appears in Collections:化學工程學系所

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