研究生: |
簡筱芳 Hsiao-Fang Chien |
---|---|
論文名稱: |
石英壓電晶體感測器應用於有機化合物與DNA作用力的研究 The Interaction between Organic Compounds and DNA Studied by Quartz Crystal Microbalance |
指導教授: |
施正雄
Shih, Jeng-Shong |
學位類別: |
碩士 Master |
系所名稱: |
化學系 Department of Chemistry |
論文出版年: | 2001 |
畢業學年度: | 89 |
語文別: | 中文 |
論文頁數: | 137 |
中文關鍵詞: | 石英壓電晶體感測器 、DNA 、有機化合物 、作用力 |
英文關鍵詞: | QCM, DNA, organic compounds, interaction |
論文種類: | 學術論文 |
相關次數: | 點閱:264 下載:0 |
分享至: |
查詢本校圖書館目錄 查詢臺灣博碩士論文知識加值系統 勘誤回報 |
本實驗是利用自組式CPS (carboxypropyl disulfide)/EDAC (1-ethyl-3(3-dimethylaminopropyl) carbodimide)/NHS (N-hydroxy- succinimde)修飾的單層膜與雙股螺旋DNA共價鍵結固定在石英晶片的銀電極上,固定化DNA的石英壓電晶體感測器設計來作為DNA與有機化合物間作用力研究之用。
有機化合物吸附在DNA固定的石英晶片導致石英晶片上質量增加和石英壓電晶體感測器振盪頻率下降,有機分子脫附的研究用來決定DNA與有機分子間是化學吸附或物理吸附。研究發現DNA與有機酸、醛類、pyrrole、pyridine及多環芳香族碳氫化合物(PAH)間存在有化學吸附,反之DNA與醇類或酮類間只有物理吸附存在,固定化DNA石英壓電晶體對有機分子的頻率變化大小依序為:正己酸>正戊酸>正丁酸>丙酮酸>丙酸=pyridine>丙醛=pyrrole>戊二醛=乙醛>甲醛>乙酸>甲酸,而DNA每個核甘酸與有機分子反應的鍵結數大小依序為:正己酸>丙酮醛>正戊酸>正丁酸>丙醛>乙醛>丙酸>pyrrole>pyridine>甲醛>戊二醛>乙酸>甲酸,隨著有機化合物直鏈長度的增加,會增加DNA與有機化合物間反應的鍵結數,本研究亦研究單股和雙股DNA和有機分子作用,雙股DNA和有機分子作用力顯然比單股DNA大。
本研究同時也探討了溶劑、有機化合物的濃度、溫度、pH值和不同種類的DNA對固定化DNA石英壓電晶體感測器之感應頻率變化及DNA和有機分子作用力的影響,DNA與有機酸間反應所引起的頻率變化並不會受到溶劑變化的影響,相反的,DNA與醛類反應所引起的頻率變化則會受到溶劑變化的影響,頻率變化的大小依序為:丙醇>乙醇=丙酮>甲醇>>純水中,而有機化合物的濃度與頻率變化關係的曲線發現都趨向蘭穆爾式的飽和吸附,在pH效應研究中發現當溶液pH值的增加,會加速DNA與醛類間的反應速率,可能為鹼性溶液會催化DNA鹼基N-1或胺基的反應,而當pH值小於7時,頻率變化相差不大,隨著pH值(>7)增加,會增加DNA與醛類反應的頻率變化,在溫度效應研究中發現當反應溫度升高,會大大的降低DNA與醛類的反應時間,而最好的化學吸附溫度約在37°C左右,不同種類的雙股DNA與有機酸反應時,發現頻率變化相差不大,此可能由於各種DNA的鹼基組成類似。
利用固定化DNA石英壓電晶體感測器成功地即時研究DNA與有機化合物間作用力,不需要複雜的分離步驟就可以計算出化學鍵結之有機分子的含量及DNA每個核甘酸可與有機分子反應的鍵結數。
Double-stranded deoxyribonucleic acid (dsDNA) was covalently immobilized onto a self assembled CPS (carboxypropyl disulfide) / EDAC (1-ethyl-3 (3-dimethylaminopropyl) carbodimide)/ NHS (N-hydroxy-succinimde) modified sliver electrodes on a piezoelectric quartz crystal. A piezoelectric quartz crystal sensor based on immobilized dsDNA was set up to study the interaction between dsDNA and various organic molecules.
The adsorption of organic compounds onto DNA modified quartz crystal electrodes caused the increase in the mass of quartz crystal and resulted in the decrease in the oscillating frequency of the piezoelectric crystal sensor. The desorption study was also performed to determine whether the adsorption was chemical or physical. Among various organic molecules, organic acid, aldehyde, pyrrole, pyridine and PAH (Poly-Aromatic Hybrocarbons) such as naphthalene and pyrene seemed to exhibit the chemisorption on dsDNA, while the physical adsorption was found for alcohol or ketone. The frequency shifts of the dsDNA-immobilized piezoelectric crystal sensor for various organic molecules were in the order: n-caproic acid> n-varleric acid> n-butanoic acid> methylglyoxal> n-propionic acid» pyridine> propionldehyde» pyrrole> glutaraldehyde» acetaldehyde> formaldehyde> acetic acid> formic acid and the binding numbers of organic molecules per nucleotide of DNA were in the order: n-caproic acid> methylglyoxal > n-varleric acid> n-butanoic acid> propionaldehyde > acetaldehyde> n-propionic acid> pyrrole> pyridine> formaldehyde> glutaraldehyde> acetic acid> formic acid. Binding numbers of organic molecules per nucleotide of DNA obviously increased with the longer chain length of organic molecules. Comparison of binding abilities of single strand helix (eq. ss-STDNA) and double strand helix (eq. ds-STDNA) was also made. The frequency shift of dsDNA-immobilized piezoelectric crystal sensor was higher than that of ssDNA.
The effects of solvent, concentration of organic compounds, pH value, temperature, and different kinds of DNA on the frequency shifts of the dsDNA-immobilized piezoelectric crystal sensor were also investigated and discussed. Obviously, solvent showed no effect on the frequency shifts of the dsDNA-immobilized piezoelectric crystal sensor for organic acid, on the contrary, solvent exhibited quite significant effect for aldehyde. The frequency shifts of dsDNA-immobilized piezoelectric crystal sensor for aldehyde in various solvents were in the order: propanol>ethanol»acetone>methanol>>pure water. The curve shape of frequency shifts for concentration of organic compounds seemed to trend to be langmuir saturated adsorption. Once pH of solution raised, the reaction rate between DNA and aldehyde speed up, presumably due to the base-catalyzed N-1 or amino group reaction. The frequency shift was essentially pH independent < pH 7, but ³ pH 7, the frequency shift increased with increasing pH. Once temperature of solution raised, the reaction time decreased sharply, and an optimum frequency shift was formed at 37°C. Frequency shifts of various dsDNA-immobilized piezoelectric crystal sensor for organic acid are similar which may be attributed to the similar compositions for these dsDNA.
In conclusion, DNA-immobilized piezoelectric crystal sensor can be successfully applied for in-situ study of the interaction between organic compound and DNA without complicated isolation of reactants and adducts. The amount of organic molecules for chemical binding to DNA and the binding numbers of organic molecules per nucleotide of DNA can be obtained easily.
1. 三浦謹一郎,劉文正,DNA與遺傳訊息,1996,台北市編譯館
2. Blackburn, G. J.; Gait, M. J. Nucleic Acids in Chemistry and Biology. 1990, Oxford University press, Tokyo.
3. Nelson, D. L.; Cox, M. M. Lehninger Principles of Biochemistry. Ⅲ. 2000, Worth Press, New York.
4. Campbell, M. K. Biochemistry. 1995, Saunders College Press, New York.
5. 林能傑,去氧核糖核酸(DNA)與核糖核酸(RNA) <上>,台灣醫界,1995,38,601-604
6. Blackburn, G. J.; Gait, M. J. Nucleic Acids in Chemistry and Biology. Ⅱ. 1996, Oxford University press, Tokyo.
7. Waston, J. D.; Crick, F. H. C. A structure for deoxyribose nucleic acid. Nature, 1953, 171, 737-738
8. Saenger, W. Principles of nucleic acid structure. 1984. Springer-Verlag Press, New York.
9. Crothers, D. M.; Gartenberg, M. R.; Shrader, T. E. DNA bonding in protein-DNA complexes. Prog. Nucleic Acid. Res. Mol. Biol. 1992, 208, 118-145
10. Dickerson, R. E.; Drew, H. R.; Connor, B. N.; Wing, R. M.; Fratini, A. V.; Kapka, M. L. The anatomy of A-, B-, and Z-DNA. Science, 1982, 216, 475-485
11. Kochetkov, N. K.; Boudovskii, E. I.; Sverdlov, E. D.; Shibaev, V. N. Organic Chemistry of Nucleic Acids. 1972, Plenum Press, London and New York.
12. Cohn, W. E.; Doherty, D. G. The catalytic hydrogenation of pyrimidine nucleosides and nucleotides and the isolation of their ribose and respective ribose phosphates. J. Am. Chem. Soc. 1956, 78, 2863-2866
13. Highton, R.; Murr, B. L.; Shafa, F.; Beer, M. Electro microscopic study of base sequence in nucleic acids VIII. Specific conversion of thymine into anionic osmate esters. Biochemistry, 1968, 7, 825-833
14. Khym, J. X.; Cohn, W. E. Characterizations and some chemical reactions of periodate-oxidized nucleosides. J. Am. Chem. Soc. 1960, 82, 6380-6386
15. Patel, A. B.; Brown, H. D. Selective deamination of nucleosides by 2,4-dinitrophenyl hydrazine. Nature, 1967, 214, 402-405
16. Verwoerd, D. W.; Kohlhage, H.; Zillig, W. Specific partial hydrolysis of nucleic acids in nucleotide sequence studies. Nature, 1961, 192, 1038-1040
17. Fukuhara, T. K.; Visser, D. W. Cytidine derivatives. J. Am. Chem. Soc. 1955, 77, 2393-2395
18. Wempen, I.; Doerr, I. L.; Kaplan, L.; Fox. J. J. Pyrimidine nucleosides. Ⅵ. Nitration of nucleosides. J. Am. Chem. Soc. 1960, 82, 1624-1629
19. Ma, T. H.; Harris, M. M. Review of the genotoxicity of formaldehyde. Mutat. Res. 1988, 196, 37-59
20. Vaca, C. E.; Nilsson, J. A.; Fang, J. L.; Grafstrom, R. C. Formation of DNA adducts in human buccal epithelial cells exposed to acetaldehyde and methylglyoxal in vitro. Chem.-Biol. Interact. 1998, 108, 197-208
21. Shapiro, R.; Hachmann, J. Use of synthetic polyglucose of density-gradient centrifugation of viruses. Biochemistry, 1966, 5, 2799-2807
22. Blackburn, G. J.; Gait, M. J. Nucleic Acids in Chemistry and Biology. 1990, Oxford University press, Tokyo.
23. Huber, K. W.; Lutz, W. K. Methylation of DNA by incubation with methylamine and nitrite. Carcinogenesis, 1984, 5, 403-406
24. Nouraldeen, A. M.; Ahmed, A. E. Studies on the mechanisms of haloacentronitrile- induced genotoxicity Ⅳ: In vitro interaction of haloacetonitriles with DNA. Toxicol. In Vitro. 1996, 10, 17-26
25. Wang, J.; Rivas, G.; Luo, D.; Cai, X.; Valera, F. S.; Dontha, N. DNA-modified electrode for the detection of aromatic amines, Anal. Chem. 1996, 68, 4365-4369
26. Sheweita, Salah A.; Mostafa, Mostafa H. N-nitroso compounds induce changes in carcinogen-metabolizing enzymes. Cancer Letters, 1996, 106, 243-249
27. Talaska, G.; Underwood, P.; Maier, A.; Polycyclic aromatic hydrocarbons (PAHs), nitro-PAHs and related environmental compounds. Biological markers of exposure and effects. Occupational Health and Industrial Medicine, 1997, 36. 68-75
28. Harvey,R. G.; Geacintor, N. E.; Intercalation and binding of carcinogenic hydrocarbon metabolities to nucleic acids. Accts. Chem. Res. 1988, 21, 66-73
29. 彭成鑑,壓電材料,科儀新知,1995,16,18-29
30. Lu G., Czanderna A. W., Application of Piezoelectric Quartz Crystal Microbalance. 1984. Elsevier Science. New York.
31. Ikeda, T. Fundamentals of Piezoelectricity. 1990, Oxford Sci. Publ.
32. Cavic, B. A.; Hayward, G. L.; Thompson, M. Acoustic waves and the study of biochemical macromolecules and cells at the sensor-liquid interface. Analyst, 1999, 24, 1405-1420
33. 黃錦城,壓電晶體生物感測器之原理與應用,食品工業月刊,1997,29,8-16
34. Levenson, L. L.; Cimento, N. suppl. 2. Ser. I., 1967, 5, 321
35. Martin, S. J.; Frye, G. C.; Ricco, A. J.; Senturia, D. S. Effect of surface roughness on the response of thickness-shear mode resonators in liquids. Anal. Chem. 1993, 65, 2910-2922
35. 紀培錦,晶體振盪電路分析與設計,新電子科技,1989,17,196-207
37. 袁帝文,黃柏鈞,數位邏輯設計與分析,1992,全欣科技圖書
38. 江宗達,鍾健文,IBMPC與感測器介面的探討,1994,全華科技圖書
39. Sauerbrey, G., Phys. 1959, 155,206
40. Hlaray J., Gahbault G. G. Applications of the Piezoelectric Crystal Detector in Analytical Chemistry. Anal. Chem. 1977, 49, 1890-1898
41. Chang, P.; Shin, J. S. Application of piezoelectric Ru(III)/cryptand coated quartz crystal gas chromatographic detector for olefins. Anal. Chem. Acta. 1999, 380, 55-62
42. Chang, P.; Shin, J. S. Preparation and application of cryptand-coated piezoelectric crystal gas chromatographic detector. Anal. Chem. Acta. 1998, 360, 61-68
43. Thompson, M.; Kipling, A. L.; Rajakovic, L. V.; Thickness-shear-mode acoustic wave sensors in the liquid phase. A review. Analyst. 1991, 116, 881-890
44. Chang, P.; Shin, J. S. Multi-channel piezoelectric quartz crystal sensor for organic vapors. Anal. Chem. Acta. 2000, 403, 39-48
45. Mandelis and Christofides, Physics Chemistry and Technology of Solid State Gas Sensor Devices, 1993, New York.
46. Konash, P. L.; Basiaans, G. J. Piezoelectric crystal as detectors in liquid chromatography. Anal. Chem. 1980, 52, 1929-1931
47. Bruckensien, S.; Shay, M. Experimental aspects of use of quartz crystal microbalance in solution. Electrochemical Acta. 1985, 30, 1295-1300.
48. Nomura, T.; Yanagihara, T.; Mitsui, T. Electrode-separated piezoelectric quartz crystal and its application as detector for liquid chromatography. Anal. Chem. Acta. 1991, 248, 329-335
49. Scouten, W. H.; Long, J. H. T.; Brown, R. S. Enzyme or protein immobilization techniques for applications in biosensor design. Trends in Biotechnol. 1995, 13, 178-185
50. Nakanishi, H. M.; Karube, I. A novel method of immobilizing antibodies on a quartz crystal microbalance using plasma-polymerized films for immunosensors. Anal. Chem. 1996, 68, 1695-1700
51. Fawcett, N. C.; Evans, J. A.; Chien, L. C.; Flowers, N. Nucleic acid hybridization detected by piezoelectric resonance. Anal. Lett. 1988, 21, 1099-1114
52. Okahata, Y.; Matsunobu, Y.; Ijiro, K.; Mukae, M.; Murakami, A.; Makino, K. Hybridization of nucleic acids immobilized on a quartz crystal microbalance. J. Am. Chem. Soc. 1992, 114, 8299-8300
53. Okahata, Y.; Matsunobu, Y.; Ijiro, K. Detection of intercalation behaviors of dyes in DNAs using a quartz-crystal microbalance. Sensors and Actuators B, 1993, 13-14, 380-383
54. Su, H.; Yang, M.; Kallury, K. M. R.; Thompson, M. Network analysis: Acoustic energy transmission detection of polynucleotide hybridization at the sensor-liquid interface. Analyst, 1993, 118, 309-312
55. Xu, X. X.; Yang, H. C.; Mallouk, T. E.; Bard, A. J. Immobilization of DNA on a aluminum (III) alkanebisphosphonate thin film with electrogenerated chemi- luminescent detection. J. Am. Chem. Soc. 1994, 116, 8386-8387
56. Zhao, Y. D.; Pang, D. W.; Hu, S.; Wang, Z. L.; Cheng, J. K.; Dai, H. P. DNA-modified electrodes; part 4: optimization of covalent immobilization of DNA on self-assembled monolayers. Talanta, 1999, 49, 751-756
57. Caruso, F.; Rodda, E.; Furlong, D. N.; Niikura, K.; Okahata. Y.; Quartz crystal microbalance study of DNA immobilization and hybridization for nucleic acid sensor development. Anal. Chem. 1997, 69, 2043-2049
58. Zhang, H.; Wang, R.; Tan, H.; Nie, L.; Yao, S. Bovine serum albumin as a means to immobilize DNA on a silver-plated bulk acoustic wave DNA biosensors. Talanta, 1998, 46, 171-178
59. Zhang, H.; Tan, H.; Wang, R.; Wei, W.; Yao, S. Immobilization of DNA on silver surface of bulk acoustic wave sensor and its application to the study of UV-C damage. Anal. Chem. Acta. 1998, 374, 31-38
60. Higashi, M.; Takahashi, M.; Niwa, M. Immobilization of DNA through Intercalation at self-assembled monolayers on Gold. Langmuir, 1999, 15, 111-115
61. Huang, E.; Zhou, F.; Deng, L. Studies of surface coverage and orientation of DNA molecules immobilized onto performed alkanethiol self-assembled monolayers, Langmuir, 2000, 16, 3272-3280
62. Lide, D. R. Handbook of chemistry and physics. 1993, CRC Press, London.
63. Herskovits, T. T.; Singer, S. J. Nonaqueous solutions of DNA. Denaturation in methanol and ethanol. Arch. Biochem. Biophys. 1961, 94, 99-114
64. Piskur, J.; Rupprecht, A. Aggregated DNA in ethanol solution, FEBS Letters, 1995, 375. 174-178
65. Matzea, M.; Onori, G.; Santucci, A. Condensation of DNA by monohydric alcohols, Colloids and Surfaces B: Biointerfaces, 1999, 13, 157-163
66. Herskovits, T. T. Nonaqueous solutions of DNA: factors determining the stability of the helical configuration in solution. Arch. Biochem. Biophys. 1962, 97, 474-484
67. 高文弘,周孟儒,界面化學,1983,黎明書局
68. Fraenkel-Conrea, H.; Singer, B. Nucleoside adducts are formed by cooperative reaction of acetaldehyde and alcohols: Possible mechanism for the role of ethanol in carcinogenesis, Proc. Natl. Acad. Sci. USA, 1988, 85, 3758-3761
69. McGhee, J. D.; Hippel, P. H. V. Formaldehyde as a probe of DNA structure.Ⅰ. Reaction with exocyclic amino groups of DNA bases. Biochemistry, 1975, 14, 1281-1296
70. McGhee, J. D.; Hippel, P. H. V. Formaldehyde as a probe of DNA structure.Ⅳ. Mechanism of the initial reaction of formaldehyde with DNA. Biochemistry, 1977, 15, 3276-3293
71. Leupin, W.; Chazin, W. J.; Hyberts, S.; Denny, W. A.; Wuthrich, K. NMR studies of the complex between the decadeoxynucleotides d(GCATTAATGC) and a minor-groove-binding compounds. Biochemistry, 1986, 25, 5902-5910