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研究生:陳姿穎
研究生(外文):Chen Tsu-Ying
論文名稱:聚二氟亞乙烯再生化抗體壓電感測器之應用及特性分析
論文名稱(外文):Study and Application of Poly(vinylidene fluoride) in piezoelectric Bio-Immunosensor
指導教授:施正雄施正雄引用關係
指導教授(外文):Shih Jeng-Shong
學位類別:碩士
校院名稱:國立臺灣師範大學
系所名稱:化學研究所
學門:自然科學學門
學類:化學學類
論文種類:學術論文
論文出版年:2002
畢業學年度:90
語文別:中文
論文頁數:146
中文關鍵詞:聚偏二氟亞乙烯壓電生化感測器免疫球蛋白GIR光譜
外文關鍵詞:Poly(vinylidene fluoride)piezoelectric Bio-ImmunosensorIgGIR spectra
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  • 被引用被引用:4
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本研究利用聚偏二氟乙烯(Poyl(vinylidene fluoride))當作壓電感測器的塗佈膜,探討PVDF與蛋白質間的作用力。研究發現具有非極性的a-phase PVDF薄膜會吸附較多的蛋白質,因為非極性的a-phase PVDF與蛋白質之間是以疏水性作用力互相鍵結,將蛋白質固定在PVDF薄膜上。
  本研究同時研製一固定化之抗體(anti-IgG)-a相PVDF塗佈之免疫壓電感測器,以偵測水溶液中的抗原(IgG)。結果顯示,固定後的抗體(anti-IgG)依然保有活性,可吸附及偵測到水溶液中的抗原。針對Anti-IgG的濃度及PVDF 的塗佈量作探討,發現塗佈1.1mg PVDF及注入0.025mg/ml抗體可獲得最大訊號。觀察到Anti-IgG-IgG結合成一穩定的復合體,並發現可利用Glycine-HCl打斷抗體-抗原之間的作用力,若重複再注入抗原,發現有相同反應,證實此PVDF/Anti-IgG壓電晶體感測器可用來重複偵測IgG存在,且有相當不錯的再現性。若將此PVDF/Anti-IgG保存在4℃下,可保存其有效性六天以上。同時發現本研究因為pH值、溫度會影響anti-IgG與IgG的反應,發現pH在7.2左右,溫度是在35℃下為最適條件,此PVDF-anti-IgG免疫壓電感測器對IgG的偵測下限可達146.7 ng/mL。
  本研究並觀察金屬離子和有機物質存在下,是否會影響抗體-抗原反應。研究發現在Mn2+、Cd2+存在下,並不會影響anti-IgG與IgG反應,但是Zn2+會。在有機物質方面,丙酮、丙醛、丙酸稍微會影響到anti-IgG與IgG反應,但是丙胺會使IgG完全變性,造成anti-IgG與IgG無法反應。  
另外,本研究亦探討PVDF和金屬離子作用力,利用製造PVDF薄膜的過程中填入金屬離子化合物,希望獲得具有較多b相含量的極性且具有壓電性薄膜。針對不同溶劑作研究,觀察IR光譜發現PVDF溶於80% acetone+20% DMSO會獲得b+g 的混合相。PVDF溶於80% acetone+20% DMSO中並加入不同陰離子的二價銅化合物製膜,發現在硝酸根及硫酸根的IR光譜中觀察到有較多比例的b相的產生。在PVDF填入16種金屬離子化合物的IR光譜中觀察到,Cd(NO3)2填入PVDF中時會獲得最多比例的b相。金屬離子和PVDF之間主要是靜電吸引力,當金屬帶較多的正電時(如Nb5+、Cr3+)會有較多比例的b相的產生。同時亦發現金屬離子的大小也會影響b相的產生,因為第三過渡金屬因離子半徑太大,實驗結果顯示b相的比例都不高,另外,發現加入Cd2+會大幅增加b相的比例,除了Cd2+因靜電和PVDF互相吸引外,可能因其大小剛好能吸引相隔一段的氟原子靠近,所以能獲得最多比例的b相。實驗結果顯示加入過渡金屬離子化合物都能增加b相的比例,就有機會能製成壓電膜,如此一來一極性的PVDF壓電薄膜就就能容易製作了。

The PVDF coated piezoelectric quartz crystal sensor was employed to study the interaction between PVDF and Proteins. The partially irreversible responses for these proteins were observed by the desorption study, which implied these proteins could be immobilized onto PVDF coated PZ crystals and proteins immobilized onto a phase of PVDF was better than onto a+g phase of PVDF. Proteins were immobilized onto nonpolar a phase of PVDF by hydrophobic interaction.
The antibody (anti-human IgG) immobilization on a phase of PVDF was applied in a piezoelectric crystal immunosensor for human IgG in water. The immobilized antibody was still actived that could be used to detect human IgG in water. The antibody immobilization was influenced by PVDF coating load and anti-IgG concentration; The optimum PVDF coating load was found to be 1.1mg with 0.025mg/mL anti-IgG. The strong interaction between IgG and anti-IgG was found, and thus Glycine-HCl could be used to desorb IgG form Anti-IgG. The piezoelectric immuno-sensor could bw reused for human IgG with good reproducibility. The lifetime of immobilized PVDF/anti-human IgG was about 6 days at 4℃. The interaction of anti-human IgG and human IgG was influenced by pH, temperature, and IgG concentration. Optimum pH and optimum temperature were found to be 7.2 and 35℃ respectively. The detection limit of the sensor for IgG was found to be 146.7 ng/mL. The interaction of anti-human IgG and human IgG affinity in organic solvents such as acetone, propylamin, propyl aldehyde, propyl acid was studied. The interference of ions (e.g. Cd2+, Mn2+, Zn2+, Co2+) were also investigated and discussed.
Furthermore, the interaction of PVDF and metallic compound were also investigated. PVDF films, filled with various of transition metal compounds, were prepared. The effects of crystalline and electronic structural variations were investigated by infrared analysis. The b+g phase PVDF films could be prepared by dissolving PVDF in 80% acetone+20% DMSO. The b-phase PVDF could be obtained by dissolving PVDF in 80% acetone+20% DMSO with copper salts, e.g., Copper(Ⅱ) Sulfate and Copper(Ⅱ) nitrate. The IR spectra were employed to detect PVDF with 16 transition metal ions. Among these metal ions the addition of Cadmium(Ⅱ) nitrate resulted in the greatest b-phase content PVDF. The interaction between PVDF and transition metal ions was proposed to be electrostatic attraction, and the doping of metal ions with higher charges (e.g., Nb5+andCr3+) could result in higher b-phase content. The effect of radius of metal ions was also found the formation of b-phase of PVDF. The third transition metals with quite big radii gave low content of b-phase PVDF. In contrast, the doping of Cd(Ⅱ) result in quite high content of b-phase PVDF which could be attributed to and the Cd(Ⅱ) ion can be filled in the space between two fluorine atoms quite well. Owing to the presence of transition metal ion would increase b-phase content of PVDF, so a polar and piezoelectricity PVDF film could be simply prepared.

第一章 緒論………………………………………………………………...1
1-1 聚偏二氟乙烯(PVDF) 1
1-1-2 聚偏二氟乙烯(PVDF)的結構與性質 3
1-1-3聚偏二氟乙烯的性質及應用 6
1-1-4 PVDF薄膜 7
1-1-5 PVDF紅外光譜定性與定量分析 8
1-2 生物感測器 10
1-2-1生物感測器簡介 10
1-2-2生物感測器的基本結構 10
1-2-3免疫感測器分類 11
1-2-4蛋白質結構 15
1-2-5脊椎動物體內的免疫系統 16
1-2-6抗體 17
1-2-6.1免疫球蛋白G(immunoglobin G, IgG) 19
1-2-6.2免疫球蛋白M(immunoglobin M,IgM) 20
1-2-7蛋白質的結合專一性: 22
1-2-8蛋白質固定法 25
1-3壓電晶體 28
1-3-1壓電晶體之壓電性 28
1-3-2石英振盪器 30
1-3-3石英振盪器的線路 32
1-3-4振盪頻率的量測 34
1-3-5石英微量天平(Quartz Crystal Microbalance,QCM)感測系統 36
1-3-6石英壓電感測器種類及應用 40
1-4研究動機 48
第二章 實驗部分………………………………………………………..49
2-1藥品及儀器 49
2-2 PVDF有機壓電系統 49
2-2-1石英晶體的處理 49
2-2-2 石英晶體表面塗佈液(coating solution) 50
2-2-3表面塗佈法 50
2-3 壓電感測系統 51
2-3-1 振盪線路 51
(A)石英壓電晶體靜相實驗系統 52
(B)石英壓電晶體動相實驗系統 53
2-3-2 PVDF壓電感測項目及步驟 54
2-4 PVDF與金屬離子化合物作用力研究 55
第三章 結果與討論…………………………………………………………55
3-1 PVDF與蛋白質的的吸附研究 55
3-1-1 PVDF與蛋白質(BSA)間的吸附及脫附 55
3-1-2溶劑對PVDF、蛋白質(BSA)間吸附影響 55
3-1-3 PVDF與其他蛋白質吸附研究 56
3-2 PVDF對於抗體的吸附研究 65
3-2-1塗佈PVDF晶片對抗體IgM、IgG吸附 65
3-2-2 PVDF的塗佈量效應 65
3-2-3抗體濃度效應 65
3-2-3 PVDF晶片吸附抗體的pH效應 70
3-3 PVDF-抗體 晶片與抗原反應研究 72
3-3-1 PVDF-抗體 的活性 72
3-3-2 PVDF-anti-human IgG和抗原(IgG)作用力研究 75
3-3-3 PVDF-抗體與抗原結合的溫度效應 77
3-3-4 PVDF-抗體與抗原結合的pH效應 77
3-3-5 PVDF/Anti-IgG-IgG的濃度效應 80
3-3-6抗體-抗原之吸附與脫附 82
3-3-7 PVDF-抗體與抗原反應的再現性 84
3-3-8 PVDF-抗體晶片儲存活性 84
3-3-9金屬離子的影響 87
3-3-10有機溶劑對抗體-抗原反應的影響 91
3-4 金屬離子化合物填入PVDF薄膜的IR光譜分析 97
3-4-1溶劑對PVDF相轉變的影響 97
3-2-2金屬離子與PVDF之間的作用力 100
3-2-3陰離子與結晶相轉變的關係 103
3-2-4離子化合物的濃度對於相變化的影響 106
3-2-5金屬離子化合物對於PVDF結晶相轉換的影響 110
3-2-6金屬離子化合物與溶劑影響的比較 112
第四章 結論………………………………………………………………..120
參考資料……………………………………………………………………122

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