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研究生:林欣怡
研究生(外文):Hsin-Yi Lin
論文名稱:用微接觸技術製備肌紅蛋白質的人工抗體模版
論文名稱(外文):Fabricating an artificial antibody film for detecting myoglobin — using microcontact printing method
指導教授:周澤川
指導教授(外文):Tse-Chuan Chou
學位類別:碩士
校院名稱:國立成功大學
系所名稱:化學工程學系碩博士班
學門:工程學門
學類:化學工程學類
論文種類:學術論文
論文出版年:2006
畢業學年度:94
語文別:中文
論文頁數:250
中文關鍵詞:肌紅蛋白質分子模版微接觸技術辨識性孔洞
外文關鍵詞:molecular imprintingmicro-contact printingmyoglobinrecognition binding site
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中文摘要

  肌紅蛋白質具有多種疾病的指標,尤其是急性心肌梗塞(acute myocardial infarction, AMI)早期的心肌損傷標幟物,血清肌紅蛋白質的升高的幅度和持續時間可以幫助評估梗塞部位和梗塞處的面積大小。如果血清中高達2000 ng / ml,將會引起腎臟方面的併發症,血清肌紅蛋白質濃度越高,死亡率也高,所以肌紅蛋白質的量也可預測死亡率的發生。
  目前市面上的檢驗試劑或免疫分析技術,不是只可半定量,就是需要用到不易取得且又昂貴的抗體,檢驗時不但分析的步驟繁雜,抗原抗體的穩定性也不佳,故若使用分子模版為人工抗體取代自然界中的抗體免疫分析應用於肌紅蛋白質的檢測,則期望可解決上述的問題。
  本研究主要目的在於最適化以微接觸技術製備出的肌紅蛋白質模版,希望以人工抗體的地位,取代目前市面上只可定性的檢驗試劑,或需要用到不易取得且又昂貴抗體的免疫分析技術,並探討未來發展快速又簡易的生物感測器應用的可能。
  實驗結果顯示經由ITC於非均相的滴定實驗及實際使用不同單體製備模版,可由本研究測試的單體中決定組裝肌紅蛋白質模版的最適交聯劑及功能性單體分別為TEGDMA及MMA。TEGDMA高分子膜對肌紅蛋白質有最低親和力及最低肌紅蛋白質吸附量2.37 ± 0.30×10-11 mole / cm2,而MMA與TEGDMA在1:3的條件下製備模版且UV下聚合9小時,對肌紅蛋白質有最高吸附量15.03 ± 0.89 × 10-11 mole / cm2(2645.28 ng / cm2)。當使用酸洗(AcOH)、鹼洗(NaOH)或以不同濃度的胰蛋白脢移除目標分子時,使用2 wt. % SDS和0.6 wt. % NaOH清洗條件,移除率可達72.82 %且imprinting factor為5.82。在探討模版吸附的動力機制中,可求得模版上的專一性吸附的部份Kd為3.4 × 10-7 M,辨識性孔洞n*有7.24 × 10-11 mole / cm2(即模版表面的的active sites);而非專一性吸附的phase 2的Kd2為1.355 × 10-5 M,假性孔洞量n*為9.62 × 10-10 mole / cm2。
  肌紅蛋白質模板在使用IgG、HSA、hemoglobin做競爭性吸附時,在獨立蛋白質環境下進行再吸附,Myo-MIP吸附量相於其他蛋白質的莫爾比分別為115.5、230.9及2.5。在雙成份等莫爾濃度下作競爭性分析,對肌紅蛋白質的選擇性分別為94.18 %、98.21 %及61.09 %。  
  在真實樣品下做模版吸附效能評估,當在經20倍稀釋的血清中肌紅蛋白質總濃度多了50 ng / ml,模板吸附量增加0.63 × 10-11 mole / cm2,而在未經稀釋尿液樣品中,進行再吸附,吸附的imprinting factor可達37.4 ± 3.21。
  由此可見,本研究不但成功的製備出的肌紅蛋白質模版,也證明了在真實樣品下感測的可能性,不過在模版選擇性方面,肌紅蛋白質對人體中最多含量的兩種蛋白質(IgG及HSA)選擇性皆可在高達90 %以上,對hemoglobin的選擇性卻不好,這點將會對未來的應用條件有所限制。
Abstract

Myoglobin is known to be an important biological index for the diagnosis of various diseases. Myoglobin tests are done to evaluate a person who has symptoms of acute myocardial infarction (AMI). The serum myoglobin concentration measured over a period, allows the prediction of the position and the area of myocardial infarction. In large quantities, myoglobin can damage the kidneys and break down into toxic compounds, causing kidney failure.
In general, the tests for detecting myoglobin are either indicative (i.e. rapid tests), or they need to use expensive natural antibodies (i.e. clinical immunoassays). Additionally, processes for detecting myoglobin by immunoassay in the clinical setting are usually complicated, as antibody activity is not easy to maintain. Therefore, if MIPs can serve as artificial antibodies able to replace their natural counterparts, we can look forward their application in immunoassays.
  The purpose of this research was to optimize the formation of myoglobin-imprinted polymers to be used in micro-contact printing, which has as its ultimate objective the development of a biosensor.
Comparing the Isothermal Titration Calorimeter (ITC) results, obtained in a heterogeneous titration system used to prepare the polymer films with various monomers by UV polymerization for 9 hours, TEGDMA (a crosslinking agent) exhibited the lowest and MMA (a functional monomer) exhibited the highest affinity to myoglobin. The adsorpted quantity of myoglobin on the polymer film prepared with TEGDMA was 2.37 ± 0.30×10-11 mole / cm2. With a ratio of MMA and TEGDMA of 1 to 3, the Myo-MIP appeared to have the highest adsorption quantity, 15.03 ± 0.89 × 10-11 mole/cm2 (2645.28 ng/cm2). For evaluating the removal and imprinting factors of the Myo-MIPs various wash conditions: i.e. acidic, basic, and trypsin extraction methods were investigated. Finally, an extraction solvent comprising of 2 wt. % SDS and 0.6 wt. % NaOH used at 80 ℃ for 30 min was shown to give the highest imprinting factor i.e. 5.82 with 72.82 % myoglobin removal. Scatchard binding plots showed the dissociation constant for the specific binding phase to be 3.4×10-7 M and the theoretical recognition binding site capacity to be 7.24×10-11 mole/cm2; for the non-specific binding phase Kd = 1.355×10-5 M and the non-specific recognition binding site capacity was determined as 9.62×10-10 mole/cm2.
  The selectivity binding experiments were carried out in both single protein and binary protein systems. The molar ratio of adsorbed myoglobin to IgG, HSA and hemoglobin was found to 115.5, 230.9 and 2.5 respectively. In binary competition systems, myoglobin selectivity to IgG, HSA and hemoglobin was respectively 94.18 %, 98.21 % and 61.09 %.
Rebinding the template protein in natural biological matrices, i.e. human serum diluted 20 times by phosphate buffer, showed the film to have significant uptake when 50 ng/ml myoglobin was added compared to myoglobin free controls. Furthermore, when re-binding in undiluted urine, the imprinting factor was determined as 37.4 ± 3.21.
This research has resulted in the successful imprinting of myoglobin and proved the possibility of sensing real samples. Comparing IgG and HSA uptake under competitive conditions, the selectivity of the Myo-MIP was 90 %. But the limited selectivity with respect to hemoglobin will limit application in the immediate future.
目錄
中文摘要……………………………………………… I
英文摘要……………………………………………… III
誌謝…………………………………………………… V
目錄…………………………………………………… VII
表目錄………………………………………………… XIII
圖目錄………………………………………………… XV
專有名詞對照表……………………………………… XXV
符號說明……………………………………………… XXIX
第一章 緒論…………………………………………… 1
 1-1前言……………………………………………… 1
 1-2分子辨識要素…………………………………… 2
第二章 原理…………………………………………… 6
 2-1分子模版………………………………………… 6
   2-1-1分子模版之起源與發展………………… 6
   2-1-2分子模版之原理………………………… 8
      2-1-2.1微接觸壓印技術製備分子模版
          之起源與發展…………………14
      2-1-2.2分子模版的優點……………… 17
   2-1-3製備分子模版所需之材料……………… 17
      2-1-3.1目標分子之決定……………… 17
      2-1-3.2高分子單體之決定…………… 18
          2-1-3.2-a交聯劑………………18
          2-1-3.2-b功能性單體…………19
          2-1-3.2-c預測吸附效能方式…20
      2-1-3.3起始劑之決定………………… 26
      2-1-3.4溶劑之決定…………………… 27
   2-1-4分子模版之應用………………………… 28
      2-1-4.1研究方面……………………… 28
      2-1-4.2實際產品的開發……………… 34
   2-1-5分子模版面臨之問題與未來發展……… 34
      2-1-5.1分子模版待解決的問題……… 34
      2-1-5.2分子模版未來的發展………… 36
   2-1-6分子模版之動力式推導………………… 37
 2-2儀器原理………………………………………… 38
   2-2-1研究偵測方式-螢冷光發光機制原理……38
   2-2-2等溫滴定微卡計之原理………………… 42
      2-2-2.1卡計之基本介紹……………… 42
      2-2-2.2恆溫滴定微卡計之基本介紹… 43
   2-2-3傅立葉變換紅外光譜儀之原理………… 49
   2-2-4非接觸式原子力顯微鏡之原理………… 52
   2-2-5能量分散式光譜儀(EDS)之原理………55
   2-2-6表面輪廓儀之原理……………………… 56
第三章 研究目標……………………………………… 58
 3-1肌紅蛋白質……………………………………… 58
   3-1-1肌紅蛋白質之簡介……………………… 58
   3-1-2肌紅蛋白質之結構……………………… 58
   3-1-3肌紅蛋白質的臨床意義………………… 60
 3-2蛋白質的檢測…………………………………… 67
   3-2-1目前蛋白質的檢測方式………………… 67
   3-2-2市面上肌紅蛋白質的檢測方式………… 69
   3-2-3研究肌紅蛋白質分子模版之動機與優勢 72
第四章 實驗設備與方法……………………………… 74
 4-1藥品與儀器……………………………………… 74
   4-1-1藥品……………………………………… 74
   4-1-2儀器……………………………………… 77
 4-2實驗的流程與方法……………………………… 78
   4-2-1製備分子模版前的準備工作…………… 78
      4-2-1.1分子模版之載體-玻璃基板清
          洗與改質………………………78
          4-2-1.1-a蓋玻片-玻璃基板之
              清洗與改質…………78
          4-2-1.1-b載玻片-玻璃基板之改
              質……………………79
      4-2-1.2緩衝溶液之配製……………… 82
   4-2-2製備肌紅蛋白質微接觸壓印分子模版… 83
      4-2-2.1肌紅蛋白質分子模版之壓印與
          聚合……………………………83
      4-2-2.2目標分子-肌紅蛋白質之移除…85
      4-2-2.3分子模版之再吸附…………… 86
          4-2-2.3-a單一成分之吸附……86
          4-2-2.3-b雙成份系統之吸附…87
          4-2-2.3-c真實樣品之吸附……87
      4-2-2.4吸附效能之評估……………… 88
          4-2-2.4.a螢光偵測……………88
          4-2-2.4.b冷光偵測……………89
   4-2-3其他變因探討…………………………… 93
      4-2-3.1 pH值對肌紅蛋白質分子模版的
          影響……………………………93
      4-2-3.2不同吸附濃度對分子模版的影
          響………………………………93
      4-2-3.3不同吸附時間對分子模版的影
          響………………………………93
   4-2-4蛋白質於分子模版上之定量方式……… 93
      4-2-4.1於溶液中的校正曲線………… 94
      4-2-4.2於膜上的校正曲線…………… 94
   4-2-5其他分析………………………………… 95
      4-2-5.1等溫微熱卡儀之滴定(ITC)……95
          4-2-5.1.a均相中目標分子與單
              體的作用力…………95
          4-2-5.1.b非均相中目標分子與
              單體的作用力………96
      4-2-5.2傅立葉轉換紅外光譜儀分
         (FTIR)…………………………98
      4-2-5.3非接觸原子力顯微鏡分析
         (AFM)………………………… 98
      4-2-5.4表面輪廓儀-膜厚的測定
          (alpha-step)………………98
      4-2-5.5能量分散式光譜儀的測定
         (EDS)………………………… 98
 4-3研究架構與流程………………………………… 99
第五章 實驗結果與討論……………………………… 100
 5-1實驗各變因的決定……………………………… 100
 5-2校正曲線………………………………………… 103
   5-2-1肌紅蛋白質之抗體用量決定…………… 103
      5-2-1.1二級抗體之用量決定………… 103
      5-2-1.2一級抗體之用量決定………… 106
      5-2-1.3血紅蛋白質螢光抗體之用量決
          定………………………………108
   5-2-2不同蛋白質之定量方式………………… 111
      5-2-2.1於磷酸緩衝溶液(PB)下之定
          量………………………………111
          5-2-2.1-a G型球蛋白質(IgG)
               於溶液之校正曲線112
          5-2-2.1-b人類血清白蛋白質
              (HSA)於溶液之校
              正曲線………………113
          5-2-2.1-c血紅蛋白質
              (hemoglobin)於溶
              液之校正曲線………114
          5-2-2.1-d肌紅蛋白質
              (myoglobin)於溶液
              之校正曲線…………115
      5-2-2.2於分子模版上之定量………… 116
          5-2-2.2-a G型球蛋白質(IgG)
               於模版上之校正曲
               線…………………116
          5-2-2.2-b 人類血清白蛋白質
              (HSA)於模版上之校
               正曲線……………117
          5-2-2.2-c血紅蛋白質
              (hemoglobin)於模版
              上之校正曲線………118
          5-2-2.2-d肌紅蛋白質
              (myoglobin)於模版
              上之校正曲線………119
 5-3製備肌紅蛋白質分子模版……………………… 120
   5-3-1交聯劑之選擇…………………………… 120
      5-3-1.1等溫微熱卡滴定探討不同交聯
          劑對肌紅蛋白質之影響………120
          5-3-1.1-a均相中目標分子與交
              聯劑的作用力………121
          5-3-1.1-b非均相中目標分子與
              交聯劑的作用力……122
      5-3-1.2不同交聯劑製備高分子模評估
          非專一性吸附的影響…………123
   5-3-2功能性單體之選擇……………………… 130
      5-3-2.1等溫微熱卡滴定探討不同功能
          性單體對肌紅蛋白質之影響…130
          5-3-2.1-a均相中目標分子與功
              能性單體的作用力…131
          5-3-2.1-b非均相中目標分子與
              功能性單體的作用力132
      5-3-2.2不同功能性單體製備分子模版
          評估其效能……………………134
   5-3-3功能性單體與交聯劑之間使用之比例問   
      題…………………………………………142
   5-3-4聚合時間對模版效能影響……………… 146
   5-3-5探討清洗條件對肌紅蛋白質分子模版效
      能之影響…………………………………148
      5-3-5.1使用不同濃度的NaOH及2 wt.%
          SDS溶液移除目標分子對模版
          效能的影響……………………148
      5-3-5.2使用不同濃度的AcOH及2 wt.%
          SDS溶液移除目標分子對模版
          效能的影響……………………154
      5-3-5.3使用不同濃度的胰蛋白酶溶液
          移除目標分子對模版效能的影 
          響………………………………154
   5-3-6 pH值對肌紅蛋白質分子模版效能之影
      響……………………………………… 158
      5-3-6.1不同pH下製備肌紅蛋白質之分
          子模版…………………………159
      5-3-6.2不同pH下吸附肌紅蛋白質之評  
          估…………………………… 161
      5-3-6.3 pH對模版製備及再吸附的影
           響………………………… 162
   5-3-7吸附時間對肌紅蛋白質分子模版效能之
      影響…………………………………… 168
   5-3-8吸附濃度對肌紅蛋白質分子模版效能之
      影響…………………………………… 168
      5-3-8.1飽和吸附曲線………………… 168
      5-3-8.2 Scatchard Plot…………… 169
 5-4肌紅蛋白質分子模版對干擾物之吸附………… 175
   5-4-1獨立蛋白質環境下肌紅蛋白質分子模版
      吸附效能評估………………………… 175
   5-4-2雙成分且同比例蛋白質環境下肌紅蛋白
      質分子模版吸附效能評估…………… 176
      5-4-2.1干擾物G型球蛋白(IgG)對肌
          紅蛋白質分子模版效能之影
          響…………………………… 177
      5-4-2.2干擾物人類血清白蛋白(HSA)
          對肌紅蛋白質分子模版效能
          之影響……………………… 178
      5-4-2.3干擾物血紅素(hemoglobin)
          對肌紅蛋白質分子模版效能
          的影響……………………… 179
   5-4-3於真實環境下肌紅蛋白質分子模版吸附
      效能評估……………………………… 187
 5-5分子模版薄膜之特性分析……………………… 192
   5-5-1分子模版FTIR之定性分析……………… 192
   5-5-2分子模版表面元素分析評估…………… 196
   5-5-3分子模版AFM表面影像評估…………… 199
      5-5-3.1 NIP及Myo-MIP表面影像…… 199
      5-5-3.2肌紅蛋白質粒徑評估………… 199
   5-5-4分子模版alpha-step膜厚評估………… 204
第六章 綜合討論與結論……………………………… 205
 6-1綜合討論………………………………………… 205
   6-1-1研究達成情形綜合整理………………… 205
   6-1-2交聯性與功能性單體的選擇…………… 209
      6-1-2.1最適交聯性選擇……………… 209
      6-1-2.2最適功能性單體選擇………… 209
      6-1-2.3均相與非均相ITC無法相比較
          的原因……………………… 210
   6-1-3綜合比較各清洗條件…………………… 213
   6-1-4綜合探討pH值對分子模版之影響……… 215
   6-1-5肌紅蛋白質分子模版吸附動力機制之討
      論……………………………………… 217
   6-1-6模版選擇性綜合比較…………………… 218
   6-1-7分子模版目標物單顆分子的迷思……… 220
   6-1-8肌紅蛋白質分子模版實際應用於人工
      抗體或生物感測器可行性的評估…… 221
 6-2結論……………………………………………… 224
第七章 研究改善與未來展望………………………… 226
 7-1研究改善建議…………………………………… 226
   7-1-1實驗方面………………………………… 226
   7-1-2走向應用………………………………… 227
 7-2未來展望………………………………………… 228
參考文獻……………………………………………… 230
附錄…………………………………………………… 244
參考文獻

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