肝癌(HCC)是台灣癌症十大死因排行第二的疾病，而甲胎兒蛋白(AFP)是偵肝癌重要的腫瘤標誌。本論文利用一種高靈敏度、低成本的石英晶體微天秤(QCM)取代傳統的生化分析法以偵測人體血清中微量的AFP抗原。通常以QCM為基礎的生物感測器或免疫感測器較傳統的分析系統需要的檢體較少並且反應時間較短。
本論文目前已成功地將AFP抗體鍵結於QCM的表面，進而改變QCM感測器的輸出頻率，藉由頻率變化即可測得AFP濃度。本研究探討三種表面修飾法對QCM-AFP感測器的靈敏度的影響：(1)吸附(adsorption)法，(2)胱胺酸-戊二醛(CA-GA)法及(3)胱胺酸(CA)法，另外，人體血清AFP抗體濃度變化對感測器靈敏度的影響也將在本論文中探討。
經過實驗證明，吸附法鍵結能力明顯現較其它兩種修飾法差，故不適用於後續AFP免疫感測器的研發。然而CA-GA法與CA法之抗體濃度分別於0.1 mg/mL與1.0 mg/mL時，可得到最大的AFP抗體與抗原之鍵結比；在此條件下，CA-GA法與CA法的靈敏度與線性度分別達到0.142(Hz mL mg-1)/99.8%與0.35(Hz mL mg-1)/98.2%。最後本論文使用商用AFM系統來分析CA-GA法與CA法之AFP抗原與抗體有效表面積，並且經由與QCM感測器特性比對，可發現CA法因為具有較大的有效表面積，所以比CA-GA法擁有更高的AFP抗原吸附量，亦即可獲得較大的QCM頻率變化量。

The α-fetoprotein (AFP) is an important tumor marker for detecting hepatocellular carcinoma (HCC), the second major cancer killers in Taiwan. This thesis utilized a high-sensitivity low-cost quartz crystal microbalance (QCM) to replace the conventional biochemical analysis to detect the extreme low concentration of AFP in human serum. In general, QCM-based biosensors or immunosensors require smaller volume of sample and fewer responding time than conventional analysis system.
Once the AFP antibody has been successfully adsorbed on the surface of QCM, any AFP antigen bonded with it will change the output frequency of the QCM sensor. This research investigates the influence of the mass-sensitivity of QCM-AFP sensor on three surface-modification methods: (i) Adsorption method, (ii) Cystamine-Glutaldehyde (CA-GA) method and (iii) Cystamine (CA) method. The effects of the concentration of AFP antibody on the sensitivity also will be studied.
The adsorption method shows an unstable adhesion than the other methods. The CA-GA method and CA method presents respectively the maximum binding ratio of AFP antibody/antigen pair at 0.1 mg/mL and 1.0 mg/mL AFP-antibody concentration. Under such condition, the high sensitivity and linearity of the CA-GA and CA methods equal to 0.142 (Hz mL mg-1)/99.8% and 0.35 (Hz mL mg-1)/98.2% can be demonstrated. Finally, this thesis uses a commerical AFM system to analyze the effective bonding area of the CA-GA and CA methods. The CA method shows a higher effective surface area than that of CA-GA method so it can present a higher mass-sensitivity of AFP-antigen.

摘要……………………………………………………………………... I
ABSTRACT…………………………………………………………….III
誌謝…………………………………………………………………….. IV
目錄……………………………………………………………………... V
圖目錄………………………………………………………………….. IX
表目錄………………………………………………………………….. XI
第一章 緒論……………………………………………………………..1
1-1 研究動機與目的 1
1-2 實驗方法與論文架構 2
第二章 甲胎兒蛋白與量測系統介紹………………………………….4
2-1 甲胎兒蛋白 4
2-1-1 甲胎兒蛋白與肝癌的關係 4
2-1-2 甲胎兒蛋白與孕婦、胎兒的關係 5
2-1-3 甲胎兒蛋白傳統偵測方法 6
2-1-4 甲胎兒蛋白的文獻回顧 8
2-2 免疫感測器的原理與種類 9
2-2-1 免疫感測器的原理 9
2-2-2 免疫感測器的種類 11
2-3 壓電免疫感測器 13
2-3-1 壓電效應 14
2-3-2 QCM的特性 16
2-3-3 QCM外觀與構造 18
2-3-4 QCM等效電路 19
2-3-5 QCM的理論基礎 21
2-3-6 表面修飾法 23
2-4 原子力電子顯微鏡 24
2-4-1 原子力電子顯微鏡在生物學上的優勢 25
2-4-2 原子力電子顯微鏡的基本原理 25
2-4-3 原子力電子顯微鏡的主要結構 26
2-4-4 原子力電子顯微鏡的工作模式 27
2-4-5 原子力電子顯微鏡量測參數定義 30
第三章 實驗方法與材料……………………………………………...32
3-1實驗試劑 32
3-2 實驗設備 32
3-3 實驗方法 34
3-3-1吸附法 35
3-3-2 CA-GA法 36
3-3-3 CA法 38
3-3-4 AFM的操作步驟 40
第四章 結果與討論…………………………………………………...42
4-1 不同表面修飾法對QCM免疫感測頻率響應之影響 42
4-1-1 經吸附法修飾的QCM元件之AFP質量感測結果 分析 42
4-1-2 經CA-GA法修飾的QCM元件之AFP質量感測結 果分析 45
4-1-3經CA法修飾的QCM元件之AFP質量感測結果分 析 46
4-1-4 以上三種表面修飾法於低濃度AFP抗體(0.1 mg/mL) 之比較 47
4-1-5 CA-GA法與CA法的AFP抗原反應曲線比較 48
4-1-6 QCM免疫感測器的專一性試驗 51
4-2 CA-GA與CA表面修飾法之AFP抗體層表面分析 53
4-2-1 QCM祼晶片表面粗糙度量測 53
4-2-2 經CA-GA法修飾後之AFP抗體層表面分析 55
4-2-3經CA法修飾後之AFP抗體層表面分析 58
4-2-4 CA-GA法與CA法AFM之量測結果比較 60
4-2-5 AFM表面分析結果與QCM感測器頻率響應之分析 61
第五章 結論與未來展望……………………………………………...64
5-1 結論 64
5-2 未來展望 66
參考文獻……………………………… ………………………………67

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