臺灣博碩士論文加值系統

肌酸酐 (Creatinine) 是肌酸代謝最終產物，為臨床診斷腎功能重要指標，因此血液及尿液中的肌酸酐濃度是相當重要的生理檢測項目。本研究以共功能性單體製備肌酸酐模版高分子膜披覆於電極上，並以交流阻抗式感測肌酸酐濃度。本研究先合成主要的功能性單體。以 2-amino-5-bromo-3-nitropyridine (ANBP) 為反應物，用乙烯基取代溴，合成單體 2-amino-3-nitro-5-vinylpyridine (ANVP)，再以 1H-NMR進行鑑定，確認單體正確合成。肌酸酐模版高分子薄膜之製備，乃是以肌酸酐為模版分子，加入兩種以上的功能性單體，以共單體進行合成，是由自行合成的ANVP 與 methacrylic acid (MAA)、1-vinylimidazole (1-VD) 及 4-vinylpyridine (4-VP) 等作不同的單體組合，接著加入交聯劑 ethylene glycol dimethacrylate (EGDMA)，在起始劑 2,2′-azobisisobutyronitrile (AIBN) 作用下進行聚合反應，以製備分子模版高分子 (molecularly imprinted polymer, MIP)。
本論文以交流阻抗進行連續式肌酸酐濃度感測，也探討預聚合液中各成份之比例對感測靈敏度及模印指數之影響。由實驗結果可知，功能性共單體與交聯劑之莫爾比為 1: 25 時，電極會有較佳之模印效果。MIP 電極之靈敏度以阻抗與相位角進行估算，分別為 0.68 ± 0.04 ((|Z|–|Zo|)/|Zo| / (mg/dL)) 與 0.41 ± 0.09 ((θ–θo)/θo / (mg/dL))，其阻抗與相位角分析之模印指數分別為 1.94 及 1.78。在肌酸酐選擇性實驗上，是以肌酸 (creatine) 與N-烴基丁二醯亞氨 (N-hydroxysuccinimide, NHS) 分別作為共存物及相似物。在雙成份混合液下共存物之選擇率 (creatinine/creatine)，為 76.52 (以阻抗計)；在雙成份混合液中，以阻抗變化進行估算，則相似物之選擇率 (creatinine/NHS) 分別為 7.49 (各成份濃度10 mg/dL) 及 9.53 (各成份濃度50 mg/dL)。MIP 電極重複使用性測試中，在六次重複使用後，阻抗與相位角回復率仍相當良好；另外，在重複暨保存性方面，經過二十天保存及十次使用後，其穩定性雖隨時間越長而較差，但仍具有辨識能力。綜觀以上，此模版高分子膜電極對肌酸酐具有良好辨識能力，未來能應用於臨床檢測，可更進一步朝微小化生醫感測器發展。

A novel AC impedance sensor based on molecularly imprinted polymer (MIP) has been developed for the measurement of creatinine concentration. Creatinine is a significant biomarker for the diagnosis of kidney function because it can be easily measured in urine and serum. Therefore, the measurement of creatinine became critical in clinical analysis. In this study, we used three combinations of monomers, including 2-amino-5-bromo-3-nitro-pyridine (ANVP), methacrylic acid (MAA), 1-vinylimidazole (1-VD), 4-vinylpyridine (4-VP), as functional co-monomers. In the presence of creatinine template, EGDMA (ethylene glycol dimethacrylate) as the crosslinker was added. With the initiator AIBN (2, 2′-azobisisobutyronitrile) and UV irradiation, photo-polymerization was triggered. The MIP was extracted to remove, then creatinine imprinted polymer was obtained. The results indicate sensitivities of MIP film in impedance and phase angle are calculated by 0.68 ± 0.04 ((|Z|–|Zo|)/|Zo| / (mg/dL)) and 0.41 ± 0.09 ((θ–θo)/θo / (mg/dL)), then imprinting factors in impedance and phase angle analysis are 1.94 and 1.78. The MIP electrode has excellent selectivity tests for creatinine in the presence of creatine and N-hydroxysuccinimide (NHS). Selectivity in impedance analysis for creatinine in the presence of creatine is 76.52. Additionally, selectivities in impedance analysis for creatinine/NHS are 7.49 (10 mg/dL) and 9.53 (50 mg/dL) respectively. In summary, this MIP electrode has great potential in the development of microbiosensor and for being used on clinical detection in the future.

目錄
中文摘要 I
Extended Abstract II
誌謝 V
目錄 VI
表目錄 X
圖目錄 XI
第一章 緒論 1
第二章 文獻回顧 3
2-1 生物感測器 (Biosensor) 3
2-1-1 簡介 3
2-1-2 生物感測器之分類 3
2-1-2-1 酵素 3
2-1-2-2 抗體‐抗原 4
2-1-2-3 DNA 4
2-1-2-4 微生物 4
2-1-2-5 仿生受體 5
2-2 肌酸酐 (Creatinine) 5
2-2-1 簡介 5
2-2-2 臨床檢測及疾病診斷 6
2-2-3 肌酸酐檢驗法 7
2-3 分子模版高分子 (Molecularly imprinted polymer, MIP) 8
2-3-1 簡介 8
2-3-2 發展起源 8
2-3-3 製備過程 9
2-2-3-1非共價鍵型 (Non-covalent bond) 10
2-2-3-2 共價鍵型 (Covalent bond) 10
2-3-3-3 金屬離子螯合型 (Metal coordination) 11
2-3-4 分子模版高分子之組成 11
2-3-4-1 模版分子 11
2-3-4-2 功能性單體 11
2-3-4-3 交聯劑 12
2-3-4-4 溶劑 12
2-3-4-5 起始劑 12
2-4 分子模版高分子之聚合方式 13
2-4-1 總體聚合法 (Bulk polymerization) 13
2-4-2 球珠模印聚合法 (Bead imprinted polymerization) 13
2-4-2-1 懸浮聚合法 (Suspension polymerization) 13
2-4-2-2 沉澱聚合法 (Precipitation polymerization) 13
2-4-2-3 多步驟沉澱聚合法 (Multi-step swelling polymerization) 14
2-4-3 表面模印聚合法 (Surface imprinted polymerization) 15
2-4-4 電聚合法 (Electrochemical polymerization) 15
2-5 分子模版高分子之應用 15
2-5-1 分離純化 15
2-5-2 人工抗體 16
2-5-3 光學感測之應用 16
2-5-3-1紫外‐可見光分光光度計 (UV-vis spectrophotometer) 16
2-5-3-2 螢光檢測 (Fluorescent detection) 17
2-5-3-3 表面電漿共振儀 (Surface plasmon resonance, SPR) 17
2-5-4 壓電感測之應用 (Piezoelectric sensor) 18
2-5-5 電化學感測之應用 18
2-5-6 交流阻抗分析法 (AC impedance) 19
2-5-6-1 原理簡介 19
2-5-6-2 奈奎斯特圖 (Nyquist plot) 20
2-5-6-3 模版分子之應用 21
第三章 實驗方法、材料與儀器 23
3-1 功能性單體之合成 23
3-1-1 2-amino-3-nitro-5-vinylpyridine 之合成 23
3-2 分子模版高分子薄膜之製備 23
3-2-1 金電極之製備 23
3-2-2 高分子薄膜之製備 23
3-2-3 預聚合液之配製 24
3-3 分子模版高分子薄膜之萃洗 25
3-4 電化學交流阻抗分析 (EIS) 25
3-5 交流阻抗分析與感測 27
3-6 選擇性測試 27
3-7 重複性測試 28
3-8 重複暨保存性測試 28
3-9 肌酸酐濃度之測定法 28
3-10 相關儀器分析 29
3-10-1 核磁共振光譜儀 (Nuclear magnetic resonance spectrophotometer, NMR) 29
3-10-2 傅立葉紅外線光譜儀 (Fourier transform infrared spectroscope, FT-IR) 29
3-10-3 掃描式電子顯微鏡 (Scanning electron microscopy, SEM) 30
3-11實驗藥品 31
3-12 實驗儀器 32
第四章 實驗結果與討論 33
4-1 合成功能性單體分析 33
4-1-1 功能性單體之 FT-IR 圖譜分析 33
4-1-2 1H-NMR 圖譜 35
4-1-3 單體導電性 37
4-2 模版高分子薄膜性質分析 37
4-2-1 FT-IR 圖譜分析 37
4-2-2 模版高分子薄膜 SEM 分析 40
4-3 交流阻抗分析感測 42
4-3-1 肌酸酐電化學性質 42
4-3-2 感測機制 43
4-3-3 感測實驗參數之確立 46
4-3-3-1 頻率選擇 46
4-3-3-2 萃洗液選擇 49
4-4 模版高分子薄膜之聚合參數探討 51
4-4-1 肌酸酐溶解度之影響 51
4-4-2 預聚合液體積之影響 53
4-4-3 交聯劑之影響 56
4-4-4 功能性共單體之影響 59
4-4-5 起始劑用量之影響 62
4-5 選擇性測試 65
4-6 重複性測試 70
4-7 重複暨保存性測試 72
第五章 結論 73
參考文獻 74

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