臺灣博碩士論文加值系統

蛋白質檢測對於監測健康並診斷免疫反應、心血管疾病、阿茲海默症、糖尿病和癌症等疾病至關重要。然而，傳統的檢測方法存在著低靈敏度、複雜的樣品準備、和冗長地時間分析限制等。近年來，電化學感測技術嶄露頭角，特別是分子印跡技術的無標記電化學生物感測器，其具有高靈敏度、快速檢測和實時監測等優勢。然而，分子印跡技術本身仍存在一些限制和挑戰，例如優化分子印跡聚合物 (MIP) 的合成條件、提高模板分子的印跡效率、降低檢測環境的外在干擾程度、及減少聚合物基質本身存在的非特異性結合等問題。這些因素可能會影響分子印跡感測器對目標物檢測的靈敏度和再現性。
本研究旨在利用分子印跡技術，開發具高靈敏度的蛋白質電化學感測平台，用於診斷和監測人體中的蛋白質濃度。本研究成功製備了兩種感測器：一種是基於聚苯胺 (PANI) 的感測器，用於檢測牛血清白蛋白 (BSA)；另一種是基於聚鄰苯二胺 (P(o-PD)) 的感測器，用於檢測白血球介素-6 (IL-6)。這兩種開發的感測器成本效益高，製備過程相對簡單。研究結果顯示，PANI 印跡感測器對於 BSA 的檢測具有卓越的靈敏度和可靠性，甚至在極低濃度水平下仍能檢測到 BSA。在最佳優化的條件下，該感測器對於 BSA 在磷酸緩衝鹽溶液 (PBS)、牛奶、和人血清等環境中的樣品測量極限，分別為 2.5 pg/ml、2.7 pg/ml 和 3.6 pg/ml。此外，研究發現，在使用醋酸 (AcOH) 與十二烷基硫酸鈉 (SDS) 清洗 PANI 印跡感測器的過程中，除了能夠去除 BSA 模板分子以產生印跡空腔外，SDS 分子還會在 BSA 的印跡空腔上形成刷狀薄膜，從而協同地增強對 BSA 的再結合能力。
另一方面，本研究製備的 P(o-PD) 印跡感測器對於 IL-6 的檢測也表現出良好的性能。該感測器對 PBS 和人類血清中的 IL-6 有良好的響應，檢測極限分別為 1.5 pg/ml 和 2.4 pg/ml。此外，本研究對 P(o-PD) 印跡電極進行了表面濕潤性、表面形態、表面粗糙度、化學結構和機械性能等物理化學分析。這些分析有效地證明了成功將 IL-6 模板嵌入P(o-PD) 中。綜上所述，本研究開發的 PANI 和 P(o-PD) 印跡感測器具有應用於檢測人體免疫疾病並監測患者健康的潛力。未來期望將研究方向發展為實踐定點照護檢驗 (point-of-care testing)，並進一步在實際臨床應用中獲得驗證和實踐。目標為將本研究的分子印跡感測器應用於商業化的健康監測和蛋白質檢測。

Protein detection is essential for monitoring health and diagnosing diseases, such as immune reactions, cardiovascular diseases, Alzheimer's, diabetes, and cancers. Traditional methods have limitations, including low sensitivity, complex sample preparation, and long analysis times. Electrochemical sensing technology, particularly label-free biosensors using molecular imprinting, offers advantages like high sensitivity, rapid detection, and real-time monitoring. However, there are challenges in optimizing the synthesis conditions of molecularly imprinted polymers (MIP), improving template molecule imprinting efficiency, reducing interference, and minimizing non-specific binding. These factors can affect the accuracy and reproducibility of molecularly imprinted sensors in target detection.
This study aims to develop a protein electrochemical sensing platform using MIP to detect protein concentration in human body. Two types of sensors were successfully prepared: one based on polyaniline (PANI) for detecting bovine serum albumin (BSA) and the other based on poly(o-phenylenediamine) (P(o-PD)) for detecting human interleukin-6 (IL-6). The PANI-based MIP sensor showed excellent sensitivity and reliability in detecting BSA, even at picogram levels. Under optimal conditions, the sensor achieved detection limits for BSA in phosphate-buffered saline (PBS), milk, and human serum at 2.5 pg/ml, 2.7 pg/ml, and 3.6 pg/ml, respectively. During the template molecule removal process using sodium dodecyl sulfate (SDS) in the PANI imprinting sensor, SDS not only removed the template protein molecules to create imprinted cavities but also formed a brush-like film on these cavities, which enhanced the re-binding capability for BSA.
On the other hand, the P(o-PD)-based MIP sensor developed in this study also exhibited a good performance in detecting IL-6. The sensor exhibited a good response to the IL-6 target in PBS and human serum, with a detection limit as low as 1.5 pg/ml and 2.4 pg/ml, respectively. Additionally, physico-chemical analyses of the P(o-PD) imprinting electrodes were performed, encompassing surface wettability, morphology, roughness, chemical structure, and mechanical properties. These analyses effectively demonstrated the successful embedding of the IL-6 template into the P(o-PD) matrix.
In summary, the developed PANI and P(o-PD) MIP sensors in this study have the potential for applications in detecting human immune diseases and monitoring patient health. It is anticipated that this research direction will pave the way for practical-clinical point-of-care testing, leading to the validation and implementation of the diagnostic tool in real-world clinical applications. Ultimately, this could contribute to the commercial development of health monitoring and protein detection technologies.

摘要 I
Abstract III
Acknowledgment V
Content VII
List of Figures IX
List of Tables XVII
Abbreviation 1
Chapter 1 Introduction 2
1.1 Background of research 2
1.2 Challenges of MIP technology 4
1.3 Goals of research 4
1.4 Thesis overview 5
Chapter 2 Literature Review 7
2.1 Protein biomarkers 7
2.2 Protein detection methods 8
2.3 Electrochemical sensing platform 14
2.4 Molecular imprinting technique 17
2.5 MIP-based electrochemical sensor 25
2.6 Challenges of MIP-based electrochemical sensor 27
Chapter 3 Experimental 29
3.1 Chemical and instrument 29
3.2 Preparation procedure of MIP-based electrochemical sensor 31
3.3 Surface characterization 34
3.4 Electrochemical measurements 43
Chapter 4 Results and Discussion 45
4.1 MIP-based BSA electrochemical sensor 45
4.1.1 Optimization of BSA template anchor platform 45
4.1.2 Effect of electro-imprinting cycles of PANI on imprinting BSA template 49
4.1.3 Effects of washing time on the removal of BSA template 51
4.1.4 Performance evaluation of MIP-based BSA sensors 53
4.1.5 Surface properties between MIP and NIP sensors 56
4.1.6 Application of MIP-based BSA sensors 62
4.2 MIP-based IL-6 electrochemical sensor 67
4.2.1 Optimization of IL-6 template anchor platform 67
4.2.2 Surface properties of each modified layer on IL-6 anchor platform 75
4.2.3 Effect of electro-imprinting cycles of P(o-PD) film 83
4.2.4 Surface characterization of synthesis of MIP and NIP sensors 85
4.2.5 Effects of washing time on the removal of IL-6 template 93
4.2.6 Performance evaluation of MIP-based IL-6 sensors 94
4.2.7 Application of MIP-based IL-6 sensors 99
Chapter 5 Conclusion 102
Chapter 6 Future perspective 105
Appendix: Q&A 107
Reference 126

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