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系統識別號 U0026-3007201214141300
論文名稱(中文) 利用奈米金材料/自組裝單層膜修飾微電極發展多巴胺生物感測器之應用
論文名稱(英文) Development and Application of Dopamine Biosensor Using Nano-gold Materials/Self-assembled Monolayers Modified Microelectrodes
校院名稱 成功大學
系所名稱(中) 生物醫學工程學系
系所名稱(英) Department of BioMedical Engineering
學年度 100
學期 2
出版年 101
研究生(中文) 蔡田畯
研究生(英文) Tien-Chun Tsai
電子信箱 p8897117@mail.ncku.edu.tw
學號 P88971173
學位類別 博士
語文別 英文
論文頁數 99頁
口試委員 指導教授-陳家進
口試委員-張憲彰
口試委員-葉晨聖
口試委員-王國禎
口試委員-林哲信
中文關鍵字 多巴胺  電化學感測器  微電極  奈米金粒子  奈米金樹枝  自組裝單層膜  植入式量測 
英文關鍵字 Dopamine  Electrochemical sensor  Microelectrode  Gold nanoparticle  Gold nanodendrite  Self-assembled monolayer  Implantable sensing 
學科別分類
中文摘要 帕金森氏症起因於中腦黑質體的多巴胺神經元產生缺失,並呈現出肌肉僵直、震顫和行動障礙。針對帕金森氏症的動物模型,體內多巴胺的電化學量測可提供監測多巴胺損耗之證據,但卻面臨腦中大量具有電化學活性的分子所干擾,如︰抗壞血酸和尿酸。在本研究中,我們提出利用奈米金材料與自組裝單層膜針對大電極和針狀微電極進行表面修飾,以改善多巴胺量測的靈敏性,和排除抗壞血酸及尿酸之干擾。
關於奈米金粒子/自組裝單層膜修飾微電極整合安培法量測,體外實驗已顯示白金微電極經由奈米金粒子和3-巰基丙酸修飾後,其檢測靈敏性高於傳統的Nafion修飾方法達到2.7倍。此外,奈米金粒子/3-巰基丙酸修飾微電極對於多巴胺的檢測特性包括快速反應時間(小於2秒)、低檢測極限(7 nM)、良好再現性(± 2.4% relative standard deviation)及可限制抗壞血酸的干擾與白蛋白的吸附汙染。為驗證其修飾微電極對於大鼠腦內多巴胺量測之可應用性,採用深腦電刺激的方式誘發多巴胺釋放,且調整多種刺激參數包含強度和頻率,並配合注射多巴胺回收的抑制劑,證實奈米金粒子/3-巰基丙酸修飾微電極於麻醉大鼠的紋狀體中,已成功進行植入式的多巴胺監測。
此外,關於非侵入式的多巴胺監測,於尿液檢體中進行多巴胺定量係屬於一種可行之技術,但目前的電化學技術面臨大量尿酸的干擾,因其與多巴胺具有相似的電化學特性。此研究透過整合三維結構的奈米金樹枝(gold nanodendrite)與自組裝單層膜進行金電極表面的修飾,希冀能改善檢測多巴胺的靈敏性,與排除尿酸之干擾而達到專一性檢測。針對自組裝單層膜結構中的碳鏈長度進行最佳化探討,發現奈米金樹枝/8-巰基辛酸修飾電極的差式脈波伏安法(differential pulse voltammetry)訊號中具有兩個明顯的波峰,其顯示多巴胺和尿酸的氧化電位相差約260 mV。而利用安培法的量測實驗,奈米金樹枝/8-巰基辛酸修飾電極於多巴胺濃度0.01~5 μM範圍中,具有一良好的線性關係(R2 = 0.999),並且其針對多巴胺的檢測靈敏性高於8-巰基辛酸修飾電極達到20倍,其他重要的檢測特性包括低檢測極限(20 nM)與可有效排除尿酸的干擾,如︰尿酸的添加不會產生反應電流,且尿酸的存在不會降低多巴胺的檢測靈敏性。此外,將此修飾電極成功應用於非稀釋的人體尿液檢體中進行多巴胺的量測,發現具有良好的穩定性、準確性與再現性;因此,針對尿液中的多巴胺進行檢測,本研製電極相較於其他傳統技術呈現出更多具有吸引性的特色。
英文摘要 Parkinson’s disease (PD) resulted from a deficiency of dopaminergic neurons in the substantia nigra of midbrain is characterized by muscular rigidity, tremor, and gait problems. Electrochemical detections of in vivo dopamine (DA) can provide direct evidences for monitoring the DA depletion on animal model of PD but faces substantial amount of electroactive interferences in the brain, such as ascorbic acid (AA) and uric acid (UA). In this study, we proposed that utilization of nano-gold materials and self-assembled monolayer (SAM) to modify macroelectrodes and needle-type microelectrodes for improving sensitivity of DA recording as well as repelling AA and UA interferences.
As regards Au-NP/SAM modified microelectrodes coupling with amperometric i–t measurements, in vitro tests have shown that the sensitivity of platinum microelectrode coated with Au-NP and 3-mercaptopropionic acid (MPA) was 2.7-fold that of conventional Nafion modification. Moreover, the DA sensing features of Au-NP/MPA modified microelectrodes including the fast response time (below 2 s), a low detection limit (7 nM), good reproducibility (± 2.4% relative standard deviation) as well as the resistances of AA interference and albumin adhesion were observed. In order to validate the feasibility of DA monitoring in rat’s brain, Au-NP/MPA modified microelectrodes have been successfully applied for the implantable detections in the striatum of anethetized rats with various parameters of deep brain electrical stimulations including different intensities, frequencies or inhibitor administration (e.g. nomifensine) of DA uptake.
The determination of DA in urine samples is a promising technique for the non-invasive DA monitoring, but current electrochemical techniques face substantial interferences from UA due to similar electroactive characteristics. A combination of three-dimensional gold nanodendrite (Au-DT) and SAM onto the gold electrodes was proposed to improve sensitivity and specificity to DA and alleviate UA interference. With the optimum choice of ω-mercaptoalkane carboxylic acid chain length, the differential pulse voltammetry (DPV) of Au-DT deposited electrode coated with 8-mercaptooctanoic acid (MOA) showed two remarkable voltammetric peaks representing the oxidation potentials of DA and UA with a potential separation of 260 mV. Using amperometric measurement, the responsive current of Au-DT/MOA linearly depends on DA over the range of 0.01–5 μM with a correlation coefficient of 0.999 and more importantly the sensitivity is 20-fold of the MOA coated electrode. Other important features include a low detection limit of 0.02 μM for DA at a signal to noise ratio of 3 and resistance to UA interference, showing no responsive currents for UA additions and no obvious decrease on the sensitivity of DA detections in the presence of UA. Moreover, the performances of our designed electrode, such as a good sensibility, recovery and reproducibility, were successfully validated for detecting DA in non-diluted human urine with satisfactory results. The developed electrodes present more attractive features than conventional options for the urinary DA monitoring.
論文目次 中文摘要..................................................i
Abstract................................................iii
誌謝......................................................v
Contents.................................................vi
List of Figures and Tables...............................ix

Chapter 1 Introduction....................................1
1.1. Introduction to Parkinson’s disease and treatments................................................1
1.2. Concepts of electrochemical techniques............2
1.2.1 Voltammetry.......................................3
1.2.2 Amperometry.......................................5
1.3. Significance and manipulation of DA monitoring....6
1.3.1 Immunohistological staining and functional imaging techniques................................................7
1.3.2 In vivo DA detections with microdialysis..........8
1.3.3 In vivo DA detections with electroanalytical methods...................................................9
1.4. Applications of modified techniques in DA sensing electrodes...............................................11
1.4.1 Self-assembled monolayer (SAM).................12
1.4.2 Nano-gold materials............................13
1.5. Motivation and the aims of this study............14

Chapter 2 Materials and Methods..........................18
2.1. Au-NP/MUA modified electrodes....................18
2.1.1 Electrode preparation and modification............18
2.1.2 Evaluation of sensitivity and selectivity.........19
2.2. Au-NP/SAM modified microelectrodes...............19
2.3. Characteristics of in vitro DA detections with Au-NP/SAM microelectrodes................................21
2.4. In vivo DA recording with Au-NP/MPA microelectrodes..........................................22
2.4.1 Design of micro-electrical stimulator and stimulating electrode....................................22
2.4.2 Surgical procedures and system setup for in vivo DA recording................................................23
2.5. Preparation of Au-DT/SAM modified electrodes.....25
2.6. Characteristics of in vitro and urinary DA detections with Au-DT/SAM electrodes.....................26

Chapter 3 Results and Discussion.........................28
3.1 Au-NP/MUA modified macroelectrodes..................28
3.1.1 Validation of Au-NP electrodeposition.............28
3.1.2 Characterizations of specific DA detections with Au-NP/MUA................................................31
3.1.3 Electrocatalytic effects in DA detections with Au-NP/MUA................................................33
3.2 Au-NP/SAM modified microelectrodes..................35
3.2.1 Electrochemical response of Au-NP modified microelectrodes..........................................35
3.2.2 Study of specific DA measurements with different structures of SAM........................................38
3.2.3 Sensitivity of amperometric DA detections Au-NP/MPA modified electrodes......................................40
3.2.4 Anti-fouling effect through MPA modification......42
3.2.5 Comparison for selectivity of amperometric DA detections at Au-NP and Au-NP/MPA modified microelectrodes..........................................44
3.2.6 DA recording in aCSF and in cerebral striatum of anesthetized rats........................................47
3.2.7 Effects of electrical stimulations with various conditions for DA efflux.................................48
3.3 Au-DT/SAM modified macroelectrodes..................51
3.3.1 Construction of nano-gold structures..............51
3.3.2 Characteristics of nano-gold structures...........55
3.3.3 Electrochemical characteristics of DA redox at nano-gold deposited electrodes...........................60
3.3.4 Investigation of SAM immobilization by X-ray photoelectron spectroscopy...............................61
3.3.5 Characteristics of sensitivity and selectivity for DA detections at different structures of SAM.............64
3.3.6 Electrochemical behaviors and impedances at different structures at different structures of SAM......68
3.3.7 Characteristics of amperometric DA detections with Au-DT/MOA modified electrodes............................73
3.3.8 Specific DA detections using Au-DT/MOA modified electrodes with amperometric method......................75
3.3.9 Determinations of DA in human urine samples.......77

Chapter 4 Conclusions....................................80

References...............................................81

Appendix.................................................91
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