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系統識別號 U0026-2907201516574400
論文名稱(中文) 發展無線電化學量測與光刺激系統模組應用於光基因遺傳動物
論文名稱(英文) Development of Wireless Sensing and Optical Stimulation Module for Optogenetic Animal Study
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 103
學期 2
出版年 104
研究生(中文) 劉孟君
研究生(英文) Meng-Chun Liu
學號 P86024077
學位類別 碩士
語文別 英文
論文頁數 57頁
口試委員 指導教授-陳家進
口試委員-張憲彰
口試委員-方佑華
口試委員-謝宗勳
口試委員-曲桐
中文關鍵字 快速循環伏安法  碳纖維微電極  多巴胺  光刺激  無線 
英文關鍵字 Fast-scan cyclic voltammetry  Carbon fiber microelectrode  Dopamine  Optical stimulation  Wireless 
學科別分類
中文摘要 對特定的神經細胞作專一性地光刺激,被稱為光遺傳學,其能藉由不同波長的光選擇性地控制具有光基因修飾的神經細胞。此外,光刺激對於特定細胞能提供空間上的特異性,並能緩和電刺激干擾的影響。在本研究中,我們發展一微型化模組應用於光基因遺傳大鼠,此模組包含著具無線調控刺激參數功能以興奮多巴胺神經之光基因刺激系統,並結合以快速循環伏安法之原理研發無線電化學紀錄系統即時監控位於大鼠腦內黑質體多巴胺訊號的變化。除了能無線地控制光基因刺激系統外,其能耦合光纖傳送藍光(470 nm)進入老鼠腦內進行光刺激以誘發多巴胺的釋放。對於即時監測瞬間變化的多巴胺,快速循環伏安法能提供較好的時間檢析度以及造成較小的組織損傷,由於結合碳纖維自製微電極(7 μm)。最後,所量測之局部場電位訊號以及多巴胺訊號,能經由無線模組單向傳輸至遠處的接收端。另外,藉由修飾Nafion膜於微電極表面以提升對多巴胺靈敏性和選擇性,結合快速循環伏安法進行體外試驗來驗證系統。根據多巴胺反應電流相對於多巴胺濃度的檢量線,顯示線性工作區為10 nM ~ 640 nM、相關係數為0.999、低檢測極限為20 nM (訊雜比= 3)等特點。為證實電化學紀錄系統能即時性的偵測光刺激所誘發的多巴胺,藉由不同光刺激參數刺激具有ChR2基因表現的神經母細胞瘤細胞誘發多巴胺之釋放,觀察出多巴胺反應電流訊號與刺激參數具有相關性。在動物實驗方面,應用光刺激模組進行誘發訊號,並由無線模組單向傳輸至電腦端做訊號分析。透過以上實驗可證實所設計的無線微型化感測器及光刺激模組對於光刺激誘發多巴胺以及電生理訊號的即時監測具有可行性,未來可整合成為特殊用途晶片應用於帕金森氏症動物模型的新穎治療與療效探討,以釐清皮質可塑性至多巴胺神經傳遞路徑所產生的調節性療效。
英文摘要 Optical stimulation of target neurons, known as optogenetics, is an effective method to selectively control the genetically-modified neurons using different wavelengths of light. The optical stimulation can provide spatial specificity for target cells and alleviated electrical artifacts. In this study, we develop a wireless sensing and optical stimulation module which can wirelessly control the parameters of optical stimulation for exciting dopaminergic neurons and provide fast-scan cyclic voltammetry (FSCV) for detecting the phasic dopamine (DA) release in the dorsal striatum of rat. The optical stimulator system can be remotely controlled to deliver blue light (470 nm) in the brain via optical fiber to evoke DA releasing. FSCV provides higher time resolution for measuring transient dopamine changes and causes less tissue damage by using implantable carbon fiber microelectrodes (7 μm). The measured local field potential and dopamine signals are unidirectionally transmitted from the wireless FSCV module to the host unit. Our data showed that the carbon fiber microelectrode coated with Nafion can increase the electrode sensitivity and improve the selectivity of DA detections. We also observed that the responsive current were increased linearly in proportion to DA concentrations (0.01-0.64 uM) with a correlation coefficient of 0.999, and the detection limit is 20 nM (S/N = 3). The DA sensing module was verified in in-vitro cellular level under different parameters of optical stimulation. In an in-vivo test, the wireless system delivered the blue light for exciting local field potentials (LFP) which is transmitted to the host via a wireless transmitter. The occurrence of measured LFPs was in synchronizing with the optical stimulation which was averaged to show the LFP template. The developed wireless system is proven to be a useful experimental tool for the continuous monitoring of LFPs and DA. A miniature FSCV module might be feasible using ASIC approach in the future. The proposed wireless system will be extended as an experimental platform for optogenetic stimulation of Parkinson’s disease animal model.
論文目次 中文摘要...i
Abstract...ii
致謝...iv
Contents...v
List of Figures...vii
Chapter 1 Introduction...1
1.1 Introduction of Parkinson’s disease and treatment..1
1.2 The techniques of brain stimulation...2
1.2.1 Repetitive transcranial magnetic stimulation...2
1.2.2 Transcranial direct current stimulation...3
1.2.3 Optical stimulation...4
1.3 Significance and manipulation of DA monitoring...5
1.3.1 In-vivo DA detections with microdialysis...5
1.3.2 In-vivo DA detections with electroanalytical methods...6
1.4 Approaches of electrochemical measurement...7
1.4.1 Voltammetric techniques...7
1.4.2 Fast-scan cyclic voltammetry...9
1.5 Motivation and the aims of this study...10
Chapter 2 Materials and Methods...11
2.1 Design of DA sensing module...11
2.2 Characterizations of in-vitro DA detection with carbon fiber microelectrodes...13
2.3 Validation of DA sensing under optical stimulation for in-vitro study...14
2.3.1 Electrochemical recording systems setup...14
2.3.2 Optical stimulator set up for in-vitro study...16
2.3.3 Detection of dopamine release in cellular level under optical stimulation...16
2.4 Development of wireless integrated system for the optogenetic animal...19
2.4.1 Fabrication of optrode...19
2.4.2 Wireless optical stimulator set up...20
2.4.3 Animal preparation and surgical procedure for in vivo ECoG recording...21
Chapter 3 Results and Discussion...23
3.1 Development of DA sensing module...23
3.1.1 The performance of carbon fiber microelectrode by FIB fabrication...23
3.1.2 The structure and the electrochemical current response of DA sensing...25
3.2 The characterizations of FSCV dopamine detections with bare/Nafion coated electrodes...26
3.2.1 The sensitivity of bare/Nafion coated electrodes...26
3.2.2 The selectivity of bare/Nafion coated electrodes...28
3.3 Validation of DA sensing under optical stimulation for in-vitro study...29
3.3.1 Validation of electrochemical recording system...29
3.3.2 The development of optical stimulator for in-vitro study...30
3.3.3 Detecting the extracellular dopamine under optical stimulation...31
3.4 Development of wireless integrated system for the optogenetic animal...33
3.4.1 The structure of optrode...33
3.4.2 Validation of miniature wireless sensing and optical stimulator module...34
3.4.3 Local field potential signal recording in rat...38
Chapter 4 Conclusion...39
References...40
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