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系統識別號 U0026-2706201410290500
論文名稱(中文) 應用電阻抗量測與電路模型於可攜式單細胞分析儀器系統
論文名稱(英文) Battery-powered portable single-cell analysis instrument systems using electrical impedance measurement and electrical circuit modeling
校院名稱 成功大學
系所名稱(中) 電機工程學系
系所名稱(英) Department of Electrical Engineering
學年度 102
學期 2
出版年 103
研究生(中文) 蔡松霖
研究生(英文) Sung-Lin Tsai
學號 n28981476
學位類別 博士
語文別 英文
論文頁數 74頁
口試委員 召集委員-許藝菊
口試委員-李順來
口試委員-陳明坤
指導教授-張凌昇
口試委員-楊慶隆
中文關鍵字 單細胞分析  介電泳  MEMS  細胞模型  細胞捕捉電極  可攜式阻抗分析儀 
英文關鍵字 Single-cell analysis (SCA)  Electric cell-substrate impedance sensing (ECIS)  Electrochemical impedance spectroscopy (EIS)  Dielectrophoresis (DEP)  Alternating-current electrothermal (ACET)  HeLa (human cervical epithelioid carcinoma)  Analytical modeling method  Extracellular fluid (ECF)  Finite element method (FEM)  Coplanar electrode 
學科別分類
中文摘要 生物晶片的發展漸趨成熟,越來越多的研究投注於其上。現今,生物科技的應用已經是相當地普遍。一般的生物分析著重在研究切片或活體組織。可是,光靠切片和活體組織的檢查實在無法呈現詳細的生理狀態。因此,為得到較詳細的生理資訊,研究人員已經將研究目標進一步轉向細胞的層級。傳統的細胞檢驗所檢測出細胞參數是很多細胞的平均值,無法正確地代表任何一個單一細胞的反應結果,甚至會模糊了各個細胞對同一環境可能產生的差異性。為瞭解複雜的細胞反應,必需要測量單一活體細胞。在過去數年裡,許多分析群體細胞的技術已有相當成熟的發展,而且其分析群體細胞的技術也已用在檢測像是癌症和惡性腫瘤之類的疾病。然而,在許多疾病發展的早期階段,只有非常少部份的細胞是呈現異常的狀態。但是將許多細胞一視同仁的檢查方法可能會忽略個別細胞的差異,進而使得臨床的診斷報告顯示錯誤的資料。因此,為解決這個問題,研究人員開始進行單一細胞的研究分析。分析單細胞可以確認個別細胞實際上的生理和物理機能。也因為如此,對於醫學和生物學領域而言,單一細胞的分析方法嚴然已成為主流。
細胞的特性可以利用視覺、化學或電學的方式來進行觀察與分析。在近幾年,細胞電阻抗分析技術發展相當快速,它提供了一個簡單、快速和非侵入式的方法檢測活體細胞。細胞電阻抗分析法是利用細胞的電特性,如導電度和介電常數等,來描述其生理上的狀態。而且細胞電阻抗分析可利用建立細胞模型方式來幫助我們更深入的了解細胞的各項物理和生物特性。因此,在醫療、藥品開發與環境檢測等需要即時檢測或大量資料分析應用時,細胞電阻抗分析的快速與高靈敏度特性具有相當的優勢。此外,利用MEMS技術來量測分析單一細胞阻抗的導電度與介電常數的變化可以幫助我們去瞭解細胞的狀態(是否有病變與否)。此檢測方法縮短了檢驗所需的時間,對於臨床上的快速疾病檢驗具有極大的潛力。因此,單一活體細胞晶片不僅解決了傳統方法的缺點,而使得生物學家能更進一步地了解細胞內複雜的反應過程,而有能力回答目前使用傳統方法無法回答出的一些生物問題。
有鑑於此,本論文提出一個完整的單細胞分析模型,用於探討電極造成的影響以及調查細胞於電場下的影響等基礎研究,對於暴露在電場下的細胞,也會做一詳細的模擬與分析,期望了解細胞內受影響的一些參數變化,以利後續測量細胞時的電性分析。最後利用電阻、電容與電感等電路元件,建立完整的細胞阻抗量測模型,再與獨立出來的量測電極配合形成一個完整的晶片系統。為了計算模型中的每個電路元件參數,本論文也提出了獨一無二的細胞外流體校正因子(Extracellular fluid volume (ECFV) correction factor)用以輔助計算頻率範圍1-101kHz的模型參數,進而求得每個參數的解析解。最後依照細胞特性分析出適用的頻率範圍。
然而,在針對單一細胞研究分析之前,單一細胞之控制及捕捉即成為首要之工作。目前常用的單細胞捕捉技術可大致分為機械式捕捉、光學式捕捉、與電性式捕捉,其中最為常見的就是機械式捕捉,例如使用微米等級的物理結構將細胞攔截或限制在某特定範圍都算是機械式捕捉,有著製作簡單與直觀的優勢,但物理性的結構常常需要接觸細胞,並且結構一經固定就無法改變,有時甚至還會影響到後續測量的結果。另一種技術是光學式,其原理為利用光壓控制微米大小的物體,將其移動到需要的位置,然而操作過程中使用的能量往往太強,會使被操作物體的熱量太高,容易傷害生物組織。在本研究中,使用捕獲細胞的技術是電性式,其原理是利用微電極產生非均勻電場製造電熱流與介電泳效應來引導細胞朝向特定的區域,其特點是不需接觸細胞即可進行捕捉,進行測量的區域也較少多餘的物理結構造成阻礙。
儘管單細胞電阻抗的檢測發展地相當快速,但其細胞捕捉與量測還是需要外接其他市售的大型儀器才能進行分析。因此,單細胞電阻抗分析的發展會受到儀器的限制,因為這些儀器笨重又昂貴。所以,將單細胞捕捉與檢測儀器整合成一個微小的可攜式系統對單細胞電阻抗分析會有相當大的助益。因此,本論文提出了一個完整的可攜式單細胞分析儀器系統,包含一個通用規格的5伏特鋰聚合物電池、細胞捕捉電路、阻抗量測電路、手持式顯微鏡以及細胞捕捉與量測生物晶片。對於單細胞的捕捉與測量情形,本論文利用有限元素分析軟體COMSOL來模擬細胞所處環境中的電場與電流分佈。並且分析頻率1-101kHz之間的細胞阻抗特性,依照細胞的特性,選定電壓0.4Vpp與頻率11-101kHz之間作為儀器的適用量測範圍,同時,也提出了一系列的校正方法,確保阻抗量測系統的準確度與可靠信與市售的商用阻抗分析儀相同。最後,提供了人類子宮頸癌細胞(HeLa cell)的捕捉與阻抗量測結果。
英文摘要 Impedance measurements provide basic electrical properties and are used to analyze the characteristics of electrochemical materials for biomedical applications. In the present study, the battery-powered portable instrument system for single-cell trapping and analyses is developed. A method of alternating-current electrothermal (ACET) and dielectrophoresis (DEP) are employed for the cell trapping and the method of impedance spectroscopy is employed for cell characterizations. The proposed instrument (160 mm × 170 mm × 110 mm, 1269 g) equips with a highly efficient energy-saving design that promises approx. 120 hours of use. It includes an impedance analyzer performing an excitation voltage range at a voltage range of 0.2-2 Vpp and a frequency sweep of 11-101 kHz, a function generator with the sine wave output at an operating voltage of 1-50 Vpp and a frequency of 4-12 MHz, a cell-trapping biochip, a microscope, and an input/output interface. The biochip for the single cell trapping is designed and simulated based on a finite element method (FEM). In order to improve measurement accuracy, the curve fitting method is adopted to calibrate the proposed impedance spectroscopy. The measurement results from the proposed system are compared with the results from a precision impedance analyzer. Moreover, an analytical modeling method is used to calculate the cytoplasmic resistance, cell membrane capacitance, medium resistance and medium capacitance. Many advantages are offered in the proposed integrated instrument system such as the small volume, real-time monitoring, rapid analysis, low cost, low-power consumption and portable application.
The extracellular fluid (ECF) in microfluidic devices greatly affects the accuracy of impedance measurements of cells. When a single cell is placed in large amounts of ECF, the electric current mostly passes through the ECF, not the cell. Hence, this work presents the modeling method for eliminating the effect of ECF in coplanar impedance sensors. The method is demonstrated using numerical and analytical solutions. The proposed modeling method uses fundamental formulas of circuits that include the electrical parameters of the ECF, cytoplasm, and cell membrane. Equivalent circuit models for the coplanar impedance sensor are established to simulate the impedance as well as the measured ones for excitation frequencies in the range of 11-101 kHz. For a single HeLa (human cervical epithelioid carcinoma) cell, the impedance magnitude decreases from 17.91 to 4.03 kΩ and the impedance phase increases from -72.28º to -59.04º at 0.4 Vpp in 11-101 kHz. According to the calculation result using the proposed modeling method, the cytoplasmic resistance, membrane capacitance, medium resistance, and medium capacitance of HeLa cell are 9.3 kΩ, 180.6 pF, 23.7 kΩ, and 265.6 pF, respectively. Moreover, the electric current distribution in the coplanar impedance sensor is investigated using FEM simulation software. The variation in the impedance during measurements with the simultaneous application of an alternating-current (AC) voltage amplitude of 0.4 Vpp in the fluid volume range of 9-144 μL is also studied. Many advantages such as the portable application in remote areas, the small volume, the real-time monitoring, the rapid analysis, the low cost and the low power consumption are offered in the integrated system.
論文目次 ABSTRACT (CHINESE)...III
ABSTRACT (ENGLISH)...VI
ACKNOWLEDGEMENT...IX
CONTENT...XI
LIST OF TABLES...XII
LIST OF FIGURES...XIII
CHAPTER 1 INTRODUCTION...1
1.1 Background and motivation...1
1.2 Organization of thesis...5
CHAPTER 2 FUNDAMENTAL PRINCIPLES...7
2.1 Extracellular fluid volume correction factor...7
2.2 Modeling method...8
CHAPTER 3 MATERIALS AND METHODS...13
3.1 Microelectrode fabrication and impedance measurements...13
3.2 Cell preparation...14
3.3 Finite element method simulation...15
CHAPTER 4 BATTERY-POWERED PORTABLE INSTRUMENT SYSTEMS...17
4.1 Hardware fabrication...17
4.2 Software design...25
4.3 Calibration estimation...27
CHAPTER 5 RESULTS AND DISCUSSION...29
5.1 Cell-trapping electrodes...29
5.2 Electric current distributions...33
5.3 Extracellular fluid volume correction factor...36
5.4 Impedance variation...38
5.5 Validation of proposed modeling method...40
5.6 Instrument calibration...45
5.7 Single-HeLa-cell measurements...50
5.8 System advantages and limitations...55
CHAPTER 6 CONCLUSION...60
REFERENCE...64
PUBLICATIONS...71
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