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系統識別號 U0026-0812200915072588
論文名稱(中文) 電化學式單細胞分泌即時監測系統之開發
論文名稱(英文) Development of an Electrochemical Cellular Chip for Real-time Monitoring of Single-cell Secretion
校院名稱 成功大學
系所名稱(中) 醫學工程研究所碩博士班
系所名稱(英) Institute of Biomedical Engineering
學年度 97
學期 2
出版年 98
研究生(中文) 張景裕
研究生(英文) Ching-Yu Chang
電子信箱 p8894101@mail.ncku.edu.tw
學號 p8894101
學位類別 博士
語文別 英文
論文頁數 89頁
口試委員 口試委員-鄭國順
口試委員-謝奇璋
口試委員-沈孟儒
口試委員-末永智一
口試委員-林奇宏
口試委員-楊重熙
召集委員-湯銘哲
口試委員-黎煥耀
指導教授-張憲彰
中文關鍵字 單細胞操控  單細胞檢測  微小電極  分泌性鹼性磷酸酶(SEAP)  組織胺  細胞生物電化學  胞吐作用  氧化活性物  骨髓過氧化酶(MPO) 
英文關鍵字 single-cell manipulation  reactive oxygen species (ROS)  myeloperoxidase (MPO)  exocytosis  single-cell analysis  ultramicroelectrode  microbioelectrochemistry  histamine  secreted alkaline phosphatase (SEAP) 
學科別分類
中文摘要 本研究嘗試開發一種可供單細胞操控與分析的技術,針對此一需求我們提出了微孔電極(micro well electrode, MWE)的設計概念,並以微製程技術成功製造出適合單細胞研究的微孔電極晶片。此晶片主要由白金電極、SU-8光阻層(25 微米厚)、雙面膠布(30微米厚)以及導電玻璃(Indium Tin Oxide, ITO)等組合而成,其中微孔電極的大小,係由構築在SU-8光阻層上的微孔結構所定義。藉由微孔電極與其上方導電玻璃的電壓操控,可在微孔電極開口處形成一股與管道垂直的電泳力(electrophoretic force),配合管道內的水平流力(hydraulic force),成功將單一細胞捕捉在微孔電極內。透過數值模擬,我們發現直徑30 微米且深度25微米的微孔電極,可在孔洞底部提供一深度約14微米的低流速區(low flow velocity area, LFVA),藉以安定被捕捉的細胞,使其在流動的管道中依然可以安穩停留。此一設計結合了現行主動以及被動操控的優點,成功將捕捉電位操控的時間縮短至5秒以下,進而降低了電場操控對細胞可能造成的傷害。
依據超微小電極(ultramicroelectrode, UME)理論,以及個別微孔電極在已知濃度的K3Fe(CN)6溶液下所測得的穩態電流,計算發現微孔電極的有效半徑與設計數值相符。此外,藉由導電玻璃貼附與移除後所測得之伏安圖(voltammogram)的比較,因導電玻璃所造成的電流放大效應獲得證實,但因導電玻璃與微孔電極間的距離,無法藉由雙面膠布精確控制或量測,致使所得之放大倍率與理論數值有所出入。利用具有鹼性磷酸酶(alkaline phosphatas, ALP)修飾表面的乳膠微粒(10 微米直徑),進一步驗證微孔電極對於微量催化反應的檢測能力。發現在微孔電極偏壓+0.3 V條件下,修飾微粒在含有p-aminophenylphosphate (PAPP)溶液中所測得之電流明顯較未修飾微粒高。此一結果證明微孔電極之偵測靈敏度,可滿足微量細胞分泌物質檢測之需求。
針對一般細胞可能分泌的酵素或電化學活性物質,本研究以帶有分泌性鹼性磷酸(secreted alkaline phosphatase, SEAP)轉殖基因之子宮頸癌細胞(pSEAP-HeLa)、癌化嗜鹼性球(KU-812)以及分離自人體週邊血液的嗜中性球等三種細胞模型,驗證微孔電極之單細胞檢測能力。結果顯示(1)在微孔電極偏壓+0.3 V條件下,在含有4.7 mM PAPP溶液中,觀測到pSEAP HeLa細胞分泌之鹼性磷酸酶所產生的波浪狀的電流響應。 (2)在微孔電極偏壓+0.6 V條件下,以含有145 mM 鉀離子溶液刺激KU-812細胞,成功觀測到許多閃爍電流(spike current),藉此推測此電流為該細胞因刺激所分泌之組織胺(histamine)信號。(3)以phorbol 12-myristate 13-acetate (PMA)對捕捉在微孔內的嗜中性球進行刺激,在定電位(-0.25 V)以及電位掃引分析下,觀測到嗜中性球所分泌的氧化活性物質(reactive oxygen species, ROS)。
藉由上述實驗結果證實,微孔電極在單細胞操控與檢測應用具有實用價值。同時研究中所發展的微粒子操控技術,若搭配其他具有不同功能的修飾微粒(例如:酵素、抗體或基因探針等)的使用,則可以突破現行修飾方法,在同一微晶片上進行不同物質的定址修飾。此外,若將微孔晶片整合於其他光學顯微鏡上,提供光學與電化學的量測資訊,將可發展出許多有利的單細胞(特別是懸浮型細胞)研究工具。
英文摘要 In this study, we intended to develop a chip which can be used for single-cell manipulation and analysis. A microwell electrode (MWE) which was constructed by sequential microfabrication processes was proposed to meet these requirements. With the cooperation of an Indium Tin Oxide (ITO) cover on the top of the chip, a vertical electrophoretic force was employed to trap/repel the living cells into/out the MWE with a DC voltage biased between MWE and ITO cover. Moreover, a flow channel was defined by a double-side adhesive tape, and thus a hydraulic force was used to remove the additional trapped cells around the MWE. By a cooperation of the electrophoretic and hydraulic forces, an individual cell can be accommodated inside the addressed MWE. According to the simulation results, the depth of the MWE (30 m diameter) was optimized to be 25 m, and then the trapped cell can be stabilized in a sufficient low flow velocity area (LFVA) in a continuous flow. A technique for single-cell manipulation was successfully developed.
The electrochemical characteristics of the MWE were investigated with potassium ferricyanide (K3Fe(CN)6), and then the steady-state current was measured by calculating the difference between baseline and diffusion-limit current in a sigmoid voltammogram. The effective electrode radius was estimated based on the theorem of ultramicroelectrode (UME) with a given constants, known K3Fe(CN)6 concentration, and the measured steady current. The calculated radius was found to be in a good agreement with the design value. A positive feedback phenomenon was observed when the ITO cover was attached onto the MWE, and thus an enhanced current was measured. However, the enhanced factor was not consistent with a theoretical value; the difference was supposed to the uncertainty of the tape thickness. ALP-beads (10 m diameter), with alkaline phosphatase (ALP) coating, was employed to validate the sensitivity of the MWE for trace chemical detection. The amperograms (+0.3 V vs. Ag/AgCl) of a MWE with blank and ALP-bead were compared in p-aminophenylphosphate (PAPP) solution. A larger current response was found in the amperogram of ALP-bead, and the elevated current suggested an evidence of trace catalytic reaction by the ALP-bead. The sensitivity of the MWE to a trace signal was confirmed to be applicable for the detection of single-cell secretion.
The MWE was employed for the living cell detection, and three kinds of cells including pSEAP-HeLa, KU-812, and neutrophil were tested. The related findings and results were illustrated as the followings: (1) The recombinant pSEAP-HeLa cell which can continuously secret the secreted alkaline phosphatase (SEAP) was measured in PAPP solution. The secretion activity was real-time monitored by an amperogram (+0.3 V vs. Ag/AgCl), and a wave-like current was found in the detected amperogram. The secreted SEAP was supposed to catalyze the PAPP substrate to produce the wave-like current, and provided an evidence of cell secretion. (2) KU-812, a human basophilic cell line, was employed for the exocytosis detection. The exocytosis of histamine vesicles was induced with high K+ solution, and then a spiked amperogram (+0.3 V vs. Ag/AgCl) was detected. (3) Primary neutrophils which were isolated from peripheral blood of volunteers were measured with the MWE to detect the reactive oxygen species (ROS) and myeloperoxidase (MPO) after phorbol 12-myristate 13-acetate (PMA) stimulation. Amperogram of the activated neutrophil was monitored in phosphate buffer saline (PBS) solution, and the ROS release was confirmed by the spiked current. Voltammograms and amperograms of the activated neutrophil in 3, 3’, 5, 5’-tetramethylbenzidine (TMB) solution verified the MPO release. A significant redox current was observed in the voltammogram of activated cells, and several reductive spiked current appeared in the amperogram when the MWE was biased at -0.25 V.
The MWE device presents some feasible applications for the single-cell manipulation and analysis. The manipulating technique can be employed to address the functionalized beads which were coated with enzyme, antibody, or DNA on a specific position, and then the problem of addressable modification in conventional method can be solved. Moreover, the MWE provides an effective method for the preparation of cell array. This device also demonstrates possibilities to integrate the chip with a confocal microscopy, and the combined technology might provide a simultaneous detection of intra- and extracellular species.
論文目次 Chapter 1 Introduction ..........................................................................................................1
1.1 Aim of This Study ..................................................................................................1
1.2 Single-cell Analysis ................................................................................................2
1.3 Methods for Single-cell Analysis ...........................................................................3
1.4 Strategy for Single-cell Microelectrochemistry .....................................................6
1.5 Cell Secretion .........................................................................................................8
1.5.1 Rease through the Exocytosis Mechanism .................................................8
1.5.2 Release of Membrane Permeable Species ................................................10
1.6 Microelectrochemistry Used for Cell Secretion Detection...................................12
1.7 The Manipulation of Microparticles.....................................................................15
Chapter 2 Theory and Methodologies ................................................................................17
2.1 Design of MWE....................................................................................................17
2.2 Theory of Microelectrode with a Recessed Tip....................................................18
2.3 Positive Feedback of MWE on an Unbiased Conductive Substrates ...................21
2.4 Single Bead/Cell Manipulation ............................................................................25
2.5 The Cells Used for Cell Secretion Study..............................................................27
2.5.1 HeLa Cells Transfected with Recombinant pSEAP Gene........................27
2.5.2 Detection of Histamine Release from KU-812 Cell.................................29
2.5.3 Detection of ROS and MPO Release from an activated neutrophil .........29
Chapter 3 Materials and Experiments ...............................................................................34
3.1 Equipments ...........................................................................................................34
3.2 Materials ...............................................................................................................34
3.2.1 Chemicals for Microfabrication................................................................34
3.2.2 Chemicals and Buffers for Analysis .........................................................34
3.2.3 Reagents for Cell Culture and Treatment .................................................35
3.2.4 Test Solutions ...........................................................................................35
3.3 Simulation of Flow Field inside a MWE..............................................................36
3.4 Chip Fabrication ...................................................................................................36
3.5 Validation of MWE Characteristics......................................................................38
3.6 Preparation of Functional Beads and Its Validation .............................................39
3.7 Cells Culture and Treatments ...............................................................................40
3.8 Characterization of TMB and MPO with Pt Electrode.........................................41
3.9 Single-cell Measurement by Using the MWE......................................................42
3.9.1 Measurement of pSEAP-HeLa Cell .........................................................42
3.9.2 Measurement of KU-812 Cell ..................................................................43
3.9.3 Measurement of Neutrophils ....................................................................43
Chapter 4 Results and Discussion .......................................................................................44
4.1 The Optimal MWE Design...................................................................................44
4.2 Electrical Field inside the MWE ..........................................................................48
4.3 Entrapment of Bead/Cell inside MWE.................................................................51
4.4 Electrochemical Characteristics of the MWE ......................................................53
4.4.1 Comparison of the MWE and UME Based on the Voltammograms ........53
4.4.2 Effect of Well Depth.................................................................................54
4.4.3 Current Amplification by the Redox Recycling Effect of ITO Cover......58
4.5 Validation of the Detecting Capability of MWE ..................................................60
4.5.1 Confirmation of ALP Immobilization on the Microbeads........................60
4.5.2 Electrochemical Measurement of the Single ALP Bead...........................61
4.6 Detection of SEAP Secretion from a pSEAP-HeLa Cell .....................................64
4.7 Detection of Histamine Release from the KU-812 Cells .....................................66
4.8 Detection of Neutrophil Secretion........................................................................68
4.8.1 Voltammogram of TMB/MPO Reaction ..................................................68
4.8.2 Detection of ROS Release by the Activated Neutrophil...........................72
4.8.3 Voltammogram of the Activated Neutrophils with the MWE ..................74
4.8.4 Real-time Monitoring of a Single Activated Neutrophil ..........................76
Chapter 5 Summary and Prospects ....................................................................................79
Chapter 6 Reference .............................................................................................................82
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