單一細胞濃度等級之電泳/電層析感測晶片平台開發—類神經細胞之電流應答分析

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研究生：	吳仁貴 Wu, Ren-Guei
論文名稱：	單一細胞濃度等級之電泳/電層析感測晶片平台開發—類神經細胞之電流應答分析 Development of Chip-Based Electrophoresis/Electrochromatography Platform for Amperometic Sensing and Analyses of PC-12 Cells Response at Single Cell Releasing Level
指導教授：	曾繁根 Tseng, Fan-Gang
口試委員:
學位類別：	博士 Doctor
系所名稱：	原子科學院 - 工程與系統科學系 Department of Engineering and System Science
論文出版年：	2009
畢業學年度：	98
語文別：	英文
論文頁數：	131
中文關鍵詞：	單一細胞分析、毛細管電泳/電層析晶片、多壁奈米碳管分離管柱、電動力濃縮技術、奈微米流體系統
外文關鍵詞：	Single cell analysis, Electrokinetic preconcnetration, MWCNTs nanocolumn, Micro/Nano-fluidic system, Electrophoresis / electrochromatography chip
相關次數：	點閱：4 下載：0
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近年來在單細胞檢測與分析的領域裡，已由多種類細胞間的反應分析，轉為細胞內胞器或細胞體液的反應分析。另外，也從破壞細胞本體取得樣本的方式，轉為活體細胞即時釋放檢測收集的方式。但不論採用何種方式都會遇到樣本量不足(zmole)造成濃度過低難以檢測，或是體積量太少(pL)難有適當的操控介面造成樣本流失，因此一即時濃縮與收集的技術發展便是一大關鍵。同時，唯有將操控尺度微小化以及具有高分離效率與高靈敏的檢測分析技術，方可達成單一細胞研究之目的。
故本研究主要目的為建構一可檢測至單一細胞濃度等級之晶片式細胞分子操控分析平台，用來觀察與量測在細胞受到外加的物理性與化學性的刺激後，所釋放出的訊息分子的成分與數量。研究中，利用從老鼠體內培養出的類神經細胞(PC-12 Cell)來當做研究樣本，該細胞可分化出神經細胞具有的功能性特徵(軸突與突觸)，同時在接受到外在刺激之後會釋放出神經傳導因子。與傳統必須溶解細胞取得大量生物樣本且只著重在捉取、培養或是檢測訊號的生物晶片不同的是，本晶片平台的優勢為在不須溶解細胞的情況下，發展成高分離效率與高靈敏的檢測技術之微全分析系統，並具有對極微量的生物分子進行純化、收集、濃縮、分離與檢測等多功能處理系統。
在本研究中發展了三各不同功能的晶片系統，並進行整合為一操控分析平台，分別為純化與收集晶片，濃縮與分離晶片以及檢測晶片。實驗結果顯示，利用此整合晶片平台可達成一具有高解析效能的分離理論板數(3.8×105 plants/m)、高效率的樣本濃縮能力(105 folds in 15min)以及超微量的檢測濃度極限值(1.6 zmol, 10-21)之分析系統。未來可望能夠應用於在單一細胞之間內所釋放之微量(ng-pg)蛋白質、DNA以及神經傳導因子等生物分子的操控與分析。

CONTENTS

ABSTRACT…………………………………………………………………….………..…I
ABSTRACT IN CHINESE…………………………………………………….….…….IV
ACKNOWLEDGMENTS……………………………………………………………….VI
CONTENTS…………………………………………………………………….……….VII
LIST OF FIGURES………………………………………………….……………………X
NOMENCLATURE………………………………………………..……….………….XIV

CHAPTER I
Introduction for Single Cell Analysis....………………………………………………1
1.1 Rationale for Single Cell Analysis………………………………………………….2
 1.1.1 Background and Development..………...….……………….…………...........2
 1.1.2 Specifications and Challenges………………………..………….…………....6
1.2 Single-Cell Manipulation………..…………………………………..……………...8
   1.2.1 Sampling and Filtration.………...………………….…….……..…………….8
   1.2.2 Preconcentration………………………….………………………………….11
1.3 Single-Cell Analysis……………………………………………………………….13
   1.3.1 Fluorescence Method....…………………..……………….…….…………..13
   1.3.2 Amperometry Method……………………….………………………………14
   1.3.3 Mass Spectrometry Method………….………………………………………15
CHAPTER II
Concept and Methodology of Thesis………………………………..……….………20
2.1 Construction of Sampling and Filtration……….………..………………...…...27
   2.1.1 Capillary Electrochromatography (CEC)……………………………………28
2.2 Construction of Preconcentration and Ionic Separation………….…….……..31
      2.2.1 Characterization of Nanofluidic Preconcentraton…………………………32
2.3 Construction of Restacking and Detection………………………………….……..35
      2.3.1 Mass Transfer and Diffusion Controlled Electrochemical Reaction………36
CHAPTER III
   Chip-Based Capillary Electrochromatography (μCEC) Consist of Vertically
   Aligned Carbon Nanotubes (CNTs) Nanostructured Pillars for Single Cell
   Sampling………………………………………………………………………………39
3.1 Introduction ………………………………………………………………….…40
3.2 Materials and Methods…………………………………………………………..47
3.3 Results and Discussion………………………….……...….….….……………..52
   3.3.1 Electrophoresis Characteristics of MWCNTs Array Channel……………56
   3.3.2 Capillary Electrochromatography of dsDNAs……………………………59
3.4 Conclusions.………………………………………………..…………….… 62
CHAPTER IV
   Electrostatic Sieving Control of Biomolecules in Nanofluidic Multi-Wall Carbon
   Nanotubes Array Preconcentrator for Single Cell Preconcentration………63
4.1 Introduction……………………………………………………………………..64
4.2 Materials and Methods……………………………………………………..……69
4.3 Results and Discussion…………………………………………………….…….74
 4.3.1 Concentration Polarization at the Nanoporous Size Exclusion Membrane..74
 4.3.2 Electrokinetic Concentration and Separation of Protein….…….………….79
 4.3.3 Electric Field Strength Effects on the Protein Concentration….………….83
4.4 Conclusions…………………….……………………………………...………..86
CHAPTER V
   Dual-Asymmetry Electrokinetic Flow (DAEKF) Focusing for Restacking and
   Analysis of Neurotransmitters at Single Cell Releasing Level in CEEC
   Nanochannels………………………………………………………………………....87
5.1 Introduction……………………………………………………………………..88
5.2 Materials and Methods……………………………………………………..……93
5.3 Results and Discussion…………………………………………………….…….100
   5.3.1 Simulations of the DAEKF Technique in the Nanochannel…...101
   5.3.2 Flow  Characterization  and  Analyte  Concentration  for  the
        DAEKF System………………………......................................................106
 5.3.3 DAEKF  System  for  Separation  and  Electrochemical  Detection
      of Catecholamine...…….……………………………….…………….…110
   5.3.4 Efficiency and Detection Limit of Neurotansmitters by the DEFKE CEEC
        Nanochip……….…………………………………………………………..113
5.4 Conclusions…………………….……………………………………...………..117
CHAPTER VI
Conclusion…………………………………………………………………………...118
    6.1 Overview of Dissertation …………………………………..…………………...119
    6.2 Future Direction …………………………………………….…..…..........….....121
REFERENCES………………….……………………………………………………....122





LIST OF FIGURES
    Page
Fig. 1.1    A schematic representation of the “whole cellomics” research challenge……….5
Fig. 1.2    Generic workflow for single-cell analysis on a microfluidic platform…………..5
Fig. 1.3    (A) Detection of d-Asp in the processes of sensory neurons. (B) (a) SEM images of a microinjector. (b) SEM images of the tip of microinjector (c) Electropherogram of a 280-fL sample injected from a 20 μm diameter PC12 cell obtained using a 770-nm i.d. capillary……………….……………………….10
Fig. 1.4    (A) Membrane schematic and background-corrected images of protein concentration (B) Preconcentration, elution, and separation of proteins in membrane based microchip……………………………………………………..12
Fig. 1.5    (A) Traditional Microdialysis coupled to CE bioreaction chamber with a LIF detector. (B) Electrophoretic separation of the contents of a single cell in a microchannel……………………………………………………………………17
Fig. 1.6    A traditional CE equipped with a bienzyme electrode………………………18
Fig. 1.7    Microfluidic chip integrated with CFNE. (A) Layout of the chip. (B) Configuration of electrophoresis system with an integrated electrochemical detector…………………………………………………………………………18
 Fig. 1.8  Overall design of the coupling of the microwell plate sample delivery system
        equipped with a microdevice for high-throughput separation–MS analysis……19
Fig. 2.1    Schematic of one idealized total-cell analysis device showing the various functions………………………………………………………………………...25
Fig. 2.2    Chip based Micro-Total-Analysis-System (μTAS) for single cell analysis…..25
Fig. 2.3    Fabrication process (a-f) and photographic top view of a multi-functional
        micro/nano-fluidic chip for singlecell analysis…………………………………26
Fig. 2.4    The chip design and HI-CEC for sampling and filtration of biomolecules analysis
        at single cell releasing level………..………………….………………………..30
Fig. 2.5    The chip design and nanofluidic preconcentrator for sample preconcentration and ionic separation of biomolecules analysis at single cell releasing level…..........34
Fig. 2.6    The chip design and nanofluidc redox current ampilified for sample restacking and detection of of biomolecules analysis at single cell releasing level……......38
Fig. 3.1    Schematic setups and images of μCEC chip with vertically aligned MWCNTs nanostructured pillars……………………………………………………….45
Fig. 3.2    Fabrication process (a–f) of an MWCNTs chromatographic column
for μCEC, (g) Photographic top view of the μCEC chip……………………….51
Fig.3.3 IR spectra obtained for the (a) MWCNTs, (b) O2-MWCNTs, and (c)
        PDDA-MWCNTs used for channel inner surface modifications………….55
Fig. 3.4    Influence of pH level on the electrophoretic mobility of the electroosmotic flow
        in pyrex/silicon channels with different modifying condition…………………55
Fig. 3.5    Sample bands moving through (a) the pyrex/silicon channel and (b) the MWCNTs array channel………………………………………………………..58
Fig. 3.6    The μCEC analysis of three dsDNA fragments with different lengths (1: 254 bp, 2: 360 bp, 3: 572 bp) in (a) a pyrex/silicon channel, (b) an O2-MWCNTs array channel, (c) an PDDA-MWCNTs array channel…………………………..........61
Fig. 4.1    Schematic illustration of electrokinetic transport processes (fig. 4.1b) in the microfluidic device containing an in-situ growth, controllable surface charged MWCNTs array preconcentrator (fig. 4.1.a) with a mean pore size comparable to the electrical double layer (EDL) thickness (fig. 4.1c)……………………….. 68
Fig. 4.2    Schematic setups and images of the parylene-MWCNTs preconcentrator
        in electrostatic sieving micro/nano-fluidic chip………………………………73
Fig. 4.3    Parylene-MWCNTs array electrodes were applied a critical voltage for cathodic
        electrostatic sieving is -20V. (I) polarizes field on, (II-III) polarizes field off. This
        device has both the size exclusion and the electrostatic sieving function for Rabbit IgG-FITC and FITC dye.………………………………………………..77
Fig. 4.4    Parylene-MWCNTs array electrodes were applied a critical voltage for anodic electrostatic sieving is +20V. (a) the counterion analyte (2-NBD-Glucose, negative charge), and (b) the coion analyte (Rhodamine 6G) were separatied by electrostatic sieving parylene-MWCNTs preconcentrator……………………...78
Fig. 4.5  Concentration of FITC-labeled BSA with same voltage configuration in figure 4.3. Three different initial concentration samples are presented..………………82
Fig. 4.6    10 nM FITC-labeled protein concentration profile with different applied voltages. The concentrations of protein by different applied voltages are not the same at the same product of applied voltage and time…………………………………85
Fig.5.1  The schematics of (a) flow field evolution for DAEKF in a capillary electrophoresis nanochannel (solid black arrows represent analyte flow direction, and ζ2>ζ1>ζ3>ζ0), and the distribution of analytes in (b) the CE nanochannel with DAEKF, and in (c) the traditional CE nanochannel……………………. 92
Fig. 5.2    Schematics and images of (a) the driving system for the DAEKF CEEC nanochannel chip, (b) the DAEKF and detection region (G: Field-Effect electrodes, W: working electrode, R: reference electrode, C: counter electrode, D: decoupler electrode), and (c) the nanochannel………………………………99
Fig. 5.3    Numerical simulations of the CEEC systems with (a) traditional EOF, (b)
        asymmetric EOF, (c) traditional EOF combined with field-effect control, (d)
        asymmetric EOF combined with field-fffect Control (or DAKEF).….104
Fig. 5.4    Detail flow fields of five different regions (a) are revealed in (b) for DAEKF
        system in nanochannel…………………………………………………………108
Fig. 5.5    Fluorescence image of Rhodamine B for (a) traditional EOF in a nanochannel, (b) the restacking effect by the DAEKF system in a nanochannel, in the five different regions defined in Fig. 5.4 (a)……………………………………… 109
Fig. 5.6    Detection of 1 □M dopamine and catechol in a 500 nm CEEC nanochannel under four different electrokinetic conditions A: asymmetric EOF combined with field-effect control (DAEKF), B: traditional EOF combined with field-effect control, C: asymmetric EOF, D: traditional EOF)…………………………… 112
Fig. 5.7    Electropherograms obtained using a solution of 1.25 nM Dopamine and 3.3 nM catechol in 5mM HEPES buffer in the nanochannel…………………………..115
Fig. 5.8 Demonstration that analytes present at concentrations close to the limit of detection can be analyzed in a cell culture medium. Condition: cell culture medium only, as background noise (gray line); 10 nM Dopamine and catechol in cell culture medium using the DAKEF CEEC nanochannel (black line)……..116

                                

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