Amperometric microbiosensors: an ELISA based electrochemical Estradiol sensor and all-in-one Glutamate sensors on microelectrode arrays

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研究生：	黎玉華 LE NGOC QUYNH HOA
論文名稱：	Amperometric microbiosensors: an ELISA based electrochemical Estradiol sensor and all-in-one Glutamate sensors on microelectrode arrays Amperometric microbiosensors: an ELISA based electrochemical Estradiol sensor and all-in-one Glutamate sensors on microelectrode arrays
指導教授：	曾婷芝 Ting-Chih Tseng
口試委員:	曾婷芝江志強陳崇賢陳建宏鮑致寧
學位類別：	博士 Doctor
系所名稱：	工程學院 - 化學工程系 Department of Chemical Engineering
論文出版年：	2018
畢業學年度：	107
語文別：	英文
論文頁數：	110/121
中文關鍵詞：	Biosensors 、Micromachined electrode array (MEA) 、Electroanalytical Chemistry 、Electrochemical ELISA 、Estradiol 、4-aminophenyl phosphate
外文關鍵詞：	Biosensors, Micromachined electrode array (MEA), Electroanalytical Chemistry, Electrochemical ELISA, Estradiol, 4-aminophenyl phosphate
相關次數：	點閱：207 下載：1
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五十多年來，生物感測器的發展自 Leland C. Clark教授首先提出生物感測器的構想開始，如今生物感測器已被廣泛地應用於生醫診斷、環境監測、食安控制等領域。在大多數的例子裡，生物樣品中的待測分析物通常僅微量地存在於組成複雜的溶液中，且在量測時訊號雜訊通常很高，因此，為了解決此類問題，許多系統性的方法已被用於生物感測器的設計並改善其效能，特別是用以提高感測器的靈敏度與選擇性。由於生物感測器具有低製備成本、操作便利性、高準確度等優點，因此可視為一種感測小分子分析技術之方法。
具良好感測效能與快速感測時間的微型化的生物感測器在生物科學領域的應用具有逐漸升高的重要性，微米/奈米尺寸的可植入型感測探針可降低在植入過程中對組織的損傷，也可增加對於植入點的空間精準度。此外，電化學方法與微機械加工電極元件的結合，可使感測器的快速分析與即時監測成為可能，並使其具有在經濟與成本考量上的可行性，因此，微米/奈米尺寸之生物感測器的研發並使其應用於多樣化的領域中是一種趨勢。
本研究已成功展示出一種具便利性、高選擇性、高靈敏度、快速反應，且具高穩定性的微型化電化學式生物感測器，用以偵測人類賀爾蒙雌二醇與神經傳導物質穀氨酸，並應用於體外與血清樣品的量測。本研究的範圍涵括以矽為基礎的微電極探針製備之微機械加工製程、化學性/電化學性/酵素的或免疫的電極修飾製備、感測器效能的電化學或特性分析，以及體外與血清樣品的測試。本研究的討論聚焦於感測器靈敏度、選擇性、反應時間、感測範圍、穩定性，以及樣品量測可行性的評估。最後，本研究亦建議可利用電沉積法以氧化銥修飾白金電極，用以提供未來更進一步提升感測器靈敏度與良好化學穩定性及熱穩定性之可行方案。

More than 50 years, starting from the first concept of biosensor proposed by Professor Leland C. Clark, biosensors are nowadays utilized ubiquitously in biomedical diagnosis, environmental monitoring, food safety control, etc. In most cases, the target analytes are usually in trace amounts in biological samples with complex compositions and signal noises during measurements are usually high. Thus, biosensors are designed and improved by different systematic methods to overcome these problems; especially, to increase the sensitivity and selectivity of biosensors. Biosensors have the advantages of low frabrication cost, operational converience, and high accuracy and therefore are considered as solutions for analytical techniques for the detection of small molecules.
Miniaturized biosensors with improved detection capabilities and faster response time have increasing importance to nowadays applications in life science. Implantable probes in micro/nano-sized can reduce tissue damages during the implantation process and increase the spatial accuracy targeting the implantation site. The combination of electrochemical methods and micromachined-electrode devices make rapid analysis and real-time monitoring possible with economic feasibility. Thus, the development of micro/nano-biosensors is a trend for numerous applications in diverse fields.
This research has successfully demonstrated convenient, highly selective, sensitive, responsive miniaturized electrochemical biosensors with longer stability for the detection of the human hormone β-estradiol and the neurotransmitter L-glutamate and to apply the sensors in vitro in serum studies. The scope of this research covers micromachining process for manufacturing silicon-based microelectrode probes, chemical/electrochemical/enzymatic or immunogenic modifications for sensor preparations, electrochemical and characteristic analysis for sensor performance, and in vitro and in serum tests. Discussions have been focused on evaluations towards sensor sensitivity, selectivity, response time, detection range, stability, and the feasibility for sample measurements. Further improvement of sensor selectivity with low resistivity and good chemical and thermal stabilities is suggested by electrodepositing IrOx film on Pt microelectrode to promote the electron transfer and increase the sensor stability.

TABLE OF CONTENTS

論文摘要    I
ABSTRACT OF DISSERTATION    II
ACKNOWLEDGMENT    III
INDEX OF FIGURES    VII
INDEX OF TABLES    XI
VITA    XII
PUBLICATIONS AND PRESENTATIONS    XIII
ABBREVIATIONS    XIV
CHAPTER 1. INTRODUCTION    1
1.1    BIOSENSOR OVERVIEW    1
1.2    HISTORICAL BACKGROUND OF BIOSENSING TECHNIQUES    2
1.3    COMPONENTS OF BIOSENSORS AND CLASSIFICATIONS    5
1.4    ELECTROCHEMICAL DIVICES AND PRINCIPLES    10
1.5    TECHNIQUES FOR BIOSENSOR FABRICATION    14
CHAPTER 2. A SIMPLE, SENSITIVE AND COMPACT ELECTROCHEMICAL ELISA FOR ESTRADIOL BASED ON CHITOSAN DEPOSITED PLATINUM WIRE MICROELECTRODES    21
2.1    ELECTROCHEMICAL BIOSENSORS OF ESTRADIOL FOR ESTRADIOL DETECTION    21
2.2    RESEARCH METHOD OVERVIEW    26
2.3    SENSOR FABRICATION    27
2.4    ESTRADIOL BASED ON CHITOSAN DEPOSITED Pt WIRE    32
2.5    CONCLUSION    44
CHAPTER 3. AN ARRAYED MICRO-GLUTAMATE SENSOR PROBE INTEGRATED WITH ON-PROBE SILVER/SILVER CHLORIDE REFERENCE AND COUNTER ELECTRODES    46
3.1    ELECTROCHEMICAL BIOSENSORS FOR GLUTAMATE DETECTION    46
3.2    SENSOR FABRICATION    51
3.3    FABRICATION PROCESS    52
3.4    ELECTROCHEMICAL DETECTION OF GLUTAMATE    58
3.5    CONCLUSION    70
CHAPTER 4. RECOMMENDATIONS FOR FUTURE RESEARCH – APPLICATION OF IRIDIUM OXIDE FOR BIOSENSOR FABRICATION    71
4.1    MOTIVATION    71
4.2    INTRODUCTION    71
4.3    EXPERIMENTAL    74
4.4    ELECTROCHEMICAL DETECTION OF H2O2    76
4.5    POSSIBLE PROBLEMS AND PROPOSED SOLUTIONS    79
REFERENCE    80
APPENDIX    94

INDEX OF FIGURES
Figure 1.1 The number of biosensor publications by searching on Scopus with the keyword "biosensor".    2
Figure 1.2 A schematic diagram showing the basic working principle a biosensor system [64].    6
Figure 1.3 The schematic surface area of an electrode [80].    12
Figure 1.4 Variables in an electrochemical setup [81].    14
Figure 1.5 A schematic diagram showing a basic photolithography process [50].    15
Figure 1.6 Basic steps of photolithography process.    17
Figure 2.1 Procedure for preparing and performing the electrochemical ELISA for estradiol.    27
Figure 2.2 EIS of the bare Pt electrode (■), the chitosan/Pt electrode (▼), the capture antibody/ chitosan/Pt electrode (▲), and the BSA/capture antibody/chitosan/Pt electrode (●).    33
Figure 2.3 CV diagrams obtained from testing an AP immobilized Pt wire microelectrode in the Tris buffer (pH 9.0) containing 0 mM (buffer), 1.0 mM, and 4.0 mM 4-APP; (a) −0.2 V to +0.7 V and (b) −0.2 V to +0.3 V.    35
Figure 2.4 Current response (I) vs. time (t) at +0.14 V obtained from testing the chitosan coated Pt wire microelectrode (Chitosan-Pt) and the AP/chitosan Pt wire microelectrode (AP-chitosan-Pt) in Tris buffer (pH 9.0) before and after the addition of 4.0 mM 4-APP.    36
Figure 2.5 . A representative plot of current responses of the electrochemical ELISA corresponding to different estradiol concentrations (0 pg/mL, 1.0 × 10−1 pg/mL, 1.0 × 100 pg/mL, 1.0 × 101 pg/mL, 1.0 × 102 pg/mL, 1.0 × 103 pg/mL, 1.0 × 104 pg/mL, and 1.0 × 105 pg/mL). Electrochemical measurements were performed using chitosan coated Pt electrodes after the ELISA procedure by constant potential amperometry at +0.14 V vs. Ag/AgCl in 100 mM Tris buffer (pH 9.0) under stirring condition.    38
Figure 2.6 The calibration curve of electrochemical ELISA for estradiol plotted in (a) linear scale and (b) in semi-log scale with estradiol concentration ranging from 1.0 × 10−1 pg/mL to 1.0 × 105 pg/mL (N = 3). Electrochemical measurements were performed using chitosan coated Pt electrodes after the ELISA procedure by constant potential amperometry at +0.14 V vs. Ag/AgCl in 100 mM Tris buffer (pH 9.0) under stirring condition.    40
Figure 2.7 The calibration curve of electrochemical ELISA for serum estradiol plotted in (a) linear scale and (b) in semi-log scale (N = 3). Electrochemical measurements were performed using chitosan coated Pt electrodes after the ELISA procedure by constant potential amperometry at +0.14 V vs. Ag/AgCl in 100 mM Tris buffer (pH 9.0) under stirring condition.    43
Figure 3.1(a) The schematic diagram showing the micromachining fabrication process flow (cross-section view) of the Si-based MEA probes, (b) a 4-inch silicon wafer with 164 MEA probes, (c) the picture of a released MEA probe (total length: 1.8 cm), and (d) the SEM image of the MEA probe tip.    53
Figure 3.2 (a) The schematic cross-section diagram of the sensor after all modification steps, (b) the sensor modification steps: from the bare Pt microelectrodes, preparation of the on-probe Ag/AgCl reference electrode, deposition of PPY, coating of Nafion®, to to the immobilization of GlutOx, and (c) the SEM image of a complete sensor.    55
Figure 3.3 A schematic diagram for illustrating the function of three different modification layers (the enzyme layer: GlutOx and perm-selective polymer layers: Nafion® and PPY) on a completed Glut sensor and its working principle.    57
Figure 3.4 (a). The SEM image of the deposited on-probe Ag/AgCl layer (indicated by a black arrow) on the microelectrode wire.    59
Figure 3.5 Energy-dispersive X-ray spectroscopy (EDS) for the on-probe deposited Ag/AgCl.    60
Figure 3.6 XRD patterns of AgCl nanoparticle. The concentration of metal precursors is 0.1 M.    61
Figure 3.7 The OCP data of the on-probe Ag/AgCl micro-reference electrode (Eon-probe Ag/AgCl) vs. Ag/AgCl wire reference electrode (EAg/AgCl wire) in PBS (pH =7.4) over 24 hours (N = 3). The geometric area of the on-probe Ag/AgCl micro-reference electrode and the Ag/AgCl wire reference electrode is ~8.5 × 10-5 cm2 and ~1.57 × 10-2 cm2, respectively.    62
Figure 3.8 (a) The typical current to time (I-t) results corresponding to different glutamate concentrations in PBS (pH = 7.4) at V = +0.7 V (vs. on-probe Ag/AgCl) (N = 5) with the Glut sensor (black line) and the control sensor (gray line). Each gray arrow indicates the timing of each glutamate addition. (b) The full range current to concentration plot of the integrated micro-Glut sensor probe and the calibration curve of the integrated micro-Glut sensor probe tested in PBS (pH = 7.4).    64
Figure 3.9 The typical I-t plot recording interferent current responses when common interferents were added to the system. In the test, Glut (20 μM, 40 μM), AA (250 μM, 500 μM), DA (5 μM, 10 μM), and H2O2 (20 μM, 40 μM) were injected every 100 s sequentially and the test was measured at +0.7 V (vs. on-probe Ag/AgCl) in PBS (pH = 7.4).    65
Figure 3.10 Operational stability (relative sensor sensitivity vs. number of operation(s)) of the integrated micro-Glut sensor probe during repetitive operations for 20 times (N = 5).    67
Figure 3.11 (a) The current to time (I-t) resutls corresponding to different glutamate concentrations (4.99 μM, 9.98 μM, 29.88 μM, 49.70 μM, 69.44 μM, 89.11 μM, 108.69 μM) in spiked serum samples at V = +0.7 V (vs. on-probe Ag/AgCl) (N = 5). (b) The calibration curve of the integrated micro-Glut sensor probe tested in serum.    69
Figure 4.1 The possible mechanisms by redox center of IrO2/Ir2O3 with the Pt wire (gray color) and the IrOx layer (dark gray color).    74
Figure 4.2 A schematic of the preparation of IrOx solution and electrodeposition setup.    75
Figure 4.3 SEM images of Pt bare wire sensor before electrodeposition.    76
Figure 4.4 SEM images at various magnifications showed the morphology of Pt sensor after electrodeposition (a) Pt/IrOx sensor magnified 500, (b) Pt/IrOx sensor magnified 2000.    77
Figure 4.5 (a) The typical current to time (I-t) results corresponding to different glutamate concentrations in PBS (pH = 7.4) at V = +0.7 V with the Pt sensor (black line) and the Pt/IrOx sensor (gray line). Each gray arrow indicates the timing of each glutamate addition. (b) The current to concentration plot of Pt/IrOx sensor tested in PBS (pH = 7.4) with the Pt sensor (black square) and the Pt/IrOx sensor (gray dot).    78
 
INDEX OF TABLES
Table 1.1. Historical events of biosensor development [58].    5
Table 1.2 Classification of biosensors based on the type of biological recognition system for an electrochemical transducer [60].    8
Table 1.3 Classification of biosensors according to the type of transducers [59, 60].    10
Table 1.4 Advantages and drawbacks of five basic immobilization methods [88].    20
Table 2.1 Electrochemical ELISAs for the detection of estradiol.    25
Table 3.1 Recently reported fully integrated amperometric Glut sensors.    50
Table 4.1 Application of IrOx electrode fabricated by electrodeposition technique.    73
Table 4.2 Chemical preparation for IrOx deposition and H2O2 test.    75


                                

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