根據衛生署統計，97年國內十大主要死因(除意外事故外)中，心血管疾病造成死亡的人數占總死亡人數的11.1 %，僅次於惡性腫瘤的27.3 %。因此心血管疾病是當今不容忽視的慢性疾病。如何能夠提供精準度高的檢測數據，以提供醫師正確的診斷是目前科學家最棘手的課題。有別於過去的醫學檢驗報告，大部分都需透過實驗室大型儀器設備才能檢測得知，這種方式往往是曠日費時。因此，微型化檢驗機台之開發乃是定點照護最需要之設備。尤其現今國人高度注重健康以及社會已趨向高齡化，可攜式定點照護儀即變成不可或缺的配備。本研究針對主要心血管疾病標記分子，包含C-reactive protein (CRP)、cardiac troponin I (cTnI)以及S100A1 protein (S100A1)進行研究分析，並發展出一套新型無標定生醫免疫電學感測技術。此技術透過已設計並合成之有機電誘導型化合物分子進行電極晶片表面的自我組裝修飾；再將一系列心血管疾病標記分子進行生物固定化製程；配合電學感測技術之恆電位分析法於低電壓(≦-0.5V)下進行探討，研究發現抗體抗原間結合後形成一阻抗層並造成電極表面電性改變，藉由此變化可進行抗體抗原間專一性與非專一性之量測，且可量測達到ng等級之精確度。本研究並利用導電型原子力顯微技術觀察心血管疾病標記分子表面奈米結構與力學間之作用及電性變化。結果顯示，於低電壓(≦-0.5V)下，抗體抗原間之作用力不因電壓增加而改變，而高電壓(＞-1V)下，有造成構形改變且抗體抗原間之作用力也隨之變化。再與X光結晶繞射法及螢光染色定量法之結果交叉比對，以提供此新型無標定生醫免疫電學感測技術更完整之技術架構。本技術之成功開發，有助於了解抗體抗原間之奈米結構、力學曲線與生物電性之關聯。並以此技術背景為主體，整合並研製完成微型化可攜式定點照護儀(e- Ab sensor)，結合微流道系統及可拋式免疫感測晶片，將即時顯示檢測結果，可提供醫師臨床診斷更完整且精準之檢驗數據，以縮短診斷時程並提升治癒效果。此裝置解決過去醫學檢驗使用實驗室大型儀器之困擾，提供未來居家自我看護更完善之微型化可攜式定點照護儀。未來，除了醫學檢驗可以更快速外，更進一步提供家庭、機場、醫院、車站等居家或公共場所的慢性疾病控制、用藥監測、環境稽查、食品檢驗以及毒品測試等等更完善的定點照護機制。

According to the statistics of Department of Health (DoH), the deaths caused by cardiovascular disease have 11.1 % in total number of deaths at Taiwan the year before last (2008). Currently, people have become more concerned about health and fitness. In the past, clinical diagnostic examination with blood required bulky equipments, time-consuming and expert staff in the hospital or central laboratory. Therefore, development of medical or healthcare monitoring device for detection and control of cardiovascular disease is in needed of point of care in the aging population. This work develops the novel electrical immunosensing technique for screening and analysis of major cardiovascular disease markers, including C-reactive protein (CRP), cardiac troponin I (cTnI), and S100A1 protein (S100A1). The technique consists of conductance-enhanced molecules for SAM formation, biological species immobilization and electrical immunosensing. Manipulation of the novel technique in amperometric analysis at the low voltage (≦-0.5V), the electricity or specificity were measured in antibody-antigen interactions and the accuracy achieve nanogram (ng) level. Furthermore, this work analyzed nano-mechanical structure, force and electrical distributions using conductive atomic force microscopy. The data compare with results of x-ray crystal diffraction method and enzyme-linked immunosorbent assay qualitative methods to complete the framework of this novel technique. This work integrates the novel electrical immunosensing technique to a handheld diagnostic device, called “e- Ab sensor”. The device consists of microfluidic system, immunoelectrodes and disposable cartridge. It has many benefits including rapid diagnosis, high accuracy, reliable, reproducible, affordable, robust and user-friendly, therefore, it can be performed on site. e- Ab sensor is a platform technique which can further develop for various applications, such as healthcare screening, chronic-diseases monitoring, environmental inspection, food-safety testing and bacteria or virus level determinations. e- Ab sensor could be used to quickly screen people at home, airport, hospital, and emergency clinic to control outbreaks of diseases such as SARS and H1N1 flu. However, the trends toward miniaturization and automation demand more advances in developing microfluidic system before e- Ab sensor can realize the commercial potential.

ACKNOWLEDGEMENTS...................I
ABSTRACT...................II
NOMENCLATURES...................IV
CONTENTS...................VI
FIGURES...................IX
TABLES...................XIII

CHAPTER I.
GENERAL INTRODUCTION...................1
POINT-OF-CARE...................2
BIOSENSOR...................5
TO LABEL OR NOT TO LABEL ?...................9
MARKERS OF CARDIOVASCULAR DISEASE...................11
C-Reactive Protein...................12
Cardiac Troponin Complex...................14
S100 Family...................16
SELF ASSEMBLED MONOLAYER (SAM)...................19
SCANNING PROBE MICROSCOPY (SPM)...................25
Atomic force microscopy...................25
Conductive atomic force microscopy (c-AFM)..............38
ELECTRICAL IMMUNOSENSOR...................40
Electrical Immunosensor...................40
Electrodes...................41
Electron Transfer (ET)...................47

CHAPTER II.
CHARACTERIZATIONS OF THIOPHENE-BASED MOLECULES ON ITO NANOELECTRIC FIELD USING CONDUCTING AFM...................52
ABSTRACT...................53
INTRODUCTION...................54
EXPERIMENTAL...................57
Materials...................57
Layer-by-layer deposition...................57
Various nanoelectronic fields in c-AFM...................59
RESULT AND DISCUSSION...................61
Nanostructure observation...................61
Surface wettability...................62
Electrical distribution...................64
Time course...................66

CHAPTER III.
DYNAMIC RESPONSES OF LABEL‐FREE CVD MARKERS ARE INDUCED IN VARIOUS ELECTRIC FIELDS WITH CONDUCTING AFM..............68
ABSTRACT...................69
INTRODUCTION...................70
EXPERIMENTAL...................73
Materials	...................73
CVD markers preparation...................73
Proteins coated on the substrate...................73
c-AFM and probes...................75
RESULT AND DISCUSSION...................77
Morphology of CVD markers...................77
Electrical behaviors...................80
Force analysis...................82

CHAPTER IV.
NOVEL ELECTRICAL IMMUNOSENSING TECHNIQUE OF LABEL-FREE CVD MARKERS...................86
ABSTRACT...................87
INTRODUCTION...................88
EXPERIMENTAL...................91
Materials and preparation...................91
Electrodes...................91
Calibration and Setting...................91
Sensing monitoring	...................93
Calibration curves	...................93
RESULT AND DISCUSSION...................95
Integrated System...................95
Calibration and optimization...................97
Electrical immunosensing...................99
Cross-interferences in dynamic system...................110
Coefficient of variation...................113

CHAPTER V.
DYNAMIC INTEGRATED SYSTEM...................115
INTRODUCTION...................116
DEMAND FOR HANDHELD POC DEVICES...................117
DEVICE DESIGN AND MINIATURIZATION...................118
Immunoelectrodes...................118
Biosensing Cartridge...................122
Microfluidic system...................123
Integrated Device...................124
FUTURE OF HANDHELD POC DEVICE...................127

CHAPTER VI.
CONCLUSION...................128

REFERENCES...................130

 
Figures
Fig. 1 In vitro diagnostics market............................................................................................2
Fig. 2 Schematic of biosensor.................................................................................................5
Fig. 3 Principles of biosensings ...............................................................................................6
Fig. 4 Difference between label‐free and label biosensings.....................................................9
Fig. 5 Targets for CVD detection ...........................................................................................11
Fig. 6 3D X‐ray crystal structure of CRP molecule ..................................................................12
Fig. 7 Structure of cardiac troponin‐tropomyosin complex. ..................................................15
Fig. 8 Gold‐thiolate monolayer and alkylsilane monolayer ...................................................19
Fig. 9 Schematic of SAM structure........................................................................................19
Fig. 10 Single molecule adsorbed on the substrate ...............................................................20
Fig. 11 Chemical and biological surfaces ...............................................................................21
Fig. 12 Overview of SAM preparations .................................................................................21
Fig. 13 Silanization on silicon substrates...............................................................................22
Fig. 14 Thiolation on gold substrates ....................................................................................22
Fig. 15 Defects found in SAM on substrates ..........................................................................24
Fig. 16 V‐shaped cantilever ..................................................................................................26
Fig. 17 Resolutions depend on sharp end of the probes........................................................26
Fig. 18 Four‐quadrants PSD detector ....................................................................................28
Fig. 19 Basic mechanism of AFM ..........................................................................................29
Fig. 20 Tapping mode AFM...................................................................................................30
Fig. 21 Contact mode AFM...................................................................................................32
Fig. 22 Non‐contact mode AFM............................................................................................33
Fig. 23 Phase imaging...........................................................................................................34
Fig. 24 Force vs. distance curve ............................................................................................35

Fig. 25 Basic mechanism of c‐AFM measurement .................................................................39
Fig. 26 Two‐electrodes cell ...................................................................................................43
Fig. 27 Three‐electrodes cell.................................................................................................43
Fig. 28 Direct ET tunneling mechanism.................................................................................47
Fig. 29 Electron transfer via a redox mediator “ET shuttle” ...................................................48
Fig. 30 ET on self‐assembled monolayers..............................................................................50
Fig. 31 Illustration of DL‐3TA, DL‐4TA and DL‐3TAC functionalized ITO substrates ..................57
Fig. 32 Process of contact angle meter .................................................................................58
Fig. 33 Topographic and I‐V images with c‐AFM....................................................................59
Fig. 34 AFM height and 3D topographic images of the different ITO substrates .....................61
Fig. 35 AFM height and 3D topographic images of the different ITO substrates .....................62
Fig. 36 Process of DL‐3TAC derivative conjugates with biomolocules.....................................62
Fig. 37 Contact angles analysis of wettability........................................................................63
Fig. 38 AFM height, 3D topographic and I‐V response images of different ITO substrates ......64
Fig. 39 I‐V characteristics of the different ITO substrates ......................................................65
Fig. 40 I‐V curves of DL‐3TAC functionalized in 2 and 20 hours ..............................................67
Fig. 41 Processing of c‐AFM..................................................................................................71
Fig. 42 Antibody‐antigen interactions are presented on the substrate ..................................74
Fig. 43 Topographic images and I‐V responses obtained with c‐AFM.....................................75
Fig. 44 Structure of cTnI by AFM with topographic and phase images ...................................77
Fig. 45 Structure of S100A1 by AFM with topographic and phase images ..............................78
Fig. 46 Height images and 3D topographic images of different gold substrates......................79
Fig. 47 Height images and 3D topographic images of antibody‐antigen interactions..............80
Fig. 48 Topographic images and I‐V response images of antibody‐antigen interactions .........81
Fig. 49 Antibody‐antigen interaction at various bias .............................................................82
Fig. 50 Adhesion forces of the different substrates at 0~1 voltages .......................................83

Fig. 51 Stiffness of gold blank substrate at 0~1 voltages........................................................84
Fig. 52 Adhesion force of the substrates at 0.2V ...................................................................85
Fig. 53 Stiffness of the substrates at 0.2V .............................................................................85
Fig. 54 Comparison of two electrodes and three electrodes system in amperometry ............95
Fig. 55 Current changes of ITO‐WE at various electric fields ..................................................96
Fig. 56 Current changes of Au‐WE at various electric fields ...................................................96
Fig. 57 Static system.............................................................................................................97
Fig. 58 Comparison the signals with DL‐3TAC and non‐DL‐3TAC biolinkers ............................97
Fig. 59 Cyclic voltammograms of the multilayered electrode ................................................98
Fig. 60 Signals of S100A1 markers at ‐0.2V ............................................................................99
Fig. 61 Selectivity of anti‐S100A1 antibodies with cross interferences .................................100
Fig. 62 Signals of cTnI markers at ‐0.2V...............................................................................102
Fig. 63 Selectivity of anti‐cTnI antibodies with cross interferences ......................................103
Fig. 64 Signals of CRP markers at ‐0.2V...............................................................................104
Fig. 65 Selectivity of anti‐CRP antibodies with cross interferences ......................................105
Fig. 66 Cross interferences of specific and non‐specific interactions....................................107
Fig. 67 Specific and non‐specific interactions in the dynamic system ..................................107
Fig. 68 S100A1 antigens interacted with anti‐S100A1 antibodies in the dynamic system......108
Fig. 69 Accuracy of specific interaction...............................................................................109
Fig. 70 Whole framework in the dynamic system................................................................109
Fig. 71 Cross interferences of specificity and non‐specificity in S100A1 test.........................111
Fig. 72 Cross interferences of specificity and non‐specificity in CRP test..............................112
Fig. 73 Absorptions of serum antibody‐antigen interaction using ELISA ..............................113
Fig. 74 Fabrication of immunoelectrode.............................................................................119
Fig. 75 1st immunoelectrode...............................................................................................119
Fig. 76 Ag/AgCl‐RE electrode flaked off the surface ............................................................120

Fig. 77 2nd immunoelectrode..............................................................................................120
Fig. 78 Ag/AgCl‐REs were scoured out the surface after experiment...................................120
Fig. 79 3rd immunoelectrode ..............................................................................................121
Fig. 80 Changes of three electrodes Au‐WE, Pt‐CE and Au‐RE..............................................121
Fig. 81 4th immunoelectrode ..............................................................................................121
Fig. 82 1st biosensing cartridge ...........................................................................................122
Fig. 83 2nd actual biosensing cartridge ................................................................................123
Fig. 84 3rd actual biosensing cartridge ................................................................................123
Fig. 85 Framework of the microflidic system.......................................................................123
Fig. 86 Framework of integrated system in this work..........................................................124
Fig. 87 1st novel handheld PoC devices ...............................................................................125
Fig. 88 2nd novel handheld PoC devices [e‐ Ab sensor] ........................................................125
Fig. 89 3rd novel handheld PoC devices [e‐ Ab sensor].........................................................126

TTA AAB BBL LLE EES SS
Table. 1 Some commercial handheld PoC devices for CVD markers.........................................3
Table. 2 Type of electrochemical transducers .........................................................................7
Table. 3 Combinations of head‐group and substrate.............................................................20
Table. 4 Typical properties of different conductive‐coated tips .............................................39
Table. 5 Characterization of ITO surfaces after DL‐3TA modification ......................................63
Table. 6 Conductance measurement of different ITO substrates in cAFM ..............................65
Table. 7 Roughness measurement of different ITO substrates at various applied bias............66
Table. 8 Physical parameters of the gold substrate for biomolecules interaction ...................79
Table. 9 Accuracy of anti‐S100A1 and S100A1 interactions...................................................101
Table. 10 Accuracy of anti‐cTnI and cTnI interactions ..........................................................104
Table. 11 Accuracy of anti‐CRP and CRP interactions...........................................................106
Table. 12 Current signals of specific and non‐specific interactions.......................................108
Table. 13 Cross interference test of S100A1 specificity ........................................................111
Table. 14 Cross interference test of CRP specificity .............................................................112
Table. 15 CVs S100A1..........................................................................................................114
Table. 16 CVs CRP...............................................................................................................114
Table. 17 Some features for handheld PoC diagnostic devices.............................................117

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