為發展一C反應蛋白感測晶片作為心血管粥狀硬化症與冠狀動脈疾病的臨床檢測，本研究使用原子力顯微術與雙偏極化干涉術進行C反應蛋白表面微結構的量測，並定量五聚體蛋白三維結構與x光結晶學之結果比較。另外，利用此C反應蛋白感測晶片配合雙偏極化干涉術分析，經由表面厚度、單層密度與表面濃度等結構參數，分析C反應蛋白於不同pH值條件的構形變化，結果顯示在中性環境pH值6.0到7.0為五聚體的構形；酸性環境pH值4.0下，C反應蛋白分子薄膜解離為單體的CRP；在pH值2.0時則發現部份的單體的CRP會產生聚集作用；而在鹼性環境pH值8.0和10.0下，則只存在單體的C反應蛋白結構。此發現將有助於C反應蛋白生物性功能與構形的研究。另一方面，了解抗體-抗原間的交互作用可作為生物檢測的依據，本研究藉由多單體的C反應蛋白與單株C反應蛋白抗體間的系統，計算其中蛋白動力學與結合常數的化學量論，並驗證小分子化學（鈣離子） 與C反應蛋白分子薄膜的結合試驗。最後，開發一新型的電子轉移生物結合分子OT-3C，提供電化學生物晶片的應用，研究成果更發現此化學分子可將銦錫氧化物基材的導電性提高數倍之上，大大增加C反應蛋白感測晶片的發展性。

In order to develop the C-reactive protein (CRP) sensor chips for clinical detection of atherosclerosis and coronary heart disease, we used an atomic force microscope (AFM) and a dual polarization interferometric (DPI) biosensor to probe the surface ultrastructure and to measure the dimensions of CRP. The quantitative measurements of the dimensions of the protein were basically corresponds to that previously obtained from the structure of CRP determined by X-ray crystallography. Moreover, the DPI sensor was used to investigate the structure and conformational changes by structural parameters (thickness, layer density, surface mass loading concentration) under different physiological condition. This current work revealed that the conformations of CRP monolayer in the acidic region were existed two forms include of the monomeric CRP at pH 4.0 and a “partial” subunits’ aggregation at pH 2.0. On the other hands, only compact monomeric form was observed in the pH 8.0 and 10.0 alkaline region. These informations were useful to discover the biological role in the function of homopentamer CRP. For the purposed of biosensing, understanding the interaction kinetics between a ‘homopolyvalent’ antigen (Ag) and a monoclonal antibody (Ab). A model system, which uses a monoclonal Ab against a homopentameric Ag, CRP, was presented with principle and experiments for the study of the interactions between an Ab and an Ag that had multiple identical epitopes. This allowed evaluation of the dissociation constant (KD) and of the binding stoichiometry. In the final parts, the novel electron transfer type biolinker called 5"-formyl-5-carboxylic acid-2,2',5',2'-terthiophene, OT-3C, was designed into an EC sensor chip development. The aldehyde (-CHO) and carboxyl (-COOH) head groups could provide a higher potential for biomolecules immobilization and modification of hydroxyl ITO surfaces, which conductivity increased several folds.

Table of Content

Abstract II
Acknowledgments IV
Abbreviations V
Table of Contents VI
Figures XIII
Tables XVII

Chapter I 1
General Introduction 1
1. Biosensor 2
2. C-reactive protein 3
3. Dual polarization interferometry 8
3.1 Optical sensors 8
3.2. Dual-slab waveguide interferometer 9
3.3 Operating principles 10
3.4. Resolving layer thickness and index 11
3.5. Instrumentation 11
4. Atomic Force Microscopy 13
4.1 Instrumentation 13
4.2 Principle of Operation 16
4.3 conductive Atomic Force Microscopy (cAFM) 18
Chapter II 20
Characterization of Protein Structure
Measurement of dimensions of pentagonal doughnut-shaped C-reactive
protein using an atomic force microscope and a dual polarization interferometric
biosensor 20
1. Introduction 22
2. Methods 23
2.1. AFM analysis 23
2.2. DPI analysis 23
3. Results and discussion 24
3.1. Each single CRP particle was readily distinguishable 24
3.2. CRP was a pentagonal doughnut-shaped CRP molecule 26
3.3. Measurement of the average monolayer thickness of CRP by DPI 29
4. Conclusions 31
Chapter III 32
Protein Conformational Changes
Measurement of the pH-induced conformational changes in the structure
of C-reactive protein by dual polarization interferometry 32
1. Introduction 34
2. Materials and Methods 35
2.1. Instrument - Dual Polarization Interferometry 35
2.2. Amination of DPI sensor chip 36
2.3. CRP Immobilization 38
2.4. pH-induced Conformational Change 38
3. Result and Discussion 39
3.1. Characterization of the CRP monolayer 39
3.2. Conformational properties of CRP in different pH solution 45
4. Conclusion 52
Chapter IV 55
Protein–Small Molecules Interaction
Characterization of calcium-binding structure in C-reactive protein 55
1. Introduction 56
2. Materials and Methods 56
2.1. Instrument - Dual Polarisation Interferometry 56
2.2. Surface chemistry of DPI sensor chip 56
2.3. CRP Immobilization 57
2.4. Calcium Interaction 59
3. Result and Discussion 59
3.1. Characterization of the CRP monolayer 59
3.2. Calcium-bound Structure of CRP 64
Chapter V 66
Protein–Protein Interaction Kinetic & Stoichiometry
Homopolyvalent antibody–antigen interaction kinetic studies with use of a
dual-polarization interferometric biosensor 66
1. Introduction 68
2. Materials and methods 70
2.1. Amination of DPI sensor chip 70
2.2. Surface CRP–anti-CRP interactions 71
2.3. Affinity measurements by an indirect ELISA 72
3. Theoretical considerations 72
4. Results and discussion 74
5. Conclusions 83
Chapter VI 85
Nanoelectronic Monolayer for Protein Immobilization
Surface modification of indium tin-oxide via a novel biolinker 5"-formyl-5-
carboxylic acid-2,2',5',2'-terthiophene induced nanoelectronic transfer
behavior 85
1. Introduction 86
2. Methods 87
2.1. DPI analysis 87
2.2. cAFM studies 88
3. Results and discussion 90
Chapter VII 94
Cell Investigation for Disease Determination
Surface ultrastructure and mechanical property of human chondrocyte
revealed by atomic force microscopy 94
1. Introduction 96
2. Materials and methods 98
2.1. Chemicals 98
2.2. Isolation of Human Chondrocytes and Cell Immobilization 98
2.3. Atomic Force Microscopy 99
2.4. Single-Cell AFM Measurement 99
2.5. Adhesion Force and Stiffness Measurements 101
3. Results 102
3.1. AFM Imaging of Chondrocytes 102
3.2. Force-Curve Analysis of Chondrocytes 106
3.3. Mechanical Properties of Chondrocytes 108
4. Discussion 110
5. Further Research 113
5.1 Isolation of Human Chondrocytes from Cartilages 113
5.2 Chondrocyte cells from Bone marrow 114
5.3 Micropellet Formation and Chondrogenic Induction 115
5.4 Identification of Chondrocytes within AFM 115
5.5. Mechanical measurements of Chondrocytes 117
Chapter VIII 120
General Conclusion 120
References 126
Chapter IX 140
Other Applications in AFM
Atomic force microscopy in biomolecular nanostructure measurements
and mechanical investigation 140
1. Introduction 141
2. pheochromocytoma (PC-12) cell 141
2.1 Objective 141
2.2 Methods and Instruments 141
2.3 AFM Imaging of PC-12 cells 142
2.4 Stiffness and Adhesion Force Measurements 143
3. Human sperm 145
3.1 Objective 145
3.2 Methods and Instruments 145
3.3 Cell Morphology Imaging 145
4. Organic Biolinker self-assembled monolayers (SAMs) 147
4.1 Objective 147
4.2 Methods and Instruments 147
4.3 Ultrastructure Investigation within AFM 148
4.4 Indium-tin oxide (ITO) Substrate 149
5. primary osteoblastic cells (POB) 151
5.1 Objective 151
5.2 Methods and Instruments 151
5.3 AFM Imaging of POB 151
5.4 Stiffness and Adhesion Force Measurements 152

Figures

Fig. I-1 The principle of function of a biosensor. 2
Fig. I-2 Structure of the CRP protomer. 4
Fig. I-3 Crystal structure of C-reactive protein complexed with phosphocholine (PC). 5
Fig. I-4 The structural dissociation of pentameric CRP to momnmeric subunits. 7
Fig. 1-5 Dual slab waveguide interferometer. 10
Fig. I-6 Schematic of the instrument and the physical embodiment. 12
Fig. I-7 Schematic representation of the atomic force microscope. 14
Fig. I-8 Tip-sample arrangements in the two modulation modes of AFM. 17
Fig. I-9 Illustration of conductive atomic force microscopy (cAFM). 19
Fig. II-1 Topographic images of the native CRP molecules scanned in air with AFM. 25
Fig. II-2 The nanostructure of CRP molecules scanned in vacuum with AFM. 28
Fig. II-3 Immobilization of CRP illustrated in the DPI biosensor. 30
FIg. III-1 The main components of AnaLight(R) Bio200 DPI biosensor. 36
Fig. III-2 The DPI sensor chip modification processes used for protein immobilization. 37
Fig. III-3 Immobilization of CRP on a glutaldehyde-functionalized sensor chip surface. 40
Fig. III-4 Addition of various concentrations of CRP onto a glutaldehyde-functionalized DPI sensor chip. 43
Fig. III-5 CRP conformational changes in different pH solution. 47
Fig. III-6 Structure parameters of CRP monolayer obtained from the DPI biosensor under different pH solution. 49
Fig. III-7 CRP monolayer retained a stable pentameric conformation. 51
Fig. IV-1 Raw phase data for the Ca2+ interactions of CRP molecules to a dual
waveguide sensor chip surface. 58
Fig. IV-2 Immobilization of CRP on a glutaldehyde-functionalized sensor chip surface. 61
Fig. IV-3 Addition of various concentrations of CRP onto a glutaldehyde-functionalized DPI sensor chip. 63
Fig. IV-4 The conformational changes obtained from the interaction between Ca2+ and CRP. 65
Fig. V-1 Scheme of the binding of homopolyvalent CRP Ag(s) to divalent monoclonal anti-CRP Ab(s). 70
Fig. V-2 Raw phase data for the immobilization of anti-CRP and the binding of CRP to a dual-waveguide sensor surface. 74
Fig. V-3 Immobilization of anti-CRP on a glutaldehyde-functionalized sensor chip surface. 76
Fig. V-4 Addition of various concentrations of monoclonal anti-CRP IgG onto a glutaldehyde-functionalized DPI sensor chip. 78
Fig. V-5 Binding curves of CRP to anti-CRP Ab at the silica/water interface by DPI. 81
Fig. V-6 Scatchard plot of CRP binding to monoclonal anti-CRP Ab measured by an indirect competition ELISA. 83
Fig. VI-1 The molecular structure of novel 5"-formyl-5-carboxylic acid-2,2',5',2'- terthiophene biolinker, OT-3C. 87
Fig. VI-2 Raw phase data (TM/TE) for modification of hydroxyl ITO surfaces and biomolecules immobilization were obtained from a DPI sensor chip. 88
Fig. VI-3 Illustration of the cAFM with OT-3C modified electrode. 90
Fig. VI-4 AFM/cAFM measurements in ITO and OT-3C modified ITO electrode, respectively. 91
Fig. VI-5 Characterization of the nanoelectronic properties of the bared ITO, 3-APTES activated, and OT-3C modified ITO electrode. 92
Fig. VII-1 Schematic showed a typical cycle for force measurement. 102
Fig. VII-2 Observations with AFM; a chondrocyte was isolated from OA cartilage. 104
Fig. VII-3 Topographic AFM images of single old and young chondrocytes. 105
Fig. VII-4 Force-distance curves acquired with three systems. 107
Fig. VII-5 Histograms and Gaussian distribution curves showed the differences in the adhesion forces of old and young chondrocyte cells. 109
Fig. VII-6. Histograms and Gaussian distribution curves showed stiffness measurements of old and young chondrocyte. 110
Fig. VII-7 Chondrocytes image obtained from AFM. 116
Fig. VII-8 Histograms and Gaussian distribution curves showed stiffness measurements of chondrocytes. 117
Fig. VII-9. Histograms and Gaussian distribution curves showed adhesion force measurements of chondrocytes. 119
Fig. IX-1 PC-12 cell image obtained from AFM. 142
Fig. IX-2 Histograms and Gaussian distribution curves showed stiffness measurements of dendrite and nucleus PC-12 cells. 143
Fig. IX-3 Histograms and Gaussian distribution curves showed adhesion force measurements of dendrite and nucleus PC-12 cell. 144
Fig. IX-4 Human sperm images obtained from AFM. 146
Fig. IX-5 Illustration of the alkanethiol biolinker modification on the gold surfaces. 148
Fig. IX-6 The formation of the alkanethiol biolinkers SAMs were scanned in air with AFM. 148
Fig. IX-7 The height images, 3D topographic images and thickness of alkanethiol biolinker SAMs. 149
Fig. IX-8 POB cell image obtained from AFM. 152
Fig. IX-9 Histograms and Gaussian distribution curves showed stiffness measurements of native POB and US stimulated POB. 153
Fig. IX-10 Histograms and Gaussian distribution curves showed adhesion force measurements of native POB and US stimulated POB. 154
Fig. IX-11 Comparison of the stiffness between center, side, and corner parts in the native POB and US stimulated POB cells. 155
Fig. IX-12 Comparison of the adhesion force between center, side, and corner parts in the native POB and US stimulated POB cells. 156

Tables

Table II-1 The physical measurements of CRP ultrastructure. 31
Table III-1 The structural parameters in thickness and density of CRP monolayer at the solid /liquid interface were characterized under different pH environment. 48
Table. VI-1 The conductance measurement of the bared ITO, 3-APTES activated, and OT-3C modified ITO electrode in the cAFM. 93
Table VII-1 Statistical analysis of cellular surface topographies of old and young chondrocytes. 105
Table VII-2 Comparison of adhesion forces and stiffness measurements of old and young chondrocytes 108
Table. IX-1 The topography, 3D images, and the surface parameters of ITO substrate. 150

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