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系統識別號 U0026-2505201809232200
論文名稱(中文) 掃描探針技術於生物即時、微區多重性質量測之開發
論文名稱(英文) Development of Rapid, In-situ Multi-property Characterization of Biological Materials Using Scanning Probe Microscopy
校院名稱 成功大學
系所名稱(中) 材料科學及工程學系
系所名稱(英) Department of Materials Science and Engineering
學年度 106
學期 2
出版年 107
研究生(中文) 張敬萱
研究生(英文) Alice Chinghsuan Chang
學號 N58031188
學位類別 博士
語文別 英文
論文頁數 210頁
口試委員 指導教授-劉浩志
口試委員-黃一修
口試委員-蔡佩珍
口試委員-邵佩琳
口試委員-陳三元
口試委員-傅尉恩
口試委員-李旺龍
中文關鍵字 原子力顯微技術  困難梭狀芽孢桿菌  大腸桿菌  超彈性材料  變形機制  修正之柱狀針公式 
英文關鍵字 AFM  Clostridium difficile  Escherichia coli  hyperelastic materials  deformational mechanism  modified flat-punch equation 
學科別分類
中文摘要 分子生物法與染色法為生物樣品特性檢測最為廣泛使用的方法,然而繁瑣的試片製備流程造成時間的消耗以及人力的需求、影響該領域的研究發展與臨床檢測。近年來,許多文獻提出生物生理與力學表現的關聯性;而考量細胞的尺寸以及有效的力學量測,擁有簡易的試片製備、彈性的工作環境、以及奈米級解析度等優點的原子力顯微技術(Atomic force microscopy, AFM)不僅能有效簡短生物樣品檢測所需時間,更提供試片表面為結構與多重性質等資訊。有鑑於此,本研究以 AFM 於生物樣品為主軸,進行微區、即時、多重性質之量測以連結生理與表面力學特性。

本篇論文選用的生物樣品包含小鼠的表皮組織、人體病原體困難梭狀芽孢桿菌(Clostridium difficile)與大腸桿菌(Escherichia coli)不同的基因型。以 AFM 進行表面量測,試片形貌的解析度取決於探針的針尖尺度、而材料表面不同分子組成的結構可由力學顯微模式加以觀察;結果建立三種生物材料的表面特性與其生理特性分別之關聯,更說明了 AFM 於該領域有潛力發展為快速檢測試片狀態的方法。除此之外,學生亦發現一般為 AFM 之彈性模數(elastic modulus, E)量測計算公式的選用皆取決於探針的針尖幾何形狀,而然,根據探針形狀定義試片形變的假設並不適用於細菌細胞、並且造成高達 40%的實驗誤差值。為了增進生物材料 E 值檢測的精準度,本研究更針對試片的力學特性,修改現有的計算公式以及提出具備高彈性力學行為的生物材料於奈米壓痕(nanoindentation)中獨特的變形方式;最後,學生建立了使用 nanoindentation 的標準流程圖、提供各項實驗參數選擇的方法。
英文摘要 To characterize the physiologies of biological samples, the molecular and immunological staining combining optical microscopy for mammalian tissues, mammalian cells, and microbes are probably the most common techniques. Although those conventional methods have been conventionally used, the major drawbacks including time-consumed and labor-intensive need to be solved for the effective development of research. Believing the physiologies of biological matters influence their physical properties, the atomic force microscopy (AFM) is adapted in this work for the in-situ, rapid, multi-property examination. Possessing several advantages, such as simple preparation for specimen, flexible working environments, and nanoscale resolution, the examination time could be significantly decreased from couples of days by those conventional ways to few hours by the probe-based technique.

Here, the surface characteristics of a variety kind of biological materials involving mouse skin tissues, and multiple strains of two human pathogen Clostridium difficile and Escherichia coli were focused on. The resolution of surface morphologies were determined by the sharpness of AFM tip, the detailed information of surface components could be revealed by the simultaneous mechanical mapping, and the localized mechanical behaviors of the bacteria were found to be specific to each specimen. The results gave the hint of the connection between biological physiologies and physical traits, and consequently, this method was reckoned as the promising way for the rapid examination. The tip-based contact mechanism theory, which is required for the calculation of the elastic modulus, was noticed to result in the high uncertainty in microbial samples and the real stiffness behaviors of those specimens were obscured. To obtain a reliable elastic modulus, a reference specimen was selected for multiple testing to establish a new formula fitting to such types of materials. Our proposed equation successfully improved both the precision and accuracy of sample modulus and the corresponding new deformational mechanism of the hyperelastic matters was suggested.
論文目次 Chapter 1 Introduction 1
1.1. Background 1
1.2. Motivation 3
Chapter 2 Probe-based characterization methods in biology 6
2.1. Atomic force microscopy 6
2.1.1. Working principle 6
2.1.2. Mechanical characterization of material 7
2.2. Electrochemical impedance spectroscopy (EIS) 10
2.2.1. Working principle 10
2.2.2. Electrical characterization of material 12
2.3. Application in biology 13
2.3.1. Ultrastructure imaging in biology 13
2.3.2. Mechanical characterization in biology 16
2.3.3. Electrical characterization of biological samples 19
Chapter 3 Theoretical basis for contact mechanisms 21
3.1. Models for the homogeneous materials 21
3.1.1. Hertz model 22
3.1.2. Sneddon model 23
3.1.3. Flat-punch model 24
3.1.4. Oliver & Pharr method 25
3.2. Models for the heterogeneous samples 27
3.2.1. Derjaguin-Muller-Toporov model 27
3.2.2. Johnson-Kendall-Roberts model 28
3.2.3. Maugis model 29
3.3. Protocol for the model selection 31
3.4. Hyperelastic model 33
Chapter 4 Materials and methods 35
4.1. Biological samples 35
4.1.1. Skin tissues 35
4.1.2. Clostridium difficile 38
4.1.3. Escherichia coli 40
4.1.4. Streptococcus mutans 42
4.1.5. Staphylococcus aureus 44
4.1.6. Pseudomonas aeruginosa 44
4.2. Polydimethylsiloxane 45
4.1.1. Biological-mimicking synthetic material 46
4.1.2. Specimen preparation 47
4.3. Surface modification of substrate 48
4.3.1. Electrostatic attraction 48
4.3.2. Covalent binding 49
4.3.3. Adhesive proteins 50
4.3.4. Practical application of the immobilized methods 50
4.4. Characterization protocols 52
4.4.1. Atomic force microscopy (AFM) 52
4.4.2. Nanoindentor (NI) 63
4.4.3. Compression test 66
4.4.4. Electrochemical impedance spectroscopy (EIS) 67
4.5. Statistical analysis 69
Chapter 5 Multi-properties of biological samples by AFM 70
5.1. Skin tissue 70
5.1.1. Ultrastructure of sample surface 70
5.1.2. Macromolecule detection by mechanical mapping 78
5.1.3. Macromolecular structures of the tissue layers 80
5.1.4. Conclusion of the structure-dependent tissue layers 83
5.2. Clostridium difficile strains 84
5.2.1. Effects of cultivation times on cellular morphologies 84
5.2.2. Morphological characteristics of different C. difficile strains 89
5.2.3. Mechanical characterization 95
5.2.4. Potential phenotypes of C. difficile strains 101
5.3. Escherichia coli strains 102
5.3.1. Establishment of the effective time for AFM measurements 103
5.3.2. Morphological characteristics of different E. coli strains 105
5.3.3. Surface ultrastructure by multiple mapping 106
5.3.4. Genomic-manipulating differences in morphological characteristics 110
5.3.5. Mechanical characteristics of E. coli strains 116
5.3.6. Electrical characteristics of E. coli strains 118
5.3.7. Connection of microbial multi-behavior and physiology 126
Chapter 6 Probe-dependent mechanical characterization using AFM nanoindentation 128
6.1. PDMS reference samples 129
6.2. Homogeneities of PDMS samples 131
6.2.1. Planar homogeneity of PDMS samples 131
6.2.2. Perpendicular homogeneity of PDMS samples 134
6.3. Cantilever stiffness of AFM probes 134
6.4. Characterization of cantilever sensitivity in nanoindentation 134
6.4.1. Data collection in shallow indentation 135
6.4.2. Measurement of units in the applied/detected force and distance 136
6.5. Accuracy of mechanical characterization in PDMS systems 142
6.5.1. Theoretical E of the PDMS samples 142
6.5.2. AFM nanoindentation 142
6.5.3. Practical E of the PDMS samples 147
6.5.4. Tensile test 148
6.6. Bending mechanism of probe in AFM nanoindentation 150
6.7. Selection of suitable probe for mechanical characterization 151
Chapter 7 A behavior-dependent model for hyperelastic materials 152
7.1. Hyperelastic PDMS specimen 153
7.2. Indenter-dependent contact mechanism in nanoindentation 154
7.2.1. AFM nanoindentation 154
7.2.2. NI nanoindentation 155
7.2.3. Effects of power-law of force curve on E uncertainty 157
7.3. Examination of sample heterogeneity 159
7.3.1. Surface effects in PDMS 159
7.3.2. Surface effects in other materials 162
7.3.3. Adhesion from sample surface 164
7.4. Development of a new equation in AFM nanoindentation 166
7.4.1. Power-law dependent contact mechanism model 166
7.4.2. Assessment of PDMS E 169
7.4.3. A modified flat-punch equation 170
7.4.4. Examination of new equation 172
7.5. A deformational mechanism for the hyperelastic materials 176
7.5.1. Initial contact between tip and sample surface 178
7.5.2. Break of sample surface by tip 179
7.5.3. Propagation of crack into sample 180
7.5.4. Role of spherical tip in nanoindentation 181
7.6. Selection of force-curve section for E calculation 182
7.6.1. Discontinuous force curve 182
7.6.2. Continuous force curve 183
7.7. E evaluation for the microbial samples 184
7.7.1. E calculation of bacterial samples 185
7.7.2. Theoretical E of bacterial samples 186
7.8. Selection of model for E characterization 188
Chapter 8 Conclusions 189
8.1. AFM applications on biological materials 189
8.2. Development of method for E computation 192
8.3. Revised protocol for the E evaluation by nanoindentation 193
Chapter 9 Bibliography 197
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