||Development of Rapid, In-situ Multi-property Characterization of Biological Materials Using Scanning Probe Microscopy
||Department of Materials Science and Engineering
||Alice Chinghsuan Chang
modified flat-punch equation
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。
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.
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
 R. K. Saiki, S. Scharf, F. Faloona, K. B. Mullis, G. T. Horn, H. A. Erlich, and N. Arnheim, "Enzymatic amplification of b-globin genomic sequences and restriction site analysis for diagnosis of sickle cell anemia," Science, vol. 230, no. 4732, pp. 1350-1354, 1985.
 H. Cai, J. Caswell, and J. Prescott, "Nonculture molecular techniques for diagnosis of bacterial disease in animals: a diagnostic laboratory perspective," Veterinary pathology, vol. 51, no. 2, pp. 341-350, 2014.
 M. Lekka, P. Laidler, D. Gil, J. Lekki, Z. Stachura, and A. Hrynkiewicz, "Elasticity of normal and cancerous human bladder cells studied by scanning force microscopy," European Biophysics Journal, vol. 28, no. 4, pp. 312-316, 1999.
 S. E. Cross, Y.-S. Jin, J. Rao, and J. K. Gimzewski, "Nanomechanical analysis of cells from cancer patients," Nature nanotechnology, vol. 2, no. 12, pp. 780-783, 2007.
 G. Binnig, C. F. Quate, and C. Gerber, "Atomic force microscope," Physical review letters, vol. 56, no. 9, pp. 930, 1986.
 Y.-C. Yang, "Study of biomechanical and electrochemical responses of Streptococcus mutans under stressed environment," Master thesis, Materials Science and Engineering, National Cheng Kung University, Taiwan, 2015.
 A. C. Chang, J.-D. Liao, and B. H. Liu, "Practical Assessment of Nanoscale Indentation Techniques for the Biomechanical Properties of Biological Materials," Mechanics of Materials, 2016, vol. 98, pp. 11-21, 2016.
 J. Ďurkovič, M. Kardošová, and R. Lagaňa, "Imaging and Measurement of Nanomechanical Properties within Primary Xylem Cell Walls of Broadleaves," Annals of Botany, 2014.
 X.-Z. R. Yuan, C. Song, H. Wang, and J. Zhang, Electrochemical impedance spectroscopy in PEM fuel cells: fundamentals and applications. Springer Science & Business Media, 2009.
 H.-J. Butt, E. Wolff, S. Gould, B. D. Northern, C. Peterson, and P. Hansma, "Imaging cells with the atomic force microscope," Journal of structural biology, vol. 105, no. 1-3, pp. 54-61, 1990.
 A. C. Chang, B. H. Liu, P. L. Shao, and J. D. Liao, "Structure-dependent behaviours of skin layers studied by atomic force microscopy," Journal of Microscopy, vol. 267, no. 3, pp. 265-271, 2017.
 S. Ido, K. Kimura, N. Oyabu, K. Kobayashi, M. Tsukada, K. Matsushige, and H. Yamada, "Beyond the helix pitch: direct visualization of native DNA in aqueous solution," Acs Nano, vol. 7, no. 2, pp. 1817-1822, 2013.
 B. H. Liu, K.-L. Li, W.-K. Huang, and J.-D. Liao, "Nanomechanical probing of the septum and surrounding substances on Streptococcus mutans cells and biofilms," Colloids and Surfaces B: Biointerfaces, vol. 110, pp. 356-362, 2013.
 A. Touhami, B. Nysten, and Y. F. Dufrêne, "Nanoscale mapping of the elasticity of microbial cells by atomic force microscopy," Langmuir, vol. 19, no. 11, pp. 4539-4543, 2003.
 M. Lekka, D. Gil, K. Pogoda, J. Dulińska-Litewka, R. Jach, J. Gostek, O. Klymenko, S. Prauzner-Bechcicki, Z. Stachura, J. Wiltowska-Zuber, K. Okoń, and P. Laidler, "Cancer cell detection in tissue sections using AFM," Archives of Biochemistry and Biophysics, vol. 518, no. 2, pp. 151-156, 2012.
 M. Nikkhah, J. S. Strobl, R. De Vita, and M. Agah, "The cytoskeletal organization of breast carcinoma and fibroblast cells inside three dimensional (3-D) isotropic silicon microstructures," Biomaterials, vol. 31, no. 16, pp. 4552-4561, 2010.
 Z. Zhou, C. Zheng, S. Li, X. Zhou, Z. Liu, Q. He, N. Zhang, A. Ngan, B. Tang, and A. Wang, "AFM nanoindentation detection of the elastic modulus of tongue squamous carcinoma cells with different metastatic potentials," Nanomedicine: Nanotechnology, Biology and Medicine, vol. 9, no. 7, pp. 864-874, 2013.
 M. Plodinec, M. Loparic, C. A. Monnier, E. C. Obermann, R. Zanetti-Dallenbach, P. Oertle, J. T. Hyotyla, U. Aebi, M. Bentires-Alj, R. Y. H. Lim and C.-A. Schoenenberger, "The nanomechanical signature of breast cancer," Nature Nanotechnology, vol. 7, no. 11, pp. 757-65, 2012.
 C. B. Volle, M. A. Ferguson, K. E. Aidala, E. M. Spain, and M. E. Núñez, "Spring constants and adhesive properties of native bacterial biofilm cells measured by atomic force microscopy," Colloids and Surfaces B: Biointerfaces, vol. 67, no. 1, pp. 32-40, 2008.
 B. H. Liu and L.-C. Yu, "In-situ, time-lapse study of extracellular polymeric substance discharge in Streptococcus mutans biofilm," Colloids and Surfaces B: Biointerfaces, vol. 150, pp. 98-105, 2017.
 L. Yang, "Electrical impedance spectroscopy for detection of bacterial cells in suspensions using interdigitated microelectrodes," Talanta, vol. 74, no. 5, pp. 1621-1629, 2008.
 L. R. Arias, C. A. Perry, and L. Yang, "Real-time electrical impedance detection of cellular activities of oral cancer cells," Biosensors and Bioelectronics, vol. 25, no. 10, pp. 2225-2231, 2010.
 B. H. Liu, K.-L. Li, K.-L. Kang, W.-K. Huang, and J.-D. Liao, "In situ biosensing of the nanomechanical property and electrochemical spectroscopy of Streptococcus mutans-containing biofilms," Journal of Physics D: Applied Physics, vol. 46, no. 27, p. 275401, 2013.
 Y.-H. Liu, S.-S. Liao, and B. H. Liu, "Nanoscale electrochemical characterization of solid-state electrolyte using manganese-based thin-film probe," Nanoscale, vol. 48, 2016.
 H. Hertz, "On the contact of elastic solids," Z. Reine Angew. Mathematik, vol. 92, no. 110, pp. 156-171, 1881.
 I. Johnston, D. McCluskey, C. Tan, and M. Tracey, "Mechanical characterization of bulk Sylgard 184 for microfluidics and microengineering," Journal of Micromechanics and Microengineering, vol. 24, no. 3, pp. 1-7, 2014.
 K. D. Costa, "Single-cell elastography: probing for disease with the atomic force microscope," Disease markers, vol. 19, no. 2-3, pp. 139-154, 2004.
 I. N. Sneddon, "The relation between load and penetration in the axisymmetric Boussinesq problem for a punch of arbitrary profile," International journal of engineering science, vol. 3, no. 1, pp. 47-57, 1965.
 W. C. Oliver and G. M. Pharr, "An improved technique for determining hardness and elastic modulus using load and displacement sensing indentation experiments," Journal of materials research, vol. 7, no. 06, pp. 1564-1583, 1992.
 J. Chen, "Nanobiomechanics of living cells: a review," Interface focus, vol. 4, no. 2, pp. 1-16, 2014.
 B. V. Derjaguin, V. M. Muller, and Y. P. Toporov, "Effect of contact deformations on the adhesion of particles," Journal of Colloid and interface science, vol. 53, no. 2, pp. 314-326, 1975.
 K. Johnson, K. Kendall, and A. Roberts, "Surface energy and the contact of elastic solids," in Proceedings of the Royal Society of London A: Mathematical, Physical and Engineering Sciences, 1971, vol. 324, no. 1558, pp. 301-313: The Royal Society.
 D. M. Ebenstein and K. J. Wahl, "A comparison of JKR-based methods to analyze quasi-static and dynamic indentation force curves," Journal of Colloid Interface Science, vol. 298, no. 2, pp. 652-62, 2006.
 D. Maugis and M. Barquins, "Fracture mechanics and adherence of viscoelastic solids," in Adhesion and adsorption of polymers: Springer, 1980, pp. 203-277.
 D. Maugis and B. Gauthier-Manuel, "JKR-DMT transition in the presence of a liquid meniscus," Journal of adhesion science and technology, vol. 8, no. 11, pp. 1311-1322, 1994.
 J. E. Mark, H. R. Allcock, and R. West, Inorganic polymers. Oxford University Press, 2005.
 D. C. Lin, D. I. Shreiber, E. K. Dimitriadis, and F. Horkay, "Spherical indentation of soft matter beyond the Hertzian regime: numerical and experimental validation of hyperelastic models," Biomechanics and Modeling in Mechanobiology, vol. 8, no. 5, pp. 345-358, 2009.
 R. W. Ogden, "Large Deformation Isotropic Elasticity - on the Correlation of Theory and Experiment for Incompressible Rubberlike Solids," Proceedings of the Royal Society of London Series a-Mathematical and Physical Sciences, vol. 326, no. 1567, pp. 565-584, 1972.
 T. Kaster, I. Sack, and A. Samani, "Measurement of the hyperelastic properties of ex vivo brain tissue slices," Journal of Biomechanics, vol. 44, no. 6, pp. 1158-1163, 2011.
 A. Samani and D. Plewes, "A method to measure the hyperelastic parameters of ex vivo breast tissue samples," Physics in Medicine and Biology, vol. 49, no. 18, pp. 4395-4405, 2004.
 M. M. Gibbons and W. S. Klug, "Influence of nonuniform geometry on nanoindentation of viral capsids," Biophysical journal, vol. 95, no. 8, pp. 3640-3649, 2008.
 C. Wex, S. Arndt, A. Stoll, C. Bruns, and Y. Kupriyanova, "Isotropic incompressible hyperelastic models for modelling the mechanical behaviour of biological tissues: a review," Biomedical Engineering / Biomedizinische Technik, vol. 60, no. 6, pp. 577-592, 2015.
 L. A. Mihai, L. Chin, P. A. Janmey, and A. Goriely, "A comparison of hyperelastic constitutive models applicable to brain and fat tissues," Journal of the Royal Society Interface, vol. 12, no. 110, pp. 1-12, 2015.
 M. L. Crichton, B. C. Donose, X. F. Chen, A. P. Raphael, H. Huang, and M. A. F. Kendall, "The viscoelastic, hyperelastic and scale dependent behaviour of freshly excised individual skin layers," Biomaterials, vol. 32, no. 20, pp. 4670-4681, 2011.
 P. M. Elias, "The skin barrier as an innate immune element," Seminars in immunopathology, vol. 29, no. 1, pp. 3-14, 2007.
 B. Carlson, "Integumentary, skeletal, and muscular systems," Human Embryology and Developmental Biology, pp. 153-181, 1994.
 K. S. Saladin, "Human Anatomy," 2007.
 L. Ma, C. Gao, Z. Mao, J. Zhou, J. Shen, X. Hu, and C. Han, "Collagen/chitosan porous scaffolds with improved biostability for skin tissue engineering," Biomaterials, vol. 24, no. 26, pp. 4833-41, 2003.
 S. G. Priya, H. Jungvid, and A. Kumar, "Skin tissue engineering for tissue repair and regeneration," Tissue Engineering Part B: Reviews, vol. 14, no. 1, pp. 105-18, 2008.
 F. Groeber, M. Holeiter, M. Hampel, S. Hinderer, and K. Schenke-Layland, "Skin tissue engineering--in vivo and in vitro applications," Advanced Drug Delivery Reviews, vol. 63, no. 4-5, pp. 352-66, 2011.
 J. S. Orringer, S. Kang, T. M. Johnson, D. J. Karimipour, T. Hamilton, C. Hammerberg, J. J. Voorhees, and G. J. Fisher, "Connective tissue remodeling induced by carbon dioxide laser resurfacing of photodamaged human skin," Archives of Dermatology, vol. 140, no. 11, pp. 1326-1332, 2004.
 P. L. Shao, J. D. Liao, T. W. Wong, Y. C. Wang, S. Leu, and H. K. Yip, "Enhancement of Wound Healing by Non-Thermal N2/Ar Micro-Plasma Exposure in Mice with Fractional-CO2-Laser-Induced Wounds," PLoS One, vol. 11, no. 6, pp. 1-15, 2016.
 C. P. Kelly, C. Pothoulakis, and J. T. LaMont, "Clostridium difficile colitis," New England Journal of Medicine, vol. 330, no. 4, pp. 257-262, 1994.
 D. E. Voth and J. D. Ballard, "Clostridium difficile toxins: mechanism of action and role in disease," Clinical microbiology reviews, vol. 18, no. 2, pp. 247-263, 2005.
 S. A. Kuehne, S. T. Cartman, J. T. Heap, M. L. Kelly, A. Cockayne, and N. P. Minton, "The role of toxin A and toxin B in Clostridium difficile infection," Nature, vol. 467, no. 7316, pp. 711-713, 2010.
 C. W. Knetsch, M. P. M. Hensgens, C. Harmanus, M. W. van der Bijl, P. H. M. Savelkoul, E. J. Kuijper, J. Corver, and H. C. van Leeuwen, "Genetic markers for Clostridium difficile lineages linked to hypervirulence," Microbiology, vol. 157, no. Pt 11, pp. 3113-3123, 2011.
 Y.-C. Wu, J.-J. Lee, B.-Y. Tsai, Y.-F. Liu, C. -M. Chen, N. Tien, P.-J. Tsai, and T.-H. Chen, "Potentially hypervirulent Clostridium difficile PCR ribotype 078 lineage isolates in pigs and possible implications for humans in Taiwan," International Journal of Medical Microbiology, vol. 306, no. 2, pp. 115-122, 2016.
 F. C. Tenover, T. Åkerlund, D. N. Gerding, R. V. Goering, T. Boström, A.-M. Jonsson, E. Wong, A. T. Wortman, and D. H. Persing, "Comparison of strain typing results for Clostridium difficile isolates from North America," Journal of Clinical Microbiology, vol. 49, no. 5, pp. 1831-1837, 2011.
 A. Goorhuis, D. Bakker, J. Corver, S. B. Debast, C. Harmanus, D. W. Notermans, A. A. Bergwerff, F. W. Dekker, and E. J. Kuijper, "Emergence of Clostridium difficile infection due to a new hypervirulent strain, polymerase chain reaction ribotype 078," Clinical Infectious Diseases, vol. 47, no. 9, pp. 1162-1170, 2008.
 A. A. Monteiro, R. N. Pires, S. Persson, E. M. Rodrigues Filho, and A. C. Pasqualotto, "A search for Clostridium difficile ribotypes 027 and 078 in Brazil," Brazillian Journal of Infectious Diseases, vol. 18, no. 6, pp. 672-674, 2014.
 B.-Y. Tsai, W.-C. Ko, T.-H. Chen, Y.-C. Wu, P.-H. Lan, Y. -H. Cheng, Y.-P. Hung, and P.-J. Tsai, "Zoonotic potential of the Clostridium difficile RT078 family in Taiwan," Anaerobe, vol. 41, pp. 125-120, 2016.
 S. L. Stubbs, J. S. Brazier, G. L. O'Neill, and B. I. Duerden, "PCR targeted to the 16S-23S rRNA gene intergenic spacer region of Clostridium difficile and construction of a library consisting of 116 different PCR ribotypes," Journal of Clinical Microbiology, vol. 37, no. 2, pp. 461-3, 1999.
 H. Kurka, A. Ehrenreich, W. Ludwig, M. Monot, M. Rupnik, F. Barbut, A. Indra, B. Dupuy, and W. Liebl, "Sequence similarity of Clostridium difficile strains by analysis of conserved genes and genome content is reflected by their ribotype affiliation," PLoS One, vol. 9, no. 1, pp. 1-11, 2014.
 S. Janezic, M. Ocepek, V. Zidaric, and M. Rupnik, "Clostridium difficile genotypes other than ribotype 078 that are prevalent among human, animal and environmental isolates," BMC Microbiology, vol. 12, pp. 1-8, 2012.
 D. Griffiths, W. Fawley, M. Kachrimanidou, R. Bowden, D. W. Crook, R. Fung, T. Golubchik, R. M. Harding, K. J. M. Jeffery, K. A. Jolley, R. Kirton, T. E. Peto, G. Rees, N. Stoesser, A. Vaughan, A. S. Walker, B. C. Young, M. Wilcox, and K. E. Dingle, "Multilocus sequence typing of Clostridium difficile," Journal of Clinical Microbiology, vol. 48, no. 3, pp. 770-778, 2010.
 J. B. Kaper, J. P. Nataro, and H. L. Mobley, "Pathogenic Escherichia coli," Nature Reviews Microbiology, vol. 2, no. 2, pp. 123-140, 2004.
 S.-H. Huang, C. Wass, Q. Fu, N. V. Prasadarao, M. Stins, and K. S. Kim, "Escherichia coli invasion of brain microvascular endothelial cells in vitro and in vivo: molecular cloning and characterization of invasion gene ibe10," Infection and Immunity, vol. 63, no. 11, pp. 4470-4475, 1995.
 T. Ikeda and H. Sandham, "Prevalence of Streptococcus mutans on various tooth surfaces in Negro children," Archives of oral biology, vol. 16, no. 10, pp. 1237-1240, 1971.
 M. Vatankhah-Varnosfaderani, W. F. M. Daniel, M. H. Everhart, A. A. Pandya, H. Liang, K. Matyjaszewski, A. V. Dobrynin. and S. S. Sheiko, "Mimicking biological stress-strain behaviour with synthetic elastomers," Nature, vol. 549, no. 7673, pp. 497-501, 2017.
 Z. Wang, "Polydimethylsiloxane mechanical properties measured by macroscopic compression and nanoindentation techniques," Master thesis, Mechanical Enginnering, University of South Florida, USA, 2011.
 R. L. Meyer, X. Zhou, L. Tang, A. Arpanaei, P. Kingshott, and F. Besenbacher, "Immobilisation of living bacteria for AFM imaging under physiological conditions," Ultramicroscopy, vol. 110, no. 11, pp. 1349-1357, 2010.
 C.-C. Wu, T.-M. Pan, C.-S. Wu, L.-C. Yen, C.-K. Chuang, S.-T. Pang, Y.-S. Yang, F.-H. Ko, "Label-free detection of prostate specific antigen using a silicon nanobelt field-effect transistor," International Journal of Electrochemical Science, vol. 7, no. 5, pp. 4432-4442, 2012.
 L. Calabri, N. Pugno, C. Menozzi, and S. Valeri, "AFM nanoindentation: tip shape and tip radius of curvature effect on the hardness measurement," Journal of Physics: Condensed Matter, vol. 20, no. 47, pp. 1-7, 2008.
 M.-S. Kim, J.-H. Choi, J.-H. Kim, and Y.-K. Park, "Accurate determination of spring constant of atomic force microscope cantilevers and comparison with other methods," Measurement, vol. 43, no. 4, pp. 520-526, 2010.
 P. J. Cumpson, C. A. Clifford, and J. Hedley, "Quantitative analytical atomic force microscopy: a cantilever reference device for easy and accurate AFM spring-constant calibration," Measurement Science and Technology, vol. 15, no. 7, pp. 1337-1346, 2004.
 H. J. Butt, P. Siedle, K. Seifert, K. Fendler, T. Seeger, E. Bamberg, A. L. Weisenhorn, K. Goldie, and A. Engel, "Scan speed limit in atomic force microscopy," Journal of microscopy, vol. 169, no. 1, pp. 75-84, 1993.
 N. A. Burnham, X. Chen, C. S. Hodges, G. A. Matei, E. J. Thoreson, C. J. Roberts, M. C. Davies, and S. J. B. Tendler, "Comparison of calibration methods for atomic-force microscopy cantilevers," Nanotechnology, vol. 14, no. 1, pp. 1-6, 2002.
 W. C. Young and R. G. Budynas, Roark's formulas for stress and strain. McGraw-Hill New York, 2002.
 J. E. Sader and L. White, "Theoretical analysis of the static deflection of plates for atomic force microscope applications," Journal of Applied physics, vol. 74, no. 1, pp. 1-9, 1993.
 O. Tabata, K. Kawahata, S. Sugiyama, and I. Igarashi, "Mechanical property measurements of thin films using load-deflection of composite rectangular membranes," Sensors and actuators, vol. 20, no. 1-2, pp. 135-141, 1989.
 C. J. Drummond and T. Senden, "Characterisation of the mechanical properties of thin film cantilevers with the atomic force microscope," in Materials Science Forum, vol. 189, pp. 107-114, 1995.
 P. Walsh, A. Omeltchenko, R. K. Kalia, A. Nakano, P. Vashishta, and S. Saini, "Nanoindentation of silicon nitride: A multimillion-atom molecular dynamics study," Applied physics letters, vol. 82, no. 1, pp. 118-120, 2003.
 A. Khan, J. Philip, and P. Hess, "Young’s modulus of silicon nitride used in scanning force microscope cantilevers," Journal of Applied Physics, vol. 95, no. 4, pp. 1667-1672, 2004.
 J. L. Hutter and J. Bechhoefer, "Calibration of atomic‐force microscope tips," Review of Scientific Instruments, vol. 64, no. 7, pp. 1868-1873, 1993.
 C. V. Heer, Statistical mechanics, kinetic theory, and stochastic processes. Elsevier, 2012.
 P. R. Saulson, "Thermal noise in mechanical experiments," Physical Review D, vol. 42, no. 8, p. 2437, 1990.
 H.-J. Butt, B. Cappella, and M. Kappl, "Force measurements with the atomic force microscope: Technique, interpretation and applications," Surface science reports, vol. 59, no. 1, pp. 1-152, 2005.
 R. W. Stark, T. Drobek, and W. M. Heckl, "Thermomechanical noise of a free v-shaped cantilever for atomic-force microscopy," Ultramicroscopy, vol. 86, no. 1, pp. 207-215, 2001.
 D. A. Walters, J. P. Cleveland, N. H. Thomson, and P. K. Hansma, "Short cantilevers for atomic force microscopy," Review of Scientific Instruments, vol. 67, no. 10, pp. 3583-3590, 1996.
 M. A. Beckmann, S. Venkataraman, M. J. Doktycz, J. P. Nataro, C. J. Sullivan, J. L. Morrell-Falvey, and D. P. Allison, "Measuring cell surface elasticity on enteroaggregative Escherichia coli wild type and dispersin mutant by AFM," Ultramicroscopy, vol. 106, no. 8-9, pp. 695-702, 2006.
 C. J. Sullivan, S. Venkataraman, S. T. Retterer, D. P. Allison, and M. J. Doktycz, "Comparison of the indentation and elasticity of E. coli and its spheroplasts by AFM," Ultramicroscopy, vol. 107, no. 10-11, pp. 934-942, 2007.
 C. B. Volle, M. A. Ferguson, K. E. Aidala, E. M. Spain, and M. E. Núñez, "Quantitative changes in the elasticity and adhesive properties of Escherichia coli ZK1056 prey cells during predation by bdellovibrio bacteriovorus 109J," Langmuir, vol. 24, no. 15, pp. 8102-10, 2008.
 S. R. Cohen and E. Kalfon-Cohen, "Dynamic nanoindentation by instrumented nanoindentation and force microscopy: a comparative review," Beilstein journal of nanotechnology, vol. 4, no. 1, pp. 815-833, 2013.
 D. M. Ebenstein and L. A. Pruitt, "Nanoindentation of biological materials," Nano Today, vol. 1, no. 3, pp. 26-33, 2006.
 I. D. Johnston, D. K. McCluskey, C. K. L. Tan, and M. C. Tracey, "Mechanical characterization of bulk Sylgard 184 for microfluidics and microengineering," Journal of Micromechanics and Microengineering, vol. 24, no. 3, pp. 1-7, 2014.
 J. F. Rubinson and Y. P. Kayinamura, "Charge transport in conducting polymers: insights from impedance spectroscopy," Chemical Society Reviews, vol. 38, no. 12, pp. 3339-3347, 2009.
 R. R. Anderson and J. A. Parrish, "The optics of human skin," Journal of Investigative Dermatology, vol. 77, no. 1, pp. 13-19, 1981.
 R. L. Bronaugh, R. F. Stewart, and E. R. Congdon, "Methods for in vitro percutaneous absorption studies II. Animal models for human skin," Toxicology and applied pharmacology, vol. 62, no. 3, pp. 481-488, 1982.
 J. G. Marks and J. J. Miller, Lookingbill and Marks' principles of dermatology. Elsevier Health Sciences, 2013.
 H. M. Sanders, M. Iafisco, E. M. Pouget, P. H. H. Bomans, F. Nudelman, G. Falini, G. de With, M. Merkx, G. J. Strijkers, K. Nicolay, and N. A. J. M. Sommerdijk, "The binding of CNA35 contrast agents to collagen fibrils," Chemical communications, vol. 47, no. 5, pp. 1503-1505, 2011.
 E. D. Congdon, J. Edson, and S. Yanitelli, "Gross structure of the subcutaneous layer of the anterior and lateral trunk in the male," American Journal of Anatomy, vol. 79, no. 3, pp. 399-429, 1946.
 B. R. MacIntosh, P. F. Gardiner, and A. J. McComas, Skeletal muscle: form and function. Human Kinetics, 2006.
 G. C. Gurtner, S. Werner, Y. Barrandon, and M. T. Longaker, "Wound repair and regeneration," Nature, vol. 453, no. 7193, pp. 314-321, 2008.
 C. Xia, Q. Meng, L.-Z. Liu, Y. Rojanasakul, X.-R. Wang, and B.-H. Jiang, "Reactive oxygen species regulate angiogenesis and tumor growth through vascular endothelial growth factor," Cancer research, vol. 67, no. 22, pp. 10823-10830, 2007.
 J. Buján, G. Pascual, C. Corrales, V. Gómez‐Gil, N. Garcia‐Honduvilla, and J. M. Bellón, "Muscle‐derived stem cells used to treat skin defects prevent wound contraction and expedite reepithelialization," Wound repair and regeneration, vol. 14, no. 2, pp. 216-223, 2006.
 S. Amini‐Nik, D. Glancy, C. Boimer, H. Whetstone, C. Keller, and B. A. Alman, "Pax7 Expressing Cells Contribute to Dermal Wound Repair, Regulating Scar Size through a β‐Catenin Mediated Process," Stem Cells, vol. 29, no. 9, pp. 1371-1379, 2011.
 H. Ju, S. Zhao, D. S. Jassal, and I. M. Dixon, "Effect of AT1 receptor blockade on cardiac collagen remodeling after myocardial infarction," Cardiovascular research, vol. 35, no. 2, pp. 223-232, 1997.
 A. J. Bailey, T. J. Sims, Le Lous, and S. bazin, "Collagen polymorphism in experimental granulation tissue," Biochemical Biophysical Research Communications, vol. 66, no. 4, pp. 1160-1165, 1975.
 R. A. Stabler, R. A. Stabler, L. F. Dawson, E. Valiente, M. D. Cairns, M. J. Martin, E. H. Donahue, T. V. Riley, J. G. Songer, E. J. Kuijper, K. E. Dingle, and B. W. Wren, "Macro and micro diversity of Clostridium difficile isolates from diverse sources and geographical locations," PLoS One, vol. 7, no. 3, pp. 1-12, 2012.
 M. Reil, M. Erhard, E. J. Kuijper, M. Kist, H. Zaiss, W. Witte, H. Gruber, and S. Borgmann, "Recognition of Clostridium difficile PCR-ribotypes 001, 027 and 126/078 using an extended MALDI-TOF MS system," European Journal of Clinical Microbiology & Infectious Diseases, vol. 30, no. 11, pp. 1431-1436, 2011.
 A. C. Chang and B. H. Liu, "Identification of Characteristic Macromolecules of Escherichia coli Genotypes by Atomic Force Microscope Nanoscale Mechanical Mapping," Nanoscale research letters, vol. 13, no. 1, pp. 1-6, 2018.
 A. Gillis, V. Dupres, G. Delestrait, J. Mahillon, and Y. F. Dufrêne, "Nanoscale imaging of Bacillus thuringiensis flagella using atomic force microscopy," Nanoscale, vol. 4, no. 5, pp. 1585-1591, 2012.
 U. B. Sleytr and T. J. Beveridge, "Bacterial S-layers," Trends in Microbiology, vol. 7, no. 6, pp. 253-260, Jun 1999.
 P. C. Gufler, D. Pum, U. B. Sleytr, and B. Schuster, "Highly robust lipid membranes on crystalline S-layer supports investigated by electrochemical impedance spectroscopy," Biochimica et Biophysica Acta - Biomembranes, vol. 1661, no. 2, pp. 154-165, 2004.
 A. I. Shevchuk, G. I. Frolenkov, D. Sánchez, P. S. James, N. Freedman, M. J. Lab, R. Jones, D. Klenerman, and Y. E. Korchev, "Imaging proteins in membranes of living cells by high-resolution scanning ion conductance microscopy," Angewandte Chemie, vol. 45, no. 14, pp. 2212-2216, 2006.
 U. B. Sleytr, B. Schuster, E. M. Egelseer, and D. Pum, "S-layers: principles and applications," FEMS Microbiology Reviews, vol. 38, no. 5, pp. 823-864, 2014.
 R. P. Fagan and N. F. Fairweather, "Biogenesis and functions of bacterial S-layers," Nature Review Microbiology, vol. 12, no. 3, pp. 211-222, 2014.
 C. Schäffer and P. Messner, "Surface-layer glycoproteins: an example for the diversity of bacterial glycosylation with promising impacts on nanobiotechnology," Glycobiology, vol. 14, no. 8, pp. 31-42, 2004.
 N. Ilk, E. M. Egelseer, and U. B. Sleytr, "S-layer fusion proteins--construction principles and applications," Current Opinion in Biotechnology, vol. 22, no. 6, pp. 824-831, 2011.
 F. L. Lederer, T. J. Günther, J. Raff, and K. Pollmann, "E. coli filament formation induced by heterologous S-layer expression," Bioengineered Bugs, vol. 2, no. 3, pp. 178-181, 2011.
 M. S. Walters and H. L. Mobley, "Identification of uropathogenic Escherichia coli surface proteins by shotgun proteomics," Journal of Microbiological Methods, vol. 78, no. 2, pp. 131-135, 2009.
 Y. Xie, V. Kolisnychenko, M. Paul-Satyaseela, S. Elliott, G. Parthasarathy, Y. Yao, G. Plunkett III, F. R. Blattner, and K. S. Kim, "Identification and characterization of Escherichia coli RS218-derived islands in the pathogenesis of E. coli meningitis," Journal of Infectious Diseases, vol. 194, no. 3, pp. 358-364, 2006.
 L. Yang and R. Bashir, "Electrical/electrochemical impedance for rapid detection of foodborne pathogenic bacteria," Biotechnology Advances, vol. 26, no. 2, pp. 135-150, 2008.
 A. Kadier, S. Kalil, P. Abdeshahian, K. Chandrasekhar, A. Mohamed, N. F. Azman, W. Logroño, Y. Simayi, and A. A. Hamid, "Recent advances and emerging challenges in microbial electrolysis cells (MECs) for microbial production of hydrogen and value-added chemicals," Renewable and Sustainable Energy Reviews, vol. 61, pp. 501-525, 2016.
 H. He, D. C. Chang, and Y.-K. Lee, "Nonlinear current response of micro electroporation and resealing dynamics for human cancer cells," Bioelectrochemistry, vol. 72, no. 2, pp. 161-168, 2008.
 V. A. Shepherd, "The cytomatrix as a cooperative system of macromolecular and water networks," Current Topics in Developmental Biology, vol. 75, pp. 171-223, 2006.
 P. B. Lillehoj, C. W. Kaplan, J. He, W. Shi, and C. M. Ho, "Rapid, electrical impedance detection of bacterial pathogens using immobilized antimicrobial peptides," Journal of Laboratory Automation, vol. 19, no. 1, pp. 42-9, 2014.
 N. K. Simha, H. Jin, M. L. Hall, S. Chiravarambath, and J. L. Lewis, "Effect of indenter size on elastic modulus of cartilage measured by indentation," Journal of biomechanical engineering, vol. 129, no. 5, pp. 767-775, 2007.
 Z. Wang, A. A. Volinsky, and N. D. Gallant, "Nanoindentation study of polydimethylsiloxane elastic modulus using Berkovich and flat punch tips," Journal of Applied Polymer Science, vol. 132, no. 5, pp. 1-7, 2015.
 A. C. Chang and B. H. Liu, "Modified flat-punch model for hyperelastic polymeric and biological materials in nanoindentation," Mechanics of Materials, vol. 118, pp. 17-21, 2017.
 T. Wang, "The mechanical properties of hydrogel with CNTs and PDMS by nanoindentation," Master of Science, Department of Mechanical Engineering, Tatung University, Taiwan, 2011.
 G. D. Shockman and J. Barren, "Structure, function, and assembly of cell walls of gram-positive bacteria," Annual Reviews in Microbiology, vol. 37, no. 1, pp. 501-527, 1983.
 T. J. Beveridge, "Structures of gram-negative cell walls and their derived membrane vesicles," Journal of bacteriology, vol. 181, no. 16, pp. 4725-4733, 1999.
 P. Eaton, J. C. Fernandes, E. Pereira, M. E. Pintado, and F. Xavier Malcata, "Atomic force microscopy study of the antibacterial effects of chitosans on Escherichia coli and Staphylococcus aureus," Ultramicroscopy, vol. 108, no. 10, pp. 1128-1134, 2008.