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研究生:李昆霖
研究生(外文):Kun-LinLi
論文名稱:變異鏈球菌於微區之結構,生物力學,與電性之分析
論文名稱(外文):Analysis of Structure, Biomechanical, and Electrical Properties on Local Positions of Streptococcus mutans Biofilm
指導教授:劉浩志
指導教授(外文):Bernard Hao-Chih Liu
學位類別:碩士
校院名稱:國立成功大學
系所名稱:材料科學及工程學系碩博士班
學門:工程學門
學類:材料工程學類
論文種類:學術論文
論文出版年:2012
畢業學年度:100
語文別:英文
論文頁數:60
中文關鍵詞:變異鏈球菌原子力顯微鏡掃描阻抗顯微鏡電子束沉積
外文關鍵詞:Streptococcus mutansatomic force microscopescanning impedance microscopeelectron beam induced deposition
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變異鏈球菌(Streptococcus mutans)在人類的齟齒中扮演著重要的角色,藉由與蔗糖合成所分泌出的胞外基質(extracellular polysaccharide)來形成生物膜並侵蝕牙齒表面的組織。為了瞭解變異鏈球菌之生物膜與胞外基質的物理性質,本實驗使用原子力顯微鏡對生物膜進行不同狀態下之結構與機械性質的量測。原子力顯微鏡具有奈米尺度的針頭,使其在微區的量測中能夠有著靈活的運用。
過去的研究中,針對變異鏈球菌的分析主要著重於不同種類之變異鏈球菌的表面形貌以及粗糙度分析,或是以蔗糖培養不同時間下,針尖與變異鏈球菌表面間的吸附力差異,於其他機械性質的部分著墨甚少。因此在本實驗中,我們針對變異鏈球菌在Z-ring (或隔膜),細胞壁,兩菌間的連結處以及胞外基質在不同的成長時間下以及脫水前後進行力學性質的量測,藉由得到的力與距離的曲線關係圖,分析與探討彈力常數以及結構與力的關係。此外,我們利用掃描阻抗顯微鏡 (Scanning Impedance Microscopy)進行電性的量測,從得到的阻抗圖譜,我們可以觀察生物膜在不同狀況下的組成差異。
在本研究中所得到的彈性係數,我們觀察到細胞壁有著最高的彈性係數,Z-ring次之,其後為兩細菌間的連結處以及胞外基質。在得到的數據中,我們發現連結處及胞外基質有著相近的彈性係數,藉此可以推斷兩者間在組成或結構上存在著相似性。此外隨著成長時間的增加以及酒精的脫水作用,彈性係數也會隨之增加。在Z-ring進行力曲線的量測時,探針針頭在拉回時的曲線由於細胞內蛋白鍵結的拉伸出現了週期性鋸齒狀的行為,在脫水過後,由於細胞結構變得較為剛性導致此現象會變得更加明顯。藉由接觸式掃描模式以及單點掃描的功能,我們觀察到變異鏈球菌的隔膜處存在著由生物反應 (metabolism)所引起的週期性擺動,而此行為在脫水後雖然有減弱之跡象但依舊存在著,代表細菌中某些代謝反應在脫水後仍會進行著。
在實驗的過程中,我們發現到探針針尖的大小也會影響量測隔膜時所觀察到的行為。因此利用電子束沉積法 (Electron Beam Induced Deposition)的方式長出具有高深寬比的針尖。在成長的過程中,我們利用了靜電吸附的方式將氧化鋁的奈米顆粒吸附在探針上作為針尖成長前導物,並且成功的長出了長度約200奈米的針尖。利用所長出的針進行掃描,我們得到了較一般商業探針更佳解析度的影像。同時,我們在連續的電子束沉積過程中,觀察在不同順序下,所長出的針在主要成分上的變化。(附錄B)

Streptococcus mutans plays an important role in human dental caries. The S. mutans secretes extracellular polysaccharide (EPS) by synthesizing with sucrose to form the biofilm and erode the tooth tissues (enamel). For the purpose of characterizing the physical properties of S. mutans biofilm, we utilized the atomic force microscope (AFM) to measure the structure and biomechanical properties. An AFM combined with a nanoscale tip apex, allows a high flexibility in probing low-dimensional area.
In previous researches, analyses on S. mutans were focused on the surface morphology and roughness of different strains, or the tip-cell adhesion force under variant sucrose treatment time; however, other biomechanical properties have not been well discussed. Therefore, we made a mechanical measurement on four distinct regions: Z-ring (septum), cell wall, the interconnecting area between two bacteria, and EPS under different biofilm cultivation with/without dehydration. In addition, we use the scanning impedance microscope (SIM) to characterize the differences in the composition and structure between different biofilm statuses.
We observed that the cell walls have the highest elastic modulus, followed by Z-ring, interconnects, and EPS. The elastic moduli of the EPS region and of the interconnecting region between two cells are very similar, suggesting that the chemical composition and molecular structure of these regions might be similar. It is also noticed that the elastic moduli increase with increasing growth time, and the elastic moduli also increase after dehydration treatment due to stiffened cell wall. The force-displacement curve of Z-ring area shows a distinct serrated behavior, which becomes more pronounced after dehydration. We suppose that the serrated force-displacement curve was caused by the unfolding behavior of the cell protein on the septum area; the dehydration treatment stiffened cell wall in septum, thus the magnitude of force-displacement serration was increased. We further investigated the active metabolism in the septum of S. mutans by in situ detection of the biomechanical fluctuations via an AFM dot scan under contact mode. The pulsation patterns varied with biofilm growth time, and the detection of weaker patterned pulsation existence on dehydrated S. mutans suggests unknown cell metabolism still active after dehydration.
In the process of measuring the biomechanism on the septum, we noticed that the force-displacement curve shows distinct behaviors by using different tip diameter and shape. Hence, we utilize the electron beam induced deposition (EBID) to produce a high-aspect-ratio tip with a view to measuring the biomechanical properties on the septum. We use the electrostatic pick up manipulation to adsorb the alumina nanoparticles on the AFM tip apex for providing the precursor of EBID tip fabrication, and successfully produce a needle-like tip with a length of 200nm. By scanning with the fabricated EBID tip, we obtained a better resolution image than commercial AFM probe. Meanwhile, in the continuous EBID tip fabrication process, we observed the composition varied with the tip creating sequence. (Appendix B)

摘要 i
Abstract iii
Acknowledgement v
Contents vii
List of Tables x
List of Figures xi
Nomenclature xiv
Chapter 1 1
Introduction 1
1.1. Streptococcus mutans 1
1.2. Atomic Force Microscopy (AFM) 3
1.2.1. Background 3
1.2.2. Basic Principles 5
1.2.2.1. Contact (DC) mode 7
1.2.2.2 Tapping (AC) mode 7
1.2.2.3 Non-contact mode 8
1.3. Biomechanical and motion analysis with AFM 8
1.3.1 Force Spectroscopy 8
1.3.2. Dot scan in Contact mode 11
1.4. Scanning Impedance Microscopy 12
1.5. Motivation 12
Chapter 2 14
Experimental 14
2.1. Streptococcus mutans culture 14
2.2. Measurement on Biomechanical Properties 14
2.2.1. AFM image and nanoindentation on S. mutans 14
2.2.2. Measurement on cell pulsation 16
2.3. Analysis method for Biomechanical properties 17
2.3.1. Hertz model 17
2.3.2. Fast Fourier Transform 19
2.4. Electrical properties measurement 20
Chapter 3 21
Analysis on S. mutans 21
3.1. S. mutans morphology 21
3.2. Elastic properties 22
3.3. The force-displacement behavior in each area 26
3.4. Pulsation on septum 33
3.5. Electrical properties of S. mutans biofilm 36
Chapter 4 38
Conclusions and Future works 38
4.1. Conclusions 38
4.2. Future works 39
References 40
Appendix A 45
Electrostatic manipulation 45
A.1. Electrostatic pick up manipulation with AFM 45
Appendix B 47
Electron beam induced deposition 47
B.1. Introduction and principle 47
B.2. Nanofabrication of EBID AFM tip 49
B.2.1. Electrostatic manipulation on Alumina nanoparticles 49
B.2.2. E-beam-induced-deposition 50
B.3. Fabrication and Composition Analysis 51
B.4. Scan capability test 56
B.5. TEM analysis 58
B.6. Conclusions 60
B.7. Future works 60


[1]W. J. Loesche, ROLE OF STREPTOCOCCUS-MUTANS IN HUMAN DENTAL DECAY, Microbiological Reviews, vol. 50, pp. 353-380, Dec 1986.
[2]S. Hamada and H. D. Slade, BIOLOGY, IMMUNOLOGY, AND CARIOGENICITY OF STREPTOCOCCUS-MUTANS, Microbiological Reviews, vol. 44, pp. 331-384, 1980.
[3]R. H. Selwitz, et al., Dental caries, Lancet, vol. 369, pp. 51-59, Jan 2007.
[4]S. Pichoff and J. Lutkenhaus, Tethering the Z ring to the membrane through a conserved membrane targeting sequence in FtsA, Molecular Microbiology, vol. 55, pp. 1722-1734, Mar 2005.
[5]S. Pichoff and J. Lutkenhaus, Unique and overlapping roles for ZipA and FtsA in septal ring assembly in Escherichia coli, Embo Journal, vol. 21, pp. 685-693, Feb 2002.
[6]C. A. Hale and P. A. J. deBoer, Direct binding of FtsZ to ZipA, an essential component of the septal ring structure that mediates cell division in E-coli, Cell, vol. 88, pp. 175-185, Jan 1997.
[7]D. W. Adams and J. Errington, Bacterial cell division: assembly, maintenance and disassembly of the Z ring, Nat Rev Micro, vol. 7, pp. 642-653, 2009.
[8]L. W. Hamoen, et al., SepF, a novel FtsZ-interacting protein required for a late step in cell division, Molecular Microbiology, vol. 59, pp. 989-999, Feb 2006.
[9]S. Ishikawa, et al., A new FtsZ-interacting protein, YlmF, complements the activity of FtsA during progression of cell division in Bacillus subtilis, Molecular Microbiology, vol. 60, pp. 1364-1380, Jun 2006.
[10]Y. F. Dufrene, Atomic force microscopy, a powerful tool in microbiology, Journal of Bacteriology, vol. 184, pp. 5205-5213, Oct 2002.
[11]P. J. Eaton and P. West, Atomic force microscopy. Oxford ; New York: Oxford University Press, 2010.
[12]G. Binning, et al., SURFACE STUDIES BY SCANNING TUNNELING MICROSCOPY, Physical Review Letters, vol. 49, pp. 57-61, 1982.
[13]V. J. Morris, et al., Atomic force microscopy for biologists, 2nd ed. London
Singapore ; Hakensack, NJ: Imperial College Press ;
Distributed by World Scientific Pub., 2010.
[14]A. San Paulo and R. Garcia, Tip-surface forces, amplitude, and energy dissipation in amplitude-modulation (tapping mode) force microscopy, Physical Review B, vol. 64, pp. art. no.-193411, Nov 2001.
[15]F. J. Giessibl, Forces and frequency shifts in atomic-resolution dynamic-force microscopy, Physical Review B, vol. 56, pp. 16010-16015, Dec 1997.
[16]J. Liang and G. Scoles, Nanografting of alkanethiols by tapping mode atomic force microscopy, Langmuir, vol. 23, pp. 6142-6147, May 2007.
[17]B. H. Liu and D. B. Chang, Simulation-aided design and fabrication of nanoprobes for scanning probe microscopy, Ultramicroscopy, vol. 111, pp. 337-341, Apr 2011.
[18]D. J. Muller and Y. F. Dufrene, Atomic force microscopy as a multifunctional molecular toolbox in nanobiotechnology, Nature Nanotechnology, vol. 3, pp. 261-269, May 2008.
[19]G. Guhados, et al., Measurement of the elastic modulus of single bacterial cellulose fibers using atomic force microscopy, Langmuir, vol. 21, pp. 6642-6646, Jul 2005.
[20]S. E. Cross, et al., Nanomechanical analysis of cells from cancer patients, Nature Nanotechnology, vol. 2, pp. 780-783, Dec 2007.
[21]D. Alsteens, et al., Structure, cell wall elasticity and polysaccharide properties of living yeast cells, as probed by AFM, Nanotechnology, vol. 19, Sep 2008.
[22]A. Touhami, et al., Nanoscale mapping of the elasticity of microbial cells by atomic force microscopy, Langmuir, vol. 19, pp. 4539-4543, May 2003.
[23]G. Francius, et al., Detection, localization, and conformational analysis of single polysaccharide molecules on live bacteria, Acs Nano, vol. 2, pp. 1921-1929, Sep 2008.
[24]S. Sen, et al., Indentation and adhesive probing of a cell membrane with AFM: Theoretical model and experiments, Biophysical Journal, vol. 89, pp. 3203-3213, Nov 2005.
[25]G. Lee, et al., Nanospring behaviour of ankyrin repeats, Nature, vol. 440, pp. 246-249, Mar 2006.
[26]D. J. Muller, et al., Controlled unzipping of a bacterial surface layer with atomic force microscopy, Proceedings of the National Academy of Sciences of the United States of America, vol. 96, pp. 13170-13174, Nov 1999.
[27]M. Sotomayor, et al., In search of the hair-cell gating spring: Elastic properties of ankyrin and cadherin repeats, Structure, vol. 13, pp. 669-682, Apr 2005.
[28]A. E. Pelling, et al., Local nanomechanical motion of the cell wall of Saccharomyces cerevisiae, Science, vol. 305, pp. 1147-1150, Aug 2004.
[29]A. E. Pelling, et al., Time dependence of the frequency and amplitude of the local nanomechanical motion of yeast, Nanomedicine: Nanotechnology, Biology and Medicine, vol. 1, pp. 178-183, 2005.
[30]C. M. Ruan, et al., Immunobiosensor chips for detection of Escherichia coli O157 : H7 using electrochemical impedance spectroscopy, Analytical Chemistry, vol. 74, pp. 4814-4820, Sep 2002.
[31]F. Patolsky, et al., Precipitation of an insoluble product on enzyme monolayer electrodes for biosensor applications: Characterization by faradaic impedance spectroscopy, cyclic voltammetry, and microgravimetric quartz crystal microbalance analyses, Analytical Chemistry, vol. 71, pp. 3171-3180, Aug 1999.
[32]S. E. Cross, et al., Atomic force microscopy study of the structure-function relationships of the biofilm-forming bacterium Streptococcus mutans, Nanotechnology, vol. 17, pp. S1-S7, Feb 2006.
[33]S. E. Cross, et al., Nanomechanical properties of glucans and associated cell-surface adhesion of Streptococcus mutans probed by atomic force microscopy under in situ conditions, Microbiology-Sgm, vol. 153, pp. 3124-3132, Sep 2007.
[34]S. O. Jensen, et al., Cell division in bacillus subtilis: FtsZ and FtsA association is Z-ring independent, and FtsA is required for efficient midcell Z-ring assembly, Journal of Bacteriology, vol. 187, pp. 6536-6544, Sep 2005.
[35]J. K. Singh, et al., SepF Increases the Assembly and Bundling of FtsZ Polymers and Stabilizes FtsZ Protofilaments by Binding along Its Length, Journal of Biological Chemistry, vol. 283, pp. 31116-31124, Nov 2008.
[36]M. Osawa, et al., Reconstitution of contractile FtsZ rings in liposomes, Science, vol. 320, pp. 792-794, May 2008.
[37]H. Liu, et al., Growth of single-walled carbon nanotubes from ceramic particles by alcohol chemical vapor deposition, Applied Physics Express, vol. 1, Jan 2008.
[38]I. U. Vakarelski, et al., Penetration of living cell membranes with fortified carbon nanotube tips, Langmuir, vol. 23, pp. 10893-10896, Oct 2007.
[39]R. E. J. Sladek, et al., Treatment of Streptococcus mutans biofilms with a nonthermal atmospheric plasma, Letters in Applied Microbiology, vol. 45, pp. 318-323, Sep 2007.
[40]A. Touhami, et al., Atomic force microscopy of cell growth and division in Staphylococcus aureus, Journal of Bacteriology, vol. 186, pp. 3286-3295, Jun 2004.
[41]B. Alberts, Molecular biology of the cell, 5th ed. New York: Garland Science, 2008.
[42]S. Decossas, et al., Atomic force microscopy nanomanipulation of silicon nanocrystals for nanodevice fabrication, Nanotechnology, vol. 14, pp. 1272-1278, Dec 2003.
[43]D. Fotiadis, et al., Imaging and manipulation of biological structures with the AFM, Micron, vol. 33, pp. 385-397, 2002.
[44]D. J. Muller, AFM: a nanotool in membrane biology, Biochemistry, vol. 47, pp. 7986-7998, Aug 2008.
[45]B. Mokaberi and A. A. G. Requicha, Compensation of scanner creep and hysteresis for AFM nanomanipulation, Ieee Transactions on Automation Science and Engineering, vol. 5, pp. 197-206, Apr 2008.
[46]T. Junno, et al., CONTROLLED MANIPULATION OF NANOPARTICLES WITH AN ATOMIC-FORCE MICROSCOPE, Applied Physics Letters, vol. 66, pp. 3627-3629, Jun 1995.
[47]J. E. Kim and C. S. Han, Use of dielectrophoresis in the fabrication of an atomic force microscope tip with a carbon nanotube: a numerical analysis, Nanotechnology, vol. 16, pp. 2245-2250, Oct 2005.
[48]H. W. Lee, et al., The effect of the shape of a tip's apex on the fabrication of an AFM tip with an attached single carbon nanotube, Sensors and Actuators a-Physical, vol. 125, pp. 41-49, Oct 2005.
[49]S. J. Randolph, et al., Focused, nanoscale electron-beam-induced deposition and etching, Critical Reviews in Solid State and Materials Sciences, vol. 31, pp. 55-89, 2006.
[50]W. F. van Dorp, et al., Approaching the resolution limit of nanometer-scale electron beam-induced deposition, Nano Letters, vol. 5, pp. 1303-1307, Jul 2005.
[51]K. Edinger, et al., Novel high resolution scanning thermal probe, Journal of Vacuum Science & Technology B, vol. 19, pp. 2856-2860, Nov-Dec 2001.
[52]J. D. Beard, et al., An atomic force microscope nanoscalpel for nanolithography and biological applications, Nanotechnology, vol. 20, Nov 2009.
[53]J. D. Beard and S. N. Gordeev, Large flexibility of high aspect ratio carbon nanostructures fabricated by electron-beam-induced deposition, Nanotechnology, vol. 21, Nov 2010.
[54]P. Boggild, et al., Fabrication and actuation of customized nanotweezers with a 25 nm gap, Nanotechnology, vol. 12, pp. 331-335, Sep 2001.
[55]S. Bauerdick, et al., Direct wiring of carbon nanotubes for integration in nanoelectromechanical systems, Journal of Vacuum Science & Technology B, vol. 24, pp. 3144-3147, Nov-Dec 2006.
[56]J. D. Beard and S. N. Gordeev, Fabrication and buckling dynamics of nanoneedle AFM probes, Nanotechnology, vol. 22, Apr 2011.
[57]K. L. Klein, et al., Single-crystal nanowires grown via electron-beam-induced deposition, Nanotechnology, vol. 19, Aug 2008.
[58]I. Utke, et al., Density determination of focused-electron-beam-induced deposits with simple cantilever-based method, Applied Physics Letters, vol. 88, Jan 2006.
[59]W. Ding, et al., Mechanics of hydrogenated amorphous carbon deposits from electron-beam-induced deposition of a paraffin precursor, Journal of Applied Physics, vol. 98, Jul 2005.
[60]I. Utke, et al., Focused-electron-beam-induced deposition of freestanding three-dimensional nanostructures of pure coalesced copper crystals, Applied Physics Letters, vol. 81, pp. 3245-3247, Oct 2002.
[61]Z. Q. Liu, et al., Nanofabrication of tungsten supertip by electron-beam-induced deposition, Physica E-Low-Dimensional Systems & Nanostructures, vol. 29, pp. 702-706, Nov 2005.
[62]Z. Q. Liu, et al., The growth behavior of self-standing tungsten tips fabricated by electron-beam-induced deposition using 200 keV electrons, Journal of Applied Physics, vol. 96, pp. 3983-3986, Oct 2004.
[63]F. Cicoira, et al., Electron beam induced deposition of rhodium from the precursor RhCl(PF3)(2) (2): morphology, structure and chemical composition, Journal of Crystal Growth, vol. 265, pp. 619-626, May 2004.
[64]M. Shimojo, et al., Electron beam-induced deposition using iron carbonyl and the effects of heat treatment on nanostructure, Applied Physics a-Materials Science & Processing, vol. 79, pp. 1869-1872, Dec 2004.
[65]M. Wendel, et al., Sharpened electron beam deposited tips for high resolution atomic force microscope lithography and imaging, Applied Physics Letters, vol. 67, pp. 3732-3734, Dec 1995.
[66]K. Mitsuishi, et al., Electron-beam-induced deposition using a subnanometer-sized probe of high-energy electrons, Applied Physics Letters, vol. 83, pp. 2064-2066, Sep 2003.
[67]A. Champi, et al., Thermal expansion coefficient of amorphous carbon nitride thin films deposited by glow discharge, Thin Solid Films, vol. 420–421, pp. 200-204, 2002.

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