變異鏈球菌是造成蛀牙的主要因素之一，此種菌藉由代謝口腔內的碳水化合物而形成一種高黏度的胞外基質(extracellular polysaccharide, EPS)黏附於牙齒上並形成生物膜層疊。變異鏈球菌在代謝養份之後將降低周遭環境酸鹼值並侵蝕牙齒琺瑯質，其胞外基質形成的生物膜亦會對牙齦產生不良影響進而使人患上牙周病。進行本實驗的主要目的為藉由阻抗分析技術找出偵測細菌活性與生物膜生長時數的方法。阻抗量測所得之結果可協助我們監控水管或下水道系統中生物膜黏附的厚度變化，在未來將可以進一步推廣至醫學應用並降低口腔牙周病及齲齒的機率。
在本實驗中，我們使用了三軸探針座與原子力顯微鏡(atomic force microscope, AFM)兩種不同系統量測變異鏈球菌的阻抗變化。原子力顯微鏡主要是用於微區阻抗量測(local impedance measurement, LIM)，能夠選取局部區域擷取試片阻抗訊息能夠使我們更為瞭解單一菌體的電學特性。接著我們提出一生物等效電路並以電路中各個電學元件模擬變異鏈球菌與生物膜與細胞膜特質。配合生物等效電路對阻抗圖譜做曲線擬合我們將得到模擬的各電路元件數值。
由所得結果我們比較不同量測系統下的阻抗數據並分析活體菌種與失去活性菌種的阻抗結果。根據模擬數據的變化趨勢提一理論：菌體與胞外基質在平面方向連結的方式可分別視為電容及電阻的串聯；而菌體與胞外基質在垂直方向的堆疊可分別視為電容及電阻的並聯。
使用三軸探針座與原子力顯微鏡系統量測阻抗頻譜，皆可觀察到生物膜厚度的變化，但使用三軸探針座時應不易控制下壓力道將壓縮生物膜試片，造成高估生物膜厚度及菌體顆粒數，其探針電極也因針頭半徑較大 (大於1 μm)，偵測到的細胞膜內部電阻值並不均勻。原子力顯微鏡能精準控制力道，並能成功的估算生物膜厚度的變化以及偵測到細胞膜内部阻抗為穩定且均勻的數值，又因為所使用的探針其針尖半徑小 (小於30 nm)，能夠使得量測的電極接觸電容值一致。
由各元件模擬數值當中，我們觀測到代表胞外基質的電阻值隨著電極距離增加與試片厚度減少而有增大的趨勢，主要源自於胞外基質與菌體在試片中分布不均。而在原子力顯微鏡系統量測下，因電極間距較小(導電探針與鋁基版)，所得到的胞外基質電阻值小於三軸探針座量測的數值。針對失去活性的菌體，胞外基質電阻值將微高於活體菌種，歸因於失去活性的細胞不再代謝養份使得周圍環境不再呈現酸性，且生物膜中水通道(water channel)的水份及其他導電物質也會流失，因此導電度比起活體菌種來得低。
我們同時試著量化並估算單一細胞的細胞膜電容值，觀測到失去活性的細胞有較低的細胞膜電容值，表示導電度降低，此現象是源自於細胞膜中控管離子進出的蛋白質因失去活性而停止離子的運輸而造成失去活性的細胞其導電度較低。

Streptococcus mutans is one of the main reasons that cause tooth decay. By metabolizing carbohydrate, S.mutans emits slime (extracellular polysaccharide, EPS) which adheres to teeth and forms layers of biofilm. Periodontal disease occurs due to low pH environment created by S.mutans, and this environment gradually erodes teeth enamel. Our goal here is to detect the living status and culture time for S.mutans biofilm using impedance analysis. This technique will greatly asist us monitor biofilm thickness on water pipe or sewer system. In the future, it can further apply to stomatology to lower the possibility of periodontitis and dental caries.
In our experiment, micropositioners and an atomic force microscope (AFM) system are for impedance measurement. AFM was for local impedance measurement (LIM) owing to its ability to choose a specific location for impedance measuring and helped us gain insight of the electrical property of a single cell. A biological equivalent circuit was demonstrated, and electrical components simulated characteristics of S.mutans EPS and cytoplasmic membrane. With the help of equivalent circuit, we obtained simulation data for each component.
From our results, we observed how measuring methods affects impedance results, and numerical differences between living and devitalized cells were analyzed. According to simulation results, a theory was proposed that cells and EPS connecting to each other in planar direction could be simulated respectively as capacitor and resistor in series; while cells and EPS piled up in lateral direction could be simulated respectively as capacitor and resistor in parallel.
Both impedance measuring systems were able to detect variation of biofilm thickness; however, force-controlling was not precise for micropositioner, and cell numbers and biofilm thickness were overestimated due to cell compression. The large radius (larger than 1μm) of probe tip for micropositioner also resulted in unstable and non-uniform resistance inside cytoplasmic membrane. Impedance measurement using AFM successfully estimated biofilm thickness as well as stable and uniform resistance inside cytoplasmic membrane. Moreover, AFM probe which had small radius tip (within 30 nm) led to a stable and consistent electrode double layer capacitance.
From numerical results for each component, we found that EPS resistance increased with increasing electrode spans and decreasing biofilm thickness due to non-uniform distribution of EPS and cells across the sample. Owing to shorter electrode spans, the EPS resistance in AFM system was smaller. As for devitalized sample, EPS resistance was higher and the conductivity was lower because cells ceased to create a low pH environment, and water with other conductive mater evaporated from water channels inside biofilm.
The cytoplasmic membrane capacitance for each cell was quantified, and the devitalized sample had lower capacitance and higher impedance. This phenomenon was caused by malfunction of membrane proteins and the termination of proton transportation.

[1] D. Ajdic, W. M. McShan, R. E. McLaughlin, G. Savic, J. Chang, M. B. Carson, et al., "Genome sequence of Streptococcus mutans UA159, a cariogenic dental pathogen," Proceedings of the National Academy of Sciences of the United States of America, vol. 99, pp. 14434-14439, Oct 29 2002.
[2] E. Johansson, R. Claesson, and J. W. V. van Dijken, "Antibacterial effect of ozone on cariogenic bacterial species," Journal of Dentistry, vol. 37, pp. 449-453, Jun 2009.
[3] S. Hamada and H. D. Slade, "Biology, immunology, and cariogenicity of streptococcus-mutans," Microbiological Reviews, vol. 44, pp. 331-384, 1980.
[4] W. J. Loesche, "Role of streptococcus-mutans in human dental decay," Microbiological Reviews, vol. 50, pp. 353-380, Dec 1986.
[5] S. K. Roberts, G. X. Wei, and C. D. Wu, "Evaluating biofilm growth of two oral pathogens," Letters in Applied Microbiology, vol. 35, pp. 552-556, 2002.
[6] K. Todar, Todar's online textbook of bacteriology: University of Wisconsin-Madison Department of Bacteriology, 2006.
[7] J. C. Weaver, K. T. Powell, R. A. Mintzer, H. Ling, and S. R. Sloan, "The electrical capacitance of bilayer membranes: The contribution of transient aqueous pores," Bioelectrochemistry and Bioenergetics, vol. 12, pp. 393-404, 1984.
[8] S. H. White, "A Study of Lipid Bilayer Membrane Stability Using Precise Measurements of Specific Capacitance," Biophysical Journal, vol. 10, pp. 1127-1148, 1970.
[9] A. I. Cano and J. A. Llobet, Contributions to the measurement of electrical impedance for living tissue ischemia injury monitoring: Universitat Politècnica de Catalunya, 2005.
[10] C. Margo, J. Prado, M. Kouider, A. Rouane, and M. Nadi, Bioimpedance spectroscopy for cell characterization in the 50Hz-5MHz frequency range, 2006.
[11] A. Lasia, "Electrochemical impedance spectroscopy and its applications," in Modern aspects of electrochemistry, ed: Springer, 2002, pp. 143-248.
[12] A. A. Volkov and A. S. Prokhorov, "Broadband dielectric spectroscopy of solids," Radiophysics and quantum electronics, vol. 46, pp. 657-665, 2003.
[13] R. Parsons, Modern Aspects of Electrochemistry vol. 1, 1954.
[14] A. J. Bard, Electroanalytical Chemistry. New York: MARCEL DEKKER, 1966.
[15] F. Silva, C. Gornes, M. Figueiredo, R. Costa, A. Martins, and C. M. Pereira, "The electrical double layer at the BMIM PF6 ionic liquid/electrode interface - Effect of temperature on the differential capacitance," Journal of Electroanalytical Chemistry, vol. 622, pp. 153-160, Oct 15 2008.
[16] S. S. Zhang, K. Xu, and T. R. Jow, "EIS study on the formation of solid electrolyte interface in Li-ion battery," Electrochimica Acta, vol. 51, pp. 1636-1640, 2006.
[17] W. Franks, I. Schenker, P. Schmutz, and A. Hierlemann, "Impedance characterization and modeling of electrodes for biomedical applications," Ieee Transactions on Biomedical Engineering, vol. 52, pp. 1295-1302, Jul 2005.
[18] T. Kim, J. Kang, J. H. Lee, and J. Yoon, "Influence of attached bacteria and biofilm on double-layer capacitance during biofilm monitoring by electrochemical impedance spectroscopy," Water Research, vol. 45, pp. 4615-4622, Oct 2011.
[19] L. Y. Chuanmin Ruan, and Yanbin Li, "Immunobiosensor Chips for Detection of Escherichia coli O157:H7 Using Electrochemical Impedance Spectroscopy," Analytical Chemistry, vol. 74, pp. 4814-4820, 2002.
[20] R. A. Millikan and E. S. Bishop, Elements of electricity: a practical discussion of the fundamental laws and phenomena of electricity and their practical applications in the business and industrial world: American Technical Society, 1917.
[21] G. Binnig, "Atomic Force Microscpoe," Phys Rev Lett, vol. 56, pp. 930-3, 1986.
[22] A. Touhami, B. Nysten, and Y. F. Dufrene, "Nanoscale mapping of the elasticity of microbial cells by atomic force microscopy," Langmuir, vol. 19, pp. 4539-4543, May 2003.
[23] M. Benoit and H. E. Gaub, "Measuring cell adhesion forces with the atomic force microscope at the molecular level," Cells Tissues Organs, vol. 172, pp. 174-189, 2002.
[24] M. Arnoldi, M. Fritz, E. Bauerlein, M. Radmacher, E. Sackmann, and A. Boulbitch, "Bacterial turgor pressure can be measured by atomic force microscopy," Physical Review E, vol. 62, pp. 1034-1044, Jul 2000.
[25] Q. Zhong, D. Inniss, K. Kjoller, and V. B. Elings, "Fractured polymer/silica fiber surface studied by tapping mode atomic force microscopy," Surface Science Letters, vol. 290, pp. 688-692, 1993.
[26] 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, 2013.
[27] A. E. Pelling, S. Sehati, E. B. Gralla, J. S. Valentine, and J. K. Gimzewski, "Local nanomechanical motion of the cell wall of Saccharomyces cerevisiae," Science, vol. 305, pp. 1147-1150, 2004.
[28] A. E. Pelling, S. Sehati, E. B. Gralla, and J. K. Gimzewski, "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.
[29] G. Agarwal and T. M. Nocera, "Atomic Force Microscopy," The Nanobiotechnology Handbook, p. 369, 2012.
[30] P. Xie, P. Gu, and J. J. Beaudoin, "Contact capacitance effect in measurement of ac impedance spectra for hydrating cement systems," Journal of Materials Science, vol. 31, pp. 144-149, Jan 1 1996.
[31] R. O'Hayre, G. Feng, W. D. Nix, and F. B. Prinz, "Quantitative impedance measurement using atomic force microscopy," Journal of Applied Physics, vol. 96, pp. 3540-3549, Sep 2004.
[32] L. J. Yang and Y. B. Li, "AFM and impedance spectroscopy characterization of the immobilization of antibodies on indium-tin oxide electrode through self-assembled monolayer of epoxysilane and their capture of Escherichia coli O157 : H7," Biosensors & Bioelectronics, vol. 20, pp. 1407-1416, Jan 2005.
[33] C. L. Enloe, E. Garnett, J. Miles, and S. Swanson, Physical Science: What the Technology Professional Needs to Know, illustrated ed. vol. 7: John Wiley & Sons, 2000.
[34] J. Ushida, M. Tokushima, M. Shirane, A. Gomyo, and H. Yamada, "Immittance matching for multidimensional open-system photonic crystals," Physical Review B, vol. 68, Oct 15 2003.
[35] W. H. Mulder, J. H. Sluyters, T. Pajkossy, and L. Nyikos, "Tafel current at fractal electrodes - connection with admittance spectra," Journal of Electroanalytical Chemistry, vol. 285, pp. 103-115, Jun 11 1990.
[36] J. B. Jorcin, M. E. Orazem, N. Pebere, and B. Tribollet, "CPE analysis by local electrochemical impedance spectroscopy," Electrochimica Acta, vol. 51, pp. 1473-1479, Jan 20 2006.
[37] D. Klotz, Characterization and Modeling of Electrochemical Energy Conversion Systems by Impedance Techniques: KIT Scientific Publishing, 2012.
[38] A. Conde, A. Duran, and M. de Damborenea, "Polymeric sol-gel coatings as protective layers of aluminium alloys," Progress in Organic Coatings, vol. 46, pp. 288-296, Jun 2003.
[39] E. Barsoukov and J. R. Macdonald, Impedance Spectroscopy: Theory, Experiment, and Applications: Wiley, 2005.
[40] C. Ho, I. D. Raistrick, and R. A. Huggins, "Application of ac techniques to the study of lithium diffusion in tungsten trioxide thin-films," Journal of the Electrochemical Society, vol. 127, pp. 343-350, 1980 1980.
[41] C. J. Newton and J. M. Sykes, "A galvanostatic pulse technique for investigation of steel corrosion in concrete," Corrosion Science, vol. 28, pp. 1051-1074, 1988.
[42] R. E. J. Sladek, S. K. Filoche, C. H. Sissons, and E. Stoffels, "Treatment of Streptococcus mutans biofilms with a nonthermal atmospheric plasma," Letters in Applied Microbiology, vol. 45, pp. 318-323, Sep 2007.
[43] E. M. Decker, I. Dietrich, C. Klein, and C. v. Ohle, "Dynamic Production of Soluble Extracellular Polysaccharides by Streptococcus mutans," International Journal of Dentistry, 2011.
[44] H. F. Jenkinson and R. J. Lamont, "Streptococcal adhesion and colonization," Critical Reviews in Oral Biology & Medicine, vol. 8, pp. 175-200, May 1997.
[45] J. R. Macdonald, J. Schoonman, and A. Lehnen, "Applicability and power of complex nonlinear least squares for the analysis of impedance and admittance data," Journal of Electroanalytical Chemistry and Interfacial Electrochemistry, vol. 131, pp. 77-95, 1982.
[46] V. F. Lvovich, Impedance Spectroscopy: Applications to Electrochemical and Dielectric Phenomena: Wiley, 2012.
[47] K. G. Murty, Linear programming: Wiley, 1983.
[48] G. B. Dantzig and M. N. Thapa, Linear Programming: 1: Introduction: Springer, 1997.
[49] G. B. Dantzig and M. N. Thapa, Linear Programming: 2: Theory and Extensions: Springer, 2003.
[50] T. Hill and P. Lewicki, Statistics: Methods and Applications : a Comprehensive Reference for Science, Industry, and Data Mining: StatSoft, 2006.
[51] T. Fei, "Foodborn Pathogens Detection With Nanoporous Anodic Aluminum Oxide Membrane Based Biosensor," Master Degree, Department of Health techonology and Informatics, Hong Kong Polytechnic University, 2012.
[52] J. Nalbandian, M. L. Freedman, J. M. Tanzer, and S. M. Lovelace, "Ultrastructure of Mutants of Streptococcus mutans with Reference to Agglutination, Adhesion, and Extracellular Polysaccharide," Infection and Immunity, vol. 10, pp. 1170-1179, November 1, 1974.
[53] R. M. Donlan, "Biofilms: Microbial Life on Surfaces," Emerging Infectious Diseases, vol. 8, pp. 881-890, 2002.
[54] 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, p. 275401, 2013.
[55] G. Thedei Jr., D. P. S. Leitao, M. Bolean, T. P. Paulino, A. C. C. Spadaro, and P. Ciancaglini, "Toluene permeabilization differentially affects F- and P-type ATPase activities present in the plasma membrane of Streptococcus mutans," Brazilian Journal of Medical and Biological Research, vol. 41, pp. 1047-1053, Dec 2008.