胞外聚合物 (Extracellular polymeric substances，EPS) 具有形成生物膜或者與其他物質自組裝的功能，而EPS的黏性 (stickiness) 決定聚集體的黏附效率，影響含碳物質的沉降。在本文研究中，我們使用了一種全新的方法，利用磁鑷子系統施加機械力 (〜15 pN) 對EPS的黏性進行定量，以評估EPS分子之間的相互作用。本研究選擇由兩種藻類 (Amphora sp.、Emiliania huxleyi) 和藻菌 (Sagittula Stellata) 所分泌的non-attached EPS作為主要的實驗模型，各別量化三種EPS的黏性後，接著改變水環境的鹽度，以觀察不同鹽度下EPS黏性的變化，最後添加EDTA捕獲溶液中的二價陽離子，評估疏水作用對EPS黏性的貢獻。此外，我們使用相同方法量測異質EPS之間的黏性，觀察不同種類EPS碰撞後的黏附現象，並且添加三種聚苯乙烯奈米顆粒，評估海洋中塑膠微粒對EPS黏性產生的影響。期許透過此一高通量定量EPS黏性方法的開發，對EPS的性質有更深入的了解，並將其廣泛應用於環境研究中。

Extracellular polymeric substances (EPS) have the function of forming biofilms or self-assembling with other substances, and the stickiness of EPS determines the adhesion efficiency of aggregates, affects the sedimentation of carbonaceous substances. In this study, we used a new method to quantify the stickiness of EPS by using the magnetic tweezers system and applying designed mechanical force (approximately 15 pN) to evaluate the interaction between EPS molecules. Non-attached EPS were extracted from two kinds of algae (Amphora sp., Emiliania huxleyi) and algae bacteria (Sagittula Stellata) were selected as the experimental models. After quantifying the stickiness of these EPS respectively, the salinity of the solution was decreased to observe the change of stickiness under different salinity. Finally, EDTA was added to capture the divalent cations in the solution to evaluate the contribution of hydrophobic effect to the stickiness of EPS. In addition, we used the same method to measure the stickiness between heterogeneous EPS to observe the adhesion phenomenon of different kinds of EPS after collision, and added polystyrene nanoparticle further to evaluate the environmental impacts on EPS assembly. It is expected that through the development of this high-throughput quantitative stickiness measurement method, we can further understand the properties of EPS and this developed method will be widely used in environmental research.

摘要-----------------------------------i
Abstract------------------------------ii
目錄----------------------------------iii
圖表目錄--------------------------------v
Chapter 1 文獻回顧-------------------1
1.1 胞外聚合物 (EPS)--------------------1
1.1.1 功能與重要性----------------------1
1.1.2 實驗模型--------------------------2
1.2 EPS黏性與其對海洋生態影--------------2
1.3 EPS間的物理化學作用------------------4
1.4 其他測量黏性之方法-------------------5
1.5 磁鉗系統----------------------------6
1.6奈米塑料 (Nanoplastic)---------------7
1.7 實驗設計與目的-----------------------7
Chapter 2 材料與方法-----------------9
2.1 藥品與儀器--------------------------9
2.2 磁鉗系統磁力分析--------------------10
2.2.1 修飾Lambda DNA-------------------10
2.2.2 磁珠修飾--------------------------11
2.2.3 DNA錨定實驗-----------------------12
2.2.4 軌跡與磁力分析--------------------14
2.3 EPS黏性實驗-------------------------15
2.3.1 EPS保存處理-----------------------15
2.3.2 修飾磁珠--------------------------15
2.3.3 樣品腔室製作----------------------16
2.4 確認磁珠表面吸附EPS------------------17
2.4.1 WGA染色--------------------------17
2.4.2 以SEM觀察磁珠表面-----------------17
2.5 分析黏性----------------------------18
2.6 鹽度改變對黏性的影響-----------------19
2.7 二價陽離子對EPS黏性的影響-------------19
Chapter 3 結果與討論-----------------20
3.1 磁鉗系統之磁力分析-------------------20
3.1.1 DNA長度測量-----------------------20
3.1.2磁珠軌跡---------------------------21
3.1.3 磁力分析--------------------------21
3.2 EPS黏性實驗-------------------------22
3.2.1 吸附EPS的磁珠表面------------------22
3.2.2 測量不同磁力下EPS-EH的黏性----------23
3.2.3 同質EPS間的黏性--------------------25
3.3 表面材質影響EPS黏性------------------27
3.4 異質EPS間的黏性----------------------29
3.5 鹽度改變對黏性造成的影響--------------31
3.6 二價陽離子對EPS黏性的影響-------------33
3.7 塑膠微粒對EPS黏性的影響---------------34
Chapter 4 結論-----------------------37
參考文獻--------------------------------38
附錄------------------------------------42
附錄一、使用光鑷子測量Lambda DNA的輪廓長度
附錄二、程式編碼

1. Santschi, P.H., et al., Can the protein/carbohydrate (P/C) ratio of exopolymeric substances (EPS) be used as a proxy for their ‘stickiness’ and aggregation propensity? Marine Chemistry, 2020. 218: p. 103734.
2. Long, R.A. and Azam, F., Antagonistic interactions among marine pelagic bacteria. Applied and environmental microbiology, 2001. 67(11): p. 4975-4983.
3. Xu, C., et al., Optimized isolation procedure for obtaining strongly actinide binding exopolymeric substances (EPS) from two bacteria (Sagittula stellata and Pseudomonas fluorescens Biovar II). Bioresource technology, 2009. 100(23): p. 6010-6021.
4. Zhang, S., Xu, C., and Santschi, P.H., Chemical composition and 234Th (IV) binding of extracellular polymeric substances (EPS) produced by the marine diatom Amphora sp. Marine Chemistry, 2008. 112(1-2): p. 81-92.
5. Bramucci, A.R. and Case, R.J., Phaeobacter inhibens induces apoptosis-like programmed cell death in calcifying Emiliania huxleyi. Scientific reports, 2019. 9(1): p. 1-12.
6. Ding, Y.X., et al., Amphiphilic exopolymers from Sagittula stellata induce DOM self-assembly and formation of marine microgels. Marine Chemistry, 2008. 112(1-2): p. 11-19.
7. Alldredge, A.L. and McGillivary, P., The attachment probabilities of marine snow and their implications for particle coagulation in the ocean. Deep Sea Research Part A. Oceanographic Research Papers, 1991. 38(4): p. 431-443.
8. Ducker, W.A. and Mastropietro, D., Forces between extended hydrophobic solids: Is there a long-range hydrophobic force? Current Opinion in Colloid & Interface Science, 2016. 22: p. 51-58.
9. Bhaskar, P. and Bhosle, N.B., Microbial extracellular polymeric substances in marine biogeochemical processes. Current Science, 2005: p. 45-53.
10. Mühlenbruch, M., et al., Mini‐review: Phytoplankton‐derived polysaccharides in the marine environment and their interactions with heterotrophic bacteria. Environmental microbiology, 2018. 20(8): p. 2671-2685.
11. Mague, T., et al., Extracellular release of carbon by marine phytoplankton; a physiological approach 1. Limnology and Oceanography, 1980. 25(2): p. 262-279.
12. Myklestad, S., et al., Rate of release of extracellular amino acids and carbohydrates from the marine diatom Chaetoceros affinis. Journal of Plankton Research, 1989. 11(4): p. 763-773.
13. Flemming, H.C. and Wingender, J., The biofilm matrix. Nature reviews microbiology, 2010. 8(9): p. 623-633.
14. Verdugo, P., Marine microgels. Annual Review of Marine Science, 2012. 4: p. 375-400.
15. Ackleh, A.S., Hallam, T.G., and Muller-Landau, H.C., Estimation of sticking and contact efficiencies in aggregation of phytoplankton: The 1993 SIGMA tank experiment. Deep Sea Research Part II: Topical Studies in Oceanography, 1995. 42(1): p. 185-201.
16. Xu, C., et al., Chemical composition and relative hydrophobicity of microbial exopolymeric substances (EPS) isolated by anion exchange chromatography and their actinide-binding affinities. Marine Chemistry, 2011. 126(1-4): p. 27-36.
17. Umemura, M., Yuguchi, Y., and Hirotsu, T., Hydration at glycosidic linkages of malto-and cello-oligosaccharides in aqueous solution from molecular dynamics simulation: Effect of conformational flexibility. Journal of Molecular Structure: THEOCHEM, 2005. 730(1-3): p. 1-8.
18. Frank, B.P. and Belfort, G., Polysaccharides and sticky membrane surfaces: critical ionic effects. Journal of Membrane Science, 2003. 212(1-2): p. 205-212.
19. Ribeck, N. and Saleh O.A., Multiplexed single-molecule measurements with magnetic tweezers. Review of Scientific Instruments, 2008. 79(9): p. 094301.
20. Smith, S.B., Finzi, L., and Bustamante, C., Direct mechanical measurements of the elasticity of single DNA molecules by using magnetic beads. Science, 1992. 258(5085): p. 1122-1126.
21. Kriegel, F., et al., Probing the salt dependence of the torsional stiffness of DNA by multiplexed magnetic torque tweezers. Nucleic acids research, 2017. 45(10): p. 5920-5929.
22. Lipfert, J., Hao, X., and Dekker, N.H., Quantitative modeling and optimization of magnetic tweezers. Biophysical journal, 2009. 96(12): p. 5040-5049.
23. Dulin, D., et al., High spatiotemporal-resolution magnetic tweezers: Calibration and applications for DNA dynamics. Biophysical journal, 2015. 109(10): p. 2113-2125.
24. Cnossen, J.P., Dulin, D., and Dekker, N., An optimized software framework for real-time, high-throughput tracking of spherical beads. Review of Scientific Instruments, 2014. 85(10): p. 103712.
25. Kim, K. and Saleh, O.A., A high-resolution magnetic tweezer for single-molecule measurements. Nucleic acids research, 2009. 37(20): p. e136-e136.
26. Velzeboer, I., Kwadijk, C., and Koelmans, A., Strong sorption of PCBs to nanoplastics, microplastics, carbon nanotubes, and fullerenes. Environmental science & technology, 2014. 48(9): p. 4869-4876.
27. Rossi, G., Barnoud, J., and Monticelli, L., Polystyrene nanoparticles perturb lipid membranes. The journal of physical chemistry letters, 2014. 5(1): p. 241-246.
28. Chaiet, L. and Wolf, F.J., The properties of streptavidin, a biotin-binding protein produced by Streptomycetes. Archives of biochemistry and biophysics, 1964. 106: p. 1-5.
29. Lee, G.U., Kidwell, D.A., and Colton, R.J., Sensing discrete streptavidin-biotin interactions with atomic force microscopy. Langmuir, 1994. 10(2): p. 354-357.
30. Janissen, R., et al., Invincible DNA tethers: Covalent DNA anchoring for enhanced temporal and force stability in magnetic tweezers experiments. Nucleic acids research, 2014. 42(18): p. e137-e137.
31. Satterthwait, A.C. and Jencks, W.P., Mechanism of the aminolysis of acetate esters. Journal of the American Chemical Society, 1974. 96(22): p. 7018-7031.
32. Reddy Paluvai, N., Mohanty, S., and Nayak, S., Mechanical and thermal properties of sisal fiber reinforced acrylated epoxidized castor oil toughened diglycidyl ether of bisphenol A epoxy nanocomposites. Journal of Reinforced Plastics and Composites, 2015. 34(18): p. 1476-1490.
33. Huan, Y., et al., Slide cover glass immobilized liquid crystal microdroplets for sensitive detection of an IgG antigen. RSC advances, 2017. 7(60): p. 37675-37688.
34. Kyaw, H.H., et al., Self-organization of gold nanoparticles on silanated surfaces. Beilstein journal of nanotechnology, 2015. 6(1): p. 2345-2353.
35. Lee, C.Y., et al., Structure and DNA Hybridization Properties of Mixed Nucleic Acid/Maleimide− Ethylene Glycol Monolayers. Analytical chemistry, 2007. 79(12): p. 4390-4400.
36. Koniev, O. and Wagner, A., Developments and recent advancements in the field of endogenous amino acid selective bond forming reactions for bioconjugation. Chemical Society Reviews, 2015. 44(15): p. 5495-5551.
37. Zhang, Z., Magnetic tweezers: Actuation, measurement, and control at nanometer scale. 2009, The Ohio State University.
38. Sarkar, R. and Rybenkov, V.V., A guide to magnetic tweezers and their applications. Frontiers in Physics, 2016. 4: p. 48.
39. Yu, Z., et al., A force calibration standard for magnetic tweezers. Review of Scientific Instruments, 2014. 85(12): p. 123114.
40. Johnson, K.C., et al., A multiplexed magnetic tweezer with precision particle tracking and bi-directional force control. Journal of Biological Engineering, 2017. 11(1): p. 1-13.
41. Assi, F., et al., Massively parallel adhesion and reactivity measurements using simple and inexpensive magnetic tweezers. Journal of applied physics, 2002. 92(9): p. 5584-5586.
42. Allemand, J., et al., pH-dependent specific binding and combing of DNA. Biophysical journal, 1997. 73(4): p. 2064-2070.
43. Miller, H., et al., Single-molecule techniques in biophysics: a review of the progress in methods and applications. Reports on Progress in Physics, 2017. 81(2): p. 024601.
44. Lionnet, T., et al., Single-molecule studies using magnetic traps. Cold Spring Harbor Protocols, 2012. 2012(1): p. pdb. top067488.
45. Huhle, A., et al., Camera-based three-dimensional real-time particle tracking at kHz rates and Ångström accuracy. Nature communications, 2015. 6(1): p. 1-8.
46. Pimprikar, P., et al., Influence of biomass and gold salt concentration on nanoparticle synthesis by the tropical marine yeast Yarrowia lipolytica NCIM 3589. Colloids and Surfaces B: Biointerfaces, 2009. 74(1): p. 309-316.
47. Diener, A., et al., Control of focal adhesion dynamics by material surface characteristics. Biomaterials, 2005. 26(4): p. 383-392.
48. Yong-Xue, D., et al., Spontaneous assembly of exopolymers from phytoplankton. TAO: Terrestrial, Atmospheric and Oceanic Sciences, 2009. 20(5): p. 7.
49. Chen, C.S., et al., Effects of engineered nanoparticles on the assembly of exopolymeric substances from phytoplankton. PLoS One, 2011. 6(7): p. e21865.