臺灣博碩士論文加值系統

水膠複合物在生物材料上的應用在這幾年一直備受矚目。水膠材料的新穎變化，將有助於多功能材料的發展，其中以生物化學檢測、組織工程、藥物釋放為大宗。本篇研究是利用萘乙酸為基礎合成出水膠，並研究它在生物醫學感測中的應用。實驗樣品以水相合成方法製備2-Naphthylacetic acid- L-Phenylalanine- L-Phenylalanine- L-O-Phosphor- L -Tyrosine (Nap-FF-Yp)。在水解反應中，磷酸水解酵素會將Nap-FF-Yp的磷酸根切下，使得超分子堆疊形成膠體。研究中首先對水膠形成的條件進行探討，例如樣品濃度、酸鹼值、溫度及反應時間。為了讓凝膠化系統同時產生顏色變化，我們在接著在Nap-FF-Yp中混入奈米金形成一複合材料，並且分析了奈米金加入後對凝膠化的影響。利用此一特性，我們建構出鹼性磷酸水解酵素活性的檢測系統。
此系統的作用機制是由於凝膠化產生奈米纖維網狀交聯，對奈米金形成立體空間障礙，進而抑制奈米金的聚集反應。其中凝膠化的反應速率主要來自酵素活性強弱。因此當酵素加入後，不但形成水膠，同時依照酵素活性不同會產生紅-藍的顏色變化。依照成膠、變色兩種關鍵指標，就可以推斷出磷酸水解酵素的活性。

Hydrogels are an important class of nanocomposite biomaterials, which have attracted great deal of attention in recent years for their application in biochemical analysis, tissue engineering and drug delivery. The present study was focused on the synthesis of a novel hydrogel based on Napthyl acetic moiety for its use in biomedical sensing. In this direction, liquid phase synthesis strategy is employed to synthesize 2-Naphthylaceticacid-L-Phenylalanine-L-Phenylalanine-L-O-Phosphor-L-Tyrosine (Nap-FF-Yp). During the hydrolysis reaction, phosphate group in Nap-FF-Yp was cleaved by alkaline phosphatase, leading π-π stacking of supramolecular occurred and finally turned into hydrogels. The conditions of hydrogelation such as concentration, pH value, temperature and reacting time were first optimized for its further biomedical application. To establish a colorimetric and gelation sensor, this hydrogel was mixed with AuNPs to form a novel composite material. The effects of combining AuNPs to hydrogel are also investigated. Finally, we constructed a detecting system to explore the activity of alkaline phosphatase.
In our sensing system, hydrogelation plays an important role during the response time. We suggested that the sensing mechanism is related to the hydrogelation phenomenon which is resulted from the reaction between phosphatase and Nap-FF-Yp. The hydrogelation caused supramolecular nanofibers getting thicker and the generating steric hindrance prevent AuNPs from aggregation. Meanwhile, the reaction rate of hydrogelation depends on the activity of alkaline phosphatase. Thus, the addition of alkaline phosphatase into Nap-FF-Yp and AuNPs composite material in TBS leads to hydrogel formation with immediate color changes from wine-red to blue. The color change of the chemosensor has been successfully verified in this study for its further practical application in biomedical field. By means of the combination of sol-to-gel transition and colorimetric assays, activity of alkaline phosphatase could be inferred easily and rapidly.

Chapter 1: Introduction ………………………………………………………………1
1.1 Overview of Biosensors ……………………………………………………1
1.2 Naked-Eye Detection for Biomedical Diagnosis …………………………2

Chapter 2: Literature Review and Motivation …………………………………………4
2.1 Alkaline Phosphatase ………………………………………………………4
2.1.1 Overview of Alkaline ………………………………………………4
2.1.2 Diagnosis Application of Alkaline Phosphatase ……………………6
2.1.3 Examples of Other Applications Using Alkaline Phosphatase ……6
2.1.4 Alkaline Phosphatase Detection by Using Different Methods ……11
2.2 Composite Materials from Hydrogels and Nanoparticles …………………16
2.2.1 Hydrogels …………………………………………………………16
2.2.1.1 Supramolecular Hydrogels ………………………………16
2.2.1.2 Detecting Application of Supramolecular Hydrogels ……19
2.2.1.3 Sol-Gel Transition for Detection …………………………21
2.2.1.4 Hydrogel Based on Diphenylalanine Peptide ……………24
2.2.2 Gold Nanoparticles ………………………………………………..32
2.2.2.1 Introduction of Gold Nanoparticles ………………………32
2.2.2.2 Alkaline Phosphatase Detection by Gold nanoparticles …35
2.2.3 Hydrogels Conjugated with Nanoparticles ………………………..37
2.3 Motivation …………………………………………………………………39

Chapter 3: Experiment ………………………………………………………………43
3.1 General ……………………………………………………………………43
3.2 Materials and Reagents ……………………………………………………43
3.3 Instruments ………………………………………………………………49
3.4 Experimental Methods ……………………………………………………51
3.4.1 Synthesis of O-Phospho-L-Tyrosine ………………………………52
3.4.2 Synthesize Supramolecular Hydrogel Compound Nap-FF-Yp …53
3.4.3 Prepare the Nap-FF-Yp Hydrogel at Different pH Value …………56
3.4.4 Nap-FF-Yp Hydrogel in Presence and Absence of Mg2+ …………56
3.4.5 Nap-FF-Yp Hydrogel in Different Buffer Solution ………………57
3.4.6 Prepare the Hydrogel with and without 37℃ Warming …………57
3.4.7 Prepare the Hydrogel in Different Concentration of Zn2+ …………58
3.4.8 Synthesis of Gold nanoparticles …………………………………58
3.4.9 Nap-FF-Yp Hydrogel Mixed with Gold Nanoparticles ……………60
3.4.10 Gelation with AuNPs Using Various Concentration of ALP ………60
3.4.11 Sample Preparation for Chemical and Property Analysis …………60

Chapter 4: Results and discussions …………………………………………………62
4.1 Synthesis and Characterization …………………………………………62
4.1.1 Characterization of O-Phosphor-L-Tyrosine ………………………62
4.1.2 Characterization of Nap-FF-Yp Compound ………………………63
4.1.3 Characterization of Gold Nanoparticles …………………………67
4.2 Nap-FF-Yp Hydrogelation Triggered by ALP ………………………….68
4.2.1 Gelation at Different pH Value ……………………………………69
4.2.2 Gelation in the Presence and Absence of Mg2+ ……………………70
4.2.3 Gelation in Different Buffer Solution ……………………………71
4.2.4 Gelation at 37℃ Warming ………………………………………73
4.2.5 Gelation in Different Concentration of Zn2+ ………………………74
4.3 Nap-FF-Yp Hydrogelation with Gold Nanoparticles ……………………75
4.3.1 The Steric Effect between Nap-FF-Yp and AuNPs ………………76
4.3.2 The Color Changes by Different Concentration of AuNPs ………77
4.3.3 Mechanism Verifying by Other Methods …………………………80
4.3.3.1 Verifying the Sol-to-Gel Transition ………………………80
4.3.3.2 Verifying the Colorimetric Assay by UV-Vis ……………83

Chapter 5: Conclusions ………………………………………………………………85


Reference ……………………………………………………………………………86

1. E. Bakker and Y. Qin, Electrochemical Sensors. Analytical Chemistry, 2006. 78(12): p. 3965-3984.
2. K. L. Haas and K. J. Franz, Application of Metal Coordination Chemistry to Explore and Manipulate Cell Biology. Chemical Reviews, 2009. 109(10): p. 4921-4960.
3. S. K. Kim, et al., Chemosensors for Pyrophosphate. Accounts of Chemical Research, 2008. 42(1): p. 23-31.
4. A. Heller, Electrical Wiring of Redox Enzymes. Accounts of Chemical Research, 1990. 23(5): p. 128-134.
5. L. C. Clark and C. Lyons, Electrode Systems for Continuous Monitoring in Cardiovascular Surgery. Annals of the New York Academy of Sciences, 1962. 102(1): p. 29-45.
6. Y. Lin, et al., Glucose Biosensors Based on Carbon Nanotube Nanoelectrode Ensembles. Nano Letters, 2004. 4(2): p. 191-195.
7. G. A. Urban, Micro-and Nanobiosensors—State of the Art and Trends. Measurement Science and Technology, 2009. 20(1): p. 012001.
8. A. Gao, et al., Enhanced Sensing of Nucleic Acids With Silicon Nanowire Field Effect Transistor Biosensors. Nano Letters, 2012. 12(10): p. 5262-5268.
9. S. Zeng, et al., Nanomaterials Enhanced Surface Plasmon Resonance for Biological and Chemical Sensing Applications. Chemical Society Reviews, 2014. 43(10): p. 3426-3452.
10. L. He, et al., Colloidal Au-Enhanced Surface Plasmon Resonance for Ultrasensitive Detection of DNA Hybridization. Journal of the American Chemical Society, 2000. 122(38): p. 9071-9077.
11. F. Fan, et al., Novel Genetically Encoded Biosensors Using Firefly Luciferase. ACS Chemical Biology, 2008. 3(6): p. 346-351.
12. J. J. Lavigne and E. V. Anslyn, Sensing a Paradigm Shift in the Field of Molecular Recognition: From Selective to Differential Receptors. Angewandte Chemie International Edition, 2001. 40(17): p. 3118-3130.
13. E. P. Marbach and M. H. Weil, Rapid Enzymatic Measurement of Blood Lactate and Pyruvate Use and Significance of Metaphosphoric Acid as a Common Precipitant. Clinical Chemistry, 1967. 13(4): p. 314-325.
14. W., H. Werner Rey, and W. H, Properties of a New Chromogen for Determination of Glucose in Blood According to God/Pod-Method. Zeitschrift fur Analytische Chemie Fresenius, 1970. 252(2-3): p. 224-&.
15. H. Stockbridge, R. Hardy, and C. Glueck, Photometric Determination of Cholesterol (CHOD-PAP Method). Journal of Laboratory and Clinical Medicine, 1989. 114: p. 142-151.
16. P. C. Ray, Size and Shape Dependent Second Order Nonlinear Optical Properties of Nanomaterials and Their Application in Biological and Chemical Sensing. Chemical Reviews, 2010. 110(9): p. 5332-5365.
17. K. Saha, et al., Gold Nanoparticles in Chemical and Biological Sensing. Chemical Reviews, 2012. 112(5): p. 2739-2779.
18. H. Jans and Q. Huo, Gold Nanoparticle-Enabled Biological and Chemical Detection and Analysis. Chemical Society Reviews, 2012. 41(7): p. 2849-2866.
19. J. Liu, Oligonucleotide-Functionalized Hydrogels as Stimuli Responsive Materials and Biosensors. Soft Matter, 2011. 7(15): p. 6757-6767.
20. C. Ren, et al., Self-Assembling Small Molecules for the Detection of Important Analytes. Chemical Society Reviews, 2014. 43(21): p. 7257-7266.
21. P. A. Gale and T. Gunnlaugsson, Preface: Supramolecular Chemistry of Anionic Species Themed Issue. Chemical Society Reviews, 2010. 39(10): p. 3595-3596.
22. N. Kameta, M. Masuda, and T. Shimizu, Two-Step Naked-Eye Detection of Lectin by Hierarchical Organization of Soft Nanotubes into Liquid Crystal and Gel Phases. Chemical Communications, 2015. 51(31): p. 6816-6819.
23. E. E. Kim and H. W. Wyckoff, Reaction Mechanism of Alkaline Phosphatase Based on Crystal Structures: Two-Metal Ion Catalysis. Journal of Molecular Biology, 1991. 218(2): p. 449-464.
24. A. Garen and C. Levinthal, A Fine-Structure Genetic and Chemical Study of the Enzyme Alkaline Phosphatase of E. coli I. Purification and Characterization of Alkaline Phosphatase. Biochimica et Biophysica Acta, 1960. 38: p. 470-483.
25. M. Harada, et al., Inorganic Pyrophosphatase Activity of Purified Bovine Pulp Alkaline Phosphatase at Physiological pH. Journal of Dental Research, 1986. 65(2): p. 125-127.
26. https://en.wikipedia.org/wiki/Alkaline_phosphatase#cite_note-pmid13666805-3.
27. T. Horiuchi, S. Horiuchi, and D. Mizuno, A Possible Negative Feedback Phenomenon Controlling Formation of Alkaline Phosphomonoesterase in Escherichia Coli. Nature, 1959. 183: p. 1529 - 1530.
28. M. H. Du, et al., Crystal Structure of Alkaline Phosphatase from Human Placenta at 1.8 Å Resolution Implication for a Substrate Specificity. Journal of Biological Chemistry, 2001. 276(12): p. 9158-9165.
29. D. Goltzman, D. Miao, and L. Martini, Alkaline Phosphatase. Encyclopedia of Endocrine Diseases, ed. L. Martini, Academic Press/Elsevier, San Diego, 2004: p. 164-169.
30. K. Ooi, et al., High‐Molecular Intestinal Alkaline Phosphatase in Chronic Liver Diseases. Journal of Clinical Laboratory Analysis, 2007. 21(3): p. 133-139.
31. G. Ramaswamy, et al., Serum Levels of Bone Alkaline Phosphatase in Breast and Prostate Cancers with Bone Metastasis. Indian Journal of Clinical Biochemistry, 2000. 15(2): p. 110-113.
32. G. Rao and L. Morghom, Correlation between Serum Alkaline Phosphatase Activity and Blood Glucose Levels. Enzyme, 1985. 35(1): p. 57-59.
33. N. Ehrhart, et al., Prognostic Importance of Alkaline Phosphatase Activity in Serum from Dogs with Appendicular Osteosarcoma: 75 cases (1990-1996). Journal of the American Veterinary Medical Association, 1998. 213(7): p. 1002-1006.
34. https://labtestsonline.org/understanding/analytes/alp/tab/test.
35. H. T. Pruessner, Detecting Celiac Disease in Your Patients. American Family Physician, 1998. 57(5): p. 1023-34, 1039-41.
36. C. Manoil, J. J. Mekalanos, and J. Beckwith, Alkaline Phosphatase Fusions: Sensors of Subcellular location. Journal of Bacteriology, 1990. 172(2): p. 515-518.
37. C. A. Brennan, et al., A Molecular Sensor System Based on Genetically Engineered Alkaline Phosphatase. Proceedings of the National Academy of Sciences, 1995. 92(13): p. 5783-5787.
38. B. Prakash, et al., Development of a Sensitive Enzymeimmunoassay (EIA) for Progesterone Determination in Unextracted Bovine Plasma Using the Second Antibody Technique. Journal of Steroid Biochemistry, 1987. 28(6): p. 623-627.
39. E. Engvall and P. Perlmann, Enzyme-Linked Immunosorbent Assay (ELISA) Quantitative Assay of Immunoglobulin G. Immunochemistry, 1971. 8(9): p. 871-874.
40. https://en.wikipedia.org/wiki/ELISA.
41. A. L. Ghindilis, et al., Immunosensors: Electrochemical Sensing and Other Engineering Approaches. Biosensors and Bioelectronics, 1998. 13(1): p. 113-131.
42. D. A. Palmer, T. E. Edmonds, and N. J. Seare, Flow Injection Immunosensor for Theophylline. Analytical Letters, 1993. 26(7): p. 1425-1439.
43. M. Tarasevich, Bioelectrocatalysis, in Comprehensive Treatise of Electrochemistry. 1985, Springer. p. 231-295.
44. J. Kulys, V. Razumas, and A. Malinauskas, Electrochemical Oxidation of Catechol and p-Aminophenol Esters in the Presence of Hydrolases. Journal of Electroanalytical Chemistry and Interfacial Electrochemistry, 1980. 116: p. 11-24.
45. H. T. Tang, et al., p-Aminophenyl Phosphate: An Improved Substrate for Electrochemical Enzyme Immnoassay. Analytica Chimica Acta, 1988. 214: p. 187-195.
46. C. G. Bauer, et al., Zeptomole-Detecting Biosensor for Alkaline Phosphatase in an Electrochemical Immunoassay for 2, 4-Dichlorophenoxyacetic Acid. Analytical Chemistry, 1996. 68(15): p. 2453-2458.
47. J. Ambler, D. Arnold, and A. Green, A Study of the Alkaline Phosphatase Activity in Some Commercial Quality Control Sera with p-Nitrophenyl Phosphate and Phenyl Phosphate Substrates. Clinica Chimica Acta, 1970. 27(2): p. 350-353.
48. www.sigmaaldrich.com/catalog/product/sigma/p7998?lang=en®ion=TW.
49. M. A. Arnold, Enzyme-Based Fiber Optic Sensor. Analytical Chemistry, 1985. 57(2): p. 565-566.
50. P. Fanjul-Bolado, M. González-Garcı́a, and A. Costa-Garcı́a, Voltammetric Determination of Alkaline Phosphatase and Horseradish Peroxidase Activity Using 3-Indoxyl Phosphate as Substrate: Application to Enzyme Immunoassay. Talanta, 2004. 64(2): p. 452-457.
51. V. F. Ximenes, et al., Facile Chemiluminescent Method for Alkaline Phosphatase Determination. Analytica Chimica Acta, 1999. 402(1): p. 99-104.
52. Y. Liu and K. S. Schanze, Conjugated Polyelectrolyte-Based Real-Time Fluorescence Assay for Alkaline Phosphatase with Pyrophosphate as Substrate. Analytical Chemistry, 2008. 80(22): p. 8605-8612.
53. S. Ito, et al., Highly Sensitive Electrochemical Detection of Alkaline Phosphatase. Analytica Chimica Acta, 2000. 424(1): p. 57-63.
54. N. M. Sangeetha and U. Maitra, Supramolecular Gels: Functions and Uses. Chemical Society Reviews, 2005. 34(10): p. 821-836.
55. K. Ishikawa, et al., Soft Nanotubes with a Hydrophobic Channel Hybridized with Au Nanoparticles: Photothermal Dispersion/Aggregation Control of C60 in Water. Advanced Functional Materials, 2013. 23(13): p. 1677-1683.
56. T. Shimizu, M. Masuda, and H. Minamikawa, Supramolecular Nanotube Architectures Based on Amphiphilic Molecules. Chemical Reviews, 2005. 105(4): p. 1401-1444.
57. J. Boekhoven and S. I. Stupp, 25th Anniversary Article: Supramolecular Materials for Regenerative Medicine. Advanced Materials, 2014. 26(11): p. 1642-1659.
58. X. Du, J. Zhou, and B. Xu, Supramolecular Hydrogels Made of Basic Biological Building Blocks. Chemistry–An Asian Journal, 2014. 9(6): p. 1446-1472.
59. N. Kameta, et al., Supramolecular Nanotube Hydrogels: Remarkable Resistance Effect of Confined Proteins to Denaturants. Chemistry of Materials, 2009. 21(24): p. 5892-5898.
60. S. Yamaguchi, et al., Cooperation between Artificial Receptors and Supramolecular Hydrogels for Sensing and Discriminating Phosphate Derivatives. Journal of the American Chemical Society, 2005. 127(33): p. 11835-11841.
61. M. Winson, et al., Dye Displacement Assay for Saccharide Detection with Boronate Hydrogels. Chemical Communications, 2009(5): p. 532-534.
62. J. H. Holtz and S. A. Asher, Polymerized Colloidal Crystal Hydrogel Films as Intelligent Chemical Sensing Materials. Nature, 1997. 389(6653): p. 829-832.
63. L. M. Bonanno and L. A. DeLouise, Integration of a Chemical‐Responsive Hydrogel into a Porous Silicon Photonic Sensor for Visual Colorimetric Readout. Advanced Functional Materials, 2010. 20(4): p. 573-578.
64. M. D. Cathell, et al., Structurally Colored Thiol Chitosan Thin Films as a Platform for Aqueous Heavy Metal Ion Detection. Biomacromolecules, 2007. 9(1): p. 289-295.
65. C. Cheng, et al., A New Synthesis Route of Hydrogel through Bioinspired Supramolecular Approach: Gelation, Binding Interaction and In Vitro Dressing. ACS Applied Materials & Interfaces, 2015.
66. H. Jung, et al., 3D Tissue Engineered Supramolecular Hydrogels for Controlled Chondrogenesis of Human Mesenchymal Stem Cells. Biomacromolecules, 2014. 15(3): p. 707-714.
67. S. Kiyonaka, et al., Semi-Wet Peptide/Protein Array Using Supramolecular Hydrogel. Nature Materials, 2004. 3(1): p. 58-64.
68. I. Yoshimura, et al., Molecular Recognition in a Supramolecular Hydrogel to Afford a Semi-Wet Sensor Chip. Journal of the American Chemical Society, 2004. 126(39): p. 12204-12205.
69. J. Li, et al., Self-Assembled Supramolecular Hydrogels Formed by Biodegradable PEO–PHB–PEO Triblock Copolymers and α-Cyclodextrin for Controlled Drug Delivery. Biomaterials, 2006. 27(22): p. 4132-4140.
70. G. Chen and A. S. Hoffman, Graft Copolymers That Exhibit Temperature-Induced Phase Transitions Over a Wide Range of pH. Nature, 1995. 373(6509): p. 49-52.
71. K. Y. Lee, et al., Controlled Growth Factor Release from Synthetic Extracellular Matrices. Nature, 2000. 408(6815): p. 998-1000.
72. J. A. Rowley, G. Madlambayan, and D. J. Mooney, Alginate Hydrogels as Synthetic Extracellular Matrix Materials. Biomaterials, 1999. 20(1): p. 45-53.
73. Z. Yang, et al., Conjugates of Naphthalene and Dipeptides Produce Molecular Hydrogelators with High Efficiency of Hydrogelation and Superhelical Nanofibers. Journal of Materials Chemistry, 2007. 17(9): p. 850-854.
74. S. S. Babu, V. K. Praveen, and A. Ajayaghosh, Functional π-Gelators and Their Applications. Chemical Reviews, 2014. 114(4): p. 1973-2129.
75. R. Narayanaswamy and O. S. Wolfbeis, Optical Sensors: Industrial, Environmental and Diagnostic Applications. Vol. 1. 2004: Springer Science & Business Media.
76. S. M. Narayanaswamy and O. S. Wolfbeis, Optical Biosensors. Chemical Reviews, 2008. 108(2): p. 423-461.
77. Z. Yang and B. Xu, A Simple Visual Assay Based on Small Molecule Hydrogels for Detecting Inhibitors of Enzymes. Chemical communications, 2004(21): p. 2424-2425.
78. M. Reches and E. Gazit, Casting Metal Nanowires within Discrete Self-Assembled Peptide Nanotubes. Science, 2003. 300(5619): p. 625-627.
79. X. Yan, P. Zhu, and J. Li, Self-Assembly and Application of Diphenylalanine-Based Nanostructures. Chemical Society Reviews, 2010. 39(6): p. 1877-1890.
80. D. M. Ryan and B. L. Nilsson, Self-Assembled Amino Acids and Dipeptides as Noncovalent Hydrogels for Tissue Engineering. Polymer Chemistry, 2012. 3(1): p. 18-33.
81. A. Mahler, et al., Rigid, Self‐Assembled Hydrogel Composed of a Modified Aromatic Dipeptide. Advanced Materials, 2006. 18(11): p. 1365-1370.
82. A. M. Smith, et al., Fmoc‐Diphenylalanine Self Assembles to a Hydrogel via a Novel Architecture Based on π–π Interlocked β‐Sheets. Advanced Materials, 2008. 20(1): p. 37-41.
83. V. Jayawarna, et al., Three-Dimensional Cell Culture of Chondrocytes on Modified Di-Phenylalanine Scaffolds. Biochemical Society Transactions, 2007. 35(3): p. 535-537.
84. Y. Zhang, et al., Versatile Small-Molecule Motifs for Self-Assembly in Water and the Formation of Biofunctional Supramolecular Hydrogels. Langmuir, 2010. 27(2): p. 529-537.
85. Z. Yang, G. Liang, and B. Xu, Supramolecular Hydrogels Based on β-amino Acid Derivatives. Chemical Communications, 2006(7): p. 738-740.
86. Z. Yang, G. Liang, and B. Xu, Enzymatic Hydrogelation of Small Molecules. Accounts of Chemical Eesearch, 2008. 41(2): p. 315-326.
87. Z. Yang, et al., Enzymatic Formation of Supramolecular Hydrogels. Advanced Materials, 2004. 16(16): p. 1440-1444.
88. Z. Yang, et al., In Vitro and In Vivo Enzymatic Formation of Supramolecular Hydrogels Based on Self‐Assembled Nanofibers of a β‐Amino Acid Derivative. Small, 2007. 3(4): p. 558-562.
89. S. Guo and E. Wang, Synthesis and Electrochemical Applications of Gold Nanoparticles. Analytica Chimica Acta, 2007. 598(2): p. 181-192.
90. M. De, P. S. Ghosh, and V. M. Rotello, Applications of Nanoparticles in Biology. Advanced Materials, 2008. 20(22): p. 4225-4241.
91. W. K. Leutwyler, S. L. Bürgi, and H. Burgl, Semiconductor Clusters, Nanocrystals, and Quantum Dots. Science, 1996. 271(5251): p. 933-937.
92. M. M. Alvarez, et al., Optical Absorption Spectra of Nanocrystal Gold Molecules. The Journal of Physical Chemistry B, 1997. 101(19): p. 3706-3712.
93. T. K. Sau and C. J. Murphy, Room Temperature, High-Yield Synthesis of Multiple Shapes of Gold Nanoparticles in Aqueous Solution. Journal of the American Chemical Society, 2004. 126(28): p. 8648-8649.
94. Liao, H.G. Liao, et al., Shape‐Controlled Synthesis of Gold Nanoparticles in Deep Eutectic Solvents for Studies of Structure–Functionality Relationships in Electrocatalysis. Angewandte Chemie, 2008. 120(47): p. 9240-9243.
95. Y. Sun and Y. Xia, Shape-Controlled Synthesis of Gold and Silver Nanoparticles. Science, 2002. 298(5601): p. 2176-2179.
96. M. Daniel and D. Astruc, Gold nanoparticles: Assembly, Supramolecular Chemistry, Quantum-Size-Related Properties, and Applications Toward Biology, Catalysis, and Nanotechnology. Chemical Reviews, 2004. 104(1): p. 293-346.
97. Y. Choi, N. H. Ho, and C. H. Tung, Sensing Phosphatase Activity by Using Gold Nanoparticles. Angewandte Chemie International Edition, 2007. 46(5): p. 707-709.
98. C. Ruan, W. Wang, and B. Gu, Detection of Alkaline Phosphatase Using Surface-Enhanced Raman Spectroscopy. Analytical Chemistry, 2006. 78(10): p. 3379-3384.
99. C. Volkringer, et al., Hydrothermal Crystallization of Three Calcium-Based Hybrid Solids with 2, 6-Naphthalene-or 4, 4′-Biphenyl-Dicarboxylates. Crystal Growth and Design, 2007. 8(2): p. 685-689.
100. A. K. Gaharwar, N. A. Peppas, and A. Khademhosseini, Nanocomposite Hydrogels for Biomedical Applications. Biotechnology and Bioengineering, 2014. 111(3): p. 441-453.
101. S. Goenka, V. Sant, and S. Sant, Graphene-Based Nanomaterials for Drug Delivery and Tissue Engineering. Journal of Controlled Release, 2014. 173: p. 75-88.
102. X. Zheng, et al., Magnetic Nanocomposite Hydrogel Prepared by ZnO-Initiated Photopolymerization for La (III) Adsorption. ACS Applied Materials & Interfaces, 2014. 6(22): p. 19840-19849.
103. S. Zhang, et al., A Self-Assembly Pathway to Aligned Monodomain Gels. Nature Materials, 2010. 9(7): p. 594-601.
104. M. Ma, et al., Aromatic− Aromatic Interactions Induce the Self-Assembly of Pentapeptidic Derivatives in Water to Form Nanofibers and Supramolecular Hydrogels. Journal of the American Chemical Society, 2010. 132(8): p. 2719-2728.
105. P. F. Alewood, et al., A Simple Preparation of O-phospho-L-Tyrosine. Synthesis-Stuttgart, 1983(1): p. 30-31.
106. H. Wang, et al., Gold Nanoparticle-Based Colorimetric and “Turn-On” Fluorescent Probe for Mercury (II) Ions in Aqueous Solution. Analytical Chemistry, 2008. 80(23): p. 9021-9028.
107. E.M. Rivera-Muñoz, Hydroxyapatite-Based Materials: Synthesis and Characterization. 2011: INTECH Open Access Publisher.
108. L. K. Tamm and S. A. Tatulian, Infrared Spectroscopy of Proteins and Peptides in Lipid Bilayers. Quarterly Reviews of Biophysics, 1997. 30(04): p. 365-429.
109. http://www.sigmaaldrich.com/Graphics/COfAInfo/SigmaSAPQM/SPEC/P7/P7640/P7640-BULK________SIGMA____.pdf.
110. R. L. Dean, Kinetic Studies with Alkaline Phosphatase in the Presence and Absence of Inhibitors and Divalent Cations. Biochemistry and Molecular Biology Education, 2002. 30(6): p. 401-407.
111. H. Fernley and P. Walker, Studies on Alkaline Phosphatase. Biochemical Journal, 1967. 104: p. 1011-1018.
112. D. A. Byers, H. N. Fernley, and P. G. Walker, Studies on Alkaline Phosphatase. European Journal of Biochemistry, 1972. 29(2): p. 197-204.
113. F. J. Olorunniji, et al., Cofactor Interactions in the Activation of Tissue Non-Specific Alkaline Phosphatase: Synergistic Effects of Zn2+ and Mg2+ Ions. Biokemistri, 2007. 19(2).
114. Y. Pan, et al., A Versatile Supramolecular Hydrogel of Nitrilotri Acetic Acid (NTA) for Binding Metal Ions and Magnetorheological Response. Journal of Materials Chemistry, 2011. 21(19): p. 6804-6806.
115. M. F. Bush, et al., Effects of Alkaline Earth Metal Ion Complexation on Amino Acid Zwitterion Stability: Results from Infrared Action Spectroscopy. Journal of the American Chemical Society, 2008. 130(20): p. 6463-6471.