Novel Sol-Gel Nanoporous Materials, Nanocomposites and Their Applications in Bioscience

A Thesis Submitted to the Faculty of Drexel University by Zhengfei Sun in partial fulfillment of the requirements for the degree of Doctor of Philosophy September 2005

Copyright 2005 Zhengfei Sun. All Rights Reserved.

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Dedications This dissertation is dedicated to my parents, Mr. Chongzhen Sun and Mrs. Xiuqing Nie for their encouragement, support and love.

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Acknowledgments In retrospect as I approach the completion of my doctorate, I feel a deep gratitude towards many people for their assistance and support. I would like to express my genuine gratitude to each of them, although it would be impossible for me to name all. First of all, I would like to sincerely thank my advisor, Dr. Yen Wei, for his tremendous time and effort spent in leading, supporting and encouraging me during the last five years. His passion for challenges has given me inspiration; his knowledge of science has given me guidance; his perseverance in research has given me confidence. Without his help and effort, it would be impossible for me to even get close to this point. I want to express my gratitude to all the committee members in my candidacy examination and/or my dissertation defense, Dr. Anthony Addison, Dr. Jean-Claude Bradley, Dr. Joe Foley, Dr. Susan Jansen-Varnum, Dr. Caroline Schauer, Dr. Sally Solomon and Dr. Jian-Min Yuan for their time and valuable suggestions. Special thanks are due to the committee chair, Dr. Anthony W. Addison for insightful discussions on many topics, including my oral proposal and research. I am also grateful to Dr. Sally Solomon, Dr. Caroline Schauer and Dr. Jian-Min Yuan for their detailed comments on my oral proposal and thesis. I would like to thank many of my collaborators. I specially thank Dr. Jian-Min Yuan for his valuable suggestion and assistance, especially in protein folding and amyloid aggregation projects. I also would like to thank Dr. Karl Sohlberg for giving me the opportunities to work with him on an important project. I would also thank Dr. Reinhard Schweitzer-Stenner for his wonderful insights on many of my research works, especially on the project of aggregation of Amyloid peptide. I also thank Dr. Solomon Praveen for

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his help in dental materials projects and suggestions in thesis writing. Many thanks are due to Dr. Thomas G. Spiro of Princeton University and his student Dr. Gurusamy Balakrishnan on assistance in Raman spectroscopy. I am grateful to many people for their selfless support in many areas. I especially acknowledge Dr. Patrick Loll for his assistance in circular dichroism spectroscopy, Dr. Guoliang Yang for AFM studies, Ms. Edith Smith for her kindly help and coordination in obtaining the chemicals and instruments for our research work. Many thanks are due to my friends in the Department of Chemistry who make my life here memorable. I thank Dr. Shuxi Li, Dr. Qiuwei Feng, Dr. Shan Cheng, Dr. Hua Dong, Dr. Houping Yin, Ms. Alpa Patel, Mr. Yi Guo, Dr. Jim Tu, Ms. Stephanie Schuster for their collaboration, discussions and help during these years. I also want to thank all the professors, staff and students in Department of Chemistry, Drexel University for making the Department such a joyful working and studying environment. Finally, I am greatly grateful to my parents, Mr. Chongzhen Sun and Mrs. Xiuqing Nie, for their continuous encouragement and unconditional love throughout in my life.

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Table of Contents Table of Contents................................................................................................................ 6 List of Tables .................................................................................................................... 15 List of Figures ................................................................................................................... 17 Abstract ............................................................................................................................. 24 Chapter 1: An Overview to Nanoporous Sol-Gel Materials............................................. 27 1.1 1.2 Introduction.................................................................................................. 27 Fundamentals of Sol-Gel Process ................................................................ 29 1.2.1 Sol-Gel Reactions .................................................................................... 30 1.3 1.4 1.5 Nanoporous Sol-Gel Materials from Surfactant Templated Pathways........ 31 Nanoporous Sol-Gel Materials from Nonsurfactant Templated Pathway ... 35 Characterizations of Nanoporous Materials................................................. 37 1.5.1 1.5.2 1.5.3 1.6 1.7 1.8 Gas sorption measurement.................................................................... 37 X-Ray Diffraction (XRD) ..................................................................... 40 Electron Microscopy............................................................................. 41

Organic/Inorganic Hybrid Materials by Sol-Gel Approach......................... 42 Sol-Gel Encapsulation of Biomolecules ...................................................... 45 References.................................................................................................... 49

Chapter 2: Rigid Matrix Artificial Chaperon (RMAC) Mediated Refolding of Cytochrome c .................................................................................................................... 63 2.1 Introduction......................................................................................................... 63 2.1.1 Fundamentals of Protein Folding Unfolding ........................................... 63 2.1.2 Chaperone and Protein Folding ............................................................... 64

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2.1.3 Rigid Matrix Artificial Chaperone (RMAC) ........................................... 65 2.1.4 Why Cytochrome c? ................................................................................ 67 2.1.5 Analytical Methods to Monitor Cytochrome cs Folding Unfolding ...... 68 2.1.5.1 Fluorescence Spectroscopy........................................................... 68 2.1.5.2 Circular Dichroism (CD) .............................................................. 69 2.2 Experimental ....................................................................................................... 71 2.2.1 Materials .................................................................................................. 71 2.2.2 Encapsulation of Unfolded Cc into Silica Matrix.................................... 71 2.2.3 Removal of Templates ............................................................................. 73 2.2.4 Characterizations of Nanoporous Silica Matrix....................................... 73 2.2.5 Fluorescence Spectroscopy of Encapsulated Cc...................................... 73 2.2.6 Circular Dichroism of Cc......................................................................... 74 2.2.7 Fourier Transform Infrared Spectroscopy (FTIR) of Silica Matrix......... 75 2.2.8 UV-Vis Spectroscopy (UV) of Encapsulated Cc..................................... 76 2.2.9 Attempts of Making Silica Thin Film...................................................... 77 2.3 Results and Discussion ....................................................................................... 78 2.3.1 Characterization of Silica Matrix............................................................. 79 2.3.2 FT-IR Spectroscopy on Silica Matrix ...................................................... 80 2.3.3 Fluorescence Spectroscopy of Encapsulated Cc...................................... 80 2.3.4 Leakage Tests........................................................................................... 81 2.3.5 CD Spectroscopy ..................................................................................... 82 2.4 Conclusion .......................................................................................................... 83 2.5 Acknowledgement .............................................................................................. 83

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2.6 References........................................................................................................... 83 Chapter 3: Using Resonance Raman Spectroscopy to Study Folding Unfolding Behavior of Encapsulated Heme Proteins in Silica Matrix with Controlled Pore Sizes .................. 99 3.1 Introduction......................................................................................................... 99 3.1.1 Resonance Raman Spectroscopy and Protein Folding............................. 99 3.1.2 Heme Protein ......................................................................................... 102 3.1.2.1 Cytochrome c and its Resonance Raman Spectroscopy ............. 102 3.1.2.1.1 Marker Band Region........................................................ 103 3.1.2.1.2 Fingerprint Region ........................................................... 104 3.1.2.2 Hemoglobin (Hb) and Its Resonance Raman Spectra................. 104 3.1.2.3 Myoglobin (Mb) and Its Resonance Raman Spectra .................. 105 3.1.2.4 Protein Encapsulation ................................................................. 108 3.2 Experimental ..................................................................................................... 109 3.2.1 Materials ................................................................................................ 109 3.2.2 Encapsulation of Cc into Silica Matrix by Using Urea as Template ..... 109 3.2.3 Encapsulation of Cc into Silica Matrix by Using Glucose as Template 110 3.2.4 Encapsulation of Hb into Silica Matrix by Using Glucose as Template 111 3.2.5 Encapsulation of DeoxyMb into Silica Matrix by Using Glucose as Template ......................................................................................................... 112 3.2.6 Resonance Raman Spectroscopy ........................................................... 113 3.2.7 Characterizations of Nanoporous Silica Matrix..................................... 114 3.2.8 Fourier Transform Infrared Spectroscopy (FTIR) of Silica Matrix....... 114 3.3 Results and Discussion ..................................................................................... 115

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3.3.1 Characterization of Silica Matrix........................................................... 115 3.3.1.1 Ccg series .................................................................................... 115 3.3.1.2 Ccu series .................................................................................... 116 3.3.2 Resonance Raman Spectroscopy of Ccu series ..................................... 117 3.3.3 Resonance Raman Spectroscopy of Ccg series ..................................... 119 3.4 Conclusions....................................................................................................... 123 3.5 Acknowledgements........................................................................................... 124 3.6 References......................................................................................................... 124 Chapter 4: A Novel Method to Study Aggregation of Amyloid 1-42 - A Key Peptide Associated with Alzheimers Disease............................................................................. 146 4.1 Introduction....................................................................................................... 146 4.1.1 Alzheimers Disease and Amyloid ..................................................... 146 4.1.2 The Aggregation of A peptides............................................................ 148 4.1.3 Detection of A aggregation.................................................................. 149 4.1.3.1 The Thioflavine T Fluorescence Assay ...................................... 149 4.1.3.2 The Congo Red Birefringence Assay ......................................... 150 4.1.3.3 Negative staining of amyloid fibrils for TEM ............................ 150 4.1.4 Bioencapsulation of A peptide in silica matrix with controlled pore size ......................................................................................................................... 151 4.2 Experimental ..................................................................................................... 152 4.2.1 Materials ................................................................................................ 152 4.2.2 Redissolving of A peptide in their monomeric state ........................... 153 4.2.3 Bioencapsulation of A1-42 in silica matrix with controlled pore size... 154

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4.2.4 pH changing of suspension by adding NaOH........................................ 155 4.2.5 Fluorescence Spectroscopy Study.......................................................... 156 4.2.5.1 Steady State Fluorescence Spectroscopy .................................... 156 4.2.5.2 Time Scale Fluorescence Spectroscopy...................................... 157 4.2.6 Congo Red Birefringence Assay............................................................ 157 4.2.6.1 Preparation of the Staining Solution ........................................... 157 4.2.6.2 Incubation of Silica Biogel Samples in Buffer Solution............. 157 4.2.6.3 Mounting Silica Biogel Samples on Glass Slides....................... 158 4.2.7 Negative staining of amyloid fibrils for TEM ....................................... 158 4.3 Result and Discussion ....................................................................................... 159 4.3.1 Characterization of Silica Matrix........................................................... 159 4.3.2 Steady State Fluorescence Spectroscopy ............................................... 159 4.3.2.1 Abata42 Series Samples.............................................................. 160 4.3.3 Time Scale Fluorescence Spectroscopy................................................. 162 4.3.4 Congo Red Birefringence Assay............................................................ 164 4.4 Conclusion ........................................................................................................ 165 4.5 Acknowledgements........................................................................................... 166 4.6 References......................................................................................................... 167 Chapter 5: Fabrication of Poly (2-hydroxyethyl methacrylate)-Silica Nanoparticle Hybrid Nanofibers via Electrospinning....................................................................................... 182 5.1 Introduction....................................................................................................... 182 5.1.1 Organic-Inorganic Nanocomposite: Opportunities to Advanced Materials ......................................................................................................................... 182

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5.1.2 Electrospinning ...................................................................................... 184 5.2 Experimental ..................................................................................................... 186 5.2.1 Materials ................................................................................................ 186 5.2.2 Synthesis of the Hybrid Material ........................................................... 187 5.2.3 Set-up of Electrospinning Apparatus ..................................................... 188 5.2.4 Electrospinning of PHEMA-Silica Hybrids........................................... 189 5.2.5. Instrumentation and Characterization ................................................... 189 5.2.5.1 FTIR Spectroscopy ..................................................................... 189 5.2.5.2 Thermal Gravimetric Analysis (TGA)........................................ 190 5.2.5.3 SEM and TEM ............................................................................ 190 5.3 Results and Discussion ..................................................................................... 190 5.4 Conclusion ........................................................................................................ 195 5.5 Acknowledgements........................................................................................... 196 5.6 References......................................................................................................... 196 Chapter 6: Synthesis and Characterization of Dental Composite Containing Nanoporous Silica as Fillers................................................................................................................ 211 6.1 Introduction....................................................................................................... 211 6.1.1 Resin Matrix........................................................................................... 211 6.1.2 Filler System .......................................................................................... 212 6.1.3 Coupling Agent...................................................................................... 213 6.1.4 Limitations of Coupling Agent .............................................................. 213 6.1.5 Porous Filler without Coupling Agent................................................... 214 6.1.6 Non-surfactant Templated Sol-Gel Process........................................... 215

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6.2 Experimental ..................................................................................................... 216 6.2.1 Materials ................................................................................................ 216 6.2.2 Preparation of Nanoporous Silica Filler ................................................ 216 6.2.3 Characterization of Nanoporous Silica Filler ........................................ 217 6.2.4 Preparation of Dental Resin ................................................................... 218 6.2.5 Silanization of Non-Porous Silica Particles........................................... 218 6.2.6 Preparation of Dental Composite........................................................... 219 6.2.7 Evaluation of Mechanical Properties ..................................................... 220 6.2.8 Aging Test.............................................................................................. 220 6.3 Results and Discussion ..................................................................................... 220 6.3.1 BET Analysis ......................................................................................... 221 6.3.2 Compression Testing ............................................................................. 221 6.3.2.1 Comparison of Non-Porous, Nanoporous and Neat Resin Materials ................................................................................................................. 221 6.3.2.2 Comparison of Heat Treatment Temperature Effects on Nanoporous Filler ................................................................................... 222 6.3.2.3 Aging Test................................................................................... 223 6.4 Conclusion and Future Works .......................................................................... 224 6.5 Acknowledgements........................................................................................... 226 6.6 References......................................................................................................... 226 Chapter 7: Dense Packing of Vinyl Modified Silica Nanoparticles and Its Potential Application as Low Shrinkage Dental Materials ............................................................ 235 7.1 Introduction....................................................................................................... 235

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7.2 Experimental ..................................................................................................... 238 7.2.1 Materials ................................................................................................ 238 7.2.2 Separation of Nanoparticle from OG100-31.......................................... 239 7.2.2.1 Reduced Pressure Distillation ..................................................... 239 7.2.2.2 Ultracentrifuging......................................................................... 239 7.2.3 Atomic Force Microscopic (AFM) Measurements................................ 241 7.2.4 Thermal Gravimetric Analysis (TGA)................................................... 241 7.2.5 FTIR Spectroscopy ................................................................................ 241 7.2.5.1 Solid Sample ............................................................................... 241 7.2.5.2 Liquid Sample............................................................................. 241 7.2.6 Isolation of Silica Nanoparticles............................................................ 242 7.2.7 Preparation of Dental Resin ................................................................... 242 7.2.8 Preparation of Dental Composite........................................................... 242 7.2.9 Evaluation of Mechanical Properties ..................................................... 244 7.3 Results and Discussion ..................................................................................... 245 7.4 Conclusion and Future Work ............................................................................ 248 7.5 Acknowledgements........................................................................................... 249 7.6 References......................................................................................................... 250 Chapter 8: Summary and Conclusions............................................................................ 259 8.1 Nanoporous Materials and Their Application in Bioscience............................ 259 8.2 Organic-Inorganic Hybrid Nanocomposites ..................................................... 262 Appendix A: Supplemental Data of Chapter 2 ............................................................... 266 Appendix B: Supplemental Data of Chapter 4. .............................................................. 268

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Appendix C: Supplemental Data of Chapter 5. .............................................................. 273 Appendix D: Fabrication of a New Type Molecularly Imprinted Polymer Membrane Sensor for Atrazine ......................................................................................................... 279

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List of Tables Table 2- 1. Pore parameters of water-extracted Ccu series prepared at various urea concentrations ................................................................................................... 89

Table 3- 1 Pore parameters of water-extracted Ccu series prepared at various urea concentrations ................................................................................................. 129 Table 3- 2 Pore parameters of water-extracted Ccg series prepared at various urea concentrations. ................................................................................................ 130

Table 4- 1 Summary of porous parameters of Abeta series samples after removel of templates. ........................................................................................................ 172 Table 4- 2 Relative fluorescence intensity of immobilized Amyloid 1-42 and free at difference pH............................................................................................... 173

Table 6- 1 BET analysis of mesoporous fillers at different fructose concentration. ......................................................................................................................... 230 Table 6- 2 The pore parameters of the nanoporous silica fillers after template removal of fructose by water extraction and heat treatments at different temperatures.................................................................................................... 231 Table 6- 3 Comparison of compressive properties of composites with different types of fillers. The number of specimens tested is given in parenthesis. ............... 232

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Table 6- 4 The effect of aging (in water at 37o C) on the compressive properties of composites prepared using mesoporous and SiO2 fillers. The number of specimens tested is given in parenthesis......................................................... 233

Table A-1 Data of relative difference, (IU-IT)/IT, in fluorescence intensity between unfolded and refolded Cc for the samples with increasing pore size up to free Cc in solution. ................................................................................................. 266

Table B- 1 Data of time scale fluorescence study of Abeta42 series samples when the pH value jumping from 2.35 to 7.02......................................................... 272

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List of Figures Figure 1- 1 Sol-gel process and their products. (www.chemat.com/ html/solgel.html) .......................................................................................................................... 58 Figure 1- 2 Diagrams of sol-gel reactions: (a) Hydrolysis reaction; (b) Condensation reaction.............................................................................................................. 59 Figure 1- 3 Possible mechanisms for formation of MCM-41: (1) liquid crystal phase initiated and (2) silicate anion initiated. 20,21..................................................... 60 Figure 1- 4 IUPAC classification of physisorption isotherms.16 .............................. 61 Figure 1- 5 IUPAC classification of hysteresis loops.16 ........................................... 62 Figure 2- 1 Schematic diagram of a folding energy landscape. Denatured molecules at the top of the funnel might fold to the native state by a myriad of different routes, some of which involve transient intermediates (local energy minima) whereas others involve significant kinetic traps (misfolded states). For proteins that fold without populating intermediates, the surface of the funnel would be smooth.23 ........................................................................................................... 90 Figure 2- 2 Illustrations of artificial chaperones assisting protein folding. .............. 91 Figure 2- 3 N2 adsorption-desorption isotherm at 196C. ...................................... 92 Figure 2- 4 BJH pore size distributions for the sol-gel material synthesized in the presence of 0-50% wt% of urea. ....................................................................... 93 Figure 2- 5 Relationship between BJH average pore diameter, pore volume and amount of urea template used in the synthesis. For Ccu0 (wt%=0), pore diameter was taken as 1.7 nm. .......................................................................... 94 Figure 2- 6 Representative IR spectra of (a) Ccu50 before template extraction; (b) Ccu0 before template extraction; (c) Cc50 after template extraction. .............. 95

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Figure 2- 7 Plot of relative difference, (IU-IT)/IT, in fluorescence intensity between unfolded and refolded Cc for the samples with increasing pore size up to free Cc in solution. ................................................................................................... 96 Figure 2- 8 Circular dichroism in far UV range at 25 oC of (A) unfolded cytochrome c (dashed line) in 9M urea and native cytochrome c (solid line) and (B) cytochrome c entrapped in Ccu0 (dashed line) and cytochrome c entrapped in Ccu50 (solid line) after washing out urea. All the curves are normalized by concentrations of Cc in solution or suspension but not convert to mean residue ellipticity because of strong light scattering in suspension............................... 97 Figure 2- 9 A cartoon presentation of cytochrome c refolding upon removal of urea template/denaturant by water extraction. (a) When pore size is large, Cc refolds to its native state. (b) When pore size is small, Cc cannot refold to its native state. The small dots represent urea molecules................................................. 98

Figure 3- 1 Diagram of a typical heme group......................................................... 131 Figure 3- 2 Deconvoluted resonance Raman spectra of the various heme coordinated forms of Cc.25 .................................................................................................. 132 Figure 3- 3 N2 adsorption-desorption isotherm of Ccg at 196C.......................... 133 Figure 3- 4 BJH pore size distributions for Ccg samples synthesized in the presence of 0-60 wt% of glucose. .................................................................................. 134 Figure 3- 5 N2 adsorption-desorption isotherm of Ccu at 196C.......................... 135 Figure 3- 6 BJH pore size distributions for the Ccu samples synthesized in the presence of 0-50% wt% of urea. ..................................................................... 136

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Figure 3- 7 Low frequency region of Resonance Raman Spectra of Ccu series and native cytochrome c samples at pH 7.4. ......................................................... 137 Figure 3- 8 Relationship between relative intensity (I567/I418) and BJH average pore diameter. For Ccu0 (wt%=0), pore diameter was taken as 1.7 nm................. 138 Figure 3- 9 Relationship between relative intensity (I397/I418) and BJH average pore diameter. For Ccu0 (wt%=0), pore diameter was taken as 1.7 nm................. 139 Figure 3- 10 Resonance Raman spectra of Ccu50 and Ccu0 at pH 7.0 and pH 3.5. ......................................................................................................................... 140 Figure 3- 11 Resonance Raman spectra of Ccu50 and Ccu0 at different temperatures.................................................................................................... 141 Figure 3- 12 Resonance Raman spectra of Ccg series samples when first immersed in water. (a: in high frequency region; b: in low frequency region) ............... 142 Figure 3- 13 Resonance Raman spectra of Ccg series samples when immersed in 9M urea solution. (a: in high frequency region; b: in low frequency region) ....... 143 Figure 3- 14 Resonance Raman spectra of Ccg series samples when washed out urea and reimmersed in water for 24 hours. (a: in high frequency region; b: in low frequency region) ............................................................................................ 144 Figure 3- 15 Ratio of intensity of peak 1503 cm-1 over peak 1494 cm-1 for Ccg series samples in different conditions. ...................................................................... 145

Figure 4- 1 Model of alternate aggregation pathways.33......................................... 174 Figure 4- 2 Molecular structure of Thioflavin T..................................................... 175

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Figure 4- 3 Relationship between average pore diameter and amount of DMA template used in the synthesis......................................................................... 176 Figure 4- 4 (a) Relative fluorescence intensity of immobilized Amyloid 1-42 at different pH..................................................................................................... 177 Figure 4- 5 Time scale fluorescence study of Abeta42 series samples when the pH value jumping from 2.35 to 7.02..................................................................... 178 Figure 4- 6 The aggregation of A1-42 peptides into amyloid fibrils typically begins with a lag phase in which no aggregation is observed. During this time, the entropically unfavorable process of initial association occurs. Once the aggregation process begins and a critical nucleus is formed, the aggregation proceeds rapidly into amyloid fibrils (solid line). The lag phase, however, can be overcome (dotted line) by the addition of a pre-formed nucleus (i.e., an aliquot of solution containing pre-formed fibrils). This schematic represents the nucleationpolymerization kinetics for amyloid fibril formation.39 ........... 179 Figure 4- 7 Microscope images of control silica samples without A1-42 inside: (a) under ordinary light (b) under polarized light................................................. 180 Figure 4- 8 Microscope images of Abeta42-50 samples with encapsulated A1-42 inside after incubation in pH 7.1 buffer for 2 hours: (a) under ordinary light (b) under polarized light. ...................................................................................... 181

Figure 5- 1 Schematic diagram to show polymer nanofibers by electrospinning... 202 Figure 5- 2 The picture of the set-up of electrospinning apparatus in our group. .. 203

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Figure 5- 3 FTIR spectra of pure PHEMA electrospun fiber and hybrid electrospun fiber. ................................................................................................................ 204 Figure 5- 4 TGA spectra of pure PHEMA electrospun fiber and hybrid electrospun fiber. ................................................................................................................ 205 Figure 5- 5 SEM picture of hybrid electrospun fiber when the molecular weight of hybrid material was not high enough.............................................................. 206 Figure 5- 6 SEM (a) and TEM (b) pictures of hybrid electrospun fiber................. 207 Figure 5- 7 EDX analysis of hybrid electrospun fiber. (The white cross in the SEM picture indicates the detection spot)................................................................ 208 Figure 5- 8 SEM pictures of electrospun hybrid fiber under different composition of solvents. .......................................................................................................... 209 Figure 5- 9 SEM pictures of electrospun hybrid fiber in solutions with different concentrations. ................................................................................................ 210

Figure 6- 1 The effect of filler heat treatment on the compressive modulus of post cured composites............................................................................................. 234

Figure 7- 1 FTIR spectra of (a) HEMA monomer; (b) dry silica nanoparticles modified with vinyl groups on surface; .......................................................... 252 Figure 7- 2 FTIR spectra of silica nanoparticle sediments after ultracentrifuge (a) with EtOH as solvent; (b) with acetone as solvent. ........................................ 253 Figure 7- 3 FTIR spectrum of the new dental composite with vinyl modified silica nanoparticles as inorganic fillers. ................................................................... 254

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Figure 7- 4 TGA spectra of silica nanoparticle sediments after ultracentrifuge (a) with EtOH as solvent; (b) with acetone as solvent. ........................................ 255 Figure 7- 5 TGA spectrum of the new dental composite with vinyl modified silica nanoparticles as inorganic fillers. ................................................................... 256 Figure 7- 6 Picture of the new dental composite with vinyl modified silica nanoparticles as inorganic fillers. For this particular sample, the loading percentage is 51 wt%. ..................................................................................... 257 Figure 7- 7 AFM picture of nanoparticle sediments after ultracentrifuge. ............. 258

Figure A- 1 Representative plot of free Cc in its refolded and unfolded state. ...... 267

Figure B- 1 Representative fluorescence spectra of free A 1-42 in buffer with different pH value. .......................................................................................... 268 Figure B- 2 Representative fluorescence spectra of Abeta42-0 in buffer with different pH value. .......................................................................................... 269 Figure B- 3 Representative fluorescence spectra of Abeta42-30 in buffer with different pH value. .......................................................................................... 270 Figure B- 4 Representative fluorescence spectra of Abeta42-50 in buffer with different pH value. .......................................................................................... 271

Figure C- 1 SEM pictures of electrospun hybrid fiber when the ratio of DMF to EtOH in solvent mixture was 20:80................................................................ 273

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Figure C- 2 SEM pictures of electrospun hybrid fiber when the ratio of DMF to EtOH in solvent mixture was 30:70................................................................ 274 Figure C- 3 SEM pictures of electrospun hybrid fiber when the ratio of DMF to EtOH in solvent mixture was 40:60................................................................ 275 Figure C- 4 SEM pictures of electrospun hybrid fiber when the ratio of DMF to EtOH in solvent mixture was 60:40................................................................ 276 Figure C- 5 SEM pictures of electrospun hybrid fiber when the ratio of DMF to EtOH in solvent mixture was 70:30................................................................ 277 Figure C- 6 SEM pictures of electrospun hybrid fiber when the ratio of DMF to EtOH in solvent mixture was 80:20................................................................ 278

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Abstract Sol-Gel Nanoporous Materials, Hybrid Nanocomposites and Their Applications in Bioscience Zhengfei Sun Advisor: Yen Wei

Transparent, nanoporous silica materials have been prepared successfully via the acid-catalyzed hydrolysis and condensation of tetramethyl orthosilicate using the nonsurfactant templated sol-gel process. The synthetic conditions have been systematically studied and optimized. The effects of template and synthetic process, especially template removal steps on pore structure, have been investigated. The composition and pore structures were thoroughly characterized with various spectroscopic and microscopic methods such as IR, TGA, SEM, TEM and BET. The obtained nanoporous materials usually exhibit high surface area, large pore volume and narrowly distributed pore diameter. The porosity can be fine tuned to a certain extent simply by adjusting the template concentration. The convenient synthesis, as well as the distinctive structure and physical-chemical properties, render these sol-gel materials suitable for a wide range of potential applications, such as chemical and biological sensors, catalysts, drug delivery and functional coatings. Because of excellent biocompatibility of this novel sol-gel technology, the method was used to study behaviors of encapsulated biospecies in silica matrix within a confined space. We call such nanoporous materials rigid matrix artificial chaperone because they mediate protein folding process in many aspects like a chaperone. In the studies included in this thesis, cytochrome c, hemoglobin, myoglobin as well as amyloid peptide were encapsulated in sol-gel nanoporous silica matrix with controlled pore size.

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Then their folding-unfolding and aggregation behaviors were investigated by a variety of analytical methods, such as fluorescence spectroscopy, circular dichroism and resonance Raman spectroscopy. It was found that the size of pore had great effects on the folding and aggregation process of those encapsulated biospecies. In the second part of this thesis, the synthesis, processing and characterization of hybrid nanocomposites and their applications in dental materials are described. Two approaches have been developed to achieve homogenous hybrid nanocomposites: the first approach involves the use of vinyl modified silica nanoparticle as inorganic component and the other one employs nanoporous silica particle as inorganic filler. The materials synthesized by both of these two approaches demonstrated promising potential in dental applications.

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Chapter 1: An Overview to Nanoporous Sol-Gel Materials

1.1 Introduction In the past few years, nanomaterials have attracted tremendous international interest, investment and effort both in scientific research and in industrial development because of their potential applications in various fields. 1-4 Nanoporous materials are one of subset of nanomaterials. With their unique porous structure in nanometer dimensions, they can be used in various applications such as ion exchange, separation, catalysis, sensor, biological molecular recognition and purification. 5-15 According to the International Union of Pure and Applied Chemistry (IUPAC) 16 porous materials can be classified into three categories: 1) micropores are smaller than 2 nm in diameter; 2) mesopores are between 2 to 50 nm; 3) macropores are larger than 50 nm. But this definition is somewhat conflict with the more broadened definition of nanoporous materials. The term of nanoporous currently refers to the class of porous materials having pore diameters between 1 and 100 nm. It is noted that nanoporous materials actually encompass some micro and macro porous materials and all mesoporous materials. 1 Nanoporous materials can bring us many interesting unique properties. The high surface area to volume ratio, large surface area and porosity, versatile surface composition and properties enable the nanoporous materials to be used widely in applications, such as catalysis, chromatography, separation, sensing and so on. Furthermore, nanoporous materials, especially inorganic nanoporous materials, which are made of mostly metal oxides, are usually non-toxic, inert, chemically and thermally

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stable, so they have wide applications where biocompatibility or thermal stability requirements are essential. Sol-gel technology has been used extensively in the synthesis of nanoporous materials as illustrated in Figure 1-1.17-19 The overall sol-gel process, as the name implies, usually involves two stages: precursors initially form high molecular weight but still soluble oligmeric intermediates, a sol, and the intermediates further link together to form a three-dimensional crosslinked network, a gel. The precursors for a sol-gel reaction could be either inorganic salts or organic compounds, such as metal alkoxides. The synthesis of M41-S family brought a great breakthrough in the research of nanoporous materials.20-23 The first example in this family is MCM41, which was developed in the early 1990s by researchers at Mobil Corporation.20 In this approach, surfactant molecules were used as template to direct mesophase formation during sol-gel process, thus high surface areas (> 1000m2 g-1), tunable, uniform and long-range ordered porous structure (2-10 nm) can be achieved after removal of the templates. Afterwards, this method has been extended to the synthesis of a wide range of porous silica materials, as well as many other metal oxides species, like alumina, titania, zirconia etc.24-27 A novel non-surfactant pathway to synthesize nanoporous materials via sol-gel process has been developed in our group.28-32 In this method, instead of using surfactant molecules as a template, we chose non-surfactant molecules, such as glucose, fructose and urea, etc, to direct the mesophase formation during the sol-gel process. This method turned out to be an effective way to synthesize nanoporous materials with high surface area ( e.g. 1000 m2/g) and pore volume ( e.g. 0.5-1.0 cm3/g), as well as pore size in the range of 2-12 nm with narrow size distributions. Most importantly, this process has been

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proved to be convenient, mild and biofriendly method. In our group, this approach has been exploited to immobilize enzymes and other biological substances for biosensing and biocatalysis applications.33-36 A main part of my research work has been focused on further developing this novel non-surfactant template sol-gel technology and their applications in bioscience, especially in study of protein folding unfolding and peptide aggregation.37-40 Besides that, the synthesis of novel nanostructured organic-inorganic hybrid materials based on the sol-gel process have also been done.41,42 For the convenience of further discussion, some background knowledge, like sol-gel process, surfactant and non-surfactant pathways to nanoprous materials and fundamental of enzyme immobilization will be introduced in the following sections. Due to the importance of establishment of structure-properties relationship in porous materials, major characterization methods will also be discussed with emphasis on gas adsorption measurements.

1.2 Fundamentals of Sol-Gel Process In general, sol-gel process involves a transition of a system from a liquid "sol" (mostly colloidal) into a solid "gel" phase. The starting materials used in the preparation of the "sol" are usually inorganic metal salts or metal organic compounds such as metal alkoxides. The first metal alkoxide was prepared from SiCl4 and alcohol by Ebelmen in 1844, who found that the compound gelled on exposure to ambient environment. In a typical sol-gel process, the precursor is subjected to a series of hydrolysis and polymerization reactions to form a colloidal suspension, or a "sol". When the "sol" is cast into a mold, a wet "gel" will form. With further drying and heat-treatment, the "gel" is

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converted into dense ceramic or glass particles.43,44 The whole process is illustrated in Figure 1-2.

1.2.1 Sol-Gel Reactions The precursors for a sol-gel reaction could be either inorganic salts or organic compounds, known as metal alkoxides. Compared with inorganic salts, reactions of alkoxide precursors have easily controlled reaction kinetics and generate byproducts of alcohols and water, which can be readily removed during the drying process. Tetraethoxysilane (TEOS), the most widely used alkoxide precursor, is used as an example below to illustrate the sol-gel process. In the first step, TEOS is hydrolyzed by mixing with water:OC2H5C2H5OOHHO

Si

OC2H5 + 4 H2O

SiOH

OH + 4 C2 H5 OH

OC2H5

In this reaction, either an acid or a base can serve as catalyst. The silanols, i.e., hydrolyzed TEOS undergoes further condensation reaction forming siloxane bonds, that is the second step of the sol-gel process:

Si

OH

+ HO

Si

Si

O

Si

+ H2 O

or

Si

OR

+ HO

Si

Si

O

Si

+ ROH

30

Linkage of additional Si(OH)4 tetrahedra occurs as a polycondensation reaction and eventually results in a three dimensional SiO2 network. The water and alcohol byproduct molecules generated from the reactions remain in the pores of the silicas three dimensional networks. They can also, as most often done, be removed during the reaction. The hydrolysis and polycondensation reactions happen at numerous sites within the TEOS and water mixture upon mixing. When sufficient interconnected Si-O-Si bonds are formed in region they respond cooperatively as colloidal particles, e. g. a sol. The size of the sol particles and the crosslinking within the particles depend upon the pH and [H2O]/Si(OR)4 ratio in the solution. Since sol is a relatively low-viscosity liquid, it can be cast into any shape according to its applications. With time, the colloidal particles and condensed silica species link together to become a three-dimensional network. The physical characteristics of the gel network depend greatly on the size of particles and extent of crosslinking prior to gelation. At the gelation step, the viscosity increases sharply and the system losses its fluidity to form a solid-like object resulting in the general shape of the mold. The ultrastructure and texture of a gel are established at the time of gelation. Subsequent processing such as aging, drying can also contribute to the forming of a gel ultrastructure.

1.3 Nanoporous Sol-Gel Materials from Surfactant Templated Pathways MCM-41 was the first example of nanoporous sol-gel material synthesized through surfactant templated pathway in the early 1990s.20,21 Since then, tremendous attention and effort have been made in this research field. This surfactant-templated

31

method proved to be an extremely useful strategy to synthesize nanoporous materials with the following characteristics: High surface areas (> 1000 m2g-1); Tunable, uniform pore sizes (2-10 nm); Long-range ordered pore structures; Structural stability and so on. Due to those interesting properties, nanoporous materials synthesized by surfactant templated pathways have been studied for various applications, such as heterogeneous catalysis, separation, optics, electronics and sensing. The synthesis process always involves two conceptually simple steps: first forming the surfactant/inorganic mesophase and second removing the surfactant template molecules from the mesostructure after metal oxide framework formation. But the actual kinetics are much more complicated. A variety of parameters can affect the mesophase formation, such as solvent, temperature, pH value, aging time, initial precursor/water/catalyst ratio, etc. Surfactants are bifunctional molecules that contain both hydrophilic and hydrophobic groups.45,46 As a result of their amphiphilic nature, surfactants can associate into supramolecular arrays. For example, cetyltrimethylammonium bromide (CH3(CH2)15N(CH3)3+Br-) in water will self-assemble into spherical micelle forms incorporating about 90 molecules. In the micelle, hydrophilic ends point toward outside and form an outer surface, while hydrophobic ends point toward center. The concentration of surfactant molecules in solution is the key factor to determine the extent of micellization, the shape of the micelles, and the aggregation of micelles into liquid

32

crystals. When the concentration of surfactant is very low, these molecules distribute individually in the solution or absorb at the interfaces. With the concentration going up beyond the critical micelle concentration (CMC1), the separated surfactant molecules form small, spherical aggregates (micelles). At higher concentrations (CMC2), those micelles can coalesce to form elongated cylindrical micelles. If the concentrations increase, liquid-crystalline (LC) phases will form. Initially, rod like micelles aggregate to form hexagonal close-packed LC arrays. As the concentration increases, cubic bicontinuous LC phases form, followed by LC lamellar phases.23 Though several mechanisms have been proposed to explain the formation of mesophase during surfactant-templated sol-gel process with slightly differences, the liquid-crystal templating (LCT) mechanism suggested by Beck, et al. includes many of the proposed mechanisms.20,21 In LCT theory, two possible pathways were proposed. In the first, the liquid-crystal phase is intact before the silicate species are added; in the second, the addition of the silicate results in the ordering of the subsequent silicateencased surfactant micelles. The difference between the two pathways rises from changes in surfactant properties, depending on the surfactant concentrations in water and the presence of other ions. This theory is illustrated in Figure 1-3. Dependent on their end group chemistry and charge, the surfactant can be classified into three categories: Anionic surfactant: the hydrophilic end group carries a negative charge, e.g. sulfate47 (CnH2n+1OSO3- ( n = 12, 14, 16, 18), sulfonates47 (C16H33SO3- and C12H25C6H4SO3-Na), phosphates47,48 (C12H25OPO3H2, C14H29OPO3K), and carboxylic acids (C17H35COOH and C14H29COOH);

33

Cationic surfactant: the hydrophilic end group carries a positive charge, e. g. alkylammonium salts, such as (CnH2n+1(CH3)3NX, n = 6 (nonmesophase), 8, 9, 10, 12, 14, 16, 18, 20, 22; X = OH/Cl, OH, Cl, Br, HSO4, and CnH2n+1(C2H5)3N, n = 12, 14, 16, 18), gemini surfactants47 [CmH2m+1(CH3)2N-CsH2s-N(CH3)2CmH2m+1]Br2, m= 16, s = 2-12) cetylethylpiperidinium salts47 (C16H33N(C2H5)(C5H10)+); and bichain salts47 (dialkyldimethylammonium); Nonionic surfactant: the hydrophilic group is not charged; examples include primary amines49 (CnH2n+1NH2) and poly(ethylene oxides),50 octaethylene glycol monodecyl ether (C12EO8), and octaethylene glycol monohexadecyl ether (C16EO8).51 Six templating pathways have been identified: S+ I-, S- I+, S+ X- I+, S- X+ I-, S-I, and S0 I0,49,52,53 where S is the surfactant, I is the inorganic phase, and X is the mediating ion. For example, in S+ I- type, a cationic surfactant is chosen and the pH is set such that the inorganic precursors will carry negative charge. Beside silicate, the surfactant-templated pathway can be applied in synthesis the other periodic porous materials. Various inorganic oxide frameworks, some of which may have important technological applications, have been realized, including silica doped with Al,20,21,54-56 Ti,49,57 V,58 B,59 Fe,60 Mn,61 Ga,62 and transition metal and main-group materials based on tungsten oxide,63 antimony oxide,63 titanium oxide,47 zirconium oxophosphate,64 and zirconium oxide, vanadium oxide,63 vanadium phosphate,65 and tantalum oxide.66 In addition, a large number of lamellar mesophases have been synthesized including those based on Si, Zn, Pb, Fe, Mg, Mn, Co, Ni, Al, and Ga oxides and Sn, W, and Mo sulfides.47,67

34

1.4 Nanoporous Sol-Gel Materials from Nonsurfactant Templated Pathway Even though the surfactant templated pathway has proved to be successful for synthesis of nanoporous materials, some drawbacks have prevented their application in some areas. As an example, some cationic surfactants are toxic and expensive; the synthesis is often achieved under harsh conditions during reactions or the templated removal processes, such as high temperature and pressure, strongly acidic or basic media. For some applications, especially in bioscience and biotechnology, those drawbacks are fatal. To solve these problems, a novel nonsurfactant templating pathway has been developed in our group.28,29 Instead of using surfactant molecules as template to direct the nanoporous structure formation, non-surfactant small molecules are applied in this new method to function as template molecules. Those small nonsurfactant molecules include glucose, fructose, maltose, urea, dibenzoyl-L-tartaric acid (DBTA), etc..28-32,42,68-80 In general, the nonsurfactant pathway starts with the sol-gel reactions of inorganic precursors, e.g., tetraethyl orthosilicate (TEOS) for silica, in the presence of a nonsurfactant compound, e.g. glucose. Upon gelation and drying, nonporous, transparent and monolithic solids can be obtained. The template removal can be easily achieved by simple solvent extraction, which means template molecules can be washed out from silica matrices and leave nanoporous structure. As described above, the nonsurfactant templating pathway has several important advantages over surfactant-templating pathway. First, the whole process can be done at room temperature. Because the interactions between non-surfactant molecules and inorganic phase are much weaker than surfactant/inorganic phase, the removal of

35

templates can be easily accomplished by solvent extraction at room temperature, avoiding high temperature calcination step in the surfactant-templated pathway. Second, many non-surfactant template molecules, like glucose, fructose and maltose are non-toxic and biofriendly. That makes applications in bioscience and biotechnology feasible. Third, during the whole non-surfactant templated process, no acid, alkaline or even organic solvent is necessary at the point when biological substances are introduced. All these characteristics of non-surfactant templating pathway provide a convenient and effective way to prepare nanoporous materials for various applications, especially in biological applications. The nanoporous materials synthesized via the non-surfactant templating pathway share some characteristics with those from surfactant templating pathway. They have high surface area (e.g. 1000 m2/g) and pore volume (e.g. 0.5-1.0 cm3/g) as well as pore size in the range of 2-12 nm with narrow size distributions, which are all similar to surfactant templated materials. But they do not have long ordered range of nanoporous structure, discernable packing or orientation of the nanopores/channels. In fact, the porous structures inside non-surfactant templated samples are made of interconnected channels of regular diameters. Indeed, such a worm-hole like structure can be observed in the TEM images of all nanoporous materials prepared via the non-surfactant templating pathway. Though the long range ordered structure is missing from the materials, it is interesting to point out that may be advantageous for many applications, such as catalysis or sensing, because the nanoporosity is accessible from all directions. The mechanism of forming mesophase through the non-surfactant templating pathway is still not very clear. After investigating nearly 100 template compounds, we

36

found only those compounds with highly polar functional groups can serve as the templates. Because the size of pores are much bigger than the single non-surfactant templated molecule, those small molecules must work in some aggregation or assembly form when function as the template. We believe that strong polar interactions and hydrogen bonding between the non-surfactant molecules or their aggregations and inorganic phase may play an important role in directing the mesophase formation.

1.5 Characterizations of Nanoporous Materials Reliable characterization methods are very important for developing nanoporous materials. Various structural parameters, such as surface area, pore size, pore volume and pore size distribution are needed to evaluated nanoporous materials. Gas adsorption, Xray diffraction, electron microscopy are among the most important experimental methods used.

1.5.1

Gas sorption measurement It has long been known that a porous solid can take up a relatively large volume

of condensable gas. Because surface area and porosity of the porous materials play complementary roles in adsorption phenomena, measurements of adsorption of gases can be made to yield information as to surface area and the pore structure of a solid. The term of adsorption was first introduced by Kayser in 1881 to connote the condensation of gases on free surfaces; while the term absorption refers to the phenomenon where gas molecules penetrate into the mass of the absorbing solid. Since it

37

is sometime difficult, impossible or irrelevant to distinguish between these two terms, the wider term sorption which embraces both types of phenomena is uesd.81 When a highly dispersed solid is exposed in a closed space to a gas or vapor at some definite pressure, the solid begins to adsorb the gas, resulting in a gradual reduction in the gas pressure. After some time, the gas pressure would become constant. The amount of adsorbed gas can be calculated from the decrease of pressure by application of the gas laws. The amount of adsorbed gas per gram of solid depends on the equilibrium pressure p, the temperature T, and also on the nature of the gas and of the solid. For a given gas adsorbed on a given solid, maintained at a fixed temperature: X = f (P)T, gas, solid If the gas is below its critical temperature, i.e. if it is a vapor, the alternative form: X = f (P/P0)T, gas, solid These two equations are expressions of the adsorption isotherm, which can be defined as the relationship at constant temperature, between the adsorbed and the equilibrium pressure of the gas and can be used to determine the general pore structure of a porous solid. The adsorption isotherm is usually constructed point-by-point by the admission to the adsorbent of successive charges of gas with the aid of a volumetric dosing technique and application of the gas laws. Though there are recorded tens of thousands of adsorption isotherms, the majority of those isotherms can be grouped into six classes as illustrated in Figure 1-4:16

38

Type I isotherms are given by microporous solids having relatively small external surfaces, the limiting uptake being governed by accessible micropore volume rather than by internal surface area. Type II isotherms are commonly forms of isotherms obtained with a non-porous or macroporous solid. It demonstrates unrestricted monolayer-multilayer adsorption. The point B labeled in the graph normally means the conversion form monolayer adsorption to multilayer adsorption. Type III isotherms are not common. In this case, the adsorbate-adsorbate interaction plays an important role. Type IV isotherms have a characteristic hysteresis loop, which is associated with capillary condensation taking place in mesoporous solids. The initial part of the Type IV isotherm is attributed to monolayer-multilayer adsorption since it follows the same path as the corresponding part of a Type II isotherm. Type IV isotherms are given by many mesoporous materials. Type V isotherms are also not common ; they are related to the Type III isotherms in that the adsorbate-adsorbate interactions are weak, but is obtained with certain porous adsorbents. Hysteresis appearing in the multilayer range of physisorption isotherms is usually associated with capillary condensation in mesopores structures.16,82,83 There are four types of hysteresis loops as shown in Figure 1-5: H1 loop is always associated with pores with regular shape and narrow size distribution; H2 loop is especially difficult to interpret: it was originally attributed to a difference in mechanism between condensation and evaporation processes occurring in pores with narrow necks and wide bodies before, but

39

now it is believed that the role of network effects must play an important role too; H3 loop, which does not exhibit any limiting adsorption at high P/P0, is observed with aggregates of plate-like particles and H4 loop is often assigned narrow slit-like pores. The Brunauer-Emmett-Teller (BET) gas adsorption method has become the most widely used standard procedure for the determination of the surface area of finely-divided and porous materials. The BET equation can be described in the linear form: P 1 (C 1) P = a + a n (P0 P ) n m C nm C P0aa where n a is the amount adsorbed at the relative pressure P/P0 and n m is the

monolayer capacity.

1.5.2

X-Ray Diffraction (XRD) X-ray diffraction is one of the cornerstones of twentieth century science.84 It has

been widely used to characterize sol-gel nanoporous materials. X-rays are relatively short wavelength, high energy electromagnetic radiations. After they are generated from a source and focused into a fine beam, the X-ray can be shined on a solid sample. Though various interactions happen between X-ray and the inspected matter, such as heat conversion, photoelectric effect, fluorescence, auger electron production and Compton scattering, the most important mechanism of X-ray absorption in matter, which leads to the phenomenon of diffraction is coherent scattering. It is analogous to a perfectly elastic collision between a photon and an electron. The photon changes direction after colliding with the electron but transfers one of its energy to the electron. The result is that the scattered photon leaves in a new direction but with the same phase and energy as that of

40

the incident photon. Structural information can therefore be deduced from the knowledge of scattering intensity and angle.85 For example, approximated as the repeating distance in the porous materials, the sum of a pore diameter and a pore wall thickness can be estimated base on the d spacing calculated from the Bragg equation.

n = 2d sin where is the wavelength, d is the separation between planes and is the diffraction angle. In the case of nanoporous materials with regular pore diameter and wall thickness, the d spacing is the sum of the pore diameter and wall thickness.

1.5.3

Electron Microscopy Electron microscopic observations can provide a straightforward evaluation on

pore size, shape and distribution in the nanoporous materials.86-89 Generally, electron microscopes use a beam of highly energetic electrons to examine objects on a very fine scale. This examination can yield the following information: Topography: The surface features of an object or "how it looks", its texture; Morphology: The shape and size of the particles making up the object; Composition: The elements and compounds that the object is composed of and the relative amounts of them; Crystallographic Information: How the atoms are arranged in the object; There are two types of electron microscopy: The transmission electron microscope (TEM) was the first type of electron microscope to be developed and is patterned exactly on the light transmission microscope except that a focused beam of electrons is used instead of light to "see through" the specimen; the first scanning electron

41

microscope (SEM) debuted in 1942. Its late development was due to the electronics involved in "scanning" the beam of electrons across the sample. For mesoporous materials, TEM is the most useful method to provide direct observation of pore structures and parameters.

1.6 Organic/Inorganic Hybrid Materials by Sol-Gel Approach Hybrid materials formed by the combination of inorganic materials and organic polymers are attractive for the purpose of creating high-performance or high-functional polymeric materials.90-95 Both pure organic polymer and pure inorganic materials have their own advantages and disadvantages. Generally, organic polymer materials possess of the following merits, light weight, good flexibility and excellent moldability. However they usually lack of hardness and strength; on the other hand inorganic materials, such as silica glass are often in the different ways: they have good mechanical and thermal stability but sometime too brittle. So, if the homogeneous combination of inorganic and organic moieties in a single-phase material can be achieved, this material may provide unique possibilities, which combined advantages of both organic and inorganic materials and let us to tailor the mechanical, electrical, and optical properties with respect to numerous applications. Sol-gel technology, which is mainly based on inorganic polymerization reactions, is an important way to synthesize organic/inorganic hybrid materials,79,91,94,96,97 because of its unique low temperature processing characteristic, providing opportunities to let organic and inorganic phases mix well and incorporate with each other at temperatures under which the organic phase can survive. Since the last few decades, the preparation,

42

characterization and application of those organic/inorganic hybrid materials based on solgel process are a fast growing research field in materials science. By using the sol-gel technology, the hybrid nanocomposite hybrid materials provide some new and interesting properties which conditional macroscale composites do not have. For example, unlike conventional composites with inorganic or organic phase domains at millimeter or micrometer scale, the most of hybrid materials by sol-gel process are nanoscopic, with phase domain size in nanometer scale. Therefore, they are often optically transparent. Furthermore, via sol-gel process, various kinds of bonds between organic and inorganic phases can be introduced in the system and consequently enhance the interactions between these two phases. Dependent on structural differences, organic/inorganic hybrid materials can be grossly divided into two classes:92,93 Class I: The organic phase is physically embedded inside the inorganic matrix. The synthesis of this class of materials is usually carried by formation of inorganic network backbone in presence of preformed organic phase, like prepolymer. Thus, only weak bonds can be formed between these two phases. Class II: The organic and inorganic phases are covalent bonded. In this approach, the inorganic precursors must carry functional groups, which can react with organic phase during or after sol-gel process. Based on sol-gel technology, there are several different synthetic techniques by incorporating various starting inorganic and organic components with varied molecular structures:

43

(1)

Organic groups are introduced into hybrid network by using low molecular weight organoalkoxysilanes as one or more of precursors for sol-gel reaction.98-100

(2)

Organic/inorganic hybrid materials can be also be formed via the cocondensation of functionalized polymers with metal alkoxide, such as trialkoxysilyl groups. So, covalent bonding can be established between inorganic and organic phases.101,102

(3)

In situ formation of inorganic phase domain within organic polymer matrix can be another way to synthesize hybrid materials.103-105 Those inorganic species with homogenous particle size in nanometer scales can be obtained by using sol-gel reaction of the inorganic precursors.

(4)

Just opposite to method (3), organic phase domain can be formed by either infiltrating reformed oxide gels with polymerizable monomer or mixing the polymers with metal alkoxide in a common solvent.106

(5)

Simultaneous formation of inorganic and organic phases together provides another way to synthesize hybrid materials. For example, triethoxysilance RSi(OR)3 where R is a polymerizable group like epoxy group, can be used as a precursor in sol-gel reaction, so by either photo- or thermal- initiation, organic network can be formed within inorganic network.100,107

To date, only a few sol-gel hybrid materials have been commercialized, but the future of these materials is promising. A larger number of potential applications have appeared, such as scratch and abrasive-resistant coatings,108,109 electrical and nonlinear

44

optical (NLO) materials,110,111 contact lens,112 reinforcement of elastomers and plastic,113115

catalyst and sensor materials,116,117 porous supports, adsorbents etc.

1.7 Sol-Gel Encapsulation of Biomolecules Though sol-gel science has been developed for many decades, bioencapsulation via sol-gel technology was not realized until 1980s.118,119 Since then, research on this field thrives and a large number of examples of bioencapsulation by sol-gel technology have emerged out.120-132 In comparison with conventional bioimmobilization methods, in which biospecies are often covalently bonded to organic polymer matrix, the sol-gel bioencapsulation methods have the following advantages: (1) Inorganic matrix synthesized by sol-gel process usually has much better thermal and chemical stability than organic polymer system, which can allow the sol-gel bioencapsulated system working in an elevated temperature and harsh environment. (2) The synthesis of sol-gel materials can be done at room temperature. That makes directly encapsulation of temperature sensitive biomolecules feasible. (3) The surface area and porosity of sol-gel materials can easily be controlled so the suitable pore size can be designed, so leakage of biomolecules is reduced while penetration of small required reagent molecules is not prohibited. (4) The good optical transparency of most of sol-gel materials enable their usages with optical requirements, such as optical sensors.

45

Since the first example of sol-gel bioencapsulation introduced by Avnir and coworkers,133 several types of sol-gel matrices have been developed to be used as substrates: Inorganic sol-gels: The pure inorganic xerogels, such as aluminum, titanium, zirconium and tin oxides as well as their mixed oxides with silica, are always hard, transparent glasses with microporous structure. They are chemically robust, but limited by their brittleness and too small pore size, which prevents small molecule diffusion through the matrix.134 Organically modified silica sol-gels (Ormosils): In this category, organic groups, from simple alkyl, alkenyl, and aryl to those additionally bearing amino, amido, carboxy, hydroxy, thiol, and mixed functionalities as well as nicotinamides, flavins and quiniones, can be grafted on precursor silanes. Thus after sol-gel reactions, those organic functional groups are attached on the silica matrix by stable Si-C bonds. Because of those groups, tailorable properties, such as hydrophilic, hydrophobic, ionic as well as H-bonding capacities can be achieved in the silica matrix. However, the optical transparency and stability are lower than inorganic sol-gels.134 Hybrid sol-gels: Amino- or hydroxyl- functional polymers, such as polymethy silane, polyurethane, polyacrylate, and polyphosphazene are mixed with alkoxysilane during the sol-gel reaction. After polymerization, hybrid organic-inorganic structure can be formed in the silica matrix to provide good mechanical properties and variable hydrophilic-hydrophobic balances. But they are often not optically transparent and in some case only are available as hydrogels. 135,136 Reinforced/filled composite sol-gels: To improve the mechanical properties and processing behavior of the sol-gel materials, some nano-or micro-particles, such as

46

graphite powder, fume silica, clays, cellulose and so on, can be incorporated inside the sol-gel silica.120,121,137 In addition, some active metal filler like gold, palladium, platinum can be used when conducting and redox-active materials are desired. In our group, a novel one-step direct bioencapsulation via nonsurfactanttemplated sol-gel process has been developed. This bioencapsulation is achieved by direct introduction of bioactive substances to nonsurfactant-templated sol-gel reactions prior to the system gelation with pH adjusted to near neutral and partial removal of organic byproducts. In this method, instead of using surfactant as template molecules, nonsurfactant small molecules, such as glucose, fructose, maltose, etc, are chosen to function as template. This small change in the sol-gel process makes a big difference in bioencapsulation in the following ways: (1) Unlike surfactant molecules, especially some ionic surfactant molecules, which are toxic and expensive, the small nonsurfactant molecules used as templates are biocompatible, low cost and harmless to most biological substances. (2) Because of the positive or negative charges carried by surfactant molecules, it is very difficult to remove surfactant molecules after the silica matrix is formed. Normally a high temperature up to 1000 0C is needed to burn surfactant template molecules away from silica matrix. In the application of bioencapsulation, this method does not work since no biospecies can survive at that high temperature. Whereas, nonsurfactant molecules have high solubility in water or other solvents,

47

thus by simply solvent extraction at room temperature, nonsurfactant molecules can be removed and leave desired porous structure. (3) The porous structure of silica matrix, such as pore size, surface area and pore size distribution, can be controlled by adjusting the amount of nonsurfactant template added into sol-gel reaction. As the result, desired pore size can be designed to entrap biological substances inside the matrix without leakage, while still permit the small molecules, such as reacting reagents and byproducts, penetrating through the matrix. (4) As discussed in (3), biological substances such as enzymes and proteins can be physically entrapped inside the pores instead of covalently bonded to matrix. Bonding enzyme to the substrate usually needs modifications of enzyme and reaction between enzyme and substrate, which are always tedious works and may involve protein denaturing. In this new method, without chemical reaction between enzyme and silica matrix, the lost of enzyme activities during encapsulation step can be neglected. Bioencapsulation has been applied in a variety of fields. Applications of solgel derived biomaterials include selective coatings for optical and electrochemical biosensors, stationary phases for affinity chromatography, immunoadsorbent and solid-phase extraction media, solid-phase biocatalysts and controlled release agents. Besides, the bioencapsulation, especially our new method by which protein can be entrapped in its native state, may also be used in basic biochemistry studies, such as protein folding unfolding and peptide aggregations.

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1.8 References

1. 2. 3. 4. 5. 6. 7. 8. 9. 10. 11. 12. 13. 14. 15. 16.

17.

Lu, G. Q. & Zhao, X. S. Nanoporous materials- an overview. Series on Chemical Engineering 4, 1-13 (2004). Rao, C. N. R., Mueller, A. & Cheetham, A. K. Nanomaterials - An introduction. Chemistry of Nanomaterials 1, 1-11 (2004). Kear, B. H. & Skandan, G. Overview: status and current developments in nanomaterials. International Journal of Powder Metallurgy (Princeton, New Jersey) 35, 35-37 (1999). Tolles, W. M. & Rath, B. B. General overview on nanomaterials. Nanomaterials: Synthesis, Properties and Applications, 545-553 (1996). Lin, Y.-W., Huang, M.-F. & Chang, H.-T. Nanomaterials and chip-based nanostructures for capillary electrophoretic separations of DNA. Electrophoresis 26, 320-330 (2005). Zecho, T. 31 pp ((Future Camp GmbH, Germany). Application, 2004). Martin, C. R. & Mitchell, D. T. Nanomaterials in analytical chemistry. Analytical Chemistry 70, 322A-327A (1998). Boennemann, H. & Nagabhushana, K. S. Chemical synthesis of nanoparticles. Encyclopedia of Nanoscience and Nanotechnology 1, 777-813 (2004). Ledoux, M. J., Vieira, R., Pham-Huu, C. & Keller, N. New catalytic phenomena on nanostructured (fibers and tubes) catalysts. Journal of Catalysis 216, 333-342 (2003). Lu, G. Q. Nanomaterials in Catalysis. (Proceedings of a Workshop from the Chemeca 2000 Conference held 9-12 July 2000 in Perth, Australia.) [In: Catal. Today, 2001; 68(1-3)] (2001). Wang, Z. L. Nanomaterials for nanoscience and nanotechnology. Characterization of Nanophase Materials, 1-11 (2000). Shi, J., Zhu, Y., Zhang, X., Baeyens, W. R. G. & Garcia-Campana, A. M. Recent developments in nanomaterial optical sensors. TrAC, Trends in Analytical Chemistry 23, 351-360 (2004). Mahadevan, V. & Sethuraman, S. Nanomaterials and nanosensors for medical applications. ICASE/LaRC Interdisciplinary Series in Science and Engineering 9, 207-228 (2003). Walcarius, A. Electrochemistry of silicate-based nanomaterials. Encyclopedia of Nanoscience and Nanotechnology 2, 857-893 (2004). Ong, K. G. & Grimes, C. A. Magnetostrictive nanomaterials for sensors. Encyclopedia of Nanoscience and Nanotechnology 5, 1-27 (2004). Sing, K. S. W., Everett, D. H., Haul, R. A. W., Moscou, L., Pierotti, R. A., Rouquerol, J. & Siemieniewska, T. Reporting physisorption data for gas/solid systems with special reference to the determination of surface area and porosity (Recommendations 1984). Pure and Applied Chemistry 57, 603-19 (1985). Brinker, C. J., Wallace, S., Raman, N. K., Sehgal, R., Samuel, J. & Contakes, S. M. Sol-gel processing of amorphous nanoporous silicas: Thin films and bulk.

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18. 19. 20. 21.

22. 23. 24. 25. 26.

27. 28. 29. 30. 31. 32.

Access in Nanoporous Materials, [Proceedings of a Symposium on Access in Nanoporous Materials], East Lansing, Mich., June 7-9, 1995, 123-139 (1995). Dave, B. C., Dunn, B. & Zink, J. I. 21 pp (Dep. Materials,California Univ.,Los Angeles,CA,USA., 1995). Lakshmi, B. B., Patrissi, C. J. & Martin, C. R. Sol-gel template synthesis of semiconductor oxide micro- and nanostructures. Chemistry of Materials 9, 25442550 (1997). Kresge, C. T., Leonowicz, M. E., Roth, W. J., Vartuli, J. C. & Beck, J. S. Ordered mesoporous molecular sieves synthesized by a liquid-crystal template mechanism. Nature (London, United Kingdom) 359, 710-12 (1992). Beck, J. S., Vartuli, J. C., Roth, W. J., Leonowicz, M. E., Kresge, C. T., Schmitt, K. D., Chu, C. T. W., Olson, D. H., Sheppard, E. W. A new family of mesoporous molecular sieves prepared with liquid crystal templates. Journal of the American Chemical Society 114, 10834-43 (1992). Ciesla, U. & Schuth, F. Ordered mesoporous materials. Microporous and Mesoporous Materials 27, 131-149 (1999). Raman, N. K., Anderson, M. T. & Brinker, C. J. Template-based approaches to the preparation of amorphous, nanoporous silicas. Chemistry of Materials 8, 1682-1701 (1996). Zhang, W. Rare earth stabilization of mesoporous alumina molecular sieves assembled through an N DegI Deg pathway. Chemical Communications (Cambridge), 1185-1186 (1998). Bagshaw, S. A. & Pinnavaia, T. J. Mesoporous alumina molecular sieves. Angewandte Chemie, International Edition in English 35, 1102-1105 (1996). Alberius, P. C. A., Frindell, K. L., Hayward, R. C., Kramer, E. J., Stucky, G. D. & Chmelka, B. F. General predictive syntheses of cubic, hexagonal, and lamellar silica and titania mesostructured thin films. Chemistry of Materials 14, 3284-3294 (2002). Yang, P., Zhao, D., Margolese, D. I., Chmelka, B. F. & Stucky, G. D. Generalized syntheses of large-pore mesoporous metal oxides with semicrystalline frameworks. Nature (London) 396, 152-155 (1998). Wei, Y., Jin, D. L., Ding, T. Z., Shih, W. H., Liu, X. H., Cheng, S. Z. D. & Fu, Q. A non-surfactant templating route to mesoporous silica materials. Advanced Materials (Weinheim, Germany) 10, 313-316 (1998). Wei, Y., Xu, J., Dong, H., Dong, J. H., Qiu, K. & Jansen-Varnum, S. A. Preparation and physisorption characterization of D-glucose-templated mesoporous silica sol-gel materials. Chemistry of Materials 11, 2023-2029 (1999). Pang, J.-B., Qiu, K.-Y., Xu, J., Wei, Y. & Chen, J. Synthesis of mesoporous silica materials via nonsurfactant urea-templated sol-gel reactions. Journal of Inorganic and Organometallic Polymers 10, 39-50 (2000). Pang, J.-B., Qiu, K.-Y. & Wei, Y. A new nonsurfactant pathway to mesoporous silica materials based on tartaric acid in conjunction with metallic chloride. Chemistry of Materials 13, 2361-2365 (2001). Zheng, J.-Y., Pang, J.-B., Qiu, K.-Y. & Wei, Y. Synthesis of mesoporous silica materials via nonsurfactant templated sol-gel route by using mixture of organic

50

33.

34. 35. 36.

37. 38.

39. 40. 41.

42.

43. 44. 45. 46. 47.

compounds as template. Journal of Sol-Gel Science and Technology 24, 81-88 (2002). Wei, Y., Xu, J., Feng, Q., Lin, M., Dong, H., Zhang, W.-J. & Wang, C. A novel method for enzyme immobilization: direct encapsulation of acid phosphatase in nanoporous silica host materials. Journal of Nanoscience and Nanotechnology 1, 83-93 (2001). Wei, Y., Xu, J., Feng, Q., Dong, H. & Lin, M. Encapsulation of enzymes in mesoporous host materials via the nonsurfactant-templated sol-gel process. Materials Letters 44, 6-11 (2000). Ping, G., Yuan, J.-M., Sun, Z. & Wei, Y. Studies of effects of macromolecular crowding and confinement on protein folding and protein stability. Journal of molecular recognition : JMR 17, 433-40 (2004). Wei, Y., Xu, J., Feng, Q., Lin, M., Dong, H., Zhang, W. J. & Wang, C. A novel method for enzyme immobilization: direct encapsulation of acid phosphatase in nanoporous silica host materials. Journal of nanoscience and nanotechnology 1, 83-93 (2001). Ping, G., Yuan, J.-M., Sun, Z. & Wei, Y. Studies of effects of macromolecular crowding and confinement on protein folding and protein stability. Journal of Molecular Recognition 17, 433-440 (2004). Sun, Z., Balakrishnan, G., Nielsen, S. B., Yuan, J.-M., Spiro, T. G., Wei, Y. & Venkatesh, S. A study of cytochrome c folding and unfolding in rigid silica matrices with controlled pore size by resonance Raman spectroscopy. Abstracts of Papers, 228th ACS National Meeting, Philadelphia, PA, United States, August 22-26, 2004, BIOL-069 (2004). Ping, G., Yuan, J. M., Vallieres, M., Dong, H., Sun, Z., Wei, Y., Li, F. Y. & Lin, S. H. Effects of confinement on protein folding and protein stability. Journal of Chemical Physics 118, 8042-8048 (2003). Wei, Y., Sun, Z., Zheng, J., Dong, H., Yuan, J. & Ping, G. Rigid matrix artificial chaperone (RMAC)-mediated refolding of heme protein. PMSE Preprints 87, 252-253 (2002). Sun, Z., Li, S., Patel, A. C., Wang, C. & Wei, Y. Fabrication of poly(2hydroxyethyl methacrylate)-silica nanoparticle hybrid nanofibers via electrospinning. Abstracts of Papers, 228th ACS National Meeting, Philadelphia, PA, United States, August 22-26, 2004, INOR-582 (2004). Patel, A. C., Sun, Z. & Wei, Y. Sol-gel synthesis and characterization of silver nanoparticles in D-glucose-templated mesoporous silica materials. Abstracts, 36th Middle Atlantic Regional Meeting of the American Chemical Society, Princeton, NJ, United States, June 8-11, 270 (2003). Brinker, C. Sol-gel science Academic Press, New York, N. Y. (1990). Hench, L. L. Sol-Gel Silica: Properties, Processing and Technology Transfer Noyes, Westwood, N. J.(1998). Rosen, M. J. Surfactants and Interfacial Phenomena (1995). Myers, D. Surfactant Science and Technology (1988). Huo, Q., Margolese, D. I., Ciesla, U., Demuth, D. G., Feng, P., Gier, T. E., Sieger, P., Firouzi, A., Chmelka, B. F. Organization of organic molecules with inorganic

51

48. 49. 50. 51. 52. 53.

54. 55. 56. 57.

58. 59. 60. 61. 62.

molecular species into nanocomposite biphase arrays. Chemistry of Materials 6, 1176-91 (1994). Antonelli, D. M. & Ying, J. Y. Synthesis of hexagonally packed mesoporous TiO2 by a modified sol-gel method. Angewandte Chemie, International Edition in English 34, 2014-17 (1995). Tanev, P. T. & Pinnavaia, T. J. A neutral templating route to mesoporous molecular sieves. Science (Washington, D. C.) 267, 865-7 (1995). Bagshaw, S. A., Prouzet, E. & Pinnavaia, T. J. Templating of mesoporous molecular sieves by nonionic polyethylene oxide surfactants. Science (Washington, D. C.) 269, 1242-4 (1995). Attard, G. S., Glyde, J. C. & Goltner, C. G. Liquid-crystalline phases as templates for the synthesis of mesoporous silica. Nature (London) 378, 366-8 (1995). Behrens, P. Voids in variable chemical surroundings: mesoporous metal oxides. Angewandte Chemie, International Edition in English 35, 515-18 (1996). Huo, Q., Margolese, D. I., Ciesla, U., Feng, P., Gier, T. E., Sieger, P., Leon, R., Petroff, P. M., Schueth, F. & Stucky, G. D. Generalized synthesis of periodic surfactant/inorganic composite materials. Nature (London, United Kingdom) 368, 317-21 (1994). Luan, Z., Cheng, C.-F., Zhou, W. & Klinowski, J. Mesopore Molecular Sieve MCM-41 Containing Framework Aluminum. Journal of Physical Chemistry 99, 1018-24 (1995). Borade, R. B. & Clearfield, A. Synthesis of aluminum rich MCM-41. Catalysis Letters 31, 267-72 (1995). Luan, Z., He, H., Zhou, w., Cheng, C.-F. & Klinowski, J. Effect of structural aluminum on the mesoporous structure of MCM-41. Journal of the Chemical Society, Faraday Transactions 91, 2955-9 (1995). Corma, A., Navarro, M. T. & Perez Pariente, J. Synthesis of an ultralarge pore titanium silicate isomorphous to MCM-41 and its application as a catalyst for selective oxidation of hydrocarbons. Journal of the Chemical Society, Chemical Communications, 147-8 (1994). Reddy, K. M., Moudrakovski, I. & Sayari, A. Synthesis of mesoporous vanadium silicate molecular sieves. Journal of the Chemical Society, Chemical Communications, 1059-60 (1994). Sayari, A., Danumah, C. & Moudrakovski, I. L. Boron-Modified MCM-41 Mesoporous Molecular Sieves. Chemistry of Materials 7, 813-15 (1995). Yuan, Z. Y., Liu, S. Q., Chen, T. H., Wang, J. Z. & Li, H. X. Synthesis of ironcontaining MCM-41. Journal of the Chemical Society, Chemical Communications, 973-4 (1995). Zhao, D. & Goldfarb, D. Synthesis of mesoporous manganosilicates: Mn-MCM41, Mn-MCM-48 and Mn-MCM-L. Journal of the Chemical Society, Chemical Communications, 875-6 (1995). Cheng, C.-F., He, H., Zhou, W., Klinowski, J., Goncalves, J. A. S. & Gladden, L. F. Synthesis and characterization of the gallosilicate mesoporous molecular sieve MCM-41. Journal of Physical Chemistry 100, 390-6 (1996).

52

63. 64. 65. 66. 67. 68. 69. 70.

71. 72. 73. 74. 75. 76.

77.

Luca, V., MacLachlan, D. J., Hook, J. M. & Withers, R. Synthesis and characterization of mesostructured vanadium oxide. Chemistry of Materials 7, 2220-3 (1995). Ciesla, U., Schacht, S., Stucky, G. D., Unger, K. K. & Schueth, F. Formation of a porous zirconium oxo phosphate with a high surface area by a surfactant-assisted synthesis. Angewandte Chemie, International Edition in English 35, 541-3 (1996). Abe, T., Taguchi, A. & Iwamoto, M. Non-Silica-Based Mesostructured Materials. 1. Synthesis of Vanadium Oxide-Based Materials. Chemistry of Materials 7, 1429-30 (1995). Antonelli, D. M. & Ying, J. Y. Synthesis and Characterization of Hexagonally Packed Mesoporous