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«A Thesis Submitted to the Faculty of Drexel University by Zhengfei Sun in partial fulfillment of the requirements for the degree of Doctor of ...»

-- [ Page 1 ] --

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.

2

Dedications

This dissertation is dedicated to my parents, Mr. Chongzhen Sun and Mrs. Xiuqing

Nie for their encouragement, support and love.

3

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 β 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 β peptide. I also thank Dr. Solomon Praveen for 4 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.

–  –  –

Table of Contents

List of Tables

List of Figures

Abstract

Chapter 1: An Overview to Nanoporous Sol-Gel Materials

1.1 Introduction

1.2 Fundamentals of Sol-Gel Process

1.2.1 Sol-Gel Reactions

1.3 Nanoporous Sol-Gel Materials from Surfactant Templated Pathways........ 31 1.4 Nanoporous Sol-Gel Materials from Nonsurfactant Templated Pathway... 35 1.5 Characterizations of Nanoporous Materials

1.5.1 Gas sorption measurement

1.5.2 X-Ray Diffraction (XRD)

1.5.3 Electron Microscopy

1.6 Organic/Inorganic Hybrid Materials by Sol-Gel Approach

1.7 Sol-Gel Encapsulation of Biomolecules

1.8 References

Chapter 2: Rigid Matrix Artificial Chaperon (RMAC) – Mediated Refolding of Cytochrome c

2.1 Introduction

2.1.1 Fundamentals of Protein Folding Unfolding

2.1.2 Chaperone and Protein Folding

–  –  –

2.1.4 Why Cytochrome c?

2.1.5 Analytical Methods to Monitor Cytochrome c’s Folding Unfolding...... 68 2.1.5.1 Fluorescence Spectroscopy

2.1.5.2 Circular Dichroism (CD)

2.2 Experimental

2.2.1 Materials

2.2.2 Encapsulation of Unfolded Cc into Silica Matrix

2.2.3 Removal of Templates

2.2.4 Characterizations of Nanoporous Silica Matrix

2.2.5 Fluorescence Spectroscopy of Encapsulated Cc

2.2.6 Circular Dichroism of Cc

2.2.7 Fourier Transform Infrared Spectroscopy (FTIR) of Silica Matrix......... 75 2.2.8 UV-Vis Spectroscopy (UV) of Encapsulated Cc

2.2.9 Attempts of Making Silica Thin Film

2.3 Results and Discussion

2.3.1 Characterization of Silica Matrix





2.3.2 FT-IR Spectroscopy on Silica Matrix

2.3.3 Fluorescence Spectroscopy of Encapsulated Cc

2.3.4 Leakage Tests

2.3.5 CD Spectroscopy

2.4 Conclusion

2.5 Acknowledgement

–  –  –

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

3.1.1 Resonance Raman Spectroscopy and Protein Folding

3.1.2 Heme Protein

–  –  –

3.1.2.1.1 Marker Band Region

3.1.2.1.2 Fingerprint Region

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

3.2 Experimental

3.2.1 Materials

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

3.2.6 Resonance Raman Spectroscopy

3.2.7 Characterizations of Nanoporous Silica Matrix

3.2.8 Fourier Transform Infrared Spectroscopy (FTIR) of Silica Matrix....... 114

3.3 Results and Discussion

–  –  –

3.3.1.1 Ccg series

3.3.1.2 Ccu series

3.3.2 Resonance Raman Spectroscopy of Ccu series

3.3.3 Resonance Raman Spectroscopy of Ccg series

3.4 Conclusions

3.5 Acknowledgements

3.6 References

Chapter 4: A Novel Method to Study Aggregation of Amyloid β1-42 - A Key Peptide Associated with Alzheimer’s Disease

4.1 Introduction

4.1.1 Alzheimer’s Disease and Amyloid β

4.1.2 The Aggregation of Aβ peptides

4.1.3 Detection of Aβ aggregation

4.1.3.1 The Thioflavine T Fluorescence Assay

4.1.3.2 The Congo Red Birefringence Assay

4.1.3.3 Negative staining of amyloid fibrils for TEM

4.1.4 Bioencapsulation of Aβ peptide in silica matrix with controlled pore size

4.2 Experimental

4.2.1 Materials

4.2.2 Redissolving of Aβ peptide in their monomeric state

4.2.3 Bioencapsulation of Aβ1-42 in silica matrix with controlled pore size... 154

–  –  –

4.2.5 Fluorescence Spectroscopy Study

4.2.5.1 Steady State Fluorescence Spectroscopy

4.2.5.2 Time Scale Fluorescence Spectroscopy

4.2.6 Congo Red Birefringence Assay

4.2.6.1 Preparation of the Staining Solution

4.2.6.2 Incubation of Silica Biogel Samples in Buffer Solution............. 157 4.2.6.3 Mounting Silica Biogel Samples on Glass Slides

4.2.7 Negative staining of amyloid fibrils for TEM

4.3 Result and Discussion

4.3.1 Characterization of Silica Matrix

4.3.2 Steady State Fluorescence Spectroscopy

4.3.2.1 Abata42 Series Samples

4.3.3 Time Scale Fluorescence Spectroscopy

4.3.4 Congo Red Birefringence Assay

4.4 Conclusion

4.5 Acknowledgements

4.6 References

Chapter 5: Fabrication of Poly (2-hydroxyethyl methacrylate)-Silica Nanoparticle Hybrid Nanofibers via Electrospinning

5.1 Introduction

5.1.1 Organic-Inorganic Nanocomposite: Opportunities to Advanced Materials

–  –  –

5.2 Experimental

5.2.1 Materials

5.2.2 Synthesis of the Hybrid Material

5.2.3 Set-up of Electrospinning Apparatus

5.2.4 Electrospinning of PHEMA-Silica Hybrids

5.2.5. Instrumentation and Characterization

5.2.5.1 FTIR Spectroscopy

5.2.5.2 Thermal Gravimetric Analysis (TGA)

5.2.5.3 SEM and TEM

5.3 Results and Discussion

5.4 Conclusion

5.5 Acknowledgements

5.6 References

Chapter 6: Synthesis and Characterization of Dental Composite Containing Nanoporous Silica as Fillers

6.1 Introduction

6.1.1 Resin Matrix

6.1.2 Filler System

6.1.3 Coupling Agent

6.1.4 Limitations of Coupling Agent

6.1.5 Porous Filler without Coupling Agent

6.1.6 Non-surfactant Templated Sol-Gel Process

–  –  –

6.2.1 Materials

6.2.2 Preparation of Nanoporous Silica Filler

6.2.3 Characterization of Nanoporous Silica Filler

6.2.4 Preparation of Dental Resin

6.2.5 Silanization of Non-Porous Silica Particles

6.2.6 Preparation of Dental Composite

6.2.7 Evaluation of Mechanical Properties

6.2.8 Aging Test

6.3 Results and Discussion

6.3.1 BET Analysis

6.3.2 Compression Testing

–  –  –

Nanoporous Filler

6.3.2.3 Aging Test

6.4 Conclusion and Future Works

6.5 Acknowledgements

6.6 References

Chapter 7: Dense Packing of Vinyl Modified Silica Nanoparticles and Its Potential Application as Low Shrinkage Dental Materials

7.1 Introduction

–  –  –

7.2.1 Materials

7.2.2 Separation of Nanoparticle from OG100-31

7.2.2.1 Reduced Pressure Distillation

7.2.2.2 Ultracentrifuging

7.2.3 Atomic Force Microscopic (AFM) Measurements

7.2.4 Thermal Gravimetric Analysis (TGA)

7.2.5 FTIR Spectroscopy

7.2.5.1 Solid Sample

7.2.5.2 Liquid Sample

7.2.6 Isolation of Silica Nanoparticles

7.2.7 Preparation of Dental Resin

7.2.8 Preparation of Dental Composite

7.2.9 Evaluation of Mechanical Properties

7.3 Results and Discussion

7.4 Conclusion and Future Work

7.5 Acknowledgements

7.6 References

Chapter 8: Summary and Conclusions

8.1 Nanoporous Materials and Their Application in Bioscience

8.2 Organic-Inorganic Hybrid Nanocomposites

Appendix A: Supplemental Data of Chapter 2

Appendix B: Supplemental Data of Chapter 4.

–  –  –

Appendix D: Fabrication of a New Type Molecularly Imprinted Polymer Membrane Sensor for Atrazine

–  –  –

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

Table 3- 1 Pore parameters of water-extracted Ccu series prepared at various urea concentrations

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

Table 4- 1 Summary of porous parameters of Abeta series samples after removel of templates.

Table 4- 2 Relative fluorescence intensity of immobilized Amyloid β 1-42 and free at difference pH

Table 6- 1 BET analysis of mesoporous fillers at different fructose concentration.

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

Table 6- 3 Comparison of compressive properties of composites with different types of fillers. The number of specimens tested is given in parenthesis................ 232

–  –  –

composites prepared using mesoporous and SiO2 fillers. The number of specimens tested is given in parenthesis

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.

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

–  –  –

Figure 1- 2 Diagrams of sol-gel reactions: (a) Hydrolysis reaction; (b) Condensation reaction

Figure 1- 3 Possible mechanisms for formation of MCM-41: (1) liquid crystal phase initiated and (2) silicate anion initiated. 20,21

Figure 1- 4 IUPAC classification of physisorption isotherms.16

Figure 1- 5 IUPAC classification of hysteresis loops.16

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

Figure 2- 2 Illustrations of artificial chaperones assisting protein folding............... 91 Figure 2- 3 N2 adsorption-desorption isotherm at –196°C.

Figure 2- 4 BJH pore size distributions for the sol-gel material synthesized in the presence of 0-50% wt% of urea.

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.



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