«Kinetic Investigations of Thiolate Protected Gold Nanoparticles: Protein Interactions, Electron Transfer, and Precursor Formation By Brian N. Turner ...»
Kinetic Investigations of Thiolate Protected Gold Nanoparticles: Protein Interactions,
Electron Transfer, and Precursor Formation
Brian N. Turner
Submitted to the Faculty of the
Graduate School of Vanderbilt University
in partial fulfillment of the requirements
for the degree of
Doctor of Philosophy
Professor David E. Cliffel
Professor John A. McLean
Professor James E. Crowe, Jr.
Professor David W. Wright In Memory of Sharon Rose Turner and Ruth Shelton Turner A special thanks to Mom, Dad, April, Eric, Gramps, Keith, and Patti Lovingly dedicated to Mary Pat ii
ACKNOWLEDGEMENTSAcknowledgements of people who helped along the way are included at the end of each chapter, but I would like to reserve this section to thank people who helped in unique ways.
As for my committee, I will open with Dr. David Cliffel. I have always appreciated your knowledge and creativity in generating new ideas. I have also appreciated the freedom I have had to go in my own direction with research that seems very unique to this lab. Also, I really appreciate your humanistic approach to leaves of absence, and never once interfering in personal business. This was especially appreciated as many personal crises and family crises seemed to continually arise over the last six years, and I am not sure I would have been able to deal with them appropriately in a lot of other situations. On a lighter note, it was a pleasant surprise to work for someone who would have the group over to play strategy games, volleyball, and generously provided wonderful food to a somewhat larger research group. Finally, thanks for having the patience to allow me to work here over a six year period, all the while providing financial support.
Dr. Wright, I have always secretly appreciated your no-holds-barred attitude, and tellit-like-it-is approach to committee meetings. I think this is a very important trait when it comes to making sure that the science we practice is legitimate. My favorite part about your approach is your intensity about the science because I believe that it should be exciting and meaningful to the researchers instead of just a means to a job. Until I saw
design really is in terms of practical potential and elegance.
Dr. McLean, I would like to thank you for some of the most interesting science conversations I have had during my stay here. My personal favorite was the discussion about how to weigh an electron accurately. I always looked forward to your classes, because you have a unique gift of making complicated topics seem approachable and exciting. I also enjoyed hanging out with you socially in Atlanta during Pittcon. We will have to meet at another conference and play that shuffleboard game again.
Finally, Dr. Jim Crowe, until I met you, I had not met somebody who works on such a high level in their field that is as approachable as you are. Additionally, I appreciate that you are always very thoughtful with your questions.
It would take me way too long to thank everybody individually in my research group for sitting through my practice exams and the like. Thank you to everyone, as it was much appreciated.
Graduate school can be a difficult time, so I would like to thank Bill Evans, Vanessa Phelan, Dr. Victor Ghidu, Easton Selby, Danielle “Watkins” Kimmel, the volleyball crew, the soccer crew, the old Magic the Gathering crew (ignore that), and the current strategy gaming crew for keeping things light when times were not. I would also like to thank all of my old friends back home and elsewhere for staying in touch, offering encouragement, and giving me an excuse to leave town every here and again, especially Mike Pateras, Lora Robinson, Greg Raffio, Alicia DiMarco, Mykola Bilokonsky, and Ryan Stolte-Sawa.
suffice it to say that when things got tough here, I would not have continued along if not for your encouragement and belief. I love you all.
And Mary Pat, I just want you to know that I would not have finished this if you hadn’t pushed me. And Dad, I never would have gotten into this in the first place without your constant support and help.
I would like to acknowledge the following sources of funding: the National Institutes of Health Sections on General Medicine [NIH/NIGMS (R01 GM 076479)] and Allergies and Infections Disease- Southeastern Regional Center for Excellence in Biodefense [NIH/NIAID/SERCEB (U54 AI057157)], the Defense Threat Reduction Agency, the Research Coporation, the Vanderbilt University Graduate School and their Travel Grant Program (which provided for trips to Pittcon 2007 and ACS National Meeting Fall 2010), Oak Ridge National Laboratory Center for Nanophase Materials Science and the Vanderbilt University Department of Chemistry Teaching Assistant Program.
LIST OF TABLES
LIST OF FIGURES
INTRODUCTION: SYNTHESIS AND CHARACTERIZATION OF VARIOUSI.
THIOLATE PROTECTED GOLD NANOPARTICLES AS PREPARED BY
Functionalization of Thiolate Protected Gold Nanoparticles.......5 Characterization Methods for MPCs
Instrumentation for Routine Nanoparticle Characterization...... 14
LINEAR EPITOPE MAPPING APPROACH TO LOCATE BINDING “HOTII.
SPOTS” IN THE CONFORMATIONAL ANTIGENIC SITE A OF HUMANRESPIRATORY SYNCYTIAL VIRUS (HRSV) FUSION PROTEIN (F)......... 15 Introduction
Human Respiratory Syncytial Virus
Quartz Crystal Microbalance (QCM)
Formation of peptide SAMs
ELISA Stepwise linear epitope mapping of the full length RSV F Antigenic Site A
GOLD NANOPARTICLES PRESENTING PEPTIDES AS BIOMIMCSIII.
OF HRSV F PROTEIN ANTIGENIC FUNCTION
Nanoparticles Presenting Peptides as Antigenic Mimics........... 54 Quartz Crystal Microbalance Immunosensing
Synthesis of Au216Tiopronin129 Nanoparticles
Synthesis of Au118Tiopronin71 Nanoparticles
Synthesis of Au696Tiopronin265 Nanoparticles
Place Exchange to Form Series 3 Peptide-Nanoparticle Conjugates
QCM Immunosensor Construction and Detection of HRSV..... 67 Adaptation of the QCM Immunosensor to Detect Peptide Presenting Gold Nanoparticles
Results and Discussion
QCM Immunosensor for HRSV
Development of Peptide Presenting Gold Nanoparticles........... 73 Detection of Peptide Presenting Gold Nanoparticles with the QCM Immunosensor for HRSV
SECM OF “WIRED” GOLD NANOPARTICLES TO DETERMINEIV.
ELECTRON TRANSFER KINETICS: PROOF OF CONCEPT FORPOTENTIAL COMPONENTS IN NANOMOLECULAR CIRCUITS............... 87 Introduction
Electrochemical Properties of Gold Nanoparticles
Molecular Wire Molecules
Scanning Electrochemical Microscope
Synthesis of Au1120 Dodecanthiolate690
Synthesis of Au228Octanethiol92
KINETIC INVESTIGATION OF TIOPRONIN PROTECTED GOLDV.
NANOPARTICLE PRECURSOR FORMATION
UV-Visible Spectroscopy Kinetics Experiments
Results and Discussion
SUMMARY OF CONCLUSIONS AND SUGGESTIONS FOR FUTUREVI. WORK
Summary of Conclusions
Suggestions for Future Work
Nanoparticle Precursor Formation
A. THERMAL GRAVIMETRY-MASS SPECTROMETRY AND
ELEMENTAL ANALYSIS OF TIOPRONIN PROTECTED GOLDNANOPARTICLES
D. PURIFICATION AND CHARACTERIZATION DATA FOR PEPTIDESUSED IN THIS STUDY OR OTHERWISE DESIGNED AND PURIFIED..... 158
ADDENDUM TO “COLOR MY NANOWORLD” EXERCISE USED ATE.
THE VANDERBILT SUMMER ACADEMY
1. Summary of reported HRSV escape mutations in HRSV F antigenic site A............. 21
2. Tabulated characterization data for peptides purified by this author.
3. Peptides designed to be evaluated by the QCM step-wise linear epitope mapping method.
4. Peptides evaluated against palivizumab in an ELISA assay.
Student’s t-tests for peptide ELISA
6. Peptides that were conjugated to nanoparticles in this study.
7. Nanoparticle mimics studied with the QCM HRSV immunosensor.
8. Summary of mimic A detection at various concentrations, along with sensor characterization data
9. electron transfer kinetic constants determined for Au228Octanethiol92-nPEPEPSn.... 123 Rate data for the reaction of Au(III)Cl4 - with tiopronin in MeOH at unfixed ionic 10.
11. Rate data at fixed [RSH] and [Au(III)] with variable [H+] (adjusted with perchloric acid/MeOH solution) and [Cl-] (adjusted with sodium chloride/MeOH solution)... 145
12. Rate data for conditions designed to test saturation kinetics of the tiopronin/tetrachloroaurate system.
13. Elemental analysis-calculation compared to two different TGA-TEM calculations of molecular formula.
14. Characterization data for peptides synthesized by this author.
15. Modified Brust reaction scheme for polar ligands.
16. Examples of thiolate ligands used in the synthesis of water soluble Au MPCs............4
17. Scheme of the solution phase place-exchange reaction.
18. The stoichiometry of incoming to exiting ligand is 1:1.
19. Chart of the different relative rates at which place-exchange and (possibly) migration occur
The non-covalent interaction based place-exchange reaction.49
21. Cartoon schematic of an HRSV virion.
22. HRSV F protein, showing the head, neck, and stalk structure.
23. ELISA study of synthetic peptides from Rutledge and coworkers
24. QCM graph illustrating the binding of BSA to bare gold and PZ to a mixed SAM of peptide 2 and tiopronin.
25. QCM graph illustrating the binding of BSA to bare gold and PZ to a mixed SAM of peptide 8 and tiopronin.
26. QCM graph illustrating the binding of BSA to bare gold and PZ to a mixed SAM of peptide 10 and tiopronin.
27. Results of the optimized peptide ELISA
28. A conceptual interpretation of PZ approaching the synthetic peptide epitope........... 47
29. Motavizumab peptide epitope of HRSV F (gray) interacting with the light chain (blue) and heavy chain (green).
30. Comparison of the glutathione (top) and tiopronin (bottom) ligands
31. 10-amino acid HA peptide on a 3-D gold tiopronin nanoparticle
32. Loop presenting MPCs (left) shown compared to the native protein
34. Detection of HRSV with the QCM-PZ immunosensor.
35. Detection and control experiments for the QCM-PZ immunonosensor.
36. H NMR, with double water-gate solvent suppression, of a peptide-nanoparticle conjugate
37. Detection of Au696Tiopronin255Peptide(3-F)10 by the QCM immunosensor with and without PZ.
38. Representative QCM mass versus time curves at various concentrations of mimic A during the detection step.
39. Plot to determine the time constant for mimic A binding to the Palivizumab sensor. 80
40. Plot of individual time constants determined for each concentration.
41. Plot to determine binding coefficients using the four lowest concentration samples.. 83
42. Nanoparticle-bipyridine redox switch
43. Cowpea mosaic virus-gold nanoparticle-molecular wire conjugate molecular electronic nanosensor.
44. Square wave voltammogram of AuHexanethiolate nanoparticles.
45. The molecular wire ligand
46. Current-voltage profiles of molecular wires sandwiched between gold wires........... 97
47. Read-write computing system with gold electrodes sandwiching molecular wires.... 98
48. SECM as a useful imaging technique in biological and materials applications........ 101
49. SECM experimental design
50. SECM approach curves of AuAlkanethiolate nanoparticles
Proof of concept experiments to confirm whether “wired” AuDodecanethiol 51.
nanoparticles (left) exhibit faster electron transfer than their “unwired” precursors (right)
52. MALDI-IM mass spectrum of AuDodecanethiolate nanoparticles functionalized with the molecular wire.
SECM approach curve with 1V Pt UME through larger “wired” AuDodecanethiolate 54.
Comparison of 1H NMR spectra of dodecanethiol gold nanoparticles synthesized by 55.
the Brust method (blue), and by the method of Rowe and coworkers (red)............. 120
56. SECM approach curve through Au228Octanethiol90PEPEPS2, Au228Octanethiol90PEPEPS2.6, and Au228Octanethiol92.
57. Nanoparticle and ring complex structures
Time resolved UV-Vis spectra of AuCl4 - undergoing reduction by tiopronin.......... 134 58.
59. UV-Vis region containing the saddle at 289 nm for the gold(III)/tiopronin system. 135