Platelet Adhesion to Various Surfaces Studied by On-Line Acoustic Wave Sensor

140 Pages·2000·4.59 MB·English

Platelet Adhesion to Various Surfaces
Studied by On-Line Acoustic Wave Sensor
by
Zeynep Morel
A thesis is submitted in conformity *th the requirements
for the degree of Master of Science
Department of Chemistry
University of Toronto
@ Copyright by Zeynep Morel
1999
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Platelet Adhesion to Various Surfaces
Studied by On-Line Acoustic Wave Sensor
M.Sc. Thesis, 1999
Zeynep Mord
Department of Chemistry, University of Toronto
Platelet adhesion to bare gold, collagen and bovine serum albumin (BSA)-coated
devices was studied by ihickness-shear-mode (TSM)a coustic wave anal ysis in the liquid
phase. Collagen and BSA-coated sensors were characterized by X-ray photoelectron
spectroscopy (XPS). BSA prevented adhesion of platelets to the bare gold sensor and the
responses of such devices were surface energy-dependent. Washed platelet suspensions
showed lower frequency shifts than platelet-rich-plasma (PRP) and platelet-poor-plasma
(PPP) on al1 studied surfaces. Kinetics and extent of platelet adhesion to collagen is
dependent on the type of collagen used. PRP monitored on collagen-coated sensors at pH 3.4
showed very high frequency changes and scanning electron microscopy indicated
intermediate changes of platelet structures taken at different points on the frequency profile.
The well-known Sauerbrey equation has been demonstrated to be inadequate to explain the
frequency changes for the operation of TSM acoustic wave sensors operated in liquid phase.
"The important thing is not to stop questioning. Curiosity has its own
reason for exisling. One cannot help but be in awe when he contemplates
the mysteries of eternity, of life, of the marvelous structure of realiw It is
enough if one tries merely to comprehend a Little of this rnystery every day.
Never lose a holy curiosity. "
--
Albert Einstein
iii
Acknowledgements
1 would like thank my supervisor, Professor Michael Thompson for providing me the
opportunity to work in the fascinating field of sensors. This work would not have been
possible without his enthusiasm, support and encouragement.
1 am pleased to acknowledge the generous help of many other people in this project.
Several interesting and invaluable discussions with Ms. M. Mody and Dr. J. Freedman from
St. Michael's Hospital. Toronto and Dr. M. Rand from The Hospital for Sick Children,
Toronto about blood platelets have been highly valuable to me and my thesis. I am also
gratehil to Ms. M. Mody, for her platelet preparation and helpful suggestions.
Assistance of the XPS analysis by Dr. Lân Bùi and SEM by Dr. David Stone are
greatly acknowledged. My special thanks go to Mr. Ani1 Deisingh and Dr. David Stone for
the critical reading of this thesis and many valuable suggestions. 1 would like to thank Dr.
Biljana tavie, Mr. Shakour Ghafoun and Ms. Michelle Furtado, Ms. Nardos Tassew, Ms.
Emma-Louise Lyle and Ms. Rosa Nisman for their valuable help and fnendship. 1 also would
like to thank other members of the Chernicd Sensors Group, past and present, for the
fnendly atmosphere in the group.
1 would like to express my sincere appreciation to my friends from Turkey and
Canada. This work could not have been completed without their love, support and
understanding. Special thanks to those who have proven our friendships can exist beyond
time and distance. We will be friends for life.
1 am grateful to my parents, Mrs. Sevimser Morel and Mr. Osman Morel for their
love, support and ailowing some of my goals to be achieved. This thesis is dedicated to
parents, nurnerous teachers and friends.
Table of Contents
Abstract
Quotation
Ac knowledgements
Table of Contents
List of Tables
List of Figures
1. Introduction
1.1. Platelets
1.2. Platelet A dhesion
1.3. Collagen
1.4. Platelet Adherion to Collagen
1.5. nMory of the TSM Sensor Rosponse
1.6. The TSM Sensor in the Gas Phase
1.7. Operation of the TSM sensor in the Liquid Phase
1.8. Butter-Van-Dyke (8V D) Equivalent Circuit Mode1
1.9. BVD Equivaknt Circuit Elements
1.S . 1. Motionai Resistance (Rm)
7.9.2. Motional Inductance (Lm)
1.9.3. Motional Capacltance (Cm)
1.9.4. Electroststic Capacitanco (Co)
1.10. C dA dhesion studied by TSM
1.11. Platelet Adhesion to Collagen monitomd by TSM Sensors
1.12. Research Objectives
2. Experimental
2. 1. Reagents
2.2. Procedures
2.2.1. Surface Treatment
2.2.2. Platelet Preparation and Fixation
2.2.3. Flow Cytometric Evaluation of PRP and Platelet Samples
2e2.4. Impedance Measurements
2.3. Apparatus
2.3.1. TSM Sensors
2.3.2.N etwork Analyser
.
2.3.2.1 Flow Injection Analysis (FIA) Technique
2.3.2.2.F low Cell
2.3.3. Contact Angle Measumments
2.3.4. Xiey Photoelgctron Spectroscopy (XPS)
2.3.5. Ught Microscopy
2.3.6. Confocal Microscopy
2.3. 7. Scanning Electron Microscopy (SEM)
3. Results and Discussion
3.1. Platelet Adherence to the TSM Sensor
3.1.1. Efîbct of Platelet Count on Adhesion of Platelets 54
3.1.2. Etfect of Antic08g~lantSo n Adhesion of Platelets 57
3.1.3. Effec? of Platelet Activation on Adhesion of Platelets 60
3.1.4. Effect of POEI on Adhesion of Platelets 60
3.1.5. PlateIet=rich-plasmao n Hydrophobie T MS ensor 63
3.2. Platelets on BSA-coated TSM Sensor 63
3.2.1. Bovine Serum Albumin (BSA) on the TSM Sensor 66
3.2.2. Adhesion of Platelets to BSA-eoated TSM Sensors 66
3.2.3. Adhesion of PRP and PPP to BSA coated TSM Sensors 70
3.2.4. Effect of Bumr on Adhesion of Platelets to BSA coated TSM Sensors 72
3.3. Platelets on collagen-coated TSM Sensors 79
3.3.1. Collagen on the TSM Sensor 79
.
3.3.1.1 Adsorption of Collagen Type IV on the TSM Sensor 79
3.3.1.2. Adsorption of Collagen type I on the TSM Sensor 83
3.3.1.3. Adsorption of Collagen Hom on the TSM Sensor 87
3.3.2. Adhesion of Platelets to Collagen-coated TSM Sensors 87
3.3.3. Adhesion of PRP and PPP to Collagen-coated TSM sensors 93
3.3.4. Effect of pH on Adhesion of Platelets to Collagen-coated TSM
Sensors 101
3.3.5. Effect of Collagen Receptor Blocking on Adhesion of Platelets 112
4. Conclusions 117
vii
5. Future Work
References
Appendix
.*.
Vlll
".
Table 1 : Platelet Defects and Disorders 6
Table 2: Collagen Types, Classes and Tissue ~istributions~~. 10
Table 3: Liquid Phase Theories of TSM Acoustic Wave Sensors. 16
Table 4: Platelet counts and observed frequency changes for the adhesion of platelets to the
hydrophobic gold TSM device. 56
Table 5: Elemental compositions of carbon (C), nitrogen (N), oxygen (0)a nd gold (Au) of
bare gold, BSA-coated and collagen-coated TSM sensors. 68
Table 6: Cornparison of the series frequency responses of PRP and PPP on BSA-coated
surfaces. Tyrode's buffer used as the carrier of the fiow system. 78
Table 7: Comparison of observed frequency changes with washed platelet suspensions, PRP
and PPP on collagen type 1, type IV and Hom-coated TSM sensors. 98