Immobilization and Characterization of Physisorbed Antibody Films Using Pneumatic Spray as ...

140 Pages·2015·6.37 MB·English

University of South Florida
Scholar Commons
Graduate Theses and Dissertations Graduate School
January 2013
Immobilization and Characterization of
Physisorbed Antibody Films Using Pneumatic
Spray as Deposition Technique
Jhon J. Figueroa
University of South Florida, [email protected]
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Figueroa, Jhon J., "Immobilization and Characterization of Physisorbed Antibody Films Using Pneumatic Spray as Deposition
Technique" (2013).Graduate Theses and Dissertations.
http://scholarcommons.usf.edu/etd/4889
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Immobilization and Characterization of Physisorbed Antibody Films Using Pneumatic

Spray as Deposition Technique

by

Jhon Figueroa

A dissertation submitted in partial fulfillment
of the requirements of the degree of
Doctor of Philosophy
Department of Chemistry
College of Arts and Sciences
University of South Florida

Major professor: Rudy Schlaf, Ph.D.
Xiao Li Ph.D.
Li-June Ming Ph.D.
Abdul Malik Ph.D.

Date of approval:
November 14, 2013

Keywords: Biosensors, avidin-biotin bridge, surface morphology, antibody
Immobilization

Copyright © 2013, Jhon Figueroa
DEDICATION

This dissertation is dedicated principally to God who gave the strength to achieve
the unachievable. I want to honor Him in all that I did and in all that I would do after this
stage. I also dedicate this to my dear wife Ruth Mary who was able to accept all the hard
conditions that brought the challenge of pursuing a PhD degree. I have to recognize you
nena as inherent part of this achievement and thank you for your commitment, support,
appreciation, patience, love and overall thank you for being my friend, I love you.
ACKNOWLEDGMENTS

The first person that I want to acknowledge is Dr. Schlaf who with his guidance,
advice, trust and support has helped me to complete all the requirements to achieve this
dissertation. There are many words to express feelings, but two can summarize well what
I feel towards him: gratitude and admiration for what he has done through these years.

I also want to thanks the committee members for their time and guidance making
it possible to achieve this dream.

I would like to thanks Dr. Lim and his team in the Advanced Biosensor
Laboratory for all the technical support given to me and especially to Sonia Magana who
has been an invaluable asset to this project and an incomparable source of knowledge;
thank you Sonia for all the fructiferous scientific discussions.

Thank you to all the members of the surface science laboratory for their support
and unconditional help especially Eric Tridas and Daniel Gomez. Finally, I want to thank
my parents, family, church members and friends who in one way or another were a
crucial part of this achievement by their patience and understanding.
TABLE OF CONTENTS

LIST OF TABLES ............................................................................................................. iv

LIST OF FIGURES .............................................................................................................v

LIST OF ABBREVIATIONS ..............................................................................................x

ABSTRACT….. ................................................................................................................ xii

CHAPTER 1. INTRODUCTION AND FUNDAMENTALS……………... ......................1
1.1 Outline and motivation .......................................................................................1
1.2 Immobilization methods. ....................................................................................3
1.3 Pneumatic spray. .................................................................................................6
1.4 Proteins, structure and importance. ...................................................................11

CHAPTER 2. EXPERIMENTAL METHODOLOGY......................................................16
2.1 Analysis performed by fluorescent microscopy ..............................................16
2.1.1 Visualization of antibody and bacteria patterns ...............................16
2.1.2 Specificity, shelf life, capture efficiency and sensitivity test. ............19
2.2 Characterization of antibody thin films. ..........................................................23
2.2.1 Ellipsometry and Ultraviolet visible spectroscopy. ..........................23
2.2.2 Attenuated total reflection Fourier transform infrared
(ATR-FTIR) ................................................................................................27
2.2.3 Atomic force microscope (AFM) .......................................................30
2.2.4 X-ray photoemission spectroscopy (XPS). ........................................33
2.2.5 Contact angle (wetting properties of a film). ....................................38
2.3 Experimental set up..........................................................................................41
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2.3.1 Materials ...........................................................................................41
2.3.2 Immobilization methods (pneumatic spray and avidin-
biotin bridge) .............................................................................................43
2.3.3. Reproducibility and visualization of patterns of
immobilized antibody. ................................................................................45
2.3.4 Testing capture efficiency, specificity and shelf life of
immobilized antibody. ................................................................................46
2.3.5 Testing sensitivity of immobilized antibody films. ............................47
2.3.6 Equipment (ellipsometry, UV/vis spectroscopy and ATR-
FTIR). .........................................................................................................49

CHAPTER 3: ANTIBODY IMMOBILIZATION USING PNEUMATIC SPRAY:
COMPARISON WITH THE AVIDIN-BIOTIN BRIDGE IMMOBILIZATION
METHOD ..................................................................................................................53
3.1 Introduction ......................................................................................................53
3.2 Results ..............................................................................................................56
3.3 Discussion ........................................................................................................66
3.4 Conclusions ......................................................................................................72

CHAPTER 4. CHARACTERIZATION OF FULLY FUNCTIONAL SPRAY-ON
ANTIBODY THIN FILMS ...............................................................................................73
4.1 Introduction ......................................................................................................73
4.2 Results ..............................................................................................................76
4.2.1 Ellipsometry, UV-Vis spectroscopy and ATR-FTIR..........................76
4.2.2 AFM, XPS and contact angle measurements ...................................79
4.2.3 Capture cell performance and fluorescent microscopy ....................85
4.3. Discussion .......................................................................................................87
4.3.1 Surface morphology, physical characteristics and
capture activity...........................................................................................88
4.3.2 Chemical and mechanical properties ...............................................91
4.3.3 Film thickness and growth ................................................................93
4.4 Conclusions ......................................................................................................95
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REFERENCES ..................................................................................................................96

APPENDIX A: COPYRIGHT APPROVAL ...................................................................108

APPENDIX B: PUBLICATION 1: ANTIBODY IMMOBILIZATION USING
PNEUMATIC SPRAY: COMPARISON WITH THE AVIDIN-BIOTIN BRIDGE
IMMOBILIZATION METHOD .....................................................................................112

APPENDIX C: PUBLICATION 2: CHARACTERIZATION OF FULLY
FUNCTIONAL SPRAY-ON ANTIBODY THIN FILMS ..............................................122

iii
LIST OF TABLES

Table 1. Assays on sprayed slides to determine the relationship of deposition time
and capture cell counts. Captured E. coli O157:H7 cell counts on glass slides
pneumatically sprayed with goat anti- E.coli O157:H7 IgG at different deposition
times. ..................................................................................................................................86

iv
LIST OF FIGURES

Figure 1. Schematic of attachment through intermediate layer immobilization
method. Goat-anti-E coli O157:H7antibody and donkey anti-goat reporter
antibody were used for the immunoassay. ...........................................................................5

Figure 2. Schematic of pneumatic spray immobilization of antibody on glass
surface. Goat-anti-E coli O157:H7antibody and donkey anti-goat reporter
antibody were used for the immunoassay. ...........................................................................6

Figure 3 Schematic of pneumatic spray process during deposition of antibody
solution on glass surface. A suggested multilayer formation of pneumatic spray
antibody films with randomly oriented antibody. ................................................................8

Figure 4. Low flow concentric pneumatic nebulizer DS5 ...................................................9

Figure 5. Representative diagram. Effects on the sprayed area by changing the
distance between the nebulizer and substrate ....................................................................11

Figure 6. . Antibody basic structure. Each heavy (H) part has a constant (C) and a
variable (V) section (VH, CH). The light chain has also two parts one constant
and one variable (VL, CL). The chains are linked by disulfide bonds (s-s).
Antibody main two regions are fragment antigen binding (Fab) and fragment
crystallizable (Fc)...............................................................................................................15

Figure 7. Diagram of the basic components of a fluorescence microscope. ......................17

Figure 8. Representative sample of fluorescent biomarkers on antibody and
bacteria. Images of E. coli O157: H7. The left image shows GFP- E. coli O157:
H7 (green particles) immobilized on an AF647 conjugated anti-E. coli O157: H7
IgG antibody film. The right image shows E. coli O157: H7 immobilized on goat
anti-E. coli O157: H7 IgG after being incubated with Rhodamine red conjugated
donkey anti goat-E. coli O157:H7 IgG. .............................................................................18
v
Figure 9: Screen shot of DIME 1.31, setting parameters for the images to be
analyzed. ............................................................................................................................21

Figure 10. Representative sample of HLAB 5000 images. Left image, the blue
(background) and yellow (region of interest) rectangles were used to calculate
SNR and measure intensities. Right image, patterns created by pneumatic spray of
unlabeled goat anti- E. coli O157:H7 plus detector antibody AF647 labeled
Donkey Anti-Goat. .............................................................................................................22

Figure 11. Schematic of Rudolph null ellipsometer used for these experiments...............24

Figure 12. UV/vis representative absorption spectrum of goat anti-E.coli O157:H7
IgG in PBS solution. ..........................................................................................................26

Figure 13. Schematic of attenuated total reflection system. The refractive index
(n) of the zinc selenide crystal is higher than the refractive index of the sample. .............28

Figure 14. Two amino acids forming an amide bond (peptide bond). ...............................29

Figure 15. Stretching vibration mode of carboxylic group in the amide bond
(Amide I vibration). ...........................................................................................................30

Figure 16. AFM image of immobilized antibody on glass surface. (A)
Immobilization of anti-E. coli O157:H7 IgG using pneumatic spray method, rings
from droplets formed during the spray can be noticed on the surface after drying.
(B) Immobilization of anti-E. coli O157:H7 IgG using avidin-biotin bridge
method, large particles form due to aggregation of avidin. ...............................................32

Figure 17. Basic schematic for a surface spectroscopy equipment. For the XPS the
primary beam is X-ray photons and the detected secondary beam are electrons. .............34

Figure 18. Representative image of a X-ray photoelectron survey spectrum. Three
different films were deposited by pneumatic spray on silicon wafer: from bottom
to top, silicon surface as reference, a PLA film, a film of anti E. coli antibody
deposited on a PLA film and the top survey is a film of anti E. coli antibody
deposited on silicon wafer. The presence of antibody (protein) is confirmed by the
N1s peak in the top two surveys. .......................................................................................36
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