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Application of J-Aggregate Monolayers in Silica Encapsulated
SERS Nanoprobes for Immunophenotyping of B-cell
Malignancies
by
Byron Song
A thesis submitted in conformity with the requirements
for the degree of Masters of Science
Department of Laboratory Medicine and Pathobiology
University of Toronto
© Copyright by Byron Song 2015
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Application of J-Aggregate Monolayers in Silica Encapsulated SERS
Nanoprobes for Immunophenotyping of B-cell Malignancies
Byron Song
Masters of Science
Department of Laboratory Medicine and Pathobiology
University of Toronto
2015
Abstract
Immunophenotyping is indispensable in studying B-cell malignancies as the cell surface markers
on malignant cells provide critical information for the diagnosis, treatment decisions and
prognosis of the disease. Surface Enhanced Raman Spectroscopy (SERS) nanoprobes as an
alternative optical label have been explored as they may overcome limitations of fluorescent
labels through their naturally sharper spectral bands and resistance to photobleaching. In this
project, we demonstrate the successful labelling of lymphoma cells with rituximab-anti-CD20
functionalized silica-encapsulated J-aggregate SERS gold nanoparticles – these particles
represent the brightest of their kind thus far. Additionally, we demonstrate that the Raman signal
on cells labelled with our particles is not affected by treatment with two hematological stains:
hematoxylin and methylene blue; although two others: Giemsa and eosin mask the Raman
spectra with intense fluorescence. These results support the potential of simultaneously using
hematological stains with SERS nanoprobes for visualizing B-cells.
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Acknowledgments
I would like to express my appreciation and gratitude towards my graduate committee members:
Dr. Sarah Keating, Professor Gilbert Walker, and supervisor Dr. Chen Wang who have helped
me tremendously in my research and obtaining my degree. I would also like to give special
mention to all of my colleagues and lab mates at Walker Labs and important friends and family
that have inspired and supported me along the way.
I am grateful for Dr. Sarah Keating who has continually provided me support at every committee
meeting both with regards to my research as well as my academic and career directions. You’ve
made me excited for what the future holds in my endeavors outside of research.
Professor Gilbert Walker has been an absolutely cornerstone individual in my academic career
over the last six years. Aside from your invaluable role in introducing me to the captivating field
of SERS and as my professor for Physical Chemistry, you have been an amazing mentor and role
model. My tremendous respect for you as a scientist is only exceeded by my regard for your
character and leadership qualities as you’ve made myself and others feel so comfortable and
welcome around you. Despite your extremely busy schedule you seem to manage to understand
and take considerations for all those you work with while also maintaining a wonderful family –
with whom I’m glad to have met.
To my supervisor Dr. Chen Wang, I cannot thank you enough for all that you’ve taught me.
You’ve given me a great sense of independence in my research yet have always been there when
needed. During this project, I’m truly appreciative of the many times you’ve gone above and
beyond in your role as my supervisor and how much valuable insight you’ve imparted onto me
not only with regards to research but also with developing into a successful academic that can
deal with diverse challenges. These are lessons I won’t soon forget and will continually view you
as my mentor for the rest of my academic career. I’ve thoroughly enjoyed working with you and
the many talented individuals you’ve introduced me to.
To all those I’ve worked with in Walker Labs since first year of undergrad, you have all provided
me with wonderful conversation, memories from all the various excursions with the group, and a
great work environment with each passing day. In particular I want to express gratitude to Colin
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Zamecnik and Christina MacLaughin who have both been great role models: Colin for the many
interesting conversations on life, and Christina MacLaughin who’s been a tough but fair teacher.
Special thanks to Christopher Walters and Brandon Gagnon who have been a pleasure to work
and hang out with. You guys have always been really friendly and helpful and I’m glad to have
had you two as colleagues.
Last but not least, I want to thank my mom, dad and grandma. Your support has been consistent
through the good and the bad times. I have always counted on you guys to get me through the
greatest challenges but be there to share my important successes as well. Whatever my future
stores and wherever it may take me, you three will always be my rock.
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Table of Contents
Acknowledgments .......................................................................................................................... iii
List of Abbreviations ..................................................................................................................... ix
List of Figures ................................................................................................................................. x
1 Chapter 1 - Introduction ............................................................................................................. 1
1.1 Thesis Overview ................................................................................................................. 1
1.2 B-Cell Malignancies ........................................................................................................... 2
1.2.1 Lymphoid Neoplasm Classification ........................................................................ 2
1.2.2 Introduction to B-Cell Malignancies ...................................................................... 3
1.2.2.1 Normal B-Cell Development .................................................................... 3
1.2.2.2 B-Cell Malignancies ................................................................................. 4
1.2.2.3 Chronic versus Acute B-cell Malignancies .............................................. 5
1.2.2.4 Mature B-Cell Lymphoma Classification ................................................. 6
1.2.2.5 Hodgkin Lymphoma ............................................................................... 11
1.2.3 Diagnostic and Prognostic Approaches ................................................................ 12
1.2.3.1 Immunophenotyping ............................................................................... 12
1.2.3.2 Morphology ............................................................................................ 15
1.2.3.3 Molecular Genetics ................................................................................. 16
1.3 Use of SERS Nanoprobes for Optical Application ........................................................... 17
1.3.1 Limitations of Immunofluorescence ..................................................................... 17
1.3.2 SERS Alternative to Immunofluorescence ........................................................... 18
1.3.3 SERS Theory ........................................................................................................ 20
1.3.3.1 Surface Plasmons .................................................................................... 20
1.3.3.2 Localized Surface Plasmon Resonance .................................................. 21
1.3.3.3 Raman and Rayleigh Scattering ............................................................. 24
1.3.3.4 Electromagnetic Enhancement of SERS ................................................ 26
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1.3.3.5 Chemical Enhancement of SERS ........................................................... 28
1.3.4 Biological Delivery ............................................................................................... 28
1.3.4.1 Lipid Encapsulation ................................................................................ 28
1.3.4.2 Antibody Conjugation ............................................................................ 29
2 Chapter 2 - Study Design and Methods ................................................................................... 33
2.1 Research Objectives .......................................................................................................... 33
2.1.1 Objective 1 - Demonstrate Successful Application of J-Aggregate Monolayers in the Labelling of Lymphoma Cells using SERS Nanoprobes ............................ 33
2.1.1.1 Hypothesis for Objective 1 ..................................................................... 33
2.1.2 Objective 2 - Demonstrate Successful Application of Silica Encapsulation in the Labelling of B-cell Lymphoma Cells using J-Aggregate Monolayer SERS Nanoprobes ........................................................................................................... 34
2.1.2.1 Hypothesis for Objective 2 ..................................................................... 34
2.1.3 Objective 3 - Demonstrate the Effect of Hematological Staining on the Silica Encapsulated J-Aggregate Monolayer SERS Nanoprobes in Lymphoma Cell Labelling ............................................................................................................... 34
2.1.3.1 Hypothesis for Objective 3 ..................................................................... 34
2.2 General Instrumentation Methods ..................................................................................... 34
2.2.1 Raman Spectroscopy ............................................................................................. 34
2.2.2 Dark Field Microscopy ......................................................................................... 35
2.2.3 UV-Vis Microscopy .............................................................................................. 37
2.2.4 B-cell lymphoma cell line and cell culture ........................................................... 37
2.3 General Sample Preparation Methods .............................................................................. 38
2.3.1 Materials ............................................................................................................... 38
2.3.1.1 Lipids ...................................................................................................... 38
2.3.1.2 SERS Particle Synthesis Reagents ......................................................... 38
2.3.1.3 Conjugation Chemistry Reagents ........................................................... 38
2.3.1.4 Miscellaneous ......................................................................................... 38
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2.3.1.5 Cell Culturing ......................................................................................... 39
2.3.2 Lipid Preparation .................................................................................................. 39
2.3.3 Lipid Encapsulation .............................................................................................. 40
2.3.4 Raman Functionalization of Particles ................................................................... 40
2.3.5 Antibody Conjugation ........................................................................................... 41
2.3.5.1 EDC-NHS ............................................................................................... 41
2.3.5.2 Maleimide ............................................................................................... 41
2.3.5.3 Post-Functionalization ............................................................................ 41
2.3.6 Cell Labelling ........................................................................................................ 41
2.3.7 Dark Field Imaging ............................................................................................... 42
2.3.8 Raman Microscopy and Raman Mapping ............................................................. 43
3 Chapter 3 - Results ................................................................................................................... 44
3.1 Objective 1 - Application of J-Aggregates for Cell Labelling .......................................... 44
3.1.1 Introduction to J-Aggregate Monolayers .............................................................. 44
3.1.2 S0271 .................................................................................................................... 45
3.1.3 Particle Preparation ............................................................................................... 48
3.1.3.1 TMAT Synthesis ..................................................................................... 50
3.1.4 Cell Labelling ........................................................................................................ 50
3.2 Objective 2 - Application of Silica-Encapsulated Nanoparticles for Cell Labelling ........ 52
3.2.1 Silica Encapsulation .............................................................................................. 52
3.2.2 Silica Encapsulation Procedure ............................................................................. 55
3.2.3 Comparison of EDC-NHS and Maleimide Conjugation ...................................... 56
3.3 Objective 3 - Application of Hematological Stains .......................................................... 58
3.3.1 Hematological Stains ............................................................................................ 58
3.3.2 Effect of Hematological Stains on Raman Spectra ............................................... 58
3.3.3 Cell Labelling Experiments .................................................................................. 62
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4 Chapter 4 - Discussion and Future Work ................................................................................. 67
4.1 Discussion of Objective 1 Results .................................................................................... 67
4.2 Discussion of Objective 2 Results .................................................................................... 67
4.3 Discussion of Objective 3 Results .................................................................................... 68
4.4 Summary of Thesis and Future Directions ....................................................................... 69
5 Chapter 5 – References ............................................................................................................ 71
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List of Abbreviations
CLL – Chronic Lymphocytic Leukemia
ALL – Acute Lymphoblastic Leukemia
DLBCL – Diffuse Large B-Cell Lymphoma
NHL – Non-Hodgkin Lymphoma
SERS – Surface Enhanced Raman Spectroscopy
SPR – Surface Plasmon Resonance
SPP – Surface Plasmon Polariton
LSPR – Localized Surface Plasmon Resonance
AuNP – Gold Nanoparticle
CD – Cluster of Differentiation
UV-Vis – Ultraviolet-Visible
SUV – Small Unilamellar Vesicle
EDC – 1-ethyl-3-(-3-dimethylaminopropyl) carbodiimide
NHS – N-hydroxylsuccinimide
TMAT - thiocholine
FITC – fluorescein isothiocyanate
PE – phycoerythrin
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List of Figures
Figure 1 Visual representation of Localized Surface Plasmon Resonance (LSPR). Retrieved from
(Stiles, Dieringer, Shah, & Van Duyne, 2008) ............................................................................. 22
Figure 2 Visualization of different types of light scattering. A) Rayleigh; B) Stokes; C) Anti-
Stokes. ........................................................................................................................................... 24
Figure 3 Chemical Reaction Mechanism for EDC-NHS to produce an amide bond between
carboxylic acid and primary amine. .............................................................................................. 32
Figure 4 Visual representation of dark field microscopy set-up using a dark field patch stop to
block transmitted light. Image retrieved from:
http://en.academic.ru/pictures/enwiki/68/Dark_Field_Microscope.png; accessed on July 2014. 36
Figure 5 Graph depicting absorption spectra of S0271 dye at differing concentration in H2O
solution. Chemical structure presented in top-right corner. .......................................................... 46
Figure 6 UV-Vis spectra of S0271 in H2O solution. Dye in monolayer form. Peaks are labelled.
....................................................................................................................................................... 47
Figure 7 UV-Vis spectra of S0271 in sodium chloride solution. Dye in J-aggregate form with J-
aggregate peak labelled. ................................................................................................................ 47
Figure 8 Schematic depicting the main steps in synthesizing the SERS nanoprobes. First the J-
aggregate monolayer is allowed to form (yellow – S0271 dye molecules; black line – TMAT
linker molecule). Then a silica layer is produced if the particle is to be silica encapsulated.
Finally, the resultant nanoparticle is given a lipid layer. .............................................................. 49
Figure 9 Thiocholine (TMAT) chemical structure ....................................................................... 50
Figure 10 Dark field image depicting LY8 lymphoma cells labelled with Rituximab conjugated
(via EDC-NHS chemistry) lipid-encapsulated SERS nanoprobes with S0271 J-aggregate dye.
Magnification is 100x. .................................................................................................................. 51
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Figure 11 Raman spectra obtained with 532 nm laser from LY8 lymphoma cells labelled
Rituximab conjugated (via EDC-NHS chemistry) lipid-encapsulated SERS nanoprobes with
S0271 J-aggregate dye. Laser was used at 5% power for 5 accumulations of 5 seconds each. ... 51
Figure 12 Raman spectra obtained of lipid encapsulated SERS nanoprobe depicting intense
fluorescence spectra masking Raman peaks. ................................................................................ 53
Figure 13 Raman spectra obtained of silica encapsulated SERS nanoprobe depicting fluorescence
quenching. ..................................................................................................................................... 53
Figure 14 UV-Vis spectrum of lipid-encapsulated SERS nanoprobe. Surface plasmon peak at
533nm and J-aggregate peak at 561nm are labelled. .................................................................... 54
Figure 15 UV-Vis spectrum of silica encapsulated SERS nanoprobe. Surface plasmon peak at
537nm and J-aggregate peak at 569nm are labelled. .................................................................... 55
Figure 16 Dark-field image of LY8 cells labelled with antiCD20+ EDC-NHS conjugated SERS
nanoprobes at 100X magnification. .............................................................................................. 57
Figure 17 Dark-field image of LY8 cells labelled with antiCD20+ Maleimide conjugated SERS
nanoprobes at 100X magnification. .............................................................................................. 57
Figure 18 Dark-field image of LY8 cells incubated with control antiCD20- SERS nanoprobes. 58
Figure 19 In-depth analysis of SERS nanoparticles incubated in solution with hematoxylin
followed by removal of hematoxylin by centrifugation. Top: no encapsulation of SERS
nanoprobes; Middle: lipid encapsulated SERS nanoprobes; Bottom: dual-encapsulated (silica and
lipid) nanoprobes; Left: control condition (no hematoxylin); Center: low concentration of
hematoxylin (50µL stain per 1mL SERS probes); Right: high concentration of hematoxylin (1mL
stain per 1mL SERS particles). 532nm laser at 5% power for 1 second exposure with 1
accumulation. ................................................................................................................................ 59
Figure 20 Raman spectra of dual encapsulated (lipid and silica) SERS nanoprobes incubated at
ratio of 1mL to 50µL of hematoxylin. 532nm laser at 5% power for 1 second exposure with 1
accumulation. ................................................................................................................................ 60
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Figure 21 Raman spectra of dual encapsulated (lipid and silica) SERS nanoprobes incubated at
ratio of 1mL to 50µL of methylene blue. 532nm laser at 5% power for 1 second exposure with 1
accumulation. ................................................................................................................................ 61
Figure 22 Raman spectra of dual encapsulated (lipid and silica) SERS nanoprobes incubated at
ratio of 1mL to 50µL of Giemsa. 532nm laser at 5% power for 1 second exposure with 1
accumulation. ................................................................................................................................ 61
Figure 23 Top: Raman mapping of signal-to-base line intensity of dual peaks bounded by the two
vertical lines in the Raman spectra (Bottom) of the brightest spot on the map. Bottom: Raman
spectra of the brightest spot on the Raman map (top) taken with 532 nm laser at 5% laser
intensity for 1 second with 1 accumulation. Experimental sample is LY8 cells labelled with
antiCD20+ silica-encapsulated SERS nanoprobes. ...................................................................... 62
Figure 24 Top: Raman mapping of signal-to-base line intensity of dual peaks bounded by the two
vertical lines in the Raman spectra (Bottom) of the brightest spot on the map. Bottom: Raman
spectra of the brightest spot on the Raman map (top) taken with 532 nm laser at 5% laser
intensity for 1 second with 1 accumulation. Experimental sample is LY8 cells labelled with
control antiCD20- silica-encapsulated SERS nanoprobes. ........................................................... 63
Figure 25 Top: Raman mapping of signal-to-base line intensity of dual peaks bounded by the two
vertical lines in the Raman spectra (Bottom) of the brightest spot on the map. Bottom: Raman
spectra of the brightest spot on the Raman map (top) taken with 532 nm laser at 5% laser
intensity for 1 second with 1 accumulation. Experimental sample is LY8 cells labelled with
antiCD20+ silica-encapsulated SERS nanoprobes stained with hematoxylin. ............................. 64
Figure 26 Top: Raman mapping of signal-to-base line intensity of dual peaks bounded by the two
vertical lines in the Raman spectra (Bottom) of the brightest spot on the map. Bottom: Raman
spectra of the brightest spot on the Raman map (top) taken with 532 nm laser at 5% laser
intensity for 1 second with 1 accumulation. Experimental sample is LY8 cells labelled with
antiCD20+ silica-encapsulated SERS nanoprobes stained with methylene blue. ........................ 65
Figure 27 Microscopy images of LY8 cells incubated with silica-encapsulated SERS nanoprobes
with different hematological stains applied; Top: No stain; Middle: Hematoxylin stain; Bottom:
Methylene Blue stain. Left: Darkfield image of labelling with antiCD20+ SERS nanoprobes;
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Middle: Brightfield image of labelling with antiCD20+ SERS nanoprobes; Right: Darkfield
image of labelling with control antiCD20- SERS nanoprobes. .................................................... 66
1
Chapter 1 - Introduction 1
1.1 Thesis Overview
Chapter 1 - Introduction: This chapter is separated into three distinct sections with the first being
the thesis overview. Subsequently, the background knowledge pertaining to this project is
divided into two main sections.
The first section will discuss B-cell malignancies, the class of diseases that are the subject and
inspiration for this project. This sub-chapter will cover a summary of the lymphoid neoplasm
classification system that has changed over time as well as briefly describe several of the most
important subtypes of B-cell malignancies in terms of their distinctive features, disease
progression, as well as diagnostic and prognostic factors. The latter part of this sub-chapter will
go over the three most important techniques used to describe these diseases clinically:
immunophenotyping, morphology studies, and molecular genetics.
The second section will explain the fundamental physical concepts behind Surface Enhanced
Raman Spectroscopy (SERS) as a well-studied optical phenomenon. The potential use of SERS
for diagnostic application will be supported with discussion regarding limitations of
immunofluorescence. Additional background will be provided on lipid encapsulation and
antibody conjugation techniques that functionalize SERS nanoparticles for immunophenotyping.
Chapter 2 – Study Design and Methods: This chapter will describe how the background
information in the introductory chapter ties in to the direction of research that led to this project.
The project is described in terms of three objectives and this structure is used to format the
results and discussion section of this thesis.
Also next section of the chapter describes the four pre-dominant instrumentation or techniques
used in this thesis are Raman spectroscopy, dark field spectroscopy, UV-Vis spectroscopy and
cell cultures. Background information and specifics regarding the devices used will be provided
in this chapter.
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The last section of this chapter will go over all the reagents used to perform the experiments in
this thesis. This chapter also describes the general procedures that were used to synthesize and
functionalize the SERS nanoprobes, to label the lymphoma cells with the SERS nanoprobes, and
to obtain data: dark field images, Raman spectra, Raman mapping images.
Chapter 3 - Results: This chapter describes the experiments and results organized into the three
subsections for the three objectives of this project. Additional background information and
methodology pertaining specifically to an objective is also provided in the relevant subsection.
Chapter 4 – Discussion and Future Work: The first part of this chapter describes the conclusions
derived from the experimental results organized into the three objectives of this project. The
latter part provides a general summary of this project and possible directions of further
experimentation both in the short and long term.
1.2 B-Cell Malignancies
1.2.1 Lymphoid Neoplasm Classification
Lymphoid neoplasms cover a wide range of diseases that are classified under Hodgkin
lymphomas, non-Hodgkin lymphomas (NHLs), lymphocytic leukemias and plasma cell
neoplasms (Kumar, Abbas, & Aster, 2013). Traditionally, the terms lymphoma and leukemia
were used to respectively describe neoplasms originating in lymph nodes in contrast to those
originating in bone marrow and peripheral blood. While the site in which the tumor resides is
important for classification, the type of cell involved in the neoplasm is more clinically
significant. Furthermore, all lymphoid neoplasms can eventually spread to lymph nodes and
other tissues and have overlapping clinical behaviour, so distinguishing between them is done
with greater certainty using the morphologic and molecular characteristics of the tumor cells.
Molecular diagnostic techniques emphasize detecting lineage-specific antigens and markers of
cell differentiation.
While there are many subtypes of lymphoid neoplasms, acute lymphoblastic leukemia, chronic
lymphocytic leukemia, follicular lymphoma, mantle cell lymphoma, diffuse large B cell
lymphomas, Burkitt lymphoma, multiple myelomas, and Hodgkin lymphoma make up about
90% of the diagnoses (Kumar, Abbas, & Aster, 2013).
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Lymphoid neoplasms cause problems in patients by disrupting normal immune function. This
can present itself either as deficiency of immune responses as well as autoimmunity (Kumar,
Abbas, & Aster, 2013). Depending on the type of lymphoid neoplasm, the disease can have a
significantly different clinical presentation and behaviour. Making precise diagnoses of
lymphoid malignancies however, has always been a major challenge both clinically and in their
study (Shaffer, Rosenwald, & Staudt, 2002).
As knowledge and understanding of lymphoid neoplasms increased, new divisions had to be
made while older distinguishing factors became irrelevant. For example, the previous model
placed little emphasis on the distinction between T cell and B cell lymphomas; the B-cell
description of marginal zone lymphoma became redundant as there are no T-cell marginal zone
neoplasms. Classical Hodgkin lymphoma and diffuse large B-cell lymphoma, a subtype of non-
Hodgkin lymphoma was seen to have significant biological overlap and the overall designation
of non-Hodgkin lymphoma was abandoned (Jaffe, 2009). These changes and others were part of
a significant re-haul of the lymphoma classification system reflected in the 4th edition of the
WHO Classification of Tumors of Hematopoietic and Lymphoid Tissues (Swerdlow, et al., 2008).
Since then, lymphoid neoplasms has been classified into mature B cell neoplasms, mature T- and
NK-cell neoplasms, Hodgkin lymphoma, histiocytic and dendritic cell neoplasms, and
posttransplantation lymphoproliferative disorders (PTLDs). The focus of this thesis will be on B-
cell neoplasms (Jaffe, 2009).
1.2.2 Introduction to B-Cell Malignancies
1.2.2.1 Normal B-Cell Development
B cells are lymphocytes that are important for the humoral component of the adaptive immune
system. Like all blood cells, B cells originate from the hematopoietic stem cell within bone
marrow. Along with T lymphocytes and NK cells, B lymphocytes derive from the multipotent
common lymphoid progenitor cell. B lymphocytes express several lineage-specific receptors
such as CD20 and CD19 and, most importantly, a B cell receptor for detecting and binding
foreign antigens (Kumar, Abbas, & Aster, 2013).
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B cells play a key role in the humoral immunity of the adaptive immune system. Their primary
functions are to produce and secrete antibodies, act as antigen-presenting cells (APCs) and
release cytokines. During the development of naive B cells, their B-cell receptor gene undergoes
V(D)J or somatic recombination to produce a wide variety of antigen-specific receptors and
various maturation processes select for the most appropriate variations. This random
reassortment of immunoglobulin loci produces a large pool of antigen-specific receptors while
the maturation processes eliminate the receptors that have a high propensity for self-recognition.
These processes typically occur during development in the bone marrow before the B cells are
released into circulation. Terminal differentiation as well as amplification of a B cell clone
occurs when its B cell receptor finds its target antigen and receives further signals from T helper
cells; this usually happens in secondary lymph organs such as lymph nodes. This transforms the
naive B cell into either a plasma cell or memory cell where the former produces large amounts of
soluble antibodies and the latter live in dormancy for an extended period of time until it re-
encounters its target antigen and activates a secondary immune response (Kumar, Abbas, &
Aster, 2013).
The antibodies produced by B cells develop greater affinity to their targeted antigen through a
process called affinity. This is facilitated through a programmed process called somatic
hypermutation where the immunoglobulin loci coding for the variable region of the antibody
responsible for antigen specificity undergoes a high degree of mutation.
1.2.2.2 B-Cell Malignancies
B cell maturation involves two periods of regulated genomic instability: somatic hypermutation
of the B-cell receptor as well as immunoglobulin class-switching. While these are natural
processes of humoral immunity aimed at improving antibody diversity, they are mistake-prone
which make them likely attributable for the mutations and chromosomal translocations involved
in lymphoid neoplasms (Shaffer, Rosenwald, & Staudt, 2002). It has been demonstrated that
most B cell lymphomas have undergone these events. In nearly all cases, the B-cell receptor gene
arrangement precedes the malignant transformation leading to a clonal marker of lymphoid
neoplasms traceable by its antigen receptor (Kumar, Abbas, & Aster, 2013).
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The clonality of B-cell neoplasms is a means of distinguishing them from polyclonal, reactive
processes. Since B-cells rearrange receptor genes randomly and assemble immunoglobulins with
either a kappa or lambda light chain in a single cell, an abnormally skewed ratio of kappa to
lambda light chain antibodies would be indicative of the presence of a B cell clone (Kumar,
Abbas, & Aster, 2013).
1.2.2.3 Chronic versus Acute B-cell Malignancies
Acute lymphoblastic leukemia (ALL) or lymphoblastic lymphoma are tumours composed of
lymphoblasts which appear early on in the cell differentiation and are consequently highly
aggressive tumours. They comprise 80% of childhood leukemias with most cases being B-cell in
origin (Kumar, Abbas, & Aster, 2013).
Diagnosis usually begins with a complete blood count which reveals an elevated white blood cell
count. A higher white blood cell count would be associated with a worse prognosis (Collier,
1991). Blast cells are also observed on blood smear in majority of the cases. A bone marrow
biopsy can be used to confirm ALL (Longo, Fauci, Kasper, Hauser, Jameson, & Loscalzo, 2011).
Since it is sometimes difficult to find blasts in peripheral blood, marrow examination is more
effective in revealing the malignancy through having blasts comprise more than 25% of the total
cell population. This high degree of proliferation also suppresses normal marrow function and
the development of normal hematopoetic cells. Consequently, this leads to symptoms such as
anemia-related fatigue, suppressed immune function, and bleeding secondary to
thrombocytopenia as erythrocytes, leukocytes, and platelets are all affected (Kumar, Abbas, &
Aster, 2013).
In contrast, most chronic B-cell malignancies are considered disease of more mature B-cell
neoplasms as they involve cells that are past the precursor B-cell stage. Many of these are
chronic and indolent in that the disease course is longer and the symptoms may not be as severe.
This is not always the case as some mature B-cell malignancies are aggressive, such as Burkitt
lymphoma/leukemia (Kumar, Abbas, & Aster, 2013).
The general trend is that indolent B cell malignancies like chronic lymphocytic leukemia (CLL)
and follicular lymphoma allow for long term survive but are unlikely to be cured. Aggressive B
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cell malignancies like the aforementioned ALL, and diffuse large B cell lymphoma (DLBCL)
require intensive treatment and are highly symptomatic but have the potential to be cured
completely.
1.2.2.4 Mature B-Cell Lymphoma Classification
1.2.2.4.1 Diffuse Large B-Cell Lymphoma
Diffuse large B cell lymphoma (DLBCL) is the most common type of NHL in adults (30-40%
cases (Shaffer, Rosenwald, & Staudt, 2002). The median age of presentation is 60 (Kumar,
Abbas, & Aster, 2013), although patients of any age can be diagnosed with this disease. DLBCL
also constitutes 15% of childhood lymphomas.
Like other mature B cell tumors, these malignant cells express markers such as CD19, CD20,
and usually surface IgG. Distinguishing cell surface markers include CD10 which is variably
expressed. DLBCL is generally a very heterogeneous disease both morphologically and
molecularly: 1/3 of cases have a BCL6 abnormality and 1/5 of cases have a BCL2 chromosomal
translocation (Shaffer, Rosenwald, & Staudt, 2002).
DLBCL is an aggressive B cell lymphoma that can be rapidly fatal if left untreated. Intensive
chemotherapy as well as anti-CD20 immunotherapy can lead to complete remission in 60-80%
of patients where about half of these will appear cured. Otherwise, high-dose chemotherapy and
stem cell transplantation are treatment options. Overall 5-year survival for adults is 58%
(Akyurek, Uner, Benekli, & Barista, 2011).
Patients typically present with a symptomatic, rapidly enlarging mass at one of several sites. The
site is often extra-nodal and the tumor can actually appear in virtually any organ or tissue
although the brain and GI tract are most frequently seen. Unlike the more indolent lymphomas
such as follicular lymphoma, there is rarely liver, spleen, or bone marrow involvement at
diagnosis.
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1.2.2.4.2 Follicular Lymphoma
The second most common NHL is follicular lymphoma (Shaffer, Rosenwald, & Staudt,
2002)(Kumar, Abbas, & Aster, 2013). The neoplastic cells are always B cell in origin and
express CD19, CD20, and CD10 but usually do not express CD5.
More than 85% of these tumors have a characteristic (14;18) chromosomal translocation fusing
the BCL2 gene on chromosome 18 to the IgH locus on chromosome 14. This leads to BCL2 over
expression that promotes tumor survival. In about 1/3 of the cases there are also loss-of-function
mutations in genes coding for histone acetyl transferases suggesting that epigenetic changes may
also be involved in the tumorigenesis (Kumar, Abbas, & Aster, 2013).
Unlike DLBCL, follicular lymphoma has an indolent disease course and treatment generally isn’t
performed until significant symptoms arise. It is generally incurable except in the rare cases of
early stage follicular lymphoma since the majority of follicular lymphomas are only detected at
late-stage (Shaffer, Rosenwald, & Staudt, 2002).
The 10-year survival rate for follicular lymphoma is 73% (Shaffer, Rosenwald, & Staudt, 2002).
1.2.2.4.3 Mantle Cell Lymphoma
Mantle cell lymphoma (MCL) one of the rare NHLs and is composed of cells resembling the B
cells found in the mantle zones that surround normal germinal follicles (Shaffer, Rosenwald, &
Staudt, 2002). It constitutes approximately 4% of all NHLs and occurs mainly in men older than
50 years of age.
Tissue biopsies show lymph nodes affected in a diffuse or vaguely nodal pattern. Tumor cells are
usually slightly larger than normal lymphocytes and have an irregular nucleus, inconspicuous
nucleoli and scant cytoplasm. Less commonly, the cells are larger and morphologically resemble
lymphoblasts. The malignancy is usually first found in the bone marrow and in peripheral blood
in 20% of cases.
This disease usually has bone marrow and GI tract involvement with the latter manifesting as
polyposis in the intestinal lining. Colonoscopies have consequently been a routine part of
evaluation of MCL.
8
Cytogenetics show an association with a t(11;14) translocation that results in overexpression of
cyclin D1, a regulator of cell cycle progression (Kumar, Abbas, & Aster, 2013) (Shaffer,
Rosenwald, & Staudt, 2002).
Diagnosis generally requires stained slides of a surgically removed part of a lymph node.
Immunophenotype is CD5+ in 80% of cases with CD5+ and CD10- being the most common case
(Barekman, Aguilera, & Abbondanzo, 2001).
MCL has a 5 year survival rate of 70% except in the case of advanced stage disease where it
drops to 50% (Herrmann, et al., 2009). It is generally treated with normal chemotherapy
(Shaffer, Rosenwald, & Staudt, 2002).
1.2.2.4.4 Marginal-Zone Lymphoma
Marginal-zone lymphoma (MZL) is an indolent malignancy that occurs in organs that lack
organized lymphoid tissue such as the stomach, salivary glands, lungs and thyroid glands
(Shaffer, Rosenwald, & Staudt, 2002).
The most common subtype, gastric mucosal-associated lymphoid tissue (MALT) lymphoma is
associated with H. pylori infection and complete remission can be achieved in 70% of early-
stage patients through removal of the infection (Shaffer, Rosenwald, & Staudt, 2002).
1.2.2.4.5 Burkitt’s Lymphoma
Burkitt’s lymphoma is predominantly a B cell malignancy with tumor cells that express surface
IgM, B cell markers CD19 and CD20, and the germinal center B cell markers CD10 and BCL6
(Kumar, Abbas, & Aster, 2013). This disease is relatively rare and accounts for 2.3% of all B-
cell malignancies (Turgeon, 2012).
There is an endemic form associated with Epstein-Barr virus (EBV) infection in regions with
chronic malaria that is believed to reduce resistance to EBV (Kelly, et al., 2013).
Burkitt’s lymphoma is well characterized by a c-MYC translocation predominantly to
immunoglobulin heavy chain (IgH) locus described as t(8;14)(q24;32), although other variants
with involving c-MYC exist (Shaffer, Rosenwald, & Staudt, 2002).
9
1.2.2.4.6 Chronic Lymphocytic Leukemia
Chronic lymphocytic leukemia is the most prevalent form of leukemia in North America
(Shaffer, Rosenwald, & Staudt, 2002). More than 2000 Canadians are newly diagnosed each year
and around 600 Canadians died from the disease in 2011 (Society, 2011). It is an incurable
disease with most treatment regimens aimed at prolonging survival (Chiorazzi & Rai, 2005). At
the same time, chronic lymphocytic leukemia is often an indolent disease with patients living
relatively normal life spans (Chiorazzi & Rai, 2005).
Diagnostic criteria for CLL critically depends on blood B-cell count (> 5000/uL), morphology
small lymphocytes, and flow cytometric immunophenotyping (Hallek, et al., 2008).
Chronic lymphocytic leukemia is a cancer of B lymphocytes wherein a malignant clone
proliferates and expands (Welinder, Ahsberg, & Sigvardsson, 2011). Diagnosis of the disease
happens through regular blood tests wherein the presence of lymphocytes in peripheral blood
exceeds 5x109 cells/L (Dillman, 2008) (Nguyen, Nguyen, Rutledge, Zhang, Wang, & Walker,
2010). Subsequently, the B lymphocytes are tested through flow cytometry to determine if they
are clonal (Nguyen, Nguyen, Rutledge, Zhang, Wang, & Walker, 2010).
Small lymphocytic lymphoma (SLL) is generally understood to be identical to CLL aside from
the latter clinically having a peripheral blood lymphocyte count exceeding 5000 cells/uL
granting it a more leukemic appearance (Hallek, et al., 2008).
Blood smears reveal small, mature lymphocytes with a narrow border of cytoplasm and a dense
nucleus lacking discernible nucleoli and having partially aggregated chromatin (Hallek, et al.,
2008).
Expectedly, these cells express the B cell markers CD19, CD20, and CD23 along with surface
immunoglobulin heavy and light chains. CD5 is an important marker associated with CLL along
with another B cell neoplasm, mantle cell lymphoma. CLL has characteristically reduced CD20
and CD79b expression (Kumar, Abbas, & Aster, 2013) (Hallek, et al., 2008).
Half of reported CLL cases have been shown to have karyotypic abnormalities including trisomy
12 and deletions on chromosomes 11, 13, and 17 (Kumar, Abbas, & Aster, 2013). Activating
10
mutations in the Notch1 receptor usually predicts a worse outcome. Unlike other B cell
neoplasms, chromosomal translocations are infrequent.
1.2.2.4.7 Multiple Myeloma
Multiple myeloma is a malignancy of plasma cells which are B-cells that have been activated,
terminally differentiated, and secrete antibodies. The disease predominantly involves bone
marrows and oftentimes presents with lytic lesions throughout the skeletal system.
It is one of the most common hematologic tumors with an incidence rate of 20, 000 new cases
diagnosed each year in the United States. The median age at diagnosis is 70 (Kumar, Abbas, &
Aster, 2013).
Multiple myeloma is clinically distinct from pre-myeloma states such as monoclonal
gammopathy of undetermined significance (MGUS) by presence of end-organ damage such as
hypercalcemia, renal failure, anemia, and bone marrow lesions in conjunction with the clonal
plasma cell making up >10% of the cells in a bone marrow biopsy (Kumar, Abbas, & Aster,
2013).
Since these are clonal antibody secreting cells, they produce identical antibodies that share the
same constant region and consequently, immunoglobulin class. The most frequently produced
myeloma immunoglobulin class is IgG which occurs in 60% of the cases; IgA occurs at around
20-25% of the cases. In another 15-20% of cases, the plasma cells produce only free light chains
which are described as Bence Jones proteins that get excreted through urine due to their low
molecular weight. More often though, both complete immunoglobulins as well as Bence Jones
proteins are produced by the malignant cells (Alexanian, Barlogie, & Dixon, 1990).
Various translocations of IgH and cyclin D1, cyclin D3, c-MAF, MMSET (multiple myeloma
SET-domain protein) or fibroblast growth factor receptor 3 (FGFR3) are associated with multiple
myeloma (Shaffer, Rosenwald, & Staudt, 2002).
A peripheral blood smear from a myeloma patient can reveal red cell aggregates termed
Rouleaux due to the presence of monoclonal immunoglobulin in the serum.
11
With treatment, the median survival time for patients with multiple myeloma is around 4 to 6
years. Stem cell transplantation is an option for patients under the age of 65; otherwise standard
chemotherapy is given as well as supportive therapy for end-organ symptoms (Kumar, Abbas, &
Aster, 2013).
1.2.2.5 Hodgkin Lymphoma
Hodgkin lymphomas and non-Hodgkin lymphomas both typically originate in lymphoid tissues.
Unlike most NHLs, Hodgkin lymphomas start off in a single lymph node or cluster of lymph
nodes and spread in a contiguous fashion (Kumar, Abbas, & Aster, 2013). Hodgkin lymphoma
is distinguished by the presence of distinctive Reed-Sternberg giant cells that are thought to be of
B-cell origin. Its behavior and clinical treatment are considerably different than those of Non-
Hodgkin lymphomas. Hodgkin lymphoma accounts for 10% of lymphoid malignancies (Shaffer,
Rosenwald, & Staudt, 2002).
Hodgkin’s lymphoma is diagnosed by the finding of characteristic tumor cells. . The Reed-
Sternberg cells in most subtypes of Hodgkin’s lymphoma express CD15 and CD30 and test
negatively for CD45 and CD20.
The disease occurrence shows two peaks: the first in young childhood and the second in those
over 55 years old (Kumar, Abbas, & Aster, 2013). Hodgkin’s lymphoma has a cure rate of 80%
(Shaffer, Rosenwald, & Staudt, 2002) with most common subtype, nodular sclerosis Hodgkin
lymphoma having the greatest prognosis and the lymphocyte depleted Hodgkin lymphoma
having the worst prognosis.
The most common symptom of Hodgkin lymphoma is enlargement of one or more lymph nodes
or lymphadenopathy. Lymph node excision biopsy is generally used for a definitive diagnosis.
This test helps reveal complete or partial effacement of the lymph node architecture by large
often bi-nucleated Reed-Sternberg cells mixed in with variable proportions of lymphocytes,
histiocytes, eosinophils, and plasma cells.
Staging of the disease is also based on how many lymph node groups are affected. Most
frequently this involves nodes of neck and shoulders (80-90% of the time) and chest lymph
12
nodes or mediastinal masses are often detected through a chest radiogram (Kumar, Abbas, &
Aster, 2013).
The general treatment for Hodgkin lymphoma is a combination chemotherapy regiment with a
greater number of cycles for later-stage neoplasms.
1.2.3 Diagnostic and Prognostic Approaches
1.2.3.1 Immunophenotyping
Immunophenotyping is a technique used to analyze cell surface protein expression. The
technique involves the use of antibodies targeting surface proteins of a population of cells
commonly in the form of a tumor biopsy or cell suspension. Differentiation of cells can be made
through appropriate selection of antibodies and the process can analyze thousands of cells per
second when performed with a flow cytometer.
Immunophenotyping is an important tool for the specific diagnosis of leukemias. Analysis of cell
surface protein expression allows for identification of the cell lineage and determination of
malignancy which both help in the specific diagnosis of disease.
Clonality is an important concept in lymphoproliferative disorders. If the neoplasm is clonal,
essentially all the malignant cells carry the same genetic abnormality. In application, this is
reflected by the expression of the same surface antigens including a specific re-arrangement of
the immunoglobulin gene or T cell receptor gene in B-cells and T-cells respectively. For
example, clonality most easily established through clonal pattern of immunoglobulin gene
rearrangement restricted by either kappa or lambda immunoglobulin light chains (Hallek, et al.,
2008).
The stage of B-cell development in which the tumor cells presents as is important for its clinical
behavior and responsiveness to therapy. An important caveat to consider is that we have to
consider the stage in which the oncogenic mutations and chromosomal translocations occurred
may not coincide with the stage in which the malignant cells arrested in development (Shaffer,
Rosenwald, & Staudt, 2002).
13
Immunophenotyping cell surface markers also have prognostic value. For example, CLL with
ZAP-70 or CD38 expression has worse prognosis (Hallek, et al., 2008).
1.2.3.1.1 Cell Surface Markers
Cell surface markers are proteins that are expressed on the surface of cells and the important
targets of immunphenotyping. Here we use CLL as a primary example to describe how cell
surface marker expression can be used to analyze the disease.
Differentiation and maturation of all blood cells originate from the hematopoetic stem cell in a
process that marks stages of different lineages with specific cell surface markers. Markers such
as CD19, CD20, CD21 and CD 23 are generally expressed in CLL (Hulkkonen, Vilpo, Hurme, &
Vilpo, 2002) (Belov, Vega, Remedios, Mulligan, & Christopherson, 2001).
Terminal deoxynucleotidyl transferase (TdT) is an enzyme present in pre-B and pre-T cells in
over 95% of cases. Presence of CD19 over CD3 allows the distinction of the malignancy as pre-
B cell type as opposed to pre-T cell type. TdT+ immature B cells are characteristic of ALL but
unlikely to be found in mature B cell malignancies like CLL.
The presence of aberrant surface proteins on a B lymphocyte clone that is not appropriate for its
lineage or maturation state is often indicative of a disease state. The same is true for the absence
of a surface receptor that should be expressed in reference to the B lymphocytes lineage or
maturation stage.
In CLL, malignant B-cells are also differentiated from normal B-cells by having comparatively
lower expression levels of CD20 and CD79b (Dillman, 2008). Malignant lymphocytes are also
known to have low expression of the B-cell receptor (Dillman, 2008) as well as CD22 molecule
(1-28). CLL cells share a phenotype closer to mature antigen-experienced B lymphocytes
(Damie, et al., 2002).
Cell surface marker expression can help differentiate CLL from a similar but far more rare B-cell
malignancy prolymphocytic leukemia (PLL) because PPL doesn’t express CD5 50% of the time
and have high CD20 and CD79b which CLL has characteristically low levels of. Another similar
14
disease, mantle cell lymphoma expresses CD5 but not CD23 which CLL does (Hallek, et al.,
2008).
In CLL, CD5+ expressing B cell is of diagnostic significance. In follicular lymphoma we find
BCL2+ mature B cells that express surface Ig as well as CD10+, which suggests a strong
association with the germinal center B cell stage (Shaffer, Rosenwald, & Staudt, 2002). Marginal
zone lymphoma presents with CD5-, CD10-, mature B cells with surface Ig (Kumar, Abbas, &
Aster, 2013).
1.2.3.1.2 Immunofluorescence
Currently, the predominant clinical strategy for immunophenotyping of leukemia and
lymphomas involve the use of fluorescent dyes conjugated to antibodies for evaluating cell
surface expression of the targeted antigen (Naeim, Rao, & Grody, 2008).
These molecules possess fluorochromes that are essentially chemicals that absorb
electromagnetic energy from an excitation source and subsequently emit longer wavelength
photons through a phenomenon called fluorescence (Naeim, Rao, & Grody, 2008). The released
photons can be detected and produce a characteristic spectra specific to the fluorochrome. Two
widely used fluorochromes are fluorescein isothiocyanide (FITC) and phycoerythrin (PE) that
produce fluorescence bands with emission peaks at 520 and 576 nm respectively (Naeim, Rao, &
Grody, 2008).
1.2.3.1.3 Flow Cytometry
Flow cytometry is a laser-based biotechnology used for high throughput multiparametric analysis
of particles, predominantly cells in fluid. Essentially, the cells of interest pass as a single file
stream through flow cytometer that subjects the sample to multiple lasers. Any elastically
scattered light or fluorescence can be detected and resolved to generate detailed analysis of the
cell population.
Light scattering provide it with general capabilities of analyzing cell size and internal
complexity. When applied with immunofluorescence, it can be used to analyze a wide array of
cellular components both on the cell surface as well as within the cell (Brown & Wittwer, 2000).
The principles of high through-put immunophenotyping using flow cytometric analysis currently
15
uses high quality and diverse fluorochromes conjugated to antibodies against cluster of
differentiation (CD) molecules.
Flow cytometry is currently the predominant clinical method for immunophenotyping patient
lymphocytes in blood (Welinder, Ahsberg, & Sigvardsson, 2011) (Craig & Foon, 2008)(Jennings
& Foon, 1997). International consensus is that use of flow cytometric immunophenotyping is
indicated for all cytopenias, elevated leukocyte counts, presence of atypical or blast cells in
peripheral blood, marrow, or bodily fluids, plasmacytosis, as well as presence of tissue masses
(Jennings & Foon, 1997).
1.2.3.2 Morphology
Lymph node biopsies, bone marrow biopsies, and peripheral blood smears can be examined for
typical morphological changes that are useful for diagnosing and staging neoplasms.
In ALL we typically see lymphoblasts with irregular nuclear contours, condensed chromatin,
small nucleoli, and scant, granular cytoplasm. In CLL blood smears reveal small lymphocytes
mixed with variable numbers of large activated cells. Lymph nodes may be enlarged and
diffusely effaced. In biopsies we frequently observe small “cleaved” cells mixed with large cells
in the case of follicular lymphoma. In mantle cell lymphoma, small to intermediate-sized
irregular lymphocytes growing in a diffuse pattern can be observed. Diffuse large B cell
lymphoma presents with variable morphology that resembles large germinal center B cells and
has a diffuse growth pattern. In Burkitt’s lymphoma, tissue morphology shows intermediate-
sized round lymphoid cells with several nucleoli. They have a diffuse apoptosis associated
growth pattern that produces a “starry sky” appearance. Multiple myeloma is often associated
with a microcytic anemia in which peripheral blood smear shows smaller erythrocytes due to
bone marrow problems in end stage disease. Rouleaux formation, or the clumping of red blood
cells is also a prevalent feature in blood smears of Multiple Myeloma patients (Kumar, Abbas, &
Aster, 2013).
Grading of ALL follows the first stage presents with small cells with minimal cytoplasm, the
second stage has large nucleoli and abundant cytoplasm, and the third stage presents as a mature
B cell leukemia. Follicular lymphoma has three grades: small, mixed small and large, and large;
as determined by the overall tumor cell sizes. Hodgkin lymphoma prognosis depends more on
16
the stage of spread as it follows a contiguous pattern from lymph nodes to lymph nodes.
Presence of a large mediastinal mass is usually indicative of a worse prognosis and later stage
disease (Kumar, Abbas, & Aster, 2013).
Histology stains are also applied to tumor biopsies or blood cell suspensions to study and
diagnose cancer. These stains contain a mixture of dyes that introduce contrast to enhance optical
microscopy images. Furthermore, the dyes are differentially absorbed by cellular contents and
tissue structures which aid in the morphology analysis.
In the study of leukemia and lymphocma cells in blood and bone marrow, Giemsa, hematoxylin,
eosin, and methylene blue are several commonly used hematological stains. Giemsa is a mixture
of methylene blue, eosin, and Azure B used commonly for staining in peripheral blood smears
and bone marrow biopsies. It allows for the differentiation of blood cell types as erythrocytes
stain pink, platelets stain a lighter pink, lymphocytes and monocytes stain blue, and leukocytes
stain magenta. Specifically, Giemsa works through binding the phosphate groups of DNA which
gives it other uses such as G-banding of chromosomes and diagnosing blood borne pathogen
diseases such as Malaria (Shapiro & Mandy, 2007). Usage of hematoxylin and eosin (together
known as H&E stain) is a principal staining technique in histology. This allows for red staining
of acidic cellular components and counterstaining of basic cellular components blue. This is
useful in peripheral blood smears as making distinct the nuclear components of cells from the
cytoplasm can help identify neoplastic chromatin structures as well as draw distinctions from
different cell types, particularly granulocytes.
1.2.3.3 Molecular Genetics
Many B cell malignancies are characterized by specific oncogenic chromosomal translocations
that we can detect by performing cytogenetics using a technique called interphase fluorescence
in-situ hybridization (Swerdlow, et al., 2008)(Hallek, et al., 2008)(FISH)(Willis & Dyer, 2000).
More complicated genetic abnormalities require techniques that target specific mutations.
Typical chromosomal translocations that lead to B-cell malignancies involve the changing of a
gene’s normal regulatory sequence with a different one; many of these translocations involve the
IgH locus (Willis & Dyer, 2000). Two examples are the t(14;18) translocation connecting the
BLC2 gene with the immunoglobulin heavy-chain (IgH) locus that is highly predictive of
17
follicular lymphoma, and the t(11;14) translocation connecting cyclin D1 to the same IgH locus
that is predictive of mantle-cell lymphoma (Shaffer, Rosenwald, & Staudt, 2002). The t(8;14)
translocation connecting c-MYC to IgH is implicated in endemic Burkitt lymphoma (Shaffer,
Rosenwald, & Staudt, 2002). The t(4;14)IgH translocations would only occur very rarely in CLL
or ALL (Willis & Dyer, 2000).
As mentioned previously, the two major points of B-cell differentiation with genetic
recombination are during somatic hypermutation and class-switching. Most non-Hodgkin B-cell
lymphomas possess immunoglobulin genes that suggest they are post somatic hypermutation. An
exception to this rule, mantle-cell lymphoma would thus be considered as originating from a pre-
germinal center stage (Shaffer, Rosenwald, & Staudt, 2002).
Cytogenetics can provide indicators towards prognosis that generally predicts better outcomes
than hypoploidy in ALL (Kumar, Abbas, & Aster, 2013). DLBCL which has been traditionally a
very molecularly and morphologically heterogeneous disease has been subtyped into
classifications made distinct through gene-expression profiling with clinical prognostic value
(Shaffer, Rosenwald, & Staudt, 2002)(Lenz, et al., 2008)(Thieblemont, et al., 2004) (Alizadeh, et
al., 2000). CLL with del(17p) have inferior prognosis and greater resistance to standard
chemotherapy (Hallek, et al., 2008) (Grever, et al., 2007)(Döhner, et al., 1995).
1.3 Use of SERS Nanoprobes for Optical Application
1.3.1 Limitations of Immunofluorescence
Several limitations of immunofluoresence has led to the efforts from a wide variety of scientific
disciplines in developing biocompatible nano particles for cellular labeling, imaging, and
targeted therapy (Faulds, Barbagallo, Keer, Smith, & Graham, 2004). Firstly, the fluorochromes
produce wide spectral bands that have the propensity to interfere with another making it difficult
to use multiple fluorochromes (Faulds, Barbagallo, Keer, Smith, & Graham, 2004). Secondly,
fluorochromes are sensitive to photobleaching upon prolonged light exposure. Histological stains
also interfere with fluorescence signals making it difficult to perform a combined analysis with
both techniques.
18
1.3.2 SERS Alternative to Immunofluorescence
One promising alternative employs the optical phenomenon of Surface Enhanced Raman
Scattering (SERS) to produce nanoparticles with bright and distinct spectra used for
immunophenotyping. This technique depends on significant amplification of Raman scattering,
which is inherently weak with approximately 1 in 106 photons incident to a Raman-active
molecule undergoing a vibrational transition to produce either a Stokes or anti-Stokes Raman
shifted photon. To produce the necessary enhancement, Raman-active molecules that produce
distinct Raman spectra are excited in close proximity to a plasmonic substrate which amplify the
signal by up to 1014 to 1015 fold (Qian, et al., 2008). This allows for Raman scattering to be
effective in the detection of single molecules (Qian, et al., 2008).
Several strategies have been previously used to apply plasmonic substrates, predominantly of
gold and silver metal composition, to amplify Raman signals. Most of these involve
chemisorbing or physisorbing the dye as close to the metal surface as possible to maximize the
enhancement effect.
The plasmonic substrate enhances the Raman signal through surface plasmon resonance (SPR).
Surface plasmon polarities (SPPs) are oscillations of conduction electron density that propagate
in a direction parallel to the interface between a metal and a dielectric (Raether, 1988) (Shalaev
& Kawata, 2007). When this interface is stimulated by photons with a frequency that matches the
natural frequency of the oscillations, this resonance is an SPR.
Localized surface plasmon resonance (LSPR) refers to SPR in nanoscale substrates wherein
resonance produces a highly localized near-field amplitude enhancement. The field and
enhancement effects rapidly decay with distance away from the substrate. SERS is an
application of LSPR to enhance the signal of Raman-active molecules that are placed spatially in
close proximity to the substrate via surface adsorption. This will be explained in further detail in
the following section of this thesis.
The nanoscale of the SERS probes used is also advantageous for biological study both in vitro
and in vivo as they are small enough to enter and bind to eukaryotic cells. In this thesis, spherical
gold nano particles are used as they are readily commercially available and have been previously
demonstrated in biomedical diagnostic imaging (Kneipp, Kneipp, Rajadurai, Redmond, &
19
Kneipp, 2009)(Shah, et al., 2008)(Qian, et al., 2008). We use 60nm citrate-coated gold nano
particles that have a resonance wavelength of 534nm. We then subsequently functionalize them
for cell labeling.
While nano particles are specifically explored in this thesis, the literature suggests numerous
other structures such as nano rods (Nikoobakht & El-Sayed, 2003), nano shells (Oldenburg,
Averitt, Westcott, & Halas, 1998), nano cages (Skrabalak, Chen, Au, Lu, Li, & Xia, 2007), as
well as many others (Kumar, Pastoriza-Santos, Rodríguez-Gonzále, García de Abajo, & Liz-
Marzán, 2008) (Wang, Brandl, Le, Nordlander, & Halas, 2006) (Shankar, Rai, Ankamwar,
Singh, Ahmad, & Sastry, 2004) (Grzelczak, Pérez-Juste, Mulvaney, & Liz-Marzán, 2008)
(Kairdolf, Smith, Stokes, Wang, Young, & Nie, 2013) (Liu, et al., 2007) (Mornet, Vasseur,
Grasset, & Duguet, 2004) (Yang, 2014) (Li, Barnes, Bosoy, Stoddart, & Zink, 2012) (Elsabahy
& Wooley, 2012) have been explored. It is worthwhile to note that plasmonic substrates
composed of noble metals and noble metal alloys and different compositions have also been
explored in addition to different structures (West, Ishii, Naik, Emani, Shalaev, & Boltasseva,
2010).
Manipulating the surface structure and composition of these substrates allow for tuning the
optical resonance to a desired frequency. Material composition also effect the substrates’
scattering efficiency in contrast to its absorption efficiency wherein the former serves better for
diagnostic purposes and the latter allows for conversion of light to heat that may be explored for
therapeutic purposes.
SERS nanoprobes offer several advantages over fluorescence-based labelling, the current
predominant strategy for biological studies and clinical diagnostics (MacLaughlin, Parker,
Walker, & Wang, 2013). Firstly, SERS nanoprobes do not photobleach which allows them to
retain signal upon exposure to histological staining or prolonged illumination that would quench
fluorescence signals. Additionally, the sharp bands characteristic of Raman spectra allow for the
potential of multiplexing multiple SERS probes with different reporters. When multiple spectra
with peaks found in similar wavelength regions are analyzed, these sharp peaks (approximately
1nm FWHM) allow them to be distinguished from one another which would be otherwise
difficult using fluorescence spectra.
20
1.3.3 SERS Theory
In 1977, it was concluded through usage of a silver electrode with a Raman reporter molecule
produced an enhanced Raman signal through a separate phenomenon distinct from mere
increases in surface area, reporter concentration, or incident light intensity (Jeanmarie & Van
Duyne, 1977) (Albrecht & Creighton, 1977). Specifically, this was a 106-fold intensity
enhancement of the normal Raman scattering cross-section.
Today the understanding is that both an electromagnetic enhancement mechanism as well as a
chemical enhancement mechanism contribute to SERS (Stiles, Dieringer, Shah, & Van Duyne,
2008).
1.3.3.1 Surface Plasmons
A surface plasmon polariton (SPP) is a collective oscillation of conduction electrons between a
metal and a dielectric. As the electron density shifts along the interface of the two materials,
electromagnetic waves arise at the surface in a perpendicular direction. While these can be
produced through providing energy to the material by electrons and photons, the use of photons
is most relevant in our discussions.
There is a natural restorative force produced by the positive nuclei that establishes a natural
frequency for the oscillating electrons. Surface plasmon resonance (SPR) is established when the
incident light has a matching frequency to that of the oscillating electrons. Since the surface
plasmon propagates along the boundary of the metal and dielectric or external medium, the
oscillations are also very sensitive to gratings, roughness, as well as other molecules in close
proximity to the surface. These shift the plasmon resonance condition and can be detected by
varying the wavelength of incidence light used or the angle at which it hits the surface (Willets &
Van Duyne, 2007). Doing so has allowed SPR to be manipulated in a vast array of applications
aimed at biological and chemical sensing (Haes, Chang, Klein, & Van Duyne, 2005) (Yonzon,
Zhang, & Van Duyne, 2003) (Englebienne, 1998) (Raschke, Kowarik, Franzl, Sönnichsen, Klar,
& Feldmann, 2003).
21
1.3.3.2 Localized Surface Plasmon Resonance
Localized Surface Plasmon Resonance (LSPR) is a phenomenon important to metal
nanoparticles that resonate with the frequency of the incident light. Since the particles used are
actually smaller than the wavelength of electromagnetic radiation used, which is commonly in
the visible and near-infrared for Raman applications, the plasmon oscillates around the particle
instead of being propagated along a surface. The LSPR wavelength is also highly sensitive to
changes in the dielectric environment so shape, size and material have all been demonstrated to
influence it (Kelly, Coronado, Zhao, & Schatz, 2003) (Knobloch, Brunner, Leitner, Aussenegg,
& Knoll, 1993) (Miller & Lazarides, 2005).
Importantly, there is an enhancement of the Raman spectra of the molecule due to the LSPR of
the metal particle. This is because the LSPR provides a significant enhancement of the
electromagnetic near field of the particle (Guillot & de la Chapelle, 2012). Since the
electromagnetic field is enhanced this way by a factor of 10 (Stiles, Dieringer, Shah, & Van
Duyne, 2008) and Raman scattering intensity scales approximately with E4, the Raman
enhancement by the LSPR is described as 104.
The LSPR corresponds to a particular wavelength that can be predicted by classical Mie theory
which describes the scattering of light by spherical particles (Guillot & de la Chapelle, 2012).
Since we are dealing with a nanoparticle which has a size that significantly smaller than the
incident light wavelength (radius/wavelength < 0.1), Mie’s solution is simplified to a quasistatic
approximation. In this approximation, the incident electric field can be treated as spatially static
relative to the particle simplifying the electron density oscillation of the particle to a dipole
plasmon (Long, 1977) (Kreibig & Vollmer, 1995) (Kerker, 1969). In other words, the electric
field around the nanoparticle is treated as uniform (Stiles, Dieringer, Shah, & Van Duyne, 2008).
22
Figure 1 Visual representation of Localized Surface Plasmon Resonance (LSPR). Retrieved
from (Stiles, Dieringer, Shah, & Van Duyne, 2008)
With this approximation, the solution for magnitude of the electromagnetic field outside the
particle Eout is:
In this solution, r is the radial distance which demonstrates in this equation that field
enhancement decays with r-3. This demonstrates the enhancement at the near field exponentially
decaying with distance. X, y, and z are the Cartesian coordinates; alpha is the metal polarizability
that is defined as:
with ‘a’ as the radius of the spherical particle and g is defined as:
23
In this equation, ɛin and ɛout are the dielectric constants of the metal nanoparticle and the external
environment respectively. This also demonstrates that the maximum enhancement occurs when
the denominator of g approaches zero in the condition:
Since the real component of the metal nanoparticle dielectric constant is wavelength dependent,
the condition describes the enhancement maximum as being dependent on the physical properties
and composition of the metal nanoparticle in relation to the external environment and
wavelength of incident light.
The full extinction spectrum for a given nanoparticle under the aforementioned quasistatic
approximation is function E(λ):
Where ɛr and ɛi are the real and imaginary components that make up ɛin. Note that χ is a factor
related to the shape of the particle. For spherical nanoparticles, χ is 2 (Stiles, Dieringer, Shah, &
Van Duyne, 2008).
As described previously, the field enhancement around the metal nanoparticle decays with r-3.
Considering the E4 approximation also described previously, the overall distance dependence of
the Raman scattering intensity should scale with r-12. Taking into account surface area of the
particle scaling with r2, one should experimentally observe r-10 distance dependence (Stiles,
Dieringer, Shah, & Van Duyne, 2008):
In this relationship, I-SERS is the intensity of the Raman mode, ‘a’ is the average size of the
field-enhancing features of the surface, and r is the distance from the surface to the adsorbate.
24
1.3.3.3 Raman and Rayleigh Scattering
Raman scattering is the inelastic scattering of photons caused by incident light exchanging
energy with a molecule. In most interactions, photons undergo a process elastic scattering known
as Rayleigh scattering in which the frequency and wavelength of the released photon is
unchanged from that of the incident photon.
However, around 1 in 10 million photons that are scattered undergo an energy transition wherein
the released photon is of lower energy in the case of a Stokes shift or higher energy in the case of
an anti-Stokes shift. In the former, the material absorbs some of the energy of the photon before
re-emission and in the latter, the material loses energy to the released photon. These Raman
scattering interactions are visualized in the figure below.
Figure 2 Visualization of different types of light scattering. A) Rayleigh; B) Stokes Raman;
C) Anti-Stokes Raman
The difference in energy between the vibrational states of the molecule is related to the
frequency of the photon released in accordance to the Bohr frequency rule with Planck’s constant
(h) as a co-efficient.[10]
∆𝐸 = ℎ𝑣
The scattered photons can form a Raman spectrum that shows the intensity of scattered light as a
function of the Raman shift which is the change in frequency from the incident light. The
25
majority of the Raman shifts would be Stokes as opposed to anti-stokes as the ground vibrational
state of the molecule is most stable and also the most populated.
Compared to fluorescence which also takes in incident light and releases them at shifted
frequencies, the major difference is that Raman scattering does not require a specific frequency
of incident light. Consequently, Raman spectra intensities are a function of relative frequencies
instead of absolute. The nature of the spectra is also different, conferring several advantages to
Raman spectroscopy that are previously mentioned.
The classical electrodynamic theory describes the phenomena which produces both Raman and
Rayleigh scattering (Long, 1977). It predicts a significantly higher rate of Rayleigh scattering
over Raman scattering as well as a linear dependence Raman scattering intensity and the
intensity of the incident light and concentration of molecule.
Light scattering results from an induced molecular dipole moment that is produced through the
interaction of the molecule and incident electromagnetic radiation. The induced dipole moment
is both dependent on the polarizability of the molecule as well as the intensity of the incident
electromagnetic field (Stiles, Dieringer, Shah, & Van Duyne, 2008).
P is the induced dipole moment, alpha is the polarizability tensor, and Ē is the magnetic field of
the incident electromagnetic wave. Considering that the electric field is oscillating, we treat it as
a sinusoidal electric field that induces a dipole moment that is also oscillating. It is this
oscillating dipole moment that produces the emitted photons (García-Vidal & Pendry, 1996).
This gives us the relationship:
E0 is the electromagnetic field produced by the electromagnetic wave of frequency ‘v’. This
results in the previous definition of P being described as:
26
Since the polarizability of the molecule can be changed by vibration within the molecule, we
describe it as a Taylor expansion that includes a component for static polarizability, α0 and a
component representing the molecular polarizability as a function of a vibrational mode Q:
Q represents a normal mode of vibration inherent to the molecule with frequency vvib and
amplitude Q0. We can express dQ as:
Combining these equations produces a relationship for the induced oscillating dipole:
This can be simplified further using a trigonometric identity into:
Of these three terms in the equation, the first represents the Rayleigh scattering and the second
two represent the Raman scattering. The term subtracting the vibration frequency: vvib, is the
Stokes shift and the term adding it is the Anti-Stokes shift.
1.3.3.4 Electromagnetic Enhancement of SERS
Kerker and colleagues have described a first-order approximation to evaluate the enhancement of
radiation from a dipole plasmon as in the case of metal nanoparticle LPSR (Kerker, Wang, &
Chew, Surface enhanced raman-scattering (SERS) by molecules adsorbed at spherical particles,
1980)(Wang & Kerker, 1981):
27
All these fields represented by E are associated with a specific frequency. E0 is the incident field
intensity and Eout is the intensity of the scattered light. The radially averaged intensity for Eout of
a metal sphere is (Stiles, Dieringer, Shah, & Van Duyne, 2008):
Where ‘g’ describes the average field enhancement over the surface of the particle. The purpose
of the approximation by Kerker and colleagues is to account for the Raman stoke-shifted
frequencies as represented by E′out. This equation simplifies to:
where the g′ is associated with the field enhancement of the Stoke-shifted frequency. In cases
where the Stoke shift is small, we treat g′ and g being at approximately the same wavelength thus
producing the relationship that EF scales with g4. This theoretical treatment of SERS describes a
104-105 enhancement of Raman scattering intensity attributable to the LPSR and electromagnetic
mechanisms.
From a practical standpoint, we experimentally measure the enhancement factor by comparing
the intensity at a single excitation wavelength between a SERS intensity and a normal Raman
intensity:
In this equation ISERS is the surface-enhanced Raman intensity which we divide by Nsurf, the
number of Raman molecules bound to the metal nanoparticle producing the signal. INRS is the
normal Raman intensity obtained from a solution where a particular volume of Raman molecules
are excited and Nvol represents the number of Raman molecules in that volume. A detailed
28
procedure for measuring SERS EFs experimental has been described by McFarland et al.
(2005)(McFarland, Young, Dieringer, & Van Duyne, 2005)
1.3.3.5 Chemical Enhancement of SERS
There is a chemical enhancement factor of 102 that is caused by resonance Raman scattering
through which is a charge-transfer resonance that occurs between the metal and Raman-active
molecule (Stiles, Dieringer, Shah, & Van Duyne, 2008). This electrochemical mechanism was
initially observed when Raman resonances were obtained by varying the applied potential.
Charge-transfer between the Raman-active molecule and the conduction metal band in either
direction is responsible for this effect (Lombardi & Birke, 2009).
1.3.4 Biological Delivery
1.3.4.1 Lipid Encapsulation
The gold nano particles received require additional surface modification for stability and
biocompatibility. The colloidal state of particles is extremely sensitive to ion charge, easily
losing monodispersity and aggregating in any buffer environment resembling a biological
system. While gold nano particles are selected for their inertness and low toxicity (Tam, Scott,
Voicu, Wilson, & Zheng, 2010), residuals from the reduction reaction used to synthesis the
particles have unwanted interactions with biological systems. Lipid encapsulation is used to
maintain the colloidal stability of the SERS nanoprobes and make them compatible for biological
targeting.
The use of phospholipid bilayers to form spherical vesicles called liposomes had been
demonstrated to be successful for the in vivo drug delivery of doxorubicin, a chemotherapy drug
(Al-Jamal & Kostarelos, 2011). This was the first clinically approved nanoparticle drug to use
this delivery method, and subsequently liposomes became widely used for drug delivery and
there exists a large body of literature exploring its use (Ip, MacLaughlin, Gunari, & Walker,
2011) (Hofheinz, Gnad-Vogt, Beyer, & Hochhaus, 2005) (Wu, Liu, & Lee, 2006). Liposomes
also best mimic cellular membranes in comparison to other methods such as polyethylene glycol
or silica coating (Tam, Scott, Voicu, Wilson, & Zheng, 2010).
29
The liposomes are self-assembling and the formulation of lipid components can be easily
designed to serve specific purposes. Surface charge, phase transition temperature, chemical
functionalization, and steric interactions can be modulated through formulation design. Even the
size, shape, and surface charge can be manipulated due to the wide varieties of lipids with
different head groups. The fluid nature of lipids also confers the ability for liposomes to have
increased permeability across barriers in vivo (Ceccheli, et al., 1999). The liposome used in this
thesis is functionalized with reacting groups to facilitate antibody conjugation and is described in
detail in the methods section. Furthermore, the phospholipid coating allows the maintenance of
colloidal stability and monodispersity of the gold nanoparticles especially in the presence of an
ionic environment that would otherwise lead to aggregation of unprotected gold nanoparticles.
The lipid encapsulation process involves sonicating polydispersed multilamellar vesicles
(MLVs) in the presence of silica-encapsulated 60 nm gold nanoparticles with J-aggregate Raman
dye monolayer physisorbed to the gold surface via linker molecule. The sonication is expected to
produce small unilamellar vesicles (SUVs) around the gold nanoparticles (Woodbury,
Richardson, Grigg, Welling, & Knudson, 2006).
The design of the lipid formulation is based loosely around a ternary lipid bilayer mixing DOPC,
sphingomyelin and cholesterol in a 2:2:1 molar ratio as investigated by McLaughlin et al.
(2011)(Ip, MacLaughlin, Gunari, & Walker, 2011) Such designs focus on emulating the cell
membrane model system that has been demonstrated to remain stable for several weeks and
survive through harsh acidic and high ionic strength suspension conditions. DPSE functionalized
with either a maleimide or carboxylic acid group replaced a portion of the DOPC used while a
percentage of polyethylene glycol terminated DOPC was used to improve colloidal stability.
1.3.4.2 Antibody Conjugation
Monoclonal antibodies were functionalized to SERS nano probes in this thesis to crosslink with
surface receptors on target cells. Compared to other targeting molecules such as DNA, antibody
fragments, aptamers and affibodies, Antibodies have a number of advantages, most notably their
high affinity for their target antigen which is on the order of 10-9M (Alberts, 2008). Furthermore,
antibodies are widely available commercially and designed to specifically target various
30
antigens. SERS nanoprobes take advantage of using this method of targeting that has already
been demonstrated in fluorescence probes.
1.3.4.2.1 Rituximab Monoclonal Antibody
In this project, we specifically use rituximab, a chimeric monoclonal antibody that specifically
targets CD20, a cell surface receptor primarily found on the surface of B cells. Rituximab is
widely available as targeted therapy towards chronic lymphocytic leukemia as well as other B-
cell predominant hematological cancers and B-cell predominant autoimmune disorders (Gürcan,
Keskin, Stern, Nitzberg, Shekhani, & Ahmed, 2009). Rituximab’s targeting and effects are
widely studied due to its prevalence in the clinical setting which validates its use as a targeting
moiety for B cell lymphoma cells as used in this thesis (Eisenberg, 2005)(Scheinfeld, 2006).
As a therapeutic, Rituximab works by binding specifically to CD20 positive cells leading to their
elimination through induction of apoptosis, activating the complement system leading to lysis via
formation of a membrane attack complex (MAC), and lysis by NK cells via antibody-dependent
cell-mediated cytotoxicity (ADCC) (Gürcan, Keskin, Stern, Nitzberg, Shekhani, & Ahmed,
2009).
Monoclonal antibodies are produced to bind a single epitope of the target antigen – Rituximab’s
fragment, antigen binding (Fab) region is designed to recognize a four amino acid sequence on
an extra-cellular loop of CD20 (Gürcan, Keskin, Stern, Nitzberg, Shekhani, & Ahmed, 2009).
Since our lymphoma cell lines are of B-cell origin, we expect a properly Rituximab
functionalized SERS nanoprobe to bind to the cells through Rituximab-CD20 cross-linking.
1.3.4.2.2 Conjugation Chemistry
Conjugation of targeting proteins to SERS labels can be done through a variety of chemical
reactions. The three most widely used approaches are sulfhydryl coupling to maleimide, carboxyl
to amine conjugation, and alkyne to azide chemistry (Biju, 2014).
EDC/NHS coupling and maleimide coupling were both used in this project for antibody
conjugation.
31
The former converts carboxylic acid (-COOH) groups to primary amine-reactive esters, which
are perfect for crosslinking to antibodies (Grabarek & Gergely, 1990). This technique has been
widely used for the attachment of proteins to other optical molecules such as fluorophores. The
reaction first uses EDC (1-ethyl-3-(-3-dimethylaminopropyl) carbodiimide) hydrochloride to
form an unstable O-acylisourea intermediate. The second species used, N-hydroxylsuccinimide
(NHS) in its soluble sulfonate form, Sulfo-NHS offers a reactionary pathway or the intermediate
to avoid reverting back to its acid form via hydrolysis, and instead convert to another primary
amine-reactive form, the N-Hydroxysuccinimide ester. This ester is then replaced by an amine
group on the antibody forming a covalent bond that bridges the lipid layer of the SERS nano
probe to the antibody.
A 2:5 molar ratio of EDC and sulfo-NHS were added to the particles after lipid encapsulation
(Biju, 2014). The EDC crosslinking occurs most efficiently in an acidic environment around pH
4.5 although neutral pH phosphate buffer conditions still facilitate the reaction with lower yield.
Sulfo-NHS is the soluble analog of N-hydroxysuccinimide (NHS) which we preferentially used
due to the reaction occurring in solution. While use of EDC alone is capable of producing
successful crosslinking, the intermediate is prone to hydrolysis as an alternate pathway over
forming a stable bond with the primary amine group of the targeting protein. Sulfo-NHS
provides an alternative pathway for the intermediate to form a dry-stable amine-reactive
intermediate that then reacts with the primary amine to form the bond.
32
Figure 3 Chemical Reaction Mechanism for EDC-NHS to produce an amide bond between
carboxylic acid and primary amine.
Retrieved from: http://www.piercenet.com/method/carbodiimide-crosslinker-chemistry;
Accessed on July, 2014.
In maleimide conjugation chemistry, the maleimide group can form a stable thioether linkage
with sulfhydryl groups in pH environments between 6.5 and 7.5 (Ryazanova, Winkler, Friedhoff,
Viryasov, Oretskaya, & Kubareva, 2011). This bond is irreversible in that it cannot be cleaved by
reducing agents.
For both reactions, centrifugation is performed several times both before the conjugation
procedure. Since there are excess lipids with functional groups, it is important that they are
removed as they will compete for binding with reagents as well as the antibodies themselves.
Excess free floating reagents in the case of EDC-NHS cross-linking are also removed in an
additional centrifugation step before the antibodies are added.
Afterwards, the antibodies and SERS labels are allowed to react overnight (12-16 hours) at 4ºC.
Centrifugation and re-suspension in buffer has is then performed to remove excess unbound
33
antibodies. The presence of conjugated proteins as well as changes in surface charge upon NHS-
ester formation seem to give the antibody labelled particles the propensity to irreversibly adhere
and coat the centrifuge tubes used in this step in several experiments. As a pre-caution and
solution, 2k-PEG lacking terminal thiol is added to the suspensions in a 2% w/v ratio. As well, a
phosphate buffer solution of 0.01g/100mL bovine serum albumin is also stored in the centrifuge
tubes overnight at 4ºC to reduce particle adhesion. The solution is rinsed out of the tubes and the
antibody conjugated particles are transferred into them with the 2k-PEG before undergoing the
aforementioned centrifugation steps.
Chapter 2 - Study Design and Methods 2The introductory chapter of the thesis has described the importance of immunophenotyping as a
diagnostic as well as prognostic technique for B-cell malignancies. It has also provided
background on SERS nanoprobes and how and why they fare well as potential tools for
immunophenotyping. Our study follows along the current direction of the field in producing
better SERS nanoprobes in terms of signal strength and robustness particularly in a biological
environment while also exploring the myriad of different methods of approaching particle
functionalization.
2.1 Research Objectives
2.1.1 Objective 1 - Demonstrate Successful Application of J-Aggregate Monolayers in the Labelling of Lymphoma Cells using SERS Nanoprobes
Zamecnik et al. (2013) has synthesized a class of SERS nanoprobes using cyanine dyes that form
J-aggregate monolayers as Raman reporters (Zamecnik, Ahmed, Walters, Gordon, & Walker,
2013). These particles were shown to have extremely bright signals in solution but have yet to be
tested in a biological setting. Naturally, for this project the next step is to functionalize these
particles for cell labelling.
2.1.1.1 Hypothesis for Objective 1
As SERS nanoprobes generally require generic procedures for functionalization, we
hypothesized the successful application of these particles to LY8 lymphoma cells while retaining
their exceptional signal strength.
34
2.1.2 Objective 2 - Demonstrate Successful Application of Silica Encapsulation in the Labelling of B-cell Lymphoma Cells using J-Aggregate Monolayer SERS Nanoprobes
Within the lab, experiments have been performed to further improve on the signal strength of the
J-Aggregate SERS Nanoprobes as well as devise a solution to inconsistent fluorescence issues
with these particular particles. Silica encapsulation was shown to be a method of achieving both
of these goals and can be considered the next upgrade in our particle synthesis.
2.1.2.1 Hypothesis for Objective 2
We hypothesized that using a successful protocol designed for the first objective with minor
adjustments should produce successful cell labelling with these silica-encapsulated particles.
2.1.3 Objective 3 - Demonstrate the Effect of Hematological Staining on the Silica Encapsulated J-Aggregate Monolayer SERS Nanoprobes in Lymphoma Cell Labelling
For this objective, we investigate the potential for combining immunophenotyping techniques
with the use hematological stains through the robustness of SERS signals. Four common
hematological stains: Giemsa, eosin, hematoxylin, and methylene blue will be applied to our
silica-encapsulated J-aggregate monolayer SERS nanoprobes both in solution and post-cell
labelling.
2.1.3.1 Hypothesis for Objective 3
We hypothesized that our particles should be able to continue to produce strong SERS signals
when exposed to the stains.
2.2 General Instrumentation Methods
2.2.1 Raman Spectroscopy
To produce and capture Raman emission spectra, these experiments used a Renishaw inVia
inverted confocal Raman microscope built around a Leica DMI6000 automated epifluorescence
microscope. The confocal detection optics confer additional sensitivity and improve the signal-
to-noise ratio of obtaining the Raman spectra (Schlücker, 2009) (Schlücker, Schaeberle,
Huffman, & Levin, 2003) (Dieing, Hollricher, & Toporski, 2010).
35
Taking advantage of the LSPR, Raman spectroscopy requires use of laser wavelengths that
match closely with the resonance energy level of the plasmonic material. We discuss later the
necessity of matching it further with the resonance of the Raman molecule exciton in the case of
J-aggregates. The microscope was equipped with 3 solid state excitation sources providing
incident laser wavelengths of 532nm, 638nm, and 785nm. 532nm was most relevant for our
purposes.
Scattered light is collected through a 1024x256 deep depletion silicon RenCam CCD (charged-
coupled decide) detector.
2.2.2 Dark Field Microscopy
Dark field light microscopy is an illumination technique through adapting the traditional light
microscope to enhance contrast. This is done using an annular diaphragm or dark field patch stop
that blocks transmitted light so that only an outer ring of illumination persists that focuses the
sample at a high angle as can be seen in the figure below. Additionally, the numerical aperture
(NA) of the condenser must be greater than that of the objective. The key concept here is that the
angle of incidence of illumination must always exceed the angle of collection of the objective
(relative to the normal of the substrate platform). This way, only the light that is scattered by the
sample is collected by the objective while the directly transmitted light simply misses the lens.
This technique is both inexpensive and effective for providing high contrast images without
using stains that could otherwise produce artifacts or damage live cells.
36
Figure 4 Visual representation of dark field microscopy set-up using a dark field patch stop
to block transmitted light. Image retrieved from:
http://en.academic.ru/pictures/enwiki/68/Dark_Field_Microscope.png; accessed on July
2014.
This type of imaging is particularly effective in the application of SERS nanoprobes as the gold
nanoparticles exhibit Rayleigh scattering allowing them to be easily viewed through dark field
microscopy. Consequently, dark field has become increasingly popular for imaging studies using
metal nanoparticles (Hutter, et al., 2010)(Aaron, Travis, Harrison, & Sokolov, 2009) (Fairbairn,
Christofidou, Kanaras, Newman, & Muskens, 2013)(Badireddy, Wiesner, & Liu, 2012). Whereas
the size of the particles themselves are smaller than the standard diffraction limit of light
microscopy, their large light scattering cross sections allow them to be visualized through dark
field microscopy (Jain, Lee, El-Sayed, & El-Sayed, 2006). The particles themselves present as
clusters of green scattered light that is red-shifted based on higher degrees of particle clustering
and density. Other factors that affect the color of light scattering are the index of refraction of the
medium, the incident light wavelength in relation to the localized surface plasmon and the size of
the actual particle (Wax & Sokolov, 2009) (Schultz, Smith, Mock, & Schultz, 2000) (Jain,
Huang, El-Sayed, & El-Sayed, 2008) (Kreibig & Vollmer, The Optical Properties of Metal
Cluster, 1995).
37
Dark field images were collected using an inverted Nikon TE2000 microscope using an oil
immersion condenser and 100X objective lens with an adjustable NA range of 0.5-1.25. The
condenser has a dark field stop inside to facilitate the dark field effect. Scattered light is collected
through a DSFi1 CCD camera.
2.2.3 UV-Vis Microscopy
UV-Vis spectroscopy was performed on a Varian Cary 5000 UV-vis-NIR spectrophotometer.
Distilled water was used as a blank and samples were placed in a 1 cm path-length black wall
cuvette. The spectra obtained were used to confirm the gold plasmon speak shift (generally 536
to 538 nm) as well as to characterize the J-aggregate peak (approximately 561 nm). Undesired
aggregation of particles is also indicated by further red-shifted gold plasmon peaks – lack of this
indicated monodispersity of particles.
2.2.4 B-cell lymphoma cell line and cell culture
The B cell cancer model used in this thesis is a human B cell lymphoma line (LY8). Cells
suspensions are grown in culture medium with 10% fetal bovine serum. They are kept in a
37°C incubator with 5% CO2 atmosphere.
All cell culturing and experimentation steps were performed in a sterilized environment under
fumehood.
38
2.3 General Sample Preparation Methods
2.3.1 Materials
The following materials were used without further purification unless specified:
2.3.1.1 Lipids
1,2-dioleoyl-sn-glycero-3-phosphocholine (DOPC), 1,2-dioleoyl-sn-glycero-3-
phosphoethanolamine-N-[methoxy(polyethylene glycol)-2000] (DOPC:PEG), 1,2-distearoyl-sn-
glycero-3-phosphoethanolamine-N-[carboxy(polyethylene glycol)-2000] (DPSE-PEG), 1,2-
distearoyl-sn-glycero-3-phosphoethanolamine-N-[maleimide(polyethylene glycol)-2000]
(DPSE:Mal), and ovine cholesterol (Chol) were purchased from Avanti Polar Lipids. These were
used for making the liposomes we use to lipid-encapsulate nanoparticles.
2.3.1.2 SERS Particle Synthesis Reagents
60 nm citrate-coated gold nanoparticles are purchased from Ted Pella Inc.
Thiocholine (TMAT) is synthesized from acetylthiocholine in the lab. Protocol is described in
later section of thesis.
4-(2-hydroxyethyl)piperazine-1-ethanesulfonic acid) (HEPES) was obtained from Sigma
Aldrich.
2.3.1.3 Conjugation Chemistry Reagents
Acetylthiocholine, N-(3-dimethylaminopropyl)-N’-ethylcarbodiimide hydrochloride (EDC), N-
hydroxysulfosuccinimide sodium salt (Sulfo-NHS) is purchased from Sigma Aldrich.
Rituximab is obtained from Hoffman-La Roche.
PEG2k is obtained from Sigma Aldrich
2.3.1.4 Miscellaneous
Bovine serum albumin (BSA) is purchased from UofT MedStore.
39
Water was purified using a Millipore Milli-Q water system to 18.2 MΩ cm.
Glassware used was cleaned through a process of four sonication steps with water and soap
followed by three organic solvents ethanol, methanol, and chloroform. These solvents were
obtained through Lash Miller Chemistry lab.
Polyvinylpyrrolidone (PVP), Tetraethyl orthosilicate (TEOS), Poly(allylamine
hydrochloride)(PHP) were obtained from Sigma Aldrich
2.3.1.5 Cell Culturing
LY8, flasks, culture medium, index matching media, glass slides, cover slips, formaldehyde,
hemotoxyline, giemsa, methylene blue, eosin, Fetal bovine serum (FBS) were used.
2.3.2 Lipid Preparation
Lipid preparation: Lipids were prepared as per the protocol reported by Ip et al. (2011)(Ip,
MacLaughlin, Gunari, & Walker, 2011)
One-dram vials were cleaned through a four-solvent process to ensure no contaminants were
present when the lipids were added. This process involved placing the vials in a bath treated
through 30 minutes of sonication each under soap water, methanol, ethanol, and then chloroform.
The vials are then dried through 15 minute airing with Argon gas under fume hood and then
desiccated under vacuum overnight. The vials were then backfilled with argon, capped and
stored.
Lipids used for mixture preparation were obtained either in crystallized or chloroform solvated
forms. For the formulations used, DOPC, DOPC:PEG, cholesterol, and the functionalized DPSE
with either carboxylic acid group or maleimide group were added and solvated in a 3:1
chloroform to methanol solution.
The preparation used loosely followed in Ip et al. (2011)(Ip, MacLaughlin, Gunari, & Walker,
2011) to recapitulate a lipid bilayer resembling a natural cell membrane. Specifically the mole
ratios were 48% DOPC, 32% cholesterol, 13% DOPC:PEG, with the remaining 7% composing
40
of either DPSE:COOH or DPSE:Maleimide. The total molar quantity was 0.03mmol dissolved in
5mL of 3:1 chloroform to methanol ratio and this is distributed into 10 one-dram vials to a final
molar content of 0.003mmol per vial. These vials are then tried through 13 minutes airing under
Argon gas, capped, and stored in -20°C freezer until further use.
2.3.3 Lipid Encapsulation
Lipid encapsulation was performed on both silica-encapsulated and non-silica-encapsulated Au
NPs. Lipid formulations previously prepared freezer stored through the protocol in the previous
section are warmed to room temperature and then redissolved in 40 mmol HEPES buffer heated
to 50°C. Each 0.003mmol of lipids is solvated with 3mL of the buffer. The solution is then
heated to 50°C in a water bath for 30 minutes with periodic mixing every 10 minutes.
Subsequently, the lipids are added to Au NPs at a volume ratio of 1:1. These Au NPs have
already been SERS functionalized with TMAT and S0271 through a procedure described in the
section on J-aggregate monolayers in this thesis. For particles to be prepared to have both silica
and lipid encapsulation, they will have been silica encapsulated prior to this step.
The Au NPs are previously prepared to be in a distilled water solution so that the addition of
lipids in 40 mmol HEPES buffer should bring the combined buffer concentration to 20 mmol.
The lipids are gently added to the Au NPs while they are being sonicated to ensure a gentle
exposure to the buffer solution. The mixture is then sonicated for 60 minutes at 50°C at which
point it appears clear, indicating that small unilamellar vesicles have formed.
The solution is then washed through two centrifugation steps at 3500 G for 11 minutes and
resuspended in 20 mmol HEPES to remove excess lipid as the lipid formulations are expected to
be in excess.
2.3.4 Raman Functionalization of Particles
60 nm gold nanoparticles are made into Raman SERS-active nanoprobes upon addition of S0271
and linker molecule TMAT. The detailed synthesis protocol is described in the next section of
this thesis.
41
2.3.5 Antibody Conjugation
Rituximab anti-CD20 is the monoclonal antibody used in these experiments.
Two different conjugation methods are used: EDC-NHS and maleimide reactions.
2.3.5.1 EDC-NHS
In the former 2.5 µg of EDC and 2.5 µg are first solubilized in 1X PBS buffer and then added
one after the other to lipid encapsulated particles with the necessary DPSE:COOH lipid in its
formulation. The reaction is allowed to take place over 15 minutes under stirring. Afterwards, the
particles are washed at 3500 G for 11 minutes remove excess reagents. Then 5ug of rituximab
diluted in 1X PBS is added to the mixture and incubation is allowed to proceed overnight at 4°C.
2.3.5.2 Maleimide
In maleimide antibody conjugation, lipid encapsulated particles with the necessary
DPSE:Maleimide lipid in its formulation are solvated in 20mmol HEPES solution adjusted if
necessary to have a pH range between 6.5-7.5 to facilitate the conjugation reaction. 5µg of
rituximab diluted in 1X PBS is added and incubation is allowed to proceed overnight at 4°C.
2.3.5.3 Post-Functionalization
After overnight incubation in both EDC-NHS or maleimide facilitated antibody conjugation
reactions, the particles are transferred to centrifuge tubes that have treated with 1.5 mL of 10%
BSA overnight solvated in 1X PBS overnight. 150µL of 2k-PEG is also added to the mixture to
reduce particle sticking to the tube from centrifugation.
Particles are centrifuged at 3500 G for 11 minutes twice, resuspended in 20 mmol HEPES and
then stored at 4°C until use.
2.3.6 Cell Labelling
LY8 B cell lymphoma cells are transferred to 10mL centrifuge tubes and spun at 500 G with for
5 minutes and resuspended in 1X PBS buffer twice. A whole blood cell count is performed to
obtain the concentration of the lymphoma cells. The cells are then added to particles at an
42
approximate ratio of 200,000 cells per 1mL for non-silica encapsulated particles and 200,000
cells per 0.1mL for silica encapsulated particles. The discrepancy is due to an issue with the extra
size and density of silica-encapsulated particles which make it difficult for unbound particles to
be washed from the cells and bound particles. It was determined experimentally that the
particular particle-to-cell ratio was the best middle ground between having enough particles
bound to cells while minimizing free-floating particles in solution.
The cell-particle mixture is incubated at room temperature for 30 minutes with periodical re-
mixing of contents.
After incubation is complete, the cell-particle mixtures are spun at 500 G and resuspended in
PBS twice. For non-silica encapsulated particles they are spun for 5 minutes, and for silica-
encapsulated particles they are spun for 30 seconds. This discrepancy is again for the same
reason of difficulties in separating unbound dense silica-encapsulated particles from the rest of
the solution.
The washed mixtures are then cytocentrifugated at about 200 G for 5 minutes onto glass slides
cleaned with ethanol.
After cytocentrifugation, the slides are treated with 4% formaldehyde solvated in 1X PBS for 5
minutes in order to fix the cells. Afterwards they may be treated with appropriate hematological
stain; detailed procedure for staining is described in the hematological stain cell experiments
section of the thesis. Finally, index matching media is added to the slide before a cover slip is
placed on and the slide is allowed to dry overnight. The purpose of this is to improve contrast in
dark field images.
2.3.7 Dark Field Imaging
Dark field images were obtained as a means to evaluate SERS nanoprobe labelling of lymphoma
cells. The high contrast provided by the dark field as well as elimination of transmitted light by
the technique allowed for the easy visualization of natural Rayleigh scattering of the gold
nanoparticles. Since we used 60 nm gold nanoparticles with a plasmon peak approximately at
536 nm, these generally appear as green dots. This is technically an indirect visualization of the
43
nanoparticles are their inherent size would otherwise be undetectable under the 100X
magnification used to capture the images.
This Rayleigh scattering effect can also be used as a means of evaluating the degree of particle
aggregation as aggregated particles have a red-shift in their surface plasmon and can
consequently appear as yellow to red dots depending on the degree of aggregation. However, this
is not always the case as it can be confounded by other scenarios with similar effects: for
example single particles in close proximity to one another can still produce this red-shifted effect
and this can be the result of their antibody-targeted receptors being in close proximity to another
on the cell surface.
In preparation of slides for dark-field imaging, B-lymphoma cells are incubated with SERS
particles and cytocentrifuged onto microscope slides. 4% Formaldehyde allows fixing of the
cells but also seems to help visualize the overall location of the cells in the dark field image.
Image matching media is added to enhance contrast and reduce background scattering and a
cover slip is placed on top. The slide is allowed to dry for a day before imaging.
2.3.8 Raman Microscopy and Raman Mapping
Raman microscopy was used to evaluate the SERS nanoprobes both in solution as well as when
used to label cell slides. In the former case, the particles are placed in a microplate and in the
latter, the slides have been prepared in the same way as in the case of dark-field imaging. Since
the 60 nm nanoparticles have an absorption peak indicative of the LSPR at around 536 nm and
the Raman molecule we use has an absorption peak at around 561 nm in J-aggregate form (this is
explained in the next section in greater detail), we use the closest available laser wavelength of
532 nm to produce the greatest wavelength resonance effect for a bright SERS signal.
For the purpose of analyzing SERS nanoprobe labelled cell slides, simply obtaining one Raman
spectra provides a qualitative indication of the presence of particles but no indication on how
well the particles bind specifically to the lymphoma cells.
Using the Renishaw Wire 3.0 acquisition software, we can perform a technique described as
Raman mapping that allows precise excitement of a grid surrounding a selected section of the
slide. Parameters can also be adjusted for the distance between each measurement in two
dimensions to a maximum resolution of 1µm between measurements. The software additionally
44
allows for the section to be simultaneously visualized through bright field light microscopy. The
result is a 2x2 grid of Raman scattering spectra overlaying a bright field image of the cell slide.
The resultant spectra can be analyzed through various means to produce a heat map showing
where the spectrum is strongest according to various parameters – our analyses look for heat
maps as a function of signal-to-baseline intensity centered on a distinct Raman peak.
Ideally, we look for a lack of signal where there are no lymphoma cells and a presence of strong
signal where the cells are. In addition to binding specificity, this can also ideally reveal localized
receptor clustering based on signal strength.
The specific analysis used in this thesis for producing a Raman heat map is a signal-to-base line
peak strength around a prominent double peak near a Raman shift of 1100 cm-1.
Chapter 3 - Results 3
3.1 Objective 1 - Application of J-Aggregates for Cell Labelling
3.1.1 Introduction to J-Aggregate Monolayers
The overall optic enhancement through SERS phenomena using plasmon nanomaterials has been
demonstrated to range quite significantly and in many cases be significantly greater than the
approximate 106-1011 enhancement factor predicted by the mechanisms described earlier. Many
experiments have shown enhancements as high as 1014 (Moskovits, 2005) (Krug, Wang, Emory,
& Nie, 1999) (Kneipp K. , Kneipp, Itzkan, Dasari, & Feld, 1999). This has inspired researchers
to investigate methods of producing even bright signals for ultrasensitive analytical strategies
employing SERS (Moskovits, 2005).
While the plasmonic material is the predominant factor in SERS enhancement, the Raman active
molecule itself is believed to also provide significant contributions that explain why certain ones
are better enhanced than others in SERS experiments. While one explanation is the charge-
transfer resonance described earlier, resonances within the molecule itself are believed to also be
a contributing factor (Limbardi 2008). Rhodamine 6G (Zhao, Jensen, Sung, Zou, Schatz, & Van
Duyne, 2007), cytochrome c (Sivanescan, Kalaivani, Fischer, Stiba, Leimkuhler, & Weidinger,
2012), porphyrin and derivatives (Murphy, Huang, & Kamat, 2011) (Haes, Zou, Zhao, Schatz, &
Van Duyne, 2006) have all demonstrated further enhancement of the SERS signal through a
45
method of coupling to plasmonic nanostructures. One important concept here is that dyes with
absorption peaks close to the incident light wavelength produce a greater SERS spectra through
an effect described as Surface-enhanced resonance Raman scattering (SERRS) (Doering & Nie,
2003) (Kneipp K. , Kneipp, Itzkan, Dasari, & Feld, 1999).
In this experiment, we take advantage of the epsilon near zero (ENZ) used to further amplify the
SERS signal as used in Zamecnik et al. (2013) (Zamecnik, Ahmed, Walters, Gordon, & Walker,
2013). In their study, they achieved brighter SERS nanoprobes through a wavelength matching
approach to couple the excitation source with the resonances of both the plasmonic nanoparticle
as well as a J-aggregate Raman-active dye molecule.
The type of Raman active molecule used in their experiments is called a J-aggregate which is a
well-studied resonant excitonic species (Würthner, Kaiser, & Saha-Möller, 2011). These were
first discovered by Scheibe et al. (1937) (Scheibe, 1937) and Jelley (1936) (Jelley, 1936) whom
independently observed that in aqueous solutions, a dye molecule called pseudoisocyanine
chloride had an absorption maximum red-shift with increases in dye concentration. Furthermore,
the band becomes more intense and sharp. Now the term J-aggregates applies to dyes that have
the ability to aggregate at higher concentrations with resultant red-shifting and narrowing of their
absorption band.
Cyanine dyes like the ones used by Zamecnik et al. (2013) (Zamecnik, Ahmed, Walters, Gordon,
& Walker, 2013) are prime examples of J-aggregates and their ability to self-assemble in
monolayer form on plasmonic nanoparticles and couple its exciton with the surface plasmon for
a significantly enhanced Raman signal is demonstrated in their study.
In this experiment, we apply J-aggregates monolayers in a SERS probe designed to label
lymphoma cells in vitro. It is important to note that experiments in this thesis use gold
nanoparticles for their biocompatibility over silver nanoparticles.
3.1.2 S0271
In this experiment, we use S0271, a thiacyanine dye that self-assembles into a J-aggregate
monolayer with a collective exciton that resonates closely with the surface plasmon of the gold
nanoparticle (Zamecnik, Ahmed, Walters, Gordon, & Walker, 2013). Its absorption spectra can
46
be seen below to portray the formation of the J-aggregate with concomitant shifting and
narrowing of the absorption peak at around 561 nm.
Figure 5 Graph depicting absorption spectra of S0271 dye at differing concentration in
H2O solution. Chemical structure presented in top-right corner.
Experimentally, we’ve also demonstrated this is the case as the low concentration solution of
S0271 produced two main peaks at 472 and 502 nm. In the higher concentration solution where
the J-aggregate formed, there is one major peak at 561 nm instead. The overall absorption of this
J-aggregate peak is also significantly stronger than the peaks of the thiacyanine dye in monomer
form.
47
Figure 6 UV-Vis spectra of S0271 in H2O solution. Dye in monolayer form. Peaks are
labelled.
Figure 7 UV-Vis spectra of S0271 in sodium chloride solution. Dye in J-aggregate form
with J-aggregate peak labelled.
UV-Vis absorption spectra showing this absorption peak from a J-aggregate monolayer formed
on a gold nanoparticle are in a subsequent section comparing lipid and silica encapsulation.
502nm
472nm
-‐0.1
0
0.1
0.2
0.3
0.4
0.5
0.6
0.7
0.8
561nm
-‐0.5
0
0.5
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1.5
2
2.5
3
48
3.1.3 Particle Preparation
60 nm citrate-coated gold nanoparticles are stored at -4ºC prior to use. For every 1 mL of Au
NPs, 5uL of 1mM S0271 dye and 75 uL of 1mM TMAT. S0271 dye is the thiacyanine Raman
active dye molecule. TMAT binds covalently to the gold nanoparticle surface through a
sulfhydryl group and provides a positively charge on its other end to facilitate electrostatic
adsorption of the negatively charged S0271. S0271 dye is solvated in methanol and TMAT is
solvated in distilled water.
First, S0271 is added to the gold nanoparticles followed by addition of TMAT and the solution is
allowed to stir overnight to allow formation of the J-aggregate monolayer. It has been observed
that the reverse order of addition consistently causes particle aggregation and that may be due to
the surface charge being changed by TMAT as it displaces the citrate coating of the gold
nanoparticles. The negatively-charged citrate coating maintains nanoparticle monodispersity by
providing a charge repulsion that is disrupted by TMAT binding. This doesn’t happen if the dye
is added likely due to a shielding effect from the negatively charged dye molecules.
The reaction is allowed to proceed overnight with continuous stirring to allow proper formation
of the J-aggregate monolayer around the gold nanoparticle. After this stage, we can refer to the
particle as a SERS nanoparticle.
Subsequently, the particles are lipid encapsulation through the procedure mentioned previously.
In the case of silica-encapsulated nanoparticles which will be discussed in further detail in a later
section, a silica encapsulation step is performed before lipid encapsulation. For non-silica
encapsulated particles, there is merely a lipid layer above the J-aggregate monolayer.
49
Figure 8 Schematic depicting the main steps in synthesizing the SERS nanoprobes. First
the J-aggregate monolayer is allowed to form (yellow – S0271 dye molecules; black line –
50
TMAT linker molecule). Then a silica layer is produced if the particle is to be silica
encapsulated. Finally, the resultant nanoparticle is given a lipid layer.
After the liposome is formed and excess lipids and other reagents are washed away through the
centrifugation steps, the particles are conjugated to rituximab monoclonal antibodies through the
procedures mentioned previously either via EDC-NHS or maleimide chemistry.
3.1.3.1 TMAT Synthesis
Figure 9 Thiocholine (TMAT) chemical structure
A procedure similar to that of Peng et al. (2009) was used to synthesize TMAT from
acetylthiocholine (Peng, et al., 2009). 500 mg of acetylcholine was solvated in a solution
consisting of 4 mL of 37% HCl and 15 mL of absolute ethanol. The acid hydrolysis reaction was
allowed to proceed over the course of 7 hours with stirring while being refluxed at 100ºC. A
rotary evaporator is used to remove excess solvent and recrystallization was performed in a
solvent system of 0.5 mL distilled water, 5mL isopropanol, and 25 mL ether to remove
impurities. The recrystallization was done using a chilled ice bath for 20 min and the resultant
white product was isolated through filtration. The isolate containing 90% pure TMAT is then
desiccated overnight and stored in a container backfilled with argon gas at -20ºC until further
use.
3.1.4 Cell Labelling
The rituximab bound lipid-encapsulated J-aggregate SERS nanoparticles were used to label LY8
lymphoma cells through a procedure described in an earlier section of this thesis.
51
Figure 10 Dark field image depicting LY8 lymphoma cells labelled with Rituximab
conjugated (via EDC-NHS chemistry) lipid-encapsulated SERS nanoprobes with S0271 J-
aggregate dye. Magnification is 100x.
Figure 11 Raman spectra obtained with 532 nm laser from LY8 lymphoma cells labelled
Rituximab conjugated (via EDC-NHS chemistry) lipid-encapsulated SERS nanoprobes
with S0271 J-aggregate dye. Laser was used at 5% power for 5 accumulations of 5 seconds
each.
52
3.2 Objective 2 - Application of Silica-Encapsulated Nanoparticles for Cell Labelling
3.2.1 Silica Encapsulation
Silica encapsulation has been a method of improving SERS nanoparticle stability as an
alternative to lipid encapsulation. (references) To investigate brighter SERS nanoparticles,
Küstner, B.et al. (2009) demonstrated silica-encapsulation of gold nanoparticles that were
labelled with a self-assembled monolayer (SAM) of Raman labels which conferred greater signal
enhancement (Küstner, et al., 2009). Their reasoning, also described in subsequent experiments
by Scütz, M. et al. (2014), was that high chemical and mechanical stability of silica shells was
particularly advantageous when used in conjunction with SAMs that provide high signal
intensities dependent on complete surface coverage and uniform molecular orientation (Schütz,
Salehi, & Schlücker, 2014).
Lipid encapsulation has been sufficient thus far for maintaining particle stability of the SERS
probes used within this thesis. However, the J-aggregate monolayer which is also an SAM, has
had issues with fluorescence in response to laser excitation to the extent where it overwhelms the
Raman scattering. This is demonstrated in the figure below characteristic of a lipid-encapsulated
J-aggregate SERS nanoprobe that has a Raman spectra overwhelmed by fluorescence.
53
Figure 12 Raman spectra obtained of lipid encapsulated SERS nanoprobe depicting intense
fluorescence spectra masking Raman peaks.
In contrast, silica encapsulation of the J-aggregate monolayer SERS nanoprobe demonstrates
consistently the complete quenching of the previously observed fluorescence. There is also some
further signal enhancement that may have to do with the lipid encapsulation not maintaining the
structure of the SAM well enough.
Figure 13 Raman spectra obtained of silica encapsulated SERS nanoprobe depicting
fluorescence quenching.
It is generally understood that resonant energy transfer (RET) occurs between organic dye
molecules and plasmonic surfaces (Dulkeith, Morteani, Niedereichholz, Klar, & Feldmann).
This prevents the dye from fluorescing which explains the fluorescence quenching effect of
plasmonic materials. This phenomenon has been well studied for metal films but less so in the
case of nanoparticles (Imahori, et al., 2000) (Andrew & Barnes, 2000).
The liposomes used to encapsulate the nanoparticles likely do not pack as densely as a silica
shell. Since fluorescence quenching depends mainly on the proximity of the dye molecule to the
metal surface, there may be free floating dye molecules in the space between the SAM and lipid
layer that are too far from the metal surface to be quenched; they may be the main contributing
factor to the fluorescence. It is important to note that since the SAM is held onto the gold
54
nanoparticle only through electrostatic interactions prior to lipid encapsulation, centrifugation as
a means to remove unbound or excess dye and TMAT is not performed.
While the use of a TMAT linker molecule does create a gap between the dye molecule and the
metal surface in contrast to SERS probes that have directly adsorbed dyes, this is probably not a
significant factor contributing to fluorescence as there have been reproducible lipid-encapsulated
J-aggregate SERS nanoprobes that do not fluoresce.
It is still unclear exactly why the silica shell completely quenches the fluorescence so
consistently, but it is likely due to its denser packing around the SAM.
The UV-Vis absorption spectra of lipid-encapsulated versus silica-encapsulated J-aggregate
SERS nanoprobes are below. In the lipid-encapsulated sample, the first peak at 533 nm
corresponds to the surface plasmon of the gold nanoparticle while the 561 nm peak corresponds
to the J-aggregate of S0271. There is a slight shift of these peaks in the silica-encapsulated
sample to 537 nm and 569 nm respectively. The increased size and density of the particle is the
probably factor involved in this shift.
Figure 14 UV-Vis spectrum of lipid-encapsulated SERS nanoprobe. Surface plasmon peak
at 533nm and J-aggregate peak at 561nm are labelled.
561nm 533nm
0
0.2
0.4
0.6
0.8
1
1.2
55
Figure 15 UV-Vis spectrum of silica encapsulated SERS nanoprobe. Surface plasmon peak
at 537nm and J-aggregate peak at 569nm are labelled.
3.2.2 Silica Encapsulation Procedure
The procedure for silica encapsulation used in these experiments adhered closely to protocols
described by Pastoriza-Santos et al. (2006), and Küstner, B.et al. (2009) 10 mg of
poly(allylamine hydrochloride)(PAH) and 17.5 mg of NaCl were dissolved in 5 mL of water and
then sonicated for 30 minutes (Pastoriza-Santos, Pérez-Juste, & Liz-Marzán, 2006)(Küstner, et
al., 2009). J-aggregate monolayer labelled gold nanoparticles were synthesized in the same
fashion as described in previous sections but are added to the PAH/NaCl mixture instead of
undergoing lipid encapsulation. In particular, we first dilute 5 mL of the particles in 5 mL of
distilled water before adding it to the PAH/NaCl mixture post-sonication and do so in a drop-
wise fashion for a minute.
This mixture is left to stir vigorously for 4 days at room temperature. Then it is centrifuged at
3500 G for 11 minutes, the supernatant is removed, and the pellet is resuspended in 5 mL of
distilled water.
A separate solution is made using 20 mg of polyvinylpyrrolidone-3500 (PVP-3500) dissolved in
5 mL of distilled water. The PAH-functionalized particles are added to the PVP-3500 in a drop-
wise fashion for two minutes.
569nm 537nm
0
0.2
0.4
0.6
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1
1.2
56
The mixture is left to stir vigorously for 2 days at room temperature. Then it is centrifuged at
3500 G for 11 minutes, the supernatant is removed, and the pellet is resuspended in 200µL of
distilled water.
The PAH/PVP-functionalized particles are added to 1 mL of isopropanol in a drop-wise fashion
over 3 seconds. Then it is immediately transferred to 460 µL of water already stirring.
A separate solution composed of 1.375 mL isopropanol and 54.9 µL of 33% aqueous ammonium
hydroxide is then added to the stirring solution of PAH/PVP-functionalized particles.
A separate solution composed of 397 µL isopropanol and 1.94 µL tetraethoxyorthosilicate
(TEOS) is then added to the mixture. This is left to stir further overnight (12 hours).
The particles that are now silica-encapsulated are centrifuged at 3500 G for 11 minutes and re-
suspended in water.
The silica-encapsulated J-aggregate SERS nanoprobes are then lipid encapsulated and antibody-
conjugated with rituximab in the same way we have done for non-silica encapsulated J-aggregate
SERS nanoprobes. The details of this have been explained in earlier sections of the thesis.
3.2.3 Comparison of EDC-NHS and Maleimide Conjugation
As mentioned in the earlier section describing cell labelling, a challenge with the increased
density and size of the silica-encapsulated nanoparticles was the difficulty in removing unbound
particles from cells and bound particles using centrifugation methods.
Thus, the centrifugation times post-incubation were reduced and the ratio of particles to cells was
decreased tenfold. While this reduced unbound free-floating particles, there was naturally also
less particles bound to the cells.
We also explored an alternative antibody cross-linking reaction, maleimide conjugation to
improve on the cell labelling. Thus, comparisons were made between silica-encapsulated
particles conjugated to rituximab by maleimide versus EDC-NHS conjugation. As seen in the
following figures, maleimide conjugation produced significantly higher amounts of particle-to-
cell binding as observed in dark field images.
57
Figure 16 Dark-field image of LY8 cells labelled with antiCD20+ EDC-NHS conjugated
SERS nanoprobes at 100X magnification.
Figure 17 Dark-field image of LY8 cells labelled with antiCD20+ Maleimide conjugated
SERS nanoprobes at 100X magnification.
58
Figure 18 Dark-field image of LY8 cells incubated with control antiCD20- SERS
nanoprobes.
3.3 Objective 3 - Application of Hematological Stains
3.3.1 Hematological Stains
One important challenge in using fluorescent dyes for immunophenotyping is the propensity that
fluorochromes to be photobleach and be damaged by the use of hematological stains and other
harsh chemical environments. Hemotological stains such as Giemsa and H&E staining are
important staples in analyzing lymphomas through morphological analysis of biopsies as well as
characterizing disease through peripheral blood smears.
In this section we look at the capabilities of our SERS nanoprobes to maintain their Raman
signals in the presence of these stains so that they can be used in a combined test of cell surface
marker analysis as well as cell morphology analysis for lymphoma cells.
3.3.2 Effect of Hematological Stains on Raman Spectra
The first experiment was to test the SERS nanoprobes capabilities of withstanding hematological
dyes in solution. We performed tests with hemotoxylin, Giemsa, methylene blue, and eosin on
three SERS nanoprobe types: unprotected, lipid encapsulated, and dual encapsulated with both
lipid and silica.
59
To perform these experiments, first the SERS nanoprobes were synthesized. Then varying
concentrations (none, low (50uL per 1mL gold), and high (1mL per 1mL gold) of hematological
stain were added to the particles and incubated for 30 minutes. Then the solutions were
centrifuged at 3500 G for 11 minutes and resuspended in distilled water. The number of
centrifugation steps varied from 1 to 5 based upon how many washes it took to allow for a clear
resuspension that indicated most of the stain was washed out. In many cases, the high
concentration treatment still retained strong opaqueness from the stain after 5 washes.
These samples were then excited with 532nm laser light to obtain the resultant Raman spectra.
Figure 19 In-depth analysis of SERS nanoparticles incubated in solution with hematoxylin
followed by removal of hematoxylin by centrifugation. Top: no encapsulation of SERS
nanoprobes; Middle: lipid encapsulated SERS nanoprobes; Bottom: dual-encapsulated
(silica and lipid) nanoprobes; Left: control condition (no hematoxylin); Center: low
concentration of hematoxylin (50µL stain per 1mL SERS probes); Right: high
concentration of hematoxylin (1mL stain per 1mL SERS particles). 532nm laser at 5%
power for 1 second exposure with 1 accumulation.
60
This set of figures demonstrates that significant signal is lost when the SERS nanoprobes are
treated with hematoxylin if there is no dual-encapsulation by both silica and lipid. It is important
to note however that in the unprotected and lipid encapsulated SERS nanoprobes, the spectra
without stain already had a strong fluorescent background for reasons mentioned in the previous
section. Thus, the Raman peaks were already relatively difficult to discern even before the
application of hematological stain. It is possible that the unprotected and lipid encapsulated
SERS nanoprobes already have too much signal-to-noise, that the stain doesn’t actually reduce
the signal.
Importantly, the signal remains strong and unaffected by the hematoxylin stain in the dual-
encapsulated condition.
Figure 20 Raman spectra of dual encapsulated (lipid and silica) SERS nanoprobes
incubated at ratio of 1mL to 50µL of hematoxylin. 532nm laser at 5% power for 1 second
exposure with 1 accumulation.
61
Figure 21 Raman spectra of dual encapsulated (lipid and silica) SERS nanoprobes
incubated at ratio of 1mL to 50µL of methylene blue. 532nm laser at 5% power for 1
second exposure with 1 accumulation.
Figure 22 Raman spectra of dual encapsulated (lipid and silica) SERS nanoprobes
incubated at ratio of 1mL to 50µL of Giemsa. 532nm laser at 5% power for 1 second
exposure with 1 accumulation.
As demonstrated by these experiments, only hematoxyline and methylene blue treatments did not
significantly reduce the signal of dual-encapsulated SERS nanoprobes. Giemsa and Eosin both
eliminated the Raman signal entirely. Note that the spectrum of Eosin treatment on the SERS
nanoprobes could not be obtained as the fluorescence background was so strong it immediately
oversaturated the detector.
One important consideration is that the stains are incredibly hard to remove from solution. Thus,
it is uncertain as to whether the Raman signal is lost or simply masked by the fluorescence of the
62
stain. In the experiments with the incompatible stains, there was always a presence of strong
fluorescence as opposed to a complete lack of overall signal. This is elaborated on further in the
discussion section.
3.3.3 Cell Labelling Experiments
Figure 23 Top: Raman mapping of signal-to-base line intensity of dual peaks bounded by
the two vertical lines in the Raman spectra (Bottom) of the brightest spot on the map.
Bottom: Raman spectra of the brightest spot on the Raman map (top) taken with 532 nm
laser at 5% laser intensity for 1 second with 1 accumulation. Experimental sample is LY8
cells labelled with antiCD20+ silica-encapsulated SERS nanoprobes.
63
Figure 24 Top: Raman mapping of signal-to-base line intensity of dual peaks bounded by
the two vertical lines in the Raman spectra (Bottom) of the brightest spot on the map.
Bottom: Raman spectra of the brightest spot on the Raman map (top) taken with 532 nm
laser at 5% laser intensity for 1 second with 1 accumulation. Experimental sample is LY8
cells labelled with control antiCD20- silica-encapsulated SERS nanoprobes.
64
Figure 25 Top: Raman mapping of signal-to-base line intensity of dual peaks bounded by
the two vertical lines in the Raman spectra (Bottom) of the brightest spot on the map.
Bottom: Raman spectra of the brightest spot on the Raman map (top) taken with 532 nm
laser at 5% laser intensity for 1 second with 1 accumulation. Experimental sample is LY8
cells labelled with antiCD20+ silica-encapsulated SERS nanoprobes stained with
hematoxylin.
65
Figure 26 Top: Raman mapping of signal-to-base line intensity of dual peaks bounded by
the two vertical lines in the Raman spectra (Bottom) of the brightest spot on the map.
Bottom: Raman spectra of the brightest spot on the Raman map (top) taken with 532 nm
laser at 5% laser intensity for 1 second with 1 accumulation. Experimental sample is LY8
cells labelled with antiCD20+ silica-encapsulated SERS nanoprobes stained with methylene
blue.
66
Figure 27 Microscopy images of LY8 cells incubated with silica-encapsulated SERS
nanoprobes with different hematological stains applied; Top: No stain; Middle:
Hematoxylin stain; Bottom: Methylene Blue stain. Left: Darkfield image of labelling with
antiCD20+ SERS nanoprobes; Middle: Brightfield image of labelling with antiCD20+
SERS nanoprobes; Right: Darkfield image of labelling with control antiCD20- SERS
nanoprobes.
These results show that the stains that were shown to be compatible with our SERS nanoprobes
in the earlier non-cellular experiments remain compatible when applied to lymphoma cells
labelled with the nanoprobes.
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Chapter 4 - Discussion and Future Work 4
4.1 Discussion of Objective 1 Results
The results from these experiments show the successful targeting of LY8 lymphoma cells
expressing CD20 cell surface markers via rituximab functionalized SERS nanoprobes employing
S0271 J-aggregate dye. To date, this is the first successful use of J-aggregate monolayer dyes in
labelling lymphoma cells.
Compared to other types of SERS nanoprobes, J-aggregate monolayers theoretically have a far
stronger enhancement factor and this has been demonstrated prior to antibody functionalization
(Zamecnik, Ahmed, Walters, Gordon, & Walker, 2013).
One recurrent disadvantage, however, is the strong fluorescence background produced by the
S0271 dye that often masks the desired Raman peaks. While several samples of SERS probes
have been synthesized without presence of the background, this is fairly inconsistent. Thus, the
next step in improving the design of these particles is to develop a way to mask the fluorescent
background.
4.2 Discussion of Objective 2 Results
These experiments demonstrate that dual-encapsulation of SERS nanoprobes with silica and
lipids offers both a stronger Raman signal as well as consistently quenches fluorescent
backgrounds compared to only lipid encapsulated SERS nanoprobes. This is both demonstrated
with particles in solution as well as after cell-labelling as will be shown in the next section of the
thesis.
It has also been demonstrated that there is an additional challenge presented by using silica-
encapsulated particles for cell studies as the density of the particles make it difficult to separate
unbound particles from the cells and bound particles. The strategies used in these experiments
were to reduce centrifugation times and lower the particle-to-cell ratio which showed success.
However, there are certainly many other separation techniques that can be attempted in future
experiments.
68
Usage of maleimide conjugation chemistry has also been demonstrated to be far more successful
than EDC-NHS conjugation chemistry for the purposes of our cell labelling studies using the
specific labelling probes investigated in these experiments.
4.3 Discussion of Objective 3 Results
These experiments mainly demonstrate a proof of concept that under in vitro conditions of
lymphoma cell-line labelling with hematological staining, the dual-encapsulated J-aggregate
monolayer SERS nanoprobes retain intensely bright Raman scattering signals.
While only two of the four stains used did not interfere with the SERS spectra, we postulate
important trends from the results: it appears that the stains that are incompatible do not
significantly interfere with the SERS nanoprobe or its spectra directly. We have demonstrated
that there are stains that have no significant effect on the SERS signal as exemplified by
hemotoxylin and methylene blue so there is certainly the potential for combining SERS probe
immunophenotyping with morphology studies of lymphoma cells.
If we assume the predominant reason eosin and Giemsa were incompatible with the SERS
nanoprobes is fluorescence of the stains overwhelming the Raman signal and believe the Raman
signal remains intact underneath that noise, we can hypothesize ways to develop and use Raman
nanoprobes in which there won’t be interference. The resonance wavelengths of plasmonic
nanoparticles are tunable through both the size and metal composition. Similarly, since we are
interested in wavelength matching, we can tune the resonance wavelength of dyes through choice
of molecule. If we match those to an incident laser wavelength that does not excite the stain, we
may have produced a compatible SERS nanoprobe.
In the specific case of methylene blue, we can see in the non-cell experiments that the spectra
begin to rise right near the end of the Raman spectra. This also suggests the possibility that stains
that fluoresce can still be compatible if their emission spectra does not coincide with the relevant
Raman peaks.
69
4.4 Summary of Thesis and Future Directions
This project has demonstrated that J-aggregate monolayers previously shown to enhance SERS
signals to produce exceptionally bright nanoprobes can be successfully applied as an antibody-
conjugated SERS nanoprobe for binding specifically to cell surface markers in in
vitro lymphoma cancer models.
Silica encapsulation is a necessary part of the particle synthesis to quench fluorescence, further
strengthen the Raman signal, and improve robustness. We have shown that, although some
challenges arise from the increased size and density of the particle itself in addition to the dual
encapsulation with another liposome layer, the silica encapsulated SERS nanoprobes confer all
the aforementioned advantages to the same in vitro experiments. Having evidence that SERS
particles with this degree of signal enhancement can successfully be used to label lymphoma
cells gives great promise to the practical use of SERS nanoprobes in actual diagnostic settings.
Furthermore, the experiments with hematological stains demonstrate that our particles can retain
their signal strength when treated with certain stains and consequently have the potential to be
used in cell labelling experiments using said stains. This is both demonstrated in solution and on
actual cell slides in the case of hematoxylin and methylene blue. While this could not be shown
with Giemsa and eosin, our results suggest a general pattern regarding what circumstances allow
for compatibility of SERS nanoprobes and particular stains: the absence of Raman signal is
likely an indirect effect caused by masking by an overpowering fluorescence of the dye. If this is
true, such incompatibilities can be circumvented by designing particles with resonance
wavelengths outside of the range in which the stain molecule is also excited. This idea could be
tested through tuning SERS nanoprobes resonant wavelengths to be far from the absorbance
spectras of the stains using different metalloid compositions in conjunction with cyanine dyes
with J-aggregate capabilities that also resonate at that wavelength.
There are many other cyanine dyes that exist that can produce the J-aggregate effect and it is
certainly worthwhile expand the repertoire of SERS nanoprobes, especially if they consistently
produce such strong Raman signals. This can allow multiplexing using gold nanoparticles with
different J-aggregate reporter molecules that resonate around the laser wavelength of 532 nm but
70
can also include the previously mentioned metalloid nanoparticles that have greater flexibility in
terms of resonant frequencies and consequently applicable dyes.
The usage of Raman mapping and production of signal heat maps opens the possibility of
analyzing cell surface markers at a higher resolution: specifically, characterizing receptor
distribution and clustering over the cell surface. This can potentially allow us to study lipid rafts
which are signaling microdomains and also be used to study the changes in receptor location in
response to certain signaling pathways.
Overall, this project generated the data that may serves as a stepping stone in the continual
development of SERS nanoprobes for immunophenotyping. It has shown J-aggregate
monolayers in conjunction with silica encapsulation as the proper direction if we are to produce
signals strong enough to compete other optical labels. Small changes can be explored to further
improve on this model such as direct silica-to-antibody conjugation to bypass the need of the
additional liposome layer that may be redundant. Application of these particles to tumor models
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