Nanoencapsulation of quantum dot nanoparticles in biological labeling

SYNTHESIS OF QUANTUM DOT-POLYMER
NANOPARTICLES FOR BIOLOGICAL LABELING

HUANG NING
(B.Eng)

A THESIS SUBMITTED
FOR THE DEGREE OF MASTER OF SCIENCE
GRADUATE PROGRAM IN BIOENGINEERING
NATIONAL UNIVERSITY OF SINGAPORE
2005


Acknowledgements

The successful completion of this thesis would not have been possible without
the help of a handful of people whom I would like to thank here. First of all, I have
been indebted in many ways to my thesis supervisor, Dr Zhang Yong, whose patience
and kindness have been invaluable to me. I would also like to thank Dr. Yang
Xiaotun, research fellow of our lab. This project has benefited tremendously from
their diligent guidance and sound advice. I am also grateful to the many laboratory
officers whose assistance has been indispensable; in particular, Miss Satinderpal Kaur
of the Biochemistry Lab and Miss Xu Xiaojing of the TEM. My fun-loving friends in
the group have also made my stint in the lab a truly enjoyable and memorable one.
Lastly, I would like to acknowledge the immense support as well as strong
encouragement of my family and close friends that accompanied me along the way.

ii


Summary

Quantum Dots (QDs) have attracted considerable interest in luminescence tagging
due to their unique optical and electronic properties. The ideal optical properties of
QDs offer the possibility of using them as fluorescent probes in biological staining
and diagnostics. But some problems associated with QDs, for example, poor water
solubility, poor biocompatibility and chemical stability in physiological media, limit
their use in biomedical applications. Though some efforts have been made to prepare
QD-tagged microbeads, the sizes of the beads are above 100 nm, which make them
not suitable for labeling of subcellular components. In this work, fluorescent QDs
were incorporated into polystyrene nanoparticles using emulsion polymerization
method and particle sizes were controlled. The nanoparticles have carboxyl groups on
the surfaces that can be used for further attachment of biomolecules. After surface
modification with folic acid, the intracellular delivery of the nanoparticles into NIH3T3 and HT-29 cells was investigated using confocal microscope. The QD encoded
nanoparticles are suitable for staining or labeling of subcellular components or
intracellular measurements due to their very small size.

Keywords: Quantum dots; Nanoparticles; Polystyrene; Surface modification

iii


Table of Contents

Page
Acknowledgements

ii

Summary

iii

List of figure

vii

Chapter 1 Introduction

1

1.1

Introduction

1

1.2

Aims and Objectives

3

Chapter 2 Literature Review

4

2.1

Introduction to Fluorescent Labeling

4

2.2

Fluorescent Materials

6

2.2.1

Flurescent Proteins

6

2.2.2

Organic Fluorophores

8

2.2.3

Lanthanide Chelates

12

2.2.4

Nanoparticles

15

2.3

2.2.4.1 Optically Active Metal Nanoparticels

15

2.2.4.2 Quantum Dots

16

2.2.4.3 Latex Nanospheres

22

Reference

23

iv


Chapter 3 Materials and Methods

32

3.1

Chemicals

32

3.2

Synthesis of CdSe

33

3.3

Synthesis of CdSe/ZnS

34

3.4

Synthesis of Polystyrene Encapsulated CdSe/ZnS Nanoparticles

35

3.4.1

35

3.4.2

Synthesis of Polystyrene Encapsulated CdSe/ZnS with COOH
Group on the Surface
Surface Modification with Folic Acid

36

3.5

Cell Culture

37

3.6

Characterization

38

3.6.1

Fluorescent Microscopy

38

3.6.2

Confocal Laser Scanning Microscopy

38

3.6.3

Transmission Electron Microscopy (TEM)

38

3.6.3

Fourier Transform Infrared (FT-IR)

39

3.6.4

UV Spectrometry

39

3.6.5

Fluorescent Spectrometry

39

3.7

Reference

41

Chapter 4 Experimental Results and Discussions

42

4.1

Synthesis of CdSe

42

4.2

Synthesis of CdSe/ZnS

47

4.3

Synthesis of Polystyrene Particles

50

4.4

Surface Modification of the Nanoparticles with Folic Acid

55

4.5

Intercellular Delivery of Nanoparticles

58

v


4.6

Reference

63

Chapter 5 Conclusions

69

Appendix

71

vi


List of Figures

Page
Fig. 2.1

Wild type GFP chromophore, consisting of a cyclized tripeptide
made of Ser65, Tyr66, and Gly67

7

Fig. 2.2

The structure of dicarbocyanines dyes

9

Fig. 2.3

Structure of thiazole orange (TO)

10

Fig. 2.4

Structure of oxazole yellow (YO)

10

Fig. 2.5

Structure of Cy 3 bis functional dye

11

Fig. 2.6

Structure of Cy 5 monofunctional dye

11

Fig. 2.7

Structure of representative chelates

14

Fig. 3.1

The schematic of the synthesis of CdSe

33

Fig. 3.2

Diagram of synthesis of polystyrene/quantum dot nanocomposites
conjugated with carboxyl

35

Fig. 4.1

TEM image of CdSe

43

Fig. 4.2

The absorbance spectrum of CdSe

43

Fig. 4.3

The emission spectrum of CdSe

44

Fig. 4.4

The compared emission spectra of samples collected at different
reaction time

45

Fig. 4.5

Comparison of emission spectra of the quantum dots at different
coating stage

48

Fig. 4.6

TEM image of CdSe/ZnS

49

Fig. 4.7

TEM images of PS@CdSe/ZnS micro/nano particles of different

sizes

52

Fig. 4.8

The optical (a) and fluorescence (b) images of PS@CdSe/ZnS
micro-sized particles

52

vii


Fig. 4.9

TEM image of nano-sized PS@CdSe/ZnS

53

Fig. 4.10 FT-IR spectra of (a) PS@CdSe/ZnS with COOH on the surface
and (b) PS@CdSe/ZnS without surface modification

54

Fig. 4.11 FTIR spectra of (a) PS@CdSe/ZnS nanoparticles, (b) pure folic
acid and (c) folic acid modified PS@CdSe/ZnS nanoparticles

57

Fig. 4.12 Confocal images of NIH-3T3 cells after cultured with (a)
unmodified and (b) folic acid modified nanoparticles and(c, d)
their corresponding bright field images

58

Fig. 4.13 Confocal images of HT-29 cells after cultured with folic acid
modified nanoparticles for (b) 1 hours and (c) 3 hours, and(a) the
bright field images.

59

Fig. 4.14 Bright field and confocal images of HT-29 cells after cultured
with folic acid modified nanoparticles. The bright field image was
taken after culture for 30 mins and the confocal images were
taken every 30 mins

61

Fig. 4.15 Bright field and confocal images of HT-29 cells after cultured
with unmodified nanoparticles. The bright field image was taken
after culture for 30 mins and the confocal images were taken
every 30 mins

61

viii


Chapter 1. Introduction

Chapter 1. Introduction

1.1 Introduction
Semiconductor quantum dots (QDs) have good potential for use as fluorescent
probes in biological staining and diagnostics. Compared with conventional organic
fluorophores, QDs have a strong fluorescence emission and narrow, symmetric
emission spectrum, and are photochemically stable. QDs also exhibit a wide range of
size-tunable colors and a series of different-colored dots can be activated using a
single laser. The ideal optical properties of QDs offer the possibility of using them to
tag biomolecules in ultra-sensitive biological detection based on optical coding
technology. Some techniques have been developed to incorporate QDs into polymer
beads, to solve the problems relating to QDs’ surface chemistry such as water
solubility, biocompatibility, chemical stability in physiological media, etc., or to pack
different combinations of QDs and produce QD encoded polymer beads; for example,
create QD bar codes and the use of six colors and 10 intensity levels can theoretically
encode one million biomolecules. However, the QD encoded polymer beads that have
been reported so far are above 100 nm, therefore they are very useful for multiplexed
bioassays, but not suitable for staining or labeling of subcellular components or
intracellular measurements due to the relatively big size of the beads. In addition, it is
difficult to produce uniform beads and to control the number of QDs incorporated.
These drawbacks severely limit their applications in biological labeling.

1


Chapter 1. Introduction

In this work, luminescent CdSe/ZnS QDs were incorporated into polystyrene
(PS) nanoparticles grafted with carboxyl groups using emulsion polymerization

method and separation of nanoscale QD encoded PS particles (30 nm) were
performed through centrifugation at high speed in viscous solution. The nanoparticles
were further surface modified with folic acid and their intracellular delivery into NIH3T3 and HT-29 cell lines was investigated using confocal laser scanning microscope.
The longevity of QDs allows us to track the intracellular delivery of the nanoparticles
over a certain time period.

2


Chapter 1. Introduction

1.2 Aims and Objectives
Based on the previous literatures, it is envisaged that hydrophobic quantum dots
can be rendered water-soluble by surface modification with thiolates or encapsulation
into hydrophilic polymeric nanoparticles. The encapsulation of quantum dots into
polymer nanoparticles has some advantages over the surface modification with
thiolates.

In this work, our goal is to prepare nano-sized quantum dot-polymer
nanocomposites, which are water soluble and chemically stable, and have functional
groups for further attachment of biomolecules, to study the intracellular delivery of
nanoparticles. There are two specific aims:

Specific aim 1: To prepare CdSe/ZnS-polystyrene nanoparticles with carboxylic
groups on the surface. The nanoparticles should be water soluble and some
biomolecules can be attached to the surface of the nanoparticles via the reaction with
carboxylic groups.

Specific aim 2: To attach folic acid to the nanoparticles and to study their
intracellular delivery. The nanoparticles modified with folic acid are expected to

target cancer cells with folate receptors expressed on the cell membrane. The delivery
process will be studied using confocal fluorescence microscope.

3


Chapter 2. Literature Review

Chapter 2. Literature Review

2.1 Introduction to Fluorescent Labeling
The demands to analyze biomolecules such as polypeptide, proteins and nucleic
acid result in some biological analytical systems, for example, biosensors. However,
organism lacks sensitive detectable signals. Researchers have begun to find a new
way and an idea of introducing labels into analytes has been proposed. American
scientists Yalow and Berson first discovered radioimmunoassay (RIA) [1]. Though
RIA has many advantages, such as their sensitivity and wide application, it does have
drawback such as the side-effect of radioactive isotope to the health of the operators
and the limitation of its lifetime [2]. Therefore, new techniques emerged, among
which fluorescent labeling is the most widely used [3, 4].

Fluorescence is the property of some atoms and molecules. It is a process in
which a fluorophore absorbs a suitable-energy light quantum (a photon) to raise an
electron from an occupied orbital to a higher energy vacant orbital, followed by the
electron returning back to the original ground state energy level, and emitting a
quantum of light with an energy corresponding to the energy difference between the
excited state and the ground state level [5]. In short, the atoms or molecules absorb
light of a particular wavelength and after a brief interval, termed the fluorescence
lifetime, to emit light at longer wavelength. Fluorescence is proportional to the
amount of light absorbed and quantum yield.

4


Chapter 2. Literature Review

Fluorescent labels represent one of the most widely developing fields in biology
and medicine. Their application potential is enormous. Labels are dyes that are
environmentally sensitive and can be used as molecular reporters. The information
about what is happening in their molecular environment can be derived from their
fluorescence signal and their location inside cells can be determined from fluorescent
microscopy images [6-10]. The availability of sensitive and selective fluorescent
probes for living cells has opened new horizon in cell biology. With the aid of the
modern microscopy, fluorescent labeled organelles and molecules can be visualized
and measured.

The key problem is to specifically target the molecules or cells by the
appropriate label. There are some materials that can be utilized as fluorescent labels.

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Chapter 2. Literature Review

2.2 Fluorescent Materials
2.2.1 Fluorescent Proteins
The most popular member of fluorescent proteins family is Green fluorescent
protein (GFP) which was originally isolated from the light-emitting organ of the
jellyfish Aequorea Victoria by Shimomura et al. in 1962 [11] as a companion protein
to aequorin, the famous chemiluminescent protein from Aequorea jellyfish. In a

footnote to their account of aequorin purification, it is said that “a protein giving
solutions that look slightly greenish in sunlight through only yellowish under tungsten
lights, and exhibiting a very bright, greenish fluorescence in the ultraviolet of a
Mineralite, has also been isolated from squeezates.” This description of the discovery
of GFP solutions is still accurate. The same group soon published the emission
spectrum of GFP, which peaked at 508 nm [12].

The discovery and development of GFP, a -barrel-shaped protein that contains
an amino-acid triplet (Ser-Tyr-Gly) that undergoes a chemical rearrangement to form
a fluorophore [13], have enabled the study of protein localization, dynamics and
interactions in living cells [14]. Virtually any protein can be tagged with variants of
GFP. The currently known GFP variants may be divided into seven classes based on
the distinctive component of their chromophores: 1), wild-type mixture of neutral
phenol and anionic phenolate; 2), phenolate anion; 3), neutral phenol; 4), phenolate
anion with stacked¼-electron system; 5), indole; 6), imidazole; and 7), phenyl. Each
class has a distinct set of excitation and emission wavelengths [14].

6


Chapter 2. Literature Review

Figure 2.1 Wild type GFP chromophore, consisting of a cyclized tripeptide made of
Ser65, Tyr66, and Gly67.

Biological applications of GFP may be mainly in two areas, being a tag or an
indicator. In tagging applications, the majority to date, GFP fluorescence can reflect
levels of gene expression or subcellular localizations. As an indicator, GFP
fluorescence can also be modulated post-translationally by its chemical environment
and protein-protein interactions [14]. The first proposed application of GFP was to

detect gene expression in vivo [15]. The most successful application is using GFP as
a genetic fusion partner to host proteins. By genetic engineering, GFP can be fused as
a tag to the protein of interest, often without altering the function of the protein. As
GFP is spontaneously fluorescent, chimeric GFP fusions give the great advantages
that they can be expressed in situ by gene transfer into cells. The resulting chimera
often remains parent-protein targeting and function when expressed in cells [14].

The fluorescent proteins have been used as tools in numerous applications,
including as markers to track and quantify individual or multiple protein species [16],
as probes to monitor protein-protein interactions [14, 17, 18], and as biosensors to
describe biological events and signals [14, 17]. Moreover, they have provided an
important new perspective to investigate protein function.

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Chapter 2. Literature Review

Besides the bright side of the fluorescent proteins, their molecular mass (~26
kDa for GFP) might be problematic, due to interference with expression and folding
of the labeled protein, and with interactions between molecules. A further problem is
the potential aggregation of the fluorescent proteins, which impedes any cellular
application and maybe leads to cellular toxicity [19].

2.2.2 Organic Fluorophores
Organic fluorophores is amongst the earliest types of fluorescent labels used in
biology. In the past decades many fluorescence dyes have been developed to label
biomolecules. Among them, flouresceins, rhodamines, trimethine cyanines (Cy3), and
pentamethine cyanines (Cy5) are available commercially. In the early times, the most
popular fluoresceins are fluoresein-5-isothiocyanate (FITC) and rhodamine 6G.

However, because of their low Stokes shift, short fluorescent lifetime, and the similar
emission wavelength to that of the background, these conventional fluorophores are
limited in use and hard to characterize.

Cyanine dyes are widely used for application due to their versatile properties.
Their absorption range can readily be predicted [20-24]; their fluorescence properties
make them useful as laser dyes [25], as mode-locking dyes and as fluorescence
probes in molecular biology [26, 27]. Their respective structures are shown in figure
2 [28-33]. The range of their maximum absorption is between 600-800 nm, some
even exceed 800 nm. When in aqueous solution, their quantum yields are very low.
After binding to the analytes, the system has changes in the absorption wavelength

8


Chapter 2. Literature Review

and emission wavelength. The important change is the increased lifetime. Up to now,
these reagents have been successfully used in the field of immunoassay and have
received some attention in the area of separation science [34, 35].

Cyanine dyes can be divided into two classes: one which include the
asymmetric cyanines thiazole orange (TO), oxazole yellow (YO), a dimer of both TO
and YO (TOTO, YOYO) and polymethine dyes such as Cy 3 and Cy5.

Figure 2.2 The structure of dicarbocyanines dyes.

In 1992 Rye et al. [36] first described two novel, highly fluorescent dimeric
symmetric cyanine dyes namely the thiazole orange dimer (TOTO) and the oxazole
yellow dimer (YOYO; an analog of TOTO). Asymmetric cyanines like TO and YO

do not fluoresce in solution but possess intense fluorescence when bounded to nucleic
acids [37]. For example, Cyanine dimers significantly enhanced their fluorescence on
binding to dsDNA 1100-fold for TOTO and 3200-fold for YOYO [36, 38].The
fluorescent intensity is believed to arise when the rotation around the bond between

9


Chapter 2. Literature Review

the aromatic systems is restricted, which closes a channel for non-radioactive decay.
TO has an impressive fluorescence enhancement of about 3000-fold upon binding to
nucleic acids [39]. However, its affinity for nucleic acids is relatively low.

Figure 2.3 Structure of thiazole orange (TO).

Figure 2.4 Structure of oxazole yellow (YO).

Polymethine dyes are a large class of compounds, which have been extensively
used in a wide range of applications, from lasers [40] and photography [41] to
diagnostic by fluorescent detection [42]. The most general structure of these dyes is
characterized by a conjugated (polymethine) chain terminated at each end by a
heterocyclic moiety. In general, polymethine dyes have poor resistance to light
compared with other fluorophores, but many structural modifications improving
lightfastness and resistance can be made [43]. That is typically obtained by the
insertion of a stabilizating cyclic unit, for instance chlorobenzene, in the conjugating
chain of the fluorophore.

10

Chapter 2. Literature Review

Polymethine dyes are highly fluorescent and they absorb and emit light mostly
in the visible region of the optical spectrum, as a function of the chain length and of
the terminating moieties. It is known that, at first, elongation of the polymethine chain
increases the fluorescence quantum yields, but, as it grows further, it causes a
decrease of the efficiency [44].

Polymethine dyes like Cy3 and Cy5 have been widely and routinely used in the
labeling of biological compounds such as antibodies, nucleic acids, lipids and other
amino groups-containing materials [45, 46] and a popular choice of fluorescent
probes in microarray technology [47]. The Cy 3 dye is an orange fluorescing cyanine
that produces a signal easily detected using a fluorescein filter set while the Cy 5 dye
produces an intense signal in the far red region of the spectrum.

Figure 2.5 Structure of Cy 3 bis functional dye.

Figure 2.6 Structure of Cy 5 monofunctional dye.

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Chapter 2. Literature Review

However, organic fluorophores suffer the disadvantage of generally having a
small Stokes shift, short life, poor stability and their fluorescence is always rapidly
quenched. Efforts have also been made to synthesize new classes of cyanine dyes
with more desirable and better fluorescent properties.

2.2.3 Lanthanide Chelates
Lanthanide chelates have attracted great interest because of their important roles
in the study of luminescent materials and biological systems. In general, excellent
luminescence properties of lanthanide chelates are attributed to the intramolecular
energy transfer between the ligands and chelated metal, that is, the absorption of
excitation energy mainly take place in the organic part of chelate ligands (“antenna
effect”) instead of the bound central lanthanide ion [48].

Lanthanide chelates with aromatic carboxylic acids are frequently used as
structural and functional probes in biological systems [49, 50]. The sensitized
luminescence of lanthanide ions by salicylic acid plays an important role in the
analyses of trace salicylic acid and its derivatives in biological systems, and in
fluorimetric immunoassay [51-53]. On the other hand, they are a kind of potential
luminescent materials for further application. There has been a growing interest in the
study of the luminescence property of such chelates.

The specific physical and chemical properties of lanthanides which make them
so useful in studies of biochemical systems are dependent on their electronic structure.

12


Chapter 2. Literature Review

The electrons of the 4f shell of the lanthanide atoms and their trivalent ions are
shielded by electrons from higher shells (5s, 5p) and thus protected from the influence
of the environment. Owing to this, lanthanides in different chemical combinations
show the same spectroscopic properties (related to the 4f electronic transitions) as
their free ions in the gas state. The electronic transitions in the 4f shell are responsible
for the characteristic absorption and luminescence spectra of very narrow bands

(small half-intensity width) and the long lifetime of the excited states (the order of
milliseconds).

The most important role in biochemical studies falls to Eu(III) and Tb(III) ions,
because of the suitable energy gap between the lowest emission level and the ground
state. The Gd(IlI) ion is characterized by a highest excited state (6P7/2) which lies
above the excited states of the majority of ligands.

Figure 2.7 shows four prototypical luminescent lanthanide probes. All contain
an organic chromophore, which serves as an antenna or sensitizer, absorbing the
excitation light and transferring the energy to the lanthanide ion. An antenna is
necessary because of the inherently weak absorbance of the lanthanide (1 M-11cm-1,
or 104--105M-1cm-1 smaller than conventional organic fluorophores). The complexes
also contain a chelate that serves several purposes, including binding the lanthanide
tightly, shielding the lanthanide ion from the quenching effects of water, and acting as
a scaffold for attachment of the antenna and a reactive group, the latter for coupling
the chelate complex to biomolecules.

13


Chapter 2. Literature Review

Figure 2.7 Structure of representative chelates.

Their spectral characteristics include millisecond lifetime, sharply spiked
emission spectra, large Stokes shifts, high quantum yield, and good solubility. These
properties make them useful alternatives to radioactive probes and to organic
fluorophores, particularly where there are problems of background autofluorescence
[54, 55], and as donors in luminescence resonance energy transfer experiments [5658].

However, the fluorescent properties of lanthanides are dependent on their ability
to form complexes with the chelating agents. Commonly used chelating agents are
restricted to EDTA, DTPA and -diketonates. Recent development in the research of
lanthanides as fluorophores has yielded new classes of lanthanide complexes with
better properties.
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Chapter 2. Literature Review

2.2.4 Nanoparticles
The integration of nanotechnology with biology and medicine is expected to
produce major advances in medical diagnostics, therapeutics and bioengineering [59].
With the development of this nanoscience, the nanomaterials have numerous
applications. Many papers have reported nanometer-sized luminescent particles
linked to DNA or proteins as the detection probe. Typically, the wet-chemical
preparation of the nanoparticles is carried out in the presence of stabilizing agents
(often citrate, phosphanes, or thiols) which bind to the atoms exposed at the surface of
the nanoparticles. This capping leads to a stabilization and prevents uncontrolled
growth and aggregation of the nanoparticles. In the case of a labile capping layer such
as citrate, biomolecules can be linked directly with the nanoparticles. The
nanoparticles are usually highly sensitive which makes single-molecule detection
(SMD) possible. Three types of nanoparticles are potentially useful as singlemolecule probes, in particular, latex nanospheres, optically active metal nanoparticles
and luminescent quantum dots. Using nanoparticles as probes in bioanalysis offers
several potential advantages. First, suspensions of nanoparticles do not appreciably
scatter light. Second, the low background results in low detection limits. In addition,
nanoparticles form more stable suspensions and are therefore less susceptible to selfagglomeration.

2.2.4.1 Optically Active Metal Nanoparticles

15


Chapter 2. Literature Review

It is found that direct adsorption of proteins, such as enzymes, onto bulk metal
surfaces frequently results in denaturation of the protein and loss of bioactivity. In
contrast, when such proteins are adsorbed onto metal nanoparticles, bioactivity is
often retained. Crumbliss [60] found that they could adsorb redox enzymes to
colloidal gold without loss of enzymatic activity. In addition, Natan and co-workers
[61] found that cytochrome c retained reversible cyclic voltammetry when deposited
onto 12 nm diameter gold particles attached to a conductive substrate. These results
demonstrate another unique feature of metal nanoparticles-biocompatibility.

The optical active metal nanoparticles are widely used in the technique of
surface enhanced Raman spectroscopy (SERS). This concept was first introduced by
Van Duyne et al. [62], showing that attomole mass sensitivity could be achieved
using micrometer-sized sampling areas. The key to using SERS as an analytical
technique is to obtain a reproducible and uniform surface roughness. Natan’s group
has studied SERS surfaces prepared by self-assembling gold and silver nanoparticle
on glass and other substrates. Such surfaces have shown excellent reproducibility, for
both different locations on a single surface and different but identically prepared
surfaces [63]. If binding with biological molecules, use of Au nanoparticle tags leads
to a more than 1000-fold improvement in sensitivity [64].

2.2.4.2 Quantum Dots
Current enhanced security and health concerns worldwide have placed a large
burden on the scientific community to rapidly develop numerous biodetection assays.
16

Chapter 2. Literature Review

One priority is in developing and delivering rapid, robust, simple and efficient assays
for detecting pathogens in various media to provide not only environmental
monitoring but also assurance of food and water supplies [65]. These assays and their
reagents will need to be stable in many environments for long periods of time. The
ability to screen for multiple analytes in a single assay offers many advantages to
answer this demand, including higher throughput compared with single-target
systems, decreasing sampling errors, and easy inclusion of internal controls, as well
as savings in reagents and consumables. Fluoroimmunoassay using conventional
fluorophores can be multiplexed to screen for multiple analytes simultaneously [66].
However, when conventional organic dyes are used, multicolor analysis of more than
one or two colors is often complicated by the requirement of an elaborate excitation
and detection scheme and challenging data collection and analysis [67, 68].

Colloidal semiconductor nanocrystals, often referred to as “quantum dots” or
“QDs”, have the potential to overcome problems encountered by organic fluorescent
dyes in certain fluorescent tagging applications by combining the advantages of high
photobleaching threshold, good chemical stability, and readily tunable spectral
properties. Colloidal quantum dots used most often to date in fundamental or applied
studies are spherical nanocrystals with core sizes that vary between 15 and 120 Å in
diameter [69-71]. Their optical and electronic properties are dominated by carrier
confinement (electron/hole), which results in size dependence of their optical
properties, including light absorption, photoluminescence (PL), electroluminescence
(EL) [72-76], and cathodoluminescence (CL) [77, 78]. Nanocrystals composed of

17