Rational design of nanoparticles for biomedical imaging and photovoltaic applications ... › smash › get › diva2:414548 › FULLTEXT01.pdf · 2011-05-03 · Turro, Marlin Halim,

Rational design of nanoparticles for biomedical imaging and photovoltaic applications

Haiyan Qin

秦海燕

Doctoral Thesis

Theoretical Chemistry and Biology

Royal Institute of Technology

Stockholm, Sweden 2011

ISSN 1654-2312 ISBN 978-91-7415-973-8

Thesis for the degree of Doctor of Philosophy in Biotechnology to be presented with due permission of Royal Institute of Technology for public examination and criticism on Tuesday May 24th in FB52, Albanova University Center, KTH, Stockholm

Copyright © Haiyan Qin Stockholm 2011

Department of Theoretical Chemistry and Biology School of Biotechnology Royal Institute of Technology SE-106 91 Stockholm Sweden

Tryck: Universitetsservice US AB

To my family…

i

Abstract

This thesis aims to rationally design nanoparticles and promote their applications in biomedical imaging and photovoltaic cells.

Quantum dots (QDs) are promising fluorescent probes for biomedical imaging. We have fabricated two types of MSA capped QDs: CdTe/ZnSe core/shell QDs synthesized via an aqueous method and CdTe QDs via a hydrothermal method. They present high quantum yields (QYs), narrow emission band widths, high photo- and pH-stability, and low cytotoxicity. QD-IgG probes were produced and applied for labeling breast cancer marker HER2 proteins on MCF-7 cells.

For the purpose of single molecule tracking using QDs as fluorescent probes, we use small affibodies instead of antibodies to produce QD-affibody probes. Smaller QD-target protein complexes are obtained using a direct immunofluorescence approach. These QD-affibody probes are developed to study the dynamic motion of single HER2 proteins on A431 cell membranes.

Fluorescence blinking in single QDs is harmful for dynamic tracking due to information loss. We have experimentally studied the blinking phenomenon and the mechanism behind. We have discovered an emission bunching effect that two nearby QDs tend to emit light synchronously. The long-range Coulomb potential induced by the negative charge on the QD surface is found to be the major cause for the single QD blinking and the emission bunching in QD pairs.

We have studied the in vitro cytotoxicity of CdTe QDs to human umbilical vein endothelial cells (HUVECs). The QDs treatment increases the intracellular reactive oxygen species (ROS) level and disrupts the mitochondrial membrane potential. The protein expression levels indicate that the mitochondria apoptosis is the main cause of HUVCEs apoptosis induced by CdTe QDs.

Gold nanorods (GNRs) are scattering probes due to their tunable surface plasmon resonance (SPR) enhanced scattering spectrum. In order to control the yield and morphology of GNRs, we have systematically studied the effects of composition and concentration in the growth solution on the quality of the GNRs produced via a seed-mediated method. The aspect ratios of GNRs were found to be linearly depended on the concentration ratio of silver ions and CTAB. The high quality GNRs obtained were adsorbed to COS-7 cell membranes for dark field imaging.

We have rationally designed two types of QDs by wave function engineering so as to improve the efficiency of QD-sensitized solar cells. A reversed type-I CdS/CdSe QD confines excitons in the shell region, whereas a type-II ZnSe/CdS QD separates electrons in the shell and holes in the core. Their absorbed photon-to-current efficiencies (APCE) are as high as 40% and 60% respectively.

In conclusion, rationally designed nanoparticles are proven a high potential for applications as probes in biomedical labeling, imaging and molecule tracking, and as sensitizers for photovoltaic cells.

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Contents

Abstract ............................................................................................................................... i

Contents .............................................................................................................................iii

List of papers .......................................................................................................................v

Contributions by the candidate .......................................................................................... vii

List of abbreviations ........................................................................................................... ix

Chapter 1. Introduction ................................................................................................... 1

Chapter 2. Background .................................................................................................... 3

2.1. Nanoparticles ................................................................................................................... 3

2.2. Quantum dots and their optical properties ....................................................................... 3 2.2.1. What are QDs? ............................................................................................................................. 3 2.2.2. Chemical components and classification of QDs ......................................................................... 4 2.2.3. Quantum confinement of QDs ..................................................................................................... 5 2.2.4. Optical properties: QDs versus organic dyes ............................................................................... 6 2.2.5. Blinking properties of single QDs ................................................................................................. 8

2.3. Quantum dots for bioimaging applications ..................................................................... 10 2.3.1. QD bio-probes ............................................................................................................................ 11 2.3.2. Cell cultures ................................................................................................................................ 12 2.3.3. Staining cells with QD probes .................................................................................................... 13 2.3.4. Fluorescence microscopy for bioimaging .................................................................................. 13 2.3.5. In vitro imaging ........................................................................................................................... 14 2.3.6. In vivo imaging ........................................................................................................................... 16 2.3.7. Cytotoxicity of QDs..................................................................................................................... 16

2.4. Quantum dots for single molecule tracking application .................................................. 17 2.4.1. Comparation of FRAP, FCS, FRET and SPT ................................................................................. 17 2.4.2. SPT using QDs for cell membrane protein diffusion ................................................................. 19

2.5. Gold nanorods for bioimaging applications .................................................................... 22 2.5.1. Optical properties of GNRs ........................................................................................................ 22 2.5.2. Dark field microscopy................................................................................................................. 24 2.5.3. SPR enhanced Rayleigh scattering bioimaging using GNRs ...................................................... 25

2.6. Quantum dots for photovoltaic application .................................................................... 26 2.6.1. Solar cells .................................................................................................................................... 26 2.6.2. QD-sensitized solar cells ............................................................................................................ 27 2.6.3. QD-sensitized solar cells versus dye-sensitized solar cells ....................................................... 27

Chapter 3. Experimental design ..................................................................................... 29

3.1. Bioimaging using quantum dots (Paper I and II) .............................................................. 29 3.1.1. Water-soluble QDs ..................................................................................................................... 29 3.1.2. Cell culture for fixed cell labeling .............................................................................................. 30

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3.2. Single molecule tracking using quantum dots (Paper III) ................................................. 31 3.2.1. Cell culture for single molecule tracking ................................................................................... 31 3.2.2. Living cell labeling strategy for single molecule study by QDs ................................................. 31

3.3. Blinking of single quantum dots (Paper IV) ..................................................................... 32

3.4. Cytotoxicity of quantum dots (Paper V) .......................................................................... 32 3.4.1. Cell culture for QDs cytotoxicity study ...................................................................................... 32 3.4.2. Apoptosis induced by QDs ......................................................................................................... 32

3.5. Gold nanorods for dark field bioimaging (Paper VI) ........................................................ 33 3.5.1. Control synthesis of GNRs .......................................................................................................... 33 3.5.2. Cell culture selection for GNRs labeling .................................................................................... 33

3.6. Quantum dot-sensitized solar cells (Paper VII and VIII) ................................................... 34

Chapter 4. Results and discussion .................................................................................. 35

4.1. Bioimaging using quantum dots (Paper I and II) .............................................................. 35 4.1.1. Properties of aqueous synthesized CdTe/ZnSe QDs ................................................................. 35 4.1.2. Properties of hydrothermal synthesized CdTe QDs .................................................................. 36 4.1.3. Detection of HER2 on MCF-7 cells using QD bio-probes ........................................................... 37

4.2. Single molecule tracking using quantum dots (Paper III) ................................................. 38

4.3. Blinking of single quantum dots (Paper IV) ..................................................................... 39

4.4. Cytotoxicity of quantum dots (Paper V) .......................................................................... 40

4.5. Gold nanorods for dark field bioimaging (Paper VI) ........................................................ 41

4.6. Quantum dot-sensitized solar cells (Paper VII and VIII) ................................................... 43

Chapter 5. Conclusions and future perspectives ............................................................. 47

Acknowledgements ........................................................................................................ 51

References ....................................................................................................................... 53

v

List of papers

This thesis is based on the following papers that will be referred to by their roman numbers:

I. Tao Fu, Hai-Yan Qin, Hua-Jun Hu, Zhi Hong, Sailing He Aqueous Synthesis and Fluorescence-Imaging Application of CdTe/ZnSe Core/Shell Quantum Dots with High Stability and Low Cytotoxicity Journal of Nanoscience and Nanotechnology, 2010, 10(3), pp 1741-1746

II. Hai-Yan Qin, Tao Fu, Ming Yan, Zhijun Ning, Sailing He, Hjalmar Brismar and Hans Ågren Highly Stable and Low Toxic CdTe Quantum Dots Synthesized via a Hydrothermal Method for Cell Imaging Applications Submitted manuscript

III. Hai-Yan Qin, Torbjörn Gräslund, and Hjalmar Brismar Quantum Dot-Affibody Fluorescence Probes for Single Molecule Tracking in Living Cell Membranes In manuscript

IV. Hai-Yan Qin, X.-J. Shang, Z.-J. Ning, Tao Fu, Z.-C. Niu, H. Brismar, H. Ågren, and Y. Fu Time Correlation Analysis of Blinking from Individual Colloidal CdSe/CdS and CdSe/ZnS Quantum Dots In manuscript

V. Ming Yan, Yun Zhang, Kedi Xu, Tao Fu, Haiyan Qin, Xiaoxiang Zheng An in vitro Study of Vascular Endothelial Toxicity of CdTe Quantum Dots Toxicology, 2011, 282(3), pp 94-103

VI. Hai-Yan Qin, Tao Fu, Zhijun Ning, Hans Ågren, Hjalmar Brismar, and Sailing He Controlling Yield and Morphology for Gold Nanorods in a Seed-Mediated Synthesis Method for Cell Imaging Advances in Optoelectronics and Micro/Nano-Optics (AOM), 2010 OSA-IEEE-COS, 3-6 Dec. 2010, pp 1-4 The best student paper award

VII. Zhijun Ning, Haining Tian, Haiyan Qin, Qiong Zhang, Hans Ågren, Licheng Sun and Ying Fu Wave-Function Engineering of CdSe/CdS Core/Shell Quantum Dots for Enhanced Electron Transfer to a TiO2 Substrate The Journal of Physical Chemistry C, 2010, 114 (35), pp 15184–15189

VIII. Zhijun Ning, Haining Tian, Chunze Yuan, Ying Fu, Haiyan Qin, Licheng Sun, Hans Ågren Solar Cells Sensitized with Type-II ZnSe-CdS Core/Shell Colloidal Quantum Dots Chemical Communications, 2011, 47(5), pp 1536-1538

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Additional papers not included in this thesis:

IX. Guang S. He, Hai-Yan Qin, Qingdong Zheng Rayleigh, Mie, and Tyndall Scattering of Polystyrene Microspheres in Water: Wavelength, Size, and Angle Dependences Journal of Applied Physics, 2009, 105(2), 023110

X. Guang S. He, A. Ping Zhang, Qingdong Zheng, Hai-Yan Qin, Paras N. Prasad, Sailing He, Hans Ågren Multifocus Structures of Ultrashort Self-Focusing Laser Beam Observed in a Three-Photon Fluorescent Medium IEEE Journal of Quantum Electronics, 2009, 45(7), pp 816 - 824

XI. Guang S. He, Ken-Tye Yong, Hai-Yan Qin, Qingdong Zheng, Paras N. Prasad, Sailing He, Hans Ågren Stimulated Rayleigh-Bragg Scattering From a Two-Photon Absorbing CdSe-CdS-ZnS Quantum-Rods System: Optical Power Limiting and Phase-Conjugation IEEE Journal of Quantum Electronics, 2008, 44(9-10), pp 894-901

XII. Guang S. He, Hai-Yan Qin, Qingdong Zheng, Paras N. Prasad, Steffen Jockusch, Nicholas J. Turro, Marlin Halim, Dalibor Sames, Hans Ågren, Sailing He Dynamic Properties and Optical Phase Conjugation of Two-photon Pumped Ultrashort Blue Stimulated Emission in a Chromophore Solution Physical Review A, 2008, 77(1), 013824

XIII. Haiyan Qin, Xuan Li, Su Shen Novel Optical Lithography Using Silver Superlens Chinese Optics Letters, 2008, 6(2), pp 149-151

XIV. Tao Fu, Hai-Yan Qin, Wenjiang Li, Sailing He A Simple Method to Self-assemble Two-dimensional and Three-dimensional Superlattices of Gold Nanoparticles Materials Research Society Symposium Proceedings, 2007, 1056E, 1056-HH03-01.

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Contributions by the candidate

Paper I, II and III: The candidate contributed to the experimental design, did the major part of experiments, data analysis and manuscript writing.

Paper IV: The candidate contributed to the experimental design, did the major part of experiments and preliminary data analysis. The candidate also participated in manuscript writing.

Paper V: The candidate contributed to the quantum dot synthesis and characterization, results discussion, and parts of manuscript writing and modification.

Paper VI: The candidate did parts of the experiments including optical characterization, cell preparation, treatment and imaging. The candidate also wrote most of the manuscript.

Paper VII and VIII: The candidate participated in the experiments, data analysis and discussion.

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List of abbreviations

APCE Absorbed photon to current efficiency

AR Aspect ratio

CB Conduction band

CBD Chemical bath deposition

CTAB Cetyltrimethylammonium bromide

DSCs Dye-sensitized solar cells

ECs Endothelial cells

EGF Epidermal growth factor

EGFR Epidermal growth factor receptor

FCS Fluorescence correlation spectroscopy

FRAP Fluorescence recovery after photobleaching

FRET Förster resonance energy transfer

FWHM Full width at half maximum

GNPs Gold nanoparticles

GNRs Gold nanorods

HER2 Human epidermal growth factor receptor 2

HUVECs Human umbilical vein endothelial cells

IgG Immunoglobulin G

IPCE Incident photon to current efficiency

MEG Multiple exciton generation

MPA Mercaptopropionic acid

MSA Mercaptosuccinic acid

NIR Near infrared

NKA Sodium-potassium ATPase

NPs Nanoparticles

PBS Phosphate buffered saline

PEG Polyethylene glycol

PS Phosphatidylserine

x

QDs Quantum dots

QDSCs Quantum dot-sensitized solar cells

QY Quantum yield

ROS Reactive oxygen species

SPR Surface plasmon resonance

SPT Single particle tracking

TBP Tri-n-butylphosphine

TCZ Transient confinement zone

TOP Tri-n-octylphosphine

VB Valence band

Chapter 1. Introduction

During the past decade, nanotechnology has evolved at a very fast pace. Nanoparticles, which play a starring role in nanotechnology, are currently applied or being evaluated for applications in many emerging research fields. Perhaps the most important of these is the biomedical field, where nanoparticles offer new tools and approaches for significantly improving screening, diagnosis and therapy. Another important filed is nanophotonics, where nanoparticles hold immense potential for high performance devices of luminescence, solar cells, and display panels.

Although some nanoparticles by now have a long history for fabrication and applications, many challenges remain to be addressed, from the synthesis methods, property characterization, and the understanding of the physics or chemistry behind the various applications.

This thesis involves experimental studies of design, property characterization, and applications of quantum dots (QDs) and gold nanorods (GNRs). The goals of this thesis are:

⎯ Develop new QDs with high stability and low toxicity for biomedical imaging; ⎯ Improve the experimental strategy for single bio-molecule tracking studies using QDs as

fluorescent bio-probes; ⎯ Investigate the mechanism of fluorescence blinking in individual QDs; ⎯ Evaluate and reveal the in vitro cytotoxicity of QDs; ⎯ Produce high quality GNRs for biomedical imaging via systematical studies of the synthesis

method; ⎯ Enhance the performance of QD-sensitized solar cells (QDSCs) by QD wave-function

engineering.

The thesis also summarizes the research progresses of other groups in the field, and outlines the experimental designs, major results and conclusions of the papers involved.

Chapter 2 summarizes the quantum confinement effect and optical properties of QDs, reviews their applications in bioimaging and single molecule tracking applications. The fluorescence blinking phenomenon and cytotoxicity of QDs are highlighted. Moreover, Chapter 2 also introduces the optical properties of GNRs and their bioimaging applications, and QDs for photovoltaic applications as well. Chapter 3 describes the experimental considerations and designs for all papers involved in this thesis. Chapter 4 summarizes and discusses the experimental results. Lastly, Chapter 5 concludes

2 Chapter 1. Introduction

all the works and discusses the future perspectives for QDs and gold nanoparticles in biomedical and photovoltaic applications.

Chapter 2. Background

2.1. Nanoparticles

One nanometer is one billionth of a meter. Nanoparticles (NPs) are small particles in the nanoscale.

The first observation of nanoparticles can be traced to the beginning of the 20th century. In 1914 Richard Adolf Zsigmondy published a book containing studies of gold sols and other nanomaterials with size down to 10 nm 1. Meanwhile, he was the person that introduced the unit of nanometer for characterizing particle size. “There‘s Plenty of Room at the Bottom” was the title of the first ever lecture on nanotechnology, given by Richard Feynman at the annual meeting of the American Physical Society at Caltech in 1959 2. The tremendous potential suggested by Feynman and others was though not being realized until the late 20th century, for instance, quantum dots were not discovered until 1982 3, fullerenes in 1985 4, and carbon nanotubes in 1991 5.

Nanoscale is a magical region of the length scale. Unique effects occur while materials shrink to this scale, for example surface and interface effects, small size effects, quantum size effects, macroscopic quantum tunneling effects and so on. As a result, nanoparticles exhibit many unique physical properties such as low melting point, high hardness and strength, and special optical and electrical properties 6. According to their chemical composition, nanoparticles can be classified into organics (e.g. polystyrene nanospheres, poly (methyl methacrylate) NPs, polyamidoamine NPs), inorganic nonmetals (e.g. quantum dots, silicon NPs, carbon nanotubes and fullerenes), metals (e.g. gold NPs, GNRs, silver NPs) or complexes (e.g. organically modified silicates (ORMOSILs), metal-organic framework (MOF) NPs).

This thesis involves studies of properties of quantum dots and gold nanorods, and their applications.

2.2. Quantum dots and their optical properties

2.2.1. What are QDs?

Quantum dots (QDs) are nanocrystals of semiconductor materials in a size ranging from several nanometers to tens of nanometers, comprising 100 to 100,000 atoms. QDs are also known as colloidal semiconductor nanocrystals or artificial atoms. They are usually composed of atoms from groups II-VI, III-V or IV-VI elements in the periodic table. As their physical size is in the same order or

4 Chapter 2. Background

smaller than the Bohr radius of an electron-hole pair, QDs are under a quantum confinement in all three dimensions. As a result, QDs have unique property differences from individual molecules or bulk solids 7.

In 1982, Louis E. Brus from Bell Laboratories firstly discovered QDs, which is a point that marks the beginning of QD development 3. The term “Quantum Dot” was introduced by Mark Reed at Texas Instruments as late as 1988 8.Thereafter, advance in the research of QDs was steadily growing during the past two decades. QDs were introduced for photovoltaic applications by B. O’Regan and M. Gratzel in 1991 9. In the same year, Weller et al. described and studied the quantum confinement effects of QDs 10. Bawendi et al. developed a novel and reproducible synthesis route for monodisperse QDs 11. Soon after, Alivisatos’ 12 and Nie’s 13 groups used QDs as fluorescent probes for biological imaging in 1998.

2.2.2. Chemical components and classification of QDs

QDs with various physical structures and chemical components have been synthesized and used in different application areas.14 The most typical kinds of QDs in common use are classified in Table 2.1. Most QDs reported can be classified by their composition and structure: single-component, binary-component, ternary-component (alloys) and core/shell structure. Single-component QDs are not wildly used so far due to their poor monodispersion, low stability, low purity and sometimes uncontrollable synthesis procedure. Simple and mature synthesis methods made binary-component QDs popular in the early period. Alloy QDs, with three or more chemical components, have uniform or gradient composition. They are reported to have superior optical properties compared to others, for example large Stokes shift and higher quantum yield (QY) 15-17. Binary-component QDs can be used as cores for further synthesis into core/shell QDs. The shell isolates the core of the QD from the surrounding environment and passivates its surface by reducing the dangling bonds. Therefore, the stability and optical properties of QDs are unprecedentedly optimized by the core/shell structure.

Table 2.1 Classification of quantum dots in common use.

Single-component Si 18, 19, Ge 20 etc.

Binary-component

II-VI CdS 21, CdSe 22, CdTe 23, ZnSe 24 etc.

III-V GaN 25, InP 26 etc.

IV-VI PbS 27, 28, PbSe 27, 28 etc.

Ternary-component (Alloy) CdHgTe 15, 16, CdZnSe 17 etc.

Core/shell structure Core/shell CdSe/ZnS 29, 30, CdSe/CdS 31, 32, CdTe/CdSe 33, 34 etc.

Core/shell/shell CdSe/CdS/ZnS 35, 36, CdSe/ZnSe/ZnS 36, CdTe/CdS/ZnS 37 etc.

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Figure 2.1 Emission of QDs can cover the whole visible and near IR range.(Reprinted by permission of Nature)38

According to the spatial distribution of the charge carriers, core/shell structures are divided into type-I and type-II. As shown in Figure 2.2, the core of type-I QD, e.g. CdSe/ZnS QDs and CdSe/CdS QDs has a lower conduction-band (CB) and a higher valence-band (VB) compared to its shell. Both charge carriers are confined in the core. In contrast, both the CB and VB of the core are higher, e.g. CdTe/CdSe QDs, (or lower, e.g. CdSe/ZnTe QDs) than those of the shell in type-II QDs. One of the charge carriers is confined to the core and the other confined to the shell. Thus type-II QDs have a smaller band gap, which corresponds to a broader absorption spectrum and a longer emission wavelength 33. Type-II QDs have been applied in bioimaging applications for their suitable size and NIR emission.

Figure 2.2 A scheme of the band gap structures for type-I and type-II QDs.

The semiconductor QD is capped by an outer layer of surfactants, which provides sufficient repulsion between the particles to avoid aggregation. QDs coated with long-chain unsaturated fatty acid or amine (e.g. oleic acid or oleylamine), and organic phosphate (e.g. tri-n-octylphosphine (TOP) and tri-n-butylphosphine (TBP)) can easily disperse in organic solutions. Whereas, short chain mercaptoacetic acid molecules (e.g. mercaptopropionic acid (MPA) and mercaptosuccinic acid (MSA)), polyether compound (e.g. PEG) and dendrimer modified quantum dots are water-soluble and essential for many biological applications.

2.2.3. Quantum confinement of QDs

When material size reduces to the nanoscale, many “strange phenomena” occur. For example, nanomaterials possess higher hardness, ductility and toughness, and lower melting points than the corresponding bulk materials 39. Nevertheless, QDs received a lot of attention for a different unique phenomenon - the above mentioned “quantum confinement effect”. In bulk semiconductor

Core Shell

+

-

+

-

Core Shell

CB1

VB1

VB2

CB2

VB2

CB2CB1

VB1

Type-I QDsBandgap = CB1-VB1

Type-II QDsBandgap = CB2-VB1

0 0

-V-V


materials, an electron can be excited from the valence band up to the conduction band leaving a hole in the valence band when a photon with appropriate energy is absorbed. Due to Coulomb attraction, the electron and hole cannot move independently and form an exciton. The exciton Bohr radius (rB) estimates the size of the exciton or the distance between the electron and the hole. For example, the rB values of bulk CdS, CdSe and CdTe are 2.8, 6.1 and 10.0 nm approximately 40. Reducing the size of a semiconductor material to a size approaching its rB value, the exciton is not free to move in the material anymore and suffers from quantum confinement. The case here is similar to the “Particle-in-a-Box” model 41. Consequently, the continuous energy bands in bulk semiconductor materials split into discrete energy levels in the QDs. The band gap of bulk semiconductor is a fix value, whereas for QDs it increases with a reducing size (Figure 2.3). Therefore, the emission wavelength of a QD, which is relative to the band gap, is tunable by its size.

Figure 2.3 When the size of semiconductor material decreases to the nanoscale, the continuous energy bands of the bulk material split to discrete energy levels. The smaller the particle size, the greater separation between the adjacent energy levels and the higher band gap it has.

Figure 2.4 Emission mechanism of QDs. An electron jumps from the valence band to the conduction band and leaves a hole in the valence band, while excited by a photon with appropriate energy. The electron is not stable in the conduction band and falls back to the valence band, and emits a photon with lower energy.

2.2.4. Optical properties: QDs versus organic dyes

Fluorescent organic dyes have a long history that can be traced back to one and a half centuries ago. They did not become popular for applications in biological or medical fluorescence imaging until the 1940s when Coons and Kaplan firstly introduced a method to label antibodies with fluorescent dyes42,

Band gap

Band gap

QD QDBulk semiconductor

material

Band gap

Ener

gy

Conduction band

Valence band

hγ0

hγ1

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43. Thousands of dyes have been prepared and many of them are nowadays widely applied in immunofluorescence technique. For instance, DAPI is usually used for nucleus and chromosome staining, nile red for lipids and calcein for biofilms. However, even today some essential problems remain and limit their applications. Firstly, the absorption spectra of organic dyes are discrete bands with full widths at half maximum (FWHMs) of 30 to 100 nm. As a result, one can hardly observe the fluorescence from different dyes with the same excitation wavelength, thus switching of light source or wavelength is required. Secondly, the asymmetric emission spectra tailing to the long wavelength side render the cross-talk between different dyes. Thirdly, their small Stokes shifts cause self absorption and are detrimental to their QYs. More to the point, most organic dyes suffer from photobleaching, which limits their applications to single molecule analysis and long term imaging.

Nowadays, QDs become an important fluorescent probe in biological applications since they have many superior optical properties compared to organic dyes. QDs have very broad excitation spectra which make it possible to visualize multi-color labeling simultaneously by single wavelength excitation. Due to the narrow and symmetric emission spectra of QDs, the overlap of emission bands can be considerable reduced. Moreover, as mentioned above, the emission spectra are tunable in the whole visible and near infrared (NIR) spectral regions by adjusting the size or the chemical components of the QDs. Therefore, a series of QDs with the same chemical components but different emission spectra can be used for multi-color labeling. The large Stokes shift of QDs minimizes the self-absorption effect and helps to increase the QYs. For imaging applications, it also improves the signal-to-noise ratio by simply separating the emission signal from the excitation signal. A single QD has higher fluorescence intensity than a single organic dye molecule. This makes QDs an alternative for single particle and single molecule studies. Furthermore, the fluorescence lifetime of QDs is generally 20 to 50 nanoseconds, whereas that of organic dyes is only several nanoseconds, which is similar to that of the self-fluorescence from biological components. As a result, the endogenous fluorescence background can be easily cut off while using QDs for fluorescence lifetime imaging. Last but not least, the photostability of QDs is orders of magnitude higher than that of organic dyes. Hence QDs are proper fluorescent probes for long term (hours or even days) imaging or tracking.

The outstanding optoelectronic properties of QDs have also attracted a lot of attention for photovoltaic applications. QD-based solar cells are potential alternates to the traditional dye-based solar cells. QDs have much broader absorption spectra compared to organic dyes, so that they can absorb the solar spectrum more efficiently. More importantly, many QDs, such as PbSe28, 44, 45, PbS28, PbTe46 and CdSe45, have a unique feature called multiple exciton generation (MEG) or carrier multiplication. MEG is a process of generating two or more excitons while absorbing one high energy photon. As a result, the maximum theoretical efficiency of QDs-based solar cells can be boosted to 42%47, which is far beyond that of the traditional solar cells (30%)48. However, the MEG property of QDs is still under debate and the mechanism behind remains unclear. In addition, QDs-based solar cells have a longer service life for the higher stability of QDs compared to organic dyes-based solar cells.


Figure 2.5 Absorbance (dash line) and emission (solid line) spectra of organic dyes and QDs. (a) CdS QDs dispersed in toluene (thick line) versus Coumarin 1 dissolved in ethanol (thin line); (b) CdTe/CdS QDs dispersed in water (thick line) versus Nile red dissolved in dioxane (thin line). Original spectra data of Coumarin 1 and Nile red is from Ref 49.

2.2.5. Blinking properties of single QDs

Fluorescence intermittency, or blinking, is a phenomenon that the emission signal of individual fluorophores irregularly switches on and off with continuous excitation. Blinking was observed in many different fluorophores, for example quantum dots,50, 51 quantum rods,52 organic molecules53 and fluorescence proteins54.

The irregular blinking of QDs complicates the data analysis process of single QD tracking, since the dark periods of QD cause information loss for continuous particle tracking.55 The blinking phenomenon also reduces the QYs of QDs and is detrimental to their applications in Förster resonance energy transfer (FRET) microscopy, organic light-emitting diodes (OLED), solar cells and so on.

Figure 2.6 Fluorescence intensity of a single CdSe/ZnS QDs (QD655 from Invitrogen) over 2000 frames at 20 Hz and an exposure time of 20 ms. The data were recorded with a fluorescence microscope system equipped with a mercury lamp, a filter set (excitation FF01-435/40-25, emission FF01-655/15-25, beam splitter FF510-Di01-25x36) and a CCD camera. Fluorescence images of “Off” and “On” states (right) were corresponding to the fluorescence fluctuation trajectories (left) at 45.00 s and 87.80 s. Image size: 2 μm × 2 μm.

0

0.5

1

0 10 20 30 40 50 60 70 80 90 100

Fluo

resc

ence

Inte

nsity

(a.u

.)

Time (s)

“Off” state

“On” state

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Experimental as well as theoretical studies of blinking properties of QDs are still in a very early stage. Experimentally, under continuous irradiation, the blinking phenomenon of single QDs can be recorded by a high-speed CCD camera or through time-resolved single-photon counting. The fluctuation of the QD intensity over time can be obtained by integrating the intensity or photon numbers within a fixed time bin. The durations of “on” and “off” states can be varied from microseconds up to seconds or even minutes as shown in Figure 2.6. Many statistical results have shown that the probability distribution for “on” and “off” interval times follows a power law of ∆ ∝ ∆ 51, 56. The exponent m depends on many factors such as the composition and structure of the QDs, excitation intensity and wavelength, and also the environment.

The mechanism behind the blinking phenomenon of QDs remains unclear so far. There is a recent consensus that QD fluorescence blinking can be ascribed to the nonradiative Auger recombination process in a charged or ionized QD 57, 58. Charge carriers or excitons are generated while the QD absorbs photons with sufficient energy. As shown in Figure 2.7 (a), the charge carriers can escape through direct quantum mechanical tunneling or Auger ejection, and get trapped at the QDs surface or in the surrounding matrix, which leads to ionization of the QDs. The Auger recombination process is predominant for it is much faster than the radiative recombination process for excitons in QDs 59. In an Auger recombination process, the energy released from the exciton pair recombination is absorbed by the redundant charge carrier and increasing its energy (Figure 2.7 (c)). The “off” state lasts until the QD regains the ejected charge carrier and restores to electro-neutral (Figure 2.7(b)).

Figure 2.7 Mechanism behind single QDs blinking. (a) Schematic illustration of QDs ionization process, (b) radiative recombination for “on” state (middle) and (c) nonradiative Auger recombination for “off” state.

Increasing efforts have been made to suppress the blinking phenomenon of QDs and pursue non-blinking QDs. One way is to save QDs from ionization. Dubertret’s 60 and Hollingsworth’s 61 groups obtained nearly non-blinking QDs by fabricating “giant” shells out of the core QDs to deeply confine the charge carriers in the core. Another way is to shorten the fluorescence lifetime of QDs so as to prevent Auger recombination. Krauss’ group 62 reported a novel kind of QDs with a radially graded alloy structure. Due to their gradually changing potential energy function, these QDs have a much shorter fluorescence lifetime, which is shorter than the Auger recombination time. The novel gradient alloy QDs emit fluorescence despite being charged and present a truly non-blinking feature.

+ -

“On” state

+ -

“Off” state

hγ

Radiative recombination Nonradiative Auger recombination

+

Conduction band

Valence band

Trap statehγ0 hγ1

excitation emission

tunneling--

(a) (b) (c)


Nevertheless, blinking is not always bad. According to the Rayleigh criterion, the resolution of an optical system is roughly not better than half the wavelength of the observed light, that is, two to three hundred nanometers for visible lights. It is impossible to distinguish between single QDs and clustered QDs from their fluorescence images, despite the nanoscale size of a QD. Due to an average effect of the emission fluctuation, blinking behavior is absent for a cluster of QDs. Hence, the blinking behavior is an important criterion to judge whether a fluorescence signal is from a single QD or a QD cluster.

2.3. Quantum dots for bioimaging applications

Immunofluorescence is a technique that uses fluorescent bio-probes to specifically recognize and stain biomolecule targets within cells, and then uses fluorescence microscopy to observe the fluorescent signals from the sample.

The application of QDs as biological fluorescent probes is one of the most important and rapidly developing interfaces of nanotechnology. In 1998, Alivisatos’ 12and Nie’s 13groups first reported water soluble QDs respectively for Hela cells and 3T3 cells labeling in the same issue of Science. Since QDs have a lot of advantages over organic dyes, the potential of QDs for biological labeling and imaging attracts a lot of attention and with a research that has increased exponentially during the past decade. Today QDs have been successfully used as bio-probes for gene technology, biomolecule labeling and tracking, cell targeting and tracking, in vivo imaging, tumor diagnosis and therapy, etc.

QDs for bioimaging applications need to satisfy many requirements. Firstly, the QDs should have high biocompatibility, which means high water-solubility and low cytotoxicity. Secondly, high QY is essential in order to get a high signal-to-noise ratio for imaging. Thirdly, high photostability is important for long term dynamic process studies. Furthermore, some additional properties of QDs are required for some special applications. For example, a narrow FWHM of the emission spectrum is crucial for multi-color labeling; near infrared (NIR) emission is necessary for in vivo studies, in order to avoid the red absorption from hemoglobin and green auto-fluorescence from tissues and organs, and fit to the NIR transparent window between 700 and 950 nm for mammalian bodies 63.

Figure 2.8 Size of a surfactant-capped QD comparing with other nano- or micro-scale biological objects.

Atom0.3-3 Å

GFP4 nm

IgG14 nm

Virus20-400 nm

Bacterium0.5-10 µm

Animal cell10-100 µm

Surfactant

Quantum dot

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2.3.1. QD bio-probes

QDs can be prepared as bio-probes by conjugating to biomolecules and subsequently labeling the biological objects. Different conjugation strategies have been developed for QDs with different surface functional groups 64, 65.

Figure 2.9 Schematic diagram showing reactions of conjugating carboxyl-QDs to amine-containing biomolecules.

Figure 2.10 Schematic diagram showing reactions of conjugating amine-QDs to sulfhydryl-containing biomolecules.

QD-COOH

EDC

Sulfo-NHS

Biomolecule

QD-NHS

QD-BiomoleculeProbe

QD-EDC

QD-NH2

Sulfo-SMCC

QD-Maleimide

DTT

Antibody

Partly reduced antibody

QD-antibody probe


One of the most commonly used bioconjugation methods is offered by a zero-length crosslinking agent EDC (1-ethyl-3-[3-dimethylaminopropyl]carbodiimide hydrochloride) to conjugate carboxylic acid functional QDs (QD-COOH) and primary amine-containing biomolecules 66. As shown in Figure 2.9, the QD-COOH firstly reacts with EDC and forms an amine-reactive O-acylisourea intermediate. As to stabilize this intermediate, sulfo-NHS (N-hydroxysulfosuccinimide) is added to form a more stable form of the amine-reactive sulfo-NHS ester. With the present of amine-containing biomolecules, QDs are finally conjugated with biomolecules with a stable amide bond. The major advantage of this method is that proteins generally contain primary amine groups and therefore no pretreatment of proteins is needed. However as a drawback, conjugation can occur to different bonding sites randomly on the protein. For example, an antibody is apt to lose its ability to bind to the antigen if the bonding site to the QD is too close to its antigen-binding site.

Another bioconjugation technique is applied for conjugating amine-QDs to sulfhydryl-containing biomolecules using a heterobifunctional crosslinking agent sulfo-SMCC (sulfosuccinimidyl 4-[N-maleimidomethyl]cyclohexane-1-carboxylate) 66, 67. QD-NH2 reacts with sulfo-SMCC forming a stable amide bond. The QD-maleimide subsequently reacts with sulfhydryl-biomolecules and forms a stable thioether bond. For this method, antibodies need to be reduced to antibody fragments with exposing sulfhydryl groups by DTT (dithiothreitol) or cysteamine before conjugation. The sulfhydryl groups on the antibody fragments are sufficiently far away from the antigen binding sites so that it would not influence the succedent immunolabeling process.

Many other approaches have been developed for covalent conjugating QDs to biomolecules 65. For example, EGS (ethylene glycolbis(succinimidylsuccinate)) or DSP (dithiobis(succinimidyl propionate)) can be used to conjugate QD-NH2 and biomolecules containing -NH2 groups. BMME (bis(maleimido methyl)) can be used to conjugate QD-SH and biomolecules containing –SH groups.

Besides, noncovalent conjugate strategies have also been reported. Histidine-tagged biomolecules can bind to Ni-NTA (nickelnitrilotriacetic acid) modified QDs by chelation 68. The stable bound of streptavidin to biotin can used to conjugate biotinylated proteins and streptavidin coated QDs 69, 70.

2.3.2. Cell cultures

Cell cultures include primary cells and cell lines. Cells taken from an organism are called primary cells. After seeded onto culture plates and treated with appropriate media, most primary cells can grow and reproduce for several passages and then die. Some types of cells get over this period and become immortal cell lines.

According to whether attached to the surface of the culture plates and flasks or suspended in the culture medium, cells are divided into adherent cells and suspension cells. Adherent cells are relatively static and facile for imaging studies.

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2.3.3. Staining cells with QD probes

Two strategies are commonly applied for staining cells with QD bio-probes: direct immunofluorescence and indirect immunofluorescence. As shown in Figure 2.11, the direct immunofluorescence method involves the use of primary antibody-QD probes to recognize and bind to the antigenic biomolecules. The indirect method is a two step labeling process. Primary antibodies are first bonded to the antigenic biomolecules and then secondary antibody-QD probes are used to label the primary ones. A secondary antibody is an antibody against, and able to recognize and label the primary ones.

There is an even higher sensitive indirect immunofluorescence approach called “Indirect complement immunofluorescence” method. In this method, antibodies are bonded to many biotin molecules which are conjugated with QD-streptavidin probes.

The indirect method has a lower specificity but higher sensitivity than the direct method. One primary antibody can bind to several secondary antibodies and which leads to amplification of fluorescence signals. The indirect method has another merit while labeling many different target antigenic molecules. The indirect method only requires a small amount of QD conjugated secondary antibodies. For example, a secondary antibody raised against goat IgG (Immunoglobulin G) can be used to label any primary antibody raised in goat. On the contrary, with the direct method, conjugations of QDs to each kind of primary antibodies are needed.

Figure 2.11 Different strategies of immunofluorescence techniques using QD bio-probes.

2.3.4. Fluorescence microscopy for bioimaging

Fluorescence microscopy is an optical technique to observe and image the fluorescent signals from samples with fluorescence or labeled by fluorophores. The specimen is normally illuminated from above and fluorescent signals are collected from the top. Therefore, this microscopy technique is also called epi-fluorescence microscopy. As shown in Figure 2.12, according to the absorption spectrum of the fluorophores on the specimen, the excitation light with a suitable wavelength is

QD

Membrane molecule

Cell membrane

Intracellular

Extracellular

QD

Secondary antibody

QD

Biotin

Streptavidin

Primary antibody

Direct Immunofluorescence

Indirect Immunofluorescence

Indirect Complement Immunofluorescence


selected by an excitation filter from the white light emitted by a mercury lamp. Thereafter, the light is reflected by the dichroic beam splitter and focused by the objective to illuminate the sample. The fluorescent light from the sample passes through the diachronic beam splitter and finally reaches the detector. Since the dichroic beam splitter is not perfect, a barrier filter can be used to block all remnants and only transmit the fluorescent light.

Basing on the principle of fluorescence microscopy describe above, various advanced microscopy techniques are developed to meet specific purposes, for example, confocal microscopy, total internal reflection fluorescence microscopy (TIRF), two-photon microscopy, near-field microscopy, stimulated emission depletion microscopy (STEP) and so on. Confocal microscopy is one of the most widely applied fluorescence microscopy techniques for biological fluorescence imaging. Unlike in the conventional fluorescence microscope, only a single point of the specimen is illuminated in the confocal microscope system. A scanning procedure is performed to the whole imaging area in order to produce an image of the specimen. Confocal microscopy has two advantages in that it improves the resolution and can provide a 3D image by scanning along the axial direction. In addition, STEP is a new super resolution scanning microscopy that overcomes the diffraction limit. A second doughnut shape pulse illuminates the specimen immediately after the first excitation pulse, and de-excites the outer area of the illumination spot. Hence STED presents a much higher resolution than a confocal microscope does.

Figure 2.12 An illustration showing the principle of fluorescence microscopy.

2.3.5. In vitro imaging

In vitro studies are performed in an artificial environment outside the living organism. QD fluorescent probes are very effective for in vitro cellular labeling and imaging. As one of the most important breakthroughs, Wu et al. 71 successfully used immunoglobulin G (IgG) or streptavidin conjugated QDs to specifically label different subcellular targets in both fixed and live cells, and tissue sections, in 2003. The targets included the breast cancer marker HER2 on cell membranes, actin and microtubule fibers in the cytoplasm, and antigens inside the nucleus (Figure 2.14). To date, a variety of

Detector

Objective

Specimen with red fluorophore

Dichroic beam splitter

Barrier filter

Eyepiece

Excitation filter

Light source

Excitation light

Emission light

Haiyan Qin 15

intracellular components have been successfully labeled with QDs, including nuclei 71, 72, mitochondria 73, actin filaments 71, microtubules 71, vesicles in cytoplasm74-76, perinuclear recycling endosomal compartments 77 and intracellular proteins (e.g. martalin 78, cytokeratin 79 and matrix metalloproteinase 80). Cell membrane proteins as important media for the cells to connect the surrounding environment have also been studied using QD bio-probes, for example, serotonin transport receptors of neurotransmitters on cell membrane 81, glycine receptors on surface of spinal nerve cells 55, breast cancer marker HER2 receptor 71, glycoprotein transporter 79, and so on.

Figure 2.13 Structure of an animal cell.

Figure 2.14 (A) Staining of HER2 on sections of mouse mammary tumor tissue with QD 630-streptavidin (red). (B) Staining of actin filaments on 3T3 mouse fibroblast cells with biotinylated phalloidin and QD 535-streptavidin (green). (C)The microtubules were labeled with mouse anti-α-tubulin antibody, anti-mouse IgG-biotin, and QD 535-streptavidin (green). (D) HER2 on the surface of SK-BR-3 cells was stained green with mouse anti-HER2 antibody and QD 535-IgG (green). The nucleus of a 3T3 cell was stained with ANA (anti-nuclear antigen), anti-human IgG-biotin, and QD 630-streptavidin (red) for (C) and (D). Scale bar, 8, 24, 10 and 50 μm for (A) through (D) respectively. (Reprinted by permission of Nature) 71

Plasma membrane

Microtubules

Free ribosome

Vesicle

Golgi complex

Smooth endoplasmic

reticulum

Peroxisome

CentrioleNuclear pore

Rough endoplasmic

reticulum

Mitochondrion

NucleolusChromatin

Ribosome

NucleusLysosome

A B C D


2.3.6. In vivo imaging

In vivo studies, as opposed to in vitro studies, are performed in living organisms, such as animal models and clinical treatments. Near infrared (NIR) QDs are remarkably superior for in vivo imaging applications due to their highly stability and penetration in tissue.

For most in vivo experiments, QDs are directly intravenously injected into the living animal and then delivered to other parts of the body by blood circulation. The in vivo distribution of QDs is a key concern in QD toxicity studies. Dubertret et al. 82 (Figure 2.15 (A)) and Prasad et al. 83reported studies of both short-term and long-term body distribution of QDs after injected into mice82, 83. Bawendi et al. 84 and Chan et al. 85 studied the renal clearance of QDs in vivo.

The achievement in sentinel lymph node and vascular mapping by QDs in vivo targeting and imaging is remarkable. Frangioni and coworkers 86 imaged lymph nodes using type-II NIR QDs and Kosaka and coworkers 87 reported a real-time multicolor lymph nodes mapping in mice (Figure 2.15 (B)). Similar studies were also performed in rats 88 and pigs 89. Chan et al. 90 first applied peptide-QD conjugates to target receptors on blood vessels in 2002. Soon after Webb et al. 91 demonstrated two-photon fluorescence imaging of small blood vessels (Figure 2.15 (C)). In 2007, Campbell et al. 92 extended the technique to image chick chorioallantoic membrane vessels.

Another important part of in vivo imaging studies using QDs is tumor imaging. In 2004, Gao et al. 93demonstrated the targeting and imaging of a prostate tumor using QDs in a live mouse model (Figure 2.15 (D)). This work was followed up by many in vivo imaging studies of tumor models, for example murine squamous cell tumors in C3H mice 94, subcutaneous U87MG human glioblastoma tumors in athymic nude mice 95, and orthotopic pancreatic tumor in nude mice 96.

Figure 2.15 (A) In vivo imaging of QDs distribution in mouse after 90 min intravenous injection. (Reprinted by permission of ACS) 82 (B) Multicolor in vivo imaging of lymph node in a mouse labeled by QDs. (Reprinted permission of JID) 87 (C) In vivo imaging of small blood vessels in a mouse labeled by QDs. (Reprinted by permission of Science) 91 (D) In vivo targeting and imaging of a prostate tumor in a live mouse using QD bio-probes (right) and a control using a healthy mouse (left). (Reprinted by permission of Nature) 93

2.3.7. Cytotoxicity of QDs

The bulk forms of most nanomaterials are low toxic or even not toxic themselves, for example GNRs, which are gold in the nanoscale, and carbon nanotubes, which are carbon in the nanoscale.

A B C D

Haiyan Qin 17

Nanoparticles, however, are more or less toxic due to their large surface area and small sizes. It is even worse in the case of QDs. Because many of the compositions of the common used QDs are known to be highly toxic themselves, such as cadmium, selenium and lead. There are a lot of concerns for QD toxicity and consequently researchers are cautious regarding biological and medical applications of QDs.

Most of the recent evaluations of the toxicity of QDs are in short term using in vitro or in vivo assays. Among the most commonly used for in vitro studies is MTT (3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide) assay 97-99, which is a colorimetric method evaluating the viability of cells. The SDH (succinatedehydrogenase) from mitochondria of living cells can reduce MTT to formazan, which is insoluble purple precipitate and subsequently redissolved in DMSO (dimethyl sufloxide). By measuring the absorbance of the solution at 490 nm wavelength, the number of living cells can be determined. Besides, other assays are also involved, such as colonigenic assay 72, LDH (lactate dehydrogenase) assay 100, 101, cell attachment assay 99 and etc. For in vivo studies, QDs are normally intravenously injected into the tail vein of mice or rats. In vivo imaging provides information about the QD migration and accumulation in different internal organs of animal body after different circulation time 102-104.

The toxicity of QDs is still a very uncertain and complicated problem today. On one hand, the physical and chemical properties are different from QD to QD. Their toxicity is influenced by their composition, structure, size, surface coating, charge, concentration, and even synthesis method. ZnS shells protect QD cores from releasing cadmium ions and significantly reduce their toxicity 98, 105, 106. PEG, surface coatings for most commercial QDs, is superior to MPA, MAA, MUA, TOPO and many other surface coatings which are found to be toxic 97, 107. A small size or positive charge on the surface of the QD facilitate the cellular internalization 108. On the other hand, assessments from different studies presented in the literature are hard to compare due to the diversity of experimental methods and the absence of testing standards 109, 110.

2.4. Quantum dots for single molecule tracking application

2.4.1. Comparation of FRAP, FCS, FRET and SPT

Until now, several techniques basing on fluorescence microscopy have been developed to study the dynamic behaviors of biomolecules. The most widely used ones are fluorescence recovery after photobleaching (FRAP), fluorescence correlation spectroscopy (FCS), Förster resonance energy transfer (FRET) and single particle tracking (SPT).

FRAP

FRAP technique was developed during the 1970s by Axelrod and coworkers 111. In order to study the lateral diffusion of molecules in two-dimension using FRAP, the sample is labeled by fluorophores first. A small area of this label is rapidly photobleached by flashing with an intense light. Thereafter, the bleached fluorophores binding with the favorite molecules diffuse out of this area and the unbleached ones diffuse in, as shown in Figure 2.16 (A). It becomes a uniform mixture eventually. Therefore, the fluorescence intensity curve of this dark area will gradually increase and a uniform


value will be restored. The recovery kinetics depends on the speed at which the fluorophores move from surrounding regions to the bleached area, and provides information regarding the mobility of the labeled molecules.

FRAP protocol can be applied with most standard confocal microscopes. Thus it becomes one of the most common techniques of molecule mobility studies. However FRAP cannot provide information about single molecule behaviors, but only measures average behaviors of population of molecules. FRAP has a low resolution comparing to other techniques.

FCS

FCS technique was invented by Magde et al. in 1972 112. In FCS, a small volume of the fluorophores labeled sample is illuminated and the fluorescence signal recorded along with time. As molecules diffusing in and out of the measurement volume, the fluorescence intensity recorded fluctuates up and down (Figure 2.16 (B)). A subsequent correlation analysis of this fluctuation data against time can provide information of the molecule diffusion properties.

FCS measures concentrations and diffusion coefficients of molecules, and molecule to molecule interactions. FCS has limitations such as it is inapplicable for immobile targets and the subsequent data analysis is complicated.

FRET

FRET technique was invented and named after Theodor Förster in 1948 113. Besides Förster resonance energy transfer, FRET has many other names such as fluorescence resonance energy transfer and resonance energy transfer (RET). The FRET technique is based on the theory of nonradiative energy transfer between two fluorophore molecules depending on the distance between them. Shown in Figure 2.16 (C), the distance between two molecules can be calculated according to the changes of fluorescence intensities of molecules.

FRET is one of the highest resolution techniques to measure the distance between two different fluorophores or molecules labeled with fluorophores. False-negative results tend to occur in FRET.

SPT

SPT is one of the most direct approaches to study the diffusion properties of single molecules. Molecules labeled with fluorophores are observed and their locations or coordinate information over time are recorded directly using high resolution microscopy, showing in Figure 2.16 (D). Through analyzing their trajectories, the diffusion properties, such as diffusion coefficient and confinement zones can be determined.

SPT provides information of individual molecule mobility, such as diffusion coefficients, confinement zones, dwell time and etc. It has both high spatial and temporal resolution while applied with microscopy equipping high-speed CCD (charged coupled device) cameras. SPT technique prefers solution samples with low labeling density and target molecules with low mobility.

Haiyan Qin 19

Figure 2.16 Four fluorescence microscopy techniques for studying the dynamic behaviors of biomolecules: (A) FRAP, (B) FCS, (C) FRET and (D) SPT.

2.4.2. SPT using QDs for cell membrane protein diffusion

SPT using QDs

SPT using organic dyes or fluorescent proteins in cells suffers from photobleaching which limits its applications in long-term tracking. SPT using fluorescent polymer beads has another drawback of the beads’ large sizes which tend to influence the behavior of the target molecules. The introduction of QDs into SPT improves the technique in many terms. QDs can directly and simply conjugate with a variety of biological molecules. The highly brightness of QDs makes high speed particle tracking possible and so as favors the studies of fast moving targets. The highly photostability of QDs enables long-term SPT for minutes and even hours. Moreover it is possible to simultaneously track different molecules labeled by QDs with different emission wavelengths. In addition, QDs are much smaller than fluorescent polymer beads, however still larger than organic dye molecules or most biological molecules. Thus interference from the QDs is still non-ignorable. The emission blinking is another disadvantage of QDs for SPT, which causes information missing during tracking or even targets loss. So far, scientists give consistent efforts to develop non-blinking QDs in smaller sizes and therefore make SPT using QDs an ultrasensitive technique with high spatial and temporal resolutions.

Time

Inte

nsity

Time

Inte

nsity

Energy transfer

Wavelength Wavelength

Inte

nsity

Inte

nsity

C

A

D

B


Figure 2.17 Cell membrane structure.

Cell membrane and membrane proteins

Cell membrane is a semipermeable membrane of lipid bilayer, which houses and protects the cytoplasm and organelles from the surroundings. Small lipophilic non-polar molecules (e.g. O2, CO2, N2 and benzene) can pass through the lipid bilayer by diffusion; whereas most uncharged polar molecules (e.g. amino acids, nucleic acids, carbohydrate and proteins) can hardly pass through; and ions are completely blocked.

As shown in Figure 2.17, the cell membrane is similar to a two-dimensional homogeneous viscous fluid. Lipids and proteins anchored on the membrane can move by Brownian diffusion. This is described by the famous Fluid-Mosaic Model, which was proposed by Singer and Nicolson in 1972 114. More recently Murase et al. improved this model by compartmentalizing the membrane into domains 115. These domains can be classified into obstacles (e.g. barriers, fences and pickets), protein-protein complexes and lipid rafts.

Cell membrane proteins are protein molecules penetrating (integral membrane proteins) or attaching to (peripheral membrane proteins) the cell membrane. Enzymes are proteins under extensive research. They catalyze a variety of reactions and help to produce substances for cell functions. Many membrane proteins participate in the signal transduction between or within cells. For example, membrane receptors can bind to signal molecules in extracellular fluid and trigger the reactions in cytoplasm. Transport proteins, including carrier proteins and channel proteins, control substances entering and leaving the cell and so as to maintain the ion concentration in cells. A carrier protein can specifically bind to one or several kinds of molecules and carry them across the cell membrane. The transportation performed by carrier proteins is normally active transport. It carries substances from a low concentration site to a high concentration site and consumes energy. As a

Extracellular environment

Cytoplasm

Protein channel(Transport protein)

Lipid bilayer

Phospholipids

Phospholipids

Fatty acid tails

GlycoproteinGlycolipid

Peripheral proteinIntegral protein

Carbohydrate

Alpha-helix proteinIntegral protein

Haiyan Qin 21

contrast, channel proteins perform passive transportation. A channel protein can change the permeability of the cell membrane and specifically allows one or several certain kinds of molecules going through the cell membrane freely by diffusion following the ion concentration gradient. Structural proteins are normally fibrous proteins. They play an important role in maintaining the size and the structure of cells.

Tracking membrane proteins with SPT

SPT is a very important technique for mapping the dynamic structure and organization of cell membrane. Monitoring the behavior of membrane proteins in a molecular scale can help to elucidate the roles of lipid rafts, the mechanisms behind protein-protein interactions and cell signaling.

Dahan et al. first demonstrated single receptor tracking using QDs in live cells in 2003 55. Soon after, Lidke et al. used single QD tracking for the real time study of EGF/HER signal transduction in live cells 116. Dahan et al. also presented a broad applicable protocol 70, 117, 118 in detail including conjugation between QDs and biomolecules, cell labeling, microscopy setting up, imaging and tracking, and data analyzing methods. Softwares for analyzing experimental data from SPT were released for free by Marguet et al. 119 and Jaqaman et al. 120 Until now, the dynamic behavior of many membrane proteins has been studied using single QD tracking, including glycine receptor 55, erbB/HER receptor 116, AMPA receptor 121, 122, GABA receptor 123, Eag1 channel 124, potassium channel 125, CFTR channel126 , aquaporin-1 127 and -4 128 proteins, and so on.

There are still two challenges for SPT using QDs: three-dimensional (3D) molecule tracking and intracellular tracking. 3D tracking requires fast layer by layer scanning along z direction using confocal microscopy. Enhancing the brightness or QY of QDs, or improving the sensitivity of detector can help to fasten the scanning speed. For intracellular tracking, the internalization of QDs into cytoplasm requires QDs with smaller sizes, for example less than 4 nm.

Figure 2.18 Single QD tracking erbB/HER receptor in A431 cells. (b) are magnified images indicated in the last panel of (a). Scale bars, 5 μm. (Reprinted permission by Nature Publication Group) 116


2.5. Gold nanorods for bioimaging applications

Gold nanoparticle is among the nanomaterials that have the longest history. For many centuries, gold nanoparticles have been used as the red color staining of glasses and decorations for their bright and variable colors. In the British Museum, the Lycurgus cup created in the 4th century AD, stained with gold and silver dust, shows fantastic colors with different light incident angles. The first scientific investigation of gold nanoparticles was in 1857. Michael Faraday prepared a “gold sol” which was a colloid dispersion of gold nanoparticles as known today.129 He recognized that the colloid dispersion made of different particle sizes appeared various colors. No progress was made until almost half century later, Richard Adolf Zsigmondy 130 and Theodor Svedberg 131 improved Faraday’s synthesis method. Meanwhile Gustav Mie attempted to explain the colors of gold nanospheres by solving Maxwell’s equations for the interaction between electromagnetic field and matter 132, and Richard Gans further improved this theory to apply to rod or ellipsoid shape particles 133. Thereafter increasing studies have been conducted on the synthesis and properties of gold nanoparticles. The applications of gold nanoparticles nowadays cover a wide range from photonics to biomedicine.134

2.5.1. Optical properties of GNRs

Metals in nanoscale present significant different optical properties as semiconductors do, but in a very different way. The mean free path for electrons, for example in gold, is about 50 nm. While a light interacts with a gold nanoparticle in a size much smaller than this distance, surface effects become dominate. As shown in Figure 2.19, the conduction band electrons in the particle are driven back and forth to the surface by the oscillating electromagnetic field of incident light. This resonant dipole oscillation with the same frequency of incident light is called the surface plasmon resonance (SPR). This resonance can amplified the light field and increase light intensity by several orders of magnitude. As a result, SPR band exists in the spectra of the metal nanoparticles, such as absorption spectrum, fluorescence spectrum, Rayleigh scattering spectrum and Raman scattering spectrum, possess peaks corresponding to the resonance frequency. Similar to QDs, the optical properties of metal nanoparticles are found to be dependent on the particle size, shape, and dielectric constants of both the particle and the surrounding environment.

Figure 2.19 Size dependent localized plasmon resonance (oscillations of the conduction electrons) in metal nanospheres. Accordingly, a peak appears in the extinction spectrum of the metal nanospheres dispersion.

Extin

ctio

n

Wavelength

E E

-- ---

+ +++ + -- ---

+ +++ +

Haiyan Qin 23

The metal nanospheres has one SPR band, which only slightly red-shifts while enlarging the particle size. Metal particles with an anisotropy shape, such as gold nanorods (GNRs), can possess very different optical properties. For a rod-shaped nanoparticle, the conduction band electrons can oscillate resonantly with the external electromagnetic field along either the transverse direction or the longitudinal direction depending on the polarization of the incident light, as shown in Figure 2.20. As a result, two SPR bands, the transverse band and the longitudinal band, appear in the extinction spectra of metal nanorods. Similar to the SPR band in metal nanospheres, the transverse band in metal nanorods can hardly shift while changing their sizes; whereas the longitudinal band is found to shift in a wide wavelength range while varying the aspect ratio of rods. The tunable optical properties of metal nanorods make them a promising candidate for various applications.

Figure 2.20 Size dependent localized plasmon resonance (oscillations of the conduction electrons) in metal nanorods along both transverse and longitudinal directions. Accordingly, two peaks appear in the extinction spectrum of the metal nanorods dispersion.

Extinction is a combine effect of absorption and scattering. While exposing a metal nanoparticle, a certain part of incident light is absorbed by the particle and converted into heat immediately. This refers to the photothermal property of metal nanoparticles. The rest of light is scattered into all directions by the particle. The likelihood of a particle absorbs or scatters the incident light can be quantitatively described by the absorption cross-section or scattering cross-section . According to Mie and Gans’ theories, the and for a metal nanoparticle in an ellipsoid shape can be derived as follows 135:

≅ 23 + 1 − +

≅ 89 − ++ 1 − +

and the total extinction cross-section is = +

Extin

ctio

nWavelength

Transverse band

Longitudinal bandE E

-- ---+ + + + +

+ + + + +-- ---

-- -

+ ++

E E

+ ++

-- -


here λ is the wavelength of the incident light; V is the particle volume; and are the real and imaginary components of the particle dielectric constant. It is important to note that the dielectric constants for metals are highly wavelength dependent and can be written as . is the depolarization factor in the direction. represents for three axes of the ellipsoid a, b and c. For

spheres, the depolarization factors along three axes are equal, that is = = = . For a

rod-shaped particle with a >> b = c, ≅ and = = , where R is the aspect ratio

equal to .

From the theoretical results above, one can find that both and reach a maximum value

when + = 0. At the corresponding wavelength satisfied this equation, SPR occurs.

Although the scattering or absorption spectra for metal particles are difficult to measure directly, their scattering and absorption properties are indicated by the extinction spectrum, which can be measured very easily.

Several noble metal nanorods has SPR band falling into the visible and NIR range, such as gold, silver and copper, are potential candidates for SPR enhanced scattering imaging applications. Among them, GNRs are superior and got much more attention than others. On one hand, GNRs with various sizes and respect ratios can be easily obtained by a simple and repeatable synthesis method. On the other hand, gold is more chemical or photo stable than others.

2.5.2. Dark field microscopy

The scattering signals from a specimen can be collected and imaged by dark field microscopy. The dark field imaging system is a conventional bright field imaging system with a ring-shaped mask. As shown in Figure 2.21, the ring-shaped mask blocks all the paraxial rays from the light source. The off-axial rays illuminate the specimen but cannot directly enter the objective, whereas the scattering lights radiate to all directions and are partly collected by the objective. Using a dark field microscope, highly scattering particles in a size smaller than the diffraction limit can be detected. This does not overcome the diffraction limit but attributes to the high contrast providing by the dark background.

Figure 2.21 An illustration showing the principle of dark field microscopy.

Light source Ring-shaped mask

Condenser lens

Specimen with scattering substants

Objective

Illuminating light

Scattering ight

Haiyan Qin 25

2.5.3. SPR enhanced Rayleigh scattering bioimaging using GNRs

GNRs, similar to QDs, can be prepared to bio-probes, applied to stain biological targets, and recorded by their enhanced Rayleigh scattering signals instead of fluorescence signal for QDs. As bio-probes for imaging applications, GNRs have several advantages over QDs. First, the longitudinal SPR band of GNRs is tunable for a wider wavelength range from visible to mid-infrared 136, whereas the fluorescence for QDs can hardly beyond NIR. Second, the scattering of GNRs does not suffer from photobleaching or blinking as the fluorescence of QDs does. Third, GNRs are higher biocompatible. The toxicity of GNRs is mostly from the cetyltrimethylammonium bromide (CTAB) surfactant but not from the particles themselves 137. The CTAB surfactant nevertheless can be modified or replaced by other surfactants which have higher biocompatibility.

GNRs have been applied as SPR enhanced scattering probes to cellular labeling and imaging. For example, El-Sayed et al. 138 and Sokolov et al. 139 applied anti-EGFR antibodies conjugated GNRs to in vitro label EGFR receptors in tumor cells (Figure 2.22). Furthermore, Prasad et al. 140 used GNRs to stain Transferin receptors and monitored the receptor-mediated uptake into Hela cells. Oyelere et al. 141 applied GNRs in nucleus targeting. In addition, combined with the photothermal property, GNRs can distinguish the malignant cells out of nonmalignant cells and then killed them by heat 140.

Figure 2.22 Scattering images of anti-EGFR-GNRs incubating with (A) HaCaT nonmalignant cells, (B) HSC malignant cells and (C) HOC malignant cells. (Reprint permission by ACS) 140

However, GNRs have two fatal weaknesses which limit their applications. One is their relatively large size which is normally tens of nanometers in length. The large particle size limits the application of GNRs in single molecule tracking and reduces the spatial imaging resolution. The other weakness is the wide FWHM of their SPR band. The FWHM increases from about 50 nm to more than 200 nm while the enhanced Mie scattering peak shifts from visible to NIR region 136. Thus cross talk from particles with different scatting colors likely occurs and consequently it is difficult to apply GNRs for multicolor scattering imaging.

A B C


2.6. Quantum dots for photovoltaic application

Nowadays, the energy shortage is one of the stringent challenges faced by mankind. Fossil fuels, including oil, gas and coal, are the dominant energy sources. However, according to the current proved reserves and consumption rates, non-renewable resources of the earth were predicted to be exhausted in the near further, for example oil in one and a half centuries, gas and coal in decades. Recently, the post-earthquake nuclear crisis in Japan prompted the reassessment of the safety of nuclear energy and affected the energy policies of many countries. More and more efforts are being turned to push the clean, safe and renewable energy sources.

2.6.1. Solar cells

Solar energy is one of the most promising renewable energy sources in the world. The solar energy that reaches the planet in an hour is about 4.4 × 10 , which is closed to the total energy consumption of the whole world in a year. A solar cell or a photovoltaic cell is a device converting solar energy into electricity. In 1883, an American inventor Charles Fritts built the first solar cell 142. However, as more than a century has passed, solar photovoltaics is still holding a very small portion of the global energy consumption (0.04 % in 2005).

The first generation solar cells are p-n junction crystalline silicon cells. A p-n junction contains a p-type semiconductor layer, which is Si doped with boron or aluminum to provide an abundance of holes, and an n-type semiconductor layer, which is doped with phosphorus to provide extra free electrons. In a p-n junction, electrons flow from the n-type side to the p-type side to equalize the Fermi energy of both sides, and form a potential barrier. While illuminated with sunlight, electrons from the n-type side, excited by photon energy, overcome the potential barrier and flow to the p-type side. Thus an external potential is formed to power a load. The silicon cells are holding a share of about 85% of the global solar cell market. Despite of their high efficiency, silicon solar cells have poor prospects due to their high manufacture cost.

The second generation solar cells are based on the thin-films of semiconductors. They include amorphous silicon cells, polycrystalline silicon, CdTe cells on glass, Cu(In,Ga)Se2 alloy cells and so on. This is a low cost manufacturing strategy. However the efficiencies of thin-film solar cells are low comparing to the first generation solar cells. The thin-film cells are now holding a market share about 15%.

The third generation solar cells, aiming in high efficiency and low cost, are not based on the traditional charge carries separation by p-n junctions. Unlike the first and second generations, the theoretical efficiency of the third generation solar cells is not limited by the so-called Shockley-Queisser limit of 30 % 48. They include nanocrystal solar cells, dye-sensitized hybrid solar cells, and polymer solar cells and so on. The third generation solar cells are still in the research stage but considered to be a promising low-cost high-efficiency alternative to the commercial solar cells.

Haiyan Qin 27

2.6.2. QD-sensitized solar cells

A QD-sensitized solar cell (QDSC) is a variation of a dye sensitized solar cell (DSC) belonging to the third generation solar cells. A typical structure of QDSCs is shown in Figure 2.23. Unlike the traditional silicon solar cells, in which both free electrons and the electric field that separates charges are provided by the silicon, QDSCs separate these two functions. In QDSCs, the QDs provide photon excited electrons; whereas the TiO2 nanoparticle pack and the electrolyte provide the electric field. TiO2 nanoparticles adsorbing QDs are deposited on an ITO glass. A liquid redox electrolyte fills between the TiO2 nanoparticle pack and the platinum cathode. While exposed to sun light, electrons are generated in the QDs, flow to the TiO2 particles, and then the ITO anode. The electrons loose energy after passing through the external circuit and back to the platinum cathode. After passing through the redox electrolyte solution, electrons finally recombine back to the QDs.

Figure 2.23 Principle of the QD-sensitized solar cells.

2.6.3. QD-sensitized solar cells versus dye-sensitized solar cells

As sensitizers for solar cells, QDs absorb sunlight covering a broad wavelength range with high efficiency. On one hand, a QD can absorb a much broader range of the solar spectrum due to its much wider absorption band compared to that of an organic dye. On the other hand, the high charge mobility of QDs 143 allows to utilize a film of QD sensitizers to absorb light in QDSCs instead of only using a monolayer of dye sensitizers in DSCs. Moreover, the carrier multiplication process occurring in QDs further enhance the efficiency of the QD sensitizers. Carrier multiplication, or so called multiple exciton generation, is an inverse Auger process that one photon creates two or more excitons through impact ionization 144. In addition, QDSCs are not only highly photostable but also of low cost and facile to manufacture.

ITO

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Chapter 3. Experimental design

3.1. Bioimaging using quantum dots (Paper I and II)

3.1.1. Water-soluble QDs

An ideal QD for bioimaging application purpose is supposed to have the following features: water soluble, small size, red or NIR emission, high QY, high stability, low cytotoxicity, narrow FWHM and facile to produce. Due to the mature synthesis methods for oil-phase QDs, a traditional way to obtain aqueous QDs is to transfer from oil-phase QDs by ligand exchange or ligand coating. However, the properties of the aqueous QDs obtained are unsatisfactory for many biomedical imaging applications. The ligand exchange process significantly decreases the QY and stability of the QDs. An extra ligand coating added on the surface of oil-phase QDs unfavorably increases the particle size. Moreover, the transferring methods themselves are very complicated. To overcome all these problems and meet the application requirements, we proposed CdTe/ZnSe core/shell QDs to be synthesized via an aqueous method and CdTe QDs via a hydrothermal method.

Figure 3.1 Structures of MSA capped CdTe/ZnSe core/shell QDs and MSA capped CdTe QDs.

CdTe/ZnSe core/shell QDs synthesized via aqueous method (Paper I)

CdTe QDs are promising bio-probes since their emission peak can be tuned to red or even NIR region ascribing to the narrow bulk band offset (1.475 eV) they have. ZnSe is not popular as shell material

Aqueous synthesis method

CdTe CdTe

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30 Chapter 3. Experimental design

for CdTe QDs, but has two major merits over other commonly used shell materials such as CdS, HgTe and ZnS. First, zinc ions are much less toxic than cadmium ions or mercury ions. Second, ZnSe has a much smaller lattice parameter mismatch to CdTe (13.4%) than most other materials, for example, ZnS to CdTe is 18%.

MSA, which is a small molecule, can not only provide the QDs with a small size, but also greatly enhance the stability of the QDs in weak acid environments. This is because MSA has a high dissolubility in acid environments similar to some other surfactants, e.g. MAA and TGA. More importantly, each MSA has two carboxyl groups while other surfactants normally have only one. The primary and secondary ionization processes of these two carboxyl groups equip the QDs with a pH buffer surrounding. Hence MSA capped QDs present much higher pH stability than those capped with other small molecules. We directly synthesized QDs in aqueous phase based on the epitaxial growth of ZnSe shells on the CdTe core with MSA as surfactants.

CdTe QDs synthesized via hydrothermal method (Paper II)

To further decrease the size of the QDs, stripping away the shells is one of the most effective ways. Without the protection from the shell structure, QDs are more unstable and very toxic for cells. Applying the hydrothermal method for QDs synthesis is one solution to this problem.

We report results from a hydrothermal synthesis of CdTe QDs with MSA as surfactants. The hydrothermal method is a modification of the traditional aqueous synthesis. Besides all the advantages of the traditional aqueous method and using MSA as surfactants, the hydrothermal method also benefits from the relatively high synthesis temperature. Defects, which are harmful for QY and stability of the QDs, can be significantly reduced under high temperature. Over all, CdTe QDs synthesized via the hydrothermal method capped with MSA are highly stable, low toxic and in small size.

3.1.2. Cell culture for fixed cell labeling

QD-IgG probes have been fabricated for HER2 protein labeling in MCF-7 breast cancer cells. MCF-7 is a female breast cancer cell line isolated in 1970 by the Michigan Cancer Foundation. The MCF-7 cell line is a typical in vitro model expressing HER2 (human epidermal growth factor (EGF) receptor 2) proteins 145. HER2 is a remarkable breast cancer marker. Overexpression of HER2 promotes disease recurrence and malignancy of mammary epithelial cells 146. It is a significant attempt to in vitro label and detect the HER2 proteins on cell membrane using QD bio-probes.

In order to obtain strong fluorescence signals, the indirect immunofluorescence technique (shown in Figure 2.11) is performed for fixed cell staining. The reaction process to conjugate carboxyl-QDs with IgG molecules using NHS is similar to Figure 2.9. Conjugation efficiency remains high enough in our experiments while skipping the QD activation step using EDC.

Haiyan Qin 31

3.2. Single molecule tracking using quantum dots (Paper III)

3.2.1. Cell culture for single molecule tracking

A-431 cell line was established from an epidermoid carcinoma in the vulva of an 85 year old female patient. A-431 cell line expresses high levels of epidermal growth factor receptor (EGFR) on their membranes. Thus it is a very good model to study the physiological properties of EGFR. The overexpression of EGFR has been associated with many types of cancers. HER2 is an important member of the EGFR family. Activation of the HER2 is believed to involve dimerization and also interaction with other subtypes of EGFRs. It is important to have a close study of HER2 dynamics in living cells.

3.2.2. Living cell labeling strategy for single molecule study by QDs

Most of the current single biomolecule tracking studies reported have used the indirect immunofluorescence strategy or the indirect complement immunofluorescence strategy, as shown in Figure 2.11. The final fluorescence labeling obtained are big complexes including target molecules, primary antibodies, secondary antibodies (or biotins and streptavidin) and QDs. There are some obvious shortcomings of these strategies for single molecule tracking. One IgG secondary antibody can conjugate to several QDs. While labeling with QD-IgG probes, one primary antibody can also bind to several IgG proteins. The target molecules labeled with such large labeling complexes can hardly be located in a high spatial resolution by monitoring the QDs position. Moreover, the labeling complexes are also heavy compared to the target molecule, and may affect the movement of the target molecule.

Our QD bio-probes for single molecule tracking are prepared using the direct immunofluorescence strategy to conjugate QDs with primary affibodies. An affibody is a small and simple artificial protein that mimics the functions of an antibody. The size of an affibody molecule is only 6 kDa, which is one twenty-fifth of the size of an antibody. The use of both the direct immunofluorescence strategy and affibodies ensures that the labeling of target molecules with QDs is one-to-one, and minimizes the influence from the fluorescent probes on the target molecules. In addition, unlike antibodies, affibodies can tolerate environments with wide pH range and high temperature.

Figure 3.2 Label membrane molecules with QD-affibody bio-probes for single molecule tracking.

QD

Membrane molecule

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Intracellular

ExtracellularAffibody


3.3. Blinking of single quantum dots (Paper IV)

Many efforts are underway to produce nonblinking QDs and to understand the blinking mechanism for single QDs. Ionization of QDs is considered to be a dominating factor for the dark state. According to this consensus, QDs with thick shells or gradient compositions have been fabricated to downplay ionization and therefore to suppress emission blinking. Nonradiative Auger recombination caused by ionization has also been a popular explanation for the dark state of single QDs. However, the Auger recombination rates in QDs are several orders of magnification faster than the blinking rates in QDs. Eliminating the nonradiative Auger recombination process, the long-range Coulomb potential becomes one of the more suspected causes.

In order to study the long-range Coulomb potential effect on single QD blinking, we have a close look at the blinking phenomenon on QD pairs. Two types of negative charged water-soluble QDs, CdSe/CdS QDs and CdSe/ZnS QDs, were selected for experiments. Small droplets of diluted QD samples were added on clean cover glasses and dried in air. QD pairs selected for study should meet the following two conditions. First, the spatial distance between the two QDs should range from hundreds of nanometers to several micrometers. Second, there should be no other QDs present around within 10 µm of the QD pair, so that any influence from other QDs can be ruled out. The time correlation analysis of the two nearby QDs can help to identify the influence from the long-range Coulomb potential on single QD blinking.

3.4. Cytotoxicity of quantum dots (Paper V)

3.4.1. Cell culture for QDs cytotoxicity study

Since the mechanism behind the cytotoxicity of QDs remains unclear, scientists are still hesitant to apply QDs in the clinic. In most in vivo studies or clinical applications QDs are directly injected into vascular intravenously. Thus the vascular endothelial cells suffer first from the toxic damage caused by QDs.

We study the vascular endothelial toxicity of CdTe QDs using an in vitro model of HUVECs. Primary HUVECs are human umbilical vein endothelial cells isolated directly from human umbilical veins. HUVECs are widely used for endothelial physiological studies including macromolecule transport, blood coagulation and vasculogenesis, and pathologic processes.

3.4.2. Apoptosis induced by QDs

A high quality QD with little toxic ion leaking is selected for studying their endothelial toxicity, in order to eliminate the toxicity from heavy metal ions. The cell viability, which is depending on the dose of QDs treated to HUVECs, is investigated using a MTT assay and calcein ester green fluorescence assay. The QD-induced apoptotic cell death is measured by an Annexin-V apoptosis detection kit. The intracellular reactive oxygen species (ROS) increases and the ROS-induced mitochondrial membrane potential loss is likely to be the major causes for cell apoptosis. To further

Haiyan Qin 33

reveal the mechanism behind, we measured the expressions of Bcl-1, Bax, cytochrome c, caspase 9 and caspase 3 proteins, which play important roles in cell apoptosis via a mitochondrial pathway. In a mitochondrial apoptosis pathway, the balance between anti-apoptosis protein Bcl-2 and pro-apoptosis protein Bax is broken first. This results in a mitochondrial membrane potential Δψm dissipation and thereby mitochondrial fragmentation. Subsequently cytochrome c will leak from the mitochondria to the cytoplasm, and thereafter activates the mitochondrial death pathway. The released cytochrome c binds to pro-caspase 9 and cleaves it into active caspase 9. The caspase 9 subsequently cleaves and active pro-caspase 3 into caspase 3, which finally introduces apoptosis.

3.5. Gold nanorods for dark field bioimaging (Paper VI)

3.5.1. Control synthesis of GNRs

Gold nanorods (GNRs) are promising bioimaging agents due to their superior features such as extremely high biocompatibility, tunable scattering color, and most importantly no photobleaching or blinking of their scattering signal. However, an important feature to be desired from GNRs as bio-probes is a narrow FWHM of their scattering spectrum. The scattering light with the broad spectrum from GNRs is hard to distinguish from the scattering signals from cellular organelles or dirt. The broad FWHM of the scattering spectrum has two causes. First, GNRs are produced with low yield. Along with the production of nanorods, many particles with other shapes are also produced, such as triangles, circular disks, wires and so on. Second, GNRs with different morphologies, referring to aspect ratios of the rod-shape, are produced simultaneously. Gold nanoparticles with different sizes and shapes hold different SPR wavelength, and thus altogether cause a very broad SPR enhanced scattering spectrum. Separating GNRs from other shapes or those with undesired sizes or aspect ratios is very difficult and time consuming. It is desperately needed to produce GNRs with high yield and controllable morphology.

We control the yield and morphology for GNRs in a seed-mediated synthesis method. The seed-mediated synthesis method is based on the work by Perez-Juste et al 147. The synthesis procedure contains two steps: producing gold nanoparticle seeds and seeds growing to rods. We systematically studied the dependence of GNRs growth on silver ions and CTAB concentrations in the growth solution. As a demonstration, we apply the high quality GNRs to unspecifically stain COS-7 cells.

3.5.2. Cell culture selection for GNRs labeling

COS-7 is one of the two COS cell lines from African green monkey kidney fibroblast cells. COS-7 cells are widely used as target cells for transfection, which is to artificially modify the genes by introducing nucleic acids into cells. Staining and imaging of COS-7 cells using GNRs is a convincing demonstration illustrating the great potential of GNRs as bio-probes.


3.6. Quantum dot-sensitized solar cells (Paper VII and VIII)

Although the QD-sensitized solar cells (QDSC) have many advantages over traditional solar cells, the weakness of low efficiency slows its commercialization process. The efficiency of solar cells depends on three factors: the absorption of solar lights, the electron injection efficiency and the electron recombination rate. We rationally designed two novel types of QDs as sensitizers to improve the efficiency of QDSCs considering these three terms.

The first one is the reversed type-I CdS/CdSe QD shown in Figure 3.3 (a). The CdS/CdSe QD is formed with a barrier material as the core and a well material as the shell. The photogenerated electrons are confined mostly in the shell, where the ground electron state is mainly distributed. Compared to the type-I core/shell QD which confines photogenerated electrons in the core, the electrons are more easily extracted from the reversed type-I QDs to the TiO2 electrode. Furthermore, a thin layer of CdS is grown on the QDs-TiO2 film (QD adsorbed TiO2 electrode) in order to reduce the recombination between electrons from the TiO2 electrode and holes from the redox electrolyte.

The second type of QDs we proposed is type-II ZnSe/CdS core/shell QDs. As shown in Figure 3.3 (b), the optimal CB is located in the CdS shell and the optimal VB is located in the ZnSe core. Therefore the bandgap shrinks and the absorption spectrum broadens. More importantly, the electrons are confined in the shell of QDs, which favors the electron extraction from QDs to TiO2 electrode. A layer of ZnS is adsorbed on the QD-sensitized electrode to reduce the charge recombination between TiO2 electrode and electrolyte.

Figure 3.3 Schematic representation of reversed type-I QD-sensitized solar cells and type-II QD-sensitized solar cells.

CB

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Chapter 4. Results and discussion

4.1. Bioimaging using quantum dots (Paper I and II)

4.1.1. Properties of aqueous synthesized CdTe/ZnSe QDs

CdTe/ZnSe core/shell QDs were obtained by the aqueous synthesis method. The ZnSe coating shifted both the absorption and fluorescence spectra of the core CdTe QDs to longer wavelengths as shown in Figure 4.1. As the ZnSe became thicker, the surface defects reduced and the QY of the QDs increased. However the QY met a maximum value when emission reached 587 nm and started to decrease for thicker ZnSe shells. This is because when the shell layer overgrows the surface defects are formed in the shell layer. Another reason is the QY measurement error caused by emission wavelength mismatch between QDs and the reference, which is Rhodamine 6G (QY = 95% in ethanol 148). The narrow and steady FWHMs of the fluorescence spectra indicated that no ZnSe QD was produced and no degradation of the CdTe cores occurred.

Figure 4.1 Optical properties of CdTe/ZnSe QDs. (a) A digital image of the CdTe/ZnSe QD solution illuminated by a UV lamp. (b) Temporal evolution of absorption (dashed line) and fluorescence (solid line) spectra of CdTe core and CdTe/ZnSe QDs. (c) QYs and FWHMs of CdTe/ZnSe QDs versus emission peak wavelength.

Transmission electron microscopy (TEM) images confirmed that the QD samples were highly uniform and monodispersed. From the high resolution TEM (HRTEM) images (Figure 4.2 (a)), the size of CdTe/ZnSe QDs with 610 nm emission was measured to be 4.6 nm with σ = 15%. The clear lattice fringes manifested the high crystallinity of the QDs. The selected area electron diffraction (SEAD)

36 Chapter 4. Results and discussion

pattern and X-ray diffraction (XRD) result indicated that the QDs had a cubit zinc blende structure. The energy dispersive X-ray spectrum (EDX) confirmed the compositions of CdTe/ZnSe QDs.

The robust CdTe/ZnSe QDs had much higher photostability comparing to the CdTe core QDs as shown in Figure 4.2 (b). With continuous illumination from a 1.5 mW Xe-lamp at 488nm, CdTe QDs lost 50% of their emission intensity within 13 min, while CdTe/ZnSe QDs maintained their original emission intensity for more than 150 min. The CdTe/ZnSe QDs also presented extremely low cytotoxicity (Figure 4.2 (c)). MCF-7 cells were incubated with CdTe/ZnSe QDs at an extremely high concentration of 310 μg/mL, which was 1 to 2 orders of magnitude of the normal concentration for immunocytochemistry applications. As many as nearly 80 % of the MCF-7 cells survived after 24 hr and more than 65% even survived after 48 hr.

Figure 4.2 (a) HRTEM image of CdTe/ZnSe QDs. (b) Photostability of CdTe/ZnSe QDs compared with CdTe QDs. (c) Cytotoxicity of CdTe/ZnSe QDs using a MCF-7 cells as an in vitro model.

4.1.2. Properties of hydrothermal synthesized CdTe QDs

The fluorescence peak of the CdTe QDs synthesized via the hydrothermal method was tunable from about 500 nm to nearly 700 nm by continuously increasing the particle size (Figure 4.3 (a), (b) and (c)). The hydrothermal method is a fast QD synthesis approach. Under an environment temperature of 180 ⁰C, red emission QDs were obtained within 70 min. As shown in Figure 4.3 (d), a high QY of 64% of QDs with 570 nm emission was obtained. The QY value maintained a high level beyond 50% for QDs with the emission peak located between 555 nm and 600 nm. According to the HRTEM image, the size of CdTe QDs with 590 nm emission was 3.8 nm in diameter. The XRD result indicated that the hydrothermal synthesized CdTe QDs had a cubic zinc blende structure.

The CdTe QDs synthesized via the hydrothermal method presented extremely high pH stability, high photostability and low cytotoxicity. No emission peak wavelength shift occurred while adjusting the pH value of the QDs solution from pH 5 to pH 11. As shown in Figure 4.4 (a), more than 85% emission intensity remained for 24 hr and 65% for 48 hr in environments with all pH values measured. Under continuous illumination by a halogen lamp, the CdTe QDs synthesized via traditional aqueous method lost 50% of their fluorescence intensity within 15 min, while the fluorescence intensity of CdTe QDs synthesized via hydrothermal method, in contrast, continued to increase (Figure 4.4 (b)). The illumination induced emission enhancement is probably due to the elimination of surface defects on the QDs. In a cell viability study using MTT assay and MCF-7 cells, almost no cells survived while

Haiyan Qin 37

incubated with 100 μg/mL aqueous synthesized CdTe QDs for 24 hr. In sharp contrast, 62.8% cells survived after 24 hr incubated with hydrothermal QDs in the same conditions, and 28.6% even survived after 48 hr incubation.

Figure 4.3 Optical properties of CdTe QDs. Digital images of QD solutions (a) under sunlight and (b) illuminated by a UV lamp. (c) Temporal evolution of absorption (dash line) and fluorescence (solid line) spectra. (d) QYs and FWHMs versus emission peak wavelengths.

Figure 4.4 The (a) pH stability, (b) photostability and (c) cytotoxicity of hydrothermal synthesized CdTe QDs.

4.1.3. Detection of HER2 on MCF-7 cells using QD bio-probes

Both aqueous synthesized CdTe/ZnSe QDs and hydrothermal synthesized CdTe QDs were conjugated with IgG secondary antibodies forming QD bio-probes. MCF-7 cells as an in vitro model were incubated with primary anti-HER2 antibodies. Subsequently the QD bio-probes were applied to specifically label the HER2 breast cancer marker in the MCF7 cell membrane, shown in Figure 4.5.

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Figure 4.5 HER2 proteins of MCF7 cells were incubated with anti-HER2 antibodies and QD-IgG bio-probes. (a) Confocal fluorescence microscopic image, CdTe/ZnSe QDs; (b) normal fluorescence microscopic image, CdTe QDs. Scale bar, 10 μm.

4.2. Single molecule tracking using quantum dots (Paper III)

After optimizing the imaging system, single QDs with blinking were observed and recorded in high spatial and temporal resolution. The FWHM of the intensity profile (spot size) of a single QD was about 500 nm. The temporal resolution was 7 Hz for frames with 256 × 256 pixels. A low concentration staining of HER2 proteins on the cell membrane was obtained by treating the cells with a low concentration (< 2 nM) of QD bioprobes. More than 95% of the fluorescence spots observed were blinking. This confirmed that the direct immunofluorescence strategy highly reduced the occurrence of two or more QDs conjugating to one target protein. The individual blinking QDs which had no crosstalk from others were selected for studies (Figure 4.6 (a) and (b)).

Figure 4.6 Single HER2 protein tracking using QD bio-probes. (a) A white filed image of an A431-cell. The white lines show the profiles of the cell membrane and nucleus membrane; (b) A fluorescence image of the selected area identified with a red rectangle in (a); Examples of HER2 proteins interact with TCZs (c), (d) and (e).

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Haiyan Qin 39

The trajectories of individual HER2 proteins on the membrane of MCF-7 cells were analyzed by Matlab programming and a “SpotTracker” plug-in on ImageJ 149. In addition to the 2-D random walks, the HER2 proteins exhibited confined diffusion. Some features of the dynamic behavior of the HER2 proteins and the properties of the transient confinement zones (TCZ) for HER2 proteins were demonstrated. The TCZs were in a size of 200 nm to 400 nm in diameter. The dwell time of HER2 proteins in a TCZ was ranging from seconds to minutes. The diffusion coefficient of single HER2 proteins marked by QD-affibody probes was about 3.5 × 10-10 cm2/s. Three modes of movements (Figure 4.6 (c)-(e)) could be identified: (1) molecules fall in and get trapped in a TCZ; (2) molecules move from one TCZ to another; and (3) molecules visit the same TCZ repeatedly.

4.3. Blinking of single quantum dots (Paper IV)

We have experimentally studied the phenomenon of emission blinking from negatively charged single CdSe/CdS QDs and CdSe/ZnS QDs. A spectacular bunching effect was discovered in the emission from two nearby QDs. Two QDs, separated from each other less than 1 μm, tend to emit synchronously, as shown in Figure 4.7. The time correlation effect was shown to slowly disappear while increasing the distance between the two QDs.

Figure 4.7 A typical fluorescence image of a QD pair (left) and their emission intensity fluctuations along with exposure time (right).

We proposed that the influence from the long-range Coulomb potential provided by the negative charge at the QD surface was the main cause for blinking in single QDs and bunching effect in QD pairs. According to the simulation results, the hole states of the negative charged QD were attracted towards the charge on the particle surface. As a result, the photogenerated hole and electron were separated spatially and prohibited radiative recombination. The tunneling rate between the two spatial positions inside the QD and in the negative charge region was calculated to be in the order of


103 Hz. This simulation result agreed with the QDs blinking rate observed in experiments. Furthermore, without the influence of the Coulomb potential from the surface charge, the spontaneous emission rate in a QD was calculated to be much lower than the stimulated emission rate. Therefore direct stimulated emission is not responsible for the observed emission bunching effect in QD pairs. However, considering a situation that the hole state splits into two parts, one inside the QD and the other close to the negative charge, stimulated emission induced by the other QD nearby became dominant. That is to say, when the “off” QD is stimulated by the emission from the QD nearby, the hole which has been attracted to the QD surface tunnels back into the QD. Thereafter, the hole and the electron recombine and the “off” QD switches to “on” state.

4.4. Cytotoxicity of quantum dots (Paper V)

The aqueous synthesized CdTe QDs decreased the cell viability and increased the cell apoptosis of HUVECs in a dose-dependent manner. With 24 hr treatment, the TC50 value (concentration of CdTe QDs causes 50% cell death) was 10.60 μg/mL, which was approximately 24 nM (Figure 4.8 (a)). As a pro-apoptosis marker, the phosphatidylserine (PS) membrane translocation indicated a rapid apoptosis response of the HUVECs incubated with CdTe QDs. Since the quantity of Cd2+ ions degrading from the CdTe QDs was extremely small, cell death and apoptosis observed were caused by QDs, and not by the heavy metal ions.

The intracellular ROS level increased in a QD dose dependent manner for 12 hr incubation. However, with 24 hr QD incubation, the ROS level fell back to a level close to or even lower than the control sample without QDs treatment. These results illustrated that the ROS responded earlier than and had an important effect on the cell apoptosis process. The high intracellular oxidative stress led to mitochondria dysfunctions and morphological changes. As the QDs concentration increased, more and more cells presenting disruption of mitochondrial membrane potential Δψm were observed. In the meantime, the morphology of mitochondria changed from a tubular shape to a granular shape. The experimental results for cells treated with QDs and the ROS scavenger NAC together suggested that ROS-induced mitochondria impairment was the main cause for the QD-induced cell apoptosis.

Following the mitochondrial apoptotic pathway in HUVECs, we applied the immunoblotting technique to evaluate the expression levels of pro-apoptotic Bax and anti-apoptotic Bcl-2 proteins, leakage of cytochrome c in cytoplasm, the expression levels of pro-/cleaved-caspase-9 and pro-/cleaved-caspase-3. The increase of Bax expression and decrease of Bcl-2 expression induced the release of cytochrome c from mitochondria (Figure 4.8 (b)). Subsequently, initiator caspase-9 and effector caspase-3 were proteolytic activated (Figure 4.8 (c)). Hereafter, the activated caspases would cleave cellular targets and eventually lead to cell apoptosis. General caspase inhibitor Z-VAD-FMK and specific caspase-3 inhibitor AC-DEVD-CHO effectively suppressed the QD-induced cell apoptosis. In all, the cell apoptosis induced by CdTe QDs is a caspase-dependent process through a mitochondrial pathway, which is restored in Figure 4.9.

Haiyan Qin 41

Figure 4.8 Cytotoxicity of aqueous synthesized CdTe QDs on HUVECs. (a) Cell viability measured by MTT assay. (b) Cytochrome c (green) leakage from mitochondria (red) after 24h QDs treatment. Scale bar, 50 μm. (c) CdTe QDs induced activation of mitochondrial apoptotic pathways.

Figure 4.9 A proposed model showing the mechanism of QD-induced apoptosis effects in HUVECs.

4.5. Gold nanorods for dark field bioimaging (Paper VI)

In a seed-mediated synthesis method, the yield and morphology of GNRs produced were controlled by adjusting the CTAB and silver ion concentrations in the growth solution. At a high concentration of CTAB (0.1 M), the aspect ratio (AR) of GNRs increased linearly with silver ion concentration [Ag+]

Control

10 μg/mL QDs

Bax

Bcl-2

Pro-caspase-9

Cleaved-caspase-9

Pro-caspase-3

Cleaved-caspase-3

β-actin

CdTe QDs (10 μg/mL)

(a) (b) (c)

cytoplasm

QD


from 20 to 100 μM (Figure 4.10 (a)-(c)). A maximum AR value, or longitudinal band absorption peak, occurred at [Ag+] of 120 μM. An empirical equation of AR = 2.73 + 0.018 × Ag

was found in this case. The value of [Ag+] is here in the unit of μM.

At a lower CTAB concentration, between 0.1 and 0.04 M, the AR and yield of GNRs were also found to be highly dependent on the [Ag+] in the growth solution. While further reducing the CTAB concentration, the yield and morphology of the GNRs became more sensitive to the [Ag+] in the growth solution. At a CTAB concentration of 0.02 M, gold nanoparticles with irregular shapes were obtained while [Ag+] exceeded 40 μM. At a [Ag+] value of 80 μM, the longitudinal band peak disappeared indicating no rod-shaped particles were formed. The TEM image confirmed that only gold nanospheres were produced in this case.

In a GNR growth process, CTAB micelles were firstly formed in the growth solution, as suggested by the “soft template mechanism” 150. The CTAB micelles were templates for controlling the growth of GNRs. A part of CTAB reacted with Ag+ ions provided by AgNO3 forming Ag-Br-surfactant complexes. These Ag-Br-surfactant complexes were supposed to be a critical factor to control the size and shape of CTAB micelles. However, the mechanism behind is still unclear. Therefore, the yield and morphology of GNRs were effectively controlled by adjusting the concentrations of CTAB and Ag salt in the growth solution. Moreover, because the Ksp of AgBr (3.3 × 10-13) is much lower than that of AgCl (1.0×10-10), the formation of Ag-Br-surfactant complexes is prior to that of Ag-Cl-surfactant complexes. While increasing the concentration of Ag salt to a certain level, not only Ag-Br-surfactant complexes but also a small amount of Ag-Cl-surfactant complexes occurred in the growth solution. We speculated that the occurrence of Ag-Cl-surfactant complexes could be a critical factor affecting the yield and morphology of the GNR growth.

In addition, a high quality GNR sample produced under the controllable seed-mediated synthesis method was applied for unspecifically staining on COS-7 cells. The GNRs were adsorbed on the surface of COS-7 cells and presented bright SPR enhanced scattering color under dark field with white light illumination. As shown in Figure 4.10 (d), the bright yellow scattering signal from the GNRs was easily distinguished from the white scattering signal from cell organelles.

Figure 4.10 (a) A digital image of GNRs solutions under sunlight. (b) A TEM image of GNRs with longitudinal band absorption at 805 nm. (c) Absorption spectra of GNRs solutions synthesized at CTAB concentration of 0.1 M and five different [Ag+] of 20, 40, 60, 80 and 100 μM. (d) A dark field image of COS-7 cells stained with GNRs.

Haiyan Qin 43

4.6. Quantum dot-sensitized solar cells (Paper VII and VIII)

A series of reversed type-I CdS/CdSe core/shell QDs were produced for QDSCs applications. As the simulation results show, see Figure 4.11, the electron wave function distributes largely in the core region of the type-I CdSe/CdS QDs and shows no change while increasing the shell thickness. In contrast, the electron wave function of the reversed type-I CdS/CdSe QDs is sensitive to the shell thickness. The wave function distributes largely in the shell region in a reversed type-I QD with a thick shell. As located in the shell region instead of in the core region, photon generated electrons can easily transport from the QDs to the TiO2 electrode.

Figure 4.11 Ground states of electron in CB and the corresponding wave-function distribution in the (a) type-I CdSe/CdS QDs and (b) reversed type-I CdS/CdSe QDs.

Figure 4.12 APCE spectra of reversed type-I CdS/CdSe, type-I CdSe/CdS, and CdSe QDSCs (a) without and (b) with a layer of CdS adsorbed on the TiO2 film.

Type-I QD Reversed Type-I QD


In order to reduce the charge recombination between the QDs and the redox electrolyte, a layer of CdS was adsorbed onto the QDs-TiO2 film by chemical-bath deposition (CBD). With this CdS layer, the absorption spectra of the QDs-TiO2 film red shifted and the absorbance was greatly increased in the wavelength region of 400 to 500 nm. Therefore the solar light absorption efficiency of the designed QDSCs was enhanced.

According to the fluorescence lifetime of the QDs, the charge transfer rate constant (Ket) of the CdS/CdSe QDs-TiO2 structure was estimated 151 to be 0.3575 ns-1, which was much larger than that of the CdSe/CdS QDs-TiO2 or CdSe-TiO2 structures. This result further confirmed that the reversed type-I QDs could significantly enhance the electron injection efficiency of the QDSCs. Furthermore, a thicker shell favored the Ket value of the reversed type-I QD, because the electron wave function distributed more in the shell region in this case. However, the opposite was the case for type-I QDs. Since the electron wave function distributed mainly in the core region, the thicker the shell was, the lower Ket the type-I QD had.

Both normal and reversed type-I QDSCs were manufactured for incident photon to current efficiency (IPCE) and absorbed photon to current efficiency (APEC) measurements. Without the CdS CBD layer, the IPCE and APCE values for CdS/CdSe QDSCs were 25.2% and 33.4%, respectively, which are significantly higher than those for CdSe/CdS or CdSe QDSCs. The APCE value for CdS/CdSe QDSCs with CdS CBD layer was even as high as 60.2%.

In addition, two type-II ZnSe/CdS core/shell QD samples with different shell thicknesses were synthesized for QDSCs application: ZnSe/CdS-1 QDs (shell, 0.65 nm) and ZnSe/CdS-2 QDs (shell, 0.95 nm). As shown in Figure 4.13 (a), both absorption and fluorescence spectra of ZnSe/CdS QDs were red-shifted by increasing the CdS shell thickness. The wavelength of absorption maximum for ZnSe/CdS QDs could reach 580 nm, which was much longer than that for the CdS or ZnSe QDs. This ascribed to the separate CB and VB optimal edges in the type-II QDs. In this structure, the absorption spectrum could be broadened without decreasing the CB level as type-I QDs do. Along with an increasing shell thickness, the fluorescence lifetime of the ZnSe/CdS QDs was increased significantly. A thicker shell favored the charge separation in type-II QDs, since the electrons and holes were located separately in the core and the shell.

Figure 4.13 Type-II ZnSe/CdS QDSCs. (a) Absorption and fluorescence spectra of ZnSe, ZnSe/CdS-1, and ZnSe/CdS-2 QDs. (b) ICPE spectra of ZnSe/CdS-1 and ZnSe/CdS-2 QDSCs. Inset: absorption spectra of ZnSe/CdS-1 and ZnSe/CdS-2 adsorbed on the TiO2 film.

(a) (b)

Haiyan Qin 45

ZnSe/CdS QDSCs with a layer of ZnS adsorbed on the QDs-TiO2 film were fabricated for test. As shown in Figure 4.13 (b), the ZnSe/CdS-2 QDSC had an IPCE of 28% at 410 nm. Taking the absorption spectrum into consideration, the APCE of the QDSC was as high as 40% at 410 nm. Under 100 mW/cm2 AM 1.5 irradiation, the open circuit voltage and short circuit current of the ZnSe/CdSe-2 QDSC were 0.44 V and 2.29 mA/cm2. However the QDSC had a low fill factor of 0.27. Further design of the electrolyte or the whole solar cell structure can probably harness more of the potential of type-II QDs as QDSC sensitizers.

Chapter 5. Conclusions and future perspectives

This thesis focuses on improving the properties of nanoparticles and developing their applications in biomedical imaging and photovoltaic devices. It includes highly stable and low toxic QDs for in vitro imaging applications (Paper I and II), single molecule tracking on living cell membranes (Paper II), single QD blinking property and mechanism study (Paper IV), in vitro endothelial toxicity of QDs (Paper V), GNRs with high yield and controllable morphology for cell imaging (Paper VI), and QDs for QD-sensitized photovoltaic devices (Paper VII and VIII).

The properties of aqueous synthesized CdTe QDs for bioimaging applications have been highly improved by coating with a ZnSe shell and MSA surfactant. The emission of MSA capped CdTe/ZnSe possesses, with narrow FWHM and high QY, can be shifted to as long as 630 nm. Comparing to CdTe QDs with the same surfactant and obtained with the same synthesis method, CdTe/ZnSe QDs present higher photostability and lower cytotoxicity, which are significant properties for biomedical imaging applications. Furthermore, in order to obtain aqueous QDs with a small particle size for single molecule tracking and intracellular labeling applications, we have produced MSA-capped CdTe QDs via hydrothermal method. Comparing with aqueous synthesis CdTe QDs, hydrothermal method is not only a more efficient synthesis method, but also provides CdTe QDs with higher stability over a wide pH range, higher photostability and lower cytotoxicity. As demonstration of biomedical imaging applications, both aqueous synthesized CdTe/ZnSe QDs and hydrothermal synthesized CdTe QDs were conjugated with IgG proteins forming fluorescent bio-probes. Subsequently, the QD-IgG bio-probes were applied to detect the breast cancer marker HER2 on the surface of fixed MCF-7 cancer cells. All results above suggest that both the aqueous synthesized MSA-capped CdTe/ZnSe QDs and hydrothermal synthesized MSA-capped CdTe QDs are promising candidates as fluorescent probes for biomedical applications.

Besides fixed cell labeling and imaging, the dynamics behavior of single proteins in living cell membranes have been studied using QD bio-probes. Taking the advantages of the direct immune-fluorescence strategy and the small size of affibody molecules, individual HER2 molecules were labeled by single QD probes. According to the trajectories of the HER2 proteins on the A431 cells, modes of TCZs and characterizing parameters such as dwell time and diffusion coefficient of HER2 proteins have been identified. This advanced single molecule tracking protocol provides an efficient way for single biomolecule labeling and tracking studies.

48 Chapter 5. Conclusions and future perspectives

We have discovered experimentally an emission bunching effect of two single QDs located close to each other. This bunching effect became more pronounced while the distance between two QDs decreased to less than 1.0 μm. We proposed that the ionization of the QDs and the resulting long-range Coulomb potential are the major causes for the fluorescence blinking in the single QDs and the bunching effect observed in the QD pairs. Simulations using quantum mechanical theory support our hypothesis and give numerical results that agree with the experimental data.

The cytotoxicity of QDs and the mechanism behind provide crucial information to predict the potential of clinical applications of QDs. Since QD bio-probes are normally injected intravenously into in vivo animal models or humans in clinic, vascular ECs are the first to suffer from the toxicity of QDs. In our studies, the in vitro cytotoxicity of aqueous synthesized CdTe QDs on HUVECs includes the following features: PS translocation, intracellular ROS increase, mitochondrial dysfunction, cytochrome c release, and caspases activation. The experimental results illustrate that the endothelial apoptosis induced by CdTe QDs proceed through a typical mitochondria apoptosis path way. This suggests a potential risk of cardiovascular diseases for clinical applications of QDs. Nevertheless, drugs such as NAC present an efficient prevention against HUVECs apoptosis induced by QDs. Besides decreasing QD toxicity by rational design of the QDs themselves, discovering drugs to protect the cells from QD induced apoptosis would be an alternative promising direction.

GNRs are another potential type of nano-probes for biomedical imaging applications. GNRs with high yield and controllable morphology are required for narrowing down the FWHM of their SPR enhanced scattering spectrum. We have systematically studied the quality of GNRs fabricated via the seed-mediated synthesis method. The concentration ratio of silver ions to CTAB in the growth solution was found to be the key factor for controlling the yield and morphology of GNRs produced. While the CTAB concentration being fixed, the aspect ratio of GNRs increased linearly along with the increase of silver ions concentration in the growth solution until the silver ions became overdosed. Dark field scattering images of GNRs on glass slides and adsorbed on living cells confirmed the high quality of the GNRs obtained by this controllable synthesis.

Rational design of QDs, which act as sensitizers in QDSCs, is the key approach to enhance the efficiency of QDSCs. Promoting high electron extraction efficiency and low charge recombination rate are ways to achieve this goal. We have proposed a reversed type-I CdS/CdSe QD for QDSCs which confines electrons and holes mainly in the shell region. This facilitated the electrons extract from the QDs to the TiO2 electrode. Adsorbing a layer of CdS to the QDs-TiO2 film highly suppressed the charge recombination between the QDs and the redox couple. It was worth to notice that the APCE of this reversed type-I QDSCs reached 60%. Moreover, another novel QDSC using type-II ZnSe/CdS QDs was also proposed. In a type-II QD, the photo generated electrons and holes are located separately in the shell and core regions. This unique structure enables the QDSCs a broader absorption spectrum and more efficient electron extraction efficiency simultaneously. Similar to the first design, a layer of ZnS adsorbed to the QDs-TiO2 film further increased the QDSCs efficiency by reducing the charge recombination between QDs and redox electrolyte. The APCE of type-II ZnSe/CdS QDSCs was about 40% at 410 nm.

Haiyan Qin 49

Future perspectives:

(1) Designing aqueous QDs with small particle sizes, high biocompatibility, high photo- and pH- stability will gain more and more attention because of the applications in single molecule tracking and intracellular or even intra-nucleus labeling.

(2) Compared with heavy metal ion QDs (e.g. CdTe based QDs), InP QDs, providing very low toxicity, seem to be the most useful QDs for biomedical applications in the future. Besides, emission of InP QDs covers almost the whole visible region from 400 to 750 nm. A key challenge for InP QDs is to find another phosphorus source material to take the place of the current TMSP (tris(trimethylsilyl)phosphine) phosphorus source, which is highly flammable.

(3) The development of multi-functional QDs defines a new important direction. For example, Mn doped ZnSe QDs, which are red emitting and magnetic, could be applied for cancer cell imaging and also hyperthermia therapy, while being conjugated to a cancer marker antibody and exposed to an external magnetic field.

(4) Seeking for QDs synthesis methods which are more environmental friendly and universal is always an important topic for QDs researches.

(5) Nonblinking QDs remain an important research goal. They open up new opportunities for dynamic single biomolecule tracking and QD devices with high efficiency. Understanding the QDs blinking mechanism is the first step to achieve nonblinking QDs.

(6) In vitro and in vivo toxicity of nanoparticles in various cell types and in animal models is in urgent need of study.

(7) GNRs are excellent hyperthermia agents for photodynamic therapy, whereas they are inferior for biomedical imaging. It is a promising strategy to utilize two or more optical properties of GNRs together for bioimaging, such as the SPR enhanced scattering and Raman scattering, one- and two-photon fluorescence.

(8) Besides optimizing the QDs photoelectric properties, the structure or components of QDSCs also require optimal design. For example, re-select redox couples according to the QDs properties.

Acknowledgements

This thesis would not be possible without supports, encouragements and love from my supervisors, colleagues, family and friends. I would like to express my special appreciation to the following people:

First and foremost, my supervisor Prof. Hans Ågren not only for giving me the opportunity to study and work in his group but also being a great mentor.

My co-supervisor Prof. Hjalmar Brismar for letting me join his groups in both cellphysics, KTH and Astrid Lindgren Children Hospital, KI, sharing his knowledge in science and his great ideas.

Prof. Sailing He in Zhejiang University, China for leading me into the world of scientific research.

Prof. Paras N. Prasad in the State University of New York (SUNY), at Buffalo, USA for giving me the opportunity to visit his excellent group for more than one year, and also for opening my eyes to the fantastic nano-bio-photonics world.

People in Department of Theoretical Chemistry and Biology, KTH: Ying Fu and Zhijun Ning for their encouragement, the great collaboration and inspiring discussions. Xiangjun Shang for the interesting discussion and the pleasant collaboration. And also Kai Fu, Zhihui Chen, Xiao Cheng, Xing Chen, for their kindly helps and taking care of me always.

Members, past and present, in Cellphysics, KTH: Aman Russom, Björn Önfelt, Sergey Zelenin, Marina Zelenina, Thomas Frisk, Bruno Vanherberghen, Padideh Kamali-Zare, Jacob Kowalewski, Gordon Zwartz, Sahar Ardabili, Karolin Guldevall, Elin Forslund, Ali Khorshidi, Jonas Hansson and others for being patient to teach me a lot of experimental skills and for very interesting discussions. The study and research became so much fun with them.

Prof. Anita Aperia in Astrid Lindgren Children Hospital, KI for her helpful ideas and discussions. Other colleagues in Astrid Lindgren Children Hospital: Xiaoli Liu, Zuzana Špicarová, Thomas Liebmann, Markus Kruusmägi, Lena Scott, Alexander Bondar and Nina Illarionova, for all their helps and nice discussions.

I especially thank Guang S. He in SUNY for teaching me so many practical experimental skills and most importantly how to look for, think, and solve scientific problems. Also other colleagues and friends in SUNY: Gaixia Xu, Jun Qian, Qingdong Zheng, Ken-Tye Yong, Indrajit Roy, Wing-Cheung Law, Hong Ding, Aping Zhang, Rui Hu. My life in Buffalo would be very boring without them.

Members from COER both in China: Zhanghua Han, Fei Wang, Xianyu Ao and Jun She; and in Sweden: Pu Zhang, Zhangwei YU, Hu Jun, Zhechao Wang, Ning Zhu, Mingyu Li, Yongbo Tang, Biao Chen and Shuai Zhang. There are so many funs to have you around.

I also thank Prof. Antonio Barragan and Romanico Arrighi in SMI, KI for the interesting collaborations.

Special thanks to my dearest friend Athanasia Christakou. Thanks for the fantastic Greek food, olives and olive oils. I enjoyed every minute with her. Also Ida Iranmanesh. I can never forget the wonderful time we spent together.

52 Acknowledgements

Thanks to Mei Fang for always taking care of me and listening to my boring complaining.

Shaoliang Zheng for his kindly advices and helpful suggestions.

My best friend Xinying Zhang for helping me to apply funding from CSC to support my study.

Most importantly, I sincerely thank my dearest family for their endless love, understanding and encouragement during my study and life for all these years. They are my father Shuquan Qin, my mother Shuzhen Huang and my sister Yuyan Qin. I would express my warmest welcome to our new family member, my brother-in-law Penggao He.

I would like to express the fond memory to my late grandma Jiukuan Zhong, who has meant a lot to me.

Finally, and most of all, I would like to express my gratitude to Tao Fu, the best gift in my life, for his love, understanding, encouragement. You lead me walk into not only the fantastic nano-world but also your colorful life.

Haiyan Qin

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