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Spatially resolved photoluminescence spectroscopy of quantum dots

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Title:
Spatially resolved photoluminescence spectroscopy of quantum dots
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English
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Dybiec, Maciej
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University of South Florida
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Tampa, Fla
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Nanocrystal
Biomarker
Nanotechnology
Bioconjugation
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Dissertations, Academic -- Electrical Engineering -- Doctoral -- USF
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bibliography   ( marcgt )
theses   ( marcgt )
non-fiction   ( marcgt )

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Abstract:
ABSTRACT: Recent advancements in nanotechnology create a need for a better understanding of the underlying physical processes that lead to the different behavior of nanoscale structures in comparison to bulk materials. The influence of the surrounding environment on the physical and optical properties of nanoscale objects embedded inside them is of particular interest. This research is focused on the optical properties of semiconductor quantum dots which are zero-dimensional nanostructures. There are many investigation techniques for measuring the local parameters and structural characteristics of Quantum Dot structures. They include X-ray diffraction, Transmission Electron Microscopy, Wavelength Dispersive Spectroscopy, etc. However, none of these is suitable for the study of large areas of quantum dots matrices and substrates. The existence of spatial inhomogeneity in the quantum dots allows for a deeper and better understanding of underlying physical processes responsible in part icular for the observed changes in photoluminescence (PL) characteristics. Spectroscopic PL mapping can reveal areas of improved laser performance of InAs/InGaAs quantum dots structures. Establishing physical mechanisms responsible for two different types of spatial PL inhomogeneity in InAs/InGaAs quantum dots structures for laser applications was the first objective of this research. Most of the bio-applications of semiconductor quantum dots utilize their superior optical properties over organic fluorophores. Therefore, optimization of QD labeling performance with biomolecule attachment was another focus of this research. Semiconductor quantum dots suspended in liquids were investigated, especially the influence of surrounding molecules that may be attached or bio-conjugated to the quantum dots for specific use in biological reactions on the photoluminescence spectrum. Provision of underlying physical mechanisms of optical property instability of CdSe/ZnS quantum dots used for biologi cal applications was in the scope of this research. Bioconjugationand functionalization are the fundamental issues for bio-marker tagging application of semiconductor quantum dots. It was discovered that spatially resolved photoluminescence spectroscopy and PL photo-degradation kinetics can confirm the bioconjugation. Development of a methodology that will allow the spectroscopic confirmation of bio-conjugation of quantum dot fluorescent tags and optimization of their performance was the final goal for this research project.
Thesis:
Dissertation (Ph.D.)--University of South Florida, 2006.
Bibliography:
Includes bibliographical references.
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Mode of access: World Wide Web.
Statement of Responsibility:
by Maciej Dybiec.
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Title from PDF of title page.
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Document formatted into pages; contains 143 pages.
General Note:
Includes vita.

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oclc - 179636099
usfldc doi - E14-SFE0001767
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Spatially Resolved Photolumines cence Spectroscopy of Quantum Dots by Maciej Dybiec A dissertation submitted in partial fulfillment of the requirements for the degree of Doctor of Philosophy Department of Electrical Engineering College of Engineering University of South Florida Co-Major Professor: Sergei Ostapenko, Ph.D. Co-Major Professor: Stephen Saddow, Ph.D. Andrew Hoff, Ph.D. Chris Ferekides, Ph.D. Sarath Witanachi, Ph.D. Tatyana Zhukov, Ph.D. Date of Approval: October 20 2006 Keywords: nanocrystal, biomarker, nanotechnology, bioconjugation, tag Copyright 2006, Maciej Dybiec

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Dedication To my wife Aleksandra, without he r all this would not be possible.

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Acknowledgments I would like to thank my major professor Dr. Sergei Ostapenko for his help, guidance and patience during my research work that led to this PhD, and for all other support that I have rece ived. I would also like to thank Dr. Stephen Saddow, who was of great help and shared his expertise with me while motivating me to work harder and obtain better study results-for this I am very grateful. I wanted to thank the many ot hers who have lent their support during my course work period, especially Dr. Tatyana Zhukov. Without her experience and support the biology portion of these experiments woul d not have been as successful. Many fruitful discussions with Dr. Ta tyana Torchynskaya and Professor Nadia Korsunkaya contributed a great deal to this work and I want to thank them in this letter as well. And last but certainly not le ast, I would like to tha nk Igor Tarasov. He had the patience to work and aid me in many aspect s of photoluminescence segment of this work.

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i Table of Contents List of Tables iii List of Figures iv Abstract x 1. Introduction 1 1.1. Scope and motivation 1 1.2. QD photoluminescence 6 1.3. Research plan 9 1.4. Summary 10 2. Electronic Structure of Quantum Confined Systems 12 2.1. Introduction 12 2.2. Electronic structure of quantum dot s as zero dimensional systems 13 2.3. Quantum dots in compound semiconductors 22 2.3.1. Quantum dots in group III-V element systems 22 2.3.1.1. Growth modes and methods 22 2.3.1.2. Properties of III – V quantum dots 26 2.3.1.3. III – V QD applications 28 2.3.2. Quantum dots in group II–VI element systems 30 2.3.2.1. Growth modes and creation techniques 30 2.3.2.2. Properties of II-VI quantum dots 32 2.3.2.3. II – VI QD applications 34 2.4. Biomarkers 36 2.5. Summary 40 3. Quantum Dots Experiments 42 3.1. InAs/InGaAs QD samples 42 3.2. CdSe/ZnS QD samples 44 3.2.1. Bio-conjugation substrates 46 3.3. Spatially resolved PL spectro scopy experimental details 49 3.3.1. Hardware description 49 3.3.2. PL mapping measurements 50 3.4. Bio-conjugation confirmation techniques 53 3.4.1. Enzyme Linked ImmunoSorbent Assay (ELISA) technique 54 3.4.2. Protein microarray technique 56 3.5. Summary 59

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ii 4. Results and Discussion 61 4.1. PL spectroscopy of InAs/InGaAs quan tum dots for laser applications 61 4.1.1. Growth temperature investigations – PL spectroscopy 61 4.1.2. Growth temperature investigations – PL mapping 67 4.1.3. Cladding layer composition investig ation – experimental results 75 4.1.4. Spatial inhomogeneity of full size QD wafers – experimental results 81 4.1.5. Spatial PL inhomogeneity of full size QD wafers – discussion 87 4.2. PL spectroscopy of CdSe/ZnS quantum dots for bioapplications 91 4.2.1. Photo-induced enhancement of th e PL intensity introduction 91 4.2.2. Photo-induced enhancement of th e PL intensity – experimental results 92 4.2.3. Photo-induced enhancement of th e PL intensity – discussion 101 4.2.4. PL spectroscopy of bio-conjugated QD’s 107 4.2.4.1. PL mapping of ELISA plates 107 4.2.4.2. Bioconjugation with micro array technique 112 4.2.4.3. Spectral shift of QD luminescence caused by bioconjugation 118 4.3. Summary 124 5. Conclusions and Recommendations 126 5.1. Recommendation for further research 128 References 130 Bibliography 143 About the Author End Page

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iii List of Tables Table 3.1 QDot.com samples description 45 Table 3.2 Biomarkers molecules chos en for bio-conjugation experiments 46 Table 3.3 ELISA plate types 47 Table 3.4 Lasers used in the PL experiments 49

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iv List of Figures Figure 1.1 Comparison of the emission and absorption spectra of QD (continuous line) and organic dye (dotted line) (curves with the shaded area are the absorption spectra) 4 Figure 1.2 Schematic representation of the basic photoluminescence mechanisms for bulk materials 8 Figure 2.1 Densities of stat es versus energy for bulk, 2D, 1D and 0 dimensional structures 14 Figure 2.2 Sketch of a quantum box embedded in a matrix 15 Figure 2.3 Two dimensional schematic band diagrams of (a) quantum wire with non discrete energy levels and two different size QD’s with (b) large and (c) small characteristic di mension showing the origin of different photon energies resu lting from the size of the QD 17 Figure 2.4 Assumed geometry of the (a) pyramidal and (b) lens shaped dots for which wave functions were calculated by Williamson [30] as shown in Figure 2.5 19 Figure 2.5 Top view of the calculated el ectron and hole wave functions squared (space occupation probability) for InAs QDs geometries shown in Figure 2.4 after Williamson [30]. The light and dark areas represent 20% and 60% charge density 20 Figure 2.6 Schematic diagram of three pos sible growth modes after [Bauer]: (a) – Frandk-van der Merwe, (b) – Volmer-Weber, (c) – StranskiKrastanov, which is the mode responsible for QD growth 24 Figure 2.7 Schematic of the Turing growth mode (after Temmyo) [48] Note the formation of “nanodisk” inside the nanocrystals 25 Figure 2.8 Schematic diagram of a typica l core-shell nanoparticle used for biotagging. This figure represents the scheme with the highest quantum efficiency design, in some cases the core is protected only by singlelayer shell [7] 31

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v Figure 2.9 Schematic representation of the QD bound to a biomarker (molecules are drawn to scale) – af ter Jaiswal [97]. Single QD in the center is attached to the detector antibodies (IgG proteins) via linker molecules (PEG). Note that not all linker molecules are utilized 37 Figure 3.1 Schematic structure of multi quantum well and quantum dot “sandwich” of InAs/InGaAs sample s. These samples were prepared by Dr. Andreas Stintz from Center of High Technology Materials at the University of New Mexico 43 Figure 3.2 Schematic representation of the bioconjugated quantum dot (after Invitrogen, Inc) [11] 45 Figure 3.3 Background signal from differen t ELISA plates as a function of wavelength 48 Figure 3.4 Photoluminescence setup for room temp measurements of InAs QD’s 50 Figure 3.5 Photoluminescence setup for liquid ELISA experiments 51 Figure 3.6 Photoluminescence setup for low temperature PL mapping 52 Figure 3.7 ELISA concentration curve vs. optical signal. Lin ear part (shaded area) constitutes the limits of detection for given biomarker concentration range 53 Figure 3.8 “Sandwich” immunoreaction sche me for the direct ELISA approach 54 Figure 3.9 Direct sandwich ELISA pr ocedure (after Chemicon.com) [116] 56 Figure 3.10 Printing scheme in the micro array technique 57 Figure 3.11 Basic microarrays immuno reactions after Schena [117] 58 Figure 4.1 PL spectra of the QD structures #684 (grown at 470 C) at various intensity points (from lo west to highest) measured at 300 K (a) and 80 K (b) 62 Figure 4.2 PL spectra of the QD structures #685 (grown at 490 C) at various intensity points measured at 300 K (a) and 80 K (b) 62 Figure 4.3 PL spectra of the QD structures #687 (grown at 510 C) at various intensity points measured at 300 K (a) and 80 K (b) 63

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vi Figure 4.4 PL spectra of the QD structures #689 (grown at 525 C) at various intensity points measured at 300 K (a) and 80 K (b) 63 Figure 4.5 PL spectra of the QD structures #698 (grown at 535 C) at various intensity points measured at 300 K (a) and 80 K (b) 64 Figure 4.6 PL spectra of the QD ensemb le measured at different excitation power with the highest one of 1.0 kW/cm2 (Structure #689 measured at 77K) 65 Figure 4.7 Ground state PL band inte nsity dependence vs. temperature (Excitation power density 650 W/cm2) (Structure #689) Two distinct regions were observed, labeled I and II indicating two different activation energies 66 Figure 4.8 (a) Map of PL intensity at 0.99 eV (0.25 mm step size, 200 m excitation spot diameter, (b) map PL intensity at GS max position, and (c) map of peak energy positi ons (0.5 mm step size) at ~90 W/cm2 power density. Arrow in (b) corresponds to spectroscopic line scans presented in Figure 4.9 68 Figure 4.9 PL spectra measured at 80K (a ) and 300K (b) at various PL intensity points on QD structure #689, (see Fig. 4.8(b)). (~90 W/cm2 power intensity) 69 Figure 4.10 Ground state PL intensity vs. GS maximum position measured at room temperature and 90 W/cm2 power intensity on QD structures grown at different temperatures 70 Figure 4.11 Experimental peak positions fo r GS, first, and second ES vs. GS peak energy measured at 80 K and excitation power intensity 650 W/cm2 71 Figure 4.12 PL intensity mapping at room temperature performed at 1.02 eV on sample #1360 (a) and at 1.044 eV on sample #1361 (b) Shading bars represent the PL intensity variation in both samples. Arrows indicate orientation of the maximum PL in tensity gradient from the water center to the periphery 76 Figure 4.13 PL spectra at room temperat ure measured at different spots along the arrows in Fig. 4.12 on samples 1360 (a) and 1361 (b).Curve (1) corresponds to the central area of the samples; curve (2) corresponds to the periphery 77

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vii Figure 4.14 PL intensity vs. PL maximu m dependencies measured at room temperature on 155 points across the entire wafer as shown in Fig. 4.12 78 Figure 4.15 Distribution of PL intensity ma xima at 80K and 300K for the sample #1361. Note the PL intensity increase for 80K with the rising peak maxima energy, and opposite decrease of PL with peak maxima increase for 300K 80 Figure 4.16 PL intensity mapping at room temperature performed at 1.008 eV on the QD #1718 structure with In /Ga composition x = 0.10, Mapping step 0.2 mm 82 Figure 4.17 PL intensity mapping at room temperature performed at 1.010 eV on the QD #1719 structure with In /Ga composition x = 0.15. Mapping step 0.5 mm 82 Figure 4.18 PL intensity mapping at room temperature performed at 1.035 eV on the QD #1720 structure with In /Ga composition x = 0.20. Mapping step 0.5 mm 83 Figure 4.19 PL intensity mapping at room temperature performed at 1.06 eV on the QD #1721 structure with In /Ga composition x = 0.25. Mapping step 0.5 mm 83 Figure 4.20 The average PL peak intens ities for all four investigated QD structures 84 Figure 4.21 Typical normalized PL spectra corresponding to the spots with highest PL intensities on four investigated InxGa1-xAs QD structures with variable In composition in the capping layer 85 Figure 4.22 PL spectra at room temperature measured at different intensity spots (# 1719 In content x=15%) 87 Figure 4.23 PL maxima peak position vs it’s intensity (#1719 In content x = 15%) 89 Figure 4.24 PL spectra of the CdSe/Z nS QDs measured at different temperatures: 80 K, 140 K, 210 K a nd 300 K. Note the blue shift as the sample temperature decreases 93 Figure 4.25 PL maximum versus temperature variation (points) and fitting with the Varshni equation (solid line ) using parameters: Eg(0) = 1.9845 eV, = 0.00081, = 567.2 94

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viii Figure 4.26 The PL intensity variation of the 655 nm luminescence band at room temperature and different la ser power densities (W cm 2): (1) 500; (2) 20, (3) 0.2. The PL intensity is normalized using a multiplication factor of 25 in curve (2) and 2500 in curve (3) 95 Figure 4.27 (a) Cycling kinetics of lig ht-induced PL enhancement and dark recovery at room temperature; (b) time dependence of sample illumination, using 325 nm HeCd laser. Points A, B, C and D illustrate gradual increase of the PL intensity at the beginning of the consecutive enhancement curve. The inset shows two consecutive kinetics revealing time constants of (1) 9.4 min and (2) 20.6 min 97 Figure 4.28 The PL temperature depende nce before and after (a) the NRE process and (b) the RE process. Cu rves (1) and (3) correspond to the initial state measured on two diff erent groups of samples. Curve (2) corresponds to the NRE state, and cu rve (4) to the RE state. Solid lines show linear fit to extract the activation energy of the Tquenching 99 Figure 4.29 Model of the light-induced PL enhancement illustrating different states of the QD sample: (a) initia l state at the be ginning of PL kinetics. The pot ential barrier A is reduced by the electric field generated by charging the electr on trap (D+) and hole trap (A ) in the dark; (b) photo-enhanced state when the luminescence increased due to the recharging of traps acco rding to equation (3); (c) final state after relaxation of the revers ible enhancement process. It is suggested that the non-reversib le process is driven by photochemical reactions. PL transitions ar e depicted by the dashed arrows 104 Figure 4.30 PL intensity vs. dilution ratio, QD 525 diluted in Tris (DAKO) buffer [139] 107 Figure 4.31 ELISA imunoreaction attach ment “sandwich” diagram for streptavidin coated wells. This scheme was used to attach the QD (fluorescent label) to the bi otin-avidin bridge as shown 108 Figure 4.32 Map of four ELISA wells with different concentration of antigen CA125. 300 (a), 200 (b) 100 (c) a nd 0 Units/ml (the unit of measurement for this marker is femtomole per milligram of tumor) (d). Map was collected at 655 nm (1.89 eV), for the OC125 (250 u/ml) – CA125 (antibody – antigen ) complex labeled with QD655 quantum dots. Mapping step 0.2 mm 109 Figure 4.33 Averaged over the well bottom area PL intensity signal for OCCA125 complex from maps shown in Figure 4.32 110

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ix Figure 4.34 (a) Map of ELISA plate with different amounts of CA125 antigen (at 655 nm); numbers shown above each well at the map represents averaged PL intensity in each well, (b) description of the given wells content 112 Figure 4.35 Micro array immuno reaction “sandwich” diagram for nitrocellulose coated substrate 115 Figure 4.36 A glass substrate can hold up to couple hundred spots of different antibodies or their concentrations The spots are printed as shown schematically 115 Figure 4.37 Example of fluorescent microscope image (mag. 2.5x) of the small part of an array printed from micro array printer (QD655 + IL10 complex) with 100 m spot diamet er (a), spots description (b) 116 Figure 4.38 Calibration curve of IL10 antig en concentration detected with the QD665 label and the micro array technique 117 Figure 4.39 PL intensity maps of two drops/spots on Si substrate – conjugated (a) and non-conjugated (b). Spectra were measured at each point of respective map (QD655 + anti-Interleukin10) 118 Figure 4.40 Spectra measured on ever y map spot presented in Fig 4.39 119 Figure 4.41 Normalized spectra that meas ured on every map spot presented in fig 4.39 (single spectra are plotted on top of each other to show shift for the whole group – width of each curve is caused by single spectra spread for given sample) 120 Figure 4.42 Peak position for all spectra measured over conjugated and nonconjugated spots. The average peak separation “shift” is 4nm for QD655+IL10 complex vs. pure QD655 121 Figure 4.43 Relative shift of nonconjuga ted QD’s vs. conjugated to IL10 antibody for 605, 655 and 705 QD’s 122

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x Spatially Resolved Photoluminesce nce Spectroscopy of Quantum Dots Maciej Dybiec ABSTRACT Recent advancements in nanotechnology create a need for a better understanding of the underlying physical proces ses that lead to the diffe rent behavior of nanoscale structures in comparison to bulk materials. The influence of the surrounding environment on the physical and optical properties of nanos cale objects embedded inside them is of particular interest. This research is focu sed on the optical propert ies of semiconductor quantum dots which are zero-dimensional na nostructures. There are many investigation techniques for measuring the local parameters and structural characteristics of Quantum Dot structures. They include X-ray diffr action, Transmission El ectron Microscopy, Wavelength Dispersive Spectrosc opy, etc. However, none of these is suitable for the study of large areas of quantum dots matrices and substrates. The existence of spatial inhomogeneity in the quantum dots allows for a deeper and better understandin g of underlying physical processes responsible in particular for the observed changes in photoluminescence (P L) characteristics. Spectroscopic PL mapping can reveal areas of improved laser pe rformance of InAs – InGaAs quantum dots

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xi structures. Establishing physical mechanisms responsible for two different types of spatial PL inhomogeneity in InAs/InGaAs quant um dots structures for laser applications was the first objective of this research. Most of the bio-applications of semic onductor quantum dots utilize their superior optical properties over orga nic fluorophores. Therefore, op timization of QD labeling performance with biomolecule attachment was another focus of this research. Semiconductor quantum dots suspended in liqui ds were investigated, especially the influence of surrounding molecules that may be attached or bio-conjugated to the quantum dots for specific use in biological reactions on the photoluminescence spectrum. Provision of underlying physical mechanisms of optical property inst ability of CdSe/ZnS quantum dots used for biological applications was in the scope of this research. Bioconjugation and functionaliza tion are the fundamental issu es for bio-marker tagging application of semiconductor quantum dots. It was discovered that spatially resolved photoluminescence spectroscopy and PL photo-de gradation kinetics can confirm the bioconjugation. Development of a methodology that will allow the spectroscopic confirmation of bio-conjugation of quantum dot fluorescent tags and optimization of their performance was the final goal for this research project.

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1 1. Introduction “The principles of physics, as far as I can see, do not speak against the possibility of maneuvering things atom by atom.” Richard Feynman, 1959 [1] 1.1. Scope and motivation Nanotechnology since its beginnings c onnected many areas of research. Nowadays its an already establish field of science that interconnects mechanics, electronics, optics and other fields of scien ce with one thing in common the nanometer sizes of final structures [1, 2]. Today we are able to control material processes that allow us to create particles and structures on the nanometer scale, even allowing the manipulation of single atoms. In nanotechno logy not only size matters but the shape, chemical and bio surface properties and physi cal properties of these small structures determine their use and applications. Since nanoparticles have large volume to surface area ratio most of their propert ies are determined by the phys ical and chemical reactions that take place at the surface. The possibi lity of using these surface properties and a virtually inexhaustible way to modify it has drawn the at tention of scientists and

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2 engineers. Study of the physical chemistry of materials of a size in between the bulk and isolated molecules is a major trend no wadays even though the discovery of semiconductor nanocrystallites was made in 19 32 [3]. The focus is on nanometer size fragments of semiconducting inor ganic solids which contain fr om a handful to thousands of atoms. Fragments of ma tter of these sizes are ofte n large enough to exhibit a crystalline core but too small to have deve loped solid state electronic and vibrational band structure. One may say that for matte r on this scale they are neither bulk nor molecular in structure. In this range of sizes properties of solid state bulk begin to form from the molecular level but the clear band stru cture has yet to form. In addition to being interesting from a fundamental science viewpoint, materials in this size range may have a number of technological applications. For example, semiconductor nanocrystallites may have large optical nonlinearities which coul d be useful in optic al devices. Organized arrays of little crystallites may have mechanical, optical, el ectrical, and thermal properties quite different from the bulk. In this work Qu antum Dot (QD) matrices were investigated that are used for laser applications and the spatial inhomogeneity of their optical parameters was studied. In the majority of cases the observed sp atial non-uniformity is contributed to non uniform size distribution of the QD’s, but there are other factors that come into play apart from this, such as the capping layer inhomogeneity, different electron hole wave function overlapping for di fferent QD shapes, different shape/strain conditions in real samples, just to list few examples. A deeper photoluminescence study of such structures is still needed a nd is a major part of this research. Quantum Dot lasers are of particular interest for their advantages over the quantum well laser such as: narrower gain sp ectra, lower threshold currents and weaker

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3 temperature dependence. All of th ese spring from the very narrow -function-like density of states for the carriers confined in the QD’s [4]. Strong quantum confinement in all three dimensions creates discrete carrier ener gy spectra. Transitions between the electron and hole levels in the QD are analogous to thos e between the discrete levels of individual atoms. For this reason, QD’s have generated much interest as a new cl ass of artificially structured materials with tunable (through varying composition and sizes) energies of discrete atomic-like states that are ideal for use in laser [5, 6]. One of the most common applications of QD’ s apart from lasers is in fluorescence tagging, where they are being developed to replace molecular dyes. These range from biological to environmental applications. In these applications when the tagging substance is irradiated with light it absorb s the light and then re-emits at a different wavelength. The presence of the tagging subs tance can then be easily identified by detection of the characteristic emission sp ectrum. For example, injecting a tagging substance into a particular biological cell makes it possible to identify that cell amid other cells from its fluorescence signature. There are several reasons why QD’s ha ve advantages over organic fluorophores [2, 7, 8]. The first of these is that QD’s can absorb a wide band of light for their excitation, but they emit in a very narrow band. In contrast, most molecular dyes can absorb only a very narrow band of wavelength, so most of the illu minating light is not used. This is illustrated in Figure 1.1.

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4 Figure 1.1 Comparison of the emission a nd absorption spectra of QD (continuous line) and organic dye (dotted line) (c urves with the shaded area are the absorption spectra) Also, these molecules emit in a much wi der band of wavelengths compared to QD’s. As a result, to distinguish separate features one needs to use very different molecular dyes, each with its own required excitation wavelengths. In the case of QD’s the emission wavelength depe nds on the size. So, QD’s made of the same semiconductor material, but of different sizes, can all be excited by the same light source (provided above band-gap energy is used), but then th ey emit distinctly different wavelengths. Emission is very efficient in Quantum Dots and doesn’t decrease so rapidly with time under UV illumination as in organic tags [9]. Quantum dots have large molar extinction coefficient value[10], t ypically on the order of 0.5-5 x 10-6 M-1cm-1 [11] which Wavelength [nm] PL Intensity [arb units]

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5 means that quantum dots are capable of absorb ing excitation photons very efficiently; the absorption rate of QD’s is approximately 1050 times faster than organic dyes [12]. The higher rate of absorption is directly correlat ed to the quantum dot brightness and it has been found that QD’s are 10-20 times bright er than organic dyes [9, 10, 13], allowing highly sensitive fluorescence imaging. Their photo stability over long periods of ti me is one of the key factors that put them as the best fluorophores so far. In co mparison to organic dyes that bleach after a couple of minutes under a standard confocal microscope, QD’s can last for several hours under same illumination conditions [14]. Another feature of QD’s that makes them a good candidate for tagging purposes is that their tagging property is controllable With proper chemistry these objects can be attached to specific biomolecules that perfor m specific tasks, such as anti-gene and antibody recognition, for example. This is in contrast to traditionally used molecular tags that have well defined binding charac teristics. As a result a par ticular fluorophore tag may or may not bind with a given molecule or surface of interest. QD’s have a surface that can bind with a variety of molecules, they can be prepared (functionalized) so as to attach to well defined targets. A fourth feature of QD’s that makes them desirable for taggi ng is their nonlinear optical behavior. Through frequency-doubling no nlinear optical materi als can absorb two photons of a longer wavelength and create a single photon of shorter wavelength. But most optical materials have a small non-li nearity. QD’s are made with large nonlinear properties that have allowed researchers to employ them for deep noninvasive imaging. Although the quantum efficiency is many tim es lower than without frequency up

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6 conversion, it is still possibl e to detect QD’s due their hi gh extinction coefficients. One example of this principle is that this "fluorescence" allows the imaging of the blood vessels/tumors without opening the tissue [15]. Though these features make QD’s a better choice over organic fluorophores there is not much known about the interaction of quantum dots with surrounding molecules in terms of photoluminescence properties. Bio-co njugation is nothing ot her than attaching proteins to the surface of a quantum dot either covalently, electrostatically, or with the use of a linker molecule, (through ionic inte raction for example). In fact all these interactions come down to electrostatic forces at some level. This has an effect on the interaction of a particular quantum dot with the electroma gnetic radiation, either with absorption or emission and some signs of that are suspected to appear in photoluminescence spectra. 1.2. QD photoluminescence Photoluminescence is a process in whic h a chemical compound absorbs a photon with a wavelength in the ra nge of visible or UV electr omagnetic radiation, thus transitioning to a higher electr onic energy state, and then radiates a photon back out, returning to a lower energy state. The pe riod between absorption and emission is typically extremely short, on the order of 10 nanoseconds. Under special circumstances, however, this period can be exte nded into minutes or hours [16]. Ultimately, available chemical energy states and allowed transitions between states (and therefore wavelengths of light preferentially absorbed and emitted) are

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7 determined by the rules of quantum mechanic s. A basic understandi ng of the principles involved can be gained by studying the electron configurations and mo lecular orbitals of simple atoms and molecules. More complicat ed molecules and advanced subtleties are treated in the field of computational chemistry The simplest photoluminescent processes are resonant radiations, in which a photon of a particular waveleng th is absorbed and an equi valent photon is immediately emitted. This process involves no significant inte rnal energy transitions of the chemical substrate between absorption and emission and is extremely fast, on the order of 10 nanoseconds. More interesting processes occur when th e chemical substrate undergoes internal energy transitions before re-emitting the energy from the absorption event. The most familiar such effect is fluorescence, which is also typically a fast process, but in which some of the original energy is dissipated so that the emitted light is of lower energy than that absorbed.

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8 Figure 1.2 Schematic representation of th e basic photoluminescence mechanisms for bulk materials Photoluminescence in QD is governed by th e same mechanisms as in the bulk material, major difference is that all the energy levels inside the quantum dots are strongly quantized due to small dimensions of QD. Direct consequences of this quantization are very sharp emission spectral lines ( function – like for a single QD), that are in general broadened only by the QD size distribution. h 1 > h 2 > h 3

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9 1.3. Research plan This research is divided into two major part s. In the first part InAs self assembled QD’s embedded in InGaAs matrices are bei ng investigated. Spatial photoluminescence (PL) scanning of the 5x10 mm samples is performed followed with the full 50mm in diameter wafers investigations of the same structures. All samples contain InAs quantum dots stacked in “sandwich” like structures placed in between capping and wetting layers respectively, apart from the spatial distributi on of PL intensity, In composition of capping layers is investigated as well as the influen ce of the growth temperature for the In islands creation phase. The second part is focused on the photol uminescence investigations of CdSe/ZnS core/shell quantum dots dispersed in liquids and conjugated to different biomolecules for biomedical applications. The influence of bi oconjugation on the PL features of these QD is of major interest. Four ma jor sizes of quantum dots were studied with the major peak emissions around: 525, 605, 655 and 705 nm. Fo r the conjugation experiments four different biomolecules were chosen with fu rther aim to be app lied in early cancer detection. These were: interleukin 10 (IL 10), cancer antigen 125 (CA125), prostate specific antigen (PSA) and Osteopontin. All of these biomolecules are being widely used as ovarian cancer biomarkers. Initial expe riments were done to show PL intensity dependence vs. pure QD concentration; thes e were followed with wet enzyme-linked immunosorbent assays (ELISA) measurements of conjugated QD a nd finally with PL measurements of dried QD-conjugate spots prin ted with the micro array technique. Time dependent and maxima spectral position stud ies were performed in order to detect

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10 bioconjugation. The sample preparation was done in two ways, the initial samples for wet ELISA experiments involved QD’s preconjugat ed by the supplier (Invitrogen) [11], while in the micro array part the conjugation was done at Moffitt Cancer Center at the University of South Florida. 1.4. Summary Photoluminescent features of quantum dots in III-V (InAs) and II-VI (CdSe) element groups are the main focus of this re search. The spatial PL intensity distribution with connection to quantum dot paramete rs, capping layer composition and growth temperature influence for 50mm wafers is st udied. InAs QD are of great importance for optoelectronic applications in the infrared (1.3 m) part of the electromagnetic spectrum. Unique optical properties of quantum dots structures in colloidal solutions make them suitable for medical applications as fl uorescent markers. CdSe/ZnS QDs have been studied for photoluminescent signatures of possible biomolecules attachment (bioconjugation). The practical implications of any PL feature that is not relaying on the averaged intensity as a measure of bioconj ugation would be of great importance for bio applications. Increased sensitivity of any nowadays applicable cancer tests is very desirable and motivation for this type research is clear. In the short term the ability to detect bioconjugation with some characteri stic spectral features would prove another advantage of QD’s over organic fluorophores and save time and money before any further bio experiments would have to be done.

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11 The objectives of the research were: Establish mechanisms responsible for sp atial photoluminescence inhomogeneity of InAs/InGaAs quantum dot stru ctures for laser applications. Establish mechanisms of optical properti es instability of Cd Se/ZnS quantum dots used for biological applications. Develop a methodology that will allow sp ectroscopic confirmation of the bioconjugation of quantum dots fluorescent tags to bio-molecules and optimize their performance in different working environments including substrates and solutions.

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12 2. Electronic Structure of Qu antum Confined Systems 2.1. Introduction Semiconductor quantum dots have emerged as one of the first applications of nanotechnology [17-19]. Quantum dot applicat ions have been pr imarily in nonlinear optics and in the Q-switching of lasers [4 -6, 20, 21]. Though, the quantum dot laser has yet to enter the mainstream, it is recognized as a key application of QD’s materials. Their specific optical properties made them very good fluorescence particles used for tagging in the biomedical field which so far is the s econd most popular app lication. Among possible applications being discussed is quantum computing, [22] whic h is a very promising field and their use in photovoltaic’s technology [23] is another one. However, the full impact of quantum dot technolo gy is still to come. Discovery of the yellow or red color made in the early 1930’s [3] of some silicate glasses was attributed to nanom eter size crystal inclusions of CdSe and CdS, but it took almost fifty years to link co lored glasses to the energy stat es determined by the quantum confinement in embedded CdS or CdSe na noparticles [24]. The idea to use quantum confinement in thin layers of materials was discussed since the early 1960’s and the theory of electron motion in a crystal w ith superimposed periodic potential’s was developed at that time [25]. The first quantum structures to appear were quantum wells

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13 (QWs) that were formed as a sandwich of ve ry thin 5-7 nm [26] thin layers of narrow band gap semiconductors located between wider band gap semiconductor material. Quantum wires and quantum dots (QDs) are, respectively, low dimensional structures that follow this trend of development – from one dimensional (QW) down to “zero dimensional“ (QD) structures. 2.2. Electronic structure of quantum dots as zero dimensional systems The band structure of a bulk semiconductor material is a result of the superposition of the wave functions and energy states of all the atoms that constitute the crystal. In the case of a quantum dot the 3D structure is comprised only of a couple of hundred to a couple of thousand atoms; their sp ecific properties are due to the fact that the size of the QD is smaller or comparable to the bulk Bohr exciton radius. The very strong quantum confinement effects come into play to form the band structure of a single QD with very discrete energy states. QD’s confine carriers (e lectron – hole pairs) in all three dimensions, accordingly they are called “zero dimensional structures” while they are effectively 3D structures. The band structur e of a single quantum dot has very distinct allowed energy levels, and due to this property, quantum dots are sometimes called artificial atoms since they are very similar to atomic states. Figure 2.1 shows the density of electron states function vs energy for all possible quantum confinement arrangements.

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14 Figure 2.1 Densities of states versus en ergy for bulk, 2D, 1D and 0 dimensional structures The density of states for the three-dimensi onal system (bulk semiconductor) has the form: 2 / 1 2 / 3E E dE d dE dN (1) for a two-dimensional system (qua ntum well) is a step function, E E ii iE dE d dE dN 1 (2) for a one-dimensional system (quantum wire) has a peculiarity, E i E ii iE E dE d dE dN 2 / 1 2 / 1 (3) and for a zero-dimensional system (quantum dot) has the shape of -peaks i ii E iE E dE d dE dN (4) Above, i are i-th discrete energy levels inside well, wire and QD respectively, is a Heaviside step function, and is the Dirac-delta function.

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15 When considering the energy sp ectrum of a zero-dimensional system, we have to study the time-independent Sc hrdinger equation (5) with the co nfining potential, which is a function of all three sp atial coordinates and confines the electron in all three directions: z y x E z y x z y x V m , , , 22 2 (5) Where is Planck’s cons tant divided by 2 is the Laplacian op erator, m is mass of the particle, V(x,y,z) is the potential and (x,y,z) is the wavefu nction. The simplest potential V(x, y, z) of this type is: box box the the outside inside 0 z) y, V(x, Figure 2.2 Sketch of a quan tum box embedded in a matrix Where boundary cond itions on the box walls are 0 < x < Lx, 0 < y < Ly, 0 < z < Lz; (see Fig. 2.2) For th is case, one can write down th e solutions to the Schrodinger equation (5) immediately:

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16 z y x z y x n n nL xn L xn L xn L L L z y x3 2 1 ,sin sin sin 8 ,3 2 1 (6) 2 2 3 2 2 2 2 2 1 2 2 ,23 2 1z y x n n nL n L n L n m E (7) where nl, n2, n3 =1, 2, 3... Of fundamental im portance is the fact that En1,n2,n3 is the total electron ener gy, in contrast with quantum wells and wires, in wh ich the solution for the bound state in a quantum well and wire (sha ded areas in Fig 2.3 (a)) yields only the energy spectrum associated with transverse confinement. Another unique feature is the presence of three discrete quantum numbers resulting directly from the existence of the three directions of quanti zation. Thus we obta in threefold discrete-energy levels and wave functions localized in all three dimensions of th e quantum box. Generally, all energies are different, i.e., the levels ar e not degenerate. However, if two or all dimensions of the box ar e equal or their ratios are integers, some levels with different quantum numbers coincide. Such a situation result s in degeneracy: tw ofold degeneracy if two dimensions are equal and sixfold degenera cy for a cube. This discrete spectrum in a quantum box and th e lack of free-electron propag ation are the ma in features distinguishing quan tum boxes from quantum wells and wires. As is well known, these features are typical for atomic systems. The quantum dot itself doesn’t constitute an object that has the quantum confinement ability of an electron. Quantum dots may exist as small crystal inclusions but in order to confine the electron or (hole) in all three dimensions its potential depth must exceed the minimum energy state of the lowest order particle, U0min, where:

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17 2 2 2 min 08 ml U (8) According to equation (7), the size of the quantum dot plays a fundamental role in the emission and absorption spectra originating from electron transitions between discrete photonic energy levels, En, which are system levels. With decreasing quantum dot size the discrete energy states become more separate d in energy resulting in a blue shift of the emission spectrum. Figure 2.3 Two dimensional schematic band diagrams of (a) quantum wire with non discrete energy levels and two differ ent size QD’s with (b) large and (c) small characteristic dimension show ing the origin of different photon energies resulting from the size of the QD (a) (b) (c) e2 e1 e0 h0 h1 h2 e1 e0 h0 h 1

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18 For this simple approach not all of the phys ical properties of the quantum dot are fully accounted for. In some materials the sp ace dependence of the effective mass in heterostructures might lead to a strong interdepende nce of the longitudinal and transverse motion in systems with heterointerfaces such as quantum wells [27] quantum wires [28] and quantum dots [29]. The actual shape of th e quantum dot plays also an important role since the electron and hole wave functions have different spatial di stributions and they overlap differently in QD’s of different sh apes (see Figure 2.5). In most cases quantum dots have pyramidal or lens – like shapes, especially in the systems grown on substrates and nearly spherical shape in a liquid suspension form. One of the long standing problems that ha s held back the development of accurate models for the energy states in semiconductor qu antum dot heterostructur es is the need to accurately determine the size, shape and composition of quantum dot samples. This problem is further compounded by the fact that while it is possible to determine the shape of dots using atomic force mi croscopy (AFM) before they ha ve been capped with a GaAs "barrier", it is believed that the capping process itself induces the diffusion of gallium into the dots and diffusion of indium from the dots into the surr ounding matrix [26]. Hence, AFM data for the size and shape of uncapped dots is of only limited use in evaluating the quality of theoretical models for capped dots. The earliest quantum dot samples were believed to contain pure InAs, pyramidal structures, with facets (see Fig. 2.4) forming 45 angles between the facets and the base. Following this interpretation of the structure, early calculations were also performed assuming a pyramidal geometry. More recent characterization using cross sectional TEM and STM measurements of capped dots, has predicted that a more realistic dot geometry and composition is a lens

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19 shaped InxGa1-xAs quantum dot [30]. Williamson presented results of calculations for the resulting energy states, excitonic band gaps a nd Coulomb matrix elements in lens shaped InxGa1-xAs quantum dots embedded in GaAs matri ces in a similar configuration to the samples investigated during this disserta tion research. Figure 2.4 Assumed geometry of the (a) pyramidal and (b) lens shaped dots for which wave functions were calculated by Williamson [30] as shown in Figure 2.5 (a) (b)

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20 Figure 2.5 Top view of the calculated elec tron and hole wave func tions squared (space occupation probability) for the InAs QD geometries shown in Figure 2.4 after Williamson [30]. The light and dark areas represent 20% and 60% charge density

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21 In the case of quantum boxes or dots the electron energi es are discrete and the density of states function is simply a set of delta-shaped peaks: v vE E E ) ( (9) where v = (nl, n2, n3) (E) the density of states For an idealized system, the peaks are very narrow and infinitely high, as illust rated in Figure 2.1 (d) In fact, interactions between electron s and impurities as well as collisions with phonons bring about a broadening of the discrete levels and, as a result, the peaks for physically realizable systems have finite amplitudes and widths. Nevertheless, the major trend of sharpening the spectral de nsity dependences as a resu lt of lowering the system dimensionality is a dominant effect for near-perfect stru ctures at low temperatures. There are many other reported works in th e literature [19, 30-35] devoted to the theoretical considerations of the quantum dot band structure for many different QD material compositions and shapes For the particle in a spherical potential well there are theoretical models in the literature proposed by Efros [31, 35]. For lens-like and pyramidal shapes the electronic structure was calculated by Williamson [30] and Grundmann et al [36]. Elastic strain/stress at the hetero-structu re interface also contri butes to the optical properties of QD’s. More stressed interfaces ar e prone to have more defects that directly influence the optical output [37]. Impurity atom s and native crystal structural defects also cannot be neglected. For quantum dot ensemble s the size distribution is responsible for the inhomogeneous broadening of the emissi on spectra, creating additional factors that must be taken into account duri ng the PL study of quantum dots.

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22 2.3. Quantum dots in compound semiconductors 2.3.1. Quantum dots in group III-V element systems Semiconductor quantum dots in III – V systems have been reported in the literature for over two decades. Their first disc overy was made by the failure to grow thin layers of InAs over GaAs substrates attemp ted by Goldstein, et al. [38]. It was observed that after some critical thickness the planar growth mode jumps to 3D nucleation growth forming small InAs islands over the surface. Since then, quantum dots have been continuously reported in variety of III-V ternary and binary systems [20, 39-43]. 2.3.1.1. Growth modes and methods The classification of the well known th ree growth modes dates from 1958, when Ernst Bauer wrote a much quoted review papers in Zeitschrift fur Kristallographie [44, 45]. The Layer-by-Layer, or Frankvan der Me rwe, growth mode arises because the atoms of the deposit material are more strongly attracted to the substrate compared to each other. In the opposite case, where the deposit atoms are more strongly attached to each other than they are to the substrate, th e Island, or Volmer-Weber mode, results. An intermediate case, the Layer-plus-Is land, or Stranski-Krastanov [46] (“S-K”) growth mode is when the initial layer form ation is followed by 3D island nucleation after

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23 the initial layer thickness reaches some critical value. In this case, layers form at first and then it is more favorable for the whole syst em (from the thermodynamical point of energy conservation) to switch into 3D island creation mode. Bauer was the first to systematize these growth modes in terms of surface energies. If we deposit material A on B, we get layer growth if A < B + *, where is the interface energy, and vice -versa for island growth. The S-K mode arises because the interface energy increases as the layer thickness increases; typically this layer is strained to fit the inter-atomic distance to the substrate. Pseudom orphic growth is the term used when it fits exactly. In the island growth m ode, the adatom concentration on the surface is small at the equilibrium vapor pressure of the deposit; no depos it would occur at all unless one has a large supersaturation. In layer growth, the equilibrium vapor pressure is approached from below, so th at all the processes occur at undersaturation. In the S-K mode, there are a finite number of layers on the surface in equilibrium. The new element here is the idea of a nucleation barrier The existence of such a ba rrier means that a finite supersaturation is required to nucleate the de posit. For Quantum Dots the creation of the S-K mode is the predominant one.

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24 Figure 2.6 Schematic diagram of three possi ble growth modes after [Bauer]: (a) – Frank-van der Merwe, (b) – Volmer-Web er, (c) – Stranski-Krastanov, which is the mode responsible for QD growth There is also a fourth growth mode ca lled the Turing mode [47], it was found in 1993 by Temmyo [48] and occurs in a st rained InGaAs/AlGaAs semiconductor heterostructure system on a GaAs substr ate as a novel phenomenon leading to the formation of well-ordered arrays of nanocryst als with built-in InGaAs strained quantum disks with diameters ranging between 30 and 150 nm. The built-in quantum disks within the nanocrystals can exhibit a strong, narro w line photoluminescence emission at room temperature. Turing instability is responsible for their rearrangement phenomenon and selforganization. It also suggests the existenc e of a novel fourth grow th mode on the high Miller index faces of III–V semiconductors. (a) (b) (c)

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25 Figure 2.7 Schematic of the Turing growth mode (after Temmyo) [48]. Note the formation of “nanodisk” inside the nanocrystals Among the possible deposition techniques Molecular Beam Epitaxy (MBE) and metalorganic chemical vapor deposition (M OCVD) are the most widely employed. The MBE method utilizes solid phase sources for Ga Al and In, as well as Ga and As and Sb. For the N source a nitrogen plasma or a mmonia serve as major sources [40, 49, 50]. The first reported MOCVD grown QD were performed by Dimitriev et al. [51] who grew GaN QD’s directly on 6H-SiC. This work was followed by Tanaka and others [52-54] and eventually led to a new met hod called the anti-surf actant growth mode. There are a few other techniques reported in the literature for III-V QD creation. Goowin et al. [55] used reactive lase r ablation of pure Ga in a high purity N2 atmosphere. Nanocrystalline films were created by a sputte ring technique of Ga As material on quartz substrates [56] and by the means of pyr olysis (chemical decomposition of organic materials by heating in the absence of oxygen or any other reagents) reported by Wells et al. [57, 58].

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26 2.3.1.2. Properties of III – V quantum dots In the InGaAs/GaAs systems the 2D to 3D growth mode transition was used during the initial stages of growth of highl y strained InGaAs on GaAs to obtain quantumsized 3D (i.e. QD) structures. These dislocation-free islands (dots) of InGaAs had a size of 30 nm in diameter and 10% size disper sion. Photoluminescence spectra displayed a maximum emission at 1.2 eV [59] which is w hy this type of QD is widely used in optoelectronics for the IR part of the spectrum. For the InGaAs/AlGaAs system arrays of vertically aligned quantum dots in a matrix have been investigated. In this syst em it was shown that in creasing the band gap of the matrix material makes it possible to increase the localizat ion energy of quantum dots relative to the edge of the matrix band [ 20], as well as the states of the wetting layer. The wetting layer is the residue after coati ng a surface with layers of atoms under high temperature during MBE creation of quantum dots, which can interfere with the stimulation of the dot growth influencing th e final size and shape. QD’s of this type reported in literature have PL maxima around 1.3 eV for 5-7 nm QD sizes [60]. The InAlAs/AlGaAs system has a visible luminescence obtained from ensembles of defect-free, InxAl(1x )As islands of ultra small dimensions embedded in AlyGa(1y )As cladding layers grown by the MBE technique [17]. Structural and optical properties of InAlAs quantum dots (QD) were studied as a function of the InAs mole fraction (x). Decreasing of x resulted in a sequential disappearance of the bound electron and hole states in these QDs [61].

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27 In the InP/InGaP system the transitio n towards three-dimensional Stranski– Krastanow (SK) island growth occurs before the second monolayer of InP is formed and vertical alignment of grow n QD’s also occurs improving the overall photoluminescence properties [62]. The width of the emission p eak at half maxima value is 41 meV for a single layer of QD’s while for triple layers it is reduced to 26 meV [42]. Reported PL maximum is 1.757 eV for InP QD used for laser applications, which are 5-7 nm in lateral dimension [41]. InAs/InAlAs displays a room temperature photoluminescence (PL) emission at 0.7 up to 0.8 eV [63]. Molecular beam epitaxy was used to grow InAs self-assembled quantum dots in InAlAs on an InP substrate [64]. For InAs/InGaAs the InAs dot size has been found to be 3–4 times larger than in an InAlAs matrix for the SK growth mode, but the lateral QD’s densit y is about an order of magnitude smaller [64]. Low-temper ature photoluminescence (PL) of the InAs/InGaAs quantum dots is characterized by a narrow (35 meV) PL line as compared to that of InAs/InAlAs quantum dots (170 meV). Quantum dot formation increases the carrier localization energy as compared to qua ntum well structures with the same InAs thickness in a similar manner for both InAs/I nGaAs and InAs/InAlA s structures [64]. In the InAs/InP: in III-V element bina ry combination two different modes of island size and spatial distri bution have been identified. For a deposit of 1.5 and 1.8 monolayers, the islands are about 7 nm high and randomly distributed. Above 2 monolayers, they are about five times smalle r in volume and locally self-organized, with a typical distance of 40 nm independent of the island density [65] The peak wavelength

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28 from the InAs QDs can be continuously t uned from above 0.77 down to 0.73 eV at room temperature [66]. InP grows pseudomorphically on GaP for 3 ML before island crystallization is observed by reflection high-en ergy electron diffraction (R HEED), following a typical Stranski–Krastanov growth mode [67]. Photoluminescence and cathodoluminescence spectroscopy reveal la rge inhomogeneous broadening with the emission peak centered at 1.7 eV for room temp PL [68]. Finally there is the InAs/GaP system. For the growth of InAs on GaP, threedimensional diffraction peaks are observed af ter 0.9 ML of InAs have been deposited, indicating a Volmer–Weber gr owth mode [67]. A maxima of the PL emission is in infrared range around 1.6 eV [68]. 2.3.1.3. III – V QD applications The major use for III-V quantum dots is in the solid state la sers for the next generation of high-speed opti cal communication devices[20, 69] The atomic-like density of states improves optical performance of se miconductor lasers resulting in low-threshold lasing, high-temperature operation, low chirp ( frequency change due to dispersion) [4], and improved high-speed modulation. In pa rticular, recent development of well controlled high-quality self-assembled QDs contributes to the development of new optical devices such as QD semiconductor op tical amplifiers [70] (SOA) and Quantum Dot laser LED’s [21] for the infrared region.

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29 Apart from obvious optical applications there have been attempts made in quantum computing based on these specific op tical properties [22]. Quantum computing is based mostly on dot to dot interaction that can be easily controlled via their separation distance. For example in the simplest case of two quantum dots that exhibit weak coupling there are two interba nd resonances at frequencies 1 and 2 if the system is singly excited. It is thus possible by using ul tra fast two color laser pulses (centered at 1 and 2 + ) to create a photon absorption at 2+ conditional to the occupancy of the QD-pair by an electron-hole create d by the absorption of a photon at 1; hence the possible use of the QD pair for simple optical logic operations. Quantum dots have been also applied in the 3rd generation (low cost, high efficiency) of solar cells in the photovoltaic industry [23] The first advantage derived from the use of quantum dots stems from their tunable bandga p. High currents and voltages are desired for efficient solar-elect ric conversion. Thus, there exists an optimum bandgap that corresponds to the highest possible solar-electri c energy conversion. Quantum dots provide a much more exact me thod of matching the ba ndgap of the solar cell material to the optimum bandgap than tr aditional semiconductor materials for energy conversion, resulting in greater efficienci es. Secondly, in cont rast to traditional semiconductor materials which are often rigid in form, quantum dots can be molded into a variety of different forms. This formati on processes may allow for the creation of ordered 3-D arrays with inter-quantum dot spacing sufficiently small such that strong electronic coupling occurs and minibands are formed to allow for long-range electron transport for better electron extraction [23].

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30 2.3.2. Quantum dots in group II – VI element systems Semiconductor QD’s in II-VI systems were first discovered as nanosize crystal inclusions in glass matrices [3]. The pr operties of bulk II-VI materials have been investigated for over 40 years [71, 72], while nanosize II-VI struct ures have received much attention as synthesis methods have been recently developed for CdSe that leads to an unprecedented degree of monodispersity (the state of uniformity in molecular weight/size of all molecules of a substance) and crystalline order [73], allowing detailed investigations of the size-depende nt optical absorption and emission. For these systems the most common na nocrystallites are cadmium binary compounds such as CdO, CdS, CdSe and Cd Te along with zinc compounds ZnO, ZnSe, ZnS (used only as the shell material) and ZnTe and mercury co mpounds HgS and HgTe. 2.3.2.1. Growth modes and creation techniques The majority of nanocrystallites of II-V I semiconductors are embedded into glass or oxide matrices [74] apart from colloidal solutions where the QD’s are suspended in liquids. The same techniques fo r III-V quantum dots also apply to II-VI nanostructures especially MBE epitaxy [37, 40, 52, 63-65, 75, 76], though the majority of commercially available QD products are creat ed using colloidal particle growth in a solvent media. These include: creation from supersaturated gl ass solution [77], an arrested precipitation technique [78], pyrolisis [79], and aqueous coll oidal growth [80, 81].

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31 Compared to self-organized dots, nanocry stallites suffer from poor interfaces as far as their optical properti es are concerned. They are usually smaller (hence display larger size quantization) and easier to produce, but their surface problems have detrimental consequences on their emission pr operties such as the “blinking effect” [82, 83]. For biological tagging applications CdSe/C dTe (core) nanocrystals are the most popular. They are covered with a wide-gap Zn S or CdS (shell) cappi ng layer providing a barrier for quantum confinement and also improved quantum yield and photo stability. Figure 2.8 Schematic diagram of a typical core -shell nanoparticle used for bio-tagging. This figure represents the scheme with the highest quantum efficiency design, in some cases the core is protec ted only by single-layer shell [7] Most of the quantum dots for fluorescen t imaging typically possess a core-shell structure where the core is fluorescent. The nan oparticle shell is to protect the core from photobleaching and to improve the dispersibil ity in aqueous media. The shell structure Core Intermediate shell Outermost shell

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32 can also be designed to obtain appropria te surface functional groups for attaching biomolecules. In some cases the core is en capsulated by multiple shell structures for improved detection capabilities with fluorescent and magnetic detection done simultaneously [80]. 2.3.2.2. Properties of II – VI quantum dots Although CdO is often used to produce CdS and CdTe quantum dots as the cadmium source, there are very few reports of the QD’s created from CdO itself. Radi et al [81] reports CdO quantum dots incorporated in polyacrylamide th at were synthesized by adding an aqueous suspension of cadmium oxide in acrylamide of bisacrylamide copolymer. The size ranges between 2 and 3 nm [84, 85]. For MBE growth of CdS coherently strained layers on ZnS or ZnSe substrates of the SK formation type of QD is observed [ 79]. It was found recentl y that CdS QDs could be grown with either circular or rectangul ar shapes depending on the growth conditions forming a 3D spherical lens as well as te trahedral shapes [72] These CdS quantum structures show efficient photoluminescence and optical gain in the deep blue to ultraviolet spectral range [71]. It has been found that in wide -gap II-VI systems (CdS and CdSe mostly) quantum structures with small monolayer fluctuations result in such a strong localization of excitons that the exciton binding en ergy reaches energies around 100 meV. CdS QD’s usually range in size between 2 and 10 nm [74]. For colloidal methods of creation the size di spersion can be as small as 5% for these type QD’s [80].

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33 CdSe QD’s were the first to become highly luminescent at room temperature with quantum yields of 30-50% when grown as core/shell structures [13]. CdSe/ZnS composite quantum dots with CdSe usually have cores ranging in diameter from 23 to 55 [86]. The narrow photoluminescence (FWHM 40 nm) from these composite dots spans most of the visible spectrum from the blue through the red. For CdSe as well as for CdTe the photoluminescence intermittenc y effect is observed [35, 72, 87]. Self-assembled CdTe quantum dots form when grown by molecular beam epitaxy on ZnTe substrates [75]. They also form QD structures in glass matrices. Formation of QDs usually starts after de position of 1.5–2.5 monolayers of CdTe for the MBE method. The resulting dots have a typical diameter of 2 nm and a planar density of 1012 cm-2 [75]. The photoluminescence spectra consist of two emission lines. The high-energy line originates from excitonic recombination in a wetting layer which is required for their formation while the low-energy emission PL ba nd is assigned to recombination in CdTe quantum dots [88]. The activation energy of the thermal quenching of QD related PL emission was found to be equal to 47 meV [75]. ZnSe quantum dots have been reported as colloidal synthesize d nanoparticles with an arrested precipitation t echnique [77], or more recently they are prepared from a supersaturated glass solution [78]. Coati ng ZnSe quantum dots with a ZnS monolayer yields a remarkable enhancement in the PL quantum efficiency at room temperature without affecting the spectral distribution. The result suggest that pa ssivation of surface states, along with an increased localization of the holes in the core ZnSe layer, gives rise to a high luminescence quantum yield [18] The controlled growth process affords tunable sample sizes with ba nd-edge fluorescence size-tunab le between 2.8 and 3.4 eV is

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34 obtainable at room temperature with quantum yields between 20% and 50% relative to Stilbene 420 [77]. Nanocrystals of ZnTe in collo idal form were reported by Jun et al [89] though this material is mostly used as a host matrix for other telluride QD’s. The resulting nanocrystals are relatively large (length 500–1200 nm, widt h 30–100 nm). Their optical properties were investigated w ithout isolation or characteriza tion of the nanocrystals. In fact, there have not been any reports on isol ated ZnTe nanocrystals below 10 nm in size. There are not too many reports about Hg S type of QD as they are mostly mentioned in theoretical works. Though collo idal mercury sulfide QDs synthesized at room temperature have been reported [90]. HgS material also serves as a host material for epitaxial growth in a CdS/HgS heterostructur e of nanometer dimensions, prepared by wet chemistry methods [90]. Kershaw et al. [88] reports an aqueous colloidal growth technique of HgTe QD’s to form quantum dots with a broad, str ong fluorescence in the infrared (~1.0–1.5 eV) region. The reported quantum efficiency wa s high, around 44%, when pumped in the visible (~2.54 eV), and the excited state lifetime was around 130 ns, making the material interesting as an optical amplifier medium. 2.3.2.3. II – VI QD applications II-VI semiconductor QD’d have found appli cation in optoelectronics where their optical properties are widely employed in light-emitting diodes [91] and solid-state lasers [4]. For laser applications ZnSe is very pr omising QD material, with a room temperature

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35 bulk band gap of 2.7 eV (460nm), and has long b een a material of choice for blue diode lasers [77]. Creating ZnSe QD’s will allow em ission even further into the ultra-violet region. More recently developed colloidal na nostructures including core-shell QD’s. Elongated rods [92] and mixed semiconductor quantum dots [93] have been used in biosensing [94] and bio-labeling [12] where they are used as fluo rophore substitutes. Their role is to provide a fluorescent signa l that allows the detection and tracking of specific bio-molecules that QD’s are attached to. More advanced bio-labeling technique s involve mixing semiconductor QD’s allowing for multicolor optical coding for biol ogical assays (so called multiplexing) [93]. This has been achieved by embedding differe nt-sized quantum dots into polymeric microbeads at precisely controlled ratios, which makes them ideal fluorophores for wavelength-and-intensity multiplexing. The use of 10 intensity levels and 6 colors could theoretically code one million nucleic acid or protein sequences. Imaging and spectroscopic measurements indicate that the QD-tagged beads are highly uniform and reproducible, yielding bead identification ac curacies as high as 99.99% under favorable conditions [93]. One of the interesting applications of II-VI QD’s is their use as temperature sensors [95]. The steady-state photoluminescen ce properties of cad mium selenide QD’s are strongly dependent on temperature in the range from 100 to 315 K. The PL intensity from these QD’s matrices increases by a factor of ~5 when the temp erature is decreased from 315 to 100 K, and the peak of the emi ssion band was blueshifted by 20 nm over the same range. This dependence is linear and re versible (–1.3% per C) for temperatures

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36 close to ambient conditions. The significant temperature dependence of the luminescence, combined with its insensitivity to oxygen quenching, establishes these QD’s as optical temperature indicators for temperature-sens itive coatings making an another possible application. 2.4. Biomarkers A biomarker is a biological parameter/mate rial (gene, metabolite or protein) that is indicative of a physiological or pathological state. For this bei ng an overall definition of a biomarker in ex-vivo as well in in-vivo cancer detection techniques the biomarker is a substance used as an indicator of a biol ogic state. It can be any kind of molecule indicating the existence (past or presen t) of living cells, proteins, etc [96]. In biology and medicine, a biomarker can be a substance whose detection indicates a particular disease state (for exampl e, the presence of an antibody may indicate an infection or cancer). Such a biomarker can be native to the body. A biomarker can also be used to indicate exposure to various e nvironmental substances in epidemiology and toxicology. In genetics, a biomarker (identifie d as a genetic marker) is a fragment of a DNA sequence that is associated with, changes su sceptibility to disease, or causes disease [96].

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37 Figure 2.9 Schematic representation of the QD bound to a biomarker (molecules are drawn to scale) – after Jaiswal [97]. Singl e QD in the center is attached to the detector antibodies (IgG proteins) via linker molecules (PEG ). Note that not all linker molecules are utilized For the purpose of this research four an ti-bodies, anti-gene bi omarker groups were selected: IL-10, CA125, PSA (Prostate Speci fic Antigen) and Osteopontin. These antibodies target their specific anti-genes and serve the purp ose of early stage ovarian, prostate and other cancer detection. The overall conjugate scheme is presented in Fig 2.9 with the QD and antibodies drawn approximately to scale. Ovarian cancer causes more deaths each year among women than any other gynecologic cancer. For this type of cancer survival rates are 30% for patients that are diagnosed with advanced (past stage Antibodies (IgG) protein Linker molecules (PEG) Quantum Dot ~40 nm

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38 III) disease. However, 5-year survival rates are less than 20%. In contrast 80-90% 5-year survival rates are associated with patients diagnosed with Stage I or II ovarian cancer. These statistics provide the primary rationale to improve ovarian cancer screening and early detection [98]. Stages in cancer development are as follows: Stage I the cancer has not spread past the tissue or organ where it started. Stage II there is some local and regiona l spread of the cancer, sometimes to lymph nodes. Stage III there is extensive local and re gional spread of the cancer, usually to draining lymph nodes. Stage IV the cancer has spread (metastasized) beyond the regional lymph nodes to distant parts of the body. Interleukins (ILs, IL10) are a large group of cytokines (proteins) that are produced mainly by leukocytes, although some are produ ced by certain phagocytes and auxiliary cells. ILs have a variety of func tions, but most of their functi on is to direct other immune cells to divide and differentiate [99, 100]. E ach IL acts on a spec ific, limited group of cells through a receptor (protein on the cell membrane or within the cytoplasm or cell nucleus that binds to a specific molecule [ 101]) specific for that IL. Human IL10 (which is used in this research) is a non glycosylated polypeptide consisting of 160 amino acids. There is 73% homology between the human and mouse IL10 proteins that we have used in our conjugation experiments, however, the human IL10 acts on both human and mouse target cells, while the mouse IL 10 has species-specific activity.

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39 Cancer antigen 125 (CA125) is a protei n found on the surface of many ovarian cancer cells and it also has b een chosen for bioconjugation is our experiments. It can be found in other cancers and in small amounts within normal tissue. CA125 is used as a tumor marker as indicator of some types of cancer, espe cially ovarian cancer [102]. Studies are being done to determine whether CA125 can be used as a screening test for ovarian cancer. Most often, the CA125 test is used to help determine the effectiveness of treatment for ovarian cancer or to help di agnose the recurrence of ovarian cancer [103]. CA125 is a high-molecular-wei ght glycoprotein that is recognized by the monoclonal antibody OC125 and is elevated in most wome n with ovarian cancer. CA125 is the most extensively studied biomarker for possible use in the early detection of the disease [103]. This antibody stains the membranes of epith elial cells in most non-mucinous epithelial ovarian carcinomas. Normal ovarian epithelium, breast, gastroinetestinal tract, liver and skin tissue are negative when exposed to this antibody [104]. Prostate Specific Antigen (PSA) is a molecule produced by the normal prostate and secreted in large amounts in the male semen and is al so normally present in minute quantities in the male bloodstream. It is now well known and accepted that PSA levels in the blood are elevated by several disease proc esses in the prostate, most importantly by prostate cancers. This molecule bel ongs to a group of kallikreins (class of peptidases/enzymes that cleave peptide bonds in proteins [105]) that are a subgroup of serine proteases having divers e physiological functions. This is another biomarker which is used in our bioconjugation experiment s. Growing evidence suggests that many kallikreins are implicated in carcinogenesis and some have potential as novel cancer and other disease biomarkers [106] This gene is one of the fifteen kallikrein subfamily

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40 members located in a cluster on chromosome 19 [107]. Serum leve l of this prot ein, called PSA in the clinical setting, is useful in the diagnosis and monitoring of prostatic carcinoma. Alternate splicing of this gene generates two transcri pt variants encoding different isoforms (version of a protein w ith some small differences in amino acid sequences, folding etc.). Additional transcript variants have been described, but it is unclear if these transcripts are normally expre ssed or if they are specific to benign or malignant tumors [108]. Osteopontin is the last biomarker empl oyed and bioconjugated by us. It is a glycoprotein identified in osteoblasts (cells that build and reshape bones). The prefix of the word "osteo" indicates that the protein is expressed in bone, where it is one of the extra cellular structural proteins that constitute the organic part of bone. This protein is composed of ~300 amino acids and is rich in acidic residues: 30-36% is either aspartic or glutamic acid [109]. Osteopontin is synthesized by a variet y of tissue types including preosteoblasts, osteoblasts, osteocytes, extrao sseous cells in the inne r ear, brain, kidney, placenta, odontoblasts, some bone marrow cells, macrophages, smooth muscle, and endothelial cells [109]. Osteopon tin is over expressed in a variety of cancers, including lung cancer, breast cancer, co lorectal cancer, stomach cancer, ovarian cancer, melanoma and mesothelioma [110]. 2.5. Summary Size-tunable optical proper ties have become a hallmark of quantum dots and related nanostructures. These properties are currently under intensiv e study for potential

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41 use in optoelectronics, high density memo ry, quantum-dot lasers, and lately for biosensing [12] and biolabeling [95]. Recent advances have led to the development of colloidal nanostructures incl uding core-shell quantum dots and other non-spherical nanoparticles, as well as quantum-dot qua ntum-well (QDQW) heterostructures, and mixed semiconductor dots [93]. Sometimes t uning of electronic, optical, and magnetic properties by changing the particle size coul d cause problems in many applications such as nanoelectronics, superlatti ce structures, and biological labeling and there are first reports about adjusting emission wavelength without changing the pa rticle size but its composition [111]. There are stil l not many reports about the influence of the surrounding molecules on their photoluminescence proper ties, along with the spatial la rge area investigations of these parameters fluctuations. This research is focused on the non uni formity of the QD optical parameters across the QD matrices to investigate and expl ain possible mechanism of it. This is an important piece of information for the manufact ures of self assembled QD samples since most of the sample properties come from th e growth condition fluc tuations, like capping layer composition, temperature of growth, annealing etc. The other part is devoted to the infl uence of the surrounding molecules on the optical properties of the II-VI QD systems fo r bio medical applications as fluorescent tags. The spectral features induced by bi omolecules proximity may have the large influence on the use of QD’s as the labels for given biomarker detection. The specific sample details along with the experimental se tups are described in the next chapter.

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42 3. Quantum Dots Experiments 3.1. InAs/InGaAs QD samples This set of samples contains InAs/InGaAs self-assembled quantum dots on GaAs substrates grown in the Stranski–Krastanow (S-K) growth mode. These samples were prepared by Dr. Andreas Stintz from Ce nter of High Technol ogy Materials at the University of New Mexico. St ructures were created on GaAs at different growth temperatures from 470 to 535 C. Solid-source molecular beam epitaxy (MBE) in a V80H model reactor was used for growth of three In As self organized QDs layers inserted into In0.15Ga0.85As/GaAs Multi Quantum Wells – there are three QD/QW structures placed on top of each other (Fig 3.1). The Stranski -Krastanov [46] (S-K) growth mode is responsible for the self assembled creation of QD’s in this case. In the growth process the initial layer formation is followed by the 3D island nucleation after the initial layer thickness reaches some critical value (2.4 ML for InAs). The InGaAs/GaAs structures were grown under As-stabilized conditions at different substrate temperatures: 470 C (#684), 490 C (#685), 510 C (#687), 525 C (#689) and 535 C (#698), during the deposition of the InAs active regions and InGaAs wells and 590-610C for the remaining layers of the structure [112, 113] The dot density (2.5-5.3) 1010/cm2 was determined by

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43 AFM measurement of surface roughness on a para llel wafer with open QDs that have not been overgrown by the QW and cladding layers. Figure 3.1 Schematic structure of multi quantum well and quantum dot “sandwich” of InAs/InGaAs samples. These samples were prepared by Dr. Andreas Stintz from Center of High Technology Material s at the University of New Mexico In addition to growth temperature ch anges, the effect of cladding layer composition was also studied. A set of simila r samples grown at 590-610C with varied InGaAs cladding layer was measured. These samples had an InxGa1-xAs layer composed of x=0.1 (10%) (#1361), 15% (#1360) to 20 % (#1363), respectively. They were created using the molecular beam epitaxy technique. Each sample was composed of three InAs self-organized QD arrays embedded into external In0.15Ga0.85As/GaAs strained multi quantum wells. AFM studies of sister sample s without overgrown capping layers showed that individual QD’s were of 15 nm in base diameter and approximately 7 nm in height. The in-plane density was 7-10 x 1010 cm-2. Physical dimensions of this set of samples was 1 x 2 cm approximately and they were cutouts from larger 2 inch wafers cut in the way that each sample contained the wafe r periphery and some inner area portion. QD formation in S-K growth mode InAs In.15Ga.85As In.15Ga.85As

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44 For the spatially resolved PL studies of InAs QD’s the whole 50mm wafer was used. The experimental set of samples was cr eated in a similar way to the previous 13XX sample series. All investigated QD structures had a In0.15Ga0.85As buffer layer under the wetting layer. A variable parameter similar to the 13XX series was the In-composition of the InxGa1-xAs capping layer, which covers the InAs QDs. Four different compositions of the cap layer were studied with x = 0.10 (#1718), 0.15 (#1719), 0.20 (#1720) and 0.25 (#1721). Sister samples with exposed QD layers showed that the individual dots were of 15 nm in base diameter and approximately 7 nm in height. The me asured in-plane dot density was 3–5 x 1010 cm-2. 3.2. CdSe/ZnS QD samples QD nanocrystal samples used in this pa rt of the research are fluorophores — substances that absorb photons of excitati on light, and re-emit photons at a different wavelength. All samples used in this part of the experiments were purchased from the Quantum Dot Corp. (now Invitr ogen, Inc.) All quantum dots ha d a CdSe core of different diameter and ZnS shell as illust rated in Fig 3.2. This core/she ll material is further coated with a polymer shell that allows the material to be conjugated to bi ological molecules and to retain their optical properties.

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45 Figure 3.2 Schematic representation of the bioconjugated quantum dot (after Invitrogen, Inc) [11] Some of the samples were purchased already conjugated to linker molecules (Table 3.1). The number in th e product label represents th e peak position of the photoluminescence spectrum in nanometers (e.g, QD 525 has a peak wavelength of 525 nm, etc.). Table 3.1 QDot.com samples description Qdot.com product Conjugation Volume / Concentration QD 525 F(ab’)2 fragment conjugate 50 l at 10 mM QD 655 Streptavidin conjugate 50 l at 10 mM QD 525 (conjugation kit) Pure non conjugated 250 l at 10 mM QD 565 (conjugation kit) Pure non conjugated 250 l at 10 mM QD 605 (conjugation kit) Pure non conjugated 250 l at 10 mM QD 655 (conjugation kit) Pure non conjugated 250 l at 10 mM QD 705 (conjugation kit) Pure non conjugated 250 l at 10 mM 10 -15 nm Core CdSe ShellZnS Polymer coating Bio-molecules

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46 These QD nanocrystals are nanometer-scale (roughly protein-sized ) atom clusters as illustrated in Fig 3.2 which is approxima tely drawn to scale. For the non conjugated quantum dots, conjugation was performed acco rding to supplied conjugation protocols [114] with the following bio-molecules: Table 3.2 Biomarkers molecules chos en for bio-conjugation experiments Biomolecule type Supplier Additional info OC 125 (mouse antihuman OC125, mouse IgG) DakoCytomation (Carpinteria, CA) stock concentration -679 mg/L, code M3519 CA 125 (cancer antigen 125) Fitzgerald Industries (Concord, MA) stock concentration 1000000 U/ml Interleukin 10 (anti human IL10, rat IgG2a) Serotec (Raleigh, NC) stock concentration 1 mg/ml, clone JES3-12G8, code MCA2250 Recombinant human IL10 Serotec (Raleigh, NC) stock concentration 1mg/ml, code (PHP047A) PSA (anti prostate specific antigen, mouse IgG1), Chemicon International (Temecula, CA ), stock concentration 5.14 mg/ml, code MAB4082 Osteopontin (anti human Osteopontin, mouse IgG) Assay Designs (Ann Arbor, MI), stock concentration 1mg/ml, code 905-078 3.2.1. Bio-conjugation substrates For the bioconjugation experiments diffe rent substrates were used. Surface chemistry for ELISA plates is basically the same i.e. binding of the capture antibodies is

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47 done via biotin-streptavidin bridge or dire ct protein immobilizat ion on the aldehyde surface. However the substrate material of the plate can be optimized to reduce the intensity of the background photoluminescence signal, which superimpose with the PL from the QD’s. For the photoluminescence measurements background signal elimination is a critical step allowing low PL intensit y study to be performed. These are the ELISA substrates that have been tested and used in the experiments: Table 3.3 ELISA plate types ELISA plate type Manufacturer FluoroNunc Flat Bottom Maxi Sorp NUNC Black Bio-Bind Assembly streptavidin coated Thermo electron White microtiter plate Thermo Labsystems Flat bottom immuno plat e VWR international Transparent bottom immuno plate VWR international

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48 Figure 3.3 Background signal from different ELI SA plates as a function of wavelength The best substrates for PL measuremen ts should not have any signal in the spectral range of interest; in our case this is almost all of the visible part of the spectrum from 400-750 nm. A bulk single crystal Si wa fer with the band gap of 1.1 eV at room (1100 nm) is a suitable material for a substrate. 400450500550600650700 0 100 200 300 400 ELISA Background comparison HeCd (325nm) excitaion room tempIntensity [au]Wavelength [nm] White microtiter plate (Thermo Labsystems) Transparent bottom immuno plate (VWR) Flat bottom immuno plate (VWR) FluoroNunc Flat Bottom Maxi Sorp (NUNC) Black Bio-Bind Assembly streptavidin coated (Thermo electron) Si substrate

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49 3.3. Spatially resolved PL spectro scopy experimental details 3.3.1. Hardware description Various lasers were used as the excitation sources in the PL experiments. In Table 3.4 their specifications is presented: Table 3.4 Lasers used in the PL experiments Laser type Emission wavelength Output power Manufacturer/Model HeCd (cw) 325 nm 50 mW Coherent Inc. HeCd series 74 Ar+ (cw) 488 nm 514 nm 50 100 mW 50 100 mW Coherent Inc. Innova 70 AlGaAs laser diode (pulsed) 804 nm (10 nm FWHM) 150 mW (in pulse) Spectra Diode Labs SDL 800 The photoluminescence signal was disper sed with a 0.5 m SPEX-500M grating spectrometer possessing a reciproc al dispersion of 3.2 nm/mm (2nd order) with a 600 lines/mm diffraction grating. The dispersed si gnal was registered w ith either an aircooled photomultiplier (Electron Tubes) in th e spectral range of 400 800 nm or a liquid nitrogen cooled Ge detector (North Coast Scientific Corp.) in the range of 700 – 1700 nm. A mechanical chopper modulated the exc itation light of the CW laser with 82 Hz frequency. AC signal from the detectors wa s fed to Lock-in amplifier EG&G Model 5209 and collected by a computer. A schematic of the PL setup for InAs QD measurement is shown on Figure 3.4

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50 Figure 3.4 Photoluminescence setup for r oom temp measurements of InAs QD’s 3.3.2. PL mapping measurements The PL mapping experiment was done w ith the use of an X-Y computer controlled moving stage (Velme x 8300) with 10 m step precision and (Klinger CC 1.2) for 1 m resolution maps. Liquid phase ELISA measurements were performed with the modified optics scheme as shown in figure 3.5. In this scheme th e vertical optics alignment had to be used in order to keep the liquid in place. An a dditional mirror with a hole in the center for the excitation laser beam allowed for PL signa l collection from horizontally placed ELISA plates.

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51 Figure 3.5 Photoluminescence setup for liquid ELISA experiments Low temperature measurements were carri ed out in a closed cycle He cryostat (RMC cryosystems 22CB Cryogenics Inc.) al lowing temperature va riation from 10 to 350 K. The small dimensions of the cryostat head allowed for the low temperature mapping measurements with the expe rimental setup shown on Figure 3.6.

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52 Figure 3.6 Photoluminescence setup for low temperature PL mapping Mapping of the glass slides prep ared with micro array techni que was done with the use of our experimental setup and with the use of the optical confocal microscope (Nikon Eclipse 800) in parallel. Micro array spots were printed with a BioRobotics MicroGrid microarrayer from Genomic Solutions (Ann Arbor, MI).

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53 3.4. Bio-conjugation confirmation techniques To ensure that our quantum dots have been conjugated to biomolecules we employed two different techniques to detect bioconjugation occurrence. Use of these techniques allows us to detect the amount of a given biomarker via integrated PL intensity measurements. The well known inte nsity vs. concentration dependence for ELISA and micro array approach is shown in Fig 3.7. We utilized it in order to confirm that bioconjugation to QD’s ha s taken place, if the overall QD PL signal follows it we may assume the bioconjugation was successful. Figure 3.7 ELISA concentration curve vs. op tical signal. Linear part (shaded area) constitutes the limits of detection fo r given biomarker concentration range Concentration Optical signal

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54 3.4.1. Enzyme Linked ImmunoSorbent Assay (ELISA) technique The ELISA technique is used for the detec tion of a given biomolecule (antigen in our case) by means of the attachment of a sp ecifically recognizing molecule that has been labeled with a fluorescent label. ELISA comb ines the specificity of antibodies with the sensitivity of simple enzyme assays, by using antibodies or antigens coupled to an enzyme or directly labeled secondary antibody. Sandwich ELISA assays are one of the most useful immunoassays, this is a twoantibody “sandwich” (Fig 3.8) This assay is us ed to determine the antigen concentration in unknown samples [115]. The ELISA is a fa st and accurate technique, and if the purified antigen standard is available, the assay can determine the absolute amount of antigen in an unknown sample. What we have uti lized here is the Elisa method to validate our bioconjugation procedure. Figure 3.8 “Sandwich” immunoreaction sche me for the direct ELISA approach Fluorescent label Detector antibody Antigen Capture antibody

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55 The sandwich requires two antibodies that bind to epitopes (conjugation sites on the protein surface) that do not overlap on the antigen. This can be achieved with either two monoclonal antibodies (mAb antibodies ar e produced by one type of immune cell or it’s clones) that recognize discrete sites or one batch of affinity -purified (different antibodies that recognize this specific antigen with 100% specificity) polyclonal antibodies [115]. To utilize this assay, one antibody (the “capture” an tibody) is purified and bound to a solid phase typically attached to the bo ttom of the plate. Antigen is then added and allowed to complex with the bound antibody. Unbound products are then removed with a wash, and a labeled secondary antibody (the de tection antibody) is allowed to bind to the antigen, completing the sandwich. The assay is then quantized by measuring the amount of the labeled secondary anti body bound to the matr ix, either via optic al density change or the fluorescence intensity of the attached la bel. Major advantages of this technique are that the antigen does not need to be purified prior to use, and that these assays are very specific. However, one disadvantage is that not all antibodies can be used. Monoclonal antibody combinations must be qualified as “matched pairs” meaning that they can recognize separate epitopes on the antigen so they do not hinder each other’s binding.

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56 Figure 3.9 Direct sandwich ELISA pr ocedure (after Chemicon.com) [116] 3.4.2. Protein microarray technique Protein Microarray is the methodology to detect the existence of a given biomolecule with the use of the immunoreactions technique. In it’s basic assumption it is similar to the ELISA technique although it is more advanced in this aspect with use of much less sample volume. The major difference is that the proteins are printed by a needle on the substrate surface instead of be ing deposited from the solution (Fig 3.10). The printed spots are usually 100 to 200 microns in diameter what utilizes very small amounts of sample and allows the printing of large spot numbers on relatively small areas. The reaction speed for sm aller volumes is also faster and allows one to measure relatively smaller quantities of antibody th an ELISA, though it requires much better scanning techniques. The following processing and wash steps are similar to the ELISA procedure shown in Fig. 3.9. Sandwich ELISA

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57 Figure 3.10 Printing scheme in the micro array technique Mark Schena [117] author of the firs t paper demonstrating the usefulness of protein microarrays, describes them as anal ytical devices that possess four distinct characteristics: (a) microscopic target elements or spots, (b) planar substrates, (c) rows and columns of elements and (d) specific bindi ng between microarray target elements on the substrate and probe molecules in the solu tion [117]. It is a miniaturized assay (spots are usually in the range 0.1 – 0.5 mm in diamet er) [117] where each spot contains “bait” molecules (antibody in prot ein / antibody microarrays), which are probed with an unknown biological sample containing analytes of interest [118]. By processing the microarrays with a detector antibody tagged with a fluorescen t label, each spot produces a fluorescent signal proportional to the analyte of interest present in the solution and the captured/bound to the “bait” molecule [118].

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58 Applications of functional protein microarrays include: expression profiling for identification and quantitation of proteins present in the solution; protein-protein interactions for examination of the binding activity and binding part ners of proteins across the entire proteome; drugs for iden tification of drug activ ity, targets, crossreactivity, and diagnostics to measure prot eins expressed in serum samples [117]. Figure 3.11 Basic microarrays immu no reactions after Schena [117] Production and use of microarrays consists of the following steps: printing and immobilization of capture antibodies on a f unctionalized surface (usually glass slide covered with poly-L-lysine, aldehyde, epoxy or nictrocellulose) [ 119]; incubation with the sample, detection with fluorescent probe – QD in our case, image capture and analysis. The most sensitive method for prot ein microarrays processing is the “sandwich assay” (Figure 3.8) based on the Elisa tech nique. It utilizes two antibodies that simultaneously bind to the same antigen. One of them called “capture antibody” is responsible for immobilization of antigen ont o the slide surface. The other one called “detector antibody” which is fluorescently labe led, is attaching to already immobilized

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59 antigen from the other side producing a fluor escent signal. Averaged intensity of the fluorescent signal is the repres entation of biomarker con centration in the solution. Protein expression profiling, protein-prot ein binding, drug interactions, protein folding, substrate specificity, enzymatic activ ity, and the interactions between proteins and nucleic acids are among the many a pplications of protein microarrays. For bio conjugation confirmation the prot ein-protein interaction was of our interest. Having quantum dots c onjugated to a detector antibody we are able to check this linkage by their immobilization through a “san dwich” reaction on the substrate surface and scanning for a luminescence signal. 3.5. Summary Samples of InAs quantum dots were prepared using MBE technique. Measurements mostly reflect a non uniformity of this process. This allowed us to investigate hard to control growth parameters such as a planar QD size distribution. Study of QD size influence on the optical properties was done along with other growth parameters that were changed on purpose like growth temperature. All of these samples represent multi-quantum well st ructures with very strong ca rrier confinement in the QD layer itself – there are no si gns of the surrounding quantum wells even for very high excitation intensities. Indirec tly this may suggest that our samples are of very high QD quality with deep enough QD potential for room temp laser applications. CdSe quantum dots for biological applications were purchased from commercially available sources; hence in dept h details of their cr eation parameters are

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60 not known, manufacturers assure the 5% distribution size among one QD species but not all of the purchased QD material actually show ed repeatable PL results, especially for different batches of the same product. Bio – conjugation tec hniques allowed for indirect determination of bio-molecule attachment a nd further spectroscopic investigations of conjugated vs. non-conjugated QD’s. For repeat ability reasons we decided to perform bioconjugation ourselves without purchasing already pre-conjugated QD’s for which we did not have a pure reference QD sample. All wet samples experienced substantial scattering, and as a direct consequence, we st arted to work with the dried residues of the ELISA procedures. Since ELISA is very depe ndent on the washing procedures and initial well functionalization, the human factor played a huge role in sample preparation. Use of the microarray printer with the automated prin ting procedure was the next step in order to increase the sample quality. For the characte ristic spectral feat ures of biomolecule attachment we used drops of dried conjuga te samples on a Si substrate for better background separation. The “strength” of the conjugation was investigated with the use of all the fractions from conjugation expe riments and appropriate sample collection during each step of this procedure.

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61 4. Results and Discussion 4.1. PL spectroscopy of InAs/InGaAs quant um dots for laser applications PL spectroscopy of InAs Quantum dots was carried out on two sets of samples. The initial one that contained small pieces of full size wafers approximately 1x2 cm in size that showed already some spatial i nhomogeneity and the full set of whole 50mm diameter wafers for larger area scans. Spectroscopic mapping was carried out to investigate the influence of growth temper ature as well the cladding layer composition for the two subsets of samples respectively. 4.1.1. Growth temperature investi gations PL spectroscopy The temperature of the substrate during QD growth was varied in the range of 470 up to 535C. For QD’s grown at 470C only one PL band was observed with a higher value of full width at half maximum (FWH M) of 50-60 meV at 80 K (Fig. 4.1 b) with respect to higher temp samples. With an increase of the QD growth temperature the integrated PL intensity of the investigated structures increased and the PL spectrum changed (Fig. 4.1 to 4.5).

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620.951.001.051.101.15 0.0 0.2 0.4 0.6 0.8 1.0 (a)Sample #684 at 300KIntensity [au]Energy [eV] 0.951.001.051.101.15 0.0 0.2 0.4 0.6 0.8 1.0 (b)Sample #684 at 80KIntensity [au]Energy [eV] Figure 4.1 PL spectra of QD structure #684 (grown at 470 C) at various intensity points (from lowest to highest) measured at 300 K (a) and 80 K (b) 0.900.951.001.051.101.15 0.0 0.2 0.3 0.5 0.7 0.8 1.0 (a)Sample #685 at 300KIntensity [au]Energy [eV] 0.951.001.051.101.151.20 0.0 0.1 0.3 0.4 0.6 0.7 0.9 1.0 (b)Energy [eV]Intensity [au]Sample #685 at 80K Figure 4.2 PL spectra of QD structure #685 (grown at 490 C) at various intensity points measured at 300 K (a) and 80 K (b)

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630.900.951.001.051.101.15 0.0 0.2 0.3 0.5 0.7 0.8 1.0 (a)Sample #687 at 300KIntensity [au]Energy [eV] 0.951.001.051.101.151.20 0.0 0.2 0.4 0.6 0.8 1.0 (b)Sample #687 at 80KIntensity [au]Energy [eV] Figure 4.3 PL spectra of QD structure #687 (grown at 510 C) at various intensity points measured at 300 K (a) and 80 K (b) 0.900.951.001.051.101.15 0.0 0.2 0.4 0.6 0.8 1.0 (a)Sample #689 at 300KIntensity [au]Energy [eV] 0.951.001.051.101.15 0.0 0.2 0.4 0.6 0.8 1.0 Sample #689 at 80K(b)Intensity [au]Energy [eV] Figure 4.4 PL spectra of QD structure #689 (grown at 525 C) at various intensity points measured at 300 K (a) and 80 K (b)

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640.900.951.001.051.101.15 0.0 0.2 0.4 0.6 0.8 1.0 (a)Sample #698 at 300KIntensity [au]Energy [eV] 0.951.001.051.101.15 0.0 0.2 0.4 0.6 0.8 1.0 Intensity [au](b)Energy [eV] Sample #698 at 80K Figure 4.5 PL spectra of QD structure #698 (grown at 535 C) at various intensity points measured at 300 K (a) and 80 K (b) The variation of PL band intensities versus excitation laser power density presented in Figure 4.6 indicate s that the low energy PL band can be attributed to the carrier recombination between ground states (GS) and the higher energy PL band and appears at an excitation density > 50 W/cm2, indicating that it is an optical transition via the first-excited state (1ES) in the QDs. Th e FWHM of the GS band for the two band PL spectra is 35-38 meV at 80 K that is typica l for high quality InAs QD structures [36]. PL spectra measured at different excitation power densities at 77 K are presented in Fig. 4.6 for the QD structure #689 with the QD laye r grown at 525 C. Up to 3 consecutive excited state PL transitions are observed at the highest excitation laser power density.

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651.001.051.101.151.201.251.30 0.0 0.2 0.4 0.6 0.8 1.0 6 5 4 3 2 1PL Intensity [arb units]Energy [eV] 1 1000 W/cm22 500 W/cm23 160 W/cm24 100 W/cm25 30 W/cm26 10 W/cm2 Figure 4.6 PL spectra of the QD ensemble m easured at different excitation powers with the highest power density of 1.0 kW/cm2 (Structure #689 measured at 77K) This structure exhibited the highest PL intensity at 12 K temperature and an excellent resolution of the ground and excited PL bands. The spectra reveal a set of PL bands with peak energies of 1.110, 1.160, 1.207, 1.249, and 1.281 eV as can be typically observed on a QD ensemble having a good homogeneity [39, 120]. Three former PL bands are well resolved and are close to Ga ussian in shape. The variation of PL band intensities versus excitation power indicat es that the low-energy PL band can be attributed to the ground state (G S) of the QD’s. The higher-en ergy PL bands appear at an excitation power density exceeding 100 W/cm2, indicating the optical transitions are via the excited states (1ES-4ES). The deconvol ution procedure using Gaussian bands was Energy [eV] PL intensity [arb units]

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66 applied to these spectra, which has shown that the half width of the three lowest energy PL bands (GS, 1ES, and 2ES) are equal to 39, 31, and 28 meV, re spectively. The energy separation between GS and ES bands is no t equidistant and equa l to 50, 47, 42, and 32 meV, which indicate that the studied QDs could not be characterized by a harmonic oscillator potential [121]. Ground state PL in tensity dependence vers us temperature for the QD ensemble presented in Fig. 4.7 has been measured for an ex citation power density of 650 W/cm2 for the structure #689 as well. 24681012 103104105106 II I Eact = 36 meV Eact = 323 meVPL Intensity [arb units]1000/T [K-1] Figure 4.7 Ground state PL band intensity depend ence vs. temperature (Excitation power density 650 W/cm2) (Structure #689). Two distin ct regions were observed, labeled I and II, indicating two different activation energies

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67 Two different slopes (labeled as I and II in Fig 4.7) of PL intensity dependence are clearly seen on the temperature curve: ( I) the first slope is in the low-temperature range 80–250 K before the main thermal que nching process (II) starts. The activation energies of these processes were estimate d by analyzing the temp erature dependence of the PL intensity using the Arrhenius plot (Fi g. 4.7). The estimated activation energies are 36 (I) and 323 (II) meV. In this case, we can interpret the PL intensity thermal quenching of the first (I) slope as the carrier thermal escape processes from the QD levels. Taking into account that QDs in this experiment ar e excited by the light with energy quanta of 2.41 eV, which are effectively absorbed in Ga As and wetting layers the small activation energy of 36 meV can also be associated w ith thermal carrier escape from wetting layers to the GaAs layer where it is possible for them to recombine via nonradiative channels. The second 323 meV thermal activ ation energy is larger th an the electron-hole binding energy in this type of QD, which typically ranges from 100 to 150 meV [112, 113]. Thus, we attribute this high thermal activatio n energy of 323 meV to the activation of nonradiative recombination centers. 4.1.2. Growth temperature investigations PL mapping PL mapping at the GS state energy was al so performed for this sample. The PL intensity in the QD sample s was measured at 80 and 300 K and shows long-range inhomogeneity across the wafer area, which is accompanied by a spectral shift of the PL maximum. We explored this effect in more detail using the sp ectroscopic PL mapping technique where the PL spectrum is record ed and analyzed at each sample point.

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68 Figure 4.8 (a) Map of PL intensity at 0.99 eV (0.25 mm step size, 200 m excitation spot diameter), (b) map PL intensity at GS max position, and (c) map of peak energy positions (0.5 mm step size) at ~90 W/cm2 power density. Arrow in (b) corresponds to spectroscopic line scans presented in Figure 4.9 In Figure 4.8 (a) the room-temperature PL map measured on QD structure #689 at energy of 0.99 eV close to the principal PL maximum is shown. Notice the logarithmic scale specified in the contrast bar of Fig. 4.8( a). Two orders of magn itude variation in the PL intensity signal was observed. This can be seen from the low PL intensity in the central part of the structure to the high PL intensity at the sample periphery area. To establish the origin of su ch a strong PL inhomogeneity, spectroscopic PL mapping

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69 measurements were performed. At first the ground state PL scanning spectra were measured at 300 K in highand low-intensity areas of the QD structure. Low-intensity regions at 300 K are characterized by the “blu e” shift of the PL spectrum compared to high intensity areas, as shown in Figure 4.9(b). 1.001.051.101.15 80K(a)5 4 3 2 1 PL intensity [arb units]Emission Energy [ev]0.900.951.001.051.101.15 5 4 3 2 1300K(b) Emission energy [eV]PL intensity [arb units] Figure 4.9 PL spectra measured at 80K (a) and 300K (b) at various PL intensity points on the QD structure #689, (see Fig. 4.8(b)). (~90 W/cm2 power intensity) The PL peak position shifted from 0.98 up to 1.02 eV with a threefold decrease in PL intensity. Maps of the maximum positions and PL intensities at these maxima were plotted from measured spectra in Figs. 4.8(b) and 4.8(c). In correla tion with previously described spectral behavior, low-intensity points (dark areas in Fig. 4.8(b)) are characterized by a higher energy position of the principal maxi mum (white areas in Fig. 4.8(c)). We observe a clear co rrelation between PL intensity and PL maximum measured across entire sample areas on four di fferent QD structures #685, #687, #689 and #698 (Fig. 4.10).

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70 0.940.960.981.001.02 100101 T=300K PL Intensity [arb units]Energy [eV] #685 (490 oC) #687 (510 oC) #689 (525 oC) #698 (535 oC) Figure 4.10 Ground state PL intensity vs. GS maximum position measured at room temperature and 90 W/cm2 power intensity on QD structures grown at different temperatures Spectroscopic PL mapping was performed across the entire sample’s area. In the semi-logarithmic plot of the ground state PL intensity versus GS energy position, this trend can be fitted with a linear dependen ce as presented in Fig. 4.10 Scanning PL study at 80K was performed and compared with ro om-temperature data along the same line scans. The GS PL peak position shifts at 80K to the higher energy by 100 meV due to a band-gap energy increase. Concu rrently, a trend of the PL maximum versus PL intensity at 80 K is inverted compared to the room -temperature data, i.e., higher intensities correspond to higher PL en ergy peaks (Fig. 4.9(a)).

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71 PL mapping of the ground state and multiple excited states was performed at 80 K at the excitation power density of 650 W/cm2. For PL spectra measured at high excitation, the three lowest energy PL bands (GS, 1ES, 2ES) were well resolved, which allows us to perform spatially resolved mapping of their peak positions across the sample. Figure 4.11 presents the variation of GS, 1ES, and 2ES energies versus corresponding GS maximum. Figure 4.11 Experimental peak positions for the GS, first, and s econd ES vs. GS peak energy measured at 80 K and excita tion power intensity of 650 W/cm2 The energy separations vary across the sample. These separations are 55.5 (GS1ES) and 45.0 (2ES-1ES) meV for the low ener gy GS optical transition at 1.090 eV and it decreases monotonically to 50.9 (GS-1ES) and 31.5 (2ES-1ES ) meV for the high-energy GS optical transition at 1.129 eV. GS Peak energy [eV] PL peak energy [eV]

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72 The long-range GS PL intensity varia tion across the QD structure, which is accompanied by the PL peak shift at 300K (Figs. 4.8, 4.9(b), and 4.10), is presented. In our cladding layer investigations (see S ection 4.1.2) it’s shown that there are two mechanisms exhibiting PL intensity spatial va riation in the InAs QD structures. The first is attributed to inhomogeneous distribution of non radiative (NR) defects across the QD structures. In this case, scanning PL inte nsity variation was not accompanied by a PL spectral shift. The second mechanism is re lated to QD parameter changes along the area of high quality QDs. In the last case, the PL intensity variation is accompanied by a shift of the PL maximum. Our results show that in the investigated InAs QD structures the second mechanism of PL inhomogeneity along the structure area is present. The ground state PL intensity (IPL) is directly proportional to the excitation light power density and the internal quantum efficiency, which can be presented as = R/ ( R+ NR), where R and NR are radiative recombina tion (R) and non-radiative recombination (NR) rates, respectively. From the GS PL temperature dependence (Fig. 4.7) it follows that RNR at 300 K due to thermal quenching of the GS radiative recombination. In this case, the value can be substituted by the following: = R/ NR. For a QD ensemble the emission rate is given as: ) (1 R p i e i N i Rf fD (10) where e if and p if are the occupation proba bilities for electrons and holes at ground state levels given by the Fermi-Dirac distribution functions: 1 ,1 / exp kT E f fp n p n p e (11)

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73 where n,p are the quasi-Fermi-levels for the con duction and valence bands, respectively, measured from the QD band edges, En,p are the quantized energy le vels of an electron and a hole in the conduction and valence bands of the QD, measured from the QD band edges, ND is QD density, and R is the electron-hole radiative recombination time [122]. At low excitation light intensity (90 W/cm2), used during GS PL scanning, which is well below the GS saturation intensity, we can present the occupati on probabilities using Maxwell-Boltzmann distribution functions kT E f fp n p n p e/ exp ,, (12) Taking into account that excitation light power is not changed during the GS PL scanning experiment, we can assume that n,p are constant along the scanning line and the PL intensity variation occurs due to parameters the En,p only. In this case, fe exp( En/kT) and fp exp( Ep/kT). The energy levels En,p can be presented as En=Ece locE and Ep=Evp locE, where Ec,v are the conduction and valence-band offsets at the QDnarrow-gap region heteroboundary, measur ed from the QD band edges, and e locE and p locE are the binding energies of the electron and hole located at the GS levels. We assumed that the values of Ec,v and the QD density, ND, do not change signif icantly along the PL scanning line and as a result the variation of fe,p in the PL scanning experiment can be presented as fe exp( e locE/kT) and fp exp( p locE/kT). In this case, the GS radiative emission rate is changing along the PL sca nning line at room temperature as follows: kT E h kT E kT E E kT E Eex bin GS QD GS QD GS InGaAs GS p loc e loc R maxexp exp exp exp (13)

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74 where InGaAs GSE is the energy gap between the GS electron-hole levels in the narrow-gap In0.15Ga0.85As layer and QD GSE is the energy gap for the GS electron-hole levels in the QD. Here we are taking into account that the GS optical transition energy (GShmax) is the difference between QD GSE and the exciton binding energy ex binE in the QDs. Exciton binding energies were computed as a function of QD size using the eight-b and kp approach and are estimated as 19.5 and 22.5 meV for QDs with a base size of 16 and 13.4 nm, respectively [123]. Thus ex binE value changes ( ex binE 2 meV) versus QD parameters are small in comparison with GS optical transition energy variation (GShmax 1.09–1.25 eV) (Fig. 4.11). Finally, we could ob tain scanning PL intensity va riation at room temperature very close to the dependence IPL exp( GShmax/kT), as is demonstrated by the fitting line in Fig. 4.10. The long-range variation of QD electron and hole localiz ation (binding) energies across the sample area in general can be attributed to the following: QD size changes as a result of inhomogeneous temperature fields across the wafer or InAs layer thickne ss inhomogeneity during QD growth, elastic stress variation across the sample due to layer composition variation We suppose that the decrease of GS elect ron-hole binding energies across the scanning area is the result of the long-range varia tion of an average dot size in the QD ensemble from the periphery toward the sample center. This is exhibited as the “blue” shift of the PL maximum at 300 K (Fig. 4.9(b)). This effect lead s, at the sample center, to shallower QD localized states (i.e., smalle st electron and hole bi nding energies) and a

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75 higher probability of the carrier thermal escape, which reduces their room-temperature PL intensity. To confirm our assumption and to study this effect in more detail, we performed scanning luminescence at 80 K along the same line scans as for 300 K. For the low temperature PL scanning measurement, we avoid carrier thermal escape process from the QDs. As we mentioned above, a trend of the PL maximum versus PL intensity at 80 K is inverted compared to the room-temperature data. The two-fold rise of PL intensity corresponds to a shift of the PL peak to higher en ergies from 1.05 up to 1.09 eV (Fig. 4.9(a)). 4.1.3. Cladding layer composition invest igation experimental results The cladding layer composition study was performed for four different concentrations of In in the InxGa1-xAs layer. In Fig. 4.12 are s hown two maps of the PL intensity measured on samples 1360 (x=0.15) and 1361 (x=0.1) at the energy of the principle PL maxima, 1.020 and 1.044 eV, re spectively. White contrast on the maps represents higher PL intensity. Besides indivi dual dark regions, which were caused by the sample holders in the MBE system, we observe on both samples a gradual up to five fold reduction of the PL intensity from the wafe r center toward the periphery. These samples were parts of larger 50 mm diameter wafers.

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76 Figure 4.12 PL intensity mapping at room te mperature performed at 1.02 eV on sample #1360 (a) and at 1.044 eV on sample #1361 (b) Shading bars represent the PL intensity variation in both samples. Arrows indicate orientation of the maximum PL intensity gradient from the water center to the periphery Arrows drawn on the maps exhibit line-s cans of the data points with the most significant PL variation, where the follo wing spectroscopic and temperature study was performed. In Figure 4.13 room-temperature PL spect ra are presented m easured along these line-scans in both samples, which show quite different features. Specifically, in sample #1360 a principal PL maximum maintains the spectral position at 1.020 eV within the band of 10 meV, while reducing the PL inte nsity by more than a factor of two. In contrast, sample 1361 exhibits a gradual “blue” ener gy shift of the maximum starting at the wafer’s center at 1.044 eV and approach ing 1.110 eV at the periphery, which matches a threefold degradation of the PL intensity. These observations are statistically confirmed PL Intensity [arb. units]

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77 by measuring the PL spectra across the entire sample area as illustrated in Figure 4.14 for a set of 115 data points measured in the scanning mode. 0.951.001.051.101.151.20 0.0 0.2 0.4 0.6 0.8 1.0 (2) (a)(1)PL intensity [arb.units]Energy [eV] 0.951.001.051.101.151.20 0.0 0.2 0.4 0.6 0.8 1.0 (2) (1)(b)PL intensity [arb.units]Energy [eV] Figure 4.13 PL spectra at room temperature m easured at different spots along the arrows in Fig. 4.12 on samples 1360 (a) and 1361 (b). Curve (1) corresponds to the central area of the samples; curv e (2) corresponds to the periphery It was noticed that the lu minescence in sample #1360 ha s, at room temperature, the highest intensity averaged across the wa fer compared to samples 1361 and 1363; 3.2 times and 1.2 times, respectively. Sample #1360 shows also the narrowest half width of the PL maximum of 37–42 me V (Fig. 4.13) compared to 73–79 in #1361. It exhibits an additional peak at 1.08 eV, which was prev iously observed on high-quality QD samples and attributed to the lumine scence through the excited states of the holes in QD [112, 113]. These facts indicate that the electronic quality of the QD structure is superior in sample #1360 with an x=0.15 composition of the cap layer. It is worth noticing that sample #1360 also partially shows features of sample #1361 at the low PL intensity periphery region. This is illustrated in Fig. 4.14 as data points with a “blue” shift of the maximum accompanied by PL degradation. These data points in both samples have

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78 identical slope in the PL maximum versus PL intensity dependence (see the dashed lines in Figure 4.14). 1.001.041.081.12 5 10 15 20 #1360 (15% In) #1361 (10% In) PL intensity [arb.units]PL maximum [eV] Figure 4.14 PL intensity vs. PL maximum depe ndencies measured at room temperature on 155 points across the entire wafer as shown in Fig. 4.12 We can interpret the spectroscopic PL mapping results on the InAs/InGsAs QDs grown with different composition of the cap InGaAs layer, as two separate physical mechanisms realized in various samples. In sample #1361, the size of the dots decreases gradually from the center of the wafer toward the sample periphery. This is exhibited as a “blue” shift of the PL maximum. This effect leads at the wa fer periphery to shallower QD localized states (i.e., smallest electr on and hole binding energy), poorer carrier localization and, as a consequence, a higher probability of carrier thermal ionization, which reduces their room-temperature PL intensity. It is possible also, that in the smaller quantum dots, carrier wave functions penetr ate more into the barriers surrounding the dot

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79 and overlap more with nonradiativ e point defects. In this case the intensity of the PL for small QDs should decrease faster with incr easing temperature. Regarding sample #1360, the maximum position of the 1.02 eV PL band is maintained, and the drop of the PL intensity can be rela ted to an inhomogeneous distri bution of nonradiative centers, competing with QD luminescent transitions. We notice again that the first mechanism is also evident in this sample in areas with lo w PL intensity. To confir m and explore each of the mechanisms in more detail, we performed scanning luminescence at 80 K along the same line-scans as in Fig. 4.12. In sample #1360, we again obs erve a constant PL peak position, which is now shifted to the higher energy by 80 meV due to the band-gap increase. For sample #1361, the PL peak positio n shifts at 80 K to higher energy by 110 meV. At the same time, a trend of the PL maximum versus PL in tensity at 80 K is inverted compared to the room-temperature data, i.e., higher intensities correspond to higher PL peak energy (Fig 4.15).

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801.041.061.081.101.141.161.18 0.0 0.2 0.4 0.6 0.8 1.0 PL Intensity [arb units]Energy [eV] 300K 80K Figure 4.15 Distribution of PL intensity maxima at 80K and 300K for the sample #1361. Note the PL intensity increase for 80K with the rising p eak maxima energy, and opposite decrease of PL with peak maxima increase for 300K This experiment confirms the model of the QD size distribution in sample #1361. In fact, the high-energy PL bands corres pond to the smallest size QDs, where the electron–hole wave functions strongly overlap and, due to this, the matrix element for optical recombination transitions is relativel y large. The opposite is valid for low-energy PL bands and large QD size. At 80 K, when the thermal ionization of the QD levels is negligible, this factor governs the high PL inte nsity of the small-size dots. At elevated temperatures PL of the small QDs is quenche d, first due to thermal ionization of trapped carriers followed by PL quenching of the large QD emission. We extracted activation energies of the PL temperature quenching fr om 80 to 300 K in both samples from both the high and low PL intensity regions. Th ese values fall in th e range of 260–280 meV in

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81 all samples, which is larger than the electron–hole binding energy in the QD, which typically ranges from 50 to 100 meV [112, 113] This large thermal activation energy was attributed to nonradiative recombination cente rs. Earlier it was show n that increasing the In content in the InxGa1 xAs capping layer changes the en ergy of the QD ground state, which was revealed by the PL peak shifts from 1.03 to 0.95 eV without changing its full width at half maximum (FWHM) [124]. In our experiment the PL peak also shifts to lower energy with increasing the parameter x both in high PL intensity area (from 1.04 to 1.01 eV) and in low PL intensity area (from 1.10 to 1.01 eV) in all structures. But we have also seen the change of the FWHM for the ground state PL line as a f unction of the parameter x. 4.1.4. Spatial PL inhomogeneity of full size QD wafers – experimental results PL intensity maps of the 50 mm diam eter InAs/InGaAs wafer are shown in Figures 4.16 through 4.19. They were measured on samples with InxGa1-xAs composition in capping layer equal to x = 0.10, 0.15, 0.20 and 0.25 and at the energy of the principal PL maximum, which are 1.008, 1.010, 1.035 and 1.06 eV, respectively.

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82 Figure 4.16 PL intensity mapping at room temperature performed at 1.008 eV on QD structure #1718 with In/Ga compos ition x = 0.10, Mapping step 0.2 mm Figure 4.17 PL intensity mapping at room temperature performed at 1.010 eV on QD structure #1719 with In/Ga compos ition x = 0.15, Mapping step 0.5 mm

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83 Figure 4.18 PL intensity mapping at room temperature performed at 1.035 eV on QD structure #1720 with In/Ga compos ition x = 0.20, Mapping step 0.5 mm Figure 4.19 PL intensity mapping at room temperature performed at 1.06 eV on QD structure #1721 with In/Ga compos ition x = 0.25, Mapping step 0.5 mm

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84 We observe on all samples a gradual reductio n of the PL intensity from the wafer center towards the periphery. This effect wa s confirmed by correcting the intensity maps on the shift of the PL maximum from point to point (intensity change is not connected with spectral shift). It was expected that the PL intensity of the QD structure with InxGa1xAs composition in capping layer of x = 0.15 (#1719) was much higher in comparison with the x = 0.25 (#1721) structure. The average PL peak intensities over the whole sample area for all four investigated QD structures are shown in Fig. 4.20. 0 50 100 150 200 250 10152025 Indium composition [%]Averaged PL intensity [arb units] Figure 4.20 The average PL peak intensities for all four investigated QD structures Indium composition [%] Averaged PL intensity [arb units]

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85 Figure 4.21 Typical normalized PL spectra corresponding to the spot s with highest PL intensities on four investigated InxGa(1-x)As QD structures with variable In composition in the capping layer In Figure 4.21 we present normalized PL spectra for all four investigated QD samples. The structures with x = 0.10 a nd 0.15 are characterize d by a double-band PL spectra with the half-width of the first principal maxi mum of 30–36 meV. These halfwidth values are typical for ground state (GS) emission of high quality QD structures [69, 125].The PL spectrum exhibits an additiona l peak at 1.06 eV, which was previously observed on high-quality QD samples and attr ibuted to the luminescence through the excited states in the QD [112, 113]. With an increase of In composition from x = 0.10 to 0.15 the PL peak shifts into the low en ergy spectral range from 1.015 up to 1.007 eV. Then for QD structures with InxGa1-xAs composition equal to x = 0.20 and 0.25 the x20 x2 x2 In0.25Ga0.75As In0.20Ga0.80As In0.15Ga0.85As In0.10Ga0.90As

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86 direction of the PL peak shift changes a nd the PL peaks shift into the higher energy spectral range up to 1.030 and 1.054 eV, respectively. With an increase of In/Ga compositi on in the capping layer (x = 0.20 and 0.25) the shape of the PL spectrum changes as we ll and the PL intensity falls off (Figs. 4.20 and 4.21). The half-width of the PL band in creases up to 80 meV wh ich indicates higher QD size dispersion for the structure with x = 0.25 (Fig. 4.21). We conclude that the electronic quality of the QD structure is supe rior in the sample with x = 0.15 composition of the capping layer. Room temperature PL spectra for sample x=0.15 (#1719) measured at different intensity points are shown in Figure 40.The Principal PL maximum exhibits a gradual ‘‘blue’’ energy shift of the maximum starti ng at the wafers center at 1.007 eV and approaching 1.037 eV at the periphery, which ma tches a two-fold degr adation of the PL intensity.

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870.951.001.051.101.151.20 0.0 0.2 0.4 0.6 0.8 1.0 300KPL intensity [arb units]Energy [eV] Figure 4.22 PL spectra at room temperature m easured at different in tensity spots (# 1719 In content x = 15%) 4.1.5. Spatial PL inhomogeneity of full size QD wafers – discussion As we have shown earlier [section 4.1.3] the scanning PL results on the InAs/InGaAs QD structures indicate two sepa rate physical mechanisms of PL intensity inhomogeneity. The first mechanism is not accom panied by a PL spectrum shift. In this case a drop of the PL intensity can be relate d to an inhomogeneous distribution of the QD density across the wafer and (or) of the dens ity of nonradiative centers, competing with QD luminescent transitions. The second mech anism of the PL intensity change is accompanied by a PL spectrum shift and deals with the QD size variation along the QD structure.

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88 We interpret the spectroscopic PL ma pping on the InAs/InGaAs QD’s 50mm samples grown with different composition of th e capping InGaAs layer as a result of the dot size decreasing from the center of the wafer toward the sample periphery in a similar way to the samples from section 4.1.3. This is exhibited as a ‘‘blue’’ shift of the PL maximum. This effect leads at the wafer peri phery to shallower QD localized states (i.e. smallest electron and hole binding energy), poorer carri er localization and, as a consequence, a higher probabil ity of carrier thermal ioniza tion, which reduces their room temperature PL intensity. It is possible that in the smaller quantum dots, carrier wave functions penetrate more into the barriers surrounding the dot and overlap more with nonradiative point defects. In this case, the intensity of the PL for smaller QDs should decrease faster with increasing temperature. Room temperature PL intensity variation across the wafer can be described by the dependence: kT h IGS PL maxexp (14) Where IPL is the PL intensity, GShmax is the energy of the ground state, k is Boltzmann constant and T is temperature. This is demonstrated by the fitting line in Fig. 4.23.

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891.011.021.031.04 0.5 0.6 0.7 0.8 0.9 1.0 Peak intensity [arb units]Peak energy [eV] Figure 4.23 PL maxima peak position vs. its intensity (#1719 In content x = 15%) Earlier it was shown that increasing the In content in the InxGa1-xAs capping layer (from 0.10 up to 0.30) changes the energy of the QD ground stat e, which was revealed as the PL peak shift from 1.03 to 0.95 eV without variation of the band half-width [124]. The last effect was explained [124] as the result of stress decreasing on the InAs/InGaAs interface. In our experiment fo r QD structures with x = 0. 10 and 0.15 the PL peak shifts by the same way in the low energy spectral si de. But for QD structures with x = 0.20 and 0.25 the PL peak shifts in the opposite dire ction. This difference in the PL spectral behavior may be related to the difference in investigated QD structures. In all our QD structures below the wetting layer there is an In0.15Ga0.85As buffer layer. In Ref. [124] the InAs QD layers were grown directly on GaAs layers. Moreover, th e thickness of capping InxGa1-xAs layers in [124] was decreased with th e parameter x increasing with the aim to prevent approaching of the critical thickness a nd the start of the stress relaxation process

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90 in QD structures [126]. In investigated QD structures the reason for PL peak position shifts into the ‘‘blue’’ side with a variation of InxGa1-xAs composition (x = 0.15, 0.20 and 0.25) can be related to partial InAs QD deco mposition at higher elastic stress conditions and to partial elastic stress relaxation in QD structures with x > 0.15 [127]. In the investigated QD structures the thickness of the InxGa1-xAs capping layers was the same in all structures and equal to 7 nm —the height of the QDs. This InxGa1-xAs thickness for QD structures with x > 0.15 is more than criti cal for the nucleation of misfit dislocations at the InGaAs/GaAs interface [ 126]. Actually this may be th e reason of partial stress relaxation in QD structures for x > 0.15 wi th the appearance of dislocation and nonradiative recombination centers. The later as sumption can be confir med by a decrease in the PL intensity of the QD structures with the parameter x equal to 0.20 and 0.25 presented in Fig. 4.18 and 4.19. Note we made the assumption above that the density of QDs along the line that we measured the spect ra on the wafer does not change essentially and the main reason for the PL intensity ch ange is the QD size dist ribution. Actually, as was shown in Ref. [128] for the AFM investigation of the same type of uncapped InAs QDs grown on the In0.15Ga0.85As buffer layer, the density of QDs across the wafer changes not more than 50%. It is really not an essential variation in comparison with the observed twoto four-fold magnitude variat ion in the PL intensity signal at room temperature.

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91 4.2. PL spectroscopy of CdSe/ZnS quant um dots for bioapplications 4.2.1. Photo-induced enhancement of th e PL intensity – introduction The stability and efficiency of QD luminescence is a critical aspect for the technology. Under intense laser or electron be am illumination, a strong degradation of the luminescence intensity was observed and at tributed to the photo-ionization of nanocrystals and subsequent tr apping of the ejected el ectrons in the surrounding semiconductor matrix [87]. Therefore, even in capped QDs with wide-gap semiconductors as the barrier or those em bedded into a ZnS matrix, photo-degradation occurs. On the other hand, it was documente d previously that the luminescence and photocurrent intensity are increased under lig ht illumination in the bulk CdS and CdSe crystals [71, 129]. This was identified as light-induced defect reactions caused by donor– acceptor pair dissociation, assigned to a photo-chemical process. A similar effect of photo-induced PL enhancement was noted in th e glassy close-packed films of CdSe QDs covered with a ZnS film [87]; however, the process was not studied in detail. A reversible PL intensity photo-enhancement (‘photobri ghtening’) was also observed at low temperatures in CuI and CuBr nano-crystals embedded into a glass or polymer matrix and attributed to photo-generated carrier trapping and their rele ase when the temperature was increased [130, 131]. The experimental study of the PL photo-enhancement effect in pure and bio-conjugated CdSe/ZnS core-shell quantum dots was performed. While we specifically focus on the pure QDs the light-induced PL enhan cement is also observed in bio-conjugated samples.

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92 4.2.2. Photo-induced enhancement of the PL intensity – experimental results PL spectroscopy was performed between 80 K and room temperature using a 50 mW HeCd laser line at 325 nm and a 200 mW Ar+ laser line at 488 nm as the excitation source. Laser power density varied by use of calibrated neutral density filters and was focused down to a 100 m spot. For low intensity measurements the laser beam was unfocused with an approximately 1.5 mm lase r spot diameter at the sample surface. Commercially available CdSe/ZnS polymer coated quantum dots from Quantum Dot Corp. [11] were used in this experime nt. A sample of the Qdot 655 Goat F(ab’)2 antiMouse IgG conjugate in a form of a mm-size spot was dried on a polished surface of a crystalline silicon substrate to achieve a lo w level of scattered light. One dried spot contained 2 l of QD bio-conjugate diluted with a phosphate buffer (PBS) in a 1:50 volume ratio. Bio-conjugated samples contained Qdot 655 F(ab’)2 complex fragments conjugated to OC125 detector antibodies th at recognize the CA125 anti-gene molecule, used in the early stage detection of ovarian cancer. Some experiments were done on the QD F(ab’)2 OC125 bioconjugate structure before attachment to the CA125 anti-gene molecule. The PL spectrum of the CdSe/ZnS quantum dots in the range of 0.73–3.54 eV (350–1700 nm) exhibits only one prominent lumi nescent band with the maximum at 1.89 eV (655 nm) and half-width of 90 me V at room temperature (Figure 4.24).

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931.851.901.952.002.05 0.0 0.2 0.4 0.6 0.8 1.0 300K 210K 80K 140KPL intensity [arb. units]Energy [eV] Figure 4.24 PL spectra of CdSe/ZnS QDs meas ured at different temperatures: 80 K, 140 K, 210 K and 300 K. Note the blue shift as the sample temperature decreases When the temperature is decreased, the PL maximum shows a narrowing and ‘blue’ shift following the temperature band-gap variation of the bulk CdSe, which is described in Figure 4.25 by a solid line using the Varshni equation [132]. ) ( ) 0 ( ) (2 T T E T Eg g (15)

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9450100150200250300 1.90 1.91 1.92 1.93 1.94 1.95 1.96 1.97 1.98 Varshni parameters: Eg(0) = 1.9845 = 0.00081 = 567.2h [eV]Temp [K] Figure 4.25 PL maximum versus temperature variation (points) and fitting with the Varshni equation (solid line) us ing parameters: Eg(0) = 1.9845 eV, = 0.00081, = 567.2 We concentrate here on the transient char acteristic of the luminescence which is exhibited as a variation of the PL intensit y versus sample exposure with the excitation laser. To explore the transient, the kinetic curves of the PL intensity versus exposure time, I(t), are measured at different power de nsities of the 325 nm HeCd laser excitation. In Figure 4.26, we present luminescence kinetics of the QDs diluted in the 1:50 ratio in PBS measured at the maximum of the PL spectrum of 655 nm.

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95 Figure 4.26 PL intensity variation of the 655 nm luminescence band at room temperature for different laser power densities (W-cm 2): (1) 500; (2) 20, (3) 0.2. The PL intensity is normalized using a multiplication factor of 25 in curve (2) and 2500 in curve (3) At the highest laser power density of 500 W cm 2 (laser spot focused down to 100 m), we observe a strong photo-quenching, when the luminescence intensity degrades by a factor of 3 from its maximum value within 15 min (curve 1). At lower excitation power, it is recognized that this photo-quenching follo ws an initial PL increase, hereafter photoenhancement, as shown on curve (2). When th e laser power density is reduced further, the enhancement kinetic starts to dominate quenching, and with laser power of a few W cm 2 it is possible to clearly separate the enhancement part as illustrated by curve (3). We explore in this study the enhancement kineti c only, which is strongly motivated as a potential means to improve the quantum ef ficiency of the bi oconjugated QDs. The following observations were depicted on th e basis of the transient PL study. (1) PL

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96 photo-enhancement amplitude can be quite substantial, spanning the range from 10% up to fivefold with respect to the initial lumi nescence intensity. (2) The enhancement effect is observed both at 325 nm (H eCd) and 488 nm (Ar+) laser excitation. The enhancement rate is increased (time constant reduced) at hi gher excitation power density as illustrated in Figure 4.26. (3) If the sample subjected to UV exposure was held in the dark for a definite time, the enhancement effect can either be recovered, which is assigned to reversible enhancement (RE) and the kinetics can be repeated again, or the effect can exhibit the non-reversible enhancement (NRE) and show no recovery at room temperature for at least overnight sample storage. Typically RE and NRE occur simultaneously. Both RE and NRE kinetics are thermally activated, meaning that they are substantially slowed down when the temper ature decreases. Specifically, the RE time constant ( RE) yields tens of minutes at 300 K a nd its transient kinetic is no longer observed below 240 K. The PL spectrum measur ed at room temperature before and after the enhancement completed shows no noticeable variation of the peak position and the half-width. Cycling the light-induced PL e nhancement and dark recovery leads to a cycle-by-cycle permanent increase of the PL intensity as illustrated in Figure 4.27.

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97 Figure 4.27 (a) Cycling kinetics of light-induced PL enhancement and dark recovery at room temperature; (b) time dependen ce of sample illumination, using 325 nm HeCd laser. Points A, B, C and D illustrate gradual increase of the PL intensity at the beginning of the consecutive enhancement curve. The inset shows two consecutive kinetics revea ling time constants of (1) 9.4 min and (2) 20.6 min Time [min] PL Intensity [arb. units] 250 200 150 100 50 0 0 30 60 90 120 150

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98 This suggests that both RE and NRE occur in parallel. We emphasize the significant features of both processes. Th e NRE amplitude is e xponentially increased with illumination time yielding the time constant of approximately 180 s at room temperature. This is indicated by points A, B, C and D in Figure 4.27. We observed that a time constant of the RE process is graduall y increased from one cycle to the consecutive cycle. Generally, it is complicated to describe RE kinetics with a single exponent at room temperature due to a superposition with the NRE process. A single exponential curve is observed either at the very fi rst PL enhancement when comple te recovery occurs, or after multiple enhancement–recovery–enhancement cycles when the NRE process is completed. The recovery time constant is cl ose to 30 min. It does not change between various cycles and increases at lower temperatures. The QD samples can be classified into two different groups according to the PL transient features. Group 1 samples show neglig ible RE kinetics and the entire transient is controlled by the NRE process. Group 2 sa mples exhibit both RE and NRE kinetics possessing comparable amplitudes. We we re unable to realize samples where RE dominates over NRE. This sample selec tion allowed separating the input of both processes in temperature-dependent lumines cence. Additional insight and complementary data on the photo-enhancement effect yield th e PL intensity temper ature dependences, I(T). They were measured using three differe nt sample states: (F ig. 4.27 point A) the initial state, prior to any PL enhancement, (Fig. 4.27 point B) the RE state, when the reversible process is saturated while the NRE is negligible and (Fig. 4.27 point C) the NRE state, when the non-revers ible effect is completed. To assess the (Fig. 4.27 point A) state, the sample was cooled down to 80 K in the dark. The spectral PL maximum was

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99 acquired within 60 s at a low laser excitation of 20 mW cm-2. After this, the excitation light was turned off until a c onsecutive higher temperature poi nt was stabilized and the PL spectrum sweep was performed again. We intentionally increased the dark holding time between individual temperatures to ensu re that the RE process, which may start during the PL measurement, was diminished. These precautions allowed for minimization of the PL enhancement effect. State (Fig. 4.27 point B) was achieved on group 2 samples after the very first room-temperature PL enhancement is saturated using a fast sample cooling down to 80 K with UV light on. In this case, the relaxation is terminated and the RE effect is not erased. Finally, state (Fig. 4.27 point C) was measured in group 1 samples after long-term ~30 min illumination at room temperature to saturate the NRE processes. Figure 4.28 The PL temperature dependence before and after (a) the NRE process and (b) the RE process. Curves (1) and (3) correspond to the initial state measured on two different groups of sa mples. Curve (2) corresponds to the NRE state, and curve (4) to the RE st ate. Solid lines show linear fit to extract the activation en ergy of the T-quenching

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100 The dependence I(T) before and after comp leting the NRE is presented in figure 4.28(a) by curves 1 and 2, respectively. At the initial (Fig. 4.27 point A) state, the PL intensity decreases or quenches when the temp erature increases from 80 to 300 K. This is attributed to a thermally ac tivated release of photo-genera ted carriers from the CdSe quantum dot levels over the potential barrier a nd recombination at nonr adiative defects in the ZnS, polymer or at interfaces. The Arrhen ius plot, log(I) versus 1/T, reveals the activation energy of PL quenching in the (Fig. 4.27 point A) state A = 33 meV. It is important that above 250 K we observe the PL increase which is attributed to the NRE process. After NRE occurred (curve 2 Fig 4.28(a)), the PL incr eased over the entire temperature range from 80 to 300 K; howev er, the largest changes are observed at temperatures of the steepest quenching in state A, between 180 and 250 K. By completing the NRE (Fig. 4.27 point C state), th e sharp PL quenching range is shifted to higher temperatures while the activ ation energy valu es range from C = 75 to 90 meV (different values in different samples). We may conclude that the photo-stimulated NRE process leads to a noticeable increase of the potential barrier for confined carriers to escape the quantum dot. In Figure 4.28(b), we compare I(T) depe ndences before and after RE occurred. Comparing with NRE curves in Figure 4.28(a), we noted that the region of PL increase region is shifted towards lower temperatures and a minimum of the PL intensity is observed now at 200 K. In this case, however, the PL increase is not related to the RE process, which occurs at higher temperatur es. Above 250 K, the PL is quenched again with the activation energy of 45 meV corresponding to the (Fi g. 4.27 point B) state of the

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101 sample. After RE occurred, the PL enhancemen t is mostly expressed in the range of Tquenching, similar to the NRE case. 4.2.3. Photo-induced enhancement of th e PL intensity discussion When thermally released from the QD levels, photo-generated carriers can annihilate at other recombination centers or can be captured by traps located at interfaces, in the barrier or surrounding polymer shell. When both electrons and holes are released, the competition of other recombination centers dramatically reduces the PL intensity exhibited as temperature quenching (Figure 4.28). The carrier trapping may lead to the appearance or neutralization of the local elec tric field or exhib it Auger recombination leading to PL reduction [87]. The trap rechar ging may cause an increase of the potential barrier height for carriers to be released from the QD levels, which would enhance the PL output. Concurrently, one would expect tim e-dependent processe s for establishing equilibrium of rates between carrier gene ration, recombination and trapping. The PL transient may show a characteristic time much longer than the PL re combination time due to dynamic carrier trapping [133]. This is a ttributed to the fact that photo-generated carrier trapping reduces recombination flow at the QD levels. When majority traps are recharged at low temperatures, the PL intensity approaches a stationary level. This mechanism was proposed to explain PL photobrightening in CuI quantum dots [131]. We note that the mechanism described strongly depends on the ratio of the recombination rate in the QDs ( PL 1) and capture rate at traps ( cap 1). If these rates are comparable, the PL intensity may show a substantial increase during the transient. However, in this case

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102 the transient time is short, on the order of the recombination time in the QDs ( PL). In the opposite case when PL 1 >> cap 1, the PL transient is slow, but as a consequence the PL enhancement effect is negligibly small. Th is simple analysis allows ruling out the transient mechanism of the PL enhancement in our case. The following observations are critical and form a basis to interpret the effect and specify a mechanism of the photostimulated enhancement of the QD luminescence intensity. PL increase occurs after a direct absorption of the 488 nm laser light in the CdSe core rather than in the ZnS she ll or the polymer, that are transparent at this wavelength. Similarly this is true for the 325 nm HeCd laser considering the negligible thickness of the ZnS and polymer transparency at this wavelength. Both processes of RE and NRE are activated in the range of the PL temperature quenching. The activation energy of the PL quenching is increased after the photoenhancement process is completed, ch anging from 33 to 45 meV for RE and 33 to 75–90 meV for NRE. These data allow us to formulate the following statements. The increase of the PL intensity is observed at the temperature ra nge of the PL quenching while the quenching decreases after the PL enhan cement. Both RE and NRE processes are accompanied by an escape of carrier(s) from the quantum dot leve ls. Therefore, the experimentally observed PL increase is caused by a reduction of the carrier flow over pote ntial barriers to nonradiative centers. This reduced flow is a result of the incr eased potential ba rrier for photogenerated carriers to escape the dot and recombin e non-radiatively. It is obvious that

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103 defects and traps outside the well in ZnS layers or at interfaces play an important role in both RE and NRE processes due to the fact th at carriers are released from the QD states. It is known that carrier trapping leads to re charging of the traps and can result in the compensation of the local electric field around the QDs. This may cause a PL increase opposite to the PL quenching under light or elec tron beam irradiation attributed to the Auger recombination that can be realized af ter carrier trapping [ 87]. Alternatively, the light exposure may stimulate photo-chemic al reactions studied widely in II–VI compounds [134, 135]. Therefore, the activation energy of the PL enhancement can be attributed either to the potential barrier in crease for carriers to escape the quantum well or, alternatively, to thermal activation of the photo-chemical react ion. The activation energy can be assessed from the kinetic curves measured at different temperatures. However, our attempts to perform this failed in accuracy due to the superposition of RE and NRE. On the other hand, we can estimate a change of the activation energy of the RE using the following analysis. According to th e experiment (insert in Figure 4.27), the time constant of RE is increased by a factor of 2–6 in different samples after completing NRE. This allows an estimate of the variation of the activation energy i ndependently. The time constant, of a temperature-activated process is related to the activation energy ( ) and absolute temperature as follows: T kB exp0 (16) where kB is the Boltzmann constant. An estimation of increase, = 2 1, can be performed from the ratio of time constants, 1/ 2, of the photo-enhanced kinetics measured at two different states of the sample, as follows:

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104 T k T kB B exp exp2 1 2 1 (17) This estimation yields a value of = 17–45 meV, which is in a reasonable range for the barrier variation from 33 meV to (75–90) meV accessed from the PL temperature quenching curves measured at different sample states presented in figure 4.28. Thus, we conclude that the activation en ergy of the RE process is determined by the height of the barrier for carriers to escape the QD. We can briefly discuss the origin of processes leading to the photo-stimulated increase of th e QD’s potential barrier as illustrated in Figure 4.29. Figure 4.29 Model of the light-induced PL enha ncement illustrating different states of the QD sample: (a) initial state at the beginning of PL kinetics. The potential barrier A is reduced by the electric field generated by charging the electron trap (D+) and hole trap (A ) in the dark; (b) photo-e nhanced state when the luminescence increased due to the recharging of traps according to equation (3); (c) final state after relaxation of the reversible enhanc ement process. It

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105 is suggested that the n on-reversible process is driven by photo-chemical reactions. PL transitions are depicted by the dashed arrows As was mentioned, the processes can be attributed to the recharging of the interface trapping states and cha nge of the local electric fiel d at the QD, or alternatively, to the atomic bond restructuring classified as photo-chemical processes in II–VI compounds. The QD’s surface charge can reduce the exciton PL intensity. This can be a result of the exciton ionization in the exte rnal electric field as observed in the photoconductivity study of close-pack ed glassy solids of colloidal CdSe QDs [136]. In the opposite case, the compensation (n eutralization) of the surfac e charge would lead to the PL increase due to the stabi lization of the exc iton, increasing its binding energy, and reducing the PL thermal quenching (Figure 4.29(b )). We emphasize that if only one type of photo-generated carrier is captured and the other left on the QD level, the Auger mechanism would reduce the PL intensity as was predicted in [137] and observed experimentally in [87]. Theref ore, we postulate that both the electron and hole after being released from the QD states must be capture d by spatially separated and charged donor (D+) and acceptor (A ) defects, correspondingly. This lead s to the neutralization of these defects and reduction of the surf ace charge (i.e. electric fiel d), and as a c onsequence an increase of the exciton PL intensity. The following reactions desc ribe this process: D+ + e D0 ; A + h A0. (18) This is illustrated in Figure 4.29 as the QD energy diagram when the trap-related electric field is active ((a) and (c)) or comp ensated (b). It is conceivable that the RE process is caused by a trap recharging which can be reversed due to a temperature-

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106 activated release of carriers from the trap s (Figure 4.29(c)). Similarly, the NRE effect may also be accounted for by carrier capture at deeper trapping states In this case, the recovery could occur at higher temperat ures. This, however, was not observed experimentally; heating the sample above 320 K leads to a strong PL degradation and eliminates following photo-enhancement. We s uggest tentatively that NRE is linked to a photo-chemical reaction of the bond restru cturing. Alternativel y, the described RE process can also be attributed to a phot o-chemical process of light-induced trap elimination followed by their temperature-activ ated recovery [138]. Experimentally, it was observed that when temperature is below 200 K luminescence degradation is noticeable, caused by the Auger process due to enhanced non-radiative recombination after capture of light-generated carriers by traps [87]. When the temperature increased above 200 K these traps are emptied, which results in a PL increase due to suppression of the Auger process (figure 4.28(b), curve 3). This is evident due to a ‘dip’ at 200 K in the T-dependence. It is important that the dip is completely removed from the T-dependence after RE was performed (curve 4). We can postulate that a reversible photochemical reaction occurs due to light absorption in QDs above 200 K, which leads to the trap elimination. By holding the sample at room temp erature in the dark, recovery of the traps and the dip are seen again.

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107 4.2.4. PL spectroscopy of bio-conjugated QD’s 4.2.4.1. PL mapping of ELISA plates The first step to perform Enzyme-Li nked Immunosorbent Assay ELISA [115] experiments was to establish dilution curves fo r a given marker. In our case we started to dilute pure QDs to determine the limits of de tection for our system. Stock solutions of purchased QDs (non conjugated) were dilute d with the antibody diluting buffer (TRIS Buffered Saline from DAKO Corp.) [139] in the volume ratios in the range from 1:20 down to 1:2000. Figure 4.30 presents the ini tial concentration measurements. CdSe quantum dots are detectable in the dilutio n ratio of 1:2000 on the background of ELISA plates (Flat bottom immuno plate, VWR international) [140]. 1:101:501:2001:1000101102103104 Log (PL Intensity) [arb units]Dilution ratio Figure 4.30 PL intensity vs. dilution ratio of QD 525 diluted in a Tris (DAKO) buffer [139]

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108 The PL mapping of whole ELISA wells wi th the full immuno reaction shown in Fig 4.31 was performed. Used wells had their surface covered with biotin (biotinolated) in order to attach capture antibodies fo r our detection scheme (see Fig 4.31(a)). Figure 4.31 ELISA imunoreaction attachment “sandwich” diagram for streptavidin coated wells. This scheme was used to attach the QD (fluorescent label) to the biotin-avidin bridge as shown After attaching capture antibodies from the liquid phase, wells were rinsed to remove any unbound capture antibodies (Fig 4.31 (b)). The next step was the addition of known amounts of antigen to prove that conjugation took place. If the conjugation reaction follows the assumed scheme the averaged PL intensity should reflect the different amounts of antigen (Fig 4.31 (c)). The last step was to add labeled (conjugated) quantum dots with the specific detector anti body in order to “see” the presence of bound antigen molecules. (a) (b) (c) (d)

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109 Figure 4.32 Map of four ELISA wells with di fferent concentrations of antigen CA125. (a) 300, (b) 200 (c) 100 and (d) 0 Un its/ml (the unit of measurement for this marker is femtomole per milligram of tumor). Map was collected at 655 nm (1.89 eV), for the OC125 (250 u/ml) – CA125 (antibody – antigen) complex labeled with QD655 quantum dots. Mapping step 0.2 mm Figure 4.32(d) shows the ELISA plate and we ll with zero antigen content which is called the negative control. The purpose of th is control is to prove that there are no residues of labeled detector antibodies left. The absence of antigen in the immuno reaction chain will prevent attaching tagged de tector antibodies to the ELISA wells, what is reflected in the PL intensity map of Fi g 4.32(d). The non uniformity of the PL intensity at the bottom of the wells may be contribute d to wash and drying step artifacts that attribute greatly to the accuracy of this a pproach and has to be taken into account during averaging and background removal in orde r to obtain a usef ul signal (smallest noise/signal ratio). (a) (b) (c) (d) 300U/ml 2 00U/ml 1 00U/ml neg control

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110 101001000 10 100 Area averaged Log (PL intensity) [arb units]CA125 [Units/ml] Figure 4.33 Averaged over the well botto m area PL intensity signal for OC-CA125 complex from maps shown in Figure 4.32 The averaged PL intensity dependence shows a linear dependence of the PL signal vs. antigen concentrati on. This linear part of the standard curve (curve that represents given marker signal PL in our cas e vs. its concentration) shows that we are above the limit of detection for this given marker, since we d on’t see the lowe r non linear part of this curve. We can still decrease this biomarker concentration for this ELISA. A classical standard ELISA curve representing th e optical density or extinction coefficient versus concentration is shown in chapter 3 Fig 3.7. This curv e consists of a linear region when the detection is most ac curate and two saturation region s at very high and very low concentrations establishing limits of detection for a given ELISA.

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111 This experiment was repeated with larger number of samples of different antigen concentration for many times without any success in improving the calibration curve for different antibody-antigen complexes used (IL10, PSA, M11 and CA125). Figure 4.34 shows an example of the ELISA map of 12 we lls at 655 nm that no correlation between antigen concentration and PL intensity signal can be found. Unfortunately the wells that were meant to be negative c ontrols show the highest PL in tensity which allow some to draw a conclusion that the actual QD presence inside the wells has nothing or at least very little to do with the actual bioconjugation and only rand om QD residue is giving a signal. The search for spectral features of bi oconjugation has to be carried on the samples with proven immuno reaction what leads us to another approach for the conjugation confirmation technique, employing a microarra y which is described in next section.

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112 Figure 4.34 (a) Map of ELISA plate compos ition with different amounts of CA125 antigen (at 655 nm); numbers shown a bove each well at the map represents averaged PL intensity in each well, (b) description of the well content 4.2.4.2. Bioconjugation with micro array technique The micro array technique that we have employed is very si milar to the ELISA approach when it comes to the immuno reaction scheme. The major difference is the ( a ) ( b ) PL intensity [arb. units] F E D C B A 2 1

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113 substrate and the method of a ttaching the capture antibody. Th e antibody is printed on the functionalized substrate surface (w ith nitrocellulose or aldehyde s) and it’s confined to a spot of 100 200 m in diameter. A major adva ntage of this approach is that it uses a very small amount of sample (up to a couple of l) and the spots can be printed very densely allowing for many different reacti on schemes to occur over relatively small areas. Wash steps in the same condition for th e whole slide basically eliminate any wash artifacts that were present in previous ELISA ’s. Each spot is printed with the different needle making this process very efficient and fast for different antibodies and their concentrations. A single microscope glass s lide can have up to a couple of hundred spots printed in the single pass of micro array printer. For the micro array experiment we prepared samples by conjugation of nanocrystals to antibody specific to the sele cted marker – IL10 in this case, and the second one by use of streptavidin coated quantum dots and biotinylated detector antibody. The same biotin-avidin bridge was us ed this time to conjugate the QDs to the detector antibody Monoclonal capture (clone JES3-9D7) rat anti-human Interleukin-10 antibody, detector (clone JES3-12G8) rat anti-human Interleukin-10 antibody, and recombinant human Interleukin-10 were pur chased form Serotec (Raleigh, NC) [141]. Microarray nitorcellulose co ated glass slides were purchased from Whatman (Sanford, ME) [142] and a Quantum Dot 655 antibod y conjugation kit was purchased from Invitrogen (Carlsbad, CA) [11]. The conjugatio n of detector rat anti-IL10 antibody to QD655 was performed according to the provided pr otocol [114]. Six arrays of capture rat anti-IL10 antibody at 0.5mg/ml (1:2 dilution in printing buffer (TeleChem International, Sunnyvale, CA)) [143] were printed on nitrocellulose coat ed glass slides After spotting

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114 the slides were placed in a box at a temper ature of 4C overnight. The next morning the slides were rinsed with PBS (Sigma-Aldric h, Germany) [144] and blocked with blocking buffer (Whatman; Sanford, ME) [142] for one hour. After rinsing again with PBS the slides were incubated for 2 hours with hum an IL-10 solutions in PBS at 500 ng/ml (2 arrays), 1000 pg/ml (2 arrays) and 100 pg/ml (2 arrays) and th en rinsed again with PBS. Half of the arrays were incubated with de tector rat anti-IL10 antibody conjugated to QD655 (20nM) and the remaining half with bi otinylated rat anti-IL10 antibody for 1 hour with gentle rocking. Following incubation, th e slides were rinsed with PBS and three arrays, previously incubated with biotinylat ed detector antibody, we re incubated for 30 min with streptavidin QD655 (Invitrogen; Ca rlsbad, CA) [11], diluted (1:50, 20nM) with Tris–Buffered Saline (Dako; Carpinteria, CA) [139]. Slides were fi rst rinsed with PBS then water and centrifuged dry at 2500 rpm for 3 minutes. The slides were then imaged under a fluorescent microscope (Nikon Ec lipse E800) [145] equipped with Qdot655 filters and qualified using custom designed software which utilizes computerized dynamic analysis system (CDAS) for classifyin g microarrays to measure the features on spots: such as area and in tensity profiles [146, 147].

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115 Figure 4.35 Micro array immuno reaction “sandwich” diagram for nitrocellulose coated substrate Figure 4.36 A glass substrate can hold up to c ouple hundred spots of different antibodies or their concentrations. The spots are printed as shown schematically These samples were read by a fluore scence confocal microscope. A mercury lamp was used as the excitation source and a set of band pass filters and a dichroic mirror served as the means to determine the excitation and emission wavelength parameters. For Su b strate (a) Su b strate(b) Su b strate(c) Su b strate (d)

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116 the QD 655 samples an excitation low pass filte r with the cut off wavelength of 460 nm was used and the band pass emission filter of 650 nm with the 20 nm band width. The example of a fluorescent im age is shown in Figure 4.37. Figure 4.37 Example of fluorescent microscope image (mag. 2.5x) of the small part of an array printed from micro array pr inter (QD655 + IL10 complex) with 100 m spot diameter (a), spots description (b) We have also performed a protein microa rray assay with streptavidin coated QD and biotinylated detector antibody to detect human IL10 over the rang e of concentrations from 500 ng/ml to 500 fg/ml. Utilizing the “sandwich” assay technique (Fig 4.35) we have used a nitrocellulose co ated glass substrate, rat anti-human IL10 (IgG1, JES3-9D7) capture antibody, biotinylated rat anti-hum an IL10 (IgG2a, JES3-12G8) detector antibody, recombinant human IL10 and strept avidin coated QD655 as a label (Fig 4.35 (d)). (b) (a)

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117 10-410-310-210-1100101102103101102 ELISA detection limit Log (PL Intensity) [arb units]IL10 concentration [ng/ml] Figure 4.38 Calibration curve of the IL10 antigen concentrat ion detected with the QD 665 label and the micro array technique The recorded PL intensities for the micro array slide showed much more repeatable calibration with over 4 orders of magnitude of the useful linear ra nge. (see Fig 3.7 for the corresponding ELISA concentra tion curve in chapter 3). The same experiments were repeated on a Si substrate for the PL spectroscopic measurements. The Si substrate was used as a very low bac kground for UV excitation of our samples (see section 3.2).

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118 4.2.4.3. Spectral shift of QD luminescen ce caused by bio-conjugation With the proven and repeatable bioconj ugation of quantum dots we performed detailed spectroscopic maps of dried sample spots on a Si surface with larger spot sizes than micro array diameter, around 2 mm. Th e drying process itself introduced some non homogeneity, (Figure 4.39) but this time we investigated the spectroscopic signatures of bioconjugation and the overall sample PL intensity was of secondary importance. Figure 4.39 PL intensity maps of two drops/spots on a Si substrate – (a) conjugated and (b) non-conjugated. Spectra measured at each point of respective map (QD655 + anti-Interleukin10) These maps have spectra measured at ever y single spot, the spatial resolution is 0.2 mm for 2 mm diameter spots which yields approximately 80 spectra per spot. The conjugated spot which contai ns the QD655 + IL10 complex pr oven to be conjugated in the previous section while the non conjugated spot is just a drop of pure quantum dot solution as it was purchased from the manufacturer. (a) (b)

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119620630640650660670680690 Non conjugated Conjugated Wavelength [nm]PL Intensity [arb units]0 30 60 90 120 150 180 210 240 Figure 4.40 Spectra measured on ever y map spot presented in Fig 4.39 Normalized spectra from Fi gure 4.40 are shown in Figure 4.41, it is clearly seen that the conjugated complex ha ve their spectra shifted on average by 4 nm (Figure 4.42) in comparison to non conjugated ones. The shif t is towards a shorter wavelength “blue” shift for conjugated quantum dots. Its existe nce was confirmed with repetition of the conjugation scheme with identical parameters and the “blue” shift was observed in all repeated experiments. We believe the surround ing molecules that are attached to the QD are responsible for this result, although more experiments need to be done to investigate the mechanisms involved.

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120620630640650660670680690700 0.0 0.2 0.4 0.6 0.8 1.0 Non Conjugated ConjugatedPL Intensity [arb units]Wavelength [nm] Figure 4.41 Normalized spectra measured on every map spot presented in Fig 4.39 (single spectra are plotted on top of each other to show shift for the whole group – width of each curve is caused by single spectra spread for given sample)

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121010203040506070 650 652 654 656 658 660 662 664 ~4 nm Maxima PL position [nm]Consecutive spectra number QD 655 conjugated QD 655 non-conjugated Figure 4.42 Peak position for all spectra measured over conjugated and nonconjugated spots. The average peak separatio n “shift” is 4 nm for QD655+IL10 complex vs. pure QD655 The shift was also observed for other QD sizes (different emission wavelength maxima) namely 605 (4 nm in diameter) and 705 (7 nm in diameter) [148]. Experiments were carried out in a similar manner as for the 655 QDs. The spots were dried out on a Si surface and mapped with 0.2 mm spatial resolu tion. Spectra were recorded at each spot and their maxima position and relative shif t vs. non conjugated QDs is presented in Figure 4.43.

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122010203040506070 600 620 640 660 680 700 17 meV 20 meVPL Maxima position [eV]39 meV 5 nm 7 nm 15 nm Consecutive spectra #PL Maxima position [nm]2.05 2.00 1.95 1.90 1.85 1.80 1.75 Figure 4.43 Relative shift of nonconjugated QD’s vs. conjugated to IL10 antibody for 605, 655 and 705 QD’s The spectral shift is different for QDs of different sizes; it is larger for quantum dots of big diameter (see Figure 4.43). The PL intensity kinetics unde r UV light was also measured for these samples, which was quite similar for non-conjugated and conjugated QD samples.

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123 The spectral shift observed on bio-co njugated QDs with antigens may be attributed to the existence of an external local electric field that is imposed by the surrounding biomolecules (Stark effect). Bio-conjugation occurs in most cases by covalently bound bio-molecules attached to the polymer coating layer [148] or through ionic interaction with a linkage molecule [93]. The electric fi eld of the polarized covalent bio-molecule or charged linkage molecule can be strong enough due to close proximity to the QD core of a few nanometers. The electric field has a strong effect on the lower lying hole states of the exciton which has been shown by Klimov and Jankovics [149, 150] and it affects to less extent the electron states. Such a feature of the Star k effect on QD states has strong implications on the op tical properties and influen ces the electron-hole binding energy in the exciton through le vels splitting via the Stark effect. For a quantum dot with the single non degenerate state the Stark effect will result only in an energy level shift. Any change in the overall electr ic field will shift the exciton levels of the dot for optical transitions either though band splitting (shi fting) or decreasing the barrier of the confining potential. For different potential barrier heights disc rete energy levels are also different. The Stark effect was studied in CdSe/ZnS core shell quantum dots embedded into quartz matrix under external electric field [151]. The authors documented a “blue” PL shift of 60 meV of the pr incipal PL peak under external negative bias providing electric field 300 kV/m. This sp ectral shift is close to what was experimentally observed in our study. It is also inte resting that the authors of Re f [151] also observed that the internal electric field actually decreases w ith increasing QD size [86] In our experiment we observed the opposite effect as presented in Fig. 4.43, which can be explained by the enlarged surface area of the larger size quantum dots that allow more bio molecules to

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124 come into contact resulting in a stronger to tal electric field imposed on the QD levels. The labeling factor for our experiments (the amount of biomolecules attached to a single QD) is close to 3 and can be increased in larger QDs as was illustrated in Fig 2.9. 4.3. Summary In conclusion to the first part of this research, we observed in this study that InAs/InGaAs QD structures suffer from an inhomogeneity of QD parameters. Scanning PL spectroscopy revealed two distinctive m echanisms for such an inhomogeneity. The first is a variation of the QD size across th e wafer and the second is attributed to an uneven spatial distribution of nonradiative def ects. Both cases can be carefully tailored by a selection of the QD structural parameters and growth regimes. For the biomedical applications of quant um dots we have two major observations. First is the PL output incr ease of CdSe quantum dot s called photo-stimulated enhancement. Both reversible and non-reversible PL enhancement are observed and documented. The kinetic curves and temp erature dependences suggest that the enhancement effect is attributed to a light-induced increase of the potential barrier for electron–hole escape from the QD levels. Th is increase can be affected by the conjugation of the QDs with bio-molecules a nd can be used to improve the accuracy and sensitivity of the QD luminescence tags. So far, however, this has not been confirmed. Second is that application of QD-probes significantly increased assay sensitivity, particularly for IL-10 concen tration in normal plasma (not detectable with standard ELISA assays). Additional potential increase in the sensitivity is possible due to a

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125 spectral shift that has been attributed to a change in electronic QD structure due to bioconjugation, changing or filtering the sp ectral range of signal collection only for conjugated quantum dots will further increase th e signal to noise ratio. Conclusions from this research and further experiments are de scribed and proposed in the next chapter.

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126 5. Conclusions and Recommendations As the result of this PhD research project, the mechanisms behind the spatial inhomogeneity of InAs/InGaAs QD’s have been established, a photo-enhancement phenomena was observed on CdSe QD’s and a po ssible explanation provided, and finally the existence of a spectral shift in bioc onjugated QD is discovere d and statistically confirmed as a means to detect bioconjugation. It was revealed that the optimal temper ature range for InAs QD growth in InGaAs/GaAs laser structures is 490 510 C. InAs/InGaAs QD structures suffer from an inhomogeneity of QD parameters. Scanning PL spectroscopy revealed two distinctive mechanisms for such an inhomogeneity. The fi rst is a variation of the QD size across the wafer and the second is attributed to an uneven spatial distribution of nonradiative defects. Both cases can be car efully tailored by a selection of the QD structure parameters and growth regimes. Capping layer composition studies show that there is optimal In content for these layers in InAs/InGaAs systems. For the capping layer of InxGa1-xAs it was shown that with a change of the para meter x from 0.10 to 0.25 the ground state PL peak maximum shifts from 1.010 to 1.054 eV. The reason for this PL peak position shift has been attributed to partial InAs QD decomposition at higher elastic stress conditions and to partial stress relaxation in QD structures with x > 0.15. The photoluminescence at 20 K

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127 and scanning PL spectroscopy at 80 and 300 K of the ground and multiple excited states in InAs/InGaAs QDs have been investigated al ong with the influence of the excited state energy trends versus average ground state energy variations (or QD sizes) in the QD ensemble. In the course of this study, the mechanis m of optical instability in CdSe/Zns quantum dots and a model of a possible ex planation was proposed. Photo-stimulated enhancement of the CdSe/ZnS quantum dot luminescence intensity was observed, classified and explained. Both reversible a nd non-reversible PL enha ncements have been documented. The kinetic curves and temp erature dependences suggest that the enhancement effect can be attributed to a light-induced increase of the potential barrier for electron–hole escape from the QD levels. This increase can be affected by the conjugation of the QDs with bio-molecules a nd can be used to improve the accuracy and sensitivity of QD luminescence tags. Finally, it was discovered and statistical ly confirmed that bioconjugation can be spectroscopically identified by the existenc e of the spectral shift in a principal luminescence band. A blue shift was observed for different sizes of quantum dots and different types of biomolecule s attached to them suggesti ng that this feature may be utilized as a bioconjugation marker, althoug h the absolute value of the shift, in connection with bioconjuga tion, is not fully understood.

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128 5.1. Recommendation for further research Further experiments to confirm the proposed photo enhancement model are advised for other QD structures; i.e. with a different shell layer. Different relative barrier heights or other passivation conditions ma y provide additional information about the observed increase in PL intensity. It is also suggested to study other sizes of quantum dots for photo enhancement since weaker locali zed excitons in larger QD may not always be trapped by nearby traps giving additional in sights into the validity of proposed model. It is suggested that the further inve stigation of bioconjugation on the optical properties of quantum dots as fluorescent labels will take into account time resolved PL properties (intensity, maxima position). The in fluence of the surrounding environment on the sample should also be investigated, i.e. the influence of nitrogen/oxygen and even the pressure since part of the presented measur ements were carried out in an evacuated cryostat showing some influence of pressure. The bioconjugation should be performed on a larger set of detector molecules in order to develop a full array of spectral features proving or disproving successful bioconjuga tion. The bioconjugation should also be checked for the usefulness of the created biomarker after successful quantum dot attachment that is detected via spectroscopi c means; the presence of QD may hinder the biological activity of detecting biomolecule wh ich may not be directly connected with the characteristic PL features but must be taken into account.

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129 The results of this work were published in the following journals: 1. M. Dybiec, L. Borkovska, S. Ostapenko, T.V. Torchynska, J. L. Casas Espinola, A. Stintz, K. J. Malloy (2006). "Photolumin escence scanning on InAs/InGaAs quantum dot structures." Applied Surface Science. 2. M. Dybiec, S. Ostapenko, T. V. Torchyns ka and E. Velasquez Losada (2004). "Scanning photoluminescence spectroscopy in InAs/InGaAs quantum-dot structures." Applied Physics Le tters 84(25): 5165-5167. 3. M. Dybiec, S. Ostapenko, T. V. Torchynska, E. Velsquez Losada, P. G. Eliseev, A. Stintz, and K. J. Malloy (2005). "Phot oluminescence mapping on InAs/InGaAs quantum dot structures." Physi ca Status Solidi. (c) 2(8): 2951–2954. 4. N E Korsunska, M. Dybiec, L Z hukov, S Ostapenko and T Zhukov (2005). "Reversible and non-reversible photo-enchan ced luminescence in CdSe/ZnS quantum dots." Semiconductor Science a nd Technology 20(8): 876-881. 5. T. V. Torchynska, M. Dybiec and S. Ostapenko (2005). "Ground and excited state energy trend in InAs/InGaAs quant um dots monitored by scanning photoluminescence spectroscopy." Pysical Review B 72: 195341.

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About the Author Maciej Dybiec received his bachelor de gree in Mechatronics at International Faculty of Engineering of Technical Universi ty of Lodz (Poland). His bachelor diploma work was dedicated to inves tigation of diamond coatings fo r biomedical applications. In January 2000, Maciej jo ined the Master’s program in Biomedical Enginneering department of Technical Univer sity of Lodz doing most of his thesis research on the scholarship at Forschungszentrum Fr Mikros trukturtechnik at Univ ersity of Wuppertal (Germany). He successfully defended his thesis “Plasma electron density measurements by the microwave resonance cavity technique” in September 2002. In January of 2003 he join ed research group of Pr of. Sergei Ostapenko at University of South Florida to study the phot oluminescence properties of quantum dots as a Ph.D. candidate. His Ph.D. work was accomp lished in September 2006 and in October 2006 Maciej defended his dissertation. Now he works for Semiconductor Diagnostics, Inc. in the research and development department located in Tampa, FL.