Semiconductor Quantum Dots for Biological Applications

CHAPTER 26 Semiconductor Quantum Dots for Biological Applications Beate S. Santos,1,3 Patrícia M.A. Farias,2,3 and Adriana Fontes2,3 1

Departamento de Ciências Farmacêuticas – CCS – Universidade Federal de Pernambuco-Recife – PE, Brazil, CEP: 50670-901; 2 Departamento de Biofisica e Radiobilogia – CCB – Universidade Federal de Pernambuco-Recife – PE, Brazil, CEP: 50670-901; 3 Research group on Nanostructures and Biological Interfaces (NIB)

26.1 Introduction

Nanophotonics, the fusion of biophotonics and nanotechnology, is an emerging multidisciplinary field that describes nanoscale optical science and technology. It deals with the interaction of light with matter in the nanometre-size scale. Nanomaterials constitute a major area of nanophotonics. Materials can be scaled down for many orders of magnitude, from macroscopicto nanoscopic-size scale. For many semiconductor materials, the nanometric-size region confers huge alterations in some physical–chemical properties, if compared with the properties of the same material in the macroscopic-size scale. By manipulating size and structure of nanometrical semiconductor particles, one can, for instance, perform changes in their optical properties. These unique characteristics of semiconductor nanoparticles occur due to the fact that they are in the quantum confinement regime. These nanoparticles are called quantum dots (QDs). QDs are semiconductor three-dimensional nanoparticles, with typical dimensions ranging from nanometres to tens of nanometres. A QD is often described as an artificial atom because the electron is dimensionally confined just like in a real atom and similarly it shows only discrete energy levels. The energy levels of QDs can be probed by optical spectroscopy techniques as well as in atoms. In other words, it is possible to excite these semiconductor nanocrystals, generate optical signals and use their optical properties. For example, it is possible to use QDs to generate fluorescence. On the other hand, in contrast to atoms, the energy spectrum of a QD can be engineered by controlling size, shape and strength of the confinement potential. QDs of the same material, but with different sizes, can emit light of different colours. As energy is related to wavelength (or colour), this means that the optical properties of the particle can be finely tuned depending on its size. Thus, particles can be produced in order to emit or absorb specific wavelengths, merely by controlling their size. The larger the dot, the more towards to the red end (lower energy) of the spectrum is its corresponding light emission. The smaller the dot, the more towards to the blue end (higher energy) it is. The light emission is directly related to the energy levels of the QD. Quantitatively speaking, the band gap energy that determines the energy of the fluorescent light is inversely proportional to the square of the size of the quantum dot. Larger quantum dots have more energy levels which are more closely spaced. The ability to change optical properties by tuning the size of the quantum dot is advantageous for many applications. Basically, for this reason, quantum dots have found applications for solar

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cells [1], diodes and lasers [2], detectors [3], switches [4], telecommunication [5] and also as fluorescent biological labels [6, 7]. Although the first works using quantum dots of CdS and CdSe in telecommunications arose in the 1980s, the first biological applications of quantum dots as fluorescent labels for biological application was first reported in literature in 1998 simultaneously by Bruchez et al. [8] and Chan et al. [9]. Both groups used CdSe quantum dots coated with silica and mercapto-acetic acid layers, respectively. These layers constitute the functionalization shell, which was labelled by covalent coupling of specific biomolecules to the QDs’ surface. This reaction step is called bioconjugation, resulting in QD bioconjugates, which are ready to interact with biological systems. Subsequently, many authors reported the labelling of whole cells and tissue sections, using several different experimental procedures for modification of the QDs’ surface [10–12]. Since the first results reported in 1998, the use of semiconductor quantum dots for biological purposes has increased exponentially. Currently, fluorescent semiconductor quantum dots became a promising class of materials in the labelling of biological systems, as well as in the diagnostics of pathologies, such as different kinds of cancer [13]. Fluorescent semiconductor quantum dots have been used mainly for bioimaging, using conventional or confocal fluorescence microscopy to study cellular biology and also for optical diagnostics. However, they can also be used in any experiment that is conventionally performed with fluorescent dyes, such as: flow cytometry, fluorescence resonance energy transfer (FRET) and fluorescence lifetime measurements (FLIM), as well as in two-photon/multiphoton microscopy [14, 15]. Biological applications of fluorescent semiconductor quantum dots will be further detailed. One of the fundamental goals in biology is the comprehension of the complex spatio-temporal interplay of biomolecules from the cellular to the integrative level. To study these interactions, researchers commonly use fluorescent labelling for both in vivo/in vitro cellular or tissular imaging and assay detection. Fluorescence provides a unique method for the investigation of basic physical properties of biological structures. The high sensitivity of fluorescence, combined with the advances in measurement techniques, permits detection of ultra-small quantities of specific compounds present in biological systems. In modern biological analysis, many kinds of fluorescent organic dyes are used. However, with each passing year, more flexibility is being required of these dyes, and the traditional dyes are simply unable to meet the necessary standards at times. The intrinsic photophysical properties of organic and genetically encoded fluorophores, which generally have broad absorption/emission profiles and low photobleaching thresholds, have limited their effectiveness in long-term imaging and “multiplexing” (simultaneous detection of multiple signals) without complex instrumentation and processing. To this end, quantum dots have quickly filled in the role and showed that their unique properties could overcome these issues and therefore be superior to traditional organic dyes on several counts. Compared to organic fluorophores, the major advantages of interest to biologists offered by fluorescent semiconductor quantum dots are the following: (a) Narrower emission bands compared to organic dyes, symmetric luminescence spectra (full width at half-maximum 25–40 nm) spanning the UV to near-infrared (as exemplified in Fig. 26.1) and large effective Stokes shifts. Thus, the complication in simultaneous quantitative multiplexing detection, posed by cross-talks between different detection channels from spectral overlap, is significantly reduced. (b) Longer emission lifetimes (hundreds of nanoseconds) compared to organic fluorophores, thus allowing, for example, one to utilize time-gated detection to suppress autofluorescence, which has a considerably shorter lifetime. (c) Higher brightness (due to the high-fluorescence quantum yields) as well as photostability (in general, no photodegradation is observed for a time interval of many hours) which leads to a high resistance to photobleaching (1000 smaller than in organic dyes) and exceptional resistance to photo- and chemical degradation (Fig. 26.2). As a consequence, single QDs can easily be detected in living cells, and their localization can be monitored over minutes to days. (d) Compared with molecular dyes, two properties in particular stand out: the unparalleled ability to tune fluorescent emission spectra as a function of the nanoparticle’s size and the broad excitation spectra, which allow excitation of mixed quantum dot populations at a

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Fluorescent organic dyes

400

Emission

500 600 Wavelength (nm)

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400

Emission Excitation

500 600 Wavelength (nm)

700

Comparative excitation and emission spectra of quantum dots and fluorescent organic dyes.

Emission intensity

Figure 26.1

Intensity

Intensity

Excitation

Wavelength

Figure 26.2 Fluorescent emission as a function of size of the quantum dots. The larger the dot, the more towards to the red end of the spectrum is its corresponding light emission.

single wavelength far removed (100 nm) from their respective emissions bands (refer to Fig. 26.1). (e) Larger two-photon cross-sections allowing, for example, in vivo imaging at greater depths [16]. Quantum dots used in biological applications are exclusively obtained by colloidal synthesis methodologies, which result in colloidal suspensions, in which the solid nanocrystals are dispersed into a liquid phase. Several energetic parameters have to be taken into account in order to control the growth of the particles, as well as to maintain the stability, and the conditions to make them feasible for biological labelling. The major problems and challenges in the use of quantum dots in biological systems are: (a) Surface-induced quenching of emission efficiency due to the high surface area of the nanocrystals. This requires surface passivation by encapsulation or by using core–shell techniques, as will be discussed in the next sections. (b) To be able to prepare quantum dots of different sizes, in order to have distinct emission spectra and to be able to control their size dispersion, a parameter closely related to the width of the emission spectra. (c) Dispersibility of the quantum dots in biological media. The nanocrystals need to be biologically compatible, either lipid soluble or mainly water soluble. Some researchers ascribe the use of silica layer encapsulation [8], others use synthetic routes in aqueous medium which produce quantum dots with hydrophilic surfaces [13, 17].

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(d) The colloidal dot suspensions also need to be isotonic and in physiological pH media to be compatible with the intra-cellular/tissular medium. (e) Labelling and functionalizing the quantum dots to target specific cells and other biological systems. This step consists in a great challenge for this area. (f) The quantum dots in general, bearing from 4000 to 6000 atoms, are much bigger than the usual dyes, so they have to be internalized in the biological systems using specific ways. All these boundary conditions make the application of semiconductor quantum dots for biological labelling purposes an emerging and important field which demands a lot of effort towards interdisciplinary expertise, and promises to have a potential impact for novel nanomedical applications on disease diagnosis, therapy and prevention, which can lead to fundamental changes in health care now and in the future. In the next sections there will be presented and discussed a short review of the experimental procedures which yield water-soluble fluorescent quantum dots. There will also be presented some applications of these QDs as biological labels as well as potential tools for cancer diagnostics.

26.2 Creating a fluorescent biolabel out of semiconductor nanocrystals

For an optimal performance in biological imaging (and competitive to the commercial organic fluorophores), semiconductor quantum dots are being developed in order to optimize their luminescent, surface and chemical stability properties. These conditions result in a very complex multilayered chemical assembly where the nanocrystal core determines its emission colour, the passivation shell determines its brightness and photostability and the organic capping determines its stability and functionality. A schematic representation of what this “hybrid bio/organic/inorganic nanostructured assembly” looks like is shown in Fig. 26.3.

Semiconductor core Passivation shell Stabilizing or capping layer Organic functional layer Targeting biomolecule

Figure 26.3 Schematic representation of a semiconductor quantum dot adapted for biolabelling purposes. Each part of this hybrid bio/organic/inorganic assembly is designed to optimize its functionality.

Nanostructured semiconductor systems received increased attention in the early 1980s and are still being optimized to render good quality nanoparticles. Two independent research fields brought to existence stable semiconductor nanocrystals (10 nm). It was expected that lowering the size of semiconductor particles would increase their oxidation/reduction performance in catalysis and, on the other hand, it was also expected that semiconductor materials in the nanometre size range would show quantum confinement properties. In order to test this hypothesis, researchers such as the groups of A. Henglein, M. Grätzel and L. Brus adapted methodologies based on colloidal chemistry developed in the 19th century. They produced large quantities of selected semiconductor materials, mainly CdS and ZnS which, aside from a large surface area, also possessed unique optical and electrical properties. The theoretical model of quantum confinement regime was successfully applied in the description of their behaviour. Brus, Efros and

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Ekimov published the first papers describing three-dimensional size quantization effects using effective mass approximations [18, 19]. In order to control the growth, chemical nature and surface of the particles, the bottom-up technique based on colloidal chemistry was and still is the primary choice for the preparation and control of large numbers of these assemblies. After the initial success in the obtention of QDs, their photophysical processes are currently being extensively studied. There are four basic steps in the assembly of these complex chemical biofunctional semiconductor nanoparticles and each one must be developed to optimize its performance in biological imaging: ● ●

●

●

The semiconductor core particle growth in a solution containing its chemical precursors and stabilizing molecules. The passivation procedure of the core particle by introducing a capping layer of a higher semiconductor band gap. As defect structures on surface can alter the total number of photons emitted, this step is fundamental for the optimization of their fluorescence quantum yield. The solubilization of the particles in aqueous solution. This step is necessary for methodologies where the core–shell particles are obtained in organic solvents and must be extracted to aqueous media. Surface functionalization of the particles in order to direct their interaction with biomolecules or biological systems.

Synthetic routes for the obtention of colloidal II–VI nanoparticles (2–20 nm) were extensively investigated and several reviews in this field have been published [20–26]. Colloidal methods involve the controlled formation (due to its low solubility product, Kps) of a crystalline precipitate from a solution containing its ionic precursors. Just as in the crystal growth kinetics from supersaturated solutions, there are three main processes which determine the formation and aging of these particles: (a) The nucleation stage in which the first aggregates are formed in the mother solution. (b) The growth of these nuclei by consuming the species present in the mother solution to form primary crystalline particles. (c) The aging of the primary particle dispersion during which changes in shape, structure, size, agglomeration and flocculation may take place. The success observed for the colloidal methods in the obtention of nanostructured crystals relies on the ability to stop the crystal growth process immediately after nucleation begins. This is accomplished by controlling the equilibrium between the solid crystalline precipitate and the solvated metal ions in solution and this can be experimentally achieved by selecting an appropriate reaction temperature and solvent. Another crucial point to be observed in the chemical stability of these colloidal systems is the occurrence of long-term growth processes, mainly the diffusion growth and the Ostwald ripening, which end up producing bigger agglomerates which may lead to an irreversible flocculation and phase separation. These processes can be controlled by using appropriate stabilizing conditions, mainly by rendering particles with charged surfaces and/or sterically built systems using polymers. This surface stabilization may also have an additional purpose: the nanocrystal solubilization in water media. In the next sections the evolution of the currently known methodologies for the obtention of soluble surface passivated semiconductor nanocrystals using colloidal chemistry techniques is described. Special attention is given in section 26.2.4 to the functionalization procedures which are used to optimize the chemical association of the nanocrystal with a specific targeting biomolecule. 26.2.1 How did people work out how to control the particles down to the nanoscale size range? The evolution of the synthetic procedures

The first methods that successfully described II–VI semiconductor nanocrystals reported the preparation of CdS and ZnS particles in the 3–6 nm range [27–30]. This was accomplished by

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applying an arrested precipitation technique at room temperature involving either the injection of the metal salt into a solution of ammonium, sodium sulphide or the injection of H2S(g) in a Cd2 containing solution, both bearing a suitable reaction solvent (water, methanol, 2-propanol or acetonitrile) in the presence or not of a stabilizing agent, such as polyphosphate and thioglycerol [28] or AOT (sodium bis(2-ethylhexyl) sulphosuccinate) reverse micelle systems [31, 32]. Figure 26.4 shows two representative transmission electronic micrographs of CdS nanocrystals obtained from the reaction of Cd2 ions with H2S(g) in the presence of either polyphosphate chains [NaPO3]n or mercaptoacetic acid as the stabilizing agents. The Cd:S ratio was the same in both cases. The size dispersion of the polyphosphate-stabilized CdS nanocrystals is 15% while for the thiol stabilized ones it is 10%. The polyphosphate method for the preparation of CdS (and ZnS, (CdZn)S, HgS) particles is still used for the preparation of highly stable, fluorescent and biocompatible colloidal aqueous systems. On the other hand, the stabilization of the colloidal suspensions was longer for larger chains than for smaller capping molecules.

(a)

(b)

Figure 26.4 Transmission electronic microscopic images of CdS nanocrystals obtained in water, at room temperature, using Cd(ClO4)2 and H2S (1:1) as the precursors and two different stabilizing agents: (a) mercaptoacetic acid, bar  40 nm and (b) polyphosphate anions, bar  50 nm.

The precipitation technique in aqueous medium was extended by Weller and his group to other semiconductor materials such as CdSe and HgTe by using different stabilizers (also referred to as capping agents) such as those containing amine and thiol groups [33–35] similar to a previously described synthesis by Nosaka et al. [36]. They reported the synthesis of gram-scale water redispersible stable powders showing luminescence quantum yields up to 50% and high photostability. Although all these previously obtained systems present simpler methodologies and good optical properties, they show a poor degree of crystallinity, with high concentration of surface defects and large size variations (relative standard deviation 15%) requiring size-selective precipitation procedures [37, 38] or chromatographic gel separation [39] to obtain narrower-size distributions. The size-selective precipitation method is based on the size-dependent solubility of the nanocrystals. Adding controlled amounts of a non-solvent to the colloidal suspension will lead to the precipitation of the large nanocrystals, which can then be separated by either filtration or centrifugation. Such a procedure may be carried out consecutively to obtain a set of nanocrystal fractions with different diameters. A similar procedure may in principle be applied again to each fraction to further narrow the size distribution [37, 40]. In 1988, Steigerwald [41] reported the preparation, in micelle systems, of II–VI nanoparticles which were then coated with covalently bound phenyl groups. This first report brought up the possibility of organometallic rather than inorganic precursors. Later on Bawendi and collaborators [42] also used inverse micelle systems to prepare similar nanoparticles, which after annealing were dispersed in Lewis base solvents such as trialkylphosphines/phosphine oxides. The 1990s showed an extensive diversification of these new methodologies driven by the potential application of these materials in non-linear optics. High-quality CdSe nanocrystals became

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available using high-temperature crystal formation in organometallic solvents. Knowing that a great number of divalent transition ions show strong coordination ability towards various solvent molecules such as amine and phosphine containing molecules [43], forming low-dimensional structures referred to as chalco(genido)metalates [44], researchers directed these reactions towards the obtention of II–VI nanocrystals. In 1993 Bawendi and collaborators [37] introduced the “organometallic TOP/TOPO” procedure by synthesizing highly luminescent CdSe quantum dots with nearly perfect crystal structures and narrow-size variations (relative standard deviation 5%). This route was based on the pyrolysis of organometallic reagents (e.g. dimethylcadmium – Cd(CH3)2 – and bis(trimethylsilyl)selenium) by injection into high boiling point coordinating solvents (like tri-n-octylphosphine oxide (TOPO) and tri-n-octylphosphine (TOP)). A series of organometallic routes was described aimed at the obtention of II–VI nanocrystals, mainly CdSe, but also CdS, CdTe and ZnSe [45, 46]. CdSe nanoparticles showed narrower-size dispersion, consequently a greater control on the amplitude of emission colours (ranging from violet to red). The central point in this methodology is the control of the nucleation and growing steps of the particles performed at high temperatures (T  300°C) for an extended period of time (ranging from minutes to hours, depending on the desired particle size) [47]. In this process, smaller nanocrystals are broken down, and the dissolved ions are transferred to larger crystals. The rate of this “ripening” process is dependent on both the temperature and the amount of the limiting reagent [47, 48]. Continuous injection of precursor solutions into the CdSe reaction mixture at 300°C also produces larger nanocrystals. As a matter of fact, when using different TOP:TOPO ratios for the preparation of ZnSe, Hines and Guyot-Sionnest commented that the reaction could render nanocrystals so small that they could not be isolated by standard solvent/non-solvent precipitation techniques, neither could they precipitate as large aggregates [46]. The authors suggested that these difficulties arose from TOPO binding too strongly and TOP too weakly to Zn. This new organometallic route succeeded in obtaining homogeneous nanocrystals with a small size dispersion but the fluorescence quantum yields were still relatively low (10%). Moreover, it was observed that the TOP/TOPO system showed better growth control for CdSe nanocrystals not larger than 4.5 nm (i.e. in the strong quantum-confinement size regime, with first exciton absorption peak 600 nm) [49]. Currently the organometallic procedures are still the most popular choice for the development of a variety of colloidal nanocrystals in non-aqueous solutions although the main chemicals used are highly toxic, expensive, pyrophoric and explosive (the systems are heated above TOPO’s flash point: 350°C). To improve this methodology, some researchers tried to introduce other coordinating systems in the reaction solution, such as amines which were a logical choice for ligands of intermediate strength. Amines are slightly weaker bases than phosphine oxide and long-chain alkylamines have much higher boiling points (hexadecylamine, bp: 330°C). In addition, the less sterically hindered amine creates a larger capping density, which probably increases the surface passivation. As such, dodecylamine (DDA) and hexadecylamine (HDA) have been successfully used to cap the surface of CdSe nanocrystals [49, 50–53]. Another feature of these organometallic methodologies is the possibility of the nanocrystal’s final shape control (e.g. obtaining nanorods) by altering the ratio of the surfactants (e.g. TOPO and hexylphosphonic acid, HPA) [53–55]). Peng and Peng [49], motivated by the instability and the low reproducibility of the optical properties of the nanocrystals obtained by the organometallic route, developed a series of methodologies based on CdO, CdCO3 and Cd(acetate)2 in fatty acid solutions aimed at the substitution of the pyrophoric Cd(CH3)2. They argumented that this cadmium precursor actually decomposes in hot TOPO and generates insoluble metallic cadmium precipitates. Studying a different set of experimental conditions and precursors these authors suggested that the existence of any anion of a strong acid, either in the form of the cadmium precursor or as an added cadmium ligand, made it impossible to form high-quality CdSe nanocrystals in the current systems. Thiols, which bind strongly to cadmium, were found to inhibit the nucleation process. Using this procedure they reported a size range of nearly monodisperse CdSe crystals, from about 1.5 nm to above 25 nm, a much broader range than that achieved by the original organometallic method. Peng and Peng [49] further reported that the temporal evolution of the size and size distribution of CdSe nanocrystals in fatty acid systems were quite reproducible, although the reaction

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rates were fast. They considered this to be due probably to the more controllable nucleation step initiated by cadmium carboxylates less active than Cd(CH3)2 used in the traditional organometallic approach. This phenomenon implies that the control of the nucleation process may be the key step towards a fully controllable synthesis. Moreover, the authors state that in practice, the fatty acid systems are not recommended to synthesize small nanocrystals because of their fast growth rates. In the late 1990s two rather different alternative methodologies were also described for the synthesis of selenides and tellurides. In the first one the chalcogenide precursors were produced in situ by using reducing agents such as KBH4, which converts Se2 and Te2 to Se2 and Te2, respectively [56, 57]. Wang and collaborators proposed a simple solution synthesis for pure quantum dots of M chalcogenides (M  Bi, Cu, Cd, Sn, Zn; chalcogenide  S, Se) by providing the in situ reduction of S or Se in the presence of KBH4 and the corresponding metal salt at room temperature in strong basic solvents. They showed that the solvent significantly influenced the quality of the final product, yielding small uniform nanoparticles (4–6 nm) in the case of ethylenediamine and a mixed metal/chalcogenide precipitate with poor crystallinity and low yield in the case of pyridine. In fact, pyridine is known to provide stable capping through the N atom, but its low boiling point suggests limitations as a growth solvent. Recently, amine-capped PbSe nanoparticles of tunable sizes and shapes were also obtained with this method [58]. As in the previous methods, the main limitation is the difficulty to achieve narrow-size distributions and high crystallinity. The second methodology, proposed for large-scale production, involves the application of ultrasound (formation and implosive localized hot spots induced by acoustic cavitations) on chemical reactions [59, 60]. Zhu et al. reported the preparation of spherical ZnSe nanoparticles of average sizes of 3, 4, and 5 nm by reacting Zn(acetate)2 and selenourea in water followed by sonication with a high-intensity ultrasonic probe under inert atmosphere for a determined period of time [60]. Pb and Cu selenide nanoparticles were also obtained by using the corresponding acetates. A recent search for “greener” and simpler procedures that could produce semiconductor nanocrystals directly in aqueous media, aimed at bioapplications, readapted the thiol stabilized CdTe synthetic methods originally reported by Rogach et al. [33, 34] and the one reported by Nosaka et al. for the synthesis of CdS [36]. By using different approaches Gaponik et al. [38], Zhang et al. [61], and Menezes et al. [17] prepared highly luminescent CdSe or CdTe nanoparticles directly in water. Gaponik reported the dissolution of Al2Te3 in an acidic solution to render the Te2 precursor ions as H2Te(g), while, Zhang et al. [61] and Menezes et al. [17] used NaBH4 in aqueous solution to reduce Te to Te2. This reduction process generates Na–Te–Te–Na a stable intermediate complex which will be converted to CdTe after injection of the metal precursor complexed with a thiol molecule in water under inert atmosphere. These colloidal systems render particles in the 2–6 nm range and show luminescence after a certain period of time suggesting a slow kinetic surface passivation process [17]. This observation will be discussed in the next section. Regarding the inherent toxicity of these systems for in vivo applications, for example, it was recently reported by Pradhan et al. [62] that the synthesis of pure and doped ZnSe QDs as an alternative for CdSe nanocrystal aimed at the obtention of a less toxic labelling material which could be envisioned for a safe use in in vivo experiments and diagnostics. On the other hand, to avoid the inconvenient autofluorescence observed in biological systems when these are excited in the UV blue region of the spectrum, lower band gap II–VI semiconductor QDs are also being converted into biolabels. These systems when quantized present the onset of the absorption and emission bands in the near-infrared region (700–1300 nm). As an example, Kumar and Jahkmola reported on the RNA-mediated fluorescent PbS nanoparticles as novel tools for biophotonic applications [63]. Also a recent report of DNA-directed semiconductor quantum dot synthesis described highly optically emissive PbS nanocrystals [64]. Furthermore, Hinds et al. [65] were able to synthesize infrared emitting PbS QDs (4 nm) stabilized with Guanine-triphosphates (GTP). The authors systematically investigated how nucleotide functionalities (base, sugar and phosphate) influenced nanoparticle growth. They proposed a set of rules for using nucleic acids as ligands in order to profit from the natural biorecognition properties of DNA and programmable templates for nanoparticle synthesis.

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In summary, much work has been done in the synthesis of II–VI nanostructured semiconductor compounds in the past three decades. Still, researchers are looking for the ideal preparation methodology, aimed at particles with fewer surface defects and small size dispersion using less expensive, non-toxic, less risky and simpler experimental conditions which can be extended to a large-scale production. The need to improve their surfaces in order to increase their fluorescence quantum yields, for example, prompted a close look at the QDs surfaces. The next section comprises the processes used to overcome this problem. 26.2.2 Still some problems: quantum dots have imperfect surfaces! The passivation process

The observed fluorescence in semiconductor nanoparticles is produced upon the recombination of the charge carriers which are generated by light absorption. The first colloidally obtained nanocrystals showed a very low fluorescence quantum yield (ϕ  1%). The non-radiative processes involved in semiconductor nanocrystals are said to have the same physico-chemical nature as those observed in bulk semiconductor materials [66]. Taking into account the high number of surface atoms compared to bulk atoms, it was suggested that the main contribution for this was that the prepared colloidal particles had a lot of surface defects (shallow and deep traps) where radiationless recombination of the charge carriers occurred. A schematic representation of these defects is illustrated in Fig. 26.5.

CB Shallow traps Eg

Shallow traps Deep traps

VB Bulk

QD

Figure 26.5 A schematic representation of deep and shallow traps (originated from structural defects) claimed to be responsible for the radiationless processes in bulk and quantized semiconductor particles.

Soon it was realized that if the defect sites, most probably resulting from dangling bonds, were located at the surface of the colloidal particles, there was a chance to chemically modify these sites. Several studies on the chemical influence on the spectroscopic properties of semiconductor quantum dots were reported. The fluorescence intensity (and also the fluorescence spectrum pattern) of CdS nanocrystals was shown to be drastically increased by certain surface modification procedures such as exchanging the aqueous solvent by alcohol [31], adsorbing triethylamine in low concentrations [31], and covering the surface with cadmium hydroxide or silver sulphide [28]. By coating the CdS nanocrystals with a layer of Cd(OH)2 the resulting system is a core semiconductor coated by a shell of another semiconducting material possessing a higher band gap. The resulting assembly was later called a core–shell system. The increased luminescence quantum yields of the core–shell particles is explained by preventing from the photogenerated excitons spreading over the entire particle, forcing them to recombine while being spatially confined to the core. This luminescence enhancement was taken as an indication of the formation of the proposed structure due to the difficulty at that time to investigate such a thin chemical layer (just a few atomic monolayers). Figure 26.6 illustrates schematically the band gap offset created in the interface of the CdSe core (Eg  1.71 eV, wurtzite) and the ZnS shell (Eg  3.68 eV, zinc-blende) during the passivation process.

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e

CdSe ZnS

h ZnS

CdSe

ZnS

Figure 26.6 Schematic representation of the energy band gap offset between the CdSe core and the ZnS shell during the surface passivation procedure.

Using the synthetic methods described in the previous section, several core–shell semiconductor nanoassemblies have been prepared since the CdS/Cd(OH)2 CdS/Ag2S [67], CdS/ZnS [68], CdS/HgS [69], CdSe/ZnS [70–73], CdSe/CdS [50], CdTe/CdS [17, 38, 61]. The capping of II–VI nanocrystals with long chain organic surfactants was also utilized to passivate surface atoms, but at room temperature the luminescence quantum yield was as high as 10% with a very long fluorescence lifetime and some non-band edge luminescence [74, 75]. The deposition of a second layer onto the particle’s surface represents the chemical growth of another crystalline phase. Two main problems may arise during this process: (i) formation of single particles in the colloidal suspension instead of the second layer growth and (ii) imperfect growth patterns or even alloying of the second layer due to a large or too small crystal mismatch, respectively. This last structural problem either may lead to unstable chemical assemblies presenting a large quantity of surface defects or may alter the original optical properties of the core itself. The first difficulty is overcome by controlling the experimental parameters during the formation of the second layer. Very low concentrations of the capping precursors and a fast crystallization rate are recommended. Dabbousi et al. [71] reported that the growth of ZnS shell on CdSe particles was accomplished, without the precipitation of ZnS, by dropwise adding diethylzinc and hexamethyldisilathiane (as Zn and S precursors) in equimolar amounts to vigorously stirred CdSe colloidal solutions held at temperatures between 140 and 220°C, depending on the core sizes. ZnS has a wider band gap than CdSe (Eg  3,91 eV and 1.71 eV, respectively [76, 77]) and, in fact, the authors report an enhancement of 10 to 50% of the quantum yield of CdSe nanocrystals after this passivation procedure. The thickness of the capping layer was monitored by small angle X-ray scattering (SAXS) measurements indicating 0.65 monolayers to 5.3 monolayers. The authors also suggest, by correlating the structural and optical properties, that the ZnS layer tends to generate structural defects when more than 1.3 monolayers are grown, decreasing the luminescence quantum yield. The second difficulty is the possible chemical alloy between the core and shell layers which would influence the core optical properties. By choosing capping layers with different crystal structures or even with same crystal structure but possessing different bond lengths one prevents this possibility. For example, the growth of wurtzite-type CdS on wurtzite CdSe nanocrystals shows a lattice mismatch of 3.9% which is small enough to allow epitaxial growth while still preventing alloying. The band gap of CdS (Eg  2.50 eV [76, 77]) is greater than the CdSe band gap (Eg  1.71 eV [76, 77]) promoting a quantum yield enhancement [50]. On the other hand, in CdSe/ZnS core–shell nanostructures the Zn–S bond length is about 12% larger than Cd–Se [77]. This chemical mismatch that would prevent the growth of flat heterostructures in epitaxial growth is believed to be relaxed in nanocrystals with short facets [71] explaining the chemical stability and excellent optical properties of these core–shell nanoparticles. Regarding the optical properties of the core after passivation, several reports on different core–shell systems show that there is no observable modification of the original properties of the core semiconductor nanocrystal. As an example, Fig. 26.7a shows the absorption spectra of CdS nanoparticles of d  6 nm prior and after passivation with a Cd(OH)2 layer. In this methodology the surface of the CdS nanocrystals is loaded with hydroxyl groups by increasing the pH of the suspension up to 10.5 [28, 78], followed by a dropwise addition of a low-concentration solution of Cd2. The increase of the fluorescence intensity may be monitored

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Incresing (Cd2)

Absorbance (a.u.)

Emission intensity

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CdS CdS-OH 200

300 400 500 Wavelength (nm)

600

450 500 550 600 650 700 Wavelength (nm)

Figure 26.7 (a) Absorption spectra of a polyphosphate stabilized CdS (6 nm) suspension prior to and after the passivation procedure. (b) Monitoring the emission intensity increase as the cadmium hydroxide layer is being deposited onto the particles’s surface.

by emission spectroscopy (as observed in Fig. 26.7). The quantum yield increases to 20% and the mechanism suggested is that the excess of OH will convert SH surface states into S2, which in turn will bind to the excess Cd2 ions. The cadmium-rich surface will then associate to the polyphosphate anions present. The passivation on the thiol-capped cubic CdTe nanocrystals prepared in aqueous solution [61, 79–81] was brought up in a different form. It was mentioned in the previous section that the photoluminescence of the colloidal suspensions of CdTe nanoparticles (2–6 nm) developed slowly after its synthesis take up to several days to become readily observable even in daylight. This suggested a slow kinetic mechanism of surface defect supression. Rockenberger et al. [79] studied these systems using extended X-ray absorption fine structure measurements (EXAFS) and observed that the particles possessed CdS bonds although they were larger than the bulk value for the CdS bond. Later Bouchert et al. [81] and Zhang et al. [61] investigated and reported on the nature of the surface of these particles by photoelectron spectroscopy (XPS). Zhang et al. suggested that the carboxyl groups of the mercapto-carboxylic acid stabilizers coordinated to the surface of the CdTe particles have a great influence on both their photoluminescence and stability by substituting some Te2 ions with oxygen of the carboxyl group. They also observed CdS bonds, which they suggested result from the decomposition of the thiol groups. On the other hand, Bouchert et al. showed strong evidence for Cd–SR bonds near the surface and no indications for the presence of Te atoms at the surface. They suggested that this was the result of the hydrolysis of the thiol molecules which ended up incorporated onto the particle’s surface, substituting Te2 ions and satisfying Cd2 dangling bonds. Figure 26.8 shows a structure model of the CdS layer growing on the surface of the cubic CdTe particle. Thinking of a structural match one may observe that both the core and the shell possess cubic crystalline structures. A reasonable extension of the thiol hydrolysis process would end up creating a CdS shell, which in turn possesses a higher band gap, and would be responsible for the passivation of the CdTe surface defects. Once the particles are optimized to show a high fluorescence quantum yield they may be turned into fluorescent biolabels by adapting their surface to be (i) water soluble and (ii) biocompatible. In the next sections further modifications of the nanocrystal’s surface are discussed. 26.2.3 How to render QDs soluble (hydrophilic surface) for biological applications. Solubilization of the nanoparticles

In order to interact with biological systems there are two forms for the QDs to present themselves: solubilized in aqueous medium or encapsulated inside lipophylic carrying systems, such

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O C

alkyl

Sulphur Cadmium Tellurium

Figure 26.8 A schematic model of a cubic CdS layer growing on the surface of a cubic CdTe lattice from the hydrolysis of the thiol alkyl carboxilate capping molecules.

as micelles. Although the general properties of the nanocrystalline surface appear to be understood, the exact surface chemistry involved in such processes remains to be elucidated. While the QDs prepared in aqueous media such as the polyphosphate-and/or thiol-capped CdS, CdSe and CdTe nanocrystals have inherent solubility, QDs obtained by the organometallic route must overcome the aqueous incompatibility problem by altering the nature of their surface. Among the different techniques developed for surface modification, the four most general methods described in the literature [82] are: the surface-exchange procedure, the silanization of the nanocrystals, the formation of a hydrophilic interface with amphiphilic molecules and the micellization of the nanocrystals. Figure 26.10 represents three of these methods. The surface exchange of the hydrophobic surfactant molecules (e.g. TOPO) for bifunctional binding water-soluble molecules [9, 83] has been employed using a wide variety of species. These bifunctional linkers (such as HS–alkyl–COOH molecules) act in similar fashions to solubilize the phosphine-capped nanocrystals and to provide functional groups (e.g. carboxylic acid and amine) which may be used, in a posterior step, to conjugate to biomolecules using wellestablished protocols. This capping procedure yields gram quantities of water-soluble QDs, but the thiol ligands are not completely stable. Slow desorption of thiol–alkyl–acid molecules and the possibility of intermolecular reactions resulting in disulphides decreases the capping layer and leads to the flocculation of the particles [82]. Moreover, these particles show an overall decrease in their fluorescence quantum yields [84]. Another approach is the silanization with organosilicone molecules containing NH2 or SH functional groups [8, 84–88]. A silica/siloxane shell is formed on the surface by the induced hydrolysis of the silanol groups. Polymerizing silanol groups help to stabilize the nanocrystals against flocculation but only small amounts (milligram quantities) can be prepared per batch [24]. Also, residual silanol groups on the nanocrystal’s surface often lead to precipitation and gel formation at neutral pH. A third possibility is to apply phase-transfer methods using amphiphilic molecules that act as detergents for solubilizing the QDs coated with hydrophobic groups [17, 26]. This method has been particularly advantageous in allowing the retention of the native surfactant molecules, which appear to increase the stability and fluorescence efficiency over those samples where the native layer has been replaced with a bifunctional binding molecule. Encapsulating QDs and their initial ligands with macromolecules such as polymers or lipids can preserve the emission quantum efficiency, but generally adds a large volume to the nanocrystals, resulting in a final size that may be greater than desired. This may diminish imaging sensitivity by decreasing the number of QDs that can be attached to a target. For in vivo imaging, bulky nanocrystals may have limited accessibility to target systems.

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Silica encapsulation O -S-alkyl-SI-OO

H I-O l-S y k al H -SSI-O lkyl-S-a -S-alky l-SI-OH -Salk ylSIOH

Ligand exchange

O

H

I-O

l-S

-al ky

O -S-alkyl-C

H

H -a SI-O -S lkyl-S-a -S-alkyl-SI-OH -Salk yl-S SI-a OH lk yl -S I-O H

OH SIylalk -S-S-alkyl-SI-OH

-S

OH SIylalk -S-S-alkyl-SI-OH -S-alk yl-SIOH

-S-a lkyl-S SI-O -a H l k yl -S I-O H

Hydrophobic interaction

I-O

-S

yl lk

Figure 26.9 Representation of three different experimental modifications described in the literature to produce water-soluble QDs.

Two different solubilization approaches based on the micellization of the hydrophobic nanostructures were recently reported by Dubertret and collaborators and also by Korgel and Monbouquete. By encapsulating individual CdSe/CdS QDs in phospholipid block–copolymer micelles Dubertret et al. overcame the solubility problems and also demonstrated a longer stability of the particles in a living body [89]. Moreover, by adapting DNA to the nanocrystal micelles, these systems acted as in vitro fluorescent probes to hybridize to specific complementary sequences. Korgel and Monbouquete successfully used phosphatidylcholine vesicles to prepare mixed core and layered (Zn,Cd)S and (Hg,Cd)S nanocrystals [90]. 26.2.4 How to target the QDs to biological systems. The functionalization step

The synthesis, passivation and stabilization steps for the obtention of core–shell fluorescent semiconductor quantum dots prepared for biological labelling are generally succeeded by a functionalization procedure. The term functionalization (also known as organic capping and ligand conjugation) refers to the chemical modifications of the quantum dot’s surface to render biocompatible systems feasible for binding with specific biomolecules which will target the QDs to specific sites in the biological systems. This process is thermodynamically favoured due to the highly

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active chemical surfaces of the nanocrystals, either presenting dangling bonds at the passivation shell or active binding groups in the stabilizing species. There are several different experimental routes for this functionalization [91–94]. The functionalizing agents may be small organic molecules (e.g. thiol molecules [38], nucleotides [95, 96], carbohydrates [97], polyphosphates [98]), or larger biomolecules (e.g. proteins [7, 99], biotin [100], avidin [101], polyethylene-glycol [102], streptavidin, RNA [63], DNA [103], glucose oxidase and IgG). Usually the combination of small and large molecules will promote adequate modifications on the inorganic QDs’ surfaces, providing functional groups that will intermediate the QD/cell interaction [104]. For a schematic illustration of a functionalized core–shell semiconductor quantum dot, see Fig. 26.10. Figure 26.11 represents, as an example, the reaction steps involved in the obtention of fluorescent CdS/Cd(OH)2 QDs, functionalized with polyethyleneglycol (PEG), a biocompatible polymer.

Passivation shell Functionalization shell QD (core)

Figure 26.10

A functionalized core–shell quantum dot is schematically represented here.

H2S Stirring 2

CD3 (NaPO3)n

CdS

OH

PEG

pH  10.5 CdS/Cd(OH)2

CdS/Cd(OH)2/PEG

Figure 26.11 Synthesis, surface passivation and functionalization: reaction steps involved in the obtention of fluorescent CdS/Cd(OH)2 QDs, functionalized with polyethylene-glycol (PEG).

In order to promote an adequate functionalization procedure, every single modification performed in the colloidal suspension conditions (e.g. temperature, pH, composition) has to take into account the fact that the colloidal suspension must remain stable. Another important feature which cannot be neglected is the maintenance of the QDs characteristic luminescence. Moreover, the choice of the adequate functionalizing agent is someway driven by the desired QD function/ application in the bio-system. More complex functionalization procedures may employ conjugation protocols adapted from those used in the binding of biomolecules and organic fluorophores. The functionalization of proteins (which represents the chemical nature of a great amount of biofunctional molecules, such as antibodies, antigens, enzymes, growth factor molecules, etc.) to QDs may be performed

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in different forms, which will depend on the protein type and the nature of the nanocrystal’s capping surface. Some described methods are: (i) the condensation of amine groups of the protein to carboxyl groups of the QDs’ surface by using EDC, 1-ethyl-3-(3-dimethylaminopropyl) carbodiimide as a chemical activator; (ii) direct binding to the QD surface using thiolated peptides or polyhistidine (HIS) residues; and (iii) adsorption or non-covalent self-assembly using engineered proteins [91, 105]. Optimal functionalization techniques should also be able to keep the quantum dots’ fundamental properties for periods of time longer than several weeks. An example described in the literature refers to “engineered-protein” functionalized quantum dots which retain their quantum efficiency and offer longer shelf life [91]. These systems were also further functionalized with multiple functional groups without decreasing their quantum efficiency [93]. It has been shown that the optical properties of semiconductor nanocrystals are very sensitive to certain capping molecules attached to their surfaces. Some stabilizing agents or hydrophobic surfactant molecules used in the solubilization procedure decrease the overall fluorescence quantum yield and the explanation for this effect remains in the “electronic blinding capacity” of the passivation shell [61, 92, 106, 107]. This has been demonstrated in the case of mercaptoacetic acid-treated QDs where the quantum efficiency was drastically reduced [93, 94]. However, for some probing applications this feature is taken as an advantage, especially if the change in fluorescence is generated by resonance energy transfer (FRET) to target analyte molecules [91]. This opens up other applications in biosensing, where the extension of this FRET process can be controlled by the thickness of the passivation shell and the distance to the binding species. The choice of the functionalizers will provide low or high specificity concerning the association of the functionalized QDs with biomolecules. As already mentioned in the previous section, thiol–alkyl–COOH ligands (such as mercapto-acetic acid) used as stabilizing agents in the synthesis of CdTe QDs in aqueous medium also act as functionalizing agents and provide surface carboxylate groups, making the QDs feasible to react with primary amines expressed by cell surface proteins. This kind of reaction produces relatively stable amides [107]. Glutaraldehyde (at very low concentrations), thiol-containing molecules and polyethylene-glycol can be cited as efficient functionalizing agents, in an increasing order concerning specificity in the bioconjugation process [91]. 26.2.5 Bioconjugation of the QDs

The term bioconjugation is used to denominate the interaction between the functionalized quantum dots and the molecules present in a biological system. A more general definition includes also the conjugation of functionalized QDs with biomolecules produced by the bio-system and dispersed in a liquid medium (e.g. the conjugation of circulating antibodies in the human serum). As mentioned previously, QDs are prepared to bind chemically (covalent bonds) or physically (adsorption phenomena) to molecules present in the bio-system. The nature of the functionalizing groups will, in theory, define the nature of the interaction with the biological system. For instance, if the target biomolecule is expressed in the cell membrane then most probably a superficial labelling will be observed. Depending on the kind of interaction observed one may identify different internalization mechanisms related to the bioconjugation process. In specific biomolecules recognition the QDs will bind specifically to the target molecule [94, 95]. Biotinylated antigens, for example, interact strongly with QDs functionalized with avidin or streptavidin molecules [94, 99] rendering great specificity labelling. The QDs may be functionalized with molecules that interact specifically to species presented in distinct cellular organelles/structures, this is exemplified by the cell cytoskeleton microtubules labelled with CdSe/ZnS quantum dots functionalized with anti-tubulin [92]; another example is the CdS/Cd(OH)2 polyphosphate stabilized QDs which were directed to high consuming energy internal sites of living leishmania parasites [98]. When the functionalizing groups are not specific, the interaction may occur indistinctively with all the similar molecules and in this case the QDs are internalized by a natural process known as endocytosis and end up bound to internal cell compartments [13, 109]. Endocytosis is

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the process whereby cells engulf vitamins and nutrients from their outside surroundings. The nanocrystals can also be artificially internalized by inserting them using micromanipulators [91]. In fixed cells the membrane pores are increased and the QDs can pass through these channels easily without any other kind of internalization. Various covalent and non-covalent strategies employing crosslinkers [7, 13, 91–94, 109– 115] have been developed for conjugating biomolecules to the QDs’ surfaces. Biomolecules can be linked to the QDs via functional groups such as –COOH, –NH2 or –SH, which may be present at their modified surface. These groups may be provided by functionalizing compounds such as protein A, avidin, streptavidin, glucose oxidase horseradish peroxidase, and IgG [13, 114–116]. Figure 26.12 summarizes the steps discussed up to this section, concerning the preparation and use of fluorescent semiconductor quantum dots for biological labelling purposes.

Quantum dot core

Passivation shell

Figure 26.12

Functionalizing agent

Conjugated biomolecule

A schematic representation of a passivated, functionalized and bioconjugated quantum dot.

For living cells labelled with quantum dots, the bioconjugation step may be processed into the cell culture medium [7, 13] or even in another appropriate medium, such as saline solution. The resulting systems to be used for bioconjugation must be compatible with cellular/tissular conditions, i.e. they must be used in physiological pH, present low toxicity, be isotonic with the intracellular (or intra-tissular) medium, and the resulting QDs must remain hydrophilic and retain their characteristic fluorescence. In the next section some applications of QDs in biolabelling procedures are presented.

26.3 Applications

Since the first reports published in 1998 [8, 94] concerning the application of quantum dots as biolabels, much progress has appeared in this field. There are several reports which indicate the fast improvement on conjugating these nanostructures to biological molecules, cells, microorganisms and tissues. It is worthwhile mentioning some representative examples: ● ● ● ● ● ●

Multiplexed biological detection and imaging [24, 117, 118]. Sensing trace analytes in biological samples [119]. Tools for rapid and sensitive diagnosis of viruses [120]. Molecular characterization in combination with optical imaging to detect the progression of precancerous lesions [121]. Nanoparticles for targeting in vivo [122]. Cervical, breast and brain cancer diagnostics [13, 109], as well as many other applications in life sciences.

Multiplexed biological detection and imaging, using the unique properties of photostability and especially the narrower emission spectra of QDs, have been applied for fluorescence in situ hybridization (FISH) to study molecular biology. For instance, Chan et al. used FISH and QDs for monitoring mRNA transcripts [107]. This work demonstrated an increased sensitivity of FISH using QDs in comparison with organic fluorophores which can facilitate the ultrasensitive

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simultaneous study of multiple mRNA and protein markers in tissue cultures and histological sections. The feasibility of in vivo targeting by using QDs has being explored, for instance, by Akerman et al. using ZnS-capped CdSe nanoparticles [11]. They showed that ZnS-capped CdSe QDs coated with a lung-targeting peptide accumulate in the lungs of mice after i.v. injection. Two other kinds of peptides direct the QDs specifically to blood and lymphatic vessels in tumours. They also show that QDs with polyethylene glycol prevent non-selective accumulation of QDs in reticuloendothelial tissues. These studies encourage the synthesis of nanostructures associated with drug delivery systems. After this brief overview of biological applications of quantum dots, this section will now focus on and discuss some results obtained by our research group: (i) determination of the antigenA expression in living red blood cells; (ii) obtention of non-linear microspectroscopy in an optical tweezers system – application to living macrophage cells marked with quantum dots – and (iii) cancer diagnostics. 26.3.1 CdS quantum dots for red blood cells antigen – A labelling

Core–shell CdS/Cd(OH)2 quantum dots obtained in aqueous medium were successfully used as efficient fluorescent labels for living human red blood cells. The aim of this investigation was to precisely determine the antigen-A expression in subgroups of group A erythrocytes. Luminescent CdS nanoparticles were functionalized via a one-pot cross-linking glutaraldehyde procedure. These functionalized nanoparticles were conjugated to monoclonal antibody anti-A for 5 hours. Living human red blood cells, before the contact with QDs, were diluted in 0.9% saline solution, centrifuged and separated from the liquid phase. The resulting conjugate’ QDs/  anti-A were incubated with human erythrocytes of blood groups A, A 2 and O for 30 minutes at 37°C. Prior to visualization, the samples were centrifugated for 2 minutes (3000 rpm) in saline buffer solution and washed. A schematic diagram of the conjugation of the QDs/anti-A as well as of their interaction with red cell membrane antigen is depicted in Fig. 26.13. Functionalization and conjugation steps were performed in an aqueous medium at physiological pH. Antigen-A CDS

Anti-A antibody

Red cell membrane

Figure 26.13 antigen-A.

Schematic diagram for the specific conjugation QDs/anti-A interaction with red cell membrane

The cells conjugated with the quantum dots were characterized by confocal laser scanning microscopy. The conjugates’ QDs/anti-A intensely marked group A erythrocytes, showing different intensities of luminescence for the A2 group investigated, and did not show any luminescence for group O erythrocytes [7]. Figure 26.14 shows respectively a confocal image obtained for QDs/anti-A marking A erythrocytes and for QDs/anti-A marking O. The lack of emission of the type O erythrocytes is explained by the absence of anti-A binding, indicating the absence of antigen-A. These results show the high potential for the use of CdS/Cd(OH)2 semiconductor luminescent nanoparticles as fluorescent labels for red cells. The specificity of the QDs’ conjugation in the erythrocyte cell membrane opens up the possibility of using this methodology as a quantitative

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Figure 26.14 erythrocytes.

Microscopic confocal images obtained for QDs/anti-A marking living A (left) and O (right)

tool to investigate the distribution and expression of alloantigens in red blood cells. The results obtained show the high potential presented by these new fluorescent labels for living cells. 26.3.2 Non-linear microspectroscopy in an optical tweezers system – application to cells marked with quantum dots

Optical tweezers have been used as a tool to manipulate and measure mechanical properties at the cellular level [123]. Adding linear and non-linear spectroscopic capacity to the optical tweezers would allow one to observe real time biochemical reactions of captured living cells. The dynamic, mechanical and spectroscopic information of triggered events can clarify biological events such as cell infection by parasites. Here we present a set-up consisting of an optical tweezers combined to a non-linear microspectroscopy system that was used to perform scanning microscopy and observe spectra from two-photon excited (TPE) luminescence of trapped living single cells conjugated with quantum dots (CdS and CdTe) [124, 125]. Confocal microscopy using TPE luminescence of living cells with infrared lasers has the advantages of high resolution in the vertical direction, the absence of damage caused by heating and larger light penetration revealing underneath structures [126]. Using this combined set-up it is also possible to obtain images and spectra of hyper-Rayleigh also known sometimes in the literature as second harmonic generation (SHG). The combined system used a cw Nd:YAG laser (for trapping) and a femtosecond Ti:sapphire laser (for TPE luminescence and hyper-Rayleigh). The laser beams were focused through 100 oil immersion in a microscope. The back-scattered spectroscopic signals were collected with the 30 cm monochromator-equipped CCD. For TPE luminescence and hyper-Rayleigh a short pass colour filter was used to transmit the visible and cut the infrared. In these first experiments image scanning was performed with a mechanical translation stage. The luminescent CdS nanoparticles were functionalized with glutaraldehyde solution while the CdTe QDs were functionalized with mercaptoacetic acid (AMA). The CdTe and also CdS were obtained using synthetic routes in aqueous medium which produce quantum dots with hydrophilic surface in a water solution [7, 17]. After testing the performance of the system, we obtained TPE luminescence spectra and also hyper-Rayleigh images and spectra for trapped and non-trapped cells and particles. Figure 26.15 shows the image from TPE luminescence of macrophages conjungated with CdS QDs. It was shown that a system of optical tweezers combined with non-linear spectroscopy is capable of analysing trapped single particles and living cells from the visible to the infrared, and also to perform spectroscopic image reconstruction. The acquired spectra and images include

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1

0 Intensity

Figure 26.15

5 µm

Image from TPE luminescence of a living macrophage cell conjungated with CdS/Cd(OH)2 QDs.

hyper-Rayleigh and TPE luminescence of trapped and non-trapped particles. This system demonstrated that it is possible to trigger and observe chemical reactions and mechanical properties in real time of living trapped microorganisms in any neighbourhood in combination with quantum dot fluorescent markers which show several advantages over the conventional fluorophores. 26.3.3 Quantum dots as fluorescent bio-labels in cancer diagnostics

Quantum dot applications in the investigation of neoplastic processes (which may give rise to cancer) constitute a topic of interest with many questions still waiting for precise answers. In the pursuit of sensitive and quantitative methods to detect and diagnose cancer, nanotechnology has been identified as a field of great promise. Hydrophylic quantum dots at physiological pH conditions have the potential to expand conventional protocols used for cancer diagnostics, which need previous tissue/cell fixation, and extend to investigate living cellular and tissular neoplastic mechanisms in real time. Some results concerning the application of water-soluble colloidal semiconductor quantum dots for the purpose of diagnostics in living cells are presented here. The fluorescence was used as a primary tool in order to explore and differentiate the labelling of the samples. Tissues and cells conjugated with QDs were analysed by laser scanning confocal microscopy. All the images were collected with the same acquisition parameters for comparison. In order to confirm the presence of QDs inside the cells, some of the conjugated systems were also characterized by transmission electronic microscopy (TEM). This kind of measurement complements the fluorescence analysis, as they show where the QDs were internalized in the cell. The images obtained show that the nanocrystals accumulate near the nuclear envoltorium. Figure 26.16 shows a representative

Figure 26.16 Transmission electronic microscopy image of a glioblastoma-labelled cell, in which the highest QDs concentration is nearby the nuclear envoltorium (arrows).

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TEM image of core–shell CdS/Cd(OH)2 quantum dots functionalized with glutaraldehyde (QD/ Glut) and conjugated in vitro with live human glioblastoma cells. The cells showed no signs of damage after the conjugation procedure and maintained their integrity even after 5 days of incubation time, demonstrating the low toxicity of the QDs for in vitro studies. In the first application, the quantum dots (CdTe/CdS or CdS/Cd(OH)2 at physiological pH) were functionalized with a 0.01% glutaraldehyde solution and then incubated with living healthy and neoplastic cells (glial, glioblastoma and cervical) and tissues (breast) in culture medium [13]. For the purpose of diagnostics with living cells, the CdS/Cd(OH)2 presented the best results, maintaining high levels of luminescence as well as high stability in biological media. The measurements were performed for different time intervals in order to monitor the time evolution of the interaction between the QDs and the cells. Figures 26.17 and 26.18 show confocal microscopy images and the corresponding fluorescence intensity maps for the time evolution (1–3 min incubation time) of the interaction of healthy and neoplastic glial cells. In the fluorescence intensity maps, the dark grey regions correspond to the absence of fluorescence, while the lighter but more intense grey corresponds to regions of highest fluorescence intensities, respectively.

1

1 min

0 Intensity

3 min

Figure 26.17 Time evolution of the fluorescence pattern of healthy glial cells incubated with QDs/Glut. Confocal microscopy images (left) and the corresponding fluorescence intensity maps (right).

1

1 min

0 Intensity

3 min

Figure 26.18 Time evolution of the fluorescence pattern of neoplastic glial (glioblastoma) cells incubated with QDs/Glut. Confocal microscopy image (left) and the corresponding fluorescence intensity maps (right).

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For all kinds of cells analysed, the time evolution of the interaction clearly reveals different labelling patterns as well as different fluorescence intensities. It can also be noticed that the QD/ Glut easily interacts with both healthy and neoplastic cells. As the glutaraldehyde is a homofunctional bidentade ligand, it establishes hemi-acetal interaction with the QDs’ outer shell at the same time as it binds to cell proteins by Schiff ’s base interactions. As a final application, we report the use of QDs/Glut conjugated to the concanavalin-A lectin (Con-A) to investigate cell alterations regarding carbohydrate profile in human mammary tissues diagnosed as fibroadenoma (a benign type of mammary tumour). The Con-A lectin is a biomolecule which binds specifically to glucose/mannose residues present in the cellular membrane. The bioconjugated particles were incubated with normal tissue sections of fibroadenoma tissue sections. The tissue sections were deparafinized, hydrated in graded alcohol and treated with a solution of Evans Blue (EB) in order to avoid autofluorescence. One class of targeting biomolecules that is commonly used with organic fluorophores are the lectins. Lectins are structurally diverse carbohydrate-binding proteins of non-immune origin that agglutinate cells and recognize carbohydrates in oligosaccharides and glycoconjugates. They have been used in the medical and biological fields. In histochemistry, lectins with different carbohydrate specificity can provide a sensitive detection system for changes in glycosylation and carbohydrate expression that may occur during embryogenesis, growth and disease. Tumour lectinology has so far shown cytochemical and histochemical differences between normal and transformed tissues such as mammary [127, 128] and brain [129]. Quantitative and qualitative changes in glycoconjugates of cell membranes play significant roles in the development and progression of pathologies, including neoplasias. Higher or weaker and even the absence of staining patterns between normal and transformed tissues suggests a dearrangement of secretory mechanisms. The observation that, in general, the more anaplastic the cell becomes the more intense is its staining, seems to indicate that the site and nature of cell surface glycoconjugates are altered. In addition, tissue factors may influence and induce differentiation/dedifferentiation reflected in the different lectin binding patterns [128]. Figure 26.19 depicts schematically how the bioconjugate QD–lectin interacts with the carbohydrate residues of the glycoprotein on the cell membrane. QD Lectin

Carbohydrates

Glycoprotein

Cell membrane

Figure 26.19

Scheme of the cell membrane carbohydrate residues labelled with QDs-lectin.

In the bioconjugation of quantum dots with concanavalin A, the pH of the suspension containing the quantum dots was decreased to 7.2 with 0.1 M HClO4. QDs were functionalized with a 0.1% glutaraldehyde solution (100 μL) to 5 mL of QD suspension (QD/Glut). Finally, Con-A (Sigma, 1 mg/mL) was incubated with QD/Glut (1013 nanoparticles/mL) for 2 h at 25°C in 100 mM phosphate buffer pH 7.2 containing 150 mM (w/v) NaCl (reaction buffer). The specimens used were, mammary normal and transformed tissues, diagnosed as fibroadenoma,

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obtained from the Tissue Bank of the Sector of Pathology of the Keizo Asami Immunopathology Laboratory at the Federal University of Pernambuco, Brazil. In lectin histochemistry, sections (4 μm) of specimens were deparaffinized in xyline and hydrated in graded alcohols (100–70%). Tissue slices were treated with a 0.1% (w/v) trypsin solution for 2 min at 35°C and then incubated with a solution of Con-A–QD (50 μg/mL) and QD/Glut, prepared or not with Evans Blue solution (5 mg%). The samples were prepared in sets described as follows: (1) NEB (normal tissue sections treated with EB); (2) NEB–Con-A–QD (normal tissue sections treated with EB and conjugated to CdS/ Cd(OH)2/Glut/ConA); (3) FIB–EB (fibroadenoma tissue sections treated with EB); (4) FIB–EB– QD (fibroadenoma tissues treated with EB and conjugated to CdS/Cd(OH)2/Glut QDs); and (5) FIB–EB–Con-A–QD (fibroadenoma tissues treated with EB and conjugated to CdS/Cd(OH)2/Glut/ ConA QDs). Analysing the confocal microscopic images of the fibroadenoma and normal tissue samples in the 500–535 nm range the following observations were made: (a) FIB–EB tissues presented a very faint luminescence; (b) FIB–EB–QD tissue samples showed a slightly bright luminescence in the green region. This emission showed a homogeneous pattern which is expected for a non-specific labelling which can be the result of the occurrence of covalent attachment between glutaraldehyde and the amino acid residues, such as lysine in cell proteins, through Schiff bases; (c) in FIB–EB–Con-A–QD tissue samples all the samples where brightly luminescent and showed a specific labelling corresponding to the internal structures of the mammalian ducts; (d) norm-EB tissue samples presented no detectable luminescence; (e) NEB–Con-A–QD tissue samples showed luminescence and also showed selected structures brighter than the overall tissue sample (data not shown). Figure 26.20 presents the fluorescence pattern of the normal and fibroadenoma tissues obtained under the same acquisition parameters.

1a

1b

1c

1d

1e

1f

Figure 26.20 Human mammary tissue diagnosed as fibroadenoma (Fib). (1a) Fib treated with Evan’s Blue solution (EB); (1b) Fib incubated with QDs diluted in EB (FIB–EB–QD); (1c) Fib incubated with Con-A–QD conjugate diluted in EB (FIB–EB–Con-A–QD). (1d), (1e) and (1f) are the intensity maps of (1a), (1b) and (1c), respectively. The bright pattern observed in (1c) is related to regions of high glucose/mannose expression in the tissue.

In summary, the fluorescence intensity of QD–Con-A stained tissues showed different patterns which reflect the carbohydrate expression of glucose/mannose in fibroadenoma when compared to the detection of the normal carbohydrate expression. The pattern of inespecific labelling of the tissues with QD-Glut is compared to the targeting driven by the Con-A lectin. These results, although preliminary, show a very promising tool in the detection of tumour differentiation in human mammary tissues. It is important to note that in both applications, due to the differences that rise in neoplastic processes (such as metabolic rates and regimen, cell membrane permeability and fluidity), the fluorescence intensities and patterns are quite different for healthy and neoplastic cells. The neoplastic mechanisms almost always result in cancer. These results show that a simple procedure of synthesis, functionalization and incubation with healthy and neoplastic cells of quantum dots at physiological conditions may represent a potential tool for fast and precise diagnostics of different kinds of cancer.

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