ABSTRACT
Apart from these intrinsic and unique quantum confinement properties, many of the QDs photophysical properties are competitive or superior to the ones of organic dyes. First, QDs are characterized by very high molar extinction coefficients, often exceeding 106 M-1 . cm-1 at the first excitonic absorption peak, and even much higher in the blue and UV regions of the optical spectrum [7]. Moreover, they absorb photons from a large range of wavelengths thanks to their wide and quasi-continuous UVvisible absorption spectra, allowing the excitation of different-sized QDs by a single light source [5], whereas organic dyes have a narrower absorption window, each of them needing to be excited with a specific wavelength. Second, QDs emit their fluorescence in the form of a narrow and symmetric emission peak, when emission peaks from organic dyes are usually broader and asymmetric, most of the time red-tailed (Fig. 6.1 shows Rhodamine 6G as an example of an organic dye). QDs are also brighter than organic dyes thanks to their efficient absorption, and an extreme photostability makes this brightness stable in time. QDs were found to be 100 times more resistant against photobleaching than Rhodamine 6G [8], and 380 and 3800 times more photoresistant than AlexaFluor and fluorescein isothiocyanate (FITC), respectively [9]. QDs fluorescence, being due to the decay of an exciton, also has a
much longer lifetime compared with organic dyes, typically ranging from 5 ns to hundred of nanoseconds [10]. (b)(a)
These differences of the optical and physico-chemical properties between QDs and conventional organic fluorophores have proved to be of great importance in biotechnology [3,4]. The possibility to excite multicolor QDs with a single light source opens breakthrough perspectives in such a field as biomedical imaging as biomedical imaging, where signal multiplexing is of considerable importance, especially for applications to parallel and high-throughput analysis of multiple biological parameters or simultaneous detection of numerous biomarkers [11,12]. In addition, brightness and stability
of QDs are of direct interest, since these properties are a necessary requirement for a fluorophore to be adapted to biological conditions, especially in single-molecule measurements [10,13,14], and in experiments of long-term tracking of different biological processes in living systems [15]. Finally, their long fluorescence lifetime allows for an enhanced contrast in time-gated biological imaging by reducing the contribution of the autofluorescence background commonly observed in biological conditions [16]. Owing to their size being similar to the one of biological macromolecules such as antibodies and nucleic acids, QDs’ advantages as a biolabel have indeed found a use in several biotechnology fields, through an integration of nanotechnology and biology that constituted a major advance in the relatively new field of nanobiotechnology [3,4]. At the heart of this integration lies the ability to build a physical interface between biocompatible QDs and biological objects through bioconjugation [17].QDs biocompatibility is achieved mostly through QDs’ synthesis itself followed, when necessary, by modifications of their surface functionalities. Biocompatible QDs may be synthesized in water (e.g. CdTe QDs) or using a synthesis in organic solvents followed by different surface chemistry approaches to make them water soluble (e.g. CdSe QDs) (see Chapter 2). When made biocompatible, QDs exhibit multiple crucial properties, such as a long-term water solubility, presence of accessible functional groups in order to make bioconjugation possible, lack of perturbation of the conjugated biomolecule and finally biological innocuousness [18]. Among the various water solubilization techniques, ligand exchange, in which hydrophobic ligands on the QDs surface are replaced by bifunctional molecules like cysteine or cysteamine, with an end able to connect with QDs and the other one being both hydrophilic and reactive to biomolecules, is widely used. It is also possible to create a hydrophobic interaction between these hydrophobic ligands and the hydrophobic end of an amphiphilic polymer, the hydrophilic end being responsible for water solubility and providing functional groups for bioconjugation [5,8,18,19]. Silanization, a variant of ligand exchange in which QDs are coated with silica-based compounds in order to make them water soluble has also received attention, as did electrostatic attraction and nanobead-based conjugation [11,20]. Several techniques have been explored to link
such biocompatible QDs with biomolecules. Among them can be cited passive adsorption, electrostatic interaction or standard covalent cross-linking approaches. The latter technique takes advantage of the hydrophilic surfactant shells reactive groups to realize the conjugation, through, for example, 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide (EDC) coupling of carboxylic acid-functionalized QDs or bifunctional cross-linkers coupling with thiol-containing ligands such as 4-(N-maleimidomethyl)-cyclohexanecarboxylic acid N-hydroxysuccinimide ester (SMCC) for amine-functionalized QDs [17,21]. The avidin-biotin system has also been described in the context of QDs bioconjugation with biomolecules, even if covalent linkages are preferred when size is an important factor [22,23].Hybrid materials made of biocompatible QDs and biomolecules result directly from these techniques and have been used in a very broad range of biological domains. Indeed, since the first use of QDs as biological labels by Bruchez et al. and Chan et al. [8,24], who managed to make CdSe QDs water soluble and to conjugate them with biomolecules, the number of biological studies involving QDs have increased dramatically [12,18,25,26]. Until now, the most prominent QDs for life science applications have been CdSe QDs surface-passivated with semiconductor material of wider bandgap, mostly ZnS, although CdTe, InP and InGaP QDs have also attracted some attention [27]. Other available cores such as ZnS, ZnSe, PbS, CdS and PbSe are not as common in biological applications [18]. Biological applications of QDs generally include sensing, imaging and the development of biomimetic systems. QDs have been used in the field of biosensing for immunoassays [28,29], nucleic acid detection [22], Förster resonance energy transfer (FRET)–based assays for the detection of various molecules such as proteases [30,31] and optical encoding for high-throughput screening [11]. QDs have also contributed to bioimaging through molecular and cellular labeling and cell tracking [15,32], singlemolecule fluorescence methods [10,26] or immuno-histochemistry [33]. The involvement of QDs in biomimetic systems has also been part of QDs’ role in nanobiotechnology [3,34,35].In this chapter an overview of CdTe QDs biological applications is provided. After describing the CdTe QDs themselves with respect to life science applications, notably by comparing them with other
biologically compatible QDs, various techniques of conjugation of these CdTe QDs with biomolecules will be presented. Applications of the resulting hybrid materials consisting of CdTe QDs and biomolecules will then be detailed for several domains of biology. 6.2 Cadmium Telluride Quantum Dots
The factors that influence the compatibility of QDs with their use in life science, for example, water solubility, presence of accessible functional groups, lack of perturbation of the conjugated biomol-ecule and biological innocuousness [18], are mostly dependent on synthesis, surface passivation and surface chemistry of the QDs. To date, the majority of studies involving an application of QDs in life sciences were realized with core/shell CdSe/ZnS QDs and CdTe QDs [27,36]. CdSe/ZnS QDs are normally synthesized according to a method initially developed by Murray and coworkers which is based on the pyrolysis of an organometallic reagent at a relatively high-temperature, using surfactants such as tri-n-octylphosphine oxide (TOPO) or hexadecylamine (HDA) [37]. Although this procedure has been improved multiple times, for example, by the replacement of the precusor dimethyl cadmium by the more stable and less toxic CdO [38], it still inherently produces QDs that are hydrophobic and that have to be made water soluble to avoid their precipitation or aggregation in biological conditions. The subsequent water solubilization steps can sometimes be time consuming and difficult and can impair QDs’ stability [39].
