Biological Applications of Fluorescent Quantum Dots

Colloidal quantum dots are semiconductor nanoparticles, which are dispersed in a solvent. Quantum dots have peculiar properties, dependant on their size and material. In particular, they are fluorescent. Because of their broad excitation spectra, narrow emission spectra, tunable emission peaks, and reduced photobleaching they are interesting candidates for labeling. This article gives some examples on how they have been used so far in (cell-)biological applications.
Colloidal semiconductor nanoparticles are crystalline clusters of a few 100 to a few 1000 atoms and have thus the size of a few nanometers (Fig. 1). These particles are fluorescent and the wavelength of their fluorescence depends strongly on their size. The color of the fluorescence can be tuned by the size of the particles and the material and ranges from the UV to the IR (Fig. 2). Since they have broad absorption spectra they can be exited at any wavelength shorter than their emission-wavelength. Excited semiconductor nanocrystals emit light with narrow emission spectra (< 30 nm fwhm).
A further exceptional property of these quantum dots is their reduced tendency to photobleach in comparison with biological dyes. Thus they are of great interest for all kind of labeling studies. For a general introduction see Reference [1].

Solubility in water solutions
Colloidal semiconductor nanocrystals of different materials, such as CdS, CdSe, CdTe, and CdSe/ZnS, can be routinely grown in solution by several approaches. A common synthesis route that yields highly monodisperse nanoparticles is the thermal decomposition of organo-metallic precursors in presence of stabilizer molecules [1]. The nanoparticles obtained in this way are coated by a monolayer of nonpolar molecules (the stabilizer molecules) and are thus not soluble in aqueous solutions. As almost all biological reactions take place in aqueous solution, it is necessary to make these nanocrystals water-soluble. Several methods exist for converting hydrophobic nanoparticles into hydrophilic ones. Most of them rely on the exchange of the stabilizer molecules. However, recently a new procedure has been introduced, in which the hydrophobic nanoparticles are embedded in an amphiphilic polymer-shell. The particle is caged by the hydrophobic part of the polymer and the hydrophilic parts point towards solution and ensure in this way water-solubility. The polymer-coating method is very general and can be applied as well to fluorescent semiconductor (e.g. CdSe/ZnS) as to magnetic (e.g. CoPt3, Fe2O3) nanoparticles [2].
Toxicity and Bio-conjugation
Although the core of the semiconductor nanoparticles contains toxic materials (e.g. cadmium), their effect on living cells seems to be relatively small. This can be explained by the fact that the toxic core is embedded in a variety of different shells, which prevent its contact to the environment. Quantum dots ingested by cells stay inside the cells for long periods (> 1 week) and seem not to influence their behavior. However, the critical concentrations still have to be determined. To use nanoparticles for labeling experiments it is necessary to functionalize their surface to achieve specific interactions between the fluorescent particles and the molecules of interest (Fig. 3). Conjugation of quantum dots with biological molecules is well established and has been demonstrated for a whole variety of molecules, including peptides, proteins, the lectin wheat germ agglutinin, or antibodies, and DNA. As colloidal quantum dots have equal or even bigger physical dimensions than biological molecules, the preservation of the functionality of quantum dot-labeled molecules cannot be taken for guaranteed. However, in most reports, no or only marginal effects of the quantum dot labeling on the molecular functionality have been reported.
Labeling of cell-compartments
The fluorescence labeling of compartments inside cells involves the problem of introducing the antibody-quantum dot conjugates into the cells. As antibodies conjugated to classic organic fluorophores cells have to be fixed and permeated. The so-treated cells are then typically incubated with primary antibodies and after a washing step with quantum dot-conjugated secondary antibodies, which target the primary antibody. Since the direct conjugation of antibodies to quantum dots is laborious, often biotinylated secondary antibodies are used, which are finally labeled by streptavidin-quantum dot conjugates via the biotin – streptavidin interaction. Another benefit of this strategy is, that a quantity of different biotinylated antibodies is commercially available and can easily be linked to the streptavidin-quantum dot conjugates (Fig. 4, [3]). Maybe the biggest advantage of using quantum dots instead of conventional fluorophores for this type of experiments is the possibility to image many different colors in parallel due to the sharp emission spectra without red-tail.
Labeling of membrane-proteins
The labeling of surface compartments of living cells is possible without permeating the cell membrane, as the quantum dots do not need to enter the cell. The Rosenthal group has for example imaged the location of serotonin transporter proteins in this way (Fig. 5, [4]). Another example for this technique is the detection of human epidermal growth factor receptor 2 (Her2) on the surface of breast cancer cells. The biggest advantage of quantum dot labeling of living cells is the reduced tendency to photobleach. In this way the dynamics of surface bound proteins can be investigated over extended periods of time. Such time-resolved experiments have been recently performed [5, 6]. Dahan et al. traced the diffusion of a neurotransmitter receptor along the cell membrane, and Lidke et al. followed the endocytotic pathway of the erbB1-receptor for the epidermal growth factor (EGF). These experiments demonstrate very well the potential of quantum dots for time resolved studies of single membrane-bound proteins.
Dubertret et al. injected quantum dots into a single cell at several stages of Xenopus embryos [7]. As the nanoparticles distribute among the daughter cells after cell division, the progeny of the injected cells could be visualized until the late tadpole stage of the embryos. When one cell from a two-cell embryo was injected, the quantum dot fluorescence was confined to the offspring of the injected cell, i.e. only the half of the embryo was fluorescently labeled. Quantum dots offer several advantages for such fate-mapping studies (Fig. 6). First, they are relatively biocompatible, and second, long-term studies are possible because of their reduced tendency to photobleach.
Nanocrystals and living cells
When cells are exposed to quantum dots they start to ingest the nanoparticles in a nonspecific way. The authors of a variety of publications agree, that the nanoparticles are internalized via endocytosis, are incorporated in vesicles, and are transported to regions near the nucleus. Semiconductor nanoparticles can also be used in an indirect way to follow the pathway of cells. Cells were cultured on a substrate coated with a homogenous layer of a marker, in this case colloidal gold. As cells migrate on the marker substrate, they crawl across the layer, ingest the marker, and leave behind a trail free of marker, which is called phagokinetic track. The trails in the layer of colloidal gold were visualized by dark-field microscopy. Recently this method has been revived by using fluorescent semiconductor nanoparticles as marker. In this way the homogeneity of the layers could be improved and now phagokinetic tracks can be imaged with fluorescence microscopy. In particular a two-dimensional in vitro invasion assay has been realized with this method. Phagokinetic tracks were recorded with different cell lines [8]. Two distinct behaviors of cancer cells have been observed, which can be used to further categorize these cells. Some cancer cell lines demonstrated fibroblastic behavior and created long fluorescent-free trails as they migrate across the quantum dot layer, whereas other cancer cells left only small clear zones of varying sizes around their periphery. In this way it is possible to discriminate between noninvasive and invasive cancer cell lines, by using the size of the zone cleared of nanoparticles per cell as an indicator for the cell’s invasiveness: The bigger the area cleared of nanocrystals per cell, the more invasive the cell is.
Conclusions
Well-established syntheses of nanoparticles in nonpolar solvents allow for having colloidal particles of different materials with controlled size distribution. These particles can be rendered water-soluble by several procedures and they can be conjugated with different biological molecules. It has been demonstrated that such water-soluble nanoparticles can be biocompatible. In particular, colloidal semiconductor nanoparticles, called quantum dots, have interesting fluorescent properties. Due to their improved photo-stability real-time observation of ligand-receptor interaction, time-resolved single molecule studies and visualization of molecular transportation in living cells have been demonstrated with quantum dots. In the near future the use of hybrid structures of different nanoparticle materials could be used to target specific cells and for drug-delivery applications. For example, a combination of the fluorescence of semiconductor nanoparticles with the magnetic properties of cobalt-platinum nanocrystals could be useful to direct drugs, which are attached to this construct, towards regions of interested in magnetic fields, while at the same time monitoring the process using fluorescence microscopy.