Thomas M. Jovin is chairman of the Department of Molecular Biology, Max Planck Institute for Biophysical Chemistry, D-37070 Göttingen, Germany tjovin@gwdg.de
Two reports demonstrate the specific labeling of cellular constituents with fluorescent quantum dot probes conjugated covalently or electrostatically to antibodies and streptavidin.
The phrase "less is more," coined by Mies van der Rohe to describe mid-20th-century architecture, epitomizes the task facing researchers engaged in developing new types of probes for imaging applications. The challenge is to identify or devise the smallest probes possible that exhibit the highest selectivity, sensitivity, spectral versatility, stability, and capacity to penetrate cells and organelles. In recent years, quantum dots have come to the fore (for excellent and timely reviews, see refs. 1 and 2) as promising alternatives to chemical fluorophores3 and visible fluorescent proteins4. Now, four papers published in this issue5,
6 and elsewhere7,
8 demonstrate the successful application of bioconjugated quantum dots for labeling cells and macromolecular constituents of cells.
Despite the interest in quantum dots for use in imaging applications, it has been difficult to meet the requirements for reproducible fabrication and to overcome fundamental difficulties posed by the biological milieu. These include first, prevention of quenching of quantum dot emission in an aqueous environment; second, elaboration of strategies for the conjugation or adsorption of molecules of interest; third, suppression of nonspecific binding and aggregation; and fourth, provision for biological inertness (that is, absence of cellular toxicity). Of all these problems, the first has been particularly vexing; to use the vernacular associated with the whimsical banking institutions of contemporary Argentina, incompletely coated quantum dots tend to function as photon "corralitos" (photons in, no photons out).
The paper by Jaiswal et al.5 describes a generalized approach for conjugation of quantum dots by an electrostatic mechanism (Fig. 1A, B) and their subsequent use on fixed and live cells in numerous cell biological applications. In a second report, Wu et al.6 achieve immunofluorescent labeling of cells using quantum dots (4−10 nm in diameter) coated with a hydrophobically modified polyacrylic acid (Fig. 1C) and conjugated to immunoglobulin G and to streptavidin so as to achieve the specific labeling of cell surface, cytoskeletal, and nuclear proteins. Together with other recent reports featuring the use of quantum dots bearing co-adsorbed peptides and polyethylene glycol7 or encapsulated in phospholipid block−copolymer micelles8, these publications demonstrate that appropriately designed quantum dots are very photostable and nontoxic, and exhibit high selectivity with little background from nonspecific binding. In other words, the limitations cited above and previously9 have been overcome: quantum dots are certifiably useful.
Figure 1. Three strategies for bioconjugation to quantum dot probes.
The quantum dot consists of a CdSe core passivated by a ZnS shell. The surface cap is negatively charged, with carboxylate groups from either DHLA or an amphiphilic polymer (40% octylamine modified polyacrylic acid). Proteins are conjugated to the DHLA−quantum dots electrostatically either (A) directly or (B) via a bridge consisting of a positively charged leucine zipper peptide (zb) fused to recombinant protein G (PG). The latter binds to a primary antibody (Ab) with specificity for the intended target. In (C), antibodies, streptavidin, or other proteins are coupled covalently to the polyacrylate cap with conventional carbodiimide chemistry. (A, B) from Jaiswal et al.5; (C) from Wu et al.6
The quantum dots used in the present studies were colloidal inorganic semiconductor nanocrystals consisting of a CdSe core and a ZnS cap. The absorption spectra of such particles are very broad, extending from the ultraviolet to a cutoff wavelength in the visible spectrum, the position of which is dictated by size (larger size results in longer wavelengths) and by the composition of the core. Emission is confined to a narrow band (typically 20−40 nm full width at half maximum) likewise centered at a wavelength characteristic of the particle size. In combination, these spectral properties—unmatched by any known organic dye fluorophore—permit the systematic generation of probes that have different biochemical specificities and can be excited and detected simultaneously. Quantum dots are rendered water compatible by providing a shell of functionalized silica or linkers, such as mercaptoacetic acid, dihydrolipoic acid (DHLA)5, or modified polyacrylic acid6, for conjugation to macromolecules and ligands (Fig. 1).
In the experiments of Jaiswal et al., HeLa cells labeled by endocytosis of quantum dots coated with DHLA retained the internalized quantum dots and continued to grow for more than a week. The absence of deleterious effects was also confirmed by the unaltered cAMP signaling of quantum-dot-labeled Dictyostelium discoideum. The authors used two approaches for the specific labeling of live cells (Fig. 1A, B): the first involves the use of biotinylated primary antibodies and quantum dots conjugated to avidin, and the second is based on conjugation of the primary antibody to the quantum dot via a recombinant protein G−peptide construct or an avidin bridge. The researchers' data indicate that different cell populations labeled with quantum dots of distinctive emission colors (520 and 570 nm) can be readily distinguished. Nonspecific binding seems to be minimal.
In the second paper, Wu et al. demonstrated the successful targeting of quantum dots to a cell surface receptor (Her2, the orphan receptor tyrosine kinase often overexpressed in human breast cancer), cytoskeletal components (microtubules, actin filaments), and nuclear antigen. All three cellular compartments could be accessed in cultured cells as well as in tissue sections. Quantum dots of different colors (535 nm and 630 nm) were used simultaneously and also in combination with an Alexa Fluor fluorophor. The latter was considerably less photostable than the quantum dots, the emission of which remained virtually unaltered over minutes of exposure.
The virtues and applications of quantum dots are best assessed in the general context of fluorescent and luminescent probes of molecular structure and function. For real-time observation of macromolecules and compartments of living cells under the fluorescence microscope, expression systems are generally preferred over microinjection of molecules labeled in vitro. The tags currently available for genetic fusion with target proteins include the vast array of visible proteins derived from marine organisms4, single-chain antibodies specific for permeable fluorophores10, and small peptide sequences incorporating a tetracysteine motif recognized by an biarsenylated heterocyclic probe4. It remains to be established whether it is possible to incorporate into such strategies quantum dots or other molecular clusters (such as phycobiliproteins11) or nanostructures (such as up-converting phosphors12) exhibiting strong fluorescent signals and high photostability.
Other questions not touched on in the current studies also need to be addressed. Does the finite size of quantum dots impede their access to targets in confined cellular compartments or multicomponent molecular complexes? Do the "Alice in Wonderland" properties of quantum dots as quantum structures (such as blinking) limit their utility as quantitative probes? Are quantum dots useful for fluorescence lifetime (FLIM) or fluorescence resonance energy transfer (FRET) measurements of molecular association and conformation? And can quantum dots be excited in general by multiphoton techniques?
The good news is that these promising reagents are finally becoming available to the general scientific community from commercial sources, a process that may be accelerated by the introduction of alternative quantum dot cores and shells13. We can anticipate that the vision of high-throughput screening, highly multiplexed bioassays, nanoengineering, and other exotic applications of quantum dots1,
2 will also soon become a reality.
