by Zong-huan Li and Hong-lei Chen

Quantum dots are a class of nanoparticles with excellent optical and chemical properties that are applied widely in the biomedical imaging areas. Here we briefly introduce what quantum dots are and how they can be used as labels for molecular detection and live cell imaging. We also conclude by focusing on the advantages and the drawbacks of quantum dots.

Over the past few decades, molecular imaging has been greatly developed. The label used is one of the most important components in order to achieve successful imaging. With the advancement of nanotechnology and nanoscience, Quantum dots (QDs) are emerging as inspiring labels in biomedical fields. QDs, as inorganic fluorescence which overcomes most of drawbacks of conventional dyes, are excellent labels that are used for detecting molecules and for live cell imaging, as well as in many other biomedical fields. QDs have been widely used in almost every biomedical area including molecular detection, live cell imaging, imaging in vivo and their clinical application. In this review, we mainly discuss the first two areas and their advantages and drawbacks.

What are QDs?

QDs, also named fluorescent semiconductor nanocrystals, have a three-layer structure that is composed of two kinds of heavy atom from the II/VI or III/V group elements [1]. Due to the difference in the number of the atoms, QDs are of different sizes ranging from 2 nm to 10 nm.

Generally, as mentioned above, a single QD is comprised of three layers, namely the core, shell and polymer coating [2]. Cadmium selenide (CdSe) QDs are very common. The primary structure of this QD is just a CdSe core, which is unsuitable for biomedical research because of toxicity and low quantum yield. After capping the core with a ZnS shell, the toxicity is reduced but not removed and the quantum yield is enhanced dramatically. QDs with hydrophobic ligands synthesised in organic environments are only soluble in nonpolar solvents. To enable further applications in biomedicine, QDs must be soluble in physiological conditions. Several strategies, for example ligand exchange, amphiphilic polymer coatingsand silica encapsulation enable a third layer to provide good solubility.

This three-layered structure is still not sufficient. For their wide applications in biomedicine, QDs need to be modified by specific molecules so that they can monitor the relevant biological processes in real time. After conjugation with molecules using non-covalent or covalent methods, QDs show a higher quantum yield and reduced aggregation, as well as better biological activity.

3 Molecular detection

Protein

With the completion of the human genome project, the postgenomic era is investigating the structure and function of each protein as well as their interactions. QDs provide an efficient way of clearly visualising these processes. By conjugating QDs with a certain protein, for example antibody, peptide or other small molecule, the QD-conjugates can target the specific protein [3]. The events occurring on the protein can then be illustrated distinctly.

There are so many techniques that can be used to detect proteins, for example immunohistochemistry (IHC), Western blot and fluorescence resonance energy transfer (FRET). However, there are several shortcomings of these conventional methods. To compensate for the disadvantages of these techniques, researchers have combined them with QDs. Compared with conventional IHC, the combination of QDs with IHC is a cheap, time-saving and high-throughput method. We focus on the study of caveolin-1, a protein distinctively expressed in different cancers including cervical cancer, tongue squamous cell carcinoma and non-small cell lung carcinoma, where the combination of IHC and QDs have yielded significant results.

Not only single molecular imaging, but multicolour imaging can also be carried out with QDs. Different sizes of QDs with different emitation wavelengths can be excited by a single excitation wavelength, making it possible for multiple targets to be detected simultaneously without signal overlap. Investigations of different proteins and their interactions are greatly accelerated, so three-, four-, five- and six-colour real time imaging has also been successfully developed.

FRET is a highly sensitive way to monitor protein-protein interactions, receptor-ligand binding, DNA hybridisation and sequencing, as well as conformation changes of protein and oligonucleotide [2]. Because of their broad absorption spectra and narrow emission spectra, QDs are excellent FRET donors and make the FRET process handy. QDs can also be modified as acceptors in FRET, but they are not ideal and studies are limited.

Nucleic acid

For the FRET technique mentioned above, conjugated QD-based fluorescence in situ hybridisation (FISH) is another common method used to examine nucleic acids in biology. The target DNA or mRNA can be detected specially using modified QDs with a chain of complementary base sequences.

We mainly focus on DNA or RNA detection in different viruses associated with cancer. High-risk HPV in cervical cancer, oral squamous cell carcinoma and EBV in nasopharyngeal and gastric carcinoma have been detected by QD-based FISH and conventional ISH. Compared with conventional ISH, QD-based FISH has advantages including long-lasting fluorescence, strong emission, improved sensitivity and excellent specificity. After conjugation with mRNA, QDs are microinjected into nuclei to track route and further analyse possible function (4). By this method, both DNA and mRNA detection can be easily carried out.

Live cell imaging

QDs are a novel kind of fluorescent label with broad ranges of applications in live cell imaging. Complex biological processes are basically a network of different molecules and their interactions. Different QDs are designed to tag different sites or molecules in live cells to further understanding.

There are types of protein which are located on the live cell membrane and act as receptors or signal transductors. These are important windows in order for cells to communicate with the surrounding environment and need to be investigated in depth. In order to label surface protein on the membrane efficiently, QDs need to be conjugated to biotin ligase. Then specific antibodies are linked to QD conjugates. For example, QDs conjugated with EGF can selectively bind with EGF receptors on the cell membrane, monitoring the interaction in real time.

Conjugated QDs show great potential for labeling molecules in the cytoplasm and the nucleus. For successful subcellular imaging, when what matters most is their relatively large size compared with the biomolecules, there are two ways of getting QDs into cells, namely endocytic uptake and independent of endocytosis. QDs taken up by endocytosis are trapped in vesicles and move restrictively along the cytoskeleton towards the nucleus. QDs are only used to monitor live cells and their migration route. To illustrate the microenvironment in the cytoplasm, it is important to label molecules in the cytoplasm using invasive techniques, such as scape loading, microinjection and electroporation. Using these methods, QDs enter the cytoplasm efficiently and facilitate the labeling of specific molecules. But the drawback is that cells are injured and can die easily, and the culture environment must be optimum.

Advantages and drawbacks

Compared with conventional dyes or fluorescent proteins, QDs have many advantages which are determined by their special composition and structure. Briefly, there are six main points as follows:

(1) Size-dependent emitting wavelengths. By controlling the number of dots in each QD, the emitting wavelengths differ as the diameter changes.

(2) Broad excitation wavelengths, narrow emitting wavelengths and large Stokes shift. With this property, QDs can be excited by any wavelength while emit a narrow band of wavelength. Different diameters of QDs can be excited by a single excitation light, which is the theoretical basis of multicolour imaging. A large Stokes shift (difference between peak absorption and peak emission wavelengths) means nearly no spectrum overlap between the excited state and ground state, with reduced autofluorescence which increases sensitivity.

(3) High fluorescence intensity. Because of the quantum effect, QDs have high fluorescence yield, which makes single molecular imaging possible.

(4) Long fluorescence lifetime. QDs have a fluorescence lifetime range from 10 to 50 ns. This property enables QDs to be bright and stable enough for long time imaging in vitro or in vivo.

(5) High resistance to photobleaching. By unique design and modification strategies, QDs have higher stability compared with conventional dyes, in accordance with their longer fluorescence lifetime, which enables QDs to be ideal biosensors.

(6) High specificity. Tagging QDs with antibodies, peptides or other molecules, QD-conjugates can target specific sites or molecules in biological processes.

Although QDs have so many unique properties and have been used widely in biomedicine, they are not flawless. To be the ideal probes in biomedical imaging, the labels must be simple, safe and specific. The strategies of synthesis and modification of QDs are complex, which limits their further application. Furthermore, QDs are much larger than biomolecules. So it is not easy for QDs to get into cells or nuclei. Without intervention, QDs get into cells or nuclei mainly by endocytosis, which means QDs are trapped by vesicles and transported to the peri-nuclear area through thecytoskeleton. The signals from this non-specific binding make the detection of low concentrations difficult. In addition, QDs are prone to aggregation which limits their further application in subcellular imaging.

With labels used in biomedicine, one of the parameters that must be noted is safety. QDs composed of heavy atoms are potentially toxic. The toxicity of QDs is associated with many physicochemical parameters including composition, size, concentration, contact time, shape, charge, surface coating and so on. The toxicity of QDs results from the heavy atoms and free radicals generated during excitation [2]. The CdSe core is toxic to cells. Through coating with a ZnS shell, their toxicity is obviously reduced but not eliminated. The toxicity of QDs is size-, time- and dose-dependent [5]. QDs with smaller size are more prone to get into cells than larger QDs, so larger QDs are less toxic. With the time prolonged and the concentration increased, the toxicity of QDs becomes evident. Thus large QDs used in research with low concentration in a short time may cause no detectable harm to the cell. QD metabolism and degradation within the human body is still largely unknown; QD use is limited for clinical applications. There is still a long way to go to increase applications of QDs in the clinic.

Conclusion

QDs as novel fluorescent labels have been widely used in biomedicine. Compared with conventional dyes, QDs have many unique properties. It is ideal to image with QDs for tracking a single molecule and for long-term imaging. In a sense, QDs are very important for molecular imaging but will never replace conventional dyes. To expand their use in the clinic, the toxicity must be eliminated.

Acknowledgements

This work was supported by grants from the National Natural Science Foundation of China (No. 30900652).

References

1. Alivisatos AP et al. Quantum dots as cellular probes. Annu Rev Biomed Eng 2005; 7:55-76.

2. Li ZH et al. Bioconjugated quantum dots as fluorescent probes for biomedical imaging. J Nanosci Nanotechnol 2011; 11:7521-36.

3. Rosenthal SJ et al. Biocompatible quantum dots for biological applications. Chem Biol 2011; 18:10-24.

4. Ishihama Y et al. Single molecule tracking of quantum dot-labeled mRNAs in a cell nucleus. Biochem Biophys Res Commun 2009; 381:33-8.

5. Michalet X et al. Quantum dots for live cells, in vivo imaging, and diagnostics. Science 2005; 307:538-44.

The authors

Zong-huan Li, Hong-lei Chen*

Department of Pathology

School of Basic Medical Science

Wuhan University

Wuhan 430071

P. R. China

*chenhlwhu@gmail.com