Applications of quantum dots in medical diagnostics
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What are Quantum Dots[1]
Quantum dots are fluorophores—substances that absorb photons of light, then re-emit photons at a different wavelength. These nanochrystals consist of a core of a semiconductor material, enclosed within a shell of another semiconductor that has a larger spatial gap. The most common QD core is composed of CdSe with a shell of a high bandgap material such as ZnS. The size of a typical QD vary 2-10 nm in diameter, a size that allows them to interact with biomolecules on a one-to-one basis. Monodisperse QDs such as CdSe are usually synthesized by injection of liquid precursor into hot (300 C) nonpolar coordinating organic solvent (tri-n-octylphosphine oxide and hexadecylamine) and the size and shape of QDs are controlled by the temperature and crystal growth duration as well as ligand molecules employed in their synthesis. The outer semiconductor shell ZnS is epitaxially grown around the core.
Operation of QDs
When QDs are hit by light they absorb a photon with higher energy than that of the band gap (the separation between electronic energy levels of a material) of the composing semiconductor and release a new photon with a lower energy and longer wavelength. An incoming photon is absorbed from the ground state of the material (E0) and causes the material to go into an excited energy state (E1) (up arrow). The energy between E0 and E1 is discrete and any excess energy the incoming photon was carrying will be dissipated as heat or vibrations (diagonal arrow). As the system relaxes back to its ground state (down arrow) it will release a new photon that will have less energy and a longer wavelength than the original photon. The emission band width for QDs is 20-40 nm.
Preparation and functionality
When preparing QDs it is important to keep in mind the physical properties it will get as a finished compound. For instance, biocompatibility, which is the ability of a material to perform with an appropriate host response in a specific application. It is also defined as the quality of not having toxic or injurious effects on biological systems. Examples of how to make QDs biocompatible are silanization, (covering the surface with silane like molecules) and surface exchange with bifunctional molecules. Another strategy is to encapsulate the QD in for instance within phospholipids. The functionality of QDs is achieved by giving the surface layer (ZnS) different characteristics. These characteristics enable the QDs to adapt to the desired application by conjugating to a recognition moiety, like antibodies, peptides, oligonucleotides or aptamers. The functionality depends on the biomolecule of interest
Use of Quantum dots in in vitro nanodiagnostics
Certain biological molecules can recognize and bind to other molecules with extremely high selectivity and specificity; key and lock. This is how QDs recognize their target.
Single particle detection
Because of the photostability and the brightness of the QDs you can study a particle over a long period of time. This can be very useful in diagnostic. The first detection of neurotransmitter acetylcholine was done by using QDs.
Immunohistochemistry
Immunohistochemistry refers to the process of localizing proteins in cells of a tissue section exploiting the principle of antibodies binding specifically to antigens in biological tissues. Lack of or defect proteins can cause disease and detection of these can be useful. Some of the early and most successful uses of QDs were in immunofluorescence labeling of fixed cells and tissues. Labeling of mortalin (heat shock protein) using QD showed different staining patterns between normal and cancer cells. Immunohistochemical staining is widely used in the diagnosis of abnormal cells such as those found in cancerous tumors. Specific molecular markers are characteristic of particular cellular events such as cell division or cell death (apoptosis) Testing of QDs showed that they are much better and brighter fluorescents signal compared to organic dye and that QDs survive typical tissue-mounting procedures. QDs for immunohistochemical labeling are much more sensitive than conventional dyes, where better signal was obtained than to enzymatic signal amplification methods.
Immunoassays
Immunoassay is a biochemical test that measures the concentration of a substance in a biological liquid using the reaction of an antibody or antibodies to its antigen. This can be very useful in diagnostic. Scientists developed a flour immunoassay for the detection of prostate specific antigen(PSA).[2] As you can see on the figure you can use that the intensity is proportional to the concentration. PSA detection was achieved in both solid and liquid phases and visualization of individual molecules was also possible. With use of QD you can also perform multiplex immunoassay using QDs with different size. Different size means different color. The excitation of all the QDs were done by using a single wavelength and the signals was detected simultaneously. This is much simpler than using several different organic fluorophores, were you need different excitation wavelengths.
Nucleic acid detection[3]
QDs can also be used in fluorescence in situ hybridization as labels for detection of DNA. This use of QDs overcomes the two problems of classic organic fluorescent labeling: Cleavage DNA molecules due to photo bleaching and subsequent formation of free radicals. They also allow two color determination of the orientation of a single molecule.
Detection of genetic polymorphism
QDs can be used for simultaneous detection of unknown multiple single nucleotide polymorphism (SNPs). A single-nucleotide polymorphism is when a DNA molecule differs in one base pare compared to a different one. Here different QDs were conjugated to each of the four nitrogenous bases. Sequentional addition of four QDs linked monobase results in their specific binding, via base pairing, to complementary sites in the target. By using electrochemical methods you can detect them in a current versus potential plot.
Imaging
Imaging is a powerful tool in medical diagnostics, allowing us to see inside the body in a simple and pain free way. Organic fluorophores have so far been used to label the part of the body that is to be investigated. The development of QDs now makes it possible to get better and more detailed images. The emission spectra from the QDs depend only on their size.[5] QDs with small diameters (ca 13nm) emits in the blue area, while QDs with larger diameters (ca 24nm) emits in the red area. All though different sized QDs emit varying wavelengths, they can be excited simultaneously by one wavelength. By using QDs of different sizes at the same time we are able to make multicolor images. This is possible because QDs have narrow and intense emission bands, so the signals do not overlap.
By attaching different biomolecules, we can assure that the QDs accumulate in the organs or cells of interest. The binding of biomolecules would also make imaging of specific cellular organelles possible, such as the nucleus, mitochondria and the cytoskeleton. By combining QDs of different size with the right biomolecules, detailed images can be produced. This makes it easier to study complex biological systems, and could be important during diagnostics.
Most research done on QDs have been in vitro, but QDs have been used for in vivo imaging. This was done in mice expressing human cancer. QDs with tumor-targeting antibodies were injected into the mice, and the resulting images showed clearly where the tumor was located. Small QDs can be used to get images of the lymph nodes and cancer cells during metastases. This is very useful in diagnostics, when it is determined if the cancer cells have spread to the lymphatic system.[6] There is still some research left before QDs can be used for imaging in humans.
The QDs does not only emit visible light, but also near-infrared.[7] This makes imaging of deeper tissue possible, where light scattering is a problem. Water-soluble QDs have been injected into mice skin and adipose tissue. These tissues have high light scattering properties, but in spite of this the QDs could easily be seen using multiphoton microscopy. Their high photostability makes QDs suitable for imaging for longer periods of time. The water-soluble QDs were stable for over 9 months, indicated by fluorescence correlation spectroscopy.
In the last few years the QDs have been made even smaller.[8] This makes them able to enter smaller parts of the body. These QDs were injected into the bloodstream of mice, and they did not just follow the bloodstream; they escaped it, moving toward tissue. It seems like QDs have unlimited potential, when it comes to colors, stability and navigable ability. The development of QDs has benefits that will further improve imaging, which is so important for medical diagnostics.
Therapy
QDs also have therapeutic applications. The most promising application is drug delivery. QDs can be loaded with drugs, and with suitable biomolecules attached they reach their specific target. The QDs will release their content in response to something in the environment, specific for the target. Photodynamic therapy uses singlet oxygen, excited from a photosensitizer activated by light, to degrade cellular components, for example in cancer cells. QDs can be used in this treatment by being the photosensitizer itself, or they can be the energy donor, activating another photosensitizer. The nature of QDs makes us able to monitor different components in a cell culture over extended periods of time. This is used for example in drug discovery, where possible drugs are monitored simultaneously.
Technical limitations in use of QDs[10]
An increased size of QDs due to functionalizations (up to 100nm) limits their ability to reach targets within multi-component molecular complexes. Inherent properties of the QDs themselves may also limit their use as for instance the CdSe/Zns QDs, that do not emit in the infrared region which in turn makes them unsuitable for whole blood analysis. Still they can be used in serum testing and other body fluids.
Toxicity concerns
The basic components of QDs are in fact quite toxic, and are therefore capped in hydrophilic coating. Problems with the coating can lead to the leakage of for instance cadmium ions which are very toxic to humans. Dispite these limitations QDs have a large potential in medical diagnostics.
References
- ↑ From diagnostics to therapy: Prospects of quantum dots; Hassan M.E Azzazy, Mai M.H Mansour, Steven C. Kazmierczak (2007)
- ↑ Härmä H, Soukka T, Lövgren T. Europium nanoparticles and timeresolved fluorescence for ultrasensitive detection of prostate-specific antigen. (2001)
- ↑ Detection of single DNA molecules by multicolor quantum-dot end-labeling (2005)
- ↑ www.aist.go.jp/aist_e/aist_today/2006_21/pict/p22_2.png
- ↑ Applications of nanomaterials inside cells; Jinhao Gao, Bing Xu (2008)
- ↑ Quantum Dots Could Guide Surgeons (NIBIB, Health & Education, February 2004) (http://www.nibib.nih.gov/)
- ↑ Applications of nanomaterials inside cells; Jinhao Gao, Bing Xu (2008)
- ↑ Smaller Quantum Dots Improve In Vivo Imaging (http://nano.cancer.gov/news_center/nanotech_news_2006-02-21b.asp)
- ↑ www.nano.org.uk/news/july2008/1476.jpg
- ↑ From diagnostics to therapy: Prospects of quantum dots; Hassan M.E Azzazy, Mai M.H Mansour, Steven C. Kazmierczak (2007)
