March 6, 2006
Self-Illuminating Quantum Dots Eliminate Need for Light Source in Imaging
An important limitation in the use of quantum dots for in vivo imaging applications has been the need to shine a light directly on them in order to detect where they have gone in the body. Now, however, researchers at Stanford University’s Center for Cancer Nanotechnology Excellence have developed a quantum dot-protein conjugate that generates its own light on the spot. This new self-illuminating quantum dot not only eliminates the need for an external light source, but also increases the sensitivity of in vivo imaging applications.
Reporting its work in the journal Nature Biotechnology, a team led by Stanford colleagues Jianghong Rao, Ph.D., and Sanjiv Gambhir, M.D., Ph.D., describes how it uses bioluminescent light generated by an engineered enzyme to trigger the normal fluorescent light signal from a typical quantum dot. This enzyme, attached via a chemical linker to the quantum dot, generates light when it catalyzes a chemical reaction between a specific organic molecule known as coelenterazine and oxygen. Both the enzyme and coelenterazine come from the marine organism known as the Sea Pansy, which is found frequently along the Florida coast, among other places.
To form the self-illuminating quantum dots, the researchers started with Sea Pansy luciferase, an enzyme related to a similar protein that fireflies use to generate their bioluminescent mating signal. The investigators chose this particular luciferase because the blue light that it generates closely matches the optimal absorption frequency of polymer-coated cadmium selenide/zinc sulfide core-shell quantum dots. They also developed a variant of this protein that is more stable in serum and more efficient at producing light. This stability is critical, since the protein coupled to the quantum dot must be stable in the harsh environment of the animal serum.
When the researchers mixed the enzyme-quantum dot hybrids with coelenterazine, they observed the emission of blue light, the result of enzyme-generated bioluminescence, and red light, from the quantum dot. When they repeated this test in serum, blue light emission nearly vanished – hemoglobin in serum absorbs any stray blue light efficiently – but the quantum dot’s red light emission were barely affected. This result shows that the quantum dot can effectively produce light through bioluminescence resonance energy transfer (BRET).
As a first proof of principle experiment, the researchers first injected the hybrid quantum dots into the shoulder of a mouse, followed by an injection of coelenterazine into the mouse’s tail. As a control, the researchers also injected the modified enzyme without a linked quantum dot into the opposite shoulder. Fluorescence imaging readily detected the hybrid, but not the enzyme. Subsequent experiments with four different hybrids, each with the same enzyme but with a quantum dot that emits a unique color of light, showed that the four quantum dots could be easily distinguished within the animal.
In a final experiment, the researchers added a peptide known as R9 to the surface of the quantum dots. The addition of the R9 peptide was intended to increase the efficiency of cellular uptake, and in fact, this construct was efficiently taken up by glioma cells. The labeled cells, when injected into mice, were readily spotted using fluorescence imaging. Because the addition of R9 had no effect on light emission, it should be possible to also add tumor-targeting agents to these hybrids.
These new class of nanoparticles should allow marked improvements in sensitivity and multiplexing in applications with living subjects. These improvements should allow many more applications than currently possible with quantum dots for imaging cancer specific targets.
This work, supported in part by the National Cancer Institute, is detailed in a paper titled, “Self-illuminating quantum dot conjugates for in vivo imaging.” An abstract of this paper is available through PubMed.