Wu X, Liu H, Liu J, Haley KN, Treadway JA, Larson JP, Ge N, Peale F, Bruchez MP.
Immunofluorescent labeling of cancer marker Her2 and other cellular targets with semiconductor quantum dots.
Nat Biotechnol. 2003 Jan;21(1):41-6.
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Biology is at a wonderfully exciting, but vexing point. We have the sequences for all of the human genes. However, the business end of the body occurs [is run by] the products of the genes. Often, to understand how things are functioning, or what happens when they do not function properly, we need to be able to follow these gene products. Many diseases are diseases in trafficking of proteins, i.e., the protein is properly made but goes to the wrong place in a cell, where it may serve another function or may be processed abnormally. Often, it is even more important to know where a protein is being made, where it is going in response to a stimulus, where is it not going, than to know how much protein is being made. For the past 20 years, my lab has focused on such issues as protein targeting and transport. A nice summary of work on protein targeting and transport appears in Gunter Blobel's Nobel Address at the Nobel Web site.
To study the movement of a protein we need to study it in a living cell. One of the best ways to do that is to label it with something that allows it to be seen with a light microscope (electron microscopes require "fixing" cells, and trying to study protein movement in a "fixed" cell would be like trying to study blood flow in a cadaver). Two of the more powerful approaches have been to chemically attach a fluorophore or to synthesize the protein of interest fused together with the GFP protein from jellyfish so it would glow, or fluoresce. Alas, all these fluorophores have a few limitations: first, they bleach (fade away) very quickly, often in seconds. Second, they emit light over a wide region of the light spectra, thus it is usually possible only to resolve one or two proteins at the same time.
Quantum dots have been suggested to be a panacea for many of the problems of imaging multiple proteins. However, there have been a few major limitations:
1) Quantum dots have been studied by physicists in organic solvents. In water, they die.
2) It has been difficult to develop techniques for linking the quantum dots to probes so the specific molecules could be followed in the cell.
3) It was not known if quantum dots would damage the viability of the cells.
The current manuscript (one of six that we have in press) is the result of a few years’ work in which we have tried to develop quantum dots as a tool that can be used by the wider biological community. In the manuscript:
1) We demonstrate that we can selectively and specifically label proteins in living cells with quantum dots. This is extremely important. In a field of cells, a few were selectively transfected to express a reporter protein that was fused to GFP. Only those cells expressing the jellyfish protein were labeled with the quantum dots. All other attempts have failed to show specificity.
2) We demonstrate that it is possible to do continuous imaging of cells labeled with quantum dots through their entire development with no adverse effects. We followed Dictyostelium (the common amoeba) for 14 hours through their whole life cycle, and we followed human HeLa cells for 12 days.
3) We demonstrate that it is possible to follow multiple quantum dot probes simultaneously, fulfilling a long-anticipated goal for these markers.
The processing of APP is a critical problem in our attempts to understand Alzheimer’s. Thus, the interest of the Alzforum is quite relevant to the work at hand.
It was demonstrated a few years ago that QDs had the potential to become a new and better class of fluorescent labels with advantages over conventional organic dyes. However, since some key technical problems remained to be solved, these advantages were not fully demonstrated in real applications, and QDs were not available for scientists who didn't have a chemistry lab to make QDs.
Recently we have made a breakthrough in generating QD conjugates. In the article, we demonstrated the advantages of QDs (high intensity, photostability, multiplex flexibility) in real applications with specimens ranging from fixed tissue sections to live cells. In addition, we reported the first true multiplexed detection of specific cellular targets with QDs conjugated to streptavidin and IgGs.
Our new quantum dot technology allows us to generate QDs at a commercial scale and sell QD conjugates (see www.qdots.com for products information). QD-based probes will have applications in immunocytochemistry, pathological diagnosis, live cell imaging, and multiple target analysis in many biological and biomedical applications. QDs have potential for in-vivo biomedical imaging. For example, QDs can be conjugated to antibodies or drugs, and the specificity of the antibodies and drugs can be monitored in vivo after they are injected into the circulation system.