Biosensors Environmental Dyes Quantum Dots Remote Detection
In order to understand the location and fate of cells in live animals, we have been developing a number of methods for bulk-labeling of cell populations using quantum dots. To date, these methods include incubation of streptavidin conjugates with biotinylated polyarginine, conjugation to transferrin and cholera toxin B, and passive cellular labeling with cationic and anionic quantum dots. Each of these methods can be applied to a range of distinct cell types, though the actual performance depends on the particular cellular behavior. These labels can be incorporated into cells at high levels, and partitioned through multiple generations into daughter cells. In addition to a number of different cell lines, we have successfully labeled muscle-derived stem cells (mouse and human) and bone-marrow-derived stem cells (mouse) using QDs conjugated to cholera toxin B. The cholera toxin B subunit binds to cell surfaces and is internalized, but, without the A subunit, does not cause the toxic effects of the whole cholera toxin.
As shown in Figure 1, the internalized conjugates are in small, uniform vesicles. As with the amino- and polyarg-QDs, it is necessary only to add the cholera toxin-conjugated QDs to cells in the presence of normal serum. The cholera-toxin conjugates are rapidly bound and internalized into endosomes. As Figure 2 shows, the cholera toxin B conjugates are dispersed into many small endosomes; these conjugates, like the PEG-streptavidin QDs, give more uniform labeling than the original polyarginine-streptavidin conjugates. These conjugates are still a work in progress; we are testing methods for conjugation, levels of substitution, and best storage conditions. It is noteworthy that QDs substituted with an average of one molecule of cholera toxin B per QD are still internalized by cells, though more slowly that the polysubstituted QDs. We do not yet know whether this level of substitution results in one monomer per QD, one pentamer per every fifth QD or something in between.
Labeled cells may be used directly, or are flow-sorted to remove any unlabeled cells and to prepare uniformly labeled cell populations. Our laboratory is equipped with a B-D FACS Vantage SE that allows rapid sorting and distribution into individual wells of multiwell plates. Our current research is directed to understanding the loading of quantum dots into cells by these methods, as well as the fate of loaded cells during normal cell growth, division and ultimately death.
We have shown that different surface chemistries have a dramtic effect on the serum lifetime of quantum dots. We have also used these quantum dots to map lymphatic circulation. Video 1 shows passage of quantum dots injected at the base of the tail into the lymphatic system. Note the pulsating flow and passage through the lymphatics to label the inguinal and brachial nodes.
