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LLNL s Daniele Gerion on Getting Quantum Dots into the Nucleus

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At A Glance

Name: Daniele Gerion

Position: Postdoctoral research staff, Lawrence Livermore National Laboratory

Background: Postdoc, University of California and Lawrence Berkeley National Laboratory — 1999-2002; PhD, Swiss Federal Institute of Technology — 1995-1999

Lawrence Livermore National Laboratory and Lawrence Berkeley National Laboratory have an ongoing research collaboration in the area of semiconductor nanocrystals — better known as quantum dots — as reagents in a wide variety of biological assays. LLNL postdoc Daniele Gerion, along with colleague Fanqing Chen at LBNL, have discovered a way to get the rather large but optically promising q-dots into the nuclei of living cells — work that is detailed in a recent issue of Nano Letters [Vol. 4, No. 10; pp. 1827-1832]. Gerion began his work with q-dots working in the lab of Paul Alivisatos, who co-founded Hayward, Calif.-based biotech Quantum Dot. Last week, Gerion discussed with Cell-Based Assay News the evolution of his research career, the evolution of q-dots as biological assay reagents, and his recent research efforts in this area.

How did you come to work with quantum dots for biological applications? Do you have a bio background?

Not at all. Actually, my PhD was in physics. But I went to Berkeley for a postdoc, and I worked in the group of Paul Alivisatos. That’s how I got into the area of quantum dots. When we looked for a collaboration, we got in contact with [Fanqing] Chen. He’s the biologist, he’s in the life science division, but the interest of the group is in everything related to DNA. So we started off working in DNA microarrays, and things like that, and then we got interested in the process of DNA damage; specifically, wondering if we could monitor it inside living cells. So that’s pretty much how we started.

So is there more to it now than just detecting DNA damage in living cells, or is that the primary focus?

It still needs to be developed a little further, but that is the direction we’d like to go in.

Quantum dots are still difficult to get inside cells for live cell assays, but your group has taken it a step farther and gotten them into the nucleus of live cells. Can you comment on the difficulty this presents?

Yes, it’s still very difficult, and I don’t have a magic answer to that. The reason, we think, that they went inside is … you have to work on the surface composition, what is on the surface of the q-dots. And the q-dots that we used have a nuclear localization sequence. It seems that this sequence does two things: One, it brings them into the cells, and two, it drives them to the nucleus. We have worked with other types of surface preparations. For instance, we put other types of peptides [on the surface]. And for certain types of peptides, the q-dots don’t enter the cell. But for other types of peptides, we do. And it’s not clear to me now why that happens, or what is the sequence of the peptides that allows transfection. So to answer your question — yes, I think it’s pretty much still not an easy task.

So this will still be some time before even getting them into cells is perfected.

Exactly, and I think we want to have it be very robust, so I think we would need to go even one step back and start to understand all the processes. Because putting a peptide on them and finding them in the cell, you say, ‘Wow, that’s cool!’ But we don’t really know why that happens.

How about the size of quantum dots as compared to the cell? They are much larger than fluorescent tags, so does this have any negative effect on an assay?

This is another very important debate, and I think that no one knows. There are two issues. First, how do the q-dots alter the processes in cells? Nobody knows. The only thing that’s been done so far is to take the q-dot, and bring them inside cells. And when you put this inorganic stuff inside cells, you look at the survival rate of the cells — if they are still alive after, say, two weeks. And the q-dots don’t kill the cells. But how do they affect various processes in great detail — this is not known. This is something that we’re looking at with Fanqing Chen. They have a microarray facility, and I think they are studying the expression levels of cells that have been incubated with quantum dots to see if there is over-expression.

How do you get the quantum dots into cells right now — standard transfection methods?

In the paper we published we did it with electroporation. The reason we did it like that was that it was the fastest way to get all the cells transfected. When we used lipofectin, for instance, we saw that the q-dots were partially quenched, that some weird thing was going on. So we decided not to go that way.

The optical advantages of q-dots compared to other types of tags have been well documented. Do you think that the instrumentation has been evolving fast enough to take advantage of these optical properties?

I think it needs to evolve more. That’s something that I think could be developed further, because appropriate microscopes are still not something that you can get very easily. For instance, we are building one at Livermore, but we built it from scratch. It’s still easier to go to Berkeley, where they have a few of these microscopes.

So there aren’t really many tailor-made instruments for q-dot assays?

There are more and more, but it’s not something that you can just go to a website and buy. Most of the fluorescent microscopes that you can buy are adapted for different types of dyes. But dyes are probably slightly different from q-dots.

What is the potential of quantum dots in cell-based drug discovery?

That’s something that we were thinking to investigate, but we haven’t started yet. I think the big advantage is that you can very quickly look at where the quantum dot ends up in the cell. You just look at the fluorescence, and it’s very clear where they go. I don’t know if it’s possible or not, but if you can link a drug to the q-dot, and if the q-dot does not perturb cellular processes too much, then the drug might go in certain places. So that’s something we’re very interested in. If you think about a q-dot, they’re about 10 or 12 nanometers, and there is a lot of space on the surface to put different molecules. So you can feasibly put on some drugs, but at the same time you can put on some targeting peptides. That might be a very simple way to target very specific organelles with certain drugs. How you might do that in reality is not really clear, but this is an area to explore.

Being that this is a collaboration between Livermore and Lawrence Berkeley, there is some government interest here. Do you think it’s main interest is drug discovery?

I think it will be, but the interest that Livermore and Berkeley — with Livermore being a military lab — is different, and they might be interested in sensing for pathogens and those types of things. For them, targeting the nuclei of cells might not be the top priority. Down the line, though, it’s interesting, because the same platform can be used for very different things, potentially. But I don’t think that we are ready to go right into sensing with these, and so these experiments teach us a lot on surface modification and other aspects.

What’s next for this research collaboration?

We still dream to see DNA damage happening live. But we still need to develop the dots a bit. One of the issues you mentioned earlier is that of size. And one of the ways we are doing these q-dot-peptide conjugates is through a strepavidin-biotin bridge. And I think we need to get rid of this bridge if we want to get higher nuclear targeting efficiency. But that requires some chemistry to link the peptides, and I think that’s something we will want to look at in the future. I think there is a lot that physicists and chemists can bring to biology, and vice versa.

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