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Todd Giorgio Discusses the Use of Nanotechnology in Flow Cytometry

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At A Glance

Name: Todd Giorgio

Position: Associate Professor, Biomedical Engineering and Chemical Engineering, Vanderbilt University

Background: Assistant Professor, Chemical Engineering, Vanderbilt — 1987-1993; PhD, Chemical Engineering, Rice University — 1982-1987

What is different about the high-throughput flow cytometry platform you described in your recent publication [Comb Chem High Throughput Screen, 2004 Mar; 7(2):141-51] and traditional flow cytometry?

I guess there are really two aspects that we’re trying to promote in this particular work, and one aspect of that is the quantitative nature of the work. And so, one of the things we feel is important for flow cytometry is the ability to, for example, quantitate the number of receptors that bind to a cell. In fact, we have a second paper, which is due to appear in Annals of Biomedical Engineering in about a month or two, that is basically a similar strategy but really emphasizes the quantitative nature. And so we’re looking for particular receptors, in that case, on HeLa cells, and have discovered that a very common receptor is present on HeLa cells that hadn’t been discovered before — so that’s kind of interesting.

So I think the quantitative nature of what we’re doing is important for the screening — if you have just a few molecules that bind, it may not be sufficient to elicit the response you’re looking for, whereas more molecules might be better. So that’s one aspect.

And then the other aspect I think we’re moving toward with this is to use more colors by using quantum dots. So we see a real opportunity in moving toward a nanotechnology kind of basis for the screening. So what you see [in the most recent paper] is more of a proof-of-concept more than anything else. But we see that we’ll be able to get more colors and screen bigger libraries using quantum dots.

I’m familiar with quantum dots, but I’m not sure about how close they are to being used frequently in cell-based assays. How would you be using them in your applications?

Just as you would have to functionalize the fluorescent microparticles in [the most recent paper], you’d have to functionalize the quantum dots with libraries of molecules that you’re interested in. So one of the early steps will be the chemistry that will allow you to surface-functionalize the dots — and we’re doing that now. We don’t have anything published on it yet, but we have a number of studies that are now ongoing as well as a bunch that are written in the form of grant applications that show that you can synthesize different surface functionalizations on quantum dots to get them to bind to cells and actually report the number of dots bound to cells. Because the dots are so small, we’re going to be able to do a lot of different kinds of screening — more powerful screening. You know, frankly, functional screening using the microspheres in our work is really limited for ligands that have a high surface concentration on the cells, because these microspheres are one to three or four microns, and you just get steric hinderance at some point, especially with receptors with high surface concentrations. But with a quantum dot, which is down around 12 or 15 nanometers, even after surface functionalization, which is very, very small, we should be able to interrogate all of the possible receptors on the cell surface, perhaps all at once.

Do you think that quantum dots are poised to eventually replace or complement traditional fluorescent particles? How do you think they will fall into line with current fluorescent labeling techniques?

Certainly there are some applications for which quantum dots will be immediately superior, if you take advantage of either their narrow wavelength characteristics, or if you take advantage of their exceedingly bright nature or high quantum efficiency, or if you take advantage of the lack of photobleaching. Any of those things make quantum dots an appealing initial choice over organic fluorophores right now.

Where quantum dots really haven’t yet been the match for organic fluorophores is simply the number of colors that is commercially available. So there are a few, but what we’d really like to see are a bigger panel of available colors, and especially dots that reach down into the near infrared, where biological samples of greater thickness might be interrogated. So we’re really looking forward to that kind of thing, but I think eventually we’ll see a lot more applications for quantum dots. I think there are some right now that are very, very compelling, and so I think it’s a very exciting area to be involved in. Vanderbilt has actually started a brand new nanotechnology institute, called the Vanderbilt Institute for Nanoscale Science and Engineering, which can be found on the web by looking for VINSE on the Vanderbilt website. We just finished a rather large renovation of a floor of the building which includes a couple thousand square feet of continuous clean-room space, and includes a wet lab for biological-related manipulations, and administrative space as well. So, it’s a very exciting time for nanoscale manipulations here, and we’re just moving into that facility right now. And that’s another reason locally that we’re interested in quantum dots, because we’ll actually have soon the capability of manufacturing — in small levels — some very custom characteristics.

What was your immediate impetus for designing this flow cytometry platform?

Really, it was a very specific tool for use in our lab to do some screening. What we discovered, when we really gave it some thought, is that of all of the screening tools that are out there, they tend to either be very expensive in that they require unique hardware that isn’t useful for anything but screening, which makes it difficult for small laboratories to use, or they’re kind of clunky and not very fast and useful, except for very small libraries. So we saw flow cytometry as a very ubiquitous piece of instrumentation — almost every medical center has a flow cytometry core, and many investigators can afford flow cytometry. And so it was easy access to the hardware that made us think: ‘Well this is a reasonable idea.’ And then the second thing was that the surface chemistry of these microparticles are very commonly available. You can pick a surface chemistry and get a library of microparticles. You can imagine doing this in a 96-well plate, for example, where each well has a different microparticle but the same surface chemistry, and if you have a library, you can put each element of that library one per well, mix them all together, put them on the cells, and do kind of modest-level cell-based screening in your own laboratory. So that’s where we’re heading for this particular technology — something that individual investigators or small groups can do reasonably high-throughput screening with, with no real investment, provided that they have a cytometer.

We actually started doing some combinatorial peptidomimetics, and we’re still working on that a little bit, but the plan was to use this technology to screen some combinatorial peptidomimetics for some biological functions.

Can you be more specific about the screening you are doing?

I guess I shouldn’t talk about the exact plan, but the screening was a modest library of combinatorial peptidomimetics, and the idea was to look for peptides that we had validated the function of, but to look for peptidomimetic analogs that retained function but also had a longer half life and biological integrity. So you probably get the idea that the peptidomimetics would be more robust in a biological system, but of course after you do the peptidomimetic manipulation, you have no idea a priori whether they’re going to retain function.

What is the importance, in your opinion, of using cell-based assays as opposed to biochemical assays?

I think there are applications that are perfectly suited for each of those kinds of platforms. For cell-based screening, we see this moving more towards a peptidomimetic kind of biological-based assay — looking for surface expression of proteins on cells that might be induced by outside influences or genotype/phenotype kinds of changes. And so it’s almost — on a small scale — trying to do proteomics on individual cells. And so, in addition to getting the average values, which many other tools allow you to get — the average binding over a cohort of, say, a million cells — we actually get the distribution because we measure each of the cells individually by flow cytometry. And so what that allows you to do is identify subpopulations of cells that might be behaving differently than the overall population. And that small subpopulation, if you had mixed it with all the cells and averaged the signal out, might get swamped — you might never have known it was there. And so by using flow cytometry, we can identify subcohorts of big populations and see how they respond differently from the main population, and that’s one of the cool things in general about flow cytometry.

What’s next for your laboratory?

We’re continuing to move toward the nanotechnology kinds of applications, and in particular we’re looking for nanoparticles, including quantum dots and other things that have surface functionalizations that give them some activity. So rather than simply trying to, say, screen molecules by applying a molecule to them, we’re actually having them do some response. You might imagine that they could respond to, say, an enzyme, and in response to that enzyme reveal a binding ligand. With that kind of thing, you might be able to get multiple functions on a single particle. The advantage there is that the functions could be co-localized on the particle, so I think we’re headed in that direction. And I think that there will be some very interesting technology if you build that on a quantum dot, because then you can track that, you can image it, and you can see some of these changes in function in real time.

But this particular [paper] is a real combination of inspiration and chemistry. And chemistry is the hardest part, I think, of the whole synthesis — to try to make quantum dots or nanoparticles with the right functionalization, and confirm that you’ve got the right function and it works, and it’s a slow process right now, because I think everybody’s kind of finding their way to efficient ways to do that. I don’t think we’re there yet in terms of the efficiency, but we certainly can make very complicated, surface-functionalized nanoparticles and show that the work. Now the next step is: ‘How do we do this so that other people can use them, and how can we get these into the hands of other investigators so that they can screen biological events?’