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BU s Bennett Goldberg on How SSFM Can Improve DNA Hybridization

Bennett Goldberg
Center for Nanoscience and Nanobiotechnology, Boston University

Name: Bennett Goldberg

Title: Director, Center for Nanoscience and Nanobiotechnology, Boston University

Professional Background: Following a post-doctoral appointment at the Massachusetts Institute of Technology and the Francis Bitter National Magnet Lab, Goldberg joined the physics faculty at Boston University in 1989. He currently is a professor of physics, professor of electrical and computer engineering, and a professor of biomedical engineering at BU. Since 2004, he has served as director of BU's Center for Nanoscience and Nanobiotechnology, as well as chairman of the university's physics department.

Education: 1987 — PhD, physics, Brown University; 1984 — MS, physics, Brown University; 1982 — BA, Harvard College

A team of researchers from Boston University has developed a method to enable more precise measurement of the location of a fluorescent label in a DNA layer. Published last month in Proceedings of the National Academy of Sciences, the paper, entitled "DNA Conformation on Surfaces Measured by Fluorescence Self-Interference," explores a method that can provide insight into the shape of DNA molecules attached to a microarray surface, and information on how surface-bound DNA molecules conform.

The technique, called spectral self-interference fluorescence microscopy, maps the interference spectrum from a fluorophore label located on a layered reflecting surface into a position with sub-nanometer accuracy, and it is the hope of paper co-authors that the method may significantly improve the efficiency of DNA hybridization and microarray technology and thus impact emerging clinical and biotechnological fields.

To learn more about the new method and its possible ramifications, BioArray News spoke with Bennett Goldberg, a co-author on the paper and the director of BU's Center for Nanoscience and Nanobiotechnology.

How did your team at the Center for Nanoscience and Nanobiotechnology get involved in this project?

I am a condensed-matter experimentalist, a physicist by training, although I have a joint appointment in electrical engineering as well as biomedical engineering. I have been collaborating with an engineer for many years, and about six or seven years ago we started to look at questions that were beginning to be relevant in microscopy and location of fluorophores. They were beginning to be relevant for people way outside our area of expertise. So originally we got talking to [BU Center for Advanced Biotechnology co-director and acting chief scientific officer at Sequenom] Charlie Cantor, who has done a lot of work in biomolecular systems, and we started to work together in trying to figure out if we could remove the signal from fluorophores bound close to a surface, so that you could completely get rid of non-specific binding as a source of signal for any kind of target analyte system.

He said that one of the biggest problems in the industry is that you get non-specific binding, and most of that is associated with non-specific binding to the surface, not in all target probe systems. And so we had expertise in waveguides, and in evanescent fields and in near-field microscopy, and in a variety of optical techniques from the physics and engineering side. We worked on it for a couple of years and were able to develop several approaches. We started to work on resonant cavity systems with the idea that you could sort of take your field profile and tune it in the vertical direction.

And so, the idea started to [coalesce] that we were interested in slicing, if you will. What we wanted to do was be able to look at the 10 nanometers that are, say, 100 nanometers above the surface or 200 or a micron and a half. We wanted to take those very well-defined vertical regions above an array or above a surface, and interrogate them without the influence of any other region.

And so one way to do that is to create a cavity, and that cavity has a standing wave in it, just like in any resonant structure — anything with two mirrors in them — and then shift that around. But we also wanted to do this in a simple sense. So myself, and [co-authors] Selim Ünl and Anna Swan, developed the idea of a buried mirror structure inside a flat substrate and having that provide the cavity that we needed. In a sense, people have been working for a long time on the idea of of just quenching the fluorescence with its own enhancement or destructive interference. And that has been known for 40 years. But what we did was create a large spacer layer so we could look inside these different layers, but using the spectral component.

So you came at this from an interdisciplinary approach?

Well, the student who is the first author on this paper is Lev Moiseev, and he is an interesting guy. He has a PhD from our molecular and cell biology program at Boston University, but he was almost entirely funded by and did his research in a physics and engineering lab. He was advised by Charlie Cantor in BU's Center for Advanced Biotechnology, but he learned all of the fundamental optics and physics of fluorophores and resonant cavities and electromagnetic theory, and incorporated both the basic physics and all of the DNA biology as part of his thesis. So from my point of view, he's the perfect interdisciplinary student of the future. He is somebody who can move freely from molecular biology all the way towards new platforms and tools being developed by engineers and physicists.

And, in my opinion, one of the most exciting things about nanoscience and nanobiosciences, is that over the last decade we've seen a real convergence, a convergence of physics and chemistry and engineering and biology and molecular biology and medical science, where people are really applying new tools, and seeing at the length scale that's relevant for biological systems.

There's a lot of work being done where we now have control and can fabricate things at that length scale. We can provide manipulation, and now we can start to answer questions that are really relevant to biologists and medical scientists about what is happening in membranes and what is happening intracellularly in a dynamic way.

Now, I know that I am never going to have enough background, or maybe I will, but I don't now anyway, to really understand what are the most relevant biological questions. On the other hand, if I can really work with somebody who does, I can create tools or measurement approaches that may help answer those questions.

One of these tools is self-interference fluorescence microscopy. Is that a new idea, or is it an older technology that has been updated to work in this arena?

I think it is a new idea, but that it is a significant modification of an existing approach. We are always developing things that rest on past achievements, but it has some new aspects to it. It is built out of this idea of fluorescence-interference contrast microscopy to determine the position of cells or membrane proteins above a surface and things like that. What we've done is advance the technology to use what we call a spectral self-interference. So, instead of just looking at the total signal, whether it goes up or down in intensity based on interference, we look at the spectral oscillations, and we can do that because we have a longer path length in the interference. So if you have a short path length, then you'd see the entire spectrum go up and down, light and dark, But if you have a long path length, in the difference between the direct and the reflected light, it means that you start to see the fringes. It's a newer application of using the spectral component to determine the precise position. By using the spectral component you can look at emitters that are buried that are not just on the surface.

How would this technique work with existing technologies like ellipsometry and white light reflectivity?

Ellipsometry measures the complex surface reflectivity of a multilayer stack. And so if you know the indices, or the thickness of your materials, and you have enough information, you can determine those thicknesses. But you couldn't tell me, if at one of those buried interfaces, where an emitter was located. White light reflectivity is similar, and will tell me the thickness of a protein layer on a surface or something like that. But again, that can't tell me the position of buried emitters.

We are complementary to ellipsometry and white light, but we have this additional benefit that if you want to know within DNA or cellular membranes, you can combine WL and ellipsometry, which tell me about the thickness of the layers, to a precise location of, say, where a protein is on a membrane, by tagging it.

So this can be done with any biomolecule printed on a surface…

Sure, and we can even do it if it is not even on a surface. It's not restricted to that. But it is basically using this spectral interference signature to determine precise location.

Who benefits from this technology?

I think a number of people will benefit from it. Generally, in microarray technology, there's still an enormous amount of variability in microarray response. One of the reasons the technology is not yet [widely] clinically approved, is that it's hard to get consistent responsivity and consistent and quantifiable data out of microarrays. Here's a way of determining some precise information about hybridization in microarrays that you couldn't get before. So this may be able to help quantify microarray response.

It's also not restricted to DNA. We are working on different kinds of arrays and things like that.

Have any companies shown interest in your work?

It hasn't happened yet, but maybe that's because we're physicists. I don't know. We are starting to explore that. The first thing is to develop the technique and present the data that proves it. The next step is to work with companies that would be interested in that [technique]. We are talking to several companies, the normal players that you would expect, but nothing has developed out of that. We would like to work with people who are interested in all sorts of arrangements on surfaces. Hopefully we will be able to provide them with really quantifiable and relevant data.

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