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Lasers make biological assays easier

Zapping off tiny regions of samples removes the need for more tedious extraction methods.

For a more than a decade, Akos Vertes, professor of chemistry as well as biochemistry and microbiology at George Washington University in Washington, DC, has directed his research effort toward refining techniques for obtaining tiny samples of biological materials and subjecting them to analysis by mass spectrometry.

In 2007 he and his team devised an ingenious system whose elaborate name—Laser Ablation Electrospray Ionization (LAESI)—belies its essentially straightforward nature. Compared to previous methods, LAESI frees researchers from tedious laboratory preparation and in vacuo analysis, and allows them instead to sample areas a few tens of microns across on living organisms like plant leaves.

The technique attracted the interest of Protea Biosciences, a company based in Morgantown, West Virginia, which develops novel technology for identifying and assaying biomolecules. Protea, with assistance from Vertes, developed a commercial LAESI machine, and their collaboration continues on other fronts. For Vertes, this has been an ideal arrangement: His research career continues apace, and his contact with the business world benefits his graduate students.

Akos Vertes in his lab. CREDIT: William Atkins/ The George Washington University

Akos Vertes in his lab. CREDIT: William Atkins/ The George Washington University

LAESI developed out of an earlier technique known as MALDI (Matrix Assisted Laser Desorption/Ionization), in which a biological sample is prepared by immersion in a solution that, after evaporation, leaves the organic material embedded in a crystalline matrix. Then, under vacuum, a UV laser blasts off a small piece of the sample, and the ionized debris is sent into a mass spectrometer. One disadvantage of of MALDI is that the preparation procedure is finicky and can subtly alter the chemical structure of the test material. Another is that matrix molecules may end up in the mass spectrometer along with the sample.

Vertes and his colleagues first came up with the laser ablation part of LAESI. They used a laser tuned to a strong hydroxyl absorption line to eject small plumes of material from water-containing biological materials—which in practice means anything except bone, hair, and teeth. “You don’t need to prep, so you can use the technique on a living plant leaf without uprooting it,” Vertes says. However, only a very small proportion—less than 0.01%—of the ablated material was ionized, so the amount of material that could be steered into the mass spectrometer was tiny.

To solve for that drawback, the researchers added the electrospray step, a technique that dates back to Lord Rayleigh’s calculations of how large an electrically charged liquid droplet can be before Coulombic forces blow it apart. Vertes and his team worked out a new theoretical analysis of liquids sprayed from an electrified nozzle, and performed high-speed imaging experiments on the strings of droplets—and droplets breaking into smaller droplets—that an electrospray produces. Ultimately, they developed a way to send the plume of laser-ablated material into an electrospray so as to ionize it efficiently for entry into a mass spectrometer. The spray liquid is typically a 50–50 mixture of methanol and water, with a trace of acetic acid to promote ionization. “It’s about as gentle an ionization method as possible,” says Vertes. “It adds little kinetic energy to the plume, and doesn’t fragment it much.”

The first paper on LAESI—Vertes pronounces it “lazy”—caused a stir when it appeared in 2007, mainly because it demonstrated that researchers could get a very small sample size with a focused laser, and do so on samples in situ. To date, it has garnered 233 citations. The technique also needs little fine-tuning, Vertes says: “The spray grabs the plume and sends ions into the mass spec, so the relative placement of the components is not critical.”

Those virtues made the LAESI technology eminently commercializable, and George Washington University was keen on promoting patenting and commercialization of research findings. The head of Protea visited the Vertes lab “out of the blue” when the team was working on the LA part of LAESI but hadn’t yet added the ES. He was interested in the work and stayed in touch, Vertes recalls, and when his team devised the full LAESI protocol they quickly put in a patent application. Protea then took out a license to develop the technology. Their system made its debut in 2010.

The Vertes group and Protea continue to work together on 10 other patented inventions to date, with another 12 pending. Some are refinements of LAESI, others move into different areas. Vertes involves his graduate students in these efforts: “It’s part of their education; they learn not just about publishing new work but get to know the patenting and commercialization processes.” All but one of his patents have student co-inventors, which he describes as a matter of fairness. “They are doing the day-to-day work.”

Further developments

One current graduate student, Laine Compton, is working on a method to increase the separation between the test sample and the point of entry into the mass spectrometer by flowing the plume from laser ablation into a tube, with the electrospray ionization occurring at the other end. In her setup, the separation is about half a meter, but she sees no reason why it couldn’t be a couple of meters. This would give LAESI still more flexibility, allowing it to be used on large and oddly-shaped samples. Compton says that she didn’t come to the lab expecting to be involving in patenting but has found that “the marriage between research and commercialization is part of the attraction” of working there. And she says she’s open to the idea of moving into the commercial rather than academic world after she graduates.

Another student, Amy Li, is building a precision control system with a sample mounted on a steerable platform and observed by high resolution cameras so that areas as small as single plant cells, measuring from 30 to 100 μm across, can be picked out for laser ablation. Her interest in imaging technology led her to the project, and while she’s focused for now on pure research, she’s been impressed with the lab’s track record in commercialization.

Vertes himself is pushing to take LAESI to finer levels of precision with a technique that places a sample on an array of more than two million silicon nanoposts grown on a 500 × 500-μm2 wafer. Shining an ultraviolet laser on the arrays causes the nanoposts to heat up in a way that can be precisely controlled by adjusting the incident angle and polarization of the UV light. Individual cells can then be disrupted so that their contents can be assayed.

Vertes has used this method to investigate how yeast cells respond to environments with varying oxygen contents, something that had been studied for yeast colonies but not for single cells. It’s important to understand individual cell responses because cells are not identical, he says. Anticancer drugs, for example, are developed by looking at the response of large numbers of cells, but it may be the outliers in the cell population of an actual tumor that determine how the disease responds to the drug.

Vertes says that, while he has considered starting his own company, he is glad he chose to work with Protea. He doesn’t think it’s possible to be a conscientious academic and run a company too. Besides, he says, he has friends who’ve started companies, “and they have a lot more gray hair than me.”

David Lindley is an author and editor based in Alexandria, Virginia. His most recent book is Uncertainty: Einstein, Heisenberg, Bohr, and the Struggle for the Soul of Science (Doubleday, 2007).

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