Top Raman

As two soldiers approach a remote base near the Pakistan-Afghanistan border after a long trek through hostile territory, a laser beam zigzags across unusual-looking badges on their vests. The beam is emanating from a Raman instrument that checks the men for worrisome substances. If the troops have been exposed to pathogens or chemical warfare agents while on patrol, an alarm will sound before they can reach the base, and their comrades will rush to decontaminate them.

That vision was put forth by Jason Guicheteau at last month’s ACS meeting in Washington, D.C. For several years now, he and colleagues at the Army’s Edgewood Chemical Biological Center, located on Aberdeen Proving Ground, in Maryland, have made a concerted effort to develop systems that can detect poisons, pathogens, and explosives from a distance using surface-enhanced Raman spectroscopy (SERS). Their work is one of many programs to adapt the analytical technique for defense applications.

SERS can be remarkably sensitive, yielding spectra even from individual microbes. And it shows promise as a tool in fields ranging from explosives detection to medical diagnostics. The military hopes to use the unique SERS spectra of analytes to detect explosives, chemical weapons, and whole pathogens. The same technology could prove equally useful in hospitals, where rapid methods to diagnose infections are needed. At the moment, however, SERS remains primarily the domain of experts. But perhaps not for long: Products for defense and medical applications could hit the market over the next several years, once a few technical hurdles have been overcome, according to several speakers at a meeting session sponsored by the Division of Colloid & Surface Chemistry.

The technique dates back to a curious observation in the late 1970s: As a molecule comes very close—within a few angstroms—to a roughened noble metal surface, the intensity of its Raman spectrum is greatly enhanced. Now, researchers understand the phenomenon well enough to use it.

At Edgewood Chemical Biological Center, for example, Guicheteau and his colleagues have evaluated SERS’s ability to identify pathogens and its potential to detect chemical warfare agents, including the nerve gas soman. Guicheteau said that his lab can detect some analytes from meters away. The goal is to reach even farther.

Thanks to advances in nanofabrication, imaging, Raman hardware, and theoretical work, SERS has flourished in the past decade, according to Martin Moskovits, a chemistry professor at the University of California, Santa Barbara. His group has championed the concept that SERS is a plasmonic phenomenon, meaning that the emission of photons from an analyte is tied to the resonance of electrons within a nearby metal structure. That insight has helped other teams design SERS substrates, materials that greatly intensify the Raman spectra of molecules on their surfaces. George C. Schatz of Northwestern University and Mikael Käll of Sweden’s Chalmers University have developed theoretical techniques that reliably predict the enhancement of a substrate based on its composition and geometry. But many questions about those materials can be answered only by experimental work.

Synthesis of materials with high enhancement properties is among the fastest moving fronts of SERS research. Chemists have made countless patterns and particles with silver and gold and have tested their ability to enhance Raman emission. Much of that work has been supported by a Defense Advanced Research Projects Agency (DARPA) basic science and technology project. The forward-thinking agency invests in emergent technologies that might be of use to the military.

DARPA has been funding research to understand what controls a material’s so-called enhancement factor, or the extent to which it intensifies an analyte’s Raman emission. Then scientists can “make big surfaces that have big enhancement factors,” said Michael Natan, chief executive officer of Oxonica Materials, a Mountain View, Calif.-based firm that’s commercializing SERS materials.

One difficulty is that every lab has its own way of calculating enhancement factors; in general, this is done by taking the ratio of Raman signal intensity for a molecule on a surface compared with its intensity in the absence of that support material. Different calculations make comparing new substrates difficult, not to mention controversial.

Natan, however, views the controversy as a distraction, arguing that there are more important problems to solve. “The single biggest issue is the development of coatings on SERS active surfaces that preserve the SERS activity and afford some level of selectivity” for the analyte of interest, Natan said. “That is the problem with SERS. For SERS to advance, it needs organic chemists” to develop such coatings, he added.

In addition to a substrate’s enhancement factor, researchers also consider how consistent that effect is across the substrate’s surface and from batch to batch.

Among the most consistent and highly enhancing materials are nanospheres coated in silver films, or AgFON. Chemistry professor Richard P. Van Duyne and his colleagues at Northwestern first started working with these substrates more than a decade ago, and his group has been refining them ever since. Van Duyne’s team is among the six groups of academics working on the DARPA project. The AgFON surfaces have met every milestone set by the agency so far.

AgFON substrates are made from 600-nm silica nanospheres coated in a thin layer of silver. Recently, the Northwestern team learned that the SERS enhancement of AgFON depends on rodlike protrusions from the silver surface that are far smaller than the spheres themselves. That revelation sends a clear message to other teams working on SERS substrates that they should pay attention to the nanostructures of their materials on many different length scales.

DARPA is also supporting the efforts of bioengineering professor Luke P. Lee of UC Berkeley and his collaborators at Lawrence Livermore National Laboratory. Using ion beam lithography, Lee’s team has developed gold patterns that resemble eagle beaks. Those materials have enhancement factors and consistency comparable with that of AgFONs. DARPA hopes that Lee and his rivals will be able to mass-produce 6-inch wafers of their exotic materials.

Although the search for better materials continues, researchers can obtain informative spectra from biological analytes with nearly any SERS substrate. For laboratory applications, they need not use the most exceptional substances.

Lawrence D. Ziegler, a chemical physics professor at Boston University, has developed some clever tactics for automatically processing the spectral fingerprints of pathogens: By creating a bar code based on the second derivative of each spectrum, he greatly simplifies the data and allows reliable automated processing. Each of the 50 microbes that he has identified has had a distinctive signature.

This work was originally funded by the Department of Defense, Ziegler said. The 9/11 terrorist attacks and the letters filled with Bacillus anthracis spores in the fall of 2001 raised awareness of biological attacks in the U.S. So the government increased its support of research “to develop rapid, specific, and point-of-care techniques for sensitive pathogen detection and identification,” he explained.

Ziegler also pointed out that the most distinctive parts of pathogens are on their outer surfaces, and thus a technique like SERS, which reports the composition of these outer layers, is quite effective. It has worked out well for other researchers, too.

For example, Richard A. Dluhy, a chemistry professor at the University of Georgia, has developed arrays of silver nanorods that are remarkably consistent from batch to batch. He has used them to construct several SERS-based viral assays that could soon make their way into the clinic.

“In our work on respiratory syncytial virus [RSV], we showed that we could use the raw SERS spectrum of the virus in combination with multivariate statistical techniques to identify individual virus strains,” Dluhy said. “In fact, our SERS method could even readily distinguish one RSV strain—called ΔG—that differed from the wild-type RSV strain, the A2 strain, by the deletion of only a single gene in the virus genome.”

Dluhy has licensed some of his work to the Georgia-based start-up company Argent Diagnostics. The Centers for Disease Control & Prevention recently awarded Argent a Small Business Innovation Research grant to develop the technology for measles tests.

The diagnostic approaches pioneered by Guicheteau, Ziegler, and Dluhy are forms of direct detection. In those assays, SERS spectra provide rich molecular information about the surfaces of the pathogens themselves. But those spectra are often muddied by background signals. Making direct assays work reliably in complicated mixtures such as blood is still a challenge.

When direct detection is unworkable, indirect detection is an attractive alternative. Highly sensitive assays based on indirect detection make use of SERS tags—gold or silver nanoparticles coated with organic reporter molecules—rather than fluorophores. The assays do not provide any spectral information about the analytes, but their unique emission signatures shine brightly in unprocessed samples. SERS tags have other benefits as well: With their narrow spectral peaks and intense emissions, they can outperform fluorescent dyes as optical labels, they are ideal for multiplexing—running many tests in the same tube—and they allow chemists to build tests with remarkable sensitivity.

At the University of Utah, chemistry professor Marc D. Porter has developed immunoassays that use SERS tags as optical labels. His lab is close to beating the gold-standard test for Johne’s disease, which costs the cattle industry nearly $500 million each year. Porter’s group has also developed SERS assays for detecting herpesvirus, Escherichia coli bacteria, and the prostate-specific antigen used as a prostate cancer marker. “We’re working to drive limits of detection to extremely low levels,” Porter said.

Porter is a cofounder of several SERS-related start-ups, including Concurrent Analytical, a Hawaii-based diagnostics company that’s commercializing Raman technology, and Nanopartz, a Salt Lake City firm that sells a wide variety of gold labels, including rods, wires, and modification kits. Most of the tags for Porter’s immunoassays come from Nanopartz. He sees a tremendous opportunity in developing multiplex SERS tests that can operate in a single sample without any preparation.

Products using SERS are heading to market as academics and start-ups slay the last few technical dragons that remain in the way of commercialization.

Emission enhancement is one still-unoptimized technical detail. “Intense SERS is only observable when the analyte is in very close proximity to an appropriately nanostructured metal substrate normally fabricated out of silver or gold,” UC Santa Barbara’s Moskovits said. For SERS to be useful in the real world, chemists must find clever ways to bring an analyte to a region of a SERS substrate that has a particularly high enhancement factor—a so-called hot spot. Alternatively, they must design materials that self-assemble around the analyte and create such a hot spot in the process.

To that end, Moskovits and his collaborators developed a microfluidics system that draws gaseous analytes onto a SERS substrate. The system detects explosive vapors from a distance, and it has been licensed by a Santa Barbara, Calif.-based start-up company, SpectraFluidics, which aims to make smoke-detector-style ­sensors.

Put any SERS tag into human serum or foodstuffs, and it will be fouled by natural substances that can obscure visible light, throw off its emission spectrum, and otherwise ruin assays. Oxonica has developed a line of SiO₂-encapsulated SERS tags that are so robust they can be used in nightmarishly complex media—from ground-up spinach to blood to peanut butter. The tags, whose gold cores are coated with an organic reporter layer and then encapsulated in silica, can be excited at near-infrared wavelengths, at which few materials absorb. This construction allows them to function as fluorophore replacements in murky solutions. Fluorescence-based assays rarely work in turbid mixtures.

“The signal is generated from inside the particle, making the particles both exceptionally durable and insensitive to external environmental parameters that typically complicate assay development,” Natan said.

That attribute is both a virtue and a drawback. Because the gold particles are insulated from their environment, their emission cannot be perturbed by contaminants. At the same time, they don’t provide spectral information about analytes, just a rather strong spectral signature.

Oxonica has screened a vast array of organic molecules that can act as reporters. Each reporter gives rise to a unique spectral fingerprint that can serve as an identification code for a batch of tags. “There are literally thousands of reporter molecules with unique spectra,” Natan said. “This makes it very simple to develop multiplexed tests.”

The company has developed some remarkably versatile assays by using those reporter groups as the middle layer in its SiO₂-coated nanoparticles. Becton, Dickinson & Co., better known as BD, have licensed this technology for in vitro diagnostics, according to Natan, and it could be available in the next few years.

Natan is convinced that SERS will make its way to the point of care. He is quick to point out that handheld Raman spectrometers, such as the ReportR system from Wyoming-based firm DeltaNu, are widely available for sale to researchers and law enforcement personnel. Devices like that could be used to provide the readout from all sorts of medical tests based on SERS.

Despite this progress, SERS has not been adopted by many scientists outside the SERS research community. If uninitiated biotech researchers decided to do a SERS experiment tomorrow, they would find not only that few substrates are commercially available but also that the learning curve is steep. Most SERS protocols require a great deal of experience. Right now there is too much confusion about which substrates to use and not enough simple protocols that could allow uninitiated researchers to do SERS experiments with ease.

Perhaps when all the kinks are worked out, a wide range of analytical devices will realize the full potential of a technology that has been more than 40 years in the making. And then when disaster strikes, it will be time for SERS and rescue.