Understanding the functions of proteins is fundamental to cell biology and chemistry. It can help us identify the mechanisms of disease and can contribute to the discovery of new drugs by shedding light on how proteins interact with one another or with the drugs in question. Hatice Altug, an investigator in the Boston University Photonics Center, has been working to develop biodetection systems with which to study the structures of proteins as well as protein-protein interactions. Conventionally, functional studies of proteins involve complicated techniques including x-ray crystallography, nuclear magnetic resonance and electron paramagnetic resonance. Infrared absorption spectroscopy, on the other hand, offers a direct path for functional studies by tracking changes in the vibrational fingerprints of the molecular structure. Researchers have reported an infrared absorption spectroscopy technique with which to study the structures of proteins and protein-protein interactions by measuring vibrational signatures in real time. The technique offers significantly enhanced sensitivity with respect to similar methods. The investigators therefore began to explore the technique. “Infrared absorption spectroscopy is actually quite powerful,” Altug said, “and it gives you vibrational signatures in real time, which is important in looking at protein functionality and their interaction processes.” But for all its strengths, she added, the technique is “not very sensitive.” Enhanced sensitivity In the Nov. 17, 2009, issue of PNAS, Altug and her team – graduate student Ronen Adato and postdoctoral fellow Ahmet Ali Yanik, both in Altug’s lab; professor of biomedical engineering and physics Shyamsunder Erramilli; research professor of physics Mi K. Hong; and Tufts University bioengineers David Kaplan, Fiorenzo Omenetto and Jason Amsden – reported a novel means to enhance the sensitivity of the technique. Previous attempts have involved chemically prepared or roughened metal surfaces, a method known as surface-enhanced infrared absorption. But with this approach, signal enhancement factors have been limited to the 10 to 100 range and are not repeatable. The researchers addressed these limitations using tailored nanoantennas made of tiny plasmonic gold particles 1.2 µm long and 200 nm wide, initially achieving 103- to 104-fold signal enhancements. But this was still not sufficient to study proteins at zeptomole levels. “We did some more thinking,” Altug said, “and we came up with the technique in which we arranged the antennas in a very certain way to achieve a strong collective plasmonic response reinforced with photonic resonances.” Using this approach, dubbed collectively enhanced infrared absorption spectroscopy, the team significantly boosted the absorption signal that it could measure. Yanik and Adato demonstrated up to a 105 enhancement of signals from single protein monolayers of silk fibroin, detecting 300 zeptomoles of proteins for the entire array, corresponding to only 145 molecules for each antenna. The researchers are exploring metals with which to boost the collective plasmonic enhancement mechanism still further, hoping to increase the signal intensity by an order of magnitude. “We would like to get down to 50 or 30 molecules per antenna,” Altug said. “Ultimately, we would like to get down to a single molecule, but we have to come up with new structures and fabrication schemes to increase the enhancement even more.”