Recent advances in surface-enhanced Raman spectroscopy have positioned the technology as a potential alternative to fluorescence-based ELISAs, with work underway in several academic and industry labs to adapt it for use in proteomics research and protein-based diagnostics.
The technique, which was discovered in the mid-1970s, relies on the inelastic scattering of monochromatic light to detect changes in analytes' molecular state, and is potentially more sensitive and easier to multiplex than conventional ELISAs. However, several issues, most significantly poor reproducibility, have kept it from being widely adopted for protein detection.
Primarily, the reproducibility challenge has been a materials-synthesis problem, said Marc Porter, professor of analytical chemistry at the University of Utah. Detection via surface-enhanced Raman signaling, or SERS, is done on special surfaces — typically on those made of metal nanoparticles — to enhance the normally weak Raman signal. To achieve high reproducibility across experiments, manufacture of these surfaces must be similarly reproducible.
The technique was discovered in the mid-1970s, but "it's really just been a research phenomenon because the ability to repeat or reproducibly get the same [level of] enhancement has been a challenge," Porter told ProteoMonitor.
Recently, however, his lab has been able to bring the method's variability down into the 5- to 10-percent range and has additional preliminary data that suggests it may be even lower, he said.
Porter's lab conducts its SERS work on thin films of gold onto which it attaches a layer of antibodies. As in a typical ELISA, that layer of antibodies is then exposed to the antigenic sample of interest, allowing for binding.
The researchers then add gold nanoparticles coated in both the secondary antibodies and reporter molecules that give off a signal when read by a Raman spectroscope.
With materials from Loveland, Colo.-based nanotech company NanoPartz, Porter said he believes his lab has "figured out how to reproducibly manage the [Raman] enhancement."
"NanoPartz makes gold nanoparticles with a very tight control over particle size, and by being able to reproducibly create particles of a well-defined size and shape, you've now got something that will give you a consistent enhancement," he said.
"We're also able now to reproducibly coat the particles with a consistent amount of the Raman label and a consistent amount of antibody," Porter added. "So now we can begin to argue that we can manage the SERS enhancement [in a way] that competes with purifying a fluorophore and hooking it onto an antibody for a fluorescence-based assay.
"There are a couple of different [research] groups going after this in a couple different ways," he said. "This is what's been limiting [adoption], and now I think we're going to see [SERS] become an important player."
Another entity exploring SERS as a protein detection platform is Scottish biotech firm Renishaw Diagnostics, which has ongoing studies looking at using the technique to detect protein biomarkers tied to the infection of chronic wounds.
Originally named D3 Technologies, Renishaw in 2007 was spun out of Glasgow's University of Strathclyde with the aim of commercializing IP developed there that uses SERS to detect DNA.
That year, the company purchased the analytical business unit of Mesophotonics, obtaining its silicon- and gold-based SERS substrate Klarite. According to David Eustace, the company's project leader for development, Renishaw plans to launch DNA diagnostics using the Klarite product by the end of 2011 and hopes to develop protein detection assays within the next several years.
"The DNA diagnostic products are the first generation," Eustace told ProteoMonitor. "But we know that we need to be able to do protein assays in two, three, four years' time."
Renishaw began using SERS to detect protein biomarkers with funding from the Scottish business-development organization Scottish Enterprise. At the annual Pittcon meeting in March, Eustace gave a presentation on the research, which he said has thus far focused on proteins like metalloproteases and inflammatory markers.
The work is still in the proof-of-concept phase, he noted, but the ultimate goal is to develop "a point-of-care device, or a near-patient care device that will carry out essentially [SERS-based] ELISA assays for multiplex target detection."
Currently, Bruker, Thermo Fisher Scientific, and Horiba are among the leading instrument vendors making Raman spectroscopes that could be used for such applications.
Eustace cited Chicago-based Sword Diagnostics as a company that offers Raman-based ELISAs. He noted, however, that Sword's product is a simplex assay, and that one of SERS' main advantages over traditional ELISAs is its potential for multiplexing.
Raman scattering features are 10 to 100 times narrower than fluorophores and chemophores, which allows for easier multiplexing, according to Porter. That's potentially useful "in that in one well we can look for a bunch of different [analytes] whereas with ELISA each well looks basically at just one thing," he added.
Although his lab has thus far multiplexed only four analytes, "if you look at the literature, we think we could go up to 30 on one address," he said.
According to Eustace, "clinicians need to save time when doing diagnostic assays, so if you can do a one-well assay for multiple targets, that will be an attractive product."
SERS' other main advantage is increased sensitivity. Renishaw's platform, Eustace said, currently detects proteins at the picogram-per-mL level. Meantime, Porter said his lab's SERS technique offers a 20-fold improvement in sensitivity over conventional ELISAs, and he said he anticipates it will eventually boost assay sensitivity by as much as 200-fold.
It can possibly go even higher than that. In February, a team of researchers at Princeton led by electrical engineering professor Stephen Chou published a study in Optics Express detailing a new SERS platform that Chou said is three orders of magnitude more sensitive than SERS substrates on the market today.
His lab is currently testing the system for protein-biomarker work, he told ProteoMonitor, adding that it's "clearly one of the really great applications" for SERS technology.
Porter is likewise putting his lab's SERS system to use for biomarker research. Working with the US Food and Drug Administration's Critical Path Initiative, he's building a multiplex panel that could be used to predict when the bacteria responsible for tuberculosis will move from a latent to active state.
His lab is also looking at biomarkers to detect Aspergillus infections – increasingly a problem for patients on immunosuppression regimes after procedures like bone marrow or organ transplants.
Additionally, he's begun a collaboration with oncologists at the University of Utah to explore SERS as a biomarker discovery tool, using it in combination with antibody arrays to look for protein markers tied to pancreatic cancer.
"What we like about SERS is that it's very extensible," he said. "You change your antibody and you're off to the races."
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