ELISA meet SERRS

Surface-enhanced resonance Raman scattering could be used to quickly spot the telltale signs of prostate cancer at the molecular level, according to a British team. This is the first use of a well-known ELISA colorimetric substrate as a SERRS marker.

Writing in the current issue of Analyst, Ross Stevenson, Andrew Ingram, and Duncan Graham of the Centre for Molecular Nanometrology at the University of Strathclyde, who have collaborated with Hing Leung and Donald McMillan of the Division of Cancer Sciences and Molecular Pathology, at University of Glasgow, Glasgow, describe details of the new method.

Their approach combines the power of SERRS with the potency of enzyme-linked immunosorbent assay, or ELISA. ELISA is a fundamental modern technique in biology.

ELISA is commonly used in medical diagnostics and other areas to detect various immunological marker chemicals in biopsy samples. Put simply, extract from the biopsy or serum is fixed in a test plate and washed with a solution containing antibody specific to the suspect antigen. If the target antigen is present in the sample, then the antibody will bind to the target.

However, to be useful as a detection method, a prior step is required. This involves attaching an enzyme to the antibody that can produce a detectable signal on antigen binding. In the present example, the simplest horseradish peroxidise was used. A  fluorescent marker is added as a reagent that is transformed by the presence of the enzyme. This provides a signal that can be picked up spectroscopically should antigen binding have occurred in the sample. ELISA thus reveals the presence of the antigen in question in the sample.

The trouble with using fluorescence spectroscopy in ELISA is that only a single fluorescent probe can be used at a time because multiple signals would swamp each other in the spectra.

SERRS, in contrast, requires no fluorescence marker and instead can utilise gold nanoparticles in the enhancement of scattering signals. This can thus be used to exploit the 10¹⁴ enhancement seen with conventional Raman. Graham and colleagues excited the sample at 514.5 nanometres and saw Raman shifts at 1442 and 1405 wavenumbers.

The team explains that using commercially available gold nanoparticles offer greater biocompatibility over the alternative material, silver. They also point out that the relative inertness of gold particles compared to silver means they are less likely to precipitate out of solution in unwanted side reactions. One putative advantage of silver over gold would be if there were more significant enhancements. However, the team has demonstrated that signals obtained with silver are not significantly stronger than with gold, which implies that the advantages of gold over silver outweigh the marginal enhancement benefits.

This approach could ultimately allow SERRS to be useful for detecting several antigens at once, each specific to a different antibody. In proof of principle experiments, Graham and his colleagues have focused on just prostate specific antigen and were able to detect picograms per millilitre of this cancer marker in a patient serum sample.

The new ELISA-SERRS hybrid technique quickly reveals the presence of elevated levels of prostate specific antigen in a sample of the patient's blood serum and could preclude more invasive investigations for men suspected of having prostate cancer.

Previously, SERRS had proved itself in DNA detection. With fluorescently labelled oligonucleotides can produce detection limits much lower than standard fluorescence methods. Other researchers have looked at enzyme activity measurements using SERRS for enzymes such as lipases, alkaline phosphatase, proteases, and peroxidise, the team adds.

Other teams have also reported SERRS in immunoassays with labelled nanoparticles and antibody conjugates, glass-coated dye-labelled nanoparticles conjugates, and silver-stained fluorescently labelled antibodies.

"However," the team says, "very few methods reporting SERRS as a readout technique for an enzyme linked immunosorbent assay (ELISA) have been reported."

"The [current] technique is amenable over a wide range of concentrations and lends itself to future multiplexing analysis," the researchers explain. They add that, "With single-molecule detection possible and potential for multiplexing analysis that outstrips other mainstream spectroscopic techniques, the reliability and low-costs allow SERRS to compete with existing analytical techniques over a wide range of applications."