Sensitive SERS beats ELISA

Scientists in South Korea have developed a new magnetic approach to immunoassay detection of important biological marker compounds and antigens using surface-enhanced Raman spectroscopy (SERS) of hollow gold nanospheres. The technique is not only much faster than standard assays but up to 1000 times more sensitive.

Hyangah Chon, Sangyeop Lee, and Jaebum Choo of Department of Applied Chemistry, Hanyang University, Ansan, and Sang Wook Son of the Department of Dermatology, Korea University Hospital, Seoul, South Korea, have developed a technique that overcomes the problem of slow immunoreaction caused by the diffusion-limited kinetics on a solid substrate because all of the reactions occur in solution.

The team points out that immunoassays can be rapid and inexpensive in clinical diagnostics, biological sensors, and food safety tests. Enzyme-linked immunosorbent assays (ELISA) and surface plasmon resonance (SPR) spectroscopy are commonly used for detecting antigens or complementary antibodies. Fluorescent labels can be applied to help identify antigen-antibody interactions in immunoassays.

Advances in metal nanoparticle science and the emergence of quantum dots, however, are providing analysts with a putative alternative to fluorescent dyes in the form of tuneable nano-tags that offer even more precise results. The application of SERS in immunoassays using antibody-conjugated metal nanoparticles is also now on the increase.

A popular approach involves immobilising polyclonal antibodies (PAb) on a solid substrate and then adding a solution of suspect antigen and monoclonal antibody (mAb)-conjugated metal nanoparticles one after the other and carrying out the SERS experiment. However, this specific approach has several limitations, not least the fact that all components of the assay must be immobilised, in air, and washed repeatedly; something many proteins cannot endure.

Chon and colleagues have now used magnetic beads as supporting substrates for the formation of an immunocomplex. The reagents can then be immobilised easily within a microtube by placing the tube on a magnetic bar. This avoids many of the standard immobilisation steps and also benefits from the immunoreaction taking place in solution rather than in the solid phase.

"Our technique overcomes the slow immunoreaction problems caused by the diffusion-limited kinetics on a solid substrate because the reaction occurs in solution," the researchers explain, "As a result, the assay time is greatly reduced to less than 1 hour if properly designed metal nanotags are prepared."

It is those metal nanotags in the form of hollow gold nanospheres that provide the hook for the SERS experiments. They offer strong enhancement effects from individual particles because hot spots can be localized on the pinholes in the hollow particle structure.

In order to validate their novel approach to SERS immunoassay, the team has tested the method on the well-known lung cancer marker, carcinoembryonic antigen (CEA). This biochemical is not usually found in the lungs of healthy individuals but is present at low concentration in heavy smokers.

The researchers were able to determine the presence of this marker in solution at a concentration as low as 1-10 picograms per millilitre. "This value is about 100 to 1000 times more sensitive than ELISA," the researchers claim, "and the assay time took less than 1 h, including washing and optical detection steps."

Their proof of principle not only bodes well for developing the approach for other biomarkers but could also have clinical application for assaying CEA itself. Other researchers have shown that CEA is raised in the serum of people with colorectal, gastric, pancreatic, lung, thyroid, and breast carcinomas, compared with healthy individuals. Testing for CEA is therefore often used to identify recurrences of cancerous tissue after surgical re-section, the team says.

Current cut-off values for CEA are well below the limits of detection for conventional analysis however, at 2.5 nanograms per millilitre for non-smokers and 5.0 nanograms per millilitre for smokers. "A sensitive immunoassay technique to detect a sub-nanogram per millilitre level is required to satisfy detection of this cut-off level," the researchers say. Their comparison of SERS-based detection and ELISA results for CEA markers reveals a significantly higher sensitivity for the magnetic SERS method.