Scientists have developed an enzyme-linked immunosorbent assay (ELISA) technology for detecting blood biomarkers that generates an eye-visible color change reaction and, they claim, is up to 10 times more sensitive than traditional ELISA or PCR-based techniques. The researchers at Imperial College London (U.K.) hope their plasmonic ELISA could form the basis of a qualitative diagnostic test suitable for use in countries where sophisticated equipment isn’t available.
In a conventional sandwich ELISA, the target molecule in a sample binds to a capture antibody that is immobilized on a substrate. The bound target molecule is then recognized and bound by a primary antibody, essentially generating a sandwich of target between two antibodies. Detection is carried out using an enzyme-linked secondary antibody that binds to the primary antibody, and converts a substrate into a colored reporter. The intensity of the color indicates the concentration of the target. While the results can be visible to the naked eye if the target is present at high enough concentrations, in practice the color intensity is quantified by measuring the absorbance using a plate-reader.
In the new technology developed by Molly M. Stevens, Ph.D., and Roberto de la Rica, Ph.D., the enzyme bound to the secondary antibody is harnessed to control the growth of gold nanoparticles in the presence of hydrogen peroxide. If there is no analyte present, and thus the enzyme-linked secondary antibody doesn’t bind to the primary antibody, the reduction of gold ions in the presence of hydrogen peroxide occurs at a fast rate, and near-spherical nonaggregated gold nanoparticles are generated, which causes the solution to turn a red color. If the analyte is present, the hydrogen peroxide is used up in the presence of catalase enzyme, and so gold nanoparticle growth is slowed, and clumps of nanoparticles form. In this event, the solution turns blue.
The team tested plasmonic ELISAs designed to detect either prostate specific antigen (PSA), or the HIV-1 capsid antigen p24, using blood samples from human patients and controls. Results from both sets of studies showed the technique to be highly sensitive, and generated a color-change reaction that was easily visible to the naked eye, even at concentrations of target that wouldn’t generate a positive result using traditional ELISA or nucleic acid-based approaches.
And while plasmonic ELISA is qualitative, rather than quantitative, this potential drawback is offset by the sensitivity of the technique, the Imperial team claims. “Our approach affords for improved sensitivity, does not require sophisticated instrumentation, and is ten times cheaper, which could allow more tests to be performed for better screening of many diseases,” Dr. Stevens claims.
The potential to use the technique as an alternative to gold standard nucleic acid-based tests for detecting HIV is particularly encouraging, the researchers state in their published paper in Nature Nanotechnology. “The detection of p24 holds great promise for the diagnosis of HIV infection in laboratories with fewer resources where expensive nucleic acid-based tests may not be available...The methodology proposed here could potentially be adapted for the detection of any analyte as long as antibodies directed against it were available, therefore making plasmonic ELISA a versatile tool for the detection of other clinically relevant molecules using the naked eye.”
Drs. Stevens and de la Rica describe the plasmonic ELISA technology in a paper titled “Plasmonic ELISA for the ultrasensitive detection of disease biomarkers with the naked eye.”