Anton Simeonov Ph.D. National Institute of Health

Researchers provide an assay chemistry that allows for a direct visual scoring of an ELISA result to detect protein analytes at ultralow concentrations.

Ultrasensitive detection often goes hand-in-hand with expensive instrumentation. The report by de la Rica and Stevens* serves as a testimony that this does not always have to be the case: the authors provide an assay chemistry that allows for a direct visual scoring of an enzyme-linked immunosorbent assay (ELISA) result to detect protein analytes at ultralow concentrations.

While the traditional capture and detection antibodies were unchanged, the authors introduced a variation in how the detection signal was generated: instead of utilizing chromogenic substrate (which only carries a very limited potential for generating detectable colored product), the team coupled the reporter enzyme reaction with the generation of intensely colored gold nanoparticles, termed plasmonic ELISA (Figure 1). Furthermore, it was discovered that the structure of the nanoparticles, and hence their color, changed dramatically as a function of the concentration of hydrogen peroxide substrate for the catalase reporter (Figure 2): at H2O2 concentrations of 120 μM and above, the nanoparticles form regular uniform spheres of red color, while at lower H2O2 levels (achieved through the increased action of the reporter catalase enzyme at higher analyte concentrations) the nanoparticles formed irregular aggregates that were blue in color.

Figure 1. Schematic representation of the sandwich ELISA format used here and two possible signal generation mechanisms. In sandwich ELISA, the target molecule is anchored to the substrate by capture antibodies and recognized by primary antibodies. In the present work, the enzyme is linked to the immunocomplex through interactions between enzyme-decorated streptavidin and biotinylated secondary antibodies. (a) In conventional colorimetric ELISA, enzymatic biocatalysis generates a colored compound. (b) In plasmonic ELISA, the biocatalytic cycle of the enzyme generates colored nanoparticle solutions of characteristic tonality. S, substrate; P, product; NP, nanoparticle.

Using this approach, the team evaluated prostate specific antigen and the HIV-1 capsid antigen p24 as model low-abundance biomarkers of broad interest and demonstrated that the new system permitted a visual scoring of samples containing as low as 10−18 g/mL analyte. The overall simplicity of the system should prompt further research in this area to ultimately productize the plasmonic ELISA reagents.

Figure 2. Generation of colored solutions for detection with the naked eye. (a) In the presence of hydrogen peroxide, gold ions are reduced. (b) High concentrations of hydrogen peroxide favor the formation of non-aggregated, spherical nanoparticles that give rise to a red solution. (c) When the concentration of hydrogen peroxide decreases, for example due to the biocatalytic action of the enzyme catalase, aggregates of nanoparticles are formed and this turns the solution blue.

*Abstract from Nature Nanotechnology 2012, Vol. 7: 821–824

In resource-constrained countries, affordable methodologies for the detection of disease biomarkers at ultralow concentrations can potentially improve the standard of living. However, current strategies for ultrasensitive detection often require sophisticated instruments that may not be available in laboratories with fewer resources. Here, we circumvent this problem by introducing a signal generation mechanism for biosensing that enables the detection of a few molecules of analyte with the naked eye.

The enzyme label of an enzyme-linked immunosorbent assay (ELISA) controls the growth of gold nanoparticles and generates colored solutions with distinct tonality when the analyte is present. Prostate specific antigen (PSA) and HIV-1 capsid antigen p24 were detected in whole serum at the ultralow concentration of 1 × 10−18 g/mL. p24 was also detected with the naked eye in the sera of HIV-infected patients showing viral loads undetectable by a gold standard nucleic acid–based test.

Anton Simeonov, Ph.D., works at the NIH.

ASSAY & Drug Development Technologies, published by Mary Ann Liebert, Inc., offers a unique combination of original research and reports on the techniques and tools being used in cutting-edge drug development. The journal includes a “Literature Search and Review” column that identifies published papers of note and discusses their importance. GEN presents here one article that was analyzed in the “Literature Search and Review” column, a paper published in Nature Nanotechnology titled “Plasmonic ELISA for the ultrasensitive detection of disease biomarkers with the naked eye.” Authors of the paper are de la Rica R and Stevens MM.

Previous articleExperts Push for Drug Development Reform
Next articleCrackdown on Biopharma Payments to Doctors