Abstract
Colorimetric enzyme-linked immunosorbent assay utilizing 3′-3-5′-5-tetramethylbenzidine(TMB) as the chromogenic substrate has been widely used in the hospital for the detection of all kinds of disease biomarkers. Herein, we demonstrate a strategy to change this single-color display into dual-color responses to improve the accuracy of visual inspection. Our investigation firstly reveals that oxidation state of 3′-3-5′-5-tetramethylbenzidine (TMB2+) can quantitatively etch gold nanoparticles. Therefore, the incorporation of gold nanoparticles into a commercial TMB-based ELISA kit could generate dual-color responses: the solution color varied gradually from wine red (absorption peak located at ~530 nm) to colorless, and then from colorless to yellow (absorption peak located at ~450 nm) with the increase amount of targets. These dual-color responses effectively improved the sensitivity as well as the accuracy of visual inspection. For example, the proposed dual-color plasmonic ELISA is demonstrated for the detection of prostate-specific antigen (PSA) in human serum with a visual limit of detection (LOD) as low as 0.0093 ng/mL.
Highlights
The discrimination of colors in-between red and blue are still a big challenge with the naked eyes
We demonstrate another alternative to conduct plasmonic enzyme-linked immunosorbent assay (ELISA) based on enzyme mediated etching of gold nanoparticles
The concentration of TMB2+ was proportional to the concentration of antigen in the assay samples, which was the basis of the commercial ELISA for quantitative determination of the concentration of proteins
Summary
The discrimination of colors in-between red and blue are still a big challenge with the naked eyes. Color variations between red and blue result in continuous changes of the absorption spectral peaks, which is inconvenient to be monitored with a microplate reader for the quantification of the analytes. During the whole detection process, the absorption spectra of the solution only shows two peaks; one located at ~530 nm, which corresponds to the wine red solution; the other located at ~450 nm, which corresponds to the yellow solution. These two distinct absorption peaks are to be detected with the microplate reader. Comparing with the conventional ELISA, this dual color plasmonic ELISA can effectively improve the accuracy of visual inspection
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