No abstract

We report a quantitative evaluation of the choice of reporters for multiplexed surface-enhanced Raman spectroscopy (SERS). An initial library consisted of 15 reporter molecules that included commonly used Raman dyes, thiolated reporters, and other small molecules. We used a correlation matrix to downselect Raman reporters from the library to choose five candidates: 1,2-bis(4-pyridyl)ethylene, 4-mercaptobenzoic acid, 3,5-dichlorobenzenthiol, pentachlorothiophenol, and 5,5′-dithiobis(2-nitrobenzoic acid). We evaluated the ability to distinguish the five SERS reporters in a dipstick immunoassay for the biomarker human IgG. Raman nanotags, or gold nanostars conjugated to the five reporters and anti-human IgG polyclonal antibodies were constructed. A linear discriminant analysis approach was used to evaluate the separation of the nanotag spectra in mixtures of fixed ratios.

Surface enhanced Raman spectroscopy (SERS) has been attractive for enhancing the sensitivity of lateral flow immunoassays (LFA). A format that has enabled specific detection of biomarkers is to use Raman reporter molecules linked to gold nanoparticles (NPs), which are conjugated to antibodies specific for the target of interest. Many factors such as the NP and Ab properties and the method of signal readout impact the sensitivity of a SERS based immunoassay. To understand how to optimize assay sensitivity, we studied SERS readouts of multiplexed sandwich immunoassays for the zika and dengue non-structural protein 1 (NS1) biomarkers as a test case. We investigated the effect of NP shape on the SERS enhancement of the reporter molecules 1,2-bis(4-pyridyl)ethylene (BPE) and 4-mercaptobenzoic acid (MBA). We also performed SERS imaging of test lines to map the spatial distribution of signal in test lines on the nitrocellulose. Finally, we used a modified least squares analysis to differentiate reporter contributions.