Digital SERS Protocol Using Au Nanoparticle-Based Extrinsic Raman Labels for the Determination of SARS-CoV-2 Spike Protein in Saliva Samples

Surface-enhanced Raman scattering (SERS)-based immunoassays have several advantages, such as high sensitivity and multiplex capabilities; they are emerging as a potential avenue for early disease diagnosis and screening. Here, we focused on SERS-based heterogeneous immunoassays, in which the number of extrinsic Raman labels (ERLs) at the sensor surface is related to the concentration of the intended target. The ERLs are made of gold nanoparticles and are constructed to be selective to the target and to boost the signal of a Raman reporter. However, as the concentration of the target biomarker decreases, the number of ERLs per unit of area (mm(2)) also decreases, leading to a small number of ERLs being probed within an exciting laser spot. This poor sampling adds to the large intensity variations inherent to the SERS effect, resulting in a loss in the linearity between the SERS signal and the marker/target analyte concentration. This characteristic has rendered SERS-based immunoassays unreliable for quantification at low bioanalyte concentrations. We propose the use of a digital quantification protocol to overcome this problem. A SERS-based sandwich immunoassay was developed for the detection of the SARS-CoV-2 S1-S2 spike protein in saliva. A conventional data analysis that relates SERS intensities to concentration was compared to the digital protocol for the same dataset. The digital SERS assay presented an LOD of 6.3 ng.mL(-1 )or 34.9 pM and an LOQ of 19.0 ng.mL(-1) or 105.7 pM within a 95% confidence level. These metrics show an 11-fold improvement to the conventional data