Abstract
This paper examines the impact of the sampling error caused by the small size of the focused laser spot when using surface-enhanced Raman scattering (SERS) as a quantitative readout tool to analyze a sandwich immunoassay. The assay consists of a thin-film gold substrate that is modified with a layer of capture monoclonal antibodies (mAbs) and extrinsic Raman labels (ERLs) that consist of gold nanoparticle cores (60 nm diameter) coated with a monolayer of a Raman reporter molecule and a layer of human IgG mAbs to tag the captured antigen. The contribution of sampling error to the measurement is delineated first by constructing and analyzing an antigenic random accumulation model; this is followed by an experimental study of the analysis of an assay substrate using two different laser spot sizes. Both sets of findings indicate that the analysis with a small laser spot can lead to a sampling error (i.e., undersampling) much like that found when the size of a measured soil sample fails to accurately match that of a larger, more representative sample. That is, the smaller the laser spot size, the larger probable deviation in the accuracy of the measurement and the greater the imprecision of the measurement. Possible implications of these results with respect to the general application of SERS for quantitative measurements are also briefly discussed.
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