Mining the salivary proteome with grating-coupled surface plasmon resonance imaging and surface plasmon coupled emission microarrays

Abstract

Biological indicators have numerous and widespread utility in personalized medicine, but the measurement of these indicators also poses many technological and practical challenges. Blood/plasma has typically been used as the sample source with which to measure these indicators, but the invasiveness associated with sample procurement has led to increased interest in saliva as an attractive alternative. However, there are unique issues associated with the measurement of saliva biomarkers. These issues are compounded by the imperfect correlation between saliva and plasma with respect to biomarker profiles. In this manuscript, we address the technical challenges associated with saliva biomarker quantification. We describe a high-content microarray assay that employs both grating-coupled surface plasmon resonance imaging and surface plasmon-coupled emission modalities in a highly sensitive assay with a large dynamic range. This powerful approach provides the tools to map the proteome of saliva, which in turn should greatly enhance the utility of salivary biomarker profiles in personalized medicine.

Figure 1. Indirect Fluorescent Immunoassay using Surface Plasmon coupled emission (SPCE)

In this SPCE assay diagram, capture antibodies have been printed on a gold sensor chip which has a holographic diffraction grating. Analyte capture was detected by recirculating an analyte-specific biotinylated secondary antibody and streptavidin-Alexa Fluor 647 over the chip. Fluorophores on the chip were then excited using an LED light source, and a CCD camera detected the resulting fluorescence. In addition to the standard fluorescent excitation shown above, the interaction of the surface plasmon increases the directional fluorescent emission which amplifies this fluorescent signal ~80-fold.

Figure 2. SPCE Microarray Image using a Saliva Sample

The SPCE microarray image was taken after exposure to human saliva doped with 14 recombinant human protein biomarkers of interest and a cocktail of fluorescently labeled secondary antibodies. Six cytokines were not included in the saliva sample to act as negative controls. Regions of interest (ROIs) are printed in groups of three for each biomarker. Isotype controls, diluent control (PBS), and positive control ROIs are included in the image.

Figure 3. Saliva does not affect the IL-2 limit of detection by SPCE

Recombinant human IL-2 was diluted in protease inhibitor treated saliva and PBS (1:1) (●) or PBS alone (▲) and detected using an indirect sandwich assay. Points indicate the mean fluorescence detected from 3 ROI microspots. Error bars indicate standard deviation.