Enhancement of Fluorescence-Based Immunoassay for Point-of-Care Testing Using the Plasmonic Nanopatch Metasurface

Fluorescence-based methodologies have been used extensively for biosensing and to analyze molecular dynamics and interactions. An emerging, promising diagnostic tool are fluorescence-based microarrays due to their high throughput, small sample volume and multiplexing capabilities. However, their low fluorescence output has limited their implementation for in vitro diagnostics applications in a point-of-care (POC) setting. Here, by integration of a sandwich immunoassay microarray within a plasmonic nanogap metasurface, we demonstrate strongly enhanced fluorescence enabling readout by a fluorescence microarray even at low sensitivities. We have named this plasmonic architecture the plasmonically enhanced D4 (PED4) assay. The immunoassay consists of inkjet-printed capture and fluorescently labeled detection antibodies on a polymer brush which is grown on a gold film. Colloidally synthesized silver nanocubes (SNCs) are placed on top of the brush through a polyelectrolyte layer and interacts with the underlying gold film creating a nanogap plasmonic structure supporting local electromagnetic field enhancements of ~100-fold. By varying the thickness of the brush between 5 and 20 nm, a 151-fold increase in fluorescence and a 14-fold improvement in the limit-of-detection (LOD) is observed for the cardiac biomarker B-type natriuretic peptide (BNP) compared to the unenhanced assay, paving the way for a new generation of point-of-care clinical diagnostics.

To move the PED4 towards a single step point of care test (POCT), SNCs are conjugated with a secondary antibody that attaches specifically to the detection antibody. This allows SNCs to deposit on the surface without the need of a charged polyelectrolyte layer. In addition, multiplexing capabilities are demonstrated in this plasmonic platform where NT-proBNP, Galectin-3, and NGAL are simultaneously detected and fluorescently enhanced. Microfluidics integration and use of a low-cost detector is also demonstrated.