Biosensor platform for parallel surface plasmon-enhanced epifluorescence and surface plasmon resonance detection

For the first time, plasmonic diffraction grating is implemented for the combined direct and epifluorescence-based readout of assays in real-time. This plasmonic structure serves for grating-coupled surface plasmon resonance (SPR) monitoring of molecular binding-induced changes in the refractive index. In parallel, it allows to simultaneously perform plasmonically enhanced fluorescence (PEF) analysis of affinity binding of molecules that are labeled with fluorophores. This configuration offers facile readout of e.g. magnetic nanoparticle-enhanced assays which is not possible with more conventional Kretschmann geometry. The performance characteristics of this combined approach are discussed by using two types of assays. In the first assay a fluorophore-labeled protein with a medium molecular weight of 55 kDa was affinity captured on the plasmonic sensor grating. The associated PEF signal in this assay showed a signal-to-noise ratio that was 140-fold higher compared to that of the SPR detection channel. In the second assay, extracellular vesicles were detected by using antibodies against CD81 attached to the plasmonic grating. These vesicles were pre-concentrated by their coupling to magnetic nanoparticles with cholera toxin B chain. This assay exploited magnetic nanoparticles as labels enabling rapid collection of analyte at the sensor surface and for the enhancement of the SPR sensor response. In this case the label-free SPR detection channel outperformed the fluorescence-based detection, as the SPR signal-to-noise ratio was 2.4-fold higher than that of PEF.