UV to NIR Surface Plasmon Coupled and Metal-Enhanced Fluorescence Using Indium Thin Films: Application to Intrinsic (Label-less) Protein Fluorescence Detection

Recently, the surface plasmon coupled fluorescence (SPCF) phenomenon has become a rapidly progressive area of research in the field of analytical biosciences to understand biomolecular kinetics and binding events at interfaces. 10 SPCF spectroscopy is a more sensitive analytical tool with respect to the closely related surface plasmons resonance (SPR) technology. 13 Surface plasmons are coherent collective oscillating electrical charges between a metal dielectric interface. When a thin metal film is illuminated by radiation in a suitable angle through a prism, the surface plasmons start to resonate with an incident photon frequency, thereby reducing the reflected light intensity at that particular angle by absorbing resonant photons. The SPR angle is sensitive to the refractive index of the sample above the metal, distal from the glass prism. The origin of this sensitivity is the evanescent field from the plasmons, which penetrates several nanometers into the sample. However, binding events for a very low concentration of analyte typically cannot produce enough change in the SPR angle and so conversely, the SPR technique is hinged on the ability to detect very small changes in angle. This sensitivity limitation with the SPR technique can be overcome by detecting fluorescence excited by the evanescent field associated with surface plasmons. There are various reports on the utilization of SPCF in bioassays. These include studies for DNA hybridization, immunoassays, ultrafast assays, and protein detection, etc. In all of these reports, one of the biological assay components is linked or positioned close to the metal surface, and their interactions with the biomolecule of interest in the presence of a third fluorophore-labeled biomolecule is monitored via the change in SPCF intensity from the back of the prism. Subsequently, the concentration of the biomolecule of interest is directly calculated from the SPCF intensity. In surface plasmon fluorescence spectroscopy (SPFS), the excitation of the fluorophores can be achieved directly from the sample side (reverse Kretschmann (RK) configuration) or through a prism side (Kretschmann (KR) configuration). In the RK configuration, fluorophores are directly excited by the incident excitation source, and the resulting fluorescence emission induces and couples to surface plasmons. In the KR configuration, the excitation light entering through the prism generates the surface plasmons in the metal film and excites fluorophores within a certain distance (typically 10 to 200 nm) from the surface. Below