< Back to previous page

Publication

Spectral and Angular Filtering for On-Chip Near-Field Fluorescence Sensing

Book - Dissertation

Supercritical angle fluorescence (SAF) is a near-field collection technique that has surface sensitivities similar to near-field excitation techniques like total internal reflection fluorescence (TIRF) and waveguide-based excitation. SAF is emitted by fluorophores that are a few hundred nanometres away from an interface, above the critical angle, into the higher index material and decreases exponentially with increasing distance from the interface. SAF accounts for up to 40% of the total fluorescence emitted by a fluorophore.Although a lot of research has used SAF for biosensing and microscopy, the angular dependence of SAF on both the surface and bulk fluorescence contributions hasn't been experimentally studied. Therefore, we first present an experimental technique that measures the surface selectivity of SAF in the presence of bulk fluorophores. Two different fluorophores were used. One was bound to the surface and the other was suspended in the bulk of the solution. The spectrum was measured at discrete points in the back focal plane (BFP) and the contribution of the two fluorophores was extracted from it. The results show the highest signal-to-noise ratio when all of the SAF is collected above the critical angle of 61.3° because of the higher signal intensity. However, for experiments where bulk exclusion is important, we observe the highest signal-to-bulk ratio at angles above 68° for a glass-water interface. Understanding the dependence of bulk exclusion on the collection angles of a SAF biosensor allows it to be optimized for different applications.Following experimental characterization of the bulk exclusion capabilities of SAF, we present a novel SAF transduction technology that is capable of performing biosensing in a small form factor while being as sensitive as the well plate based fluorescent immunoassay (FIA). It features a monolithically fabricated interference filter that blocks excitation photons and undercritical angle fluorescence (UAF) from the bulk of the solution, while allowing SAF photons from surface bound molecules to pass through. This allows us to fabricate a SAF biosensor on a chip using existing complementary metal oxide semiconductor (CMOS) manufacturing techniques. To demonstrate the sensitivity of this technology, the filter was fabricated using amorphous silicon and silicon dioxide and a real-time wash-free protein A immunoassay was performed on it. The results show that our novel SAF biosensor is comparable in performance to the traditional microwell plate based FIA, the current gold standard. The novel SAF biosensor achieved this with a measurement time of only 100 seconds, a greater dynamic range and while providing information on the binding kinetics of the analyte. We believe this technology has the potential to create biosensor products in a wide range of form factors, from large expensive high multiplex devices to small handheld point-of-care solutions.
Accessibility:Open