Plasmonically amplified bioassay - Total internal reflection fluorescence vs. epifluorescence geometry.
Simone Hageneder1, Martin Bauch2, Jakub Dostalek1
1Biosensor Technologies, AIT-Austrian Institute of Technology, Muthgasse 11, 1190 Vienna, Austria.
Talanta
|June 5, 2016
Summary
Plasmonic amplification significantly boosts fluorescence signals in bioassays. While epifluorescence (EPF) with gratings offers higher signal enhancement than total internal reflection fluorescence (TIRF), both achieve similar low detection limits for interleukin-6 (IL-6).
Area of Science:
- Optics and Photonics
- Biophysics
- Materials Science
Background:
- Fluorescence readout is crucial for bioassays.
- Plasmonic amplification offers enhanced sensitivity.
- Comparing epifluorescence (EPF) and total internal reflection fluorescence (TIRF) configurations is important.
Purpose of the Study:
- Investigate and compare plasmonic amplification in EPF and TIRF configurations for bioassay fluorescence readout.
- Evaluate the impact of different plasmonic geometries on signal enhancement and detection limits.
- Demonstrate the application for analyzing biomarkers like interleukin-6 (IL-6) and surface affinity reaction kinetics.
Main Methods:
- Implemented plasmonic amplification in EPF using crossed gold diffraction gratings.
- Employed Kretschmann geometry for attenuated total reflection (ATR) in TIRF configuration.
- Utilized identical assays, surface architectures, and optical setups for both configurations.
Main Results:
- Simulations predicted a 10^2 fluorescence signal amplification for EPF via surface plasmon-enhanced excitation and coupled emission.
- EPF geometry showed 4-times higher fluorescence intensity than TIRF for IL-6 immunoassay.
- Both configurations achieved an identical low detection limit of 0.4pM for IL-6.
Conclusions:
- Crossed gold diffraction grating in EPF offers superior fluorescence signal amplification compared to TIRF's Kretschmann geometry.
- Lower background fluorescence in TIRF compensates for weaker signal enhancement, leading to comparable detection limits.
- Plasmonic amplification is effective for sensitive biomarker detection and kinetic analysis in bioassays using both EPF and TIRF.
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