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Supercritical angle fluorescence (SAF) sensing offers a wash-free alternative for immunoassays. This study shows increasing the SAF collection angle improves surface sensitivity, crucial for accurate fluorescence detection.

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Area of Science:

  • Biomedical Engineering
  • Optics and Photonics
  • Analytical Chemistry

Background:

  • Fluorescent immunoassays often require wash steps to remove unbound molecules, complicating the process.
  • Supercritical Angle Fluorescence (SAF) sensing is a promising wash-free alternative to Total Internal Reflection Fluorescence (TIRF) for sensitive detection.
  • Limited experimental data exists on how SAF collection angles affect surface sensitivity (Signal to Background Ratio - SBR).

Purpose of the Study:

  • To investigate the impact of collection angle on SAF surface sensitivity.
  • To develop a novel technique for simultaneously discriminating free and bound fluorophores.
  • To optimize SAF sensing for enhanced signal-to-background ratio in fluorescence detection.

Main Methods:

  • Developed a novel technique combining angular and spectral information collection.
  • Utilized a multimode fiber connected to a spectrometer to probe the spectrum at multiple back focal plane positions.
  • Calculated the SBR by measuring the intensity difference between free and bound fluorophores.

Main Results:

  • Demonstrated that increasing the SAF collection angle enhances surface sensitivity (SBR).
  • Observed a trade-off: higher angles decrease overall signal intensity while improving SBR.
  • Validated findings through comprehensive full-field 3D simulations.

Conclusions:

  • The study provides crucial experimental insights into optimizing SAF sensing parameters.
  • Increasing SAF collection angles is a viable strategy to improve surface sensitivity for fluorescence-based assays.
  • The developed technique enables simultaneous discrimination of free and bound fluorophores, advancing wash-free immunoassay development.