Chromophore-driven optical path reduction as an indirect inner filter effect mitigation strategy in fluorescence...

Area of Science:

• Biochemistry
• Analytical Chemistry
• Spectroscopy

Background:

• Microplate readers are crucial for high-throughput fluorescence measurements.
• The Inner Filter Effect (IFE) significantly impacts fluorescence assay accuracy.
• Secondary IFE (sIFE) limits measurements in high optical density samples.

Purpose of the Study:

• To develop a practical method for mitigating secondary IFE (sIFE) in microplate fluorescence assays.
• To enable accurate fluorescence quantification in samples with high absorbance.
• To expand the dynamic range for fluorescence measurements in microplate formats.

Main Methods:

• A strategy was developed to enhance primary IFE (pIFE) using a strongly absorbing chromophore.
• This confines fluorescence generation to a shallow surface layer, reducing sIFE.
• The method involves mixing samples with a fixed amount of absorber before measurement.

Main Results:

• Accurate fluorescence measurements were achieved even at emission optical densities up to 79 absorbance units/cm.
• Linear fluorescence-concentration responses (R² > 0.99) were confirmed over a broad dynamic range.
• Minimal signal loss was observed at very high analyte concentrations.

Conclusions:

• The presented method effectively mitigates sIFE in microplate fluorescence assays.
• It offers a robust, physical solution without the need for mathematical corrections.
• This approach expands the utility of microplate readers for high-density fluorophore measurements.