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Inner filter effect corrections for protein fluorescence measurements in microplates using variable z-focus position

Inner filter effect (IFE) can often be an important attributing factor to the non-linear response of the relative fluorescence intensity signal with respect to the analyte concentration. This effect is due to the partial reabsorption of the excitation and/or emission radiation. The apparent fluorescence intensity and spectral distribution are functions of the sample’s optical density as well as the geometrical parameters of the sample illumination. A commonly used and simple IFE correction method proposed by Lakowicz1 relies on the absorbance measurements of the excitation and emission wavelengths. Most modern microplate readers have the ability to measure fluorescence intensities at different positions along the z-axis (perpendicular to the sample well). This feature was used to perform the IFE corrections for the solutions of the human serum transferrin protein, as well as for the solutions of a well known non-protein fluorophore quinine sulfate. The proposed IFE correction method does not require separate absorbance measurements. Advantage of the variable z-focus method is that it can also be performed in the more affordable non-transparent microtiter plates.

fluorescence, inner filter effect, microplates, transferrin

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