engleski

NINFE - A NEW CORRECTION METHOD FOR INNER FILTER EFFECT IN MICROPLATES

Inner filter effect (IFE) arises from the absorption of excitation and/or emission light during fluorescence measurements. Therefore, fluorescence intensities are proportional to fluorophore concentration only in a limited, dilute range. Numerous mathematical models and experimental techniques have been used to extend the range of linear response of the fluorescence signal versus concentration, but most are inappropriate for measurements in microplates. A common method for IFE correction requires separate absorbance measurements and can only be used in flat-bottomed transparent microplates. Here we present a Numerical INner Filter Effect (NINFE) method that takes advantage of the variable position of the optical element perpendicular to the sample well (z-position) available in modern microplate readers. By using at least two fluorescence measurements at different z-positions, the linear range can be extended with NINFE. We have successfully applied the presented method to various plate well geometries, including the flat-bottom and round-bottom 96-well microplates (transparent, white, and black) and PCR microplates. NINFE corrections have been performed for protein solutions (BSA and human transferrin) and fluorescent amino acids (tyrosine and tryptophan) to demonstrate that this correction method is applicable to biological systems. Current investigations are aimed at integration with real-time PCR measurements.

fluorescence ; plate readers ; 96-well microplates

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