Naslov
Inner filter effect correction for fluorescence measurements in non-transparent microplates

Autori
Friganović, Tomislav ; Šakić, Davor ; Weitner, Tin

Vrsta, podvrsta i kategorija rada
Sažeci sa skupova, sažetak, ostalo

Skup
48th IUPAC World Chemistry Congress ; 104th Canadian Chemistry Conference and Exhibition (CCCE 2021)

Mjesto i datum
Online, 13.08.2021. - 20.08.2021

Vrsta sudjelovanja
Poster

Vrsta recenzije
Međunarodna recenzija

Ključne riječi
fluorescence, inner filter effect, microplates

Sažetak
Fluorescence spectroscopy implemented in microplate readers has revolutionized technical, biological and medical sciences, offering a multi-sample approach for binding studies, quenching, and cell-based assays. The apparent fluorescence intensity and spectral distribution are dependent upon the optical density of the sample, and the precise geometry of sample illumination. One of the biggest limitations of the method is the inner filter effect (IFE), where only a narrow concentration range with diluted samples provides a linear response between the sample concentration and the measured emission. Specifically, at an absorbance of A = 0.06, a relative error in recorded fluorescence intensity is about 8%, and this difference further increases to 12% at A = 0.1, and 38% at A = 0.3, thus significantly reducing the applicable sample concentration range. Numerous mathematical methods have been proposed for IFE correction, but to our knowledge, none are directly applicable to measurement in non-transparent microplates. A simple and approximate method for IFE correction of observed fluorescence proposed by Lakowicz is currently extensively used for correcting IFE-related artifacts in fluorescence measurements. An important disadvantage of this method is that the sample absorbance at both excitation and emission wavelengths need to be measured independently. Modification of sample geometry featured in modern microplate readers has been used to develop a novel numerical method for IFE correction. We have experimentally verified that the dependence of the corrected fluorescence on the fluorophore concentration is linear (R2 > 0.999) with no more than 1.3 % deviation from the ideal Beer-Lambert dependence for all samples up to Aex ~ 2, without the need for separate absorbance measurements. Importantly, we have experimentally verified that the method is suitable for fluorescence measurements in non-transparent microplates which are much more affordable than the UV/Vis-transparent microplates.

Izvorni jezik
Engleski

Znanstvena područja
Kemija

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