Friganović Tomislav, Weitner Tin
Faculty of Pharmacy and Biochemistry, University of Zagreb, Ante Kovačića 1, 10000 Zagreb, Croatia.
Anal Chem. 2023 Sep 5;95(35):13036-13045. doi: 10.1021/acs.analchem.3c01295. Epub 2023 Aug 22.
The fluorescence attenuation caused by the absorption of the excitation and/or emission light is called the Inner Filter Effect (IFE) and can lead to a nonlinear fluorescence concentration response. In this article, we propose the AddAbs (Added Absorber) method, which counterintuitively corrects IFE by increasing the absorbance of the sample. In this method, an equal amount of a highly absorbing chromophore is added to each sample to compensate for the nonuniform quenching caused by different fluorophore concentrations. The AddAbs method was able to provide a linear fluorescence response ( > 0.999) for very concentrated fluorophore solutions with extreme IFE over more than 97% of the concentration range with less than 1% deviation in calibration slope. The true limit for the AddAbs method with respect to fluorophore concentration was apparently not reached and could be higher than measured ( > 33.94). The IFE-corrected data are obtained by a single fluorescence measurement per sample without additional mathematical procedures. The method also does not require absorbance measurements, so it can be performed in non-transparent microplates with similar results. In addition, preliminary measurements indicate that the method is also suitable for measurements in standard cuvettes using a fluorimeter with a 90° angle setup.
由激发光和/或发射光的吸收所引起的荧光衰减被称为内滤光片效应（IFE），并可能导致非线性荧光浓度响应。在本文中，我们提出了添加吸收剂（AddAbs）方法，该方法通过增加样品的吸光度来反直觉地校正IFE。在这种方法中，向每个样品中添加等量的高吸收发色团，以补偿不同荧光团浓度引起的不均匀淬灭。对于具有极端IFE的非常浓缩的荧光团溶液，AddAbs方法能够在超过97%的浓度范围内提供线性荧光响应（>0.999），校准斜率偏差小于1%。AddAbs方法在荧光团浓度方面的真正极限显然未达到，可能高于所测量的值（>33.94）。通过对每个样品进行单次荧光测量即可获得经IFE校正的数据，无需额外的数学程序。该方法也不需要吸光度测量，因此可以在不透明微孔板中进行，结果相似。此外，初步测量表明，该方法也适用于使用90°角设置的荧光计在标准比色皿中进行测量。
