FluoroQuest™ Fluorescence Quantum Yield Determination Kit *Optimized for Bioconjugates*

Experimental considerations

The most reliable method for recording fluorescence quantum yield is the comparative method of Williams et al. It involves the use of well characterized standard samples with known fluorescence quantum yield values. Essentially, solutions of the standard and test samples with identical absorbance at the same excitation wavelength can be assumed to be absorbing the same number of photons. Hence, a simple ratio of the integrated fluorescence intensities of the two solutions (recorded under identical conditions) will yield the ratio of the quantum yield values. Since the fluorescence quantum yield for the standard sample (Φ_s) is known, it is trivial to calculate the fluorescence quantum yield for the sample (Φ_x).

The standard reference should be chosen to have certain absorbance at the excitation wavelength of choice for the test sample, and, if possible, emit in a similar region to the test sample. Standard 10 mm path length fluorescence cuvettes are sufficient for running the fluorescence measurements. For small amount samples, either a microplate reader or nanodrop device can be used. The absorbance in the 10 mm cuvette should be <0.2. Above this level, non-linear effects may be observed due to inner filter effects, and the resulting quantum yield values may be inaccurate.

This kit is only designed for a one-point quick estimation of the fluorescence quantum yield of a biological conjugate. For more accurate deamination of fluorescence quantum yield there are many factors that have a significant effect on fluorescence quantum yields.

Make 1 mM stock solutions of the selected standard and test samples using a proper solvent (such as water or DMSO).

Table 1. Kit components with respective quantum yield in water and similar wavelength dyes for reference standard selection.

1. Record the absorbance spectra of the selected standard and test samples (0.1 to 5 µM) using the diluted solutions prepared from their stock solutions with an aqueous buffer (such as PBS or TRIS).
   
   
2. Select the excitation wavelength based on the absorption maxima. We recommend to use this following formula to select the desired excitation wavelength:
        Desired excitation(nm) = shorter absorption maximum* – 50nm
   *Selected from either the reference standard or test sample. For example, if your test sample has absorption maximum at 500 nm, and the selected reference standard has the absorption maximum at 490 nm, you should select 440 nm (490 nm  - 50 nm) as excitation wavelength. 
   
   
3. Adjust the concentrations of the selected reference standard and test samples to have the same absorbance (between 0.2 to 0.8) at the selected excitation wavelength. Note: It might be easier to choose the isosbestic point of the reference standard and test sample absorption spectra.
   
   
4. Dilute both the selected reference standard and test sample by 10 times (must be diluted by the same proportion) using the same buffer or pure water.
   
   
5. Record the fluorescence spectra of the same diluted solutions in the 10 mm fluorescence cuvette using a fluorescence spectrophotometer or a microplate reader that can generate corrected fluorescence spectrum. Note: All the fluorescence spectra must be recorded with the same instrument settings. Changing instrument settings between samples will invalidate the quantum yield measurement.