If fluorescence based detection is used for estimation of enzyme kinetics, is it still possible to use Beer Lambert law? Will it still be valid since detection is not based upon absorbance by a product of enzyme, activity, but that of the fluorescence of a probe?
The Beer-Lambert law applies to absorbance, not fluorescence. However, in the same way that absorbance is directly proportional to the concentration of the chromophore, fluorescence intensity is directly proportional to fluorophore concentration..up to a point. Because a fluorophore is also a chromophore, it can absorb light. The absorption of the excitation light and/or the emitted light by the fluorophore reduces the observed fluorescence intensity if the fluorophore concentration is high enough. This is called the "inner filter effect," and it follows the Beer-Lambert Law. Because of the inner filter effect, a plot of fluorescence intensity versus fluorophore concentration will eventually deviate from linearity at a high enough fluorophore concentration. And, as expected based on the Beer-Lambert law, the linearity of this plot can be extended to a higher concentration range by shortening the path length through the sample.
Hi Vasundhra,
To do this it's usually more typical to set up a standard curve of fluorescence intensity against known concentrations of whatever you are testing first, so that you can then plot your absorbance values on that curve and work out.
Hope this helps.
Mark
Adam has given you an excellent answer. The only thing I would add is that it is important to think about whether your reaction is making the fluorophore or removing it. As John mentions, fluorescence is a tempting option for NADH because of the sensitivity, and life is simpler when you are making NADH than when you are removing NADH - because in one case you don't have to worry about hte inner filter effect and in the other case you absolutely do. To deal with that problem, as well as Adam's solution of cutting the length of the light path, there is also the option of calibrating, so that you apply a different proportionality factor depending on whether you are starting with 50 micromolar than if you are starting with 30 micromolar NADH.
No it does not pertain to fluorescence. On the other hand, you can obtain a standard curve for fluoresce under conditions of your assay and use that to quantify your data.
As usual Adam has presented the perfect answer. A standard curve of fluorescence signal against concentration is a relatively simple way of testing the effective concentration range over which reliable estimates can be made.
A proper selection of the parameters such as excitation and emission wavelengths, the type of cell, front face setup can extent the range of linear dependence of the signal to concentration of the fluorescent analyte. As mentioned by others, the functional dependence must be experimentally verified for the whole range of concentrations before.
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