Hi! I am getting a higher fluorescence value for a lower concentration Umblifero solution than compared to a higher concentration solution.
I am using the 0.001, 0.0008, 0.0006,0.0004, and 0.0002 M umpliferon aqueous solution.
I am taking the fluorescence readings in a transparent 96 well plate.
Hi Joshi, From looking at your concentration range, the lowest concentration you have is 0.0002M = 200 uM. When it comes to the fluorescence analysis world, this is a very high probe concentration. We typically use concentrations 10 uM or lower for analysis work. Also the fluorescence spectra is directly proportional to the concentration ONLY in dilute solutes. At higher concentrations tis can not be achieved due to various factors. Most likely at higher concentrations your fluorophore may undergo aggregation caused quenching (ACQ) which could leave to a quenching of fluorescence and you may observed the lower fluorescence in concentrated samples compared to the dilute sample. Is there anyway you can conduct the assay by using 100 fold dilution of the whole concentration gradient and see whether you still have the issue?
Also, in your absorbance data, do you see any unusual changes in the absorbance spectra when concertation increases?
Let me know how it goes and Good Luck!!
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For fluorescence measurements, you should use black plates to avoid crosstalk between wells, and interference from fluorescence of the plastic.
In general, coumarins have a high quantum yield (> 0.6). So lower conc. should be applied. In order to exclude interfering effects such as aggregation and self-quenching, it is generally recommended to use concentrations below 10 -5 M. In addition, no transparent wells should be used for the measurement.
Adam B Shapiro ,
Thanks, Adam for your response.
As I don't have the opaque plates, I used the 96 well transparent plate. I didn't get much cross-talk between the weels, maybe because I have higher fluorescence intensity (21336.4615 to 209754.1585). My problem gets solved by using the diluted solution in which I got linearity in the range of 2uM to 60uM.
@Tobis Werner
Thanks for the response. It's true the coumarin solution has shown linearity in lower concentrations only. I got the linearity in the range of 2 to 60uM.
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