I'm performing a serial dilution of Alizarin Red S and a boronic acid. Individually they are not fluorescent, but once complexed, the resulting solution is fluorescent.
Despite having issues with my fluorometer's plate reader, where different wells will give different fluorescent intensities despite having same volume and concentration in them, I got somewhat of a trend showing as conentration of the BA decreases, as does the fluorescence.
However, my blank, which is Alizarin Red S in pH 7.4 sodium phosphate buffer shows higher fluorescence (I believe due to Raman scattering) than the majority of those wells that should actually have a fluorescent compound present. What is the best way I can reduce this? I cannot subtract this well from the others as it will give a negative value in a few of the cases.
Will I have to use more alizarin red S and more boronic acid to make a more strongly fluorescent solution?
The well-to-well variation is a concern that should be sorted out first. If you are using microtiter plates for this experiment, this variation may be the result of the substances sticking to the wells. My suggestion is to include 0.01% Triton X-100 detergent in the buffer. This will coat the hydrophobic plastic surface, creating a hydrophilic layer to prevent the dye from sticking to the plate.
I don't think that the amount of Raman scattering from the solvent should be affected by the dye and boronic acid at the concentrations employed, but the measurement of it may be. With high concentrations of an absorbing substance (namely the dye), you may be getting a significant inner filter effect, causing a reduced detection of the Raman signal. To measure the inner filter effect, you need a fluorescent compound that can be measured at the same wavelength as the Raman signal. Measure the fluorescence of this compound at each Alizarin concentration to generate a correction factor for the inner filter effect at that wavelength.
Hi Adam B Shapiro,
Having a bit of a nightmare with the manufacturers of the machine. The plate reader module that is used with the fluorometer has been repaired many times. In the past the laser did not correctly sit over the wells, but it is supposedly fixed, so I can try the Triton X method you mentioned, thank you.
The dye, Alizarin Red S, is used in this assay at 9 µM in each well, would this be considered a "high" concentration of the absorbing substance? I'll look to measure the inner filter effect with a suitable fluorescent compound, thank you for your help.
I don't think the extinction coefficient of Alizarin Red at about 560 nm is high enough to cause a substantial inner filter effect at 9 µM for the path-length of a microtiter well.
Adam B Shapiro No I wouldn't have thought so either. Measuring the fluorescence of just the sodium phosphate buffer I am using gives higher "fluorescence" than the same volume of buffer containing 9 µM ARS, similar to the result seen in the OP photo. Lowering the excitation wavelength to a value that sufficiently moves the emission wavelength of the Raman scattering of the buffer unfortunately means the excitation wavelength is now too low to allow effective fluorescence of the ARS-BA complex too.
Would you recommend any other tricks or tips to ensure a valid fluorescence assay? Haven't tried the Triton X-100 yet, but will look to do it ASAP.
Is there an effect of the dye on the meniscus shape? Buffer alone will have a flat meniscus in a plastic plate. If the dye causes the meniscus to have a concave shape, it could affect the measurement of the Raman fluorescence. Detergent will equalize the meniscus, so that all wells will have the same curvature.
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