I want to make gossypetin (a flavonol, a type of flavonoid) a fluorescent molecule for the purpose of bioimaging works. Very common way is to conjugate FITC with gossypetin to make it fluorescent but the molecular structure of the conjugated product will become big. I am trying to keep the molecular structure as close as possible to gossypetin. I am thinking of diazotization (though other easy processes are also welcome) of gossypetin, but not sure which molecule to conjugate with it so that it can be excited by the laser sources in the range of say 400 nm to 700 nm wavelength. Also, is there a way to theoretically predict the excitation and emission wavelength of a molecule before it is practically done? Thank you in anticipation.
The longer the wavelength you wish to use for excitation, the larger the fluorophore will have to be. To keep the size of the addition to the gossypetin molecule to a minimum, keep the excitation wavelength as short as possible. Consider even using UV excitation.
Perhaps gossypetin is already fluorescent when excited at UV wavelengths. To find out, first take a UV absorbance spectrum with a spectrophotometer (using a quartz cuvette) to find the wavelength of maximal absorbance. Then use a spectrofluorometer to scan the emission spectrum of a dilute solution (e.g. 1 µM) when using excitation at the wavelength of maximal absorbance. (Also scan a solvent blank to correct for Raman scattering from the solvent.)
Bear in mind that if you add a fluorophore to this small molecule, its behavior is likely to change significantly.
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