I am conjugating a drug on gold nanoparticles. The unbound drug absorbs well between 350-400 nm. The naked gold nanoparticle absorbs between 250-500 nm. After conjugation of the drug to the nanoparticle, the UV features of the drug are skewed and blue shifted. I thought of creating a standard curve of the plain drug and calculate the amount of drug on the nanoparticle by correlation. However, because of this aforementioned problem, I do not know if it is a good idea to use the same technique.
The drug also shows fluorescence. If I take an excitation scan of the nanoparticle keeping fluorescence at 500 nm constant, the excitation spectra matches to that of unbound drug. Also exciting at 350 nm, the fluorescence spectra of the nanoparticles matches with that of the drug. Gold nanoparticles has absolutely no contribution in the fluorescence spectra. I proceeded to make a standard curve of fluorescence with different concentrations of the drug and calculate the amount of drug on the nanoparticle by correlation of fluorescence intensities. Is fluorescence a correct technique to use for quantification of the amount of drug? Any help would be appreciated.
It may be a good approach to verify that there is drug on the nanoparticles. However, for quantification, you need to assume that the quantum yield in DMSO is the same as the quantum yield in water/nanoparticle conjugate, which is a huge leap of faith.
If the absortion has changed because of adsorption to the SERS particle, the the fluorescence has also probably been changed, and even quenched, so your fluorescence protocol may be measuring the unbound molecules.
When you conjugates drug with gold nanostructure, in UV-Visible spectroscopy it shows blue shift it probably means gold nanoparticles involves (hydrophobic interaction) with drug, In fluorescence spectroscopy you can find out number of binding sites of drug on gold nanoparticles , bio-molecular quenching constant, and various binding forces (like Vander Waals interaction, hydrogen bonding) etc between drug and gold nanoparticles.
Thank you Hugh and Bipin for your answers. As far as I know there are no unbound particles. I must mention here that the drug is insoluble in water so the measurements for the plain drug was done in DMSO but the nanoparticles are a suspension in water. I believe that might lead to a change in absorption spectra. But my main question will it still be right to use any of these techniques to quantify the amount of drug on the nanoparticles?
It may be a good approach to verify that there is drug on the nanoparticles. However, for quantification, you need to assume that the quantum yield in DMSO is the same as the quantum yield in water/nanoparticle conjugate, which is a huge leap of faith.
Yes, you can use fluorescence spectroscopy for this. Fluorescence data have a property called "second-order advantage" which enables the construction of second-order multivariate calibration models with excitation-emission matrices (EEMs) in the presence of unknown interfering. For example, you can use PARAFAC-MLR or nPLS for this. If you use a univariate calibration approach, you may have contributions of interactions/interfering in the signal.
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