If I am loading fluorescent dye of 200ul and nanoparticle of 200ul both are in PBS. The dye conc. is (20 mg of dye in 1 ml of PBS) with a nanoparticle concentration of 3.5 mg/ml. How I will get to know about the loaded amount of dye with nanoparticles.
Depending on different factors (nature of dye, nature of NP), one approach may be to centrifuge down the nanoparticles, wash and centrifuge a few times to remove all non-incorporated fluorescent dye. Then resuspend and measure UV-Vis absorbance. However, problem is that the absorption coefficient of dye in Nanoparticles cannot be expected to remain the same as in solution. Alternatively, you could try to release the dye decidedly by dissolving the dye loaded NPs in a medium that will extract the dye, ideally in a solvent in which you know the absorption coefficient. Then you can simply calculate the amount.
In accordance with the previous answer, the UV-vis spectra of the composite and the free dye under the same concentration regime must be taken. After the proper washing, when the dye has to be released, the photon count measurement will directry lead to the amount of dye absorbed.
After loading of dye, the dispersion can be centrifuged to collect the dye-loaded particles followed by washing with solvent twice to remove the surface adsorbed dye. The dye content in the supernatant can be determined by UV−vis against the established calibration curve. Substracting the dye content in supernatant from the initial amount of dye will provide you with the amount of dye loaded into nanoparticles.
Thank you all for your kind suggestions...
I got the point and follow the same.
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