Determination of the ratio of fluorophore/nanoparticle for fluorescence-labelled nanoparticles

Optical analysis based on fluorescence labeling has been extensively used for the selective tagging of a wide range of biomedical important targets or for sensing purposes. Fluorescent nanoparticles (NPs) offer interesting properties as labels, as they can be also used as active labels that change their properties upon changes in the environment, such as pH- or distance-dependent fluorescence. In case NPs are not intrinsically fluorescent, they can be made fluorescent by attaching fluorophores to their volume and/or surface. Dye-labelled NPs can produce a highly amplified optical signal compared to a single dye molecule, as there are many dye molecule attached to each NP, providing a great improvement in analytical sensitivity. However, an appropriate control to quantify the fluorophore/NP ratio is required to succeed in the preparation of quantitative platforms matching the required application. Here a methodology to determine such parameter, the fluorophore/NP ratio, is presented. The methodology combines data obtained from UV/Vis absorption spectroscopy for determination of the dye concentration and inductively coupled plasma-mass spectrometry (ICP-MS) analysis for determination of the NP concentration. To validate the approach, it has been applied to the analysis of different sets of fluorophore-NP conjugates prepared using diverse fluorescent dyes (i.e. fluorophores with different structures and emissions) and several types of NPs (i.e. PbS QDs, Au NPs and FePt NPs). The fluorophore-NP conjugates hereby were designed to incorporate the dye directly into an amphiphilic polymer coating. The developed methodology allows for quantification of fluorophore-NP coupling, and therefore, opens up the possibility of selecting controlled conjugates.