Counting the number of proteins coupled to single nanoparticles

We implemented amine coating of lanthanide-ion doped oxide luminescent nanoparticles and coupling to a protein labeled with an organic fluorophore. We exploited the stepwise photobleaching of the organic fluorophores and their initial emission to count the number of proteins coupled to single nanoparticles. We thus precisely measured the distribution of the protein-nanoparticle ratio and showed that its maximum is different from the average ratio determined from ensemble measurements. The accurate quantification of the biomolecule-particle coupling opens up the possibility of selecting finely controlled conjugates.