Selection of the Optimal Chromatography Medium for Purification of Quantum Dots and Their Bioconjugates

Photoluminescent quantum dots (QDs), due to their unique optical properties and capacity for conjugation with biomolecules, are widely used in biomedicine. However, numerous byproducts of bioconjugation may seriously influence the interaction of these nanoprobes and their targets. The use of size exclusion chromatography (SEC) for the separation of QDs and byproducts of bioconjugation is rather challenging because of the difference in the chemical and physical nature of nanoparticles and biomolecules, which makes the choice of stationary phases for SEC a complicated task. Here we propose a detailed protocol for SEC purification of water-soluble CdSe/ZnS QDs and QD conjugates using Sephadex resins with different porosities and investigate the efficiency of SEC purification of these materials as exemplified by poly(ethylene glycol) derivatives serving as QD-stabilizing ligands, as well as two types of small biomolecules, bis-netropsin and 4,5,9-trisubstituted acridine. We demonstrate that even multiple SEC cycles using the popular prepacked Sephadex G25 columns do not provide efficient purification of QDs, whereas Sephadex G100 and G200 are much more efficient after a single SEC run because of the optimal peak resolution and preservation of the colloidal stability of QDs. Our results show that the use of less common chromatographic media in the group of Sephadex resins allows efficient purification of QD bioconjugates from contaminants for their subsequent use in bioimaging or diagnostics. The proposed SEC protocol can be adapted for purification of not only CdSe-based QDs but also other types of water-soluble nanocrystals with similar sizes and surface properties.