AF4-UV/VIS-MALS-ICPMS/MS for the characterization of the different nanoparticulated species present in oligonucleotide-gold nanoparticle conjugates

In-depth characterization of functionalized nanomaterials is still a remaining challenge in nanobioanalytical chemistry. In this work, we propose the online coupling of Asymmetric Flow Field-Flow Fractionation (AF4) with UV/Vis, Multiangle Light Scattering (MALS) and Inductively Coupled Plasma-Tandem Mass Spectrometry (ICP-MS/MS) detectors to carry out, in less than 10 min and directly in the functionalization reaction mixture, the complete characterization of gold nanoparticles (AuNPs) functionalized with oligonucleotides and surface -modified with polyethylene glycol (PEG). AF4 separation provided full separation of the bioconjugates from the original AuNPs while P/Au and S/Au ICP-MS/MS ratios in the bioconjugate fractographic peaks could be used to compute the corresponding stoichiometries, oligonucleotide/AuNP and PEG/AuNPs. MALS detection clearly showed the coexistence of two distinct nanoparticulated populations in the bioconjugation mixture, which were demonstrated to be different not only in size but in functionality as well. The major bioconjugate population showed lower hydrodynamic ratios (18 nm) with higher and steadier oligonucleotides/AuNPs (92) and PEG/AuNPs (2350) stoichiometries, in comparison to the minor abundant population (54 nm, 51 and 1877, respectively). Moreover, the ratio between the absorbance signals measured at 520 nm and 650 nm reflects a lower AuNP aggregation in the major (10.5) than in the minor (4.5) population. Results obtained prove the benefits of a detailed characterization to find out if subsequent purification of functionalized AuNP-oligonucleotides is required to design more efficiently their final bioanalytical application.