Complex analysis of concentrated antibody-gold nanoparticle conjugates' mixtures using asymmetric flow field-flow fractionation.

Conjugates of gold nanoparticles (GNPs) with antibodies are powerful analytical tools. It is crucial to know the conjugates' state in both the concentrated and mixed solutions used in analytical systems. Herein, we have applied asymmetrical flow field-flow fractionation (AF4) to identify the conjugates' state. The influence of a conjugate's composition and concentration on aggregation was studied in a true analytical solution (a concentrated mixture with stabilizing components). GNPs with an average diameter of 15.3±1.2nm were conjugated by adsorption with eight antibodies of different specificities. We found that, while the GNPs have a zeta potential of -31.6mV, the conjugates have zeta potentials ranging from -5.8 to -11.2mV. Increased concentrations (up to 184nM, OD520=80) of the mixed conjugate (mixture of eight conjugates) did not change the form of fractograms, and the peak areas' dependence on concentration was strongly linear (R2 values of 0.99919 and 0.99845 for absorption signal and light scattering, respectively). Based on the gyration (Rg) and hydrodynamic (Rh) radii measured during fractionation, we found that the nanoparticles were divided into two populations: (1) those with constant radii (Rg=9.9±0.9nm; Rh=14.3±0.5nm); and (2) those with increased radii from 9.9 to 24.4nm for Rg and from 14.3 to 28.1nm for Rh. These results confirm that the aggregate state of the concentrated and mixed conjugates' preparations is the same as that of diluted preparations and that AF4 efficiently characterizes the conjugates' state in a true analytical solution.