Mapping the relationship between total and functional antibodies conjugated to nanoparticles with spectrally-resolved direct stochastic optical reconstruction microscopy (SR-dSTORM).

Antibody-functionalized nanoparticles (NPs) have shown numerous benefits in drug delivery and biosensing, improving the specificity of cell targeting and analyte detection, respectively. However, one of the main challenges is the lack of control over antibody orientation on the NP surface. Popular and easy conjugation strategies, such as carbodiimide-based conjugations, lead to a random orientation of antibodies on the NPs, compromising ligand functionality and contributing to undesired biological effects and reduced target recognition. While new methods for more controlled NP functionalization have been proposed, there is a lack of techniques that can elucidate the orientation of the antibodies at the single-particle level to determine the conjugation outcome and, therefore, the NPs' potential in selective targeting. Here, spectrally-resolved direct stochastic optical reconstruction microscopy (SR-dSTORM), an optical super-resolution technique, is introduced to quantify the relationship between total and functional NP conjugated cetuximab antibodies at the single-particle level. An evident single-particle heterogeneity in total and functional cetuximab is observed, leading to particles with different functional : total ratios. Additionally, the results indicate that the functional : total ratio of cetuximab highly depends on the conjugated cetuximab concentration. Overall, SR-dSTORM represents a direct approach for the NP structure-functionality relationship quantification, providing a platform to improve antibody-conjugated NPs characterization and facilitating their rational design.