Physical Changes of Biomacromolecules upon Covalent Surface Immobilization

Immobilization of large biomacromolecules is often required for analytical quantification and physicochemical characterization. However, immobilization can alter the structure and size of the particles being studied. Here, two exosomes (derived from HEK-293 and MDA-MB-231 cells) and three viral particles (Suid herpesvirus 1 (SuHV), xenotropic murine leukemia virus (XmuLV), and porcine parvovirus (PPV)) were immobilized to different covalent chemistries to understand how surface chemistry influences particle deformation during immobilization. The surface chemistries explored were: (i) NHS (N-hydroxysulfosuccinimide) and EDC (1-ethyl-3-(3-(dimethylamino)propyl) carbodiimide hydrochloride), and (ii) poly l-lysine (PLL) and glutaraldehyde (GA). Morphological changes in biomolecules following immobilization were quantified by measuring the height-to-diameter (H/D) ratios attained from atomic force microscopy (AFM) topographic images. These observations were further supported by complementary size and morphology analyses using dynamic light scattering (DLS) and liquid phase transmission electron microscopy (TEM). NHS/EDC chemistry consistently resulted in more significant particle flattening than PLL/GA, as evidenced by lower average H/D ratios across all biomacromolecules. Greater flattening effects were observed on the soft lipid envelope of exosomes as compared to viruses, due to differences in structural rigidity. Both immobilization chemistries resulted in a lower H/D ratio in tumor-derived MDA-MB-231 exosomes compared to nontumor-derived HEK-293 exosomes, likely due to the known softer mechanical properties of tumor-derived exosomes. Furthermore, immobilization of the enveloped viruses SuHV and XMuLV with NHS/EDC exhibited flattening effects and lower H/D ratios. Immobilization of nonenveloped PPV resulted in a low H/D ratio on NHS/EDC, which was likely due to particle aggregation rather than deformation. These findings provide valuable guidance for selecting appropriate surface chemistries for nanoscale biointerface studies and offer implications for surface-based diagnostics, high-throughput biosensing, and nanomaterial functionalization.