# Physical Changes of Biomacromolecules upon Covalent Surface Immobilization

## TL;DR

This paper studies how different surface chemistries affect the shape of biomacromolecules like exosomes and viruses when they are immobilized for analysis.

## Contribution

The study reveals that NHS/EDC chemistry causes more flattening of soft biomacromolecules compared to PLL/GA.

## Key findings

- NHS/EDC immobilization leads to greater flattening of exosomes and enveloped viruses compared to PLL/GA.
- Tumor-derived exosomes show more flattening than non-tumor-derived ones due to softer mechanical properties.
- Aggregation, not deformation, likely causes low H/D ratios in nonenveloped PPV on NHS/EDC.

## Abstract

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.