# Integrating Protein Resistance and Bioconjugation in P(OEGMA-co-MAA) Brushes for Biosensing and Cell Culture: ToF-SIMS Profiling and Antibody Characterization

## TL;DR

Researchers developed antifouling bioactive surfaces using polymer brushes that allow controlled protein attachment for biosensing and cell culture.

## Contribution

A novel method to synthesize copolymer brushes with adjustable protein bioconjugation and minimized nonspecific adsorption.

## Key findings

- P(OEGMA-co-MAA) brushes with x = 0.25 showed maximum bioconjugation and low nonspecific adsorption.
- ToF-SIMS confirmed uniform copolymer composition and IgG immobilization within the brush.
- Brushes conjugated with fibronectin enhanced biocompatibility for human fibroblast cell culture.

## Abstract

To provide antifouling
bioactive surfaces for biosensing and cell
culture, we synthesized and characterized copolymer brush interfaces
with minimized nonspecific adsorption combined with adjustable high-capacity
bioconjugation of functional protein and examined the interfacial
protein state that determines its biological activity. Brushes were
fabricated using surface-initiated atom transfer radical polymerization
with silicon-grafted chains copolymerized from 2-(2-methoxyethoxy)­ethyl
methacrylate (OEGMA) and methacrylic acid (MAA) taken in different
proportions. For all P­(OEGMA1–x
-co-MAA
x
) coatings (0
≤ x ≤ 1), X-ray photoelectron spectroscopy
revealed the molar fraction x of MAA in the brush
equal to that of the reaction mixture. Time-of-flight secondary ion
mass spectrometry (ToF-SIMS) showed a copolymer composition that is
uniform with depth in the brush coatings, confirming a successful
random copolymerization. Bioconjugation of immunoglobulin G antibody
(IgG) within the brushes, enabled by the activation of MAA segments
with 1-ethyl-3-(3-dimethylaminopropyl)­carbodiimide and N-hydroxysuccinimide (EDC/NHS), was examined together with
nonspecific IgG adsorption to the nonactivated brushes using ToF-SIMS
and fluorescence microscopy. Protein loading was controlled by copolymer
composition and protein solution concentration. The optimal composition x = 0.25 was selected for the brushes with maximum bioconjugation
(∼0.4 g/cm3) and low nonspecific adsorption. Protein
loads per brush volume were estimated from ToF-SIMS depth profiles
that evidenced IgG immobilization within the brush. For all P­(OEGMA1–x
-co-MAA
x
) brushes with conjugated IgG antibody, the coatings
with x = 0.25 provided the highest amount of bound
antigen with an antigen binding ratio higher than that of the PMAA
coatings. This observation was related to the different interfacial
antibody states in both coatings (determined by the residue involvement
in the coupling with the MAA segments and the dominant antibody orientation),
which were investigated with multivariate principal component analysis
of ToF-SIMS data. Finally, human fibroblast cell culture showed the
biocompatibility of the developed copolymer brush coatings, further
promoted by brush conjugation with fibronectin.