Temperature-modulated interactions between thermoresponsive strong cationic copolymer-brush-grafted silica beads and biomolecules

Abstract

Thermoresponsive polymer brushes have attracted considerable research attention owing to their unique properties. Herein, we developed silica beads grafted with poly(N-isopropylacrylamide (NIPAAm)-co-3-acrylamidopropyl trimethylammonium chloride (APTAC)-co-tert-butyl acrylamide (tBAAm) and P(NIPAAm-co-APTAC-co-n-butyl methacrylate(nBMA)) brushes. The carbon, hydrogen, and nitrogen elemental analysis of the copolymer-grated silica beads revealed the presence of a large amount of the grafted copolymer on the silica beads. The electrostatic and hydrophobic interactions between biomolecules and prepared copolymer brushes were analyzed by observing their elution behaviors via high-performance liquid chromatography using the copolymer-brush-modified beads as the stationary phase. Adenosine nucleotides were retained in the bead-packed columns, which was attributed to the electrostatic interaction between the copolymers and adenosine nucleotides. Insulin was adsorbed on the copolymer brushes at high temperatures, which was attributed to its electrostatic and hydrophobic interactions with the copolymer. Similar adsorption behavior was observed in case of albumin. Further, at a low concentration of the phosphate buffer solution, albumin was adsorbed onto the copolymer brushes even at relatively low temperatures owing to its enhanced electrostatic interaction with the copolymer. These results indicated that the developed thermoresponsive strong cationic copolymer brushes can interact with peptides and proteins through a combination of electrostatic and temperature-modulated hydrophobic interactions. Thus, the developed copolymer brushes exhibits substantial potential for application in chromatographic matrices for the analysis and purification of peptides and proteins.

Keywords: Chromatography; Electrostatic interaction; Hydrophobic interaction; Polymer brush; Thermoresponsive polymer.

Graphical abstract

Fig. 1

Schematic illustration of the thermoresponsive, strong, cationic copolymer brushes with two types of hydrophobic monomers and their interactions with peptides and proteins. (A) Scheme for the preparation of the P(NIPAAm-co-APTAC-co-tBAAm) brush on silica beads. (B) Scheme for the preparation of the P(NIPAAm-co-APTAC-co-nBMA) brush on silica beads. (C) Temperature-modulated interactions between the prepared polymer brush and small acidic molecules, peptides, and proteins in a high-performance liquid chromatography column.

Fig. 2

Chromatogram of the adenosine nucleotides from the thermoresponsive-copolymer-brush-grafted silica beads. (A) P(NIPAAm-co-APTAC-co-tBAAm)-brush-grafted silica-bead-packed column (NI-QA5-tB20), (B) P(NIPAAm-co-APTAC-co-nBMA)-brush-grafted silica-bead-packed column (NI-QA5-nB10). Peaks 1: AMP, 2: ADP, 3: ATP. The mobile phase is a 66.7 mM phosphate-buffered solution.

Fig. 3

Temperature-dependent elution behavior of the insulin from the prepared bead–packed column. (A) P(NIPAAm-co-APTAC-co-tBAAm) brush–grafted silica bead–packed column (NI-QA5-tB20), (B) P(NIPAAm-co-APTAC-co-nBMA) brush–grafted silica bead–packed column (NI-QA5-nB10).

Fig. 4

Temperature-dependent elution behavior of insulin chain A (A) and (C) and chain B (B) and (D) from the prepared-bead-packed column. (A) P(NIPAAm-co-APTAC-co-tBAAm) brush–grafted silica bead–packed column (NI-QA5-tB20), (B) P(NIPAAm-co-APTAC-co-BMA) brush–grafted silica bead–packed column (NI-QA5-nB10).

Fig. 5

Temperature-dependent elution behavior of γ-globulin from the prepared-bead-packed column. (A) P(NIPAAm-co-APTAC-co-tBAAm) brush–grafted silica bead–packed column (NI-QA5-tB20), (B) P(NIPAAm-co-APTAC-co-BMA) brush–grafted silica bead–packed column (NI-QA5-nB10).

Fig. 6

Temperature-dependent elution behavior of albumin from the prepared-bead-packed column. (A) P(NIPAAm-co-APTAC-co-tBAAm) brush–grafted silica bead–packed column (NI-QA5-tB20) and (B) P(NIPAAm-co-APTAC-co-BMA) brush–grafted silica bead–packed column (NI-QA5-nB10). The mobile phase is a (1) 66.7 mM phosphate-buffered solution (pH: 7.0) or a (2) 33.3 mM phosphate-buffered solution (pH: 7.0).

Fig. 7

Peak area of the eluted albumin from the prepared-bead-packed column. (A) P(NIPAAm-co-APTAC-co-tBAAm) brush–grafted silica bead–packed column (NI-QA5-tB20) and (B) P(NIPAAm-co-APTAC-co-BMA) brush–grafted silica bead–packed column (NI-QA5-nB10).