Fabrication of low-fouling, high-loading polymeric surfaces through pH-controlled RAFT

Abstract

Low-fouling and high-loading surfaces are increasingly important for biosensing and blood purification technologies. Selective and efficient target binding from complex media can be achieved with poly(carboxybetaine) (pCB) surfaces that consist of a dense brush layer to resist non-specific protein adsorption and a sparse "mushroom" upper layer for high-density capture agent immobilization (i.e. high-loading). We developed pH-controlled surface-reversible addition-fragmentation chain-transfer (S-RAFT) polymerization to simplify fabrication of multi-modal, low-fouling and high-loading pCB surfaces without the need for quenching or re-initiation steps, toxic transition metals or light irradiation. Multi-modal polymer layers were produced through partial polymer termination by temporarily raising the pH to aminolyse a fraction of dormant chain transfer agents (CTAs); remaining polymer chains with intact CTAs continued uninterrupted extension to create the "mushroom" upper layer. The multi-modal pCB surfaces were low-fouling towards proteins (<6.7 ng cm^-2), and macrophages. Compared to mono-modal brush surfaces, multi-modal pCB surfaces were high-loading with 5-fold greater capture agent immobilization (e.g. antibody) and 4-fold greater target binding (e.g. biotin-fluorescein).

Fig. 1. Schematic for the synthesis of bimodal pCB layers via pH-controlled S-RAFT for enhanced capture agent immobilization on low-fouling surfaces. (A) Surface functionalized with a monolayer of RAFT CTA. (B) Synthesis of the dense pCB layer at pH 4.5 in the presence of a protonated primary amine, butylamine (pK_a of 10.5). (C) Temporary increase in pH (4.5 to 11) to deprotonate butylamine for the partial aminolysis of immobilized CTA. (D) pH returned to 4.5 for continued polymerization to yield the lower density, high loading pCB layer; pH 4.5 re-protonates butylamine to prevent further CTA aminolysis. (E) Compared to monomodal architectures, bimodal pCB architectures yield greater surface densities of accessible carboxylic acids for carbodiimide immobilization of capture agents such as antibodies.

Fig. 2. GPC analysis of multimodal distributions from solution polymerization of pDMAPMA using pH-controlled RAFT. pH time courses during polymerizations are plotted on the right. (A) GPC analysis of two different pDMAPMA polymerizations with partial CTA aminolysis at 2 (red) or 4 h (blue), respectively, by temporarily raising the pH for 5 min. GPC analysis prior to pH raising (dashed lines) demonstrated that pH raising is required for multimodal distributions. (B) pDMAPMA polymerization with sequential 6 min partial CTA aminolysis at 2 and 9 h resulted in three distinct M_w populations highlighted in blue, red and green.

Fig. 3. Dependence of pH 11 time interval on pCB's bimodal M_w distributions in the presence of butylamine. GPC traces (red line) of bimodal pCB polymerizations conducted as follows: (1) pH was held at 4.5 for 1 h; (2) pH was temporarily raised to 11 for (A) 5, (B) 30 and (C) 60 min, respectively; and, (3) pH was returned to 4.5 for a total polymerization time of 24 h. Dashed lines represent deconvolution of GPC data as Gaussian distributions to separate high (yellow) and low (blue) M_w populations; the black dashed line represents the sum of the deconvoluted high and low M_w populations. (D) Calculated proportions of high and low M_w polymer populations from deconvolution. Control experiments without butylamine are presented in Fig. S3.

Fig. 4. Surface characterization of functionalized silicon wafers by WCA and ellipsometry. (A) Static WCA and representative photographs of 3 μL water droplets on wafers functionalized with aminosilane, CTA, and 1 and 2 layer pCB coatings. Hydrophobic CTA increased the WCA contact, whereas increasing pCB content decreased the WCA. The WCA of 1 layer pCB was greater than 2 layer pCB due to differences in layer thickness. (B) Modeled layer thickness of native oxide, APTES, CTA, and 1 and 2 layer pCB coatings from spectral ellipsometric measurements. Statistics performed by one-way ANOVA with Bonferroni's multiple comparison test (mean ± standard deviation p < 0.05 (*), and p < 0.001 by (***)).

Fig. 5. Immobilization of bevacizumab confirmed the greater loading capacity of bimodal pCB wafers. Fluorescently labelled bevacizumab was immobilized using EDC/NHS chemistry on 1 (brush) and 2 layer (bimodal) pCB wafers. After quantification by fluorescent microscopy, the 2 layer pCB architecture was confirmed to have a higher loading capacity than 1 layer pCB. P < 0.01 (**), by Student's t-test, mean ± standard deviation.

Fig. 6. Bimodal, two layer, pCB wafers modified with avidin have greater biotin capturing efficiency. (A) Schematic for the modification of pCB surfaces with avidin and subsequent biotin-fluorescein capture. (B) Representative fluorescent micrographs (4× magnification) and micrograph fluorescent intensity from 1 and 2 layer pCB surfaces with and without avidin that were exposed to biotin-fluorescein. (C) Surface fluorescence quantification of biotin-fluorescein bound to avidin modified surfaces (n = 5 for 1 layer pCB, n = 6 for 2 layer pCB, mean ± standard deviation, p < 0.05 (*), Student's t-test). Background signal was removed by measuring fluorescence of avidin-free surfaces after exposure to biotin-fluorescein.

Fig. 7. One and two layer pCB surfaces were equally low-fouling when exposed to serum. (A) Non-specific protein adsorption onto pristine silicon wafers and wafers functionalized with single layers of pDMAPMA and pCB, and bimodal pCB when incubated in CBS for 24 h; cationic pDMAPMA and anionic SiO₂ surfaces were included as a positive fouling control. No adsorbed protein was detected on 1 or 2 layer pCB (detection limit of the assay was 6.7 ng cm⁻²); protein adsorption was therefore comparable to non-fouling surfaces.. (B) After 48 h of culturing, relative amounts of adhered macrophages on surfaces pre-exposed to serum for 24 h was quantified. Cell counts were determined from fluorescent micrographs; numbers were normalized to the tissue culture plastic (TCP) control. (C) Representative fluorescent micrographs of adhered macrophages stained with HOESCHT and calcein AM, scale bars = 200 μm.