Highly Efficient One-Step Protein Immobilization on Polymer Membranes Supported by Response Surface Methodology

Abstract

Immobilization of proteins by covalent coupling to polymeric materials offers numerous excellent advantages for various applications, however, it is usually limited by coupling strategies, which are often too expensive or complex. In this study, an electron-beam-based process for covalent coupling of the model protein bovine serum albumin (BSA) onto polyvinylidene fluoride (PVDF) flat sheet membranes was investigated. Immobilization can be performed in a clean, fast, and continuous mode of operation without any additional chemicals involved. Using the Design of Experiments (DoE) approach, nine process factors were investigated for their influence on graft yield and homogeneity. The parameters could be reduced to only four highly significant factors: BSA concentration, impregnation method, impregnation time, and electron beam irradiation dose. Subsequently, optimization of the process was performed using the Response Surface Methodology (RSM). A one-step method was developed, resulting in a high BSA grafting yield of 955 mg m^-2 and a relative standard deviation of 3.6%. High efficiency was demonstrated by reusing the impregnation solution five times consecutively without reducing the final BSA grafting yield. Comprehensive characterization was conducted by X-ray photoelectron spectroscopy (XPS), Fourier-transform infrared spectroscopy (FTIR), and measurements of zeta potential, contact angle and surface free energy, as well as filtration performance. In addition, mechanical properties and morphology were examined using mercury porosimetry, tensile testing, and scanning electron microscopy (SEM).

Keywords: electron beam; polymer membrane; radiation-induced graft immobilization; response surface methodology; serum albumin; surface modification.

FIGURE 1

Scheme of the general method and proposed reaction mechanism. (A) The RIGI process solely utilizes a polymer membrane and an aqueous solution of the modifying compound (here: BSA). The membrane is impregnated with the aqueous solution. In case of hydrophobic polymers, a pre-wetting step has to be included. Finally, irradiation with electron beam and washing is performed. (B) Simplified scheme of the proposed reaction mechanism according to recent studies (Schmidt et al., 2021). Electron beam irradiation results in the formation of active species such as PVDF mid-chain radicals, and water radiolysis products, primarily solvated electrons, OH radicals, and H radicals. Due to its low abundance (ω ≤ 2%), the radiation chemistry of the protein is of minor importance. Immediately, water radiolysis products react with the solute, e.g., via H abstraction, to form protein radicals. Finally, radical recombination reactions lead to covalent coupling between protein molecules and polymer chains.

FIGURE 2

Interaction plots for correlations between RSD and the 2FI BJ (temperature ∙ drying). Plots were obtained for following settings: β_BSA = 10 g L⁻¹, t = 10 min, D = 200 kGy, no EtOH, with pre-wetting step, and (A) without shaking; or (B) with shaking.

FIGURE 3

Contour plots of response R1, BSA GY, as a function of BSA mass concentration in the impregnation solution, irradiation dose, and impregnation time, t, for 2-steps method at (A) t = 0.1 min; (B) t = 10 min; and for 1-step method at (C) t = 0.1 min; (D) t = 10 min.

FIGURE 4

3D response surface plots for (A) optimized 1-step method (t = 2.7 min); and (B) optimized 2-steps method (t = 5.7 min). The flags highlight the predicted BSA grafting yield after numerical optimization via desirability function.

FIGURE 5

Investigation of membrane surface hydrophilicity for PVDF-Ref and PVDF-g-BSA (optimized 1-step method). (A) contact angle measurement using water and CH₂I₂; (B) surface free energy with polar and dispersive components for raw and pressed samples; and (C) dynamic attenuation curve of water contact angle with drop age for BSA-grafted sample (given are three decay rates of contact angle referred to the initial value). Please note, the reference showed no change in contact angle within 10 s.

FIGURE 6

PVDF surface characterization of modified and reference samples. (A) Zeta potential measurements; and (B) FTIR spectra in transmission and ATR mode.