Layer-by-layer adsorption of protonated poly(allylamine) (PAH) and deprotonated poly(N,N-dicarboxymethylallyl amine) (PDCMAA) yields thick films with a high density of iminodiacetic acid (IDA) ligands that bind metal ions. When film deposition occurs at pH 3.0, PAH/PDCMAA bilayer thicknesses reach 200 nm, and Cu2+ binding capacities are ~2.5 mmoles per cm3 of film. (PAH/PDCMAA)10 films deposited at pH 3.0 are 4- to 8-fold thicker than films formed at pH 5.0, 7.0, or 9.0, presumably because of the low charge density on PDCMAA chains at pH 3.0. However, with normalization to film thickness, all films bind similar amounts of Cu2+ from pH 4.1 solutions of CuSO4. In μm-thick films, equilibration of binding sites with Cu2+ requires ~4 h due to a low Cu2+ diffusion coefficient (~2.6×10−12 cm2/sec). Sorption isotherms determined at several temperatures show that Cu2+ binding is endothermic with a positive entropy (binding constants increase with increasing temperature), presumably because metal-ion complexation involves displacement of both a proton from IDA and water molecules from Cu2+. (PAH/PDCMAA)10 films retain their binding capacity over 4 absorption/elution cycles and may prove useful in metal-ion scavenging, catalysis, and protein binding.
Membrane adsorbers rapidly capture tagged proteins because flow through membrane pores efficiently conveys proteins to binding sites. Effective adsorbers, however, require membrane pores coated with thin films that bind multilayers of proteins. This work employs adsorption of polyelectrolytes that chelate metal ions to create functionalized membranes that selectively capture polyhistidine-tagged (His-tagged) proteins with binding capacities equal to those of high-binding commercial beads. Adsorption of functional polyelectrolytes is simpler than previous membrane-modification strategies such as growth of polymer brushes or derivatization of adsorbed layers with chelating moieties. Sequential adsorption of protonated poly(allylamine) (PAH) and carboxymethylated branched polyethylenimine (CMPEI) leads to membranes that bind Ni(2+) and capture ∼60 mg of His-tagged ubiquitin per mL of membrane. Moreover, these membranes enable isolation of His-tagged protein from cell lysates in <15 min. The backbone amine groups in CMPEI likely increase swelling in water to double protein binding compared to films composed of PAH and the chelating polymer poly[(N,N-dicarboxymethyl)allylamine] (PDCMAA), which has a hydrocarbon backbone. Metal leaching from PAH/CMPEI- and PAH/PDCMAA-modified membranes is similar to that from GE Hitrap FF columns. Eluates with 0.5 M imidazole contain <10 ppm of Ni(2+).
In principle, incorporation of comb-like block copolymers in multilayer polyelectrolyte films can both increase film thickness relative to coatings containing linear polymers and provide more swollen films for increased sorption of proteins. In the absence of added salt, alternating adsorption of 5 bilayers of protonated poly(allylamine) (PAH) and comb-like poly(2-hydroxyethyl methacrylate)-graft-poly(acrylic acid) (PHEMA-g-PAA) leads to ∼2-fold thicker coatings than adsorption of PAH and linear PAA, and the difference in the thicknesses of the two coatings increases with the number of bilayers. Moreover, the (PAH/PHEMA-g-PAA)n films sorb 2- to 4-fold more protein than corresponding films prepared with linear PAA, and coatings deposited at pH 3.0 sorb more protein than coatings adsorbed at pH 5.0, 7.0, or 9.0. In fact changes in deposition pH and addition of 0.5 M NaCl to polyelectrolyte adsorption solutions alter protein sorption more dramatically than variations in the constituent polymer architecture. When deposited from 0.5 M NaCl at pH 3, both (PAH/PHEMA-g-PAA)5 and (PAH/PAA)5 films increase in thickness more than 400% upon adsorption of lysozyme. These films contain a high concentration of free –COOH groups, and subsequent deprotonation of these groups at neutral pH likely contributes to increased protein binding. Lysozyme sorption stabilizes these films, as without lysozyme films deposited at pH 3 from 0.5 M NaCl desorb at neutral pH. Films deposited at pH 9 from 0.5 M NaCl are more stable and also bind large amounts of lysozyme. The high binding capacities of these films make them attractive for potential applications in protein isolation or immobilization of enzymes.
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