Temperature-Controlled Hybrid Hydrogels for Reversible and Selective Zinc(II) Removal from Minimal Culture Media

Zinc ions (Zn²⁺) play vital roles in living organisms, and Zn²⁺-deficient systems provide valuable platforms for studying zinc-dependent biological processes. Protein-functionalized materials offer advantages in constructing such systems by combining the physical removability of a solid-phase matrix with the high selectivity of zinc-binding proteins. Poly-(N-isopropylacrylamide) (PNIPAM) hydrogel serves as a promising matrix by enabling temperature-controlled reversible adsorption. The zinc-binding domain of the Zap1 protein (Zap1zf12) is well suited as the protein component owing to its Zn²⁺ specificity, dual binding sites, and PNIPAM-compatible structure. Here, we integrated Zap1zf12 into PNIPAM, generating a thermoresponsive hybrid hydrogel (PNIPAM-co-Zap1zf12) for selective Zn²⁺ removal. PNIPAM-co-Zap1zf12 was constructed through rational cross-linking sites design in Zap1zf12, optimized protein modification, and refined hydrogel synthesis. This material exhibited reversible adsorption in response to biocompatible temperature shifts (37 °C capture, 25 °C release), allowing regeneration without competitive chelators. It achieved a Zn²⁺ removal efficiency of 98.4 ± 7.3% (from 804.1 ± 41.9 nmol/L to 12.6 ± 5.8 nmol/L), and was successfully applied in various minimal culture media for selective Zn²⁺ depletion. Therefore, PNIPAM-co-Zap1zf12 extends the applicability of the hybrid hydrogels by integrating dual-site zinc-binding proteins and enabling selective Zn²⁺ adsorption. Moreover, it offers an effective approach for generating zinc-deficient conditions.