Ni(II)-Transformed BPP Hydrogel: An In Situ Generated Adsorbent for High-Affinity Capture of His-Tagged GFP

Green fluorescent protein (GFP) is widely utilized as a fluorescent probe in biomedical research, yet its high-purity separation remains challenging. Traditional purification methods often involve complex procedures, high costs, and environmental concerns. In this study, a dual-functional bacterial cellulose-based hydrogel (BPP) was successfully fabricated via graft copolymerization of acrylic acid (AA) and acrylamide (AM) onto the BC framework. The resulting hydrogel exhibited a highly hydrated three-dimensional network with outstanding mechanical properties and swelling rate (compressive strength: 1.54 MPa; 300 compression cycles at 80% strain: 90% stress retention). The maximum adsorption capacity of BPP for Ni(II) was 315.9 mg/g (T = 55 °C) and effectively suppressed metal ion leaching while generating abundant coordination-active sites for selective GFP capture (228.9 mg/g). Notably, the captured GFP retained high fluorescence activity and could be efficiently eluted using imidazole buffer, with a recovery rate of 61.1%. Spectroscopic analyses (FT-IR and XPS) revealed a sequential adsorption mechanism involving strong chelation of Ni(II) by polymeric carboxyl and amino groups, followed by Ni(II)-mediated specific coordination with His-tagged GFP. This work demonstrates a sustainable polymer-based strategy that integrates heavy metal ion immobilization with high-value protein purification, highlighting the potential of multifunctional cellulose hydrogels for advanced bioseparation and the design of resource-oriented materials.