Surface-Exposing Nickel Ion-Based Ni/Ni _<i>x</i> P _<i>y</i> Magnetic Microspheres for Stable and Efficient Separation and Purification of Histidine-Rich Proteins

Histidine-rich proteins (His-proteins) are vital biomolecules with diverse physiological roles. However, their purification from complex biological samples remains challenging. Here, we successfully prepared Ni/Ni_<i>x</i>P_<i>y</i> composite nanospheres with different phosphating degrees, featuring surface-exposing Ni²⁺ properties for efficient separation and purification of His-proteins. The phosphated products exhibited a spherical morphology, reduced cytotoxicity, and good biocompatibility. The optimal Ni/Ni_<i>x</i>P_<i>y</i> demonstrated excellent adsorption and separation capacity for His-tagged bovine hemoglobin (BHb), which is attributed to their Ni²⁺ surface-exposing capability and magnetic properties, achieving a adsorption capacity of 2130.4 mg/g under the tested conditions. While the adsorption sites for His-proteins are enhanced, the separation process is simplified. SDS-PAGE analysis confirmed the selective adsorption of Ni/Ni_<i>x</i>P_<i>y</i> for His-proteins in a BSA/BHb mixed solution. The Ni/Ni_<i>x</i>P_<i>y</i> spheres demonstrated good stability and recyclability, maintaining an 85% adsorption efficiency after five cycles. In addition, His-proteins can be efficiently separated and purified from complex fetal bovine serum. Therefore, this work reports an effective surface-exposing Ni²⁺ approach to enhance the efficiency of protein separation and purification with the potential to expand applications in diagnostics and therapeutics.