# Novel NTA-Ni2+ Agarose-Based Microspheres: Structural Features and Chromatographic Capacity

## TL;DR

This study develops new agarose-based microspheres for protein purification, showing high efficiency and structural stability.

## Contribution

A novel one-step method for preparing NTA-Ni2+ agarose-based microspheres with optimized structural and chromatographic properties.

## Key findings

- Optimal preparation conditions achieved a span value of 0.50684 for uniform microsphere size.
- NTA-Ni2+ ABM showed high binding capacity for His-tagged proteins (15.2 ± 0.8 mg/mL).
- SEM, AFM, DSC, and FTIR confirmed structural stability and uniform cross-linking network.

## Abstract

The design and optimization of immobilized metal affinity chromatography (IMAC) media are crucial to enhancing the purification efficiency of recombinant proteins. In this study, the agarose-based microspheres are prepared by using a three-factorial Box–Behnken design followed by NTA-Ni2+ agarose-based microspheres (ABM) preparation by the “one-step” crosslinking of epichlorohydrin (ECH)–nitrilotriacetic acid (NTA) to efficiently couple the NTA ligand to the surface of the matrix. After preparation, various sophisticated techniques, including SEM, AFM, DSC, FTIR, and SDS-PAGE, were used to analyze the morphological structure, thermal stability, and chemical composition of NTA-Ni2+ ABM. The optimal conditions are identified as an emulsifier PP concentration of 8.12 wt%, a stirring speed of 1624.46 rpm, and an oil-phase temperature of 53.86 °C, giving a span value (Y) of 0.50684. SEM, AFM, DSC, and FTIR results showed that the fabricated NTA-Ni2+ ABM were structurally stable and had a uniform cross-linking network for up to 8 h of coupling reaction time. The performance results showed that the beads had a high binding capacity for His-tagged proteins (15.2 ± 0.8 mg/mL), and SDS-PAGE results demonstrated the efficient purification ability for target proteins. These findings provide the theoretical basis and a practical solution for the rational design and application of IMAC medium.