High-throughput fabrication of monodisperse magnetic PAM-PEG microspheres via high-aspect-ratio droplet microfluidics for efficient and selective his-tagged protein purification

The development of chromatographic media with uniform particle size, high mechanical strength, and specific binding capacity is crucial for efficient protein purification. However, conventional methods often suffer from polydisperse particle sizes and low magnetic content, limiting their separation efficiency and scalability. Here, we report a scalable strategy integrating high-aspect-ratio (HAR) multi-channel droplet microfluidics with a robust physicochemical modification approach to fabricate monodisperse polyacrylamide-polyethylene glycol (PAM-PEG) magnetic microspheres. The optimized HAR microfluidic chip enabled high-throughput production of droplets with a coefficient of variation (CV) as low as 3.98%. Subsequent incorporation of PEG-modified Fe₃O₄ nanoparticles endowed the microspheres with superparamagnetic properties (saturation magnetization: 40.9 emu/g) and enhanced mechanical resilience. Through a sequential surface functionalization process involving alkaline hydrolysis, EDC/NHS activation, nitrilotriacetic acid (NTA) grafting, and Ni²⁺ chelation, the resulting PAM-PEG@Fe₃O₄-PEG@NTA-Ni²⁺ microspheres achieved a high nickel loading capacity of 50.17 μmol/mL. These microspheres exhibited a specific protein adsorption capacity of ∼44 mg/mL and demonstrated superior selectivity in capturing His-tagged proteins from complex biological matrices, outperforming commercial counterparts in both purity and non-specific binding reduction. This work presents a promising platform for next-generation bioseparation media with high throughput and tailored functionality.