Droplet Microfluidics‐Assisted Fabrication of Magnetite Nanoparticle Hybrid Microgels for Facile Protein Immobilization

We introduce droplet microfluidics-fabricated hybrid microgels composed of biotinylated acrylamide that is crosslinked with N,N'-methylenebis(acrylamide). These microgels are further functionalized with streptavidin and loaded with magnetite nanoparticles (MNPs). During microfluidic processing, MNPs remain dispersed in an aqueous methylcellulose solution for over 4 h due to increased viscous drag, enabling stable droplet formation. In contrast, the MNPs sediment within a few minutes in pure aqueous solution. The resulting multifunctional hybrid microgels facilitate straightforward protein immobilization under mild conditions, for example, utilizing enzymes conjugated with streptavidin or recombinant fusion proteins of magnetite-binding proteins. Simultaneously, our microgels enable the recovery of immobilized proteins through magnetic separation of the microgels from solution. The quantity of immobilized proteins can be regulated independently by varying the amount of coupled biotin or encapsulated MNPs. Using microgels containing different quantities of coupled biotin, we demonstrate binding of a streptavidin-conjugated fluorescent dye and horseradish peroxidase. To confirm the availability of MNPs for magnetite-binding proteins, a fusion protein of the magnetite-binding protein Mad10 and super-folder green fluorescent protein (sfGFP) was immobilized, and its fluorescence was detected.