Hydrogel particle-based protein display enabled by particle-templated emulsification.

Protein display technology enables high-throughput screening and plays an important role in protein discovery and engineering. Conventional in vivo display methods face challenges such as inefficient gene transformation and complex cell proliferation dynamics, while in vitro display methods are often limited to affinity-based selection and suffer from expression bias due to homogeneous reaction conditions. Here, we present a hydrogel particle-based protein display method enabled by particle-templated emulsification. This approach uses functionalized polyacrylamide hydrogel particles as isolated microreactors, incorporating DNA primers for genotype immobilization and Ni-NTA groups for capturing histidine-tagged protein phenotypes. Displayed proteins are synthesized via cell-free protein expression within isolated droplets, overcoming the limitations of in vivo cell culture and enabling compartmentalized screening, which is challenging in conventional homogeneous in vitro systems. Using particle-templated emulsification, single hydrogel particles can be rapidly encapsulated with individual DNA templates into isolated water-in-oil droplets within 30 seconds, without the need for specialized instrumentation. Up to 10(9) particles can be emulsified in a standard 50 ml conical tube. Compared to conventional droplet microfluidics, particle-templated emulsification achieves higher single-particle encapsulation and improved one-to-one particle-DNA pairing efficiency, reducing reagent consumption and minimizing DNA library loss caused by improper pairing. Digital PCR and cell-free protein expression are sequentially performed within droplets, with both the amplified DNA and the expressed protein immobilized on the same particle, thereby establishing a stable genotype-phenotype linkage. This method eliminates the need for cell handling, enables compartmentalized functional screening, and provides a fast, scalable, and user-friendly workflow for protein display, offering strong potential in directed evolution and protein engineering.