Microcompartmentalized Cell-Free Protein Synthesis in Hydrogel μ-Channels

The rapid demand for protein-based molecules has stimulated much research on cell-free protein synthesis (CFPS); however, there are still many challenges in terms of cost-efficiency, process intensification, and sustainability. Herein, we describe the microcompartmentalization of CFPS of superfolded green fluorescent protein (sGFP) in alginate hydrogels, which were casted into a μ-channel device. CFPS was optimized for the microcompartmentalized environment and characterized in terms of synthesis yield. To extend the scope of this technology, the use of other biocompatible materials (collagen, laponite, and agarose) was explored. In addition, the diffusion of sGFP from the hydrogel microenvironment to the bulk was demonstrated, opening a promising opportunity for concurrent synthesis and delivery of proteins. Finally, we provide an application for this system: the CFPS of enzymes. The present design of the hydrogel μ-channel device may enhance the potential application of microcompartmentalized CFPS in biosensing, bioprototyping, and therapeutic development.