Cell-Free Protein Synthesis in Porous Parylene Scaffolds

Cell-free protein synthesis (CFPS) has emerged as a promising platform for point-of-care (POC) biosensing due to its programmability, rapid response, and ability to operate without living cells. Integrating CFPS into solid-state materials is essential for enabling long-term storage, portability, and spatial multiplexing required for practical POC applications. While previous efforts have demonstrated CFPS integration onto two-dimensional substrates such as paper, embedding CFPS machinery within a three-dimensional matrix that preserves its activity remains a significant challenge. Here, we propose a solid-state CFPS system comprised of a parylene-based scaffold loaded with CFPS machinery, i.e., CFPS in porous parylene (CinPP). The CinPP is fabricated through ice-templated chemical vapor deposition (CVD). We further demonstrate protein synthesis that is readily activated upon rehydration. The CinPP platform supports protein expression after prolonged storage and enables spatially localized and programmable expression of distinct fluorescent proteins. Furthermore, selective reconstitution of CFPS machinery with target-responsive genetic circuits demonstrates its potential as a modular sensing platform. This approach provides a versatile solid-state CFPS material that can be extended to programmable biosensors, portable diagnostic devices, and on-demand protein production systems.