Fabrication optimization of a miniaturized array device for cell-free protein synthesis

Cell-free protein synthesis (CFPS) is an attractive alternative to cell-based protein expression systems because of its advantages including speed, simplicity, and adaptability to various formats. However, two major obstacles exist that have been preventing it from being widely used. One is high cost and the other is low protein synthesis yield. We report here a miniaturized CFPS device that addresses these challenges. The cost saving was achieved by miniaturization, which reduced the reagent consumption by two orders of magnitude. The protein synthesis yield was enhanced by prolonging CFPS reactions through continuous supply of reactants (e.g. nutrients and energy components). The reactants were contained in a feeding solution, which was replenished through a nanoporous membrane and microchannel. The design of the miniaturized device was optimized by running continuous-exchange CFPS in devices with a variation in the type of membrane, the size of the exchange interface, and the volume ratio of the reaction solution to the feeding solution. The effects of these design variations on the protein synthesis yield have been studied. Furthermore, the design was expanded into a 96-unit device that can produce a large number of proteins simultaneously, enabling high-throughput proteomics applications.