Ultra-high density protein spots achieved by on chip digitalized protein synthesis

This is the authors' abstract. We don't add key points for this paper.

Current methodologies for arraying proteins using cell-free protein synthesis on a chip have spatial limitations that prevent reaching ultra-high density necessary for high throughput analysis. To circumvent this, we developed an on-chip method based on microcompartmentalization of protein synthesis. Proteins are synthesized in arrayed micrometer scale chambers from confined DNA template molecules. On-chip protein expression is highly efficient and the method can be used with a minimal amount of template i.e. single DNA molecules to perform digitalized cell-free protein synthesis (d-CFPS). A functionalized surface at the floor of the tightly sealed microchambers enables direct capture of expressed proteins. A density of 10⁴ spots per mm² was achieved, which represents a gain by more than 3 orders of magnitude over conventional methods. This technique of forming such densely arrayed small protein spots is the first step towards the development of a general method that would allow fabrication of ultra-high density protein arrays for high-throughput analysis.