A Multiplexed, Electrochemical Interface for Cell-Free Synthetic Gene Networks

The emerging field of cell-free synthetic biology holds great promise for point-of-need applications, with sensing and diagnostics being especially compelling. Cell-free reaction-based molecular diagnostics are biosafe, cost effective and portable, which makes them ideal for de-centralized and global use. Thus far, these types of diagnostics have relied on optical reporter outputs (e.g. fluorescent, colorimetric proteins). While these optical systems perform well, they have limited bandwidth for multiplexed reporting, which could be tremendously useful for diagnostics. Recognizing this potential, we have engineered a scalable platform that allows cell-free gene circuit-based sensors to generate distinct and multiplexed electrochemical signals in a single drop. The electrochemical signal is achieved through DNA-conjugated methylene-blue chemistry and nucleic acid modifying enzymes. A large library of these enzymes has been screened for performance and paired with functionalized nanostructured microelectrodes to produce up to ten orthogonal signals that can operate in parallel. This reporter system was demonstrated using toehold switch riboregulators to detect multiple colistin antibiotic resistance genes as well as ligand-mediated gene-circuits to detect small molecules. This technology bridges the gap between gene-circuits and electronics providing a reporter platform in systems where parallel signaling, electronic device pairing, and software-mediated analysis could provide advantages.