Cell-free systems, which can express an easily detectable protein with a DNA or mRNA template without constraints of living cells, are attractive as foundations for biosensors. Moreover, by encapsulating them in lipid bilayer membranes (such as liposomes) like natural cells, these systems can avoid the adverse effects of surrounding expression inhibitors.

In order for a cell-free system-based sensor to recognize a target molecule and subsequently express a reporter protein (e.g. glowing protein), target-to-protein signal transduction must be introduced. A promising candidate for this purpose is a riboswitch, a molecule-responsive gene-regulatory sequence. When it is fused to a reporter protein gene, it can regulate (repress or promote) the protein expression in response to a specific molecule. Although the diversity of natural riboswitches (or that of their target molecules) is limited, a riboswitch responsive to a user-defined molecule can be artificially created. In fact, some natural or synthetic upregulating riboswitches (each fused to a reporter gene) have been used with cell-free systems to create artificial cell-based sensors for detecting membrane-permeable targets.

However, all such sensors reported to date are based on prokaryotic cell-free systems and therefore do not function well at room temperature. There are also problems with riboswitch design that make it difficult to expand the variety of target molecules.

We thus here utilized a eukaryotic cell-free system (wheat germ extract), which functions over a wide range of room temperature, and a highly modular synthetic riboswitch (upregulating one) that functions efficiently there. For the latter, we can change the target specificity simply by replacing the target recognition domain in the riboswitch based on our unique rational design method. In fact, we created three types of synthetic riboswitches (each for one of three target drugs) and fused each to the corresponding gene encoding a reporter protein (green, red, or bule glowing protein). We then encapsulated one of the fusions with wheat germ extract in natural cell-sized liposomes to create artificial cells, each glowing with an intensity dependent on the concentration of its target outside and a color specific to the target. In addition, due to their high orthogonality, a cocktail of these artificial cells allowed for simultaneous detection of the three targets at room temperature.