"Double gating"--a concept for enzyme-responsive imaging probes aiming at high tissue specificity.

A new archetype of activatable imaging probe is described that responds in an off-on mode with a fluorescent signal only after being consecutively converted by two different enzymes. Pre-pro-proteins are a well-established concept employed by Nature, but "pre-pro-fluorescent" molecular probes have been unknown until now, even though they promise to furnish precious extra information and added specificity over habitual probe technologies. Our prototype probe discriminates cells that express both active β-d-galactosidase and leucine amino-peptidase from those which lack one of these enzymes thanks to the release of a precipitating fluorescent tag.