respectively (Scheme 4). Using this rationale, we investigated the application of the more sensitive probe 6, due to its higher quantum yield, towards monitoring polyP digestion by PPX. PPX (2.9 mg/mL, 40 mM Tris-acetate, 10 mM Mg(OAc)2, 30 mM NH4OAc, 0.2 mM EDTA at pH= 8) was added to an aqueous solution of polyphosphates of varying chain length (2-10 mM, for polydisperse polyphosphates the concentration is based on Pi units) and incubated at 37 0 C for 21 hr. The enzyme reaction mixture as well as the control (no PPX) were added separately to a solution of 2.5 µM of 6 (in 10 mM HEPES buffer at pH= 7.4), and the ensuing fluorescence intensity at 434 nm was measured as the mean of three replicates in a 96 well-plate (details in SI section 5). When the control mixture (only polyP8, no PPX) was incubated with probe 6, a clear increase in emission was observed. However, no significant increase in the emission intensity was observed when incubated with the PPX reaction mixture, because of the digestion of polyP8 by PPX to 6 Pi units and one PPi (Fig. a,b). PolyP digestion was also monitored for polydisperse polyphosphates (polyP22, polyP60, and polyP130). A significant decrease in fluorescence emission after digestion was observed (Fig. ). It is apparent that for polyP8 the difference of emission between control and PPX digestion is lower than that obtained for longer polyPs. This can be rationalized by the fact that digestion of polyP8 results in a higher PPi:Pi ratio as compared to the digestion of longer chain-length polyPs that release less PPi overall. Since the sensor shows no response to Pi, but a small response to PPi, the larger difference can be rationalised.