The optimized PUB module is a highly robust system capable of detecting (pyro-)phosphate over a remarkable concentration range by exploiting principles of thermodynamic control. While previous nucleoside phosphorylase-based systems have used irreversible phosphorolytic steps (either via product removal or by rendering the reverse direction kinetically unfeasible), the PUB module employs a reversible phosphorolysis, which yields a significantly simplified system in terms of required materials. As such, the PUB module utilizes the tight thermodynamic control of the phosphorolysis of 1a (K = 0.16) to detect multiple equivalents of phosphate per nucleoside (Fig. ). Using modifications of previously published equations, the extent of conversion in an equilibrium state of this transformation (as monitored spectroscopically via the A304/A288 ratio) provides the phosphate concentration in solution with high accuracy and reproducibility. In practice, this means that 100 µM to >5 mM phosphate can readily be detected with standard spectro- The PUB module employs pyrimidine nucleoside phosphorolysis under alkaline conditions, d UV absorption spectra obtained during phosphorolysis of 1a at pH 9, e Spectroscopic properties of 1a and 1b at pH 9, f pH-dependence of the spectroscopic properties, g Continuous reaction monitoring of the 1a→1b transformation via isosbestic point normalization, h Thermodynamic control of the phosphorolysis of 1a enables the detection of multiple equivalents of phosphate in various amino acid buffer systems, i Extinction differences enable detection of low phosphate concentrations, j Non-linear response of A to phosphate due to thermodynamic control, k Phosphate can be detected accurately over concentrations spanning more than three orders of magnitude, l Enzymatic hydrolysis of pyrophosphate to orthophosphate, m-o Detection of pyrophosphate via thermodynamic control following hydrolysis, p Thermal stability of the IPP from Geobacillus stearothermophilus, q Eyring plots of IPP-catalyzed pyrophosphate hydrolysis, revealing curvature due to an activation heat capacity change, r Phosphatase-catalyzed hydrolysis of organophosphates, s Michaelis-Menten kinetics with selected model substrates obtained via high-throughput experimentation. PUB = phosphate detection via UV spectroscopic monitoring of 5-bromouridine phosphorolysis, NB = nucleobase, NP = nucleoside phosphorylase, Rib1P = ribose-1-phosphate, eq. = equivalent, IPP = inorganic pyrophosphatase, DMSO = dimethylsulfoxide, K = equilibrium constant. All data are freely available online. scopic equipment, with continuous and discontinuous monitoring approaches giving essentially identical results in a high-throughput setup. Notably, this approach can be carried out in 1 mL cuvettes in a photometer, 200 µL multiwell plates in a plate reader or even in 1 µL droplets assessed with a nanophotometer. Lower phosphate concentrations down to around 2 µM can be detected by providing 1a in excess and measuring absorbance differences between a baseline and a near-equilibrium state (Figs. and). The latter can be fitted with first-order kinetic assumptions for an accurate assessment of phosphate content up to at least 200 µM (see the SI for details, equations and workflow). Collectively, this enables the quantification of phosphate over a concentration range spanning nearly four orders of magnitude (Fig. ). While we recommend analysis at pH 9 for optimal performance, the PUB module can also be employed at lower pH values, albeit with reduced sensitivity due to smaller ∆ values (Fig. ; please also see the SI section "Additional considerations"). The extension of this methodology by an inorganic pyrophosphatase (IPP, hydrolyzing pyrophosphate to orthophosphate, Fig. ) enables the quantification of pyrophosphate in a fully analogous fashion, employing identical reaction conditions (Figs. ).