Metal complexation chemistry used for phosphate and nucleotide determination: an investigation of the Yb3+-pyrocatechol violet sensor.

Metal complexation reactions can be used effectively as sensors to measure concentrations of phosphate and phosphate analogs. Recently, a method was described for the detection of phosphate or ATP in aqueous solution based on the displacement by these ligands of pyrocatechol violet (PV) from a putative 2:1 (Yb3+)2PV complex. We have not been able to reproduce this stoichiometry and report this work in order to correct the coordination chemistry upon which sensor applications are based. In our work, colorimetric and spectrophotometric detection of phosphate was confirmed qualitatively (blue PV+Yb3+; yellow+Pi); however, the sequence of visual changes on the titration of PV with 2 equiv. of Yb3+ and back titration with ATP as described previously could not be reproduced. In contrast to the linear response to Pi that was reported previously, the absorbance response at 443 or 623 nm was found to be sigmoidal using the recommended 2:1 Yb3+:PV solution (100 microM:50 microM, pH 7, HEPES). Furthermore, both continuous variation titration and molar ratio analysis (Job plot) experiments are consistent with 1:1, not 2:1, YbPV complex stoichiometry at pH 7 in HEPES buffer, indicating that the deviation from linearity is produced by excess Yb3+. Indeed, using a 1:1 Yb3+:PV ratio produces a linear response in DeltaAbs at 443 or 623 nm on back titration with analyte (phosphate or ATP). In addition, speciation analysis of the Yb-ATP system demonstrates that a 1:1 complex containing Yb3+ and ATP predominates in solution at microM metal ion and ATP concentrations. Paramagnetic 1H NMR spectroscopy directly establishes the formation of Yb3+-solute complexes in dilute aqueous solution. The 1:1 YbPV complex can be used for the colorimetric measurement of phosphate and ATP concentrations from approximately 2 microM.