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

Abstract

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 µM:50 µM, 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 ΔAbs 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 µM metal ion and ATP concentrations. Paramagnetic1H 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 ~2 µM.

Dual colorimetric and luminescent assay for dipicolinate, a biomarker of bacterial spores

“…3). 12b,14,17 In addition, an absorbance titration curve fitted nicely to a 1:1 binding model (Fig. 3) and provided an association constant of (4.2 ± 1.4) × 10 4 M −1 in 10 mM HEPES buffer, pH 7.0, 295 K.…”

“…11 Although straightforward, this method is quite insensitive, so we decided to develop a new, more sensitive assay. We were drawn to literature reports of dye displacement assays that employ binary mixtures of visible 12 or fluorescent 13 dyes and lanthanide cations. Specifically, independent research groups have shown that a complex of Yb 3+ and pyrocatechol violet (PV) can be used to optically detect phosphate and polyphosphate anions in buffered aqueous solution.…”

“…Specifically, independent research groups have shown that a complex of Yb 3+ and pyrocatechol violet (PV) can be used to optically detect phosphate and polyphosphate anions in buffered aqueous solution. 12,14 The phosphate anions displace the PV dye from the Yb 3+ and produce a color change from blue to yellow. This precedence led us to consider the dye displacement assay that is illustrated in Fig.…”

Complexation of bisphosphonates with ytterbium(III): Application of phosphate and ATP detection assay based on Yb3+–pyrocatechol violet

“…Herein we describe the coordination chemistry of bisphosphonates with Yb 3+ which, providing the first example of bisphosphonate detection using a ligand displacement reaction. [13]…”

“…[16] Simple mononuclear metal ion complexes have also been reported for phosphate and organic phosphate detection. [13, 14] Mononuclear metal ion systems based on displacement assemblies have the advantage of simplicity, but the affinity for phosphate is lower because only one coordination center is involved. In the case of pyrophosphate, this advantage is smaller, because the formation constants for simple single metal-pyrophosphate complexes [17] are similar to the formation constants for dinuclear metal-pyrophosphate complexes.…”