Chemosensors and competition sensing assays

The development and application of a synthetic fluorescent chemosensor for citrate and a competition sensing assay for glucose-6-phosphate based on a synthetic receptor are described. Chemosensor 2.0 was effective for determining citrate concentrations at the micromolar level in a highly competitive medium. The chemosensor is based on a mixed ligand system. Fluorescence spectroscopy studies determined citrate coordination with 2.0 modulated the fluorescence quenching of a Cu(II) ion bound in the 1,10-phenanthroline cleft of 2.0. Binding constants were determined using a modified Rose and Drago general equation. Chemosensor 2.0 bound Cu(II) with a binding constant of 3.4 x 104 M-1 in a buffered aqueous methyl alcohol solution at a pH of 7.4. Additionally, citrate was bound by 2.0 with a binding constant of 3.9 x 106 M-1 under the same experimental conditions as the Cu(II) binding experiment. The cooperative nature of the receptor-metal-citrate mixed ligand system was established through a cyclic equilibria analysis. When Cu(II) ions and citrate were both in solution, the binding constants for 2.0 with Cu(II) and citrate both doubled to 6.4 x 10 4 M-1 and 8.6 x 106 M -1 respectively.; Our competition-sensing assay was effective for determining glucose-6-phosphate concentrations at micromolar levels in a highly competitive medium. The competition assay was assembled around receptor 3.0, which positions boronic acid groups complementarily to the hydroxyl and phosphate functionality of glucose-6-phosphate. Additionally, the phosphate functionality can be involved in an electrostatic attraction with three protonated secondary amines of 3.0. The indicator in our competition assay (5-carboxyfluorescein) was bound by 3.0 with a binding constant of 3 x 10 2 M-1 under Benesi-Hildebrand conditions. The equilibrium between 5-carboxyfluorescein, 3. 0, and the 5-carboxyfluorescein- 3.0 complex produced a spectrophotometric response. Introduction of glucose-6-phosphate to the indicator-receptor ensemble resulted in a change of this equilibrium state. Analysis of spectrophotometric: data related to this change in equilibrium produced a 6 x 103 M -1 binding constant between glucose-6-phosphate and 3.0 .