Fig. 4. Quantification of divalent metal ions in buffer solution (Tris, pH 7.4, 150 mmol L^–1) at 24 °C. (a) Chelation mechanism of o-acetanisidide with divalent metal ions. (b) Fluorescence readouts of o-acetanisidide (λ _ex/λ _em: 485/528 nm) in the presence mono/divalent metal ions in aqueous solutions (n = 3). (c) The effect of pH on fluorescence readouts of o-acetanisidide (25 μmol L^–1) at constant Mg²⁺ and Ca²⁺ ion concentrations (100 mmol L^–1) in aqueous solutions (n = 3). (d) Fluorescence intensity readouts of Mg²⁺ and Ca²⁺ ions (100 mmol L^–1) as the concentration of o-acetanisidide were varied from 3–50 μmol L^–1 (n = 3). (e) Quantification of 16-fold diluted Ca²⁺ ions on G1 matrix at a constant o-acetanisidide concentration (25 μmol L^–1) (n = 6). Insets show the quantification of non-diluted Ca²⁺ ions on G1 matrix (n = 3). Shadows in Fig. 4c and e show the physiological pH and Ca²⁺ ion concentration range. Error bars represent standard error of the mean.