| Summary: | A novel acylhydrazone-based fluorescent sensor <b>NATB</b> was designed and synthesized for consecutive sensing of Al<sup>3+</sup> and H<sub>2</sub>PO<sub>4</sub><sup>−</sup>. <b>NATB</b> displayed fluorometric sensing to Al<sup>3+</sup> and could sequentially detect H<sub>2</sub>PO<sub>4</sub><sup>−</sup> by fluorescence quenching. The limits of detection for Al<sup>3+</sup> and H<sub>2</sub>PO<sub>4</sub><sup>−</sup> were determined to be 0.83 and 1.7 μM, respectively. The binding ratios of <b>NATB</b> to Al<sup>3+</sup> and <b>NATB</b>-Al<sup>3+</sup> to H<sub>2</sub>PO<sub>4</sub><sup>−</sup> were found to be 1:1. The sequential recognition of Al<sup>3+</sup> and H<sub>2</sub>PO<sub>4</sub><sup>−</sup> by <b>NATB</b> could be repeated consecutively. In addition, the practicality of <b>NATB</b> was confirmed with the application of test strips. The sensing mechanisms of Al<sup>3+</sup> and H<sub>2</sub>PO<sub>4</sub><sup>−</sup> by <b>NATB</b> were investigated through fluorescence and UV–Visible spectroscopy, Job plot, ESI-MS, <sup>1</sup>H NMR titration, and DFT calculations.
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