A spirooxazine derivative, PheSPO (3,3-dimethyl-1-phenethylspiro[indoline-2,3′-naphtho[2,1- b ][1,4]oxazine]), as a dual-sensing probe for Cu 2+ and Fe 3+ was synthesized, and its structure was confirmed by 1 H NMR, 13 C NMR, HRMS, and single-crystal X-ray diffraction. The results reveal that the PheSPO probe is selective to both Cu 2+ and Fe 3+ through distinct colorimetric responses in acetonitrile. The sensing performance of PheSPO toward Cu 2+ was investigated, and upon addition of Cu 2+ , an instant change in color from colorless to bright yellow with a strong absorption band at 467 nm was observed. Due to a dual-sensing behavior, PheSPO also exhibits a unique response toward Fe 3+ that can be discovered from a color change from colorless to red at an absorption wavelength of 514 nm. Based on spectroscopic analyses and density functional theory calculations, the 1:1 stoichiometric complexation of PheSPO with the targeted metal ions was proposed and the binding constants of 1.95 × 10 3 M –1 for Cu 2+ and 1.29 × 10 3 M –1 for Fe 3+ were obtained. In addition, the detection limits of PheSPO for Cu 2+ and Fe 3+ were 0.94 and 2.01 μM, respectively. To verify its applicability in real samples, PheSPO was further explored for quantitative determination of both Cu 2+ and Fe 3+ in spiked drinking water. The results showed that the recoveries of Cu 2+ and Fe 3+ examined using the PheSPO probe were found comparable to those obtained from atomic absorption spectroscopy. Moreover, the PheSPO strip test was developed, and its utilization for qualitative detection of Fe 3+ in real rice samples was demonstrated.

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