Heavy metal ions are extremely poisonous and cause long-term harm to living organisms. Among these ions, mercury is the most toxic metal and has no notorious purpose in the human body. In this regard, an elegant azomethine thiazole compound AM1 was synthesized, and it was found to be highly sensitive to three-way detection of mercury ions with detection limits of 0.1126 × 10–9 M (FL) and 0.64 × 10–6 M (UV–vis). AM1 highlighted the capability to detect mercury ions through the colorimetric method, the fluorometric method, and via the naked eye in three-way detection. In addition, the structure of AM1 was confirmed by single-crystal X-ray diffraction studies and crystallized in a monoclinic crystal system with a P21/c space group, and it shows numerous noncovalent interactions in the crystal packing. The high sensitivity of AM1 to Hg2+ ions was imputed to the quenching mechanism and was estimated by Fourier transform infrared (FT-IR) spectroscopy, nuclear magnetic resonance (1H-NMR), high-resolution mass spectrometry (HRMS), ultraviolet–visible (UV–vis) absorbance, fluorescence (FL) emission, Job’s plot, B-H plot, and DFT calculation. Naked eye color change of AM1 solution to yellow and turn-off FL by the addition of mercury ion is due to complex formation. In addition to mercury ions, the sensor displayed a new absorption peak at around 240 nm. Furthermore, an AM1-coated test strip is used as the solid support sensor, and real-time detection of Hg2+ ions in the HeLa cell line by fluorescence microscopy is performed.
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