As environmental contamination caused by metal ions and anions coming from industrial sites becomes significantly more serious, the necessity of analysis methods to detect them is also growing [[1], [2], [3], [4], [5], [6]]. Heretofore, diverse analytical methods have been developed, like voltammetry, inductively coupled plasma atomic emission spectrometry, and atomic absorption spectrometry [[7], [8], [9], [10]]. Even though these methods are sensitive and selective to detect analytes, they have drawbacks like sophisticated and complicated processes, the need for skilled experts, and too costly equipment [[11], [12], [13], [14]]. On the other hand, colorimetric chemosensors have the advantage of being simple enough to be applied to onsite kits and using inexpensive equipment compared to other analysis methods, as well as having excellent sensitivity and selectivity [[15], [16], [17], [18]]. Consequently, colorimetric chemosensors that detect metal ions and anions are being actively developed.

Copper plays an important role in living organisms and is also a major environmental pollutant introduced by industry and agriculture [[19], [20], [21], [22]]. Additionally, it participates in redox regulation as an essential cofactor for metalloenzymes such as cytochrome C [23,24]. However, excessive concentrations of copper in the body can cause various diseases of the nervous system, including Alzheimer's, Menkes, and Wilson's diseases [[25], [26], [27]]. For this reason, the Environmental Protection Agency (EPA) has set a concentration limit of 20 μM for copper in drinking water [28,29]. Meanwhile, sulfide is the chief material of amino acids and is involved in various physiological functions like apoptosis, angiogenesis, neuromodulation, regulation of inflammation, and vasodilation [[30], [31], [32]]. Nevertheless, immoderate accumulation of sulfide can be toxic and cause critical health damage, such as Down's syndrome and diabetes [33,34]. As a result of its varied biological toxicity, the permitted sulfide in freshwater, according to the World Health Organization (WHO), is 14.7 μM [35]. Interestingly, it is well known that copper has a high affinity for sulfide, thereby forming a very stable copper‑sulfur complex [36,37]. Therefore, we can expect to develop an efficient chemosensor that detects copper and sulfide sequentially through metal ion displacement by using the affinity of copper and sulfide.

Numerous colorimetric chemosensors have been designed using reagents containing functional groups that cause intramolecular charge transfer (ICT) [[38], [39], [40]]. Particularly, the nitro group (-NO2) can work as an electron acceptor within the molecule due to its strongly withdrawing property and as a chromophore attributed to ICT [41,42]. For that reason, 5-nitrosalicylaldehyde was chosen as the electron acceptor and chromophore. Moreover, reported sequential chemosensors for copper and sulfide have the demerit of being challenging to use in water due to their low water solubility [31,37,38,40,43]. To compensate for such shortcomings, 1-(hydrazinocarbonylmethyl) pyridinium chloride (HPC), quaternary ammonium acetohydrazide, was selected as a moiety that would improve the solubility of the molecule in water [44]. In addition, the condensation reaction of HPC with aldehyde generates a kind of Schiff base called hydrazone. The structure would provide an excellent binding site for metal ions as it has π electrons in the CN bond [40,45]. Therefore, we expected that a compound having both HPC and -NO2 moieties may be potentially water-soluble and cause unique colorimetric variations for copper. It would also be possible to sequentially detect copper ions and sulfide by their strong affinity.

In this context, we present a water-soluble colorimetric chemosensor, NHOP ((E)-1-(2-(2-(2-hydroxy-5-nitrobenzylidene)hydrazineyl)-2-oxoethyl)pyridin-1-ium) chloride), for sequential detection of Cu2+ and S2− in pure water. NHOP could differentiate Cu2+ by undergoing a color change from pale yellow to colorless. Furthermore, NHOP-Cu2+ changed back to a pale yellow color in the presence of S2− through the demetallation process. The detecting properties of the NHOP for Cu2+ and NHOP-Cu2+ for S2− were substantiated by UV–vis titrations, Job plots, ESI-MS, and DFT calculations.