Naked-Eye Chromogenic Test Strip for Cyanide Sensing Based on Novel Phenothiazine Push–Pull Derivatives

Monitoring and detection of cyanide are of crucial interest as the latter plays versatile roles in many biological events, is ubiquitous in environment, and responsible for several acute poisoning and adverse health effects if ingested. We describe herein the synthesis and characterization of novel phenothiazine-based push–pull chromogenic chemosensors suitable for naked eye cyanide sensing. Indeed, specific detections were achieved for cyanide with a LOD of ca 9.12 to 4.59 µM and, interestingly, one of the new chemosensors has also revealed an unprecedented affinity for acetate with a LOD of ca 2.68 µM. Moreover, as proof of concept for practical applications, a paper test strip was prepared allowing its use for efficient qualitative naked eye cyanide sensing.


Introduction
Anionic species are a major class of structures involved in many physiological, biological, chemical, and environmental events and processes [1]. Owing to their central roles, the design and synthesis both of artificial and bioinspired probes of anion is of crucial interest for their detection in real samples [2]. Among them, cyanide constitutes one of the most toxic anionic species because it is able to readily bind hemoglobin's iron and interferes with many vital processes [3][4][5]. Nevertheless, despite its harmful effects, it is still widely used in mining or metallurgical industries and in plastic production plants. Thus, industrial accidents or ineffective effluent treatments are the main sources of large cyanide spills into water and environment. According to the WHO, cyanide concentration should not exceed 1.9 µM in drinking water [6]. In addition, accidental ingestion from the cyanogenesis of cyanogenic glycosides present in stone fruit kernels (bitter almonds, apricot), cassava, sorghum, linseed, etc. is also responsible of acute poisoning [7] requiring the development of efficient and reliable sensing tools.
During the last decades, important efforts have been devoted to developing efficient analytical techniques such as ion chromatography coupled with pulse amperometric detection [8] or capillary electrophoresis [9] but they require sophisticated equipment, expert manpower, and are time-consuming. In addition, a preconcentration or a filtering step is usually needed, acting as a drag in real-time detection or analysis at the point-of-need. To overcome these issues, scientists have strived to design and develop optical probes for fast cyanide sensing [2,10]. In peculiar, naked eye techniques based on colorimetric sensors are more suitable for in situ sensing and have drawn deep attention due to their low-cost instrumentation, ease-of-use, nondestructive determination, specificity, and selectivity.
To this aim, supramolecular chemistry based on noncovalent interactions has aroused particular interest in anion sensing [11]. Indeed, a host-guest recognition induces some photophysical changes associated with a detectable optical output signal. Based on their structures and mode of action, those chromogenic/fluorogenic chemosensors are categorized in different categories, namely, two-or three-component, multicomponent, and inorganic-organic hybrid, which have been exploited and used in cyanide dissolved in 100 mL of dichloromethane and washed with 60 mL of distillated water (3×). The organic phase was dried with MgSO 4 and the solvent was removed. The crude was further purified by silica column chromatography using CH 2 Cl 2 /C 6 H 12 (3:2) as eluent (Rf = 0.53). The grayish green solid was washed with diethyl ether to obtain 69.1 mg (yield 26%). 1
Optical properties of the synthetized compounds 6-8 have been investigated by absorption spectroscopy in acetonitrile at a concentration of ca 1.40 × 10 −5 M. All compounds exhibit a broad and intense band at low energy related to the internal charge transfer (ICT) taking place from the electron-rich phenothiazine unit to the electron withdrawing unit. The ICT band is red-shifted from 6 to 8 evidencing the higher electron affinity for the 1,3-bis(dicyanomethylidene)indane acceptor, which is corroborated with the electrochemical data (Table 1, Figure 1). Thus, compound 6 exhibits an ICT band centered at 410 nm (ε = 86,830 M −1 ), while for 7 the ICT is centered at 514 nm (ε = 67,470 M −1 ) and 8 at 531 nm (ε = 43,290 M −1 ). Table 1. Spectroscopic a and electrochemical b data for phenothiazine-based chemosensors 6-8. Optical properties of the synthetized compounds 6-8 have been investigated by absorption spectroscopy in acetonitrile at a concentration of ca 1.40 10 −5 M. All compounds exhibit a broad and intense band at low energy related to the internal charge transfer (ICT) taking place from the electron-rich phenothiazine unit to the electron withdrawing unit. The ICT band is red-shifted from 6 to 8 evidencing the higher electron affinity for the 1,3bis(dicyanomethylidene)indane acceptor, which is corroborated with the electrochemical data (Table 1, Figure 1). Thus, compound 6 exhibits an ICT band centered at 410 nm (ε = 86830 M −1 ), while for 7 the ICT is centered at 514 nm (ε = 67470 M −1 ) and 8 at 531 nm (ε = 43290 M −1 ).  The electrochemical behavior of the phenothiazine-based chromophores 6-8 has been evaluated by cyclic voltammetry in acetonitrile and the data are compiled in Table 1. The cyclic voltammograms (CV, Figure 2) of compounds 6-8 present one reversible oxidation peak at 0.90, 0.85, and 0.83 V, respectively, vs. Ag/AgCl associated to the formation of the radical cation on the nitrogen atom of the phenothiazine ring and two successive irreversible oxidation peaks, at similar values around 1.50 V and 1.42 V (as a shoulder) for the three compounds. Those are ascribed to the formation of the radical cation on the sulfur atom of the phenothiazine ring and the aromatic ring. In the negative potential region, an irreversible wave centered at −1.01, −0.57, and −0.39 V was observed for compounds 6, 7, and 8, respectively, which is assigned to the reduction of the electron-withdrawing groups. As observed, the reduction strongly depends on the nature and strength of the acceptor. For each compound, only the first oxidation exhibits the higher potential variation due to conjugation of the nitrogen atom with the acceptor moiety. These behaviors also suggest that the phenothiazine ring is not fully conjugated with the whole π-conjugated systems and may adopt an orthogonal orientation, confirmed by standard DFT calculations (Table 2, vide infra). Furthermore, the electrochemical bandgaps (Eg elec ) reveal similar characteristics and are in good agreement with the optical bandgaps reflecting a linear decrease in the HOMO-LUMO gap while the acceptor strength increases. tion due to conjugation of the nitrogen atom with the acceptor moiety. These behaviors also suggest that the phenothiazine ring is not fully conjugated with the whole π-conjugated systems and may adopt an orthogonal orientation, confirmed by standard DFT calculations (Table 2, vide infra). Furthermore, the electrochemical bandgaps (Eg elec ) reveal similar characteristics and are in good agreement with the optical bandgaps reflecting a linear decrease in the HOMO-LUMO gap while the acceptor strength increases.     Computational DFT theoretical studies at the DFT level of the chromophores 6-8 were performed using the (B3LYP/6-31G(d,p)) basis set (Table 2) [40]. All HOMO levels are located along the phenothiazine ring system, whereas the LUMO levels are mainly located on the acceptor and the π-conjugated spacer. The energy values of the calculated frontier orbitals can be correlated with the cyclic voltammetry experiments for the HOMO levels while the LUMO levels appear at higher energies than expected. These main differences could be explained by the fact that in the excited state, the compounds exhibit a higher polarization due to the ICT, which is more stabilized by solvation effects in CH3CN giving lowered LUMO energy levels.
The anion binding properties of the compounds 6-8 were investigated by UV-visible spectroscopy with several anions such as PF6 − , HSO4 − , Cl − , Br − NO2 − , CH3CO2 − , and CN − as tetrabutylammonium salts in CH3CN. Even in polar solvent, these anionic guests interact with the chemosensors 6-8 leading to different spectroscopic changes that are structuredependent and more specifically to the nature of the acceptor unit. Spectroscopic changes are associated to batho-, hyper-, or hypochromic effects of the ICT band. The optical changes and their amplitude as well as the presence of isosbestic points are ascribed to the ability of chemosensors to interact more or less specifically with a given anion.
The three push-pull phenothiazine derivatives exhibit different behaviors, as shown in Figures 3-5. Hence, compounds 7 and 8 displayed the most significant spectroscopic changes while compound 6 did not undergo any changes regardless of the anion tested ( Figure 3). In addition, net disparities emerged between 7 and 8 ( Figures 4 and 5) depend-   Computational DFT theoretical studies at the DFT level of the chromophores 6-8 were performed using the (B3LYP/6-31G(d,p)) basis set (Table 2) [40]. All HOMO levels are located along the phenothiazine ring system, whereas the LUMO levels are mainly located on the acceptor and the π-conjugated spacer. The energy values of the calculated frontier orbitals can be correlated with the cyclic voltammetry experiments for the HOMO levels while the LUMO levels appear at higher energies than expected. These main differences could be explained by the fact that in the excited state, the compounds exhibit a higher polarization due to the ICT, which is more stabilized by solvation effects in CH3CN giving lowered LUMO energy levels.
The anion binding properties of the compounds 6-8 were investigated by UV-visible spectroscopy with several anions such as PF6 − , HSO4 − , Cl − , Br − NO2 − , CH3CO2 − , and CN − as tetrabutylammonium salts in CH3CN. Even in polar solvent, these anionic guests interact with the chemosensors 6-8 leading to different spectroscopic changes that are structuredependent and more specifically to the nature of the acceptor unit. Spectroscopic changes are associated to batho-, hyper-, or hypochromic effects of the ICT band. The optical changes and their amplitude as well as the presence of isosbestic points are ascribed to the ability of chemosensors to interact more or less specifically with a given anion.
The three push-pull phenothiazine derivatives exhibit different behaviors, as shown in Figures 3-5. Hence, compounds 7 and 8 displayed the most significant spectroscopic changes while compound 6 did not undergo any changes regardless of the anion tested ( Figure 3). In addition, net disparities emerged between 7 and 8 ( Figures 4 and 5) depend-   Computational DFT theoretical studies at the DFT level of the chromophores 6-8 were performed using the (B3LYP/6-31G(d,p)) basis set (Table 2) [40]. All HOMO levels are located along the phenothiazine ring system, whereas the LUMO levels are mainly located on the acceptor and the π-conjugated spacer. The energy values of the calculated frontier orbitals can be correlated with the cyclic voltammetry experiments for the HOMO levels while the LUMO levels appear at higher energies than expected. These main differences could be explained by the fact that in the excited state, the compounds exhibit a higher polarization due to the ICT, which is more stabilized by solvation effects in CH3CN giving lowered LUMO energy levels.
The anion binding properties of the compounds 6-8 were investigated by UV-visible spectroscopy with several anions such as PF6 − , HSO4 − , Cl − , Br − NO2 − , CH3CO2 − , and CN − as tetrabutylammonium salts in CH3CN. Even in polar solvent, these anionic guests interact with the chemosensors 6-8 leading to different spectroscopic changes that are structuredependent and more specifically to the nature of the acceptor unit. Spectroscopic changes are associated to batho-, hyper-, or hypochromic effects of the ICT band. The optical changes and their amplitude as well as the presence of isosbestic points are ascribed to the ability of chemosensors to interact more or less specifically with a given anion.
The three push-pull phenothiazine derivatives exhibit different behaviors, as shown in Figures 3-5. Hence, compounds 7 and 8 displayed the most significant spectroscopic changes while compound 6 did not undergo any changes regardless of the anion tested ( Figure 3). In addition, net disparities emerged between 7 and 8 ( Figures 4 and 5) depend- Computational DFT theoretical studies at the DFT level of the chromophores 6-8 were performed using the (B3LYP/6-31G(d,p)) basis set (Table 2) [40]. All HOMO levels are located along the phenothiazine ring system, whereas the LUMO levels are mainly located on the acceptor and the π-conjugated spacer. The energy values of the calculated frontier orbitals can be correlated with the cyclic voltammetry experiments for the HOMO levels while the LUMO levels appear at higher energies than expected. These main differences could be explained by the fact that in the excited state, the compounds exhibit a higher polarization due to the ICT, which is more stabilized by solvation effects in CH3CN giving lowered LUMO energy levels.
The anion binding properties of the compounds 6-8 were investigated by UV-visible spectroscopy with several anions such as PF6 − , HSO4 − , Cl − , Br − NO2 − , CH3CO2 − , and CN − as tetrabutylammonium salts in CH3CN. Even in polar solvent, these anionic guests interact with the chemosensors 6-8 leading to different spectroscopic changes that are structuredependent and more specifically to the nature of the acceptor unit. Spectroscopic changes are associated to batho-, hyper-, or hypochromic effects of the ICT band. The optical changes and their amplitude as well as the presence of isosbestic points are ascribed to the ability of chemosensors to interact more or less specifically with a given anion.
The three push-pull phenothiazine derivatives exhibit different behaviors, as shown in Figures 3-5. Hence, compounds 7 and 8 displayed the most significant spectroscopic changes while compound 6 did not undergo any changes regardless of the anion tested ( Figure 3). In addition, net disparities emerged between 7 and 8 ( Figures 4 and 5) depend- Computational DFT theoretical studies at the DFT level of the chromophores 6-8 were performed using the (B3LYP/6-31G(d,p)) basis set (Table 2) [40]. All HOMO levels are located along the phenothiazine ring system, whereas the LUMO levels are mainly located on the acceptor and the π-conjugated spacer. The energy values of the calculated frontier orbitals can be correlated with the cyclic voltammetry experiments for the HOMO levels while the LUMO levels appear at higher energies than expected. These main differences could be explained by the fact that in the excited state, the compounds exhibit a higher polarization due to the ICT, which is more stabilized by solvation effects in CH3CN giving lowered LUMO energy levels.
The anion binding properties of the compounds 6-8 were investigated by UV-visible spectroscopy with several anions such as PF6 − , HSO4 − , Cl − , Br − NO2 − , CH3CO2 − , and CN − as tetrabutylammonium salts in CH3CN. Even in polar solvent, these anionic guests interact with the chemosensors 6-8 leading to different spectroscopic changes that are structuredependent and more specifically to the nature of the acceptor unit. Spectroscopic changes are associated to batho-, hyper-, or hypochromic effects of the ICT band. The optical changes and their amplitude as well as the presence of isosbestic points are ascribed to the ability of chemosensors to interact more or less specifically with a given anion.
The three push-pull phenothiazine derivatives exhibit different behaviors, as shown in Figures 3-5. Hence, compounds 7 and 8 displayed the most significant spectroscopic changes while compound 6 did not undergo any changes regardless of the anion tested ( Figure 3). In addition, net disparities emerged between 7 and 8 ( Computational DFT theoretical studies at the DFT level of the chromophores 6-8 were performed using the (B3LYP/6-31G(d,p)) basis set (Table 2) [40]. All HOMO levels are located along the phenothiazine ring system, whereas the LUMO levels are mainly located on the acceptor and the π-conjugated spacer. The energy values of the calculated frontier orbitals can be correlated with the cyclic voltammetry experiments for the HOMO levels while the LUMO levels appear at higher energies than expected. These main differences could be explained by the fact that in the excited state, the compounds exhibit a higher polarization due to the ICT, which is more stabilized by solvation effects in CH3CN giving lowered LUMO energy levels.
The anion binding properties of the compounds 6-8 were investigated by UV-visible spectroscopy with several anions such as PF6 − , HSO4 − , Cl − , Br − NO2 − , CH3CO2 − , and CN − as tetrabutylammonium salts in CH3CN. Even in polar solvent, these anionic guests interact with the chemosensors 6-8 leading to different spectroscopic changes that are structuredependent and more specifically to the nature of the acceptor unit. Spectroscopic changes are associated to batho-, hyper-, or hypochromic effects of the ICT band. The optical changes and their amplitude as well as the presence of isosbestic points are ascribed to the ability of chemosensors to interact more or less specifically with a given anion.
The three push-pull phenothiazine derivatives exhibit different behaviors, as shown in Figures 3-5. Hence, compounds 7 and 8 displayed the most significant spectroscopic changes while compound 6 did not undergo any changes regardless of the anion tested ( Figure 3). In addition, net disparities emerged between 7 and 8 (Figures 4 and 5 Computational DFT theoretical studies at the DFT level of the chromophores 6-8 were performed using the (B3LYP/6-31G(d,p)) basis set (Table 2) [40]. All HOMO levels are located along the phenothiazine ring system, whereas the LUMO levels are mainly located on the acceptor and the π-conjugated spacer. The energy values of the calculated frontier orbitals can be correlated with the cyclic voltammetry experiments for the HOMO levels while the LUMO levels appear at higher energies than expected. These main differences could be explained by the fact that in the excited state, the compounds exhibit a higher polarization due to the ICT, which is more stabilized by solvation effects in CH 3 CN giving lowered LUMO energy levels.
The anion binding properties of the compounds 6-8 were investigated by UV-visible spectroscopy with several anions such as PF 6 − , HSO 4 − , Cl − , Br − NO 2 − , CH 3 CO 2 − , and CN − as tetrabutylammonium salts in CH 3 CN. Even in polar solvent, these anionic guests interact with the chemosensors 6-8 leading to different spectroscopic changes that are structure-dependent and more specifically to the nature of the acceptor unit. Spectroscopic changes are associated to batho-, hyper-, or hypochromic effects of the ICT band. The optical changes and their amplitude as well as the presence of isosbestic points are ascribed to the ability of chemosensors to interact more or less specifically with a given anion. The three push-pull phenothiazine derivatives exhibit different behaviors, as shown in Figures 3-5. Hence, compounds 7 and 8 displayed the most significant spectroscopic changes while compound 6 did not undergo any changes regardless of the anion tested ( Figure 3). In addition, net disparities emerged between 7 and 8 ( Figures 4 and 5) depending on the nature of the analyte. In both cases, trivial or no interaction are seen for PF 6 − , HSO 4 − , Cl − , and Br − , whereas with NO 2 − , CH 3 CO 2 − , and CN − , the main spectral modifications are attained. Moreover, chromophore 7 interacts only the cyanide anion while 8 gives a colorimetric response with NO 2 − , CH 3 CO 2 − , and CN − .        Thus, optical properties of compound 6 remain unchanged regardless of the tested anions, even with an excess. On the contrary, the addition of 1 equivalent of cyanide anion to chromophore 7 induces the formation of the adduct product 2-(2-((4-(10H-phenothiazin-10yl)phenyl)(cyano)methyl)-3-oxo-2,3-dihydro-1H-inden-1-ylidene)malononitrile. 7.CN − ( Figure 5) associated with a striking hypochromic effect and a hypsochromic shift, highlighting the nucleophilic conjugate addition of the cyanide on the β-vinylic carbon of the p-conjugated system as depicted in Figure 6, and altering the push-pull effect in agreement with previously reported results on similar systems [33]. Thus, optical properties of compound 6 remain unchanged regardless of the tested anions, even with an excess. On the contrary, the addition of 1 equivalent of cyanide anion to chromophore 7 induces the formation of the adduct product 2-(2-((4-(10H-phenothiazin-10-yl)phenyl)(cyano)methyl)-3-oxo-2,3-dihydro-1H-inden-1-ylidene)malononitrile. 7.CN − ( Figure 5) associated with a striking hypochromic effect and a hypsochromic shift, highlighting the nucleophilic conjugate addition of the cyanide on the β-vinylic carbon of the p-conjugated system as depicted in Figure 6, and altering the push-pull effect in agreement with previously reported results on similar systems [33]. In the case of chromophore 8, the recognition mode appears to be different as a bathochromic shift of 561 nm, 571 nm, and 576 nm is observed for NO2 − , CN − , and CH3CO2 − anions, respectively. In addition to the red shift of the maximum of absorption, a fine vibronic structure appears for the ICT band. This behavior is in favor of the presence of anion-π interactions related to the π-acceptor strength [41][42][43] instead of nucleophilic conjugate addition, as seen for chromophore 7 (Figure 7). Interestingly, the strongest bathochromic shift is observed for the unexpected CH3CO2 − anion. This result suggests a greater affinity of 8 towards the acetate anion compared to the others even if its pKb (9.25) is two-fold higher than the pKb (4.60) of cyanide. To confirm this hypothesis, competitive titration assays were conducted with 8.CH3CO2 − by the concomitant addition of 1 equivalent of the most competitive NO2 − and CN − anions ( Figure 8). The absorption spectrum of 8.CH3CO2 − remained unchanged, testifying to the stronger specificity of 8 for the acetate anion. Similar results were obtained when CH3CO2 − was added to a solution of the complex 8.CN − or of 8.NO2 − (Figure 9). In addition, experiments conducted between the complex 8.CN − and NO2 − demonstrate the higher ability of 8 to interact with the cyanide anion, corroborating the observed spectroscopic changes and following the pKb range (pKb = 10.85 for nitrite anion). For the other anions having a pKb higher than ~11 (pKb (HSO4 − ) = 17, pKb (Cl − ) = 20, pKb (Br − ) = 22, pKb (PF6 − ) > 22 [44]), no interaction is observed. Nevertheless, not only must the pKb be taken into consideration to explain the trend, but also the symmetry, geometry, and size of the anions. On the basis of these results, a series of affinity can be settled as follows for chromophore 8: In the case of chromophore 8, the recognition mode appears to be different as a bathochromic shift of 561 nm, 571 nm, and 576 nm is observed for NO 2 − , CN − , and CH 3 CO 2 − anions, respectively. In addition to the red shift of the maximum of absorption, a fine vibronic structure appears for the ICT band. This behavior is in favor of the presence of anion-π interactions related to the π-acceptor strength [41][42][43] instead of nucleophilic conjugate addition, as seen for chromophore 7 (Figure 7). Thus, optical properties of compound 6 remain unchanged regardless of the tested anions, even with an excess. On the contrary, the addition of 1 equivalent of cyanide anion to chromophore 7 induces the formation of the adduct product 2-(2-((4-(10H-phenothiazin-10-yl)phenyl)(cyano)methyl)-3-oxo-2,3-dihydro-1H-inden-1-ylidene)malononitrile. 7.CN − ( Figure 5) associated with a striking hypochromic effect and a hypsochromic shift, highlighting the nucleophilic conjugate addition of the cyanide on the β-vinylic carbon of the p-conjugated system as depicted in Figure 6, and altering the push-pull effect in agreement with previously reported results on similar systems [33]. In the case of chromophore 8, the recognition mode appears to be different as a bathochromic shift of 561 nm, 571 nm, and 576 nm is observed for NO2 − , CN − , and CH3CO2 − anions, respectively. In addition to the red shift of the maximum of absorption, a fine vibronic structure appears for the ICT band. This behavior is in favor of the presence of anion-π interactions related to the π-acceptor strength [41][42][43] instead of nucleophilic conjugate addition, as seen for chromophore 7 (Figure 7). Interestingly, the strongest bathochromic shift is observed for the unexpected CH3CO2 − anion. This result suggests a greater affinity of 8 towards the acetate anion compared to the others even if its pKb (9.25) is two-fold higher than the pKb (4.60) of cyanide. To confirm this hypothesis, competitive titration assays were conducted with 8.CH3CO2 − by the concomitant addition of 1 equivalent of the most competitive NO2 − and CN − anions ( Figure 8). The absorption spectrum of 8.CH3CO2 − remained unchanged, testifying to the stronger specificity of 8 for the acetate anion. Similar results were obtained when CH3CO2 − was added to a solution of the complex 8.CN − or of 8.NO2 − (Figure 9). In addition, experiments conducted between the complex 8.CN − and NO2 − demonstrate the higher ability of 8 to interact with the cyanide anion, corroborating the observed spectroscopic changes and following the pKb range (pKb = 10.85 for nitrite anion). For the other anions having a pKb higher than ~11 (pKb (HSO4 − ) = 17, pKb (Cl − ) = 20, pKb (Br − ) = 22, pKb (PF6 − ) > 22 [44]), no interaction is observed. Nevertheless, not only must the pKb be taken into consideration to explain the trend, but also the symmetry, geometry, and size of the anions. On the basis of these results, a series of affinity can be settled as follows for chromophore 8: Interestingly, the strongest bathochromic shift is observed for the unexpected CH 3 CO 2 − anion. This result suggests a greater affinity of 8 towards the acetate anion compared to the others even if its pK b (9.25) is two-fold higher than the pK b (4.60) of cyanide. To confirm this hypothesis, competitive titration assays were conducted with 8.CH 3 CO 2 − by the concomitant addition of 1 equivalent of the most competitive NO 2 − and CN − anions ( Figure 8). The absorption spectrum of 8.CH 3 CO 2 − remained unchanged, testifying to the stronger specificity of 8 for the acetate anion. Similar results were obtained when CH 3 CO 2 − was added to a solution of the complex 8.CN − or of 8.NO 2 − (Figure 9). In addition, experiments conducted between the complex 8.CN − and NO 2 − demonstrate the higher ability of 8 to interact with the cyanide anion, corroborating the observed spectroscopic changes and following the pK b range (pK b = 10.85 for nitrite anion). For the other anions having a pK b higher than~11 (pK b (HSO 4 − ) = 17, pK b (Cl − ) = 20, pK b (Br − ) = 22, pK b (PF 6 − ) > 22 [44]), no interaction is observed. Nevertheless, not only must the pK b be taken into consideration to explain the trend, but also the symmetry, geometry, and size of the anions. On the basis of these results, a series of affinity can be settled as follows for chromophore 8:  The selectivity of chromophore 7 was also investigated by UV-vis spectroscopy (Figure 10). Upon the addition of putative competitive anions, the spectrum of 7.CN − remained unchanged, highlighting the fact that the addition of the cyanide is irreversible and unaffected by the presence of other ions in the medium. This indicates that the chemosensors are selective and specific to cyanide over the other anions. The same behaviors are attained when chromophore 7 is mixed with 1 eq. of CN − and 1 eq. of each anion. : . ⁻ + 1 eq. PF₆-: . ⁻ + 1 eq. HSO₄⁻ : . ⁻ + 1 eq. Cl⁻ : . ⁻ + 1 eq. Br⁻ : . ⁻ + 1 eq. NO₂⁻ : . ⁻ + 1 eq. CH₃CO₂⁻ : . ⁻  The selectivity of chromophore 7 was also investigated by UV-vis spectroscopy (Figure 10). Upon the addition of putative competitive anions, the spectrum of 7.CN − remained unchanged, highlighting the fact that the addition of the cyanide is irreversible and unaffected by the presence of other ions in the medium. This indicates that the chemosensors are selective and specific to cyanide over the other anions. The same behaviors are attained when chromophore 7 is mixed with 1 eq. of CN − and 1 eq. of each anion. : . ⁻ + 1 eq. PF₆-: . ⁻ + 1 eq. HSO₄⁻ : . ⁻ + 1 eq. Cl⁻ : . ⁻ + 1 eq. Br⁻ : . ⁻ + 1 eq. NO₂⁻ : . ⁻ + 1 eq. CH₃CO₂⁻ : . ⁻ The selectivity of chromophore 7 was also investigated by UV-vis spectroscopy ( Figure 10). Upon the addition of putative competitive anions, the spectrum of 7.CN − remained unchanged, highlighting the fact that the addition of the cyanide is irreversible and unaffected by the presence of other ions in the medium. This indicates that the chemosensors are selective and specific to cyanide over the other anions. The same behaviors are attained when chromophore 7 is mixed with 1 eq. of CN − and 1 eq. of each anion.
Job-plots were performed in CH3CN and indicate, for the studied anions, the formation of either a complex [1:1] for 8.CN − and 8.NO2 − (as exemplified in Figure 11) or a complex [1:2] for 8.CH3CO2 − . Association constants Ki were determined by solving the nonlinear equations given in literature [45]. For instance, for complex  [G], where ε corresponds to the molar extinction coefficient for each species. Hence, the determination of the association constants K1 gives the following trend K CH3CO2− >> K CN− > K NO₂− (Table 3), which is in good agreement with the experimental optical changes observed in UV-Vis spectra. In addition, based on the equilibrium constants and ε, we are able to predict a reasonable titration theoretical spectrum ( Figure 12) matching with the experimental data. Association constants K i were determined by solving the nonlinear equations given in literature [45]. For instance, for complex  (where H corresponds to the host, G the guest, and the subscript 0 denotes the initial concentration for each species). The total absorbance is given by the equation , where ε corresponds to the molar extinction coefficient for each species. Hence, the determination of the association constants K 1 gives the following trend K CH 3 CO 2 − >> K CN− > K NO 2 − (Table 3), which is in good agreement with the experimental optical changes observed in UV-Vis spectra. In addition, based on the equilibrium constants and ε, we are able to predict a reasonable titration theoretical spectrum ( Figure 12) matching with the experimental data.

Wavelenght (nm)
: . ₃ ₂⁻ + 1 eq. CN⁻ + 1 eq. NO₂⁻ (theoretical) : . ₃ ₂⁻ + 1 eq. CN⁻ + 1 eq. NO₂⁻  According to the calculations, only 7.2% of CH 3 CO 2 − remains free in solution vs. 26.8% and 36% for CN − and NO 2 − , respectively. Both experimental and calculation findings pinpoint that 8 interacts more strongly with CH 3 CO 2 − than CN − and NO 2 − . Finally, as proof of concept for practical applications, a paper test strip was prepared as follows: a white paper strip was dipped into a 10 −3 M solution of 7 in CH 3 CN for 1 min, then air-dried. After this process, the paper took on a red wine color tint. The immersion of the latter into a solution of CN − anions colorized the test strip in orange, whereas for the others (PF 6 − , HSO 4 − , Cl − , Br − NO 2 − , CH 3 CO 2 − ), the strip's color remained unchanged. The same behavior was observed when the paper test strip was immersed in a solution containing all anions together ( Figure 13). These results clearly demonstrate the possibility of using this strip as qualitatively naked eye sensing of cyanide in complex solutions. The method is very cheap and does not require any calibration for qualitative detection.
Finally, as proof of concept for practical applications, a paper test strip was prepared as follows: a white paper strip was dipped into a 10 −3 M solution of 7 in CH3CN for 1 min, then air-dried. After this process, the paper took on a red wine color tint. The immersion of the latter into a solution of CN − anions colorized the test strip in orange, whereas for the others (PF6 − , HSO4 − , Cl − , Br − NO2 − , CH3CO2 − ), the strip's color remained unchanged. The same behavior was observed when the paper test strip was immersed in a solution containing all anions together ( Figure 13). These results clearly demonstrate the possibility of using this strip as qualitatively naked eye sensing of cyanide in complex solutions. The method is very cheap and does not require any calibration for qualitative detection.

Conclusions
In summary, we reported herein the straightforward synthesis and characterization of novel phenothiazine derivatives possessing remarkable sensitivity and selectivity for cyanide (LOD = 9.12 µM) over other anions. The sensing mechanism is based on the irreversible nucleophilic addition of the cyanide leading to an irreversible change of the color. This behavior makes it a system of choice for naked eyes sensing devices (Table 4) based on the absorption properties since most of the phenothiazine-based chemosensors developed up to now have been used as fluorescent probes. Interestingly, by changing the strength of the acceptor, the mechanism is switched into an anion-p interactions recognition mechanism associated to almost a two-fold increase in the LOD for cyanide detection reaching a value of 4.59 µM. In addition, the enhancement of the acceptor strength has allowed the possibility to selectively detect nitrite and acetate. Experimental data demonstrated that the highest binding ability is achieved for the acetate anion even in presence of nitrite and cyanide with a LOD of 2.68 µM. A paper test strip was efficiently prepared and used to sense qualitatively cyanide anions in solutions. Further studies are in progress striving for new chemosensors suitable for sensing applications as well as their implementation in electronic devices such as field effect transistors [46]. Table 4. Literature survey of optical phenothiazine-based chemosensors used in cyanide sensing associated with their LOD. a Fluorescence, b absorption.

LOD Solvent [Ref]
allowed the possibility to selectively detect nitrite and acetate. Experimental data demonstrated that the highest binding ability is achieved for the acetate anion even in presence of nitrite and cyanide with a LOD of 2.68 µM. A paper test strip was efficiently prepared and used to sense qualitatively cyanide anions in solutions. Further studies are in progress striving for new chemosensors suitable for sensing applications as well as their implementation in electronic devices such as field effect transistors [46]. Table 4. Literature survey of optical phenothiazine-based chemosensors used in cyanide sensing associated with their LOD. a Fluorescence, b absorption.

LOD Solvent [Ref]
1.56 µM b DCM [22] 9.80 10 −2 µM DMF/Tris-HCl buffer 1:99 v/v 10 mM, pH 9.3 [29] 6.70 10 −2 µM a DMSO/H2O (9:1) [23] 3.21 10 −3 µM a CH3CN [26] 3.06-3.20 10 −3 µM a CH3CN [30] 13.00 µM a CH3CN [24] 3.20 10 −3 µM a CH3CN: water 3.21 × 10 −3 µM a CH 3 CN [26] tation in electronic devices such as field effect transistors [46]. of nitrite and cyanide with a LOD of 2.68 µM. A paper test strip was efficiently prepared and used to sense qualitatively cyanide anions in solutions. Further studies are in progress striving for new chemosensors suitable for sensing applications as well as their implementation in electronic devices such as field effect transistors [46].  13.00 µM a CH 3 CN [24] of nitrite and cyanide with a LOD of 2.68 µM. A paper test strip was efficiently prepared and used to sense qualitatively cyanide anions in solutions. Further studies are in progress striving for new chemosensors suitable for sensing applications as well as their implementation in electronic devices such as field effect transistors [46]. 3.20 × 10 −3 µM a CH 3 CN: water (9:1) [31] of nitrite and cyanide with a LOD of 2.68 µM. A paper test strip was efficiently prepared and used to sense qualitatively cyanide anions in solutions. Further studies are in progress striving for new chemosensors suitable for sensing applications as well as their implementation in electronic devices such as field effect transistors [46]. of nitrite and cyanide with a LOD of 2.68 µM. A paper test strip was efficiently prepared and used to sense qualitatively cyanide anions in solutions. Further studies are in progress striving for new chemosensors suitable for sensing applications as well as their implementation in electronic devices such as field effect transistors [46]. and used to sense qualitatively cyanide anions in solutions. Further studies are in progress striving for new chemosensors suitable for sensing applications as well as their implementation in electronic devices such as field effect transistors [46]. and used to sense qualitatively cyanide anions in solutions. Further studies are in progress striving for new chemosensors suitable for sensing applications as well as their implementation in electronic devices such as field effect transistors [46]. Table 4. Literature survey of optical phenothiazine-based chemosensors used in cyanide sensing associated with their LOD. a Fluorescence, b absorption.

Data Availability Statement:
The data presented in this study are available on request from the corresponding author.