Chromogenic and fluorogenic multianalyte detection with a tuned receptor: refining selectivity for toxic anions and nerve agents

Abstract

In the present study, a chemical probe was finely tuned for the highly selective and sensitive chromogenic and fluorogenic detection of toxic anions and a nerve agent. Studies on the binding sites, signaling units and spacers led to a receptor molecule, i.e., 2,7-bis-[3-(4-cyanophenyl)thiourea] fluorene 2, which detected multiple analytes such as fluoride, cyanide, and a tabun mimic. The optical responses obtained from all the three analytes were quite distinct from one another. More importantly, we have been successful in demonstrating the highly specific chromo-fluorogenic detection of a tabun mimic over other chemical warfare agents and reactive and non-reactive electrophilic interferents. In an attempt to mimic real-life scenarios, the applicability of our protocol was further extended to demonstrate the presence of a tabun mimic on solid surfaces, in the gas phase and in a spiked soil sample.

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Introduction

The design and development of molecular sensors based on the indicator–spacer–receptor (ISR) paradigm have always been at the forefront of research in supramolecular chemistry.¹ By tuning the components of the ISR approach viz. signaling unit (indicator), spacer and binding site (receptor), many chromogenic and fluorogenic sensors have been successfully developed for wide ranges of cations, anions and neutral analytes.² Following this approach, we have been working on various strategies for the detection of chemical species, particularly anions,³ and neutral analytes.⁴ In our research, fine tuning of the chemical probes has led to the selective detection of desired analytes.³ Now, we aim to design a single probe molecule for the detection of multianalytes such as fluoride, cyanide and tabun.

Among the anions, fluoride and cyanide occupy a central position because of their applications and harmful effects to human health and the environment. Fluoride is primarily used to prevent dental cavities and in the treatment of osteoporosis.⁵ Contrary to this, excessive use of fluoride causes fluorosis⁶ and nephrotoxic changes in both humans and animals, and can lead to urolithiasis. Cyanide is considered to be the most toxic anion that binds heme cofactors to inhibit the terminal respiratory chain enzyme cytochrome c oxidase, which ultimately lead to death.⁷ Furthermore, both the anions are also the hydrolysis products of nerve agents such as sarin, soman, and tabun.⁸ Therefore, the chemosensor ensembles that respond to fluoride and cyanide is highly desired for the detection of nerve agents for military/homeland security purposes. Nerve agents⁹ (Fig. 1) represent the extremely toxic class of organophosphorous compounds and chemical warfare agents (CWAs) that can lead to death within minutes if inhaled due to the inhibition of acetylcholinesterase enzyme (AChE).¹⁰ Tabun (GA) is the first of G-series nerve agents along with GB (sarin), GD (soman) and GF (cyclosarin). In the history including recent event in Syria (2013), nerve agents have been used in offensive ways against civilian/military populations by terrorists or rogue nations. Therefore, the chromogenic and fluorogenic detection of these deadliest chemical agents by a ‘single probe’ will be the most desirable, that can detect their presence even in the environment samples.

Fig. 1 Nerve agents and their mimics.

Many reports have appeared in the literature describing the detection of fluoride,¹¹ cyanide¹² and tabun mimic^4a,4d,13 using single molecule single analyte detection approach. To the best of our knowledge, there is no such chemosensor which can detect and differentiate both anions as well as nerve agent tabun with naked eye and by fluorescence methods simultaneously. In fact, the interference of both anions from each other as well as from other anions is commonly observed.¹⁴ Because cyanide being nucleophilic also have some ability to form hydrogen bonding whereas fluoride also behave as a base apart from making hydrogen bond with binding site of the receptor thus inducing optical signals.

However, some efforts have been directed to detect and differentiate F⁻ and CN⁻. For instance, Jiang and coworkers explored a phenolic Schiff based chemosensor which detect fluoride by colorimetric method and cyanide by fluorogenic method.¹⁵ Ratiometric determination of both the anions by fluorescence and/or absorbance method was also achieved by Wang and coworkers.¹⁶ Furthermore, Bhattacharya and coworkers have successfully developed a probe which differentiate both the anions but show interference from the other anions.¹⁷

Recently, extensive research has been directed towards the development of chromogenic and fluorogenic sensor for CWAs, particularly for nerve agents.¹⁸ In most of the cases, the focus remains on the detection of sarin and its simulants diisopropyl fluorophosphate (DFP) or diethyl chlorophosphate (DECP).¹⁸ But no method is selective, and frequently gives false positive signals even with less toxic and non-toxic OP-compounds or electrophilic agents such as sulfonyl chlorides, thionyl chloride, anhydrides and acid chlorides.¹⁹ Unfortunately, colorimetric and/or fluorescence detection methods for tabun are nearly non-existent. Recently, we have initiated a programme on the detection of chemical warfare agents and showed detection successfully using various approaches.⁴ In tabun, P–CN group possess a unique chemistry and is also responsible for its deadliest nature. This chemistry has been exploited in our research to achieve the selectivity over other possible interferents.^4a,4d In this paper, now we move a step further to report a highly selective, sensitive ‘turn-on’ chromogenic and fluorogenic sensor for tabun along with fluoride and cyanide anions.

Results and discussion

Design and synthesis

Chemical sensing can be achieved by the rational design of a receptor having binding site, signaling unit and spacer. A fine tuning of these components in a chemical probe can lead to the selective and sensitive detection of the target analytes. In recent years, we also have been strategically tuning these components in the ditopic receptors (Fig. 2a) to achieve detection of toxic analytes such as fluoride, cyanide and tabun. Under this paradigm, a single receptor was used for the detection of single analyte. Now we propose a receptor molecule that can offer the detection of three analytes viz. fluoride, cyanide and tabun, thus following a single molecular multianalyte detection approach. A multi-analyte detection is generally employed using single molecular sensor²⁰ or by cross-reactive sensor array²¹ which triggers different responses in presence of different analytes. It is quite promising but a challenging task due to unique molecular design combined with possibility of interferences from other analytes. Most of multianalyte recognition is focused on sensing of cations,²² and very rarely for anions and neutral analytes.²³ Such a chemosensor is highly coveted in terms of convenience and economy. Therefore, there is growing interest in developing a novel single probe for multianalyte detection, particularly anions and neutral analytes.

Fig. 2 Generic structure of ditopic receptors.

A fluorene-based receptor having thiourea as binding site and three signaling unit nitrophenyl/cyanophenyl/trifluoromethyl phenyl viz. 2,7-bis-[3-(4-nitrophenyl)thiourea] fluorene (1), 2,7-bis-[3-(4-cyanophenyl)thiourea] fluorene (2), and 2,7-bis-[3-(4-trifluoromethyl phenyl)thiourea] fluorene (3) were prepared and screened. Receptor 1 was found to be very selective for cyanide with better sensitivity than earlier one^4a but failed to detect fluoride. Furthermore, no fluorescence response was observed due to the presence of nitrophenyl substituents (a fluorescence quencher). Receptors 2 and 3 were explored further to detect fluoride, cyanide, and tabun. A change in the spacer from diphenyl sulfide to fluorene leads to dramatic changes in the selectivity, sensitivity and optical properties of the receptor. Fluoride and cyanide will interact with thiourea fragment either through H-bonding or deprotonation to induce the optical changes in signaling units and fluorene, which play a dual role as a spacer as well as signaling unit.

The approach for tabun sensing is based on the detection of its hydrolyzed product i.e. cyanide. Notably, the hydrolysis rate of this agent in water/air is not instant, however, it can be increased substantially in basic medium.⁷ But in basic medium, the receptor itself can respond due to the presence of thiourea moieties. Therefore, the first step was to explore a ‘selective nucleophile’ that can react with tabun to discharge cyanide instantaneously. While exploring a nucleophile, we should keep in mind that nucleophile should be non-responsive to the probe itself. It means that it must be non-basic in nature and should not form strong hydrogen bond with thiourea moiety. In recent report, fluoride anion was proven to react selectively with tabun and giving no response with probe.^4d Here, we observed (vide infra) fluoride anion itself offers the optical responses with 2, hence was not considered. Various nucleophiles such as pyridine, DBU, imidazoles, oximes, cyanate, thiocyanate, iodide, bromide, were screened but none responded to tabun as well as to the probe. We finally explored tetrabutylammonium azide as a nucleophile of choice, which reacts selectively with tabun to release cyanide without responding to 2 (Fig. 3).

Fig. 3 Schematic representation of tabun mimic detection.

Receptor 2 and 3 were easily prepared in a single step using commercially available 2,7-diaminofluorene. The reaction of 2,7-diaminofluorene was carried out with 4-cyanophenyl isothiocyanate and trifluoromethylphenyl isothiocyanate in acetone to give 2 and 3 in 90% yields (Scheme 1), respectively. The receptor 2 and 3 was adequately characterized by FTIR, NMR and HRMS (see ESI† for spectra).

Scheme 1 Synthesis of receptors 2 and 3.

Colorimetric and UV absorbance responses

In validating the proposed idea of multianalyte detection, at first we aimed to demonstrate the chromogenic and/fluorogenic response of receptors 2 and 3 with various anions. To investigate this, 2 was allowed to interact with F⁻, CN⁻, Cl⁻, Br⁻, AcO⁻, I⁻, HSO₄⁻, H₂PO₄⁻, NO₃⁻, BF₄⁻, SO₄²⁻, ClO₄⁻, SCN⁻, N₃⁻, and OCN⁻ in the form of tetrabutylammonium salts in THF : DMSO (9 : 1). Upon addition of 3.0 equivalents of these anions (1.86 mM) to 2 (0.62 mM), only F⁻ and CN⁻ showed responses as compared to the other anions. F⁻ gave fluorescent green color, while CN⁻ showed dark yellow color instantaneously. The other anions showed no response with 2 as shown in Fig. 4a. The UV absorbance studies of 2 with various anions were carried out in THF : DMSO (99 : 1) solution. The absorption spectra were recorded by titrating 2 at 15.7 μM with the anions (0.186 mM). Fig. 5a shows that upon addition of CN⁻ to 2, there is a bathochromic shift from 316 nm and 338 nm to 434 nm (A^o = 0.4) with the appearance of new ICT band at 274 nm and 432 nm. However, F⁻ caused red shift from 316 nm and 338 to 434 nm (A^o = 0.17) (Fig. 5b). As it can be seen from Fig. 5d that upon addition of TBA–N₃ to 2 showed no significant change in absorbance study.

Fig. 4 Naked-eye and fluorescence responses (at 365 nm under UV lamp) of 2 (THF : DMSO solutions (9 : 1), v/v) after interaction with anions/chemical agents: (a) color changes upon addition of 3.0 equiv. of TBA salt of various anions (1.86 mM) into 2 (0.62 mM): Left to right: 2, 2 + CN⁻, 2 + F⁻, 2 + DCNP and 2 + interferents (other anions/agents); (b) fluorescent response of 2 (0.15 mM) upon addition of F⁻ (left, 0.45 mM) and DCNP (right, 0.45 mM); (c) color changes upon addition of 3.0 equiv. of TBA salt of anions (1.86 mM) into THF : DMSO solutions (9 : 1), v/v of 3 (0.62 mM): Left to right: 3, 3 + CN⁻, 3 + F⁻, 3 + DCNP.

Fig. 5 Changes of UV-vis absorption of 2 (15.7 μM) in THF : DMSO solutions (99 : 1) upon the subsequent addition of 10 μM of (a) F⁻ (b) CN⁻ (c) DCNP and (d) N₃⁻.

Diethyl cyanophosphonate (DCNP) was chosen as a tabun simulant due to its less toxicity but similar chemical reactivity. This has been frequently used as the most reliable tabun simulant, therefore we conducted all the relevant studies using this mimic. Another advantage of using DCNP as its simulant is that both agent and mimic have almost same molar mass (DCNP: 163.11 and tabun: 162.13), thus our study will be applicable to the real agent without any change in the concentrations or other experimental settings. DCNP (1.86 mM) in presence of TBA–azide (1.86 mM) in THF was allowed to interact with 2 (0.62 mM). The colorless solution of 2 turns yellow immediately indicating the presence of DCNP. Various potent interferents that frequently interfere in tabun detection were also studied. The interferents such as acetyl chloride, acetic anhydride, phosphoryl chloride, diethyl chlorophosphate, diisopropyl fluorophosphate, bis(2-chloroethyl)ether, sarin soman and Vx were treated with 2 under the identical conditions, but we did not observe any changes in the optical properties. In the UV absorbance experiments, aliquots of DCNP (0.186 mM) and TBA–N₃ (0.186 mM) were treated with 2 (15.7 μM) in THF : DMSO (99 : 1) solution. The initial UV-vis spectra shifted bathochromically from 316 nm and 338 nm to another band at 398 nm and 411 nm (Fig. 5c). As evident from the visual inspection that no interference was observed from the interferent under study (as mentioned above) even in the absorbance spectra. This shows a selective detection of DCNP over the reactive and non-reactive electrophilic agents that too within the CWAs category. To observe the effect of TBA–azide on 2, an interaction study was conducted, but we did not find any change in the color or absorbance spectra with the analyte. This confirms that any change in the optical properties of 2 is due to the presence of different analytes, and not due to TBA–azide.

Similarly, 3 was also interacted with 03 equivalents of all the anions and DCNP along with its interferents. In this case, fluoride, cyanide and tabun also gave similar responses, and no interferences with other analytes (Fig. 4c). It shows poor color intensity and absorbance when compared with the results of 2 with the analytes (Fig. 4a). Even upon excess addition of these analytes to 3 did not enhance the response, hence was not considered for further study. The response of receptor 2 in terms of selectivity and sensitivity was found to be better than 3. It is simply because of the difference in the electron withdrawing property of cyanophenyl and trifluoromethyl phenyl group, which due to ICT, show better response. These results are consistent with the previously reported one. Hence, we chose receptor 2 and demonstrated detection with all the analyte under study.

NMR interaction studies

The binding interactions between the receptor 2 and analytes such as F⁻, CN⁻, and DCNP (in the presence of TBA–N₃) were evaluated using NMR spectroscopy (Fig. 6–8). The studies were performed in CDCl₃ : DMSO-d6 (95 : 5) at room temperature. The NHs and CH₂ protons of fluorene fragment were observed at δ 9.35 ppm and δ 3.83 ppm respectively. Upon addition of incremental amount of 0.2 equiv. of CN⁻, the peaks due to NH lost it resolution and finally disappeared completely with 0.4 equiv. This indicates that interaction which occurred between 2 and CN⁻ was through a fast exchange process than the kinetics of cyanide exchange on NMR time scale. Hence, we could not track the changes occurring upon the interaction. This further led us to evaluate the same interaction studies in pure DMSO-d6 solvent. Before the addition of CN⁻, both NH peaks appeared at δ 10.22 and δ 10.18 ppm. Upon addition of incremental amount of 0.2 equiv. of CN⁻ to 2, an upfield shift was observed and simultaneously NH peaks began to lose resolution resulting in the broadening of peaks which disappeared finally upon addition of 1.4 equiv. This may be happening due to the deprotonation of NH proton attached to cyanophenyl fragments. Thus, another NH proton falls in shielding zone of ring current of the triple bond which is in fast exchange process with deprotonated NH proton, so upfield chemical shift occurs. With CN⁻, no significant change was observed in case of aromatic and CH₂ protons of 2.

Fig. 6 Partial NMR spectra of 2 with (a) TBA–CN in CDCl₃ : DMSO-d6 (95 : 5) and (b) TBA–CN in DMSO-d6.

Fig. 7 Partial NMR spectra of 2 with (a) TBAF in CDCl₃ : DMSO-d6 (95 : 5) and (b) with DCNP (in the presence of TBA–N₃) in CDCl₃ : DMSO-d6 (95 : 5).

Fig. 8 Partial NMR spectra of 2 with TBA–OH in CDCl₃ : DMSO-d6 (95 : 5).

Similarly, F⁻ and DCNP (in presence of TBA–N₃) were also allowed to interact with 2. Upon addition of 0.2 equiv. of the analytes, a downfield shift of NH peaks was observed that loses resolution and the peaks finally disappeared. The change in the chemical shift of aromatic and methylene protons of fluorene was also observed. Aromatic protons showed a downfield shift while in case of methylene protons, it was an upfield shift. The downfield shift with both the analytes anions reflects the establishment of hydrogen bonding between analyte and NH group of 2. Simultaneously, anions also produce the polarization in CH bond of aromatic via through-space effect, where partial positive charge created on the proton causes deshielding effect and produces downfield shift.

Both the process enhances the electron density on fluorene ring system. This enhancement of electron density through-bond propagation generates a shielding effect on methylene proton and produces an upfield shift. This change in aromatic and methylene peaks was quite significant with F⁻ as compared to DCNP. With fluoride, the change in chemical shift of aromatic and CH₂ protons was found to be δ 0.5 and δ 0.66 ppm, respectively while with DCNP it was δ 0.4 and δ 0.35 ppm, respectively. This is simply because fluoride is more electronegative than CN⁻, hence is able to pronounce more effect.

The completion and stoichiometry of reaction between DCNP and TBA–N₃ was confirmed by ³¹P NMR. TBA–N₃ was reacted with DCNP in CDCl₃ at room temperature. The peaks due to DCNP at δ −21.8 ppm began to disappear with the appearance new peaks at δ −0.61 ppm due to the formation of diethyl phosphorazidate and upon addition of one equivalent, it disappeared completely. To support the NMR data, diethyl phosphorazidate was synthesized from reaction of diethyl chlorophosphate and sodium azide in acetone²⁴ and recorded the ³¹P NMR of pure sample. The value was found to be δ −0.7 ppm that is similar with that of the formed in the reaction mixture. However, in the reaction mixture, we also observed small peaks at −13.0 and −0.15 ppm due to the formation of diethyl pyrophosphate and diethyl phosphate in traces amount due to the presence of moisture in the solvent.

Fluorescence studies

The fluorescence behavior of 2 was examined with all the three analytes and their possible interferents. The response was observed in THF : DMSO (9 : 1) under UV lamp at 365 nm and spectrofluorometer. Upon addition of all anions (0.45 mM) to 2 (0.15 mM) only F⁻ showed blue fluorescence emission, while other anions remain silent. Addition of CN⁻ gave dark yellow color which was non-fluorescent. In case of DCNP (0.45 mM), 2 gave green fluorescence emission on excitation at 365 nm while other interfering agents such as DFP, DECP, CEES, sulfonyl chloride, phosphoryl chloride, acetyl chloride, and acetic anhydride did not respond. Fluorescence titrations between 2 and F⁻ and DCNP were also performed using THF : DMSO (99 : 1) solution. The receptor 2 at 2.0 μM was titrated with F⁻ (10 μM) and DCNP (20 μM) and both showed large turn-on fluorescence when excited at 316 nm. The fluorescence intensity at 385 nm increases upon subsequent addition of F⁻, while in case of DCNP, two emission bands were observed at 356 nm and 472 nm, and the intensity became maximum at saturation point. The spectra are shown in shown in Fig. 9 and 10.

Fig. 9 Fluorescence emission changes (ex 316 nm) of 2 (2.0 μM) upon the subsequent addition of TBAF (10 μM) in THF : DMSO solutions (99 : 1).

Fig. 10 Fluorescence emission changes (ex 316 nm) of 2 (2.0 μM) in THF-DMSO (99 : 1, v/v) upon the subsequent addition of DCNP (20 μM) in THF : DMSO solutions (99 : 1).

Mechanism

The visual and UV-vis absorbance/fluorescence changes of 2 with F⁻, CN⁻ and DCNP can be explained on the basis of internal charge transfer (ICT) from donor to the acceptor. Both the nitrogen of thiourea fragments in 2 acts as a donor whereas 4-cyanophenyl and fluorene fragments act as the acceptor. Thiourea substituent on interaction with anionic species through either H-bonding or deprotonation enhances the electron density at both the nitrogen that upon excitation undergoes ICT to both the electron acceptor substituents resulting in a hypsochromic shift in its absorption and emission spectrum. F⁻ interact with both NH protons of thiourea through H-bond formation as well as exhibit electrostatic interaction with aromatic ring of fluorene and thus showed optical responses via ICT. As a result, fluorene substituents regain its fluorescence. However, CN⁻ caused deprotonation of a NH proton adjacent to 4-cyanophenyl group, which is more acidic in nature as compared to other N-atom, and this resulted into the formation of non-fluorescent dark yellow color. In this process, CN⁻ deprotonates NH group forming HCN which remains attached with the same nitrogen. Another NH to which fluorene is attached, no longer participates in interaction, thus making it non-fluorescent. It was remarkable to note that CN⁻ obtained from TBA–CN and DCNP responded differently in either visual response including UV-vis and fluorescence or NMR spectroscopy. We proposed that CN⁻ obtained from TBA–CN deprotonate NH of thiourea immediately because of being naked. While CN⁻ obtained from the reaction between DCNP and TBA–N₃, first makes a hydrogen bonding with both NH protons of thiourea and weak electrostatic interaction with aromatic protons of fluorene. This happens almost in a similar fashion as in case of F⁻. To support our hypothesis, we allowed interaction with large excess of DCNP (10 equiv.) and found that at a high concentration, formation of dark yellow color with no fluorescence takes place, thus behaving in a similar way as naked cyanide. This indicates that NH proton closer to cyanophenyl fragment was deprotonated.

To gain better understanding of the mechanism of interaction, 2 was further interacted with a standard solution of tetrabutylammonium hydroxide (a base). A yellow color appeared with green fluorescence (λ_ex: 365 nm), fairly similar as in case of DCNP, was observed. As can be seen from NMR spectrum (Fig. 7), this change was same as in the case of F⁻ and DCNP i.e. downfield shift of NHs and aromatic protons while upfield shift for methylene protons. When 2 was allowed to interact with large excess (10 equiv.) of TBA–OH, the solution became dark yellow with the loss of fluorescence, hence similarity with the result observed with CN⁻. These studies further support our proposed mechanism for all the analyte–receptor interaction. Thus, on the basis of our experimental observation, the proposed complex formation between 2 and CN⁻, F⁻, OH⁻ and DCNP is depicted in Fig. 11.

Fig. 11 Probable complex formation of 2 with (a) CN⁻ (b) F⁻, OH⁻ and DCNP.

The inclusion of detection of F⁻ and CN⁻ along with tabun mimic in the present study has served three purposes; (1) it provides a single platform for multi-analyte detection of anions and neutral analytes, (2) F⁻ and CN⁻ could be discriminated easily with no interference from each other, and (3) the interaction study with both anions employing various spectroscopic techniques has helped us to understand the mechanism for the detection of tabun mimic. One major advantage of this protocol is that tabun detection has an edge over previous reported method⁴ as it provides a turn-on chromogenic and fluorogenic detection. The greatest advantage of ‘turn-on’ fluorescence sensor related to ‘turn-off’ sensors is the ease of detecting low-concentration contrast relative to a “dark” background, which reduces the possibility of false positive signals and enhances the sensitivity.

Analytical applications

Encouraged by these results, we took a step further to explore the presence of analytes in the various matrices. It is evident that tabun can be delivered by aerial spraying, rockets, bombs, or artillery shells. Unfortunately, it is chemically stable and environment persistent (for a week)⁹ which further aggravate its menace. In view of this, the presence of DCNP was determined on many surfaces (concrete and vegetation), in the soil and vapor phase for the development of on-field sensing methods.

For the detection on the solid surfaces, a drop of DCNP was placed on the surface and allowed to stand for 1 min. The liquid was swiped from surface by a filter paper, and this paper was extracted with 1.0 mL of THF. This solution was allowed to react with receptor 2 containing TBA–azide in 1.0 mL of THF. The reaction took place upon mixing the solution, which turn yellow immediately. A blank experiment, when there was no agent was also performed. This showed the absence of analyte as no color change occurred in the solution.

To determine the presence in soil samples, 5 μL of DCNP was spiked in 2.0 g of soil. The spiked and unspiked soil samples were allowed to stand for five minutes, and then were extracted separately with THF (1.5 mL). The dirt solution was filtered and filtrate was treated with 2 (15 μM) and TBA–azide (1.0 mM). The yellow color appeared immediately with the DCNP-spiked sample while no change was observed with unspiked sample (Fig. 12a). The detection in vapor phase was carried out using gas generation assembly (ESI,† S6). 10 μL of the agent was placed in a glass vessel covered with heat jacket. Glass vessel is heated at 60 °C, vapors formed flow with nitrogen gas which is directly dipped into a solution containing 2 and TBA–N₃. Formation of yellow color indicate the presence of tabun mimic in vapor form (Fig. 12b).

Fig. 12 Chromogenic response of receptor 2 with DCNP (a) in spiked soil sample. (b) in gas phase (c) minimum detection limits of DCNP with 2 with naked eye and its response under UV lamp (365 nm) (d) colorimetric changes in the three vials containing CN⁻ (vial 1), DCNP along with CN⁻ (vial 2) (33 mol%) and DCNP (vial 3).

For the realization of CWA sensor in the real life situation, it is mandatory for us to determine the minimum detection limit using our protocol by naked-eye under visible and UV light with good visibility and confidence. Three test solutions of DCNP at concentration of 0.092 mM, 0.184 mM and 0.27 mM were allowed to interact with 2 and we observed quite visual yellow color with conc. 0.27 mM. This solution under UV-lamp gives blue emission at 365 nm. The LOD at 270 μM with good visibility, as can be seen in Fig. 12c, is found to be much less than what can cause any hazards to human health and national security. The relative toxicities of tabun by inhalation and through skin per individual were found to be 200 (LC₅₀ mg min m⁻³) and 4000 (LD₅₀ in mg) respectively. The sensitivity of present method was shown to be even much better than our previous one.^4e Furthermore, using present method, the detection can be achieved at even lower concentration than determined (25 μM) by UV absorbance, but is not discussed purposely. As we are targeting to implement our method for field trial and to avoid any practical problems, color appearance should be reasonably good.

While working on the detection of tabun, it is most likely that cyanide or other basic anions are present in the environment and may interfere in the agent detection. To avoid such situation, the presence of cyanide must be distinguished from tabun detection. For instance, identification of the solutions of three unknown vials containing CN⁻ (vial 1), DCNP along with CN⁻ (vial 2) (33 mol%) and DCNP (vial 3) was realized, which may be present together in the environment samples (Fig. 12). In such case, the three samples are allowed to react with the receptor; vial containing cyanide (vial 1) will turn yellow, while in case of vial 2 and 3, initially no change will occur (Fig. 12d), upon addition of TBA–N₃, the solution in vials 2 and 3 turns yellow instantaneously. With vial 2, the solution becomes yellow similarly as in case of vial 3 (not shown here). This is simply because TBA–N₃ releases cyanide from DCNP in vial 2 and 3 that produces identical results. Following this exercise, even tabun mimic can be discriminated from cyanide or other basic anions easily.

Conclusion

We have reported a multianalyte detection of fluoride, cyanide and tabun mimic (DCNP) using single molecular probe. A fine tuning of the binding sites, spacers and signaling units lead to a receptor which was shown to be highly selective for fluoride and cyanide over other anions. Apart from this, both the anions could also be differentiated from each other. Having established the protocols for anions sensing, we further explored for the chromogenic and fluorogenic detection of nerve agent tabun mimic. The method showed selective and sensitive detection as no interference within the CWAs or less toxic and non-toxic OP-compounds or other potent interferents such as sulfonyl chlorides, thionyl chloride, anhydrides, acid chlorides, and CEES was observed. For the analysis of environmental samples and to demonstrate the possibility for the practical applications, the presence of DCNP on surface, in spiked soil sample and in the gas phase was also successfully detected. This study will also be highly useful for detecting nerve agents' residues in the environment sample during the proficiency test conducted by Organization for the Prohibition of Chemical Weapons (OPCW) with aim to eliminate the chemical weapons.

Experimental section

General methods

All reagents for synthesis were obtained commercially and were used without further purification. Solvent such as tetrahydrofuran (THF), dimethylsulfoxide (DMSO), and acetone were purchased from S D Fine Chem. Ltd., India and dried as per the standard methods before using. In titration experiments, all the anions in the form of tetrabutylammonium (TBA), diethyl cyanophosphonate and other substrates were purchased from Sigma-Aldrich, USA and Fluka and stored in a vacuum desiccator. UV-vis spectra were recorded on a Thermo Spectronic spectrophotometer equipped with quartz cuvette (path length = 1 cm, at 25 °C). Fluorescence measurements were carried our using a Perkin Elmer, LS-55, UK. IR spectra were recorded on a Perkin-Elmer model BXII FTIR spectrophotometer using KBr pellets. NMR was recorded on Bruker 400 MHz spectrometer using trimethylsilane (TMS) as an internal standard.

Synthesis of 2,7-bis-[3-(4-cyanophenyl)thiourea] fluorene 2 and 2,7-bis-[3-(4-trifluoromethylphenyl)thiourea] fluorene 3

The solution of 2,7-diaminofluorene (0.5 g, 0.005 mol) and 4-cyanophenyl isothiocyanate or 4-trifluoromethylphenyl isothiocyanate (0.8 g, 0.01 mol) in dry acetone (25 mL) was stirred at room temperature for 2 h. The precipitate of the products 2 and 3 formed were filtered through sintered funnel and then washed thoroughly with cold acetone in order to remove any substrate impurity.

Receptor 2. Accurate HRMS (m/z): calculated for C₂₈H₂₀N₆OS₂ (M + 1) 517.1269. Found 517.1269; ¹H NMR (400 MHz, DMSO-d₆) δ_H: 10.22 (s, 2H, NH), 10.18 (s, 2H, NH), 7.437.83 (m, 14H), 3.93 (s, 2H) ¹³C NMR (100 MHz, DMSO-d₆) δ_C: 179.28, 144.26, 143.56, 137.74, 137.59, 132.69, 122.79, 122.42, 120.55, 119.76, 19.11, 105.31, 36.56; IR (ν/cm⁻¹). 3241, 3068, 2240, 1615, 1535, 1505, 1342, 1299, 1252, 833, 679.

Receptor 3. Accurate HRMS (m/z): calculated for C₂₉H₂₀F₆N₄S₂ (M + 1) 603.1112. Found 603.1098; ¹H NMR (400 MHz, DMSO-d₆) δ_H: 10.15 (s, 2H, NH), 10.12 (s, 2H, NH), 7.83–7.44 (m, 14H), 3.93 (s, 2H) ¹³C NMR (100 MHz, DMSO-d₆) δ_C: 180.0, 143.9, 138.16, 136.13, 126.06 (q), 125.99, 124.68 (q), 123.98, 123.35, 123.25, 121.02, 120.16, 113.4, 37.0; IR (ν/cm⁻¹). 3181, 3022, 1615, 1539, 1505, 1328, 1256, 839, 717.

Acknowledgements

We thank to Dr Lokendra Singh, Director, DRDE, Gwalior for his keen interest and encouragement. We also thank Dr Sanjay Upadhyay for his support and technical discussion.

Notes and references

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Footnote

† Electronic supplementary information (ESI) available: ¹HNMR, ¹³CNMR and HRMS of spectra of receptors of 2 and 3. See DOI: 10.1039/c6ra07080a

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