Rapid ‘naked eye’ response of DCP, a nerve agent simulant: from molecules to low-cost devices for both liquid and vapour phase detection

Abstract

A rhodamine-based new chemosensor, RHM, has been designed and synthesized. It reacts selectively with the organophosphate compound, DCP, a well-known nerve gas simulant (both in the liquid and vapour phase). Moreover, RHM offers the potential for detection by the naked eye for color and fluorescence change. The sensing phenomenon is supported by DFT calculation.

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After the gas attack on the Tokyo subway in 1995, the detection of “nerve gases” gained importance, and after the recent events in Syria it has become particularly essential. The Nobel peace prize for 2013 was awarded to the Organisation for the Prohibition of Chemical Weapons (OPCW) for its extensive efforts to eliminate chemical weapons. Use of these gases in terrorist attacks is somewhat undetectable; moreover, since they are colourless gases, their presence is not usually noticed until after they have already been inhaled. Taking advantage of this, the Aum Shinrikyo group released Sarin gas in Tokyo, leading to thousands being affected and 12 deaths.¹ Tabun (GA), sarin (GB), soman (GD) and cyclosarin (GF) are known nerve gases, which are chemically active organophosphates and can inactivate acetylcholinesterase (AChE), a critical central-nervous enzyme^2–5 via permanent modification of the catalytically essential serine residue in the enzyme active site,⁶ when inhaled⁷ or absorbed through the skin. This triggers rapid and fatal consequences such as paralysis of the central nervous system⁸ and eventually death. The effectiveness of nerve agents stems from their amazing toxicity; a lethal dose can be as little as 0.70 mg for a normal 70 kg person. Thus, due to the moderately straightforward access to terrorists in present times, nerve gases are considered one of the most important and lethal classes of chemical warfare agents⁹ available and are a serious threat to countrywide and universal safety.

Due to the tremendously hazardous nature of nerve agents, diethyl chlorophosphate (DCP) (Scheme 1) has been broadly used in laboratories as a safe simulant since it displays a parallel reactivity to nerve agents, such as sarin, soman, and tabun, yet it lacks their toxicity. Systems that have been utilized to detect nerve agents include enzyme-based biosensors,^10,11 interferometry,¹² surface acoustic waves,^13,14 electrochemistry,¹⁵ and mass spectrometry.^16,17

Scheme 1 Some nerve agents and their simulants.

There is some restrictions on applying a predictability approach such as lack of portability or storage/stability issues that limit their success in some circumstances. The challenge to the scientific community is to discover methods and devices that are compact, transportable and capable of real time detection. Sensors based on the chemical species, i.e. chemosensors, are beneficial from this point of view as they involve widely used instruments and allow the opportunity to sense nerve agents with the naked eye. Chemosensors that exhibit notable fluorescence emission properties are attractive due to the highly sensitive, quick, simple and real-time monitoring of the fluorescence or change of color in the presence of nerve agents, which have been extensively investigated.¹⁸

Several chromogenic/fluorogenic and carefully planned probes,^18d,e,g,j,m in particular, fluorophores, were covalently linked with fluorescence quenchers. After reaction of nerve agents with those quenchers, the recovered fluorescence is strong. Some probes react with nerve agent mimics to directly affect either detectable species^18l,n or to form intermediates, which could further undergo intramolecular transformations in situ to generate detectable species.^18c,l,m,n

Historically, nonfluorescent and colorless rhodamine derivatives with spirolactam moiety have been extensively utilized to detect metal ions via a reversible ring-opening of the spirolactam, which gives rise to a highly fluorescent and colored rhodamine fluorophore.¹⁹

Herein, we synthesized a rhodamine B derivative as the fluorogenic and chromogenic probe for the detection of diethyl chlorophosphate (DCP), a nerve agent simulant (Scheme 1) in the liquid and vapour phase. The probe for the rhodamine B derivative is N-(rhodamine B)-lactam-di-amino maleonitrile (RHM), which is easily prepared from rhodamine B (Scheme 2). Rhodamine B is treated with an excess amount of POCl₃ at high temperature to produce rhodamine B acid chloride, which is then coupled with diamino maleonitrile in dry DCM to give the desired product (details of the procedure and spectra are given in the ESI†).

Scheme 2 Synthetic route of the receptor (RHM).

In order to avoid the interference from inorganic acids, such as HCl or HBr, we decided to use a slightly basic system to study the binding of the nerve gas mimics. Note that sarin and soman generate HF upon hydrolysis;²⁰ therefore use of an organic base in the solvent system for the sensing process is also necessary to show that the sensors interact with the nerve gas and not the nerve gas degradation products. We have used 3% Et₃N in DCM as the solvent for this process. The compounds were also tested with DMMP (dimethoxy methyl phosphate) to study the importance of the leaving group (Cl) of DCP in the binding with sensors.

The photophysical properties of the receptor RHM were investigated by monitoring the absorption and fluorescence behavior on the addition of nerve agent simulant DCP and its nonreactive analogue DMMP; some common metal ions such as Co²⁺, Cu²⁺, Hg²⁺; some reactive oxygen species (ROS), i.e. NaOCl, H₂O₂, ^tBuOOH; two common acid chlorides, i.e. benzoyl chloride, acetyl chloride; and some common pesticides, i.e. BHC, chlorothalonil and phorate in DCM (3% Et₃N v/v).‡

When we evaluated the changes in the absorbance of the receptor after treatment with DCP, the intensity of the absorption bands of the receptor at 558 nm increased rapidly (Fig. 1), indicative of a clean conversion of the receptor RHM into the RHM–DCP adduct. Thus, because of the strong binding of the oxygen and the nitrogen group present in RHM to DCP, the spirolactam ring opens to reproduce the pink color of rhodamine itself, which is absent in the addition of other species (Fig. 1). As the absorption band at 558 nm appears with a high absorption value, naked-eye detection of the nerve agent is possible (Fig. 1). Thus, without using any instrument we can sense DCP using a solution of the receptor.

Fig. 1 UV-vis absorption titration spectra of RHM (c = 1 × 10⁻⁵ M) in the presence of 2.0 equiv. DCP (c = 2 × 10⁻⁴ M) in DCM (with 3% Et₃N) with the naked-eye color change (left) and absorption spectra of RHM after the addition of 2.0 equiv. each of the guest species (right).

In the emission spectroscopy, the receptor exhibits an emission band arising at 582 nm, which shows a remarkable enhancement during the gradual addition of DCP (Fig. 2). Moreover, the binding isotherm maintains a good linearity with the addition of 1–8 μM of DCP solution towards RHM (Fig. 3). The detection limit of the nerve agent was found to be 0.2 μM based on K × Sb1/S, where Sb1 is the standard deviation of blank measurements and S is the slope of the calibration curve (ESI†), suggesting that RHM is operable well below the reported lethal dose.²¹

Fig. 2 Fluorescence titration spectra of RHM (c = 1 × 10⁻⁵ M) in the presence of 2.0 equiv. DCP (c = 2 × 10⁻⁴ M) in DCM (with 3% Et₃N) with the naked-eye color change under UV-light (inset).

Fig. 3 Binding isotherm of DCP with RHM at 582 nm (with the addition of 1 μM to 8 μM) from the titration spectra. The complete binding isotherm is shown in the inset.

The observed changes in the fluorescence emission are shown in a bar graph (Fig. 4). It seems that the fluorescence enhancement was derived from the more widespread formation of the RHM–DCP adduct with the rhodamine-based receptor, RHM. During the complex formation, the spirolactam ring of the receptor is opened up and displays the fluorescence emission of rhodamine itself upon the gradual addition of DCP (0–2.0 equiv.).

Fig. 4 Emission intensity of RHM after the addition of 2.0 equiv. each of the guest species as a bar diagram (right).

To determine the probable pathway of the sensing process (Scheme 3), the reaction solution was analyzed by mass spectrometry (Fig. 5). A major peak situated at 623.2573 was recognized, which is consistent with the theoretical molecular weight of the final compound 1 (MW: 623.2536), with the less intense peak at 668.2873 (2 + H⁺), confirming formation of the intermediate compound 2 in the examined system.

Scheme 3 Probable reaction mode of RHM towards DCP.

Fig. 5 Mass spectra of a mixture of RHM with DCP.

DFT calculations using DFT/B3LYP/6-31G* as a basis set level also support the sensing phenomenon with the formation of the RHM–DCP adduct, i.e. 1 from RHM. The highest occupied molecular orbital (HOMO) of the product, i.e. 1, is much more stable than RHM by about 0.10284 a.u. The HOMO–LUMO energy gap also decreases from 0.12392 a.u. to 0.10338 a.u. with the formation of the RHM–DCP adduct, i.e. 1, from RHM (Fig. 6).

Fig. 6 HOMO–LUMO energy levels and interfacial plots of the orbitals for RHM (a) and its DCP adduct (i.e. 1) (b) with the energy minimised structure.

Interestingly, the sensing phenomena is very rapid and completed in about 8 minutes, i.e. the receptor RHM rapidly reacts with DCP to give the opened-up form of rhodamine, which gives the characteristic color and fluorescence (Fig. 7).

Fig. 7 Time dependent fluorescence intensity spectra of RHM with the addition of 2 equiv. DCP at a time.

It is noteworthy to mention that for the rest of the species, except DCP, neither a pink coloration nor large emission intensity is observed, demonstrating that the sensor is inert towards the investigated metal ions, ROS and the other organo phosphorus compound, DMMP. Interestingly, the inertness of the receptor RHM towards DMMP confirms that ‘Cl’, being a good leaving group in the reaction condition, is the main reason for the higher reactivity of the DCP.

Provoked by its high sensitivity towards DCP, the practical application of RHM was also examined. Test strips were prepared to detect DCP both in its liquid and in its vapour phase. These test strips confirmed the clear color changes under sunlight (Fig. 8a and b) and also under the UV lamp (Fig. 8b). Thus, the capability of naked-eye detection of the nerve gas simulant in both liquid and vapour phases using coated sticks makes RHM unique and a superior proposition over previous studies.

Fig. 8 (a) Change in the TLC plate (coated with RHM) in the presence of DCP vapour and (b) change in the TLC plate (coated with RHM) in the presence of DCP solution in sunlight (up) and under UV-light (below).

In conclusion, our designed receptor RHM can detect DCP selectively with a high level of sensitivity with a unique mechanism, and the mechanism was corroborated by mass spectrometry. Moreover, the reaction-based sensing phenomenon was also supported by DFT calculations. Furthermore, test strips were also prepared for facile detection of both the vapour and liquid phase of DCP with the naked eye.

Acknowledgements

We thank DST and CSIR (Govt. of India) for their financial support. A.M. acknowledges CSIR and S.P. acknowledges UGC for providing fellowships.

Notes and references

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Footnotes

† Electronic supplementary information (ESI) available: Synthetic procedure, time dependent intensity and spectral data are available. See DOI: 10.1039/c4ra01060d

‡ DCP and other pesticides are handled very carefully.

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