Detection Papers with Chromogenic Chemosensors for Direct Visual Detection and Distinction of Liquid Chemical Warfare Agents

Home , Blister agent, Diphosgene, Tabun (nerve agent)

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Article Detection Papers with Chromogenic Chemosensors for Direct Visual Detection and Distinction of Liquid Chemical Warfare Agents

Vladimír Pitschmann 1,2, Lukáš Matˇejovský 3,* , Kamila Lunerová 4, Michal Dymák 4, Martin Urban 4 and Lukáš Králík 4

1 Oritest spol. s r.o., Nábˇrežní 90/4, 150 00 Prague, Czech Republic 2 Faculty of Biomedical Engineering, Czech Technical University in Prague, nám. Sítná 3105, 272 01 Kladno, Czech Republic 3 Faculty of Environmental Technology, University of Chemistry and Technology, Technická 5, 166 58 Prague, Czech Republic 4 National Institute for Nuclear, Chemical and Biological Protection, Kamenná 71, 262 31 Milín, Czech Republic * Correspondence: [email protected]

Received: 4 June 2019; Accepted: 9 July 2019; Published: 12 July 2019

Abstract: This work provides a summary of our results in the area of the experimental development of detection paper for the detection of liquid phase chemical warfare agents (drops, aerosol), the presence of which is demonstrated by the development of characteristic coloring visible to the naked eye. The basis of the detection paper is a cellulose carrier saturated with the dithienobenzotropone monomer (RM1a)–chromogenic chemosensor sensitive to nerve agents of the G type, blister agent lewisite, or choking agent diphosgene. We achieve a higher coloring brilliance and the limit certain interferences by using this chemosensor in the mix of the o-phenylendiamine-pyronine (PY-OPD). We prove that the addition of the Bromocresol Green pH indicator even enables detection of nerve agents of the V type, or, nitrogen mustards, while keeping a high stability of the detection paper and its functions for other chemical warfare agents. We resolve the resistance against the undesirable inﬂuence of water by providing a hydrophobic treatment of the carrier surface.

Keywords: chemosensors; colorimetric detection; drops and aerosol detection; chemical warfare agents

1. Introduction Even though the Chemical Weapons Convention came into operation in 1997, and currently 193 countries participate (as of May 2018), the risk of chemical weapons proliferation and the threat of using them is still present: They can be used in local wars, civil wars, or in terrorist attacks [1,2]. The active components of the chemical weapons are the chemical warfare agents (CWA), the combat employment of which depends to a significant extent on their state of matter. The most dangerous CWAs that have lethal effects are included within nerve agents, blister agents, and partially within choking agents. Most of them are in the liquid state, which enables their dispersion in the form of vapors, or variously dispersed liquid aerosols, with the potential of easy penetration not only through airways, but through the skin as well, resulting in serious percutaneous poisoning. Some of these CWAs are highly persistent, therefore they can contaminate the terrain for a long time, depending on actual climatic conditions and on the terrain characteristics [3]. The chemical structure of the most significant liquid CWA is shown in Figure1. Some of their physical and toxic properties are provided in Table1.

Chemosensors 2019, 7, 30; doi:10.3390/chemosensors7030030 www.mdpi.com/journal/chemosensors Chemosensors 2019, 7, x FOR PEER REVIEW 2 of 12

Chemosensorssignificant 2019liquid, 7, 30CWA is shown in Figure 1. Some of their physical and toxic properties are provided2 of 12 in Table 1.

O O O N O P P P P O O F F O F O CN

GB GD GF GA

O O P P N N O S O S

VX RVX

Cl Cl Cl Cl Cl Cl N N N

HN-1 HN-2 HN-3 Cl

O Cl Cl Cl Cl Cl S As Cl Cl O Cl Cl HD L(L-1) DP

Figure 1. ChemicalChemical structure structure of chemic chemicalal warfare agents (CWA). TableFor the 1. detectionThe summary of settling of tested liquid CWAs, particles their physical of the and CWA toxic propertiesaerosol cloud, and reactions or droplets with detectionor spray of a papers ( no response, + distinct response, ++ strong response). liquid CWA− on the surface of the terrain, objects, machines, and persons, simple technical means are usually used, based on visual evaluation (by naked eye) of color changes. The color change results LD50, mg, Colour Response Boiling Volatility, fromCWA the solubility of organic dyes, color changeLiquid, of pH indicator, or from the reaction with specific mg/m3 [25 C] RM1a RM1a/PY-OPD RM1a/PY-OPD/BCG chromogenicPoint agents. [◦C] The organic◦ dyes,Percutaneous pH indicators, and chromogenic agents are applied to the filtrationGA paper, 248 fabric, or another 497 flat porous 1500 carrier, modified+ to the form ++ of detection strips, + papers GB 150 18,700 1700 ++ ++ + and/or tapes. GD 198 3930 350 + ++ ++ GFIn the past, 228 the main focus 898 was given to 350 the detection+ of sulfur mustard, ++ a typical poison ++ with percutaneousVX 292effect (that is why 12.6 it is called a 5“blister agent”). As an example, there was the++ detection − − paperRVX saturated 300 with 1-( 10.5p-nitrophenylazo)-2-naphtylamine, (20 ◦C) 0.7–7 providing a red color with++ the sulfur − − HD 218 906 1400 mustard. Later, this dye was replaced with a less toxic compound− and used − not only for the − detection HN-1 192 2230 1400 ++ of sulfur mustard, but also for the detection of nerve agents.− Another example − is the detection paper HN-2 177 3490 1400 ++ 2+ −2+ − saturatedHN-3 with 257 the complex of 120 Crystal Violet 1400 with Hg and Cu salts, providing a dark violet color − − − withL the droplets 196 of the sulfur 3860 mustard, due 1400 to the release++ of the original ++ dye [4], or the ++ detection paperDP with 4,4 127′-bis(N,N-dimethylamino)benzophenone 47,700 (20 ◦C) Unknown and+ mercuric ++chloride, providing ++ a red color with the sulfur mustard [5].

Chemosensors 2019, 7, 30 3 of 12

For the detection of settling liquid particles of the CWA aerosol cloud, or droplets or spray of a liquid CWA on the surface of the terrain, objects, machines, and persons, simple technical means are usually used, based on visual evaluation (by naked eye) of color changes. The color change results from the solubility of organic dyes, color change of pH indicator, or from the reaction with specific chromogenic agents. The organic dyes, pH indicators, and chromogenic agents are applied to the filtration paper, fabric, or another flat porous carrier, modified to the form of detection strips, papers and/or tapes. In the past, the main focus was given to the detection of sulfur mustard, a typical poison with percutaneous effect (that is why it is called a “blister agent”). As an example, there was the detection paper saturated with 1-(p-nitrophenylazo)-2-naphtylamine, providing a red color with the sulfur mustard. Later, this dye was replaced with a less toxic compound and used not only for the detection of sulfur mustard, but also for the detection of nerve agents. Another example is the detection paper saturated with the complex of Crystal Violet with Hg2+ and Cu2+ salts, providing a dark violet color with the droplets of the sulfur mustard, due to the release of the original dye [4], or the detection paper with 4,40-bis(N,N-dimethylamino)benzophenone and mercuric chloride, providing a red color with the sulfur mustard [5]. The introduction of nerve agents to military equipment, and the real threat of using them, also calls for the introduction of a simple means for their detection in the liquid phase. An example is the detection paper impregnated with the Bromocresol Green pH indicator, providing a blue color in contact with the VX agent. At present, multicomponent detection papers are used, intended not only for the detection of nerve agents, but also for the detection of sulfur mustard, with the option of their distinction. They are usually self-sticking paper sheets impregnated with a mixture of organic dyes, which dissolve in liquid CWA and provide characteristic colors. The Dye Red E (CAS: 60033-00-3) is used for the detection of sulfur mustard, the Dye Green EDA (CAS: 5833-18-1) is used, as an example, for the detection of nerve agents of the V type, and the Disperse Yellow 23 (CAS: 6250-23-3) dye is used for the detection of G type nerve agents, providing red, green, and yellow colours [4,6]. The low selectivity of the known methods of the detection of liquid CWAs, based on the solubility of organic dyes or pH indicators, is a significant disadvantage. Chemical reactions are more selective, but the selection of chromogenic agents, suitable for practical applications, was very limited in the past. However, during the last two decades, a lot of original works were published, referring particularly to chromogenic chemosensors for the G type nerve agents, featuring a significant change of color as well as good selectivity and sensitivity; these chemosensors are based on hydroxyl-activation, N-activation, and contain metal ions (coordination compounds), or possibly form reactive polymers [7–19]. Many publications deal with new chemosensors for sulphur mustard [20–24]. The issue is that most of these chemosensors have only been verified using simulants (e.g., diethyl chlorophosphate (DCP), 2-chloroethyl ethyl sulfide (CEES), etc.), moreover, they have not been applied on the detection papers for the detection in the liquid phase. The goal of this text is to report the results of the experimental development of detection papers for the detection of CWAs in liquid phase, using the already known chemosensor based on the dithienobenzotropone [9], in combination with the chemosensor based on the o-phenylenediamine-pyronin [11], and the Bromocresol Green pH indicator. The article provides the method of detection papers preparation, their response to real CWAs, chemical interferences and stability during storage and operational ingestion. Basically, they are simple, robust, sufficiently selective, and reliable detection systems for most of the potential liquid CWA, with the exception of the sulfur mustard. Chemosensors 2019, 7, 30 4 of 12

2. Experimental Section

2.1. Chemicals and Equipment For the preparation of the detection papers, we used dithienobenzotropone monomer (RM1a), o-phenylendiamine-ronine (PY-OPD), both synthesized in VŠCHT Prague, Czech Republic (as per References [9,11]), Bromocresol Green (BCG), colophon resin and chloroform (all of them made by Sigma-Aldrich, St. Louis, MO, USA). We used cellulose ﬁltration papers as carriers, with the nominal basis weight of 85 g/m2 (Whatman, Kent, UK) and 120–125 g/m2 (Filtrak, Bärenstein, Germany). As for the CWA test we used tabun (GA), sarin (GB), soman (GD), cyclosarin (GF), VX, RVX (“Russian VX”), sulfur mustard (HD), nitrogen mustard (HN-1, HN-2, HN-3), lewisite (L-1, L-2, L-3), and diphosgene (DP), all of them provided by the Military Research Institute, Brno, Czech Republic. For the research tasks, we used DCP and CEES simulants, both made by Sigma-Aldrich. All CWAs were prepared and tested in facilities declared within OPCW (Organization for the Prohibition of Chemical Weapons). Certiﬁed protection equipment was used during all experiments, and all waste was treated using chloramine solution in 10% NaOH in the water-ethanol mixture (1:1) prior to disposal. For the objective measurement of selected parameters (detection paper ageing), we used a portable tristimulus colorimeter LMG 173 (Dr. Lange, Dusseldorf, Germany), which utilizes the color CIE-Lab system. Spectrophotometric measurement (absorption spectra) were performed using a spectrophotometer Aquamate (Thermo Spectronic, Cambridge, UK).

2.2. Preparation of the Detection Paper We used three basic variants of the detection paper (RM1a, RM1a/PY-OPD, RM1a/PY-OPD/BCG). Sheets of ﬁltration paper with a size of 100 50 mm were immersed in impregnation solution for 30 s, × consisting of the following:

(a) 30 mg RM1a in 100 mL of chloroform (RM1a), (b) 30 mg RM1a and 15 mg PY-OPD in 100 mL of chloroform (RM1a/PY-OPD), (c) 30 mg RM1a, 15 mg PY-OPD, 15 mg BCG in 100 mL of chloroform (RM1a/PY-OPD/BCG); the improved variant was enhanced with the addition of colophon resin (950 mg).

The impregnated ﬁltration papers were dried in open air for 60 min and stored in a hermetically closed package and kept away from direct sunlight.

2.3. Testing the Detection Paper Functions Droplets of test substances (5 µL) were applied using a micropipette on the detection paper and were evaluated visually (by naked eye) for color change at the point of exposure. Each test was repeated 3 using 2 lots of real CWA (the same manufacturer, diﬀerent production dates). All results × were documented using a photographic camera within 1 min after exposure.

2.4. Testing of Stability For the purpose of the stability testing, the detection papers were stored in the laboratory in the open air, at a temperature of 21–23 ◦C, for a period of 6 months. Papers were visually monitored on a regular basis once a week for surface color change without any exposure to the CWA droplets and for their function—color change after the exposure to the CWA droplets. Thermal stability was proved with part of the samples. The samples were stored in a drying cabinet at a temperature of 80 ◦C for three weeks. On a regular basis, the unexposed surface color change was monitored visually using the tristimulus colorimeter (CIE-Lab color system) following the ∆E parameter as the analytical signal [25]. The ∆E values up to 0.2 cannot be perceived by the naked eye, the values of 0.2–0.5 are very slight, 0.5–1.5 are slight, 1.5–3.0 are distinct, 3.3–6.0 are very distinct, 6.0–12.0 are strong, and above 12.0, the Chemosensors 2019, 7, 30 5 of 12 values are very strong. In addition, the function of the detection paper (visual control of the color change after CWA exposure) was tested regularly.

3. Results and Discussion

3.1. Design of the RM1a Detection Paper or RM1a/PY-OPD The first (basic) design of the novel detection paper was based on the intention of developing a detection paper with high selectivity for G type nerve agents, for which detection using existing technical means is an issue. E.g., the 3-way detection papers (M8, PDF-1, PP-3, CALID-3, and similar) provide a relatively faint yellow color with the G type nerve agents droplets (on a grey background), which is disturbed with the presence of common organic solvents and industrial chemicals; the M9 detection paper provides an even lower selectivity as we verified the selectivity of these means experimentally. By studying the latest sources, we selected, synthesized, and verified many chemosensors, potentially sensitive to alkyl/cycloalkyl-methylphosphonofluoridates. The RM1a appeared to be the most interesting, with its colorless chloroform solution, providing red color with the DCP (according to literary data), with a bathochromic shift from 330 to 580 nm [9]. The agent was not verified against real CWA, neither on a solid carrier, nor in the form of the detection paper for the detection in the liquid phase. During the experiments, we found the RM1a was not only a good chemosensor of current G type nerve agents, but also that it is very stable and functional on the cellulose carrier. Moreover, the structurally different GA (reaction on the P-CN bond), blister agent L, and choking agent DP were giving a uniquely positive response. The reaction mechanism for the alkyl/cycloalkyl-methylphosphonofluoridates is providedChemosensors in 2019 Figure, 7, x 2FOR. PEER REVIEW 5 of 12

FigureFigure 2. The 2. The dithienobenzotropone dithienobenzotropone monomer monomer (RM1a) (RM1a) reaction reaction scheme scheme with with the Gthe type G type nerve nerve agents (modiﬁed according to Referenceagents [9 (modified]). according to Reference [9]).

The extendedextended designdesign was was based based on on two two presumptions: presumptions: The The reaction reaction product product color color intensity intensity can can be increased,be increased, or, theor, colorthe color contrast contrast can becan improved be improved and theand detection the detection selectivity selectivity can be can increased. be increased. With thisWith goal, this wegoal, combined we combined the RM1a the RM1a with somewith some chemosensors chemosensors for alkylating for alkylating or acylating or acylating agents. agents. The combinationThe combination with thewith PY-OPD the PY-OPD provided provided good results. good Thisresults. chemosensor This chemosensor had been had designed been beforedesigned for thebefore detection for the of detection DCP and of phosgene, DCP and providing phosgene, yellow providing and violet yellow color and with violet them color in the with gaseous them phase,in the respectivelygaseous phase, [11]. respectively The reaction mechanism[11]. The reaction with DP mechanism comprising intramolecularwith DP comprising cyclisation intramolecular is provided incyclisation Figure3. is Examples provided ofin absorptionFigure 3. Examples spectra ofof RM1a,absorption PY-OPD spectra and of RM1a RM1a,/ PY-OPDPY-OPD (inand chloroform RM1a/PY- solution)OPD (in chloroform after addition solution) of GD are after shown addition in Figure of GD4. Theare additionshown in of Figure PY-OPD 4. featuredThe addition two advantages.of PY-OPD Atfeatured ﬁrst, the two detection advantages. paper At exposed first, the to detection droplets ofpape ther Gexposed type nerve to droplets agents of provided the G type the colornervechange agents ofprovided yellow–red, the color which change is more of suitableyellow–red, for thewhich visual is more evaluation suitable by for the the naked visual eye, evaluation compared by to thethe white–rednaked eye, color compared change to of the the white RM1a–red itself. color Secondly, changea of di theﬀerent RM1a color itself. was Secondly, created upon a different contact color with thewas liquid created DP, upon compared contact towith the the G typeliquid nerve DP, agents,compared thus to improvingthe G type thenerve detection agents, selectivity.thus improving The otherthe detection parameters selectivity. of the The detection other parameters paper have of been the detection maintained. paper The have color been response maintained. of RM1a The color and RM1aresponse/PY-OPD of RM1a detection and RM1a/PY-OPD papers to real detection CWAs are papers documented to real CWAs in Figure are5. documented in Figure 5.

Figure 3. The o-phenylendiamine-pyronine (PY-OPD) reaction scheme with diphosgene (DP) (modified according to Reference [11]).

0.8 0.8 0.7 0.7 0.6 0.6 GD Blank 0.5 0.5 RM1a RM1a 0.4 0.4 RM1a/PY-OPD RM1a/PY-OPD PY-OPD 0.3 PY-OPD 0.3 Absorbance 0.2 Absorbance 0.2 0.1 0.1 0 0 400 450 500 550 600 400 450 500 550 600 Wavelength (nm) Wavelength (nm) Figure 4. Absorption spectra of RM1a, PY-OPD and RM1a/PY-OPD (in chloroform solution) for blank and addition of soman (GD).

ChemosensorsChemosensors 2019 2019, ,7 7, ,x x FOR FOR PEER PEER REVIEW REVIEW 55 of of 12 12

FigureFigure 2. 2. The The dithienobenzotropone dithienobenzotropone monomer monomer (RM1a) (RM1a) reaction reaction scheme scheme with with the the G G type type nerve nerve agentsagents (modified (modified according according to to Reference Reference [9]). [9]).

TheThe extended extended design design was was based based on on two two presumptions: presumptions: The The reaction reaction product product color color intensity intensity can can bebe increased,increased, or,or, thethe colorcolor contrastcontrast cancan bebe improvedimproved andand thethe detectiondetection selectivityselectivity cancan bebe increased.increased. WithWith thisthis goal,goal, wewe combinedcombined thethe RM1a RM1a with with somesome chemosensorschemosensors for for alkylatingalkylating or or acylatingacylating agents. agents. TheThe combinationcombination withwith thethe PY-OPDPY-OPD providedprovided goodgood results.results. ThisThis chemosensorchemosensor hadhad beenbeen designeddesigned beforebefore forfor thethe detectiondetection ofof DCPDCP andand phosgene,phosgene, provprovidingiding yellowyellow andand violetviolet colorcolor withwith themthem inin thethe gaseousgaseous phase,phase, respectivelyrespectively [11].[11]. TheThe reactionreaction mechanismmechanism withwith DPDP comprisingcomprising intramolecularintramolecular cyclisationcyclisation is is provided provided in in Figure Figure 3. 3. Examples Examples of of absorption absorption spectra spectra of of RM1a, RM1a, PY-OPD PY-OPD and and RM1a/PY- RM1a/PY- OPDOPD (in(in chloroformchloroform solution)solution) afterafter additionaddition ofof GDGD areare shownshown inin FigureFigure 4.4. TheThe additionaddition ofof PY-OPDPY-OPD featuredfeatured two two advantages. advantages. At At first, first, the the detection detection pape paperr exposed exposed to to droplets droplets of of the the G G type type nerve nerve agents agents providedprovided thethe colorcolor changechange ofof yellowyellow––red,red, whichwhich isis moremore suitablesuitable forfor thethe visualvisual evaluationevaluation byby thethe nakednaked eye,eye, comparedcompared toto thethe whitewhite––redred colorcolor changechange ofof thethe RM1aRM1a itself.itself. Secondly,Secondly, aa differentdifferent colorcolor waswas created created upon upon contact contact with with the the liquid liquid DP, DP, compared compared to to the the G G type type nerve nerve agents, agents, thus thus improving improving theChemosensorsthe detection detection2019 selectivity. selectivity., 7, 30 The The other other parameters parameters of of the the detection detection paper paper have have been been maintained. maintained. The The 6color color of 12 responseresponse of of RM1a RM1a and and RM1a/PY-OPD RM1a/PY-OPD detection detection papers papers to to real real CWAs CWAs are are documented documented in in Figure Figure 5. 5.

FigureFigureFigure 3. The 3. 3. The oThe-phenylendiamine-pyronine o o-phenylendiamine-pyronine-phenylendiamine-pyronine (PY-OPD) (PY-OPD) (PY-OPD) reaction re reactionaction scheme scheme scheme with diphosgenewith with diphosgene diphosgene (DP) (modiﬁed (DP) (DP) according to Reference [11]). (modified(modified according according to to Reference Reference [11]). [11]).

0.80.8 0.80.8 0.70.7 0.70.7 0.6 0.60.6 0.6 GDGD BlankBlank 0.50.5 0.50.5 RM1aRM1a RM1aRM1a 0.40.4 0.40.4 RM1a/PY-OPDRM1a/PY-OPD RM1a/PY-OPDRM1a/PY-OPD PY-OPDPY-OPD 0.30.3 PY-OPDPY-OPD 0.30.3 Absorbance Absorbance Absorbance 0.20.2 Absorbance 0.20.2 0.10.1 0.10.1 00 00 400400 450 450 500 500 550 550 600 600 400400 450 450 500 500 550 550 600 600 WavelengthWavelength (nm) (nm) Wavelength (nm) Wavelength (nm) FigureFigure 4. 4. Absorption AbsorptionAbsorption spectra spectraspectra of ofof RM1a, RM1a, PY-OPD PY-OPD and andand RM1a/ RM1aRM1a//PY-OPDPY-OPDPY-OPD (in (in chloroform chloroform solution) solution) for for blank blank Chemosensorsandand addition addition 2019, 7, xof ofFOR soman soman PEER (GD). (GD). REVIEW 6 of 12

Blank GB GF GD GA

(a) VX HD L (L-1) DP DCP HN-1

Blank GB GF GD GA

(b) VX HD L (L-1) DP DCP HN-1

FigureFigure 5. 5. RM1aRM1a (a ()a and) and RM1a/PY-OPD RM1a/PY-OPD (b (b) )detection detection paper paper after after the the exposure exposure to to a a droplet droplet of of CWA. CWA.

3.2. Design of the RM1a/PY-OPD/BCG Detection Paper Another design covered the option of detecting droplets and aerosol of the V type nerve agents, representing the top of CWAs for their toxicity and persistence. We proceeded based on a simple plan: To the RM1a/PY-OPD detection paper, we incorporated (by adding to an impregnating solution) various chemosensors of amines, thiols, and aminothiols as precursors, or products of these CWA hydrolysis. We verified 5,5′-dithiobis(2-nitrobenzoic acid), 2,2′-dithiobis(5-nitropyridine), 4- chloro-7-nitrobenzofurazan, 4-chloro-5,7-dinitrobenzofurazan, and 4-nitro-2,1,3-benzothiadiazol as chromogenic reagents. In this investigation phase, the most promising results were provided by common color pH indicators, in particular by the triphenylmethane BCG indicator, providing the color change yellow (3.8)—blue (5.4). The BCG color change mechanism scheme is shown in Figure 6. Practically, the BCG incorporation to the RM1a/PY-OPD detection paper did not change its appearance, or dispositions to reactions with G type nerve agents (namely, the semi-persistent GD and GF), or, lewisite or DP, moreover, it provided an expressive blue color with the V type nerve agents. As expected, the HN-1 and HN-2 nitrogen mustards gave a response resulting in a green color (these vesicants are less basic than VX); the HN-3 nitrogen mustard did not react at all. The summary of color responses of RM1a/PY-OPD/BCG detection papers to CWA is shown in Figure 7. A summary overview of detection papers color changes, supplemented with selected physical and toxic properties, is provided in Table 1.

Figure 6. The Bromocresol Green (BCG) color change mechanism scheme.

Chemosensors 2019, 7, x FOR PEER REVIEW 6 of 12

Blank GB GF GD GA

(a) VX HD L (L-1) DP DCP HN-1

Blank GB GF GD GA

(b) VX HD L (L-1) DP DCP HN-1

Chemosensors 2019, 7, 30 7 of 12 Figure 5. RM1a (a) and RM1a/PY-OPD (b) detection paper after the exposure to a droplet of CWA.

3.2.3.2. Design Design of of the the RM1a/PY-OPD/BCG RM1a/PY-OPD/BCG DetectionDetection PaperPaper AnotherAnother design design covered covered the the option option of detecting of detecting droplets droplets and aerosol and aerosol of the ofV type the Vnerve type agents, nerve representingagents, representing the top of the CWAs top of for CWAs their for toxicity their toxicityand persistence. and persistence. We proceeded We proceeded based on based a simple on a plan:simple To plan: the ToRM1a/PY-OPD the RM1a/PY-OPD detection detection paper, paper, we weincorporated incorporated (by (by adding adding to to an an impregnating solution)solution) various various chemosensors chemosensors of of amines, amines, thiols, thiols, and and aminothiols aminothiols as precursors, as precursors, or products or products of these of CWAthese CWAhydrolysis. hydrolysis. We verified We veriﬁed 5,5′-dithiobis(2-nitrobenzoic 5,50-dithiobis(2-nitrobenzoic acid), acid), 2,2′-dithiobis(5-nitropyridine), 2,20-dithiobis(5-nitropyridine), 4- chloro-7-nitrobenzofurazan,4-chloro-7-nitrobenzofurazan, 4-chloro-5,7-dinitrobenzofurazan, 4-chloro-5,7-dinitrobenzofurazan, and and 4-nitro-2,1,3-benzothiadiazol 4-nitro-2,1,3-benzothiadiazol as chromogenicas chromogenic reagents. reagents. In Inthis this investigation investigation phas phase,e, the the most most promising promising results results were were provided provided by by commoncommon color color pH pH indicators, in in particular by by the triphenylmethane BCG BCG indicator, indicator, providing the colorcolor change yellow (3.8)—blue (5.4).(5.4). TheThe BCGBCG colorcolor changechange mechanismmechanism scheme scheme is is shown shown in in Figure Figure6. 6.Practically, Practically, the the BCG BCG incorporation incorporation to the to RM1a the/ PY-OPDRM1a/PY-OPD detection detection paper did pa notper change did not its appearance,change its appearance,or dispositions or dispositions to reactions to with reactions G type with nerve G agentstype nerve (namely, agents the (namely, semi-persistent the semi-persistent GD and GF), GD or, andlewisite GF), oror, DP, lewisite moreover, or DP, it moreover, provided anit provided expressive an blue expressive color with blue the color V typewith nervethe V agents.type nerve As agents.expected, As theexpected, HN-1 the and HN-1 HN-2 and nitrogen HN-2 nitrogen mustards mu gavestards a response gave a response resulting resulting in a green in a colorgreen (these color (thesevesicants vesicants are less are basic less basic than VX);than theVX); HN-3 the HN-3 nitrogen nitrogen mustard mustard did did not not react react at all. at all. The The summary summary of ofcolor color responses responses of of RM1a RM1a/PY-OPD/BCG/PY-OPD/BCG detection detection papers papers to to CWA CWA is is shown shown inin FigureFigure7 7.. AA summarysummary overviewoverview ofof detection detection papers papers color color changes, changes, supplemented supplemented with selected with selected physical physical and toxic and properties, toxic properties,is provided is in provided Table1. in Table 1.

Chemosensors 2019, 7Figure,Figure x FOR PEER6. 6. TheThe REVIEW Bromocresol Bromocresol Green Green (BCG) (BCG) color color change change mechanism mechanism scheme. 7 of 12

GA GB GD GF VX RVX DP DCP

HN-1 HN-2 HN-3 L-1 L-2 L-3 HD CEES

FigureFigure 7. Response of the RM1a/PY-OPD/BCG RM1a/PY-OPD/BCG detection detection paper paper to to CWA. CWA.

3.3. SulphurTable 1. Mustard The summary Tests of tested CWAs, their physical and toxic properties and reactions with detection papers (− no response, + distinct response, ++ strong response). During the development of the detection paper, we also studied the option of using new specific chromogenic/fluorogenic reagents, potentiallyLD sensitive50, mg, to sulfur mustard.Colour Based Response on the literature, we Boiling Volatility, synthesizedCWA and verified the reagents based onLiquid, rhodamine-thioamide [21RM1a/PY-], benzothiazoleRM1a/PY- (RHBT) [22], Point [°C] mg/m3 [25 °C] RM1a and pyronine (DPXT) [24]. We used solutionsPercutaneous of reagents in common organicOPD solventsOPD/BCG (methanol, ethanol,GA chloroform)248 for the impregnation 497 of the 1500 cellulose carrier, + and after ++ drying, we exposed + it to dropletsGB of sulfur150 mustard (HD) 18,700 and simulant (CEES). 1700 However, ++ we did not ++ succeed in obtaining + any positiveGD color response.198 3930 350 + ++ ++ GF 228 898 350 + ++ ++ VX 292 12.6 5 − − ++ RVX 300 10.5 (20 °C) 0.7–7 − − ++ HD 218 906 1400 − − − HN-1 192 2230 1400 − − ++ HN-2 177 3490 1400 − − ++ HN-3 257 120 1400 − − − L 196 3860 1400 ++ ++ ++ DP 127 47,700 (20 °C) Unknown + ++ ++

3.3. Sulphur Mustard Tests During the development of the detection paper, we also studied the option of using new specific chromogenic/fluorogenic reagents, potentially sensitive to sulfur mustard. Based on the literature, we synthesized and verified the reagents based on rhodamine-thioamide [21], benzothiazole (RHBT) [22], and pyronine (DPXT) [24]. We used solutions of reagents in common organic solvents (methanol, ethanol, chloroform) for the impregnation of the cellulose carrier, and after drying, we exposed it to droplets of sulfur mustard (HD) and simulant (CEES). However, we did not succeed in obtaining any positive color response.

3.4. Stability In practice, the detection papers for liquid CWAs are usually stored in closed packages, at a temperature of −40 to +40 °C (at a temperature of 60 °C up to one month maximally), and they should be functional (operationally usable) at a temperature of −40 to +60 °C [26]. The samples of RM1a, RM1a/PY-OPD and RM1a/PY-OPD/BCG detection papers, stored at a common laboratory temperature, did not show any changes in their appearance or loss of functions during the full six months. At the same time, we tested the heat stability with the goal of verifying the detection paper resistance against short-term extreme temperatures, which might occur both during storage and during operational usage. The change of detection papers appearance exposed to a temperature of 80 °C is shown in Figure 8. The charts show that a more significant darkening of the combined systems occurred during the

Chemosensors 2019, 7, 30 8 of 12

3.4. Stability In practice, the detection papers for liquid CWAs are usually stored in closed packages, at a temperature of 40 to +40 C (at a temperature of 60 C up to one month maximally), and they − ◦ ◦ should be functional (operationally usable) at a temperature of 40 to +60 C[26]. The samples − ◦ of RM1a, RM1a/PY-OPD and RM1a/PY-OPD/BCG detection papers, stored at a common laboratory temperature, did not show any changes in their appearance or loss of functions during the full six months. At the same time, we tested the heat stability with the goal of verifying the detection paper resistance against short-term extreme temperatures, which might occur both during storage and during operational usage. Chemosensors 2019, 7, x FOR PEER REVIEW 8 of 12 ChemosensorsThe change 2019, of7, x detection FOR PEER papersREVIEW appearance exposed to a temperature of 80 ◦C is shown in Figure8 of8 .12 The charts show that a more signiﬁcant darkening of the combined systems occurred during the ﬁrst first 24 h, then their appearance was stabilized. In the case of the RM1a detection paper, a slow 24first h, then24 h, their then appearance their appearance was stabilized. was stabilized. In the case In ofthe the case RM1a of the detection RM1a paper,detection a slow paper, darkening a slow darkening occurred from the very beginning. Yet, the detection papers maintained their functions, as occurreddarkening from occurred the very from beginning. the very Yet,beginning. the detection Yet, th paperse detection maintained papers maintained their functions, their as functions, noticeable as noticeable on the RM1a/PY-OPD/BCG complex system example in Figure 9. onnoticeable the RM1a on/PY-OPD the RM1a/PY-OPD/BCG/BCG complex system complex example system in Figure example9. in Figure 9.

E 25 Δ E Δ 20 20 RM1a RM1a 15 RM1a/PY-OPD 15 RM1a/PY-OPD RM1a/PY-OPD/BCG RM1a/PY-OPD/BCG 10 10

5 5

0 0 0 100 200 300 400 500 0 100 200 300 400 500 Time (h) Time (h)

Figure 8. Color stability of detection papers during storage at a temperature of 80 °C (tristimulus FigureFigure 8. 8.Color Color stability stability of of detection detection papers papers during during storage storage at at a a temperature temperature of of 80 80◦ C°C (tristimulus (tristimulus colorimetric measurement of the surface color change). colorimetriccolorimetric measurement measurement of of the the surface surface color color change). change). GD VX HN-1 L-1 DP GD VX HN-1 L-1 DP

Figure 9. Functional stability of the RM1a/PY-OPD/BCG RM1a/PY-OPD/BCG detectiondetection paper after 20 days of storage at a Figure 9. Functional stability of the RM1a/PY-OPD/BCG detection paper after 20 days of storage at a temperature of 80 °C.C. temperature of 80◦ °C. 3.5. Interferences 3.5. Interferences We also paid attention to the study of interfer interferencesences of of chemical chemical substances, substances, which which are are not not CWAs, CWAs, We also paid attention to the study of interferences of chemical substances, which are not CWAs, but might be present as stabilizers,stabilizers, tactical ingredients, or might be present forfor otherother reasons.reasons. We but might be present as stabilizers, tactical ingredients, or might be present for other reasons. We veriﬁedverified the representatives of all types of organic solvents (polar, non-polar, aprotic) for causing verified the representatives of all types of organic solvents (polar, non-polar, aprotic) for causing interferences, as well as other chemicals. The The RM1a RM1a detection detection papers, papers, or, or, the the RM1a/PY-OPD RM1a/PY-OPD detection interferences, as well as other chemicals. The RM1a detection papers, or, the RM1a/PY-OPD detection papers proved themselves as being highly resistantresistant against the interferences, only the acetic acid papers proved themselves as being highly resistant against the interferences, only the acetic acid provided aa colorcolor response;response; the the circles circles at at RM1a RM1a/PY-OPD/PY-OPD were were created created due due to “reagentto “reagent elution” elution” (Figure (Figure 10). provided a color response; the circles at RM1a/PY-OPD were created due to “reagent elution” (Figure The10). RM1aThe RM1a/PY-OPD/BCG/PY-OPD/BCG detection detection papers papers were lesswere resistant less resistant against against the interference the interference with these with organic these 10). The RM1a/PY-OPD/BCG detection papers were less resistant against the interference with these organic compounds, but still much more resistant than known M8, M9, CALID-3 detectors (we organic compounds, but still much more resistant than known M8, M9, CALID-3 detectors (we verified the selectivity of these detectors experimentally). verified the selectivity of these detectors experimentally).

Chemosensors 2019, 7, 30 9 of 12 compounds, but still much more resistant than known M8, M9, CALID-3 detectors (we veriﬁed the Chemosensorsselectivity 2019 of these, 7, x FOR detectors PEER REVIEW experimentally). 9 of 12

Methanol Ethanol Ether Acetone DMSO Acetic acid

DMF Chloroform Acetonitrile CS2 Pyridine Isooctane

(a)

Toluene Water DCMP DETA

Methanol Ethanol Ether Acetone DMSO Acetic acid

DMF Chloroform Acetonitrile CS2 Pyridine Isooctane

(b)

Toluene Water DCMP DETA

FigureFigure 10. ColorColor response response (a )( RM1a,a) RM1a, (b) RM1a(b) RM1a/PY-OPD/PY-OPD to selected to selected chemicals chemicals (DMSO—dimethyl (DMSO—dimethylsulfoxide, sulfoxide,DMF—dimethylformamide, DMF—dimethylformamide, DCMP diethyl DCMP cyanomethylphosphonate, diethyl cyanomethylphosphonate, DETA—diethylenetriamine). DETA— diethylenetriamine). The influence of water is shown to be an issue. In liquid CWAs, there is no water present, so its interferenceThe influence can practically of water beis shown neglected to be from an issue. this point In liquid of view. CWAs, However, there is water no water can bepresent, present so as its a interferencemedium of othercan practically toxic substances be neglected (suchas from pesticides), this point which of view. may However, be sprayed, wa orter it can can be be present present as as a a mediumrain or fog. of other We proved toxic substances the RM1a (such and RM1a as pesticides/PY-OPD), which detection may papers be sprayed, were resistant or it can againstbe present water as adrops. rain or Unlike fog. We the proved reaction the of RM1a the RM1a and/ PY-OPDRM1a/PY-OPD/BCG, detection detection paper’s papers reaction were resistant to water against created water only drops.smaller, Unlike but relatively the reaction intensive of the green RM1a/PY-OPD/BC dots. For this reason,G, detection we modified paper’s the reac surfacetion to of water the detection created onlypaper smaller, with hydrophobic but relatively colophon intensive resin, green which dots prevents. For this the reason, contact we of chemosensorsmodified the surface (preferentially of the detectionwith BCG) paper with water,with hydrophobic but it lets the liquidcolophon CWA resin, get throughwhich prevents and does the not blockcontact their of colorchemosensors response. (preferentiallyThe disadvantage with of BCG) the colophon with water, resin but is it its lets content the liquid of an CWA acid ingredient get through (abietic and does acid), not which block a fftheirects colorthe color response. hues ofThe the disadvantage stains to a certain of the extent colophon (this wasresin apparent is its content at V agents,of an acid showing ingredient a blue–green (abietic acid),shift). which The appearance affects the ofcolor color hues stains of the before stains and to after a certain the modification extent (this ofwas the apparent RM1a/PY-OPD at V agents,/BCG showingdetection a paper blue– usinggreen theshift). colophon The appearance is documented of color in Figurestains before11. and after the modification of the RM1a/PY-OPD/BCG detection paper using the colophon is documented in Figure 11.

Chemosensors 2019, 7, 30 10 of 12 Chemosensors 2019, 7, x FOR PEER REVIEW 10 of 12

Methanol Ethanol Ether Acetone DMSO Acetic acid DMF

Chloroform Acetonitrile CS2 Pyridine Isooctane Toluene Water

(a)

DCMP DETA DCP CEES DP

Methanol Ethanol Ether Acetone DMSO Acetic acid DMF

Chloroform Acetonitrile CS2 Pyridine Isooctane Toluene Water

(b)

DCMP DETA DCP CEES DP

FigureFigure 11. 11. ColorColor response response a) RM1a/PY-OPD/BCG, (a) RM1a/PY-OPD b/BCG,) RM1a/PY-OPD/BCG/colophon (b) RM1a/PY-OPD/BCG/ colophonto selected to chemicalsselected chemicals(DMSO—dimethyl (DMSO—dimethyl sulfoxide, sulfoxide, DMF—dimethylformamide, DMF—dimethylformamide, DCMP—diethyl DCMP—diethyl cyanomethylphosphonate,cyanomethylphosphonate, DE DETA—diethylenetriamine).TA—diethylenetriamine).

3.6.3.6. Effects Effects of of Dilution Dilution OneOne of of the the basic basic characteristics characteristics of ofCWA CWA detection detection pa paperspers is that is that they they must must allow allow the thedetection detection of substancesof substances in their in their concentrated concentrated form, form, or in ormixture in mixture with withstabilizers stabilizers or thickeners. or thickeners. By this, By the this, issue the ofissue the ofdetection the detection limit limitgets significantly gets significantly simplified. simplified. You Youmight might even even say say that that the the relatively relatively lower lower sensitivitysensitivity is is rather rather an an advantage advantage for for the the detection detection of droplets, of droplets, since since many many potential potential interferences interferences are eliminatedare eliminated in this in thisway. way. Despite Despite this, this, we weverified verified the theinfluence influence of ofthe the dilution dilution of ofreal real CWA CWA (in (in chloroform)chloroform) to to the the intensity intensity of of generated generated color, color, or, or, to to the the detection detection paper paper function. function. In In Figure Figure 12, 12 ,it it is is provedproved that that the the RM1a/PY-OPD/BCG/colophon RM1a/PY-OPD/BCG/colophon detection detection paper paper (as (as the the most most complex complex of of all all designed designed detectors)detectors) provided provided a a color response eveneven afterafter thethe 100-fold 100-fold dilution dilution of of GD GD or or 1000-fold 1000-fold dilution dilution of VX.of VX.In conclusion,In conclusion, the detectionthe detection limits limits of CWAs of CWAs (nerve (ner agents,ve agents, lewisite, lewisite, DP) range DP) range from 0.001from to0.001 0.1 mgto 0.1/µL, mg/µL,which satisfieswhich satisfies the requirements the requirements for their for detection their detection in the in liquid the liquid phase phase (for example, (for example, primary primary liquid liquidaerosol aerosol cloud) cloud) in field in conditions.field conditions.

Chemosensors 2019, 7, 30 11 of 12 Chemosensors 2019, 7, x FOR PEER REVIEW 11 of 12

Con. 1:5 1:10 1:100× 1:1000× 1:10,000

(a)

(b)

FigureFigure 12. 12. InfluenceInﬂuence of of CWA CWA dilution dilution to to the the RM1a/PY RM1a/PY-OPD-OPD/BCG/colophon/BCG/colophon detection detection paper paper colour colour intensity,intensity, ( (a)) GD, GD, ( (b)) VX. VX.

4.4. Conclusions Conclusions TheThe results results of of the the experimental experimental development development of of detection detection paper paper for for the the detection detection of of CWAs CWAs in in liquidliquid phase phase showed thatthat thethe strategystrategy of of usage usage and and the the combination combination of of the the latest latest world world findings findings in thein thearea area of the of investigationthe investigation of chromogenic of chromogenic sensors sensors (RM1a, (RM1a, PY-OPD) PY-OPD) withtraditional with traditional analytical analytical agents agents(BCG) (BCG) is highly is highly effective. effective. This strategy This strategy enabled en theabled authors the authors to design to design and verify and theverify detection the detection papers papersfor a whole for a rangewhole of range potential of potent CWA,ial which CWA, might which occur might in theoccur form in the of droplets, form of droplets, or variously or variously dispersed dispersedaerosol, even aerosol, with even the possibilitywith the possibility of their color of thei distinction.r color distinction. Practically, Practically, the only the CWA only which CWA did which not didprovide not provide a positive a colorpositive response, color response, was sulfur was mustard. sulfur Greatermustard. eff ortsGreater will efforts therefore will be therefore dedicated be to dedicatedthis resolving to this this resolving issue in thethis future.issue in the future.

Author Contributions: Conceptualization, V.P. and L.M.; methodology, V.P., L.M. and M.D.; validation, V.P., Author Contributions: conceptualization, V.P. and L.M.; methodology, V.P., L.M. and M.D.; validation, V.P., L.M. and M.D.; formal analysis, L.M. and M.D.; resources, M.U., K.L. and L.K.; data curation, V.P., L.M.; L.M.writing—original and M.D.; formal draft analysis, preparation, L.M. V.P.;and writing—reviewM.D.; resources, andM.U., editing, K.L. and V.P., L.K.; L.M. data and curation, K.L.; visualization, V.P., L.M.; writing– V.P., L.M. originaland M.D.; draft supervision, preparation, V.P., V.P.; M.U. writ anding–review L.K.; project and administration, editing, V.P., V.P.L.M. and K.L.; visualization, V.P., L.M. and M.D.;Funding: supervision,The work V.P., was M.U. funded and in L.K.; the frameworkproject administration, of a security V.P. research project of the Ministry of Interior of the Czech Republic, No. VI20172019101. Funding: The work was funded in the framework of a security research project of the Ministry of Interior of the CzechConﬂicts Republic, of Interest: No. VI20172019101.The authors declare no conﬂict of interest.

Conflicts of Interest: The authors declare no conflicts of interest. References

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