sensors Article The Identification of Seven Chemical Warfare Mimics Using a Colorimetric Array Michael J. Kangas * , Adreanna Ernest, Rachel Lukowicz, Andres V. Mora , Anais Quossi, Marco Perez, Nathan Kyes and Andrea E. Holmes * Department of Chemistry, Doane University, Crete, NE 68333, USA; [email protected] (A.E.); [email protected] (R.L.); [email protected] (A.V.M.); [email protected] (A.Q.); [email protected] (M.P.); [email protected] (N.K.) * Correspondence: [email protected] (M.J.K.); [email protected] (A.E.H.) Received: 8 November 2018; Accepted: 3 December 2018; Published: 6 December 2018 Abstract: Chemical warfare agents pose significant threats in the 21st century, especially for armed forces. A colorimetric detection array was developed to identify warfare mimics, including mustard gas and nerve agents. In total, 188 sensors were screened to determine the best sensor performance, in order to identify warfare mimics 2-chloro ethyl ethylsulfide, 2-20-thiodiethanol, trifluoroacetic acid, methylphosphonic acid, dimethylphosphite, diethylcyanophosphonate, and diethyl (methylthiomethyl)phosphonate. The highest loadings in the principle component analysis (PCA) plots were used to identify the sensors that were most effective in analyzing the RGB data to classify the warfare mimics. The dataset was reduced to only twelve sensors, and PCA results gave comparable results as the large data did, demonstrating that only twelve sensors are needed to classify the warfare mimics. Keywords: warfare mimics; mustard gas; colorimetric array; principle component analysis; RGB data 1. Introduction Improving the non-presumptive detection of chemical warfare agents for soldiers in the field is a pressing need [1]. Warfare agents include chemical and explosive compounds, such as nerve agents like sarin, soman, and tabun, which are clear, colorless liquids without strong odors, and can cause loss of consciousness, seizures, and eventual death [2]. Other chemicals of war include vesicants or poisons, such as mustard gas, lewisite, phosgene, phosgene oxime, cyanide, mace, ricin, and pepper spray made of capsaicin [2]. Additionally, the detection of the precursors and degradation products of these materials can be equally important [3]. Existing technology has shown that these devices can effectively detect such analytes, but requires high-tech, expensive, and large instrumentation, which makes them less than ideal for field applications [4–7]. Colorimetric and fluorescent sensors have the potential to be effective detection systems for these analytes, but require minimal instrumentation. New sensors for these analytes have recently been reviewed [8]. One drawback of sensors is that they can be limited in the versatility or specificity of the analytes that can be identified. This can be overcome by combining multiple sensors into a sensor array, and the pattern of color changes can be used to identify and quantify a wide range of analytes [9–12]. A few sensor arrays chromofluorogenic supramolecular complexes for the chromogenic or fluorogenic sensing of nerve agents have been reported [13], as well as color changes of organophosphorus warfare mimics in the gas phase [14,15]. Exposure guidelines by the Center Disease Control suggest that mustard gas concentrations of 3.9 mg/m3 cause life-threatening effects or death. Therefore, there is a pressing need to identify sensors that can quickly detect this life-threatening agent. Herein, we examined the screening Sensors 2018, 18, 4291; doi:10.3390/s18124291 www.mdpi.com/journal/sensors Sensors 2018, 18, x FOR PEER REVIEW 2 of 9 Sensors 2018, 18, 4291 2 of 8 sensors that can quickly detect this life-threatening agent. Herein, we examined the screening of 188 colorimetric sensors, in solution, to simulate an array for the detection of 144 samples of mustard gas mimics,of 188 colorimetric 2-chloro-ethyl-ethylsulfide sensors, in solution, (0.1 M), to simulate2-2′-thiodiethanol an array (1 for M), the and detection nerve ofagent 144 samplesmimics, 0 trifluoroaceticof mustard gas acid mimics, (1 2-chloro-ethyl-ethylsulfideM), methylphosphonic (0.1acid M),(1 2-2M),-thiodiethanol dimethylphosphite (1 M), and (1 nerveM), diethylcyanophosphonateagent mimics, trifluoroacetic (1 M), acid and (1 diethyl M), methylphosphonic (methylthiomethyl)phosphonate acid (1 M), dimethylphosphite (0.1 M) (Figure 1) (1 [14– M), 16].diethylcyanophosphonate The identity and CAS (1 numbers M), and diethylof all 188 (methylthiomethyl)phosphonate sensors are located in the Supplemental (0.1 M) (Figure Information1)[ 14–16]. The(Table identity S1). and CAS numbers of all 188 sensors are located in the Supplemental Information (Table S1). S Cl HO OH S 2-2'-thiodiethanol 2-chloro-ethyl-ethylsulfide N H O P HO O O PO O O O F F F diethylcyanophosphonate dimethylphosphite trifluoroacetic acid O S O P O OH O P OH diethyl-(methylthiomethyl)-phosphonate methylphosphonic acid Figure 1. ChemicalChemical structures structures of mustard gas mimics (red) and nerve gas mimics (blue). 2. Materials and Methods All reagentsreagents werewere purchased purchased from from various various chemical chemical supply supply companies companies at technical at technical grade grade or better, or better,and were and used were as received.used as Thereceived. universal The indicator universal was indicator composed was of Vancomposed Urk’s and of Yamada’sVan Urk’s recipe, and Yamada’sand contained recipe, methyl and red,contained methyl methyl orange, red, phenolphthalein, methyl orange, andphenolphthalein, bromothymol and blue bromothymol powders in a weightblue powders ratio of approximatelyin a weight ratio 1:3:7:8 of [17 approximately]. One percent weight-by-weight1:3:7:8 [17]. One solutionspercent ofweight-by-weight all sensors were preparedsolutions byof dissolvingall sensors each were into prepared aliquots of by a solvent dissolving mixture each consisting into aliquots of acetate of buffera solvent (0.1 M,mixture pH 5), consistingethylene glycol, of acetate triethylene buffer glycol(0.1 M, monobutyl pH 5), ethyle ether,ne andglycol, glycerol, triethylene in a ratio glycol of 14:1.6:1:3.2. monobutyl The ether, sensors and ◦ wereglycerol, dissolved in a ratio by of sonication 14:1.6:1:3.2. in The a bath sensors sonicator were (30dissolvedC) for by 1 h,sonication followed in by a bath 5 min sonicator mixing (30 with °C) a probefor 1 h, sonicator, followed and by then5 min vacuum mixing filteredwith a onceprobe through sonicator, Whatman and then #1 vacuum filter paper, filtered and once through Whatman nylon#1 filter filter paper, membranes and once (0.2 throughµm). Whatman nylon filter membranes (0.2 µm). A solution of of methyl methyl phosphonic phosphonic acid acid (1 (1 M) M) wa wass prepared prepared dissolving dissolving an an appropriate appropriate amount amount of theof theanalytes analytes in milli-Q in milli-Q water water (18 (18MΩ M-cm).W-cm). Solutions Solutions of 2 ofchloroethyl 2 chloroethyl ethyl ethyl sulfide sulfide (0.1 (0.1M), 2,2 M),′ 0 thiodiethanol2,2 thiodiethanol (1 M), (1 M),diethylcyanophosphonate diethylcyanophosphonate (0.1 (0.1 M), M), dimethylphosphite dimethylphosphite (1 (1 M), M), and and diethyl (methylthiomethyl)phosphonate (0.1 (0.1 M) M) were were prepar prepareded by diluting the reagents with milli-Q water (18 M MΩW-cm).-cm). The The concentrations concentrations were were selected selected to be to near be near the solubility the solubility limit limitor 1 M. or Solutions 1 M. Solutions of 2,2′ 0 thiodiethanolof 2,2 thiodiethanol and methylphosphonic and methylphosphonic acid were acid stored were in storedthe dark in at the room dark temperature at room temperature and were replacedand were replacedapproximately approximately every three every threemonths. months. Solutions Solutions of of2-chloroethyl 2-chloroethyl ethyl ethyl sulfide, sulfide, diethylcyanophosphonate, dimeth dimethylphosphite,ylphosphite, and and diethyl (methylthiomethyl)phosphonate(methylthiomethyl)phosphonate were ◦ portioned into ~10 mL aliquots, stored at −−6060 °CC to to minimize decomposition [18], [18], and replaced approximately everyevery 33 months.months. Solutions Solutions were were allowed allowed to to thaw thaw at roomat room temperature temperature for for 1 h before1 h before use. use. Sensor (100 µL) was added to all the wells of a flat bottomed 96-well plate using a 12-channel electronicSensor pipet. (100 µL) One was hundred added microlitersto all the wells of each of a analyte flat bottomed were applied 96-well to plate rows using of the a 12-channel well plate. Thirty-twoelectronic pipet. samples One of waterhundred and microliters 16 samples of of each the 7 analytesanalyte were were applied tested to to make rows the of total the samplewell plate. size 144.Thirty-two The plate samples was scanned of water as and detailed 16 samples below. of the 7 analytes were tested to make the total sample size 144.All arrayThe plate images was (24-bit scanned color, as detailed 400 dpi) below. were collected using an Epson Perfection V700 desktop scannerAll inarray transparency images (24-bit mode. color, To eliminate 400 dpi) interferenceswere collected from using stray an light,Epson the Perfection scanner wasV700 draped desktop in scannerblack cloth. in transparency The images mode. were analyzed To eliminate with interfer ImageJences [19], andfrom the stray extraction light, the of scanner mean RGB was valuesdraped for in blackeach wellcloth. was The automated images were with analyzed a macro with [20]. ImageJ No attempts [19], and were the made extraction to correct of mean for image-to-imageRGB values for each well was automated with a macro [20]. No attempts were made to correct for image-to-image SensorsSensors 20182018, ,1818, ,x 4291FOR PEER REVIEW 3 3of of 9 8 variation by subtracting a control row, as one sensor was tested on each well plate, and our previous workvariation found by correcting subtracting images a control to be row, unnecessary as one sensor [12]. was tested on each well plate, and our previous workAll found statistical correcting analysis images was toperformed be unnecessary using the [12].