Supramolecular Sensing of a Chemical Warfare Agents Simulant by Functionalized Carbon Nanoparticles

molecules

Communication Supramolecular Sensing of a Chemical Warfare Agents Simulant by Functionalized Carbon Nanoparticles

Nunzio Tuccitto 1,2,* , Luca Spitaleri 1,3 , Giovanni Li Destri 1,2 , Andrea Pappalardo 1,3, Antonino Gulino 1,3 and Giuseppe Trusso Sfrazzetto 1,3,* 1 Department of Chemical Sciences, University of Catania, Viale A. Doria 6, 95125 Catania, Italy; [email protected] (L.S.); [email protected] (G.L.D.); [email protected] (A.P.); [email protected] (A.G.) 2 Laboratory for Molecular Surfaces and Nanotechnology–CSGI, Viale A. Doria 6, 95125 Catania, Italy 3 National Interuniversity Consortium for Materials Science and Technology (I.N.S.T.M.) Research Unit of Catania, Viale A. Doria 6, 95125 Catania, Italy * Correspondence: [email protected] (N.T.); [email protected] (G.T.S.); Tel.: +39-0957385201 (G.T.S.) Academic Editors: Alessandro D’Urso, Maria Elena Fragala and Derek J. McPhee Received: 30 October 2020; Accepted: 2 December 2020; Published: 4 December 2020 Abstract: Real-time sensing of chemical warfare agents by optical sensors is today a crucial target to prevent terroristic attacks by chemical weapons. Here the synthesis, characterization and detection properties of a new sensor, based on covalently functionalized carbon nanoparticles, are reported. This nanosensor exploits noncovalent interactions, in particular hydrogen bonds, to detect DMMP, a simulant of nerve agents. The nanostructure of the sensor combined with the supramolecular sensing approach leads to high binding constant aﬃnity, high selectivity and the possibility to reuse the sensor.

Keywords: carbon nanoparticles; chemical warfare agents; sensor; supramolecular recognition

1. Introduction Chemical warfare agents (CWAs), organophosphorus compounds developed before the Second World War, are highly toxic compounds due to their ability to irreversibly inhibit the acetylcholine esterase enzyme [1]. Today, CWAs are classified into three classes: G-type, V-type and A-type (also called Novichok) (Scheme1). Although CWAs are today prohibited in many countries, the recent attacks in the Middle East and in Europe highlight the importance to quickly detect, in real time, the presence of these agents [2,3]. Due to the toxicity levels of G- and V-type CWAs (LC50 values of Tabun and Sarin are 2 and 1.2 ppm, respectively, while Soman and VX have LC50 of 0.9 and 0.3 ppm, respectively) [4], the ability to detect low concentration values is of primary importance. Research activity is usually performed by using simulants, less toxic compounds having geometries and sizes very similar to real CWAs [5]. In particular, the dimethyl methylphosphonate (DMMP) is today widely recognized as one of the best simulants for G-type CWAs. Sensing of CWAs can be performed by using two different approaches: the “covalent approach” [6], based on a covalent reaction between the sensor and the analyte that leads to a change on a specific measurable property, or the “supramolecular approach” [7], in which the analyte is recognized by exploiting noncovalent interactions with a synthetic receptor. The covalent approach shows some limits: (i) low specificity, (ii) possibility of false-positive responses and (iii) impossibility to reuse the

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sensor. By contrast, the recent success of the supramolecular approach is due to the possibility to reuse the sensor, because of the formation of noncovalent interactions with the analyte [8–16], and the high eﬃciency (in terms of binding constant values) [17] and selectivity due to the possibility to recognize Moleculesthe analyte 2020, 25 by, x multiple interactions (multitopic approach) [18,19]. 2 of 10

O O O O P N O F P O F P O F P O N Tabun Sarin GB Soman GD Cyclosarin GF

O O N S P O N S P O

VX R-VX

R1 O

R2 P N Cl O O X Cl F General formula of a Novichok CWA

SchemeScheme 1. 1.StructuresStructures and and names names of of G-type, G-type, V-type V-type and generalgeneral formulaformula of of A-type A-type chemical chemical warfare warfare agentsagents (CWAs). (CWAs).

SensingRecently, of CWAs we reported can be on performed the first metal-free by using fluorescent two different sensor, approaches: Naphthyl-Di-AE, the “covalent able to selectively approach” [6],and based reversibly on a covalent detect DMMP reaction via thebetween multitopic the approachsensor and by exploitingthe analyte multiple that leads hydrogen to a change bonds [ 20on]. a Furthermore, very recently, we reported on the first nanosensor based on carbon nanoparticles (CNPs), specific measurable property, or the “supramolecular approach” [7], in which the analyte is able to detect very low concentration values of DMMP both in solution and in the gas phase [21]. recognized by exploiting noncovalent interactions with a synthetic receptor. The covalent approach The use of CNPs [22–24] for sensing purposes shows many interesting features, such as the high shows some limits: (i) low specificity, (ii) possibility of false-positive responses and (iii) impossibility fluorescence quantum yield [25], water solubility, low toxicity [26], low-cost synthesis [27] and the to reuse the sensor. By contrast, the recent success of the supramolecular approach is due to the possibility to functionalize their external shell [28]. possibility to reuse the sensor, because of the formation of noncovalent interactions with the analyte Taking into account these considerations, here we present the implementation of the [8–16],Naphthyl-Di-AE and the high on efficiency the surface (in of carbonterms nanoparticles,of binding constant leading values) to CNPs-Naphthyl-Di-AE [17] and selectivity(Scheme due to2 ).the possibilityThe presence to recognize of a specific the sensor analyte for by DMMP multiple (Naphthyl-Di-AE) interactions (multitopic on the nanoparticle approach) surface [18,19]. leads to the possibilityRecently, to increasewe reported the selectivity on the towardsfirst metal-free the desired fluorescent analyte target sensor, and Naphthyl-Di-AE, to follow the recognition able to selectivelyevent by and monitoring reversibly a specific detect changeDMMP in via the the optical multitopic spectrum approach of the receptor. by exploiting multiple hydrogen bonds In[20]. particular, Furthermore, the ethanolamine very recently, arms, we responsible reported on for the hydrogen first nanosensor bond formation, based are on incarbon the nanoparticlesperfect geometry (CNPs), to recognize able to detect DMMP very with low both concen arms.tration With respectvalues of to ourDMMP recent both nanoparticle-based in solution and in thesensor, gas phase having [21]. the The ethanolamine use of CNPs arms[22–24] randomly for sensing bounded purposes onto shows the nanoparticle many interesting surface features, [21], suchCNPs-Naphthyl-Di-AE as the high fluorescenceshow thequantum recognition yield group [25], (previouslywater solubility, synthesized) low toxicity having [26], the optimal low-cost synthesisconfiguration [27] and to chelatethe possibility and DMMP. to functionalize their external shell [28]. Taking into account these considerations, here we present the implementation of the Naphthyl- Di-AE on the surface of carbon nanoparticles, leading to CNPs-Naphthyl-Di-AE (Scheme 2). The presence of a specific sensor for DMMP (Naphthyl-Di-AE) on the nanoparticle surface leads to the possibility to increase the selectivity towards the desired analyte target and to follow the recognition event by monitoring a specific change in the optical spectrum of the receptor. In particular, the ethanolamine arms, responsible for hydrogen bond formation, are in the perfect geometry to recognize DMMP with both arms. With respect to our recent nanoparticle-based sensor, having the ethanolamine arms randomly bounded onto the nanoparticle surface [21], CNPs- Naphthyl-Di-AE show the recognition group (previously synthesized) having the optimal configuration to chelate and DMMP.

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Scheme 2. Synthesis of CNPs-Naphthyl-Di-AE. 2. Results and Discussion 2. Results and Discussion CNPs-Naphthyl-BrNO2 were obtained by reacting the anhydride 1 [29] in refluxing ethanol CNPs-Naphthyl-BrNO2 were obtained by reacting the anhydride 1 [29] in refluxing ethanol with amino-terminal CNPs, and they were purified by centrifugation and filtration. Reaction with amino-terminal CNPs, and they were purified by centrifugation and filtration. Reaction of of CNPs-Naphthyl-BrNO2 with a large excess of ethanolamine in solvolysis leads to the CNPs-Naphthyl-BrNO2 with a large excess of ethanolamine in solvolysis leads to the CNPs- CNPs-Naphthyl-Di-AE, which have been purified by centrifugation and dialysis. The 1H-NMR Naphthyl-Di-AE, which have been purified by centrifugation and dialysis. The 1H-NMR spectrum spectrum suggests the covalent functionalization with the ethanolamine groups, due to the presence of suggests the covalent functionalization with the ethanolamine groups, due to the presence of aromatic signals characteristic of the naphthalic core and, in the aliphatic region, of the typical pattern aromatic signals characteristic of the naphthalic core and, in the aliphatic region, of the typical pattern of ethanolamine (see Supplementary Material). of ethanolamine (see Supplementary Material). The electronic structure of the carbon nanoparticles (CNPs) functionalized with the naphthalic The electronic structure of the carbon nanoparticles (CNPs) functionalized with the naphthalic probe, CNPs-Naphthyl-Di-AE, was also investigated by X-ray photoelectron spectroscopy (XPS), probe, CNPs-Naphthyl-Di-AE, was also investigated by X-ray photoelectron spectroscopy (XPS), thus providing information on the chemical environment. XPS also gave information on the covalent thus providing information on the chemical environment. XPS also gave information on the covalent anchoring of the naphthalic sensor on the CNPs surface, appropriately functionalized with –NH2 anchoring of the naphthalic sensor on the CNPs surface, appropriately functionalized with –NH2 groups. Finally, the surface elemental composition was estimated, once the relevant atomic sensitivity groups. Finally, the surface elemental composition was estimated, once the relevant atomic factors had been taken into account [30–34]. sensitivity factors had been taken into account [30–34]. Figure1 shows the high-resolution XPS spectrum of the CNPs-Naphthyl-Di-AE in the C 1s Figure 1 shows the high-resolution XPS spectrum of the CNPs-Naphthyl-Di-AE in the C 1s binding energy region. An accurate fitting of this spectrum revealed the presence of four components at binding energy region. An accurate fitting of this spectrum revealed the presence of four components 285.0, 286.0, 286.4 and 287.7 eV, respectively. The first component (285.0 eV) is due to both aliphatic and at 285.0, 286.0, 286.4 and 287.7 eV, respectively. The first component (285.0 eV) is due to both aliphatic aromatic backbones [35,36]. The peaks at 286.0 eV and 286.4 eV are due to the C–N and C–OH groups, and aromatic backbones [35,36]. The peaks at 286.0 eV and 286.4 eV are due to the C–N and C–OH respectively [37,38]. The peak at 287.7 eV is assigned to the carbon of the amide group (–OC–NH–) [39], groups, respectively [37,38]. The peak at 287.7 eV is assigned to the carbon of the amide group (–OC– and the presence of this signal confirms the covalent functionalization of the CNPs with the naphthalic NH–) [39], and the presence of this signal confirms the covalent functionalization of the CNPs with anhydride. Moreover, the intensity ratio between these three bands at 286.0, 286.4 and 287.7 eV is 5:2:2, the naphthalic anhydride. Moreover, the intensity ratio between these three bands at 286.0, 286.4 and and this result is in agreement with the chemical structure of the CNPs-Naphthyl-Di-AE, reported 287.7 eV is 5:2:2, and this result is in agreement with the chemical structure of the CNPs-Naphthyl- in Scheme2. Finally, the peak at 293.2 eV is ascribed to π–π* shake-up satellites characteristic of the Di-AE, reported in Scheme 2. Finally, the peak at 293.2 eV is ascribed to π–π* shake-up satellites aromatic and conjugate systems. characteristic of the aromatic and conjugate systems.

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FigureFigure 1.1.Al-K Al-Kααexcited excited XPS XPS of of the the CNPs–naphthalic CNPs–naphthalic anhydride anhydride sample sample in the in C the 1s binding C 1s binding energy energy region. Figure 1. Al-Kα excited XPS of the CNPs–naphthalic anhydride sample in the C 1s binding energy Theregion. blue, The cyan, blue, magenta cyan, andmagenta dark yellowand dark lines yello referw tolines the refer 285.0, to 286.0, the 285.0, 286.4 and286.0, 287.7 286.4 components; and 287.7 region. The blue, cyan, magenta and dark yellow lines refer to the 285.0, 286.0, 286.4 and 287.7 thecomponents; green line refersthe green to the background,line refers to and the the backgr red lineound, superimposed and the tored the line experimental superimposed black profileto the components; the green line refers to the background, and the red line superimposed to the refersexperimental to the sum black of allprofile Gaussian refers components. to the sum of all Gaussian components. experimental black profile refers to the sum of all Gaussian components. FigureFigure2 2 shows shows the the XPS XPS of of CNPs-Naphthyl-Di-AECNPs-Naphthyl-Di-AE inin thethe NN 1s1s bindingbinding energyenergy region.region. TheThe NN 1s1s Figure 2 shows the XPS of CNPs-Naphthyl-Di-AE in the N 1s binding energy region. The N 1s spectralspectral profileprofile waswas fittedfitted usingusing twotwo GaussianGaussian componentscomponents atat 400.1400.1 andand 401.7 eV. TheThe firstfirst componentcomponent spectral profile was fitted using two Gaussian components at 400.1 and 401.7 eV. The first component (400.1(400.1 eV)eV) isis duedue toto thethe nitrogennitrogen ofof amineamine groupsgroups (–NH–)(–NH–) [[39].39]. TheThe peakpeak atat 401.7401.7 eVeV isis duedue toto thethe (400.1 eV) is due to the nitrogen of amine groups (–NH–) [39]. The peak at 401.7 eV is due to the nitrogennitrogen ofof thethe imideimide group group (O (O=C–N–C=O)=C–N–C=O) [[40].40]. TheThe presencepresence ofof OO=C–N–C=O=C–N–C=O group confirmsconfirms thethe nitrogen of the imide group (O=C–N–C=O) [40]. The presence of O=C–N–C=O group confirms the covalentcovalent functionalization of of the the CNPs CNPs with with the the na naphthalicphthalic core. core. The The intensity intensity ratio ratio between between the two the covalent functionalization of the CNPs with the naphthalic core. The intensity ratio between the two twobands bands at 400.1/401.7 at 400.1/401.7 eV eVis 2:1, is 2:1, and and this this result result is isin in agreement agreement with with the the chemical chemical structure of thethe bands at 400.1/401.7 eV is 2:1, and this result is in agreement with the chemical structure of the naphthalicnaphthalic anhydrideanhydride molecule.molecule. naphthalic anhydride molecule.

FigureFigure 2.2.Al-K Al-Kααexcited excited XPS XPS of ofCNPs-Naphthyl-Di-AE CNPs-Naphthyl-Di-AEin in the the N 1sN binding1s binding energy energy region. region. The blueThe blue and Figure 2. Al-Kα excited XPS of CNPs-Naphthyl-Di-AE in the N 1s binding energy region. The blue cyanand cyan lines referlines torefer the to 400.1 the and400.1 401.7 and eV401.7 components; eV components; the green the line green refers line to refers the background, to the background, and the and cyan lines refer to the 400.1 and 401.7 eV components; the green line refers to the background, redand line the superimposedred line superimposed to the experimental to the experimental black profile black refers profile to the refers sum of to all the Gaussian sum of components.all Gaussian and the red line superimposed to the experimental black profile refers to the sum of all Gaussian components. Finally,components. Figure 3 shows the O 1s peak of the CNPs–naphthalic anhydride system. The O 1s spectral profile was fitted using four Gaussian components at 532.3, 532.9, 534.4 and 537.3 eV. The lower Finally, Figure 3 shows the O 1s peak of the CNPs–naphthalic anhydride system. The O 1s energyFinally, peak, locatedFigure at3 shows 532.3 eV, the is O assigned 1s peak to of the th –OHe CNPs–naphthalic groups of both CNPs anhydride and naphthalic system. The core O [41 1s]. spectral profile was fitted using four Gaussian components at 532.3, 532.9, 534.4 and 537.3 eV. The Thespectral second profile peak was at 532.9 fitted eV using is assigned four Gaussian to the imide components (O=C–N–C at= O)532.3, group 532.9, [40 ,41534.4] and and further 537.3 confirms eV. The lower energy peak, located at 532.3 eV, is assigned to the –OH groups of both CNPs and naphthalic thelower covalent energy functionalization peak, located at 532.3 of the eV, CNPs is assigned with the to naphthalicthe –OH groups anhydride of both molecule. CNPs and Once naphthalic more, core [41]. The second peak at 532.9 eV is assigned to the imide (O=C–N–C=O) group [40,41] and thecore intensity [41]. The ratios second of thepeak first at two 532.9 peaks eV (2:2)is assigned presently to observedthe imide are(O=C–N–C=O) strongly in agreement group [40,41] with and the further confirms the covalent functionalization of the CNPs with the naphthalic anhydride molecule. expectedfurther confirms intensity the trend, covalent on the functionalization basis of the structure of the CNPs of CNPs-Naphthyl-Di-AE with the naphthalic anhydride. The higher molecule. energy Once more, the intensity ratios of the first two peaks (2:2) presently observed are strongly in peakOnce located more, atthe 534.4 intensity eV is attributed ratios of to the the first H O two molecules peaks present(2:2) presently in the air-exposed observed CNPs–naphthalic are strongly in agreement with the expected intensity trend,2 on the basis of the structure of CNPs-Naphthyl-Di-AE. agreement with the expected intensity trend, on the basis of the structure of CNPs-Naphthyl-Di-AE. The higher energy peak located at 534.4 eV is attributed to the H2O molecules present in the air- The higher energy peak located at 534.4 eV is attributed to the H2O molecules present in the air-

Molecules 2020, 25, 5731 5 of 10 Molecules 2020, 25, x 5 of 10 anhydrideexposed CNPs–naphthalic system. Besides, anhydride the 537.3 eV system. peak is Besides, attributed the to537.3 the moleculareV peak is Oattributed2 adsorbed to onthe the molecular surface ofO2 theadsorbed air-exposed on the sample surface [41 of]. the air-exposed sample [41].

Figure 3. Al-Kα excited XPS of the CNPs-Naphthyl-Di-AE sample in the O 1s energy region. The black Figure 3. Al-Kα excited XPS of the CNPs-Naphthyl-Di-AE sample in the O 1s energy region. The line refers to the experimental profile; the green line refers to the background; the Gaussian at black line refers to the experimental profile; the green line refers to the background; the Gaussian at 532.3 (blue line), 532.9 (cyan line), 534.4 (magenta line) and 537.3 eV (dark yellow line) represent the 532.3 (blue line), 532.9 (cyan line), 534.4 (magenta line) and 537.3 eV (dark yellow line) represent the four O 1s components; the red line, superimposed to the experimental profile, refers to the sum of the four O 1s components; the red line, superimposed to the experimental profile, refers to the sum of the Gaussian components. Gaussian components. Sensing properties were evaluated by UV-Vis spectroscopy. A solution of 0.05 mg/mL of Sensing properties were evaluated by UV-Vis spectroscopy. A solution of 0.05 mg/mL of CNPs- CNPs-Naphthyl-Di-AE in toluene shows a broad absorption band, characteristic of the carbon Naphthyl-Di-AE in toluene shows a broad absorption band, characteristic of the carbon nanoparticles extending all over the visible and near UV region. In addition, the band relative to nanoparticles extending all over the visible and near UV region. In addition, the band relative to the the Naphthyl-Di-AE sensor can be detected at 430 nm. This band was monitored during the sensing Naphthyl-Di-AE sensor can be detected at 430 nm. This band was monitored during4 the sensing of of DMMP. In particular, the progressive addition of DMMP (0–100 µL of a 1 10− M solution in DMMP. In particular, the progressive addition of DMMP (0–100 μL of a 1 × 10−4 M× solution in toluene) toluene) leads to a decrease of the absorbance intensity (Figure4), in agreement with the behavior leads to a decrease of the absorbance intensity (Figure 4), in agreement with the behavior observed observed by using the molecular sensor in solution [20]. The nonlinear curve fit of this data indicates a by using the molecular sensor in solution [20]. The nonlinear curve fit of this data indicates a binding binding constant affinity of log 5.55 0.30, more than one order of magnitude higher with respect constant affinity of log 5.55 ± 0.30, more± than one order of magnitude higher with respect to that of to that of the Naphthyl-Di-AE in solution (log 4.02), thus demonstrating the higher performance the Naphthyl-Di-AE in solution (log 4.02), thus demonstrating the higher performance of the of the nanosensor with respect to the molecular sensor. Notably, the calculated detection limit is nanosensor with respect to the molecular sensor. Notably, the calculated detection limit is 0.16 ppm 0.16 ppm (see Section3), lower than the LC50 values of the most common CWAs used (fluorescence (see Section 3), lower than the LC50 values of the most common CWAs used (fluorescence measurements are precluded due to the stronger emission of the carbon nanoparticle core that overlaps measurements are precluded due to the stronger emission of the carbon nanoparticle core that the emission of the naphthalic core). overlaps the emission of the naphthalic core). Selectivity is a crucial parameter for a sensor. For this reason, we tested the UV-Vis response of CNPs-Naphthyl-Di-AE to different organic analytes, in order to test the efficiency of the multitopic approach. Figure5 shows the normalized absorbance values of CNPs-Naphthyl-Di-AE observed upon the addition of 50 ppm of different analytes. We note that carbonyl compounds do not interact with our sensor. Organic phosphorous compounds, such as triethylphosphite, triphenylphosphine and phosphocholine cause a slight increase of the optical response. The presence of air (containing 24,000 ppm of water, 400 ppm CO2, 5 ppm NO and 10 ppm CO), bubbled for 30 min, does not interfere with the nanosensor. On the contrary, the addition of 1 ppm of DMMP leads to a strong optical response.

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Figure 4. UV-Vis spectra of CNPs-Naphthyl-Di-AE (0.05 mg/mL in toluene) upon progressive addition of DMMP (0–100 μL of a 1 × 10−4 M solution in toluene), inset shows the calibration curve.

Selectivity is a crucial parameter for a sensor. For this reason, we tested the UV-Vis response of CNPs-Naphthyl-Di-AE to different organic analytes, in order to test the efficiency of the multitopic approach. Figure 5 shows the normalized absorbance values of CNPs-Naphthyl-Di-AE observed upon the addition of 50 ppm of different analytes. We note that carbonyl compounds do not interact with our sensor. Organic phosphorous compounds, such as triethylphosphite, triphenylphosphine and phosphocholine cause a slight increase of the optical response. The presence of air (containing 24,000 ppm of water, 400 ppm CO2, 5 ppm NO and 10 ppm CO), bubbled for 30 min, does not interfere with the nanosensor. On the contrary, the addition of 1 ppm of DMMP leads to a strong optical response. Figure 4.4. UV-VisUV-Vis spectra spectra of CNPs-Naphthyl-Di-AEof CNPs-Naphthyl-Di-AE(0.05 (0.05 mg/mL mg/mL in toluene) in toluene) upon progressive upon progressive addition 4 of DMMP (0–100 µL of a 1 10 M solution−4 in toluene), inset shows the calibration curve. addition of DMMP (0–100 μ×L of− a 1 × 10 M solution in toluene), inset shows the calibration curve.

Figure 5. 5. NormalizedNormalized UV-Vis UV-Vis responses responses of CNPs-Naphthyl-Di-AE of CNPs-Naphthyl-Di-AE (0.05 mg/mL(0.05 mg in/ mLtoluene) in toluene) to various to

competitivevarious competitive guests: guests:50 ppm 50 ppmof acetone, of acetone, acetic acetic acid, acid, triethylphosphite triethylphosphite (P(OCH(P(OCH22CH3))33),), triphenylphosphinetriphenylphosphine (PPh (PPh3)3 )and and phosphocholine; phosphocholine; air air(bubbled (bubbled for for30 min); 30 min); and DMMP and DMMP (1 ppm). (1 ppm). Bars representBars represent the initial the initial over overthe final the ﬁnalabsorbance absorbance values values at 430 at nm. 430 nm.

3. Materials and Methods

3.1. General Experimental Methods

The NMR experiments were carried out at 27 ◦C on a Varian UNITY Inova 500 MHz spectrometer 1 13 ( H at 499.88 MHz, C-NMR at 125.7 MHz, Varian-Agilent, Santa Clara, CA, USA) equipped with a pulse-ﬁeld gradient module (Z axis) and a tunable 5 mm Varian inverse detection probe (ID-PFG). UV-VisFigure measurements 5. Normalized were UV-Vis carried responses out of using CNPs-Naphthyl-Di-AE a JASCO V-630 spectrophotometer (0.05 mg/mL in toluene) (Mettler to various Toledo, competitive guests: 50 ppm of acetone, acetic acid, triethylphosphite (P(OCH2CH3)3), Novate Milanese, Italy) at room temperature. All chemicals were reagent grade (Signa Aldrich, triphenylphosphine (PPh3) and phosphocholine; air (bubbled for 30 min); and DMMP (1 ppm). Bars Buchs, Switzerland) and used without further puriﬁcation. X-ray photoelectron spectra (XPS) were represent the initial over the final absorbance values at 430 nm.

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measured at a 45◦ take-off angle relative to the surface normal with a PHI 5600 Multi Technique System (Physical Electronics GmbH, Feldkirchen, Germany, base pressure of the main chamber 3 10 8 Pa)[35,36]. Samples were excited with the Al-Kα X-ray radiation using a pass energy of × − 5.85 eV. Structures due to the Kα satellite radiations were subtracted from the spectra prior to data processing. Spectra calibration was achieved by fixing the Ag 3d5/2 peak of a clean sample at 368.3 eV; this method turned the C 1s main peak at 285.0 eV [35,36,42]. The instrumental energy resolution was 0.5 eV. The XPS peak intensities were obtained after Shirley background removal [35,36]. The atomic ≤ concentration analysis was performed by taking into account the relevant atomic sensitivity factors. The fittings of the C 1s, N 1s and O 1s XP spectra were carried out using Gaussian envelopes after subtraction of the background until there was the highest possible correlation between the experimental spectrum and the theoretical profile. The residual or agreement factor R, defined by R = [Σ(Fobs F )2/Σ(F )2]1/2, after minimization of the function Σ(F F )2, converged to the value of − calc obs obs − calc 0.03. The fitting was performed using the XPSPEAK4.1 software (free, fully featured, software for the analysis of XPS spectra written by Raymund Kwok). Samples for XPS measurement were deposited on silicon substrates.

3.2. Synthesis of CNPs-Naphthyl-BrNO2 One hundred milligrams of amino-terminal CNPs [43] was dissolved in 15 mL of absolute ethanol, and 70 mg (0.217 mmol) of 1 was added. The mixture was stirred at reflux for 24 h under nitrogen atmosphere. Solvent was removed by rotavapor, and the CNPs-Naphthyl-BrNO2 were purified by centrifugation and filtration in hot ethanol.

3.3. Synthesis of CNPs-Naphthyl-Di-AE

Twenty milligrams of CNPs-Naphthyl-BrNO2 was dissolved in 10 mL of ethanolamine, and the mixture was reﬂuxed under nitrogen atmosphere for 48 h. The excess of ethanolamine was removed under reduced pressure, and the CNPs-Naphthyl-Di-AE were puriﬁed by centrifugation and dialysis.

3.4. Procedure for UV-Vis Titrations From a guest stock solution (1.0 10 4 M) in toluene, diﬀerent volumes (0, 2, 4, 6, 8, 10, 20, 30, 50, × − 75, 100 µL) were added to the host (0.05 mg/mL in toluene), and the UV-Vis spectra were recorded at 25 ◦C. The apparent binding aﬃnities of CNPs-Naphthyl-Di-AE with DMMP were estimated by using HypSpec (version 1.1.33) [44–46], a software designed to extract equilibrium constants from potentiometric and/or spectrophotometric titration data. HypSpec starts with an assumed complex formation scheme and uses a least-squares approach to derive the spectra of the complexes and the stability constants. χ2 test (chi-square) was applied, where the residuals followed a normal distribution (for a distribution approximately normal, the χ2 test value is around 12 or less). In all of the cases, χ2 10 was found, as obtained by 3 independent measurement sets. Limit of detection was calculated ≤ by the method of the calibration curve using the formula DL = 3s/K, where s is the standard deviation of the blank and K is the slope of the calibration curve.

4. Conclusions In the present study, we report the covalent functionalization of carbon nanoparticles by a selective sensor of Chemical Warfare Agents. The presence of a specific sensor for DMMP (Naphthyl-Di-AE) on the carbon nanoparticle surface leads to the possibility to increase the selectivity towards the given analyte target and to follow the recognition event by monitoring a specific change of the optic spectrum of the receptor. The new nanosensor shows high binding affinity towards DMMP, high selectivity and a sub-ppm detection limit. The possibility to tune the molecular structure of the external shell of the carbon nanoparticles leads to a wide range of applications, including sensing applications. Studies are ongoing to obtain functionalized fluorescent carbon nanoparticles suitable for real prototypes to be commonly used. Molecules 2020, 25, 5731 8 of 10

Supplementary Materials: The following are available online. Figure S1: 1H-NMR spectrum of CNPs-Naphthyl- Di-AE in DMSO-d6; Figure S2: UV-Vis titration between CNPs-Naphthyl-Di-AE and DMMP. Author Contributions: Conceptualization, N.T. and G.T.S.; XPS analysis, L.S. and A.G.; synthesis, A.P. and G.T.S.; validation, G.L.D.; writing—original draft preparation, N.T. and G.T.S. All authors have read and agreed to the published version of the manuscript. Funding: This research was funded by the University of Catania (PIA.CE.RI 2020-2022–Linea Intervento 2). Acknowledgments: The authors thank the project “Nati4Smart” University of Catania for financial support. Conflicts of Interest: The authors declare no conflict of interest.

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