*2.4. Characterization*

To acquire quality imaging of the samples, a high-resolution transmission electron microscope (HRTEM), type TECNAI F30 G2STWIN, Fei Company, Oregon, USA, was used at 300 kV acceleration voltage and with a resolution of 1 Å. The correlation between dynamic viscosity and shear gradient of the decontamination solutions was studied to establish a model flow profile of the polymeric solution with superior film-forming characteristics. Rheological tests were performed on a Rheotest 2.1 device (Rheotest Medingen GmbH, Ottendorf-Okrilla, Germany) with coaxial cylinders at room temperature (25 ◦C) to determine the behavior of the solutions. The amount of solvent evaporated in time at different temperatures (25 ◦C, 30 ◦C, and 35 ◦C) was used to investigate the drying profile of the nanocomposite films. An ATS 120 Axis Thermobalance was used to measure the evaporation rate of 4 mL of sample for evaluation of the film-formation process. Promas software calculated the evaporation rate by weighting the sample every 150 s. FT-IR spectra were obtained using a Perkin Elmer Spectrum Two (Perkin Elmer, Waltham, MA, USA) with a Pike MiracleTM ATR modulus and a 4 cm−<sup>1</sup> resolution, from 550 to 4000 cm<sup>−</sup>1. To investigate the mechanical proprieties, polymeric films were obtained by casting method and then cut in a dumbbell shape with 75 mm overall length and a narrow section of about 25 ± 1 mm, which were subsequently subjected to tensile tests on a 710 Titan 2 universal strength-testing machine equipped with a 3000 N force cell, according to ISO 37: 2011(E). The test involves continuous observation of the length and force variation with an accuracy of ±0.2% at a speed of 8.33 mm/s. To compare the results, the mean values of each sample were plotted in a stress/strain graph. Five specimens from each sample were

subjected to tensile tests. Samples weighing approximately 25–30 mg were subjected to thermal tests, heated from 30 ◦C to 450 ◦C with a constant heating rate of 5 ◦C/min on a DTA OZM 551 Ex Differential Thermal Analysis System equipped with Meavy dedicated software. GC-MS investigations were performed on a GC Thermo Scientific Trace 1310 (Thermo Fisher Scientific, Waltham, MA, USA) gas chromatograph coupled with a TSQ 9000 triple quadrupole mass spectrometer (MS/MS) (Thermo Fisher Scientific, Waltham, MA, USA) using a TR5MS GOLD capillary column (5% phenyl 95% dimethylpolysiloxane). The injection mode used was splitless with an injector temperature of 250 ◦C and helium as carrier gas (1.5 mL/min). The temperature program started from 40 ◦C, up to 300 ◦C, with a rate of 10 ◦C/minute. Electron impact ionization (EI) mode (mass range between 40 and 650 amu) was used. The compounds were identified based on the interpretation of MS/EI fragmentation.

#### **3. Results and Discussion**

This first step of this study consisted of the synthesis of bentonite-supported metal and metal oxide nanoparticles that are suitable for decontamination applications, capable of reacting with chemical warfare agents to form non-toxic products while also neutralizing biologic agents due to their anti-bacterial properties. Thus, nanosized hydrophilic bentonite was used as support for the metal/metal oxides nanoparticles during their generation. In the decontamination process, bentonite can act as an efficient adsorbent for the contaminants, facilitating their deactivation induced by the presence of the nanoparticles. The bentonite-supported metal/metal oxide nanoparticles were dispersed in an aqueous solution of polyvinyl alcohol (**PVA**), a biodegradable polymeric matrix, which plays an essential role in holding together (and thus "binding") all the components of the decontamination solution (including the entrapped contaminant). The excellent film-forming capacity of **PVA** ensures the formation of peelable films from these decontamination solutions, which facilitates the efficient removal of contaminants from different types of surfaces.

TEM analysis was employed as the first characterization tool to confirm the generation of Cu and Ag nanoparticles in the decontamination solutions (Figure 1). From the images presented in Figure 1A–D, it can be noticed that for the samples containing 1% bentonite (SD5), the CuNPs diameter is around 20 nm, while for the SD6 (containing 1.5% bentonite), the particle size is around 40–60 nm. In the case of SD4 (0.5% bentonite), the presence of independent nanoparticles outside the clay structure was not visible by TEM. Thus, as the concentration of bentonite increased in the samples, larger nanoparticles were observed. Similar behavior was evidenced for the AgNPs (samples SD10, SD11, and SD12). The particle size of the AgNPs increased at a higher clay content. An explanation for the particle size modification can be attributed to the adjustment of the growth process due to the presence of clay, which causes smaller particles to destabilize, due to the clay's inherent electrostatic charge [52], and promotes growth. In the case of the samples containing TiO2, no visible effect on the particle size was observed; the CuNPs' particle size varied, depending on the bentonite content.

**Figure 1.** *Cont*.

**Figure 1.** TEM image of bentonite-supported metal nanoparticles employed in the decontamination solutions: (**A**,**B**) SD5-mag 100,000×, mag 50,000×; (**C**,**D**) SD6-mag 180,000×, mag 50,000×; (**E**,**F**) SD10-mag 180,000×, mag 50,000×; (**G**,**H**) SD11-mag 180,000×, mag 60,000×; (**I**,**J**) SD12-mag 180,000×, mag 29,000×.

> An important parameter that influences both the polymer solution deposition procedure and the type of surface that will undergo decontamination is represented by the dynamic viscosity of the solution. Thus, the solutions presenting a higher viscosity are suitable for application by brush technique, whereas less viscous solutions can be deposited by spray technique. A higher viscosity can also affect the mobility of the molecules influencing the rate of adsorption of the biological compounds, as well as decrease the capacity of the solution to enter the pores and cracks of the surface. Thus, the influence of each component on the viscosity of the solutions was evaluated, and the results are illustrated in Figure 2. Polyvinyl alcohol (**PVA**) has an essential role in film formation (SD1), while the addition of glycerol (SD2) improves the elasticity of the peelable films and bentonite aids in the improvement of the biological/chemical-agent retention inside the film by complexation and adsorption processes (SD3). The effect of glycerol and bentonite on the viscosity of the solution can be explained by the physical interactions that occur between the polymer and these components, such as the formation of hydrogen bonds. Considering these aspects, all the components employed can form hydrogen bonds with water or **PVA**, thus connecting the macromolecules through physical interaction. Consequently, there is an apparent increase in the molecular weight of the polymer, which leads to an increase in

the viscosity of the decontamination solution. Additionally, the variation of the dynamic viscosity of the decontamination solutions containing copper and silver nanoparticles was investigated. Compared with the control sample (SD3), it can be noticed that for all the solutions, similar dynamic viscosity values were obtained, due to the low concentration of nanoparticles.

**Figure 2.** Variation of the dynamic viscosity with shear rate for the decontamination solutions, (**a**) SD1, SD2, SD3, SD4 and (**b**) SD5, SD6, SD7, SD8, SD9, SD10, SD11, SD12.

In order to determine the time required for solvent evaporation from the decontamination solution and film formation, determination of the evaporation rate is essential. The capacity of solvent evaporation (water in this case), after solution deposition, depends on several factors: temperature, humidity, type of surface, etc. but also on the viscosity

and nature of solution constituents. To ascertain these values, parameters such as sample surface and quantity of solution were kept constant. The evaporation parameters are presented in Table S1. As the temperature is increased, the solvent evaporation rate is more predominantly influenced by the chemical composition of the solution, the interaction between the components, and solution viscosity. The measurements taken at temperatures between 25 and 40 ◦C showed that the solution's components influence the evaporation rate, which is due to the intermolecular interaction between the components and water molecules; thus, the stronger the interaction, the longer the interval required for drying.

FT-IR spectroscopy was employed to highlight the formed hydrogen bond between the polymer and glycerol, respectively, as well as the presence of bentonite in the polymer films. The results are presented in Figure 3 and detailed in Figure S1. The broad peaks around 3300 cm−<sup>1</sup> can be attributed to hydroxyl groups from the **PVA** chain, while the values in the range of 1095–1085 cm−<sup>1</sup> indicate the presence of hydrogen bonds formed between **PVA** and glycerol. The weak bands at 3028 and 2919 cm−<sup>1</sup> can be assigned to C–H stretching vibrations. The strong peak highlighted around 1031 cm−<sup>1</sup> has a double meaning: it appears due to the strong vibrations of the C–O bonds of a primary alcohol, and at the same time, it also indicates the presence of Si–O bonds due to bentonite clay. At the same time, an increase in absorbance is observed with the addition of bentonite.

**Figure 3.** FT-IR spectra of the polymer films (SD3, SD5, SD8, and SD11).

The mechanical properties of polymer films are very important for the peeling process and efficient decontamination of surfaces, which requires a high resistance but also a certain degree of elasticity. Figure 4 illustrates comparative stress-strain plots for all the nanocomposites obtained. As can be observed, the addition of glycerol led to higher stress-strain values. In the absence of glycerol, films containing only polyvinyl alcohol (SD1) are more resistant, but they are much too rigid and brittle to be used for surface decontamination. Based on the results of the tensile strength tests, it can be stated that each component modifies the mechanical properties of the films. Moreover, this aspect must be considered when formulating decontamination solutions to obtain the desired

characteristics. Thus, to obtain films easily exfoliated from the surface of interest while avoiding fracture of the composite material, careful selection of components and their ratio is required. The exact values of the mechanical parameters are given in the Table S2. As can be observed, the nanocomposites containing metallic nanoparticles also displayed good mechanical properties. The polymeric films maintained their integrity after the completion of peeling. Thus, when these materials were subjected to low stretching forces (typical for a peeling process) the nanocomposite film had enough mechanical resistance and did not break. The mechanical resistance of the nanocomposites employed for surface decontamination is afforded by the synergistic effect between the reinforcing nanoclay, the polymer matrix, and the glycerol (acting as plasticizer).

The differential thermal analysis presented in Figure 5 allowed the evaluation of the thermal characteristics of the polymer nanocomposite films. The thermal behavior of the film containing only **PVA** (**SD1**) is slightly different than that of the films containing glycerol. Thus, the peak situated at approximately 236 ◦C can be attributed to the melting of the crystalline regions/domains of **PVA**. Comparing SD2 and SD3, very small differences in terms of thermal transitions can be noticed. The first characteristic signal is endothermic for a temperature range between 70 and 150 ◦C (Figure 5A), which can be attributed to the evaporation of water trapped between the polymer chains. The shift of this peak to slightly higher values in the case of SD3 can be explained by the increased interaction due to the presence of bentonite. The second signal between 175 and 260 ◦C, also endothermic, can be attributed to the melting of the polymer (Tm), while the signals after 270 ◦C can be attributed to the polymer degradation process. Similarly, the presence of bentonite (SD3), copper nanoparticles (SD5, SD8), or silver nanoparticles (SD11) in the composition of the polymer films leads to a slight modification of the characteristic temperature response; nevertheless, the responses of all samples are within the abovementioned temperature intervals. The decrease in melting temperature could be attributed to an increase in the thermal conductivity and polymer chain mobility due to the presence of the metallic nanoparticles (Table S3).

#### *3.1. Decontamination Tests*

The decontamination tests were performed to prove and evaluating the efficacy of this BCWA decontamination method. Biological decontamination tests were performed first, using *E. coli*, *Ps. aeruginosa* and *S. aureus* (as simulants for biological agents), and they were followed by chemical decontamination tests run on one real chemical warfare blistering agent, **HD**, and one simulant for neuroparalytic agents, **DMMP**. Figure 6 illustrates the steps taken to perform the decontamination using the herein-reported film-forming solutions.

The method for biological or chemical decontamination consists of the utilization of the synthesized eco-friendly active solutions (containing bentonite-supported nanoparticles) for the degradation/neutralization and entrapment of toxic agents, followed by the exfoliation of the formed film, which contains the degradation products resulting from the neutralization of the targeted hazardous materials.

**Figure 4.** Tensile tests results for the polymer films: (**A**) SD1, SD2, SD3; (**B**) SD4, SD5, SD6; (**C**) SD7, SD8, SD9; (**D**) SD10, SD11, SD12; (**E**) SD3, SD5, SD8, SD11.

**Figure 5.** DTA thermograms for the polymer films (**A**) neat APV, SD1, SD2, SD3 and (**B**) SD3, SD5, SD8, SD11.

**Figure 6.** Decontamination using eco-friendly active nanocomposite peelable coatings: (**a**) decontamination solution, (**b**) decontamination solution is allowed to neutralize the contaminant, (**c**) dried peelable film, (**d**) peeling process and (**e**) decontamination waste.

#### *3.2. Biological Decontamination Tests*

The results obtained of decontamination tests performed on biological contaminants are further detailed. An antimicrobial activity assay generated the MIC and MBC values of the decontamination solutions, displayed in Table 4 and Figure 7A,B. Solutions SD4, SD5, and SD6 revealed the lowest antimicrobial activity against bacterial strains used in the test. Solutions SD7, SD8, and SD9 showed low antimicrobial activity against Gramnegative bacteria (*E. coli*, *Ps. aeruginosa*) and pronounced activity against Gram-positive bacteria (*S. aureus*), MBC being established in this case. Solutions SD10, SD11, and SD12 showed stronger antimicrobial activity against Gram-negative bacteria, with MBC values established, but lower than those presented for Gram-negative bacteria for solutions SD7, SD8, and SD9.


**Table 4.** Minimal inhibitory concentration (MIC) and Minimal bactericidal concentration (MBC).

\* Under the test conditions, the antimicrobial activity of the solution against the *E. coli* micro-organism could not be highlighted.

**Figure 7.** ((**A**) *S. aureus* ATCC 6538) MIC determination against *S. aureus* ATCC 6538 observed from broth microdilution assay using MH broth and resazurin (columns from left to right: NC, SD4, SD5, SD6, SD7, SD8, SD9, SD11, SD10, SD12, SD3 (Bk), PC). and (**B**) *E. coli* ATCC 8739.observed from broth microdilution assay using MH broth and resazurin (columns from left to right: NC, SD4, SD5, SD6, SD7, SD8, SD9, SD11, SD10, SD12, SD3 (Bk), PC).

The inhibition of the bacterial strain growth could be explained by specific interactions of nanoparticles with the cell envelope of micro-organisms [53]. When nanoparticles are small enough, they can penetrate membrane pores. Nanoparticles that can enter the cell membrane interact with bacterial enzymes, damaging the cell [54]. Some nanoparticles interact electrostatically with the bacterial membrane, and reactive oxygen species are generated, leading to the disruption of the membrane and DNA damages [55].

A time-kill assay was performed on *E. coli* and *S. aureus*. After the proposed contact times (2 h and 24 h) between strains and bentonite-supported nanoparticle solutions, bacterial growth was evaluated. In the case of the time-kill assay, it is observed that after 2 h of contact, all the decontamination solutions showed activity against both microorganisms. After periods longer than 2 h of contact, in the case of *E. coli* strains, an increase in the number of CFU/mL was observed. Most likely, in the case of this bacteria, nanoparticles adhered to the surface of the cells or penetrated inside the membranes and were blocked. It is known that nanoparticles containing Cu, Zn, and Ti ions bind to

negatively charged membranes (such as *E. coli*) [56]. This would explain the survival rate and resumption of the growth and division cycle. Considering that after 2 h of contact, an accelerated increase in the number of CFU/mL was observed, only the values recorded at time t1 (2 h of direct contact) were represented graphically (the results are illustrated in Table 5, Figures 8 and S3), highlighting their antimicrobial activity during the short contact period. In the case of S. aureus, the activity is more pronounced, with observed antimicrobial activity even after 24 h in the case of SD7, SD8, SD9, SD10, SD11, and SD12 (results illustrated in Figure S4). The substrate solution (BK) has a slight activity on *E. coli* but no activity on *S. aureus*.


**Table 5.** Bacterial cell population decrease (%) after 2 h of contact with decontamination solutions.

**Figure 8.** Bacterial cell population decrease (%) after 2 h of contact with decontamination solutions.

Rutala et al. [57] showed that the use of soap and water can sometimes be less efficient due to their lower microbial reduction capacity (≤80% reduction, in comparison with a phenolic disinfectant, which offers 94–95% reduction) and also due to the possibility of contamination of the soap solution. However, a few hours later, the bacterial count was nearly back to the pretreatment level [57]. In an Ayliffe et al. study [57,58], bacterial contamination of soap and water without a disinfectant increased from 10 CFU/mL to 34,000 CFU/mL after cleaning a hospital ward. If the soap solution or the mop are reused, contamination will, in fact, be transferred from one room to another.

The use of strippable coatings offers the advantage of avoiding these re-contamination incidents like the ones described above because on each contaminated surface, a new coating is formed, and after removal, it can be compacted and sealed in small containers dedicated to biological waste. As a conventional substitute for the classical soap and water, disinfectants significantly improved microbial removal when a conventional string mop was used (95% vs. 68%) [57], but using a microfiber mop instead of the conventional mop

could also prevent the possibility of transferring microbes from room to room if a new microfiber pad is used in each room. By comparing the classical decontamination methods with the advantages of the strippable coating method, coupled with the DF values obtained for our decontamination solutions, we can affirm that this new method, based on peelable films, ensures sufficiently high values of microbial reduction while bringing the advantage of consisting of eco-friendly materials.

To evaluate the efficacy of biological contaminant removal from the targeted surfaces, controlled contamination of Petri dishes with *E. coli* and *S. aureus* was performed, followed by the addition of the decontamination solution. At the end of the curing process, the obtained nanocomposite coatings were easily peed off (Figure 6), and the decontaminated surface was further investigated to evaluate the decontamination efficiency.

The surfaces of Petri dishes were contaminated with portions of 10 μL suspension of *E. coli* (5 × 103 CFU/Petri dish) and *S. aureus* (7 × 103 CFU/Petri dish). Following the application of the decontamination solutions and removal of the peelable films, the number of residual micro-organisms on the targeted surface was assessed by cultivation in culture media (MHa). The effectiveness of the biological decontamination can be expressed utilizing the decontamination factor (DF). The decontamination factor can be calculated by the following equation:

$$\text{DF} = 100 \times (\text{C}\_{\text{i}} - \text{C}\_{\text{f}}) / \text{C}\_{\text{i}} \tag{2}$$

where Ci represents the contamination level before applying the decontamination solution and Cf reflects the residual contamination [6]. Table 6 presents the DFs obtained for *E. coli* and *S. aureus*, and to facilitate comparison, Figure 9 summarizes all these values.

**Table 6.** Efficacy of removal of *E. coli* and *S. aureus* strains from surfaces.


**Figure 9.** Biological decontamination efficacy.

The efficacy of removal for the tested micro-organisms varies (93% < DF < 97%). Therefore, we can affirm that these polymeric decontamination solutions represent a useful tool for biological decontamination of surfaces. Basically, biological decontamination occurred through two mechanisms: the first one consists of the entrapment of micro-organisms in the polymeric matrix of the nanocomposite due to the excellent adsorptive properties of bentonite nanoclay; and the second one consists of the active inhibition of the activity of micro-organisms with the aid of the antimicrobial effect of the bentonite-supported Ag, Cu, and TiO2 nanoparticles present in the decontamination solutions. Therefore, even if some of the decontamination solutions did not show remarkable antimicrobial activity, they can still be successfully used for decontamination as they have a great potential for entrapping and sealing the biological contaminants inside the polymeric matrix of the nanocomposite film obtained following the evaporation of the solvent (water). The increased stability of the peelable films herein reported could ensure minimization of risks associated with biological contamination, ensuring immediate decontamination by first covering and then capturing the contaminant inside the polymeric film. The peeled nanocomposite films containing the entrapped contaminant can be further subjected to analysis for the identification and evaluation of the concentration of the contaminant.

#### *3.3. Chemical Decontamination Tests*

Chemical decontamination tests followed the biological decontamination tests. Since the biological decontamination tests showed that the solutions based on bentonite-supported silver nanoparticles displayed the best results, we employed only these solutions (SD10, SD11, and SD12) for the tests performed on real chemical warfare agents. This choice was also influenced by safety concerns, as working with real warfare agents imposes higher risks and requires specially trained personal. Thus, we tried to limit the number of experiments by employing only these three decontamination solutions, and we maintained the relevant steps for the decontamination procedure in order to obtain accurate information. For the same reasons, we also tested a simulant. Chemical decontamination of metallic surfaces measuring 10 cm<sup>2</sup> was accomplished in three stages: the first one consisted of the controlled contamination of the metallic coupons with **HD** and **DMMP** (10 mg/10 cm2), respectively; the second one consisted of applying the decontamination solution on the contaminated surface and allowing it to neutralize the toxin and to form the film by evaporation of the solvent at room temperature (25 ◦C); the last step consisted of DCM extraction of the decontaminated surface and of the peeled film. The results obtained for **HD** and **DMMP** are presented in Table 7, Figure 10. Some relevant chromatograms were selected and are shown in Figures S6–S9.

Decontamination factor was calculated according to the following equation, also described in the *Methods* section: DF = 100 × (C0 − Cf)/C0, where DF is the decontamination factor, C0 is the initial toxic concentration found on the tested metallic surface, and Cf is the final concentration found on the decontaminated metallic surface, reflecting the residual contamination (according to the area of the characteristic peak of toxin). The values obtained are illustrated in Figure 11.


**Table 7.** Evaluation of chemical decontamination efficacy with the aid of GC-MS results.

**HD**—sulfur mustard; S0\_HD—sulfur mustard blank; **DMMP**—dimethyl methylphosphonate; S0\_**DMMP** dimethyl methylphosphonate blank; M—samples extracted in DCM from the metalic surfaces after decontamination; P—samples extracted in DCM from the nanocomposite film after decontamination.

**Figure 10.** Chemical decontamination efficacy: decontamination factors.

As it can be noticed in Table 7 and Figure 10, the SD11 decontamination solution achieved the highest decontamination factor for **HD**. In the case of **DMMP**, employed as simulant for nerve chemical agents, the decontamination factors obtained were much

higher, with all decontamination solutions (SD10, SD11, and SD12) being highly efficient. It is well known that **HD** is more difficult to decontaminate due to its chemical structure, as it tends to establish stronger interactions with the metallic substrate on which it is deposited. Even so, SD11 managed to efficiently remove more than 90% from the contaminated surface. The other decontamination solutions were not so efficient, probably due to their composition. We can presume that SD10 was not able to entrap the same amount of toxin, probably due to the lower content of bentonite, which was reflected in a higher residual contamination ≈ 21.72%. On the other hand, even if SD12 had more bentonite and theoretically greater adsorptive capacity, having a slightly higher viscosity and less NP active centers (bigger NPs and lower specific surface) with decreased mobility in a more viscous media, this could have led to much lower DF values.
