*Article* **Colorimetric Paper-Based Device for Hazardous Compounds Detection in Air and Water: A Proof of Concept**

**Valeria De Matteis 1,\*, Mariafrancesca Cascione 1, Gabriele Fella 1, Laura Mazzotta <sup>2</sup> and Rosaria Rinaldi <sup>1</sup>**


Received: 7 August 2020; Accepted: 23 September 2020; Published: 25 September 2020

**Abstract:** In the last decades, the increase in global industrialization and the consequent technological progress have damaged the quality of the environment. As a consequence, the high levels of hazardous compounds such as metals and gases released in the atmosphere and water, have raised several concerns about the health of living organisms. Today, many analytical techniques are available with the aim to detect pollutant chemical species. However, a lot of them are not affordable due to the expensive instrumentations, time-consuming processes and high reagents volumes. Last but not least, their use is exclusive to trained operators. Contrarily, colorimetric sensing devices, including paper-based devices, are easy to use, providing results in a short time, without particular specializations to interpret the results. In addition, the colorimetric response is suitable for fast detection, especially in resource-limited environments or underdeveloped countries. Among different chemical species, transition and heavy metals such as iron Fe(II) and copper Cu(II) as well as volatile compounds, such as ammonia (NH3) and acetaldehyde (C2H4O) are widespread mainly in industrialized geographical areas. In this work, we developed a colorimetric paper-based analytical device (PAD) to detect different contaminants, including Fe2<sup>+</sup> and Cu2<sup>+</sup> ions in water, and NH3 and C2H4O in air at low concentrations. This study is a "proof of concept" of a new paper sensor in which the intensity of the colorimetric response is proportional to the concentration of a detected pollutant species. The sensor model could be further implemented in other technologies, such as drones, individual protection devices or wearable apparatus to monitor the exposure to toxic species in both indoor and outdoor environments.

**Keywords:** PAD; environmental monitoring; colorimetric detection; water; atmosphere

#### **1. Introduction**

In the last decades, due to the increase of industrialization activities, the release of hazardous materials in the atmosphere, water and soil has raised many concerns about their impact on living organisms [1]. Metals and heavy metals together with gaseous organic compounds are the most widespread toxic elements due to their ability to enter the living organism by different routes, such as inhalation and ingestion [2,3]. Then, they can enter the food chain, integrating into enzymatic processes with the consequence to boost various diseases and inflammation processes onset [4].

Cu(II) and Fe(II) are transition metals having a key role in several physiological pathways, such as fetal growth, brain development, cholesterol metabolism and immune function [5–7]. Cu(II) represents one of the main components of the PM 2.5 produced by the road dust emissions, allowing its easy penetration into the organisms' body [8].

In addition, the ecological risk deriving from Cu(II) exposure is a problem in European saltwater environments [9,10]. Cu(II) can be toxic to aquatic life at concentrations approximately 10 to 50 times higher than the tolerated range [11]. In addition, humans can adsorb a great amount of Cu(II) from drinking water, food, air and supplements, reaching a daily absorption of 1.85 mg [12]. In order to understand the collateral effects of the Cu(II), the US National Toxicology Program (NTP) exposed B6C3F1 mice to the five concentrations of Cu(II) (76, 254, 762, 2543, 7629 mg Cu/L) to [13] for 13 weeks observing the organs weight loss and animals death, at the higher concentrations tested. These results were consistent with another study in which the same toxicity was observed in female and male mice using 762 mg/L of Cu(II). Additionally, Fe(II) triggered adverse effects in vivo by acute toxicity induction [14]. In aquatic environments, Fe(II) boosted the growth default of aquatic organisms at a concentration of 1 mg/L [15]. In addition, in some European countries such as Lithuania, people were exposed to high levels of Fe(II) due to the contamination of groundwater that overcome the permissible limit established by the European Union Directive 98/83/EC, related to the quality of drinking water [16]. Regarding the volatile compounds pollution, NH3 is one of the major manufactured industrialized soluble alkaline gases on Earth [17]. NH3 originates from both natural and anthropogenic sources, in particular from the agricultural industry, high-density intensive farming practices as well as fertilizer applications [18]. According to the Agency for Toxic Substances and Disease Registry, the concentrations of NH3 in the environment are very variable due to its continuous recycling and its internalization in biosphere. Therefore, it is possible to find different natural NH3 levels in the soil (1–5 ppm), in air (1–5 ppb) and in water (approximately 6 ppm) [19]. The NH3 smell can be identified by humans at concentrations greater than 5 ppm; at 30 ppm and with an exposure time of up to 2 h, human volunteers underwent slight irritation, whereas strong effects were recorded up to 500 ppm [20]. However, NH3 lethality requires higher concentrations [21]. In addition to NH3**,** also some kinds of carbonyls which constitute the motor vehicle exhaust, such as C2H4O are toxic air contaminants, particularly dangerous for living organisms [22,23]. Woutersen et al. [24] used Wistar rats to study the toxic effect of C2H4O administered in air (6 h/day) at three concentrations (750, 1.500, 3.000 ppm) for more than a year. All the concentrations tested induced the increase of nasal tumors incidence with remarkable impact especially at higher concentrations. Other evidences suggested that the C2H4O administration (1.650–2.500 ppm) for more than two years (7 h/day) induced tracheal, but not nasal, tumors in Syrian golden hamsters [25]. Then, the study of these compounds in polluted areas is a key factor to control the exposure rate.

Today, several analytical techniques are available for the detection of toxic analytes. However, many of them are not affordable due to the expensive instrumentations and high reagent volumes required. On the contrary, point-of-care and easy-to-use analysis provide results in a short time, preventing the production of an elevated amount of waste [26]. In addition, they can be employed in resource-limited environments and developing countries where pollution is uncontrollable and not regulated with specific rules.

In particular, paper is the best choice to develop sustainable devices [27]; it is considered a valid alternative to traditional materials due to its ease of fabrication, satisfactory levels of sensitivity, specificity, low cost, lightweight, versatility, being easily portable and low reagent consumption requiring [28,29]. The paper-based analytical devices (PAD) can work following the principle of color change in the presence of specific target analytes [30]. The sensitivity and specificity of the assay are dependent on an interaction between the target analyte and the surface of the PAD due to the functionalization of cellulose fibers [31]. The paper surface can be functionalized by different molecules, such as chemoresponsive dyes, nanoparticles (NPs) and biomolecules (antibodies, aptamers, nucleic acids) [32–35]. Xi et al. [36] prepared a paper device based on Pb(II) metal-organic nanotubes characterized by a large {Pb14} metallamacrocycle, to detect H2S based on the fluorescence "turn-off" response. However, the fabrication of nanotubes and the general technique required specific scientific competences and elevated costs; moreover the toxicity of nanotubes, is not negligible [37]. Maity et al. [38] used perovskite halide (CH3NH3PbI3) to achieve a thin-film sensor fabricated on a

paper by a growth process able to detect NH3 gas by a color change from black to yellow. Despite the effectiveness of this device, the H3NH3PbI3 is chemically unstable and toxic for living organisms. [39]. Then, the disposal of the device could present a serious problem. In a recent work [40], a microporous cellulose-based smart xerogel bromocresol purple was used into cross-linked carboxymethyl cellulose to detect NH3 by a colorimetric response. The authors performed a freeze-drying process to obtain the xerogel with a low limit of detection.

In these PADs, the colorimetric shift can be evaluated by colorimetric assay, as a result of the interaction with the ligand. In general, the PADs sensing areas are fabricated by the printing method using a wax printer [41]. The results obtained can be directly interpreted by the naked eye together with the spectrophotometer analysis. In the last years, the use of smartphones to detect color change has been developed [42–44]. Therefore, its use showed some limitations regarding the low lighting conditions that prevent the smartphone camera exploitation [44].

In this work, we developed an effective PAD suitable to detect different contaminants, namely Fe(II) and Cu(II) cations (Fe2<sup>+</sup> and Cu2<sup>+</sup>) in water and NH3 and C2H4O vapor in air. The design and fabrication of the sensor did not require specific instrumentations. In particular, for metals detection, only a wax pen able to design the specific areas of chemical interaction was required, without the use of a wax printer. We functionalized the paper (Whatman filter paper) using different analytes capable of reacting with metallic ions and gaseous substances, allowing a specific response; the aim of this process was to develop calibration curves to correlate the obtained color to the concentrations of toxic compounds. The results were easily interpreted using a digital scanner and ImageJ. The tests achieved using intermediate concentrations suggested the sensitivity and reproducibility of the PAD, making it a powerful tool to detect hazardous materials in different mediums without the use of sophisticated technologies.

#### **2. Materials and Methods**

#### *2.1. Ammonia Detection*

#### 2.1.1. Reagents

Whatman filter paper n.1 (thickness 180 μm), ammonium hydroxide (NH4OH, 28%), hydrochloric acid (HCl), Aniline (C6H5NH2) and ammonium persulfate (NH4)2S2O8 were purchased from Merck.

#### 2.1.2. Functionalization of Whatman Paper for Reversible Ammonia Vapor Detection

The reversible colorimetric detection of gaseous NH3 was realized by coating Whatman filter paper with polyaniline (PANI) film, achieved by C6H5NH2 polymerization (2.5 g/L) in the presence of HCl (1 M) and (NH4)2S2O8 (0.125 g/L) at room temperature [45]. Briefly, (NH4)2S2O8 solution was added dropwise into the C6H5NH2 solution under stirring (1000 rpm). The two compounds were in a volume ratio of 1:1. After 3 min, half of the colorless reaction mixture was immediately added into a silicon funnel, where a piece of round filter paper (c.a 2 cm) was placed and fixed. Then, the remaining solution was slowly suction-filtered through the filter paper, and the unused volume was left in the dark for approximately 1 h. During this time, the solution slowly turned light blue. After this step, the solution was again filtered and then, the paper was carefully washed with Milli-Q water. Finally, it was left to air dry until the emerald green filter paper was achieved. The functionalized paper was exposed to different concentrations of NH3. The schematic representation of this procedure is represented in Figure 1a.

**Figure 1. Schematic NH3 (a) and C2H4O (b) paper sensor fabrication procedure**: (**a**) **1**. Half of the colorless reaction mixture was immediately suction-filtered into a silicon funnel where a filter paper (*white circle*) was placed. **2.** The remaining part of the solution was left to stand for 1 h. During this time, the solution turned light blue. **3.** The solution was filtered (II suction-filtration) through the filter paper, in order to induce the polyaniline (PANI) deposition. After several washes and air flow drying, the formation of emerald green filter paper (*green circle*) was completed. **4**. The emeraldine green filter paper turned into a blue emeraldine base (*blue circle*) as a result of NH3 vapor exposure. (**b**) **1.** The methyl red and methyl red sodium Salt were added to the mixture. The color solution turned into red-orange and was stirred for 1 h. After this time, NaOH was added, resulting in a color change to yellow. The solution was left to stand for 1 h. **2**. The solution was transferred in a petri dish and the filter paper was immersed in it for 1 h. The filter paper was dried overnight in the dark at room temperature. The formation of methyl red filter paper (*yellow circle*) was completed. **3**. The methyl red filter paper turned into red (*red circle*) as a result of C2H4O vapor exposure.

#### 2.1.3. Construction of Calibration Curve by Colorimetric Response to Ammonia Vapor

Glass vials were used to detect NH3 vapor exposure. In each vial, 10 mL of NH3 solution was added at different concentrations (100, 300 500 and 1000 ppm) to achieve a standard curve. Small PANI-deposited filter paper pieces were fixed on the necks of the vials in order to expose them to the vapor generated from the corresponding NH3 aqueous solution for a few seconds. The control was represented by pure NH3. After this time, the paper was immediately removed and analyzed by a scanner (Samsung SCX-3400 series (USB002)) acquiring the color change after NH3 vapor interaction.
