Naked-Eye Optical Recognition of Ammonia Vapor and Melamine in Water Using a Fluorophore Appended Polymer Matrix

The generation of solid-state emitters is a challenge due to the intrinsic aggregation-caused quenching feature of the fluorophores. A conformationally twisted pyridyl π-conjugate as a solid-state emitter is appended with well-known and inexpensive poly(methylmethacrylate) [PMMA] to afford a handy, portable, and reusable solid-state emitting polymer matrix. Entrapment of the probe is noticed through non-covalent interactions, resulting in a green-emitting platform. It quickly accepts a proton upon acid vapor exposure and switches emission from green to red with a significant 107 nm redshift. This shift is reversible with red to green emissions while exposed to base vapor. Thus, polymer-blended, homogeneous red-emitting pyridyl salt is employed as potential material to detect various basic vapors optically. Among different bases, naked-eye detection of essential analytes such as ammonia vapor and melamine shows potential demands. Hence, we have established an easy detection of ammonia vapor and aqueous melamine as low as 2.5 and 0.126 ppm, respectively, using this solid-state emitter that displays an emission color change with an enhancement of emission intensity even in an aqueous solution.


Introduction
Cost-effective fluorescence (FL)-based detection of various analytes has drawn huge attention due to its high sensitivity, rapid response time, robust signal-to-noise ratio, and easy accessibility [1]. Further, the application of fluorescencebased optical materials is reaching into almost every sector, including optoelectronics, biological and biomedical sciences, clinical strategy, environmental and agricultural domains, industry, and academia [2][3][4][5][6][7]. Especially, fluorescence-based techniques are emerging as a very reliable tool to monitor the analytes related to health hazards and food quality assessment [8].
Although there are uncountable sources of hazardous materials, sickness due to organic amines, especially ammonia vapor, has claimed a toll in the present scenario as it exists in different areas such as industry, mines, laboratories, etc. Ammonia is highly toxic and corrosive to the environment [9,10]. The frequent news on ammonia leakage is quite common due to corrosion of the high-pressure tankers and operational issues (seal leak). Inhalation of hazardous ammonia, even in lower concentrations, can cause severe damage to the eyes and respiratory systems [11]. Thus, easy detection of ammonia leakage is a pressing need. Typical color change of litmus paper with ammonia vapor has limitation to use only inside the plant, not outside, where a sprinkle of water droplets can spoil the paper. Further sensing the pungent smell of ammonia can create confusion in confirming the nature of the gas. Thus, a sustainable solution to detect ammonia vapor leakage by a distinguished color change, visible through the naked eye, would be a meaningful solution.
On the other hand, high nitrogen-containing amine and melamine (2,4,6-triamino-1,3,5-triazine) are used in paint, plastic, and adhesive industries [12]. Because of its high nitrogen contents, melamine is illegally used in milk powder and other protein-containing foodstuffs. Melamine is also toxic to humans on long-term consumption due to the formation of cyanuric acid via hydrolysis of melamine [13]. Hence, the recognition of melamine in food is essential. Although there are various techniques to detect such analytes, most are time-consuming and expensive [14].

3
The FL-based technique would be a simple, reliable, and handy tool for detecting melamine even in water and milk samples. Typically, the FL-based detections of amines are performed in the solution state, either by FL quenching or enhancement, governed by static/dynamic quenching or controlling the photoinduced electron transfer process [15]. Being operationally convenient, great efforts have been devoted to recognizing amine vapors using solid-state emitters with the help of expensive metal-organic frameworks [16] or conjugated functionalized polymeric backbones [17]. Developing solid-state emitters is challenging by disabling the aggregation-caused quenching (ACQ) effect. Most analytes cannot bind with the fluorophore in a solid state. Although the MOF [18] and polymeric materials [19] were excellent candidates for recognizing amine vapor, the synthesis and molecular level understandings are significantly lacking.
In this direction, we have recently designed and synthesized (Scheme 1) a thiophene-linked anthracenyl π-pyridyl conjugate conveniently as a solid-state emitter [20]. Such an emitter is suitably blended with the most inexpensive and accessible polymethyl methacrylate (PMMA) to generate an emitting polymeric platform to detect ammonia vapor (~ 2.5 ppm in Helium) and aqueous melamine solution (0.126 ppm) repeatedly. PMMA supports to produce the strong, and lightweight fluorescent material for a handy and sustainable solution to detect ammonia and melamine with decent sensitivity. This polymeric material was characterized with 1 H-NMR and Scanning Electron Microscopic (SEM) images. The emission color change upon treatment with those analytes is monitored by fluorescence spectroscopy.

Materials and General Conditions
The reactions were carried out in hot air oven-dried glassware under a nitrogen atmosphere. The aldehyde and K t OBu were purchased from Sigma Aldrich. Tetrahydrofuran (THF) was distilled with sodium metal and benzophenone. Dry dichloroethane (DCE), methanol, hexane, ethyl acetate, and hydrochloric acid (HCl) were purchased from Sisco Research Laboratories (SRL) and used without further purification. Millipore water was used for the experiments. Column chromatography was performed by dint of Silica gel bed of 230-400 mesh. Reactions were monitored by performing thin-layer chromatography on silica gel pre-coated 60 F254 plates (Merck & co.) under UV-light (~ 365 nm). The NMR spectra were recorded at an ambient temperature (ca. 20 °C) in CDCl 3 solution and calibrated against the characteristic solvent peaks (7.26 ppm in 1 H, 77.0 ppm in 13 C NMR). The chemical shifts were checked in ppm by denoting the multiplicities as s (singlet), d (doublet), t (triplet), and m (multiplet). All other experiments were carried on at room temperature (28-30 °C). The FE-SEM images were captured in the 1 μm range for each sample utilizing Apreo S with Leica Ultra Microtome anthEM UC7 (Sputter Coater).

Analytical Methods
The solid-state absorption spectra were recorded with a JASCO-500 spectrophotometer when the solid-state emission spectra were recorded with a fluorimeter (Fluorolog, HORIBA) by exciting the samples at their corresponding absorbance λ max. The photophysical studies are carried out using solid samples and also the fluorophore appended polymer matrix. The absolute quantum yield (Ф f ) was evaluated for all the solid samples with the help of a calibrated integrating sphere method withholding a fluorimeter (Fluorolog, HORIBA). The absolute errors with an error of ~ ± 2%. SEM analysis was carried out using field emission scanning electron microscopy (FE-SEM, FEI, Apreo).

Synthesis of AThio4P and AThio4PH
The anthracenyl π-conjugate AThio4P and its protonated form were synthesized following the existing literature [20,21].

Preparation and Characterization of Fluorophore Appended Polymer Matrix
The synthesized compound AThio4P is a solid-state green emitter under 365 nm UV lamp illumination. The alkaline nature of the pyridyl core with pK b = 8.8 can readily accept the proton and release it under a suitably sensitized chemical environment. Among many variations on pyridinyl N-atom position and (hetero) aryl substituents, we recently realized reversible AThio4P to AThio4PH (in response to Scheme 1 Synthesis of the probe (HCl is used to protonate) acid vapor) as a quick and sensitive solid platform to display FL-switching with a high color contrast from green (λ em = 498 nm) to orange (λ em = 606 nm) [20]. Thus, the reversible nature could identify the amine analytes instantly by a visible naked-eye FL-switching under 365 nm light.
The solid-state emitting AThio4P (5 mg, Ф f = 6.78%) is mixed with PMMA (30 mg or 60 mg) in 300 μL MeCN + 500 μL propylene carbonate (binder) and sonicated for 20-30 min. Next, the solution was laid down on a glass slide and allowed to dry at room temperature (27 °C). After 2-3 days, the dried polymer-appended fluorophore matrix was given a different shape for attractive usage. The fluorophore-blended polymer exhibits green emission at λ em = 493 nm with Ф f = 5.76%. Notably, both AThio4P: PMMA 1:6 and 1:12 mixing provided the green-emitting solid and durable platform. To use a small amount of probe, we preferred a 1:12 ratio for such detection studies. We have investigated the 1 H NMR spectrum for normal probe and compared it with PMMA + propylene carbonate mixture and the pure probe (Fig. S1). The 1 H-NMR reveals unchanged chemical shifts of the probe after the blending, indicating a weak van der Waals force involvement between the probe and the polymer. In addition, the SEM (Scanning electron microscopic)-images are captured before and after adding the probe with the polymer (Fig. 1). The surface of polymerblended fluorophore becomes more homogeneous and cleaner for the 1:12 ratio than for the 1:6 ratio (Fig. 1A, B). The fluorophores are segregated (Fig. 1Ac) on the surface after fuming with acid (vide infra). Thus, the probe is embedded with the polymer via various intermolecular weak noncovalent interactions.

Photophysical Studies in the Solid State for Ammonia and Melamine Detection
This red-emitting polymer (Ф f = 1.38%) is utilized to detect ammonia (pK b = 4.75) vapor and 10 -5 M aq. melamine (pK b = 9.0) solution. An immediate blue-shifted emission (107 nm switch) with an increment in the emission of the polymer matrix (Ф f = 2.77%) is detected by exposing ammonia gas to the acid-fumed polymer platform because the ammonia is capable of deprotonating the probe and bringing back the original form (Fig. 2a). As the pyridyl core is attached with electron-rich anthracenyl π-conjugate, a higher electron density on the pyridyl ring is speculated and, thus, becomes relatively more basic. Hence, this probe will be a stronger base than melamine, which disfavors deprotonation from the probe. However, amine groups and pyridyl nitrogen could interact with the probe's pyridyl proton and alter the emission. Thus, upon dipping the red-emitting polymer into 10 -5 M aq melamine solution, a shift of 24 nm is noticed with a resulting λ em = 576 nm and Ф f = 2.33%. However, the emission color can be easily distinguished from red to orange, as demonstrated in Fig. 2b.
Next, we made a simple strategy to recognize NH 3 vapor and examine the leakage detection. Commercially available NH 3 [~ 2.5 ppm; 5% in Helium (balance gas, v/v)] was taken in a large size balloon and attached with a 1 mL syringe with a tiny needle of 0.60 mm diameter. The ammonia passing from a balloon was controlled by releasing the gas flow from a balloon. The green-emitting polymer (Fig. 3a) is initially converted to a red-emitting one (Fig. 3b) under acid vapor. Subsequently, we allowed passing NH 3 through the syringe on the red-emitting polymer, resulting in a green-emitting solid rapidly (Fig. 3c), which was detected visually by illuminating a 365 nm UV lamp. When we exposed NH 3 on one surface of a diamond-shaped polymer, an immediate change in emission was observed, while the other side was the original red color. The SEM image of the acidified polymer revealed a microphase separation (Fig. 1c), possible due to the formation of relatively polar hydrophilic pyridinium salt. Thus, the emission is also shifted, and the protonation enhances the molecular π-conjugation, generating a red emission [20] It is pertinent to mention that AThio4PH powder could instantly recognize ammonia vapor with a 104 nm spectral blue shift from 606 nm to 502 nm (Fig. 4a). This color change is also dictated by the Commission Internationale d'Elcairage (CIE-1931) chromaticity diagram (Fig. 4b-c).
To investigate the stability of AThio4PH powder under gaseous NH 3 , AThio4PH powder was exposed to ammonia vapor continuously for 15 days in an ammonia-containing closed glass vial [18], and after 15 days the NMR spectra were recorded (Fig. S4). Upon exposure to ammonia, the formation of NH 4 Cl was identified on the surface of AThio4P and confirmed in the 1 H NMR spectra. A subtle shift of aromatic protons could be attributed to the overall change in the chemical environment. Further, the intact green emission at 505 nm was noticed for the probe, illustrating its long-term stability under ammonia exposure (Fig. S5).    Next, the red-emitting polymer was explored to detect dilute ammonia solution with a control experiment using 100% water. No significant FL-switching was observed in water (Fig. 5a), but the floated polymer matrix (Fig. 5b) could recognize 11.3 ppm aqueous ammonia solution (Fig. 5c). The polymer matrix exhibited visually recognizable emission switching once dipped into the solution for 1-2 min (Fig. 5d-f). Thus, this polymeric system is superior to the typical fluorophore-coated paper strips in detecting these important analytes in water.
Subsequently, the polymer probe was tested to detect melamine of different strengths. Upon treatment with 10 -5 M aq. melamine solution, a 24 nm blue shift (red to orange; 600 to 576 nm, Fig. 2b) occurred to signify a sort of interaction of the probe with melamine. Being a weaker base, melamine won't be able to snatch the proton from the probe, but other supramolecular interactions can change the position of the proton and, thus, the emission property. Numerous weak noncovalent interactions can alter the conformational behavior of a molecule. If there is any lack of coplanarity, a blueshifted emission will result. A quantitative analysis of the response of melamine was performed with AThio4PH powder by adding a few drops of 10 -5 M aq. melamine solution onto the AThio4PH powder. It turns into yellowish orange (606 to 572 nm) within 1-2 min, and such color-contrasting behavior gradually diminishes while decreasing the concentration of melamine. Thus, the solid AThio4PH powder could detect 10 -5 M aq melamine solution with 34 nm spectral blue shift and switch the powder's color from red to yellowish orange with ϕ f = 2.70%. The color change was still discernible at 10 -6 M and 10 -7 M aq melamine solution with 28 nm and 25 nm spectral blue shift. But the spectral shift was not impressive for 10 -8 M aq melamine solution (Figs. 6 and 7), and the reason was stated before. Hence, melamine could be detected by AThio4PH powder through the naked eye from 1.3-0.126 ppm. Notably, the food safety authorities of different countries have set certain limits of melamine content in foods varying from 50 ppb to 5 ppm to avoid health risks to their citizens [22]. The polymer blended AThio4P was acid-fumed, and the resulting red-emitting polymer matrix was tested to sense melamine in milk samples for real applications.
Further, the emission color remained unchanged upon dipping the red-emitting polymer matrix into marketavailable milk (Fig. 8). However, different concentrations of 10 -5 M, 10 -6 M, and 10 -7 M melamine-containing milk sources were prepared, and the polymer matrix was dipped into this contaminated milk. The color change was observed within a few min (2-7 min, Fig. 9) under UV light with the naked eye, but the color change was not much exciting at 10 -6 M and 10 -7 M solution. Thus, this polymeric probe became helpful in detecting melamine in food items in a convenient and economical route, avoiding expensive methods [23]. Most fluorescence-based techniques are reported in the solution state, and they need a fluorimeter to identify. This solid-state emitting polymer matrix is portable and reusable to distinguish the FL-switching easily after dipping into the solution or holding against the gas/vapor with the help of a portable UV lamp.
Precisely the melamine contains three -NH 2 groups linked to a triazine core. To understand the role of these functionalities in melamine recognition, a few more compounds with electronic diversities are screened with a powder AThio4PH probe. Only pyridine (pK b = 8.8) and aniline (pK b = 9.38) (10 -5 M solution) were initially tested and showed a similar effect of ~ 26 nm blue shifts as melamine (pK b = 9.0). When 2-aminopyridine (pK b = 7.20) solution  was exposed, only 13 nm switching was noticed (Fig. S6). The interactions between the nitrogen lone pair of pyridines and the hydrogens of the -NH 2 group could cause this outcome. Further, weaker base 4-nitroaniline (pK b = 12.99) brings the effect of a 21 nm shift. (Fig. S6 and Table S1). This study demonstrates that the change in the blue shift for melamine detection is not only guided by the basicity of the analytes. It is more likely directed through a reversible proton exchange and weak noncovalent interactions, and therefore the shifts are not that prominent compared to the response from ammonia.
The effect can be further supported by the almost autoreversible emission from powder AThio4PH while sensing 10 -5 M aq melamine solution. The FL-switching from 606 to 572 nm was noticed immediately, but gradually the emission λ max started regaining its original position and reached 585 nm within 5 min. Thus, the actual red emission can be almost retrieved within a day (Fig. 11a). Therefore, such weak interactions are highly reversible and can be regenerated. As reported earlier, melamine can exist in two isomeric forms, where the aromatic form is relatively more stable, as shown in Fig. 10 [24]. In melamine, nitrogen atoms possess superior basicity in azine (C = N) than -NH 2 groups. The lone-pair on -NH 2 is delocalized within the ring, resulting in a dense negative charge on the triazine nitrogen atoms. Thus, the pyridyl protons from the fluorescent probe would have a significant tendency to interact with azine N-atoms (See the graphics Fig. 10). Such interactions would perhaps enable a partial proton transfer between the probe and melamine through a dynamic equilibrium, leading to display of a blue-shifted switching. Even, the H-bonding interactions between the probe and -NH 2 functionality can also play a crucial role. However, only 34 nm reversible blue-shift indicates the presence of mostly weak to moderate interactions.
The word "mine" is drafted on the red-emitting platform using an aq melamine solution (10 -5 M) with the help of a wooden nib. It was visible with a slightly different orange coloration, which turned into the original red emission after a day (Fig. 11b).

Conclusions
The pyridyl-based solid-state emitting probe is blended with PMMA to afford a sturdy and handy platform to respond against acid vapor by fluorescing a decent red color. Such a red-emitting polymeric probe is identified to detect ammonia vapor and aqueous melamine. The visible color change is very much prominent for ammonia, and this red-emitting probe would be reusable for ammonia gas leakage detection, even during the rainy season. Melamine detection could also be carried out using this probe with a detectable color change. The weak noncovalent and acid-base interactions mainly govern this discernible detection.

Declarations
Ethical Approval This declaration is "not applicable".

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