Fluorene-Based Fluorometric and Colorimetric Conjugated Polymers for Sensitive Detection of 2,4,6-Trinitrophenol Explosive in Aqueous Medium.

Nitroaromatic explosives are a class of compounds that are responsible for various health hazards and terrorist outrages. Among these, sensitive detection of 2,4,6-trinitrophenol (TNP) explosive has always been highly desirable considering public health and national security. In this regard, three fluorene-based conjugated polymers (CP 1, CP 2, and CP 3) were synthesized through the Suzuki-Miyaura coupling reaction and were found to be highly sensitive for fluorescence detection of TNP with detection limits of 3.2, 5.7, and 6.1 pM, respectively. Excellent selectivity of CPs toward TNP was attributed to their unique π-π interactions based on fluorescence studies and density functional theory (DFT) calculations. The high sensitivity of CPs to TNP was attributed to the static quenching mechanism based on the photoinduced electron transfer process and was evaluated by fluorescence, UV-visible absorption, dynamic light scattering, Job's plots, the Benesi-Hildebrand plots, and DFT calculations. CPs were also used for colorimetric and real-water sample analysis for the detection of TNP explosive. Meanwhile, sensor-coated test strips were fabricated for on-site detection of TNP, which makes them convenient solid-supported sensors.

Introduction

The recent escalation in worldwide terrorist outrages has stimulated the requirement for convenient, sensitive, cost-effective, and remote detection of nitroaromatic (NAC) explosives. Increasing threats of NAC explosives to humans, animals, and the environment have raised worldwide concerns about their rapid sensing even in traces.[1] These explosives particularly 2,4,6-trinitrophenol (TNP) and 2,4,6-trinitrotoluene (TNT) are frequently released in the environment, and their common release points generally include chemical laboratories, chemical industries, mining units, and military training. Several reports revealed that long-term exposure to NAC explosives may lead to oxidation of hemoglobin (methemoglobin), anemia, and other adverse side effects on the bladder and liver.[2] Although NACs are not frequently soluble in water, they can cause detrimental effects even at their trace amounts. Among NAC explosives, TNP is among the strongest organic acids and has a more explosive nature in comparison to TNT and other analogous NACs that prompted its large-scale utilization in the military activities until World War I.[3] Keeping in view the high toxicity of TNP explosive, the Environmental Protection Agency has recognized TNP explosive as a potential carcinogen and warned about its harmful effects on the human health upon its excessive exposure.[4,5] Therefore, trace detection of TNP explosive plays a key role in public health and national security. At present, the traditional TNP detection methodologies include high-pressure liquid chromatography, electrochemical methods, surface-enhanced Raman spectroscopy, gas chromatography, and capillary electrophoresis.[6−8] However, these methodologies generally suffer from several drawbacks such as low stability, difficult on-site operation, less selectivity, high cost, and less usability.[9] Therefore, development of a cost-effective and easy-to-operate analytical method is highly demanding. Fluorescence quenching has been widely used and earned paramount significance for sensitive and selective explosives’ detection based on photoinduced electron transfer (PET), intramolecular charge transfer, and Forster resonance energy transfer (FRET). Fluorescence quenching methods carry several advantages such as real-time monitoring, quick response time, high specificity, and distinguished sensitivity.[10,11] In this context, various fluorescence quenching-based sensors such as fluorescently labeled imprinted polymers,[12] pyrene-based polymers,[13] thin nanofibrous films,[14] metal organic frameworks (MOFs),[15] fluorescent nanofibers,[16] and metallole-based copolymers[17] have been already reported for the detection of NAC explosives. Furthermore, Rong et al. designed and synthesized fluorescent graphitic carbon nitride (g-C3N4)-based nanosheets that displayed a fluorescence quenching response toward TNP explosive because of their efficient π–π and electrostatic interactions, and the detection limit of the developed nanosheets was found to be 8.2 nM.[18] Nagarkar and colleagues developed a 3D fluorescent MOF that exhibited selective detection of TNP explosive in the presence of RDX and TNT in aqueous medium. The excellent selectivity of MOFs toward TNP was associated with energy and the electron transfer process between fluorophores and TNP.[19] Nevertheless, there are several limitations related to these fluorescence sensors such as less sensitivity, poor selectivity, utilization of organic medium for fluorescence sensing, and less reusability. Therefore, it is still necessary to design and develop highly sensitive fluorescence sensors that can detect TNP explosive with high selectivity and portability, preferably in aqueous medium.

Choice of sensory materials plays a vital role in the design of highly sensitive fluorescence sensors.[20] Two general classes of organic materials have been exploited in fluorescence sensing applications. One is based on low-molecular-weight small fluorescence chemosensors while the other class consists of conjugated polymers (CPs).[21] Nonetheless, CPs display better solubility in aqueous medium, excellent photostability, and high photoluminescence quantum yield (PLQY) as compared to the small organic chemosensors.[22] Easy-to-tune structures of CPs for desired sensory applications signify their versatility for sensitive detection of NAC explosives. Most importantly, CP-based fluorescence sensors possess an additional advantage called the “molecular wire” effect. Swager explained that CPs contain highly mobile excitons in their isolated side chain or throughout the unit of CPs that enhance the interaction possibility between fluorophore (donor) and quencher (acceptor) molecules.[23] In this regard, numerous CPs have been employed for the detection of NAC compounds, particularly TNP explosive. For example, Scherf et al. designed and exploited AIE-based tetraphenylethylene (TPE)-substituted polycarbazoles for selective detection of TNB.[24] Conjugated polyfluorene (polyelectrolyte) units were synthesized by Iyer and colleagues for rapid detection of TNP explosive.[25] Mothika et al. reported three CPs that exhibited a selective fluorescence quenching response to TNT explosive.[26] However, most of these CPs depend upon variations in single-wavelength fluorescence intensity, exhibit less selectivity, and demand tiresome purification methodologies. Therefore, it still remains a challenge to develop new fluorescent materials that could improve the selectivity and sensitivity of CPs for the detection of TNP explosive.

As a part of our on-going research on the development of fluorescence sensors for sensitive detection of NACs,[27−33] fluorene-based three CPs (CP 1, CP 2, and CP 3) were designed and successfully synthesized through the Suzuki cross coupling reaction. Some of the already reported fluorene-based chemosensors suffer from aggregation-caused quenching (ACQ) because of their planarity in their design.[34] Fluorene has a rigid aromatic ring structure consisting of two peripheral phenyls fused with a five-carbon ring and possesses high thermal stability, good charge carrier mobility, wide band gaps, and excellent fluorescence quantum yield.[35,36] The active methylene (C–9) position of the fluorene core results in easy functional modification at the C–9 position, which makes fluorene suitable to be employed in fluorescent materials. All these characteristic features encouraged us to utilize fluorene as a basic unit.

Commonly, the ACQ phenomenon is responsible for low emission in aggregated or solid form of fluorescence sensors, which reduces their potential in practical applications.[37] However, fine tuning of fluorene-based sensors and wise reorganization of their substituents in space may facilitate enhancement of fluorescence emission in solid form. Advantageously, designed fluorene-based CPs (CP 1, CP 2, and CP 3) did not reduce their emission in aggregate form in aqueous medium, which provides them an additional advantage for their practical applicability. Thus, fine structural modifications in fluorene-based CPs elicit high fluorescence emission and highly sensitive picomolar-level detection of TNP explosive based on fluorescence quenching. Fluorescence quenching involves various quenching mechanisms, that is, PET, the inner filter effect (IFE), and FRET. The IFE mechanism is based on the overlapping of the excitation band of the fluorophore with the UV–visible absorption of the quencher.[38] The FRET process is determined through the extent of overlapping between fluorescence emission of the fluorophore and UV–visible absorption of the quencher.[39] FRET is relatively a long distance-dependent nonradiative energy transfer phenomenon. Furthermore, in PET, a nonfluorescent complex is formed because of direct electronic interaction between the excited fluorophore and acceptor molecule. PET is entirely dependent upon the extent of electron transfer from the lowest unoccupied molecular orbital (LUMO) level of the electron-rich fluorophore to the low-lying LUMO of the interacting electron-deficient compound.[40] In this study, fluorescence emission of newly developed CPs was significantly quenched in the presence of NACs, specifically TNP that corresponds to static quenching based on the PET process. An exquisite fluorescence quenching of CPs was observed in the presence of TNP explosive only, because of their unique π–π interaction with the detection limit down to the picomolar level (3.2 pM (CP 1), 5.7 pM (CP 2), and 6.1 pM (CP 3)). To the best of our knowledge, polymers presented in this study displayed a more sensitive and selective response toward TNP explosive in comparison to previously reported CP-based TNP sensors.[41,42] Moreover, a large Stokes shift (77–80 nm), ease of synthesis, fine solubility in various organic solvents, a large optical band gap (3.75–3.68 eV), and high quantum yield (0.51–0.63) make them superior to the already reported CPs. Characteristic features of these CPs were estimated through fluorescence spectroscopy, UV–visible absorption, density functional theory (DFT), dynamic light scattering (DLS), Stern–Volmer (SV) plots, the Benesi–Hildebrand plots, and Jobs plots. Furthermore, mechanistic insight into the fluorescence quenching process was investigated through UV–visible absorption, computational methodologies, linear SV plots, and calculations of SV rate constants. Additionally, CP-based visual detection and solid-state contact mode detection of TNP explosive have also been demonstrated. Finally, CP 1, CP 2, and CP 3 were successfully applied for on-site rapid detection of TNP explosive in industrial wastewaters.

Results and Discussion

Synthesis

The synthetic routes for preparation of monomers (5, 6, and 7) and conjugated (co)polymers (CP 1, CP 2, and CP 3) are illustrated in Scheme . Monomer (5) was synthesized using readily available 2,7-dibromo-9H-fluorene (1), 4-methoxybenzaldehyde (2), and tert-BuOK, and the reaction was carried out in ethanol. Furthermore, monomer () was synthesized for Suzuki coupling polymerization by the reaction of (1) with n-octyl bromide 3 in the presence of KOH. Similarly, the condensation reaction of 2,7-dibromo-9H-fluorene (1) with cinnamaldehyde (4) in the presence of tert-BuOK afforded the monomer (7). Synthesis of these monomers was intended to extend π-conjugation of the corresponding polymer because installment of electron-donating groups or highly conjugated suitable aromatic rings tends to tune the photophysical properties. These monomers 5, 6, and 7 were characterized through 1H and 13C NMR spectroscopy (Figures S21–S26). CP (CP 1) was synthesized in the palladium-catalyzed Suzuki coupling reaction of monomer (5) and (6) in the presence of 1,4-phenylenediboronic acid (8) and catalyst Pd(PPh3)4. CP (CP 2) was synthesized by the reaction of monomer (6), 2,5-dibromothiophene (9), and 1,4-phenylenediboronic acid (8) following Suzuki cross coupling reaction conditions. CP (CP 3) was accomplished through the same Suzuki coupling polymerization reaction using monomer (7) and 1,4-phenylenediboronic acid (8). The chemical structures of the obtained conjugated (co)polymers were rationally estimated through 1H NMR, infrared spectra, and combustion analysis as displayed in the Supporting Information (Figures S27–S31, Page no. S61–S71). The 1H NMR spectra of polymers were in good agreement with those of the basic core of the polymers. In this context, broad multiplet signals in the range of δ 8.12–6.99 ppm correspond to the aromatic protons in CP 1, a signal at δ 3.74 is an indication of the methoxy (O–CH3) unit, while broad signals in the range of δ 2.08–0.77 that attribute to the aliphatic protons confirm the synthesis of CP (CP 1). Similarly, broad multiplet signals in the range of δ 7.78–7.24 ppm correspond to the aromatic protons of CP (CP 2); more importantly, a broad spectral band in the range of δ 7.17–7.09 ppm is equivalent to the neighboring protons of sulfur (−S) atoms in the thiophene unit. Moreover, aliphatic group protons are indicated by the appearance of broad spectral signals in the range of δ 2.27–0.51 ppm that justify the synthesis of CP 2. Introduction of suitable aromatic substituents and electron-donating moieties into the CPs provides high electron-donating ability to the corresponding polymer. Therefore, these polymers can be employed for the efficient sensing of strong electron-poor compounds, that is, NAC compounds.

Scheme 1

Synthetic Strategy To Afford the Conjugated Polymers CP 1, CP 2, and CP 3

Selection of a Solvent System for Sensing Studies

However, viscosity or polarity of a solvent can affect the wavelength or fluorescence intensity of a fluorophore.[43] Fluorescence spectra of CP 1 (as a model example) were recorded in various solvents including tetrahydrofuran (THF), N, N-dimethylformamide (DMF), methanol (CH3OH), ethanol (C2H5OH), dichloromethane (CH2Cl2), and chloroform (CHCl3) to select suitable solvents for successive titration experiments. High emission of CP 1 at 510 nm was observed in DMF and THF with slightly better emission in THF (Figure S1). The extent of dissolution might have affected the emission of CP 1 in DMF. Similarly, CP 1 experienced a slight redshift in methanol (510–519 nm) and ethanol (510–518 nm) that is presumably due to the effect of solvent polarity. Highly polar solvents may shift the emission of fluorophores to a higher wavelength. Excited fluorophores observe vibrational relaxation followed by the transfer of vibrational energy to neighboring polar solvent molecules. These polar solvents undergo solvent reorientation (relaxation) that helps to stabilize and lower the excited state energy of the fluorophore.[43] Hence, less difference between excited and ground states of the fluorophore prompts the redshift (higher wavelength).[44] Furthermore, CP 1 was little less emissive in DCM and chloroform that is perhaps due to the heavy-atom (chlorine) effect.[45] Highly emissive nature of CP 1 in polar solvents indicates that the newly developed CP holds tendency to reduce π–π stacking interactions in polar solvents. Fluorene-based fluorescent derivative/polymers are usually prone to aggregate in polar solvents that could hamper their sensing applications. However, fine tuning of CP 1 (by suitable substitutions) not only increased the size and degree of π conjugation but also makes it capable to overcome these limitations. In this context, fluorescence emission of CP 1 was also checked in aggregated form in the presence of increasing concentration of water (fw) from 0 to 99%. Figure S1b revealed that the fluorescence intensity of CP 1 was not changed to a greater extent when the water fraction was increased from 0 to 60%, but slightly better and high emission was observed at fw 70% (H2O/THF (3:2, v/v)). Addition of water from 70 to 99% in THF solution of CP 1 slightly reduced its emission intensity that is probably due to the hydrophobic behavior of CP 1 at high water fractions.[46] Fluorophore molecules tend to stack together to reduce their interaction with water. Interactions due to π–π stacking are generally due to the planar geometry of compounds which prompts the nonradiative pathway.[47] Keeping these results in perspective, the H2O/THF (3:2, v/v) solvent system was selected for titration studies.

Concentration-Dependent Fluorescence Studies

Weak fluorescence emission of the CP is observed because of the IFE.[48] Spectral measurements are usually affected by the IFE when the highly concentrated solution of the fluorophore is used. The excitation beam of electromagnetic radiation is dissipated by the highly concentrated sample, and only surface molecules facing the excitation beam emit radiation strongly. Resultantly, molecules in the center of the sample remain less emissive ultimately reducing the emission of the sample. In addition to this, if the emission and excitation bands of the fluorophore overlap each other, emitted light from the center molecules of the sample might be reabsorbed by the sample itself. It is a good practice to record fluorescence spectra at various concentrations of the fluorophore to reduce the IFE. In this regard, fluorescence emission studies of CP 1, CP 2, and CP 3 were carried out at different concentrations ranging from 500 nM to 50 μM. Spectral results are presented in Figure S2. Upon a progressive increase in concentrations of CPs from 500 nM to 10 μM, fluorescence emission of all the CPs was continuously increased with maximum emission obtained at 10 μM of all CPs. Further increase in concentrations (10–50 μM) caused noticeable quenching in fluorescence emission of CP 1, CP 2, and CP 3 that is presumably due to aggregate formation at higher concentrations. These spectral results suggested that maximum fluorescence intensity was attained at 10 μM concentration that ensures the minimum IFE.

Photophysical Properties

The photophysical properties of three conjugated polymers CP 1, CP 2, and CP 3 were studied through UV–visible and fluorescence spectroscopy. UV–visible absorption spectra of CP 1, CP 2, and CP 3 in H2O/THF (3:2, v/v) displayed characteristic bands at 430, 421, and 415 nm, respectively, that originate because of π → π* transitions (Figure ). The UV–visible absorption band of CP 1 is marginally red-shifted by 9 and 15 nm in comparison to CP 2 and CP 3, respectively, that is perhaps due to efficient π-π conjugation in the basic unit of CP 1. Furthermore, a higher wavelength of CP 2 as compared to CP 3 is probably due to its long side chain that contributes toward electronic conjugation while the side alkyl chain of CP 3 renders it to lower absorption wavelength (415 nm). Figure b displays fluorescence emission spectra of CP 1, CP 2, and CP 3 at an excitation wavelength (λex) of 400 nm. All three conjugated polymers CP 1, CP 2, and CP 3 displayed strong emissions at 510, 495, and 492 nm, in aqueous solution (H2O/THF (3:2, v/v) with a large Stokes shift of 80, 74, and 77 nm, respectively. A slightly large Stokes shift displayed by CP 1 in comparison to other two polymers ensures a comparatively higher extent of its electronic conjugation. PLQY of CP 1, CP 2, and CP 3 was determined through the reported procedure[49] using fluorescein (Φ, 0.95) as a reference and calculated to be 0.51, 0.57, and 0.63 eV, respectively. Moreover, the optical band gap of CP 1 was estimated through DFT calculations and was found to be ∼3.68 eV, which is lower as compared to that of CP 2 (3.73 eV) and CP 3 (3.75 eV). Moreover, CP 1, CP 2, and CP 3 displayed solid-state emission at 351, 347, and 345 nm, respectively at an excitation wavelength of 370 nm (Figure S2). Excitation spectra of CPs are also presented in Figure S2. These photophysical observations ensure that CPs are highly emissive with potent electron donor ability. The photophysical data for CP 1, CP 2, and CP 3 are summarized in Table .

Figure 1

Comparison of UV–visible absorption (a) and fluorescence emission spectra (b) of CP 1, CP 2, and CP 3 (10 μM).

Table 1

Summary of Photophysical Properties for CP 1, CP 2, and CP 3

CP	absorbance (nm)	fluorescence emission (nm)	Stokes’ shift (nm)	quantum yield (Φ)	HOMO-LUMO gap (eV)
1	430	510	80	0.51	3.68
2	421	495	74	0.57	3.73
3	415	492	77	0.63	3.75

The frontier molecular orbitals of CP 1 found through the B3LYP/6-31G level of theory using Gaussian 09 displayed that the electronic density of the highest occupied molecular orbital (HOMO) and LUMO energy levels is particularly extended to the entire π-conjugated basic unit of CP 1.[50,51] For CP 2, the electron density was further extended to the aromatic unit of its side chain. A similar result was noticed in the optimized HOMO-LUMO energy levels of the CP 3 unit, with electron density prolonged to the side alkyl chain. These observations show that CPs are electron-rich and have the tendency to transfer their electronic charge efficiently to some electron-deficient compounds. However, the high LUMO energy level in CP 1 as compared to CP 2 and CP 3 units facilitates a stronger and faster electronic transition to interacting compounds. These results are in total agreement with the trend observed in the UV–visible and fluorescence emission properties. High emission, excellent quantum yield, and electron richness of the CPs inspired us to investigate their interactions with strong electron-deficient NAC explosives. NAC explosives particularly TNP, TNT, and 2,4-dinitrotoluene (DNT) have strong detrimental effects and are recognized as common ingredients of explosive materials.

Detection of TNP Explosive

To investigate the sensing potential of conjugated (co)polymers toward NAC explosives, fluorescence titration experiments were carried out in aqueous solution (H2O/THF (3:2, v/v)). NAC explosives include TNP, TNT, 2,4-DNT, 1,3-dinitrobenzene (1,3-DNB), nitrobenzene (NB), 2-nitroaniline (2-NA), 4-nitrotoluene (4-NT), and 4-nitrophenol (4-NP). In this context, fluorescence experiments were performed with CP 1, CP 2, and CP 3 (10 μM) against varying concentrations of TNP from 0 to 50 pM in aqueous solution (H2O/THF (3:2, v/v)) at an excitation wavelength of 400 nm. Addition of TNP (0–50 pM) to the aqueous solutions of CP 1, CP 2, and CP 3 (10 μM) leads to the significant quenching of ∼99.3, 98.7, and 97.2% in their strong emission at 510, 495, and 492 nm, respectively (Figure ). Drastic quenching in CPs in the presence of TNP is attributed to their efficient donor acceptor interaction. Moreover, these results demonstrate that CP 1 experienced a faster and efficient decrease in its emission intensity in the presence of TNP as compared to other conjugated polymers CP 2 and CP 3 that is corresponding to its high electron-donating ability.

Figure 2

Change in fluorescence emission behavior of (a) CP 1, (b) CP 2, and (c) CP 3 (10 μM) in aqueous solution (H2O/THF (3:2, v/v)) and (d) changes in the SV plot of CP 1, CP 2, and CP 3 treated with different concentrations of TNP explosive (0–50 pM).

Keeping this in view, it was anticipated that CP 1 should have high sensing efficiency. In this regard, the sensitivity of CP 1, CP 2, and CP 3 was determined by calculating their limit of detection (LOD = 3 σ/S). Herein, σ is the standard deviation and S is the slope of the calibration curve. Corresponding detection limits of CP 1, CP 2, and CP 3 for TNP were calculated to be 3.2, 5.7, and 6.1 pM, respectively, which shows excellent sensitivity of these CPs. Some literature data of detection limits for detection of TNP based on CPs are compiled in Table . The literature study reveals that conjugated polymers CP 1, CP 2, and CP 3 have better sensitivity as compared to previously reported TNP sensors based on CPs.

Table 2

Comparison of the Present Study of CPs with the Previously Reported TNP Explosive Sensors

sensing method	type of sensors and their response	LOD	Ka (M–1)	ref.
fluorescence quenching	fluorene-based CPs for detection of TNP explosive	3.2 pM	4.27 × 106	present work
fluorescence quenching	amine-based polymers for sensing of TNP	56 μM	7.75 × 104	(41)
fluorescence enhancement	tetraphenylethylene polymers used for TNP detection	0.053 mM (12 ppm)	1.64 × 102	(52)
fluorescence quenching	polyfluorene-based sensor for TNP	110 nM	5.1 × 104	(53)
fluorescence quenching	polycarbazole polymer used for sensing of TNP		2.83 × 105	(54)
fluorescence quenching	polytriazole-based polymers for detection of TNP		3.70 × 104	(55)
fluorescence quenching	triazolyl-based conjugated microporous polymer for sensitive detection of p-nitroaniline	4.2 μM	7.08 × 104	(56)
fluorescence enhancement	polydiynes used for NAC detection		3.39 × 104	(57)
fluorescence quenching	polytriazoles for sensing of NACs	510 nM	1.72 × 104	(58)
fluorescence quenching	alanine-based polymers used for detection of NACs	3.7 μM	1.6 × 104	(42)
fluorescence quenching	tetraphenylethene-functionalized acetylenes used for TNP sensing		3.09 × 104	(59)
fluorescence enhancement	CP nanoparticles used for NAC detection		2.03 × 104	(60)
fluorescence quenching	cationic conjugated polyfluorene used for TNP sensing	0.19 nM (43.53 ppt)	2.18 × 105	(25)
fluorescence quenching	poly(isoquinoline) derivatives used for TNP sensors		1.64 × 105	(61)
fluorescence quenching	cyclosiloxane-linked fluorescent polymers for TNP detection	59 ppb	1.64 × 104	(62)
fluorescence quenching	1,3,5-tri(4-formyl-phenyl)benzene used for TNP detection	0.084 μM	9.67 × 105	(63)
fluorescence quenching	polyurethane used for TNP detection	0.57 μM	2.77 × 105	(64)
fluorescence quenching	triptycene-based fluorescent polymers (azo sensor) for TNP	286 nM	1.86 × 105	(65)
fluorescence quenching	silsesquioxane polymers used for detection of TNP		3.2 × 104	(66)
fluorescence quenching	di(naphthalene-2-yl)-1,2-diphenylethane-based polymers used for TNP detection	0.181 μM	4.7 × 104	(67)
fluorescence quenching	TPE copolymers used for TNP detection		5.61 × 104	(68)
fluorescence quenching	TPE-CPs used for TNP detection		4.0 × 104	(69)
fluorescence quenching	fluorene-based sensor used for TNP detection	525 nM	6.40 × 104	(70)

The excellent sensitivity of CP 1, CP 2, and CP 3 for TNP was further determined through SV constants that were obtained through the SV eq :

Where Io and I are the fluorescence intensity of CPs before and after addition of TNP explosive, Ksv is the SV constant, and [Q] is equivalent to the concentration of the TNP. Larger Ksv values indicate higher sensitivity of conjugated (co)polymers toward detection of TNP explosive. Figure d displays the SV plot between Io/I of CP 1, CP 2, and CP 3 and different concentrations of TNP explosive (0–50 pM). In all cases, the Io/I value increased linearly with increasing concentration of TNP (0–50 pM) which demonstrates that a single process, most probably static quenching, is responsible for the quenching in the fluorescence intensity of CPs. The SV constant (Ksv) values were obtained from the linear regime of the SV plots. Thus, Ksv rate constants of CP 1, CP 2, and CP 3 were calculated to be 4.27 × 106, 3.71 × 106, and 2.13 × 106 M–1, respectively, which indicate excellent binding efficiency of CPs, particularly CP 1 with TNP explosive. Furthermore, comparison of Ksv values of CP 1, CP 2, and CP 3 with that of already reported CP-based TNP sensors presented in Table ensures the excellent sensitivity of CP 1, CP 2, and CP 3 toward TNP explosive.

The static quenching mechanism predominates in the linear regime of the SV plot while nonlinearity of the SV plot is generally attributed to the dynamic quenching process. The SV plots for changes in the fluorescence intensity of CP 1, CP 2, and CP 3 displayed an excellent linear character in the presence of increasing concentrations of TNP (0–50 pM) (Figure d). A presumable linear response in SV plots of CPs favors the involvement of the static quenching process and shows that increasing concentrations of TNP explosive bring its molecules in close proximity of CPs for the static quenching with less/no contribution from dynamic quenching. Static quenching is actually formation of intermolecular dimers between the electron-rich fluorophore (donor) and electron-deficient quencher (acceptor) to create a ground-state complex with an altered absorption spectrum.[49] However, the dynamic quenching mechanism is identical to FRET that corresponds to the transfer of energy from the excited fluorophore to the interacting NAC compound and does not alter fluorophore’s absorption spectrum.[71]

Possibility of the static quenching mechanism (ground-state complex) could be determined through UV–visible absorption of CPs before and after progressive addition of TNP explosive. Any spectral shift observed in absorbance maxima of CPs before and after addition of TNP ascertains the possibility of ground-state complex formation and consequently static quenching process. In this regard, the UV–visible absorption study of CP 1, CP 2, and CP 3 was carried out in aqueous solution (H2O/THF (3:2, v/v)) against selective TNP explosive. Upon progressive addition of TNP (0–50 pM), CP 1, CP 2, and CP 3 displayed enhancement in their absorption bands with an observable bathochromic shift of 8 (430–438 nm), 5 (421–426 nm), and 9 nm (415–424 nm), respectively. Therefore, an observable change in the peak and shapes of UV–visible absorbance spectra of all CPs against TNP explosive indicates the GS complex formation and static quenching mechanism. Absorption plots of CP 1, CP 2, and CP 3 in the presence of increasing concentrations of TNP are depicted in Figure .

Figure 3

Change in UV–visible absorption behavior of (a) CP 1, (b) CP 2, and (c) CP 3 (10 μM) in aqueous solution (H2O/THF (3:2, v/v)) upon addition of different concentrations of TNP (0–50 pM) and (d) spectral overlap between normalized absorption spectra of TNP and emission spectra of CP 1, CP 2, and CP 3.

The bathochromic shift in UV–visible absorption spectra and linearity in S–V plots of all CPs in the presence of cobound TNP probably eliminate the possibility of dynamic quenching (FRET). If the fluorophore and cobound TNP are in close proximity, then there is a chance of energy transfer (dynamic quenching) between them. It can be investigated by observing the overlap in the absorption band of TNP explosive with the emission spectra of CPs. TNP explosive absorbs UV radiation in the range of 300–420 nm[40] while CP 1, CP 2, and CP 3 displayed their fluorescence emission in the range of 450–600 nm (Figure ). Figure d reveals an insignificant overlap between UV–visible absorption of TNP and fluorescence emissions of CP 1, CP 2, and CP 3 that rules out the possibility of the dynamic quenching process. Therefore, these spectral results designate that fluorescence quenching of conjugated (co)polymers in the presence of TNP explosive occurs through the static quenching process only.

Selectivity, Reproducibility, and Response Time of CPs for TNP

The NAC explosives generally possess identical electronic and chemical structures. It is therefore essential to estimate the selectivity of CPs for a specific NAC explosive. In this regard, the fluorescence response of CPs was tested in aqueous solution (H2O/THF (3:2, v/v)) against incremental addition of other NACs including TNT, DNT, DNB, NB, NA, NT, NP, and NT. The resulting fluorescence spectra of CP 1, CP 2, and CP 3 in the presence of NAC explosives presented in Figures S3–S5 exhibited little/no decrease in their emission profile at 510, 495, and 492 nm, respectively. Upon incremental addition of TNT and DNT (0–100 nM) to aqueous solution (H2O/THF (3:2, v/v)) of CP 1 (10 μM), less fluorescence quenching (20 and 7%, respectively) was noticed as shown in Figure S3. The calculated detection limit (27.0 and 51.0 nM) and Ksv rate constants (1.6 × 103 and 7.2 × 102 M–1) of CP 1 for TNT and DNT are much less in comparison to those of TNP. Moreover, CP 1 displayed insignificant/no fluorescence quenching in the presence of remaining targeted NACs including DNB, NB, NT, NP, and NA (0–100 nM). Depending upon the calculated Ksv rate constants and LODs, the fluorescence quenching response of CP 1 toward NACs follows the order TNP > TNT > DNT > DNB = NB = NT = NP = NA. Calculated Ksv and detection limits of CP 1 for NACs are provided in Table S1. Under the same experimental conditions, addition of TNT and DNT (0–100 nM) to CP 2 resulted in 19 and 17% fluorescence quenching while 9 and 5% fluorescence quenching was noticed upon addition of DNB and NB (0–100 nM), respectively. Furthermore, CP 2 remained insensitive toward detection of NT, NP, and NA (0–100 nM) (Figure S4). Calculated Ksv and LOD of CP 2 for targeted NACs are given in Table S2. Comparison of detection limits and SV constants revealed that the quenching response of CP 2 toward NACs is in the order of TNP > TNT > DNT > DNB > NB = NT = NP = NA. Similarly, CP 3 experienced 16 and 4% decrease in its fluorescence intensity upon progressive addition of TNT and DNT (0–100 nM) while other targeted NACs did not affect the emission intensity of CP 3 (Figure S5). Moreover, calculated SV constants and detection limits of CP 3 for considered NACs are presented in Table S3 that shows that fluorescence quenching of CP 3 in the presence of NACs is in the order of TNP > TNT > DNT > DNB > NB = NT = NP = NA. Furthermore, SV constants for all targeted NACs were determined through the linear SV plots that are provided in Figure S6.

Lesser/no fluorescence quenching response of CPs (1, 2, and 3) toward NACs except TNP and TNT is probably due to insignificant molecular-level interaction through π···π or C–H···π connection between polymers and targeted NACs. The rapid decrease in the fluorescence intensity of CPs in the presence of TNP suggests efficient PET from the electron-rich CPs to the strong electron-deficient TNP explosive. Keeping all these results in perspective, the % quenching efficiency chart was plotted to estimate the selectivity of CPs toward specific NACs. Figure displays the variation in the fluorescence quenching response of CP 1, CP 2, and CP 3 in the presence of different concentrations of NACs. The highest sensitivity of CPs was noticed for TNP explosive, and they displayed a substantial % quenching efficiency of ∼99.3, 98.7, and 97.2%, respectively. These results clearly demonstrate the ultrasensitivity (to the pM level) and excellent selectivity of CPs for TNP explosive.

Figure 4

Quenching efficiency of CP 1, CP 2, and CP 3 (10 μM) treated with different NAC compounds including TNP, TNT, DNT, DNB, NB, NT, NP, and NA.

Keeping in view the selectivity of CPs toward TNP explosive, their basic units were believed to provide multiple binding sites for making an interaction with TNP. To investigate the binding stoichiometry between conjugated (co)polymers 1, 2, and 3 and TNP, Job’s plots were plotted between the emission intensity of the individual CP and increasing molar fractions of TNP (0–1.0). Figure shows that the maximum emission intensity of CP 1 and CP 2 was attained at 0.5 mole fractions of TNP. It clearly demonstrates the 1:1 binding stoichiometry of CP 1 and CP 2 with TNP that ensures their excellent sensitivity. However, CP 3 displayed 1:2 binding stoichiometry with TNP that justifies its relatively less sensitivity toward detection of TNP as compared to CP 1 and CP 2 (Figure S7). The 1:1 binding association of CP 1 and CP 2 with TNP was further proved through the excellent linear behavior (linearity coefficient, R2 = 0.998 (CP 1) and 0.997 (CP 2)) displayed by their Benesi–Hildebrand plots (Figure S8). Values of binding constants calculated through the linear region of the Benesi–Hildebrand plots (Ka = 6.71 × 105 and 6.57 × 105) justify the strong affinity between CPs (1 and 2) and TNP.

Figure 5

Job’s plot explaining the 1:1 stoichiometric associations of CP 1 (a) and CP 2 (b) with TNP explosive.

Strong binding stoichiometry of CPs with TNP explosive was further investigated by determination of their size using DLS. The average size of 10 μM CP 1 (PDI = 0.426), CP 2 (PDI = 0.376), and CP 3 (PDI =0.407) was determined to be 800, 667, and 720 nm before addition of TNP explosive (50 pM) (Figure ). A noticeable increase in the particle size was observed for the complex of TNP with CP 1 (PDI = 0.630), CP 2 (PDI =0.573), and CP 3 (PDI = 0.589), and the particle size was estimated to be 936 (CP 1-TNP), 821 nm (CP 2-TNP), and 860 nm (CP 3-TNP) (Figure ). Therefore, these results favor the formation of unsymmetrical nanoaggregates and efficient binding association in aqueous medium, where there was an observable increase in the size of nanoaggregates without any dramatic variations in their shape.

Figure 6

DLS analysis and size distribution of CP 1, CP 2, and CP 3 (10 μM) before (a, c, and e respectively) and after addition of TNP explosive (b, d, and f, respectively).

Favorable selectivity of CPs (1, 2, and 3) toward TNP was also supported through evaluating interaction energies of CP complexes with considered NACs at the Gaussian 09 program using the B3LYP/6-31G(d) basis set.[50,51] Optimized complexes of all CPs with targeted NACs are shown in Figures S9–S11. TNP@CP1, TNP@CP2, and TNP@CP3 displayed highest interaction among all other complexes that suggests their favorable selectivity toward TNP. Interaction energies of TNP@CP1, TNT@CP1, DNT@CP1, DNB@CP1, NB@CP1, NT@CP1, NP@CP1, and NA@CP1 are −31.34, −16.65, −11.16, −6.90, −5.01, −3.56, −3.21, and 2.90 kcal/mol, respectively. Calculated interaction energies (Eint) for CP 1 against NACs follow the order of TNP > TNT > DNT > DNB > NB > NT > NP > NA. Similarly, interaction energies provided by TNP@CP2, TNT@CP2, DNT@CP2, DNB@CP2, NB@CP2, NT@CP2, NP@CP2, and NA@CP2 are −27.34, −15.98, −15.36, −14.97, −9.87, −8.67, −7.54, and −2.24 kcal/mol, respectively. These calculations prompt the excellent interaction in TNP@CP2 as compared to other targeted NACs. The binding order of CP 2 toward targeted NACs is as follows: TNP > TNT > DNT > DNB > NB > NT > NP > NA. Likewise, calculated interaction energies of TNP@CP3, TNT@CP3, DNT@CP3, DNB@CP3, NB@CP3, NT@CP3, NP@CP3, and NA@CP3 are −24.43, −19.65, −8.15, −4.91, −4.17, −3.16, −3.15, and 3.67 kcal/mol, respectively. Hence, these theoretical calculations reveal the excellent selectivity of CP 1, CP 2, and CP 3 for the detection of TNP that correlates with fluorescence emission studies.

Advantageously, the fluorescence quenching response of CPs for TNP explosive is reproducible. In this regard, titration experiments were repeated, and the obtained results are presented in Figure S12. Moreover, fluorescence experiments were performed again to ensure selectivity for TNP. Figure S12 reflects less fluorescence quenching against all other NACs except TNP explosive. Moreover, CP 1, 2, and 3 displayed an excellent fluorescence quenching response toward TNP at 15, 20, and 20 s. respectively which signifies their quick response time (Figure S13).

Effect of Interferents and pH on Fluorescence Sensing

Selective detection of TNP among other NACs prompted to investigate their selectivity in the presence of some interfering compounds in the aqueous medium. In this regard, CP 1, CP 2, and CP 3 (10 μM) were added to separate TNP (20 nM) solution in the presence of 100 equivalents of other interferents including NACs, fructose, galactose, glucose, and commonly used organic solvents. Results displayed in Figures and S14 show that the presence of even high concentrations (100 equiv) of interferents has no influence on selective detection of TNP by CPs. Such a study provides a unique significance to CP-based TNP sensors in determination and detection of TNP in real-field samples.

Figure 7

Measurement of the fluorescence quenching response of CP 1 (10 μM) toward TNP explosive (20 nM) in the presence of potential interferents.

Furthermore, detection capacity of a fluorophore can be largely affected by varying the pH of a system, and ultimately, its sensitivity for a specific substance is compromised. In this regard, influence of pH upon sensing potential of CPs for TNP explosive was investigated in the pH range of 2.0–12.0. Figure S15 reveals that CP 1, CP 2, and CP 3 displayed their maximum quenching response at pH 7.5, 8.0, and 8.3, respectively.

Sensing Mechanism

The fluorescence quenching response of electron-rich fluorophores in the presence of electron-deficient NACs generally involves the PET process. In the PET process, fluorophores transfer their excited electrons to the low-lying unoccupied molecular orbitals of NAC compounds (quencher) that lead to the reduction in their fluorescence intensity (fluorescence quenching). Hence, upon considering the PET as a fluorescence quenching mechanism, selectivity for a specific NAC compound is entirely dependent upon the extent of electron transfer from the LUMO level of the electron-rich CP to the low-lying LUMO of the interacting NAC compound. Therefore, LUMO energy levels of CPs and NACs need to be considered to investigate the plausible quenching mechanism. In this case, the highest electron-deficient NAC compound with the lowest LUMO energy level provides a preferential driving force for the PET to occur. To adequately evaluate the above reasoning, the HOMO-LUMO energy levels and geometries of all CPs and targeted NACs were optimized and calculated at the B3LYP/6-31G (d) level of theory. The HOMO-LUMO energy profile of all targeted NACs (Figure ) suggests that TNP explosive holds the lowest LUMO energy among all other targeted NACs. These observations reveal that the electron from the LUMO level of CPs is most favorably transferred to the LUMO of TNP explosive that is, ultimately, reflected as the highest quenching efficiency. Furthermore, preferential selectivity and sensitivity of polymers CP 1, CP 2, and CP 3 toward TNP explosive are based on their energy band gaps. A smaller optical band gap of any fluorophore is an indication of low binding energy and high conversion rate that are rationale to a rapid fluorescence quenching response. Among CPs presented in this study, CP 1 has a lower energy gap and is expected to display a high fluorescence quenching response toward TNP explosive in comparison to other polymers. The contour plot presented in Figure favors the higher electron transfers from LUMO of CP 1 to that of TNP. The DFT results reveal that the LUMO energies of all considered NACs are −4.56 (TNP), −4.37 (TNT), −3.48 (DNT), −3.45 (DNB), −2.96 (NB), −2.78 (NT), −2.76 (NP), and −2.62 eV (NA), while the LUMO energies calculated for CP 1, CP 2, and CP 3 are −1.63, 1.73, and −1.84 eV, respectively. These results are the thermodynamic evidence of the PET process and preferential sensitivity of CPs, particularly CP 1 toward TNP explosive.

Figure 8

LUMO–HOMO energy levels of CP 1, CP 2, CP 3, and targeted NACs displaying the extent of the PET process.

Additionally, greater efficiency of the PET process demands mutual contact of the electron-rich fluorophore with a NAC compound. Favored selectivity of CPs toward TNP was also explained in this context. TNP with a greater number of electron-withdrawing groups (−NO2) is a very strong acid with a pKa value of ∼0.38. The strongly acidic hydroxyl unit of TNP simply dissociates in polar solvents (THF) and dissociated TNP makes an efficient electrostatic interaction with CPs (1, 2, and 3) that brings TNP molecules in closest proximity of CPs to facilitate the excellent electron transfer. Furthermore, TNT explosive contains three electron-withdrawing units (−NO2) and is a Brønsted-Lowry acid.

Its molecules could make π-π interactions with CPs that could bring TNT molecules close to the electron-rich polymer unit for the rapid PET process.

Visual Sensing

Visual sensing experiments were accomplished to support the selectivity of CPs toward TNP explosive. In this regard, 500 nM of targeted NACs (TNP, TNT, DNT, NB, NP, NT, DNB, and NA) was concomitantly added into the H2O/THF (3:2, v/v) solutions of CP 1, CP 2, and CP 3 (10 μM). Only when TNP explosive solution (500 nM) was mixed, the color of CPs changed significantly under UV (365 nm) and daylight. In daylight, colors of CP 1, CP 2, and CP 3 were changed to light yellow, wine red, and dark pink respectively in the presence of TNP (500 nM) (Figure ). These color variations might be ascribed to the more electron-deficient nature of TNP explosive. Furthermore, Figures and S16 show significant disappearance in the bright emission of CPs (10 μM) under UV (365 nm) after addition of TNP (500 nM). However, under identical parameters, no observable change in colors of CPs was noticed for other targeted NACs under UV (Figure ) and daylight (Figure S17).

Figure 9

Color changes of CP 1, CP 2, and CP 3 (10 μM) with addition of 500 nM of TNP explosive under daylight.

Figure 10

Color changes of CP 1 and CP 2 (10 μM) with addition of 500 nM of TNP explosive under UV (365 nm).

Practical Applications

Preparation of Sensor-Coated Test Strips

Contact mode testing of NAC explosives in real samples has attracted considerable attention in the past few years. In this regard, test strips were prepared by coating CP 1, CP 2, and CP 3 (10 μM) on the surface of TLC strips (250 μm) followed by drying them in air. Dried paper strips adsorbed with CPs displayed bright emission when irradiated at 365 nm (under UV). However, bright emission of CPs disappeared under 365 nm radiation when the spot of TNP solution (10 nM) was marked onto CPs’ adsorbed test strip (Figures and S18). However, dried test strips of CP 1, CP 2, and CP 3 (10 μM) did not respond to other NAC compounds under UV (Figures and S18). Hence, the excellent response of CP-coated test strips for TNP explosive signifies that these test strips are a convenient sensing tool and can be used for the rapid on-site detection of TNP explosive with high sensitivity and selectivity. Additionally, the reversibility test of conjugated (co)polymers was successfully carried out with ethanol and repeated by alternate addition of ethanol (1.5 mL) and TNP explosive (10 nM) consecutive three times (Figure S19). Interestingly, addition of ethanol led to the reappearance of original emission of CP 1 at 365 nm under UV. Therefore, recyclability and reversibility of CPs promote their practical utility for on-site detection of TNP explosive.

Figure 11

Test strips of CP 1 and CP 2 (10 μM) and their response to TNP and other NAC solutions (10 nM in THF) under UV radiation (365 nm).

Detection of Explosives in Industrial Effluents

Practical application of CPs for detection and quantification of TNP explosive was investigated in industrial wastewater through the spike/recovery test under the above-mentioned experimental conditions. These samples were collected from Pakistan Ordinance Factory (POF), Havelian, Abbottabad, KPK, Pakistan and were spiked with different concentrations of TNP explosive (10 and 50 pM) to determine its %recovery. These water samples were filtered first to eradicate undesired free-floating particles. TNP-spiked industrial solutions were added into aqueous solutions of CP 1, CP 2, and CP 3 (10 μM), and fluorescence emission spectra were obtained (Figure S20). The obtained spectra reveal that the fluorescence quenching response for these samples relates the pattern that has been noticed for laboratory TNP samples. Figure S20 shows that spiked TNP explosive (10 and 50 pM) was recovered >90%. The recovery determination of spiked TNP by CP 1, CP 2, and CP 3 is summarized in Tables , 4, and 5.

Table 3

Recovery of TNP Spiked in Industrial Water by CP 1

sr. no.	spiked (nM)	recovered	recovery (%)
1	10 pM	9.9 pM	99
2	50 nM	49.7 pM	99.4

Table 4

Recovery of TNT Spiked in Industrial Water by CP 2

sr. no.	spiked (nM)	recovered	recovery (%)
1	10 pM	9.7 pM	97
2	50 pM	49.3 pM	98.6

Table 5

Recovery of TNT Spiked in Industrial Water by CP 3

sr. no.	spiked (nM)	recovered	recovery (%)
1	10 pM	9.6 pM	96
2	50 pM	48.5 pM	97

Conclusions

In summary, three fluorene-based CPs were synthesized and successfully employed for fluorometric and colorimetric detection of TNP. Based on fluorescence quenching experiments, application of conjugated polymers CP 1, CP 2, and CP 3 was accomplished for selective detection of TNP with their LODs of 3.2, 5.7, and 6.1 pM and quenching constants (Ksv) 4.27 × 106, 3.71 × 106, and 2.13 × 106 M–1, respectively, that are much better than those of already reported TNP sensors. The fluorescence quenching mechanism for sensing of TNP was attributed to static quenching based on the PET process. Spectral changes in the UV–visible absorption of CPs in the presence of TNP estimate the involvement of a static quenching mechanism. Furthermore, the static quenching mechanism was evaluated by fluorescence, UV–visible absorption, DLS, Job’s plots, and DFT calculations. Moreover, sensor-coated portable test strip devices were fabricated for easy and rapid on-site detection of TNP explosive. Furthermore, CPs were successfully employed for the quantification of TNP in real-water samples.

Experimental Section

Instruments, reagents, and synthetic procedures for monomers (5, 6, and 7) and conjugated (co)polymers (CP 1, CP 2, and CP 3) are provided in the Supporting Information (SI 1–2). whereas details of fluorescence experiments, its spectral acquisition, visual sensing, determination of association constant (ka), LOD, Job’s plot, percent enhancement efficiency, and computational methodologies are provided in SI 3–11.