Heterocyclic biphenyl-based fluorochrome sensor for rapid hydrazine detection: design, synthesis, single crystal XRD, and DFT studies

Abstract

As part of our continuous research focused on enhancing sensing technologies, this article presents a series of ground-breaking fluorochromes that feature a biphenyl scaffold. Novel fluorochrome sensors are developed with various heterocyclic aldehydes via Claisen–Schmidt condensation. This condensation is performed using KOH and pyrrolidine as catalysts to provide two different methods with competitive studies. The obtained results show that KOH is a rapid catalyst (2–3 h; 71–80%), while pyrrolidine is an effective catalyst (5–6 h, 85–95%). The structures of the prepared fluorochromes are characterized using various spectral techniques and single crystal XRD. The photophysical properties of these fluorochromes are investigated using UV-vis and Fluorescence spectrophotometry in different solvent systems. Density functional theory (DFT) calculations are carried out and have a good correlation with experimental results. The obtained results for absorption, photoluminescence, and their theoretical correlation suggest that the prepared fluorochromes can be optimized for applications in optoelectronics, sensing, and bioimaging. Fluorochrome 3g, which exhibits the highest Stokes shift (129 nm) and photoluminescence (QY 0.87), is used to demonstrate the detection of hydrazine in actual water, soil, and air samples. The fluorochromes are inherently colored compounds and exhibit good photoluminescence, which is significantly quenched when hydrazine is added in very small quantities. The disappearance of the color and quenching of the photoluminescence signal are attributed to the formation of hydrodiazole via cyclization with hydrazine. A strong linear relationship for detecting hydrazine is observed over the concentration range of 1–5 μM in methanol. The limit of detection (LOD) for hydrazine is observed to be 1.1 μM with 5 μM 3g. Moreover, the color change of the fluorochrome solution from yellow to colorless can be observed by the naked eye, indicating that these fluorochromes can also be used as a colorimetric sensors for detecting hydrazine at very low concentration. Fluorochrome 3g was evaluated for its real-time detection ability over a pH range of 4–10, showing excellent efficiency in selectively detecting hydrazine among interfering analytes, and in soil and water samples. A probable mechanism for the detection of hydrazine is also established via spectral study. Additionally, this study describes a straightforward cost-effective probe-coated paper sheet for the detection of hydrazine in the environment and gives further hope for its commercial applications.

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Water impact

Trace amounts of hazardous chemicals in water can have great disastrous consequences. Efficient techniques to detect such chemicals are most essential. The present study highlights the synthesis of a highly conjugated novel heterocyclic biphenyl-based fluorochrome sensor. A very user-friendly protocol has been demonstrated for the selective and sensitive detection of hydrazine present in water and soil via a prepared fluorochrome sensor operable over a broad pH range.

1. Introduction

Hydrazine (H₂N–NH₂) is a colorless oily liquid¹ characterized by its robust chemical properties as a potent base, nucleophile, and reducing agent, and it exhibits significant reactivity that has found extensive utility in diverse industries.^1–3 Its applications span the large-scale manufacturing of premium aerospace propellants, pharmaceuticals, agrochemicals, polymers, dyes, and various other chemicals.⁴ It is used as a key component in rocket fuels and airbag production.^5,6 Moreover, it plays a crucial role in synthesising compounds like azobisformamide (AC), diisopropyl carbonate, and toluenesulfonhydrazide (TSH).⁷ In addition to these applications, hydrazine is also employed as a corrosion inhibitor in nuclear and electrical power plants (Fig. 1).⁸

Fig. 1 a) Importance and hazardous effects of hydrazine on the environment and human life. b) Sensors used for hydrazine detection.

Nevertheless, hydrazine poses a significant threat to living beings due to its high toxicity, which leads to severe air, water, and soil pollution.⁹ When ingested or absorbed through skin, hydrazine's high water solubility facilitates rapid absorption into the body, leading to serious damage to internal organs and the central nervous system, with potential carcinogenic effects.¹⁰ Therefore, many countries have established threshold limits for hydrazine in different media. For instance, the thresholds limits are 0.01 μg L⁻¹ for air, 0.1 mg m⁻³ for workshop air, 0.01 mg L⁻¹ for drinking water, and ≤10.0 μg L⁻¹ for surface water and fishery water.¹¹ The development of quick and easy signal detection tools for N₂H₄ in water can effectively mitigate these risks.¹² Common detection techniques like chromatography, electrochemistry, titrimetry, and surface enhanced Raman spectroscopy often involve complex procedures or expensive equipment.¹³

The fluorescence sensor is undoubtedly one of the best detection methods due to its inherent ability to analyze a specific target molecule in a sample with exceptional selectivity, sensitivity, convenience of operation and cost-effectiveness.^12,13 Furthermore, fluorescent probes tailored for detecting N₂H₄, both in laboratory settings and in living organisms, have been steadily advancing in recent years, achieving notable advancements and technological breakthroughs.¹⁴ Small-molecular sensors and probes can change their emission properties in reaction to bound analytes or environmental alterations, offering versatile control over emission, binding, and reactivity.¹⁵ Fluorochrome sensors are generally designed based on a system that consists of three key components: an acceptor, a donor and an anchor. The anchor can selectively interact with a specific analyte in a certain way, usually through coordination, ionic interaction, hydrogen bonding, or chemical reaction, and thereafter, the receptor can change its photophysical properties when the interactions between the acceptor and analyte occur. A spacer connecting the two components can also be introduced to optimize the sensing performance in some cases.¹⁶ Different types of interactions between analytes and anchors have been utilized in various reports, such as Lewis acid and base interactions, collision quenching, and formation/cleavage of chemical bonds¹⁷ Among these detection mechanisms, Lewis acid and base interactions are the most frequently used strategy, owing to the rapid response. The acceptors used in this situation are always electron-donating units containing nitrogen, sulphur, or phosphorus atoms.¹⁸

Various small-molecule-based sensors have been reported, including boron dipyrromethene (BODIPY),^19–21 porphyrin,²² conjugated oligopyrroles,²³ coumarin,²⁴ quinoline^25,26 fluorescein,^27,28 rhodamine,^29–31 naphthalimide,³² and carbazole.³³ Most of the fluorochrome sensors have been designed based on the deprotection or chemical transformation of a protecting group by a specific deprotecting agent or analyte.^12,34,35 For example, the fluorescent sensing system developed by Dalip Kumar et al.³⁶ involved selective deprotection of a BODIPY-based fluorescent probe in the presence of hydrazine in DMSO–water. The sensing system reported by Liqiang Yan et al.³⁷ involved a cationic ratio-type hydrazine-selective (I450/I605) fluorescent probe BCaz-Cy2 based on carbazole and hemicyanine via deprotection of an acetyl group, enabling dual-channel detection in aqueous solutions. The majority of these probes could only be utilized in neutral pH (pH ∼7) conditions or within a fixed pH range, which would limit their application in physiological conditions. Therefore, it is still necessary to explore new small-molecule fluorescent sensors to achieve extremely high selectivity and sensitivity and operable across a broad pH range.^28–32 Additionally, the development of sensors with near-infrared emission and tolerance to complex detection environments is of great interest.

In this study, aromatic N-, S-, and O-heterocyclic acceptors, like pyridine, thiophene, furan and their derivatives, occupy a prominent position and have been successfully introduced into either the main chains or side groups of the probe. When interactions between these acceptor moieties of the fluorochrome sensors and different analytes occur, the fluorescence of these probes can exhibit significant changes, such as quenching, emission blue shifts, and fluorescence enhancement, to a lesser extent. This is a result of the switching the charge density from the respective donor to the acceptor, which is indicative of intramolecular charge transfer (ICT) behaviour that extends from the molecular donor unit to the acceptor unit. Photoinduced electron transfer (PET) involves the enhancement/reduction of the conjugation or electron transfer between the probe and analytes (e.g., FRET, Scheme 1). The development of fluorochrome-based sensor systems that enable efficient and fast detection of such analytes is currently a prominent and growing area of research in the field of chemistry.

Scheme 1 General mechanistic design for heterocycle-based fluorochrome sensors.

2. Experimental

2.1 Synthesis of heterocyclic fluorochromes from 4-acetylbiphenyl (3a–3h)

4-Acetylbiphenyl (1; 0.098 g, 0.5 mmol) was dissolved in a minimum quantity of ethanol (3 mL) and stirred to form a homogenous solution in a 25 mL RBF. 2-Pyridinecarbaldehyde (2a; 47.55 μl, 0.5 mmol) was added to the stirred solution, and the resultant reaction mixture was stirred at room temperature (27 °C) for 5 min (Scheme 2).

Scheme 2 Synthetic roots of fluorochrome formation with 4 acetylbiphenyl.

Subsequently, a catalytic amount of KOH solution in ethanol (4%, 2 mL) or pyrrolidine (0.2 mL) was added dropwise. The obtained mixture was stirred at room temperature for 2–3 h (for KOH) or 6–8 h (Table 1). The completion of the reaction was monitored by TLC. The product began to precipitate out of the reaction mixture as the reaction continued.

Table 1 Process optimisation with two catalysts

Compound	Catalyst KOH		Catalyst pyrrolidine
	Time (h)	Yield	Time (h)	Yield
3a	2.0	74%	6.0	87%
3b	2.0	75%	6.0	86%
3c	2.5	78%	6.5	88%
3d	2.5	79%	6.0	89%
3e	2.0	78%	5.0	86%
3f	3.0	77%	7.5	86%
3g	3.0	73%	8.0	90%
3h	3.0	73%	7.0	92%

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2.2 Spectral characterization and single crystal XRD study

The structures of all the synthesised fluorochrome were confirmed by NMR, HRMS, and FT-IR spectral analysis (Fig. S01–S25†). The crystal of 3g (CCDC: 2374118) was selectively grown in chloroform as per the reported process and used for the single crystal XRD study. The X-ray study, including data gathering, cell refinement, and data reduction were carried out using various software packages and mathematical equations, which are discussed in the ESI.†

2.3 Photophysical properties

To find the most suitable and highly efficient fluorochrome for detecting hydrazine, the photophysical nature of the prepared molecules was studied using their stock solutions (100 μM; 5 mL) prepared in HPLC-grade methanol. The absorption maxima and emission maxima were measured via UV-visible and fluorescence spectrophotometry using a fixed concentration (5 μM). Among all the studied molecules, 3g showed the highest Stokes shift. Therefore, solvent screening for UV-visible absorption and fluorescence emission were performed with 3g (5 μM) to find the most suitable solvent.

2.4 Hydrazine detection study

In order to perform the hydrazine detection study, a stock solution of 3g (5 μM) was prepared in HPLC-grade-methanol (5 mL). Hydrazine solutions of the required concentrations (1–10 μM) were prepared by serial dilution of an 80% solution of hydrazine hydrate with HPLC-grade methanol (5 mL). Absorption and emission titrations of 3gvs. N₂H₄ were performed with a fixed concentration of 3g (5 μM) and varied concentrations of N₂H₄ in methanol (0–10 μM).

3. Results and discussion

3.1 Substrate scope of the reaction

A novel series of heterocyclic fluorochromes were synthesized via a base-catalyzed Claisen–Schmidt condensation reaction. This series of molecules were prepared by the condensation of 4-acetylbiphenyl (1) and various heterocyclic aldehydes (2a–2h). The reaction was performed in ethanol at room temperature with two different catalysts—KOH and pyrrolidine—to study their effects on isolated yield and reaction time. During this study, it was clearly observed that pyrrolidine was an effective alternative to KOH. The extended reaction time could be due to the mild basicity of pyrrolidine. To extend the scope of the developed reaction process, it was performed with a series of aldehydes, namely, simple pyridine and its derivatives (3a, 3b, 3c). All three derivatives showed excellent isolated yields (87% to 88%) under the pyrrolidine-catalyzed process, which were notably higher compared to those achieved via the KOH-catalyzed process (74% to 78%). However, the KOH-catalyzed reactions were rapid for all these aldehyde derivatives. The scope and generality of this protocol was further demonstrated by extending it to other heterocyclic aromatic aldehydes, i.e., thiophene (3d), furan (3e), imidazole (3f), and benzothiophene (3g, 3h). These derivatives also gave excellent isolated yields under the optimized reaction conditions with pyrrolidine.

3.2 Structure confirmation of prepared fluorochromes by spectral studies

The structures of all the newly prepared fluorochromes were confirmed using various spectroscopic techniques including NMR, IR, HRMS, and single crystal XRD. All the spectra and spectral data are shown and discussed in the Supporting Information (Fig. S1–S25†). In the ¹H NMR spectrum of fluorochrome 3c, two doublet peaks with a similar coupling constant of 15.4 Hz were observed at δ 9.05 and 7.90 ppm. These peaks confirmed the presence of α and β protons with trans geometry in an α,β-unsaturated carbonyl system, respectively. Two more doublets with a similar coupling constant 8.5 Hz were observed at 8.175 and 7.813, which confirmed the presence of the biphenyl ring. An additional two multiplets, corresponding to the four protons of the pyridine ring, were observed at δ 8.306–8.202 ppm. The presence of a carbonyl group was further supported by the ¹³C NMR spectrum, which featured a peak at δ 189.67 ppm along with thirteen asymmetric carbons in the aromatic region at δ 145.59–122.65 ppm. The structure of PLS 3c was also confirmed by its HRMS spectrum. The observed m/z value (286.1232) corresponded to the [M+H]⁺ ion, consistent with the calculated m/z (285.1254) for the molecular ion. The presence of a carbonyl group was additionally confirmed by the FT-IR spectrum with a band at 1730 cm⁻¹ along with aromatic C=C stretching at 1687 cm⁻¹ and C–H stretching at 3025 cm⁻¹. Finally, the structure of fluorochrome 3g was also confirmed by single crystal XRD. Similarly, the structures of all the fluorochromes 3a–3h were also confirmed using ¹H NMR, ¹³C NMR, HRMS, and FT-IR spectral analysis.

3.3 Final structure confirmation via single crystal XRD study

The structural geometry of the prepared scaffold 3g was finally confirmed using a single crystal XRD study. The XRD data showed the trans geometry of the structure of the prepared fluorochrome (Fig. 2). The crystal data and molecular refinement information are listed in Table S1† and discussed in the supporting information.

Fig. 2 (a) Single crystal ORTEP structure of 3g; (b) stacked structure formed by hydrogen bonds.

3.4 Hydrazine detection study using the photophysical properties of the prepared fluorochromes

The Stokes shift of a fluorochrome is a unique property that determines its detection efficiency.^37,38 This property is very important when there is a high auto-fluorescence background, which is commonly seen in complex biomedical specimens. Fluorochrome signals with a small Stokes shift are often supressed by the strong background auto-fluorescence, whereas fluorochrome signals with a large Stokes shift are clearly visible above the background.³⁸

Therefore, all the fluorochromes (5 μM) were investigated via a UV-vis absorption study in methanol (Fig. 3A). Fluorochrome 3g showed the highest absorption compared to all other fluorochromes. The results of the photophysical studies also revealed the largest Stokes shift of 129 nm for 3g (Table 2), which is similar to that of semiconductor QDs and very rarely observed in organic molecules. Even coumarin dichlorofluorescein and BODIPY-based scaffolds show Stokes shifts of around 58 and 21 nm, respectively.^39,40 Therefore, the hydrazine detection studies were conducted using 3g. To determine the most suitable solvent for the detection study, the photophysical properties of 3g (5 μM) in various solvents with different polarities from hexane to methanol were explored (Fig. 3B). The absorption spectra of 3g in the various solvents were similar, with the main absorption peaks at ∼352 nm. Solvent effects are more significant in polar solvents due to change in the charge density of the fluorochrome caused by the solvent, which alters the electronic distribution of the fluorochrome and results in solvatochromatic shifts.^41,42 These changes are smaller in nonpolar solvents due to the small dielectric constant possessed by the solvents. Hexane, a less-polar solvent, showed the lowest absorption peak at 335 nm (ε = 15.68 × 10⁻³ cm⁻¹ M⁻¹), and methanol, a polar solvent, showed the highest absorbance at 352 nm (ε = 19.71 × 10⁻² cm⁻¹ M⁻¹) for the fluorochrome 3g. Therefore, methanol was used as a solvent for all the hydrazine detection studies of 3g. Thereafter, a fluorescence study of all the fluorochromes (5 μM) was conducted in methanol (Fig. 4A), and fluorochrome 3g showed exponential emission at 481 nm. Therefore, the emission properties of fluorochrome 3g (5 μM) in various solvents with various polarities were also studied via fluorescence emission spectra. As shown in Fig. 4B, the fluorescence intensity and emission maxima of fluorochrome 3g gradually increase with the increase in solvent polarity from hexane to methanol.

Fig. 3 UV-vis spectra of 3a–3h (5 μM) in methanol (A) and of 3g in different solvents (B).

Table 2 Stokes shifts of the prepared fluorochromes

Code	Absorbance λmax (nm)	Emission λmax (nm)	Stokes shift (nm)
Ref. 1 Coumarin dichlorofluorescein.^39 Ref. 2 BODIPY fluorophore.^40
3a	310	367	57
3b	307	367	60
3c	309	367	58
3d	347	408	61
3e	339	420	81
3f	293	400	107
3g	352	481	129
3h	307	395	88
Ref. 1	416	474	58
Ref. 2	502	523	21

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Fig. 4 Fluorescence spectra of 3a–3h (5 μM) in methanol (A) and of 3g in different solvents (B).

The position of the fluorescence peaks shows an obvious red shift from 335 (hexane) to 481 nm (methanol), as expected for the increase of the solvent polarity. Among all the studied solvents, methanol showed very strong fluorescence emission of fluorochrome 3g. Therefore, all the studies of hydrazine detection by fluorescence emission were also carried out in methanol. UV-vis absorption and fluorescence emission titration studies with a fixed concentration (5 μM) of fluorochrome 3gvs. the addition of different concentrations of hydrazine (0 μM to 10 μM) were carried out. At an equal concentration ratio of 3g/hydrazine, the absorbance was almost at saturation (Fig. 5A). After the addition of 8 μM hydrazine, a hyperchromic shift was observed. The absorbance (0.7 au) was increased (0.9 au) compared to that of pure 3g. A significant blue shift in the absorption maximum (352 to 300 nm) was also observed, and visually, the colour changed from light yellow to colourless. This observation indicates that the 3g fluorochrome can also be used as a naked-eye probe for hydrazine. The fluorochrome 3g shows very high fluorescence emission at 481 nm, which resembles the bright green fluorescence in the fluorescent light. The gradual addition of NH₂NH₂ (1 μM to 5 μM) leads to an intense decrease in the fluorescence emission at 481 nm (λ_ex = 352 nm) (Fig. 5B). The elimination of the colour and fluorescence emission is due to the ratiometric response caused by the cyclization of hydrazine at the chalcone moiety. This leads to breakage of the π-conjugated system of 3g followed by the destruction of ICT, along with electron transfer between the probe and analytes (e.g., FRET), leading to a quick response. A very good linear relationship was observed for the detection of hydrazine within the concentration range of 1–5 μM in methanol. The limit of detection (LOD) and limit of quantification (LOQ) of 5 μM 3g for hydrazine were estimated to be 0.11 μM and 1.1 μM according to the determination formulae LOD = 3Sb/k and LoQ = 10Sb/k, respectively (where Sb is the standard deviation of the blank, and k is the slope of the calibration line). The linear fitting curves of absorbance at 352 nm and fluorescence intensities at 481 nm for 5.0 μM fluorochrome 3g in the presence of varied concentrations of N₂H₄ from 1.0–5.0 μM are given in the ESI† (Fig. S27). This study shows the sensitivity and efficiency of fluorochrome 3g for the detection of hydrazine, even at very low concentrations, with a 1 : 1 ratio. In comparison, as per the literature, hydrazine detection studies using coumarin dichlorofluorescein and BODIPY-based scaffolds required two equivalents of the probe with a 10.0 μM detection limit.^39,40 Also, the BODIPY-based fluorochrome required HEPES buffer in H₂O–DMSO solution for hydrazine detection.^39,40 This comparison shows the potency and user-friendliness of fluorochrome 3g. Moreover, the colour change of the 3g solution from yellow to colourless can be observed by the naked eye, showing that 3g can also be used as a visual colorimetric sensor for hydrazine at very low concentration. The time course plots (time vs. emission intensity and time vs. absorbance, Fig. S28†) clearly show the stationary phase in UV-vis absorption and emission within 3 min after addition of hydrazine, which makes 3g a quickly responding real-time sensor for hydrazine.

Fig. 5 Titration of 3g (5 μM) vs. hydrazine (1–10 μM) by UV-vis spectroscopy (A) and fluorescence spectroscopy (λ_ex = 352 nm) (B) in methanol.

3.5 Mechanistic investigations of detection by fluorochrome 3g

As per our earlier studies,^43–46 in order to establish the mechanism (Scheme 3) of hydrazine detection, the resultant mixture of fluorochrome 3g and hydrazine in ethanol was injected into a LC-MS. The observed molar mass clearly confirmed the formation of 1H-pyrazole (3g-NH₂NH₂), which was further supported by NMR (Fig. 6, S25†) and HRMS (Fig. S26†) analysis, as presented in the ESI.† Hydrazine is a good nucleophile, and therefore, it easily attacks the electrophilic carbonyl carbon of the α,β-unsaturated carbonyl system, resulting in the formation of imine as an intermediate. Due to its open-chain structure, the intermediate is unstable, leading to a second nucleophilic attack by the amine group on the β-carbon of the α,β-unsaturated carbonyl system, resulting in the formation of a stable cyclic 1H-pyrazole (3g-NH₂NH₂). The observed fluorescence quenching upon hydrazine binding is due to the disruption of the π-conjugation between the donor biphenyl and accepter benzothiphene moieties. This disruption stops the ICT and results in fluorescence quenching.

Scheme 3 Study of THE sensing of hydrazine by fluorochrome 3g.

Fig. 6 ¹H NMR spectra of 3g (a) and of 3g-NH₂NH₂ after the addition of hydrazine (b).

3.6 Quantum chemical calculation

In addition, DFT and time-dependent DFT calculations were performed to validate the accuracy of the proposed mechanism and optimized geometries.^47,48 The HOMO/LUMO level and the energy gap of fluorochrome 3g and its hydrazine analogue pyrazoline 3g-NH₂NH₂ were calculated with B3LYP/6-31G, as implemented in Gaussian 16, as shown in Fig. 7(A). The optimized structures of fluorochrome 3g, intermediate and 3g-NH₂NH₂ are presented in Tables S4–S6,† and the respective atom labeling is shown in Fig. 7. The UV-absorption spectra of fluorochrome 3g and 3g-NH₂NH₂ in methanol were calculated and compared with the experimental outcomes. Both results were in good agreement with the experimental analysis (Fig. 7(C)). The spontaneous reaction converting 3g to 3g-NH₂NH₂ reduces the absorption wavelength from 367 to 338 nm (hypsochromic shift). This blueshift property is attributed to the cyclization of the product in the presence of hydrazine, which leads to a change in the emission colour from yellowish to blue. The theoretically calculated UV-vis absorption maximum for 3g was found to be 367 nm with 0.67 oscillation strength (f) (3.37 eV), and the HOMO → LUMO contribution is 73%. In the case of 3g-NH₂NH₂ the maximum was found to be 338 nm with 0.82 oscillation strength (3.66 eV), and the HOMO → LUMO contribution was 92%. These results indicate that the H → L contribution increased from the reactants to the products. Further, to understand the intermediate stage, its UV-absorption maximum was theoretically calculated and found to 334 nm with 0.44 oscillation strength and a HOMO → LUMO contribution of 90%. This reveals that the H → L contribution is smaller before cyclization and increases with cyclization (73 → 90 → 92%). The electron density is mainly distributed within the biphenyl donor moiety of 3g in the HOMO level and moves to the thiophene acceptor moiety of 3g in the LUMO, which is a prevalent feature of donor-π-acceptor (D-π-A) type fluorochromes. When the host (3g) is combined with N₂H₄, the electron density in the HOMO and LUMO tend to homogenize, which can undergo ICT upon electronic excitation.^48,49 The energy gap between the HOMO and LUMO levels of fluorochrome 3g and 3g-NH₂NH₂ were 3.80 eV and 4.09 eV, respectively, indicating a blueshift of the absorption spectrum after the pyrazole formation, which coincides with the experimental results (Fig. 7(B)). The structure of the intermediate and the reaction energy were calculated as shown in Fig. 7(C) based on the fact that N₂H₄ first attacks the carbonyl group of fluorochrome 3gvia a nucleophilic addition reaction, and the detection mechanism probably involves the formation of imine as an unstable intermediate and followed by cyclization reaction to generate the 3g-NH₂NH₂. The processes of the formation of the hydrazine adduct (intermediate) and the final product (3g-NH₂NH₂) are both exothermic by −2.66 kcal mol⁻¹ and −8.68 kcal mol⁻¹, respectively. Thus, the overall process is exothermic by −11.33 kcal mol⁻¹, indicating the feasibility of the reaction. In addition, the Gibbs free energy change of the reaction was found to be negative (ΔG < 0), indicating a spontaneous process (Table S8†).

Fig. 7 (A) Geometry-optimized structures and frontier molecular orbital plots of 3g and 3g-NH₂NH₂. (B) UV-vis spectra of 3g and 3g-NH₂NH₂ in MeOH (5 μM). Inset: Colour change observed upon the addition of NH₂NH₂ (10 μM). (C) The optimized stationary structures and energy on the potential surfaces of the reactions with comparison of the calculated UV-vis spectra.

3.7 Interference study of fluorochrome 3g with different analytes

Resistance to interference is a precondition for a fluorochrome in real-time applications. To establish the real-time efficiency of fluorochrome 3g, the selectivity of the 3g towards hydrazine versus other interfering amino compounds including anions, metal ions, and biological species was studied (Fig. 8). As expected, the fluorochrome 3g as a fluorescent detector for hydrazine has exceptional discrimination and anti-interference ability against the interfering compounds studied during the experiment. A competitive study was performed to assess the selectivity and anti-interference ability of fluorochrome 3g under realistic conditions involving other interfering chemical species. Initially, the emission studies at 481 nm were conducted with solutions of 5 μM fluorochrome 3g and 5 μM of various interfering chemicals. The results obtained clearly showed the negligible effect of the interfering chemicals on the emission property of fluorochrome 3g (Fig. 8, solid column). Later, 5 μM hydrazine was added to all these solutions, and the emission at 481 nm was recorded. The observed results clearly show very high quenching of the emission property of fluorochrome 3g in the presence of interfering chemicals (Fig. 8, dotted column). This study demonstrated the fluorochrome as an exceptional optical sensor for hydrazine with great selectivity and anti-interference in the presence of all of the studied interfering chemicals.

Fig. 8 Selective preference of 3g (5 μM; 481 nm) for hydrazine versus other analytes.

3.8 Effect of pH on fluorochrome 3g emission

The pH plays a vital role in the sensing ability of fluorochromes; therefore, a study of the effect of pH on the emission properties of fluorochrome 3g (Fig. 9) was performed to determine its working pH range. The obtained results demonstrated that it has outstanding recognition capability for hydrazine in 4.0–10.0 pH range. At extremely acidic pH values below 4, the emissive nature of fluorochrome 3g is completely quenched, which could be due to the protonation of the carbonyl oxygen, which leads to disruption of the π-conjugation. In addition, at basic pH values of higher than 10, the emission property of fluorochrome 3g was also completely absent, which could be because of nucleophilic addition of a hydroxyl ion on the chalcone moiety, which leads to disruption of the π-conjugation.

Fig. 9 Effect of pH on hydrazine detection by 3g (5 μM; 481 nm).

3.9 Real-time hydrazine detection by fluorochrome 3g in soil, water and the atmosphere

All the above studies suggested that 3g could have excellent detection ability for hydrazine in real water, soil, and air samples. Therefore, the use of fluorochrome 3g for detecting hydrazine in different soils was also examined. As a part of the first experiment, 1 g of clay soil/ field soil/ sandy soil were added separately to 3g (5 μM) solutions in methanol (5 mL), mixed well, and allowed to settle for half an hour. The fluorescence emission spectra of the supernatants were measured (Fig. 10), which showed that the different soil samples had no major effect on the emission of fluorochrome 3g. This observation indicated that the substances in the different soils did not disturb the fluorescent nature of the fluorochrome 3g. As a second experiment, 1 g of each soil type (clay/field/sand) was contaminated with hydrazine (5 μM; 1 mL methanol), and the resulting pastes were allowed to dry at room temperature. Thereafter, these contaminated soil samples were added separately to 3g (5 μM) solutions in methanol (5 mL), mixed well and allowed to settle for half an hour. The fluorescence emission spectra of the supernatants were recorded and showed significant decreases in the emission of fluorochrome 3g. Finally, photographs of both the experiments were taken with a smartphone under daylight and portable UV lamp irradiation (365 nm), respectively (Fig. 10). Based on these studies showing the quick response time, profound detection capacity, resistance to interference and outstanding selectivity of fluorochrome 3g, a study for detecting hydrazine by 3g in actual water samples was also explored.

Fig. 10 Emission spectra of fluorochrome 3g (5 μM; 481 nm) after the addition of different soil samples in methanol with and without hydrazine.

As normal water contains some level of minerals including Na⁺, Cu²⁺, Mg²⁺, Fe³⁺, as well as dissolved organic substances, fluorochrome 3g was investigated for the detection hydrazine of in various samples of water, i.e. tap water, river water, and mineral water.

Initially, a solution of 3g (5 μM; 1.5 mL) in methanol was mixed with the three different water samples (2 mL of each), and their emission spectra were recorded.

The obtained results clearly showed that these water samples did not affect the emission properties of 3g. A very low but significant decrease in intensity was observed in the river water sample, which could be due to the presence of hydrazine or another similar analyte in this sample (Fig. 11). However, after addition of equal quantity of hydrazine (5 μM; 1.5 mL) to these water samples, there was large decrease in the emission of fluorochrome 3g. The recovery results are presented in supporting information Table S1† for both water and soil samples based on the emission intensity of fluorochrome 3g. The concentrations of hydrazine measured by 3g were appropriate to the concentrations added (the recovery ranged from 84% to 100%). These experiments clearly show that the probe 3g has good performance and practical value for the detection of trace amounts of hydrazine in water as well as soil samples.

Fig. 11 Emission spectra of fluorochrome 3g (5 μM; 481 nm) after the addition of various water samples in methanol with and without hydrazine.

To explore the further practical applications of fluorochrome 3g, it was also used for the detection of hydrazine in the gas phase. In this investigation, the changes in fluorochrome 3g-doped paper strips under 265 nm and 365 nm UV lamp were captured after exposure to various concentrations of hydrazine gas. As shown in Fig. 12, with increasing hydrazine concentration, the exterior of the probe-treated paper strips under 365 UV Light exhibited fluorescent emission quenching with the colour changing from bright green to no emission. The results indicated that hydrazine gas in the environment can be conveniently monitored by the naked eye.

Fig. 12 Photographs of the probe-coated filter paper upon exposure to gaseous hydrazine vapor from hydrazine aqueous solutions of different concentrations. Upper: Under 265 nm UV light; lower: under a hand-held UV lamp with an excitation at 365 nm in the dark.

To investigate reusability, the sensing paper strips were thoroughly washed with water, dried well, and then tested for their fluorescence under 365 nm excitation at. However, the fluorescent nature of the strips remained quenched and non-fluorescent. This result indicates that the mechanism of hydrazine sensing is irreversible.

4. Conclusion

This article reports the development of two comparative protocols for KOH- and pyrrolidine-catalyzed Claisen–Schmidt condensation. The results showed that KOH enables rapid reaction, while pyrrolidine affords higher yields in the synthesis of seven novel fluorochromes featuring various aromatic heterocycle scaffolds. The structures of the prepared molecules were confirmed by NMR and HRMS analysis. The structure of 3g was confirmed by a single crystal XRD study. Generally, aromatic heterocycle structures are photoluminescent moieties; therefore, the photophysical properties of the prepared molecules were investigated by UV-visible and fluorescence analysis in various polar and nonpolar solvents. Based on their photoluminescence, a selective heterocycle-based benzothiophene fluorochrome has been demonstrated for hydrazine detection using two spectroscopic techniques. The disappearance of the colour and photoluminescence signal relies on a ratiometric-responsive cyclization caused by hydrazine. The disruption of the π-conjugated system by hydrazine, resulting in the destruction of the ICT, along with electron transfer between the probe and analytes (e.g., FRET) in the fluorochrome leads to a quick response. The selectivity, anti-interference capacity (pH, organic and inorganic interferents), limit of detection and solvent-dependence of the sensing efficiency were investigated in detail. Moreover, compared with some reported sensors, the probe showed a rapid detection process, a good linearity range and a low detection limit of hydrazine (Table S2†). A possible mechanism for the response to exposure to hydrazine was also proven by a spectral study. Moreover, this study described a straightforward cost-effective probe-coated paper sheet for detecting hydrazine in environment. We believe that the outcome of this sensing probe and method will inspire the future design of real-time probes for hydrazine and other chemicals with diverse applications.

Data availability

Additional data related to the main manuscript are available in the ESI,† and single crystal XRD data are available at https://www.ccdc.cam.ac.uk/deposit/ with CCDC Number 2374118.

Author contributions

All the experiments were designed and performed by Dinkal Kasundra and Paresh Patel. The spectral study, interpretation, and correlations were done by Dinkal Kasundra and Paresh Patel. The manuscript was written by Dinkal Kasundra. The final proofreading and editing were done by Paresh Patel. Computational studies were performed and documented by Rajamouli.

Conflicts of interest

The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper.

Acknowledgements

The authors would like to acknowledge financial support from GSBTM and SHODH Scheme, Govt. of Gujarat (Ref No: 202201734). We acknowledge the NMR facility, CIC, Bharathiar University supported by DST (PURSE Phase II programme), New Delhi. HRMS facility, Indian Institute of Science Education and Research (IISER) Tirupati and Dr Arnab Dutta and group (IIT B) for the single crystal XRD Study. The authors are also thankful to Aether Industry Ltd. for mass analysis.

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Footnote

† Electronic supplementary information (ESI) available: Additional data related to the main manuscript. CCDC 2374118. For ESI and crystallographic data in CIF or other electronic format see DOI: https://doi.org/10.1039/d5ew00322a

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