Development of semicarbazide-based fluorene as an AIE-active fluorescent sensor for Cu²⁺ ions: a validation through experimental and DFT studies

Abstract

A fluorene-based fluorescent sensor, FHT, was rationally designed and synthesized, which exhibited prominent solvatochromic behavior and aggregation-induced emission (AIE) effects in aqueous medium. Upon aggregation, the FHT sensor showed an enhancement in emission intensity accompanied by a bathochromic shift, indicating the formation of J-aggregates. Owing to its unique photophysical properties, FHT was developed as a highly selective fluorescent sensor for Cu²⁺. Selective binding of FHT to Cu²⁺ resulted in significant fluorescence quenching via a photo-induced electron transfer (PET) process. The binding interactions of FHT with Cu²⁺ were studied in detail by ¹H NMR titration, liquid chromatography-mass spectrometry (LC-MS), dynamic light scattering (DLS), Fourier-transform infrared (FTIR) spectroscopy, and density functional theory (DFT) analysis. Upon interaction with Cu²⁺, the FHT sensor exhibited strong fluorescence quenching with a low detection limit of 82 nM and quenching efficiency of 97%. In addition to its solution-phase sensing capability, FHT demonstrated effective performance in the solid state and enabled naked-eye colorimetric identification of Cu²⁺. Furthermore, the FHT sensor was successfully integrated into molecular logic-gate operations, emphasizing its promise for on-site environmental monitoring and the development of molecular electronic devices.

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1. Introduction

In recent years, substantial interest has been directed toward the development of highly selective and sensitive fluorescent imaging tools for the detection of transition-metal ions, resulting in great progress in the design of metal-specific fluorescent sensors.^1,2 Among these transition metals, copper has become particularly important as a target because of its important role in a large number of biological, environmental and industrial processes.^3–5 Being a redox-active species, it is involved in enzymatic processes that are essential for life, including respiration in mitochondria, biosynthesis of neurotransmitters and antioxidant defense mechanisms.^6–9 Although it is biologically important, an excess of copper may cause disruption of the homeostasis of cells, leading to severe pathological conditions.¹⁰ Excess Cu²⁺ has been associated with Wilson's disease,¹¹ Alzheimer's disease,¹² Parkinson's disease,¹³ and other forms of neurodegenerative diseases.¹⁴ On the other hand, a deficiency of Cu²⁺ can cause anemia, poor immunity, and metabolic disorders.^15–17 In addition to biological concerns, Cu²⁺ is released into environmental water bodies through domestic, industrial, and agricultural activities, particularly through the use of copper-based pesticides employed to prevent plant diseases and enhance agricultural productivity.^18,19 As a result, reliable monitoring of Cu²⁺ levels in water is crucial, with guideline values set at 2.0 ppm by the World Health Organization and 1.3 ppm by the U.S. Environmental Protection Agency.^20,21 Thus, the accurate and sensitive detection of Cu²⁺ is essential to study its biological roles as well as for analyzing environmental and physiological samples.

The reported analytical techniques for Cu²⁺ sensing are atomic absorption spectroscopy (AAS),²² inductively coupled plasma mass spectrometry (ICP-MS),²³ gas chromatography and liquid chromatography mass spectrometry,^23,24 potentiometry,²⁵ electrochemical techniques,²⁶ colorimetry,²⁷ and spectrophotometry.²⁸ However, these analytical methods face challenges, including low accuracy, poor qualitative performance, and time-consuming and high-cost instrumentation, which prevent their practical application. In contrast, fluorescence-based sensing has been developed as a powerful method for metal-ion sensing due to its low cost, high sensitivity, low detection limits, rapid response, ease of operation, and the non-invasive nature of detection.

Considering their effectiveness and extraordinary characteristics, various fluorescence sensors, including polymers,²⁹ carbon dots,³⁰ and quantum dots,³¹ and small organic molecules such as rhodamine semicarbazide,³² coumarin-based semicarbazide,³³ Schiff-based semicarbazide derivatives,³⁴ and naphthalimide-based semicarbazide chemosensors, have been reported for sensing of Cu²⁺. However, these reported sensors also have drawbacks, such as high detection limits, synthetic complexity, poor selectivity, and poor stability. One factor that limits their performance is aggregation-caused quenching (ACQ), which arises due to the planar molecular geometries that result in the formation of strong intermolecular non-covalent interactions, particularly π–π stacking and dipole–dipole interactions.³⁵ The principle of aggregation-induced emission (AIE), which is opposite to aggregation-caused quenching (ACQ), was first reported by Tang's research group.³⁶ AIE-active luminogens are fluorophores that show negligible emission when diluted but present a remarkable enhancement in emission intensity in the aggregated or solid state.^37–39 The formation of molecular aggregates, such as J-aggregates, from a head-to-tail molecular alignment, which restricts intramolecular motions within the fluorophores and facilitates a more efficient radiative decay pathway.^40–42 In contrast, H-aggregates are formed through face-to-face stacking of molecules, resulting in strong excitonic coupling that leads to a blueshift (hypsochromic shift) in the emission spectra,⁴³ while X-aggregates exhibit a cross-stacked or twisted molecular packing pattern, leading to strong fluorescence in the solid state due to reduced non-radiative decay pathways.⁴⁴

In this study, a fluorene-based fluorescent sensor FHT was synthesized through a Schiff-base condensation reaction and subsequently characterized by NMR spectroscopy and mass spectrometry. The sensor FHT exhibited pronounced solvatochromism, and it exhibits significant changes in emission wavelength with variation in solvent polarity, which reflects changes in its electronic distribution and stabilization in excited state. Moreover, aggregation-induced emission (AIE) was also observed in the FHT sensor, which was attributed to the restricted intramolecular motions (RIR) and the formation of J-aggregates, leading to an increase in the emission intensity accompanied by a bathochromic shift. Owing to its unique photophysical characteristics, the FHT sensor is capable of selectively and sensitively detecting Cu²⁺ through a PET mechanism in a DMF/water (1 : 9, v/v) system with a calculated LOD of 82 nM and quenching efficiency of 97%. Additionally, the FHT sensor was demonstrated to have good stability in different environmental conditions, such as varying pH and temperature, and is resistant to prolonged UV exposure. The FHT sensor was synthesized in good yield, exhibited excellent photophysical properties, including solvatochromic behavior and aggregation-induced emission (AIE). It selectiviely detects Cu²⁺ in both the solution and solid phase, while demonstrate potential for applications in molecular logic-gate systems.

2. Experimental section

2.1. Materials and chemicals

Analytical-grade reagents were utilized throughout the study and were purchased from Alfa Aesar (UK), Sigma-Aldrich (USA), Oakwood Chemicals (USA), and Daejung Chemicals & Metals (Korea). Reagents, such as acetonitrile (HPLC grade), chloroform (HPLC grade), ethanol (HPLC grade), hexane (analytical grade), DMSO (analytical grade), DMF (HPLC grade), ethyl acetate (freshly distilled), acetic acid glacial (99–110%), calcium chloride (95%), sodium carbonate (99%), sodium sulfate (99%), copper(II) nitrate (99%), nickel(II) nitrate (99%), copper cyanide (99%), and mercury(II) chloride (99%), were obtained from Daejung Chemicals & Metals (Korea). Aluminum(III) chloride (99%), potassium iodide (99%), zinc(II) sulphate (99%), silver nitrate (99%), magnesium(II) bromide (99%), fluorene (99%) and thiosemicarbazide (99%) were purchased from Oakwood, USA, and ammonium nitrate was obtained from Sigma-Aldrich. Moreover, the structure of the synthesized FHT sensor was confirmed using ¹H and ¹³C{¹H} NMR spectroscopy with a Bruker Avance III spectrometer, with dimethyl sulfoxide (DMSO-d₆) employed as the solvent. Photoluminescence measurements were performed on a PerkinElmer LS-45 spectrofluorometer (Hitachi, Japan), using 340 nm as the excitation wavelength and 440 nm as the emission maximum.

2.2. Synthesis of 2-(9H-fluoren-9-ylidene)hydrazine-1-carbothioamide

The target compound 2-(9H-fluoren-9-ylidene)hydrazine-1-carbothioamide (FHT) was synthesized from the reaction between 9H-fluorenone and thiosemicarbazide by refluxing equimolar amounts (3.0 mmol) of 9-fluorenone 1 and thiosemicarbazide 2 in 10 mL of absolute ethanol with 2–3 drops of glacial acetic acid as a catalyst. Reaction progress was monitored using TLC with the R_f value of 0.45; after completion, the mixture was cooled to ambient temperature, and the precipitate was isolated by filtration. Purification by silica gel column chromatography (eluent :  n-hexane/ethyl acetate, 1 : 1) gave the desired pure product as a yellow powder (547 mg, 72% yield). The melting point of the synthesized compound FHT was 189 °C. ¹H NMR (400 MHz, DMSO-d₆) (δ, ppm): 10.84 (s, 1H NH), 8.73 (s, 1H, NH), 8.43 (s, 1H, NH), 8.04 (dd, J = 7.4, 0.9 Hz, 2H, ArH), 7.89 (d, J = 7.6, 0.9 Hz, 1H, ArH), 7.81 (d, J = 7.6, 0.9 Hz, 1H, ArH), 7.52 (td, J = 7.6, 1.0 Hz, 1H, ArH), 7.43 (dd, J = 7.4, 1.2 Hz, 2H, ArH), 7.34 (td, J = 7.5, 1.1 Hz, 1H, ArH). ¹³C{¹H} (100 MHz, DMSO-d₆) (δ, ppm): 180.61 (1 × C), 145.93 (1 × C), 141.89 (1 × C), 139.80 (1 × C), 137.08 (1 × C), 131.74 (1 × C), 130.72 (1 × C), 129.56 (1 × C), 128.57 (1 × C), 128.52 (1 × C), 127.69 (1 × C), 122.86 (1 × C), 121.15 (1 × C), 120.66 (1 × C). DEPT-135 NMR (100 MHz, DMSO-d₆): (δ, ppm): 131.74 (1 × C), 130.72 (1 × C), 128.57 (1 × C), 128.52 (1 × C), 127.69 (1 × C), 122.86 (1 × C), 121.15 (1 × C), 120.66 (1 × C).

2.3. Fluorescence experiments

For fluorescence emission experiments, an optimized concentration of 30 µM of the FHT sensor was utilized in a DMF/H₂O solvent system with a 1 : 9 (v/v) ratio. 30 µM of sensor FHT exhibited enhanced fluorescence intensity upon excitation at 340 nm. Sensor FHT exhibited a strong fluorescence emission peak at 440 nm without self-quenching or inner filter effect (Fig. S1a). A slight decrease in emission was observed with futher increase in concentration from 30 to 50 µM, likely due to the inner filter effect; therefore, an FHT concentration of 30 µM was selected for all subsequent sensing studies to ensure optimal sensitivity and reproducibility. The fluorescence behavior of the FHT sensor was also investigated by gradually changing the water content from 0 to 90% in dimethylformamide (DMF) to determine the solvent-dependent emission properties. Similarly, 1 mM stock solutions of different metal salts, such as calcium chloride, sodium carbonate, sodium sulfate, copper(II) nitrate trihydrate, nickel(II) nitrate hexahydrate, copper(I) cyanide, mercury chloride, aluminum chloride, ammonium nitrate, potassium iodide, zinc sulphate, silver nitrate, magnesium sulphate, and ammonium nitrate, were prepared, and each was added separately to the optimized FHT sensor solution in DMF/H₂O (1 : 9, v/v). In order to assess the effect of metal ions on the FHT sensor, fluorescence titrations were conducted by adding the metal ions gradually and tracking the variation in emission intensity.

2.4. Computational methodologies

Density functional theory (DFT) and similar theoretical calculations were performed with the Gaussian 09 program package.⁴⁵ GaussView 5.0, VMD, Multiwfn 3.7 and GaussSum were used to process and visualize the computational output data, to facilitate their analysis. The B3LYP functional (Becke's three-parameter exchange with Lee–Yang–Parr correlation) was employed for geometry optimization of the FHT sensor and FHT–Cu²⁺, and the ωB97XD functional was utilized for calculating the interaction energy of the FHT sensor with Cu²⁺ for long-range and unique dispersion interactions.⁴⁶ The Pople-type basis set 6-311G(d,p), corresponding to a triple-zeta level with added polarization functions, was employed to calculate the electronic structure and molecular properties of the FHT sensor, while the LanL2DZ basis set, due to its effective core potential, was utilized for FHT–Cu²⁺, being suitable for transition-metal complexes and organometallics.^47,48 These kinds of computation approaches are well known to provide a reliable description of molecular geometries and non-covalent interactions. In addition, frontier molecular orbital (FMO) and density of states (DOS) analyses based on Gaussian 09 were performed to give information about the electronic features of the interacting molecules.⁴⁹ Multiwfn and VMD were used to study the non-covalent interactions, which offered an understanding of van der Waals, electrostatic and steric repulsive interactions between the fragments.⁵⁰ This NCI analysis was conducted by investigating 2D reduced density gradient (RDG) space and 3D iso-surface displays.⁵¹ Furthermore, QTAIM analysis, according to Bader's theory, was carried out to elucidate the nature and strength of the interactions between the sensor and the analyte.⁵²

3. Results and discussion

3.1. Chemistry of the FHT sensor

The synthesis of the FHT sensor was achieved via a Schiff-base condensation reaction, wherein the carbonyl group of reactant 1 is attacked by the terminal amino group of reactant 2. This results in the production of a C=N double bond following a dehydration phase under acidic and reflux conditions. As a result, a C=N linkage forms between the fluorenone moiety and the thiosemicarbazide group, yielding the desired product (Scheme 1). The FHT sensor was characterized by ¹H, ¹³C, and DEPT-135 NMR spectroscopy. Aromatic and olefinic protons appeared between δ 7.02–7.87 ppm in the ¹H NMR spectrum (Fig. 1a). In the ¹³C NMR spectrum, peaks at δ 180.60 ppm and 145.93 ppm correspond to thio-carbonyl (C=S) and imine (C=N) carbons, respectively (Fig. S1b). The absence of a carbonyl (C=O) signal near δ 193 ppm confirmed the formation of the imine moiety in the FHT sensor. The DEPT-135 NMR spectrum showed the expected carbon signals, further confirming the successful synthesis of the FHT sensor (Fig. S1c). Detailed NMR spectra of FHT are shown in the SI.

Scheme 1 Synthetic scheme to access the fluorene-based sensor FHT.

Fig. 1 ¹H NMR spectrum recorded in DMSO-d₆ of the FHT sensor (a). Emission spectra of the FHT sensor recorded in various solvents (excitation wavelength: 340 nm; concentration: 30 µM) (b). CIE chromaticity diagram illustrating shifts in the emission maximum (c). Emission spectra of the FHT sensor across various water fractions from 0% to 90% (d). CIE chromaticity diagram of the FHT sensor at different water fractions (e). DLS analysis performed at 0% and 90% water content to evaluate the aggregation behavior of the FHT sensor (f and g).

3.2. Solvatochromism

To examine the solvatochromic emission performance of the FHT sensor, solvents of different polarities were studied. The FHT sensor was found to exhibit a significant change in the emission maximum throughout its response to the solvent environment, with 410 nm in ethyl acetate, 426 nm in chloroform, 440 nm in DMF, 443 nm in ethanol, and 455 nm in acetonitrile (Fig. 1b). It is suggested that this gradual bathochromic effect of 45 nm is indicative of increased stabilization of the excited state as compared to the ground state, and it is observed that the excited state of the FHT sensor exhibits intramolecular charge transfer (ICT) character. The ICT mechanism is facilitated by the presence of heteroatoms (N and S) and the π-conjugation structure in the FHT sensor, making the excited state more sensitive to the surrounding solvent environment. The imine (–C=N–), thiocarbamide linkage (–HN–C=S), and aromatic core facilitate a powerful intramolecular charge transfer on excitation. Therefore, the ICT in excited state is more stabilized by polar solvents, resulting in bathochromic emission.^53,54 The variation in emission wavelengths of the FHT sensor were evaluated using the Commission Internationale de l’Eclairage (CIE) chromaticity diagram, which provides a graphical representation of emission color based on chromaticity coordinates; the emission maxima are indicated with black markers situated within the blue spectral region at coordinates (0.152, 0.162) and (0.159, 0.221) (Fig. 1c).

3.3. Aggregation-induced emission behavior of the FHT sensor in aqueous media

The solubility of organic fluorophores is generally very low in highly polar solvents such as water, and they readily aggregate in aqueous solutions. These aggregates can greatly affect the fluorescence emission properties of fluorophores when they are formed or dissociated. In order to examine how aggregation influences the emission characteristics of the FHT sensor, fluorescence spectra were measured in mixtures of DMF/H₂O with different concentrations of water between 0% and 90%. The FHT sensor showed a significant photoluminescence in pure DMF, and this was gradually enhanced with increasing amounts of water (0–90%). The maximum emission intensity of the FHT sensor was observed in 90% aqueous medium, which can be attributed to the restriction of intramolecular rotations. Moreover, a bathochromic shift of 72 nm from 440 nm to 512 nm was observed upon incremental addition of water from 0% to 90% (Fig. 1d). The redshift in the emission maximum was attributed to J-aggregate formation arising from head-to-tail molecular alignment of the FHT sensor due to enhanced hydrophobic interactions aligned with Kasha's rule. This rule explained that excitonic coupling within the aggregates splits the excited states, and the corresponding alteration of the ground- and excited-state energies reduces the band gap, thereby producing a large bathochromic shift and increase in emission intensity.^55,56 The color change associated with the wavelength was also depicted in the CIE chromaticity diagram. The apparent variation in the coordinates of chromaticity from (0.208, 0.236) at 0% water content to (0.235, 0.387) at 90% water content demonstrates a clear shift in the emission wavelength (Fig. 1e). DLS analysis was used to further evaluate the particle size of the FHT sensor. The results showed an increase in the hydrodynamic diameter from 319 nm in pure DMF to 952 nm in a 90% aqueous medium, indicating that the FHT sensor tends to form aggregates (Fig. 1f and g).

4. Sensing studies

4.1. Fluorescence-based detection of Cu²⁺ using the FHT sensor

To investigate the sensing capabilities of the FHT sensor, photophysical titration studies were carried out. For this purpose, solutions of various metal cations, including Mg²⁺, Al³⁺, Fe³⁺, Cu²⁺, Hg²⁺, K⁺, Ag⁺, NH₄⁺, Ni²⁺, Zn²⁺, Cu⁺, and Na⁺, were individually added to a 30 µM solution of FHT sensor in DMF/H₂O (1 : 9, v/v). As shown in Fig. S1d, the absorption spectrum of the FHT sensor exhibited a maximum wavelength at 368 nm. Upon increasing the concentration of Cu²⁺ (0–30 µM), an enhancement in the absorption band was observed, which is attributed to a π–π* transition due to the donor–acceptor framework of the FHT sensor. To further evaluate the sensing capacity of the FHT sensor, emission studies were conducted. Fluorescence measurements showed that the emission intensity of the FHT sensor decreased significantly in the presence of 0–60 µM Cu²⁺ (Fig. 2a). A 2D linear SV plot was plotted to measure the sensitivity of the FHT sensor towards Cu²⁺ (0–60 µM), which showed a remarkable quenching efficiency (QE) of 97% and a limit of detection (LOD) of 82 nM, which is significantly smaller than those of the previously tuned Cu²⁺ chemosensors (Fig. 2b and Table S1). The calculated limit of quantification (LOQ) of 275 nM is an indication of the high sensitivity of the FHT sensor for Cu²⁺. The sensitivity of the FHT sensor was assessed by calculating the limit of detection (LOD) and quantification (LOQ) by using the formulas 3σ/S and 10σ/S, where σ represents the standard deviation of the blank (0.00432), and S is the slope of the calibration curve. The LOD and LOQ were determined to be 82 nM ± 2 nM and 275 nM ± 8 nM, with a relative error of approximately 2.75%, confirming the accuracy and reproducibility of the method. To determine the metal-ion sensing behavior of the FHT sensor, the fluorescence response was examined using a 3D Stern–Volmer plot in the presence of other competing analytes. The analysis demonstrated a marked variation in relative fluorescence intensity (I₀/I) upon the addition of Cu²⁺ within the concentration range of 0–60 µM, but negligible changes were observed in emission intensity with other metal ions, indicating the high selectivity of the FHT sensor for Cu²⁺ (Fig. S1e).

Fig. 2 Emission response of the FHT sensor to increasing concentrations of Cu²⁺ (0–60 µM) (a) inset represents the colorimetric change with and without copper. 2D Stern–Volmer plot of the FHT–Cu²⁺complex (b). Schematic of the detection of Cu²⁺ by the FHT sensor (c).

4.2. Plausible mechanistic approach

The FHT sensor contains the fluorene moiety as a fluorophore and weakly electron-withdrawing groups such as the Schiff base (–C=N–) and urea moiety (NH–CS–NH) as receptor units. In the absence of Cu²⁺, the FHT sensor exhibits pronounced fluorescence due to the lack of optimal coplanarity between the fluorene donor moiety and the receptor unit, which results in the suppression of photoinduced electron transfer (PET). Upon the addition of Cu²⁺, the FHT sensor coordinates with the metal through the nitrogen and sulfur atoms of the receptor unit forming a chelated complex. The receptor unit becomes significantly more electron-deficient and geometrically suitable, with better donor–acceptor electron coupling. This structural feature favors photoinduced electron transfer (PET) from the fluorene moiety to the receptor unit. Owing to the paramagnetic character of Cu²⁺ (d⁹ electronic configuration), the complex formation favors stronger intersystem crossing and non-radiative decay processes, which are responsible for the marked fluorescence quenching (Fig. 2c).⁵⁷ For this purpose, dynamic light scattering (DLS) analysis was performed to investigate the interaction of Cu²⁺ with the FHT sensor. Upon addition of Cu²⁺, the hydrodynamic diameter of the FHT sensor increased from 952 nm to 1424 nm, indicating the formation of a stable FHT–Cu²⁺ complex (Fig. S2a and b). To further investigate the interaction between the FHT sensor and Cu²⁺, ¹H NMR titration studies were carried out. These studies showed that a noticeable chemical shift change occurs in the ¹H NMR spectrum of the FHT sensor upon the addition of Cu²⁺. As acidic protons are highly sensitive to the ionic environment, upon addition of Cu²⁺, the local ionic environment is further altered and, most importantly, broadening of the NMR signals arises due to the paramagnetic nature of copper. The water peak observed at 3.36 ppm in the absence of Cu²⁺ is shifted downfield to 3.56 ppm after the addition of Cu²⁺, likely due to interactions between Cu²⁺ and the FHT sensor. Notably, the DMSO-d₆ solvent signal at 2.49 ppm remains unchanged, indicating that the shifts in other NMR signals result from specific interactions of the FHT–Cu²⁺ complex (Fig. 3a). In addition, FTIR experiments were conducted to study the non-covalent interactions between the FHT–Cu²⁺ complex. The presence of a characteristic band at 1642 cm⁻¹, associated with the azomethine (C=N) stretching vibration, confirmed the formation of the Schiff base. The distinct band at 1056 cm⁻¹ is attributed to the thiocarbonyl (C=S) mode of vibration, and an aromatic C–H vibration is observed at 3083 cm⁻¹. Upon coordination with Cu²⁺, the C=N, C=S, and C–H bands shifted to 1637 cm⁻¹, 1050 cm⁻¹, and 3079 cm⁻¹, respectively, indicating the non-covalent interactions between the FHT–Cu²⁺ complex (Fig. 3b). Furthermore, mass spectrometry titration experiments were performed to investigate the interaction between sensor FHT and Cu²⁺, with the m/z of [FHT + H]⁺ calculated for [C₁₄H₁₁N₃SH]⁺: 254.07; found: 254.17 and [FHT + H + K]⁺ calculated for [C₁₄H₁₁N₃SHK]⁺: 293.123; found: 293.33. Upon coordination of sensor FHT with Cu²⁺, the values of m/z of [(FHT)₂–Cu complex]⁺ was calculated for C₂₈H₂₂CuN₆S₂; calculated: 569.06, and found 569.17, respectively. The formation of this species likely arises from the reduction of Cu²⁺ during the ionization process in the ESI-MS, facilitated by the experimental conditions, possibly at higher temperatures. For d¹⁰ Cu(I) complexes, it is common for the metal ion to adopt a square planar geometry, in the given system, considering the weak Lewis basicity of nitrogen atoms and the strong affinity of the sulfur atoms for copper ion, favor that Cu²⁺ ion preferentially coordinates with two FHT molecules. The mass spectrometry titration results showed an increment in the m/z value of the FHT sensor upon interaction with Cu²⁺, thereby validating the sensing of Cu²⁺ (Fig. S3a and b).

Fig. 3 Stacked ¹H NMR titration spectra of the FHT sensor and FHT–Cu²⁺ complex (a). Stacked FTIR titration spectra of the FHT sensor and FHT–Cu²⁺ complex (b). Emission response of the FHT sensor in the presence of different competing analytes (excitation wavelength = 340 nm, concentration of FHT sensor 30 µM in 1 : 9 v/v DMF/H₂O) inset represent the solid phase sensing with and without Cu²⁺ (c).

4.3. Interference studies

To analyze the selectivity of the FHT sensor, we conducted interference studies. For this purpose, the FHT sensor (30 µM) was exposed to Cu²⁺ and other analytes with concentrations ranging from 0 µM to 60 µM, such as Mg²⁺, Al³⁺, Fe³⁺, Hg²⁺, K⁺, Ag⁺, NH₄⁺, Ni²⁺, Zn²⁺, Cu⁺, and Na⁺, and their counter anions SO₄²⁻, Cl⁻, NO₃⁻, CN⁻, I⁻, CO₃²⁻, Br⁻, and F⁻ (Fig. S4a and b). Fluorescence spectral analysis indicated that the emission response of the FHT sensor was not significantly altered in the presence of these competing species, indicating a high level of selectivity towards Cu²⁺ (Fig. 3c). Also, the sensitivity to changing environmental conditions of the FHT sensor was measured by conducting fluorescence measurements in the pH range of 3–11. It was observed that the FHT sensor exhibited stable fluorescence intensity after binding with Cu²⁺, irrespective of the pH of the solution, and this demonstrates the suitability of the sensor for applications in real-life, field-based studies. These findings indicate that the FHT sensor can easily be used to detect Cu²⁺ within a vast pH range (Fig. S5a). In order to further assess the suitability of the FHT sensor, the effect of temperature on the fluorescence response was investigated in the range of 10–60 °C using a solution containing 30 µM sensor and 60 µM Cu²⁺ in DMF/H₂O (1 : 9, v/v). The fluorescence intensity of FHT sensor remained relatively stable across this temperature range, demonstrating the suitability of the sensor for real sample applications (Fig. S5b). Research on photostability is essential for the practical use of the FHT sensor. To evaluate the possible photobleaching impact, a 30 µM solution of FHT containing 60 µM Cu²⁺ was exposed to constant high-energy UV-irradiation, and the fluorescence emission of the solution was recorded at various time intervals. The results indicated that the FHT sensor exhibited a minimal change in the intensity of fluorescence with time, thereby proving that this sensor is highly photostable and resistant to photobleaching (Fig. S6a). Response time is another significant parameter to be considered for assessing the practical applicability and stability of the FHT sensor. One way to determine this is by measuring the quenching of the FHT sensor using the addition of Cu²⁺ after varying intervals (Fig. S6b). These findings demonstrated that the sensor exhibits a fast and consistent response, making it suitable for real-time detection.

5. DFT studies

5.1. FMO and DOS analysis

The sensitivity of the FHT sensor towards Cu²⁺ was further examined by frontier molecular orbital (FMO) analysis.^58,59 A major alteration in the electronic properties of the FHT sensor was observed when it interacted with Cu²⁺. The FHT sensor was found to possess a HOMO–LUMO gap of 2.13 eV; however, when complexed with Cu²⁺, the H–L gap was significantly reduced to 0.67 eV. Further, the sensitivity was validated by the density of states (DOS) analysis. A significant reduction in the HOMO–LUMO energy gap, as well as the appearance of some more virtual orbitals, was observed. The initial energy levels of the FHT sensor at −4.89 eV and −2.76 eV shifted to −13.03 eV and −12.36 eV after formation of the FHT–Cu²⁺ complex, suggesting a significant electronic interaction and enhanced sensitivity of the FHT sensor towards Cu²⁺ (Fig. 4a and b).

Fig. 4 DOS spectrum of the FHT sensor and FHT–Cu²⁺ complex (a and b). 3D and 2D NCI representations of the FHT–Cu²⁺ complex (c and d). QTAIM analysis of the FHT–Cu²⁺ complex (e). Emission spectrum of the FHT sensor for detection of Cu²⁺ in industrial effluents (f).

5.2. NCI and QTAIM analysis

Computational analysis via Multiwfn was employed to gain insight into the nature and strength of non-covalent interactions between the FHT sensor and Cu²⁺.^60,61 These interactions primarly consists of hydrogen bonding, ion–dipole interactions, weak van der Waals forces, and steric repulsion. The nature of these interactions was further analyzed using 3D-isosurface representations and 2D-reduced density gradient (RDG) plots. The 3D isosurface of the FHT–Cu²⁺ complex provides visuals on the occurrence of the van der Waals interactions, indicated by the red patches located at the sites of interaction between the FHT sensor and Cu²⁺ (Fig. 4c). Dense green spikes in the 0.0102 (2 − R) range show that there are van der Waals interactions, and red spikes represent steric repulsion as the FHT sensor and Cu²⁺ interact (Fig. 4d). Meanwhile, a QTAIM analysis based on Bader's theory demonstrated that the FHT–Cu²⁺ complex contains 3 bond critical points (BCPs), which further validates the experimental findings of interaction between sensor FHT and Cu²⁺ (Fig. 4e). The FHT–Cu²⁺ complex has different bond critical points (BCPs) with different interactions energies; for example, BCP 1 has −1213.79, BCP 2 has −131.30, and BCP 3 has −770.51 kJ mol⁻¹ (Table S2). The FHT–Cu²⁺ complex was found to have three BCPs, indicating Cu–H, Cu–N and Cu–S interactions, which highlight the important binding sites contributing to sensor–metal coordination.

6. Practical applications

6.1. Solid-state detection and visual sensing of Cu²⁺

To evaluate the feasibility of the FHT sensor for Cu²⁺ detection, two types of solid-state sensing experiments were carried out. For this, a cotton bud was first immersed in a 30 µM FHT sensor solution and dried at 60 °C. Under 365 nm UV light, the sensor-coated cotton bud exhibited bright fluorescence, which was quenched upon the addition of a single drop of Cu²⁺, producing a distinct dark spot (Fig. 2a). For additional solid-state sensing, Whatman filter paper was similarly soaked in 30 µM FHT sensor solution and dried. It exhibits high emission under 365 nm UV light. Upon the introduction of Cu²⁺ on these strips, the fluorescence emission of the FHT sensor decreased significantly. Both substrates demonstrated strong fluorescence under UV light, highlighting their potential as practical platforms for Cu²⁺ detection (Fig. 3c). To further investigate the colorimetric behavior, the FHT sensor was subjected to visual sensing experiments in the presence of Cu²⁺. A 30 µM solution of the sensor in DMF/H₂O (1 : 9, v/v) exhibited bright fluorescence under 365 nm UV light. Upon addition of 60 µM Cu²⁺, a significant decrease in fluorescence emission was observed (Fig. 4f). The results suggest that the FHT sensor is suitable for rapid, qualitative detection of Cu²⁺ under UV irradiation.

6.2. Industrial waste detection of Cu²⁺

In the case of industrial wastewater, the concentration of Cu²⁺ in environmental samples is often high; therefore, its detection is necessary. Tests were conducted to assess the practical applicability of the FHT sensor by analyzing water samples collected from an industrial laser-cutting plant in Abbottabad, Pakistan, containing Cu²⁺ leached from copper-coated metal sheets. To minimize possible contaminants, the samples were filtered through carbon filters and then spiked with known concentrations of Cu²⁺ (0–60 µM). The spiked samples were incubated with a 30 µM solution of the FHT sensor in DMF/H₂O (1 : 9, v/v), and the fluorescence response was monitored using a spectrofluorometer (Fig. 4f and Table S3). Recovery efficiency was calculated from the quenching of the spiked and un-spiked samples and determined to be as high as 97%, indicating that the FHT sensor is suitable for the sensitive and reliable determination of Cu²⁺ in real environmental samples.

6.3. Development of logic gate systems using the FHT sensor

The observed variations in the fluorescence behavior of the FHT sensor suggest its potential application in the design and development of molecular logic devices.^62,63 In molecular logic systems, fluorescence signals are used to translate chemically encoded information. To examine the molecular logic behavior of the FHT sensor, a circuit was designed using a combination of NOT, AND, NAND, and OR gates, in which water (input-1) and Cu²⁺ (input-2) acted as the respective input signals (Fig. S7). Exposure of the FHT sensor to water was observed to result in an increase in fluorescence intensity, which was designated as an output of “1.” In contrast, when both inputs (H₂O and Cu²⁺) were either present or absent at the same time, the fluorescence intensity was reduced, leading to the output “0.” A resulting truth table was constructed to match the expected functions of logic gates, thus demonstrating the potential of the FHT sensor for applications in molecular electronics (Table S4).

7. Conclusion

The FHT sensor was successfully synthesized in good yield, and its photophysical properties were systematically characterized. A bathochromic shift of 72 nm was measured for the FHT sensor from 440 nm in pure DMF to 512 nm at 90% water content, which indicates that the observed photophysical behavior arises from aggregation-induced emission (AIE) and the formation of J-aggregates. These photoluminescence properties were utilized to develop the FHT sensor for the detection of Cu²⁺ with high selectivity and sensitivity. The FHT sensor exhibited a limit of detection of 82 nM and a quenching efficiency of 97%. Spectroscopic analyses, including ¹H NMR and FTIR titrations, led to the identification of non-covalent interactions between the FHT sensor and Cu²⁺. The selectivity of the FHT sensor was determined from interference studies for the detection of Cu²⁺ with other competing analytes, under different pH values and temperature conditions. Investigations on the photostability and response time indicate that the FHT sensor has high stability and provides fast and reliable sensing performance. Theoretical investigations, such as FMO and DOS calculations, showed the reduction of the HOMO–LUMO energy separation, as well as the emergence of new virtual orbitals, indicating the high sensitivity of the FHT sensor towards Cu²⁺. The existence and importance of non-covalent interactions in the FHT–Cu²⁺ system were further validated by NCI and QTAIM calculations, with the bond critical point parameters demonstrating their role in the sensing mechanism. These findings demonstrate the effective use of FHT sensor for Cu²⁺ detection and validate its suitability for use in the environmental and industrial monitoring of Cu²⁺. This sensor can also be applied for forensic studies and in the design of molecular logic-gate systems.

Conflicts of interest

There are no conflicts to declare.

Data availability

The supporting data have been provided as part of the supplementary information (SI). Supplementary information is available. See DOI: https://doi.org/10.1039/d6ra02022d.

Acknowledgements

The authors extend their appreciation to the University Higher Education Fund for funding this research work under the Research Support Program for Central Labs at King Khalid University through the project number CL/Health/2.

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Footnote

† These authors contributed equally to this work.

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