A rhodamine-conjugated fluorescent and colorimetric receptor for the detection of Cu²⁺ ions: environmental utility and smartphone integration

Abstract

A new rhodamine based turn on florescent probe (Z)-3′,6′-bis(ethylamino)-2-(((6-methoxy-2-oxo-1,2-dihydroquinolin-3-yl)methylene)amino)-2′,7′-dimethylspiro[isoindoline-1,9′-xanthen]-3-one (RME) was efficiently synthesized through a simple condensation reaction of 2-amino-3′,6′-bis(ethylamino)-2′,7′-dimethylspiro[isoindoline-1,9′-xanthen]-3-one and 6-methoxy-2-oxo-1,2-dihydroquinoline-3-carbaldehyde. The receptor RME is highly non-fluorescent and when copper ions (Cu²⁺ ions) are added in DMF/water (1 : 2, v/v) medium, the receptor RME exhibits a specific “turn-on” colorimetric and fluorometric response. Moreover, RME binding with Cu²⁺ ions produced a remarkable color variation that was perceptible to the human eye, changing from colorless to pink. The binding stoichiometry was 1 : 1, confirmed through theoretical DFT, Job's plot, and mass spectral analysis. RME showed good sensing capability for Cu²⁺ ions, with LOD values of 11.25 nM for ratiometric colorimetry, 1.42 nM for fluorometry, and 107 nM for smartphone-based detection. These LOD values are significantly lower than WHO's acceptable threshold of 2 μM. Additionally, developing portable techniques based on paper-based test strips and smartphone assistance allowed for qualitative and quantitative detection of Cu²⁺ ions. These portable and economical methods eliminate the need for expensive laboratory apparatus, enabling precise and affordable on-site assessments. In addition, the developed sensing protocol for the detection of Cu²⁺ ions was extended to real water samples such as tap, dam, and drinking water samples. These results demonstrated the effectiveness of RME in detecting Cu²⁺ ions in complex matrices, making it a promising tool for environmental monitoring and water quality assessment.

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1. Introduction

Industrial chemical waste streams often contain hazardous waterborne pollutants, including heavy metal ions, bacteria, viruses, and organic solvents. Because of their incapacity to break down in biological systems, heavy metals are among the substances that pose the greatest hazard to all living things, including humans. Heavy metals can therefore accumulate in tissues and cause a variety of health concerns.^1,2 It is now urgently necessary to purify contaminated water due to the increasing lack of clean water. After iron and zinc, copper is the third most abundant essential metal in the human body, playing a critical role as a nutrient in various biological systems. However, at elevated levels, it becomes toxic, making its detection crucial in chemical, biological, and environmental studies. Furthermore, plants, animals, and humans all require Cu²⁺ ions as a trace element.³ However, the absence or excess copper amounts may result in diseases like kidney and liver damage and⁴ neurotoxicity⁵ and also cause Wilson's disease, gastrointestinal disorders, hypoglycemia, liver disease, and dyslexia in babies.^6–10 Owing to the concentration-dependent nature of Cu²⁺ ions, guidelines have been released by the World Health Organization to guarantee that the concentration of Cu²⁺ ions in drinking water is kept below 2 ppm.¹¹ Anthropogenic activities such as mining, industrial processes, and agricultural practices emit Cu²⁺ ions into the air, soil, and water, increasing their concentration in our surroundings.^12–14 Cu²⁺ ions are one of the main elements required for the normal functioning of living organisms. They catalyze reactions and help transfer electrons within DNA and RNA.^15–19 It is extremely desirable and in great demand to counteract this concentration-dependent variable behavior of Cu²⁺ ions with something simple, selective, sensitive, and especially below the allowable limit.

Until now, Cu²⁺ ions had only been applied in a few widely used, very expensive instrumental methods, like atomic absorption spectroscopy (AAS),²⁰ inductively coupled plasma atomic emission spectroscopy (ICP-AES),²¹ potentiometry,²² anodic stripping voltammetry,²³ spectrophotometry, and spectrofluorimetry.²⁴ Of the methods mentioned above, spectrophotometric and fluorescence chemosensors have garnered the most interest from research groups working in a variety of fields.²⁵ For example, appropriate chemosensors for detecting heavy metal ions are critical due to the direct benefits applicable to essential areas such as the environment, industry, biology, and human health. Optical chemosensors are popular due to their great sensitivity, selectivity, and ease of fabrication. They also display metal-ion-specific fluorescence. In the past few years, fluorescent compounds with heavy metal detection capabilities have emerged as the most fascinating field of study.²⁶ Scientists and researchers primarily use colorimetric or fluorescence methods to determine the presence of heavy metal ions in their work; however, the latter have more affordable and rapid procedures that are easier to apply. In analytical chemistry, creating innovative Cu²⁺ sensors with less expensive equipment has become popular.²⁷ Because of its quick response time, good spectral resolution, and simple sample preparation, fluorescence is one of the most popular technologies.²⁸ It is noted that multiple events demonstrate how the coordination of Cu²⁺ affects the photophysical behavior of the receptor. In particular, the main reason why Cu²⁺ can be detected via CHEQ or CHEF methods is electronic changes based on charge transfer from the ligand to metal (LMCT – ligand-to-metal charge transfer).²⁹ Turn-on fluorescent sensors are favored over turn-off fluorescein-based chemosensors because low sensitivity and turn-off detection are caused by background fluorescence.³⁰

Rhodamine is the fluorophore moiety of a variety of fluorescent probes that have the most potential in creating fluorescence sensors for ions and molecules. Rhodamine conjugates exhibit a visual color shift in the presence of analytes, which allows the analytes to be quickly detected.^31–34 This “off–on” process of accuracy and efficiency of rhodamine makes it feasible for researchers to develop a large number of rhodamine derivatives for rapid recognition and visual determination of metal ions with the naked eye.^35,36 This occurs since rhodamine and rhodamine derivatives have a spirocyclic ring, which results in ring opening, causing a notable color change as well as absorbance or fluorescence alterations.^37,38 Rhodamine dyes with improved emission maxima, visible excitation, photostability,³⁹ and excellent spectroscopic properties are used as biomarkers, in industrial coloring, and as sensing probes. The rhodamine derivatives we obtained which had an open spirolactam ring exhibit a bright pink color and luminous emission due to metal ion chelation.⁴⁰ In particular, a chemosensor that possesses twin signalling capabilities that is, one that can both generate a discernible shift and increase fluorescence emission will achieve the detection objective. While the majority of described receptors show fluorescence quenching upon interaction with copper ions, only a limited number of sensors for Cu²⁺ have been shown to exhibit increased fluorescence.^41–43 The successful utilization of our compound's fluorescence in response to Cu²⁺ ions was effectively applied for live cell imaging.⁴⁴

Herein, we describe a rhodamine-based chemosensor RME that operates in the longer wavelength range with excitation and emission both as a colorimetric and a turn-on fluorescence chemosensor upon binding Cu(II) ions in the DMF/water medium. The sensing mechanism has been examined by systematic spectral and theoretical studies. The notable colorimetric changes observed in the current work were further extended to smartphone-based detection of Cu²⁺ ions. As expected, RME can specifically recognize and quantify Cu²⁺ ions from potentially interfering metal ions, with the lowest detection limit as low as 11.25 nM. Furthermore, photometric titrations verified the complexation ratio of 1 : 1 as per JOB's working diagram and the fluorescence enhancement of RME by Cu²⁺ ions. A reversible Cu²⁺ detection device was developed with the aid of EDTA, and an implication logic gate was also constructed for Cu²⁺ detection. Finally, RME was effectively applied to determine Cu²⁺ ions in real water samples and paper strip applications.

2. Experimental section

2.1. Materials and instrumentation

All the reagents and solvents were purchased from commercial sources. Rhodamine 6G and hydrazine hydrate 80% were sourced from Sigma Aldrich. FT IR spectra were measured using the BRUKER Alpha II model in ATR mode. ¹H and ¹³C NMR spectra were recorded on a Bruker Avance Neo (400 MHz) in DMSO-d₆ solvent. Alumina-coated F254 Merck plates were used in TLC to monitor the reaction progress. ESI-MS spectra were recorded on a Thermo Scientific High-Resolution Magnetic Sector MS DFS by the positive ion electrospray ionization (ESI) method. UV-visible absorbance spectra were recorded using a JASCO V-750 spectrophotometer. Fluorescence spectrum experiments are conducted using a Horiba Fluoromax Plus spectrofluorometer. Tap and drinking water samples were taken from our university campus (Karpagam Academy of Higher Education) and dam water samples were collected from Siruvani Dam, Coimbatore. Without any pretreatment, collected water samples were utilized for real sample investigations.

2.2. Synthesis of RME

The synthesis of 6-methoxy-2-oxo-1,2-dihydroquinoline-3-carbaldehyde was carried out by employing the Vilsmeier–Haack formylation procedure by using a mixture of p-anisidine and acetic anhydride to get N-(4-methoxyphenyl)acetamide which undergoes formylation to afford 2-chloro-6-methoxy quinoline-3-carbaldehyde; furthermore, this aldehyde was hydrolyzed to get 6-methoxy-2-oxo-1,2-dihydroquinoline-3-carbaldehyde by following the previous work.⁴⁵ The synthesis of Rhodamine 6G hydrazide was carried out by using Rhodamine 6G and hydrazine hydrate 80% in ethanol medium. The reaction mixture was stirred and refluxed at 90 °C for 6 h. The resulting precipitate was filtered, washed with distilled water, and left to dry. The crude product was crystallized with ethanol. The rhodamine 6G hydrazide was obtained as a pink solid.⁴⁶

The probe RME was synthesized by adding an equimolar mixture of synthesized 6-methoxy-2-oxo-1,2-dihydroquinoline-3-carbaldehyde (0.09 g, 1 mmol) and rhodamine 6G hydrazide (0.2 g, 1 mmol) in 10 mL of ethanol, and the reaction mixture was allowed to reflux at 90 °C in an oil bath. The reaction process was monitored by TLC, and the obtained precipitate was filtered and then completely washed with cold ethanol. The product was vacuum dried and taken for further steps without any purification.

2.2.1 (Z)-3′,6′-Bis(ethylamino)-2-(((6-methoxy-2-oxo-1,2-dihydroquinolin-3-yl)methylene)amino)-2′,7′-dimethylspiro[isoindoline-1,9′-xanthen]-3-one (RME). Yellow powder; yield: 80%; MP 320–325 °C; FTIR ν (cm⁻¹): 1711 (C=O), 2965 (–NH), 1646 (CH=N); ¹H NMR (400 MHz, DMSO-d₆) δppm: 11.74 (s, 1H), 8.61 (s, 1H), 8.13 (s, 1H), 7.93 (d, J = 4.0 Hz, 1H), 7.58–7.53 (m, 2H), 7.26–7.20 (m, 2H), 7.13 (s, 1H), 6.99 (d, J = 4.0 Hz, 1H), 6.35 (s, 2H), 6.22 (s, 2H), 5.06 (s, 2H), 4.36 (s, 1H), 3.76 (s, 3H), 3.15 (t, J = 8.0 Hz, 4H), 1.86 (s, 6H), 1.23 (t, J = 8.0 Hz, 6H); ¹³C NMR (100.1 MHz) δppm: 164.63, 160.68, 154.82, 152.87, 151.14, 148.24, 141.08, 134.46, 134.16, 134.12, 129.02, 127.94, 126.96, 126.42, 124.02, 123.58, 121.27, 119.78, 118.72, 116.81, 110.21, 104.97, 96.54, 65.66, 55.89, 19.02, 17.46, 14.63, 37.94. MS (ESI – M + Na) m/z 636.6. Anal. calcd For C₃₇H₃₅N₅O₄; C, 72.41; H, 5.75; N, 11.41; Found; C, 72.45; H, 5.71; N, 11.42.

2.3. General procedure for absorption and fluorescence measurements

RME stock solution (10 mM) was prepared by adding 6.4 mg in 10 mL of DMF solution and the stock solution (1 mM) of Cu²⁺ ions was prepared in double distilled water. For photometric titration experiments, a cuvette was filled with 10 μM RME dissolved in 2.5 mL of DMF/water (1 : 3, v/v). Subsequently, appropriate amounts of Cu²⁺ stock solution were blended with the reaction mixture and shaken well. After a 2 min incubation period, the UV-vis absorbance spectra were recorded in the range of 600 nm to 200 nm. A similar protocol was followed for fluorescence spectral investigations. The excitation wavelength for the fluorescence spectra was set at 415 nm, and spectrum analysis was carried out after 2 min with a 2/2 nm slit emission/excitation slid width. Absorbance/emission spectrum investigations were used in a photometric titration based technique, and specificity measurements were carried out in place of Cu²⁺ ions and other potentially interfering metal ions mixed with the reaction mixture. All the sensing-related measurements were performed a minimum of three times.

2.4. Smartphone assisted Cu²⁺ recognition

To identify Cu²⁺ in DMF–H₂O (1 : 3 v/v) solution, a smartphone-based color assist application (color Grap) was applied to digitalize the color shift of RME with Cu²⁺ that can be seen with the naked eye. In a glass tube, different quantities of Cu²⁺ ions were mixed in RME (10 μM) in a DMF/water medium. Color variations were captured on the back camera of a Vivo V23 Pro smartphone. Lastly, the Color Grab app, a freely available mobile application, digitalized the red, green, and blue (RGB) values of the photos.

2.5. Paper strip application

The first identical-size Whatman filter paper was developed to create the paper-based test strips. The Whatman filter paper was soaked in an RME-dissolved DMF/water stock solution to make RME sensor test strips and dried in air. Subsequently, RME-coated paper test strips were spiked into standard solutions of Cu²⁺ ions and dried in air. Finally, colorimetric images of the paper-based test strips were taken using a Vivo V23 Pro (108 MP camera resolution) under ambient conditions (without flash).

3. Results and discussion

3.1. Design and synthesis of the probe

The synthesis of the fluorescent receptor RME was explored and is outlined in Scheme 1via the well-known Schiff-base condensation reaction of compound 6-methoxy-2-oxo-1,2-dihydroquinoline-3-carbaldehyde and rhodamine 6G hydrazide under reflux conditions at 90 °C in ethanol medium. The rhodamine 6G hydrazide and the compound 6-methoxy-2-oxo-1,2-dihydroquinoline-3-carbaldehyde were synthesized by using the previously described technique.^45,46 The structure of the synthesized receptor, RME was attested through spectroscopic techniques like FTIR, ¹H and ¹³C NMR, and ESI-MS spectral analysis.

Scheme 1 Schematic representation for the synthesis of probe RME.

In the IR spectrum of probe RME, the characteristic peaks at 1711 cm⁻¹, 2965 cm⁻¹, and 1646 cm⁻¹ were attributed to –C=O, –NH –C=N, and functional groups respectively (Fig. S1†) The ¹H NMR spectrum of the receptor RME (Fig. S2†), the –NH proton appearing at δ 11.78 ppm as a singlet corresponds to the quinoline core, OCH₃ appeared at δ 3.76 ppm as a singlet, azomethine proton CH=N appeared at δ 8.6 ppm as a singlet, two –NH protons present in the rhodamine core appeared at δ 6.22 ppm as singlets, –CH₂ protons in the ethyl group appeared as a triplet at δ 3.15 ppm and –CH₃ protons in the ethyl group appeared as a triplet at δ 1.25 ppm of the rhodamine core. In the ¹³C NMR spectrum (Fig. S3†), the peak at δ 56.5 ppm was due to OCH₃ and the peak at δ 164.3 ppm was due to –NHC=O in the quinoline core. The –C=O carbon in the indole core appeared at δ 160.6 ppm and the two –CH₃ carbons appeared at δ 14.6 ppm and –CH₂CH₃ carbons appeared at δ 37.9, 17.4 and 19.0 ppm respectively. Furthermore, the molecular ion peak at m/z 614.6 (M + H)⁺ (ESI-MS) (Fig. S4†) confirms the structure of RME as (Z)-3′,6′-bis(ethylamino)-2-(((6-methoxy-2-oxo-1,2-dihydroquinolin-3-yl)methylene)amino)-2′,7′-dimethylspiro[isoindoline-1,9′-xanthen]-3-one.

3.2. Spectrophotometric features of ratiometric/colorimetric selectivity of the receptor RME

UV-vis absorption spectrum investigations were performed to track the photophysical action of RME beforehand and following the different additions of Cu²⁺ ions. The related results are shown in Fig. 1a. In the absence of Cu²⁺ ions, the receptor RME shows three absorbance bands at 306 nm, 337 nm, and 410 nm, respectively, in the DMF/water medium (1 : 2, v/v) medium, which corresponds to the π–π* and n–π* transitions, respectively.⁴⁷ It can be seen from Fig. 3 that after the addition of Cu²⁺ ions to the receptor RME, the peak at 306 nm starts decreasing and the peak around 337 nm starts increasing; simultaneously there is a new absorption peak emerging at 525 nm in the visible region. RME with Cu²⁺ ions has a significant color change from yellow to pink. As a result, a noticeable color variation from yellow to pink is visible to the naked eye. It is evident that the addition of additional competing anions and cations (Mg²⁺, Na⁺, Zn²⁺, Ni²⁺, Ce³⁺, Cd²⁺, Bi²⁺, K⁺, Co²⁺, Ca²⁺, Fe²⁺, Fe³⁺, HSO³⁻, Ag⁺, Pb²⁺, Cl⁻, Br⁻, Ba²⁺, I⁻, NO³⁻, and Cr³⁺) to the appropriate medium (RME) does not affect the color or spectral features of the RME receptor (inset of Fig. 1b). Additionally, ratiometric (A₅₂₅/A₄₁₀) investigation for Cu²⁺ may be accomplished utilizing these two wavelengths; other interfering anions and cations including Mg²⁺, Na⁺, Zn²⁺, Ni²⁺, Cu²⁺, Ce³⁺, Cd²⁺, Bi²⁺, K⁺, Co²⁺, Ca²⁺, Fe²⁺, Fe³⁺, HSO³⁻, Ag⁺, Pb²⁺, Cl⁻, Br⁻, Ba²⁺, I⁻, NO³⁻, and Cr³⁺ lack this capability, making RME-based ratiometric/colorimetric chemosensors extremely specific for Cu²⁺ ions (Fig. 1b).

Fig. 1 UV-vis absorption spectral profile (a) for RME (10 μM) and after the addition of Cu²⁺ ions (100 μM) and the inset represents the RMEbefore (i) and after (ii) the addition of Cu²⁺ ions into the receptor RME in DMF/water (1 : 2, v/v) medium. Bar graph (b) of RME with the addition of different metal cations and anions and the inset visualizes the notable color changes of RME solution containing different metal cations and anions in DMF/water (1 : 2, v/v) medium.

3.3. Spectrophotometric titration experiments for RME with Cu²⁺ ions

At the same time, absorbance spectral changes of RME with incremental amounts of Cu²⁺ ions were monitored in DMF/water (1 : 2, v/v) medium, and the typical equilibrium process is depicted in Fig. 2. After measuring the spectra, it was discovered that the isosbestic point was in the 442 nm region, signifying the beginning of the formation of a stable complex between the Cu²⁺ ions and the receptor RME where the continuous addition of Cu²⁺ towards the receptor manifests a clear color shift, and the translucent solution turns pink. This event unequivocally demonstrates that Cu²⁺ binding causes the RME ring-opening structure. The notable spectral changes, colorimetric alterations, and a new absorbance peak that appeared at 525 nm should be attributed to the characteristic peak for the Cu²⁺ ion induced by the open ring-closed spirolactam ring in the receptor RME.⁴⁸ According to colorimetric measurements, the aforementioned findings show that the receptor RME recognizes Cu²⁺ ions highly preferentially. To calculate the lowest limit of detection (LOD) of the ratiometric detection tactic, the calibration curve was plotted between the (A₅₂₅/A₄₁₀) and the different concentrations of Cu²⁺ ions. In the concentration range of 10 μM to 100 μM, good linear behavior was attained with a correlation coefficient of 0.99. The LOD was estimated to be 11.25 nM by using the formula LOD = 3σ/S, σ is the standard deviation of five measurements taken in the blank (0.0015) and S shows the calibration curve's slope (Fig. S5†). The determined LOD is significantly less than the maximum allowable level of Cu²⁺ ions in drinking water that has been approved by the WHO.

Fig. 2 UV-visible absorption spectra of RME (10 μM) upon adding Cu²⁺ ions (0–100 μM) into DMF/aqueous (1 : 2, v/v) medium and the inset shows the magnified view of the newly appeared peak at 525 nm.

3.4. Fluorescence emission spectroscopic studies of receptor RME with Cu²⁺ ions

To further verify the binding ability of RME with Cu²⁺ ions, emission spectral studies were carried out. The fluorescence emission titration experiment was carried out with the incremental addition of Cu²⁺ ions to the receptor RME in DMF–H₂O (1 : 2, v/v). Upon the addition of Cu²⁺ ions to the receptor RME, the emission intensity (550 nm) starts to gradually increase (approx. 14-fold) (Fig. 3) with a strong red shift in the wavelength. Due to the fluorescence resonance energy transfer (FRET) method, the probe RME showed a rise in fluorescence in the DMF/water medium when stimulated at 415 nm as the concentration of Cu²⁺ increased.⁴⁹ Under an UV lamp, when Cu²⁺ was added to the RME chemosensor, no notable color change was noticed. The paramagnetic couple Cu²⁺ interact in many ways would make sense if the resultant strong fluorescent complex between RME and Cu²⁺ ions. This strong interaction could induce the spirolactam ring of (RME) and subsequently increase the fluorescence of the xanthenyl moiety.⁵⁰ Based on the emission enhancement, the concentrations of Cu²⁺ ions were quantified. Then, the LOD value was determined by using the equation, LOD = 3σ/S, which was applied to calculate the limit of detection (LOD) of RME for Cu²⁺ ions, which is defined as a signal-to-noise ratio (S/N) of 3.^51–53 From the calibration plot, the LOD value was calculated to be 1.42 nM, which is much lower than the WHO criteria (2 ppm). Therefore, the proposed work is more appropriate for the detection of Cu²⁺ ions in drinking water using the RME receptor. Furthermore, the effect of pH on RME and RME with Cu²⁺ ions was investigated by emission spectral studies. A pH of 4–10 was accomplished by mixing NaOH or HCl with 0.01 M phosphate buffer saline (PBS). The developed receptor RME and RME with Cu²⁺ ions display the maximum emission intensity in neutral pH around 6 to 8 (Fig. S6†). Thus, we have kept the pH at 7 for all sensing experiments.

Fig. 3 Fluorescence spectra of RME solutions in the presence of Cu²⁺ ions in DMF/aqueous (1 : 2, v/v) medium.

3.5. Density functional theory calculations

A molecular-level investigation was conducted into the process of complex formation between the RME and Cu²⁺ ions using a density functional theory-based computational technique which was optimized at the B3LYP/M06-2X functional and LANL2DZ basis set and the quantum chemical calculations were done using the Gaussian 16 software package. The Cu²⁺ complex of RME, together with its optimal geometry and highest occupied molecular orbitals (HOMO) and lowest unoccupied molecular orbitals (LUMO), is displayed in Fig. 4. It is evidenced from Fig. 4 that the HOMO was dispersed across the rhodamine core and the LUMO was dispersed in the quinoline moiety. After the coordination of RME with Cu²⁺ ions, the HOMO and LUMO were dispersed in the rhodamine core.

Fig. 4 Frontier molecular orbital energy levels of the RME–Cu²⁺ complex calculated using the B3LYP/M06-2X functional and LANL2DZ basis set.

The DFT-optimized structures of the receptor RME and RME–Cu²⁺ ensemble are disclosed in Fig. S7.† From Fig. S7,† the geometry of RME–Cu²⁺ is trigonal bipyramidal, where the receptor RME coordinates with Cu²⁺ through quinoline (–C=O), imine (–CH=N) and ring-opened (–C–O) with calculated bond distances of 2.001 Å, 1.998 Å and 2.029 Å respectively (Fig. S7†). The SO₄²⁻ formed a coordination with Cu²⁺ with bond distances of 2.413 Å and 1.979 Å respectively. The HOMO–LUMO energy level gap for the complex is 4.437 eV, while the energy level gap for the HOMO and LUMO of the probe RME is 5.528 eV. Based on the previously described results, the RME–Cu²⁺ complex showed a smaller frontier orbital gap than the receptor RME, which made it more reactive and polarizable.

3.6. Reversibility experiments with RME and EDTA

Reversibility research is crucial for the growth of chemo biosensors with high degrees of specificity and sensitivity for components required for accurate and efficient analyte detection in real-world applications. Using EDTA, a chelating agent, as a coordinating receptor to ascertain the probe's reusable feature in the absorbance detection process allowed for the determination of the probe RME's reversibility towards Cu²⁺ ions. The Cu²⁺ analyte and the probe RME were first coupled experimentally to generate a complex at 550 nm in DMF–H₂O (1 : 1, v/v).

After adding EDTA, the RME–Cu²⁺ complex was found to have returned to its original state (Scheme 2). When the EDTA–Cu²⁺ complex formed, the pink hue of the RME–Cu²⁺ complex returned to the pale-yellow color of the free receptor RME. A series of investigations were conducted to test the receptor reversibility, and the results established that the probe can detect Cu²⁺ ions four times, as shown in (Fig. 5a and b). This reaction has shown a decrease in emission intensity when the Cu²⁺ ions are added to the RME + Cu²⁺ ions + EDTA combination. Nearly ∼80% of the emission intensity was stored at the end of the fourth cycle (Fig. 5b). This investigation was conducted to evaluate the sensor's regularity and dependability in identifying certain analytes. Thus, it has been demonstrated that adding an EDTA solution can reverse the RME's reaction to the Cu²⁺ ion. The receptor RME is reusable because this cycle can potentially be repeated at least four times.

Scheme 2 Plausible mechanism for the formation of the RME–Cu²⁺ complex and reversibility with a chelating agent, EDTA.

Fig. 5 Reversibility spectra (a) and cycle (b) of RME towards Cu²⁺ (100 μM) along with EDTA (100 μM).

3.7. Logic gate behavior

Using the sequence-dependent fluorescence output between EDTA and Cu²⁺ ions as chemical inputs, we have studied the fluorescence “turn on” process of the probe RME to build the molecular logic gate behavior of the current system.^44,45 In this instance, an INHIBIT logic gate behavior corresponds quite well with this receptor RME. The given scenario uses the INHIBIT logic gate belongings of the probe RME, Cu²⁺ ions, and EDTA as inputs (1 & 2) and the fluorescence peak intensity at 525 nm as the output (Fig. 6a). The logic gate is ON if the output is “1”, and this behavior can only be seen when Cu²⁺ ions are present. In three distinct scenarios, the existence of EDTA, the occurrence of both EDTA and Cu²⁺ ions, and without mixing of both EDTA and Cu²⁺ ions if the output is “0”, it indicates that the logic gate is OFF. These results implied that the behavior of the receptor RME is that of an INHIBIT logic gate.⁴⁵ There are probably several interactions between the paramagnetic Cu²⁺ ions, which results in the production of a highly fluorescent complex with RME. The spirolactam ring in RME may open as a result of this interaction, increasing the fluorescence of the xanthenyl moiety. The INHIBIT logic gate was the subject of another logic gate application carried out for the study (Fig. 6b–d). Herein, Cu²⁺ and EDTA signify input values 1 and 2, while color change (Fig. 6b), and/or A₅₂₅/A₄₁₀ (Fig. 6c), the outputs. In these systems, when both inputs are absent (0, 0), both inputs are present (1, 1), or input 2 is present but input 1 is absent (0, 1), the output of these systems is 0. In contrast, the result is 1 when input 1 is present and input 2 is not (1, 0) (Fig. 6d). This action, which results in a color shift from yellow to pink, is explained by the Cu²⁺ ions producing an enhanced absorbance band that is dependent on the concentration at 525 nm.

Fig. 6 (a) Construction of the truth table of the IMPLICATION logic circuit for output as emission turn on (b), output as colorimetric change (c), output as ratiometric features A₅₂₅/A_410, and the truth table (d). Truth table representation for the IMPLICATION logic gate.

3.8. Paper strip based applications

Because they are inexpensive to prepare, paper-strip-based chemosensors can be used in a wide range of settings, including those with limited resources and on-site monitoring applications. Numerous chemosensors based on paper offer prompt data, enabling prompt executive processing. For instance, certain organic molecules can yield outcomes in a couple of minutes, which is crucial in situations like ecological tracking or food-related investigations where speed is of the essence. In this work, Whatman filter paper was soaked in an RME-dissolved DMF/water mixture to develop paper-based sensor test strips which contain 2.5 mL of the receptor RME dissolved DMF/water mixture, and kept soaked for more than 5 min. Before applying the Cu²⁺ ion sensor, the paper-based test strips were allowed to air dry for almost eight hours. RME-deposited paper strips with incremental quantities of Cu²⁺ ions were investigated and the corresponding colorimetric photographs are illustrated in Fig. 7. Before the spiking of Cu²⁺ ions, paper strips were slight yellow in color. As the concentration of metal ions increased, the test strip's pink color got more vibrant, and as illustrated in Fig. 7, it was able to detect Cu²⁺ at concentrations as low as 20 μM using receptor RME. Additionally, the RME–Cu²⁺ complex has been applied as an organic ink. Initially, Whatman filter paper was dipped in the receptor RME (100 μM) for 5 min and then it was dried under air. This paper is yellow in color. Subsequently, a butterfly was sketched and inscribed using Cu²⁺ ions (50 μM) on the RME-coated paper strips; both the written word and the butterfly are readily visible to the naked eye. These findings demonstrate that the RME–Cu²⁺ complex was applied for (ink or marker) writing purposes on the filter paper.

Fig. 7 (a) Colour changes of the RME sensor filter paper test strips with the increasing concentration of the Cu²⁺ solution. (b) Colour changes observed upon the addition of Cu²⁺ in the stock solution of the RME-soaked paper strip.

3.9. Integration of the smartphone with the present colorimetric method

A straightforward facile to use smartphone testing may additionally validate the color variation for RME in the presence of Cu²⁺ ions in DMF/water medium, as it yields more accurate and sensitive results than optical detection. Here, the various quantities of Cu²⁺ ions are mixed distinctly with the standard solution of the probe RME (100 μM) in DMF/water medium and the corresponding color variation photograph is illustrated in the inset of Fig. 8. An existing smartphone color app (color picker) was used to resolve the red, green, and blue (RGB) primary color intensity values. This smartphone-based sensing technique primarily interprets digital photos of glass vials containing colloidal solutions using RGB values, which cover from 0 to 255. These systems are capable of translating color into RGB values using values like [0, 0, 0] for pure black and [255, 255, 255] for white.⁵³ Descriptive photos of each set of the probe RME solution with varying concentrations of Cu²⁺ ions were taken using the smartphone. By keeping a constant distance from the smartphone, the receptor RME with and without Cu²⁺ ion solutions was captured in a picture against a white background. The receptor RME solution's color gradually changed from slightly yellow to pink as the concentration of Cu²⁺ ions raised from 10 to 70 μM. The mean RGB values and Cu²⁺ ion concentrations between 10 and 70 μM were regressed linearly using the equation y = 193.35 + 1.61 [Cu²⁺ ions] with R² = 0.978. A calibration curve can be constructed using the average RGB value's linear increase with the quantities of Cu²⁺ ions as displayed in Fig. 8. From the slope of the linear calibration curve, the value of LOD is calculated to be 107 nM, which is quite lower than the tolerable limits by authorities' reports. As a result, a quick, easy, on-site, and reasonably priced method of detecting Cu²⁺ ions based on the receptor RME's color fluctuation is made possible via smartphone applications. Table S1† summarizes the collected analytical parameters of the current approach and compares them with those of previously published methods of Cu²⁺ ion detection using relevant chemical compounds. Table S1† illustrates the three distinct methods for detecting Cu²⁺ ions, which can be two to three orders extended or have comparable LOD values. It may contribute to the RME sensing probe's excellent qualities, such as its quick reaction time, lowest detection limit, selectivity, and so forth (RME).

Fig. 8 The calibration curve of the RGB color response using a smartphone and the inset shows a color variance of RME with different amounts of Cu²⁺ ions in DMF/aqueous (1 : 2, v/v) medium.

3.10. Selectivity of RME towards various metal ions

Selectivity is a crucial factor for the chemosensors. The fluorescence selectivity experiments of the synthesized receptor, RME towards potentially interfering metal cations anions including Mg²⁺, Na⁺, Zn²⁺, Ni²⁺, Cu²⁺, Ce³⁺, Cd²⁺, Bi²⁺, K⁺, Co²⁺, Ca²⁺, Co²⁺, HSO³⁻, Ag⁺, Pb²⁺, Cl⁻, Br⁻, Ba²⁺, I⁻, NO³⁻, Cr³⁺ and Cu²⁺ were tracked in DMF–H₂O (1 : 1, v/v) medium and the results are outlined in Fig. 9. As evidenced from Fig. 9, with the addition of 5-fold increasing amounts of different metal ions to the RME solution, only the Cu²⁺ ions induced a complete fluorescence enhancement of the receptor, RME (Fig. 9). The other abovementioned metal cations and anions such as Mg²⁺, Na⁺, Zn²⁺, Ni²⁺, Cu²⁺, Ce³⁺, Cd²⁺, Bi²⁺, K⁺, Co²⁺, Ca²⁺, Co²⁺, HSO³⁻, Ag⁺, Pb²⁺, Cl⁻, Br⁻, Ba²⁺, I⁻, NO³⁻, and Cr³⁺ could not induce any change in the emission intensity and colorimetric nature under daylight. These findings suggested that the receptor RME might function as a colorimetric and fluorescence chemosensor that is selective for Cu²⁺ ions. To understand the binding nature of Cu²⁺ with RME, the continuous variation method (Job's plot) was applied, in which the intensity of the band was plotted against the molar fraction of RME (Fig. S8†). The observed results suggested the formation of 1 : 1 stoichiometry between RME and Cu²⁺ during their complexation. Furthermore, the 1 : 1 binding stoichiometry was confirmed through the mass spectroscopy m/z value of 774.34 (Fig. S9†).

Fig. 9 Fluorescence spectra of RME with different metal cations and anions including Mg²⁺, Na⁺, Zn²⁺, Ni²⁺, Cu²⁺, Ce³⁺, Cd²⁺, Bi²⁺, K⁺, Co²⁺, Ca²⁺, Co²⁺, HSO³⁻, Ag⁺, Pb²⁺, Cl⁻, Br⁻, Ba²⁺, I⁻, NO³, Cr³⁺ and Cu²⁺ in DMF/H₂O (1 : 2, v/v) medium.

3.11. Practical applications

The amounts of Cu²⁺ ions in tap, dam, and drinking water samples were successfully measured using the probe RME. To find the Cu²⁺ ions in actual water samples, a conventional addition technique was used.⁵² In the drinking, dam, and tap water samples, Cu²⁺ ions were first not found. To account for a notable rise in emission intensity for triplicate observations, Cu²⁺ ions were mixed into environmental water samples. Table S2† displays the actual water research observations that were gathered. When Cu²⁺ ions were introduced into tap, dam, and drinking water samples, there was an outstanding recovery from 91.75 to 96.82%. The current method's great accuracy was suggested by its lowest relative standard deviation value (>2.21%).

4. Conclusion

In conclusion, a new rhodamine and quinoline-based simple naked eye and switch-on luminescent receptor (RME) has been efficaciously synthesized for Cu²⁺ ion recognition in DMF/water (1 : 2, v/v) medium. The spirolactam compound demonstrated outstanding specificity towards Cu²⁺ ions over other typically occurring cations in conventional DMF/H₂O medium. Upon the interaction of Cu²⁺ ions, the receptor color varied from slightly yellow to pink in color which was easily viewed by the naked eye whereas strong spectral fluctuations were noticed in absorbance and emission studies. RME showed a good sensing performance for Cu²⁺ ions with LOD 11.25 nM for ratiometric colorimetry, 1.42 nM for fluorometry, and 107 nM for the smartphone-based detection strategy, significantly lower than the tolerable limits (20 μM) by WHO. The mass spectral analysis, Job's plot, and DFT method were employed to reveal the probable 1 : 1 stoichiometric complexation mode between Cu²⁺ ions and the receptor RME. Furthermore, the developed fluorescence sensor was applied for the detection of Cu²⁺ ions in environmental water samples such as tap, dam, and drinking water samples. Additionally, practical smartphone and paper-based test strip applications were utilized to fruitfully accomplish both qualitative and quantitative Cu²⁺ ion quantification, avoiding the need for sophisticated equipment and enabling accurate and reasonably priced on-site assessment of potential Cu²⁺ ions.

Data availability

The authors ensure that all the data mentioned in this paper are available on request.

Conflicts of interest

The authors declare no conflict of interest.

Acknowledgements

This research work has been supported financially by the Department of Science and Technology (DST/TDT/TC/RARE/2022/13) dated 27.03.2023. The author also acknowledges NMR facility, CIC, Bharathiar University supported by DST (PURSE Phase II programme), New Delhi. The authors would like to extend their sincere appreciation to the Researchers Supporting Project number (RSPD2025R694), King Saud University, Riyadh, Saudi Arabia.

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Footnotes

† Electronic supplementary information (ESI) available: The NMR spectra, mass spectra, DFT theoretical data, and additional data. See DOI: https://doi.org/10.1039/d4ay01925c

‡ These authors contributed equally to this work.

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