Facile rhodamine-based colorimetric sensors for sequential detections of Cu(II) ions and pyrophosphate (P₂O₇⁴⁻) anions

Abstract

Two rhodamine hydrazine derivatives Rh1 and Rh2 with catechol and ether functionalities have been synthesized and utilized towards sequential colorimetric detections of Cu(II) and pyrophosphate (PPi) ions in CH₃CN–H₂O (v/v = 9 : 1, 5 mM Tris–HCl, pH 7.4) semi-aqueous medium. Notably, Rh1 and Rh2 are the first example of colorimetric rhodamine-based probes for the sequential detections of Cu²⁺ ion and PPi anion. Based on the significant colorimetric responses (from colorless to pink) of Rh1 and Rh2 to Cu²⁺ ions, the detection limits were estimated as low as 1.22 × 10⁻⁸ M and 8.0 × 10⁻⁷ M, respectively, which signified the utilities of designed probes towards facile detections of Cu²⁺ ions. Furthermore, upon the successive addition of PPi ion to Rh1–Cu²⁺ and Rh2–Cu²⁺ complexes, it has been altered and restored to its origin of Rh1 and Rh2 via re-coordination of PPi to Cu²⁺ ion, which was confirmed by color changes from pink to colorless. Moreover, computational DFT calculations provided more insights into HOMO–LUMO structures of rhodamine derivatives and their copper complexes. Additionally, the solid state strip-based colorimetric detections of Cu²⁺ ion were supplemented as a real time application.

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1 Introduction

Recently, owing to their environmental and biological significance, new artificial materials have emerged as efficient chemosensors towards the detection and estimation of transition metal ions.^1–3 Among them, copper ions were found to be essential and their detection has been paid greater attention over other metal ions.^4–7 In this regard, divalent copper ions are more fascinating not only as an environmental pollutant at higher concentrations but also as the third most abundant divalent metal ion in the human body.^8–12

Furthermore, copper has been suspected to cause liver and kidney damages for infants in long-term exposure. Similarly, short-term exposures to high levels of copper ion cause gastrointestinal disturbance.^13,14 Henceforth, the U.S. Environmental Protection Agency (EPA) has set a limit of copper ions in drinking water as 1.3 ppm (ca. 20 mM).¹⁵ Additionally, the average concentration of blood copper in the normal body is fixed as 100–150 mg dL⁻¹ (15.7–23.6 mM).¹⁶ However, copper can be toxic over its normal concentration that cause oxidative stresses and disorders associated with neurodegenerative diseases, including Meknes, Wilson diseases, familial amyotrophic lateral sclerosis, Alzheimer's disease and prion diseases. Therefore, the colorimetric visual detection of trace amounts of Cu²⁺ ions via simplified instrumental set up are become vital.^17–21 In this concept, the high sensitivity and selectivity are considered as a fundamental goal for organic and analytical chemists all over the world. In order to achieve the selective detection of Cu²⁺ against the background of competing analytes, many promising small molecules have been reported as Cu²⁺ sensor through colorimetric response.²²

On the other hand, phosphate-based inorganic and organic molecules play critical roles in both environment and biological systems. Among phosphate anions, pyrophosphate (PPi) is known to be biologically significant one,²³ which has been obtained as a product, during the hydrolysis of adenosine triphosphate (ATP) to adenosine monophosphate under cellular condition.²⁴ However, abnormal levels of PPi can lead to several diseases such as chondrocalcinosis or calcium pyrophosphate dihydrate (CPPD) crystal deposition problem and arthritis.²⁵ Recently, several approaches such as electro optical methods, colorimetric and fluorescence assays have been developed for the detection of PPi ions.^26–28 Among those techniques, colorimetric chemosensors are most attractive for the detection of PPi due to their selectivity, sensitivity, response time and low-cost.²⁹ Moreover, because of the strong binding affinities between metal ions and PPi, the utilization of metal ion complexes as a colorimetric chemosensor has been found to be the successful strategy for the detection of PPi ions. However, chemosensors based on color changes (by naked eye observation) for detection of PPi are found to be limited.³⁰ Hence, we tend to develop such colorimetric sensors for the detection of PPi ions.

On the consideration of photophysical properties, such as high quantum yield, photo stability, absorption and emission in the visible region, rhodamine fluorophore is seems to be most attractive one.^31–34 Further, the sensing behavior of rhodamine towards metal ions is also very interesting. Typically, rhodamine derivatives are non-fluorescent and colorless in their ring-closed spiro lactam structures, whereas the spiro lactams induced ring-opening by specific metal – give rise to strong fluorescence emissions to develop for colorimetric sensors.^35–38 Nevertheless, many rhodamine-based florescent and colorimetric chemosensors for detecting metal ions have been studied.³⁹ Colorimetric sensors are more facile due to the detection by naked eye that can be commonly used in low cost, less equipment and easy handling.^40–45 There are perceptible color changes in rhodamine derivatives, even at very small amounts of analyte concentrations. Therefore, much effort has been focused on the development of rhodamine-based sensor probes for the detections of heavy metal and anions.⁴⁶ On the other hand, direct detections of Cu(II) and PPi ions could be accomplished by regulating the acceptors of rhodamine sensor molecules to affect ICT mechanisms, which may evidence via ratiometric responses and ion induce naked-eye color changes.⁴⁷ Hence, rhodamine-based chemosensory probes for the sequential recognitions of Cu(II) and PPi ions remained highly demands. Yoon and co-workers previously demonstrated rhodamine derivative as a chemosensor for sequential detections of Al³⁺ and pyrophosphate.⁴⁸ However, to the best of our knowledge, rhodamine-based selective and sensitive sequential detections of Cu²⁺ and pyrophosphate via the ratiometric absorption spectral changes have never been explored yet.

Herein, we have successfully developed two reversible colorimetric chemosensor probes Rh1 and Rh2 for sequential detections of Cu²⁺ and pyrophosphate (PPi) ions. Dimers Rh1 and Rh2 (Fig. 1) contain two symmetrical rhodamine units (on both ends) as the fluorophore and two central cores of dihydroxyphenyl and multiple ethers groups as the chelating units, respectively. The receptor moieties together with the nitrogen and carbonyl of rhodamine serve as ionophores by providing coordinating sites based on spirocyclic ring-opening mechanisms. Additionally, the optical properties of these probes and their possible mechanisms for recognition of Cu²⁺ ion has been demonstrated by UV-vis titrations. Further, B3LYP/LANL2DZ density functional calculations were carried out to investigate the molecular orbitals and electronic excitations of these two probes. The stabilities of two probes were examined in the absence and presence of Cu²⁺ ion at wide ranges of pH values. Interestingly, both metal complexes (Rh1–Cu²⁺ and Rh2–Cu²⁺) have been utilized in excellent selective detections of PPi via colorimetric changes visualized by naked-eyes.

Fig. 1 Chemical structures of Rh1 and Rh2.

2 Experimental

2.1 Materials and instrumentations

All reagents were commercially purchased and were used without further purification. The solvents were dried by distillation over appropriate drying agents. Generally, reactions were monitored by TLC plates and flash chromatography was performed on silica gel. The ¹H and ¹³C NMR spectra were recorded on a 300 MHz spectrometer and samples were dissolved in CDCl₃ and DMSO-d₆. The chemical shifts were expressed in ppm and coupling constants (J) in Hz. Mass spectra (FAB) were obtained on the respective mass spectrometer. Elemental analysis was carried out by Elemental Varo EL. UV-vis absorption and fluorescence spectra were measured on V-670 spectrophotometer and F-4500 fluorescence spectrophotometer, respectively. The pH [1–12] buffer solutions were freshly prepared.

2.2 Preparation of metal ion solutions for sensor titrations

Rh1 and Rh2 were dissolved in CH₃CN–H₂O (v/v = 9 : 1, 5 mM Tris–HCl, pH 7.4) at 1 × 10⁻⁵ M concentration. The solutions of metal cations Li⁺, K⁺, Cs⁺, Ni²⁺, Fe³⁺, Fe²⁺, Co²⁺, Zn²⁺, Cd²⁺, Pb²⁺, Ca²⁺, Cr³⁺, Cu²⁺, Ba²⁺ and Al³⁺ were dissolved in water medium at 1 × 10⁻³ M concentration from their chloride salts. Ag⁺, Hg²⁺ and Mg²⁺ were made from AgNO₃, Hg(OAc)₂ and MgSO₄, respectively, in water medium at 1 × 10⁻³ M concentration. The solutions of all anions were prepared by dissolving the respective TBA salts of F⁻, CN⁻, HPO₄²⁻, H₂PO₄⁻, HSO₄⁻, ClO₄⁻, Br⁻, OH⁻, SCN⁻, P₂O₇⁴⁻, NO₃⁻ and OAc⁻ in deionized water (1 × 10⁻³ M). Pentamethyl diethylene triamine (PMDTA) was dissolved in CH₃CN at a concentration of 1 × 10⁻³ M.

2.3 NMR titrations

10 mmol (1 equiv.) of Rh1 and Rh2 in DMSO-d₆ were titrated with 20 mmol (2 equiv.) of Cu²⁺ in D₂O.

2.4 Computational methods

Density functional theory (DFT) calculations were employed to elucidate the Cu²⁺ interactions with Rh1 and Rh2 systems. All computations were carried out using Gaussian 09 software package.⁴⁹ Geometry optimization of the ground state structures was carried out with DFT at the B3LYP level of theory using LANL2DZ basis set in gas phase.⁵⁰ The excitation energies (up to 5 states) of the low-lying excited states and oscillator strengths were predicted using the time-dependent density functional theory (TD-DFT) with B3LYP/LANL2DZ level.

2.5 Synthetic procedures

The following compounds (1–3) were prepared according to the literature: 2,3-dihydroxyterephthalaldehyde (1),⁵¹ [2,2′-(oxybis(ethane-2,1diyl))bis(oxy)dibenzaldehyde] (2)⁵² and rhodamine hydrazide (3).⁵³ The detailed synthetic procedures are illustrated in the ESI and Scheme S1.†

2.6 Synthesis of compound Rh1

Rhodamine hydrazide 3 (0.6 g, 1.32 mmol) was dissolved in absolute ethanol (25 mL), then 3,6-diformylcatechol (1) (0.1 g, 0.66 mmol) was added and the reaction mixture was refluxed in an oil bath for 12 h. The solvent was evaporated by rotary evaporator and the crude product was purified by silica gel column chromatography using EA : hexane (1 : 1, v/v) to obtain compound Rh1 as a light pink solid. Yield: 0.32 g (50%). ¹H NMR (CDCl₃) δ: 10.62 (s, 2H), 9.16 (s, 2H), 7.95–7.93 (m, 2H), 7.51–7.45 (m, 4H), 7.12–7.10 (m, 2H), 6.57 (s, 2H), 6.47 (d, 4H, J = 3.0 Hz), 6.41 (d, 4H, J = 2.7 Hz), 6.21 (dd, 4H, J = 8.7 Hz and 2.4 Hz), 3.30 (q, 16H, J = 6.9 Hz), 1.12 (t, 24H, J = 6.9 Hz); ¹³C NMR (CDCl₃) δ: 164.3, 153.2, 152.5, 151.5, 148.9, 146.9, 133.9, 129.2, 128.3, 127.8, 124.0, 123.2, 120.8, 119.5, 108.0, 105.0, 98.1, 66.4, 44.3, 12.5; IR (KBr, cm⁻¹): 3425, 3079, 1706; MS (FAB⁺) m/z: calcd for C₆₄H₆₆N₈O₆: 1042.5; found: 1143.7 (M + H)⁺; anal. calcd for C₆₄H₆₆N₈O₆: C, 73.68; H, 6.38; N, 10.74; found: C, 72.66; H, 6.60; N, 10.75.

2.7 Synthesis of compound Rh2

Rhodamine hydrazide 3 (0.5 g, 1.26 mmol) was dissolved in absolute ethanol (25 mL), then 2,2′-((oxybis(ethane-2,1-diyl))bis(oxy))dibenzaldehyde (2) (0.2 g, 0.63 mmol) was added and the reaction mixture was refluxed in an oil bath for 12 h. The solvent was evaporated by rotary evaporator and the crude product was purified by silica gel column chromatography using EA : hexane (6 : 4, v/v) to obtained compound Rh2 as a light pink solid. Yield: 0.310 g (40%). ¹H NMR (CDCl₃) δ: 9.03 (s, 2H), 7.97 (dd, 2H, J = 1.8 Hz and 5.4 Hz), 7.85–7.83 (m, 2H), 7.48–7.44 (m, 4H), 7.17–7.09 (m, 4H), 6.84 (t, 2H, J = 7.5 Hz), 6.76 (d, 2H, J = 8.4 Hz), 6.53 (d, 4H, J = 9.0 Hz), 6.43 (d, 4H, J = 2.4), 6.22 (dd, 4H, J = 8.7 Hz and 2.4 Hz), 4.01 (t, 4H, J = 5.1 Hz), 3.82 (t, 4H, J = 5.1 Hz), 3.28 (q, 16H, J = 6.9 Hz), 1.11 (t, 24H, J = 6.9 Hz); ¹³C NMR (CDCl₃) δ: 164.9, 157.3, 153.6, 153.1, 149.8, 143.6, 133.1, 130.8, 129.3, 128.1, 127.4, 126.3, 124.4, 123.7, 121.0, 112.9, 107.9, 106.2, 97.9, 69.7, 66.2, 65.9, 44.30, 12.6; IR (KBr, cm⁻¹): 3460, 3065, 1692; MS (FAB⁺) m/z: calcd for C₇₄H₇₈N₈O₇: 1191.6; found: 1192.4 (M + H)⁺; 1214.5 (M + Na)⁺; anal. calcd for C₇₄H₇₈N₈O₇: C, 74.60; H, 6.60; N, 9.40; found C, 74.36; H, 6.97; N, 9.61.

3 Results and discussion

3.1 Colorimetric responses of sensor probes (Rh1 and Rh2) towards metal ions

Selectivity is one of the most important characteristics in analyte-selective chemosensors. Initially, Rh1 and Rh2 (10 μM) in CH₃CN–H₂O (v/v = 9 : 1, 5 mM Tris–HCl, pH 7.4) displayed no absorption bands corresponding to the rhodamine moiety. As shown in Fig. 2a and b, upon examine the selectivity of Rh1 and Rh2 towards various metal ions (4 equiv.), such as Li⁺, Ag⁺, K⁺, Cs⁺, Ni²⁺, Fe³⁺, Co²⁺, Zn²⁺, Cd²⁺, Pb²⁺, Ca²⁺, Cr³⁺, Cu²⁺, Ba²⁺, Al³⁺, Fe²⁺, Hg²⁺ and Mg²⁺, the UV-vis spectra of Rh1 and Rh2 were only affected by Cu²⁺, Fe³⁺ and Cr³⁺ ions to different extents. Among these three metal ions, upon the addition of Cu²⁺ both absorption bands of Rh1 and Rh2 had the largest changes with the absorption enhancements as large as 40 and 48 fold, respectively (Fig. S13a and b†), where the detailed explanation will be discussed later. Sequentially, the maximum UV-vis absorption intensities of Rh1 and Rh2 at 561 nm were also increased upon the addition of Fe³⁺ (13 and 20 folds, respectively) or Cr³⁺ (8 and 10 folds, respectively) ions. Whereas, a significant increase in the absorption intensity at 561 nm may be attributed to the induced spirolactam ring opening upon the addition of metal ions. The photo images exhibit a naked eye colorimetric change of Rh1 towards Cu²⁺ metal ion, where the most obvious and selective color change from colorless to pink color is observed in Fig. 3. Therefore, our careful investigations suggest that Rh1 and Rh2 could be useful as selective optical chemosensor towards Cu²⁺ than the other surveyed metal ions.

Fig. 2 UV-visible absorption spectra (a) Rh1 and (b) Rh2 (10 μM) upon the addition of several relevant metal ions in CH₃CN–H₂O (v/v = 9 : 1, 5 mM Tris–HCl, pH 7.4).

Fig. 3 Colorimetric changes (naked eye observations) of probe Rh1 (1 μM) upon the addition of various metal ions (4 equiv.).

Furthermore, to explore the possible applications of Rh1 and Rh2 as practical cation-selective chromogenic chemosensors for Cu²⁺, two competitive experiments were also carried out by monitoring the changes in UV-vis absorption intensities at 555 nm. To perform these dual metal ion experiments, Rh1 and Rh2 (10 μM) were first mixed with 4 equiv. of various metal ions including Al³⁺, Cr³⁺, Fe³⁺, Hg²⁺, Pb²⁺, Ag²⁺, Zn²⁺, Mg²⁺, Ni²⁺ and Ag⁺ ions, followed by the addition of 2 equiv. Cu²⁺ in CH₃CN–H₂O (v/v = 9 : 1, 5 mM Tris–HCl, pH 7.4) solution. In the presence of miscellaneous competitive cations, the observations of UV-vis spectra for Rh1 and Rh2 did not show any changes in contrast to single Cu²⁺ ion experiments. However, in contrast to Cu²⁺ ion, only the cases of Fe³⁺ and Cr³⁺ ions were evidenced to affect the absorption peaks to some extents, as illustrated in Fig. 4a and b, where the significant spectral and color changes in dual metal ion systems with Cu²⁺ were still observed. Therefore, both designed dimeric probes (Rh1 and Rh2) may be promising towards establishing selective colorimetric sensors for Cu²⁺ ion over the other competing metal ions.

Fig. 4 (a) Absorbance changes of Rh1 (10 μM) in CH₃CN–H₂O (v/v = 9 : 1, 5 mM Tris–HCl, pH 7.4) solution upon the addition of 4 equiv. various metal ions (black bars). The grey bars represent the absorbance changes as 2.4 equiv. Cu²⁺ ion was added to the mixture of Rh1 (10 μM) 4 equiv. Al³⁺, Cr³⁺, Fe³⁺, Hg²⁺, Pb²⁺, Ag²⁺, Zn²⁺, Mg²⁺, Ni²⁺ and Ag⁺ ions, respectively. (b) Absorbance changes of Rh2 (10 μM) in CH₃CN–H₂O (v/v = 9 : 1, 5 mM Tris–HCl, pH 7.4) solution upon the addition of 4 equiv. various metal ions (black bars). The grey bars represent the absorbance changes as 2.2 equiv. Cu²⁺ ion was added to the mixture of Rh2 (10 μM) 4 equiv. Al³⁺, Cr³⁺, Fe³⁺, Hg²⁺, Pb²⁺, Ag²⁺, Zn²⁺, Mg²⁺, Ni²⁺ and Ag⁺ ions, respectively.

The spectroscopic properties of Rh1 and Rh2 (10 μM) were investigated in a solvent system of in CH₃CN–H₂O (v/v = 9 : 1, 5 mM Tris–HCl, pH 7.4). As shown in Fig. 5a, the free chemosensor Rh1 exhibited an absorption band centered at 323 nm. Upon the addition of Cu²⁺ ion (0–2.4 equiv.) into Rh1 solution, the absorption intensity at 323 nm gradually decreased with a simultaneous new absorption band appearance at 555 nm due to the possible formation of phenoxide ion, which can be further proven by later discussion. Interestingly, the absorption intensity as well as absorption position at isosbestic point 366 nm were remained constant during the interaction of Cu²⁺ with Rh1 solution. Moreover, the absorbance values at 323 nm and 555 nm became saturated and leveled off with 2.4 equiv. of Cu²⁺ ion, and thus demonstrated the formation of a 1 : 2 complexations between the chemosensor Rh1 and Cu²⁺ ion. The binding constant K_a and the calculated detection limit (LOD) were found to be 6.18 × 10⁶ M⁻¹ and 1.22 × 10⁻⁸ M (Fig. S14a and S15a†), respectively.

Fig. 5 The absorption spectral changes of (a) Rh1 and (b) Rh2 (10 μM in CH₃CN–H₂O (v/v = 9 : 1, 5 mM Tris–HCl, pH 7.4)) solution during the sequential additions of Cu²⁺.

Similarly, free Rh2 Chemosensor was colorless as shown in Fig. 5b, when Cu²⁺ was added from 0 to 2 equiv., an absorption band at 556 nm gradually increased with respect to the concentrations of Cu²⁺ ion, which induced an optical color change from colorless to pink color. Therefore, a selective colorimetric change was observed only in the presence of Cu²⁺ ion, which was attributed to the spirolactam ring opening mechanism and multiple ether units reported in a previous literature.⁵⁴ The visible color change of Rh2 towards Cu²⁺ clearly demonstrates that Rh2 was highly sensitive to Cu²⁺. Moreover, the binding affinity of Rh2 towards Cu²⁺ was evaluated from UV-vis titration with an association constant K_a = 2.53 × 10⁵ M⁻¹ and its detection limit was estimated as 8.0 × 10⁻⁷ M (see Fig. S14b and S15b; ESI†). Furthermore, after careful investigation of the fluorescence properties of dimeric systems Rh1 and Rh2, we have established that they could not induce any fluorescence changes with all metal ions, including Cu²⁺ ion (see Fig. S16a and b; ESI†). The fluorescence quenching process was accompanied by the inherent quenching property of paramagnetic Cu²⁺ ion. The proposed binding mechanism of dimeric sensor systems was monitored by the UV-vis absorption spectra. In the absence of Cu²⁺, Rh1 and Rh2 displayed no absorbance above 400 nm, which indicated the closed spirolactam ring at this condition. Whereas, upon the addition of Cu²⁺ a new absorption band appeared at 555 nm suggested the disclosure of the spirolactam ring in the rhodamine unit. Therefore, we believe that the significant color change was solely due to the alteration of molecular structures between cyclic-spiro and ring-opening conformations of the rhodamine framework. Therefore, these observations suggested that among all metal ions only Cu²⁺ could induce the spirolactam ring opening of the rhodamine unit.

To further investigate the binding sites of dimeric systems, we did the ¹H NMR titrations of probes Rh1 and Rh2 in the presence of Cu²⁺ metal ion. To perform the experiments, Rh1 and Rh2 were dissolved in DMSO-d₆ (1 × 10⁻³ M) and Cu²⁺ solution (5 × 10⁻² M) was prepared in D₂O. After adding Cu²⁺ (0–2 equiv.) sequentially into Rh1 solution (1 equiv.), the total disappearance of –OH peak at 10.62 ppm (see Fig. S17†), was well verified the clear binding of Rh1 with Cu²⁺ ion through the phenoxide interactions (after the deprotonation of –OH due to binding with Cu²⁺). In addition, owing to the Schiff-base binding of nitrogen atom with Cu²⁺ ion, the –CH proton peak was upfield-shifted from 9.07 to 8.59 ppm. In the case of Rh2 (see Fig. 6), during the addition of Cu²⁺ (0–2 equiv.), the H_a (–CH₂) protons of the multiple ether unit (i.e., –O–CH₂–CH₂–O–) were upfield-shifted from 4.01 to 3.94 ppm. Similarly, the H_b (–CH₂) protons (with a lower polarity) at 3.77 ppm were also upfield-shifted in the above experiment. Further, the little upfield-shifts of Schiff-base protons (i.e., –N=CH–) were observed because of the complex formation between ‘N’ atoms and Cu²⁺. Hence, based on the ¹H NMR titrations, a possible mechanism is proposed in Scheme 1 to explain the binding mode of probes Rh1 and Rh2 towards Cu²⁺.

Fig. 6 ¹H NMR spectral changes of Rh2 (10 mM) in DMSO-d₆ and titrated with 0–2.0 equiv. of Cu²⁺ in D₂O.

Scheme 1 A possible proposed binding mechanism of sensor probes Rh1 and Rh2 towards Cu²⁺.

Additionally, to ensure the binding site of Rh1 and Rh2 towards Cu²⁺, the stoichiometries of Cu²⁺ complexes (i.e., Rh1–Cu²⁺ and Rh2–Cu²⁺) were determined from Job's plots (see Fig. S18a and b; ESI†) by fixing the final concentrations of Rh1–Cu²⁺ and Rh2–Cu²⁺ as 50 μM. The stoichiometries of both metal complexes were established by Job's plots between the mole fractions (X_m) and the maximum absorption changes at 553 nm and 556 nm for Rh1–Cu²⁺ and Rh2–Cu²⁺, respectively. After attaining the maximum values, the absorption peaks at 553 and 556 nm were slowly reduced from 0.6 molar fraction of Cu²⁺. Therefore, these phenomena clearly indicated the formation of 1 : 2 stoichiometric complexes for both dimeric systems towards Cu²⁺. To reconfirm the Cu²⁺ binding mode of the dimeric systems (Rh1 and Rh2), their FT-IR band (see Fig. S19a and b; ESI†) of –OH at 3425 cm⁻¹ and the ether linkage band (i.e., C–O–C) at 3460 cm⁻¹ were investigated in the presence of Cu²⁺ ion. Correspondingly, the IR bands of C=O (at 1706 and 1692 cm⁻¹) and –C–H (at 3079 and 3065 cm⁻¹) were also been evaluated for Rh1 and Rh2 dimeric systems. During the complex formation of probes Rh1 and Rh2 to Cu²⁺ ion, the above characteristic bands were affected incredibly as noted next. The characteristic bands of –OH and ether unit are became very broad along with the disappearance of –CH bands of Rh1 and Rh2 at 3079 and 3065 cm⁻¹, respectively. Further to note, the respective C=O stretching bands of Rh1 and Rh2 at 1706 and 1692 cm⁻¹ were also diminished with slight shift. The above FTIR changes were well confirmed the attachment of colorimetric probes Rh1 and Rh2 towards Cu²⁺ ion.

The reversibilities of sensor complexes Rh1–Cu²⁺ and Rh2–Cu²⁺ in CH₃CN–H₂O (v/v = 9 : 1, 5 mM Tris–HCl, pH 7.4) solutions are important for future applications. The reversible nature of the sensor complexes with Cu²⁺ ion was further carried out through UV-vis observations by adding penta-methyl ethylene-triamine (PMDTA), which is a well-known metal ion chelator. As depicted in Fig. S20a and b (ESI†), upon the addition of PMDTA (2.6 and 2.2 equiv.) to the mixtures of Rh1–Cu²⁺ and Rh2–Cu²⁺ (10 μM), the absorption bands were found to be restored immediately. Moreover, by increasing the concentration of PMDTA (0–2.6 equiv. and 0–2.2 equiv., respectively) the reversibilities of Rh1–Cu²⁺ and Rh2–Cu²⁺ with PMDTA were clearly established as shown in Fig. 21a and b (ESI†). Upon the addition of PMDTA, the absorption peaks of dimeric probes Rh1 and Rh2 were decreased gradually to characterize the reversible sensing capabilities of sensor probes (Rh1 and Rh2). This reversibility allow us to reuse of the sensor probes for effective Cu²⁺ detection.

3.2 Colorimetric responses of metal complexes (Rh1–Cu²⁺ and Rh2–Cu²⁺) towards anions

Next, we used the previously formed metal complexes Rh1–Cu²⁺ and Rh2–Cu²⁺ to examine their anion selectivities. The absorbance spectral studies of metal complexes with different anions F⁻, CN⁻, HPO₄²⁻, H₂PO₄⁻, HSO₄⁻, ClO₄⁻, Br⁻, OH⁻, SCN⁻, P₂O₇⁴⁻, NO₃⁻ and OAc⁻ were carried out in the solution condition of 10 μM in CH₃CN–H₂O (v/v = 9 : 1, 5 mM Tris–HCl, pH 7.4). The color changes for metal complexes (Rh1–Cu²⁺ and Rh2–Cu²⁺) towards various anions are demonstrated in the photograph images of Fig. 7, where no color changes were observed in all anions, except pyrophosphate (PPi).

Fig. 7 Photo-images of color changes for metal complexes (Rh1–Cu²⁺ and Rh2–Cu²⁺) towards various anions.

The addition of PPi anion turned the original pink colored solution into a colorless solution. Upon the addition of PPi (1 equiv.), not only these color changes could be easily recognized by naked-eyes, but also the UV-vis spectral changes were apparent as noticed in Fig. 8a and b. On the other hand, metal complexes didn't show any considerable spectral changes in absorption peaks during the addition of other anions (up to 4 equiv.). Therefore, the selectivity of the metal complexes (Rh1–Cu²⁺ and Rh2–Cu²⁺) towards PPi anion might be attributed to their release of Rh1 and Rh2 through the strong binding affinity of PPi to copper ion as illustrated in Scheme 2. This result indicates that both metal complexes can serve as potential candidates as “naked-eye” chemosensor for PPi detection in semi-aqueous medium. Meanwhile, we further investigated the binding properties of both metal complexes by UV-vis titration experiments. Fig. 9 showed the colorimetric changes of metal complexes (Rh1–Cu²⁺ and Rh2–Cu²⁺) 10 μM in CH₃CN–H₂O (v/v = 9 : 1, 5 mM Tris–HCl, pH 7.4). Upon the addition of increasing concentrations (1 equiv.) of PPi, the absorbance at 554 nm was decreased linearly with increasing the concentration of PPi. The binding constants between the metal complexes (Rh1–Cu²⁺ and Rh2–Cu²⁺) and PPi were calculated as 1.09 × 10⁵ M⁻¹ and 4.11 × 10⁴ M⁻¹, respectively (see Fig. S22a and b; ESI†). Furthermore, based on the results of UV-vis titrations, the detection limits of metal complexes (Rh1–Cu²⁺ and Rh2–Cu²⁺) mediated PPi sensors were estimated to be 0.389 × 10⁻⁶ M and 1.61 × 10⁻⁶ M, respectively, as shown in Fig. S23a and b (ESI†). These observations suggest that both metal complexes (Rh1–Cu²⁺ and Rh2–Cu²⁺) could be excellent chemosensors for the detection of PPi anion.

Fig. 8 (a) UV-visible spectral changes of Rh1–Cu²⁺ and (b) Rh2–Cu²⁺ (10 μM) upon the addition of several relevant anions in CH₃CN–H₂O (v/v = 9 : 1, 5 mM Tris–HCl, pH 7.4).

Scheme 2 Possible proposed sensing mechanism of metal complexes (Rh1–Cu²⁺ and Rh2–Cu²⁺) towards pyrophosphate (PPi) anion.

Fig. 9 Absorption spectral changes of (a) Rh1–Cu²⁺ and (b) Rh2–Cu²⁺ (10 μM) in CH₃CN–H₂O (v/v = 9 : 1, 5 mM Tris–HCl, pH 7.4) during the sequential additions of PPi.

For practical applications, we need to evaluate the utilizations of colorimetric sensor probes under wider pH conditions (other than the previous neutral form). As expected, the absorption peaks of un-bonded Rh1 and Rh2 (without Cu²⁺) were only observable in the ranges of pH = 2.0–5.0, suggesting that the strong protonation and spirolactam ring opening process of sensor probes occurred at certain pH ranges. In order to investigate the applications of Rh1 and Rh2 over a wide pH span of 0–12, the absorption intensities of Rh1 and Rh2 increased by adding Cu²⁺ under pH = 3.0–8.0 (see Fig. S24a and b; ESI†). In this pH zone, Cu²⁺ induced unique ring opening process in the rhodamine unit was obtained, which clearly demonstrated Rh1 and Rh2 as effective colorimetric probes towards Cu²⁺ under environmental conditions.

3.3 Theoretical calculations

To evaluate the experimental observations and provide a better insight into probes Rh1 and Rh2 along with their copper complexes, the DFT and TD-DFT calculations were carried out and summarized in Table 1. The model structures have been built for Rh1 and its copper complex as shown in Fig. 10.

Table 1 The predicted values of HOMO, LUMO, HOMO LUMO gap (HLG) and oscillator strength (f) for Rh1, Rh1 + Cu²⁺, Rh2 and Rh2 + Cu²⁺ by DFT and TD-DFT calculations

System	Tot. E^a	HOMO (eV)	LUMO (eV)	HLG^b (eV)	Excited states	f^c	Contribution
a Total energy.b HOMO–LUMO gap.c Oscillator strength.
Rh1	−3367.381623	−4.269328	−1.625269	2.64	S1	0.0475	H → L (0.69)
							H → L+1 (−0.1)
					S2	0.0146	H−1 → L (0.69)
							H−1 → L+1 (0.1)
Rh1 + Cu^2+	−3759.353208	−7.281195	−5.181813	2.10	S1	0.0001	H → L+2 (0.6)
							H → L+3 (−0.33)
							H → L+5 (0.16)
					S2	0.0477	H → L+2 (0.34)
							H → L+3 (0.61)
Rh2	−3830.768067	−4.172246	−1.302659	2.87	S1	0.011	H → L+1 (0.71)
					S2	0.009	H+1 → L+2 (0.7)
Rh2 + Cu^2+	−4223.285313	−4.172246	−8.467632	1.28	S1	0.0025	H → L+1 (0.7)
					S2	0	H+1 → L+1 (0.7)
					S3	0.0026	H+1 → L+2 (0.7)

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Fig. 10 Optimized structures of (a) Rh1 and (b) complex Rh2–Cu²⁺ at B3LYP/LANL2DZ in the gas phase.

The main absorption bands of transitions were H → L, H → L+2, H → L+1 and H → L+1 observed in Rh1, Rh1–Cu²⁺, Rh2 and Rh2–Cu²⁺, respectively. Fig. 11 shows the molecular orbital of Rh1, where the electron density of HOMO was located partially on the catechol unit and further on the oxa-anthracene unit and the LUMO was partially located on the catechol unit and further at the indole units on both sides. As shown in Fig. 12, the electron density of HOMO in complex Rh1–Cu²⁺ was located on the hydrazine units with part of Rh2–Cu²⁺ are shown in Fig. S25a and b (ESI†). The respective molecular orbitals of Rh2 and Rh2–Cu²⁺ are shown in Fig. S26 and S27 (ESI†). As shown in Fig. S26 (ESI†) in Rh2, the electron density of HOMO was located on a spiro unit, and that of LUMO was also located on a spiro unit. The electron density of HOMO has been shifted slightly towards the edge of the spiro unit in Rh2–Cu²⁺ complex. The LUMO also covered partially one Cu²⁺ unit with aza-carbonyl and benzene units of the complex. From the molecular orbitals of probes and complexes (Fig. 10, 11, S26 and S27†), the electron densities located on the same part of the HOMO and LUMO for the cases of Rh2 and Rh2–Cu²⁺, but on the different parts of HOMO and LUMO for the cases of Rh1 and Rh1–Cu²⁺. Based on our calculated molecular orbital diagram in Fig. 11, the conjugated system was broken due to the addition of Cu²⁺ which led to the stronger ICT mechanism^55,56 in the Cu²⁺ complexes. This could be the possible explanations for the experimental observations of ratiometric responses of the Cu²⁺ complexes. Furthermore, the HLG value was also reduced in complex Rh1–Cu²⁺ (2.10 eV) in contrast to that of Rh1 (2.64 eV).

Fig. 11 HOMO–LUMO structures of Rh1 at B3LYP/LANL2DZ level in the gas phase.

Fig. 12 HOMO–LUMO structures of complex Rh1 + Cu²⁺ at B3LYP/LANL2DZ level in the gas phase.

3.4 Colorimetric responses on a solid support

In analytical chemistry grade, the sensitivity of a sensing system is crucial. Normally, it remains big challenges to achieve high sensitivities in such colorimetric sensing systems. We systematically investigated the selectivities, sensitivities and reversibilities of Rh1 and Rh2 on TLC plates coated with ready-made silica. Test strips were prepared by immersing silica gel plates into a CH₃CN solution of Rh1 (10⁻⁴ M) and then were dried by a drier. As shown in Fig. 13, the strips were examined with various metal ions (10⁻³ M) by dipping into the corresponding solutions. We found that only copper ion induced a remarkable color change from colorless to pink, implying that probe was a highly selective naked-eye sensor probe towards Cu²⁺ over the other metal ions.

Fig. 13 Photographs of sensor tests on Rh1 strips in the presence of various metal ions.

The detection results of test strips containing Rh1 towards Cu²⁺ with different concentrations are also illustrated in Fig. S28.† The higher concentration of copper ion, the more apparent color change from colorless to pink, where the detectable concentration of copper ion could be as low as 4 × 10⁻⁶ M. As shown in Fig. 14, further reversibility investigation revealed that the pink color of the test strips containing complex Rh1–Cu²⁺ faded out in the presence of PMDTA (10⁻⁵ M). Therefore, we have developed the notable and reversible naked-eye sensor strips to detect low concentrations of copper ion (4 μM) in this study. Similar results regarding selectivity, sensitivity and reversibility of test strips for Rh2 were also been obtained (see Fig. S29–S31; ESI†). Therefore, test strips containing Rh1 and Rh2 can conveniently detect aqueous copper ion, which demonstrate a cost-effective detection method without using any equipments.

Fig. 14 Photographs of reversibility tests on Rh1 strips (10⁻⁴ M) in the presence of PMDTA (10⁻⁵ M). From left to right: Rh1, Rh1–Cu²⁺ and Rh1–Cu²⁺ + PMDTA.

4 Conclusions

We have developed reversible colorimetric probes Rh1 and Rh2 for the selective and sensitive sequential detections of Cu²⁺ and pyrophosphate via the ratiometric absorption spectral changes. Stoichiometry and binding mechanisms for both probes were well characterized and established by the respective spectroscopic techniques and theoretical calculations. Observations revealed a better performance of Rh1 towards the selective and effective colorimetric detection of Cu²⁺ ion at a lower concentration than that of Rh2. Reversibility may open the avenue towards cost effectiveness and understanding the role of metal ions by switching signals between structural transformations and thus for easy detections of analytes for societal demands. Detections of Cu²⁺ by the designed probes were performed in solution and on solid supports by simple titration and dipping of metal ion solutions (without utilizing any drastic conditions), which may push the limits of our designed probes to future concerning ecological problems as well as living systems. Importantly, Rh1 and Rh2 are good example of reversible rhodamine-based colorimetric probes for sequential detections of Cu²⁺ ion and PPi via ratiometric responses.

Acknowledgements

The financial supports of this project are provided by the Ministry of Science and Technology (MOST) in Taiwan through MOST 103-2113-M-009-018-MY3 and MOST 103-2221-E-009-215-MY3. Mr Parthiban Venkatesan is acknowledged for his timely help during the paper writing.

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Footnote

† Electronic supplementary information (ESI) available: NMR and mass spectra of Rh1 and Rh2 and sensor properties mentioned in the text. See DOI: 10.1039/c6ra24472f

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