A fluorescent sensor for Cu²⁺ and Fe³⁺ based on multiple mechanisms

Abstract

A new fluorescent sensor for the selective and sensitive sensing of Cu²⁺ and Fe³⁺ based on the rational control of multiple mechanisms has been developed. Sensor L is weakly-fluorescent (ϕ = 0.029) due to the C=N isomerization and PET process. Upon binding with Cu²⁺, the complex L + Cu²⁺ can give a remarkable fluorescence enhancement (ϕ = 0.182) with a limit of detection down to 1.3 × 10⁻⁸ mol L⁻¹ because of both inhibition of the C=N isomerization and PET process. However, upon binding with Fe³⁺, the original weak fluorescence of L is completely quenched (ϕ = 0.002) due to the paramagnetic quenching property. In addition, the complex L + Cu²⁺ could be further used as a new platform for the ultrasensitive detection of Fe³⁺ based on the fluorescence “turn-off” response with a limit of detection down to 2.4 × 10⁻⁸ mol L⁻¹ in aqueous solution.

---

Introduction

The development of highly selective and sensitive fluorescent sensors that can be used to detect bioactive metal ions has gained enormous importance.^1–4 Metal ions are important for a variety of fundamental biological processes, but can also cause serious environmental and health problems due to their high toxicity. Copper is a significant environmental pollutant and yet also an essential trace element that acts as a cofactor in all currently known life forms.^5–11 The limit of Cu²⁺ in drinking water has been set to 1.3 ppm (20 μM). Exposure to a high level of Cu²⁺ can cause a wide variety of symptoms (gastrointestinal disease, Wilson's disease, dyslexia, hypoglycemia, and infant liver damage).^12–17 Thus, the detection of Cu²⁺ from various sources including those in waste water outlets, electroplating waste and other metal processing industries is important. Cu²⁺ complexation is well known to induce intrinsic fluorescence quenching,^18–24 while chemosensors with fluorescence enhancement were more encouraging because of their simplicity in practical applications.^25–30 Therefore, fluorescence ‘turn-on’ chemosensors with high selectivity and sensitivity towards Cu²⁺ are highly desirable.

As an important physiologically relevant metal ion, Fe³⁺ performs a significant role in a variety of vital cell functions, and both its excess (hyperferremia) and deficiency (hypoferremia) can induce a variety of diseases.^31–34 The security limit for Fe³⁺ was restricted to 2 mg L⁻¹ by the WHO.^35,36 In the form of ferritin or hemosiderin iron is stored in liver, spleen and bone marrow of human body. However, excess of Fe³⁺ leads to increasing incidence of certain cancers and dysfunction of body organs (gastric upset, constipation, nausea, abdominalpain, vomiting and faintness), even causes hyperferremic and hypotransferrinemic disorder when Fe³⁺ accumulates in the body.^37,38 Similarly, the deficiency of Fe³⁺ causes a disease namely anaemia and functional deficits associated with anaemia (gastrointestinal disturbances and impaired cognitive function, immune function, exercise or work performance, and body temperature regulation).^39–41 In this regard, the development of chemosensor that can detect Fe³⁺ is very important to prevent and solve environmental and health problems induced by Fe³⁺.

Till now, a lot of effective fluorescent sensors had been reported which showed excellent optical response towards Cu²⁺ or Fe³⁺. However, most of them could detect only one metal ion (Cu²⁺ or Fe³⁺) in the solution, and this certainly limited their practical application.^18,19,27 For these fluorescent sensors, there was only one mechanism playing a leading role (such as charge transfer or paramagnetic quenching) in the detection process of metal ions. In contrast, the sensors which could be used as platforms to detect multiple metal ions based on the rational control of multiple mechanisms exhibited a lot of advantages, such as high efficiency, multiplicity and low cost. Unfortunately, there are few articles carefully describing the role of multiple mechanisms and giving an accurate sensor as an example for the metal ions detection. As a result, it is still in urgent need of designing an ultrasensitive fluorescent sensor for the accurate detection of Cu²⁺ and Fe³⁺ without interference from other anions based on the rational control of multiple mechanisms.

In this work, a novel fluorescent sensor (L) based on the fluorophore of benzimidazo[2,1-a]benz[de]isoquinoline-7-one-12-carbohydrazide was prepared. With the introduction of 4-oxo-4H-chromene-3-carbaldehyde as a receptor in the molecule, the fluorophore and receptor were linked by a C=N bond and the PET (photoinduced electron transfer) process was directed from the receptor to fluorophore, giving L potential ability to be used as a fluorescent sensing platform towards multiple metal ions. It was found that the ligand L was actually weak-fluorescent. After binding with Cu²⁺, a new complex, L + Cu²⁺, was formed and showed a strong fluorescence enhancement due to the inhibition of both C=N isomerization and PET process. Upon binding with Fe³⁺, the original weak fluorescence of L completely quenched due to the paramagnetic quenching property. What is more, the lighted complex L + Cu²⁺ could be further used as a new sensing platform for ultrasensitive detection of Fe³⁺ based on the fluorescence turn-off response. Base on the rational control of multiple sensing mechanisms, Cu²⁺ and Fe³⁺ could be very sensitively and selectively detected.

Experimental section

Measurements

UV-vis spectra were recorded on a Shimadzu 3100 spectrometer. Fluorescence measurements were carried out using an HITACHI Instruments F-4600 fluorescence spectrophotometer. ¹H NMR spectra were recorded on a Bruker AV III 400 MHz NMR spectrometer with tetramethysilane (TMS) as an internal standard. Infrared spectra were recorded using a Bruker Vertex 70 FT-IR spectrometer with KBr pellets.

The ¹³C CP/MAS NMR spectra were recorded on a Bruker AVANCE III 400 WB spectrometer equipped with a 4 mm standard bore CPMAS probehead whose X channel was tuned to 100.62 MHz for ¹³C and the other channel was tuned to 400.18 MHz for broad band ¹H decoupling, using a magnetic field of 9.39 T at 297 K. The dried and finely powdered samples were packed in the ZrO₂ rotor closed with Kel-F cap which were spun at 8 kHz rate. The experiments were conducted at a contact time of 2 ms. A total of 1500 scans were recorded with 3 s recycle delay for each sample. All ¹³C CP MAS chemical shifts are referenced to the resonances of adamantane (C₁₀H₁₆) standard (δ_CH2 = 38.4).

Sample preparation

All tests described in this paper were carried out at room temperature (25 °C) with distilled water. In the experiments of titration with various metal ions, the sensor was dissolved in HEPES acetonitrile–H₂O (9 : 1) buffer solution to afford the test solution (1 × 10⁻⁵ M). Stock solutions (1 × 10⁻⁵ M) of the metal salts of LiCl, NaCl, KCl, MgCl₂, CaCl₂, BaCl₂, NiCl₂, CuCl₂, ZnCl₂, CdCl₂, HgCl₂, PbCl₂, AgNO₃, MnCl₂, FeCl₃, CoCl₂, CrCl₃, SrCl₃ and AlCl₃ in water were prepared.

Theoretical calculation

Density functional theory (DFT) structural optimizations were calculated with the Gaussian 09 program.⁴² In all cases, the structures were optimized using the B3LYP functional and the mixed basis sets 6-31+G(d) and LANL2DZ. Each structure was subsequently subjected to TD-DFT calculation using the B3LYP functional. For all optimized structures, frequency calculations were carried out to confirm the absence of imaginary frequencies. The molecular orbitals were visualized and plotted with the GaussView 5.0 program.

Calculation of quantum yield

The quantum yield of sensor L was determined according to the following equation:
where ϕ is fluorescence quantum yield; F is integrated area under the corrected emission spectra; A is the absorbance at the excitation wavelength; n is the refractive index of the solution; the subscripts u and s refer to the unknown and the standard, respectively. Rhodamine B in ethanol solution was used as the standard, which has a quantum yield of 0.97.

Synthesis

7-Oxo-N′-((4-oxo-4H-chromen-3-yl)methylene)-7H-benzo[de]benzo[4,5]imidazo[2,1-a]isoquinoline-12-carbohydrazide (L). Compound 1, 2, 3 and 4 was synthesized according to our previous work.^43,44 Compound 4 (0.05 g, 0.15 mmol) was suspended in 30 mL absolutely ethyl alcohol and then 4-oxo-4H-chromene-3-carbaldehyde (0.040 g, 0.17 mmol) was added. The mixture was stirred at room temperature for 3 h. The resulting suspension was filtered, and the filter cake was washed with methanol (30 mL × 3) and got the pure compound L. Yield: 0.028 g, 42%. ESI-MS: m/z = 485.1 [M + H]. FTIR (KBr, cm⁻¹): 1718 (C=O), 1230 (C–N). ¹H NMR (400 MHz, CDCl₃): 13.11 (s, 1H), 9.08–8.71 (m, 5H), 8.52 (d, 1H), 8.46–8.27 (m, 3H), 7.95 (dd, 2H), 7.82–7.76 (m, 1H), 7.69–7.51 (m, 3H). ¹³C CP/MAS NMR: δ 175.18, 160.35, 159.29, 154.06, 147.26, 138.72–116.94. Melting point: 190–193 °C. Element analysis for C₂₉H₁₆N₄O₄ (%): C 71.87, H 3.35, N 11.60, O 13.11. Calculated C 71.90, H 3.33, N 11.56, O 13.21.

Results and discussion

Synthesis of L

L was synthesized in moderate yield according to the synthetic route shown in Scheme 1. For the preparation of L, 4-oxo-4H-chromene-3-carbaldehyde was added to the ethanol solution of compound 4 (benzimidazo[2,1-a]benz[de]isoquinoline-7-one-12-carbohydrazide), and the mixture was stirred at 50 °C for 3 hours. The chemical structures of the synthesized compounds were characterized by ¹H NMR, ¹³C CP/MAS NMR spectra, FTIR, element analysis and mass spectrum. All of the data in the spectra were in good accordance with the structure.

Scheme 1 Synthetic routes of L. Conditions: (i) CH₂Cl₂, at room temperature overnight; (ii) CHCl₃, at room temperature for 1 h; (iii) dioxane, refluxed at 70 °C for 3 h; (iv) CH₃CH₂OH, at room temperature for 3 h.

The optical properties of L towards metal ions

The fluorescence behavior of L was investigated in acetonitrile–H₂O (9 : 1) solution (0.01 M HEPES buffer at pH 7.4). As expected, L showed weak fluorescence intensity with a quantum yield (ϕ) of 0.029. As show in Fig. 1, upon addition of various metal ions (Li⁺, Na⁺, K⁺, Mg²⁺, Ca²⁺, Ba²⁺, Ni²⁺, Cu²⁺, Zn²⁺, Cd²⁺, Hg²⁺, Pb²⁺, Ag⁺, Mn²⁺, Fe³⁺, Co²⁺, Cr³⁺, Sr³⁺ and Al³⁺), only Cu²⁺ and Fe³⁺ could induce obvious changes in the fluorescence intensity. After addition of Cu²⁺, the emission band of L showed remarkable fluorescence enhancement (ϕ = 0.182) centred at 490 nm, indicating a fluorescence turn-on response. What is different, after addition of Fe³⁺, the original weak fluorescence intensity of L showed a completely quenching response (ϕ = 0.002), displaying an efficient turn-off behavior. However, with addition of various other metal ions, almost negligible changes of fluorescence intensity were induced. As shown in Scheme 2, when 10 equivalents of Cu²⁺ or Fe³⁺ were added to the acetonitrile–H₂O (9 : 1) HEPES buffer (pH 7.4) solution, L responded with a dramatic color change, from weak-fluorescence to strong-fluorescence for Cu²⁺, from weak-fluorescence to non-fluorescence for Fe³⁺, respectively. The apparent color changes could be distinguished by naked eye. Based on the fluorescence experiments, we found that L could be used as a fluorescent sensor for Cu²⁺ and Fe³⁺ in acetonitrile–H₂O buffer solution.

Fig. 1 Fluorescence spectra of L (1 × 10⁻⁵ M) in acetonitrile–H₂O (9 : 1, HEPES 0.01 M, pH = 7.4) solution in the presence of various metal ions (Li⁺, Na⁺, K⁺, Mg²⁺, Ca²⁺, Ba²⁺, Ni²⁺, Cu²⁺, Zn²⁺, Cd²⁺, Hg²⁺, Pb²⁺, Ag⁺, Mn²⁺, Fe³⁺, Co²⁺, Cr³⁺, Sr³⁺ and Al³⁺). Excitation wavelength is at 370 nm.

Scheme 2 The proposed binding mode of sensor L with Cu²⁺ and Fe³⁺.

The Cu²⁺ sensing

To get a further insight into the sensing property of L towards Cu²⁺, the fluorescence titration experiment was investigated in HEPES buffer solution at an excitation wavelength of 370 nm. As shown in Fig. 2, upon incremental addition of Cu²⁺, the fluorescence emission maximum of L at 490 nm gradually increased and reached a plateau when the concentration of Cu²⁺ was 10 equiv. Particularly, as shown in Fig. 3, the fluorescence intensity of L linearly increased as the concentration of Cu²⁺ changed from 0 to 10 equiv. By linearly fitting the changes of fluorescence as the function of concentration of Cu²⁺, we obtained the slope as 7.3 × 10⁶ for L. The limit of detection (LOD) was found to be 1.3 × 10⁻⁸ mol L⁻¹ based on LOD = 3σ/s where σ is the standard deviation of blank measurements, and s is the slope between fluorescence intensity versus Cu²⁺ concentration. The Job's method monitored by fluorescence intensities was applied to examine the stoichiometry of the L + Cu²⁺ complex, indicating a 1 : 1 stoichiometry of L to Cu²⁺ in the complex (Fig. 4). The titration results indicated that L had an excellent sensitivity towards Cu²⁺ based on the fluorescence turn-on response, and even was applicable for quantitative detection of Cu²⁺.

Fig. 2 Fluorescence spectra of L (1 × 10⁻⁵ M) in acetonitrile–H₂O (9 : 1, HEPES 0.01 M, pH = 7.4) upon titration with Cu²⁺ (1 × 10⁻⁵ M). Excitation is at 370 nm.

Fig. 3 Change ratio of L (1 × 10⁻⁵ M) in acetonitrile–H₂O (9 : 1, HEPES 0.01 M, pH = 7.4) upon titration with Cu²⁺ (1 × 10⁻⁵ M). Emission is monitored at 490 nm.

Fig. 4 Job's plot of the L in acetonitrile–H₂O (9 : 1, HEPES 0.01 M, pH = 7.4) at 25 °C. The total concentration of L and Cu²⁺ was 0.05 mM. Excitation is at 370 nm, and emission is monitored at 490 nm.

As well all known, the selective sensing of sensor towards guest in the presence of other competitive species was another important property. Thus, to study the influence of other metal ions on the formation of L + Cu²⁺ complex, competitive experiments were performed with 20.0 equiv. of other metal ions (Li⁺, Na⁺, K⁺, Mg²⁺, Ca²⁺, Ba²⁺, Ni²⁺, Zn²⁺, Cd²⁺, Hg²⁺, Pb²⁺, Ag⁺, Mn²⁺, Fe³⁺, Co²⁺, Cr³⁺, Sr³⁺ and Al³⁺) in the presence of 10.0 equiv. of Cu²⁺. As shown in Fig. 5, it was found that the fluorescence enhancement caused by the mixture of Cu²⁺ with most of other metal ions was similar to that caused by Cu²⁺ only. However, in the presence of Cu²⁺ mixed with Fe³⁺, a completely quenched fluorescence of L was observed, indicating there was interference from Fe³⁺ to the detection of Cu²⁺. The competitive experiments verified that the sensing ability of L towards Cu²⁺ was not affected by the presence of most other metal ions, and the new formation of lighted complex L + Cu²⁺ could be further used as a fluorescence quenching sensor for Fe³⁺.

Fig. 5 The fluorescence intensity of L (1 × 10⁻⁵ M) in the presence of 10 equiv. of Cu²⁺ and followed by 20 equiv. of various other metal ions (Li⁺, Na⁺, K⁺, Mg²⁺, Ca²⁺, Ba²⁺, Ni²⁺, Zn²⁺, Cd²⁺, Hg²⁺, Pb²⁺, Ag⁺, Mn²⁺, Fe³⁺, Co²⁺, Cr³⁺, Sr³⁺ and Al³⁺) in acetonitrile–H₂O (9 : 1, HEPES 0.01 M, pH = 7.4) at 25 °C. Excitation is at 370 nm, and emission is monitored at 490 nm.

The Fe³⁺ sensing

As shown in Fig. 6, the fluorescence titration experiment of L towards Fe³⁺ was investigated in HEPES buffer solution at an excitation wavelength of 370 nm. Upon incremental addition of Fe³⁺, the weak fluorescence intensity at 490 nm gradually decreased. When the concentration of Fe³⁺ reached 5 equiv., the fluorescence intensity quenched to the end. At this state, the emission spectra of L was almost a flat line parallel to the abscissa (ϕ = 0.002). Particularly, as shown in Fig. 7, upon the concentration of Fe³⁺ increased from 0 to 3.5 equiv., a good linear relationship was observed between fluorescence intensity and [Fe³⁺]. By linearly fitting the changes of fluorescence as the function of concentration of Fe³⁺, the LOD for Fe³⁺ was found to be 4.3 × 10⁻⁷ mol L⁻¹ based on LOD = 3σ/s, where σ is the standard deviation of blank measurements, and s is the slope between fluorescence intensity versus Fe³⁺ concentration. The titration results indicated that L had an excellent sensitivity towards Fe³⁺ based on the fluorescence turn-off response.

Fig. 6 Fluorescence spectra L (1 × 10⁻⁵ M) in acetonitrile–H₂O (9 : 1, HEPES 0.01 M, pH = 7.4) upon titration with Fe³⁺ (1 × 10⁻⁵ M). Excitation is at 370 nm.

Fig. 7 Change ratio of L (1 × 10⁻⁵ M) in acetonitrile–H₂O (9 : 1, HEPES 0.01 M, pH = 7.4) upon titration with Fe³⁺ (1 × 10⁻⁵ M). Emission is monitored at 490 nm.

As shown in Fig. 8, to study the influence of other metal ions on the fluorescence detection of Fe³⁺, competitive experiments were performed with 10.0 equiv. of other metal ions (Li⁺, Na⁺, K⁺, Mg²⁺, Ca²⁺, Ba²⁺, Ni²⁺, Zn²⁺, Cd²⁺, Hg²⁺, Pb²⁺, Ag⁺, Mn²⁺, Fe³⁺, Co²⁺, Cr³⁺, Sr³⁺ and Al³⁺) in the presence of 5.0 equiv. of Fe³⁺. It was found that the fluorescence quenching caused by the mixture of Fe³⁺ with other metal ions was very similar to that caused by Fe³⁺ only. The competitive experiments verified that the sensing ability of L towards Fe³⁺ was not affected by the presence of other metal ions.

Fig. 8 The fluorescence intensity of L (1 × 10⁻⁵ M) in the presence of 5 equiv. of Fe³⁺ and followed by 10 equiv. of various other metal ions (Li⁺, Na⁺, K⁺, Mg²⁺, Ca²⁺, Ba²⁺, Ni²⁺, Zn²⁺, Cu²⁺, Cd²⁺, Hg²⁺, Pb²⁺, Ag⁺, Mn²⁺, Co²⁺, Cr³⁺, Sr³⁺ and Al³⁺) in acetonitrile–H₂O (9 : 1, HEPES 0.01 M, pH = 7.4) at 25 °C. Excitation is at 370 nm, and emission is monitored at 490 nm.

As mentioned above, the formed complex L + Cu²⁺ could be further used as a new platform for ultrasensitive detection of Fe³⁺. To investigate the sensing property of L + Cu²⁺ towards Fe³⁺ in detail, the fluorescence titration experiment was carried out. As shown in Fig. 9, in acetonitrile–H₂O (9 : 1, HEPES 0.01 M, pH = 7.4) solution, the complex L + Cu²⁺ showed a strong fluorescence intensity centered at 490 nm (ϕ = 0.182). Upon incremental addition of Fe³⁺, the strong fluorescence intensity gradually decreased and completely quenched when the concentration of Fe³⁺ reached 15 equiv. As shown in Fig. 10, upon the concentration of Fe³⁺ increased from 0 to 12 equiv., the fluorescence intensity of L + Cu²⁺ linearly decreased. By linearly fitting the changes of fluorescence as the function of concentration of Fe³⁺, we obtained the slope as 2.3 × 10⁶ for L + Cu²⁺. The limit of detection (LOD) was found to be 2.4 × 10⁻⁸ mol L⁻¹ based on LOD = 3σ/s. where σ is the standard deviation of blank measurements, and s is the slope between fluorescence intensity versus Fe³⁺ concentration. The titration results indicated that the complex L + Cu²⁺ had an excellent sensitivity towards Fe³⁺ based on the fluorescence turn-off response.

Fig. 9 Fluorescence spectra of L + Cu²⁺ (1 × 10⁻⁵ M) in acetonitrile–H₂O (9 : 1, HEPES 0.01 M, pH = 7.4) upon titration with Fe³⁺ (1 × 10⁻⁵ M). Excitation is at 370 nm.

Fig. 10 Change ratio of L + Cu²⁺ (1 × 10⁻⁵ M) in acetonitrile–H₂O (9 : 1, HEPES 0.01 M, pH = 7.4) upon titration with Fe³⁺ (1 × 10⁻⁵ M). Emission is monitored at 490 nm.

The mechanism of binding mode

Based on the results of fluorescence titration experiments, we proposed two plausible binding modes for L + Cu²⁺ and L + Fe³⁺ as shown in Scheme 2. The weak fluorescence of L was likely due to multiple quenching mechanisms in the molecule (C=N isomerization and PET process). With the introduction of 4-oxo-4H-chromene-3-carbaldehyde as a receptor, the fluorophore and receptor were linked by a C=N bond. C=N isomerization was always thought as the predominant decay process of the excited states for compounds with an unbridged C=N structure, so that those compounds were often weak-fluorescent.⁴⁵ Meanwhile, the free 4-oxo-4H-chromene-3-carbaldehyde group have a lone pair of suitable energy for causing sufficient PET effect in the molecule directed from receptor to fluorophore, also resulting in a weak fluorescence of L. After binding with Cu²⁺, the C=N isomerization was inhabited and the stiffness of the molecular became better. In addition, the lone pair of receptor was occupied and the electron transfer was thermodynamically disallowed, resulting in the suppression of PET communication between receptor and fluorophore. Although Cu²⁺ was a paramagnetic cation which could always induce fluorescence quenching response, the inhibition of both C=N isomerization and PET processes played a dominant role and override the paramagnetic quenching property in the sensing process of L towards Cu²⁺, resulting in a strong fluorescence enhancement of the complex L + Cu²⁺.

What is different, Fe³⁺ was also paramagnetic with an unfilled d shell and could strongly quench the emission of fluorophore near it through electron and/or energy transfer processes. Upon binding with Fe³⁺, the weak fluorescence of L was completely quenched which could be ascribed to the ligand–metal charge transfer mechanism (LMCT).^46,47 In the complex L + Fe³⁺, the paramagnetism and unfilled d shell of Fe³⁺ could prompt the fluorophore to open a nonradiative deactivation channel and facilitate the transfer of electron and/or energy, resulting in the fluorescence quenching response of L.^48–50 Although the C=N isomerization was probably also inhibited to some extent during the binding mode, the LMCT could be firstly and quickly occurred and the strength of paramagnetic quenching property of Fe³⁺ was much stronger that could cover the other mechanisms. Similarly, for the lighted complex L + Cu²⁺, upon addition of Fe³⁺, the Fe³⁺ could instantly displace Cu²⁺ from L + Cu²⁺, resulting in the formation of a new complex L + Fe³⁺. It was suggested that in this case, the paramagnetic quenching property of Fe³⁺ played the leading role, so L + Fe³⁺ showed a completely quenched fluorescence. Because of the rapidly response and rational control of multiple mechanisms, the lightened complex L + Cu²⁺ had a limit of detection for Fe³⁺ as low as 2.4 × 10⁻⁸ mol L⁻¹.

To understand the PET mechanism underlying the experimentally observed photophysical characteristics of L upon complexation with Cu²⁺, we had investigated the structural, electronic, and optical properties using ab initio density functional theory (DFT) combined with time-dependent density functional theory (TDDFT) calculations as implemented in the Gaussian 09 package. We had adopted the hybrid B3LYP exchange and correlation functional using an effective core potential with the LANL2DZ basis set for transition-metal Cu and the 6-31+G(d) basis set for all the other elements in the calculations. As shown in Fig. 11, in L, the electron densities of the highest occupied molecular orbital (HOMO) were distributed over the whole fluorophore moiety, while those of the lowest unoccupied molecular orbital (LUMO) transferred to carbonyl group and receptor moiety. Upon excitation of L, an electron would be transferred from the 4-oxo-4H-chromene-3-carbaldehyde unit to the fluorophore, resulting in the weak fluorescence, which was corresponded with the PET process. In contrast, after binding with Cu²⁺, the orbitals were localized on fluorophore for both HOMO and LUMO. Upon excitation, the PET process was inhibited and there was no electron transfer between the receptor and fluorophore, leading to the strong fluorescence of complex L + Cu²⁺.

Fig. 11 PET mechanism: HOMO and LUMO of L and L + Cu²⁺, calculated with DFT/TDDFT at the B3LYP/6-31G(d) level using Gaussian 09.

Application in water samples

In order to investigate the practicability of L, the amounts of Cu²⁺ and Fe³⁺ in tap water were determined by our proposed fluorescence assay method. As demonstrated in Table 1, Cu²⁺ added to water samples could be accurately measured with good recovery (93.1–104.3%) and the relative standard deviation (RSD) of five measurements was less than 1.6%, indicating that probe L has potential application for quantitative detection of Cu²⁺ in real water samples. Furthermore, as shown in Table 2, the concentration of Fe³⁺ added to the water samples could also be accurately determined (92.0–108.0%), and the data revealed a good agreement between the added and the found concentrations of Fe³⁺ (RSD < 1.8%), indicating that the complex of L + Cu²⁺ could be used for quantitative detection of Fe³⁺ in real water samples.

Table 1 Determination of the Cu²⁺ concentration in water samples

Sample	Cu^2+ added (mol L^−1)	Cu^2+ recovered (mol L^−1)	Recovery (%)	RSD (%)
1	1.0 × 10^−5	0.9 × 10^−5	93.1	0.84
2	3.0 × 10^−5	3.1 × 10^−5	104.3	0.83
3	5.0 × 10^−5	4.8 × 10^−5	96.8	1.55
4	7.0 × 10^−5	7.0 × 10^−5	100.4	0.39
5	10.0 × 10^−5	10.1 × 10^−5	101.1	0.98

---

Table 2 Determination of the Fe³⁺ concentration in water samples

Sample	Fe^3+ added (mol L^−1)	Fe^3+ recovered (mol L^−1)	Recovery (%)	RSD (%)
1	1.0 × 10^−5	0.9 × 10^−5	92.0	0.76
2	3.0 × 10^−5	3.2 × 10^−5	108.0	1.22
3	5.0 × 10^−5	5.0 × 10^−5	101.6	0.89
4	9.0 × 10^−5	8.9 × 10^−5	99.4	1.71

---

Conclusions

In summary, an ideal fluorescent sensor L was designed and synthesized based on benzimidazo[2,1-a]benz[de]isoquinoline-7-one-12-carbohydrazide. In acetonitrile–H₂O (9 : 1) solution (0.01 M HEPES buffer at pH 7.4), L showed great selectivity and sensitivity towards Cu²⁺ with fluorescence turn-on response based on the inhibition of C=N isomerization and PET process. Furthermore, in the same condition, L and the lighted complex L + Cu²⁺ could be used as new sensors for ultrasensitive detection of Fe³⁺ based on the paramagnetic quenching process with a limit of detection down to 2.4 × 10⁻⁸ mol L⁻¹. The sensing mechanisms were demonstrated by DFT and TDDFT calculations by Gaussian 09 package. Because of the rational control of multiple mechanisms, L could be used as excellent selective and sensitive sensor towards Cu²⁺ and Fe³⁺, especially for the dual detection of Fe³⁺. In addition, L was successfully applied to determine the accurate amounts of Cu²⁺ and Fe³⁺ in tap water. Series of similar sensors based on this sensing strategy are preparing in our lab, and our further efforts will be focused on the investigation of optical properties and practical applications of sensors.

Acknowledgements

The authors thank Henan Sanmenxia Aoke Chemical Industry Co., Ltd. (w0920) for financial support and thank Prof. Guangyou Zhang for the experimental assistance.

Notes and references

1. B. Valeur and I. Leray, Coord. Chem. Rev., 2000, 205, 3–40.
2. S. Dey, A. Roy, G. P. Maiti, S. K. Mandal, P. Banerjee and P. Roy, New J. Chem., 2016, 40, 1365–1376.
3. K. Boonkitpatarakul, J. Wang, N. Niamnont, B. Liu, L. Mcdonald, Y. Pang and M. Sukwattanasinitt, ACS Sens., 2016, 1, 144–150.
4. K. P. Carter, A. M. Young and A. E. Palmer, Chem. Rev., 2014, 114, 4564–4601.
5. B. Halliwell and J. M. Gutteridge, Biochem. J., 1984, 219, 1.
6. S. Hu, P. Furst and D. Hamer, New Biol., 1990, 2, 544.
7. Handbook of Metalloproteins, ed. A. Messerschmidt, R. Huber, T. Poulos and K. Weighardt, John Wiley & Sons, Inc., New York, 2001, vol. 2, p. 1149.
8. H. Tapiero, D. M. Townsend and K. D. Tew, Biomed. Pharmacother., 2003, 57, 386.
9. L. I. Bruijn, T. M. Miller and D. W. Cleveland, Annu. Rev. Neurosci., 2004, 27, 723.
10. K. J. Barnham, C. L. Masters and A. I. Bush, Nat. Rev. Drug Discovery, 2004, 3, 205.
11. D. R. Brown and H. Kozlowski, Dalton Trans., 2004, 1907.
12. K. C. Ko, J. S. Wu, H. J. Kim, P. S. Kwon, J. W. Kim, R. A. Bartsch, J. Y. Lee and J. S. Kim, Chem. Commun., 2011, 47, 3165.
13. A. K. Jain, V. K. Gupta, L. P. Singh and J. R. Raisoni, Talanta, 2005, 66, 1355.
14. A. Mokhir, A. Kiel, D. P. Herten and R. Kraemer, Inorg. Chem., 2005, 44, 5661.
15. J. Liu and Y. Lu, J. Am. Chem. Soc., 2007, 129, 9838.
16. P. G. Georgopoulos, A. Roy, M. J. Yonone-Lioy, R. E. Opiekun and P. J. Lioy, J. Toxicol. Environ. Health, Part B, 2001, 4, 341–394.
17. R. V. Rathod, S. Bera, M. Singh and D. Mondal, RSC Adv., 2016, 6, 34608–34615.
18. A. Torrado, G. K. Walkup and B. Imperiali, J. Am. Chem. Soc., 1998, 120, 609.
19. P. Grandini, F. Mancin, P. Tecilla, P. Scrimin and U. Tonellato, Angew. Chem., Int. Ed., 1999, 38, 3061.
20. G. Klein, D. Kaufmann, S. SchVrch and J. L. Reymond, Chem. Commun., 2001, 561–562.
21. M. Boiocchi, L. Fabbrizzi, M. Licchelli, D. Sacchi, M. Vázquez and C. Zampa, Chem. Commun., 2003, 1812.
22. Y. Zheng, J. Orbulescu, X. Ji, F. M. Andreopoulos, S. M. Pham and R. M. Leblanc, J. Am. Chem. Soc., 2003, 125, 2680.
23. C. Roy, B. Chandra, D. Hromas and S. Mallik, Org. Lett., 2003, 5, 11.
24. S. H. Kim, J. S. Kim, S. M. Park and S. K. Chang, Org. Lett., 2006, 8, 371.
25. Q. Y. Wu and E. V. Anslyn, J. Am. Chem. Soc., 2004, 126, 14682.
26. J. V. Mello and N. S. Finney, J. Am. Chem. Soc., 2005, 127, 10124.
27. E. W. Miller, A. E. Albers, A. Pralle, E. Y. Isaco and C. J. Chang, J. Am. Chem. Soc., 2005, 127, 16652.
28. D. Y. Sasaki, D. R. Shnek, D. W. Pack and F. H. Arnold, Angew. Chem., Int. Ed., 1995, 34, 905.
29. R. Kramer, Angew. Chem., Int. Ed., 1998, 37, 772.
30. M. Royzen, Z. H. Dai and J. W. Canary, J. Am. Chem. Soc., 2005, 127, 1612.
31. B. D'Autreaux, N. P. Tucker, R. Dixon and S. Spiro, Nature, 2005, 437, 769–772.
32. J. W. Lee and J. D. Helmann, Nature, 2006, 440, 363–367.
33. H. Weizman, O. Ardon, B. Mester, J. Libman, O. Dwir, Y. Hadar, Y. Chen and A. Shanzer, J. Am. Chem. Soc., 1996, 118, 12368–12375.
34. J. P. Sumner and R. Kopelman, Analyst, 2005, 130, 528–533.
35. R. M. Roat Malone, Bioinorganic Chemistry-A Short Course, John Wiley and Sons, New Jersey, NJ, 2007.
36. I. Bertini, H. B. Gray, E. I. Stiefel and J. S. Valentine, J. Biol. Inorg. Chem., 2007, 12, 1197–1206.
37. F. Ge, H. Ye, H. Zhang and B. X. Zhao, Dyes Pigm., 2013, 99, 661–665.
38. E. D. Weinberg, Science, 1974, 184, 952–956.
39. S. Sidhu, K. Kumari and M. Uppal, J. Hum. Ecol., 2007, 21, 265–267.
40. A. R. Nissenson and J. Strobos, Kidney Int., 1999, 55, S18–S21.
41. R. K. Schindhelm, M. Schoorl and J. V. Pelt, Diabetes Care, 2012, 35, 797–802.
42. M. J. Frisch, et al., Gaussian 09 (Revision D.01), Gaussian Inc., Pittsburg, PA, 2009.
43. Z. Liu, Y. Qi, C. Guo, Y. Zhao, X. Yang, M. Pei and G. Zhang, RSC Adv., 2014, 4, 56863–56869.
44. Z. Liu, C. Peng, Y. Wang, M. Pei and G. Zhang, Org. Biomol. Chem., 2016, 14, 4260–4266.
45. J. S. Wu, W. M. Liu, X. Q. Zhuang, F. Wang, P. F. Wang, S. L. Tao, X. H. Zhang, S. K. Wu and S. T. Lee, Org. Lett., 2007, 9, 33–36.
46. Y. Ma, W. Luo, P. J. Quinn, Z. Liu and R. C. Hider, J. Med. Chem., 2004, 47, 6349–6362.
47. R. Nudelman, O. Ardon, Y. Hadar, Y. Chen, J. Libman and A. Shanzer, J. Med. Chem., 1998, 41, 1671–1678.
48. L. Yang, W. Yang, D. Xu, Z. Zhang and A. Liu, Dyes Pigm., 2013, 97, 168–174.
49. J. H. Xu, Y. M. Hou, Q. J. Ma, X. F. Wu and X. J. Wei, Spectrochim. Acta, Part A, 2013, 112, 116–124.
50. Z. X. Li, L. F. Zhang, W. Y. Zhao, X. Y. Li, Y. K. Guo, M. M. Yu and J. X. Liu, Inorg. Chem. Commun., 2011, 14, 1656–1658.

---

Footnote

† Electronic supplementary information (ESI) available: NMR, MS spectra and calculation details. See DOI: 10.1039/c6ra09535f

---