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In this paper, BT ((E)-2-(4-(4-(bis(pyridin-2-ylmethyl)amino)styryl)-3-cyano-5,5-dimethylfuran-2(5H)-ylidene)malononitrile) with strong donor–π-acceptor structure was synthesized, which showed both colorimetric and fluorescent sensing ability toward Cu2+ with high selectivity and sensitivity. Job plot and mass spectra measurement revealed a 1:1 coordination mode between Cu2+ and probe BT in ethanol/HEPES (1:4 v/v) buffer (pH 7.2) solution, and the binding constant was calculated to be 3.6×104M–1. The colour of BT solution (10µM) immediately turned from purple red to yellow and the red fluorescence was quenched obviously when a certain amount of Cu2+ was added, which enabled a dual-channel detection of Cu2+. A paper strip pre-stained with BT solution was further fabricated and it also showed excellent sensing ability toward Cu2+ with a detection limit as low as 10−6M with the naked eye, which represents better portability and operation simplicity that is favourable for on-site analysis of Cu2+ in water.
Copper (Cu), as the third-most abundant transition metal after Fe and Zn in the human body, has important physiological roles in many biological systems [1–3]. However, overloading of Cu in organisms can cause serious danger, resulting in not only liver and kidney damage, but also severe oxidative stress and neurodegenerative diseases [4–10]. Considering this issue, the maximum permissible level of Cu2+ in drinking water has been determined to be 20µM by US Environmental Protection Agency . Therefore, it is essential to provide an appropriate method for recognition and determination of Cu2+ in water. Some colorimetric and fluorescent Cu2+ probes have already been reported with high sensitivity and selectivity [12–20]; however, most of them are operated in solution, which is not convenient for on-site analysis. On the other hand, solid-state sensors have become numerous in the last several decades, such as dipstick and lateral-flow assays, which are based on the blotting of analytes onto a paper pre-stained with probes [21,22]. The best-known example is the pH strip which is widely used to enable quick colorimetric response to different pH solutions. These formats have gained great popularity due to their feasible readout, good portability and operation simplicity. As a result, developing a Cu2+ strip, which can easily respond to different Cu2+ concentrations is really meaningful and valuable. It enables detection of Cu2+ by the naked eye with only a paper substrate.
Bis(2-pyridylmethyl)amine (BPA) moiety had been used as a binding group to Cu2+ with good selectivity by Tian et al.  and Qin et al.  where their purposes were to detect pyrophosphate anion using the complex BPA–Cu2+. Because of its paramagnetic nature, Cu2+ usually leads to fluorescence quenching of the bonded fluorophore, resulting in fluorescent signal ‘turn-off’. Meanwhile, the electron-donating ability of the N atom in amine is seriously weakened when coordinated to Cu2+, leading to reduced intramolecular charge transfer (ICT) effect. As a result, the absorption spectrum of the molecule will be altered and enable colorimetric sensing of Cu2+. 2-(3-Cyano-4,5,5-trimethylfuran-2(5H)-ylidene)propanedinitrile (TCF), a well-known strong electron acceptor, can easily generate rather narrow band gap when connected to a strong electron donor with good conjugation, leading to long wavelength absorption and emission which is favourable for chemosensing due to reduced background interference. BT ((E)-2-(4-(4-(bis(pyridin-2-ylmethyl)amino)styryl)-3-cyano-5,5-dimethylfuran-2(5H)-ylidene)malononitrile) constructed by BPA and TCF moieties has been used as probe for Ni2+ in pure organic solvent CH3CN . In this paper, we found new applications of BT that showed selective response to Cu2+ both in ethanol–water solution and on a paper strip (figure 1). When coordinated with Cu2+, the electron-donating ability of aniline in BPA unit is decreased, so the push–pull character of the dye is weakened, resulting in blue-shift of the absorption spectra, which enables the colorimetric sensing of Cu2+ with the naked eye. At the same time, Cu2+ leads to fluorescence quenching of the probe, which made BT a fluorescence ‘turn-off’ sensor for Cu2+. We further loaded BT to a common filter paper to fabricate a Cu2+ strip, and successfully realized the detection of Cu2+ in the form of a paper probe.
All reagents and solvents were commercially purchased, and the solvents were used after appropriate distillation or purification. The intermediates BPA, 4-(bis(pyridin-2-ylmethyl)amino)benzaldehyde (BPA-CHO) and TCF were synthesized according to the literature [26–28]. Stock solutions of compound BT (1mM) were prepared in dimethylsulfoxide, then diluted to 10µM in ethanol/HEPES (1:4 v/v) buffer (pH 7.2). All solvents used in the test were chromatographically pure. UV–visible absorption spectra were recorded on a Schimadzu 160A spectrophotometer. Fluorescence spectra were recorded on a Hitachi F-4500 spectrometer. The pH measurements were made with a Sartorius basic pH-meter PB-10. 1H NMR spectra were recorded on Bruker Ascend 400MHz spectrometers, and 13C NMR spectra were recorded on 100MHz spectrometers. Mass spectra were recorded on an Ion Spec 4.7T FTMS instrument.
The synthesis route of BT is shown in scheme 1. BPA-CHO (0.15g, 0.50mmol), TCF (0.11g, 0.55mmol) and ammonium acetate (0.046g, 0.60mmol) were stirred overnight in the dark under argon at room temperature in a mixture of ethanol (1ml) and dichloromethane (1ml). The solution rapidly turned from pale yellow to dark red. The mixture was diluted in water, extracted with dichloromethane and dried over anhydrous NaSO4. Then the solvent was removed under reduced pressure. The desired residue was purified by column chromatography on silica gel using EtOAc/petroleum ether (1/2, v/v) as the mobile phase to afford compound BT as amorphous black solid (0.21g, 87%). 1H NMR (400MHz, CDCl3) δ [ppm]: 8.62 (d, J=4Hz, 2H), 7.67 (t, J=8Hz, 2H), 7.58 (d, J=16Hz, 1H), 7.49 (d, J=12Hz, 2H), 7.27–7.20 (m, 4H), 6.82 (d, J=12Hz, 2H), 6.74 (d, J=16Hz, 1H), 4.94 (s, 4H), 1.73 (s, 6H). 13C NMR (100MHz, CDCl3) δ [ppm]: 176.14, 174.28, 156.82, 152.75, 150.10, 148.04, 137.10, 132.11, 123.14, 122.69, 120.84, 113.27, 112.50, 111.70, 111.19, 109.74, 97.02, 95.38, 57.24, 55.27, 26.69. ESI-HRMS: [M+H]+, [C30H25N6O]+, calcd, m/z=485.20; found, m/z=485.23.
Solution of BT in ethanol/HEPES buffer was carefully prepared with a concentration of 10µM. Owing to the ICT effect from the electron donor (BPA) to the electron acceptor (TCF), BT covered a wide absorption range (figure 2a). The absorption maximum was at 558nm which made BT solution show light purple red colour. The fluorescence spectrum revealed a maximum emission at 636nm, which is red fluorescence owing to its low bandgap. And the fluorescence quantum yield of BT in ethanol/HEPES (1:4 v/v) buffer (pH 7.2) was calculated to be 0.13 using rhodamine B as a reference. To test the selectivity of BT to Cu2+ ions, 16 kinds of other metal ions including Ag+, Al3+, Ba2+, Ca2+, Cd2+, Cr3+, Fe2+, Fe3+, Hg2+, K+, Mg2+, Mn2+, Na+, Ni2+, Pb2+ and Zn2+ were chosen as contrast. Five equivalents of metal ions were added separately to BT solution, then the absorption and emission spectra were measured immediately and the results are shown in figure 2. Among the metal ions studied, only Cu2+ could change the spectra obviously with absorption maximum blue-shifted by 141nm to 417nm with sharp contrast. The colour change could be observed obviously by the naked eye (figure 2b inset). The phenomenon was ascribed to the decreased ICT effect between BPA unit and TCF unit, owing to the strongly weakened electron-donating ability of aniline N atom in BPA upon coordination to Cu2+. Although Fe3+ can make the absorption peak blue-shifted by 31nm, the colour change of solution was not so obvious in that it cannot be clearly distinguished by the naked eye. BT solution with Ni2+ showed a weak shoulder band at 417nm; however, the absorption maximum was still at 558nm and the colour of solution was not changed. These results clearly demonstrated that BT was highly selective towards Cu2+ in colorimetric method. The fluorescence changes of BT solution were also determined after addition of metal ions. As shown in figure 2c, only Cu2+ could quench the fluorescence emission of BT efficiently with fluorescence intensity decreased by 15 times. Other metal ions did not affect the fluorescence obviously, except that Ag+ showed certain disturbance. There may be some interactions between pyridine unit and Ag+, thus photon-induced electron transfer may occur and affect the fluorescence emission of BT. The influence of Ag+ or other metal ions except Cu2+ on fluorescence of BT is not so obvious that it cannot be perceived by the naked eye when the solution was irradiated by a UV lamp with light of 365nm as shown in figure 2d. Therefore, BT could also act as a fluorescence turn-off probe for Cu2+ with good selectivity.
Competition experiment was performed to further confirm the selectivity of BT toward Cu2+. As shown in figure 3a, although Fe3+ can make the absorption peak slightly blue-shift and Ni2+ showed some background absorbance, they still did not affect the colorimetric recognition ability of the probe to Cu2+. As shown in figure 3b, Cu2+ can quench the fluorescence of BT solution efficiently even if the other metal ions are present. And it is interesting to note that the coexistence of most of the metal ions except Fe3+ strengthened the recognition ability toward Cu2+, as the change in fluorescence intensity was enhanced. In general, the BT probe demonstrated good anti-interference ability when detecting Cu2+ in both colorimetric and fluorescent modes.
As the selectivity of the probe has been confirmed, to further understand the recognition nature of BT to Cu2+ ions, we investigated the coordination mode between them. The stoichiometry for the binding between BT and Cu2+ was studied by Job's plot. We maintained the total concentration (C0) of BT and Cu2+ unchanged, then altered the Cu2+ content () and recorded the absorption spectrum of each solution. The curve of absorbance at 417nm versus Cu2+ content is depicted in figure 4a. It revealed that BT–Cu2+ complex reached the maximum absorbance when the Cu2+ content was 0.5, indicating that there was most amount of BT–Cu2+ complex in solution at this ratio. In other words, BT coordinated to Cu2+ with 1:1 stoichiometry in ethanol/HEPES buffer (pH 7.2) solution. This coordination mode was further confirmed by mass spectrum of the mixed solution of BT and Cu2+ as shown in figure 4b, where the molecular ion peak of [BT–Cu2+] could be found (calcd: 547.1297; found: 547.1299).
The titration experiment was performed by adding various amounts of Cu2+ to BT solution, and both the absorption and fluorescence spectra were recorded. As shown in figure 5a, in the UV–visible spectra, the absorbance at 558nm decreased, while that at 417nm increased with the addition of Cu2+, revealing the formation of new complex between BT and Cu2+. From the normalized absorption signal response to the concentration of Cu2+ (figure 5b), the detection limit of colorimetric method was calculated to be 2.4×10−7M . In the fluorescence spectra, as shown in figure 5c, the fluorescence intensity decreased gradually with the addition of Cu2+, and the curve became smoother when more than 2 equivalents of Cu2+ was added. The fluorescence intensity was linearly related to the concentration of Cu2+ from 1 to 10µM, and the fluorescence detection limit was 1.02×10−7M that was calculated on the basis of 3σ/k (σ is the standard deviation of the blank measurement and k is the slope of a plot of the fluorescence intensity versus Cu2+ concentration). The association constant of BT–Cu2+ was calculated to be 3.6×104M−1 using Benesi–Hildebrand analysis  (see electronic supplementary material).
The performance of probe BT was tested in various pH environments. The fluorescence signal of BT solution before (F0) and after (F) addition of two equivalents of Cu2+ was separately collected. As shown in figure 6, the ratio of F0/F demonstrated obvious contrast with value more than 5 in the pH range of 5.0–8.5, manifesting that the probe can work well in this range. Too acidic an environment may protonate the aniline N and pyridine moiety which reduces the coordination ability of BPA group, and too basic an environment may reduce the free Cu2+ ions in solution and influence the sensitivity.
Probe BT showed high selectivity and sensitivity to Cu2+ in both colorimetric and fluorescent modes; however, it is not convenient enough to apply the probe in the form of solution for on-site analysis. If BT could be printed to a paper substrate to fabricate Cu2+ strip, the probe can demonstrate better portability and operation simplicity like the pH strip. Cu2+ strip was prepared by immersing a piece of tailed filter paper into BT solution (1mM) in acetone for one minute, and then the paper was dried in air. For testing the performance of the strips, they were separately immersed into various concentrations of Cu2+ each for only one second. Obviously, the colour change could be observed by the naked eye immediately even if the concentration of Cu2+ was as low as 1×10−5M (figure 7a). Fluorescence signal was also collected with the strip irradiated by a UV lamp, and the detection limit of this method can reach a level of 1×10−6M (figure 7b). Different Cu2+ concentrations could give different signal changes in both colorimetric and fluorescence modes using this paper strip, revealing that the Cu2+ strip prepared here could work well.
A BT probe for the detection of Cu2+ constructed by BPA and TCF moieties was successfully synthesized with a wide absorption range and red emission. It showed high selectivity and sensitivity toward Cu2+ in ethanol/HEPES (1:4 v/v) buffer (pH 7.2) solution in both colorimetric and fluorescent modes. A paper strip was further fabricated easily by dipping common filter paper into BT solution, and the test results also showed good recognition ability to Cu2+, which makes the BT probe more portable and convenient. As a result, real-time and naked eye detection of Cu2+ ion could be realized by using a Cu2+ strip like the pH strip, which is useful in environment monitoring and water analysis.
The authors gratefully acknowledge the assistance of Dr Zhengran Yi from Huazhong University of Science and Technology with mass spectrometric measurement.
The synthesis of intermediates and some measurement information are presented in the electronic supplementary material of this article.
D.C. conceived of the study, designed the study, carried out the molecular synthesis and drafted the manuscript; P.C., L.Z. and Y.S. participated in data analysis and the design of the study; G.L. and X.Y. carried out the statistical analyses; J.Q. coordinated the study and helped draft the manuscript. All authors gave final approval for publication.
We declare we have no competing interests.
This work was supported by the National Natural Science Foundation of China (no. 51504168), Natural Science Foundation of Hubei Province (no. 2015CFB230), and Science Foundation of Wuhan Institute of Technology (no. K201754).