Triphenylamine based lab-on-a-molecule for the highly selective and sensitive detection of Zn²⁺ and CN⁻ in aqueous solution

Abstract

Simple dual sensing of Zn²⁺ and CN⁻ was reported for the first time using the triphenylamine based lab-on-a-molecule TATP in both UV-Vis and fluorescence channels over other tested ions. Meanwhile, the sensing mechanism for both title ions was further studied using NMR titration and DFT calculations.

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The selective and sensitive detection of ions has emerged as an area of great interest in biological, chemical and environmental processes. However, most of the reported ion sensors are effective only in the selective recognition of one particular analyte due to specific structural properties. Therefore, the development of a lab-on-a-molecule^1–11 which has multiple recognition capability^12–15 in a competitive fashion is still a challenging task.

Among metal ions, Zn²⁺ is the second most abundant essential trace element in the human body.¹⁶ It plays an important mediation role in lots of physiological processes, including the regulation of gene expression and apoptosis, and acts as a co-factor in metalloenzyme catalysis and neurotransmission.¹⁷

On the other hand, cyanide is one of the most concerning anions due to its high toxicity^18–20 and reactivity. It is extensively produced in large quantities and applied in gold mining, plastic production and other industrial activities. Therefore, the detection of Zn²⁺ and CN⁻ are of particular concern in many areas. Although some probes have been reported for the detection of either Zn²⁺ (ref. 21–31 and 49–51) or CN⁻,^{32–41,52,53} to the best of our knowledge, a simple sensor that can detect both Zn²⁺ and CN⁻ competitively has rarely been reported.

As cyanide shows high activity for nucleophilic addition to double bonds, the easily constructed reaction site C=N was herein utilized for the recognition of cyanide. Further, the isomerization of C=N⁵⁴ could be used to detect metal ions via the CHEF effect.^42–44 Thus we designed a new probe TATP with well structured triphenylamine linked with C=N as the recognition receptor, which cooperated with a phenolic hydroxyl group to form another recognition site for metal ions. As a result, TATP demonstrated excellent sensing behaviour towards Zn²⁺ and CN⁻ among a series of cations and anions, compared with recently reported probes (Table 1).

Table 1 Comparison between our work and other reported probes in recent years

Probes for zinc ions			Probes for cyanide
Compound	LOD	Ref.	Compound	LOD	Ref.
TATP in our work	14 nM	—	TATP in our work	0.37 μM	—
	6.3 × 10^−11 M	22		2.8 × 10^−7 M	33
	7.25 × 10^−7 M	24		1.5 μM	34
	3.08 × 10^−7 M	24		0.478 μM	35
	0.01 μM	25		1.66 μM	35
	3.2 μM	49		3.2 × 10^−7 M	38
	4.6 μM	50		1.6 μM	52
	5.4 μM	51		37 nM	53

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As shown in Fig. 1, TATP could be readily prepared through the reduction of tris(4-nitrophenyl)amine, followed by aldimine condensation with salicylide. The structure of TATP was confirmed using NMR, mass spectroscopy and elementary analysis. A crystal of TATP (Fig. 2), suitable for single-crystal X-ray diffraction, was obtained through slow evaporation from a mixture solution (ethyl acetate : dichloromethane = 4 : 1, v/v) at room temperature, and displayed as a monoclinic crystal system with the space group P2₁/c.

Fig. 1 The synthetic route for the probe TATP.

Fig. 2 View of the crystal structure of TATP. Color code: C, gray; H, white; N, blue; O, red. The CCDC deposition number: 1476312.

Results and discussion

Selective cation sensing of Zn²⁺ using TATP

The UV-Vis and fluorescence properties of TATP were investigated in 0.1 M Tris-ClO₄ buffer solution (pH = 7.24, DMF : buffer = 1 : 2, v/v) by in the presence of the following various metal ions (as perchlorate salts): Ag⁺, Ba²⁺, Ca²⁺, Cd²⁺, Co²⁺, Cu²⁺, Hg²⁺, Ni²⁺, Pb²⁺ and Zn²⁺. Fig. S4a† shows that there was no significant variation in the absorption spectra of TATP for most of the added cations except for Zn²⁺ which induced a red shift from 412 nm to 425 nm.

From the emission properties of TATP (Fig. S4b†), it was shown to exhibit prominent fluorescence enhancement at 565 nm upon the addition of Zn²⁺, which could be attributed to the formation of a rigid system after binding with Zn²⁺, causing the chelation-enhanced fluorescence (CHEF) effect^42–44 that inhibits C=N isomerisation. This also can be verified through the small red shift of 13 nm in the UV-Vis spectra (Fig. S4a†) and the ¹H NMR titration experiment (Fig. S12†), in which the sharp peak of –OH becomes gentle, suggesting that the intramolecular hydrogen bond turns to an intermolecular hydrogen bond due to metal complex formation.

Consistent with the change in the fluorescence spectra, a solution of TATP underwent an immediate color change from colorless to yellow with Zn²⁺, indicating that TATP can serve as a ‘naked-eye’ Zn²⁺ indicator.

From the fluorescence titration profiles (Fig. 3a), the association constant for TATP–Zn²⁺ in 0.1 M Tris-ClO₄ buffer solution (pH = 7.24, DMF : buffer = 1 : 2, v/v) was determined as 1.68 × 10¹⁹ M⁻² using the Hill equation. A Job plot indicated a 1 : 3 stoichiometric complexation of TATP with Zn²⁺ (Fig. S5†). The detection limit using TATP for the analysis of the Zn²⁺ ion was determined as 14 nM. Compared to well-known Zn²⁺ sensors, TATP shows a highly selective and sensitive response to Zn²⁺ over other various cations with a nanomolar detection limit.

Fig. 3 Fluorescence properties (λ_ex = 350 nm) of TATP (10 μM) (a) upon the addition of increasing concentrations of Zn²⁺ and (b) with Zn²⁺ in the presence of other competitive metal ions in 0.1 M Tris-ClO₄ buffer solution (pH = 7.24, DMF : buffer = 1 : 2, v/v). Inset: The emission intensity of TATP at 565 nm as a function of Zn²⁺ concentration. [Xⁿ⁺ = Ag⁺ (1), Ba²⁺ (2), Ca²⁺ (3), Cd²⁺ (4), Co²⁺ (5), Cu²⁺ (6), Hg²⁺ (7), Ni²⁺ (8), Pb²⁺ (9), and Zn²⁺ (10)].

Further, the selectivity toward Zn²⁺ was ascertained through competition experiments. As shown in Fig. 3b, TATP was treated with 50 eq. of Zn²⁺ in the presence of other metal ions of the same concentration and no significant changes in the fluorescence intensities or emission wavelengths was found in the presence of other metal ions, indicating that the TATP–Zn²⁺ system was hardly affected by these coexistent metal ions. Thus, TATP can be used as a selective and sensitive fluorescence sensor for Zn²⁺ determination.

Selective anion sensing of CN⁻ using TATP

The UV-Vis and fluorescence spectra (Fig. S6a†) of TATP were investigated in 0.1 M Tris-ClO₄ buffer solution (pH = 7.24, DMF : buffer = 1 : 2, v/v) in the presence of the following various anions (as n-tetrabutylammonium salts): F⁻, AcO⁻, Cl⁻, Br⁻, I⁻, H₂PO₄⁻, PF₆⁻ and CN⁻. The selective response in the absorbance of TATP at 414 nm decreased and a new band at 512 nm emerged only in the presence of CN⁻, along with a solution color change from colorless to brown (Fig. S6b† inset), indicating an intramolecular charge transfer (ICT) mechanism^45–47 from the clear isosbestic point at 461 nm between TATP and CN⁻ upon the addition of excessive cyanide (Fig. S7†). Meanwhile, the strong emission quenching effect at 565 nm was only observed upon the addition of CN⁻ (Fig. S6b†).

The recognition properties of TATP for CN⁻ were further studied using absorption titration (Fig. S7†) and fluorescence titration (Fig. 4a) experiments. As shown in Fig. 4a, continuous emission quenching at 379 nm was observed after the addition of CN⁻. The Job plot for the binding between TATP and CN⁻ exhibited 1 : 3 stoichiometry (Fig. S9†), as depicted in Fig. 5. From the fluorescence titration data, the apparent association constant for TATP and CN⁻ was determined as 1.7 × 10³ using a nonlinear-fitting analysis of the change in the absorbance at 565 nm (R² = 0.994),⁴⁸ and the detection limit of TATP for the analysis of CN⁻ was 5 μM based on the fluorescence titration experiments, and 0.37 μM based on UV-Vis absorption, which is lower than the WHO drinking water standard of 1.9 μM.^34,38

Fig. 4 Fluorescence properties (λ_ex = 350 nm) of TATP (10 μM) (a) in the presence of increasing concentrations of CN⁻ and (b) with CN⁻ in the presence of other competitive anions in 0.1 M Tris-ClO₄ buffer solution (pH = 7.24, DMF : buffer = 1 : 2, v/v). Inset: The fluorescence intensity of TATP at 565 nm as a function of [CN⁻]. [Xⁿ⁻ = F⁻ (1), AcO⁻ (2), Cl⁻ (3), Br⁻ (4), I⁻ (5), H₂PO₄⁻ (6), and PF₆⁻ (7)].

Fig. 5 The proposed sensing mechanism of TATP.

The selectivity toward CN⁻ was further ascertained using competition experiments. As shown in Fig. 4b, TATP was treated with 50 equivalents of CN⁻ in the presence of other anions of the same concentration. Relatively low interference was observed in the detection of CN⁻ in the presence of other anions. Thus TATP can be used as a selective fluorescent sensor toward CN⁻ in the presence of most competing anions.

To make certain the sensing mechanism of TATP for CN⁻, ¹H NMR titration experiments were performed as shown in Fig. S13.† The peak of –OH at 13.20 ppm disappeared rapidly after cyanide addition due to the deprotonation effect. Meanwhile, the peak of –N=CH– at 9.00 ppm weakened and a new peak at around 10.20 ppm was raised which was considered to be –C–NH–, further proving the cyanide addition mechanism and thus demonstrating the destruction of the conjugation structure to cause the fluorescence quenching.

To get an insight into the electronic structures of TATP, TATP–CN⁻ and TATP–Zn²⁺, DFT calculations were performed using the B3LYP/6-31G(d,p) basis set to understand the intermolecular interactions between the probe TATP and the ions (Fig. 6). The highest occupied molecular orbital (HOMO) and lowest unoccupied molecular orbital (LUMO) diagrams of TATP, TATP + CN⁻ and TATP + Zn²⁺ indicate that internal charge transfer occurs during the anion recognition process.

Fig. 6 HOMO and LUMO diagrams of (a) TATP, (b) TATP + 3CN⁻ and (c) TATP + 3Zn²⁺ in the gas phase.

In the case of TATP, the main molecular orbital (MO) contribution of the first lowest excited state was determined for the HOMO → LUMO transition, which was assigned to be an ICT band. In contrast, the cyanide addition and zinc binding in TATP caused a decrease in the HOMO-to-LUMO energy gap, which substantially resulted in a bathochromic shift, which supports the absorbance at longer wavelengths.

Finally, cellular imaging of TATP in the presence of the target ions was also carried out (Fig. 7). It could be found that TATP was aggregated in a quasi-water matrix instead of being taken into cells, which weakened its reaction with the ions. Nevertheless, a slight emission enhancement could be seen in the presence of zinc and cyanide ions. Compared with the results observed in solution, the ion recognition performance of the probe TATP was not so obvious because its water solubility is not good enough to perform biological experiments.

Fig. 7 Cellular imaging of TATP.

Experimental

Synthesis

Synthesis of tris(4-aminophenyl)amine. Pd/C (10 wt%, 0.15 g) was added to a solution of tris(4-nitrophenyl)amine (1.05 g, 2.76 mmol) in EtOH (40 mL), followed by H₂NNH₂·H₂O (5.30 mL). The mixture was heated at reflux for 20 h. After immediate filtration through a Celit pad, the filtrate was cooled to below 0 °C. Light yellow crystals were obtained upon recrystallization from the filtrate above (0.73 g, 91.2%). IR (KBr) ν: 3405, 3335, 1619, 1503, 1328, 1260, 829, 723, 569, 511 cm⁻¹; ¹H-NMR (DMSO-d₆, 400 MHz, ppm) δ: 8.48 (d, 6H), 8.47 (d, 6H), 4.73 (s, 6H); ¹³C-NMR (DMSO-d₆, 400 MHz, ppm) δ: 115.65, 124.96, 139.65, 144.12. MS m/z: 291.16 [M + 1]⁺.

Synthesis of TATP. Tris(4-aminophenyl)amine (500 mg, 1.72 mmol) in EtOH (50 mL) was added to a solution of salicylaldehyde (0.90 mL). The solution was stirred under reflux conditions for 5 h in the presence of 2–3 drops of acetic acid. The precipitate was filtered, washed with cold absolute ethanol three times, and then recrystallized using ethyl acetate/chloroform (4/1, v/v) to get orange crystals, with a yield of 0.85 g (81.7%). IR (KBr) ν: 3420, 1615, 1490, 1320, 1182, 829, 756 cm⁻¹; ¹H-NMR (DMSO-d₆, 400 MHz, ppm) δ: 13.20 (s, 3H), 9.00 (s, 3H), 7.64 (m, 3H), 7.45 (d, 6H), 7.40 (m, 3H), 7.16 (d, 6H), 6.99 (m, 3H), 6.96 (m, 3H); ¹³C-NMR (DMSO-d₆, 400 MHz, ppm) δ: 116.54, 119.11, 119.41, 122.81, 124.54, 132.40, 133.00, 142.94, 145.76, 160.21, 162.00. MS m/z: 603.2 [M + 1]⁺. Anal., calcd for C₃₉H₃₀O₃N₄: C, 77.72; H, 5.02; N, 9.30. Found: C, 77.49; H, 5.11; N, 9.18.

Theoretical studies

The structural optimization and theoretical UV-Vis spectra of the probe TATP, TATP + Zn²⁺ and TATP + CN⁻ were calculated using the computer program Gaussian 09W, by applying density functional theory (DFT) and time dependent DFT (TD-DFT) methods with a hybrid functional B3LYP using the basis sets 6-31G(d,p).

Cellular imaging

The potential for cellular imaging with TATP was tested through the use of MCF-7 cancer cells. 2.0 mL of MCF-7 cells in DMEM medium at an initial density of 4 × 10⁴ cell per mL were seeded in each dish and cultured at 37 °C for 24 h under a humidified atmosphere containing 5% CO₂. A dispersion of TATP was prepared in DMEM medium (with 1% DMF) at a concentration of 10 μg mL⁻¹ (first dissolved in DMF at 1 mg mL⁻¹ and then diluted with DMEM medium to the target concentration). Cells were cultured with the dispersion of TATP for 6 h, then washed three times with PBS to remove the free materials, then cultured with a solution of Zn²⁺ (15 μg mL⁻¹) or CN⁻ (100 μg mL⁻¹) for 30 min, and then washed three times with PBS to remove the free materials. Finally, the samples were observed using confocal laser fluorescence microscopy (TCS SP5II, Leica, Germany).

Conclusions

In summary, we have successfully designed and synthesized a simple lab-on-a-molecule TATP probe, capable of sensing both cations and anions in aqueous solution. TATP exhibited excellent selectivity and sensitivity towards Zn²⁺ and CN⁻ through inducing instantaneous solution color change, visible by the naked eye, and dramatic emissive responses, even in the presence of other ions. The binding model between the host and guests was demonstrated using Job's plot, NMR titration and DFT calculations, suggesting that the use of TATP to sense Zn²⁺ and CN⁻ was achieved through metal–ligand coordination and nucleophilic attack mechanisms, respectively. The detection limits for Zn²⁺ and CN⁻ using TATP were determined as 14 nm and 0.37 μM, respectively. On the basis of the results, we believe that the receptor TATP will offer important guidance in the development of a single receptor for recognizing both cations and anions in aqueous solution.

Acknowledgements

We acknowledge financial support from the National Natural Science Foundation of China (21507005) and the Excellent Young Scholar Research Fund of Beijing Institute of Technology of China (3090012331542). We also acknowledge the assistance in the cell imaging tests from Ms Shan Sun of the Ningbo Institute of Industrial Technology, Chinese Academy of Sciences.

Notes and references

1. K. Chen, Q. Shu and M. Schmittel, Chem. Soc. Rev., 2015, 44, 136–160.
2. M. Schmittel and Q. Shu, Chem. Commun., 2012, 48, 2707–2709.
3. K. Chen and M. Schmittel, Analyst, 2013, 138, 6742–6745.
4. K. Chen and M. Schmittel, Chem. Commun., 2014, 50, 5756–5759.
5. D. C. Magri, G. J. Brown, G. D. Mcclean and A. Prasanna de Silva, J. Am. Chem. Soc., 2006, 128, 4950.
6. D. C. Magri, M. C. Fava and C. J. Mallia, Chem. Commun., 2014, 50, 1009–1011.
7. K. Chen, J. W. Bats and M. Schmittel, Inorg. Chem., 2013, 52, 12863–12865.
8. Q. Shu, B. Lars and S. Michael, Aqueous Solution, Inorg. Chem., 2012, 51, 13123–13127.
9. B. Shan, Y. Liu, R. Shi, S. Jin, L. Li, S. Chen and Q. Shu, RSC Adv., 2015, 5, 96665–96669.
10. L. Wan, Q. Shu, J. Zhu, S. Jin, N. Li, X. Chen and S. Chen, Talanta, 2016, 152, 39–44.
11. S. A. Lee, G. R. You, Y. W. Choi, H. Y. Jo, A. R. Kim, I. Noh, S. J. Kim, Y. Kim and C. Kim, Dalton Trans., 2014, 43, 6650–6659.
12. M. Schmittel and H. W. Lin, Angew. Chem., Int. Ed., 2007, 46, 893–896.
13. L. Liu and H. Lin, Anal. Chem., 2014, 86, 8829–8834.
14. K. Jiang, S. Sun, L. Zhang, Y. Wang, C. Cai and H. Lin, ACS Appl. Mater. Interfaces, 2015, 7, 23231–23238.
15. Y. Leng, S. Qian, Y. Wang, C. Lu, X. Ji, Z. Lu and H. Lin, Sci. Rep., 2016, 6, 25354–25362.
16. Y. Huang, Q. Lin, J. Wu and N. Fu, Dyes Pigm., 2013, 99, 699–704.
17. H. Wang, C. L. Sun, Y. H. Yue, F. F. Yin, J. Q. Jiang, H. R. Wu and H.-L. Zhang, Analyst, 2013, 138, 5576–5579.
18. B. Vennesland, E. E. Comm, C. J. Knownles, J. Westly and F. Wissing, Cyanide in Biology, Academic Press, London, 1981.
19. J. Y. Noh, I. H. Hwang, H. Kim, E. J. Song, K. B. Kim and C. Kim, Bull. Korean Chem. Soc., 2013, 34, 1985–1989.
20. G. J. Park, I. H. Hwang, E. J. Song, H. Kim and C. Kim, Tetrahedron, 2014, 70, 2822–2828.
21. C. Alberto Huerta-Aguilar, T. Pandiyan, P. Raj, N. Singh and R. Zanella, Sens. Actuators, B, 2016, 223, 59–67.
22. A. A. A. Aziz, S. H. Seda and S. F. Mohammed, Sens. Actuators, B, 2016, 223, 566–575.
23. R. Bhowmick, R. Alam, T. Mistri, K. K. Das, A. Katarkar, K. Chaudhuri and M. Ali, RSC Adv., 2016, 6, 11388–11399.
24. A. Jimenez-Sanchez, B. Ortiz, V. Ortiz Navarrete, N. Farfan and R. Santillan, Analyst, 2015, 140, 6031–6039.
25. G. J. Park, J. J. Lee, G. R. You, L. Nguyen, I. Noh and C. Kim, Sens. Actuators, B, 2016, 223, 509–519.
26. C. Patra, A. K. Bhanja, C. Sen, D. Ojha, D. Chattopadhyay, A. Mahapatra and C. Sinha, Sens. Actuators, B, 2016, 228, 287–294.
27. K. Ponnuvel, M. Kumar and V. Padmini, Sens. Actuators, B, 2016, 227, 242–247.
28. F. Qian, C. Zhang, Y. Zhang, W. He, X. Gao, P. Hu and Z. Guo, J. Am. Chem. Soc., 2009, 131, 1460–1468.
29. J. C. Qin, L. Fan and Z. Y. Yang, Sens. Actuators, B, 2016, 228, 156–161.
30. Y. Jin, S. Wang, Y. Zhang and B. Song, Sens. Actuators, B, 2015, 225, 167–173.
31. N. Khairnar, K. Tayade, S. K. Sahoo, B. Bondhopahyay, A. Basu, J. Singh, N. Singh, V. Gite and A. Kuwar, Dalton Trans., 2015, 44, 2097–2102.
32. E. G. Langeroudi, G. F. Petre, A. Azizi, E. Proulx and F. Larachi, J. Sep. Sci., 2011, 34, 1568–1573.
33. J. Li, W. Wei, X. Qi, G. Zuo, J. Fang and W. Dong, Sens. Actuators, B, 2016, 228, 330–334.
34. A. K. Mahapatra, K. Maiti, R. Maji, S. K. Manna, S. Mondal, S. S. Ali and S. Manna, RSC Adv., 2015, 5, 24274–24280.
35. N. Maurya, S. Bhardwaj and A. K. Singh, Sens. Actuators, B, 2016, 229, 483–491.
36. T. Noipa, T. Tuntulani and W. Ngeontae, Talanta, 2013, 105, 320–326.
37. L. Shang, L. Zhang and S. Dong, Analyst, 2009, 134, 107–113.
38. B. Shi, P. Zhang, T. Wei, H. Yao, Q. Lin and Y. Zhang, Chem. Commun., 2013, 49, 7812–7814.
39. Y. Singh and T. Ghosh, Talanta, 2016, 148, 257–263.
40. L. Wang, G. Fang and D. Cao, Sens. Actuators, B, 2015, 221, 63–74.
41. G. R. You, G. J. Park, S. A. Lee, Y. W. Choi, Y. S. Kim, J. J. Lee and C. Kim, Sens. Actuators, B, 2014, 202, 645–655.
42. M. Shellaiah, Y. H. Wu and H. C. Lin, Analyst, 2013, 138, 2931–2942.
43. J. C. Qin, T. R. Li, B. D. Wang, Z. Y. Yang and L. Fan, Spectrochim. Acta, Part A, 2014, 133, 38–43.
44. S. Kim, J. Y. Noh, K. Y. Kim, J. H. Kim, H. K. Kang, S. W. Nam, S. H. Kim, S. Park, C. Kim and J. Kim, Inorg. Chem., 2012, 51, 3597–3602.
45. N. Kumari, S. Jha and S. Bhattacharya, J. Org. Chem., 2011, 76, 8215–8222.
46. P. Xie, F. Guo, S. Yang, D. Yao, G. Yang and L. Xie, J. Fluoresc., 2013, 24, 473–480.
47. L. Li, F. Liu and H. W. Li, Spectrochim. Acta, Part A, 2011, 79, 1688–1692.
48. P. Thordarson, Chem. Soc. Rev., 2011, 40, 1305–1323.
49. S. Goswami, A. K. Das and K. Aich, Analyst, 2013, 138, 4593–4598.
50. S. Goswami, A. K. Das and B. Pankhira, Dalton Trans., 2014, 43, 12689–12697.
51. S. Goswami, S. Maity and A. C. Maity, Tetrahedron Lett., 2014, 55, 5993–5997.
52. S. Goswami, A. Manna and S. Paul, Chem. Commun., 2013, 49, 2912–2914.
53. S. Goswami, A. Manna and S. Paul, Tetrahedron Lett., 2013, 54, 1785–1789.
54. J. Wu, W. Liu and J. Ge, Chem. Soc. Rev., 2011, 40, 3483–3495.

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Footnotes

† Electronic supplementary information (ESI) available. CCDC 1476312. For ESI and crystallographic data in CIF or other electronic format see DOI: 10.1039/c6ra17354c

‡ These authors contributed equally.

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