Determination of nitrite in real food and water samples by a novel terbium-macrocycle complex

Abstract

A novel terbium-macrocycle complex (Tb-Ac) was designed and synthesized with a characteristic luminescence in aqueous solution. It showed excellent selectivity and sensitivity towards NO₂⁻ in real food and water samples, as well as living cells, in terms of reliable accuracy and practicability.

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Nitrites are widely found in the environment, foods and physiological systems.¹ They are used in our daily diets as a food additive, mainly as a synthetic preservative and antimicrobial agent. However, a number of studies have demonstrated that excessive nitrite intake could cause a series of physiological issues, such as acute nitrite poisoning, abortion, fetal intrauterine growth retardation, congenital central nervous system slowness, methemoglobinemia.^2–5 Moreover, they also showed a strong correlation to a high risk of cancers when their concentrations were higher than 4.5 mg mL⁻¹.⁶ Consequently, rapid and sensitive determination of nitrite levels is essential for monitoring human health in many aspects, such as in drinking water and daily foods, as well as clinical diagnosis.^7,8

To this end, many analytical methods have been developed for the detection of nitrite, such as spectrophotometry,^9–12 electrochemical analysis,^13–18 chemiluminescence,^19,20 etc. However, these traditional methods often require complicated and expensive instruments, and may cause the secondary pollution due to the use of high toxicity diazo coupling reagent.²¹ In contrast, fluorometric detection methods has played a pivotal role in modern sensing because they often allow for a rapid qualitative and quantitative assessment.²² Among them, lanthanide ions based luminescent materials have attracted very widespread concerns due to their unique and magnetic properties, such as large Stokes shift, high internal quantum efficiency, long luminescence time, line-like emission, and good colour purity.^23–25 However, free lanthanide ions cannot be directly excited due to their very inefficient light absorption and the low extinction coefficients. To overcome such a shortage, organic ligands containing suitable chromophores are often employed to improve the luminescence properties of lanthanides.²⁶ Once organic ligands coordinate with lanthanides ions, energy transfer will occur from ligands to the lanthanide ions, resulting in emitting their characteristic luminescence, which was defined as a well-known “antenna” effect.²⁷

In this regards, the development of organic ligands that enables to improve the luminescence properties of lanthanides is of great significance. Up to date, the most representative ligands for chelating lanthanides is macrocycle 1,4,7,10-tetraazacyclododecane, as documented in Fig. 1. However, in this case, some other organic chromophores, such as dipicolinic acid (DPA) and terpyridine (TPY), are still needed as antenna to efficiently transfer the absorbed energy to the lanthanide ions.²⁸ Although such kind of corresponding complexes have been proved with enhanced resistance to degradation, and inertness to acids and alkalis,²⁹ their thermal stabilities are still needed to be improved. Thus, there has been a remarkable demand in development of novel and effective organic ligands for lanthanide ions.

Fig. 1 The designed concepts of macrocycle ligand.

In view of this, we devote to develop a novel macrocycle based ligand, as shown in Fig. 1. Our rational design strategy is documented as follows: (1) we combine the DPA moiety and macrocycle together to make a new macrocycle-based ligand; (2) such a ligand owns both abilities of efficient ultra-violet energy transferring from DPA moiety and stable chelating for lanthanides ions; (3) the coordination number could be adjusted by inducing different chelating groups. What is more, the lanthanides complex based on this macrocycle is believed with more stable and intense luminescence that enables it be regarded as fluorescence probe in the biochemical analysis.

Based on the previous study, it has been reported that terbium complexes showed the highest luminescence quantum yield among the lanthanide complexes.^30–34 We herein synthesized a novel terbium-macrocycle complex (Tb-Ac) by using this ligand. Because of its unsaturated coordination numbers in this complex Tb-Ac, we assumed that it may be served as sensor for anions. As expected, our results indicated that Tb-Ac exhibited excellent selectivity and sensitivity towards NO₂⁻ with a low detection limit. Moreover, it has been employed in detecting concentration of NO₂⁻ in real food and water samples as well as living cells in terms of favourable accuracy and practicality.

Complex Tb-Ac was synthesized according to the routes outlined in Scheme 1. Intermediate 4 was easily obtained through the reported Fenton reaction. And then 4 was treated with triethylenetetramine in a high dilution solution to yield the macrocycle compound 3. Electrophilic substitution of 3 with N,N-dimethylacetamide gave the intermediate 2, and then 2 reacted with acryloylchloride to get the ligand 1. The subsequent chelation of 1 with TbCl₃ gave complex Tb-Ac. Finally, the resulting crude product Tb-Ac was further purified by HPLC (Fig. S1†).

Scheme 1 Synthesis of Tb-Ac. (a) Methanol, H₂SO₄; (b) methanol, H₂SO₄, H₂O₂, FeSO₄; (c) triethylenetetramine; (d) N,N-dimethylacetamide, K₂CO₃, KI; (e) acryloylchloride, TEA; (f) TbCl₃·6H₂O.

With this Tb-Ac in hand, we firstly evaluated its optical properties. Based on the absorption spectra data, it was demonstrated that the complex was chelated successfully (Fig. S2†). Using 280 nm as a suitable excitation wavelength, an intense luminescent emission was obtained with characteristic peaks at 493, 547, 586, and 624 nm (Fig. S3†). These emission peaks are attributed to the ⁵D₄ → ⁷F₆, ⁵D₄ → ⁷F₅, ⁵D₄ → ⁷F₄, and ⁵D₄ → ⁷F₃ transitions of Tb, respectively. A luminescence quantum yield (Φ) of Tb-Ac were determined to be 0.535 at room temperature by the relative comparison method with quinine bisulfate in 0.5 M H₂SO₄ (Φ = 0.60) chosen as the standard.³⁵ Moreover, both the ligand and the Tb ion showed no luminescence at all under the same condition, which means that the triplet level of the ligand matched with the excited level of Tb. These findings indicated that an efficient intramolecular energy transfer occurred from the macrocyclic ligand 1 to the Tb, resulting in emitting its characteristic luminescence through antenna effect.

Subsequently, we performed the investigation of sensing behaviour of Tb-Ac towards anions. As shown in Fig. 2, Tb-Ac (10 μM) showed an intense fluorescence in HEPES buffer solution (pH = 7.4). After addition of various analytes, only NO₂⁻ caused an obvious fluorescent quenching in its fluorescence spectra. In contrast, other anions didn't induce any distinct variations under the same condition. This result demonstrated that Tb-Ac exhibited high selectivity toward NO₂⁻, and showed a great capability of resisting the disturbance of other anions (Fig. 2b).

Fig. 2 (a) Fluorescence spectra of Tb-Ac (10 μM) upon the addition of various anions (10 equiv.) in HEPES buffer solution (pH = 7.4). (b) Fluorescence intensity of Tb-Ac (10 μM) to NO₂⁻ in the presence of 10 equiv. of other competitive analytes (1 represents Tb-Ac + NO₂⁻) (λ_ex = 280 nm).

We further conducted the fluorescence titrations of Tb-Ac towards NO₂⁻. As showed in Fig. 3a, with the gradual addition of nitrites (0–5 equiv.), the emission intensity of Tb-Ac decreased. Meanwhile, the titrations curves provided a good linear relationship between the fluorescent intensity and the concentration of nitrite with an obtained detection limit as low as 8.6 μM (Fig. S4†). We then evaluated its stability under different pH conditions. The results showed that Tb-Ac had excellent stability in a wide pH range (4–10) (Fig. 3b). These above-mentioned date indicated that Tb-Ac showed excellent selectivity and sensitivity towards NO₂⁻ that enables it to be applied to analyse a broader scope of real samples.

Fig. 3 (a) Fluorescence spectra of Tb-Ac (10 μM) upon addition NO₂⁻ (0–5 equiv.) in HEPES buffer solution (pH = 7.4). (b) Fluorescence spectra of Tb-Ac (10 µM) in the presence or none NO₂⁻ at varied pH values (λ_ex = 280 nm).

Accordingly, the sensing mechanism was further discussed. It has been reported that the nitrite is the only efficient excited state quencher of luminescence of terbium ion among many anions.^36,37 The most plausible mechanism has been established to the quenching through electron exchange energy transfer towards the T₁ state of NO₂⁻, with the involvement of particular inner-sphere complexation between the lanthanide ion and NO₂⁻ in solutions.³⁸ In addition, a redox reaction based mechanism was also proposed in which nitrite was believed to quench the Tb-centered luminescence due to its high oxidation capability towards ligands.^39,40 Furthermore, another possible coordination competition based mechanism was also estimated as nitrite possesses highly charge density that can readily coordinates with electropositive ions.³⁷ Besides the abovementioned mechanisms, in the case of our Tb-Ac, a possible mechanism based on N-nitrosamine reaction was proposed. As shown in Scheme 2, in the presence of nitrite, amide group in the ligand can readily react with nitrite to produce N-nitrosamines, causing alterations of its coordination behaviour, we believed that the recognition process can also be related to this issue.

Scheme 2 The proposed possible mechanism of Tb-Ac towards nitrite.

Next, groundwater (Yanji, China) was chose as a real sample. The concentration of NO₂⁻ in groundwater was detected by Tb-Ac through the standard titration curves. As shown in Table 1, the concentration of nitrite was determined as 1.227 mg kg⁻¹ according to the linear equation and, in all cases, the RSD for each sample for was calculated less than 10%. Moreover, to validate the accuracy of this analysis, the concentrations were also obtained from using previous straightforward method (hydrochloric acid naphthyl-ethylenediamine spectrophotometry) (Fig. S5†).⁴¹ The results were proved to be in great agreement (92%) with the methods of national standard (Table S1†). These experimental data indicated that we could detect NO₂⁻ in real water samples using Tb-Ac without any further processing procedure.

Table 1 Determination of NO₂⁻ by Tb-Ac

Sample	Intensity (493 nm)	Nitrite content (mg kg^−1)
Groundwater	663.7523 ± (2.87)	1.227 ± (0.115)
Pickles	645.16	6.81

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The concentrations of NO₂⁻ in local pickles were also investigated by using Tb-Ac. According to the standard curves of fluorescent titrations, the concentration of NO₂⁻ in real pickles was calculated as 6.81 mg kg⁻¹, which was lower than the lethal oral doses for human beings.⁴² From the standard of the Joint Food and Agricultural Organization/World Health Organization, in which the Acceptable Daily Intake (ADI) of the nitrite has been set as 0.06 mg kg⁻¹ of body weight,⁴³ therefore we can conclude that it is acceptable for a human to consume less than half a kilo pickled vegetables in daily life.

Finally, Tb-Ac was further employed in detection of nitrite in living cells. HeLa cells were co-cultured with Tb-Ac (10 μM) in Dulbecoo's modified Eagle medium with 10% fetal bovine serum under 5% humidified CO₂ for 30 min. In the control experiment, cells were further treated with nitrite (5 equiv.) for 10 min. After then, cells were washed with PBS for three times and cell imaging was conducted using confocal fluorescent microscopy. Fig. 4 showed the differential interference contrast (DIC) and fluorescent images of HeLa cells. A green fluorescence in Fig. 4b was observed, which demonstrated that Tb-Ac enabled to penetrate into cells and emitted detectable fluorescent signal in the biological environment. However, the fluorescent intensity in living cells was considerably low. It is probably due to the low cell uptake efficiency of Tb-Ac. Moreover, after pretreation of nitrite (5 equiv.), the fluorescence within cells was totally quenched. As showed in Fig. 4d, no emission signal was detected. These results indicated that Tb-Ac has great potential to detect nitrite in practical samples with preferable effectiveness and accuracy.

Fig. 4 Confocal fluorescence imaging of HeLa cells with Tb-Ac (10 μM) in the absence (b) and presence (d) of NO₂⁻ (5 equiv.), (a) and (c) represent DIC images.

In summary, we have designed and synthesized a novel terbium-macrocyclic complex (Tb-Ac). The Tb-Ac displayed a characteristic luminescence in buffer solution when excited at 280 nm. In addition, it exhibited excellent selectivity and sensitivity towards NO₂⁻ with the detection limit as low as 8.6 μM. Particularly, Tb-Ac enables to detect NO₂⁻ in real food and water samples, as well as living cells, in terms of demonstrated accuracy and practicability. By virtue of the excellent property of Tb-Ac, it could be used as a potential luminescent sensor for the recognition of NO₂⁻ in a broader practical applications.

Acknowledgements

We thank the Natural Science Foundation of China (NSFC) (grant no. 21165019) for support of this work.

Notes and references

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Footnotes

† Electronic supplementary information (ESI) available. See DOI: 10.1039/c5ra10889f

‡ These authors contributed equally to this work.

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