Research Article Received: 12 June 2014,
Revised: 27 August 2014,
Accepted: 27 October 2014,
Published online in Wiley Online Library: 16 February 2015
(wileyonlinelibrary.com) DOI: 10.1002/jmr.2442
A highly sensitive and reversible chemosensor for Hg2+ detection based on porphyrin-thymine conjugates Xiangzhu Hea, Duanguang Yanga, Hongbiao Chena*, Wei Zhenga and Huaming Lia,b* In this study, we demonstrated a highly sensitive, selective, and reversible chemosensor for Hg2+ determination. This chemosensor was synthesized by direct condensation of thymin-1-ylacetic acid with zinc tetraaminoporphyrin, which has a porphyrin core as the fluorophore and four thymine (T) moieties as the specific interaction sites for Hg2+. The probe (4T-ZnP) exhibited split Soret bands with a small peak at 408 nm and a strong band at 429 nm in a dimethylformamide/H2O (7/3, v/v) mixed solvent as well as a strong emission band at 614 nm. Upon the addition of Hg2+, the probe displayed strong fluorescence quenching due to the formation of T-Hg2+-T complexes. With the aid of the fluorescence spectrometer, the chemosensor in the dimethylformamide/H2O (7/3, v/v) mixed solvent (0.3 μM) exhibited a detection limit of 6.7 nM. Interferences from other common cations, such as Co2+, K+, Sn2+, Zn2+, Cu2+, Ni2+, Mn2+, Na+, Ca2+, Mg2+, Pb2+, and Cd2+, associated with Hg2+ analysis were effectively inhibited. Copyright © 2015 John Wiley & Sons, Ltd. Additional supporting information may be found in the online version of this article at the publisher’s website. Keywords: mercury; chemosensor; porphyrin; thymine
INTRODUCTION
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* Correspondence to: Huaming Li, College of Chemistry, Xiangtan University, Xiangtan 411105, Hunan Province, China. and Hongbiao Chen, College of Chemistry, Xiangtan University, Xiangtan 411105, Hunan Province, China. E-mail: [email protected]; [email protected] a X. He, D. Yang, H. Chen, W. Zheng, H. Li College of Chemistry, Xiangtan University, Xiangtan 411105Hunan Province, China b H. Li Key Laboratory of Polymeric Materials and Application Technology of Hunan Province, Key Laboratory of Advanced Functional Polymeric Materials of College of Hunan Province, and Key Lab of Environment-Friendly Chemistry and Application of Ministry of Education, Xiangtan University, Xiangtan 411105Hunan Province, China
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Mercury and most of its compounds are extremely toxic, which have a significant impact on the quality of environment and human health. Mercury can be absorbed through the skin and mucous membranes, and mercury vapors can be inhaled (Elinder et al., 1988; Gochfeld, 2003; Alizadeh et al., 2011; Wang et al., 2012). Even a trace amount of mercury intake can lead to acute or chronic damage to the human body, in which toxic effects include damage to the brain, kidney, and lung (Nylander et al., 1987). Moreover, mercurialism can result in several diseases, including acrodynia, Minamata disease, and Hunter-Russell syndrome, because mercury is highly reactive with selenium, an essential dietary element required by about 25 genetically distinct selenoenzymes (Cuvin-Aralar and Furness, 1991). Therefore, the development of novel methods for the determination of mercury at innocuous levels has become one of the most attractive topics because of their practical application. Traditionally, mercury ions have been determined by using spectroscopic methods such as atomic absorption spectrometry (Danet et al., 2009; Pourreza and Ghanemi, 2009; Zhang and Adeloju, 2012), atomic fluorescence spectrophotometry (Murkovic and Wolfbeis, 1997; Gao et al., 2011), inductively coupled plasma atomic emission spectrometry (Zhu and Alexandratos, 2007), and inductively coupled plasma mass spectrometry (Bushee, 1988). However, these methods require either multiple experimental steps with tedious sample pretreatments or sophisticated instrumentation and not suitable for in situ analysis. In this regard, there is a growing need for developing an efficient sensing system that should be either sensitive and reliable or simple and economical. Chemosensor provides an alternative platform for the detection
of mercury(II) ion (Hg2+) at trace level due to the ease of miniaturization, low cost, timesaving, high sensitivity, and rapid real-time monitoring (Gong et al., 2010, Hancock, 2013), in which the sensing process is often accompanied by changes in absorption or fluorescence spectra that can be precisely monitored and sometimes detected by the naked eye. Compared to the relatively well-developed Hg2+ chemosensors, fluorescent chemosensors based on Hg2+-thymine binding mode have emerged as a research area of significant importance due to the fact that thymine (T) has proven to be one of the most specific ligand for Hg2+ that can form a T-Hg2+-T complex (Miyake et al., 2006; Zhu et al., 2011). Taking advantage of the strong affinity and high selectivity of Hg2+-T binding, various fluorescent chemosensors for Hg2+ determination have been developed, in which the probing molecule simultaneously contains fluorophore and thymine moieties (Wang et al., 2005; Che et al., 2008; Liu
X. HE ET AL. et al., 2008; Liu et al., 2009a; Ma et al., 2011; Ma et al., 2012; Zheng et al., 2012). Hitherto, heptamethine cyanine (Wang et al., 2005; Zheng et al., 2012), pyrene (Ma et al., 2012), N,N′dideoxythymidine-3,4,9,10-perylene-tetracarboxylic diimide (Che et al., 2008), anthracene (Ma et al., 2011), phthalocyanine (Liu et al., 2009a), and tetraphenylethylene (Liu et al., 2008) have been adopted as the fluorophoric units. Among these reported fluorescent Hg2+ chemosensors, most of them are based on fluorescence quenching mechanism (Descaloz et al., 2003; Kim et al., 2007; Che et al., 2008; Liu et al., 2009a; Ma et al., 2011; Zheng et al., 2012). That is to say, the sensing mechanism relies on fluorescence quenching induced by molecular aggregation, which is caused by the intermolecular coordination of the sensor molecules with Hg2+ through the attached thymine moiety. However, chemosensor studies based on porphyrin-thymine conjugates have seldom been reported so far, although porphyrin is among the best known selfassociating dyes in poor solvents, and this self-aggregation is clearly reflected by changes in the absorption spectra (Liu et al., 2009a; Arai and Segawa, 2011). In this regard, porphyrins and their derivatives represent very attractive components of molecular sensors due to the fact that porphyrins are highly flexible and a number of structural changes involving different central metal ions and peripheral substituents can be introduced, which in turn is reflected by changes in the emission and absorption spectra (Fang and Liu, 2008, Yang et al., 2009, Choi et al., 2013, Bettini et al., 2014). In general, porphyrins exhibit an intense and narrow absorption band in the visible spectrum between 400 and 460 nm as well as an emission band in the red part of the spectrum (550–750 nm) (Karolczak et al., 2004). Nonoverlapping emission and absorption bands allow for the detection of emission and absorption spectroscopic changes by means of inexpensive and readily available lowresolution spectrometers. On the basis of these facts, we herein report a new porphyrinbased chemosensor for the selective and reversible determination of Hg2+ ion. This novel chemosensor, tetra-(thymin-1-ylacetamido)-porphyrin Zn(II) (4T-ZnP), was synthesized by direct condensation of thymin-1-ylacetic acid with zinc tetraaminoporphyrin. The monomeric 4T-ZnP molecule can form random aggregates through intermolecular cross-linking by the T-Hg2+-T complexes, resulting in significant fluorescence
quenching, which can reflect the concentration of Hg2+ very well. Moreover, the specific interaction of thymine with Hg2+ allows emission detection of Hg2+ with very high selectivity for 4TZnP. In this paper, we present the synthesis, characterization, and sensing behavior of 4T-ZnP in detail.
EXPERIMENTAL Reagents and apparatus Tetraaminoporphyrin Zn(II) and thymin-1-ylacetic acid were synthesized as reported (Liu et al., 2000; Yuasa et al., 2004). All solvents were purchased from a commercial source and used without further purification unless otherwise noted. The NMR spectra were recorded with a Bruker AV-400 NMR spectrometer (Billerica, MA, USA). Matrix-Assisted Laser Desorption/ Ionization Time of Flight (MALDI-TOF) mass spectra were recorded on a Bruker BIFLEX III mass spectrometer using a nitrogen laser (337 nm) and an accelerating potential of 20 kV. Ultraviolet–visible (UV–vis) spectra were recorded with a PerkinElmer LAMDA 25 UV–vis spectrometer (Waltham, MA, USA). Photoluminescence emission spectra were recorded with a PerkinElmer LS 55 luminescence spectrometer. Synthesis of 4T-ZnP 4T-ZnP was synthesized following a typical dicyclohexylcarbodiimide condensation method as shown in Scheme 1. Tetraaminoporphyrin Zn(II) (88 mg, 0.12 mmol) and thymin-1ylacetic acid (176 mg, 0.96 mmol) were dissolved in 10-mL dimethylformamide (DMF). To the solution was added 206 mg (1.0 mmol) of dicyclohexylcarbodiimide and trace 4-dimethylaminopyridine in one portion, and the reaction mixture was stirred at room temperature for 48 h. The products in DMF were precipitated from water under magnetic stirring. The crude product was purified by silica gel chromatography using tetrahydrofuran/CH3OH (9/1, v/v) as an eluent to isolate pure compound 4T-ZnP (95.7 mg, 57%). 1H NMR (400 MHz, DMSO-d6) δ (ppm): 11.40 (s, 4H, NH), 10.67 (s, 4H, NH), 8.78 (s, 8H, PyH), 8.10 (d, J =7.66 Hz, 8H, ArH), 7.99 (d, J =7.73 Hz, 8H, ArH), 7.62 (s, 4H, ArH), 4.67 (s, 8H, CH2), 1.83 (s, 12H, CH3). 13C NMR (100 MHz,
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Scheme 1. Synthesis of 4T-ZnP.
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CHEMOSENSOR FOR Hg2+ DETECTION BASED ON PORPHYRIN-THYMINE CONJUGATES DMSO-d6) δ (ppm): 165.92, 164.13, 151.03, 149.39, 142.09, 137.94, 134.25, 131.12, 123.24, 119.71, 117.43, 108.10, 50.10, 11.46. MALDI-TOF mass spectra (C72H56N16O12Zn) m/z: calcd for 1400.371, found: 1401.529 [M + H]+. General spectroscopic procedures The solutions of metal ions were prepared from CoCl2, KCl, SnCl2 · 2H2O, ZnCl2, CuCl2, (CH3COO)2Ni · 4H2O, (CH3COO) 2Cd · 2H2O, (CH3COO)2Pb · 3H2O, NaCl, Ca(NO3)2 · 4H2O, MnCl2, Mg (NO3)2 · 6H2O, and HgCl2 with DMF/H2O (7/3, v/v) mixed solvent. A solution of 4T-ZnP (2 μM or 0.3 μM) was prepared in DMF/H2O (7/3, v/v) mixed solvent. Then, 3.0 mL of the solution of 4T-ZnP was placed in a quartz cell (10.0 mm width), and the fluorescent and absorption spectra were recorded. The HgCl2 and other cations aqueous solution were introduced in portions, and the fluorescent and absorption changes were recorded at room temperature each time.
sorption spectra of 4T-ZnP are similar to that in DMF when the H2O volume fractions in the range of 0–50%, indicating monomeric 4T-ZnP in the solution. However, 4T-ZnP begins to aggregate when the content of H2O exceeds 50%. In the current study, DMF/H2O mixed solvent (7/3, v/v) is used to determine Hg2+ by 4T-ZnP (ε = 0.35 × 106 M–1 cm–1 at 429 nm). Such a mixed solvent can also dissolve Hg2+ very well, which is highly desirable for aqueous sample analysis that Hg2+ usually exist. The fluorescence emission spectra of 4T-ZnP in DMF/H2O mixed solvent (7/3, v/v) and in H2O (2.0 μM) are shown in Figure 2(a). As expected, 4T-ZnP in DMF/H2O solution exhibits strong emission bands centered at 614 and 665 nm, respectively, with a quantum yield (ΦF) of 0.085 by using 5,10,15,20-tetrakisphenylporphyrin (H2TPP) in DMF as a standard (ΦF = 0.12) (Ormond and Freeman, 2013). On the other hand, the fluorescence emission of 4T-ZnP in H2O was completely quenched upon excitation at 429 nm due to the formation of 4T-ZnP aggregates. Fluorescence detection of Hg2+
RESULTS AND DISCUSSION Synthesis and optical properties of 4T-ZnP In the present study, 4T-ZnP was synthesized by direct condensation of thymin-1-ylacetic acid with tetraaminoporphyrin Zn(II) as shown in Scheme 1. The whole synthetic route was simple and the purification was easy. The 4T-ZnP was carefully purified and characterized by spectroscopic methods, from which satisfactory analysis data corresponding to its molecular structure was obtained (Figures S1 and S2; see Supporting Information (SI)). The UV–vis absorption spectra of 4T-ZnP in different solvents are shown in Figure 1(a). As can be seen, 4T-ZnP in DMF or tetrahydrofuran shows a split Soret band with a small peak at 408 nm and a strong band at 429 nm together with two weak Q-bands at 562 and 603 nm, a typical absorption spectrum of monomeric zinc porphyrins. However, in toluene, chloroform, and water, the strong Soret band at 429 nm decreases greatly while it becomes broader, indicating that 4T-ZnP aggregates are formed in these solvents. As we know that Hg2+ as a high toxic pollutant to the environment is usually found in water, the selection of aqueous detection environment is always necessary. On the other hand, 4T-ZnP is aggregated in water but dissolves in DMF very well as mentioned above. Therefore, DMF/H2O mixed solvent was selected to determine Hg2+. The UV–vis absorption spectra of 4T-ZnP in DMF/H2O mixed solvent with different H2O fractions are shown in Figure 1(b). As can be seen, the ab-
As mentioned previously, Hg2+ is expected to bind to thymine residues of 4T-ZnP and to promote intermolecular aggregation, thus leading to a change in the emission and absorption spectroscopic signature, which can be used as a highly sensitive sensor for the determination of Hg2+. Other metal cations do not exhibit binding affinity toward thymine, so they are not expected to promote structural or spectroscopic changes of porphyrin-thymine conjugates. The effective molecular aggregation resulted in a dramatic decrease in fluorescence intensity as depicted in Figure 2(b), mainly owing to the cross-linking of 4T-ZnP by the T-Hg2+-T complexes. Clearly, the addition of 1 equiv of Hg2+ to the 4T-ZnP solution (2.0 μM) leads to about 92% of fluorescence intensity decrease (ΦF = 0.016, H2TPP in DMF as a standard), indicating the stoichiometric conversion of free 4T-ZnP molecules into the aggregation state. Indeed, after the addition of 1 equiv of Hg2+ to the 4T-ZnP solution with a relatively high concentration (100 μM), all the 4TZnP molecules were eventually precipitated out and turned out to be green flocs (inset of Figure 2(b)), leaving the rest of the solution almost colorless. The precipitates can be redissolved back to the solution simply by the addition of acid, which breaks up the T-Hg2+-T complex by protonation of the thymine moieties (Liu et al., 2009b). In addition, the absorbance spectra of 4TZnP in DMF/H2O mixed solvent (7/3, v/v, 2.0 μM) upon addition of 0–2.5 equiv of Hg2+ (Figure S3; see SI) as well as its reaction time profile (Figure S4; see SI) suggested that J aggregates of porphyrin were formed (Liu et al., 2013).
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Figure 1. (a) Absorption spectra of 4T-ZnP (2.0 μM) in different solvents. (b) Absorption spectra of 4T-ZnP (2.0 μM) in dimethylformamide (DMF)/H2O mixtures with different DMF contents. THF, tetrahydrofuran.
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Figure 2. (a) Fluorescence spectra (λex, 429 nm) of 4T-ZnP (2.0 μM) in dimethylformamide (DMF)/H2O (7/3, v/v) mixed solvent and H2O. (b) Fluores2+ cence spectra (λex, 429 nm) of 4T-ZnP (2.0 μM) in DMF/ H2O (7/3, v/v) solution responding 1 equiv of Hg ; inset b: photograph of the 4T-ZnP solution 2+ (100 μM) in the absence (left) and presence (right) of Hg (200 μM).
To obtain a highly sensitive probe for Hg2+, the sensing behavior of 4T-ZnP toward Hg2+ was investigated by fluorescence titration in DMF/H2O mixed solvent (7/3, v/v, 2.0 μM) at an excitation wavelength of 429 nm. The fluorescence titration results at room
temperature are shown in Figure 3(a). As can be seen, upon the addition of Hg2+, the fluorescence emission intensity of the probe at 614 and 665 nm was decreased gradually and was saturated at 1 equiv of Hg2+ (see Job’s plot between 4T-ZnP and Hg2+ in Figure S5; see SI). In the range of 0–4.0 μM Hg2+ concentration, the plot of the fluorescence intensity at 614 nm as a function of the Hg2+ ion concentrations shows a good linear relationship (R = 0.9987, inset of Figure 3(a)), indicating that 4TZnP can be used to detect Hg2+ concentration quantitatively. The detection limit of 4T-ZnP was determined to be 19.0 nM at a ratio of signal to noise of 3 (Chiang et al., 2008). In an attempt to lower the detection limit by decreasing the concentration of 4T-ZnP, a 0.3 μM solution of 4T-ZnP was therefore explored. It is noteworthy that effective binding between Hg2+ and the thymine ligand still exists in this concentration as determined by the binding affinity. In general, the lower the concentration of the 4T-ZnP, the less Hg2+ is required for the same percentage of fluorescence quenching (Che et al., 2008). For a 0.3 μM 4T-ZnP solution, the plot of the fluorescence intensity at 614 nm as a function of the Hg2+ ion concentrations again shows a good linear relationship (R = 0.9942, inset of Figure 3(b)) in the range of 0–0.6 μM Hg2+ concentration. The detection limit of 4TZnP was determined to be as low as 6.7 nM at a ratio of signal to noise of 3 (Chiang et al., 2008). Such a low detection limit is indeed competitive with most of the fluorescence or colorimetric sensors previously reported (Table 1). The high sensitivity thus obtained is consistent with the strong binding of T-Hg2+, for which Hg2+ binds to the thymine by replacing the proton at the secondary amine (Kosturko et al., 1974; Miyake et al., 2006; Table 1. Comparison of the present sensor with other Hg2+ sensors based on fluorophore–thymine conjugates Fluorophores
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Figure 3. (a) Fluorescence spectra (λex, 429 nm) of 4T-ZnP (2.0 μM) in dimethylformamide (DMF)/H2O (7/3, v/v) solution responding to various 2+ concentrations of Hg , 0, 0.5, 1.0, 1.5, 2.0, 3.0, 4.0, 6.0, 10.0, and 20.0 μM. In2+ set a: fluorescence intensity at 614 nm (I614) of 4T-ZnP versus Hg concentration. (b) Fluorescence spectra (λex, 429 nm) of 4T-ZnP (0.3 μM) in DMF/ 2+ H2O (7/3, v/v) solution responding to various concentrations of Hg , 0, 0.06, 0.12, 0.18, 0.24, 0.36, 0.48, 0.60, and 0.90 μM. Inset b: fluorescence in2+ tensity at 614 nm (I614) of 4T-ZnP versus Hg concentration.
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Porphyrin Pyrene Anthracene Phthalocyanine Tetraphenylethylene Heptamethine cyanine TT-PTCDI1
Detection limit
References
6.7 nM 0.1 μM 15 nM 32 nM 0.37 μM 4.9 nM 5.0 nM
This work (Wang et al., 2005) (Ma et al., 2011) (Liu et al., 2009c) (Liu et al., 2008) (Zheng et al., 2012) (Che et al., 2008)
1
N,N′-dideoxythymidine-3,4,9,10-perylene-tetracarboxylic diimide.
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2+
Figure 4. (a) Fluorescence quenching efficiencies of 4T-ZnP (2.0 μM) in dimethylformamide (DMF)/H2O (7/3, v/v) solution by Hg (10.0 μM) and other metal ions (each 10.0 μM), I0, I means the fluorescence intensity at 614 nm (λex, 429 nm) before and after the addition of metal ions. (b) Fluorescence spectra (λex, 429 nm) of 4T-ZnP (2.0 μM) in DMF/H2O (7/3, v/v) solution (black line) in the presence of a metal ions mixture (red line, each 10.0 μM) and in 2+ the presence of Hg (10.0 μM) and the metal ions mixture (blue line).
Figure 5. Reusability of 4T-ZnP (8.0 μM) in dimethylformamide/H2O (7/ 2+ 2+ 3, v/v) solution by adding Hg (16 μM), HCl (32 μM), and Hg (16 μM).
Tanaka et al., 2007). Moreover, the interfering effects of coexisting anions can be neglected (Figure S6; see SI).
addition, upon the addition of Hg2+, the significant color changes of the 4T-ZnP solution can be used for the naked-eye detection of Hg2+ with the assistance of a hand-held UV lamp as shown in Figure S10 (see SI). In contrast, other anions do not induce any significant color changes. These results further confirm that 4T-ZnP can act as an Hg2+-specific colorimetric fluorescent sensor. The fluorescence emission and quenching of 4T-ZnP can be switched immediately by adding HCl. As shown in Figure 5, the fluorescence can be recovered immediately to 97.4% by adding HCl. In the case of Na2S, similar results are observed with a fluorescence recovery of 79.2% after three cycles (Figure S11; see SI). On the other hand, the addition of EDTA to 4T-ZnP solution only causes a slow recovery of fluorescence quenched by Hg2+ (Figure S12; see SI). These results indicate that the fluorescence quenching of the sensor can be reversed by adding acid or Hg2+ precipitant and chelator. The stability of the T- Hg2+-T complex is similar to that of the EDTA-Hg2+ coordination compound (stability constant, logK, 21.8) and much lower than that of HgS (the solubility product constant of HgS in water is 4 × 10–53) (Liu et al., 2009a).
CONCLUSION Selectivity and reversibility
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In summary, we have designed and synthesized a novel fluorescent porphyrin-based colorimetric chemosensor (4T-ZnP) for mercury(II) ion detection. Based on the complexation interaction between the thymine moieties and mercury(II) ion, 4T-ZnP showed high selectivity for mercury(II) ion detection over other common cations. With the aid of the fluorescence spectrometer, the 4T-ZnP in DMF/H2O (7/3, v/v) mixed solvent (0.3 μM) exhibited a very low detection limit of 6.7 nM (S/N = 3) for mercury(II) ion, which was among the best results for mercury(II) ion sensing by the thymine-based fluorescence sensors.
Acknowledgements Financial support from the program for the National Natural Science Foundation of China (51273170), Research Fund for the Doctoral Program of Higher Education of China (RFDP) (20124301110006), the International S&T Cooperation Program of Hunan Province (2013WK3036), the Open Project of Hunan Provincial University Innovation Platform (12 K050), and the Construct Program of the Key Discipline in Hunan Province is greatly acknowledged.
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To evaluate the selectivity of probe 4T-ZnP for Hg2+ detection, fluorescence spectral changes upon addition of various cations including Co2+, K+, Sn2+, Zn2+, Cu2+, Ni2+, Mn2+, Na+, Ca2+, Mg2 + , Pb2+, and Cd2+ were studied. Each spectrum was obtained after the addition of various analytes at room temperature for 10 min. As shown in Figure 4(a), the complexation reaction between 4T-ZnP and Hg2+ gave a dramatic decrease of the fluorescence intensity at 614 and 665 nm. In contrast, no significant fluorescence spectral changes were promoted by the addition of other cations (Figures S8 and S9; see SI). The high selectivity was further tested in an extreme case as presented in Figure 4 (b), where a mixture of all the metal ions mentioned above (each at 10 μM) was added to the sensing system. As can be seen, the fluorescence intensity of the sensor was only weakly perturbed (9.7% quenching) upon the addition of mixed metal ions. However, after Hg2+ (10 μM) was added to the above mixture, the fluorescence intensity of the sensor was significantly reduced (about 93.5% quenching). Such ultrahigh selectivity for the sensor may help avoid the false positives in real applications, where detection of Hg2+ is often interfered with by other transition metal ions. In
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SUPPORTING INFORMATION Additional supporting information may be found in the online version of this article at the publisher’s web site.
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Revised: 27 August 2014,
Accepted: 27 October 2014,
Published online in Wiley Online Library: 16 February 2015
(wileyonlinelibrary.com) DOI: 10.1002/jmr.2442
A highly sensitive and reversible chemosensor for Hg2+ detection based on porphyrin-thymine conjugates Xiangzhu Hea, Duanguang Yanga, Hongbiao Chena*, Wei Zhenga and Huaming Lia,b* In this study, we demonstrated a highly sensitive, selective, and reversible chemosensor for Hg2+ determination. This chemosensor was synthesized by direct condensation of thymin-1-ylacetic acid with zinc tetraaminoporphyrin, which has a porphyrin core as the fluorophore and four thymine (T) moieties as the specific interaction sites for Hg2+. The probe (4T-ZnP) exhibited split Soret bands with a small peak at 408 nm and a strong band at 429 nm in a dimethylformamide/H2O (7/3, v/v) mixed solvent as well as a strong emission band at 614 nm. Upon the addition of Hg2+, the probe displayed strong fluorescence quenching due to the formation of T-Hg2+-T complexes. With the aid of the fluorescence spectrometer, the chemosensor in the dimethylformamide/H2O (7/3, v/v) mixed solvent (0.3 μM) exhibited a detection limit of 6.7 nM. Interferences from other common cations, such as Co2+, K+, Sn2+, Zn2+, Cu2+, Ni2+, Mn2+, Na+, Ca2+, Mg2+, Pb2+, and Cd2+, associated with Hg2+ analysis were effectively inhibited. Copyright © 2015 John Wiley & Sons, Ltd. Additional supporting information may be found in the online version of this article at the publisher’s website. Keywords: mercury; chemosensor; porphyrin; thymine
INTRODUCTION
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* Correspondence to: Huaming Li, College of Chemistry, Xiangtan University, Xiangtan 411105, Hunan Province, China. and Hongbiao Chen, College of Chemistry, Xiangtan University, Xiangtan 411105, Hunan Province, China. E-mail: [email protected]; [email protected] a X. He, D. Yang, H. Chen, W. Zheng, H. Li College of Chemistry, Xiangtan University, Xiangtan 411105Hunan Province, China b H. Li Key Laboratory of Polymeric Materials and Application Technology of Hunan Province, Key Laboratory of Advanced Functional Polymeric Materials of College of Hunan Province, and Key Lab of Environment-Friendly Chemistry and Application of Ministry of Education, Xiangtan University, Xiangtan 411105Hunan Province, China
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Mercury and most of its compounds are extremely toxic, which have a significant impact on the quality of environment and human health. Mercury can be absorbed through the skin and mucous membranes, and mercury vapors can be inhaled (Elinder et al., 1988; Gochfeld, 2003; Alizadeh et al., 2011; Wang et al., 2012). Even a trace amount of mercury intake can lead to acute or chronic damage to the human body, in which toxic effects include damage to the brain, kidney, and lung (Nylander et al., 1987). Moreover, mercurialism can result in several diseases, including acrodynia, Minamata disease, and Hunter-Russell syndrome, because mercury is highly reactive with selenium, an essential dietary element required by about 25 genetically distinct selenoenzymes (Cuvin-Aralar and Furness, 1991). Therefore, the development of novel methods for the determination of mercury at innocuous levels has become one of the most attractive topics because of their practical application. Traditionally, mercury ions have been determined by using spectroscopic methods such as atomic absorption spectrometry (Danet et al., 2009; Pourreza and Ghanemi, 2009; Zhang and Adeloju, 2012), atomic fluorescence spectrophotometry (Murkovic and Wolfbeis, 1997; Gao et al., 2011), inductively coupled plasma atomic emission spectrometry (Zhu and Alexandratos, 2007), and inductively coupled plasma mass spectrometry (Bushee, 1988). However, these methods require either multiple experimental steps with tedious sample pretreatments or sophisticated instrumentation and not suitable for in situ analysis. In this regard, there is a growing need for developing an efficient sensing system that should be either sensitive and reliable or simple and economical. Chemosensor provides an alternative platform for the detection
of mercury(II) ion (Hg2+) at trace level due to the ease of miniaturization, low cost, timesaving, high sensitivity, and rapid real-time monitoring (Gong et al., 2010, Hancock, 2013), in which the sensing process is often accompanied by changes in absorption or fluorescence spectra that can be precisely monitored and sometimes detected by the naked eye. Compared to the relatively well-developed Hg2+ chemosensors, fluorescent chemosensors based on Hg2+-thymine binding mode have emerged as a research area of significant importance due to the fact that thymine (T) has proven to be one of the most specific ligand for Hg2+ that can form a T-Hg2+-T complex (Miyake et al., 2006; Zhu et al., 2011). Taking advantage of the strong affinity and high selectivity of Hg2+-T binding, various fluorescent chemosensors for Hg2+ determination have been developed, in which the probing molecule simultaneously contains fluorophore and thymine moieties (Wang et al., 2005; Che et al., 2008; Liu
X. HE ET AL. et al., 2008; Liu et al., 2009a; Ma et al., 2011; Ma et al., 2012; Zheng et al., 2012). Hitherto, heptamethine cyanine (Wang et al., 2005; Zheng et al., 2012), pyrene (Ma et al., 2012), N,N′dideoxythymidine-3,4,9,10-perylene-tetracarboxylic diimide (Che et al., 2008), anthracene (Ma et al., 2011), phthalocyanine (Liu et al., 2009a), and tetraphenylethylene (Liu et al., 2008) have been adopted as the fluorophoric units. Among these reported fluorescent Hg2+ chemosensors, most of them are based on fluorescence quenching mechanism (Descaloz et al., 2003; Kim et al., 2007; Che et al., 2008; Liu et al., 2009a; Ma et al., 2011; Zheng et al., 2012). That is to say, the sensing mechanism relies on fluorescence quenching induced by molecular aggregation, which is caused by the intermolecular coordination of the sensor molecules with Hg2+ through the attached thymine moiety. However, chemosensor studies based on porphyrin-thymine conjugates have seldom been reported so far, although porphyrin is among the best known selfassociating dyes in poor solvents, and this self-aggregation is clearly reflected by changes in the absorption spectra (Liu et al., 2009a; Arai and Segawa, 2011). In this regard, porphyrins and their derivatives represent very attractive components of molecular sensors due to the fact that porphyrins are highly flexible and a number of structural changes involving different central metal ions and peripheral substituents can be introduced, which in turn is reflected by changes in the emission and absorption spectra (Fang and Liu, 2008, Yang et al., 2009, Choi et al., 2013, Bettini et al., 2014). In general, porphyrins exhibit an intense and narrow absorption band in the visible spectrum between 400 and 460 nm as well as an emission band in the red part of the spectrum (550–750 nm) (Karolczak et al., 2004). Nonoverlapping emission and absorption bands allow for the detection of emission and absorption spectroscopic changes by means of inexpensive and readily available lowresolution spectrometers. On the basis of these facts, we herein report a new porphyrinbased chemosensor for the selective and reversible determination of Hg2+ ion. This novel chemosensor, tetra-(thymin-1-ylacetamido)-porphyrin Zn(II) (4T-ZnP), was synthesized by direct condensation of thymin-1-ylacetic acid with zinc tetraaminoporphyrin. The monomeric 4T-ZnP molecule can form random aggregates through intermolecular cross-linking by the T-Hg2+-T complexes, resulting in significant fluorescence
quenching, which can reflect the concentration of Hg2+ very well. Moreover, the specific interaction of thymine with Hg2+ allows emission detection of Hg2+ with very high selectivity for 4TZnP. In this paper, we present the synthesis, characterization, and sensing behavior of 4T-ZnP in detail.
EXPERIMENTAL Reagents and apparatus Tetraaminoporphyrin Zn(II) and thymin-1-ylacetic acid were synthesized as reported (Liu et al., 2000; Yuasa et al., 2004). All solvents were purchased from a commercial source and used without further purification unless otherwise noted. The NMR spectra were recorded with a Bruker AV-400 NMR spectrometer (Billerica, MA, USA). Matrix-Assisted Laser Desorption/ Ionization Time of Flight (MALDI-TOF) mass spectra were recorded on a Bruker BIFLEX III mass spectrometer using a nitrogen laser (337 nm) and an accelerating potential of 20 kV. Ultraviolet–visible (UV–vis) spectra were recorded with a PerkinElmer LAMDA 25 UV–vis spectrometer (Waltham, MA, USA). Photoluminescence emission spectra were recorded with a PerkinElmer LS 55 luminescence spectrometer. Synthesis of 4T-ZnP 4T-ZnP was synthesized following a typical dicyclohexylcarbodiimide condensation method as shown in Scheme 1. Tetraaminoporphyrin Zn(II) (88 mg, 0.12 mmol) and thymin-1ylacetic acid (176 mg, 0.96 mmol) were dissolved in 10-mL dimethylformamide (DMF). To the solution was added 206 mg (1.0 mmol) of dicyclohexylcarbodiimide and trace 4-dimethylaminopyridine in one portion, and the reaction mixture was stirred at room temperature for 48 h. The products in DMF were precipitated from water under magnetic stirring. The crude product was purified by silica gel chromatography using tetrahydrofuran/CH3OH (9/1, v/v) as an eluent to isolate pure compound 4T-ZnP (95.7 mg, 57%). 1H NMR (400 MHz, DMSO-d6) δ (ppm): 11.40 (s, 4H, NH), 10.67 (s, 4H, NH), 8.78 (s, 8H, PyH), 8.10 (d, J =7.66 Hz, 8H, ArH), 7.99 (d, J =7.73 Hz, 8H, ArH), 7.62 (s, 4H, ArH), 4.67 (s, 8H, CH2), 1.83 (s, 12H, CH3). 13C NMR (100 MHz,
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Scheme 1. Synthesis of 4T-ZnP.
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CHEMOSENSOR FOR Hg2+ DETECTION BASED ON PORPHYRIN-THYMINE CONJUGATES DMSO-d6) δ (ppm): 165.92, 164.13, 151.03, 149.39, 142.09, 137.94, 134.25, 131.12, 123.24, 119.71, 117.43, 108.10, 50.10, 11.46. MALDI-TOF mass spectra (C72H56N16O12Zn) m/z: calcd for 1400.371, found: 1401.529 [M + H]+. General spectroscopic procedures The solutions of metal ions were prepared from CoCl2, KCl, SnCl2 · 2H2O, ZnCl2, CuCl2, (CH3COO)2Ni · 4H2O, (CH3COO) 2Cd · 2H2O, (CH3COO)2Pb · 3H2O, NaCl, Ca(NO3)2 · 4H2O, MnCl2, Mg (NO3)2 · 6H2O, and HgCl2 with DMF/H2O (7/3, v/v) mixed solvent. A solution of 4T-ZnP (2 μM or 0.3 μM) was prepared in DMF/H2O (7/3, v/v) mixed solvent. Then, 3.0 mL of the solution of 4T-ZnP was placed in a quartz cell (10.0 mm width), and the fluorescent and absorption spectra were recorded. The HgCl2 and other cations aqueous solution were introduced in portions, and the fluorescent and absorption changes were recorded at room temperature each time.
sorption spectra of 4T-ZnP are similar to that in DMF when the H2O volume fractions in the range of 0–50%, indicating monomeric 4T-ZnP in the solution. However, 4T-ZnP begins to aggregate when the content of H2O exceeds 50%. In the current study, DMF/H2O mixed solvent (7/3, v/v) is used to determine Hg2+ by 4T-ZnP (ε = 0.35 × 106 M–1 cm–1 at 429 nm). Such a mixed solvent can also dissolve Hg2+ very well, which is highly desirable for aqueous sample analysis that Hg2+ usually exist. The fluorescence emission spectra of 4T-ZnP in DMF/H2O mixed solvent (7/3, v/v) and in H2O (2.0 μM) are shown in Figure 2(a). As expected, 4T-ZnP in DMF/H2O solution exhibits strong emission bands centered at 614 and 665 nm, respectively, with a quantum yield (ΦF) of 0.085 by using 5,10,15,20-tetrakisphenylporphyrin (H2TPP) in DMF as a standard (ΦF = 0.12) (Ormond and Freeman, 2013). On the other hand, the fluorescence emission of 4T-ZnP in H2O was completely quenched upon excitation at 429 nm due to the formation of 4T-ZnP aggregates. Fluorescence detection of Hg2+
RESULTS AND DISCUSSION Synthesis and optical properties of 4T-ZnP In the present study, 4T-ZnP was synthesized by direct condensation of thymin-1-ylacetic acid with tetraaminoporphyrin Zn(II) as shown in Scheme 1. The whole synthetic route was simple and the purification was easy. The 4T-ZnP was carefully purified and characterized by spectroscopic methods, from which satisfactory analysis data corresponding to its molecular structure was obtained (Figures S1 and S2; see Supporting Information (SI)). The UV–vis absorption spectra of 4T-ZnP in different solvents are shown in Figure 1(a). As can be seen, 4T-ZnP in DMF or tetrahydrofuran shows a split Soret band with a small peak at 408 nm and a strong band at 429 nm together with two weak Q-bands at 562 and 603 nm, a typical absorption spectrum of monomeric zinc porphyrins. However, in toluene, chloroform, and water, the strong Soret band at 429 nm decreases greatly while it becomes broader, indicating that 4T-ZnP aggregates are formed in these solvents. As we know that Hg2+ as a high toxic pollutant to the environment is usually found in water, the selection of aqueous detection environment is always necessary. On the other hand, 4T-ZnP is aggregated in water but dissolves in DMF very well as mentioned above. Therefore, DMF/H2O mixed solvent was selected to determine Hg2+. The UV–vis absorption spectra of 4T-ZnP in DMF/H2O mixed solvent with different H2O fractions are shown in Figure 1(b). As can be seen, the ab-
As mentioned previously, Hg2+ is expected to bind to thymine residues of 4T-ZnP and to promote intermolecular aggregation, thus leading to a change in the emission and absorption spectroscopic signature, which can be used as a highly sensitive sensor for the determination of Hg2+. Other metal cations do not exhibit binding affinity toward thymine, so they are not expected to promote structural or spectroscopic changes of porphyrin-thymine conjugates. The effective molecular aggregation resulted in a dramatic decrease in fluorescence intensity as depicted in Figure 2(b), mainly owing to the cross-linking of 4T-ZnP by the T-Hg2+-T complexes. Clearly, the addition of 1 equiv of Hg2+ to the 4T-ZnP solution (2.0 μM) leads to about 92% of fluorescence intensity decrease (ΦF = 0.016, H2TPP in DMF as a standard), indicating the stoichiometric conversion of free 4T-ZnP molecules into the aggregation state. Indeed, after the addition of 1 equiv of Hg2+ to the 4T-ZnP solution with a relatively high concentration (100 μM), all the 4TZnP molecules were eventually precipitated out and turned out to be green flocs (inset of Figure 2(b)), leaving the rest of the solution almost colorless. The precipitates can be redissolved back to the solution simply by the addition of acid, which breaks up the T-Hg2+-T complex by protonation of the thymine moieties (Liu et al., 2009b). In addition, the absorbance spectra of 4TZnP in DMF/H2O mixed solvent (7/3, v/v, 2.0 μM) upon addition of 0–2.5 equiv of Hg2+ (Figure S3; see SI) as well as its reaction time profile (Figure S4; see SI) suggested that J aggregates of porphyrin were formed (Liu et al., 2013).
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Figure 1. (a) Absorption spectra of 4T-ZnP (2.0 μM) in different solvents. (b) Absorption spectra of 4T-ZnP (2.0 μM) in dimethylformamide (DMF)/H2O mixtures with different DMF contents. THF, tetrahydrofuran.
X. HE ET AL.
Figure 2. (a) Fluorescence spectra (λex, 429 nm) of 4T-ZnP (2.0 μM) in dimethylformamide (DMF)/H2O (7/3, v/v) mixed solvent and H2O. (b) Fluores2+ cence spectra (λex, 429 nm) of 4T-ZnP (2.0 μM) in DMF/ H2O (7/3, v/v) solution responding 1 equiv of Hg ; inset b: photograph of the 4T-ZnP solution 2+ (100 μM) in the absence (left) and presence (right) of Hg (200 μM).
To obtain a highly sensitive probe for Hg2+, the sensing behavior of 4T-ZnP toward Hg2+ was investigated by fluorescence titration in DMF/H2O mixed solvent (7/3, v/v, 2.0 μM) at an excitation wavelength of 429 nm. The fluorescence titration results at room
temperature are shown in Figure 3(a). As can be seen, upon the addition of Hg2+, the fluorescence emission intensity of the probe at 614 and 665 nm was decreased gradually and was saturated at 1 equiv of Hg2+ (see Job’s plot between 4T-ZnP and Hg2+ in Figure S5; see SI). In the range of 0–4.0 μM Hg2+ concentration, the plot of the fluorescence intensity at 614 nm as a function of the Hg2+ ion concentrations shows a good linear relationship (R = 0.9987, inset of Figure 3(a)), indicating that 4TZnP can be used to detect Hg2+ concentration quantitatively. The detection limit of 4T-ZnP was determined to be 19.0 nM at a ratio of signal to noise of 3 (Chiang et al., 2008). In an attempt to lower the detection limit by decreasing the concentration of 4T-ZnP, a 0.3 μM solution of 4T-ZnP was therefore explored. It is noteworthy that effective binding between Hg2+ and the thymine ligand still exists in this concentration as determined by the binding affinity. In general, the lower the concentration of the 4T-ZnP, the less Hg2+ is required for the same percentage of fluorescence quenching (Che et al., 2008). For a 0.3 μM 4T-ZnP solution, the plot of the fluorescence intensity at 614 nm as a function of the Hg2+ ion concentrations again shows a good linear relationship (R = 0.9942, inset of Figure 3(b)) in the range of 0–0.6 μM Hg2+ concentration. The detection limit of 4TZnP was determined to be as low as 6.7 nM at a ratio of signal to noise of 3 (Chiang et al., 2008). Such a low detection limit is indeed competitive with most of the fluorescence or colorimetric sensors previously reported (Table 1). The high sensitivity thus obtained is consistent with the strong binding of T-Hg2+, for which Hg2+ binds to the thymine by replacing the proton at the secondary amine (Kosturko et al., 1974; Miyake et al., 2006; Table 1. Comparison of the present sensor with other Hg2+ sensors based on fluorophore–thymine conjugates Fluorophores
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Figure 3. (a) Fluorescence spectra (λex, 429 nm) of 4T-ZnP (2.0 μM) in dimethylformamide (DMF)/H2O (7/3, v/v) solution responding to various 2+ concentrations of Hg , 0, 0.5, 1.0, 1.5, 2.0, 3.0, 4.0, 6.0, 10.0, and 20.0 μM. In2+ set a: fluorescence intensity at 614 nm (I614) of 4T-ZnP versus Hg concentration. (b) Fluorescence spectra (λex, 429 nm) of 4T-ZnP (0.3 μM) in DMF/ 2+ H2O (7/3, v/v) solution responding to various concentrations of Hg , 0, 0.06, 0.12, 0.18, 0.24, 0.36, 0.48, 0.60, and 0.90 μM. Inset b: fluorescence in2+ tensity at 614 nm (I614) of 4T-ZnP versus Hg concentration.
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Porphyrin Pyrene Anthracene Phthalocyanine Tetraphenylethylene Heptamethine cyanine TT-PTCDI1
Detection limit
References
6.7 nM 0.1 μM 15 nM 32 nM 0.37 μM 4.9 nM 5.0 nM
This work (Wang et al., 2005) (Ma et al., 2011) (Liu et al., 2009c) (Liu et al., 2008) (Zheng et al., 2012) (Che et al., 2008)
1
N,N′-dideoxythymidine-3,4,9,10-perylene-tetracarboxylic diimide.
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2+
Figure 4. (a) Fluorescence quenching efficiencies of 4T-ZnP (2.0 μM) in dimethylformamide (DMF)/H2O (7/3, v/v) solution by Hg (10.0 μM) and other metal ions (each 10.0 μM), I0, I means the fluorescence intensity at 614 nm (λex, 429 nm) before and after the addition of metal ions. (b) Fluorescence spectra (λex, 429 nm) of 4T-ZnP (2.0 μM) in DMF/H2O (7/3, v/v) solution (black line) in the presence of a metal ions mixture (red line, each 10.0 μM) and in 2+ the presence of Hg (10.0 μM) and the metal ions mixture (blue line).
Figure 5. Reusability of 4T-ZnP (8.0 μM) in dimethylformamide/H2O (7/ 2+ 2+ 3, v/v) solution by adding Hg (16 μM), HCl (32 μM), and Hg (16 μM).
Tanaka et al., 2007). Moreover, the interfering effects of coexisting anions can be neglected (Figure S6; see SI).
addition, upon the addition of Hg2+, the significant color changes of the 4T-ZnP solution can be used for the naked-eye detection of Hg2+ with the assistance of a hand-held UV lamp as shown in Figure S10 (see SI). In contrast, other anions do not induce any significant color changes. These results further confirm that 4T-ZnP can act as an Hg2+-specific colorimetric fluorescent sensor. The fluorescence emission and quenching of 4T-ZnP can be switched immediately by adding HCl. As shown in Figure 5, the fluorescence can be recovered immediately to 97.4% by adding HCl. In the case of Na2S, similar results are observed with a fluorescence recovery of 79.2% after three cycles (Figure S11; see SI). On the other hand, the addition of EDTA to 4T-ZnP solution only causes a slow recovery of fluorescence quenched by Hg2+ (Figure S12; see SI). These results indicate that the fluorescence quenching of the sensor can be reversed by adding acid or Hg2+ precipitant and chelator. The stability of the T- Hg2+-T complex is similar to that of the EDTA-Hg2+ coordination compound (stability constant, logK, 21.8) and much lower than that of HgS (the solubility product constant of HgS in water is 4 × 10–53) (Liu et al., 2009a).
CONCLUSION Selectivity and reversibility
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In summary, we have designed and synthesized a novel fluorescent porphyrin-based colorimetric chemosensor (4T-ZnP) for mercury(II) ion detection. Based on the complexation interaction between the thymine moieties and mercury(II) ion, 4T-ZnP showed high selectivity for mercury(II) ion detection over other common cations. With the aid of the fluorescence spectrometer, the 4T-ZnP in DMF/H2O (7/3, v/v) mixed solvent (0.3 μM) exhibited a very low detection limit of 6.7 nM (S/N = 3) for mercury(II) ion, which was among the best results for mercury(II) ion sensing by the thymine-based fluorescence sensors.
Acknowledgements Financial support from the program for the National Natural Science Foundation of China (51273170), Research Fund for the Doctoral Program of Higher Education of China (RFDP) (20124301110006), the International S&T Cooperation Program of Hunan Province (2013WK3036), the Open Project of Hunan Provincial University Innovation Platform (12 K050), and the Construct Program of the Key Discipline in Hunan Province is greatly acknowledged.
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To evaluate the selectivity of probe 4T-ZnP for Hg2+ detection, fluorescence spectral changes upon addition of various cations including Co2+, K+, Sn2+, Zn2+, Cu2+, Ni2+, Mn2+, Na+, Ca2+, Mg2 + , Pb2+, and Cd2+ were studied. Each spectrum was obtained after the addition of various analytes at room temperature for 10 min. As shown in Figure 4(a), the complexation reaction between 4T-ZnP and Hg2+ gave a dramatic decrease of the fluorescence intensity at 614 and 665 nm. In contrast, no significant fluorescence spectral changes were promoted by the addition of other cations (Figures S8 and S9; see SI). The high selectivity was further tested in an extreme case as presented in Figure 4 (b), where a mixture of all the metal ions mentioned above (each at 10 μM) was added to the sensing system. As can be seen, the fluorescence intensity of the sensor was only weakly perturbed (9.7% quenching) upon the addition of mixed metal ions. However, after Hg2+ (10 μM) was added to the above mixture, the fluorescence intensity of the sensor was significantly reduced (about 93.5% quenching). Such ultrahigh selectivity for the sensor may help avoid the false positives in real applications, where detection of Hg2+ is often interfered with by other transition metal ions. In
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J. Mol. Recognit. 2015; 28: 293–298
