Analytica Chimica Acta 839 (2014) 74–82
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Single-fluorophore-based fluorescent probes enable dual-channel detection of Ag+ and Hg2+ with high selectivity and sensitivity Yanlin Lv a , Lili Zhu b , Heng Liu a , Yishi Wu c, *, Zili Chen b, *, Hongbing Fu c , Zhiyuan Tian a, * a
School of Chemistry & Chemical Engineering, University of Chinese Academy of Sciences (UCAS), Beijing 100049, China Department of Chemistry, Renmin University of China, Beijing 100872, China Beijing National Laboratory for Molecular Science (BNLMS), State Key Laboratory for Structural Chemistry of Unstable and Stable Species, and Key Laboratory for Photochemistry, Institute of Chemistry, Chinese Academy of Sciences, Beijing 100190, China b c
H I G H L I G H T S
G R A P H I C A L A B S T R A C T
New type of fluorescent probe for Ag+ and Hg2+ sensing with high selectivity and sensitivity. First demonstration of single-fluorophore-based Ag+-selective TURN-ON and Hg2+-selective TURN-OFF type dual-channel fluorescence signaling system. Single-fluorophore-based probes for simultaneous determination of Ag+ and Hg2+ without crosstalk.
A new type of single-fluorophore-based fluorescent probes that displayed Ag+-selective TURN-ON and Hg2+-selective TURN-OFF type dual-channel antiphase-like fluorescence signaling features was developed, and their unique ability of enabling simultaneous determination of Ag+ and Hg2+ over other reference metal ions was confirmed.
A R T I C L E I N F O
A B S T R A C T
Article history: Received 22 December 2013 Received in revised form 2 June 2014 Accepted 5 June 2014 Available online 11 June 2014
A new type of fluorescent probe capable of detecting Ag+ and Hg2+ in two independent channels was developed in the present work. Specifically, in CH3CN–MOPS mixed solvents with CH3CN/MOPS ratio (v/ v) of 15/85, this type of probe fluoresced weakly, and the addition of Ag+ remarkably induced fluorescence enhancement of the probe. In CH3CN–MOPS mixed solvents with the percentage of CH3CN increased up to 65%, the probe was highly fluorescent and addition of Hg2+ dramatically induced the fluorescence quenching. Thus, using such single-fluorophore-based probe and tuning the polarity of the mixed solvent, Ag+, and Hg2+ can be detected in independent channels with high selectivity and sensitivity. As a result, the mutual interference usually encountered in most cases of Ag+ and Hg2+ sensing owing to the similar fluorescence response that these two ions induced, can be effectively circumvented by using the probes developed herein. ã 2014 Elsevier B.V. All rights reserved.
Keywords: Fluorescent probe Silver ions Mercury ions Dual-channel detection
1. Introduction Silver and mercury are two kinds of toxic metals that can be found throughout the ecosystems [1–3]. It is believed that the toxicity of
* Corresponding authors. E-mail addresses: [email protected] (Y. Wu), [email protected] (Z. Chen), [email protected] (Z. Tian). http://dx.doi.org/10.1016/j.aca.2014.06.010 0003-2670/ ã 2014 Elsevier B.V. All rights reserved.
silver mainly originates from the ability of silver ion (Ag+) to bind with various metabolites and sulfhydryl enzymes, leading to the inactivation of enzymes and consequent health problems [4,5]. Additionally, it is supposed that Ag+ exerts adverse effects on patients who overuse medication containing silver salts by accumulating in liver tissues [6,7]. Mercury is a highly toxic species and its toxic effects depend on its chemical form and the route of exposure [8]. Methylmercury ([CH3Hg]+), known as the most toxic form of mercury species, exerts toxic effects on the immune system, alters
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genetic, and enzyme systems, and leads to neurological damage, including coordination and the senses of touch, taste, and sight [9,10]. Other common forms of mercury, such as the salt HgCl2, have been proven to lead to the gastrointestinal tract damage and kidney failure. People are exposed to silver owing to its wide use in industrial products, such as photographic imaging agents and pharmaceutical products, and consumer products, such as toothpastes, soaps, and deodorants as antimicrobial agents. Human activities, such as alkali and metal processing, incineration of coal, and mining of gold and mercury result in persistent mercury contaminants in air, water, and soil, and therefore, increase the likelihood of exposure of people to such highly toxic metal. Thus, the development of probes possessing high selectivity for Ag+ and Hg2+ over other commonly coexistent metal ions in various media is of considerable significance for environmental protection and human health. Conventional strategies such as atomic absorption spectroscopy [11], mass spectroscopy [12,13] and optical emission spectrometry [14] based on inductively coupled plasma technique, and voltammetry [15] have been developed for silver and mercury detection. In spite of their ability of trace analysis, sophisticated apparatus and complicated sample treatment procedure involved in these techniques significantly impede their widespread and practical use. Fluorescence-based detection methods generally possess huge advantages in terms of selectivity, sensitivity, and simplicity for operation as compared to the above-mentioned conventional techniques. Considerable efforts have been devoted to the development of probes for fluorescence detection of Ag + and Hg2+ in recent years [16,17]. In terms of the overall signal processing in bio-systems and specificity of mode of action, high or analyte-enhanced emission signals is superior and analytically favored as compared to an analyte-induced quenching/ decrease of a specific fluorescence [18,19]. However, owing to the so-called “silent ions” nature of Ag+ and Hg2+, most fluorescent probes developed to date undergo nonspecific fluorescence quenching upon complexation with these two chemically closely related heavy transition metal ions via either electron/energy transfer or enhanced spin–orbit coupling processes, which is not only a disadvantage in terms of high signal output upon recognition but also a crucial impediment to discrimination between these two toxic metal ions [20–23]. In this paper, we described the synthesis of a new type of fluorescent sensor that enables simultaneous determination of Ag+ and Hg2+ over other competing metal ions with high selectivity. Specifically, such new type of sensors clearly displayed Ag+-induced fluorescence enhancement and Hg2+-induced fluorescence quenching with high selectivity over other metal ions. Such a fluorescence responding model of the sensors upon Ag+ and Hg2+ complexation intrinsically enables a dual-channel antiphase-like signaling performance and discrimination between Ag+ and Hg2+ with high contrast, which is fundamentally different from the probes selective for Ag+ and/or Hg2+ reported to date.
2. Experimental
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2.2. Instrumentations The structures of intermediate product and the target probe were characterized using a Bruker 400 MHz nuclear magnetic resonance spectrometer. UV–vis absorption spectra were recorded on Shimadzu UV2550 UV–vis spectrophotometers. Fluorescence spectra were obtained on Horiba FluoroMax4 spectrofluometer. Fluorescence lifetimes were determined using a picoseconds timeresolved fluorescence apparatus that has been described in details elsewhere [24]. 2.3. Synthesis of the target probe, (E)-2-((2-(2H-benzo[d,1,2,3]triazol2-yl)-1-methyl-1H-indol-3-yl) methylene) hydrazinecarbothioamide [25] The target probe was synthesized starting from 1-methyl-1Hindole and 1H-benzo[d,1,2,3]triazole [25]. A solution of 1-methyl1H-indole (63 mL, 0.5 mmol), 1H-benzo[d,1,2,3]triazole (30 mg, 0.25 mmol), N-iodosuccinimide (NIS) (170 mg, 0.75 mmol), and potassium carbonate (174 mg, 1.25 mmol) in 1,4-dioxane (3 mL) in a two-neck bottle was stirred at room temperature under a N2 atmosphere for 30 min. The reaction was stopped by sodium thiosulfate, and the reaction mixture was diluted with ethyl acetate followed by extraction with H2O (10 mL 3). The solvent was removed in vacuo, and the crude product was purified by silica gel chromatography (eluting with 20:1 petroleum ether:ethyl acetate) to yield 2-(1-methyl-1H-indol-2-yl)-2H-benzo[d,1,2,3] triazole (43.6 mg, 0.17 mmol, 69%). Following the general procedure, POCl3 (138 mL, 1.45 mmol) was dissolved in 2 mL DMF at 0 C, and then 2-(1-methyl-1H-indol2-yl)-2H-benzo[d,1,2,3]triazole (73.6 mg, 0.29 mmol) was added into the reaction mixture in a two-neck bottle, and the solution was heated at 70 C under a N2 atmosphere for 4 h. DMF played the role of both reactant and solvent. The reaction mixture was cooled to room temperature and diluted with ethyl acetate followed by extraction with H2O (10 mL 3). The solvent was removed in vacuo, and the crude product was purified by silica gel chromatography (eluting with 5:1 petroleum ether:ethyl acetate) to yield 2-(2Hbenzo[d,1,2,3]triazol-2-yl)-1-methyl-1H-indole-3-carbaldehyde as a pale red solid (41.4 mg, 0.15 mmol, 52%). To obtain the target product, a solution of 2-(2H-benzo[d,1,2,3] triazol-2-yl)-1-methyl-H-indole-3-carbaldehyde (25 mg, 0.08 mmol) and hydrazinecarbothioamide (9.1 mg, 0.1 mmol) in ethanol (3 mL) was refluxed under a N2 atmosphere in a two-neck bottle for 10 h. The reaction mixture was cooled to room temperature, and the target product as a yellow solid precipitated out, so that the product (21.8 mg, 0.06 mmol, 78%) was available directly through a filter operation. The whole synthetic route is illustrated in Scheme 1 and the structure of the target compound was confirmed by 1H NMR and 13C NMR. 1H NMR (400 MHz, DMSOd6, d) 11.21 (s,1H), 8.50 (d, J = 7.9 Hz,1H), 8.28 (s,1H), 8.19 (s,1H), 8.17– 8.14 (m, 2H), 7.70 (d, J = 8.3 Hz,1H,), 7.67–7.61 (m, 2H) 7.54 (s,1H), 7.47 (t, J = 7.8 Hz, 1H), 7.34 (t, J = 7.6 Hz, 1H), 3.81 (s, 3H). 13C NMR (100 MHz, DMSO-d6, d) 177.2, 144.9, 138.4, 135.9, 135.3, 128.5, 125.0, 123.7, 122.0, 122.5, 118.5, 111.1, 30.8.
2.1. Chemicals 3. Results and discussion AgNO3, HgCl2, and other metal ion inorganic salt used in this work were purchased from Shanghai Shenbo Chemical Co., Ltd., China. 1-Methyl-1H-indole, 1H-benzo[d,1,2,3]triazole, NIS (Niodosuccinimide), N,N-dimethylformamide (DMF), hydrazinecarbothioamide, and other organic reagents were purchased from Aladdin Chemistry Co., Ltd., China. All the reagents are analytical reagent grade and were used as received. All solutions were freshly prepared before use. Milli-Q ultrapure water (18.2 MV cm) was used in all experiments.
Fluorescence emission features of the as-prepared probe were investigated in several kinds of typical organic solvents miscible with water i.e., tetrahydrofuran (THF), acetone, acetonitrile (CH3CN), ethanol (EtOH), and methanol (MeOH). As shown in Fig.1A, probe in these solvents displayed fluorescence emission band in the region of 425–700 nm with peak around 525 nm. Of note, polarity of the solvents exerted remarkable effect on the fluorescence emission intensity of the probe. Specifically, fluorescence intensity of the
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Scheme 1. The synthesis route of (E)-2-((2-(2H-benzo[d] [1–3]triazol-2-yl)-1–methyl-1H-indol-3-yl) methylene) hydrazinecarbothioamide.
probe in THF was more than 46 times stronger than that of the probe in MeOH. Fluorescence emission features of the probe in binary solvent system containing CH3CN and 3-(N-morpholine) propanesulfonic acid (MOPS) with various CH3CN/MOPS ratio were also investigated with the results shown in Fig. 1B. It can be seen that emission intensity of the probe displayed obvious dependence on the solvent polarity of the mixed solvent. The target probe in CH3CN–MOPS mixed solvent with 85% MOPS clearly displayed Ag+-sensitive fluorescence emission features. Such probe solution weakly fluoresced in the region of 425–700 nm upon excitation at 400 nm and acquired a low fluorescence quantum yield (F = 0.008) when compared to rhodamine B in ethanol (F = 0.70). Upon addition of small aliquots of Ag+ to the solution, clear increase in the emission intensity was observed and such fluorescence enhancement gradually augmented upon further incremental addition of Ag+, eventually resulting in an 8-fold increase in the fluorescence intensity upon addition of two equiv. of Ag+ and a quantum yield of 0.065. Additionally, Ag+ induced fluorescence enhancement concomitant with obvious red-shift of the fluorescence spectrum, with the emission peak shifting from 525 to 550 nm, upon addition of two equiv. of Ag+ to the solution. Such features and the aforementioned polaritydependent fluorescence intensity of the probe provide a clue to the involvement of photo-induced electron transfer (PET) mechanism in the fluorescence emission of the probe [26,27]. Specifically, in polar solvent that facilitates PET processes, probe exhibited weak fluorescence due to PET-induced quenching. In contrast, in a less polar solvent system that suppressed the efficiency of electron transfer, relatively strong fluorescence was eventually observed, as demonstrated in Fig. 1 [28,29]. Additionally, binding of Ag+ to the probe blocked the PET mechanism and resulted in considerable
fluorescence enhancement, as shown in Fig. 2A. Of note, another mechanism, namely the conformational restriction of the probe upon Ag+ binding, probably involved in the Ag+-induced fluorescence enhancement [30–32]. For the target probe possessing unbridged CQN structure investigated in the present work, CQN isomerization might be the predominant nonradiative decay process of excited states [33–35], which may be inhibited by Ag + complexation to the recognition moiety linked to the fluorophore. Such inhibition of the radiationless deactivation channels was expected to enable more excited-state species to relax to the ground state via photon emission, and therefore, contributed to the enhanced fluorescence emission intensity [36]. It deserves mentioning that such speculation about the possible involvement of PET and conformational restriction found support from the transient fluorescence measurement results of the probe in the absence and presence of Ag+, which was discussed in Section 3. The inset in Fig. 2A displayed a plot of fluorescence emission intensity of the probe solution sample (7.5 mM) against the equiv. of Ag+. Based on such fluorescence titration, a detection limit of 97 nM of the as-prepared probe for Ag+ was obtained. Fig. 2B showed the changes in UV–vis absorption spectra of the probes in CH3CN– MOPS mixed solvent with 85% MOPS upon addition of various equiv. of Ag+. It can be clearly seen that upon addition of Ag+, the characteristic absorption features in the region of 250–380 nm remarkably decreases concomitant with an increase in the lowenergy band with wavelength longer than 380 nm. Such evolution features in the absorption spectra coincide with those in the fluorescence spectra of the probe upon the addition of Ag+, suggesting strong complexation of the probe with Ag+. Ion selectivity of the probe in CH3CN–MOPS mixed solvent with 85% MOPS was evaluated against other physiologically important
Fig. 1. Fluorescence emission spectra (lex = 400 nm) of probe (7.5 mM) in typical organic solvents miscible with water (A) and CH3CN–MOPS mixed solvents with different CH3CN/MOPS ratio (B).
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Fig. 2. Fluorescence emission spectra (lex = 400 nm) (A) and UV–vis absorption spectra (B) of the probe (7.5 mM) in CH3CN–MOPS mixed solvent with 85% MOPS upon addition of Ag+ (0, 0.4, 0.8, 1.2, 1.6, 2.0 equiv.). Inset in (A) plot of the corresponding fluorescence intensity (at 545-nm) of the probe versus equiv. of Ag+.
metal ions such as Zn2+, Pd2+, Ni2+, Na+, Mg2+, Li+, K+, Hg2+, Fe3+, Fe2 , Cu2+, Cr3+, Co2+, Cd2+, and Al3+. As illustrated in Fig. 3, upon addition of these reference metal ions with identical concentrations, fluorescence emission intensity of the probe solution sample was either unchanged or minimally affected compared to that of the free probe. Specifically, fluorescence intensity of the probe solution remained almost unchanged upon the addition of Cr3+, Fe3+, K+, Na+, and Pd2+. In contrast, the presence of Al3+, Cd2+, Co2+, Cu2+, Fe2+, Li+, Ni2+, and Zn2+ induced slight increase in the fluorescence intensity of the probe solution sample, with a maximum enhancement observed in the case of Zn2+, approximately 1.1-fold. In sharp contrast to the effects that these reference metal ions exert on the fluorescence emission intensity of the probe solution sample, a remarkable fluorescence enhancement up to 8-fold was observed upon the addition of Ag+, indicating a high selectivity of the as-prepared probe for Ag+ over other potential interfering metal ions. To evaluate the recognition specificity of the target probe to Ag+, competitive binding experiments of the probe with Ag+ in the presence of interference metal ions with much higher concentration were performed. Typically, fluorescence emission spectra of the probe in CH3CN–MOPS mixed solvent with 85% MOPS (7.5 mM) and the probe solution sample in the presence of five equiv. of various interference cations, Na+, K+, Li+, Co2+, Mg2+, Pb2+, Ni2+, Zn2 + , Cd2+, Fe2+, Hg2+, Al3+, Cr3+, Cu2+, and Fe3+, respectively, were acquired firstly, and then the counterpart emission spectra of the corresponding samples with subsequent addition of five equiv. of Ag+ were acquired under the identical condition. By comparing the change in the fluorescence intensity of each pair of probe samples, +
Fig. 3. Fluorescence emission intensity at 545 nm (lex = 400 nm) of probe (7.5 mM) in the absence and presence of five equiv. of various metal ions in CH3CN–MOPS mixed solvent with 85% MOPS (v/v) at 25 C.
namely, samples before and after the addition of Ag+, the response performances of the probe to Ag+ at the presence of various interference cations were obtained. It can be clearly seen from the profile shown in Fig. 4A that other reference metal ions could hardly interfere with the Ag+-induced turn-on fluorescence of the probe, definitely indicating the recognition specificity of the probe to Ag+. An exception was observed in the case of Hg2+, in which the subsequent addition of Ag+ did not induce obvious fluorescence enhancement in the presence of Hg2+. This suggests the strong binding interactions between Hg2+ and the probe, which is in line with the observed Hg2+-sensitive fluorescence as discussed in Section 3. Fig. 4B illustrates the influence of anions on Ag+-induced fluorescence increasing of the probe. Taking that the fluorescence intensity of the probe solution almost kept unchanged upon addition of Na+, as demonstrated in Fig. 4A, inorganic sodium salts containing typical anions, such as CO32 , HCO3 , H2PO4 , HPO42 , NO2 , NO3 , and Cl were used as the sources of anions in the experiment. It can be seen from Fig. 4B that the presence of these typical anions did not remarkably interfere with the fluorescence response feature of the probe to addition of Ag+. To acquire the information of Ag+-probe complexation in CH3CN–MOPS mixed solvent with 85% MOPS, Job’s plot analysis was performed. As shown in Fig. 5A, the obtained Job’s plot results presented a maximum at the 0.5 mol fraction of Ag+, indicated that the target probe forms a 1:1 stoichiometric complex with Ag+. Fig. 5B displays the fluorescence response feature of probe in CH3CN–MOPS mixed solvent with different pH value. It can be clearly seen that the factor of acidity/alkalinity of the mixed solvent exerted remarkable influence on the fluorescence response feature of probe. Specifically, it was found that the probe displayed the most sensitive response feature under the pH region of 7.2–7.4, indicating its potential application in biological detections. A unique feature of the as-prepared probe is its ability of simultaneous determination of Ag+ and Hg2+ via an antiphase-like signaling way. Fig. 6 shows the fluorescence emission features of the as-prepared probe in a CH3CN–MOPS mixed solvent with 35% MOPS in the absence and presence of five equiv. of various metal ions such as Zn2+, Pd2+, Ni2+, Na+, Mg2+, Li+, K+, Hg2+, Fe3+, Fe2+, Cu2+, Cr3+, Co2+, Cd2+, Al3+, and Ag+. It can be seen that in such low-watercontent CH3CN–MOPS mixed solvent system, addition of five equiv. of Ag+ lead to a 1.7-fold increase in the fluorescence peak, in contrast to a more than 10-fold increase in the maximum fluorescence peak of the probe in the CH3CN–MOPS mixed solvent with 85% MOPS upon the addition of five equiv. of Ag+ (Fig. 5B). Interestingly, addition of five equiv. of Hg2+ was found to result in apparent fluorescence quenching of the probe in such low-watercontent mixed solvent, with quantum yield decreasing from 0.056 to 0.008. In contrast, the fluorescence intensity of the probe displayed slight change upon addition of other metal ions such as
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Fig. 4. (A) Metal ion selectivity profiles of probe (7.5 mM): black bars, fluorescence intensities of free probe and probe in CH3CN–MOPS mixed solvent with 85% MOPS in the presence of five equiv. of interference metal ions; red bars, the counterpart fluorescence intensities of the above mentioned samples with the subsequent addition of five equiv. of Ag+. (B) The effect of anions on the fluorescence emission of probe: black bars, fluorescence intensities of free probe in CH3CN–MOPS mixed solvent in the presence of five equiv. of CO32 , HCO3 , H2PO4 , HPO42 , NO2 , NO3 , and Cl ; red bars, the counterpart fluorescence intensities of the abovementioned samples with the subsequent addition of five equiv. of Ag+. Bars represented the final integrated fluorescence response (F) over the initial integrated emission of the probe (F0). (For interpretation of the references to colour in this figure legend, the reader is referred to the web version of this article.)
Zn2+, Pd2+, Ni2+, Na+, Mg2+, Li+, K+, Fe3+, Fe2+, Cr3+, Co2+, Cd2+, and Al3 , with five equiv. of Cu2+ brought about 60% quenching. Obviously, such fluorescence-quenching-based responding manner of the probe to Hg2+ is in sharp contrast to the fluorescence-increasingbased responding way that the probe displayed to the stimulus of Ag+, inherently enabling the simultaneous determination of Hg2+ and Ag+. In contrast to the fluorescence emission features of probe in the CH3CN–MOPS mixed solvent with 85% of MOPS, probe in CH3CN– MOPS mixed solvent with 35% of MOPS strongly fluoresced in the region of 425–700 nm with maximum at 525 nm, as shown in Fig. 7A. Upon addition of small aliquots of Hg2+ to the solution sample, decrease in the emission intensity was clearly observed and such fluorescence quenching was gradually augmented upon further incremental addition of Hg2+, eventually resulting in 90% fluorescence quenching of the sample upon the addition of three equiv. of Hg2+. Such Hg2+-induced fluorescence decrease is typically attributable to the enhanced spin–orbit coupling [37,38], which is expected to facilitate intersystem crossing (isc) and consequently shorten the fluorescence lifetime and fluorescence quenching. Such speculation was supported by the fluorescence lifetime measurements of the probe in CH3CN–MOPS mixed solvent with 35% of MOPS, as discussed in Section 3. No further decrease in fluorescence intensity was observed upon increasing
+
the Hg2+ concentration above three equiv., indicating a saturated state of the probe with respect to Hg2+. Based on such fluorescence titration result, a detection limit of 142 nM of the probe for Hg2+ was eventually obtained. Fig. 7B showed the UV–vis absorption spectra of the probe in such CH3CN–MOPS mixed solvent (CH3CN/ MOPS = 65/35, v/v). It can be seen that absorption band of the probe in the region of 275–407 nm clearly decreased upon gradual addition of Hg2+, which was in good line with above mentioned Hg2 + -induced fluorescence quenching. Fig. 8A shows the result of competitive binding experiments of Hg2+ with the probe in the presence of interference cations such as Na+, K+, Li+, Co2+, Mg2+, Pb2+, Ni2+, Zn2+, Cd2+, Fe2+, Hg2+, Al3+, Cr3+, Cu2+, Fe3+, and Ag+ with much higher concentration. It can be seen that Hg2+-induced fluorescence quenching of the probe in the CH3CN–MOPS mixed solvent with 35% MOPS in the presence of these reference metal ions was either unchanged or minimally affected as compared to the case without interference cations, indicating the excellent ion selectivity of the as-prepared probe for Hg2+. An exception was observed in the case of Cu2+ acting as the interference cation, in which Cu2+ clearly induced fluorescence quenching of the probe and the subsequent addition of Hg2+ did not remarkably lead to further fluorescence quenching. This indicates that only Cu2+ competed with Hg2+ for binding with the probe and most of other metal ions did not interfere with the
Fig. 5. (A) Job’s plot of changes in fluorescence intensity (at 545-nm) at varying mole ratio of probe and Ag+, [probe] + [Ag+] = 50 mM in the CH3CN–MOPS mixed solvent with 85% MOPS at 25 C. (B) The pH-dependent fluorescence intensity response to Ag+ (five equiv.) of probe in CH3CN–MOPS mixed solvent with 85% MOPS at 25 C.
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Fig. 6. Fluorescence spectra (lex = 400 nm) of probe (10 mM) in the absence and the presence of five equiv. of various metal ions (Zn2+, Pd2+, Ni2+, Na+, Mg2+, Li+, K+, Hg2+, Fe3+, Fe2+, Cu2+, Cr3+, Co2+, Cd2+, Al3+, and Ag+) in CH3CN–MOPS mixed solvent with 35% CH3CN at 25 C.
binding of probe to Hg2+, which was in line with the observed result shown in Fig. 6. Another noteworthy feature is the competing binding of Ag+ and Hg2+ with the probe. Specifically, when Ag+ was added to the probe-containing solution as the reference metal ion, remarkable increase in the fluorescence intensity was observed as compared to the cases of other reference metal ions, which was in line with the aforementioned results of Ag+-induced fluorescence enhancement. Upon the subsequent addition of Hg2+, Ag+-induced fluorescence enhancement effect was nearly completely offset, and the final fluorescence intensity was found close to that of metal–ion free probe sample. Such result indicates a higher affinity of the probe with Hg2+ than that with Ag + , which is in accordance to the aforementioned results obtained in the CH3CN–MOPS mixed solvent with 85% MOPS (v/v) and contrary to the binding selectivity for Ag+ of the macrocyclic ligand [15] aneNO2S2 based probes that Spring and co-workers reported previously [23]. The influence of anions on the fluorescence response of the probe to Hg2+ was also investigated using inorganic sodium salts containing typical anions such as CO32 , HCO3 , H2PO4 , HPO42 , NO2 , NO3 , and Cl as the sources of the anions. As shown in Fig. 8B, the presence of these typical anions did not exert obvious influence on the Hg2+-induced fluorescence quenching feature of the probe. Job’s plot analysis was also performed to determine the binding stoichiometry of Hg2+-probe complex in CH3CN–MOPS mixed
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solvent with 35% MOPS. As illustrated in Fig. 9A, the fitted break point was close to 0.5, which indicated the formation of a 1:1 complex between the probe and Hg2+. To determine the effective pH range of the probe for Hg2+ sensing, the fluorescence emission features of the probe in such low-water-content CH3CN–MOPS buffer solution with different pH values were acquired. Fig. 9B displays the pH-dependent Hg2+-induced fluorescence quenching of probe in CH3CN–MOPS mixed solvent with 35% MOPS. It can be seen that the sensitivity of the probe to Hg2+ clearly increased upon decreasing the acidity of the solution in the weak acidic environment (pH 5.0–7). Additionally, fluorescence quenching of probe reached the maximum at pH 7.3, which was within the same pH region of physiological environment (pH 7.2–7.4). To further validate the Ag+-induced fluorescence increase and 2+ Hg -induced fluorescence quenching of the probe, picosecond time-resolved fluorescence spectroscopy technique was used to acquire the photo-physical information regarding the excited state behaviors of the probe. Picosecond time-resolved fluorescence kinetics of the probe in CH3CN–MOPS mixed solvents with various CH3CN/MOPS ratio (CH3CN/MOPS = 100/0, 85/15, 65/35, 50/50, 30/ 70,15/85, respectively) were firstly acquired. It was found that in the pure CH3CN solvent, the probe exhibited a fluorescence lifetime of 1.625 ns. Upon decreasing the percentage of CH3CN component in the mixed solvent to 85, 65, 50, 30, and 15, the fluorescence lifetime of the probe decreased to 288, 176, 127, 64, and 25 ps, respectively. Beyond doubt, such solvent-polarity-dependent fluorescence lifetime results were in line with the aforementioned steady-state fluorescence results demonstrated in Fig. 1B. Fig. 10A displays the picosecond time-resolved fluorescence decay curves of probe in CH3CN–MOPS mixed solvent with 15% CH3CN. The transient fluorescence decay curve of probe in the absence of Ag+ can be fit adequately with a mono-exponential function, presenting a fluorescence lifetime of 25 ps. In contrast, the fluorescence decay curve of the probe in the presence of Ag+ can be well-resolved into two decay components with high accuracy, yielding an average fluorescence lifetime of 284 ps. For probe in CH3CN–MOPS mixed solvent with 65% CH3CN, its transient fluorescence decay curve can be fit with a mono-exponential function and a fluorescence lifetime of 176 ps was therefore obtained (Fig. 10B). Addition of Hg2+ to the solution sample resulted in a decay curve that can be well-resolved into two decay components, yielding an average fluorescence lifetime of 126 ps. It was noted that the HWHM of IRF for the abovementioned fluorescence kinetics measurement was 8 ps. Thus, it is reasonable to conclude that addition of Ag+ led to a remarkable increase in the fluorescence lifetime of the probe while Hg2+ exerted the contrary effect, which was consistent with the Ag+-induced fluorescence enhancement and Hg2+-induced fluorescence quenching observed in the steady-state fluorescence emission
Fig. 7. (A) Changes in fluorescence emission spectra (lex = 400 nm) of probe (10 mM) in the CH3CN–MOPS mixed solvent with 35% MOPS upon addition of Hg2+ with different equivalents (0, 1.5, 2.25, 2.4, 2.5, 2.6, 2.7, 2.8, 3.5, 5.0 equiv.). Inset: plot of the corresponding fluorescence intensity at 525 nm of the probe versus equiv. of Hg2+. (B) UV–vis absorption spectra probe (10 mM) in the CH3CN–MOPS mixed solvent with 35% MOPS upon addition of Hg2+ with different equiv. (0, 0.4, 0.8, 1.2, 1.6, 2.0, 2.4, 2.8, 3.2 equiv.).
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Fig. 8. (A) Metal ion selectivity profiles of probe (10 mM): red bars, fluorescence intensity of free probe and that of probe in the presence of five equiv. of Na+, K+, Li+, Co2+, Mg2+, Pb2+, Ni2+, Zn2+, Cd2+, Fe2+, Hg2+, Al3+, Cr3+, Cu2+, and Fe3+ ions in CH3CN–MOPS mixed solvent; black bars: the counterpart fluorescence intensities of the abovementioned samples with the subsequent addition of five equiv. of Hg2+. (B) The effect of anions on the fluorescence emission of probe (10 mM): black bars, fluorescence intensities of free probe in the presence of five equiv. of CO32 , HCO3 , H2PO4 , HPO42 , NO2 , NO3 , and Cl ; red bars, the counterpart fluorescence intensities of the abovementioned samples with the subsequent addition of five equiv. of Ag+. Bars represented the final integrated fluorescence response (F) over the initial integrated emission of probe (F0). (For interpretation of the references to colour in this figure legend, the reader is referred to the web version of this article.)
Fig. 9. (A) Job’s plot of changes in fluorescence intensity (at 525-nm) at varying mole ratio of probe and Hg2+, [probe] + [Hg2+] = 50 mM in the low-water-content CH3CN–MOPS mixed solvent with 35% MOPS at 25 C. (B) The pH-dependent fluorescence response (at 525-nm) of probe in low-water-content CH3CN–MOPS mixed solvent to addition of Hg2+ (five equiv.).
Fig. 10. (A) Picosecond time-resolved fluorescence kinetics of the probe in high-water-content CH3CN–MOPS mixed solvent in the absence (black open circles) and presence (red open circles) of Ag+ (A) and the probe in low-water-content CH3CN-MOPS mixed solvent in the absence (red open circles) and presence (black open circles) of Hg2+ (B). The scattered symbols represent experimental data and the black solid lines are fits. (For interpretation of the references to colour in this figure legend, the reader is referred to the web version of this article.)
measurements. Beyond doubt, such remarkable agreement between the transient and steady-state fluorescence features of the probe provided a coherent picture of the strong interactions between the probes and Ag+ and Hg2+ in the present work. As aforementioned,
binding of Ag+ with the probe brought about conformational restriction and inhibition on the PET processes of the probe, which was expected to facilitate the radiative transition manner and consequently contribute to the remarkably prolonged fluorescence
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Fig. 11. Partial 1H NMR (400 MHz) spectral changes of probe (7.5 mM) in D2O–CD3CN in the absence and upon addition of 0.25 equiv. of HgCl2 at 25 C.
lifetime, from 25 to 284 ps. For the case of binding of Hg2+ with the probe, the complexation of Hg2+-probe facilitated intersystem crossing (isc) from the singlet excited state to the triplet excited state owing to the enhanced spin–orbit coupling, and therefore, resulted in the shortened fluorescence lifetime, from 176 to 126 ps. 1 H NMR titration experiments were also performed to gain insight into the interactions between the probe and Hg2+ and Ag+ in low-water-content and high-water-content CD3CN–D2O mixed solvent, respectively. As shown in Fig. 11, upon addition of 0.25 equiv. of Hg2+ to the probe-containing mixed solvent, the proton signal of C(H)QN (d 8.224, singlet, 1H), namely, He, was found downfield shifted to d 8.324. Such obvious downfield chemical shift suggests a deshielding effect due to removal of electron density of C(H)QN moiety upon addition of Hg2+ [39,40]. Specifically, the lone pair of nitrogen atom of the secondary aldimine was sequestrated upon binding with the positively charged Hg2+ and the electron density of the proximal CH moiety consequently decreased. In other words, addition of Hg2+ virtually exerted an electron-withdrawing effect on the chemical shift of the attaching proton in C(H)QN moiety. It is noted that the peaks of protons Ha–Hd on the benzotriazole ring were upfield shifted upon addition of Hg2+, suggesting that the triazole groups was also involved in the complexation with Hg2+ [41]. Additionally, protons Hf–Hi on the indole moiety were found upfield shifted, possibly due to their proximity to the C(H)QN-metal complex [41]. We failed to obtain the Ag+-probe binding information via 1H NMR titration experiments owing to the formation of gel-like sample upon addition of 0.25 equiv. of Ag+ into the probe-containing high-water-content CD3CN–D2O mixed solvent. 4. Conclusion In summary, we synthesized a new type of single-fluorophorebased fluorescent probes that displayed dual-channel antiphase-
like fluorescence responding features to Ag+ and Hg2+, and therefore, inherently enabled simultaneous determination of Ag+ and Hg2+. Specifically, probe in high-water-content CH3CN–MOPS mixed solvent displayed Ag+-induced fluorescence enhancement while the addition of Hg2+ led to remarkable fluorescence quenching of the probe in low-water-content CH3CN–MOPS mixed solvent. High selectivity of this type of probe for simultaneous determination of Ag+ and Hg2+ over other competing metal ions as well as sensitivity, 97 nM and 142 nM for Ag+ and Hg2+, respectively, was confirmed. It was demonstrated that this type of probes displayed its optimal Ag+- and Hg2+-sensing performance within the usual pH range of physiological environment. The steady-state and transient fluorescence measurements of the probe provided a coherent picture of the strong interactions between the probes and Ag+/Hg2+. To the best of our knowledge, this is the first demonstration of single-fluorophorebased Ag+-selective TURN-ON and Hg2+-selective TURN-OFF type dual-channel fluorescence signaling system, which will provide the basis for a new strategy for Ag+ and Hg2+ sensing over other competing metal ions with effective discrimination of Ag+ and Hg2+ with high contrast.
Acknowledgements Y. Lv, L. Zhu, and H. Liu contributed equally to this work. This work was financially supported by the National Natural Science Foundation of China (grant no. 21072224, 21173262, 21272268, and 21373218), and the “Hundred-Talent Program” of CAS to Z. Tian. References [1] P. Doudoroff, M. Katz, Critical review of literature on the toxicity of industrial wastes and their components to fish, Sewage Ind. Wastes 25 (1953) 802.
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Contents lists available at ScienceDirect
Analytica Chimica Acta journal homepage: www.elsevier.com/locate/aca
Single-fluorophore-based fluorescent probes enable dual-channel detection of Ag+ and Hg2+ with high selectivity and sensitivity Yanlin Lv a , Lili Zhu b , Heng Liu a , Yishi Wu c, *, Zili Chen b, *, Hongbing Fu c , Zhiyuan Tian a, * a
School of Chemistry & Chemical Engineering, University of Chinese Academy of Sciences (UCAS), Beijing 100049, China Department of Chemistry, Renmin University of China, Beijing 100872, China Beijing National Laboratory for Molecular Science (BNLMS), State Key Laboratory for Structural Chemistry of Unstable and Stable Species, and Key Laboratory for Photochemistry, Institute of Chemistry, Chinese Academy of Sciences, Beijing 100190, China b c
H I G H L I G H T S
G R A P H I C A L A B S T R A C T
New type of fluorescent probe for Ag+ and Hg2+ sensing with high selectivity and sensitivity. First demonstration of single-fluorophore-based Ag+-selective TURN-ON and Hg2+-selective TURN-OFF type dual-channel fluorescence signaling system. Single-fluorophore-based probes for simultaneous determination of Ag+ and Hg2+ without crosstalk.
A new type of single-fluorophore-based fluorescent probes that displayed Ag+-selective TURN-ON and Hg2+-selective TURN-OFF type dual-channel antiphase-like fluorescence signaling features was developed, and their unique ability of enabling simultaneous determination of Ag+ and Hg2+ over other reference metal ions was confirmed.
A R T I C L E I N F O
A B S T R A C T
Article history: Received 22 December 2013 Received in revised form 2 June 2014 Accepted 5 June 2014 Available online 11 June 2014
A new type of fluorescent probe capable of detecting Ag+ and Hg2+ in two independent channels was developed in the present work. Specifically, in CH3CN–MOPS mixed solvents with CH3CN/MOPS ratio (v/ v) of 15/85, this type of probe fluoresced weakly, and the addition of Ag+ remarkably induced fluorescence enhancement of the probe. In CH3CN–MOPS mixed solvents with the percentage of CH3CN increased up to 65%, the probe was highly fluorescent and addition of Hg2+ dramatically induced the fluorescence quenching. Thus, using such single-fluorophore-based probe and tuning the polarity of the mixed solvent, Ag+, and Hg2+ can be detected in independent channels with high selectivity and sensitivity. As a result, the mutual interference usually encountered in most cases of Ag+ and Hg2+ sensing owing to the similar fluorescence response that these two ions induced, can be effectively circumvented by using the probes developed herein. ã 2014 Elsevier B.V. All rights reserved.
Keywords: Fluorescent probe Silver ions Mercury ions Dual-channel detection
1. Introduction Silver and mercury are two kinds of toxic metals that can be found throughout the ecosystems [1–3]. It is believed that the toxicity of
* Corresponding authors. E-mail addresses: [email protected] (Y. Wu), [email protected] (Z. Chen), [email protected] (Z. Tian). http://dx.doi.org/10.1016/j.aca.2014.06.010 0003-2670/ ã 2014 Elsevier B.V. All rights reserved.
silver mainly originates from the ability of silver ion (Ag+) to bind with various metabolites and sulfhydryl enzymes, leading to the inactivation of enzymes and consequent health problems [4,5]. Additionally, it is supposed that Ag+ exerts adverse effects on patients who overuse medication containing silver salts by accumulating in liver tissues [6,7]. Mercury is a highly toxic species and its toxic effects depend on its chemical form and the route of exposure [8]. Methylmercury ([CH3Hg]+), known as the most toxic form of mercury species, exerts toxic effects on the immune system, alters
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genetic, and enzyme systems, and leads to neurological damage, including coordination and the senses of touch, taste, and sight [9,10]. Other common forms of mercury, such as the salt HgCl2, have been proven to lead to the gastrointestinal tract damage and kidney failure. People are exposed to silver owing to its wide use in industrial products, such as photographic imaging agents and pharmaceutical products, and consumer products, such as toothpastes, soaps, and deodorants as antimicrobial agents. Human activities, such as alkali and metal processing, incineration of coal, and mining of gold and mercury result in persistent mercury contaminants in air, water, and soil, and therefore, increase the likelihood of exposure of people to such highly toxic metal. Thus, the development of probes possessing high selectivity for Ag+ and Hg2+ over other commonly coexistent metal ions in various media is of considerable significance for environmental protection and human health. Conventional strategies such as atomic absorption spectroscopy [11], mass spectroscopy [12,13] and optical emission spectrometry [14] based on inductively coupled plasma technique, and voltammetry [15] have been developed for silver and mercury detection. In spite of their ability of trace analysis, sophisticated apparatus and complicated sample treatment procedure involved in these techniques significantly impede their widespread and practical use. Fluorescence-based detection methods generally possess huge advantages in terms of selectivity, sensitivity, and simplicity for operation as compared to the above-mentioned conventional techniques. Considerable efforts have been devoted to the development of probes for fluorescence detection of Ag + and Hg2+ in recent years [16,17]. In terms of the overall signal processing in bio-systems and specificity of mode of action, high or analyte-enhanced emission signals is superior and analytically favored as compared to an analyte-induced quenching/ decrease of a specific fluorescence [18,19]. However, owing to the so-called “silent ions” nature of Ag+ and Hg2+, most fluorescent probes developed to date undergo nonspecific fluorescence quenching upon complexation with these two chemically closely related heavy transition metal ions via either electron/energy transfer or enhanced spin–orbit coupling processes, which is not only a disadvantage in terms of high signal output upon recognition but also a crucial impediment to discrimination between these two toxic metal ions [20–23]. In this paper, we described the synthesis of a new type of fluorescent sensor that enables simultaneous determination of Ag+ and Hg2+ over other competing metal ions with high selectivity. Specifically, such new type of sensors clearly displayed Ag+-induced fluorescence enhancement and Hg2+-induced fluorescence quenching with high selectivity over other metal ions. Such a fluorescence responding model of the sensors upon Ag+ and Hg2+ complexation intrinsically enables a dual-channel antiphase-like signaling performance and discrimination between Ag+ and Hg2+ with high contrast, which is fundamentally different from the probes selective for Ag+ and/or Hg2+ reported to date.
2. Experimental
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2.2. Instrumentations The structures of intermediate product and the target probe were characterized using a Bruker 400 MHz nuclear magnetic resonance spectrometer. UV–vis absorption spectra were recorded on Shimadzu UV2550 UV–vis spectrophotometers. Fluorescence spectra were obtained on Horiba FluoroMax4 spectrofluometer. Fluorescence lifetimes were determined using a picoseconds timeresolved fluorescence apparatus that has been described in details elsewhere [24]. 2.3. Synthesis of the target probe, (E)-2-((2-(2H-benzo[d,1,2,3]triazol2-yl)-1-methyl-1H-indol-3-yl) methylene) hydrazinecarbothioamide [25] The target probe was synthesized starting from 1-methyl-1Hindole and 1H-benzo[d,1,2,3]triazole [25]. A solution of 1-methyl1H-indole (63 mL, 0.5 mmol), 1H-benzo[d,1,2,3]triazole (30 mg, 0.25 mmol), N-iodosuccinimide (NIS) (170 mg, 0.75 mmol), and potassium carbonate (174 mg, 1.25 mmol) in 1,4-dioxane (3 mL) in a two-neck bottle was stirred at room temperature under a N2 atmosphere for 30 min. The reaction was stopped by sodium thiosulfate, and the reaction mixture was diluted with ethyl acetate followed by extraction with H2O (10 mL 3). The solvent was removed in vacuo, and the crude product was purified by silica gel chromatography (eluting with 20:1 petroleum ether:ethyl acetate) to yield 2-(1-methyl-1H-indol-2-yl)-2H-benzo[d,1,2,3] triazole (43.6 mg, 0.17 mmol, 69%). Following the general procedure, POCl3 (138 mL, 1.45 mmol) was dissolved in 2 mL DMF at 0 C, and then 2-(1-methyl-1H-indol2-yl)-2H-benzo[d,1,2,3]triazole (73.6 mg, 0.29 mmol) was added into the reaction mixture in a two-neck bottle, and the solution was heated at 70 C under a N2 atmosphere for 4 h. DMF played the role of both reactant and solvent. The reaction mixture was cooled to room temperature and diluted with ethyl acetate followed by extraction with H2O (10 mL 3). The solvent was removed in vacuo, and the crude product was purified by silica gel chromatography (eluting with 5:1 petroleum ether:ethyl acetate) to yield 2-(2Hbenzo[d,1,2,3]triazol-2-yl)-1-methyl-1H-indole-3-carbaldehyde as a pale red solid (41.4 mg, 0.15 mmol, 52%). To obtain the target product, a solution of 2-(2H-benzo[d,1,2,3] triazol-2-yl)-1-methyl-H-indole-3-carbaldehyde (25 mg, 0.08 mmol) and hydrazinecarbothioamide (9.1 mg, 0.1 mmol) in ethanol (3 mL) was refluxed under a N2 atmosphere in a two-neck bottle for 10 h. The reaction mixture was cooled to room temperature, and the target product as a yellow solid precipitated out, so that the product (21.8 mg, 0.06 mmol, 78%) was available directly through a filter operation. The whole synthetic route is illustrated in Scheme 1 and the structure of the target compound was confirmed by 1H NMR and 13C NMR. 1H NMR (400 MHz, DMSOd6, d) 11.21 (s,1H), 8.50 (d, J = 7.9 Hz,1H), 8.28 (s,1H), 8.19 (s,1H), 8.17– 8.14 (m, 2H), 7.70 (d, J = 8.3 Hz,1H,), 7.67–7.61 (m, 2H) 7.54 (s,1H), 7.47 (t, J = 7.8 Hz, 1H), 7.34 (t, J = 7.6 Hz, 1H), 3.81 (s, 3H). 13C NMR (100 MHz, DMSO-d6, d) 177.2, 144.9, 138.4, 135.9, 135.3, 128.5, 125.0, 123.7, 122.0, 122.5, 118.5, 111.1, 30.8.
2.1. Chemicals 3. Results and discussion AgNO3, HgCl2, and other metal ion inorganic salt used in this work were purchased from Shanghai Shenbo Chemical Co., Ltd., China. 1-Methyl-1H-indole, 1H-benzo[d,1,2,3]triazole, NIS (Niodosuccinimide), N,N-dimethylformamide (DMF), hydrazinecarbothioamide, and other organic reagents were purchased from Aladdin Chemistry Co., Ltd., China. All the reagents are analytical reagent grade and were used as received. All solutions were freshly prepared before use. Milli-Q ultrapure water (18.2 MV cm) was used in all experiments.
Fluorescence emission features of the as-prepared probe were investigated in several kinds of typical organic solvents miscible with water i.e., tetrahydrofuran (THF), acetone, acetonitrile (CH3CN), ethanol (EtOH), and methanol (MeOH). As shown in Fig.1A, probe in these solvents displayed fluorescence emission band in the region of 425–700 nm with peak around 525 nm. Of note, polarity of the solvents exerted remarkable effect on the fluorescence emission intensity of the probe. Specifically, fluorescence intensity of the
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Scheme 1. The synthesis route of (E)-2-((2-(2H-benzo[d] [1–3]triazol-2-yl)-1–methyl-1H-indol-3-yl) methylene) hydrazinecarbothioamide.
probe in THF was more than 46 times stronger than that of the probe in MeOH. Fluorescence emission features of the probe in binary solvent system containing CH3CN and 3-(N-morpholine) propanesulfonic acid (MOPS) with various CH3CN/MOPS ratio were also investigated with the results shown in Fig. 1B. It can be seen that emission intensity of the probe displayed obvious dependence on the solvent polarity of the mixed solvent. The target probe in CH3CN–MOPS mixed solvent with 85% MOPS clearly displayed Ag+-sensitive fluorescence emission features. Such probe solution weakly fluoresced in the region of 425–700 nm upon excitation at 400 nm and acquired a low fluorescence quantum yield (F = 0.008) when compared to rhodamine B in ethanol (F = 0.70). Upon addition of small aliquots of Ag+ to the solution, clear increase in the emission intensity was observed and such fluorescence enhancement gradually augmented upon further incremental addition of Ag+, eventually resulting in an 8-fold increase in the fluorescence intensity upon addition of two equiv. of Ag+ and a quantum yield of 0.065. Additionally, Ag+ induced fluorescence enhancement concomitant with obvious red-shift of the fluorescence spectrum, with the emission peak shifting from 525 to 550 nm, upon addition of two equiv. of Ag+ to the solution. Such features and the aforementioned polaritydependent fluorescence intensity of the probe provide a clue to the involvement of photo-induced electron transfer (PET) mechanism in the fluorescence emission of the probe [26,27]. Specifically, in polar solvent that facilitates PET processes, probe exhibited weak fluorescence due to PET-induced quenching. In contrast, in a less polar solvent system that suppressed the efficiency of electron transfer, relatively strong fluorescence was eventually observed, as demonstrated in Fig. 1 [28,29]. Additionally, binding of Ag+ to the probe blocked the PET mechanism and resulted in considerable
fluorescence enhancement, as shown in Fig. 2A. Of note, another mechanism, namely the conformational restriction of the probe upon Ag+ binding, probably involved in the Ag+-induced fluorescence enhancement [30–32]. For the target probe possessing unbridged CQN structure investigated in the present work, CQN isomerization might be the predominant nonradiative decay process of excited states [33–35], which may be inhibited by Ag + complexation to the recognition moiety linked to the fluorophore. Such inhibition of the radiationless deactivation channels was expected to enable more excited-state species to relax to the ground state via photon emission, and therefore, contributed to the enhanced fluorescence emission intensity [36]. It deserves mentioning that such speculation about the possible involvement of PET and conformational restriction found support from the transient fluorescence measurement results of the probe in the absence and presence of Ag+, which was discussed in Section 3. The inset in Fig. 2A displayed a plot of fluorescence emission intensity of the probe solution sample (7.5 mM) against the equiv. of Ag+. Based on such fluorescence titration, a detection limit of 97 nM of the as-prepared probe for Ag+ was obtained. Fig. 2B showed the changes in UV–vis absorption spectra of the probes in CH3CN– MOPS mixed solvent with 85% MOPS upon addition of various equiv. of Ag+. It can be clearly seen that upon addition of Ag+, the characteristic absorption features in the region of 250–380 nm remarkably decreases concomitant with an increase in the lowenergy band with wavelength longer than 380 nm. Such evolution features in the absorption spectra coincide with those in the fluorescence spectra of the probe upon the addition of Ag+, suggesting strong complexation of the probe with Ag+. Ion selectivity of the probe in CH3CN–MOPS mixed solvent with 85% MOPS was evaluated against other physiologically important
Fig. 1. Fluorescence emission spectra (lex = 400 nm) of probe (7.5 mM) in typical organic solvents miscible with water (A) and CH3CN–MOPS mixed solvents with different CH3CN/MOPS ratio (B).
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Fig. 2. Fluorescence emission spectra (lex = 400 nm) (A) and UV–vis absorption spectra (B) of the probe (7.5 mM) in CH3CN–MOPS mixed solvent with 85% MOPS upon addition of Ag+ (0, 0.4, 0.8, 1.2, 1.6, 2.0 equiv.). Inset in (A) plot of the corresponding fluorescence intensity (at 545-nm) of the probe versus equiv. of Ag+.
metal ions such as Zn2+, Pd2+, Ni2+, Na+, Mg2+, Li+, K+, Hg2+, Fe3+, Fe2 , Cu2+, Cr3+, Co2+, Cd2+, and Al3+. As illustrated in Fig. 3, upon addition of these reference metal ions with identical concentrations, fluorescence emission intensity of the probe solution sample was either unchanged or minimally affected compared to that of the free probe. Specifically, fluorescence intensity of the probe solution remained almost unchanged upon the addition of Cr3+, Fe3+, K+, Na+, and Pd2+. In contrast, the presence of Al3+, Cd2+, Co2+, Cu2+, Fe2+, Li+, Ni2+, and Zn2+ induced slight increase in the fluorescence intensity of the probe solution sample, with a maximum enhancement observed in the case of Zn2+, approximately 1.1-fold. In sharp contrast to the effects that these reference metal ions exert on the fluorescence emission intensity of the probe solution sample, a remarkable fluorescence enhancement up to 8-fold was observed upon the addition of Ag+, indicating a high selectivity of the as-prepared probe for Ag+ over other potential interfering metal ions. To evaluate the recognition specificity of the target probe to Ag+, competitive binding experiments of the probe with Ag+ in the presence of interference metal ions with much higher concentration were performed. Typically, fluorescence emission spectra of the probe in CH3CN–MOPS mixed solvent with 85% MOPS (7.5 mM) and the probe solution sample in the presence of five equiv. of various interference cations, Na+, K+, Li+, Co2+, Mg2+, Pb2+, Ni2+, Zn2 + , Cd2+, Fe2+, Hg2+, Al3+, Cr3+, Cu2+, and Fe3+, respectively, were acquired firstly, and then the counterpart emission spectra of the corresponding samples with subsequent addition of five equiv. of Ag+ were acquired under the identical condition. By comparing the change in the fluorescence intensity of each pair of probe samples, +
Fig. 3. Fluorescence emission intensity at 545 nm (lex = 400 nm) of probe (7.5 mM) in the absence and presence of five equiv. of various metal ions in CH3CN–MOPS mixed solvent with 85% MOPS (v/v) at 25 C.
namely, samples before and after the addition of Ag+, the response performances of the probe to Ag+ at the presence of various interference cations were obtained. It can be clearly seen from the profile shown in Fig. 4A that other reference metal ions could hardly interfere with the Ag+-induced turn-on fluorescence of the probe, definitely indicating the recognition specificity of the probe to Ag+. An exception was observed in the case of Hg2+, in which the subsequent addition of Ag+ did not induce obvious fluorescence enhancement in the presence of Hg2+. This suggests the strong binding interactions between Hg2+ and the probe, which is in line with the observed Hg2+-sensitive fluorescence as discussed in Section 3. Fig. 4B illustrates the influence of anions on Ag+-induced fluorescence increasing of the probe. Taking that the fluorescence intensity of the probe solution almost kept unchanged upon addition of Na+, as demonstrated in Fig. 4A, inorganic sodium salts containing typical anions, such as CO32 , HCO3 , H2PO4 , HPO42 , NO2 , NO3 , and Cl were used as the sources of anions in the experiment. It can be seen from Fig. 4B that the presence of these typical anions did not remarkably interfere with the fluorescence response feature of the probe to addition of Ag+. To acquire the information of Ag+-probe complexation in CH3CN–MOPS mixed solvent with 85% MOPS, Job’s plot analysis was performed. As shown in Fig. 5A, the obtained Job’s plot results presented a maximum at the 0.5 mol fraction of Ag+, indicated that the target probe forms a 1:1 stoichiometric complex with Ag+. Fig. 5B displays the fluorescence response feature of probe in CH3CN–MOPS mixed solvent with different pH value. It can be clearly seen that the factor of acidity/alkalinity of the mixed solvent exerted remarkable influence on the fluorescence response feature of probe. Specifically, it was found that the probe displayed the most sensitive response feature under the pH region of 7.2–7.4, indicating its potential application in biological detections. A unique feature of the as-prepared probe is its ability of simultaneous determination of Ag+ and Hg2+ via an antiphase-like signaling way. Fig. 6 shows the fluorescence emission features of the as-prepared probe in a CH3CN–MOPS mixed solvent with 35% MOPS in the absence and presence of five equiv. of various metal ions such as Zn2+, Pd2+, Ni2+, Na+, Mg2+, Li+, K+, Hg2+, Fe3+, Fe2+, Cu2+, Cr3+, Co2+, Cd2+, Al3+, and Ag+. It can be seen that in such low-watercontent CH3CN–MOPS mixed solvent system, addition of five equiv. of Ag+ lead to a 1.7-fold increase in the fluorescence peak, in contrast to a more than 10-fold increase in the maximum fluorescence peak of the probe in the CH3CN–MOPS mixed solvent with 85% MOPS upon the addition of five equiv. of Ag+ (Fig. 5B). Interestingly, addition of five equiv. of Hg2+ was found to result in apparent fluorescence quenching of the probe in such low-watercontent mixed solvent, with quantum yield decreasing from 0.056 to 0.008. In contrast, the fluorescence intensity of the probe displayed slight change upon addition of other metal ions such as
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Fig. 4. (A) Metal ion selectivity profiles of probe (7.5 mM): black bars, fluorescence intensities of free probe and probe in CH3CN–MOPS mixed solvent with 85% MOPS in the presence of five equiv. of interference metal ions; red bars, the counterpart fluorescence intensities of the above mentioned samples with the subsequent addition of five equiv. of Ag+. (B) The effect of anions on the fluorescence emission of probe: black bars, fluorescence intensities of free probe in CH3CN–MOPS mixed solvent in the presence of five equiv. of CO32 , HCO3 , H2PO4 , HPO42 , NO2 , NO3 , and Cl ; red bars, the counterpart fluorescence intensities of the abovementioned samples with the subsequent addition of five equiv. of Ag+. Bars represented the final integrated fluorescence response (F) over the initial integrated emission of the probe (F0). (For interpretation of the references to colour in this figure legend, the reader is referred to the web version of this article.)
Zn2+, Pd2+, Ni2+, Na+, Mg2+, Li+, K+, Fe3+, Fe2+, Cr3+, Co2+, Cd2+, and Al3 , with five equiv. of Cu2+ brought about 60% quenching. Obviously, such fluorescence-quenching-based responding manner of the probe to Hg2+ is in sharp contrast to the fluorescence-increasingbased responding way that the probe displayed to the stimulus of Ag+, inherently enabling the simultaneous determination of Hg2+ and Ag+. In contrast to the fluorescence emission features of probe in the CH3CN–MOPS mixed solvent with 85% of MOPS, probe in CH3CN– MOPS mixed solvent with 35% of MOPS strongly fluoresced in the region of 425–700 nm with maximum at 525 nm, as shown in Fig. 7A. Upon addition of small aliquots of Hg2+ to the solution sample, decrease in the emission intensity was clearly observed and such fluorescence quenching was gradually augmented upon further incremental addition of Hg2+, eventually resulting in 90% fluorescence quenching of the sample upon the addition of three equiv. of Hg2+. Such Hg2+-induced fluorescence decrease is typically attributable to the enhanced spin–orbit coupling [37,38], which is expected to facilitate intersystem crossing (isc) and consequently shorten the fluorescence lifetime and fluorescence quenching. Such speculation was supported by the fluorescence lifetime measurements of the probe in CH3CN–MOPS mixed solvent with 35% of MOPS, as discussed in Section 3. No further decrease in fluorescence intensity was observed upon increasing
+
the Hg2+ concentration above three equiv., indicating a saturated state of the probe with respect to Hg2+. Based on such fluorescence titration result, a detection limit of 142 nM of the probe for Hg2+ was eventually obtained. Fig. 7B showed the UV–vis absorption spectra of the probe in such CH3CN–MOPS mixed solvent (CH3CN/ MOPS = 65/35, v/v). It can be seen that absorption band of the probe in the region of 275–407 nm clearly decreased upon gradual addition of Hg2+, which was in good line with above mentioned Hg2 + -induced fluorescence quenching. Fig. 8A shows the result of competitive binding experiments of Hg2+ with the probe in the presence of interference cations such as Na+, K+, Li+, Co2+, Mg2+, Pb2+, Ni2+, Zn2+, Cd2+, Fe2+, Hg2+, Al3+, Cr3+, Cu2+, Fe3+, and Ag+ with much higher concentration. It can be seen that Hg2+-induced fluorescence quenching of the probe in the CH3CN–MOPS mixed solvent with 35% MOPS in the presence of these reference metal ions was either unchanged or minimally affected as compared to the case without interference cations, indicating the excellent ion selectivity of the as-prepared probe for Hg2+. An exception was observed in the case of Cu2+ acting as the interference cation, in which Cu2+ clearly induced fluorescence quenching of the probe and the subsequent addition of Hg2+ did not remarkably lead to further fluorescence quenching. This indicates that only Cu2+ competed with Hg2+ for binding with the probe and most of other metal ions did not interfere with the
Fig. 5. (A) Job’s plot of changes in fluorescence intensity (at 545-nm) at varying mole ratio of probe and Ag+, [probe] + [Ag+] = 50 mM in the CH3CN–MOPS mixed solvent with 85% MOPS at 25 C. (B) The pH-dependent fluorescence intensity response to Ag+ (five equiv.) of probe in CH3CN–MOPS mixed solvent with 85% MOPS at 25 C.
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Fig. 6. Fluorescence spectra (lex = 400 nm) of probe (10 mM) in the absence and the presence of five equiv. of various metal ions (Zn2+, Pd2+, Ni2+, Na+, Mg2+, Li+, K+, Hg2+, Fe3+, Fe2+, Cu2+, Cr3+, Co2+, Cd2+, Al3+, and Ag+) in CH3CN–MOPS mixed solvent with 35% CH3CN at 25 C.
binding of probe to Hg2+, which was in line with the observed result shown in Fig. 6. Another noteworthy feature is the competing binding of Ag+ and Hg2+ with the probe. Specifically, when Ag+ was added to the probe-containing solution as the reference metal ion, remarkable increase in the fluorescence intensity was observed as compared to the cases of other reference metal ions, which was in line with the aforementioned results of Ag+-induced fluorescence enhancement. Upon the subsequent addition of Hg2+, Ag+-induced fluorescence enhancement effect was nearly completely offset, and the final fluorescence intensity was found close to that of metal–ion free probe sample. Such result indicates a higher affinity of the probe with Hg2+ than that with Ag + , which is in accordance to the aforementioned results obtained in the CH3CN–MOPS mixed solvent with 85% MOPS (v/v) and contrary to the binding selectivity for Ag+ of the macrocyclic ligand [15] aneNO2S2 based probes that Spring and co-workers reported previously [23]. The influence of anions on the fluorescence response of the probe to Hg2+ was also investigated using inorganic sodium salts containing typical anions such as CO32 , HCO3 , H2PO4 , HPO42 , NO2 , NO3 , and Cl as the sources of the anions. As shown in Fig. 8B, the presence of these typical anions did not exert obvious influence on the Hg2+-induced fluorescence quenching feature of the probe. Job’s plot analysis was also performed to determine the binding stoichiometry of Hg2+-probe complex in CH3CN–MOPS mixed
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solvent with 35% MOPS. As illustrated in Fig. 9A, the fitted break point was close to 0.5, which indicated the formation of a 1:1 complex between the probe and Hg2+. To determine the effective pH range of the probe for Hg2+ sensing, the fluorescence emission features of the probe in such low-water-content CH3CN–MOPS buffer solution with different pH values were acquired. Fig. 9B displays the pH-dependent Hg2+-induced fluorescence quenching of probe in CH3CN–MOPS mixed solvent with 35% MOPS. It can be seen that the sensitivity of the probe to Hg2+ clearly increased upon decreasing the acidity of the solution in the weak acidic environment (pH 5.0–7). Additionally, fluorescence quenching of probe reached the maximum at pH 7.3, which was within the same pH region of physiological environment (pH 7.2–7.4). To further validate the Ag+-induced fluorescence increase and 2+ Hg -induced fluorescence quenching of the probe, picosecond time-resolved fluorescence spectroscopy technique was used to acquire the photo-physical information regarding the excited state behaviors of the probe. Picosecond time-resolved fluorescence kinetics of the probe in CH3CN–MOPS mixed solvents with various CH3CN/MOPS ratio (CH3CN/MOPS = 100/0, 85/15, 65/35, 50/50, 30/ 70,15/85, respectively) were firstly acquired. It was found that in the pure CH3CN solvent, the probe exhibited a fluorescence lifetime of 1.625 ns. Upon decreasing the percentage of CH3CN component in the mixed solvent to 85, 65, 50, 30, and 15, the fluorescence lifetime of the probe decreased to 288, 176, 127, 64, and 25 ps, respectively. Beyond doubt, such solvent-polarity-dependent fluorescence lifetime results were in line with the aforementioned steady-state fluorescence results demonstrated in Fig. 1B. Fig. 10A displays the picosecond time-resolved fluorescence decay curves of probe in CH3CN–MOPS mixed solvent with 15% CH3CN. The transient fluorescence decay curve of probe in the absence of Ag+ can be fit adequately with a mono-exponential function, presenting a fluorescence lifetime of 25 ps. In contrast, the fluorescence decay curve of the probe in the presence of Ag+ can be well-resolved into two decay components with high accuracy, yielding an average fluorescence lifetime of 284 ps. For probe in CH3CN–MOPS mixed solvent with 65% CH3CN, its transient fluorescence decay curve can be fit with a mono-exponential function and a fluorescence lifetime of 176 ps was therefore obtained (Fig. 10B). Addition of Hg2+ to the solution sample resulted in a decay curve that can be well-resolved into two decay components, yielding an average fluorescence lifetime of 126 ps. It was noted that the HWHM of IRF for the abovementioned fluorescence kinetics measurement was 8 ps. Thus, it is reasonable to conclude that addition of Ag+ led to a remarkable increase in the fluorescence lifetime of the probe while Hg2+ exerted the contrary effect, which was consistent with the Ag+-induced fluorescence enhancement and Hg2+-induced fluorescence quenching observed in the steady-state fluorescence emission
Fig. 7. (A) Changes in fluorescence emission spectra (lex = 400 nm) of probe (10 mM) in the CH3CN–MOPS mixed solvent with 35% MOPS upon addition of Hg2+ with different equivalents (0, 1.5, 2.25, 2.4, 2.5, 2.6, 2.7, 2.8, 3.5, 5.0 equiv.). Inset: plot of the corresponding fluorescence intensity at 525 nm of the probe versus equiv. of Hg2+. (B) UV–vis absorption spectra probe (10 mM) in the CH3CN–MOPS mixed solvent with 35% MOPS upon addition of Hg2+ with different equiv. (0, 0.4, 0.8, 1.2, 1.6, 2.0, 2.4, 2.8, 3.2 equiv.).
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Fig. 8. (A) Metal ion selectivity profiles of probe (10 mM): red bars, fluorescence intensity of free probe and that of probe in the presence of five equiv. of Na+, K+, Li+, Co2+, Mg2+, Pb2+, Ni2+, Zn2+, Cd2+, Fe2+, Hg2+, Al3+, Cr3+, Cu2+, and Fe3+ ions in CH3CN–MOPS mixed solvent; black bars: the counterpart fluorescence intensities of the abovementioned samples with the subsequent addition of five equiv. of Hg2+. (B) The effect of anions on the fluorescence emission of probe (10 mM): black bars, fluorescence intensities of free probe in the presence of five equiv. of CO32 , HCO3 , H2PO4 , HPO42 , NO2 , NO3 , and Cl ; red bars, the counterpart fluorescence intensities of the abovementioned samples with the subsequent addition of five equiv. of Ag+. Bars represented the final integrated fluorescence response (F) over the initial integrated emission of probe (F0). (For interpretation of the references to colour in this figure legend, the reader is referred to the web version of this article.)
Fig. 9. (A) Job’s plot of changes in fluorescence intensity (at 525-nm) at varying mole ratio of probe and Hg2+, [probe] + [Hg2+] = 50 mM in the low-water-content CH3CN–MOPS mixed solvent with 35% MOPS at 25 C. (B) The pH-dependent fluorescence response (at 525-nm) of probe in low-water-content CH3CN–MOPS mixed solvent to addition of Hg2+ (five equiv.).
Fig. 10. (A) Picosecond time-resolved fluorescence kinetics of the probe in high-water-content CH3CN–MOPS mixed solvent in the absence (black open circles) and presence (red open circles) of Ag+ (A) and the probe in low-water-content CH3CN-MOPS mixed solvent in the absence (red open circles) and presence (black open circles) of Hg2+ (B). The scattered symbols represent experimental data and the black solid lines are fits. (For interpretation of the references to colour in this figure legend, the reader is referred to the web version of this article.)
measurements. Beyond doubt, such remarkable agreement between the transient and steady-state fluorescence features of the probe provided a coherent picture of the strong interactions between the probes and Ag+ and Hg2+ in the present work. As aforementioned,
binding of Ag+ with the probe brought about conformational restriction and inhibition on the PET processes of the probe, which was expected to facilitate the radiative transition manner and consequently contribute to the remarkably prolonged fluorescence
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Fig. 11. Partial 1H NMR (400 MHz) spectral changes of probe (7.5 mM) in D2O–CD3CN in the absence and upon addition of 0.25 equiv. of HgCl2 at 25 C.
lifetime, from 25 to 284 ps. For the case of binding of Hg2+ with the probe, the complexation of Hg2+-probe facilitated intersystem crossing (isc) from the singlet excited state to the triplet excited state owing to the enhanced spin–orbit coupling, and therefore, resulted in the shortened fluorescence lifetime, from 176 to 126 ps. 1 H NMR titration experiments were also performed to gain insight into the interactions between the probe and Hg2+ and Ag+ in low-water-content and high-water-content CD3CN–D2O mixed solvent, respectively. As shown in Fig. 11, upon addition of 0.25 equiv. of Hg2+ to the probe-containing mixed solvent, the proton signal of C(H)QN (d 8.224, singlet, 1H), namely, He, was found downfield shifted to d 8.324. Such obvious downfield chemical shift suggests a deshielding effect due to removal of electron density of C(H)QN moiety upon addition of Hg2+ [39,40]. Specifically, the lone pair of nitrogen atom of the secondary aldimine was sequestrated upon binding with the positively charged Hg2+ and the electron density of the proximal CH moiety consequently decreased. In other words, addition of Hg2+ virtually exerted an electron-withdrawing effect on the chemical shift of the attaching proton in C(H)QN moiety. It is noted that the peaks of protons Ha–Hd on the benzotriazole ring were upfield shifted upon addition of Hg2+, suggesting that the triazole groups was also involved in the complexation with Hg2+ [41]. Additionally, protons Hf–Hi on the indole moiety were found upfield shifted, possibly due to their proximity to the C(H)QN-metal complex [41]. We failed to obtain the Ag+-probe binding information via 1H NMR titration experiments owing to the formation of gel-like sample upon addition of 0.25 equiv. of Ag+ into the probe-containing high-water-content CD3CN–D2O mixed solvent. 4. Conclusion In summary, we synthesized a new type of single-fluorophorebased fluorescent probes that displayed dual-channel antiphase-
like fluorescence responding features to Ag+ and Hg2+, and therefore, inherently enabled simultaneous determination of Ag+ and Hg2+. Specifically, probe in high-water-content CH3CN–MOPS mixed solvent displayed Ag+-induced fluorescence enhancement while the addition of Hg2+ led to remarkable fluorescence quenching of the probe in low-water-content CH3CN–MOPS mixed solvent. High selectivity of this type of probe for simultaneous determination of Ag+ and Hg2+ over other competing metal ions as well as sensitivity, 97 nM and 142 nM for Ag+ and Hg2+, respectively, was confirmed. It was demonstrated that this type of probes displayed its optimal Ag+- and Hg2+-sensing performance within the usual pH range of physiological environment. The steady-state and transient fluorescence measurements of the probe provided a coherent picture of the strong interactions between the probes and Ag+/Hg2+. To the best of our knowledge, this is the first demonstration of single-fluorophorebased Ag+-selective TURN-ON and Hg2+-selective TURN-OFF type dual-channel fluorescence signaling system, which will provide the basis for a new strategy for Ag+ and Hg2+ sensing over other competing metal ions with effective discrimination of Ag+ and Hg2+ with high contrast.
Acknowledgements Y. Lv, L. Zhu, and H. Liu contributed equally to this work. This work was financially supported by the National Natural Science Foundation of China (grant no. 21072224, 21173262, 21272268, and 21373218), and the “Hundred-Talent Program” of CAS to Z. Tian. References [1] P. Doudoroff, M. Katz, Critical review of literature on the toxicity of industrial wastes and their components to fish, Sewage Ind. Wastes 25 (1953) 802.
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