Journal of Fluorescence

, Volume 22, Issue 3, pp 945–951

A Fluorescence Turn-on Sensor for Hg2+ with a Simple Receptor Available in Sulphide-Rich Environments

Authors

    • State Key Laboratory of Fine ChemicalsDalian University of Technology
  • Xiaojun Peng
    • State Key Laboratory of Fine ChemicalsDalian University of Technology
  • Song Wang
    • State Key Laboratory of Fine ChemicalsDalian University of Technology
  • Xiaojian Liu
    • State Key Laboratory of Fine ChemicalsDalian University of Technology
  • Honglin Li
    • State Key Laboratory of Fine ChemicalsDalian University of Technology
  • Shiguo Sun
    • State Key Laboratory of Fine ChemicalsDalian University of Technology
Original Paper

DOI: 10.1007/s10895-011-1033-x

Cite this article as:
Fan, J., Peng, X., Wang, S. et al. J Fluoresc (2012) 22: 945. doi:10.1007/s10895-011-1033-x

Abstract

Detection of Hg2+ in complex natural environmental conditions is extremely challenging, and no entirely successful methods currently exist. Here we report an easy-to-prepare fluorescent sensor B3 with 2-aminophenol as Hg2+ receptor, which exhibits selective fluorescence enhancement toward Hg2+ over other metal ions. Especially, the fluorescence enhancement was unaffected by anions and cations existing in environment and organism. Moreover, B3 can detect Hg2+ in sulphide-rich environments without cysteine, S2- or EDTA altering the fluorescence intensity. Consequently, B3 is capable of distinguishing between safe and toxic levels of Hg2+ in more complicated natural water systems with detection limit ≤2 ppb.

Keywords

Fluorescent sensor2-aminophenolHg2+Sulphide-rich environments

Introduction

Hg2+, a highly toxic heavy metal ion, seriously threatens many environmental and biological systems [1]. Today, mercury is present in daily life, such as in thermometers, batteries and electronic equipment [24]. The misuse of these products can lead to mercury leaks. Other sources such as volcanic emissions, combustion of fossil fuels, especially mining [5], also cause high concentrations of mercury in many environmental compartments [6] and a number of human health problems [2, 7]. These environmental and biological problems have prompted the development of methods for the detection and quantification of mercury, especially in situations where conventional techniques are not appropriate.

Recently, considerable efforts have been made to design Hg2+ fluorescent sensors with high sensitivity and selectivity, quick response time and easy signal detection. There are fluorescent probes based on different inorganic nanoparticles [813]. Main examples are organic molecules such as rhodamine or 4,4-difluoro-4-bora-3a,4a-diaza-s-indacene (BODIPY) based fluorescent turn-on sensors [1423], a ratiometric fluorescent probe based on FRET [24, 25], a colorimetric sensor based on ruthenium complexes [26] and chemodosimeters based on mercury ion-promoted hydrolysis [27, 28]. Since most of these sensors tended to make use of the thiophilic property of mercury to design mercury ligands, they often contain sulphur atoms in their ligands. However, some mercapto containing biomolecules in organisms could form stable complexes with Hg2+ [29], and there has been little discussion of how to avoid interference from sulfide in organisms or from sulfur-rich environments, preventing them from being applicable in natural environmental conditions.

Therefore, we are still facing the challenge for the exploration of new fluorescent turn-on probes with new, simpler ligands applicable in environmental conditions. Due to properties such as a large molar extinction coefficient (ε), high fluorescence quantum yield (Φ) and insensitivity to solvent polarity and pH, BODIPY-based dyes have been used as efficient fluorescent sensors for different analytes [3035] including our two Hg2+ fluorescent sensors B1 [36] and B2 [37]. Furthermore, 2-aminophenol has been proved to form a stable complex with Hg2+ in ethanol solution [38]. Therefore, herein we report a highly selective and sensitive fluorescence BODIPY-based turn-on sensor B3 for Hg2+, by introducing the very simple Hg2+ ligand, aminophenol, into BODIPY. In this molecule, -NH2 is on the para-phenyl substitute, which can cause more efficient photo-induced electron transfer (PET) process from nitrogen to BODIPY. The compound is easy to be obtained by two steps via compound 2 and performs well in natural environmental conditions without sulphur element interference.

Experimental

Materials and Apparatus

All the chemicals and solvents were of analytical quality. The listed cations and anions were used in addition to Hg2+ to test the specificity: Na+, K+, Ca2+, Cd2+, Co2+, Ni2+, Fe3+, Mg2+, Pb2+, Ag+, Cu2+, Cr3+ and Zn2+; NO3, CH3COO, SCN, ClO4, CO32−, H2PO4, Cl, and SO42−, respectively. All the salts were then dissolved in distilled water. NMR spectra were recorded on a VARIAN INOVA-400 spectrometer with chemical shifts reported as ppm (in CDCl3, TMS as internal standard). Mass spectral determinations were made on a HP1100LC/MSD mass spectrometer and a LC/Q-TOF MS spectrometer. Fluorescence measurements were performed on a VARIAN CARY Eclipse Fluorescence Spectrophotometer (Serial No. FL0812-M018) and the slit width was 5 nm for excitation and emission. Absorption spectra were measured on Lambda 35 UV/vis spectrophotometer. The pH measurements were recorded by PHS-SC instrument.
$$ {\Phi_{{unk}}} = {\Phi_{{std}}}\frac{{\left( {{I_{{unk}}}/{A_{{unk}}}} \right)}}{{{I_{{std}}}/{A_{{std}}}}}{\left( {\frac{{{n_{{unk}}}}}{{{n_{{std}}}}}} \right)^2} $$
(1)

The fluorescence quantum yield was determined using optically matching solutions of rhodamine6G (Φf = 0.94 in ethanol) as standard at an excitation wavelength of 500 nm, and the quantum yield is calculated using Eq. (1) [39] where Φunk and Φstd are the radiative quantum yields of the sample and the standard, Iunk and Istd are the integrated emission intensities of the corrected spectra for the sample and the standard, Aunk and Astd are the absorbances of the sample and the standard at the excitation wavelength (500 nm in all cases), and nunk and nstd are the indices of refraction of the sample and the standard solutions, respectively. Excitation and emission slit widths were modified to adjust the luminescent intensity in a suitable range. All the spectroscopic measurements were performed at least in triplicate and averaged.

Synthesis of Compound 2

2,4-dimethylpyrrole (190 mg, 2 mmol) and 3-hydroxy-4-nitrobenzaldehyde (167 mg, 1 mmol) were dissolved in dry CH2Cl2 (150 mL) under nitrogen. One drop of trifluoroacetic acid (TFA) was added, and the solution was stirred for 5 h at room temperature. After the mixture was concentrated to 30 mL, a solution of 2,3-dichloro-5,6-dicyanoquinone (DDQ, 442 mg, 2 mmol) in 10 mL of CH2Cl2 was added and stirring was continued for 15 min, followed by the addition of triethylamine (2 mL) and BF3•OEt2 (4 mL). After stirring for another 45 min, the reaction mixture was washed with 50 mL water, extracted with dichloromethane (3 × 20 mL). The extract was dried over anhydrous magnesium sulfate and then concentrated under vacuum. The product was purified by flash column chromatography using petrol ether/ethyl acetate (5:1, v/v) as eluent, yielding compound 2 as red solid (88 mg, 23%). 1H NMR(400 MHz, CDCl3), δ:10.67(s, 1H), 8.26(d, 1H, J = 8.0 Hz), 7.18(s, 1H), 6.98(d, 1H, J = 8.0 Hz), 6.02(s, 2H), 2.56(s, 6H), 1.50(s, 6H); 13C NMR (100 MHz, CDCl3), δ: 156.75, 155.42, 144.88, 142.48, 137.64, 133.65, 130.21, 126.07, 121.82, 120.37, 29.70, 14.67; TOF MS(ES): m/z calcd for 384.1331(M-H+), found: 384.1349.

Synthesis of Compound B3

Compound 2 (100 mg, 0.26 mmol) was dissolved in 10 mL of methanol. H2O (5 mL) and Fe (500 mg, 8.9 mmol) were added and the reaction mixture was heated to reflux. Hydrochloric acid in a methanol solution (2 mL, 0.6 mol L−1) was added dropwise. The solution was refluxed for 3 h until complete consumption of the starting material (TLC monitoring). After cooling to room temperature, filtration and concentration at reduced pressure, the product was purified by flash column chromatography using petrol ether/ethyl acetate (4:1,v/v) as eluent, yielding B3 as red solid (77 mg, 83%).1H NMR(400 MHz, CDCl3), δ: 6.91(d, 1H, J = 8.0 Hz), 6.62(s, 1H), 6.56(d, 1H, J = 8.0 Hz), 6.04(s, 2H), 2.48(s, 6H), 1.58(s, 6H); 13C NMR (100 MHz, CDCl3), δ: 154.99, 144.36, 143.33, 142.09, 135.43, 131.86, 125.15, 120.94, 116.84, 114.57, 29.7, 14.57; TOF MS (ES): m/z calcd for 354.1589(M-H+), found: 354.1592.

Results and Discussions

Synthese of B3

Scheme 1 outlines the synthetic route to B3. It was prepared in two steps. The TFA catalyzed condensation reaction of 3-hydroxy-4-nitrobenzaldehyde with 2,4-dimethylpyrrole gave compound 2, which was reduced to give the target product B3 [40]. Both compounds were confirmed by TOF-MS and NMR.
https://static-content.springer.com/image/art%3A10.1007%2Fs10895-011-1033-x/MediaObjects/10895_2011_1033_Sch1_HTML.gif
Scheme 1

Synthetic procedures for B3

Fluorescence Detection of Hg2+ in Ethanol-Water Solution

The fluorescence and absorption studies were conducted in ethanol/HEPES buffer (20 mM HEPES, 100 mM NaNO3, 1:1(v/v), pH = 7.2). As expected, in the absence of Hg2+, B3 exhibited a very weak and characteristic BODIPY-like absorption at 495 nm with a corresponding emission maximum at 513 nm. The fluorescence quantum yield = 0.3%, indicative of efficient photo-induced electron transfer (PET) quenching from the receptor to BODIPY fluorophore [41]. Upon addition of Hg2+, the fluorescence intensity increased by over 20-fold (Fig. 1a) without any shift in absorption spectrum (Fig. 1b). The saturation titration for B3 (inset graph in Fig. 1a) reveals a 1:1 stoichiometry for the B3-Hg2+ complex [42]. The dissociation constant, Kd = (7.78 ± 0.4) × 10−6 M, was obtained by plotting the fluorescence intensity (F/F0) against [Hg2+] [43].
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Fig. 1

Emission a and absorption b of B3 (10 μM) to different concentrations of Hg2+ (0, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 12, 14, 20 μM, the given concentrations correspond to the curves drawn from bottom to top of the images) with excitation at 495 nm in ethanol/HEPES buffer (20 mM HEPES, 100 mM NaNO3, 1:1, v/v, pH 7.2). Inset a: saturation titration of B3 (10 μM) with Hg2+

The nitrate salts of Hg2+, Na+, K+, Ca2+, Cd2+, Co2+, Ni2+, Fe3+, Mg2+, Pb2+, Ag+, Cu2+, Cr3+ and Zn2+ ions were used to evaluate the selectivity of metal ion binding properties of B3 (Fig. 2). As expected, B3 exhibited excellent fluorescence selectivity towards Hg2+ over all other alkali and alkaline earth metal ions, transition and heavy metal ions, although a slight fluorescence enhancement occurred with Ag+. The competition experiments were conducted in the presence of Hg2+ mixed with metal ions at 50 μM mentioned above (Fig. 3a). The fluorescence emission profiles were unaffected by other metal ions except for a slight quenching by Ag+ and Cu2+.
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Fig. 2

Fluorescence spectra of B3 (10 μM) in the presence of different metal ions (50 μM) in ethanol/HEPES buffer (20 mM HEPES, 100 mM NaNO3, 1:1, v/v, pH 7.2) solution. Excitation: 495 nm. Other ions: Na+, K+, Ca2+, Cd2+, Co2+, Ni2+, Fe3+, Mg2+, Pb2+, Cu2+, Cr3+ and Zn2+

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Fig. 3

a Fluorescence responses of B3 (10 μM) to Hg2+ (50 μM) in the presence of selected metal ions (50 μM) in ethanol/HEPES buffer (20 mM HEPES, 100 mM NaNO3, 1:1, v/v, pH 7.2) solution. b The fluorescence responses of B3 (10 μM) containing 50 μM Hg2+ to the selected anions (50 μM) in ethanol/HEPES buffer (20 mM HEPES, 100 mM NaNO3, 1:1, v/v, pH 7.2) solution. Excitation was provided at 495 nm and emission was integrated from 500 to 600 nm

The effect of anions must be considered when evaluating the response of fluorescent metal ion sensors. Lippard’s group has proposed that formation of an Hg-Cl bond or strong ion-pairing will influence the fluorescence turn-on degree in these systems [4446]. Lee [47] and our group [36] have also found that anions can control the fluorescence enhancement through formation of endo- or exo- metal complexes with Hg2+. So we investigated the fluorescence response of B3 toward Hg2+ in the presence of sodium salts of various anions such as NO3, CH3COO, SCN, ClO4, CO32−, H2PO4, Cl, and SO42−. None of the anions gave rise to interference (Fig. 3b) which suggest that B3 is applicable in complicated environmental samples.

It is known that the pH-insensitivity of fluorescence in near neutral and weakly acidic media is of importance for environmental and biological analyses. The common disadvantage of PET-based sensors is the interference of a proton, which also binds with the coordinate site, inhibits the PET process and enhances the fluorescence. In this case, the fluorescence intensity of free B3 reached a steady minimal value when pH > 5.0. The switching is reversible. This also demonstrates a typical PET fluorescence on/off effect. The resulting sigmoidal curve gives a pKa of B3 is 3.39 (Fig. 4). This indicates that B3 can work in near neutral and weakly acidic media, which is important for practical applications to environmental and biological analysis.
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Fig. 4

Dependence of the fluorescence intensity of free B3 on pH in ethanol/HEPES buffer (20 mM HEPES, 100 mM NaNO3, 1:1, v/v, pH 7.2) solution. [B3] = 10 μM, Excitation was provided at 495 nm and emission was integrated from 500 to 600 nm

For a fluorescent molecular sensor to be practically applicable, the detection limit is important. As seen in Fig. 5, the fluorescence intensity of the B3 solution was proportional to the amount of Hg2+ added in the range of ppb level (detection limit ≤2 ppb) indicating that B3 can detect environmentally relevant concentrations of Hg2+. B3 is much more sensitive to Hg2+ than our previous sensors B1 and B2.
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Fig. 5

The changes of fluorescence intensity of B3 (5 μM) upon addition of Hg2+ (0–12 ppb) in ethanol/HEPES buffer (20 mM HEPES, 100 mM NaNO3, 1:1, v/v, pH 7.2) solution

Fluorescence Detection of Hg2+ in Natural Water Samples

A variety of natural and anthropogenic environmental contaminants pose serious problems for human health and ecology. Environmental application presents a unique set of challenges and requires detailed studies of sensor performance in the environmental samples [48]. Therefore, we next proceeded to test the sensor on natural water samples. All these studies were conducted on pure natural water without any organic solvent. We chose samples from three different sources: the seawater from Yellow Sea (Dalian, China), pool water and tap water. The Environmental Protection Agency (U.S. EPA) standard for the limit of inorganic Hg in industrial waste water is no more than 50 ppb [49]. As shown in Fig. 6a, about 3.9-fold (SW), 4.5-fold (PW), 2.9-fold (TW)enhancement of fluorescence intensity were displayed when 50 ppb of Hg2+ was added in water with B3, respectively. Furthermore, F/F0 in natural water samples are linearly proportional to the amount of Hg2+ (Fig. 6b). The result shows that B3 is capable of distinguishing between the safe and toxic levels of Hg2+ in more complicated natural water systems.
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Fig. 6

a The linear fluorescence enhancement (F/F0) of B3 (10 μM) upon addition of Hg2+ to different natural water samples: pool water (PW, black circles), seawater (SW, red square), and tap water (TW, green triangles). The response (F) is normalized to the emission of the free B3 (F0). The samples were excited at 495 nm, and the emission intensities were recorded at 513 nm. b Fluorescence response of B3 (10 μM) to Hg2+ (50 ppb) in different natural water samples

Fluorescent Detection of Hg2+ in Sulfur-Rich Environments

It was known that cysteine could form a stable complex with Hg2+ [4447]. Therefore, for the next detection of Hg2+ in sulfur-rich environment, we investigated the effect of cysteine on the detection of Hg2+ in buffer solutions. As a comparison, we also investigated the effect of S2− and EDTA.

As shown in Fig. 7a, when added Hg2+ (50 μM) into the mixed solution of B3 (10 μM) and cysteine (50 μM), the obvious fluorescence enhancement (blue bar) was observed, which is very close to the enhancement induced by Hg2+ only (grey bar). When added S2− (50 μM) and EDTA (50 μM) into the solution of B3-Hg2+, respectively (Fig. 7b), the fluorescence enhancement only showed a slight change, indicating that the complex B3-Hg2+ was very stable and that the sensor B3 could detect Hg2+ in the sulfur-rich environment.
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Fig. 7

a Fluorescence responses of B3 (10 μM) to Hg2+ (50 μM) in the presence of cysteine (50 μM) in ethanol/HEPES buffer (20 mM HEPES, 100 mM NaNO3, 1:1, v/v, pH 7.2) solution. b Fluorescence spectral changes of B3-Hg2+ upon addition of excess S2−(50 μM) and EDTA (50 μM) ethanol/HEPES buffer (20 mM HEPES, 100 mM NaNO3, 1:1, v/v, pH 7.2) solution

Conclusions

We have demonstrated a BODIPY derivative B3 as a fluorescence turn-on sensor for Hg2+. This sensor exhibits very high selectivity and sensitivity for Hg2+ in the presence of various metal ions and anions in the aqueous solution. Moreover, it also performed well in natural conditions and sulfur-rich environments. Due to these excellent properties, B3 can be further applied for the detection of Hg2+ in really environmental samples.

Acknowledgements

This work was supported by NSF of China (21076032, 21136002 and 20923006), National Basic Research Program of China (2009CB724706), Scientific Research Fund of Liaoning Provincial Education Department (LS2010040).

Copyright information

© Springer Science+Business Media, LLC 2012