A Fluorescence Turn-on Sensor for Hg2+ with a Simple Receptor Available in Sulphide-Rich Environments
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- Fan, J., Peng, X., Wang, S. et al. J Fluoresc (2012) 22: 945. doi:10.1007/s10895-011-1033-x
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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.
KeywordsFluorescent sensor2-aminophenolHg2+Sulphide-rich environments
Hg2+, a highly toxic heavy metal ion, seriously threatens many environmental and biological systems . Today, mercury is present in daily life, such as in thermometers, batteries and electronic equipment [2–4]. The misuse of these products can lead to mercury leaks. Other sources such as volcanic emissions, combustion of fossil fuels, especially mining , also cause high concentrations of mercury in many environmental compartments  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 [8–13]. 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 [14–23], a ratiometric fluorescent probe based on FRET [24, 25], a colorimetric sensor based on ruthenium complexes  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+ , 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 [30–35] including our two Hg2+ fluorescent sensors B1  and B2 . Furthermore, 2-aminophenol has been proved to form a stable complex with Hg2+ in ethanol solution . 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.
Materials and Apparatus
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)  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
Fluorescence Detection of Hg2+ in Ethanol-Water Solution
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 [44–46]. Lee  and our group  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.
Fluorescence Detection of Hg2+ in Natural Water Samples
Fluorescent Detection of Hg2+ in Sulfur-Rich Environments
It was known that cysteine could form a stable complex with Hg2+ [44–47]. 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.
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.
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).