Mercury pollution is a global problem,and a major source of human exposure stems from contaminated waters ^{[1, 2]}. The terrific toxicity of mercury results from its high affinity for thiol groups in proteins and enzymes,leading to the dysfunction of cells and consequently causing health problems ^[3]. Inorganic mercury,including Hg(0) and Hg(II),is released into the environment through a wide variety of ways. Industrial sources of mercury include coal and gold mining,solid waste incineration,wood pulping,fossil fuel combustion,and chemical manufacturing. As the results of numerous applications in industry and agriculture,mercury has badly contaminated rivers and soil and accumulated in agricultural products and aquatic products,which causes serious environmental and health problems directly or indirectly ^[4]. The U.S. Environmental Protection Agency (EPA) standard for the maximum allowable level of inorganic mercury in drinking water is 2 ppb ^[5]. Moreover,a significant problem stemming from the ecological oxidation chemistry is that bacteria living in the sediments of aqueous environments transform inorganic Hg²⁺ into methylmercury,a potent neurotoxin that concentrates through the food chain in the tissues of fish and marine mammals ^[6]. Subsequent ingestion of methylmercury by humans from seafood and other dietary and environmental sources is connected to serious sensory,motor,and cognitive disorders. Therefore,in the interests of safety the detection of Hg²⁺ is still of great importance.

Among the available techniques to detect and quantify Hg²⁺,colorimetric chemosensors,so-called ‘‘naked eye chemosensors’’,have been developed and are attracting increasing interest ^{[7, 8]}. This sensing approach has clear potential application in the development of commercial Hg²⁺ indicators,such as paper test strips that can be evaluated visually without illumination ^{[7, 8]}. Water solubility is arguably a less important criterion for colorimetric chemosensors than for fluorescent ones,which are better suited for in vivo work ^{[9, 10]}. As long as the colorimetric indicator responds to Hg²⁺ in the presence of water and can be absorbed onto a paper strip or into a membrane or film,it has potential. Therefore,we herein communicate our studies on colorimetric Hg²⁺ detection,based on the new water-soluble PODIPY dye.

Our recent research interest lies in the novel BODIPY/aza- BODIPY family of fluorescent dyes and their application ^{[11, 12, 13]},and very recently we developed a type of PODIPY dye ^[14]. The new PODIPY 1 (Fig. 1) can be successfully synthesized by the reaction of dipyrromethene and azadipyrromethene with POCl₃ in the presence of Et₃N. The new PODIPY had photophysical properties similar to related PODIPYs/aza-PODIPYs ^{[15, 16, 17, 18, 19, 20, 21]},with a large Stokes shift and moderate fluorescence quantum yield. The dye 1 is more polar than BODIPYs,increasing its water solubility. Moreover,the interaction between the p-orbital of the phosphorus atom and the lone pair electrons of the nitrogen atom,and many resonators of the limit structure of charge separation in dye 1,may also enhance the stability in water. Based on the photophysical properties of 1,we made further efforts for the modifications in PODIPY as potential chemosensors and probes. Because of the low fluorescence quantum yield (Φ_f = 0.17) of PODIPY 1 ^[14] and the additional fluorescence quenching by detecting Hg²⁺,the investigation of emission spectra becomes meaningless and was abandoned. Delightedly,we found that PODIPY 1 can be used as a chemosensor for the colorimetric Hg²⁺ detection.

Fig. 1

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Fig. 1.Structure of PODIPY dye 1.

¹H NMR spectra were recorded on a Bruker AVANCE III 500 MHz spectrometer. ¹H NMR chemical shifts (δ) are given in ppm downfield from Me₄Si,determined by chloroform (δ = 7.26 ppm). ¹³C NMR spectra were recorded on a Bruker AVANCE III 125 MHz spectrometer. ¹³C NMR chemical shifts (δ) are reported in ppm with the internal CDCl₃ at δ 77.0 ppm as standard. ESI was measured by LCQ Deca XP. Tetrahydrofuran (THF) was freshly distilled from Na/benzophenone,n-hexane was distilled over Na,and other solvents were distilled over CaH₂. Merck silica gel 60 was used for the column chromatography. Fluorescence spectra were recorded on an F-4600 spectrophotometer. UV/Vis spectra were recorded on a UV-2550 spectrophotometer at room temperature. The pH measurement was performed with a PHS-3E pH meter. The refractive index of the medium was measured by 2W Abbe’s refractometer at 20 ℃.

2.4-Dimethylpyrrole (0.51 mL,4.9 mmol) and paranitrobenzaldehyde (300 mg,1.9 mmol) were dissolved in 20 mL of absolute CH₂Cl₂ under a N₂ atmosphere. One drop of trifluoroacetic acid (TFA) was added and the solution was stirred at room temperature overnight. When TLC monitoring (silica; CH₂Cl₂) showed complete consumption of the benzaldehyde,a solution of 2,3-dichloro-5,6- dicyano-1,4-benzoquinone (DDQ) (900 mg) in CH₂Cl₂ (10 ml) was added,and stirring was continued for 1 h. The reaction mixture was washed with water,dried over MgSO₄,filtered,and evaporated. The crude compound was purified by column chromatography on aluminum (CH₂Cl₂/n-hexane,1:1). This solid and triethylamine (2 mL) were dissolved in 50 mL of CH₂Cl₂ under air and the solution was stirred at room temperature for 10 min. POCl₃ (4 mL) was added,and stirring was continued for 1 h. The reaction was slowly quenched with crushed ice,extracted with CH₂Cl₂,and purified by recrystallization from CH₂Cl₂/n-hexane to afford 1 (171.2 mg,23.5%) as red solids. ¹H NMR (500 MHz,CDCl₃): δ 8.37 (d,2H,J = 8.0 Hz),7.56 (d,2H,J = 8.0 Hz),6.24 (s,2H),2.56 (s,6H),1.39 (s,6H). ¹³C NMR (125 MHz,DMSO-d6): δ 156.5,153.2,142.6,133.9,131.5,129.5,123.8,120.8,99.3,13.8,13.7. ³¹P NMR (202 MHz,CDCl₃): δ -50.011. FTMS-MALDI (m/z): calcd. for C₁₉H₁₉N₃O₄P: 384.1113 [M+H]⁺,found: 384.1085; calcd. for C₁₉H₁₉N₃O₂: 322.1511 [M-PO₂+H]⁺; found: 322.1525.

Stock solutions (4 × 10^-4 mol/L) of the salts of HgCl₂,Al(NO₃)₃,AgNO₃,CoCl₂,MnCl₂,PbCl₂,CuCl₂,MgCl₂,NiCl₂,FeCl₃,ZnCl₂,CaCl₂,PdCl₂,CrCl₃,KCl,and NaCl in H₂O were prepared. PODIPY 1 (1 × 10^-4 mol/L) was also prepared in CH₃CN. Test solutions were prepared by placing 40 μL of the sensor stock solution into a test tube,then adding an appropriate aliquot of each metal stock (0 - 1.0 mL) and diluting the solution to 4 mL with CH₃CN/H₂O (1/9,v/v).

Frontier molecular orbitals have been performed at the Becke3LYP (B3LYP) level of the density functional theory. The SDD basis set are used to describe Hg and P and 6-31G(d) basis set was used for all the other atoms (see Supporting information).

The sensitivity of phosphorus-containing PODIPY 1 (λ_abs = 507 nm) was first studied by the UV-visible absorption response toward various concentrations of Hg²⁺ in CH₃CN/H₂O at pH 7.2 (Fig. 2) ^[22]. As can be observed,a distinct response of 1 μmol/L PODIPY 1 to Hg²⁺ in the concentration range of 0- 20 μmol/L was discovered. When 10 equiv. of Hg²⁺ (10 μmol/L) was added,the reduction of the absorption intensity (λ_abs = 550 nm) was minimal,and a further increase in of Hg²⁺ concentration did not provide further reduction (Fig. 2). Based on a linear range for Hg²⁺ covering from 0.1 μmol/L to 3 μmol/L (inner panel of Fig. 2),the detection limit (3σ/k) was found to be 0.5 μmol/L ^[23].

Fig. 2

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Fig. 2.Absorption change of 1 μmol/L PODIPY 1 after the addition of increasing amounts of Hg2+ (0, 0.1, 0.25, 0.5, 1, 2, 3, 4, 5, 6, 8, 10, 12, 15, 18 and 20 μmol/L) in CH3CN/H2O (1:9, v/v) at room temperature. The inner panel displays the absorption intensity (λabs = 507 nm) of 1 μmol/L PODIPY 1 toward Hg2+ at 0, 0.1, 0.25, 0.5, 1, 2, 3, 4, 5, 6, 8, 10, 12, 15,18 and 20 μmol/L.

In fact,the presence of Hg²⁺,can be detected without the use of any instrumentation as illustrated in Fig. 3,with the color change from pink to violet red. The selectivity toward mercury has been demonstrated even in the presence of similar amounts of other metal ions,such as Na⁺,K⁺,Mg²⁺,Al³⁺,Cu²⁺,Fe³⁺,Zn²⁺,Ba²⁺,Ag⁺,Mn²⁺,Ni²⁺,Co²⁺,Cr³⁺,Pb²⁺ and no metal (Fig. 3).

Fig. 3

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Fig. 3. Digital picture of PODIPY 1 (1 μmol/L) in CH3CN/H2O (1:9, v/v). From left to right: Na+, K+, Mg2+, Al3+, Cu2+, Fe3+, Zn2+, Ba2+; no-metal, Hg2+, Ag+, Mn2+, Ni2+, Co2+, Cr3+, Pb2+, equivalent amounts of all the cations and Hg2+. The cation total concentration was 10 μmol/L.

Furthermore,the selectivity of the chemosensor 1 toward other metal ions was investigated with UV-visible spectroscopy. As shown in Fig. 4,PODIPY 1 was highly selective to Hg²⁺ with a remarkable absorption change. In contrast,addition of other relevant metal ions,including Na⁺,K⁺,Mg²⁺,Al³⁺,Cu²⁺,Fe³⁺,Zn²⁺,Ba²⁺,Hg²⁺,Ag⁺,Mn²⁺,Ni²⁺,Co²⁺,Cr³⁺,and Pb²⁺,caused almost no absorption change.

Fig. 4

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Fig. 4. Absorption spectra of PODIPY 1 (0.5 μmol/L) before and after the addition of metal salts (10 μmol/L) of Na+, K+, Mg2+, Al3+, Cu2+, Fe3+, Zn2+, Ba2+, Hg2+, Ag+, Mn2+, Ni2+, Co2+, Cr3+, Pb2+ in CH3CN/H2O (1:9, v/v).

Some ions,for example,Zn²⁺,Ag⁺,Fe³⁺,Ni²⁺,Cr³⁺,or Pb²⁺,often interfere with the selectivity of Hg^{2+ [24, 25]}. Therefore,to investigate the selectivity for Hg²⁺ in a complex background of potentially competing species,the absorption change of PODIPY 1 with Hg²⁺ was examined in the presence of other metal ions. From Fig. 5,we observed that there was no interference in the detection of Hg²⁺ in the presence of Zn²⁺,Ag⁺,Fe³⁺,Ni²⁺,Cr³⁺,or Pb²⁺,and the absorption spectra were almost identical to those obtained in the presence of Hg²⁺ alone.

Fig. 5

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Fig. 5. The relative absorption of PODIPY 1 and its complexation with Hg2+ in the presence of various metal ions. PODIPY 1 + Hg2+; PODIPY 1 + Hg2++Mn+, where Mn+ = Zn2+, Ag+, Fe3+, Ni2+, Cr3+, and Pb2+ ions. Conditions: 0.3 μmol/L of PODIPY 1, 5 μmol/L of Hg2+ in the presence of 5 μmol/L of other metal ions.

The recognition mechanism of the chemosensor 1 with Hg²⁺ was investigated by FT-IR spectra (Fig. 6a). 1 equiv. of Hg²⁺ with PODIPY 1 (red curve) intensified and widened the peak at the characteristic stretching frequency (1346.2/cm) ^[26] of the P=O bond of POPDIPY 1 alone (black curve). This indicated a strong polarization of the P=O bond upon efficient binding to the Hg²⁺ ion (Fig. 6b).

Fig. 6

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Fig. 6. (a) IR spectra of compound PODIPY 1 (black curve) and PODIPY 1–Hg2+ complex (red curve) in KBr disks. (b) Structure of 1–Hg2+. (For interpretation of the references to color in this figure legend, the reader is referred to the web version of this article.)

To gain insight into the response of chemosensor 1 to Hg²⁺,chemosensor 1 and the corresponding metal complex species 1- Hg²⁺ were examined by density function theory (DFT) at the Becke3LYP (B3LYP) level of the Gaussian 03 program. The SDD basis set are used to describe Hg and P,and 6-31G(d) basis set was used for all the other atoms. As shown in Fig. 7,for chemosensor 1,the HOMO are distributed at the PODIPY core,and the LUMO are localized on the whole PODIPY structure including the pnitrophenyl group in the neutral compounds. In addition,the planar BODIPY fragment is almost coplanar to the adjacent pnitrophenyl part with a small dihedral angle (4°). While for the metal complex species 1-Hg²⁺,the HOMO is distributed in the left BODIPY core,and the LUMO is mostly located at the right BODIPY unit. Furthermore,the energy gap between the HOMO and LUMO of the metal complex species 1-Hg²⁺ (2.04 eV) is smaller than that of chemosensor 1 (2.18 eV) (Fig. 7),which is in good agreement with the red shift in the absorption observed upon treatment of chemosensor 1 with Hg²⁺.

Fig. 7

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Fig. 7. Frontier molecular orbitals of 1 and 1–Hg2+ at the Becke3LYP (B3LYP) level of the density functional theory with Gaussian 03. The SDD basis set is used to describe Hg and P, and 6-31G(d) basis set was used for all the other atoms. HOMO/LUMO (eV) = -5.96/-3.78 for 1; HOMO/LUMO (eV) = -5.84/-3.80 for 1–Hg2+.

Motivated by the obvious color change of the system in metal ions,test strips were prepared by immersing filter papers in the CH₃CN/H₂Osolution of1 (1.0 × 10^-5 mol/L) and then drying themin air. When each drop of 5.0× 10^-5 mol/L different metal ions was dripped into the 1-based test strips,a clear color change from pink to brown was observed in the presence of Hg²⁺,comparing to noncolor change for the other metal ions,such as Al³⁺,Cr³⁺ Ba²⁺,Pb²⁺ Ni²⁺ Mn²⁺,Cu²⁺,Ag⁺ and free (Fig. 8). Therefore,the 1-based test strips can conveniently detect Hg²⁺ in solutions without any additional equipment. The 1-based test strips were easily fabricated and low-cost,useful in practical and efficient Hg²⁺ test kits.

Fig. 8

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Fig. 8. Photographs of test strips 1 to one drop of different metal ions. Test strips were prepared by immersing filter papers in the CH3CN/H2O (1/1, v/v) solution of 1 (1.0 × 10-5 mol/L) and then drying them in air.

PODIPY 1 can detect Hg²⁺ by the color change from pink to violet red without the use of any instrumentation. PODIPY 1 was selective to Hg²⁺ with remarkable absorption change,and the addition of other relevant metal ions caused almost no absorption change. The phosphorus-containing PODIPY 1 was sensitive to various concentrations of Hg²⁺. From the changes in Hg²⁺- dependent absorption intensity,the detection limit was found to be 0.5 mmol/L. Using PODIPY 1 there was no interference in the detection of Hg²⁺ in the presence of other ions. FT-IR indicated a strong polarization of the P55O bond in PODIPY 1 upon efficient binding to Hg²⁺. The energy gap between the HOMO and LUMO of the metal complex 1-Hg²⁺ (2.04 eV) is smaller than that of chemosensor 1 (2.18 eV),which is in good agreement with the red shift in the absorption observed upon treatment of sensor 1 with Hg²⁺. The 1-based test strips were easily fabricated and low-cost,useful in practical and efficient Hg²⁺ test kits. Further efforts for development of probes based on PODIPY/aza-PODIPY in biotechnology are ongoing in our lab.

This work was supported by the Public Research Foundation of Liaoning Province for the Cause of Science (No. 2014003009),Science and Technology Key Project of Liaoning Province (No. 2013304007),the Foundation of the Education Department of Henan Province for Science and Technology Research Projects (No. 13A150046),the Scientific Research Foundation for the Returned Overseas Chinese Scholars,State Education Ministry,and the startup funds from Shenyang University of Chemical Technology.

Supplementary data associated with this article can be found,in the online version,at http://dx.doi.org/10.1016/j.cclet.2015.07.002.