ABSTRACT

A colorimetric and fluorometric dual-model probe for mercury (II) ion was developed employing fluorescein hydrazide (FH) in ethanol-HEPES solution (1:1, v/v, pH 8.0). The probe exhibited high selectivity and sensitivity for Hg²⁺ detection using UV/Vis and fluorescence spectroscopy. Addition of Hg²⁺ caused a visual color change from colorless to coloured and a fluorescence change from colorless to bright green. Other metal ions did not interfere with the detection of Hg²⁺.

1. Introduction

The development of selective and sensitive imaging tools capable of monitoring heavyand transition-metal ions has attracted considerable attention because of the wide use of these metal ions and their subsequent impact on the environment and nature [1-3]. Mercury pollution specifically is a topic of recent concern [4-6] because mercury contamination is widespread and originates from a variety of natural and anthropogenic sources including oceanic and volcanic emission [7,8], gold mining [9], solid waste incineration, and the combustion of fossil fuels [10]. Once introduced into the marine environment, bacteria convert inorganic mercury into methylmercury, which enters the food chain and accumulates in higher organisms, especially in large edible fish [11]. Mercury can accumulate in the human body and may cause wide variety of diseases even in a low concentration, such as prenatal brain damage, kidney failure [12], serious cognitive and motion disorders, and Minamata disease [13].

Many types of mercury sensors have been developed based on small fluorescent organic molecules [14-20], proteins [21], oligonucleotides [22,23], genetically engineered cells [24], conjugated polymers [25], foldamers [26], membranes [27], electrodes [28], and nanomaterials [29-32]. Recently, considerable efforts have been made to develop a colorimetric or fluorescent molecular probe for mercury ions [33-37]. Many of these systems are based on well established and unique molecular frameworks, such as crown ethers [38,39], calix[4] arenes [40], cyclams [41], squaraines [42], 8-hydroxyquinolines [43] 1,4-disubstituted azines [44], thioureas [45], 1,3-dithiole- 2-thione [46]. Before fluorescein hydrazide or other fluorescein derivations were used as sensors for Cu²⁺ [39,40]. However, in current work, we employed fluorescein derivation (Figure 1) to design and construct colorimetric and fluorometric dual-channel assay to specifically detect Hg²⁺ the presence of a wide range of other cations and anions in ethanol-HEPES (1:1, v/v, pH 8.0) solution. It is noted that FH was used as Cu²⁺ sensor by Chen et al. in pH 7.2 Tris buffer before, which there are many differences from those in the manuscript: a) sensor conditions are different; b) the sensor targets are different; c) UVVisible spectra are different; d) the system color changes are different; e) fluorescence properties and intensity are different. These studies have demonstrated for the first time a controllable and multifunctional chemosensor in different buffer conditions. Thus, the results are significant and interesting as a new generation of chemosensors produced.

2. Experimental

2.1. Reagents and Chemicals

4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid (HEPES) was purchased from Sigma-Aldrich. FH was synthesized using a modification of a literature method. HEPES solutions were adjusted to pH 8.0 by adding

NaOH (0.1 M) to aqueous HEPES (10 mM). Cationic salts were purchased from Shanghai Experiment Reagent Co., Ltd (Shanhai, China). All the common chemicals used were of analytical grade.

2.2. Apparatus

A Mettler Toledo pH meter (Mettler-Toledo International Inc, Switzerland) was used to determined pH. The UVVisible spectra were recorded on a Cary 50 Bio UVVisible spectrophotometer (Agilent, Santa Clara, CA). Fluorescence spectra were measured using a Cary Eclipse fluorescence spectrophotometer (Agilent, Santa Clara, CA). A PO-120 quartz cuvette (10 mm) was purchased from Huamei Experiment Instrrument Plants (Shanhai, China). ¹H NMR and ¹³C NMR spectra were recorded on a Bruker DRX-300MHz NMR spectrometer (Billerica, MA). A light yellow single crystal of FH was mounted on a glass fiber for data collection. Cell constants and an orientation matrix for data collection were obtained by least-squares refinement of diffraction data from reflections with 2.04˚ - 27.4˚ for FH using a Bruker SMART APEX CCD automatic diffractometer. Data were collected at 173 K using Mo Kα radiation (λ = 0.710713 Å) and the ω-scan technique and corrected for Lorentz and polarization effect (SADABS) [50]. The structures were solved by direct methods (SHELX97) [51], and subsequent difference Fourier map and then refined on F2 using a full-matrix least-squares procedure and anisotropic displacement parameters.

2.3. Preparation of FH

FH was prepared in high yield by reacting fluorescein with hydrazine hydrate in methanol (Scheme 1) according to the literature [52,53]. An excessive hydrazine hydrate (85%, 1.2 mL) was added to a 0.35 g of fluorescein dissolved in 20 ml of ethanol, and the reaction solution was refluxed in oil bath for 8 h. A brown oily product resulted from evacuating ethanol under reduced pressure. The solid product was precipitated by adding water and recrystallized from ethanol/water mixture, producing the fluorescein hydrazide (FH) as a yellow powder with 72% yield (0.25 g). The H₂O/ethanol solution was allowed to evaporate slowly at room temperature for several days and yellow crystals suitable for X-ray crystallography were formed. ¹H NMR ,(DMSO-d₆): δ (ppm) 9.80 (s, 2H), 7.76 (m, 1H), 7.48 (m, 2H), 6.99 (m, 1H), 6.58 (s, 2H), 6.43 (d, 2H), 6.38 (d, 2H), 4.37 (s, 2H); ¹³C NMR (75 MHz, CDCl₃): δ 24.25, 33.00, 113.31, 117.96, 121.58, 121.88, 123.80, 138.69, 156.24, 196.37 (Figure S1(a)); ESI-MS m/z 347.2 [FH+H]⁺ (calcd. 347.l) (Figure S1(b)); Elemental analysis (calcd.%) for C₂₀H₁₄N₂O₄: C, 69.36; N, 8.09; H, 4.07: Found: C, 69.30; N, 8.11; H, 4.01. Crystal data for C₂₀H₁₆N₂O₅ (Figure S1(c)): crystal size: 0.22 × 0.2 × 0.1, triclinic, space group P-1 (No. 2). a = 7.5959(15) Å, b = 10.690(2) Å, c = 11.028(2) Å, α = 104.34(3)˚, β = 109.09(3)˚, γ = 99.67(3)˚, V = 789.0(4) ų, Z = 2, T = 173K, θ_max  = 25.0˚, 7521 reflections measured, 2762 unique (R_int = 0.0412). Final residual for 250 parameters and 2503 reflections with I > 2σ(I): R₁ = 0.0622, wR₂ = 0.1390 and GOF = 1.17.

2.4. General UV-Vis and Fluorescence Spectra Measurements

Since the chemosensor was not fully soluble in 100% aqueous media, ethanol was used as a solubilizing medium. FH stock solutions were prepared in ethanol. The UV-Vis and fluorescence spectra were obtained in mixed ethanol /HEPES aqueous buffer (1:1, v/v, 10 mM, pH 8.0) solution. Aqueous metal ion solutions were also prepared. Fluorescence measurements were carried out with a slit width of 10 nm.

2.5. Preparation of FH

The UV-Vis spectrum was characterized by a main band centred at 641 nm. The low detection threshold for Hg²⁺ was in the order of 10⁻⁶ - 10⁻⁵ M and at this level the colour change was very obvious. The fluorescence emission was measured for each sample by exciting at 450 nm and spectra and measuring from 475 - 700 nm. The sensitivity range for Hg²⁺ was 10⁻⁷ - 10⁻⁶ M.

3. Results and Discussion

3.1. UV-Vis Spectra

The complexation ability of FH with Hg²⁺ ion was investigated by UV-Vis absorption techniques. FH does not absorb in the range of 400 - 800 nm in a mixed solution of ethanol/HEPES (v/v = 1:1). Figure 2 shows the spectral changes of FH in ethanol/HEPES (v/v = 1:1) upon addition of various competitive metal ions, such as Hg²⁺,

Mg²⁺, Ca²⁺, Cu²⁺, Fe³⁺, Zn²⁺, Ni²⁺, Bi³⁺, Co²⁺, VO²⁺, Mn²⁺, Ba²⁺, Cd²⁺, Pb²⁺, Sn²⁺, Yb³⁺, Cr³⁺, La³⁺, Er³⁺, etc. From UV/Vis spectra (Figure 2(a)), we can clearly observe a new absorption band centered at 397, 504 and 641 nm for FH (30 µM) in the presence of 1 equiv of Hg²⁺. In contrast, other ions lead to almost no spectral changes (Figures 2(a) and (b)). In our present experiments, HgCl₂ as a Hg²⁺ source was gradually added to the ethanol /HEPES (v/v = 1:1) FH solution. Notably, the new band at 397, 504 and 641 nm appeared and concomitantly grew with increasing Hg²⁺ concentration (Figure 2(c)).

3.2. Fluorescence Spectra

The ability of FH to selectively sense Hg²⁺ was determined analysis of the fluorescence spectra obtained with 1 μM of FH in ethanol/HEPES (v/v = 1:1) in the presence of a number of cations including Mg²⁺, Ca²⁺, Cu²⁺, Fe³⁺, Zn²⁺, Ni²⁺, Bi³⁺, Co²⁺, VO²⁺, Mn²⁺, Ba²⁺, Cd²⁺, Pb²⁺, Sn²⁺, Yb³⁺, Cr³⁺, La³⁺ and Er³⁺ etc. (100 equiv to Hg²⁺, respectively). The fluorescence spectra (Figure 3) show a similar result, which is consistent with that of UVVisible spectra. Addition of 3 equiv of Hg²⁺ ion results in an obviously enhanced fluorescence at 522 nm (OFFON), with an excitation at 460 nm while other ions induce no increase in fluorescence (Figure 3(a)). More interestingly, Hg²⁺-induced fluorescence-on change for the FH is visual with a solution color change from colorless to green under illumination with a 365 nm UV lamp (Figure 3(b)). As shown in Figure 3(c), a new emission band peak appears with the fluorescence intensity increasing with increase in Hg²⁺ concentration. Both UVVis and fluorescence results indicate that FH shows a good selectivity and sensitivity toward Hg²⁺ over other competitive cations.

Furthermore, a plot of fluorescence intensity when FH is titrated with 3 µM of Hg²⁺ shows good linearity (correlation coefficient of R = 0.9985) for a Hg²⁺ concentration range of 0.25 - 3 µM (Figure 4).

3.3. pH Dependent

The above-mentioned UV-Visible light absorption occurred at a pH of 8.0, which is close to physiological conditions. At a pH of 9.0, it seems that Hg²⁺ detection is possible, and that the absorbance was affected by solution alkalinity. At all other pH conditions, no notable change in either color or UV-Visible spectrum was noted (Figure 5).

3.4. Proposed Mechanism

To provide reasonable envidence of FH sensing of Hg²⁺ ion, electrospray ionization mass spectrometry (ESI-MS) analysis was conducted (Figure S2). Mass peaks at m/z 531.2 corresponding to [Fluorescein + Hg]⁺ are clearly

observed, This provides direct evidence for the proposed response mechanism (Scheme 2). The hydrolysis complex with fluorescein anion is responsible for the above dual color and fluorescence changes.

4. Conclusion

In summary, we have demonstrated a simple Hg²⁺-selective chromogenic and fluorogenic chemodo-simeter system using a fluorescein hydrazide (FH) molecule in semi-aqueous solution. The sensor mechanism was proposed to be mercury-promoted hydrolysis procedure of

FH. This is another case of chemodosimeters as Rhodamine B hydrazide sensor for Cu (II) [44]. Visual color and fluorescence response suggests the probe’s practicability for further environmental and biological mecury ions detection.

5. Acknowledgements

The work was supported by the National Natural Science Foundation of China (No. 21072119, 21102086), the Shanxi Province Science Foundation for Youths (No. 2012021009-4), the Shanxi Province Foundation for Returnee (No. 2012-007), the Taiyuan Technology star special (No. 12024703).

REFERENCES

Supporting Information (SI)

NOTES

^*Contributed equally to this work.

^#Corresponding authors.