A Sensitive Ratiometric Fluorescent Sensor for Zinc(II) with High Selectivity

Abstract

A new fluorescent Zn²⁺ chemosensor (P1) based on a functionalized porphyrin was synthesized and characterized. P1 displayed dramatic ratiometric variations in absorption and fluorescent emission spectra upon exposure to Zn²⁺ due to the formation of a 1:1 Zn²⁺/P1 complex. The sensor also exhibited high selectivity and sensitivity toward Zn²⁺ over other common metal ions in the physiological pH range with a detection limit of 1.8 μM. The sensor showed fast response times and excellent reproducibility, thus confirming its potential applicability as a fluorescent sensor for Zn²⁺ sensing.

1. Introduction

Zinc ions (Zn²⁺), the second most abundant transition-metal ions in the human body, play diverse roles in biological processes, such as gene expression, metalloenzyme function, and neurotransmission [1–3]. Though zinc is a relatively nontoxic element, it can be toxic if consumed in large enough quantities. For example, zinc is a metal pollutant of environment, significant concentrations of which may reduce the soil microbial activity causing phytotoxic effects and it is a common contaminant in agricultural and food wastes. Consequently, the exploitation of chemosensors with high selectivity and sensitivity for detecting trace amounts of Zn²⁺ has attracted increased research interest. Given that Zn²⁺ does not produce spectroscopic or magnetic signals because of its 3d¹⁰4s⁰ electronic configuration, the fluorescence method is the primary method of choice for Zn²⁺ determination [4,5]. Several studies have reported the successful application of fluorescent sensors based on quinoline [6,7], fluorescein [8,9], coumarin [10], indole [11], naphthalimide [12], and peptides [13] in the detection of Zn²⁺. However, the development of new small molecular fluorescent sensors with extremely high sensitivity for Zn²⁺ and high selectivity over other relevant metal ions remains desirable.

Among various fluorescent sensors currently available, porphyrin and its derivatives are the first class of probes to be developed for Zn²⁺. These organic compounds exhibit good photostability, high absorption coefficients in the region from 400 nm to 450 nm (visible range), large Stokes shifts that minimize the effects of background fluorescence, and appreciable changes in spectral shift upon ligand binding [14–17]. Ratiometric fluorescence measurements have recently been conducted to detect metal ions. The method involves the observation of changes in the ratio of emissions at two wavelengths, thereby increasing the selectivity and sensitivity of determination and eliminating most or all of the possible variabilities arising from differences in instrumental efficiency and environmental effects [18–24]. In this study, we report a novel ratiometric fluorescent probe P1 for Zn²⁺ based on a porphyrin derivative (see Scheme 1). To achieve the requirement of high selectivity toward Zn²⁺, a strong chelator bearing three nitrogen atoms was incorporated into the porphyrin. These nitrogen atoms can form a cavity that can suitably contain Zn²⁺. The amine group of the chelator is a hydrophilic group, which can enhance the water solubility of the sensor [25].

2. Experimental Section

2.1. Materials and Apparatus

All chemicals were purchased from commercial suppliers. Methanol, ethanol, and acetonitrile were dried and distilled before use. N,N′-dimethylformamide (DMF) of analytical reagent grade was dried over 4 Å molecular sieves. All other chemicals were of analytical reagent grade and used without further purification, unless otherwise specified. 5-(4-Bromophenyl)-10,15,20-triphenylporphyrin (BrTPP) was synthesized using a reported procedure [26]. The ¹H-NMR spectra were obtained on a Bruker (Advance DMX500) NMR spectrometer. Mass spectra (MS) were recorded with a Bruker Esquire 3000 Plus spectrophotometer (Bruker-Franzen Analytik GmbH, Bremen, Germany). IR spectra were recorded on a spectrometer (Nicolet, Nexus-470, Madison, WI, USA) for the compounds in the solid state, prepared as KBr discs. Ultraviolet visible (UV−vis) absorption spectra were obtained using a Shimadzu UV 2450 spectrophotometer (Shimadzu, Kyoto, Japan). Fluorescence spectra measurements were performed on a Shimadzu RF-5301 PC (Shimadzu) fluorescence spectrophotometer. Both excitation and emission slits measured 3 nm. All pH measurements were made with a pH-10C digital pH meter. All titration experiments were run twice to generate reliable data.

2.2. Synthesis of P1

BrTPP (2.08 g, 3 mmol) and ethylenediamine (6.0 mL, 90 mmol) were dissolved in DMF (15 mL). Then CuSO₄·5H₂O (0.1 g, 0.4 mmol) was added to this solution. The resulting mixture was heated under reflux for 5 h. After cooling to room temperature, the solution was poured into water (100 mL). The precipitate was collected by filtration, washed with water, and dried to yield a purple powder. The product was purified by silica gel column chromatography using dichloromethane/methanol (15:1, v/v) as an eluent to afford 5-[4-(aminoethylene)amino]-10,15,20-triphenylporphyrin (ATPP). Yield: 61%. ¹H-NMR (CDCl₃, δ): 8.81 (s, 4H), 8.65 (s, 4H), 8.29 (m, 3H), 7.67–7.80 (m, 12H), 7.61–7.67 (m, 4H), 6.98 (m, 1H), 4.12 (s, 2H), 3.59 (m, 2H), 2.68 (m, 2H), −2.76 (s, 2H); MS (ESI): 673.2 [M + 1] ⁺; IR (KBr, ν) 3,306, 3,267, 3,227, 3,005, 2,926, 1,080, 899 cm⁻¹ (see Figures S1–S3 in Supplementary Material). For P1 synthesis, ATPP (0.404 g, 0.6 mmol), ortho-aminophenol (0.055 g, 0.5 mmol), and potassium iodide (0.008 g, 0.05 mmol) were added to an acetonitrile solution (30 mL). The reaction mixture was stirred and heated by refluxing for 6 h under a nitrogen atmosphere. After removal of the solvent, the crude mixture was purified by silica gel column chromatography using dichloromethane/methanol (12:1, v/v) as the eluent, thus yielding compound P1. Yield: 47%. ¹H-NMR (CDCl₃, δ): 8.83 (s, 4H), 8.70 (s, 4H), 8.31 (m, 3H), 7.61–7.71 (m, 12H), 7.57–7.59 (m, 4H), 7.06 (s, 2H), 6.74–6.92 (m, 4H), 5.32 (s, 2H), 3.94 (m, 4H), −2.81 (s, 2H); MS (ESI): 764.3 [M + 1] ⁺; IR (KBr, ν) 3,452, 3,417, 3,356, 3,030, 2,912, 1,489, 867 cm⁻¹ (see Figures S4–S6 in Supplementary Material).

2.3. Measurement Procedures

Stock solutions of 20 μM P1 and 1.0 × 10⁴ μM Zn²⁺ were prepared by dissolving P1 in absolute ethanol and ZnSO₄·7H₂O in doubly distilled water, respectively. The latter solution was further diluted stepwise to yield working solutions with concentrations ranging from 1.0 × 10³ μM to 1.0 μM. The wide-pH range solutions were prepared by adjusting 5.0 × 10⁴ μM Tris–HCl solution with HCl or NaOH solution. Solutions of other metal ions were prepared by dissolving NaCl, KCl, CaCl₂, Cd(NO₃)₂·4H₂O, Cr(NO₃)₃·9H₂O, Co(NO₃)₂·6H₂O, MgSO₄, Cu(NO₃)₂·3H₂O, Pb(NO₃)₂, MnSO₄·H₂O, FeSO₄·7H₂O and HgCl₂ in doubly distilled water (1.0 × 10³ μM).

A complex solution of Zn²⁺/P1 was prepared by adding 5.0 mL of the stock solution of P1 and 1.0 mL of the stock solution of Zn²⁺ to a 10.0 mL volumetric flask. The mixture was then diluted to 10.0 mL with Tris–HCl buffer solution (pH 7.4). The final concentrations of P1 and Zn²⁺ in the complex solutions were 10 μM and 100 μM–0.1 μM, respectively. The solution was protected from light and kept at 4 °C until use. A blank solution of P1 was prepared under the same conditions without Zn²⁺.

3. Results and Discussion

3.1. Preparation and Characterization of P1

P1 was easily synthesized with 47% yield by a two-step procedure [27,28]. The absorption spectrum of P1 displayed the characteristic transitions of porphyrin with an intense Soret band at 422 nm and four weak Q-bands at 519, 555, 594, and 650 nm (see Figure 1(a)). This result is in agreement with our finding that P1 exhibits a typical fluorescence peak at 650 nm when excited at 420 nm (see Figure 1(b)). Tetraphenylporphyrin was used as a standard (Φ_ref = 0.15) [29], and the fluorescence quantum yield (Φ_F) of P1 in N,N′-dimethylacetamide was determined to be 0.20, according to the method described by Demas and Crosby [30].

3.2. Sensing Properties of P1 to Zn²⁺

The mode of coordination of P1 with Zn²⁺ was investigated by spectrophotometric titration. Upon the gradual addition of Zn²⁺ (0.0–10.0 equiv.) to P1 (10 μM) solution, the Soret band of P1 showed a gradual bathochromic shift from 422 nm to 436 nm with an isosbestic point at 426 nm and a decrease in the Q_y(0–0) band at 519 nm (see Figure 2). This result indicates that a new species is only produced during the titration [21–24,31–36]. The changes in the UV–vis spectrum of the solutions can be interpreted as the electron rearrangement in P1 through strong Zn²⁺ coordination. To further understand the binding behavior and determine the stoichiometry of the formed complex, the Job's plot for the absorbance was determined by keeping the sum of initial concentrations of Zn²⁺ and P1 constant at 10 μM and changing the molar ratio of Zn²⁺ (X_M = ([Zn²⁺]/([Zn²⁺] + [P1])) from 0 to 1. As shown in Figure 3, a plot of absorbance at 436 nm versus X_M shows that the absorbance value is highest at a molar fraction of ca. 0.5, indicating that the complex formed between P1 and Zn²⁺ follows a 1:1 stoichiometry [37,38].

The sensing properties of P1 towards Zn²⁺ were also investigated by emission spectral titration. Figure 4 reveals that P1 could be developed as a ratiometric sensor for Zn²⁺. Upon the gradual addition of Zn²⁺ (0.0–10.0 equiv.) to a P1 solution (10 μM), the emission peak of P1 at 650 nm decreased with the concomitant formation of new peak at 610 nm. When Zn²⁺ ions were further added to the P1 solution, the ratiometric change in fluorescence spectra became evident with a clear isoemission point at 636 nm. The Zn²⁺-induced emission shift of P1 also confirmed the formation of a Zn²⁺/P1 complex. To investigate the binding behavior of P1 with Zn²⁺, ¹H-NMR spectra of P1 in the absence and presence of Zn²⁺ were recorded at room temperature as shown in Figure S7. Upon addition of 1.0 equiv of Zn²⁺, the protons signals for the H2, H3 and NH₂ groups underwent distinct downfield shifts due to the deshielding effect of the metal ion, implying that the three N atoms (labeled N1, N2, and N3 in Scheme 2) should be involved directly in Zn²⁺ coordination. In addition, the coordination of Zn²⁺ by the N2, N3 atoms promoted a downfield shift for all aromatic protons (H4-H7) in the phenyl ring of the ligating group. Thus, we can deduce a possible coordination mode between Zn²⁺ and P1, as shown in Scheme 2. The three N atoms (labeled N1, N2, and N3 in Scheme 2) participate in the coordination with Zn²⁺. The fourth ligand (X) was considered to be SO₄²⁻ by consideration of the charge balance in the solution [3,39]. Plotting the concentrations of Zn²⁺ against the variation in absorbance (ΔA), which refers to the difference in the absorbance intensities of P1 at 436 nm before and after exposure to various concentrations of Zn²⁺, yields the following equation [40]:

$$\mathit{\text{Absorbance}}=0.113\times \mathit{\text{concentration}}+0.152$$

(1)

(correlation coefficient = 0.9963). The calculated detection limit was 1.8 μM.

We investigated the time evolution of the response of P1 to 2.0 equiv. of Zn²⁺ in EtOH/H₂O solution (1:1, v/v) (see Figure S8 in Supplementary Material). The interaction of P1 with Zn²⁺ was completed in less than 2 min. Therefore, this system could be used for real-time tracking of Zn²⁺.

The optical responses of P1 to Zn²⁺ were found to be fully reversible. When ethylene diamine tetraacetic acid (EDTA) was subsequently added to the complexed solution of P1 and Zn²⁺, an absorption peak at 422 nm and a fluorescence signal at 650 nm were completely recovered. This result demonstrates that the binding between P1 and Zn²⁺ is chemically reversible and not a cation-catalyzed reaction (see Figures 2 and 4).

3.3. Effect of pH

The effects of pH on the fluorescence intensity of P1 at 610 nm in the presence of Zn²⁺ were determined in the pH range from 3.0 to 11.0 with the concentration of Zn²⁺ fixed at 10 μM (see Figure 5). The fluorescence intensity of P1 at 610 nm decreased with decreasing pH value, which may be caused by the protonation of P1. On the other hand, high pH values led to the precipitation of Zn(OH)₂, which, in turn, reduced complexation with P1. At pH values ranging from 6.5 to 8.5, acidity did not appear to affect the determination of Zn²⁺ with P1 [41]. Therefore, the response behavior of P1 is pH independent within the relevant biological pH range. These results indicate that the proposed sensor is convenient for practical applications of Zn²⁺ determination from actual samples.

3.4. Selectivity

High selectivity is a necessary feature of excellent chemosensors. The selectivity of P1 to Zn²⁺ ions and competition with other metal ions were determined by fluorescence measurements. A series of metal ion interferences (1.0 × 10³ μM) and their mixture were added to a solution of P1 (10 μM). Figure 6 indicated that only the addition of Zn²⁺ showed distinct ratiometric responses. Physiologically important metal ions found in living cells, such as Ca²⁺, Mg²⁺, Na⁺, Fe²⁺ and K⁺, were non-responsive to the probe. Most heavy and transition metal ions, such as Pb²⁺, Hg²⁺, and Cu²⁺, also showed no interference. More importantly, the addition of Cd²⁺, which is recognized as a typical competing ion of Zn²⁺ sensors [2,42,43], led to no obvious variation in the ratio of emission intensity. These results unambiguously demonstrate that the sensing of Zn²⁺ by P1 is hardly affected by common coexisting metal ions even when the concentration of these ions is 100 times higher than that of Zn²⁺ (see Figure 6 caption).

4. Conclusions

In conclusion, we have designed and synthesized a new ratiometric fluorescent chemosensor P1 that shows high selectivity, sensitivity to Zn²⁺, and the absence of interference from other competing cations, especially from Cd²⁺. P1 behaves like a tridentate ligand, in which Zn²⁺ is bound with three nitrogen atoms. Zn²⁺/P1 complexation quenches the fluorescence of porphyrin at 650 nm and induces a new fluorescence peak at 610 nm. Moreover, the chemosensor was pH independent under physiological conditions, indicating that it has valuable potential application in biological systems.