Citation

Xue-Mei Li, Rui-Rui Zhao, Yan Yang, XueWei Lv, Yu-Ling Wei, Rui Tan, Jun-Feng Zhang, Ying Zhou. A Rhodamine-based fluorescent sensor for chromium ions and its application in bioimaging[J]. Chin. Chem. Lett., 2017, 28(6): 1258-1261  

Xue-Mei Li^a, Rui-Rui Zhao^b, Yan Yang^b, XueWei Lv^b, Yu-Ling Wei^a, Rui Tan^c, Jun-Feng Zhang^c, Ying Zhou^b    

Chromium, an essential trace element in human nutrition, has great impacts on the metabolism of carbohydrates, fats, proteins and nucleic acids [1]. Chromium exists principally as metallic (Cr⁰), trivalent (Cr³⁺), and hexavalent (Cr⁶⁺) forms, and the common Cr⁺³ is essential and found in most food and nutrient supplements. Insufficient dietary intake of Cr³⁺ leads to increases in risk factors associated with diabetes, cardiovascular disease and impaired immune function [2]. However, exposure to high levels of Cr³⁺ can also have an adverse effect on normal enzymatic activities and cellular structures.

Compared with many of the current techniques for chromium detection, fluorescence sensing is a simple, safe, effective and rapid detection method [3]. Owing to the tendency of the paramagnetic Cr³⁺ to quench fluorescence emission, even a number of research groups have reported their recent achievements on Cr³⁺-targeted fluorescent sensors the reports with a "turn-on" response are still limited [4]. We previously reported a series of rhodamine derivatives for the efficient detection of Fe³⁺ [5], Hg²⁺ [6], Cu²⁺ [7], and Zn²⁺ [8]. It has been proven that rhodamine framework is an ideal mode to construct chelation-enhanced fluorescence (CHEF) off-on fluorescent probes due to its particular structural property.

In present work, we synthesized a rhodamine-based probe (1) (Scheme 1) to recognize and determine Cr³⁺ at biological pH value in aqueous solution, depending on a fluorescence "off-on" mode. 1 showed a colorimetric and fluorescent selectivity for Cr³⁺ in CH₃CN/Tris-HCl (0.01 mol/L, pH 7.4; v/v = 9:1) solution over other common physiologically important metal ions. It showed that sensor 1 was a low toxic compound, and was successfully applied in the in vivo imaging of Cr³⁺ in C. elegans.

Scheme1

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Scheme1. Synthetic route of probe 1.

To clarify the selectivity of 1 with metal ions, the interferences of various analytes were studied in CH₃CN/Tris-HCl buffer (0.01 mol/L, pH 7.4, v/v = 9:1) solutions, including Na⁺, K⁺, Li⁺, Ag⁺, Hg²⁺, Fe³⁺, Cu²⁺, Co²⁺, Pb²⁺, Ni²⁺, Cd²⁺, Zn²⁺, Cr³⁺. All UV–vis absorption spectra were recorded after three minutes upon addition of 150 equiv of each of these ions. Under optimized conditions, the UV–vis response of 1 to different tested ions is shown in Fig. 1. Compound 1 exhibits a tiny absorption band at 528 nm. Upon addition of different metal ions, only the presence of Cr³⁺ could lead to an obvious absorption increase at 528 nm, with a shoulder band at 592 nm (Fig. 1). It revealed that 1 has a good absorption selectivity towards Cr³⁺ among the tested ions. In the titration tests, with the addition of Cr³⁺, the absorbance at 528 nm increased sharply (Fig. 2a), which induced a color change from colorless to pink (Fig. 2c).

Figure 1

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Fig. 1. (a) Absorption spectra of 1 (1.0 × 10-5 mol/L) in CH3CN/Tris-HCl (0.01 mol/L, pH 7.4, v/v = 9: 1) with 150 equiv. Cr3+; (b) Absorbance of 1 (1.0 × 10-5 mol/L) at 528 nm after addition of 150 equiv. selected ions (a: blank, b: Ag+, c: Na+, d: K+, e: Li+, f: Hg2+, g: Fe3+, h: Co2+, i: Pb2+, g: Ni2+, k: Cu2+, l: Cd2+, m: Zn2+, n: Cr3+).

Figure 2

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Fig. 2. . (a) Absorption titration spectra of 1 (1.0 × 10-5 mol/L) in the presence of varying concentrations of Cr3+ in CH3CN/Tris-HCl (0.01 mol/L, pH 7.4; v/v = 9:1); (b) Absorbance of 1 (1.0 × 10-5 mol/L) at 528 nm as a function of varying concentrations of Cr3+; insert: Picture of compound 1 (left) and compound 1 upon the addition of Cr3+ (150 × 10-5 mol/L) (right).

Then, fluorescent selectivity of compound 1 was evaluated in CH₃CN/Tris-HCl buffer (0.01 mol/L, pH 7.4, v/v = 9:1) solutions with the above mentioned ions. A clear "off-on" fluorescence changes of 1 indicated the high selectivity for Cr³⁺ over the other species tested (Fig. 3a). This may be ascribed to the unique 1-Cr³⁺ binding mode and the resulting specific C-N bond breaking in rhodamine fluorophore.

Figure 3

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Fig. 3. (a) Fluorescence emission spectra of 1 (1.0 × 10-5 mol/L) in CH3CN/Tris-HCl (0.01 mol/L, pH 7.4, v/v = 9:1) with 150 equiv. of Cr3+; (b) fluorescence intensity of 1 (1.0 × 10-5 mol/L) at 556 nm after addition of 150 equiv. selected ions. (a: blank, b: Ag+, c: Na+, d: K+, e: Li+, f: Hg2+, g: Cd2+, h: Cu2+, i: Pb2+, j: Co2+, k: Zn2+, l: Ni2+, m: Fe3+, n: Cr3+).

Compound 1 displays weak fluorescence. When 150 equiv. of Cr³⁺ was added to the solution, a significant increase of the fluorescence intensity at 556 nm, which was attributed to the Cr³⁺ induced ring-opening of the spirolactam moiety, was observed. The significant fluorescence enhancement of up to 13-fold with a bright yellow-green emission was shown in Fig. 4, which suggested a higher fluorescence selectivity of 1 towards Cr³⁺ compared to the other tested ions.

Figure 4

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Fig. 4. (a) Fluorescence titration spectra of 1 (1.0 × 10-5 mol/L) in the presence of varying concentrations of Cr3+ in CH3CN/Tris-HCl (0.01 mol/L, pH 7.4, v/v = 9:1). Excitation wavelength was 495 nm; (b) Fluorescence intensity of 1 (1.0 × 10-5 mol/L) at 556 nm as a function of varying concentrations of Cr3+; insert: Fluorescence picture of compound 1 (left) and compound 1 upon the addition of Cr3+ (150 × 10-5 mol/L) (right).

To understand the binding mode-fluorescence change relationship, the binding mechanism of compound 1 with Cr³⁺ was studied. According to the literature [9], a reasonable binding mechanism was proposed (Scheme 2). The spirolactam moiety of the rhodamine group acts as a signal switcher, when 1 binds Cr³⁺, the fluorescence-off state of 1 converts to the Cr³⁺-promoted ringopened amide form with a fluorescence-on state. In this work, 1 is most likely to bind Cr³⁺ via the hydrazide and quinoline N and O atoms like other reported researches [10]. The fluorescence intensity at 556 nm was plotted as a concentration of Cr³⁺, and detection limit was calculated to be 5.6 × 10^-6 L/mol by using detection limit 3σ/k: Where σ is the standard deviation of blank measurement, k is the slope between the fluorescence intensity versus Cr³⁺ concentration [11].

Scheme2

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Scheme2. The proposed binding mechanism of compound 1 with Cr3+.

To demonstrate the feasibility of 1 for its application in in vivo imaging, Cr³⁺ imaging tests were performed in C. elegans. In our previous work, we successfully tested and evaluated the mercury probes and their fluorescence imaging applications in cells, C. elegans and zebrafish [12]. Inpresent study, the application of 1 in in vivo imaging was evaluated by visualizing the distribution of Cr³⁺ in nematodes previously incubated with various concentrations of Cr³⁺ for 3 h.

C. elegans larvae at developmental stage 4 (L4) were incubated in Petri dishes filled with M9 buffer, which contained 1 (1.0 × 10^-5 mol/L), at 20 ℃ for 2 h. Almost no fluorescence could be observed in this case (Fig. 5d). Very weak fluorescence was observed in the pretreated nematodes until the concentration of Cr³⁺ reached 75 ×10^-5 mol/L (Fig. 5e). With the dose of Cr³⁺ was increased, fluorescence emission became brighter accordingly. Green fluorescence emission color was mainly observed in the intestinal part of the pretreated nematodes (Fig. 5f). It suggested that the aggregation of exogenous Cr³⁺ mainly locates in the intestine of C. elegans. These results showed that 1 is a good bonding ligand for Cr³⁺ and it could be used for fluorescence imaging and monitoring intracellular Cr³⁺ in C. elegans.

Figure 5

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Fig. 5. Fluorescent imaging (bottom) and phase contrast (top) for Cr3+ in C. elegans. (a) with 10-5 mol/L of 1 only; (b) with 75 ×10-5 mol/L of Cr3+ for 3 h and 10mmol/L of 1 for 2 h; (c) with 150 × 10-5 mol/L of Cr3+ for 3 h and 10mmol/L of 1 for 2 h.

In conclusion, a rhodamine-based probe (1) was studied for colorimetric and fluorescent sensing of Cr³⁺ in vitro and in vivo. The addition of Cr³⁺ induced ring-opening of the spirolactam moiety in 1 and generated a distinct "off/on" fluorescent change with a solution color change. A selective significant fluorescence enhancement of up to 13-fold, with a bright yellow-green emission, was only observed in the case of Cr³⁺, among all the tested ions. Compound 1 was successfully applied in the in vivo fluorescent imaging of Cr³⁺ in C. elegans. The low toxicity, high penetrability and selectivity of 1 supported its further utility in Cr³⁺ tests of polluted environmental substances and tobacco samples.

All reagents were of analytical grade or the best grade commercially available, and were put into use without further purification. Deionized water was used throughout. Tris-HCl buffer solutions (0.01 mol/L, pH 7.4) were prepared in deionized water. Analyte solutions of the perchlorate of Na⁺, K⁺, Li⁺, Ag⁺, Co²⁺, Ni²⁺, Cu²⁺, Zn²⁺, Pb²⁺, Cd²⁺, Fe³⁺, Hg²⁺ and Cr³⁺ were prepared by dissolving the salts in distilled water to final concentrations of 0.1 mol/L.

A solution of 2-aminopyridine (188 mg, 2 mmol) and 0.3 mL trimethylaminein was stirred for 30 min at ℃ in CH₂Cl₂. Then chloroacetyl chloride (0.175 mL, 2.2 mmol) was added to the solution, and the mixture was stirred for 3 h at room temperature. After the reaction, the solution was extracted with H₂O (5 mL × 3), then the combined organic layer was concentrated under reduced pressure. The crude product was purified by column chromatography with CH₂Cl₂/EtOAC (v/v, 3:1) to get the target compound 5 (270 mg, 78.5%). ¹H NMR (500 MHz, CDCl₃): δ 8.87 (s, 1H), 8.33-8.32 (d, 1H, J = 5), 8.21-8.18 (d, 1H, J = 15), 7.77-7.72 (m, 1H), 7.09-7.13 (m, 1H), 4.21 (s, 2H).

A solution of compound 4 [6] (584 mg, 1 mmol), K₂CO₃ (276 mg, 2 mmol) and NaI (84 mg, 0.56 mmol) in 30 mL acetone was stirred for 1 h. Then compound 5 (205 mg, 1.2 mmol) was added to the stirred solution and the mixture was refluxed for 12 h under N₂ atmosphere. The solution was then cooled to 0 ℃ and adjusted to neutral condition with 1 mol/L NH₄Cl. The solution was extracted with CH₂Cl₂ (10 mL × 3), then the combined organic layer was concentrated under reduced pressure. The crude product was purified by column chromatography with CH₂Cl₂/EtOAC (v/v, 3:1) to afford the target product 1 (430 mg, 60%). ¹H NMR (500 MHz, DMSO-d₆): δ 10.72 (s, 1H), 8.76 (s, 1H), 8.32-8.28 (m, 2H), 7.98-7.96 (d, 1H, J = 10 Hz), 7.90-7.88 (d, 1H, J = 10 Hz), 7.62 (s, 1H), 7.57 (s, 1H), 7.50-7.48 (m, 3H), 7.21 (s, 1H), 7.06-7.05 (d, 1H, J = 5 Hz), 6.35 (s, 2H), 6.26 (s, 2H), 5.08-5.06 (t, 2H, J = 6.67 Hz), 5.03 (s, 2H), 3.12-3.09 (m, 4H), 1.83 (s, 6H), 1.20-1.16 (m, 6H). ¹³C NMR (125 MHz, DMSO-d₆): δ 165.19, 152.34, 151.84, 147.96, 141.75, 141.44, 133.78, 129.89, 129.45, 128.70, 128.22, 128.12, 127.33, 124.16, 123.75, 118.35, 106.67, 97.19, 66.44, 38.78, 17.10, 15.16. HRMS (ESI): calcd. for C₄₃H₄₀N₇O₄ [M + H]⁺ = 718.3136, found m/z 718.3135.

This work was supported by fund of China Tobacco Yunnan Industrial Co. (No. 2015JC05), and the Foundation of the Department of Science and Technology of Yunnan Province of China (Nos. 2013HB062, 2014HB008, 2016FB020), and the Program for Excellent Youth Talents of Yunnan University (No. XT412003).

Supplementary data associated with this article can be found, in the online version, at http://dx.doi.org/10.1016/j.cclet.2016.12.029.