A benzoxazole functionalized fluorescent probe for selective Fe³⁺ detection and intracellular imaging in living cells

Abstract

A rhodamine based 2-aminobenzoxazole functionalized probe was presented for reversible detection of Fe³⁺ driven by the coordination of Fe³⁺ to 2-aminobenzoxazole that resulted in strong fluorescence emission and color change. Fluorescent imaging in living L929 and MG-63 cells offered a promising candidate for mapping Fe³⁺ in biological samples.

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The design and development of fluorescent probes for sensitive and selective quantification of fluorescent cellular and subcellular imaging heavy and transition-metal ions in special chemical, environmental and biological samples has evolved into an emerging attractive area of particular interest in analytical chemistry and chemical biology.^1,2 Compared with the traditional methods such as atomic absorption spectroscopy and inductively coupled plasma mass spectroscopy which not only require large and expensive instruments, highly trained personnel, and tedious maintenance but also suffer from poor biocompatibility as well as environmental and biological applicability, fluorescence detection enjoys the advantages of simplicity, high spatial resolution, being non-destructive and real time monitoring.³ As a result, numbers of ideal fluorescent probes^4,5 are increasing at a high speed, especially for probes based on the spirocyclic ring-opening progress of rhodamine derivatives because of their excellent spectroscopic properties, such as long excitation and emission wavelengths, great photostability, high extinction coefficient and excellent fluorescence quantum yields.⁶

Amongst a variety of attractive metal ions, ferric iron, which is known for its chemical versatility, industrial importance and as an indispensable trace mineral for humans in the metabolism and energy transfer mechanisms of numerous biological systems,⁷ plays significant roles in our daily life, industry, environmental science and medicine as well as several crucial biochemical processes at the cellular level. The abnormal levels of Fe³⁺ have been involved in a series of human diseases, such as anemia and breathing problems, certain cancers, oxidative damage to lipids, proteins, nucleic acids and other cellular components, dysfunction of certain organs, such as the heart, kidneys, pancreas, and liver, and even neurodegenerative diseases, such as Parkinson's disease and Alzheimer's disease.^8,9 Therefore, a convenient and rapid method for the analysis of the mechanisms of how Fe³⁺ affects our metabolism and body health has important consequences in biological concerns.

In recent years, significant emphasis has been placed on the exploitation of facile, highly selective fluorescent probes for Fe³⁺ and much progress has been made.^10–12 Ali et al.¹³ reported a rhodamine-benzoxazine functionalized probe for Fe³⁺ in H₂O–MeCN (3 : 7, v/v) solution at pH 7.2 through 1 : 1 complex formation resulting in the fluorescence enhancement. Wang et al.¹⁴ synthesized a “double Schiff base” fluorescent turn-on probe for Fe³⁺ with a low detection limit of 0.27 μmol L⁻¹. Our group has also put considerable efforts in the development of promising fluorescent probes for Fe³⁺ and made several delightful progresses.¹⁵ However, compared with the number of reported Fe³⁺ probes, the probes for real biological detection are still rare, since the application of some reported probes was sometimes limited by the solubility, sensitivity, selectivity, stability and biocompatibility, especially in biological detection. Therefore, it still remains a significant task for selection of adequate probes with excellent fluorescence properties to be effectually applied in biological fluorescent imaging of Fe³⁺, particularly for selective sensing allowing for miscellaneous fluorescence signal generation and unknown side-effects in the complex biological systems.

With continuing interest in the development of environmentally and biologically applied fluorescent probes for Fe³⁺, we herein present another wonderful probe Z1 (Scheme 1) which exhibited superior properties with improved selectivity, sensitivity and biological compatibility compared to the previously reported probes since the selected functional group 2-aminobenzoxazole is considered to have potential excellent bio-medical and optical properties that can not only improve the solubility, selectivity, biocompatibility and cellular affinity but also contribute to the chelation enhanced fluorescence emission and color change with remarkably improved fluorescence stability and sustainability. The wonderful properties make the probe highly suitable for the visualization of Fe³⁺ in living cells which may provide breakthrough insight into the correlation between Fe³⁺ and related biological processes.

Scheme 1 The synthesis of probe Z1.

The absorption and fluorescence detection were conducted in PBS solution (pH = 7.4) during all the photo-property investigation to ensure that the fluorescent and absorption signal changes were caused by the coordination of Fe³⁺ to the probe since originally the hydrolytic process of Fe³⁺ can happen in aqueous media and results in the decrease of pH that induces the protonation-induced ring-opening process which may cause great interferences for the detection. This was supported by the pH responses of the probes in EtOH–H₂O (5 : 5, v/v) with pH values from 1.7 to 13.0 (Fig. 1) since both the fluorescence intensity and the absorption exhibited inconspicuous changes in the pH range of 5.0 to 8.0, suggesting that the probes were insensitive to pH in the neutral range and could work under approximate physiological conditions with negligible background fluorescence.

Fig. 1 Fluorescence intensity (a) and absorption (b) changes of Z1 (20 μmol L⁻¹) in the presence of 1.0 equiv. of Fe³⁺ in EtOH–H₂O (5 : 5, v/v, PBS, pH 7.4) solution under different pH conditions.

The EtOH–H₂O (5 : 5, v/v) mixed solution was used during all the photo-property investigation since the solution investigation suggested that other solvent systems including MeOH–H₂O, (CH₃)₂CHOH–H₂O, (CH₃)₂CO–H₂O, THF–H₂O, CH₃CN–H₂O, DMSO–H₂O, and DMF–H₂O all seemed to be inferior to EtOH–H₂O mixed solution because of the lower fluorescence intensities and absorptions upon addition of Fe³⁺ (Fig. S1–S4†). In addition, when the ethanol content of the solution varies from 0 to 100%, both the fluorescence intensity and absorption increased with the increasing concentration of ethanol in the mixed solvent (Fig. S5 and S6†). Considering the fact that the probe was designed for the detection of Fe³⁺ in biological samples, a 50% ethanol content seemed to be one of the best choices for fluorescence and absorption detection and a 10% ethanol content should be perfect for biological imaging.

Related metal ions including Li⁺, Na⁺, K⁺, Ag⁺, Sn²⁺, Mg²⁺, Ca²⁺, Cd²⁺, Mn²⁺, Co²⁺, Cu²⁺, Ni²⁺, Zn²⁺, Pb²⁺, Hg²⁺, Fe²⁺, Al³⁺, Cr³⁺ and Fe³⁺ were used to verify the selective property of Z1 in EtOH–H₂O (5/5, v/v, PBS, pH 7.4) solution (Fig. 2, S7 and S8†). Originally, the probe itself displays colourless and emits no fluorescence because the spirocyclic form of rhodamine prevailed. Notably, upon addition of Fe³⁺, an intense absorption band centered at 542 nm coupled with brilliant pink coloration and concomitantly, a strong emission band appeared around 571 nm, which was reasonably assigned to chelation enhanced fluorescence caused by Fe³⁺ which resulted in the delocalized xanthene tautomer of rhodamine into the quinoid structure. Although Al³⁺ and Cr³⁺ can also cause slight absorption and fluorescent responses, the signals were significantly inferior compared with Fe³⁺ induced spectroscopic changes, indicating the high specificity of the probe to Fe³⁺. Time dependent study showed that the Fe³⁺ induced ring-opening process completed rapidly within 20 s (Fig. S9 and S10†) and showed prolonged stability since both the absorption and fluorescence intensity showed an inconspicuous decrease for several hours. The competition experiments were then performed by adding 5.0 equiv. of the competing ions to the solutions that contained the probe and 1.0 equiv. of Fe³⁺. The results indicated that the selectivity of the probe did not significantly experience interference from the commonly coexistent metal ions.

Fig. 2 Histogram showing the fluorescence intensity and absorption for the selectivity and competition investigation of Z1 (20 μmol L⁻¹) in EtOH–H₂O (5 : 5, v/v, PBS, pH 7.4) mixed solution, λ_ex = 530 nm. The pillars in the front row represent fluorescence response of the probe to the metal ions of interest. The pillars in the back row represent the subsequent addition 5.0 equiv. metal ions to the solution containing Z1 and 1.0 equiv. of Fe³⁺, respectively.

Absorption and fluorescence titrations were then performed (Fig. 3). The results suggested that both the fluorescence intensity and absorption showed a steady and smooth increase with the increasing concentration of Fe³⁺ and about 1.0 equiv. of Fe³⁺ was required until a plateau was reached. The fluorescence quantum yield was calculated to be 0.86 by using rhodamine B as a standard. Good linear relationships were observed (Fig. S11 and S12†) between the relative fluorescent and absorption signals and the concentration of Fe³⁺ in the 0.009–20 μmol L⁻¹ range with a detection limit of 3.0 nmol L⁻¹ in EtOH–H₂O (5/5, v/v, PBS, pH 7.4) mixed solution when 20 μmol L⁻¹ of probe was used (Fig. 3, inset), which was much more sensitive to our previously reported Fe³⁺ probes with benzothiazole and fluorine groups, indicating a better prospect of application.

Fig. 3 Fluorescence intensity (a) and absorption (b) changes of probe Z1 (20 μmol L⁻¹) upon addition of Fe³⁺ (0–1.0 equiv.) in EtOH–H₂O (5/5, v/v, PBS, pH 7.4) mixed solution. Inset: changes of emission intensity at 571 nm (a) and absorption at 542 nm (b), λ_ex = 530 nm.

The stoichiometric ratio investigation through Job's plot established the formation of the 1 : 1 stoichiometry between the probe and Fe³⁺ (Fig. S13 and S14†) with a high association constant of 5.45 × 10⁴ M⁻¹, which indicated the strong coordination of probe Z1 to Fe³⁺. The mass spectrum manifested the peak at m/z 721.0986 (Fig. S24†), assigned to [Z1 + Fe³⁺ + 3Cl⁻ + H]⁺, providing powerful evidence for the binding mode of Z1 with Fe³⁺. The absorption at 1690 cm⁻¹ in IR analysis did not shift to lower frequency upon addition of Fe³⁺ (Fig. S23†), indicating that the carbonyl moiety was not involved in the coordination. In addition, the fluorescence intensity gradually decreased with the increasing concentration of ethylenediamine and completely quenched when excess ethylenediamine was added, coupled with the change of pink colour back to colourless (Fig. S15 and S16†). On the other hand, both the colour and the fluorescence appeared again when more Fe³⁺ was added to this solution (Fig. S17 and S18†). This process could take place several times, thus implying the reversible binding process between probe Z1 and Fe³⁺. According to our previous work,¹⁵ it can be supposed that Fe³⁺ bonds with the nitrogen atom of the benzoxazole moiety inducing the ring-opening process accompanied by the fluorescent changes (Scheme 2).

Scheme 2 Proposed recognition process for the fluorescent change of Z1 upon addition of Fe³⁺.

Inspired by the excellent optical properties which showed favorable potential for quantifying Fe³⁺ in biological systems, we performed the MTT assay in which mitochondrial metabolic enzyme activity is used as an indicator of cell viability to identify the toxicity of the probe. The MCF-7 cell was treated with different concentrations of Z1 and the results (Table. S1†) indicated the strong cell viability and low toxicity of Z1. The practical bio-imaging applications were then conducted by laser scanning confocal microscopy (Fig. 4). Both the cultured mouse fibroblast cells L929 and human osteosarcoma MG-63 cells displayed no intracellular fluorescence when incubated with the probe in culture medium for 30 min at 37 °C (Fig. 4(a)). However, a prominent fluorescence increase was observed in the intracellular area when 1.0 equiv. of Fe³⁺ was supplemented and incubated in the same growth medium for another 30 min at 37 °C (Fig. 4(b)). It is more encouraging that the cell morphology remained in good condition throughout the imaging experiments which was supported by the bright-field transmission images of cells (Fig. 4(c)). In addition, the overlay (Fig. 4(d)) of fluorescence and bright-field images confirmed that the fluorescent signals were localized in the perinuclear area of the cytosol. The preliminary experiments in living L929 cells and MG-63 cells demonstrated the special potential of the probe for in vivo mapping and imaging of Fe³⁺, which was supposed to contribute to understand the importance and functional mechanism of Fe³⁺ in related biological processes.

Fig. 4 Fluorescence images of mouse fibroblast cells L929 and human osteosarcoma MG-63 cells incubated with 20 μmol L⁻¹Z1 for 30 min at 37 °C (a) and then further incubated with 20 μmol L⁻¹ FeCl₃ for 30 min at 37 °C (b). (c) Bright-field transmission image of the cell treated with probe Z1 (20 μmol L⁻¹) and FeCl₃ (20 μmol L⁻¹). (d) The overlay images of (b) and (c).

Some of the recently reported rhodamine based Fe³⁺ probes (1–11)^{11d,12d,13–16} are given in Fig. S25† and some salient features including solubility, selectivity, fluorescence quantum yield, sensitivity, stability, and biocompatibility are given in Table S2.† Investigation of these probes indicated that they are all off–on type Fe³⁺ probes with a moderate detection limit and formation constants and some are applicable for imaging and monitoring Fe³⁺ ions in living cells. In comparison, probe Z1 has nothing less than those reported probes in the solubility and selectivity but preponderant in sensitivity, stability and fluorescence quantum yield by over 300-fold fluorescence enhancement upon addition of Fe³⁺ with a large binding constant of 5.45 × 10⁴, a large fluorescence quantum yield of 0.86 and a low detection limit of 3 nmol L⁻¹ through 1 : 1 binding stoichiometry. In addition, the MTT assay demonstrated strong cell viability, good biocompatibility and low toxicity of Z1 and the fluorescent imaging in L929 and MG-63 cells demonstrated that the fluorescent signals were localized in the perinuclear area of the cytosol thus indicating the cytoplasmic binding which makes Z1 a promising candidate for in vivo monitoring of Fe³⁺.

Conclusions

In summary, we developed a novel rhodamine based fluorescent probe with 2-aminobenzimidazole functional groups for specific detection of Fe³⁺. The probe was facilely synthesized from rhodamine B and displayed special selectivity, sensitivity and fluorescence quantum yield which were superior to those of our previously reported probes with 2-aminobenzothiazole and fluorine functional groups. Preliminary experiments in living L929 cells and MG-63 cells confirmed the membrane permeability and biocompatibility of the probe which would be a promising candidate for intracellular identification of Fe³⁺ thus providing significant insight into a better understanding of the importance and biological functions of Fe³⁺ in biological processes and organs.

Acknowledgements

We thank Prof. J. L. Li, Prof. S Y. Zhang and Prof. Z. Shi for their great helpful support. We are grateful for the support from the National Natural Science Foundation of China (NSFC 21572177; 21301139; 21272184; 21103137; 21103135; 21073139 and J1210057), the Shaanxi Provincial Natural Science Fund Project (No. 2015JZ003), the Shaanxi Provincial Higher Education Teaching Reform Project (No. 15BY47), the Xi'an City Science and Technology Project (No. CXY1511(3)), Scientific Research Cultivating Fund of Xi'an University of Science and Technology (No. 201619), and the Xi'an University of Science and Technology Teaching Reform Project (No. JG14052).

Notes and references

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Footnote

† Electronic supplementary information (ESI) available. See DOI: 10.1039/c6ay02660e

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