Rhodol-based fluorescent probe for Au³⁺ detection and its application in bioimaging

Abstract

A turn-on fluorescent probe of propargyl–rhodol conjugate responds selectively to Au³⁺. In the presence of Au³⁺, the chemodosimeter undergoes a remarkable change in its absorption spectrum with a 370-fold fluorescence enhancement. This reaction-based Au³⁺ chemodosimeter is highly sensitive with a detection limit of 7 ppb through an irreversible mechanism. Bioimaging of HeLa cells with this chemical probe has been successfully demonstrated.

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Gold has attracted a great deal of scientific attention due to its unique catalytic activities, biological activities and its useful photophysical properties in gold-based nanoparticles.¹ With its excellent alkynophilicity, Au³⁺ is a versatile catalyst in organic chemistry for activation of carbon–carbon triple bonds.² Gold complexes have also been used as therapeutic agents for rheumatoid arthritis, asthma and cancer.³ However, the toxicity of gold ions may cause liver, kidney and nervous system damage.⁴ Therefore, it is highly desirable to develop simple, effective and rapid Au³⁺-sensing probes that are applicable to monitor Au³⁺ in vitro and in vivo. A fluorescence-based sensing method has been developed to monitor gold species using various fluorophores including fluorescein, naphthalimide, rhodamine⁵ and BODIPY.^1a,6 However, some fluorescent sensors have cross affinity with other alkynophilic metal species such as Pd²⁺, Ni²⁺, Ag⁺, Cu²⁺, and Hg²⁺.⁷

Based on a reaction-based approach, several fluorescent sensors with an alkynyl group can selectively detect gold ions with high selectivity. For example, cyclization of coumarin–alkyne,⁸ rhodamine–propargylamide,⁹ rhodamine–alkynyl benzaldehyde,¹⁰ and rhodamine–hydroxamate alkyne conjugate,¹¹ intramolecular hydroarylation of phenyl alkynoates,¹² hydrolysis of rhodamine–acyl semicarbazide conjugate,¹³ BODIPY–pyridyl hydrazine conjugate^6e and desulfurization of thiocoumarin were reported.¹⁴ Rhodol (or rhodafluor), a hybrid structure of rhodamine and fluorescein, is a useful organic dye with excellent photophysical properties such as high extinction coefficients, quantum yields, photostability, and strong fluorescence with low excitation wavelength.¹⁵ Rhodol derivatives have been successfully used as scaffolds for fluorescence probes for various metal ions including Cu²⁺, Zn²⁺ and Hg²⁺ but not for Au³⁺.¹⁶

Herein, we present a reaction-based rhodol derivative appended with a propargyl moiety as a highly selective and sensitive turn-on fluorescent sensor for Au³⁺. We envisioned that the closed-form of the spirolactone ring would be colorless with no fluorescence emission while Au³⁺ would react with the alkyne moiety leading to colorimetric change and distinct fluorescence enhancement. The synthesis of propargyl–rhodol conjugate 1 has been achieved in one step with 79.8% yield (Scheme 1). The structure of propargyl–rhodol 1 was confirmed by ¹H and ¹³C NMR, and ESI-MS analyses.

Scheme 1 Synthesis of probe 1.

The structure of probe 1 consists of a rhodol fluorophore as a signal reporter and a propargyl unit as the Au³⁺-binding site. The propargyl–rhodol 1 displays excellent selectivity and sensitivity toward Au³⁺ in aqueous solutions and its biological application was demonstrated by bioimaging of Au³⁺ in cultured HeLa cells.

A colorless solution of 1 was non-emissive in aqueous DMSO and its UV-vis absorption showed no absorption band in the visible region (400–700 nm). The addition of HAuCl₄ (20 equiv.) to the probe solution (20 μM, 60% v/v DMSO/H₂O) resulted in a significant change from colorless to yellow with a new absorption band centered at 493 nm and a strong fluorescence emission at 526 nm. The fluorescence sensing behavior of 1 toward Au³⁺ and other analytes was further evaluated. The results showed that other competing alkynophilic metal species Ag⁺, Cu²⁺, Hg²⁺, Ni²⁺ and Pd²⁺ as well as other metal ions Li⁺, Na⁺, K⁺, Cs⁺, Ba²⁺, Ca²⁺, Mg²⁺, Zn²⁺, Co²⁺, Cd²⁺, Fe³⁺ and Pb²⁺ did not result in any apparent changes of fluorescence apart from Au³⁺ (Fig. 1).

Fig. 1 Fluorescence spectra of 1 (20 μM) upon the addition of Au³⁺ and other metal ions (10 equiv.) (60% v/v DMSO/H₂O). Inset: (a) color change observed in visible light and (b) fluorescence emission under blue-LED illumination.

For colorimetric detection, the solution of 1 changed from colorless to yellow upon the addition of Au³⁺, corresponding to a new UV-vis absorption peak at 493 nm. The ring-opening process of the spirolactone was induced by Au³⁺ to give fluorescein-like optical properties. Two other transition metal ions including Hg²⁺ and Fe³⁺ also turned the colorless solution of 1 to pink with two absorption bands at 493 and 526 nm, suggesting the presence of both fluorescein-like and rhodamine-like isomers.¹⁷ These pink solutions of 1 did not exhibit any fluorescence enhancement due to the formation of twisted intramolecular charge transfer or the fluorescence quenching effect of metal ions.¹⁸ These results suggested that 1 is a highly selective “turn-on” fluorescent sensor for Au³⁺. For the competitive experiments, UV-vis and fluorescence spectra revealed that the binding of 1 and Au³⁺ was diminished in the presence of other metal ions.

A fluorescence response of 1 was linearly enhanced with Au³⁺ in a concentration range of 20–96 μM (Fig. 2). Subsequently, the limit of detection for Au³⁺ was calculated to be 95 nM (7 ppb) (S/N = 3). For Au³⁺-selective fluorescent sensors based on xanthene derivatives, the limit of detection of 1 is lower than that of other reported sensors in a micromolar level.^5d A time-dependent plot indicated that the reaction was complete within 2 h.

Fig. 2 Fluorescence spectra of 1 (20 μM) upon the addition of Au³⁺ (0 to 420 μM) (λ_ex = 480 nm, λ_em = 526 nm, 60% v/v DMSO/H₂O). Inset: the fluorescence change plot of probe 1 in the presence of Au³⁺ (20–96 μM) in 60% v/v DMSO/H₂O. Excitation at 480 nm and maximum emission at 526 nm.

To investigate the binding mechanism of 1 and Au³⁺, 1 was incubated with Au³⁺ in aqueous acetonitrile at room temperature. After 12 h, a silica gel TLC analysis of the reaction mixture revealed a formation of a new product with higher polarity. After chromatographic purification, the chemical identity of the cyclized product 1e was established by ¹H-NMR and ESI-MS analyses. ¹H-NMR spectrum of 1e exhibited new chemical shifts at δ 1.2 and 7.7 ppm corresponding to a methyl group and the olefinic proton of the newly formed furan ring, respectively. The disappearance of the propargyl moiety in 1 was also noted: δ 2.55 and 4.70 ppm for the alkynyl and the methylene protons, respectively.

In addition, ESI-MS analysis of the reaction mixture showed an intense molecular ion of 1e at m/z = 426.1692 (calcd for C₂₇H₂₄NO₄ 426.1700). These results suggested a possible mechanism that the chelation of Au³⁺ with 1 may undergo a 5-endo-dig cyclization to form a vinyl-gold intermediate 1b, followed by protonolysis and isomerization to give the corresponding furan product 1e (Scheme 2).^9b,19 The product 1e shows a strong fluorescence enhancement at 526 nm, the fluorescein-like characteristic of the rhodol fluorophore.²⁰ The sensing mechanism of 1 with Au³⁺ is an irreversible process.

Scheme 2 A proposed mechanism of the cyclization of 1 induced by Au³⁺.

Probe 1 is highly selective and sensitive toward Au³⁺ among other metal ions and we further demonstrated biological applications of 1 in monitoring Au³⁺ in HeLa cells ​(Fig. 3). Cultured HeLa cells were incubated with Au³⁺ (100 μM) for 1 h at room temperature in Dulbecco's phosphate-buffered saline buffer (DPBS, pH 7.4) and then the treated cells were incubated with probe 1 (30 μM) for 1 h at room temperature and finally stained with DAPI nuclear staining dye. The fluorescence emission was observed by excitation at 473 nm. Bioimaging of cells incubated with probe 1 indicated the morphology of cells was not damaged in this process. HeLa cells incubated with probe 1 and Au³⁺ exhibited green fluorescence emission, suggesting that the propargyl–rhodol 1 can be utilized for monitoring Au³⁺ in biological samples.

Fig. 3 Confocal fluorescence images of probe 1 in HeLa cells. HeLa cells were incubated with probe 1 (30 μM) in DPBS buffer (pH 7.4) for 1 h and then incubated (a–d) without or (e–h) with Au³⁺ (1 mM) for 1 h at RT. (a) and (e) bright-field images; (b) and (f) fluorescence images; (c) and (g) fluorescence images of nuclei counterstained with DAPI; (d) and (h) merged images of (a–c) and (e–g), respectively.

Conclusion

A propargyl–rhodol chemodosimeter 1 was highly selective toward Au³⁺ with the limit of detection of 7 ppb. Upon the addition of Au³⁺, the colorless solution of 1 clearly changed to yellow with a strong fluorescence enhancement. The sensing mechanism was based on 5-endo-dig cyclization to give a furan-rhodol product. In addition, this chemodosimeter 1 could be employed to monitor Au³⁺ in living cells.

Acknowledgements

This work was financially supported by Mahidol University, the Thailand Research Fund, National Research University (NRU), and Center of Excellence for Innovation in Chemistry (PERCH-CIC). K. W. thanks a scholarship from the 60^th-Year Supreme Reign of His Majesty King Bhumibol Adulyadej Scholarship Program.

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Footnote

† Electronic supplementary information (ESI) available. See DOI: 10.1039/c6ra02342h

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