A highly selective and reversible fluorescent Cu²⁺ and S²⁻ probe under physiological conditions and in live cells

Abstract

A new spiropyran functionalized rhodamine derivative RB-SP2 has been synthesized and applied to detect Cu²⁺ and S²⁻. RB-SP2 was then used as an imaging probe for detection of these ions in HeLa cells at the physiological pH.

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A fluorescent chemosensor capable of sensing specific analytes has potential applications in chemistry and biology,¹ as it generally allows detection of analytes present in ultratrace quantity. Such detection of heavy metal ions is of high importance due to the high toxicity of these metal ions toward human health.² Fluorescent sensors for the detection of Cu²⁺ are actively investigated, as it is a significant environmental pollutant and also an essential element for humans.³ Meanwhile, development of selective and efficient signaling units for detection of various chemically and biologically important anions has also attained significant interest.⁴ Being one of the biologically and environmentally important anions, sulfide is largely used in industrial processes.⁵ Consequently, there are high risks for the sulfide ions to be exposed to drinking water. Sulfide can damage mucous membranes and can cause unconsciousness and respiratory problems.⁶ Therefore, development of a quick and sensitive fluorescence probes for the detection of S²⁻ and Cu²⁺ in aqueous media and in biological systems is of high interest.

Although a significant number of rhodamine-based fluorescent probes have been developed for different metal ions (Zn²⁺, Fe³⁺, Hg²⁺),⁷ a few ones have been reported for copper.⁸ In this work, a new fluorescent probe RB-SP2 (Scheme 1) is proposed. RB-SP2 is capable of detection both Cu²⁺ and S²⁻ at physiological pH.

Scheme 1 Synthesis of compound RB-SP2.

Compound RB-SP2 was readily synthesized in six steps. Condensation of 1 with 2 afforded the intermediate 3; which was then treated with intermediate 5 under the basic conditions to give the intermediate compound 6; and 7 was treated with hydrazine hydrate affording the intermediate 8, then compound 6 with 8 afforded 9 (RB-SP1), which was then treated with zinc powder under the acidic conditions to give the target compound RB-SP2 (Scheme 1). Details about the synthesis and characterization of RB-SP2 are presented in the ESI.†

The compound RB-SP2 is synthesized by reducing RB-SP1 which is composed of spiropyran and rhodamine. The N and O atoms of rhodamine can coordinate with metal ions inducing the ring-open of spiropyran, and consequently an increase in fluorescence. Considering that the Schiff base of RB-SP1 may respond to many transition metal ions, and in order to enhance the selectivity of the fluorescent probe, RB-SP1 was reduced to RB-SP2, and the next experiment has proved this point.

As evident from Fig. 1a, excitation of the initial solution of RB-SP2 at 500 nm wavelength did not show any significant emission over the range from 560 to 680 nm (orange line). This supports the fact that in absence of Cu²⁺, RB-SP2 remains in the ring-close form. Addition of Cu²⁺ to RB-SP2 solution induces a significant switch ON fluorescence response near 590 nm, with a visual display of reddish fluorescence. The switch ON responses for the emission band at ∼590 nm on binding to Cu²⁺ suggest the opening of the spirolactam ring in RB-SP2 through the coordination with Cu²⁺. The fluorescence intensity at ∼590 nm increases with increasing the Cu²⁺ concentration, with about 300% increase of its fluorescence intensity upon addition of only 10 μM Cu²⁺ ions. The minimum detectable concentration was 1 × 10⁻⁷ M. We have measured the linear equation detecting the Cu²⁺ (Fig. S1, see ESI†) which was y = 1.25x + 1.24 with a linear range from 0.1 μM to 1.0 μM, the limit of detection was calculated as 0.05 μM from the 3s method.

Fig. 1 Changes in the (a) fluorescence (λ_ex = 500 nm) and (b) UV-vis absorption spectra of RB-SP2 (10 μM) in the presence of increasing concentrations of Cu²⁺ (0–10 μM) in C₂H₅OH/aqueous PBS (1 mM, pH 7.4; 4 : 6 v/v; 1% DMSO) solutions. Inset: the Cu²⁺ concentration-dependent responses at room temperature.

UV-vis spectra recorded for RB-SP2 in C₂H₅OH/aqueous PBS indicated the maximum absorption at 324 nm (Fig. 1b), which may be attributed to the intra-molecular π–π* charge transfer (CT) transition. According to previous studies some transition-metal ions can selectively bind with suitable derivatives of rhodamine,⁹ inducing the opening of the spirolactam ring and generation of the xanthene form. This structural change is manifested in the electronic and fluorescence spectral patterns. Significant change in UV-vis spectrum was observed in the presence of Cu²⁺ (Fig. 1b). The absorption band appeared around 550 nm increases with increasing Cu²⁺ concentration from 0 μM to 10 μM (Fig. 1b), and the solution turned from colorless to pink (Fig. S2, see ESI†). It should be noted that a small adsorption band between 400 and 450 nm slightly increased with the increase of the band around 550 nm, which clearly indicated that Cu²⁺ caused the ring-open of not only RB but also SP.

We have examined mechanism using FTIR techniques. The IR spectra of RB-SP2 revealed that the peak of C=O amide bond at 1687 cm⁻¹, the characteristic stretching frequency for the C=O amide bond of the rhodamine unit, shifted to 1636 cm⁻¹ in presence of 2 equiv. of the Cu²⁺ ion (Fig. S3, see ESI†). Such shift in the stretching frequency of C=O amide bond of the rhodamine unit on binding to a metal ion has been reported earlier,^9c indicating that the amide carbonyl group is involved in the interactions with Cu²⁺. This is the key to the spiro ring-opening and fluorescence turn-on of the rhodamine dye. Meanwhile, the pronounced down field shifts of the methylene protons of RB-SP2 in the ¹H NMR spectra indicate that the binding interactions among the metal center, hydrazide and phenolate hydroxy group (Fig. S4, see ESI†). Furthermore, in order to verify Cu²⁺-triggered spiro ring-opening process, we studied mass spectrum of the RB-SP2-Cu system, which shows a molecular-ion peak at m/z 898.3, corresponding to [RB-SP2 + Cu + Cl] (Fig. S5, see ESI†). Taken these results together, a likely sensing mechanism based on the Cu²⁺-triggered spiro ring-opening process is proposed in Scheme 2. Based on the relationship between the fluorescence intensity and concentration of Cu²⁺, the binding constant was calculated to be log K = 6.11 (Fig. S6, see ESI†).

Scheme 2 Proposed mechanism for the fluorescent changes of sensor RB-SP2 upon the addition of Cu²⁺ and S²⁻.

As other metal ions may also coordinate with RB-SP2, the responses to other metal ions (Au³⁺, K⁺, Mg²⁺, Hg²⁺, Fe²⁺, Ni²⁺, Cd²⁺, Ba²⁺, Co²⁺, Mn²⁺, Na⁺, Fe³⁺, Cu⁺, Zn²⁺ and Ag⁺) were investigated (Fig. S7, see ESI†). There are not any noticeable changes in fluorescence spectrum with addition of these tested ions, suggesting that the generation of xanthene moiety is highly selective toward Cu²⁺ ions. This conclusion was further confirmed with the UV-vis absorption spectra analysis, no noticeable changes in UV-vis absorption were observed with the addition of these tested ions in RB-SP2 solution (Fig. S8, see ESI†).

The above-mentioned studies show that RB-SP2 selectively binds with Cu²⁺ to form RB-SP2-Cu complex resulting in considerable changes in the spectral properties. One may think that if the RB-SP2-Cu complex dissociates, the fluorescence would be quenched. Herein sulfide was introduced to quench the fluorescence by forming CuS. Fig. 2a shows that the fluorescence intensity of 10 μM RB-SP2-Cu decreases with increasing the concentration of S²⁻. In the presence of 20 μM S²⁻ anion the intensity of RB-SP2-Cu decreases by about 280%. Similar results are observed on the UV-vis spectral pattern (Fig. 2b) which is in a reverse direction to the spectral pattern as shown in Fig. 1b. These results further confirmed the conclusion that the RB-SP2-Cu complex results in the switch ON of fluorescence response (Scheme 2). Their corresponding fluorescence changes were also shown in Fig. S9 (see ESI†), implying the reproducibility and reversibility of this experiment.

Fig. 2 Changes in the (a) fluorescence (λ_ex = 500 nm) and (b) UV-vis absorption spectra of RB-SP2-Cu complex (adding 10 μM Cu²⁺ to RB-SP2 10 μM) in the presence of different concentrations of S²⁻ (0–20 μM) in C₂H₅OH/aqueous PBS (1 mM, pH 7.4; 4 : 6 v/v; 1% DMSO) solution. Inset: the S²⁻ concentration-dependent response at room temperature.

The spectral responses of the RB-SP2-Cu complex were also investigated in the presence of other anions including F⁻, Cl⁻, Br⁻, I⁻, CN⁻, H₂PO₄⁻, NO₃⁻, ClO⁻, ClO₄⁻, SO₄²⁻ and HSO₃⁻. These investigated anions show little fluorescence quenching effect except the S²⁻ (Fig. S10, see ESI†). The reason is that these anions cannot form stable precipitate/complex or the formed precipitate/complex is with less stability than RB-SP2-Cu. As expected, same results were observed on the UV-vis absorption spectral analysis, which also demonstrate high selectivity toward S²⁻ ions (Fig. S11, see ESI†).

The fluorescence intensity of RB-SP2 probe is independent on pH in the range of pH 5.0–9.0 either in the absence or presence of Cu²⁺ (Fig. S12 see ESI†), indicating that the probe was suitable for the detection of Cu²⁺ at physiological pH.

Based on the above experiment, it was conceived that compound RB-SP2 could be exploited for fluorescence imaging of live cells, particularly for sensitive detection of intracellular Cu²⁺. To pursue this goal, it was pertinent to assess the cytotoxic effect of compound RB-SP2 on live cells. Various concentrations of compound RB-SP2 and RB-SP2-Cu complex were thus chosen, and their cytotoxic effects on HeLa cells were ascertained following an exposure period of 24 h. The well-established MTT assay, which is based on mitochondrial dehydrogenase activity of viable cells, was adopted. It is quite evident from Fig. 3a that compound RB-SP2 does not exert any effect on the viability of HeLa cells. However, exposure of HeLa cells to 100 μM RB-SP2-Cu complex resulted in a decline in cell viability. In the presence of higher concentrations of RB-SP2-Cu complex, the cytotoxic effect was more prominent as a result of cytotoxic and antiproliferative effects of Cu²⁺ complex on cancer cells.¹⁰

Fig. 3 (a) MTT assay to determine the cytotoxic effect of compound RB-SP2 and RB-SP2-Cu complex in HeLa cells. (b) Fluorescence microscopic images of HeLa cells: (1) after treating with 10 μM RB-SP2 (under green light); (2) after adding 10 μM of Cu²⁺ (under green light) to the RB-SP2 treated cells, and (3) after adding 20 μM S²⁻ (under green light) to the (RB-SP2-Cu²⁺) treated cells.

Fluorescence microscopic studies reveals that HeLa cells treated with probe RB-SP2 alone show no fluorescence (Fig. 3b(1)). Upon incubation with 10 μM Cu(ClO₄)₂ for 1 h, a striking switch-ON fluorescence is observed inside HeLa cells, indicating the formation of RB-SP2-Cu complex (Fig. 3b(2)), as observed earlier in solution studies. Further, an intense red fluorescence was conspicuous in the perinuclear region of HeLa cells. Interestingly, sulfide sensing inside HeLa cells by RB-SP2-Cu complex could also be pursued as evident from the remarkable switch-OFF of the red fluorescence inside cells following incubation with Na₂S solution (Fig. 3b(3)). Essentially, the fluorescence microscopic analysis strongly suggested that probe RB-SP2 could readily cross the membrane barrier, permeate into HeLa cells, and rapidly sense intracellular Cu²⁺ and S²⁻. It is significant to mention here that brightfield images of treated cells did not reveal any gross morphological perturbations, which suggested that HeLa cells were viable. This finding is encouraging for future in vivo biomedical applications of the probe.

Conclusions

A highly selective and reversible fluorescent probe RB-SP2 was designed for the detection of Cu²⁺ and S²⁻ by incorporating both spiropyran and rhodamine. RB-SP2 can be employed to image Cu²⁺ and S²⁻ in living cells. Notably, the reduction of RB-SP1 to RB-SP2 guaranteed the reversible as well as selective response to Cu²⁺. This work opens an avenue for development of reversible fluorescent probe from irreversible chemo dosimeters with high selectivity.

Acknowledgements

We are grateful for the financial support from the NSFC (2009CB421601).

Notes and references

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Footnote

† Electronic supplementary information (ESI) available: Experimental details, synthesis and characterization of RB-SP2, optimized procedure. See DOI: 10.1039/c4ra03916e

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