A “turn-on” fluorescent probe used for the specific recognition of intracellular GSH and its application in bioimaging

Abstract

We have designed and synthesized a carbazole-based fluorescent probe (CZ-Nm) for the specific recognition of GSH over Cys and Hcy. The probe is almost non-fluorescent owing to the photoinduced electron transfer (PET) process from the carbazole fluorophore to the nitroolefin moiety. Upon treatment with GSH, owing to the Michael addition of GSH to the double bond of the nitroolefin moiety, the probe shows a fluorescence enhancement and absorption change. The CZ-Nm probe displays desirable properties such as acting as a “naked eye” probe, a wide linear range of 0–0.01 M, high sensitivity, and strong anti-jamming capability. More importantly, the probe can also be successfully applied to the detection of intracellular GSH with a bright fluorescence signal, good cell permeability and bio-compatibility.

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Introduction

Cysteine (Cys), homocysteine (Hcy), and glutathione (GSH) are important intracellular thiols that play vital roles in physiological and pathological events,^1–3 and abnormal levels of these thiols are closely related to certain diseases.^4,5 In particular, GSH is the most abundant intracellular thiol (1–10 mM) in mammalian and many prokaryotic cells, functioning as an essential endogenous antioxidant primarily involved in regulating cellular redox activities and preventing cell damage via scavenging free radicals and peroxides.^6,7 Aberrant levels of GSH are directly associated with numerous clinical diseases, including cancer, human immunodeficiency virus (HIV), Alzheimer's, liver damage, leukocyte loss, cardiovascular disease and others.^8,9 Accordingly, developing efficient techniques for the detection of GSH under physiological conditions is of considerable significance and has attracted a great deal of attention.^10,11 Among the various detection methods that have been reported in the past few decades,¹² optical imaging through staining with a fluorescent probe is considered to be the most convenient and efficient approach owing to its high sensitivity, good flexibility as well as apparent simplicity.^13,14 In particular, the fluorescent probe could be successfully applied to intracellular detection.^15,16

To date, numerous thiol probes have been designed and synthesized utilizing the strong nucleophilicity of the thiol group.¹⁷ Although these probes can selectively detect biothiols from other amino acids, the discriminative detection of certain types of thiols still remains a significant challenge due to their similar structure and reactivity.¹⁸ In recent years, pioneered by Strongin's group, the selective detection of Cys/Hcy over GSH by the cyclization of Cys/Hcy with aldehydes was realized.^19–22 Since then, the specific detection of Cys from Hcy/GSH and Hcy from GSH/Cys using Michael addition combined with steric and electrostatic interactions,^23,24 Cys-induced substitution-rearrangement cascade reaction^25,26 or the cyclization of Cys with acrylates^27,28 have also been developed. By comparison, the probes capable of detecting GSH from Cys/Hcy have been addressed to a relatively less extent.^29,30 To the best of our knowledge, some fluorescent probes based on organic dyes for the highly selective detection of GSH have been designed.³¹ For example, Chmielewski's group designed a seminal GSH-specific fluorescent probe based on breaking the disulfide bonds followed by intramolecular cyclization and cleavage of a neighboring carbonate bond between a rhodamine 110 derivative and GSH.³² Yang's group prepared a bis-spiropyran sensor based on supramolecular interactions for selectively detecting GSH.³³ Taking advantage of the thiol-induced addition reaction, Wang,³⁴ Huo³⁵ and Keillor³⁶ designed innovated chemical sensors to selectively detect GSH. Moreover, Yang,³⁷ Guo³⁸ and Lin³⁹ designed GSH-specific fluorescent probes using a thiol-induced substitution reaction. Therefore, it is of high interest to design GSH-specific fluorescent probes.

Among the various organic fluorophores, carbazole dye has been used as the mother molecule due to its widely accepted superiority^40,41 (e.g., high fluorescence quantum yield, good water-solubility, good absorption cross-section, high photostability, visible light absorption and a modular nature for facile functionalization). Focusing on advancing new strategies in sensing and signaling, we now construct a nitroethenyl-carbazole conjugate for both colorimetric and fluorescence “turn-on” response to GSH. In the absence of GSH, the nitroolefin moiety serves as an electron acceptor for the photoinduced electron transfer (PET) process, quenching the fluorescence of the carbazole fluorophore (Φ_F = 0.006). Upon treatment with GSH, owing to the Michael addition reaction, the double bond between the fluorophore and the quencher is broken, blocking the PET process and leading to the fluorescence recovery (Φ_F = 0.30).^42,43 Moreover, we conclude that the reaction of GSH with the nitroolefin breaks the conjugated structure of the CZ-Nm probe and inhibits the intramolecular charge transfer (ICT) between the N-ethyl moiety and the nitroolefin moiety, producing a blue shift in the fluorescence spectra, which is accompanied with an obvious colour change in the solution^44,45 (Scheme 1).

Scheme 1 The design concept for the CZ-Nm probe for GSH.

Experimental

Apparatus

¹H and ¹³C NMR spectra were obtained on a Bruker DMX-300 spectrometer operating at 400 MHz in chloroform-d. MS spectra were obtained on an Agilent 1100. UV-vis absorption spectra were recorded on a U-4100 spectrophotometer. An Edinburgh FS5 spectrofluorometer was used for fluorescence measurements. An Olympus Zeiss 710 laser scanning confocal microscope was used for obtaining fluorescence image of the cells. pH measurements were conducted using a Jingke PHS-3D digital pH-meter.

Materials

Carbazole, sodium hydride, nitromethane and amino acids were purchased from Aladdin. Iodoethane was purchased from Energy Chemical. Piperidine was purchased from Sinopharm Chemical Reagent Plant. The solvents were used as received without further purification. Distilled water was used throughout.

Synthesis

As shown in Scheme S1,† the key intermediate N-ethylcarbazole-3-carboxaldehyde was synthesized according to a literature procedure. The CZ-Nm probe was further prepared according to the well-known Knoevenagel condensation.⁴⁶ A solution of N-ethylcarbazole-3-carboxaldehyde (0.50 g, 2.24 mmol), MeNO₂ (196 μL, 6.72 mmol), and NH₄OAc (0.52 g, 6.72 mmol) in 20 mL of acetic acid was heated at reflux with magnetic stirring for 3 h. After cooling, the resulting solution was concentrated by distillation under reduced pressure, and purified by chromatography on silica gel using DCM/PE (v/v, 1 : 2) as the eluent to obtain an orange–red solid (0.53 g, 90% yield). Mass spectrometry: m/z, calcd: 266.11, found: 267.03 ([M + H]⁺). ¹H NMR (400 MHz, CDCl₃) δ (ppm): 8.27 (s, 1H), 8.23 (d, J = 13.5 Hz, 1H), 8.12 (d, J = 7.8, 0.9 Hz, 1H), 7.71 (d, J = 13.5 Hz, 1H), 7.65 (d, J = 8.6 Hz, 1H), 7.54 (t, J = 8.3 Hz, 1H), 7.48–7.42 (m, 2H), 7.32 (t, J = 8.0 Hz, 1H), 4.39 (q, J = 7.2 Hz, 2H), 1.47 (t, J = 7.3 Hz, 3H). ¹³C NMR (100 MHz, CDCl₃) δ (ppm): 142.21, 140.93, 140.52, 134.19, 126.87, 126.59, 123.73, 122.96, 122.55, 120.73, 120.67, 120.27, 109.19, 109.37, 37.92, 13.89.

General spectral measurements

A 1.0 × 10⁻³ M stock solution of the CZ-Nm probe was prepared in DMF. Stock solutions of the biologically relevant analytes (i.e. GSH, Cys, Hcy, Thr, Ser, Glu, Lys, Phe, His, Gly, Ala, Val, and Tyr) were prepared in distilled water with a concentration of 4.0 × 10⁻² M for the UV/vis absorption and fluorescence spectroscopy. In a typical experiment, test solutions were prepared by placing 40 μL of the probe stock solution into a solution of 4 mL buffered (0.1 M PBS, pH 7.4) aqueous DMF solution (H₂O/DMF = 4 : 1, v/v). The fluorescence spectra were obtained after the addition of the analytes for 30 min at room temperature (λ_ex = 360 nm).

Cell cultures

PC12 cells were seeded in a glass bottom culture dishes and grown in Dulbecco's modified Eagle's medium (DMEM) supplemented with 2.5% fetal bovine serum (FBS) and 15% horse serum at 37 °C under a 5% CO₂ atmosphere until harvesting for the future experiments. When harvesting, the DMEM was drawn out from the culture dishes, the dishes were rinsed three times with 10 mM phosphate buffered saline (PBS) and then treated with 4 mL trypsinase solution containing 0.25% EDTA for 3 min in the incubator.

Results and discussion

Conditional experiments

During the initial attempts, the CZ-Nm probe exhibited a response to GSH. Then, we investigated the influence of the fraction of water on the interaction of the CZ-Nm probe with GSH. Among various fractions of water, a combination of 0.1 M PBS buffer–DMF (pH 7.4, v/v, 4 : 1) proved to be highly efficient in the sensing process (Fig. S1†). Therefore, we chose 0.1 M PBS buffer–DMF (pH 7.4, v/v, 4 : 1) as our test system.

To test the effect of pH on the fluorescence intensity of the CZ-Nm probe in the absence and presence of GSH, GSH was prepared at different pH values (from 3.0 to 11.0 using NaOH and HCl). Fig. S2† shows the relationship between the pH and the fluorescence intensity at 420 nm. As can be seen, the CZ-Nm probe exhibited an obvious response in the range of 7.0–11.0. For the detection of the GSH in vivo, PBS buffer at pH 7.4 was selected as the general measurement condition owing to the fact that the biologically relevant pH range is 5.8–8.0, and the CZ-Nm probe could be used to detect intracellular GSH without interference.

The time dependent fluorescent responses of the CZ-Nm probe to thiols were monitored at 420 nm under optimal conditions (0.1 M PBS buffer–DMF, pH 7.4, v/v, 4 : 1). Fig. S3† shows the fluorescence intensity reached its maximum value at about 25 min. Therefore, a 25 min reaction time and a medium of 0.1 M PBS buffer–DMF (pH 7.4, v/v, 4 : 1) solution were selected in subsequent experiments to facilitate the reaction of the CZ-Nm probe with GSH.

UV-vis absorption and fluorescence spectra of CZ-Nm in response to GSH

The absorption spectra and fluorescence spectra of the CZ-Nm probe (10 μM) were explored in PBS buffer–DMF (0.1 M, pH 7.4, v/v, 4 : 1) solution in the presence of different amino acids and thiol compounds. As shown in Fig. 1, owing to the influence of the PET and ICT effect between the fluorophore and nitroolefin moiety, the CZ-Nm probe has a very weak fluorescence (Φ_F = 0.006) under a 365 nm UV lamp, which can be barely observed with the naked eye. The addition of GSH induced a variation in the solution from no fluorescence to a strong blue fluorescence (Φ_F = 0.30), and the maximum fluorescence emission peak exhibited a blue shift from 480 nm to 420 nm. However, biologically relevant analytes, such as Cys, Hcy, Thr, Ser, Glu, Lys, Phe, His, Gly, Ala, Val and Tyr, showed almost no changes in the fluorescence spectra, which indicated that the CZ-Nm probe selectively responded to GSH.

Fig. 1 (a) Fluorescence spectra of CZ-Nm (10 μM and λ_ex = 360 nm) in the presence of thiols and 10 other amino acids (500 equivalents) in PBS buffer–DMF (0.1 M, pH 7.4, and v/v = 4 : 1) for 30 min. (b) The corresponding fluorescent photographs of CZ-Nm (10 μM) in a cuvette in the presence of different analytes (500 equivalents) under a UV lamp.

Similarly, the UV-vis spectra also exhibited the response of the CZ-Nm probe to GSH. Upon the addition of GSH, the absorption peak at 420 nm decreased and a new peak appeared at 267 nm, accompanied with a colour change from yellow to colourless (Fig. 2), implying the break in the intramolecular conjugated structure of CZ-Nm. However, the absorption spectra did not change significantly in the presence of different amino acids. As shown in Fig. 3, when the concentration of GSH was increased, the absorption intensity at 420 nm was gradually decreased, and the new absorption peak at 267 nm was gradually increased. Under the same conditions, the CZ-Nm probe also showed similar responses to other thiol-containing compounds, namely, Cys and Hcy. However, owing to the response of the CZ-Nm probe to GSH (3 seconds) being much faster than that found for Cys and Hcy (5 min), exquisite selectivity for GSH can be obtained by controlling the reaction time.

Fig. 2 (a) Changes in the UV/vis absorption spectra of the CZ-Nm probe (10 μM) at 420 nm upon the addition of various thiols and amino acids (100 equivalents). (b) A photograph showing the color change in the presence of thiols and other amino acids.

Fig. 3 Absorbance spectra of the reaction solution of CZ-Nm (10 μM) in 0.1 M PBS buffer–DMF (pH 7.20; v/v, 1 : 1) solution with different concentrations of GSH.

Linearity

Under the optimized conditions, the responsive properties of the CZ-Nm probe to GSH were further investigated using fluorescence titration studies. As shown in Fig. 4, the fluorescence intensity increased up to 7-fold upon increasing the GSH concentration (0–500 equivalents). As envisioned, a good linear increase in fluorescence intensity could be observed upon increasing the concentration of GSH over a wide linear range (0–0.01 M) (Fig. 5). The regression equation is Y = 123116 + 66 972.9X (R = 0.9987). Then, we obtained a low detection limit of 6.4 μM based on 3 × δ_blank/k (where δ_blank is the standard deviation of the blank solution and k is the slope of the calibration plot). The relative fluorescence quantum yields were determined to be 0.30 using quinine sulfate dehydrate in 0.1 M H₂SO₄ as a standard. The corrected emission spectra were obtained for the quinine sulfate dehydrate standard (λ_ex = 360 nm; A (absorbance) < 0.01; Φ_F = 0.56) and calculated using the following equation.⁴⁷
where the subscripts x and s refer to the unknown and the standard, Φ stands for quantum yield, F represents the integrated area under the emission curve, A is the absorbance intensity at the excitation wavelength, λ_ex denotes the excitation wavelength, and n is index of refraction of the solution.

Fig. 4 Fluorescence emission spectra of the CZ-Nm probe (10 μM and λ_ex = 360 nm) upon increased concentrations of GSH (0–1000 equivalents) in PBS buffer–DMF (0.1 M, pH 7.4, and v/v = 4 : 1).

Fig. 5 Changes in the fluorescence intensity of the CZ-Nm probe (10 μM and λ_ex = 360 nm) against various concentrations of GSH (0–0.01 M) in PBS buffer–DMF (0.1 M, pH 7.4, and v/v = 4 : 1).

Tolerance of CZ-Nm to GSH over other interferents

A competitive experiment was implemented to analyze the influence of other amino acids and thiol compounds on the reaction of the CZ-Nm probe with GSH. As shown in Fig. 6, the change in the fluorescence emission intensity caused by GSH in the presence of background species, such as Cys, Hcy, Thr, Ser, Glu, Lys, Phe, His, Gly, Ala, Val and Tyr, was similar to that caused by GSH alone. The results indicated that the recognition of GSH using the CZ-Nm probe was barely affected by other amino acids and thiol compounds.

Fig. 6 Fluorescence intensities of the CZ-Nm probe (10 μM and λ_ex = 360 nm) in PBS buffer–DMF (0.1 M, pH 7.4, and v/v = 4 : 1) solution upon the addition of GSH (500 equivalents) in the presence of background species (500 equivalents).

Proposed mechanism

According to the literature,⁴⁸ we speculate the Michael addition reaction between the CZ-Nm probe and GSH is likely to be responsible for the fluorescence enhancement and changes in the UV-vis spectra. To investigate this mechanism, the stoichiometry of a binding event between GSH and the CZ-Nm probe was first determined. The results obtained from the Job's plot showed a 1 : 1 stoichiometry between GSH and CZ-Nm probe (Fig. S4†). Mass spectrometry also provided evidence for the reaction of GSH with nitroolefin. As shown in Fig. 7, the peak at m/z = 267.03 corresponded to CZ-Nm. And the peak at m/z = 572.16 corresponded to the reaction product between GSH and the CZ-Nm probe. Overall, all the measurements proved the occurrence of Michael addition between the CZ-Nm probe and GSH.

Fig. 7 Mass spectrometry monitoring the Michael addition between the CZ-Nm probe and GSH: (a) the mass spectrum of the CZ-Nm probe and (b) the mass spectrum of CZ-Nm + GSH.

Laser scanning confocal microscopy experiments with PC12

To further demonstrate that the permeability and the monitoring of intracellular GSH, fluorescence microscopy experiments were carried out using PC12 cells. As shown in Fig. 8b, the living PC12 cells exhibited almost no fluorescence signal without the incubation of the CZ-Nm probe in the control group with excitation at 405 nm. However, when the PC-12 cells were incubated with CZ-Nm (10 μM) for 30 min at 37 °C, a strong blue fluorescence was exhibited inside the cells (Fig. 8e). This indicated the cell permeability of the probe (transduction the cellular membrane). In another control group, the PC12 cells were pretreated with 0.5 mM thiol reactive N-ethylmaleimide (NEM, a trapping reagent of thiol species) for 30 min to consume all the free thiols in the PC12 cells, followed by treatment with the CZ-Nm probe for 30 min. The confocal microscopic images did not show a remarkable fluorescence signal (Fig. 8h). In addition, if the PC12 cells were pretreated with 2 mM GSH for 30 min followed by incubation with the CZ-Nm probe for another 30 min, a brighter fluorescence signal (Fig. 8k) can be observed than that shown in Fig. 8e. This demonstrated that the fluorescence change was dependent on the concentration of the intracellular GSH and not on that of the other related species. Thus, the CZ-Nm probe might provide a simple way to detect the intracellular GSH from Cys and Hcy, or other common amino acids.

Fig. 8 Confocal fluorescence images of PC12 cells. (a–c) PC12 cells incubated without the CZ-Nm probe. (d–f) PC12 cells incubated with the CZ-Nm probe (10 μM) for 30 min (λ_ex = 405 nm). (g–i) PC12 cells incubated with NEM (0.5 mM) for 30 min. (j–l) PC12 cells incubated with GSH (2.0 mM) for 30 min, then incubated with the CZ-Nm probe (10 μM) for another 30 min. (a), (d), (g) and (j) bright field image; (b), (e), (h) and (k) fluorescence image in the blue channel; (c), (f), (i) and (l) overlapped image.

Conclusions

In summary, we have developed a novel “turn-on” carbazole-based fluorescent probe (CZ-Nm) for the specific detection of GSH from Cys and Hcy, or other natural amino acids in a PBS buffer–DMF (pH 7.4, v/v, 4 : 1) solution. The abovementioned results show that the CZ-Nm probe with GSH has a wide linear range and low detection limit under physiological conditions. Furthermore, the confocal fluorescence microscopy imaging of PC12 cells was carried out successfully, which not only indicated the cell permeability and bio-compatibility of the CZ-Nm probe, but also demonstrated that this probe can be applied to monitor GSH in living cells.

Acknowledgements

The authors are grateful to the National Natural Science Foundation of China (21576071) and the International Science and Technology Cooperation Project of Henan Province (152102410023).

Notes and references

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Footnote

† Electronic supplementary information (ESI) available. See DOI: 10.1039/c5ra25204k

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