A diketopyrrolopyrrole-based fluorescence turn-on probe for the detection of Pb²⁺ in aqueous solution and living cells

Abstract

A diketopyrrolopyrrole-based fluorescent probe DPP-HBT bearing a benzothiazole hydrazone motif exhibited an obvious fluorescent turn-on response toward Pb²⁺ ions with a low detection limit of 2.3 × 10⁻¹⁰ M in aqueous solution. Furthermore, the imaging experiments indicated that this probe was cell-permeable and could be used to detect Pb²⁺ ions within living cells.

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Introduction

Lead(II) ions (Pb²⁺) are the most toxic metal ions among the heavy metals, and are often encountered in the environment due to their widespread use in batteries, gasoline, and pigments.¹ Lead toxicity causes various symptoms such as anaemia, muscle paralysis, memory loss, disorder of blood and mental retardation.² Moreover high exposures to lead can result in severe damage to the kidneys and brain.³ Even very low levels of lead exposure can cause neurological, reproductive, cardiovascular, and developmental disorders, which introduce particularly serious problems in children including slowed motor responses, decreased IQs, and hypertension.⁴ However, the mechanism of lead toxicity still remains incompletely resolved.⁵ Thus, the detection of Pb²⁺ in the environment, especially in biological systems, is an important and attractive area of research.

Currently, many approaches, such as atomic absorption spectrometry⁶ and inductively coupled plasma mass spectrometry,⁷ have been developed for the determination of Pb²⁺. However, most of these methods are expensive, require extensive sample preparation, long detection times, and can only measure the total Pb²⁺ content. Recently, fluorescent methods have become promising for Pb²⁺ detection because of their simplicity of operation, low cost, real-time detection, low detection limit, and intracellular detection. To date, considerable efforts have been devoted to the development of new fluorescent probes for Pb²⁺ based on peptide,⁸ protein,^9–11 DNAzyme,^12–14 and a very few small-molecule scaffolds.^5,15–23 Among these probes, small-molecular probe is one of the successes.

Notably, most of probes based on small-molecules showed fluorescence quenching upon interaction with Pb²⁺ via spin–orbit coupling or electron transfer, which was not only disadvantageous for a high signal output during detection but also undesirable for analytical purposes.^24–26 To the best of our knowledge, there were only two examples of small-molecule fluorescent turn-on probes capable of detecting Pb²⁺ within living cells up to now.^5,16 Chang et al. reported fluorescein-based fluorescent probe for detecting Pb²⁺ in living HEK cells,⁵ and Xu et al. prepared naphthalimide-based fluorescence probe for detecting Pb²⁺ in living HeLa cells.¹⁶ So, there is a great need for the development of structurally simple fluorescence turn-on probes that can detect Pb²⁺ selectively in aqueous solution and in living cells.

Recently, we have reported a series of diketopyrrolopyrrole-based fluorescent probes for high selectivity and sensitivity for cations and anions, respectively.^27–32 In continuation of our interests in diketopyrrolopyrrole-based fluorescent probes, we herein present the spectral properties and cell imaging studies of a diketopyrrolopyrrole-based fluorescent probe DPP-HBT, a “turn-on” fluorescent probe for sensitive and selective detecting of Pb²⁺ ions in aqueous solution and in living cells.

Experimental section

Measurements

¹H NMR and ¹³C NMR spectra were collected on a Bruker Avance II 400 MHz spectrometer. UV-vis spectra were recorded on a Shimadzu 3100 spectrometer. Fluorescence measurements were carried out using an Edinburgh Instruments Ltd-FLS920 fluorescence spectrophotometer.

Sample preparation

All tests described in this paper were carried out at room temperature (25 °C) with distilled water. In the experiments of titration with various metal ions, the probe was dissolved in CH₃CN/0.01 M PBS buffer (v/v, 1 : 1, pH 7.4) to afford the test solution (1 × 10⁻⁵ M). Stock solutions (1 × 10⁻⁵ M) of the metal salts of LiCl, MgCl₂, CaCl₂, NiCl₂, CuCl₂, ZnCl₂, CdCl₂, HgCl₂, PbCl₂, AgNO₃, AuCl₃, MnCl₂, FeSO₄, FeCl₃, CoCl₂, CrCl₃ and AlCl₃ in water were prepared.

General method

All UV-vis and fluorescence titration experiments were carried out at room temperature. To the 1 × 10⁻⁵ M CH₃CN/0.01 M PBS buffer (v/v, 1 : 1, pH 7.4) solution of DPP-HBT, the varying equivalents of the metal ions were added separately and spectra were recorded. Titration plots were generated by using Origin (Microcal software). The ¹H NMR was carried out in DMSO-d₆ using TMS as an internal reference standard. To the 2.0 × 10⁻³ M solution of probe DPP-HBT in DMSO-d₆ the varying equivalents of Pb²⁺ were added and the ¹H NMR spectra recorded after each addition.

Synthesis

Compound DPP-HBT was synthesized according to the reported method.³⁰

Determination of the limits of detection (LOD)

The LOD was calculated based on the fluorescence titration. Probe DPP-HBT was employed at 1 × 10⁻⁵ M. The LOD was calculated using the formula 3σ/k, where σ was the standard deviation of blank (10 samples) and k was the slope between intensity difference versus sample concentration (Fig. S4†).

Results and discussion

Sensing of Pb²⁺ ions

Compound DPP-HBT was synthesized through condensation of compound DPP-AL and 2-hydrazinobenzothiazole in refluxing ethanol (Scheme 1).³⁰ The sensing behavior of DPP-HBT towards the addition of different metal species was studied using UV-vis and fluorescence spectroscopy. As shown in Fig. S1,† the UV-vis spectrum of free DPP-HBT (10 μM) in CH₃CN/0.01 M PBS buffer (v/v, 1 : 1, pH 7.4) exhibited a single absorption band at 500 nm, which belonged to the DPP core. Addition of increasing amounts of Pb²⁺ DPP-HBT resulted in the significant increase in the intensity of absorption bands at 497, 338 and 279 nm with the color change from lavender to flesh color. The fluorescence spectra of free DPP-HBT had a very weak emission band centered at 580 nm (excitation at 475 nm) in CH₃CN solution due to nonradiative quenching processes that occurred via a standard C=N isomerization deactivation pathway. However, upon the addition of Pb²⁺, a new emission band with a maximum at 585 nm appeared and the intensity of this band gradually increased with an increasing concentration of Pb²⁺ (Fig. 1). The increase in emission intensity showed a linear relationship towards the addition of Pb²⁺ in the range of 0–2.0 μM. The emission intensity became saturated when 2 equiv. of Pb²⁺ was added, creating an approximately 46-fold fluorescence enhancement with a bright orange emission response.

Scheme 1 Synthetic route of probe DPP-HBT.

Fig. 1 Fluorescence spectra of DPP-HBT (10 μM) in the presence of increasing amount of Pb²⁺ (0 to 20 μM) in CH₃CN/0.01 M PBS buffer (v/v, 1 : 1, pH 7.4). Inset: calibration curve (λ_ex: 475 nm).

The binding stoichiometry of probe DPP-HBT and Pb²⁺ was further proved by Job's plot according to the continuous variations with a total concentration of [Pb²⁺] + [DPP-HBT] (10 μM) (Fig. S2†). The maximum fluorescence emission appeared at the ∼0.66 mol ratio of [Pb²⁺]/([Pb²⁺] + [DPP-HBT]), which indicating the 2 : 1 interaction between Pb²⁺ ions and probe DPP-HBT. The binding constants were calculated to be 1.59 × 10⁸ M⁻² for DPP-HBT (Fig. S3†). The limits of detection (LOD) of probe DPP-HBT for Pb²⁺ ions was found to be 0.23 × 10⁻⁹ M (Fig. S4†), which was well below the maximum contamination level defined by the EPA.³³ As shown in Fig. S5,† the reaction between probe DPP-HBT and Pb²⁺ was very rapid and reached completion in 5 s.

Selectivity, interference, and reversibility studies

Furthermore, the selective profile of DPP-HBT in response to other metal ions was also studied (Fig. 2). As shown in Fig. 2, only the addition of 2 equiv. of Pb²⁺ to the solution of DPP-HBT induced a significant enhancement of fluorescent intensity. The addition of Zn²⁺ to DPP-HBT caused a small increase in fluorescent intensity (ca. 15% compared to that with Pb²⁺). To our delight, no obvious spectral changes could be detected for the metal ions such as Ag⁺, Al³⁺, Au³⁺, Ca²⁺, Cd²⁺, Co²⁺, Cr³⁺, Cu²⁺, Fe²⁺, Fe³⁺, Hg²⁺, Li⁺, Mg²⁺, Mn²⁺, Ni²⁺, and Zn²⁺. Thus, the probe was highly selective towards Pb²⁺, and could be used as a fluorometric probe for Pb²⁺ by means of “turn-on” fluorescence response.

Fig. 2 Fluorescence spectra of compound DPP-HBT (10 μM) and on addition of different metal ions (20 μM) in CH₃CN/0.01 M PBS buffer (v/v, 1 : 1, pH 7.4).

As shown in Fig. 3 (blue bars), competition experiments indicated that when Pb²⁺ was added to the solution of DPP-HBT in the presence of other excess metal ions such as Ag⁺, Al³⁺, Ca²⁺, Cd²⁺, Co²⁺, Cr³⁺, Fe³⁺, Fe²⁺, Li⁺, Mg²⁺, Mn²⁺, Ni²⁺ and Zn²⁺, the emission spectra displayed a similar pattern at 585 nm to that with Pb²⁺ only. However, the co-existence of Pb²⁺ with Hg²⁺, Cu²⁺ or Au³⁺ led to an almost complete fluorescence quenching, which might be attributed to the competitive binding of Hg²⁺ or Cu²⁺ or Au³⁺ and Pb²⁺ to DPP-HBT. This phenomenon was also observed in some previous Pb²⁺ fluorescent probes.¹⁶

Fig. 3 The histograms showing the fluorescence intensities of probe DPP-HBT (10 μM) upon the addition of various metal ions in CH₃CN/0.01 M PBS buffer (v/v, 1 : 1, pH 7.4) solution. Red bars represent the fluorescence response of probe DPP-HBT to the metal ions of interest (20 μM), blue bars represent the addition of Pb²⁺ (20 μM) to the foregoing solution (200 μM). From 1 to 18: none, Ag⁺, Al³⁺, Ca²⁺, Cd²⁺, Co²⁺, Cr³⁺, Fe²⁺, Li⁺, Mg²⁺, Mn²⁺, Fe³⁺, Ni²⁺, Zn²⁺, Hg²⁺, Cu²⁺, Au³⁺ and Pb²⁺ (λ_ex: 475 nm).

For a chemical probe to be extensively employed in the detection of specific analytes, the reversibility was an important aspect. The interaction between DPP-HBT and Pb²⁺ was reversible, which was verified by the introduction of strong chelating ligand EDTA into the system containing DPP-HBT and Pb²⁺ in CH₃CN (Fig. S6†). The experiment, shown in Fig. S6a,† showed that the introduction of EDTA (2 equiv. to Pb²⁺) immediately quenched the fluorescence of DPP-HBT. When Pb²⁺ was added to the system again, the fluorescence of DPP-HBT was enhanced. This process could be repeated at least four times without loss of sensitivity, shown in Fig. S6b,† which clearly demonstrated the high degree of reversibility of the complexation/decomplexation process.

Sensing mechanism of Pb²⁺ ions

Based on the results of absorbance and fluorescence titration, we proposed a plausible binding modes for [Pb(DPP-HBT)] as shown in Scheme 2. The weak fluorescence of free DPP-HBT in CH₃CN solution was likely due to nonradiative quenching processes that occurred via a standard C=N isomerization deactivation pathway. For [Pb(DPP-HBT)], Pb²⁺ should be easy to fit into the pseudocavity formed between the N atom on C=N moiety and the S atom on benzothiazole moiety, which inhibited of C=N isomerization and resulted in fluorescence enhancement. The fourth coordination site of Pb²⁺ might be occupied by the counter anion Cl⁻ or acetonitrile molecule.

Scheme 2 Sensing process and binding mode of DPP-HBT for Pb²⁺.

To support the results obtained by spectroscopic experiments, and to obtain additional information about the coordination mode of Pb²⁺ by receptor DPP-HBT, we have performed the ¹H NMR spectroscopic analysis in DMSO solution (Fig. 4). The most significant spectral changes observed upon addition of increasing amounts of Pb²⁺ ion to a solution of the free receptor DPP-HBT were the following: (i) the H_c and H_g protons for compound DPP-HBT (Scheme 2) got significantly downshifted by 0.04 and 0.03 ppm, respectively, which indicated the coordination of Pb²⁺ ion with nitrogen atom on imine moiety and sulfur atom on thiazole ring, respectively; (ii) similarly, the H_d, H_e and H_f protons within the benzothiazole ring for compound DPP-HBT also got downshifted by ∼0.01 ppm, respectively; (iii) the H_a and H_b which were identified as the meso-aryl protons showed a slightly upfield shift by ∼0.01 ppm for compound DPP-HBT. From the magnitude of the observed ¹H chemical shifts, it could be concluded that the plausible binding mode of Pb²⁺ was the nitrogen atom on imine moiety and sulfur atom on thiazole ring (Scheme 2).

Fig. 4 Partial ¹H NMR titration spectra of probe DPP-HBT (2.0 × 10⁻³ M) upon addition of increasing amounts of Pb²⁺ ion in DMSO-d₆.

pH studies and fluorescence imaging of Pb²⁺ ions in live cells

To further investigate the detection performance of probe DPP-HBT, we carried out the sensing assay under different pH conditions. The effect of pH on the detection of Pb²⁺ by probe DPP-HBT was shown in Fig. S7.† DPP-HBT displayed stability over a pH range from 1 to 13, while remaining non-emissive over this range. DPP-HBT could sense Pb²⁺ in the most common pH ranges (pH = 5–11) (Fig. S7†). Consequently, DPP-HBT operated efficiently over a pH range, (pH 5–11), especially under physiological conditions, which was of primary importance for cell imaging studies.

In view of the excellent performance of probe DPP-HBT under physiological pH conditions, we next investigated the potential of probe DPP-HBT for tracking Pb²⁺ ions in living cells. The human lung adenocarcinoma (A549) cells incubated with only DPP-HBT (10 μM) exhibited almost no fluorescence (Fig. 5B). By contrast, the cells stained with both the DPP-HBT and Pb²⁺ showed bright fluorescence (Fig. 5B′), in good agreement with the fluorescence turn-on profile of the probe in the presence of Pb²⁺ in the solution. The cytotoxicity of DPP-HBT on A549 cells was determined by an MTT assay (Fig. S8†). Upon exposure of 10 μM DPP-PyR for 24 h, ∼93% of the A549 cells remained viable. This nullifies the possibility of any significant cytotoxic influence of DPP-PyR on the A549 cells. Therefore, probe DPP-PyR was cell membrane permeable and capable of sensing Pb²⁺ in living cells.

Fig. 5 Fluorescence images of human lung adenocarcinoma cells (A549). (A and A′) Bright field image of the cells incubated with probe DPP-HBT (10 μM) for 20 min at 37 °C; (B and B′) fluorescence image of the cells incubated with probe DPP-HBT (10 μM) for 20 min and then further incubated without ions, incubated with Pb²⁺ (20 μM) for 30 min, respectively; (C and C′) merged image of (A and A′) and (B and B′).

Conclusions

In summary, compound DPP-HBT could efficient recognize Pb²⁺ ions with fluorescence turn-on response with a detection limit of 2.3 × 10⁻¹⁰ mol L⁻¹ in aqueous solution. Confocal fluorescence images demonstrated that probe DPP-HBT was cell-permeable and could be used to monitor Pb²⁺ ions in living cells.

Acknowledgements

This work was supported by Natural Science Foundation of Shandong Province of China (ZR2015BL010), the Scientific Research Foundation of University of Jinan (XKY1416), Shandong Provincial Key Laboratory of Fluorine Chemistry and Chemical Materials, and University Young Key Teacher Home Visit by the Ministry of Education of Shandong Province.

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Footnote

† Electronic supplementary information (ESI) available. See DOI: 10.1039/c6ra07378f

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