Lysosome-Targeting Fluorescence Sensor for Sequential Detection and Imaging of Cu2+ and Homocysteine in Living Cells.

A conjugated polymer-based fluorescence sensor, namely, PTNPy, was constructed on the basis of a polythiophene scaffold coupled with dimethylpyridylamine (DPA) groups in side chains for the consecutive detection and quantification of Cu2+ and Hcy in a perfect aqueous medium. A dramatic fluorescence quenching of PTNPy by the addition of Cu2+ was observed in Tris-HCl buffer solution (2 mM, pH 7.4), demonstrating a quick (<1 min) and highly selective response to Cu2+ with a low limit of detection of 6.79 nM. Subsequently, the Cu2+-quenched fluorescence of PTNPy can be completely recovered by homocysteine (Hcy), showing excellent selectivity to Hcy over other competitive species such as cysteine and glutathione. Thanks to the low cytotoxicity and lysosomal targeting ability of PTNPy, it was further applied as an optical sensor for the sequential imaging of Cu2+ and Hcy in HeLa cells. More importantly, Hcy concentration was linearly related to the fluorescence intensity of PTNPy in living cells, demonstrating huge potential for real-time monitoring the fluctuation of Hcy levels in living cells.

Introduction

Biothiols mainly including homocysteine (Hcy), cysteine (Cys), and glutathione (GSH), as well as various metal ions such as Cu2+, Fe2+, Ca2+, and so forth, are well known to be involved in many biological processes and play key roles in human physiology.[1−5] Their levels in human plasma have already been directly or indirectly linked to many diseases.[6−11] For instance, the concentration of Hcy in body fluids is normally of 12–15 μM, and an elevated level is related to many human afflictions such as cardiovascular disease, Alzheimer’s diseases, stroke, cancer, and so on.[12,13] More importantly, it was observed that the levels of copper and Hcy simultaneously elevate in patients with cardiovascular disease.[14,15] Kang et al. demonstrated that Hcy interferes with Cu homeostasis and hampers the availability of copper to chaperones such as cytochrome-c oxidase and COX17, resulting in mitochondrial dysfunction and endothelial cell injury.[16] Jakubowski and co-workers also confirmed that copper promotes efficient demethylation of methionine (Met) to increase the Hcy levels.[17] In addition, O2– produced by complexation of Hcy with Cu2+ can clear NO and inhibit NO-related cerebrovascular diseases.[18] Therefore, the specific and simultaneous detection of Cu2+ and Hcy especially at the cellular level is of importance to understand their physiological functions and clinically diagnose the related diseases.

Fluorescence sensors are regarded as the most charming method to real-time monitor and track biological species through combining with the laser confocal imaging technology due to its simplicity, sensitivity, and high temporal and spatial resolution.[19,20] In recent decades, a mass of fluorescence sensors has been developed to identify Hcy from other amino acids, especially structurally similar mercaptoamino acids such as Cys and GSH[21−27] by taking advantage of distinctive redox property of Hcy[21−23] or nucleophilic reactions based on −SH and/or −NH2 of Hcy.[24−27] To date, however, only a few sensors can sequentially sense Cu2+ and Hcy[18,28−38] that usually operated in a mixed medium of organic solvent and water[18,28,29,31,32,34,35] and suffer from the interference from other analytes (Table S1).[28−32,34,36,37] Besides, there are rare sensors available for continuous imaging of Cu2+ and Hcy in living cells.[18] Thus, it is still urgently needed to develop bifunctional fluorescence sensors for the simultaneous detection of Cu2+ and Hcy with high sensitivity and selectivity in a prefect aqueous solution and living cells.

Thanks to high brightness, excellent light stability, and easily adjustable spectral characteristics, π-conjugated polymers have been widely used to construct sensing and imaging platforms for various bio-related analytes.[39,40] Zhang’s group developed a polythiophene derivative (PTMA) with a tridentate N-/O-containing ligand for the recognition of heavy metal ions and biothiols in aqueous solutions.[37] The addition of Co2+, Cd2+, and Cu2+ can induce a dramatic fluorescence decrease of PTMA, and subsequently, the Cu2+-quenched fluorescence can be restored by Hcy and GSH with fluorescence enhancement of 11.5 and 7.7 folds in the THF/Tris–HCl mixed solution.[37] More recently, Tang’s group reported a fluorene- and benzothiadiazole-containing conjugated polymer-based fluorescence nanosensor, allowing imaging of Hcy levels in the kidney and liver of diabetic mice.[38]

Inspired by these outstanding works and the fact that there is a strong interaction between Cu2+ and Hcy,[41−44] herein, we reported a new conjugated polymer (PTNPy, Scheme a) as a multifunctional sensor for the goal of a quick, sequential detection of Cu2+ and Hcy in a perfect aqueous medium and living cells. PTNPy possess a polythiophene backbone as a fluorophore, dimethylpyridylamine (DPA) as the chelating groups of Cu2+ ions, and quaternary ammonium salt groups in its side chains to increase its water solubility. As shown in Scheme b, the chelation of Cu2+ with the DPA groups of PTNPy led to the formation of stable PTNPy–Cu2+ complexes and thus effectively quenched the fluorescence of PTNPy due to the intrinsic paramagnetic properties of Cu2+.[45] When in situ generated PTNPy–Cu2+ complexes encountered Hcy, free PTNPy was released because of the stronger coordination interaction between Hcy and Cu2+,[38] resulting in that the fluorescence of PTNPy was restored. Based on the strategy, PTNPy can sequentially detect Cu2+ and Hcy with high selectivity. Further, PTNPy exhibited excellent biocompatibility and lysosome-targeting ability and was applied as an optical sensor for sequential imaging of Cu2+ and Hcy in HeLa cells.

Scheme 1

(a) Synthetic Route of the Sensor PTNPy and (b) Schematic Diagram of the Sequential Fluorescence Response of PTNPy toward Cu2+ and Hcy

Results and Discussion

The synthetic route of PTNPy is shown in Scheme a. PTNPy was conveniently prepared by oxidative polymerization of M1 with M2 (the molar ratio of M1 to M2 is 1:1 in feed) in the presence of FeCl3. The 1H NMR spectrum of PTNPy (Figure S1) revealed clear peaks in the range of 8.41–7.16 ppm, corresponding to the characteristic aromatic hydrogen on pyridine rings in DPA-functionalized thiophene segments, and at ∼2.0 ppm, corresponding to the specific alkyl hydrogen (a, a′, b, and b′). An integral ratio (I8.41–7.16/I2.0) of 1.07 was consistent with the theoretical value (m:n ≈ 1:2), indicative of the successful preparation of the target PTNPy. The polymer PTNPy can readily dissolve in deionized water, exhibiting strong yellow fluorescence with a fluorescence quantum efficiency (Φ) of 0.158 using rhodamine B as the reference (Φ = 0.73). The excellent water solubility of PTNPy, which meets the requirements of physiological applications, is related to the flexible substituent groups, especially quaternary ammonium salts on the side chains of polythiophenes.

The luminescence properties of PTNPy (100 μM) in the absence and presence of 15 different cations including Cu2+, K+, Na+, Ag+, Cd2+, Fe2+, Fe3+, Al3+, Hg2+, Ca2+, Co2+, Mg2+, Ni2+, Pb2+, and Zn2+ with the concentration of 100 μM in Tris–HCl buffer (2 mM, pH 7.4) was first studied. Among all tested ions, only Cu2+ can cause a remarkable fluorescence quenching of PTNPy (Figure a), suggesting the highly selective recognition ability of PTNPy toward Cu2+ ions. It was noted that just only Cu2+ can induce a slight red shift of the absorption spectra of PTNPy by around 19 nm along with a clear change of solution color from yellow to orange compared with the other tested metal ions (Figure S2), indicating a possible conformational transition of polymer backbone from random coil to planar conformation and even the formation of aggregates.[46] Therefore, the remarkable decrease in the fluorescence intensity of PTNPy in the presence of Cu2+ should be associated with two synergetic factors, that is, polymer aggregation and the paramagnetic properties of Cu2+.

Figure 1

(a) Fluorescence spectra of PTNPy (100 μM) in Tris–HCl buffer solution (2 mM, pH 7.4) in the absence and presence of various tested metal ions (100 μM). Inset: ratio of fluorescence intensity (Fi/F0) at 550 nm in the presence of various tested metal ions. (b) Fluorescence spectra of PTNPy (100 μM) in Tris–HCl buffer solution (2 mM, pH 7.4) with the amounts of Cu2+ (0–0.22 equiv). (c) Fluorescence intensity ratio (Fi/F0) at 550 nm of PTNPy at 550 nm as a function of addition of Cu2+ amounts; the linearity of peak intensity with respect to Cu2+ concentrations (inset). The data were extracted from the titration curves (b). (d) Job’s plot. F0 and Fi denote the emission intensity at 550 nm prior to and after the addition of Cu2+ ions, respectively. λex = 400 nm. Error bars represent the standard deviations of three trials.

As can be seen from Figure b, as the Cu2+ concentrations increase, the fluorescence intensity of PTNPy in the Tris–HCl buffer solution gradually decreased with a significant blue shift (∼36 nm) of the emission peak. When 0.2 equiv of Cu2+ was added, a plateau was arrived, indicating that the saturation concentration of Cu2+ was 20 μM. The Fi/F0 ratios of the fluorescence intensity of PTNPy at 550 nm were linearly proportional to [Cu2+] in the range of 0–3 μM (inset in Figure c). The calibration curves were thus obtained to determine the limit of detection (LOD) of PTNPy toward Cu2+ to be 6.79 nM according to the standard 3σ/S method.[47] Clearly, PTNPy is a highly sensitive sensor for copper ion in an aqueous medium, showing one of lowest LOD values reported among fluorescence sensors for the detection of Cu2+ (Table S1). Further, Job’s plot exhibited a 4:1 stoichiometry for the PTNPy–Cu2+ complex (Figure d) and that corresponding to the binding ratio of DPA groups to copper ions is about 5:2. Additionally, the binding constant of PTNPy with Cu2+ was estimated to be 1.42 × 105 M–1 based on the Benesi–Hildebrand equation (Figure S3), indicating strong affinity of PTNPy to Cu2+.

Since other thiol-containing amino acids and peptides besides Hcy can coordinate well with Cu2+ ions through the affinity between copper and sulfur atoms,[48−50] we further checked the spectral response of PTNPy–Cu2+ complexes to various amino acids and peptides, including Ala, Ser, Thr, Cys, Pro, Phe, Trp, Glu, His, Val, Leu, Cys–Cys, Met, Lys, Arg, and GSH. The photoluminescence spectra of PTNPy–Cu2+ complexes were separately recorded in the presence of different analytes (Figure a). As displayed in Figure a, only slight spectral changes were observed in the case of the addition of 4 equiv of other amino acids and GSH. In sharp contrast, the addition of Hcy to PTNPy–Cu2+ complexes caused a remarkable spectral changes and a significant increase of fluorescence intensity at 550 nm by about 38 times (Figure b). The fluorescence spectra of PTNPy–Cu2+ complexes in the presence of 4 equiv of Hcy seem to be identical with that of free PTNPy (Figure S4), indicating that the fluorescence recovery is related to the complete release of PTNPy from PTNPy–Cu2+ complexes due to the strong chelation of Hcy with Cu2+. Thus, PTNPy–Cu2+ complexes can selectively recognize Hcy over other sulfur-containing species such as Met and GSH in a perfect aqueous solution through the quick displacement mechanism.

Figure 2

(a) Fluorescence spectra of PTNPy–Cu2+ complexes (100 μM) in Tris–HCl buffer (2 mM, pH 7.4) in the presence of 4 equiv of different amino acids and peptides. (b) Relative fluorescence intensity (Fi/F0) at 550 nm of PTNPy–Cu2+ (100 μM) in Tris–HCl buffer (2 mM, pH 7.4) toward various tested amino acids, data were extracted from (a). (c) Fluorescence spectra of PTNPy–Cu2+ (100 μM) with the addition of various concentrations of Hcy (0–3.2 equiv) in Tris–HCl buffer (2 mM, pH 7.4). (d) Plot of (Fi – F0)/F0 at 550 nm vs concentrations of Hcy. F0 is the initial emission intensity of PTNPy–Cu2+ complexes (100 μM), and Fi is the recorded emission intensities of PTNPy–Cu2+ complexes in the presence of Hcy with different concentrations. λex = 400 nm. Error bars represent the standard deviations of three trials.

Figure c shows the fluorescence titration spectra of PTNPy–Cu2+ complexes (100 μM) in Tris–HCl buffer (2 mM, pH 7.4) in the presence of various Hcy contents from 0 to 320 μM at a λex of 400 nm. The quenched fluorescence of PTNPy–Cu2+ complexes was gradually restored with the increase of Hcy concentrations along with an obvious red shift of emission peaks by 40 nm. When the concentration of Hcy reached 3.2 equiv of PTNPy–Cu2+ complexes, the changes in fluorescence intensity became negligible, indicating the analyte–receptor saturation. Very importantly, the ratio of fluorescence intensity [(Fi – F0)/F0] depends linearly on the concentration of Hcy in the range of 0–300 μM with a correlation coefficient of 0.9990, where F0 is the emission intensity of free PTNPy–Cu2+ complexes and Fi is the recorded emission intensities of PTNPy–Cu2+ complexes in the presence of Hcy with different concentrations (Figure d). Based on the result of fluorescence titration, the LOD for Hcy can be determined to be 9.64 × 10–8 M according to the standard 3σ/S method.[47] Therefore, PTNPy–Cu2+ complexes have huge potential for the quantitative determination of Hcy under physiological pH conditions.

As we know the cellular environment is very complex and there are various anions and cations such as K+, Na+, Ca2+, Cl–, CO32–, SO42–, and so on, the interference experiments were thus performed to evaluate the practical applicability of PTNPy sensor for the sequential detection of Cu2+ and Hcy in living cells. In this study, the Tris–HCl buffer (2 mM, pH 7.4) of PTNPy (100 μM) was treated with 1 equiv of the tested ions. Cu2+ (20 μM) was then added into the above solution, which was subsequently subjected to 4 equiv of Hcy. The fluorescence intensity at 550 nm at each case was separately recorded. As shown in Figure , in the presence of 1 equiv of other various substrates, the fluorescence of PTNPy can still be effectively quenched by 0.2 equiv of Cu2+ and almost completely recovered by Hcy subsequently. These results demonstrated that PTNPy is an excellent ON–OFF type optical sensor for Cu2+ with high selectivity over other competing ions, and the fluorescence of PTNPy–Cu2+ complexes can selectively response to Hcy even in a complex environment containing various ions as tested in this work.

Figure 3

Competitive binding experiments of PTNPy (100 μM) in Tris–HCl buffer solution (2 mM, pH 7.4) with various bio-related species (100 μM) in the absence and presence of Cu2+ (20 μM) and Hcy (400 μM). 1: none, 2: Na+, 3: Zn2+, 4: Mg2+, 5: K+, 6: Fe2+, 7: Fe3+, 8: Ca2+, 9: Cl–, 10: CO32–, 11: HCO3–, 12: H2PO4–, 13: HPO42–, 14: I–, 15: NO3–, 16: OAc–, 17: P2O74–, 18: PO43–, 19: S2–, 20: S2O32–, and 21: SO42–.

The biocompatibility of PTNPy against HeLa cells was examined by a standard MTT assay (Figure S5). After 24 h of incubation, more than 94% of cells were viable at concentration ranging from 0 to 100 μM PTNPy, indicating its slight toxicity. Colocalization experiments were carried out to assess the organelle-targeting ability of the sensor. HeLa cells were co-stained with PTNPy and a lysosome-targeting dye, that is, LysoTracker Red (Figure ). PTNPy and LysoTracker Red showed a green and red fluorescence within lysosome, respectively (Figure a,b). The signal from PTNPy was well overlapped with that from LysoTracker Red with an overlap coefficient of 0.903 (Figure f). Therefore, PTNPy has good cell membrane permeability and can target the lysosome specifically. As a vital organelle, lysosome has the acidic physiological environment with pH 4.0–5.0. Thus, we further evaluated the effect of pH on the spectral characteristics of PTNPy and PTNPy–Cu2+ complexes. As shown in Figure S6, no significant changes in the fluorescence intensity of both were observed in the range of pH 4.0–8.0. These results indicated that PTNPy holds tremendous potential as a sensor to track Cu2+ and Hcy in the lysosome of living cells.

Figure 4

Colocalization of HeLa cells stained with PTNPy (20 μM) and LysoTracker Red. (a) PTNPy (20 μM) (λex = 405 nm and λem = 496–554 nm). (b) LysoTracker Red (75 nM) (λex = 559 nm and λem = 577–649 nm). (c) Merged image of (a,b). (d) Bright-field image. (e) Merged image of (c,d). (f) Pearson’s overlap coefficient distribution. Scale bars: 10 μm.

To verify the successive sensing capacity of the sensor PTNPy toward Cu2+ and Hcy in living cells, we explored their imaging applications in HeLa cells. After incubation with PTNPy (20 μM) for 30 min, HeLa cells showed bright green fluorescence (Figure a–c). The fluorescence of PTNPy was completed quenched after incubation with Cu2+ (4 μM) for 10 min (Figure d–f), demonstrating the formation of PTNPy–Cu2+ complexes within living cells. Subsequently, in situ generated PTNPy–Cu2+ complex-stained cells were treated by 100 μM Hcy, and bright green fluorescence was re-observed (Figure g–i). These results indicated that PTNPy can be effectively released from PTNPy–Cu2+ complexes when they encountered Hcy in the living cells.

Figure 5

Fluorescence images of HeLa cells after incubating with PTNPy (a–c), PTNPy + Cu2+ (d–f), and PTNPy + Cu2+ + Hcy (g–i). (a,d,g) show bright-field images. (b) HeLa cells were cultured with PTNPy (20 μM) at 37 °C for 30 min. (e) PTNPy-loaded HeLa cells were treated with 4 μM Cu2+ ion at 37 °C for 10 min. (h) PTNPy and Cu2+-loaded HeLa cells were cultured with Hcy (100 μM) at 37 °C for another 10 min. (c,f,i) show the merged images of HeLa cells. λex = 405 nm and λem = 505–605 nm. Scale bars: 20 μm.

Further, HeLa cells loaded by PTNPy and Cu2+ were cultured with Hcy at various concentrations such as 5, 10, 20, 30, 40, 50, 60, and 70 μM. After 30 min of incubation, the fluorescence images of cells were recorded (Figure ). The restored fluorescence in HeLa cells with Hcy (70 μM) showed slight changes with prolonged irradiation time (0–100 s), demonstrating the good photostability of the sensor in living cells (Figure S7). More importantly, it was noted that the restored fluorescence intensity (the average fluorescence intensity density of 20 cells) was positively proportional to the Hcy concentration within the scope of 5–70 μM (Figure i). The calibration curves with R2 = 0.957 can thus be obtained for possible quantitative determination of Hcy fluctuation in living cells.

Figure 6

Confocal laser scanning microscopy (CLSM) images of PTNPy–Cu2+-loaded HeLa cells incubated with different concentrations of Hcy. The concentrations of Hcy from (a–h) are 5, 10, 20, 30, 40, 50, 60, and 70 μM, respectively. (i) Plot of the fluorescence intensity density vs the concentration of Hcy. λex = 405 nm and λem = 505–605 nm. Error bars represent the standard deviations of three independent experiments. Scale bars: 20 μm.

Conclusions

In summary, we reported the design and synthesis of a new conjugated polymer-based fluorescence sensor (PTNPy) for the sequential detection of Cu2+ and Hcy in a 100% aqueous medium. PTNPy exhibits highly selective recognition ability toward Cu2+ ions over other metal ions by a fluorescence ON–OFF response with a low LOD of 6.79 nM for Cu2+. The in situ generated PTNPy–Cu2+ complexes can be applied as a fluorescence sensor for quantitative detection and bio-imaging of Hcy in living cells. Benefiting from excellent biocompatibility and easy structural modification of polythiophenes, this work will pave a way for the future development of multi-functional optical sensors for the simultaneous detection of different target analytes through rational molecular design.

Experimental Section

Reagents and Materials

Hcy, l-Cys, reduced GSH, and other tested amino acids were provided by Tianjin Heowns Biochemical Technology Co., Ltd. (Tianjin, China). Copper(II) chloride dehydrate was obtained from Beijing Chemical Works (Beijing, China). 3-(3-Bromo-propoxy)-4-methyl-thiophene and N,N,N-trimethyl-3-((4-methylthiophen-3-yl)oxy)propan-1-aminium bromide were synthesized according to the procedure reported in our previous work.[46] 3-(4,5-Dimethylthiazol-2-yl)-2,5-diphenyl-tetrazolium bromide (MTT) was bought from Sigma-Aldrich (St. Louis, USA). Ultrapure water (18.6 M cm at 25 °C) was freshly prepared using a Millipore filtration system. The polymer concentration was calculated according to the repeat units (RUs).

Equipment and Methods

1H NMR spectra were recorded on a Bruker AV400 using residual solvent peak as a reference. Fluorescence analysis was carried out utilizing an F-4600 fluorescence spectrophotometer (Hitachi, Japan) equipped with a 1 cm quartz cell. UV analysis was recorded on a UV-3600 UV–vis spectrophotometer (Shimadzu, Japan). The pH values were determined using a Mettler Toledo Delta 320 pH meter. Cells were viewed with laser scanning confocal microscopy (Olympus FV1000-IX81). All images were digitized and analyzed with an Olympus FV1000-ASW.

CLSM Experiments

HeLa cells were grown in RPMI 1640 medium containing 10% FBS (fetal bovine serum) under a 5% CO2 atmosphere at 37 °C. The cells were then planted on six-well plates and incubated overnight under the same conditions. After removing the medium, the cells were incubated with a solution of PTNPy in the medium (20 μM, 2 mL) for 30 min at 37 °C. The cells were rinsed with PBS three times and incubated with a solution of Cu2+ in culture media (4 μM, 2 mL) for 10 min at 37 °C. After washing with PBS three times, the medium was replaced by the medium (2 mL) with different concentrations of Hcy for 10 min at 37 °C. The cells were viewed using CLSM.