Introduction

Since graphene quantum dots (GQDs) are considered the perfect alternatives to organic dyes and inorganic quantum dos (QDs) as a promising class of bioimaging probes due to their low toxicity and good photostability, red fluorescent GQDs have been attracting much attention in terms of their deeper light penetration into the biological specimens, higher imaging contrast to short-wavelength fluorescence of the auto-fluorescence or light scattering background of specimens [1–9]. The current most effective ways of preparing red fluorescent GQDs are to develop labelled GQDs by covalent modification with organic dyes or to prepare large size GQDs in order to keep a high conjugation extent. The complicated preparation process and low quantum yields, however, are the major restrictions for their further applications [10–13]. We have reported red fluorescent GQDs with large sp2 clusters by electrolysis of graphite in K2S2O8 aqueous solution [14]. Nevertheless, it has still stood as a challenge to create efficient red emission GQDs which possesses high yields, high quantum yields, and high color purity.

Europium trivalent ion Eu(III) based luminescent complexes or their composites are of great interest for cellular imaging as they exhibit high quantum efficiency, narrow red-emission bands and high color purity [15–17]. However, their seriously hydrophobic properties and easily quenched luminescence by water molecules have prevented their widespread studies and applications. Improvement has mainly been focusing on designing water soluble chelating ligands that coordinate to Eu(III) to improve the solubility or stability of the complexes in aqueous solution and shield Eu(III) from the solvent water molecules as well [18]. In this respect, assembled of GQDs with Eu(III) complex may take effect in improving the solubility and remaining the unique optical properties of Eu(III). Previous reports have shown that neither directly coordinating GQDs to Eu(III) nor modifying GQDs with the β-diketonate complex Eu(DBM)3Phen result in a red fluorescent aqueous solution [19].

Herein, we report on a new strategy to prepare water soluble, red fluorescent GQDs and Eu(III) complex composites (GQD/DBM)3EuPhen/GQD by noncovalent functionalization of the chelating ligands DBM and Phen with GQDs first, followed by coordinating to Eu(III). Significantly, from such GQDs and Eu(III) complex aqueous solution in neutral pH, we obtained stable and high pure red fluorescence with a quantum yield as high as 15.5 %, raising the prospect of using the Eu(III) complex for bioimaging.

Experimental

Materials

High purity graphite rods were purchased from Shanghai Carbon Co Ltd. (Shanghai, China, www.shts488.cn.china.cn). Tetrabutylammonium perchlorate (TBAP) was bought from J&K Chemical (Beijing, China, www.jkchemical.com). Dimethylsulfoxide (DMSO), C2H6O (ethanol), concentrated sulphuric acid (98 %), concentrated nitric acid (68 %), NaOH, Europium(III) nitrate hexahydrate, DBM and Phen were all obtained from Aladdin-reagent (Shanghai, China, www.aladdin-reagent.com). All solvents and reagents were purchased and used without further purification. Deionized water was used in all experiments.

Characterization

The Fourier transform infrared (FT-IR) spectra were measured using a Nicolet 380 spectrograph. The morphologies of the as-prepared (GQD/DBM)3EuPhen/GQD were investigated by a JEOL JEM 2100 transmission electron microscope (TEM) with high-resolution TEM (HRTEM). Sample solution was drop-cast from solution onto a carbon-coated TEM grid and the solvent was evaporated at room temperature. X-ray photoelectron spectroscopy (XPS) was carried out by an ESCALab 250Xi electron spectrometer from Thermo Scientific using 300 W Al Kα radiation. The binding energies were referenced to the C 1 s line at 284.8 eV from adventitious carbon. X-ray diffraction (XRD) patterns were obtained by using Cu Kα radiation of a PANalytical X’Pert Pro MPD. The photoluminescence spectrum and the UV-vis absorption spectra were measured with a PerkinElmer-LS55 and a UV-2450 spectrometer. An Olympus confocal fluorescence microscope equipped with an argon ion laser and neon-helium laser was used to obtain fluorescence microscopy images, with excitation wavelengths at 405 nm and FVXBA530RIF as emission filter. The fluorescence quantum yield (QY) was measured through an absolute method by using Varian FLR025 spectrometer equipped with a 120 mm integrating sphere. An IVIS Lumina III system was used for in vivo and ex vivo imaging.

Preparation of GQDs

GQDs were prepared by electrochemical exfoliation of graphite rod and acidic oxidation of exfoliation using concentrated mineral acid. The electrolysis of the graphite rod was performed on a HDV-7C transistor potentiostat with cathode current about 10 mA in 5 mL DMSO solution which contained 0.01 M TBAP as electrolyte. The graphite rod was the working electrode, while a Pt foil used as counter electrode. After electrolysis of 3 h, the resulting black solution was washed with ethanol and then centrifuged for several times to remove DMSO and TBAP. Finally, it was dried at 75 °C in a drying oven to a black powder. 0.1 g of the prepared black powder was added into a mixture of concentrated H2SO4 (10 mL) and HNO3 (5 mL) [20]. After refluxing at 100 °C for 24  h, the mixture was cooled to room temperature and diluted with deionized (DI) water. The excessive acid was neutralized by Na2CO3. The obtained solution was further dialyzed over DI water in a dialysis bag (molecular weight cutoff: 3500 Da) for 1 week.

Preparation of (GQD/DBM)3EuPhen/GQD

Ten milligram GQDs aqueous solution (2 mL) was mixed with 0.3 × 10−3 mol DBM, 0.1 × 10−3 mol Phen ethanol solution (15 mL). Then the pH of the mixture was adjusted to 7.5 by NaOH and treated with ultrasonication for 20 min [21, 22]. Afterwards, the solution was heated to 70 °C, and 0.1 × 10−3 mol Eu(NO3)3 ethanol solution (3 mL) was added drop by drop. After complete addition, the solution was under vigorous stirring and refluxing for 8 h, brown precipitate was gotten. Following this, the resulting mixture was cooled to room temperature and left overnight. The precipitate was filtered out, repeatedly rinsed with ethanol and water for several times, and dried overnight under vacuum. Finally, (GQD/DBM)3EuPhen/GQD brown powders can be readily harvested. After being redispersed in water, the homogeneous and stable (GQD/DBM)3EuPhen/GQD aqueous solution was obtained.

Preparation of solid Eu(III) complex with dibenzoylmethane and 1,10- phenanthroline

Eu(DBM)3Phen was synthesized according to the conventional routes as follows. Briefly, 0.3 × 10−3 mol DBM and 0.1 × 10−3 mol Phen were dissolved in ethanol solution (15 mL) under stirring. A few drops of NaOH were then added to adjust the pH to 7.5. And then the mixture was heated to 70 °C and stirred vigorously, followed by addition of 0.1 × 10−3 mol Eu(NO3)3 ethanol solution (3 mL). After refluxing for 8 h, yellow precipitate was gotten. The resulting mixture was cooled to room temperature and left overnight. After being filtered out by vacuum, the precipitate was repeatedly washed with ethanol and water for several times. Finally, we obtained the yellow powder.

Universal buffer

The pH response experiment was carried out in universal buffer solution. Universal buffer solution (0.1 M citric acid, 0.1 M KH2PO4, 0.1 M Na2B4O7, 0.1 M Tris, and 0.1 M KCl) was adjusted to the corresponding pH using 37 % HCl solution and saturated NaOH solution determined using the pH meter. The concentration of universal buffer solution finally used was 10 mM.

QY measurements

An absolute method, using Varian FLR025 spectrometer equipped with a 120 mm integrating sphere, was employed to determine the QY of the sample. We conducted the test light from FLR025 spectrometer to the sphere. The QY was determined by the ratio between photons emitted and absorbed by the sample. The aqueous solution was placed in a UV quarts cuvette with a light path of 10 mm to measure its QY, while the solvent water filled in the quarts cuvette was used as a blank sample for the reference measurement. The spectral correction curve which relates to the sensitivity of the monochromator, detector, sphere coating and optics to wavelength was provided by Edinburgh Instruments.

Cellular imaging and MTT assays

The human Michigan Cancer Foundation-7 (MCF-7) cells were chosen to assess the fluorescence properties of (GQD/DBM)3EuPhen/GQD. After incubating for 24 h in Dulbecco’s modified Eagle’s medium (DMEM) (2 mL) supplemented with 10 % fetal bovine serum (FBS) at 37 °C in a humidified atmosphere of 5 % CO2 and 95 % air, the culture medium was then replaced by DMEM high glucose containing the (GQD/DBM)3EuPhen/GQD and further incubated for 2 h at 37 °C. Then the MCF-7 cells on the plate were washed with buffer (pH = 7.4) and fixed by 4 % paraformaldehyde in PBS buffer for 15 min at room temperature. After being washed three times with PBS buffer, the cells were imaged by a confocal fluorescence microscope with excitation wavelengths at 405 nm.

The MTT (tetrazolium salt reduction) assays were performed to evaluate the toxicity of (GQD/DBM)3EuPhen/GQD and were detected by Victor3 V Multilabel reader (PerkinElmer, U.S.A). Mean absorbance for drug dose was expressed as a percentage of the control untreated well absorbance and plotted vs. drug concentration. Cells seeded in 96-well plates were separately treated with different concentrations of (GQD/DBM)3EuPhen/GQD (0, 10, 20, 50, 100, 200, 400 μg mL−1) for 24 h. Inhibition of cell lines were obtained after 24  h regular treatment of (GQD/DBM)3EuPhen/GQD by MTT assay.

In vivo and ex-vivo imaging

HeLa tumor-bearing nude mice were selected as the animal model, which were approved by the China Committee for Research and Animal Ethics in compliance with the law on experimental animals. The mice were intravenously injected with the (GQD/DBM)3EuPhen/GQD (5 mg kg−1), while the not injected mice were selected as controls. At 2 h, 6 h, 10 h after injection, in vivo imaging of HeLa tumor-bearing nude mice were taken by an IVIS Lumina III system. A filter set (excitation at 420 nm and emission at 620 nm) was used for the measurement. The mice were kept on the imaging stage under anesthesia with 2.5 % isoflurane gas in an oxygen flow (0.8 L min−1) during the imaging process. Living Image Software was employed to analyze the imaging date.

At 10 h post-injection, the mice were sacrificed, the tumor and major organs such as kidney, liver, lung, spleen and heart were taken out and kept on the imaging stage to be imagined. Fluorescent observation was carried out by the IVIS Lumina III system too. Living Image Software was employed to analyze the imaging date, and an ROI was drawn around the tumor and organ section.

Results and discussion

Characterization of (GQD/DBM)3EuPhen/GQD

The strategy for the fabrication of the water soluble, high color purity red-emitting (GQD/DBM)3EuPhen/GQD is illustrated in Scheme 1. Briefly, in the first step, GQDs are prepared through the electrolysis of the graphite rod, followed by refluxing in concentrated nitric and sulphuric acid mixed solution [20]. Next, the chelating ligands DBM and Phen are connected to GQDs noncovalently to prepare DBM/GQD/Phen by mixing DBM and Phen ethanol solution with GQDs aqueous solution using ultrasonication. Finally, the homogeneous and stable (GQD/DBM)3EuPhen/GQD solution can be harvested by reacting DBM/GQD/Phen with Eu(NO3)3. The successful connection of GQDs to Eu(III) complex Eu(DBM)3Phen significantly improve its water solubility and fluorescence stability.

Scheme 1
scheme 1

The illustration of preparing (GQD/DBM)3 EuPhen/GQD

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Transmission electron microscopy (TEM) (Fig. 1) images reveal that pristine GQDs (Fig. 1a, b) and DBM/GQD/Phen (Fig. 1c, d) are uniform in size with the average diameter of 5.3 ± 0.5 and 5.5 ± 0.4 nm, respectively. The high-resolution TEM (HRTEM) images (inset of Fig. 1a, c) confirm that both the GQDs and DBM/GQD/Phen exhibit a lattice spacing of 0.21 nm, which is corresponding to (1120) lattice fringes of graphene. All these results indicate that there were no significant changes in size and morphology of GQDs after functionalization with the chelating ligands. Figure 1e and f display TEM images of (GQD/DBM)3EuPhen/GQD, exhibiting an average size about 150 ± 10.6 nm in diameter.

Fig. 1
figure 1

TEM images of a GQDs, c DBM/GQD/Phen and e (GQD/DBM)3EuPhen/GQD. HRTEM images of GQDs (inset of a) and DBM/GQD/Phen (inset of b). Size distribution of b GQDs, d DBM/GQD/Phen and f (GQD/DBM)3EuPhen/GQD

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The FT-IR spectra of (GQD/DBM)3EuPhen/GQD (curve A), Eu(DBM)3Phen (curve B), GQDs (curve C) are shown in Fig. 2 a. Compared with that of solid Eu(DBM)3Phen (curve B) and GQDs (curve C), the spectrum of the (GQD/DBM)3EuPhen/GQD exhibits peaks at 1623 cm−1 (C = C stretching), 1598 and 1457 cm−1 (C = O stretching), 1517 cm−1 (C = O and C = N stretching), 1387 cm−1 (C-O stretching), and 520 cm−1 (O-Eu stretching). The peak at 1304 cm−1 is attributed to the phenyl ring in plane C-H bending of DBM, while the peaks at 848 and 730 cm−1 are corresponding to the phenyl ring out of plane C-H bending of Phen. Together with the peaks at 1598 and 1517 cm−1 corresponding to the C = O stretching of DBM confirm that the Phen and DBM coordinate to Eu(III) successfully [23]. The typical weak peak at 1728 cm−1 appear in (GQD/DBM)3EuPhen/GQD is attributed to the C = O stretching vibration bands of GQDs, indicating that GQDs connect to Eu(DBM)3Phen. From the XPS spectrum of (GQD/DBM)3EuPhen/GQD shown in Fig. 2b, the Eu 3d and Eu 4d peaks centered at 1134, 1164 eV (Eu 3d 3/2, Eu 3d 5/2) and 142 eV (Eu 4d) are observed. The C 1 s, N 1 s and O 1 s position at 285, 401, and 531 eV, respectively. The high-resolution C1s spectrum (Fig. 2c) of (GQD/DBM)3EuPhen/GQD shows peaks centered at 284.6 eV (C = C), 285.6 eV (C-O), 286.4 eV (C = N) and 288.2 eV (C = O), while the high-resolution C1s spectrum (Fig. 2 d) of Eu(DBM)3Phen shows peaks at 284.6 eV (C = C), 285.6 eV (C-O), 286.4 eV (C = N). When DBM react with Eu(III), it would react to become enol form, and the two oxygen atoms are the same, so there is none C = O bonds in C 1 s XPS spectrum of Eu(DBM)3Phen, further evidencing that GQDs conjugated with Eu(DBM)3Phen [24, 25].

Fig. 2
figure 2

a FT-IR spectra of (A) (GQD/DBM)3/EuPhen/GQD, (B) Eu(DBM)3Phen and (C) GQDs. b XPS spectra of (GQD/DBM)3/EuPhen/GQD. The high resolution C1s XPS spectrum of (GQD/DBM)3EuPhen/GQD (c) and Eu(DBM)3Phen (d)

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X-ray diffraction (XRD) patterns of (GQD/DBM)3EuPhen/GQD (curve A), Eu(DBM)3Phen (curve B) and GQDs (curve C) are demonstrated in Fig. 3. The (GQD/DBM)3EuPhen/GQD exhibit a broad weak peak centered at 23.6 degrees corresponding to 002 planes of GQDs and several sharp peaks. The sharp peaks also appear in Eu(DBM)3Phen which are corresponding to high crystallization structure of Eu(III) complex. Furthermore, no additional peaks or peak shifts were observed in (GQD/DBM)3EuPhen/GQD, indicating that GQDs were non-covalently connected to Eu(DBM)3Phen successfully [24].

Fig. 3
figure 3

XRD patterns of (A) (GQD/DBM)3/EuPhen/GQD, (B) Eu(DBM)3Phen and (C) GQDs

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Optical properties of (GQD/DBM)3EuPhen/GQD

The UV-vis absorption spectra of (GQD/DBM)3EuPhen/GQD (curve A), GQDs (curve B) in aqueous solutions and Eu(DBM)3Phen in tetrahydrofuran (THF) solution (curve C) are displayed in Fig. 4a. The spectrum of (GQD/DBM)3EuPhen/GQD shows peaks at 230, 268 and 360 nm. The peak at 230 nm also appears in the absorption spectrum of GQDs which is assigned to the transition of graphitic sp2 domains, while the other two peaks at 360 and 268 nm are attributed to the absorptions of DBM and Phen, respectively, and these peaks also appear in the absorption spectrum of Eu(DBM)3Phen [26]. With reference to pure DBM and Phen whose typical absorption peaks are at 345 and 260 nm, their absorption peaks in (GQD/DBM)3EuPhen/GQD experience red shift of 15 and 8 nm, respectively, indicating the successful coordination of the ligands to Eu(III) [26].

Fig. 4
figure 4

a UV-vis absorption spectra of (A) (GQD/DBM)3/EuPhen/GQD and (B) GQDs in aqueous solution, and (C) Eu(DBM)3Phen in THF solution. b PL spectra of (A) (GQD/DBM)3/EuPhen/GQD aqueous solution and (B) Eu(DBM)3Phen in THF solution

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When excited at the wavelength of 365 nm, the photoluminescence (PL) spectra of both (GQD/DBM)3EuPhen/GQD aqueous solution (curve A of Fig. 4b) and Eu(DBM)3Phen THF solution (curve B of Fig. 4b) show a strong sharp emission band at 613 nm corresponding to the deactivation of the Eu(III) excited states 5D0 → 7F2, indicating that the composite solution can emit red fluorescence. In addition, in the red region the emission band consists of three weak peaks at 577, 591, and 650 nm, ascribed to the transitions of 5D0 → 7F0, 5D0 → 7F1 and 5D0 → 7F3, respectively [15, 16]. And the absolute fluorescent quantum yield of (GQD/DBM)3EuPhen/GQD aqueous solution is as high as 15.5 %.

The most distinguishing feature that sets (GQD/DBM)3EuPhen/GQD apart from other Eu(III)-based coordination complexes is its specific solubility and stable PL in aqueous solution. In our study, the aqueous solution of (GQD/DBM)3EuPhen/GQD was deposited for 1 month to examine the stability, it was found that almost no fluorescence decrease was detected after 1 month of storage at room temperature. And when the (GQD/DBM)3EuPhen/GQD aqueous solution was irradiated constantly with a 150 W Xe lamp for 12  h, the PL intensity decreased about 16 %, indicating that (GQD/DBM)3EuPhen/GQD was stable in water (Fig. 5a). This should be attributed to the large amount of GQDs connected to DBM and Phen, which may keep water molecules from coordinating to Eu(III), significantly minimizing the nonradiative quenching of Eu(III) emission [27]. The response of the (GQD/DBM)3EuPhen/GQD aqueous solution to pH is shown in Fig. 5b and c, the PL intensity increases with the pH increases from 3 to 10, which is attributed to the enhancement of intra-molecular charge transfer from DBM and Phen to Eu(III) at higher pH values. When the pH is over 10, the PL intensity decreases because of the water molecule interaction with Eu(III) [28].

Fig. 5
figure 5

a PL spectra of (GQD/DBM)3/EuPhen/GQD (excited at 365 nm) in aqueous solution (curve A) original, (curve B) deposited at room temperature for 1 month and (curve C) constantly irradiated with a 150 W Xe lamp for 12 h. b The PL spectra of (GQD/DBM)3/EuPhen/GQD (excited at 365 nm) in different pH solutions. c The influence of pH values on the PL intensity of (GQD/DBM)3/EuPhen/GQD

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The formation mechanism of water soluble (GQD/DBM)3EuPhen/GQD

In terms of the synthesis strategy, two facets of control have been found to lead for achieving a stable red PL (GQD/DBM)3EuPhen/GQD aqueous solution. First, noncovalently connecting water soluble GQDs to Eu(DBM)3(Phen) coordination significantly improves the water solubility of Eu(DBM)3(Phen). The high-resolution TEM (HRTEM) image (Fig. 6a, b) clearly shows the GQDs lattice spacing of 0.21 nm, which confirm the GQDs in (GQD/DBM)3EuPhen/GQD. Second and crucially, connecting the ligands DBM and Phen to GQDs first and then coordinating to Eu(III) play a vital role in generating the strong red PL of (GQD/DBM)3EuPhen/GQD for keeping GQDs from coordinating to Eu(III) directly, which would avoid quenching the emissions of Eu(III) [19]. We deduce that Phen and DBM connected to GQDs by π-π stack because of their conjugate π-π plane structure, while DBM may be integrated with GQDs by hydrogen bonding for its oxygen atoms.

Fig. 6
figure 6

a TEM image of (GQD/DBM)3EuPhen/GQD. b HRTEM image of measured lattice spacing of the edge

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Cell imaging and cytotoxicity of (GQD/DBM)3EuPhen/GQD

The water soluble, high quantum efficiency and high color purity red PL (GQD/DBM)3EuPhen/GQD impart them outstanding applications of biological labels. MCF-7 cells were incubated with DMEM medium supplemented with 10 % FBS at 37 °C, the culture medium was then replaced by DMEM high glucose containing the (GQD/DBM)3EuPhen/GQD (50 μg mL−1) and further incubated for 2 h at 37 °C, then washed with PBS buffer (pH 7.4) to remove the uninternalized composites. After being washed with PBS buffer, the cells were imaged by a confocal fluorescence microscope. From Fig. 7a–c, it can be seen that (GQD/DBM)3EuPhen/GQD are able to label both the cell membrane and the cytoplasm of MCF-7 (Michigan Cancer Foundation-7) cells with red emission under 405 nm excitation, which demonstrates that the translation of (GQD/DBM)3EuPhen/GQD from outside the cell membrane into the cytoplasm, not just adsorbed on the outer membrane surfaces. In addition, after several times of repeated excitation, no further change in PL intensity change can be observed.

Fig. 7
figure 7

a Confocal fluorescence microscopy images of MCF-7 cells incorporated with 50 μg mL−1 (GQD/DBM)3EuPhen/GQD at the excitation wavelength of 405 nm and (b) corresponding image under bright field. c Merged fluorescent and bright-field image. d Cell viability of MCF-7 cells incubated with various concentrations of (GQD/DBM)3EuPhen/GQD

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Figure 7d shows the viability of MCF-7 cells after incubation with the composites for 24  h by MTT experiments. No significant loss of cell viability was observed with the concentration of incubated 400 μg mL−1, indicating that the fluorescent (GQD/DBM)3EuPhen/GQD can serve as an effective biological imaging platform with little cytotoxicity.

In vivo and ex vivo imaging

The high pure red fluorescent (GQD/DBM)3EuPhen/GQD inspired us to further explore their feasibility as bioprobes in vivo. It has been reported that nanomaterials can accumulate at the tumor site by enhanced permeability and retention (EPR) effect [29]. The selected HeLa tumor-bearing nude mice were intravenously injected with (GQD/DBM)3EuPhen/GQD aqueous solution, while the not injected mice were selected as controls. As shown in Fig. 8a, after injection, the significant PL signals were clearly observed in the tumor area in comparison with other organs in 2 h, which is indicative of the (GQD/DBM)3EuPhen/GQD very promising for tumor labelling and chemotherapy. At 10 h post-injection, the mice were sacrificed. The images for ex-vivo fluorescence imaging of the internal organs from the mice also indicated that the tumor tissue showed the strongest fluorescent intensity (Fig. 8b and c), but major organs such as the kidneys, liver, lung tissues showed weak signals while the spleen and heart showed very low signals. The tumor accumulation of (GQD/DBM)3EuPhen/GQD should be due to passive targeting through EPR effect, similar to many other previously reported nanomaterials in vivo behaviour [30–32].

Fig. 8
figure 8

a In vivo imaging of HeLa tumor-bearing nude mice after injection of (GQD/DBM)3EuPhen/GQD (5 mg/kg). b Ex-vivo images of isolated organs of mice at 10 h after injection and c PL intensities of (GQD/DBM)3EuPhen/GQD from isolated organs

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Conclusion

In summary, we have successfully prepared water-soluble, high purity red-emitting graphene quantum dot-europium complex composites (GQD/DBM)3EuPhen/GQD by noncovalent functionalization of the chelating ligands DBM and Phen with GQDs first, followed by coordinating to Eu(III). The (GQD/DBM)3EuPhen/GQD integrate both the GQDs and Eu(III) complex merits in a single structure and display excellent optical properties in terms of bioimaging applications. Such composites alsoare expected to be useful in areas such as drug delivery [4, 33, 34]. Detailed follow-up work is underway in our laboratory and will be reported in due course.