Synthesis, characterization and cytotoxicity of europium incorporated ZnO–graphene nanocomposites on human MCF7 breast cancer cells

Abstract

Europium incorporated ZnO-chemically converted graphene (CCG) nanocomposites (ZEG) were synthesized by adopting a solvothermal process at 95 °C from the precursors of varying europium nitrate to zinc acetate molar ratios (R = 0.00, 0.05, 0.10, 0.15) in a fixed content of graphene oxide. Eu level (R value) in the precursors was found to play a role on tailoring the crystallite/particle size of hexagonal ZnO, as evidenced from X-ray diffraction/transmission electron microscopy analysis. The presence of chemical interaction/complexation between the oxygen functionalities of CCG and inorganic moieties (ZnO/Zn²⁺ and Eu ions) of nanocomposites were studied by FTIR, Raman, UV-Vis spectral and XPS measurements. The nanocomposites possessed meso pores as confirmed from BET nitrogen adsorption isotherm, and the sample ZEG(10) (R = 0.10) was found to possess the highest specific surface area. In spite of an UV emission at ∼385 nm, an orange emission (appeared at 595 nm) along with other visible emissions was observed from the photoluminescence spectra of nanocomposites. However, the intensity of the orange emission (λ_ex = 400 nm) was found to be maximum in the ZEG(10) sample, which produced relatively bright orange fluorescent images of human breast cancer cells (MCF7) under confocal laser scanning microscope. This indicated the internalization of the nanomaterials within the cells. As obtained from the MTT assay, the samples (R ≥ 0.10) exhibited comparatively low in vitro cytotoxicity (higher cell viability) on the cancer cells. The low cytotoxicity could be explained on the basis of the I_D/I_G (intensities of D and G bands of graphene) value of CCG, as realized from the Raman spectral measurement. The nanocomposite ZEG(10), having relatively large surface area, bright cell imaging capability and better cell viability, could be employed for cancer cell targeted optical imaging and drug delivery.

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Introduction

Nowadays, graphene based nanocomposites (NCs) have attracted greater attention from materials scientists in both the basic and applied fields of research.^1–3 Although, graphene is a 2-dimensional (2D) monolayer sheet of in-plane sp² bonded carbons having unaffected p_z orbitals, the research community^4,5 commonly refers to its compounds (e.g. graphene oxide, GO; chemically converted graphene (CCG)/reduced graphene oxide, rGO) “graphene”. In fact, the compounds could form functional NCs with polymers (e.g. polyaniline, polyvinyl alcohol),^6,7 metal/metal ions (e.g. Ag, Au, Pt/Eu ion)^8–11 and metal oxides (e.g. ZnO, TiO₂, Fe₃O₄, MnO₂, SnO₂, Co₃O₄).^12–17 Thus, an advancement of properties^6,18 (such as electrical, optical, electrochemical and mechanical) could be achieved in the NCs. This would find enormous applications^{6,13,14,19–22} (e.g. cellular imaging, bacterial toxicity, drug delivery, enzyme sensing, photothermal therapy, photocatalytic activity, Li-ion batteries, supercapacitors, photovoltaic devices) for the NCs.

ZnO is a well-known functional semiconductor oxide^23–26 having wide direct band gap (3.37 eV, at room temperature) and a large exciton binding energy (60 meV). Because of its characteristic UV emission, ZnO could be an important material for optoelectronic application.²⁵ Moreover, several visible emissions²⁶ could be observed under a single light excitation due to the formation of surface defects in ZnO. All these emissions could also be tuned through the doping/controlling of the parameters during material synthesis.^23–29 Alternatively, the optical property of the semiconductor could also be modified¹² through the formation of nanocomposite with graphene. In a basic aspect of the nanocomposite, Son et al.¹² reported that Zn²⁺ ions chemisorbed on the embryo of ZnO nanoparticles, which could react with the oxygen functional groups of GO, led to the formation of Zn–O–C bonding. In addition, the GO sheets could be exfoliated chemically in the reaction medium. On the other hand, graphene is more biocompatible³⁰ because the living cell can well adhere and proliferate on graphene sheets.³¹ The nanoparticles are generally toxic in nature but their toxicity could be mitigated to make the material biocompatible and nontoxic²⁰ through proper surface modification with graphene. It could also be noted^12,32 that the exfoliated graphene with several oxygen containing functional groups (carboxylic, hydroxyl and epoxide) could interact with suitable cations (adsorbed on the semiconductor oxide surface),¹² resulting in the formation of graphene based functional nanocomposites. However, functional groups could interact with cations and would help to generate reactive sites towards the nucleation and crystal growth of nanoparticles.³² However, in the nanocomposites, the GO could transform into chemically converted graphene (CCG) (also called reduced graphene oxide, rGO) with a lesser number³³ of oxygen containing functional groups. Sometimes, the highly toxic nanoparticles (for example, CdSe/ZnS quantum dots) could tag²⁰ with rGO, which made them potential biomaterials for applications such as in situ monitored bright fluorescence imaging and photothermal therapy of living cells. On the other hand, the presence of numerous oxygen functional groups in GO would create a more toxic effect on living cells compared to CCG/rGO.³⁴ This would be caused by chemical interactions³⁴ between the functional groups of GO and living cells. In fact, the living cells when combined with GO enhances the mitochondrial respiration rate by donating its available electrons and creating reactive oxygen species (ROS).³⁴ Although, ZnO is known to be a biocompatible²⁴ material, due to particle dissolution³⁵ in tissue culture medium, it could show toxic effects on living cells. During particle dissolution, Zn²⁺ ion shedding could damage lysosomes, perturb mitochondria, generate ROS, etc. In this context, by Fe doping, the dissolution can be reduced by the changes in particle matrix because the dopant could easily enter into the ZnO lattice.³⁵

In europium graphene composite, europium shows higher adsorption energy with a lower diffusion barrier compared to other rare earth elements.¹¹ Moreover, europium ions could form complexes with the oxygen functional groups of graphene that could be revealed by FTIR, Raman spectra and XPS measurements. In addition, the graphitic carbons could quench^11,36 the fluorescence of organic dyes. Wang et al.³⁶ synthesized three-dimensional europium-complexed rGO macroassembly by a one-pot self-assembly process. The authors reported the formation of inner-sphere surface complexes of europium ions and confirmed the formation of Eu(II) ions by XPS analysis. They also claimed that the reduction of Eu(III) to Eu(II) ions occurred due to complexation.^36,37 Although, several authors^38–40 have already reported Eu incorporated ZnO nanoparticles, very few claimed³⁸ that Eu enters into the crystal lattice. The problem of incorporation could be addressed because of the large size of europium ions compared to Zn²⁺.⁴⁰ However, it has already been reported that depending upon the incorporation⁴¹ of rare earth elements, an increase in surface defects were observed in ZnO as assessed from the photoluminescence emission spectral study.^26,41

In general, the graphene based nanocomposites could mostly be synthesised by solvothermal,^21,25 chemical deposition⁴² and microwave-assisted⁴³ processes. However, the low temperature solvothermal process could be advantageous over others. Recently, several exciting properties were observed in graphene based ternary nanocomposites (e.g. Fe₃O₄–graphene–TiO₂, rGO–porphyrin–ZnO, graphene–Fe₂O₃–polyaniline and Pt–ITO–graphene).^44–47 The composite materials could be used in photoconversion, photovoltaics, supercapacitors, etc. However, to the best of our knowledge, there are no reports on Eu incorporated ZnO–graphene (ZEG) nanocomposites that would generate a new class of ternary nanocomposite materials for emerging applications. Thus, for both basic and applied fields of research, the synthesis of graphene-based ZEG nanocomposites would be very promising.

In the present work, a systematic study was performed on the synthesis of europium incorporated ZnO-chemically converted graphene (CCG) nanocomposites (ZEG) from the precursors of varying europium nitrate to zinc acetate molar ratios (R = 0.00, 0.05, 0.10, 0.15) in a fixed content of graphene oxide by adopting a low temperature (95 °C) solvothermal process. In the nanocomposites, the influence of Eu incorporation (in terms of R value) on the crystallite/particle size of hexagonal ZnO, the presence of chemical interaction/complexation and optical properties were analyzed. Moreover, the cell imaging capability of the samples was investigating using a confocal laser scanning microscope. Finally, in vitro cell viability in terms of cytotoxicity of the nanocomposites on MCF7 breast cancer cells from MTT assay was also verified.

Experimentals

Synthesis of graphene oxide (GO)

Graphene oxide (GO) was prepared (see ESI† for details) from graphite powder following the modified Hummer's method.

Synthesis of Eu incorporated ZnO-chemically converted graphene (ZEG) nanocomposites

We adopted a facile low temperature (95 °C) solvothermal process for the synthesis of europium incorporated ZnO-chemically converted graphene (ZEG) nanocomposites using as-synthesized graphene oxide (GO), zinc acetate dihydrate (Zn(CH₃COO)₂·2H₂O, ZA, Sigma-Aldrich, ≥98%) and europium nitrate pentahydrate (Eu(NO₃)₃·5H₂O, EUN, Sigma-Aldrich, 99.9%) in dimethyl formamide (DMF, a polar aprotic solvent) medium. Four different nanocomposites were prepared by varying EUN/ZA molar ratios (R): 0.00, 0.05, 0.10 and 0.15 in the precursors, which were designated as ZEG(00), ZEG(05), ZEG(10) and ZEG(15), respectively. All the samples were prepared separately and in each preparation a fixed amount (60 mg) of the as-synthesized GO was uniformly dispersed in DMF (40 ml) by ultrasonication for about 60 min. Further, the GO dispersed in DMF was mixed with a fixed quantity of ZA (0.95 g) and a requisite amount of EUN under continuous stirring to form stable precursors for ZEG nanocomposites. Subsequently, the precursors were kept in an air oven at 95 °C up to 9 h (maximum) following X-ray diffraction results (Fig. S1, ESI†). After heating, the colour of the mixtures changed from black to grayish white, which indicated the formation of ZEG nanocomposites. The solid materials in the dispersed phase were then separated by centrifugation. In each preparation, the product was re-dispersed in ethanol and then in double distilled water followed by centrifugation for isolation from the dispersion medium. Finally, the samples were dried in an air oven at ∼55 °C. It is worth mentioning that the reduction of GO could be achieved via thermal annealing (>1000 °C), photochemical reduction and electrochemical reduction processes.³³ However, in the present work, none of the processes was adopted. Hence, we called the GO in the nanocomposites as chemically converted graphene (CCG).

Characterizations

Materials characterization

X-ray diffraction (XRD) study of the samples was performed by employing an X-ray diffractometer (Bruker D8 Advance with DAVINCI design X-ray diffraction unit) with a nickel-filtered CuK_α radiation source (λ = 1.5418 Å) in the 2θ range, 5°–80°. Transmission electron microscopy/high resolution transmission electron microscopy (TEM/HRTEM) along with TEM-EDS measurements on the samples were performed by an FEI Company (Tecnai G² 30.S-Twin, Netherlands) machine at the accelerating voltage of 300 kV. Carbon coated 300 mesh Cu grids were used for the placement of the samples. For the measurements, the samples were dispersed in methanol by ultrasonication and the dispersed nanocomposites were carefully placed on the Cu-grid. Moreover, to identify the layers of chemically converted graphene (CCG) onto the nano ZnO crystals in ZEG(00), the TEM measurements were also performed by using a JEOL JEM-2100F (FEG) high-resolution electron microscope operating at an accelerating voltage of 200 kV. FTIR spectral study was carried out by an FTIR spectrometer (Nicolet 5700, Thermo Electron Corporation, USA). For each experiment, the number of scans was fixed at 100 (wavenumber resolution, 4 cm⁻¹). Absorption spectra of the samples were recorded by a diffused reflectance method using an UV-VIS-NIR spectrophotometer (UV3600, Shimadzu, Japan) with ISR 3600 attachment. Raman spectra were measured using micro-Raman (Renishaw inVia Raman microscope). An argon ion laser with an incident wavelength of 514 nm was used as the excitation source. X-ray photoelectron spectrum (XPS) in the range of 200–1200 eV of a representative sample, ZEG(10), was carried out to determine the chemical state and chemical interaction/complexation of elements present in the sample by employing a PHI Versaprobe II Scanning XPS microprobe surface analysis system using Al-K_α X-rays (hν, 1486.6 eV; ΔE, 0.7 eV at room temperature). The pressure in the XPS analysis chamber was better than 5 × 10⁻¹⁰ mbar. The energy scale of the spectrometer was calibrated with pure (Ag) sample. The position of the C 1s peak was taken as the standard (binding energy, 284.5 eV). The room temperature photoluminescence (emissions and excitations) spectral property of the samples was measured by a Perkin-Elmer (LS55) spectrofluorimeter. The slit widths for the measurements of emission and excitation spectra were fixed at 2.5 nm and 10 nm, respectively, for all the samples. In each sample, a pellet with approximately 0.5 mm was prepared for the PL and PLE measurements. The surface area, pore size and pore volume of the nanocomposites were measured by nitrogen adsorption and desorption studies at liquid nitrogen temperature by adopting a Quantachrome (Autosorb1) machine. Prior to measurement, all the samples were outgassed in vacuum at a suitable temperature for about 4 h.

Cytotoxicity and cell imaging studies

Cytotoxicity measurement of three representative nanocomposites (ZEG(00), ZEG(10) and ZEG(15)) on human cell line MCF7 (breast cancer cell, procured from National Centre for Cell Science, Pune, India) were performed. We used Dulbecco's modified eagles medium (DMEM, St. Louis, Missouri, USA), fetal bovine serum (FBS, Carlsbad, CA, USA), PenStrep (Life Technologies, Carlsbad, CA, USA) and MTT (3-[4,5-dimethylthiazol-2-yl]-2,5-diphenyltetrazoliumbromide, Carlsbad, CA, USA) as necessary chemicals and reagents.

Cell culture

The MCF7 cells were cultured in DMEM (invitrogen) supplemented with 10% fetal bovine serum (FBS) and an antibiotic (1% penicillin/streptomycin) in 5% CO₂ at 37 °C. The cells from exponentially growing cultures were used for the experiments.

Measurement of in vitro cellular cytotoxicity, MTT assay

The MTT assay was performed to estimate the viable cells. In brief, MCF7 cells (10⁴ cells per well) were seeded on to a flat bottom 96-well plate and incubated at 37 °C in 5% CO₂. Cells were treated with or without (i.e. vehicle control) nanocomposites at varying concentrations, ranging from 5 to 200 μg ml⁻¹ and maintained for 24 h. Finally, they were subjected to MTT assay. Then, 20 μl of MTT (5 mg ml⁻¹) was added to each well. After four hours of incubation, the media was removed and the cells were dispersed in 200 μl of dimethyl sulphoxide (DMSO) solvent. Absorbance spectra of the samples were measured at 595 nm by a Thermo MULTISKAN EX plate reader (Thermo, USA). The cell viability values were expressed in percentages with respect to control values.

Confocal laser scanning microscopy (CLSM) study

Cell internalization of two representative nanocomposites (ZEG(00) and ZEG(10)) was visualized using confocal laser scanning microscopy (CLSM) at a 405 nm laser source. The MCF7 cells were cultured on a cover slide in 5% CO₂ medium at 37 °C temperature. Then, the cells were separately treated with individual nanocomposite (concentration, 100 μg ml⁻¹) as well as vehicle control (without nanocomposite) for 2 h. Finally, the cells were thoroughly washed twice by PBS and visualized under the Andor Spinning Disc Confocal Microscope at 20× magnification, and the images were captured using Andor IQ2 software.

Results and discussion

Fig. 1 displays the XRD patterns of ZEG nanocomposites and the as-synthesized graphene oxide (GO). The XRD pattern of GO shows⁴⁸ a strong diffraction (2θ) peak at 10.9° (interlayer spacing, 8.12 Å) along (002) plane in addition to a weak peak at 42.4° for (100) plane. However, the XRD peaks of GO were found to disappear gradually on increasing reaction time, and after 9 h, the peaks were not observed (Fig. S1, ESI†) from the synthesis of the ZEG(00) nanocomposite. This could suggest that a complete conversion of GO to chemically converted graphene (CCG) occurred in the nanocomposite. However, no obvious peaks⁴⁸ of CCG that might be due⁴⁹ to interaction/complexation between the organic and inorganic moieties of the nanocomposite (discussed later) were found. On the other hand, the formation of hexagonal ZnO (Fig. S1, ESI†) was observed after at least 7 h of reaction time but an appreciable intensity of XRD peaks for the oxide was observed after 9 h. Therefore, we fixed the reaction time at 9 h for the synthesis of Eu incorporated ZEG nanocomposites. In ZEG samples, the observed XRD peaks were fully matched with hexagonal ZnO (h-ZnO) [JCPDS 36-1451], and no additional diffraction peaks corresponding to europium oxide³⁸ were observed. Moreover, in the Eu incorporated samples, the XRD peak shifting compared to ZEG(00) was not visualized. In fact, there was a slight possibility of Eu incorporation in nano h-ZnO crystal lattice through the substitution of Zn by Eu. This would be^39,40 because of the larger ionic radius of europium ions (Eu³⁺, 0.95 Å or Eu²⁺, 1.17 Å)⁵⁰ compared to zinc ions (Zn²⁺, 0.74 Å).²⁶ Moreover, the small crystallite size might create an unfavorable condition that would hinder the incorporation of Eu into nano ZnO crystal.²⁶ We also calculated the crystallite size of h-ZnO from the XRD patterns at (101) plane using the Scherrer's equation.²⁶ On increasing Eu incorporation level, it was found that the crystallite size (inset, Fig. 1) increased with an exception in ZEG(10) where a lower size of ZnO in the nano regime was obtained compared to ZEG(05) and ZEG(15). Because Eu neither entered into the ZnO crystal lattice nor formed any europium oxide, it hopefully existed as a complexed (discussed later) species with the organic moiety (CCG) of the nanocomposites. In this context, very recently, Pal et al.²⁶ observed that an excess dopant would remain on the ZnO crystal surface and would suppress its grain growth at a higher doping level in Al doped nanostructured ZnO thin films by sol–gel process. In the present work, the probability of Eu ions to be present directly onto the crystal surface might not be valid because we found an increase in crystallite size in the nanocomposite, ZEG(15) with a higher level of Eu than ZEG(05) and ZEG(10). Thus, Eu incorporation into the ZnO–CCG composite would create a new structure. A possible chemical structure of ZEG nanocomposites is presented later. However, the reason for attaining relatively low crystallite size in ZEG(10) compared to ZEG(05) and ZEG(15) was unclear, and would be investigated in the future.

Fig. 1 XRD reflections of ZEG nanocomposites and as-synthesized graphene oxide. Inset shows the change in crystallite size on increasing Eu incorporation level in the precursors.

TEM results of nanocomposites are given in Fig. 2 and S2, ESI.† From the TEM images (Fig. 2a, c and e and S2†), the agglomeration of composite nanoparticles appeared to be diminishing at the higher level of Eu incorporation, and nearly monodispersed quasi spherical particles (Fig. 2e) resulted in ZEG(15). However, on increasing the Eu level, the trend in the change of the average size of particles obtained from the histograms (insets, Fig. 2c and e, S2a and b†), constructed from their respective TEM images, were found to be identical to the calculated crystallite size (inset, Fig. 1) of h-ZnO, as obtained by the XRD patterns. Moreover, the particle size obtained from TEM analysis was always higher than the calculated value according to the XRD patterns. This difference could suggest the formation of ZEG nanocomposite particles because the calculated crystallite size from XRD considered only h-ZnO but the observed nanoparticles in TEM images could be for nanomaterials consisting of inorganic (ZnO/Zn²⁺ and Eu ion) and organic (CCG) moieties (discussed later). In HRTEM images (Fig. 2b, d and f) of the nanocomposites, the lattice fringe with a regular spacing of 0.26 nm is fully consistent¹² with the interplanar distance of the (002) plane of h-ZnO. Although the XRD patterns of the nanocomposites did not show any evidence for the presence of CCG, their corresponding TEM images clearly show a sheet-like structure (Fig. 2), which could have originated from CCG layers. This sheet-like structure was the CCG layers present in the nanocomposites as also evidenced from its fast Fourier transform (FFT) patterns (inset (ii) of Fig. 2e). The FFT shows a set of spots corresponding to crystallographic planes (002) and (101) of h-ZnO along with (002) of CCG. In addition, as seen from the HRTEM image (Fig. 2b) of ZEG(00), the layers of chemically converted graphene (CCG) sheets of nearly ∼3 nm thickness on the nano ZnO crystals are clearly visible. Thus, the CCG layers could make the Eu incorporated ZnO@CCG core-shell-like structure in the nanocomposite. Irrespective of the incorporation level, it was not possible to quantify the Eu content from the TEM-EDS (insets, Fig. 2d and f) analysis of ZEG nanocomposites. This was because the characteristic peaks in EDS of Eu could be disguised³⁹ within the broad peaks of Zn and Cu.

Fig. 2 (a), (c) and (e): TEM images of ZEG(00), ZEG(10) and ZEG(15), respectively. Respective histograms show particle size distributions of nanocomposites (insets of (c) and inset (i) of (e)). HRTEM images of ZEG(00), ZEG(10) and ZEG(15) are shown in (b), (d) and (f), respectively. TEM-EDS of ZEG(10) and ZEG(15) are given in the insets of (d) and (f), respectively. Inset (ii) of (e) shows the FFT from the HRTEM (f). HRTEM (f) was taken from the selected image marked by red square, as shown in (e).

The presence of chemical interaction/complexation between the oxygen functionalities of CCG with inorganic moieties (ZnO/Zn²⁺ and Eu ions) was verified by FTIR vibrational spectra. Fig. 3 and S3† represent the FTIR spectra of ZEG nanocomposites and the as-synthesized GO. In GO, the presence³³ of hydroxylates, carboxylates and epoxides in the form of oxygen functional groups, C–OH, C–O–C, O–H (deformation) and –COOH, were distinctly observed from the appearance of vibrations located at ∼1070 cm⁻¹, ∼1225 cm⁻¹, ∼1400 cm⁻¹ and ∼1735 cm⁻¹, respectively.^11,21,51 In addition, a skeletal vibration of unoxidized graphitic domains in GO was also found at ∼1620 cm⁻¹.⁵¹ However, the intensity of all the vibrational peaks were found to decrease to a large extent in the GO derived nanocomposites, (ZEG(00), ZEG(10) and Eu–CCG). In addition, a new vibration observed at ∼1570 cm⁻¹ in ZEG(00), ZEG(10) and Eu–CCG samples were assigned to the characteristic skeletal vibrations of the graphitic domains of CCG.²¹ This result could favour the conversion of GO to chemically converted graphene (CCG) in the nanocomposites³³ and corroborated the TEM result (Fig. 2). However, the complete conversion of GO to CCG was found after at least 9 h of reaction time, which could easily be understood from the FTIR spectral study (Fig. S3, ESI†) of the representative (ZEG(00)) nanocomposites synthesized by altering reaction times. In addition, a strong vibration appearing at 1375 cm⁻¹ in Eu-CCG could be attributed to the symmetric stretching vibration because of the complexation of the –COOH group with europium ions^36,52 (Eu³⁺/Eu²⁺) (discussed later under XPS result, Fig. 5). Moreover, its asymmetric stretching component⁵² might be overlapped within the broad vibration (∼1570 cm⁻¹) of graphitic domains of CCG. Perhaps, the asymmetric and symmetric stretching vibrations⁵³ of complexed –COOH groups of CCG with Zn²⁺ from ZnO/Zn²⁺ (discussed later) would be disguised within the broad spectral regions. However, the FTIR spectra of both ZEG(00) and ZEG(10) nanocomposites showed a strong vibration at ∼460 cm⁻¹, which was not observed in europium–CCG nanocomposite (Eu–CCG), assigned^21,54 to Zn–O bond vibration. Moreover, a sharp vibration appeared at 418 cm⁻¹ in Eu–CCG that might be overlapped within the broad vibration region of Zn–O in ZEG(10), which is attributed to Eu–O bond vibration.³⁶

Fig. 3 FTIR spectra of different nanocomposites along with the as-synthesized graphene oxide.

Raman spectra of ZEG nanocomposites are displayed in Fig. 4. The Raman peaks located at 325 cm⁻¹, 434 cm⁻¹, 571 cm⁻¹ and ∼1135 cm⁻¹ were attributed to^21–25 the modes of 2E₂ (M), E₂ (high), E₁ (LO) and 2E₁ (LO) of hexagonal wurtzite ZnO, respectively. In fact, the E₂ (high) mode could be recognized as the signature of h-ZnO that was already confirmed by XRD (Fig. 1) and TEM (Fig. 2) studies. In addition, the defect band, D (intensity I_D) at ∼1350 cm⁻¹ as a breathing mode of k-point phonons in A_1g symmetry, appeared in all the nanocomposites including precursor GO.^12,25 This could strongly suggest the existence of defects in the graphene moiety.^25,49 In GO, the Raman peak observed at 1597 cm⁻¹ was assigned to the E_2g phonon of sp² carbon atoms^12,25 and was designated as graphene (G) band (intensity I_G). However, the G band was found to shift in the ZEG nanocomposites by 14 ± 2 cm⁻¹ towards the lower energy region^11,25 with respect to the band in GO, while the location of the D band remained approximately at the same position. Moreover, a systematic broadening¹¹ of the D band was observed with increasing Eu level. The peak broadening could be assessed from the calculation of half width at full maxima (FWHM) (Table S1, ESI†) of the peak at 1350 cm⁻¹. The result could support the conversion of GO to CCG, which is chemically interacted/formed complexes with inorganic moieties (ZnO/Zn²⁺ and Eu³⁺/Eu²⁺) in the nanocomposites. It could be noted that the chemical interaction/formation of complexes were also noticed from the FTIR vibrational spectra (Fig. 3 and S3, ESI†) of the ZEG nanocomposites. On the other hand, on increasing Eu level, a systematic enhancement of the intensity ratio, I_D/I_G, was found (Fig. 4 and Table S1 of ESI†) up to R = 0.10 (ZEG(10)), which remained the same in the next higher incorporation level for ZEG(15). The D band could be associated¹¹ with the complexed oxygen functionalities of CCG. Moreover, the relative increase in the D band intensity could also account for the exfoliation⁴⁶ of graphene layers that could be possible via chemical interaction/complexation with the inorganic moieties of nanocomposites. Therefore, on increasing Eu level in a fixed amount of CCG, the complexed oxygen functionalities in ZEG nanocomposites could be expected to reach a value with an optimum concentration of europium (Eu³⁺/Eu²⁺) ions. No evidence of Eu oxide formation that could support the incorporation of Eu into ZnO crystal lattice was found in the XRD patterns (Fig. 1), and thus, the europium ions should remain in the complexed species with CCG of the nanocomposites. Hence, after the Eu incorporation (R ≥ 0.10), there might not be available free oxygen functionalities. This would give rise the nearly constant I_D/I_G value of the nanocomposites. Although we explained the possible interaction/complex formation between the inorganic moieties and the oxygen functionalities in the nanocomposites, the existence of Zn²⁺and Eu²⁺/Eu³⁺ could not be determined by the Raman spectral study.

Fig. 4 Raman spectra of ZEG nanocomposites. Individual I_D/I_G value is also embedded in the figure.

XPS analysis could also be a useful tool^11,21,38 to confirm the interaction of oxygen functionalities of CCG with the inorganic moieties. Thus, the XPS measurement (Fig. 5) of a representative sample, ZEG(10), was performed to confirm the chemical state(s) of the elements as well as the chemical interactions existing between the organics, CCG, and the inorganic moieties (ZnO/Zn²⁺ and Eu³⁺/Eu²⁺ ions) of the nanocomposite. The XPS survey spectrum (Fig. S4, ESI†) shows the binding energy signals for C 1s, O 1s, Zn 2p and Eu 3d core levels. A broad signal for C 1s could be resolved (Fig. 5a) into three Gaussian peaks centered at 284.4 eV (a1), 285.4 eV (a2) and 288.8 eV (a3), which correspond to Sp²C, Sp³C and O–C–O, respectively,^22,36 from the organic moiety, CCG, in the sample. The O 1s peak (Fig. 5b) could also be decomposed into three distinct components with binding energies ∼530.1 eV (S1), 531.8 eV (S2) and 533.1 eV (S3). The S1 signal could be due to the lattice oxygen (O²⁻)²⁶ of hexagonal ZnO, whereas the higher binding energy signals of O 1s (S2 and S3) could associate with the presence of chemically interacted/complexed oxygen functional groups (epoxy/COOH/hydroxyl)³⁶ of CCG. On the other hand, the strong binding energy signals located (Fig. 5c) at 1044.3 eV and 1021.6 eV were assigned to Zn 2p_1/2 and Zn 2p_3/2 core levels,²¹ respectively. In this work, we observed a shifting (∼2 eV) of the Zn 2p signals towards higher energy compared to pure ZnO, as already reported by Zhang et al.²¹ This result could imply the presence of interaction and electron transformation between ZnO/Zn²⁺ moiety and the oxygen functionalities of CCG.²¹ However, an energy difference of 22.7 eV between the binding energy levels supported the existence of Zn²⁺ in the nanocomposite.²⁶ Although, the XRD (Fig. 1) study could not show the evidence for the presence of Eu; however, the XPS (Fig. 5d) confirmed the core level binding energies of Eu 3d. The intense doublets appearing at 1164.6 eV and 1134.7 eV could be assigned to the energy levels of Eu 3d_3/2 and Eu 3d_5/2, respectively, in Eu³⁺ species.^36,38 In addition, the existence of Eu²⁺ species was also evidenced from relatively weak binding energy signals located at 1155.4 eV and 1124.4 eV of Eu²⁺ 3d_3/2 and Eu²⁺ 3d_5/2 components, respectively.³⁶ In this synthesis, the presence of Eu²⁺ could be originated from the reduction of Eu³⁺ in the CCG environment.³⁶ Mercier et al.⁵⁵ reported a M_mX_xO_y type oxo-compound showing a higher core level binding energy compared to M_aO_b. Here, M (M = Eu) is a more electropositive element than X (X = C), and consequently, the M–O bond has more ionic character than the X–O bond. In the X-O-M bond, electrons could pull away from M and Eu²⁺ is formed as a result of diminishing the valency of Eu³⁺. Therefore, both Eu²⁺ and Eu³⁺ ions could associate with the free oxygen functional groups of CCG and would form an M_mX_xO_y type complex in the nanocomposites. Thus, it could be expected that at higher Eu incorporation level, it would reduce free oxygen functional groups of CCG. This would help to minimize the toxicity (discussed later, Fig. 9b) of nanocomposites on living cells because oxygen functionalities could easily interact with the cells.³⁴

Fig. 5 XPS data of the ZEG(10) nanocomposite: Binding energy spectra of (a) C 1s and (b) O 1s along with their Gaussian-fitted components; (c) typical XPS data for the binding energy spectrum of Zn 2p_3/2 and Zn 2p_1/2 core levels; (d) shows the binding energy spectrum of Eu 3d core levels.

In Fig. 6, UV-vis absorption spectral (measured by diffused reflectance method) results of GO and ZEG nanocomposites are given. The appearance of two prominent absorption peaks at 232 nm and ∼290 nm (broad shoulder, inset (i), Fig. 6) in GO could correspond to the π → π* and n → π* transitions of C=C and C=O bands,⁵⁶ respectively. However, in all the nanocomposites, the peak at 232 nm red-shifted to ∼270 nm, while the peak at ∼290 nm disappeared completely in the Eu incorporated nanocomposites. This result could signify⁵⁶ that strong coupling exists between the inorganic moieties (ZnO/Zn²⁺ and Eu³⁺/Eu²⁺ ions) and CCG during the conversion of GO to CCG in the nanocomposites. The present findings also supported the FTIR (Fig. 3), Raman (Fig. 4) and XPS (Fig. 5) results. It should be pointed out that in ZEG(00), a broad UV peak was also found at ∼360 nm, which shifted towards blue/red wavelength depending upon ZnO crystallite/particle size (as obtained from XRD (Fig. 1)/TEM (Fig. 2 and S2†), and demonstrated the size effect of semiconductor oxide in the nano regime.^26,57 In addition, the spectral region of 240 to 390 nm could be resolved into three Gaussian fitted components, located at 272 nm, 332 nm and 360 nm in the ZEG(10) nanocomposite. The peak at 332 nm could relate to electronic transition in the bulk ZnO (band gap, ∼3.4 eV).

Fig. 6 Absorption spectra of ZEG nanocomposites along with the as-synthesized graphene oxide. Inset (i) shows the enlarged spectrum (as marked) of graphene oxide; inset (ii) indicates the Gaussian-fitted components of the spectrum in wavelength range of 240–430 nm for ZEG(10).

Room temperature photoluminescence (PL) spectra (at different excitation wavelengths) of the ZEG nanocomposites and other materials are displayed in Fig. 7, S5 and S6.† The excitation wavelengths were primarily selected from the absorption spectral (measured by diffused reflectance method, Fig. 6) study of the nanocomposites. In this context, initially an excitation wavelength of 360 nm was chosen and a strong UV PL emission was observed at 384 nm along with several visible emissions (Fig. S5(a), ESI†) for ZEG(00) and ZEG(10) nanocomposites. The peak corresponds¹² to the characteristic UV emission of ZnO due to band-to-band transition. However, fixing the PL emission at 384 nm, the PLE spectrum was also recorded. The spectrum shows three distinct peaks (inset, Fig. 7a) at 224 nm, 240 nm and 340 nm. Hence, an excitation wavelength of 340 nm was further used, and recorded PL spectra where several distinct visible emissions (Fig. 7a) were located at ∼385 nm, ∼420 nm, ∼460 nm, ∼485 nm, ∼520 nm, ∼530 nm, ∼570 nm and ∼595 nm. It is known that except UV emission at 385 nm, all the visible emissions could relate to the intrinsic/extrinsic surface defects of nano ZnO.^26,57–59 However, several researchers are still doubtful regarding the origin of individual visible emissions. It could be noted that the formation and nature of the defects depend upon several parameters^26,58,59 (such as preparative method, doping) through which the concentration²⁶ of an individual defect could be tailored. In this work, the emission observed at 420 nm could support the presence of zinc interstitial,²⁶while the peak at 460 nm could relate to single positively charged oxygen vacancy in nano ZnO.²⁶ The existence of antisite oxygen was also supported from the observation of PL emission at 485 nm.²⁶ Moreover, a series of green emissions were observed (520 nm, 530 nm and 570 nm) in the PL spectra of the nanocomposites. The green emissions^58,59 would be originating from the different levels of oxygen vacancies existed in the nano semiconductor oxide. It was interesting to note that a new PLE peak was observed at 400 nm (inset (i) of Fig. 7b, S5(b) and (c) of ESI†) in addition to 340 nm when the PL emission was fixed at 595 nm. Hence, the excitation at 400 nm was further used to measure the PL spectra, which showed a distinct orange emission at 595 nm along with a broad emission in the spectral range of 520–585 nm (Fig. 7b). In fact, the broad emission consisted of four Gaussian fitted peaks located at 530 nm, 543 nm, 560 nm and 570 nm (inset (ii), Fig. 7b). These peaks were considered to be originated from different energy levels of neutral oxygen vacancies in ZnO.^58,59 However, the intensity of the orange emission at 595 nm could be influenced on Eu incorporation level in the nanocomposites and the highest intensity of the peak was found in ZEG(10), which is approximately twice as large as the peak intensity of ZEG(00). The origin of the orange emission would be related to interstitial oxygen in the nano semiconductor oxide.⁶⁰ Moreover, a systematic broadening of the peak was observed on increasing the Eu incorporation level. This peak broadening could be determined from the measurement of FWHM, where the ZEG(00) and ZEG(15) showed the lowest (11 nm) and the highest (12.5 nm) values of FWHM, respectively. It should mention that the Eu ions could give emissions, especially due to ⁵D₀—⁷F₀ and ⁵D₀—⁷F₁ transitions.³⁹ Therefore, broadening could be explained on the basis of increasing Eu incorporation in the nanocomposites but the highest emission intensity in ZEG(10) could certainly be an enhancement of the surface defect (interstitial oxygen) concentration. As the XPS (Fig. 5) study already confirmed the formation of Eu²⁺ ions in the nanocomposite, the Eu²⁺ ions in an optimum concentration might be playing a significant role³⁹ in the enhancement of the intensity of PL emission at 595 nm. On the other hand, the influence of CCG on the intensity of orange emission was also investigated in detail (Fig. S6, ESI†) and a positive function towards the enhancement of the defect concentration in hexagonal ZnO of the ZEG(10) nanocomposite was noticed. Thus, it could be concluded that CCG in combination with Eu could assist in enhancing the orange emission at 595 nm.

Fig. 7 (a) Photoluminescence emission spectra (λ_ex = 340 nm) of ZEG nanocomposites (inset show the photoluminescence excitation spectrum, fixing the emission at 384 nm). (b) Photoluminescence emission spectra (λ_ex = 400 nm) of ZEG nanocomposites (inset (i) shows the photoluminescence excitation spectrum, fixing the emission at 595 nm; inset (ii) describes the Gaussian-fitted components of the marked portion in the wavelength range of 520–585 nm for ZEG(10)).

Specific surface area (SSA) of ZEG nanocomposites measured by multipoint BET nitrogen adsorption isotherm (Fig. S7 and Table S2, ESI†) changed with Eu incorporation level. The highest surface area was measured in the ZEG(10) nanocomposite. It could be noted from the appearance of the hysteresis loop found in the relative pressure (p/p₀) range of 0.6–1.0 that the BET isotherms of the all the samples highlight (Fig. S7†) an IUPAC Type IV isotherm,⁶¹ indicating the presence of mesopores in the nanocomposites. We also confirmed the existence of mesopore from the calculated average pore diameter that was found in the range of 11.9 to 34.1 nm (Table S2, ESI†).

It could be believed that during the synthesis, major portions of zinc acetate (ZA) precursor would form^12,22 ZnO nanoparticles (ZNPs) in the reaction medium, and remaining ZA would generate Zn²⁺ ions, which would chemisorb¹² on the surface of the ZNPs, resulting in the formation of ZnO/Zn²⁺ moiety. Simultaneously, the GO converted into the chemically converted graphene (CCG) after an appreciable amount of reaction time (Fig. S1 and S3, ESI†) and could interact/form complexes with the inorganic moiety. On the other hand, in europium incorporated nanocomposites, no europium ions were found to enter into the ZnO crystal lattice (which might be due to self-purification process²⁶ in nano semiconductors), as evidenced from the XRD result (Fig. 1). However, these ions could interact/form complexes with the free oxygen functional groups of CCG, as confirmed by FTIR (Fig. 3), Raman (Fig. 4), XPS (Fig. 5) and UV-Vis (Fig. 6) studies. However, TEM studies (Fig. 2) confirmed the presence of CCG layers on the surface of ZnO nano crystals. Thus, in the present work, on the basis of our experimental results, a tentative chemical structure of europium incorporated ZEG nanocomposite is given in Fig. 8.

Fig. 8 A scheme illustrating the synthesis of graphene oxide from graphite powder as well as the formation of Eu incorporated ZnO-chemically converted graphene. A possible chemical structure of Eu incorporated ZnO–graphene nanocomposite is also displayed in the scheme.

Because of unique physical and optical properties of ZnO nanoparticles, these could have significant advantages in biomedical applications (e.g. cell imaging and drug delivery).^19,20 In addition, the nanoparticles (NPs) could possess particle size dependent tunable band edge emission²³ in the ultraviolet region. Moreover, these could be capable of generating visible emissions²⁶ due to the presence of several intrinsic/extrinsic surface defects. Hence, it would be possible to utilize a suitable visible emission towards the application of cell imaging. In this regard, undoubtedly many studies^24,58 have already been performed on ZnO nanoparticles. In the present work, the PL study already confirmed the existence of an orange emission at 595 nm (λ_ex = 400 nm) in the nanocomposites. Moreover, the intensity of the orange emission was found to be dependent on EUN/ZA molar ratio (R value), where the ZEG(10) sample exhibited the highest intensity over the others and proved the necessity of optimum amount of Eu incorporation in the ZnO graphene nanocomposite to obtain the high intensity of orange emission. Hence, the ZEG(10) nanocomposite could be expected to have a better cell imaging capability. We have investigated the performance of ZEG(10) compared with ZEG(00) for the human breast cancer cell (MCF7) imaging under a confocal laser scanning microscope. The DIC pictures and the fluorescent images were taken (Fig. 9a) for a fixed concentration (100 μg ml⁻¹) of ZEG nanocomposites. From the images, it was clear that the internalization of the nanomaterials within the living cells occurred. Bright cell images were found due to the orange fluorescence of the nanocomposites under the microscope. However, a relative intensity of the images (Fig. S8, ESI†) were calculated using ImageJ software and analyzed by one-way ANNOVA. It was noted that the highest intensity (Fig. S8, ESI†) was observed when the ZEG(10) nanocomposite was used, and in this case, the intensity was found to be more than two times higher (Fig. S8, ESI†) compared to the ZEG(00) nanocomposite. The intensity of the orange fluorescence also corroborated the PL results (Fig. 7b). Hence, the orange emission of the ZEG(10) nanocomposite could be useful for the study of cell imaging.

Fig. 9 (a) DIC pictures (i, iii and v) and fluorescence images (ii, iv and vi) of the MCF7 breast cancer cells under a confocal laser scanning microscope; (b) cell viability results from a MTT assay with ZEG nanocomposites of different concentrations. The error bars represent ±SD (P < 0.05).

ZnO is generally biocompatible and cost effective but nano ZnO has been classified as “extremely toxic” in the environment.³⁵ The extreme toxicity of nano ZnO could generally be mitigated to be biocompatible through coating on the surface of the nano semiconductor oxide by graphene derivatives. It could be noted that graphene possesses a large surface area, chemical purity and the possibility of easy functionalization with a suitable semiconductor²⁰ or suitable organic molecule.^6,7 Therefore, graphene based ZnO nanocomposite could be used as an alternative of organic dyes⁶² for cell imaging. However, for further improvement of the property, proper modification of nano matrix would be necessary, and the incorporation of an optimum amount of Eu would be beneficial in this respect. In the present work, a relatively better cell imaging performance (Fig. 9a) was found in ZEG(10). It was known that the toxic effect of nanocomposites on biological cells (known as cytotoxicity) could depend upon several of their characteristics.⁶³ It had already been demonstrated that nano ZnO exhibited a toxic effect in mammalian cells.³² This could be due to particle dissolution in the tissue culture medium and resulting from intracellular Zn²⁺ shedding. It was also reported that doping³⁵ with Fe could be an effective way to reduce the particle dissolution of nano ZnO for the concerned application. Thus, the change of particle matrix, as well as the rate of particle dissolution, could be reduced by Fe doping. On the other hand, graphene based semiconductor (e.g. ZnS, CdSe) NPs²⁰ were reported to have an effective cell imaging as well as high cell viability. In this work, we measured the cytotoxicity by measuring the cell viability of human breast cancer cells (MCF7) with ZEG nanocomposites (Fig. 9b). Approximately 74% viable cells were observed in ZEG(10) for 30 μg ml⁻¹ of ZEG nanocomposite in 24 h duration. However, low cell viability was observed for the ZEG(00) nanocomposite, where no Eu was incorporated. Moreover, the cell viability remained nearly the same in the ZEG(15) nanocomposite compared to ZEG(10). This could be because of the same I_D/I_G value (Fig. 4 and Table S1 of ESI†), generated due to the exfoliation³¹ of graphene layers through interaction/complexation between free organic functional groups of CCG and the inorganic moieties present in the nanocomposite, as already observed from Raman spectral study. Hence, the observation clearly indicates that an optimum concentration of Eu incorporation in the nanocomposite was necessary to obtain better cell viability. Usually, the better cell viability and cell imaging capability, as well as the relatively large surface area could make the materials an effective carrier for drug delivery.⁶⁴ In this respect, the Eu incorporated nanocomposites (R ≥ 0.10) could find application in drug delivery.

Conclusion

Europium incorporated ZnO-chemically converted graphene (CCG) nanocomposites were synthesized from the precursors of varying europium nitrate to zinc acetate molar ratios (R = 0.00, 0.05, 0.10 and 0.15) in a fixed content of graphene oxide, adopting a low temperature (95 °C) solvothermal process. The change in crystallite/particle size of hexagonal ZnO was found to be dependent on the Eu incorporation level (R value). In the nanocomposites, evidence of chemical interaction/complexation with the oxygen functionalities of CCG and inorganic moieties (ZnO/Zn²⁺ and Eu ions) was found. Although, the nanocomposite ZEG(10) (R = 0.10) showed the highest specific surface area, all the samples possessed mesopores. In addition to UV emission, an orange emission appeared at 595 nm and other visible emissions were observed from the photoluminescence spectra of the nanocomposites. However, the intensity of the orange emission was found to be maximum in the nanocomposite ZEG(10). Under a confocal laser scanning microscope, bright orange fluorescence images of human breast cancer cells (MCF7) with the ZEG(10) nanocomposite were found, indicating the internalization of the nanomaterials within the cells. The nanocomposite also exhibited comparatively low in vitro cytotoxicity on the cells. The ZEG(10) nanocomposite would be employed for cancer cell targeted optical imaging and drug delivery.

Acknowledgements

Authors are grateful to the Director, CSIR-CGCRI, Kolkata for his kind support, encouragement and permission to publish this work. The authors, SB, MG and MP thankfully acknowledge CSIR and UGC, Govt. of India for providing their Ph.D. research fellowships under NET Fellowship scheme. The authors also acknowledge the help rendered by Nanostructured Materials Division and Electron Microscopy Section for Raman and microstructural characterizations respectively. The work has been done as an associated research work of 12^th Five Year Plan project of CSIR (no. ESC0202).

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Footnotes

† Electronic supplementary information (ESI) available. See DOI: 10.1039/c4ra06243d

‡ Authors contributed equally to this work.

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