Facile surface modi ﬁ cation of nickel ferrite nanoparticles for inherent multiple ﬂ uorescence and catalytic activities

We synthesized biocompatible NiFe 2 O 4 nanoparticles (NPs) with diameters below 10 nm by coating their surface with a tartrate ligand, which interestingly give rise to photoluminescence covering the entire visible region. The analyses with various spectroscopic tools reveal that the reason behind the unique ﬂ uorescence properties of functionalized NiFe 2 O 4 NPs is ligand-to-metal charge-transfer transition from the highest occupied energy level of tartrate ligand to the lowest unoccupied energy level of Ni 2+ and d – d transitions centered over Ni 2+ ions in the NPs. These ﬂ uorescent NPs are also found to be suitable for cell imaging. Moreover, the functionalized NiFe 2 O 4 NPs show a good catalytic activity on biologically and environmentally toxic pigments, such as bilirubin and methylene blue which leads to their wide application towards therapeutics and waste water treatment. We believe that the developed multifunctional NiFe 2 O 4 NPs would stimulate the opportunities for advanced biomedical applications.


Introduction
With increasing applications, particularly in biomedical elds, 1-5 multifunctional magnetic nanoparticles (MNPs) have attracted tremendous attention due to their potential to be used in ultrasound, 6 optical and uorescence imaging combined with targeted drug delivery, 7-9 separation and purication of cells, 10 tissue repairing, 11 hyperthermia for cancer treatment 12 as they result from biocompatibility, chemical stability and controlled transport properties. The MNPs are also useful in the eld of catalysis since they are efficient for specic chemical transformations. In addition they are propitious economically and environmentally due to their elevated activity, sufficient stability, controlled separation by an external magnetic eld, low cost, and facile synthesis. [13][14][15][16][17][18][19] MNPs with intrinsic uorescence are in high demand. To be used in biomedical elds, they should possess criteria such as biocompatibility, monodispersity, dispersibility in water, and non-toxicity. The commonly used procedure for synthesizing magneto-uorescent NPs involves processes like making nanocomposites with quantum dots (QDs) or coatings with uorescent dyes. In order to make the NPs biocompatible, surface modications were carried out with biomolecules like proteins, DNA, RNA, small organic ligands, and polymers, but none of them were able to generate intrinsic uorescence. In addition, their applications towards the biomedical eld are narrowed due to chemical instability, photobleaching of uorescent dyes, and inherent toxicity of QDs (due to the presence of heavy metals like Cd 2+ , Pb 2+ ). 20 Therefore, to open up a wide applicability of the nanoparticles (NPs) in biomedical eld, a facile and proper process for surface modication is highly required. The main purpose of our present work is to design water dispersible 3d transition metal oxide based MNPs with intrinsic uorescence for their applications in cell imaging.
Our work has involved both the synthesis and development of NiFe 2 O 4 NPs as multifunctional nano-probes bearing inherent multicolour photoluminescence and their development of in vitro cell imaging. Moreover, we have utilized the developed MNPs in catalysis. Using ligand eld theory (LFT), we have assigned the electronic transitions responsible for the coloured photoluminescence of the functionalized NiFe 2 O 4 NPs. We tried the functionalized NPs as cell imaging agent and found them worthy to be used in imaging applications.

Experiment
We synthesized NiFe 2 O 4 NPs with minimal modication, by following a wet chemical method previously reported by Sun et al. 21 We heated a solution mixture of iron(III) acetylacetonate, nickel(II) acetate, diphenyl ether, oleylamine, and oleic acid to 270 C for 1 h in presence of cetyl alcohol instead of 1,2-hexadecanediol used by Sun et al. 21 The as-synthesized NiFe 2 O 4 NPs were then washed by ethanol, and subsequently collected aer centrifugation followed by removal of mother solution. Then assynthesized NiFe 2 O 4 NPs were dried by heating the NPs on a hotplate at a temperature of 80 C for 30 min. In order to functionalize the as-prepared NiFe 2 O 4 NPs with tartrate ligands, we put the as-prepared solid NPs with 0.5 M Na-tartrate solution (pH $ 7) in glass vial. Then the glass vial was placed on a vibrator rotor for vigorous vibration, and the rotation was done at room temperature for 12 h. The non-solubilised NiFe 2 O 4 NPs were ltered by passing the functionalized NPsolution mixture through a syringe lter of diameter 0.22 mm. We obtained a clear solution of tartrate-functionalized NiFe 2 O 4 NPs, called T-NiFe 2 O 4 NPs.
The X-ray powder diffraction (XRD) of as-prepared NiFe 2 O 4 NPs was carried out using Rikagu miniex II diffractometer equipped with Cu Ka radiation (at 40 mA and 40 kV) at the scanning rate of 3 per minute in the 2q range of 20-70 . A FEI Technai G 2 TF-20 eld-emission high-resolution transmission electron microscope (TEM), operated at 200 kV was used to study the size distribution, shape, morphology and to record the Energy-dispersive X-ray analysis (EDX) spectrum of the asprepared and functionalized NiFe 2 O 4 NPs. The ultravioletvisible (UV/Vis) absorbance spectra of T-NiFe 2 O 4 NPs were obtained on a Shimadzu UV-2600 spectrophotometer, using a quartz cuvette of 1 cm path length. A Horiba Jovin Yvon Model Fluorolog uorimeter was used to carry out the steady-state uorescence emission and excitation study of T-NiFe 2 O 4 NPs. Fluorescence micrographs of T-NiFe 2 O 4 NPs were captured using a Leica DM1000 LED uorescence microscope.
To be sure of the attachment of tartrate molecules to the NPs' surface, Fourier transform infrared spectroscopy (FTIR) measurements were performed using a JASCO FTIR-6300 spectrometer. For FTIR study, pellets were made aer homogeneous mixing of lyophilized T-NiFe 2 O 4 powder samples with KBr. The background was corrected using a reference KBr pellet. To estimate the loading of the tartrate functionalization, we performed thermogravimetry (TG) on the T-NiFe 2 O 4 and bare NiFe 2 O 4 NPs using PerkinElmer Diamond TG/DTA with the heating rate of 10 C min À1 . The magnetic measurements were performed using a Lake Shore Vibrating Sample Magnetometer (VSM).
For cell culture tests, cells of the human osteosarcoma cell line Cal-72 were ordered from the DSMZ (Leibniz Institute DSMZ -German Collection of Microorganisms and Cell Culture, DSMZ no. ACC 439). The cells were cultivated in Dulbecco's Modied Eagle Medium with a concentration of 10% foetal bovine serum. Aer three days the medium was removed and replaced by mixtures of culture medium and functionalized NPs at concentrations of 6, 2, and 0.5 mg mL À1 . Aer one day those mixtures were removed and the cells were xed using 3% paraformaldehyde in Phosphate Buffered Saline (PBS). Cytotoxicity was performed by watching the phenotype and condition of the cells for several hours using a high resolution optical microscope. Images of the cells along with NPs were taken using a Laser Scanning Microscope (LSM).
In the photocatalytic study, an 8 W UV lamp with a wavelength of 253.7 nm (UV-C) was used. The aqueous solution of T-NiFe 2 O 4 NPs (50 mL, containing 0.06 mg NPs) and 5 mM aqueous solution of methylene blue (MB) (pH $ 3) were uniformly mixed in a quartz cuvette for 1 h in the dark. Then, the cuvette was kept 2 cm away from the light source and the absorbance spectra of MB in presence of T-NiFe 2 O 4 NPs solution was measured periodically by Shimadzu UV-2600 UV/Vis spectrophotometer. The very rst recorded set of data of absorbance was marked as 0 minute. Aer 60 minutes of irradiation (rst cycle), we further added 5 mL methylene blue solution to the reaction mixture but the catalyst solution was not added for the second cycle. Similar procedure was maintained up to 5 cycles.
In order to determine the catalytic activity of functionalized NPs, the aqueous solution of T-NiFe 2 O 4 NPs (50 mL, containing 0.06 mg NPs) were added to 15 mL of aqueous solution of bilirubin (BR) (pH $ 7) kept in a quartz cuvette under continuous stirring in the dark. Then the absorbance spectra of BR in presence of T-NiFe 2 O 4 NPs solution were recorded at times by using UV/Vis spectrophotometer. The reusability study was carried out by the similar procedure as we did for photocatalysis up to 3 cycles.

Results and discussion
The XRD pattern as shown in Fig. 1(a) is consistent with the standard inverse spinel face centered cubic structure of NiFe 2 O 4 (JCPDS card no. 10-0325). The EDX spectrum of NiFe 2 O 4 NPs in the inset of Fig. 1(a) affirms the presence of nickel, iron and oxygen. To obtain the morphology and particle size of assynthesized NiFe 2 O 4 NPs, we carried out TEM analysis, as shown in Fig. 1(b). From Fig. 1(c), it is apparent that the NPs have a size distribution with an average diameter of 6.8 nm. The high-resolution transmission electron microscope (HRTEM) image, as shown in Fig. 1(d) depicts the highly crystalline nature of the as-prepared NiFe 2 O 4 NPs. The measured interplanar distance between lattice fringes is around 0.253 nm, which corresponds to the distance between the (311) planes of the NiFe 2 O 4 crystal lattice.
To make the NiFe 2 O 4 NPs biocompatible and waterdispersible, we have functionalized the as-prepared NPs with the aqueous solution of a small organic ligand, Na-tartrate. Aer surface functionalization, the size of the NPs was found to remain almost unchanged with an average diameter of 6.6 nm, as evident from Fig. 2(a) and the size distribution graph shown in Fig. 2(b). The HRTEM image (shown in Fig. 2(c)) of the functionalized NPs (T-NiFe 2 O 4 ) shows its crystalline nature. Interestingly, the UV/Vis absorption spectra of T-NiFe 2 O 4 NPs (pH $ 7) is found to exhibit a broad absorption feature at around 302 nm as shown in Fig. 2(d), indicates that the tartrate ligand affects the surface electronic structure of bare NiFe 2 O 4 NPs signicantly upon functionalization.
Aer obtaining knowledge from the UV/Vis spectra of functionalized NiFe 2 O 4 NPs, we have performed a photoluminescence study and have observed photoluminescence at 412 nm (low intensity) on exciting the sample at wavelength of 325 nm. To intensify the photoluminescence, we have carried out further surface modication by heating the functionalized NiFe 2 O 4 NPs solution for 42 h at 70 C and pH $ 12 which we call uorescence modied (. mod.) T-NiFe 2 O 4 NPs. They were found to exhibit another absorption band at 365 nm in addition to the band at 325 nm as shown in Fig. 2(d). The surface modication is found to result in three photoluminescence peaks with many fold increase in overall intensity, upon excitation at proper wavelengths as shown in Fig. 3(a), the normalized steady state photoluminescence emission spectra of . mod. T-NiFe 2 O 4 NPs. This is due to an increase in the strength of the coordination between the functional groups of the ligand (-COO À and -OH moieties) and the metal ion (Ni 2+ ) which leads to three more photoluminescence peaks as well as a huge increase in photoluminescence intensity of T-NiFe 2 O 4 NPs. Upon exciting with wavelengths of 325, 375, 450, and 503 nm, the NPs solution gave rise to signicant photoluminescence peaks at 412, 467, 530, and 603 nm, respectively. These bands are clearly distinguishable also in the excitation study as shown in Fig. 3(b). The uorescence micrographs of . mod. T-NiFe 2 O 4 NPs show the photoluminescent colors like blue (Fig. 3(c)), green (Fig. 3(d)) and red (Fig. 3(e)) on excitation of at 375, 450, and 515 nm, respectively, by using proper lters for the excitation wavelengths.
The LFT efficiently explains the generation of multicolour photoluminescence. Based on the LFT a ligand co-ordination with the NPs' surface plays the key role, which yields the crystal eld splitting energy (CFSE) (D) generated from d orbital splitting with a magnitude determined by the ligands and a given coordination symmetry. The strength of the metalligand s bond increases as a function of increasing basicity of the solution. As the basicity of the solution increases, the 2carboxyl and 2-hydroxyl groups become deprotonated resulting in larger interactions with the metal ions and as an outcome, CFSE (D) associated with the ligand increases which nally lead to an increased splitting between the previous degenerate d orbitals. The emission peaks at 467, 530 and 603 nm can be attributed to 3 A 2g (F) / 3 T 1g (P), 3 A 2g (F) / 3 T 1g (F) and 3 A 2g (F) / 3 T 2g (F) transitions involving d-d orbitals of Ni 2+ ions respectively, where these energy levels were obtained from the Tanabe Sugano diagram of Ni 2+ . These d-d transitions are formally spin allowed transitions. Note that d-d transitions involving only Fe 3+ are not efficient for the development of intense uorescence. 22 On the other hand, due to strong ligand to metal charge transfer (LMCT) from HOMO (Highest Occupied Molecular Orbital, centered on the ligand) to LUMO (Lowest Unoccupied Molecular Orbital, centered on the metal ions), bonding interactions between the metal and the ligand increases considerably. Therefore, the photoluminescence peak arising at 412 nm can be attributed to LMCT involving HOMO of tartrate ligand and LUMO centered over metal ion Ni 2+ . 23 The Quantum Yield (QY) is calculated by taking rhodamine B (Rh B) as the standard uorescent compound and the obtained QYs are 15.13% for the 412 nm band, 1.6% for the 467 nm band and the 0.15% for 530 nm band of . mod. T-NiFe 2 O 4 NPs.
In order to conrm the attachment of the tartrate molecules to the surface of NiFe 2 O 4 NPs, we carried out the FTIR study of bare NiFe 2 O 4 NPs, T-NiFe 2 O 4 NPs and pure tartrate ligand. As depicted in Fig. 4(a), the peak arising at 587 cm À1 is due to stretching vibration of metal-oxygen bonds in NiFe 2 O 4 . The peak at 587 cm À1 is absent in T-NiFe 2 O 4 NPs. Two sharp peaks arising at 1066 and 1112 cm À1 in the case of tartrate ligand are due to C-OH stretching modes 24 and two other peaks at 1411 and 1621 cm À1 are due to the symmetric and asymmetric stretching of the COO À . 25 In the case of T-NiFe 2 O 4 NPs, due to  the interactions between NPs surface and the functional group moieties of the ligand, all the bands are distinctly perturbed along with the band at 3399 cm À1 which arises because of the stretching vibrational modes of the O-H group. 24 This clearly indicates the involvement of -COO À and -OH group in the functionalization. thermogravimetric analysis (TGA) data of the as-prepared and tartrate functionalized Nickel ferrite measured under nitrogen atmosphere from 100 to 400 C and shown in Fig. 4(b). The amount of tartrate ligand bound to the nanoparticle surface was calculated from the TGA data and found to be $1.88 wt% of the T-NiFe 2 O 4 NPs.
The room temperature magnetic behaviour of the asprepared and T-NiFe 2 O 4 NPs were studied by VSM. Fig. 5 depicts the M-H loops of both as-prepared and functionalized NiFe 2 O 4 NPs. From Fig. 5(a) and (b) it is found that the saturation magnetization and coercivity are reduced from 37.1 emu g À1 to 11.7 emu g À1 and from 0.04 kOe to 0.02 kOe, respectively, in case of T-NiFe 2 O 4 NPs in comparison with as-prepared NiFe 2 O 4 NPs. The tartrate ligand contains both s donor (-OH) and p donor (-COO À ) groups which results in immense LMCT and spin pairing of Ni 2+ ions in T-NiFe 2 O 4 NPs, which reduces the saturation magnetization. 26 Aer efficiently incorporating the intrinsic uorescence in NiFe 2 O 4 NPs, we inspected the optical excitation of the functionalized NiFe 2 O 4 NPs in photocatalysis for waste water treatment. The functionalized NiFe 2 O 4 NPs were found to possess efficient photocatalytic properties (as shown in Fig. 6(a)). In case of the degradation of MB, a frequently used dye in textile industries and a model water-contaminant, a catalytic effect occur aer UV light irradiation. We found that the photodegradation of MB in presence of functionalized NiFe 2 O 4 NPs at pH $ 3 takes place exponentially with time following a rst-order rate equation (by tting with equation A ¼ A 0 e Àkt ) with a kinetic rate constant k ¼ 2.82 Â 10 À2 min À1 . We also carried out a reusability test of the catalyst with 70 min of time interval as shown in Fig. 6(b). We kept adding MB up to 5 cycles by keeping the concentration of the catalyst same (by not adding more catalyst aer the 1 st cycle). We measured the decomposition rate of the MB by monitoring the absorbance at 663 nm by UV/Vis spectroscopy that had led to conclude the reusability of T-NiFe 2 O 4 NPs as a catalyst. The comparative rate of degradation of MB by Na-tartrate, bare NiFe 2 O 4 NPs and T-NiFe 2 O 4 NPs is shown in Fig. 6(c).
Stimulated by the photocatalytic property of the functionalized NiFe 2 O 4 NPs on MB, we have performed similar experiment on a biologically toxic pigment, BR which is responsible for the occurrence of yellow coloration of skin in jaundice. T-NiFe 2 O 4 NPs shows a good catalytic activity in the degradation of BR without any photoexcitation at pH $ 7 at room temperature as shown in Fig. 7(a). The reaction follows rst order kinetics with the reaction rate constant k ¼ 6.5 Â 10 À3 min À1 . We investigated the recyclability of the catalytic efficiency of T-NiFe 2 O 4 for degradation of BR by adding the same amount of BR to the reaction mixture in every 60 min up to 3 cycles, keeping the concentration of the catalyst unchanged. We measured the fall in absorbance of the BR at 435 nm at regular intervals by UV/Vis spectroscopy. The plot of relative BR concentration as a function of time for up to three consecutive cycles, as demonstrated in Fig. 7(b), conrms the reusability of the T-NiFe 2 O 4 NPs catalyst. Fig. 7(c) 8 shows human osteosarcoma cells in the presence of culture medium with a NP concentration of 2 mg mL À1 . From Fig. 8(a) it can be deduced that the cells are alive and in a good condition indicating the non-toxicity of the NPs. Fig. 8(b) shows that NPs adhere to cells and uoresce under illumination with light of appropriate wavelength and intensity.

Conclusions
Through easy surface modication of NiFe 2 O 4 NPs with Natartrate ligands, we have prepared biocompatible multifunctional nanoparticles with intrinsic uorescence properties covering the whole visible region, ranging from blue, and green, to red. The NPs are found to be efficient in cell imaging using this inherent uorescent property. Additionally, the T-NiFe 2 O 4 NPs show good catalytic and photocatalytic activity in the degradation of biologically and environmentally toxic pigments [bilirubin and methylene blue] respectively. A thorough experimental and theoretical study has revealed that the LMCT transition from the highest occupied energy level of the tartrate ligand to lowest unoccupied energy levels of Ni 2+ and d-d transitions centered over Ni 2+ ions on the NPs' surface play crucial roles in the generation of multicolor uorescence from the T-NiFe 2 O 4 NPs. We trust that the development of these multifunctional T-NiFe 2 O 4 NPs will open new opportunities in the eld of diagnostics, such as bio-imaging, therapeutics and drug delivery, as well as in wastewater treatment of contaminants.

Conflicts of interest
There are no conicts to declare.