Nontoxic Tb3+-induced hyaluronic nano-poached egg aggregates for colorimetric and luminescent detection of Fe3+ ions

This study demonstrates that a luminescent Tb3+ complex with green emission can be complexed with hyaluronic (hya) to form nanoparticles. The structure of complexation is composed of a Tb(acac)2phen core with a hya surface, similar to those of the nano-poached eggs. What makes the structure unique is that Tb(acac)2phen and hya are connected by chemical bonds. To confirm their utility, we illustrate that the luminescence is rapidly and selectively quenched in the presence of Fe3+. Initial cytotoxicity experiments with human liver carcinoma cells show that the luminescent lanthanide complexes are cytotoxic, however, complexing lanthanides to hya renders them cytocompatible. The new complex integrates the advantages of superior lanthanide luminescence, the unique shape of nano-poached eggs, compatibility with aqueous systems, and cytocompatibility. Tb3+-induced hyaluronic nano-poached eggs (THNE) can, therefore, be used for Fe3+ detection in aqueous systems.


Introduction
Increasing attention has been focused on environmental and biological detection methods. 1-10 Among them, sensing and imaging technology, as the core of "luminescence", has shown special advantages in environmental monitoring and the diagnosis of diseases. [11][12][13][14][15] The design and discovery of novel sensors for transition-metal ions in aqueous medium are of great concern. 16-20 Fe 3+ is an essential micronutrient, which plays a critical role in basic physiological processes. [21][22][23] For example, ferritin is the key to life, which living cells need to survive. Iron ions of ferritin are used to form important intermediates for respiration, photosynthesis, nitrogen xation, DNA synthesis, etc. [24][25][26] On the other hand, wastewater containing iron discharged from factories, such as acid mining and cleaning rust on steel surfaces, seriously pollutes the environment. Therefore, the detection of Fe 3+ is an important research eld. [27][28][29][30][31][32] Lanthanide complexes have bright and narrow emission bands of visible light, fast response times, long uorescence lifetimes, and very low susceptibility to photobleaching. [33][34][35] The luminescence of lanthanide ions, such as Tb 3+ is weak, in part due to its low photon efficiencies. However, lanthanides can be sensitized by complexing them with organic ligands in their rst coordination sphere, resulting in the "antenna effect". When complexed to organic ligands, the d-f transitions of Tb 3+ generate intense luminescence. 36,37 The antenna effect can be used to prepare luminescent complexes with high luminescent efficiency and sharp emission spectra. However, the coordination with organic ligands generally renders lanthanides insoluble in water. Lanthanide-ligand complexation can be dynamic, enabling reversible on-off luminescence. Therefore, changes in the luminescence can be driven by any stimulus that changes the lanthanide-ligand coordination strength, or that introduces competing ligands or competing metal centers leading to cation-ligand exchange. Polymeric materials used as coordinating ligands can provide both dynamic binding and compatibilization with biological and aqueous environments. [38][39][40][41] Biological polysaccharides as ligands of luminescence sensors can modulate the luminescence properties and biological activity. 42 Hyaluronic acid is a polyanion mucopolysaccharide that consists of a basic unit of glucuronic acid and Nacetylglucosamine. It is recognized as the best moisturizing ingredient and is widely used in food, medicine and cosmetics. [43][44][45] The integration of natural biomass and luminescence material into one complex can lead to a wide variety of optimized biocompatible materials. 46 Thus, our pioneering experiment has integrated Tb 3+ superior lanthanide luminescence with the good bio-compatibility of hyaluronic acid. The critical point is that hyaluronic acid modulates both the photophysical properties, morphology and biological activity.
In this work, the original Tb 3+ -induced hyaluronic nanopoached eggs (THNE) integrates the advantages of superior lanthanide luminescence, the unique shape of nano-poached eggs, compatibility with aqueous systems, and non-toxicity, for the sensing of Fe 3+ in aqueous surroundings. The lanthanide complexes are cytotoxic but complexing the lanthanides to hyaluronic acid renders them cytocompatible. We reveal the mechanism of luminescence quenching. The luminescence property of THNE is affected by ligand-to-metal charge transfer (LMCT) states. However, the coordination between Fe 3+ and ligands can alter the LMCT effect of ligands on Tb 3+ . These properties of THNE have the potential for application in environmental detection systems.

Materials
Hyaluronic acid sodium salt from rooster comb (hya) and acetylacetone (acac) were supplied by Shanghai Macklin Biochemical Co. Ltd (Shanghai, China). Terbium chloride hexahydrate (TbCl 3 $6H 2 O) was purchased from Sinopharm Chemical Reagent Co. Ltd (Beijing, China). HepG2 was purchased from iCell Bioscience Inc (Shanghai, China). Fetal calf serum was obtained from Biological Industries (Kibbutz Beit-Haemek, Israel). MEM culture medium was purchased from Corning Incorporated (Corning, USA). MTT was supplied by Beijing Solarbio Science & Technology Co. Ltd (Beijing, China). Other chemical reagents were acquired from Shanghai Chemical Reagent Company (Shanghai, China). All water used was deionized. All chemicals mentioned above were of analytical grade.  (Table 1). We also prepared Tb(acac) 2 phen (5.0 mM, Tb 3+ ). Tb(acac) 2 phen and THNE with different concentrations were obtained. The schematic diagram of the synthesis of THNE is shown in Fig. 1.

Cell cytotoxicity experiment
Human liver carcinoma cells HepG2 were incubated on a 96well plate for 24 h. Culture medium, blank solvent ethanol-H 2 O (3 : 1, v/v), hya, Tb(acac) 2 phen (5.0 mM, Tb 3+ ), and different concentrations (1.25 mM, 2.5 mM, 5.0 mM, 7.5 mM and 10.0 mM) of the THNE were added to different wells, then the cells were cultured for 24 h. Each condition was prepared in triplicate. HepG2 cells were washed and incubated for 4 h with 100 mL MTT solution. The liquid medium was removed, then 150 mL of DMSO was added to each well, and the crystals in each well were reacted with the DMSO for 1 h. The absorbance measurements of each well were obtained at 570 nm by a multimode plate reader.

Results and discussion
We prepared THNEs with different concentrations (Table 1) in order to screen materials with excellent luminescence properties. Fig. 2a shows luminescence excitation spectra and emission spectra of THNE with different concentrations. A broad band from 310 to 380 nm with the maximum excitation wavelengths of 332 nm, 337 nm, 347 nm, 349 nm and 350 nm was observed in the excitation spectrum of THNE and could be caused by the intraligand p / p* transitions from acac, phen and hya. With the increased concentration of THNE, the maximum excitation wavelength was enhanced. The luminescence emission spectra were obtained by selecting the corresponding maximum excitation wavelength. The characteristic luminescence spectra of Tb 3+ were obtained with different concentrations of THNE (l em ¼ 547 nm). With the increase in the concentrations of THNE (less than 5.0 mM Tb 3+ ), the uorescence intensity was gradually enhanced. However, when the concentrations were more than 5.0 mM Tb 3+ , the luminescence intensity presented the opposite tendency. When the concentration was 5.0 mM Tb 3+ , the luminescence intensity was the highest. If the concentration of Tb 3+ was higher than 5.0 mM, the emission intensity of THNE was decreased. This is called concentration quenching, which is mainly due to a crossrelaxation (CR) process between Tb 3+ . This phenomenon widely occurs in Tb 3+ -induced luminescent materials, setting  a limit in the concentration of Tb 3+ to control the brightness. 47 Therefore, we selected THNE (5.0 mM Tb 3+ ) as an excellent luminescent material. Fig. 2b displays the luminescence excitation spectra of Tb(acac) 2 phen and THNE (l em ¼ 547 nm) and the luminescence emission spectra of Tb(acac) 2 phen and THNE (l ex ¼ 347 nm). THNE also contains the same characteristic emission bands. The luminescence emission spectra of Tb(acac) 2 phen and THNE do not have signicant differences in their respective emission wavelengths. These represent the band splitting character and 5 D 4 -7 F 5 transition intensities, with those of known complex compounds of Tb 3+ and the results of f-f transitions of the lanthanide ion. This means that added hya keeps the luminescence properties of the Tb complex. Compared to the luminescence intensity of Tb(acac) 2 phen, the luminescence intensity of THNE was slightly weakened. We explain why the luminescence intensity of THNE decreased in the further experimental results of XPS analysis (Fig. 3) and morphology ( Fig. 4a and b). The uorescence lifetime of THNE is approximately 235.83 ms and the quantum yield is 5.16% (Fig. 2c). The uorescence lifetime of Tb(acac) 2 phen is approximately 255.82 ms and the quantum yield is 6.38% (Fig. 2d). We also discuss the reasons aer the experimental results of XPS and TEM analysis. Fig. S1 † presents the FTIR spectra of TbCl 3 , acac, phen, hya, Tb(acac) 2 phen and THNE. As shown in the FTIR spectrum of acac, the peaks at 1732 and 1709 cm À1 characterize the absorptions of the C]O stretching vibration. The signicant peaks at 1304, and 1248 cm À1 are due to stretching vibration from C-C. The peak at 1420 cm À1 is assigned to -CH 2 bending vibration, and the -CH 3 bending vibration peak is observed at 1360 cm À1 . The peak at 1646 cm À1 of phen is assigned to the C]N stretching vibration, and the C-N stretching vibration peaks are observed at 1140 and 1090 cm À1 . The signicant peaks at 1616, 1590, 1506 and 1423 cm À1 are due to the stretching vibration of the skeleton from C]C and C]N (pyridine rings). As shown in Fig. S1 † † of hya, the absorption at 3421 cm À1 was due to the O-H stretching vibration. The trough at 1646 cm À1 features the absorption of the C]O stretching vibration. The signicant peak at 1400 cm À1 is due to the bending vibration absorption from the O-H group. The peak at 1109 cm À1 is assigned to the C-O-C stretching vibration of ethers. 48 Compared with the FTIR spectra of hya, Tb(acac) 2 phen and THNE, the peaks at 1646 cm À1 of the C]O stretching vibration, 1519 cm À1 of the pyridine ring stretching vibration, and 1400 cm À1 of O-H stretching vibration can be found in THNE. The above data indicate that THNE was prepared successfully.
To further conrm the interaction between the Tb complex and hya, XPS spectra were obtained as shown in Fig. 3. Tb(acac) 2 phen, hya and THNE were investigated by XPS. The obtained information about typical O 1s peaks are detailed in Fig. 3b, d and f. Fig. 3a shows a typical peak at 530.84 eV in the XPS spectrum of Tb(acac) 2 phen representing the O 1s. The peak at 398.87 eV is related to the N 1s of Tb(acac) 2 phen. The highresolution spectrum of O 1s (Fig. 3b) shows the peaks at 531.02 and 530.45 eV, which represent C-O and Tb-O.
Comparing the peaks of the samples in Fig. 3b and d, the binding energy of Tb-O was changed and C-O was formed, illustrating that the oxygen atom of Tb(acac) 2 phen interacted with hya. 49 Thus, Tb(acac) 2 phen and hya created chemical bonds to form THNE. The typical peak at 399.40 eV of hya represents the N 1s (Fig. 3c). The N 1s peak of THNE is 398.97 eV (Fig. 3e). XPS further proves the explanation of the luminescence change aer adding hya in Fig. 2b.
The morphology of THNE was investigated via TEM images ( Fig. 4a and b) and DLS (Fig. 4d). THNE was dropped onto copper mesh covered with carbon lm (230 mesh) and dried by infrared lamp prior to imaging by TEM. The core-shell morphological structure with cavities is shown in Fig. 4a and b. The width of the shell is about 185 nm. The structures of THNE are composed of Tb(acac) 2 phen cores with the hya surface. Interestingly, this morphology is very similar to the nanopoached eggs (Fig. 4c). The boiled egg white (hya) wraps its egg yolk (Tb(acac) 2 phen). What makes the nano-poached egg unique is that the egg white and egg yolk are connected by chemical bonds Tb-O. The size of THNE is distributed in the range of 450 nm to 1000 nm. The circular particles of THNE with a diameter of approximate 700 nm and a broad size distribution are shown in Fig. 4d. A similar structure was reported by Prof. Tang. 46 The nano-dumbbell aggregates were synthesized by combining Eu complexes with hyaluronic acid, which could effectively load drugs and sense drugs.
Hya has good water solubility and lm formation. These core-shell nanoparticles were formed during the coordination of Tb(acac) 2 phen and hya when the samples were stirred at high speed in the ethanol-H 2 O (3 : 1, v/v) system. A constant magnetic stirring of 1500 rpm was maintained for 2 h. The complexation of Tb(acac) 2 phen and hya was proved by XPS (Fig. 3). However, as we know, when Tb 3+ interacts with sensitizing ligands the "antenna effect" occurs. These ligands sensitize the luminescence of rare earth ions and enhance the luminescence intensity. This type of luminescence response can be driven by any stimuli that change the lanthanide-ligand coordination strength. Thus, the luminescence intensity of THNE should be greater than that of Tb(acac) 2 phen. Luminescence data show that the luminescence intensity of THNE is slightly weakened as compared to the luminescence intensity of Tb(acac) 2 phen (Fig. 2b). The luminescence intensity of lanthanide complexes is inuenced not only by lanthanide-ligand coordination but also the morphology. The Tb 3+ complex is wrapped by hya, which is similar to putting a non-luminous coat on the green phosphor. Although the coordination between Tb(acac) 2 phen and hya enhances the luminescence, the outer lm weakens the luminescence; therefore, the synergistic reaction of two factors leads to the decrease in luminescence intensity. Furthermore, on comparing the same type of luminescent materials, the luminescence intensity of THNE is slightly weaker than that of Tb(acac) 2 phen, and the uorescence lifetime of THNE (235.83 ms) is shorter than that of Tb(acac) 2phen (255.82 ms).
In order to investigate the detection of metal ions by THNE, the luminescence response with different metal ions was determined. The effects of the metal ions Ca 2+ , Mg 2+ , K + , Na + , Zn 2+ , Al 3+ , Mn 2+ , Cr 3+ , Ni 2+ , Cu 2+ , Co 2+ and Fe 3+ in aqueous solution on the luminescence emission of THNE were investigated. Fig. 5d shows the luminescence response of THNE with the addition of 10 mL (250 mM in THNE) of various metal ions. I 0 and I are the luminescence intensities of THNE in0 mL (0 mM) and 10 mL (250 mM in THNE) of different metal ions. THNE is much more sensitive to Fe 3+ than to the other cations. When Fe 3+ (250 mM) was added to the THNE, the luminescence intensity at 547 nm was reduced by more than 90%. When 10 mL Ca 2+ , Mg 2+ , K + , and Na + were added, the luminescence intensity remained the same. The THNE retained more than 80% of the luminescence in 10 mL Zn 2+ , Al 3+ , and Mn 2+ . When 10 mL Cr 3+ , Ni 2+ , Cu 2+ , and Co 2+ , were added, more than 40% of the luminescence intensity was retained. Among these various metal ions, THNE shows a selective luminescence response to Fe 3+ in aqueous solution. In order to visually describe the changes in the luminescence intensity when different metal ions were added, we present photos of naked-eye-visible and 365 nm UV lamp illumination color changes of THNE (5.0 mM Tb 3+ ) upon the addition of various metal ions (250 mM) in Fig. S2. † Aer adding Fe 3+ , the color of THNE changed from colorless to brown, and the green luminescence was quenched under a 365 nm UV lamp. However, on adding the other metal ions, we still observed obvious green luminescence.
To further illustrate the luminescence sensing property of THNE, a luminescence titration of THNE was executed by adding different concentrations of Fe 3+ . Luminescence spectra of THNE in the presence of different concentrations of Fe 3+ (0-300 mM) are shown in Fig. 5a. The luminescence spectra were obtained by excitation at 347 nm. The representative peak at 547 nm is shown in the luminescence spectrum of THNE. The primary emission peaks show a clear quenching behavior as Fe 3+ is added. With the concentration of Fe 3+ increasing, the luminescence intensity gradually decreased until quenched. When 300 mM of Fe 3+ was added to the THNE, the uorescence intensity at 547 nm was reduced by more than 90%. In Fig. 5a, the inset shows the ratio of the intensity (I) to the initial intensity (I 0 ), aer the addition of Fe 3+ . By tting the experimental data, an excellent curve relationship between I/I 0 and the concentration of Fe 3+ was obtained over the range from 0 mM to 300 mM. The curve equation is as follows: where a ¼ À0.054, b ¼ À0.011, and c ¼ 7.48 Â 10 À6 . These results demonstrate that THNE can be used as a sensor for Fe 3+ in aqueous systems based on a luminescence quenching response. The limit of detection (LOD) of Fe 3+ sensing was calculated using the following formula: S is the linear slope and s is the standard deviation of the intensity value of 10 blank samples. By linear tting at low concentrations of Fe 3+ (Fig. 5b), the results showed that the LOD is 1.66 mM. In order to visually observe different concentrations of Fe 3+ added into THNE, we took photos as shown in Fig. 5c. The luminescence properties of THNE are affected by ligandto-metal charge transfer (LMCT) states. 50 The O atom of hya was coordinated with Tb 3+ to form the Tb-O chemical bond, leading to the formation of the nano-poached egg structures ( Fig. 3f and  4a). With the addition of different metal ions (Ca 2+ , Mg 2+ , K + , Na + , Zn 2+ , Al 3+ , Mn 2+ , Cr 3+ , Ni 2+ , Cu 2+ , Co 2+ and Fe 3+ ), Tb 3+ , metal ions and ligands (acac, phen and hya) formed the balance of the coordination. The results of luminescence titration experiments ( Fig. 5d and S2 †) can be divided into three types. First, with the metal ions Ca 2+ , Mg 2+ , K + , and Na + , there were no luminescence responses. Compared with Tb 3+ , these metal ions have weak coordination ability with the N and O of ligands. They cannot affect the coordination of THNE. Second, with the metal ions Zn 2+ , Al 3+ , Mn 2+ , Cr 3+ , Ni 2+ , Cu 2+ and Co 2+ , the luminescence intensity was reduced. This is due to the stronger coordination ability of these metal ions than Tb 3+ with ligands. Third, the 300 mM concentration of Fe 3+ quenched the luminescence of THNE. The luminescence intensity gradually  until quenching occurred by the gradual replacement of Tb 3+ by Fe 3+ . 27,38 Based on the model of the Tb 3+ -ligands luminescent material, the Fe 3+ sensor mechanism can be summarized as follows: the coordination between Fe 3+ and ligands can alter the LMCT effect of ligands towards Tb 3+ . The stronger affinity of the ligands' N and O toward Fe 3+ reduces the efficiency of charge and energy transfer from ligands to Tb 3+ . This leads to a gradual decrease in the luminescence intensity until quenching occurs. To prove the complexation behavior of THNE with Fe 3+ , the uorescence lifetime of THNE with Fe 3+ (300 mM) is measured in Fig. 5e. The uorescence lifetime of THNE with Fe 3+ is 134.94 ms and the quantum yield is 1.19%. The uorescence lifetime of THNE with Fe 3+ is greatly weakened as compared to the value of THNE (235.83 ms) as shown in Fig. 2c. The values of s 1 0 (44.97 ms) and s 2 0 (152.12 ms) are both dramatically reduced as compared with s 1 (101.57 ms) and s 2 (266.18 ms) of THNE. These data indicate that on adding Fe 3+ to THNE, two other Tb 3+ complexes were obtained.
To prove the non-toxicity of THNE, the cell cytotoxicity experiments were conducted using human liver carcinoma cells, HepG2. Fig. 5f illustrates the culture medium (control), blank solvent ethanol-H 2 O (3 : 1, v/v) (B), 0.125 mg mL À1 hya (C1), 5.0 mM Tb(acac) 2 phen (C2) and different concentrations (S1-1.25 mM, S2-2.5 mM, S3-5.0 mM, S4-7.5 mM and S5-10.0 mM) of THNE on HepG2 cells incubated for 24 h. Toxicity is dened here as a 20% reduction in the metabolic activity measured by the MTT assay. From the gure given above, it can be seen that hya almost demonstrated nontoxicity on incubation with HepG2 cells. The sample of 5.0 mM Tb(acac) 2 phen is cytotoxic to HepG2 cells, exhibiting about a 35% reduction in cell viability. Furthermore, with the integration of the natural polysaccharide hya, the cell viability was signicantly improved. The cell viability was more than 80% aer incubation with different concentrations of THNE for 24 h, even at much higher concentrations. These results suggest that THNE is non-toxic to cells.

Conclusion
The original Tb 3+ -induced hyaluronic nano-poached eggs, which integrate the advantages of superior lanthanide luminescence, the unique shape of nano-poached eggs, compatibility with aqueous systems, and non-toxicity, were targeted toward the sensing of Fe 3+ in aqueous surroundings. The generation of the lanthanide-induced polysaccharide complexes is simple and environmentally friendly. The luminescence intensity of lanthanide complexes is inuenced not only by lanthanide-ligand coordination but also by the nanosized morphology. The advantageous properties of the luminescence sensors include high selectivity for Fe 3+ as compared to other cations, a fast response, bright and narrow luminescence emission bands, and suitability for use in aqueous systems. Future research will optimize lanthanide and polysaccharide-based nanoparticles for the detection of metal ions in aqueous and biological samples.

Conflicts of interest
The authors declare that they have no conict of interest.