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

Abstract

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.

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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.^11–15 The design and discovery of novel sensors for transition-metal ions in aqueous medium are of great concern.^16–20 Fe³⁺ is an essential micronutrient, which plays a critical role in basic physiological processes.^21–23 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 fixation, DNA synthesis, etc.^24–26 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 Fe³⁺ is an important research field.^27–32

Lanthanide complexes have bright and narrow emission bands of visible light, fast response times, long fluorescence lifetimes, and very low susceptibility to photobleaching.^33–35 The luminescence of lanthanide ions, such as Tb³⁺ is weak, in part due to its low photon efficiencies. However, lanthanides can be sensitized by complexing them with organic ligands in their first coordination sphere, resulting in the “antenna effect”. When complexed to organic ligands, the d–f transitions of Tb³⁺ 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.^38–41

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 N-acetylglucosamine. It is recognized as the best moisturizing ingredient and is widely used in food, medicine and cosmetics.^43–45 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 Tb³⁺ 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 Tb³⁺-induced hyaluronic nano-poached 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 Fe³⁺ 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 Fe³⁺ and ligands can alter the LMCT effect of ligands on Tb³⁺. These properties of THNE have the potential for application in environmental detection systems.

Experimental

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 (TbCl₃·6H₂O) 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.

Preparation of THNEs

THNE was synthesized with magnetic stirring in ethanol–H₂O (3 : 1, v/v). Tb(acac)₂phen was obtained by the complexation of TbCl₃·6H₂O, acetylacetone (acac) and 1,10-phenanthroline (phen). Here, 1 mL of different concentrations of TbCl₃·6H₂O (0.005 M, 0.01 M, 0.02 M, 0.03 M and 0.04 M) and 1 mL of different concentrations of acac (0.01 M, 0.02 M, 0.04 M, 0.06 M and 0.08 M) were mixed and stirred for 30 min at room temperature. Afterwards, the mixture was adjusted to pH 7–8 by using 0.25 M NaOH solution. Then, 1 mL of different concentrations of phen (0.005 M, 0.01 M, 0.02 M, 0.03 M and 0.04 M) was added to the solution and stirred for 2 h. The obtained Tb complexes were colorless and dissolved in ethanol. Next, 1 mL 0.5 mg mL⁻¹ hya was added to the Tb complexes and stirred for 2 h (Table 1). We also prepared Tb(acac)₂phen (5.0 mM, Tb³⁺). Tb(acac)₂phen and THNE with different concentrations were obtained. The schematic diagram of the synthesis of THNE is shown in Fig. 1.

Table 1 Preparation of THNEs with different concentrations of Tb³⁺, acac and phen (add 1 mL for each sample)

THNEs	Tb^3+	Acac	Phen	Hya
1.25 mM Tb^3+	0.005 M	0.01 M	0.005 M	0.5 mg mL^−1
2.5 mM Tb^3+	0.01 M	0.02 M	0.01 M	0.5 mg mL^−1
5.0 mM Tb^3+	0.02 M	0.04 M	0.02 M	0.5 mg mL^−1
7.5 mM Tb^3+	0.03 M	0.06 M	0.03 M	0.5 mg mL^−1
10.0 mM Tb^3+	0.04 M	0.08 M	0.04 M	0.5 mg mL^−1

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Fig. 1 Schematic diagram of the synthesis of THNE and the quenched effect by Fe³⁺.

Luminescence titration experiments

Here, 0.1 M metal chloride salt solutions (Ca²⁺, Mg²⁺, K⁺, Na⁺, Zn²⁺, Al³⁺, Mn²⁺, Cr³⁺, Ni²⁺, Cu²⁺, Co²⁺ and Fe³⁺) were prepared in water. THNE solution (5.0 mM, Tb³⁺) was prepared in ethanol–H₂O (3 : 1, v/v). For all measurements of luminescence spectra, the excitation wavelength was 347 nm and the emission slit widths were 5.0 nm. Luminescence titration experiments were performed using THNE solution (5.0 mM, Tb³⁺) with varying volumes of metal chloride salts. The concentrations of THNE and metal chloride salts were calculated from the luminescence titration experiments.

Cell cytotoxicity experiment

Human liver carcinoma cells HepG2 were incubated on a 96-well plate for 24 h. Culture medium, blank solvent ethanol–H₂O (3 : 1, v/v), hya, Tb(acac)₂phen (5.0 mM, Tb³⁺), 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 μL MTT solution. The liquid medium was removed, then 150 μL 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.

Characterization

Photoluminescence spectra were obtained using a fluorescence spectrophotometer (Hitachi Ltd., F-4500, Japan). The excitation and emission wavelengths were fixed at 347 nm and 547 nm. Fluorescence lifetimes and quantum yields were evaluated by a photoluminescence spectrometer (Edinburgh Instruments, FLS1000, UK). The IR spectra TbCl₃·6H₂O, acac, phen^.H₂O, hya, Tb(acac)₂phen, and THNE were evaluated by FTIR spectroscopy (Thermo Electron Scientific Instruments Corp., Nicolet iS5, USA). X-ray photoelectron spectra (XPS) of Tb(acac)₂phen, hya and THNE were obtained using a spectrometer (Thermo Fisher Scientific Inc., ESCALAB Xi+, USA). The morphology of THNE was characterized by transmission electron microscopy (TEM) (Hitachi Ltd., HT7700, Japan). The size of THNE was determined using a Malvern laser particle size analyzer (Malvern Instruments Ltd., Zetasizer Nano ZSP, UK). Photographs of THNE were obtained on illumination by a 365 nm UV lamp (SupFire Photoelectric Equipment Co. Ltd., UV03, China). Cytotoxicity experiments were conducted using a Multimode Reader (Tecan Group Ltd., SPARK 10M, Switzerland). Origin 7.0 software was used to present the experimental results of luminescence spectra, fluorescence lifetimes, FTIR analysis, DLS, XPS, MTT and luminescence response.

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 π → π* 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 Tb³⁺ were obtained with different concentrations of THNE (λ_em = 547 nm). With the increase in the concentrations of THNE (less than 5.0 mM Tb³⁺), the fluorescence intensity was gradually enhanced. However, when the concentrations were more than 5.0 mM Tb³⁺, the luminescence intensity presented the opposite tendency. When the concentration was 5.0 mM Tb³⁺, the luminescence intensity was the highest. If the concentration of Tb³⁺ was higher than 5.0 mM, the emission intensity of THNE was decreased. This is called concentration quenching, which is mainly due to a cross-relaxation (CR) process between Tb³⁺. This phenomenon widely occurs in Tb³⁺-induced luminescent materials, setting a limit in the concentration of Tb³⁺ to control the brightness.⁴⁷ Therefore, we selected THNE (5.0 mM Tb³⁺) as an excellent luminescent material.

Fig. 2 (a) Luminescence excitation spectra and emission spectra of THNE with different concentrations (λ_em = 547 nm, Table 1); (b) luminescence excitation spectra and emission spectra of Tb(acac)₂phen and THNE (λ_ex = 347 nm, λ_em = 547 nm); (c) fluorescence lifetime measurement of THNE; (d) fluorescence lifetime measurement of Tb(acac)₂phen.

Fig. 2b displays the luminescence excitation spectra of Tb(acac)₂phen and THNE (λ_em = 547 nm) and the luminescence emission spectra of Tb(acac)₂phen and THNE (λ_ex = 347 nm). THNE also contains the same characteristic emission bands. The luminescence emission spectra of Tb(acac)₂phen and THNE do not have significant differences in their respective emission wavelengths. These represent the band splitting character and ⁵D₄–⁷F₅ transition intensities, with those of known complex compounds of Tb³⁺ 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)₂phen, 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 fluorescence lifetime of THNE is approximately 235.83 μs and the quantum yield is 5.16% (Fig. 2c). The fluorescence lifetime of Tb(acac)₂phen is approximately 255.82 μs and the quantum yield is 6.38% (Fig. 2d). We also discuss the reasons after the experimental results of XPS and TEM analysis.

Fig. 3 (a) XPS of Tb(acac)₂phen; (b) high-resolution O 1s peak of Tb(acac)₂phen; (c) XPS of hya; (d) high-resolution O 1s peak of hya; (e) XPS of THNE; (f) high-resolution O 1s peak of THNE.

Fig. 4 (a and b) TEM images of THNE; (c) possible nano-poached egg-morphology of THNE; (d) size distribution of THNE by DLS.

Fig. S1† presents the FTIR spectra of TbCl₃, acac, phen, hya, Tb(acac)₂phen and THNE. As shown in the FTIR spectrum of acac, the peaks at 1732 and 1709 cm⁻¹ characterize the absorptions of the C=O stretching vibration. The significant peaks at 1304, and 1248 cm⁻¹ are due to stretching vibration from C–C. The peak at 1420 cm⁻¹ is assigned to –CH₂ bending vibration, and the –CH₃ bending vibration peak is observed at 1360 cm⁻¹. The peak at 1646 cm⁻¹ of phen is assigned to the C=N stretching vibration, and the C–N stretching vibration peaks are observed at 1140 and 1090 cm⁻¹. The significant peaks at 1616, 1590, 1506 and 1423 cm⁻¹ 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⁻¹ was due to the O–H stretching vibration. The trough at 1646 cm⁻¹ features the absorption of the C=O stretching vibration. The significant peak at 1400 cm⁻¹ is due to the bending vibration absorption from the O–H group. The peak at 1109 cm⁻¹ is assigned to the C–O–C stretching vibration of ethers.⁴⁸ Compared with the FTIR spectra of hya, Tb(acac)₂phen and THNE, the peaks at 1646 cm⁻¹ of the C=O stretching vibration, 1519 cm⁻¹ of the pyridine ring stretching vibration, and 1400 cm⁻¹ of O–H stretching vibration can be found in THNE. The above data indicate that THNE was prepared successfully.

To further confirm the interaction between the Tb complex and hya, XPS spectra were obtained as shown in Fig. 3. Tb(acac)₂phen, 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)₂phen representing the O 1s. The peak at 398.87 eV is related to the N 1s of Tb(acac)₂phen. The high-resolution 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)₂phen interacted with hya.⁴⁹ Thus, Tb(acac)₂phen 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 after 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 film (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)₂phen cores with the hya surface. Interestingly, this morphology is very similar to the nano-poached eggs (Fig. 4c). The boiled egg white (hya) wraps its egg yolk (Tb(acac)₂phen). 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 film formation. These core–shell nanoparticles were formed during the coordination of Tb(acac)₂phen and hya when the samples were stirred at high speed in the ethanol–H₂O (3 : 1, v/v) system. A constant magnetic stirring of 1500 rpm was maintained for 2 h. The complexation of Tb(acac)₂phen and hya was proved by XPS (Fig. 3). However, as we know, when Tb³⁺ 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)₂phen. Luminescence data show that the luminescence intensity of THNE is slightly weakened as compared to the luminescence intensity of Tb(acac)₂phen (Fig. 2b). The luminescence intensity of lanthanide complexes is influenced not only by lanthanide–ligand coordination but also the morphology. The Tb³⁺ complex is wrapped by hya, which is similar to putting a non-luminous coat on the green phosphor. Although the coordination between Tb(acac)₂phen and hya enhances the luminescence, the outer film 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)₂phen, and the fluorescence lifetime of THNE (235.83 μs) is shorter than that of Tb(acac)₂phen (255.82 μs).

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 Ca²⁺, Mg²⁺, K⁺, Na⁺, Zn²⁺, Al³⁺, Mn²⁺, Cr³⁺, Ni²⁺, Cu²⁺, Co²⁺ and Fe³⁺ in aqueous solution on the luminescence emission of THNE were investigated. Fig. 5d shows the luminescence response of THNE with the addition of 10 μL (250 μM in THNE) of various metal ions. I₀ and I are the luminescence intensities of THNE in0 μL (0 μM) and 10 μL (250 μM in THNE) of different metal ions. THNE is much more sensitive to Fe³⁺ than to the other cations. When Fe³⁺ (250 μM) was added to the THNE, the luminescence intensity at 547 nm was reduced by more than 90%. When 10 μL Ca²⁺, Mg²⁺, K⁺, and Na⁺ were added, the luminescence intensity remained the same. The THNE retained more than 80% of the luminescence in 10 μL Zn²⁺, Al³⁺, and Mn²⁺. When 10 μL Cr³⁺, Ni²⁺, Cu²⁺, and Co²⁺, were added, more than 40% of the luminescence intensity was retained. Among these various metal ions, THNE shows a selective luminescence response to Fe³⁺ 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 Tb³⁺) upon the addition of various metal ions (250 μM) in Fig. S2.† After adding Fe³⁺, 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.

Fig. 5 (a) Luminescence emission spectra of THNE in the presence of different concentrations of Fe³⁺; inset: the plot of I/I₀ versus the concentration of Fe³⁺, where I₀ and I are the luminescence intensities of THNE in 0 μM and the various concentrations of Fe³⁺. (b) The plot of 1 − I/I₀ versus the concentration of Fe³⁺. (c) Photographs of THNE with different concentrations of Fe³⁺ (A-0 μM, B-25 μM, C-50 μM, D-75 μM, E-100 μM, F-150 μM, G-200 μM, H-250 μM and I-300 μM) under the naked eye and with 365 nm UV lamp illumination. (d) Luminescence response of THNE to various metal ions with 250 μM; I₀ and I are the luminescence intensities of THNE in 0 μM and 250 μM of Ca²⁺, Mg²⁺, K⁺, Na⁺, Zn²⁺, Al³⁺, Mn²⁺, Cr³⁺, Ni²⁺, Cu²⁺, Co²⁺ and Fe³⁺, respectively (λ_ex = 347 nm, λ_em = 547 nm). (e) Fluorescence lifetime measurement of THNE with Fe³⁺. (f) Cell viability after incubation with culture medium (control), blank solvent ethanol–H₂O (3 : 1, v/v) (B), control sample 1 hya (C1), control sample 2 Tb(acac)₂phen (5.0 mM, Tb³⁺) (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 the THNE for 24 h.

To further illustrate the luminescence sensing property of THNE, a luminescence titration of THNE was executed by adding different concentrations of Fe³⁺. Luminescence spectra of THNE in the presence of different concentrations of Fe³⁺ (0–300 μM) 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 Fe³⁺ is added. With the concentration of Fe³⁺ increasing, the luminescence intensity gradually decreased until quenched. When 300 μM of Fe³⁺ was added to the THNE, the fluorescence 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 (I₀), after the addition of Fe³⁺. By fitting the experimental data, an excellent curve relationship between I/I₀ and the concentration of Fe³⁺ was obtained over the range from 0 μM to 300 μM. The curve equation is as follows:

y = e^a+bx+cx2

where a = −0.054, b = −0.011, and c = 7.48 × 10⁻⁶. These results demonstrate that THNE can be used as a sensor for Fe³⁺ in aqueous systems based on a luminescence quenching response. The limit of detection (LOD) of Fe³⁺ sensing was calculated using the following formula:

LOD = 3σ/S

S is the linear slope and σ is the standard deviation of the intensity value of 10 blank samples. By linear fitting at low concentrations of Fe³⁺ (Fig. 5b), the results showed that the LOD is 1.66 μM. In order to visually observe different concentrations of Fe³⁺ added into THNE, we took photos as shown in Fig. 5c. The pictures illustrates that the THNE sample is colorless to the naked eye and exhibits bright green luminescence under the 365 nm UV lamp illumination. After adding different concentrations (A-0 μM, B-25 μM, C-50 μM, D-75 μM, E-100 μM, F-150 μM, G-200 μM, H-250 μM and I-300 μM) of Fe³⁺, the luminescence intensity decreased gradually until quenched.

The luminescence properties of THNE are affected by ligand-to-metal charge transfer (LMCT) states.⁵⁰ The O atom of hya was coordinated with Tb³⁺ 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 (Ca²⁺, Mg²⁺, K⁺, Na⁺, Zn²⁺, Al³⁺, Mn²⁺, Cr³⁺, Ni²⁺, Cu²⁺, Co²⁺ and Fe³⁺), Tb³⁺, 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 Ca²⁺, Mg²⁺, K⁺, and Na⁺, there were no luminescence responses. Compared with Tb³⁺, 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 Zn²⁺, Al³⁺, Mn²⁺, Cr³⁺, Ni²⁺, Cu²⁺ and Co²⁺, the luminescence intensity was reduced. This is due to the stronger coordination ability of these metal ions than Tb³⁺ with ligands. Third, the 300 μM concentration of Fe³⁺ quenched the luminescence of THNE. The luminescence intensity gradually decreased until quenching occurred by the gradual replacement of Tb³⁺ by Fe³⁺.^27,38 Based on the model of the Tb³⁺–ligands luminescent material, the Fe³⁺ sensor mechanism can be summarized as follows: the coordination between Fe³⁺ and ligands can alter the LMCT effect of ligands towards Tb³⁺. The stronger affinity of the ligands' N and O toward Fe³⁺ reduces the efficiency of charge and energy transfer from ligands to Tb³⁺. This leads to a gradual decrease in the luminescence intensity until quenching occurs. To prove the complexation behavior of THNE with Fe³⁺, the fluorescence lifetime of THNE with Fe³⁺ (300 μM) is measured in Fig. 5e. The fluorescence lifetime of THNE with Fe³⁺ is 134.94 μs and the quantum yield is 1.19%. The fluorescence lifetime of THNE with Fe³⁺ is greatly weakened as compared to the value of THNE (235.83 μs) as shown in Fig. 2c. The values of τ₁′ (44.97 μs) and τ₂′ (152.12 μs) are both dramatically reduced as compared with τ₁ (101.57 μs) and τ₂ (266.18 μs) of THNE. These data indicate that on adding Fe³⁺ to THNE, two other Tb³⁺ 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–H₂O (3 : 1, v/v) (B), 0.125 mg mL⁻¹ hya (C1), 5.0 mM Tb(acac)₂phen (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 defined here as a 20% reduction in the metabolic activity measured by the MTT assay. From the figure given above, it can be seen that hya almost demonstrated nontoxicity on incubation with HepG2 cells. The sample of 5.0 mM Tb(acac)₂phen 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 significantly improved. The cell viability was more than 80% after 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 Tb³⁺-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 Fe³⁺ in aqueous surroundings. The generation of the lanthanide-induced polysaccharide complexes is simple and environmentally friendly. The luminescence intensity of lanthanide complexes is influenced not only by lanthanide–ligand coordination but also by the nanosized morphology. The advantageous properties of the luminescence sensors include high selectivity for Fe³⁺ 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 conflict of interest.

Acknowledgements

This work is financially supported by Natural Science Foundation of China (51801110), Shandong Province Natural Science Foundation (No. ZR2019BD061, SZJ71901CZ, ZR2019PD004, ZR2021QF120), Academy Local Cooperation Fund of Shandong Academy of Sciences (2018CXY-31, 2018CXY-32, 2019-CXY1), Foundation of Shandong Academy of Sciences (2019QN0036). The authors would like to thank Shiyanjia Lab (https://www.shiyanjia.com) for the XPS and MTT measurements.

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Footnote

† Electronic supplementary information (ESI) available. See https://doi.org/10.1039/d2ra03871d

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