Synthesis and design of terbium-activated CaWO₄ nanocrystals for fluorescence sensing of alkaline phosphatase and bioimaging applications

Abstract

Calcium tungstate nanocrystals CaWO₄ NCs represent an important family of inorganic oxides and are well-known for their rare-earth (RE) sensitizing properties. Although RE-doped CaWO₄ NCs have been extensively used in the optical field, their applications as a fluorescent probe is yet to be explored. In this report, inspired by their excellent luminescence properties, we demonstrate the potential of terbium-activated CaWO₄ NCs as a fluorescent probe for sensing and biological imaging applications. In our study, we successfully synthesized nano-sized water dispersible PEG coated Tb–CaWO₄ NCs via a facile one-step hydrothermal route. Next, the potential of PEG–Tb–CaWO₄ NCs as a fluorescent sensing probe was evaluated by employing a “signal-off–on” sensing strategy for the detection of a key disease biomarker (alkaline phosphatase, ALP). The proposed detection system realized sensitive detection of ALP with a low limit of detection of 0.5 U L⁻¹. Moreover, the cell imaging potential of PEG–Tb–CaWO₄ NCs was investigated using HeLa cells. PEG–Tb–CaWO₄ NCs were internalized by HeLa cells through endocytosis and showed characteristic bright green emission, demonstrating the cell imaging potential of the as-synthesized NCs. Also, the viability study confirmed the very low cell cytotoxicity of PEG–Tb–CaWO₄ NCs to HeLa cells. Overall, we highlighted the potential of PEG–Tb–CaWO₄ NCs as a promising fluorescence probe for sensing and biological imaging applications.

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1. Introduction

In the past decade, rare-earth activated nanoparticles (RE-NPs) have received extensive attention in fluorescence sensing applications.^1,2 This is due to their unique features and exceptional optical properties including tunable emission wavelengths, excellent photostability, large anti-Stokes shifts, and long lifetimes (milliseconds to microseconds).^3–5 Another interesting characteristic of RE-NPs is the host–lattice sensitization, where fluorescence occurs due to the resonant energy transfer from a host-sensitizer to a RE-activator dopant such as terbium (Tb³⁺), europium (Eu³⁺), ytterbium (Yb³⁺), gadolinium (Gd³⁺), or cerium (Ce³⁺), whose relaxation produces unique activator-dependent emission.⁶ Since RE-ions are poor in direct light absorption,^7,8 their luminescence efficiency highly depends on the efficiency of photon absorption by the host-sensitizer and resonance energy transfer to the luminescence center. Hence, careful selection of the host material is essential in designing RE-NPs with desired optical properties.

Among the wide range of RE-host materials, CaWO₄ NCs have proven to be appealing due to their excellent optical, chemical, and physical properties.^9,10 CaWO₄ NCs are built from Ca²⁺ ions and [WO₄]²⁻ luminescent groups with a coordination number of eight for Ca²⁺ and four for W⁶⁺.¹¹ They are also known for their self-activation properties, large quantum yields, and strong absorption in the UV-visible region.¹⁰ Moreover, new optical properties of CaWO₄ NCs can be obtained by doping with RE-metal ions. RE-doped CaWO₄ NCs show characteristic luminescence of RE-ions due to the efficient energy transfer from the host [WO₄]²⁻ group to the RE-luminescent center. Owing to their intriguing luminescent properties, RE-CaWO₄ NCs have been extensively studied for application in optical devices.¹²

Although RE-CaWO₄ NCs exhibit excellent optical properties and high chemical stability, their potential as a fluorescent sensing probe has rarely been reported. This might be related to the following major impediments. Firstly, traditional RE-CaWO₄ NC synthesis procedure usually involves solid-state annealing at extremely high temperatures (>500 °C), which makes it energy-intensive.^13–16 Secondly, the conventional RE-CaWO₄ NC synthesis procedure mainly results in large (micron-size) and agglomerated nanocrystals with the lack of surface tunability¹⁶ and hence they are not ideal for biological applications. Thirdly, CaWO₄ NCs show poor solubility in aqueous and physiological solutions and this further limits their solution-based sensing applications.^17,18 Hence, the above listed problems need to be resolved for enabling the practicality of this material.

In this work, we for the first time demonstrated the potential of Tb–CaWO₄ NCs as a fluorescent probe for sensing and biological imaging applications. Scheme 1 presents the synthesis procedure and applications of Tb–CaWO₄ NCs. In our synthesis procedure, nano-sized Tb–CaWO₄ NCs were synthesized by employing a facile one-step hydrothermal method at mild temperature (140 °C). Also, to increase the aqueous solubility of Tb–CaWO₄ NCs, a biocompatible hydrophilic polymer (polyethylene glycol, PEG-400) was used as a surface coating ligand. Next, the potential of PEG–Tb–CaWO₄ NCs as a fluorescent probe was examined by designing a “signal-off–on” sensing strategy for detecting the ALP biomarker. ALP is widely distributed in various organs of the human body and its abnormal level in serum could be associated with various diseases.^19,20 Hence, the development of a sensitive and selective ALP detection method helps diagnosis of many diseases. For this purpose, our PEG–Tb–CaWO₄-based probe system showed excellent sensitivity in both buffer and real serum samples. Moreover, the cell imaging potential of PEG–Tb–CaWO₄ NCs was investigated using HeLa cells. HeLa cells internalized PEG–Tb–CaWO₄ NCs through endocytosis and showed characteristic bright green emission, proving the cell imaging capability of PEG–Tb–CaWO₄ NCs. The viability study also confirmed the very low cell cytotoxicity of PEG–Tb–CaWO₄ NCs to HeLa cells.

Scheme 1 Schematic diagram showing (A) synthesis and (B) fluorescence sensing and cell imaging applications of PEG–Tb–CaWO₄ NCs.

2. Experimental section

2.1. Chemicals and reagents

Tris-hydrochloride buffer (Tris–HCl), L-ascorbic acid, and L-ascorbic acid 2-phosphate tri-sodium salt (AAP) were purchased from Aladdin Reagent Co., Ltd (Shanghai, China). Polyethylene glycol (MW, 400), bovine serum albumin (BSA), human serum albumin (HSA), alkaline phosphatase from bovine intestinal mucosa (ALP), sodium chloride (99.99%), sodium tungstate dehydrate (99.9%), and terbium(III) nitrate hexahydrate (99.9%) were purchased from Sigma-Aldrich Co., Ltd (Shanghai, China). Ammonium cerium(IV) sulfate dehydrate (99.5%), calcium chloride (99.9%), glucose oxidase (GOx), cysteine (Cys), and glutathione (GSH) were provided by Macklin Biochemical Technology Co., Ltd (Shanghai, China). Milli-Q water was used to prepare aqueous solutions. All reagents used were of analytical grade and used without any further purification.

2.2. Instruments

UV-visible absorption spectra were recorded using a UV-Vis spectrometer (UV-3600i, Shimadzu, Japan). X-ray diffraction patterns were acquired using an X-ray diffractometer (D/Max 2400 X-ray powder diffractometer, Cu Kα radiation). Fluorescence measurements were performed on a Shimadzu RF-6000 fluorescence spectrophotometer. High resolution transmission electron microscopy (HR-TEM) images and energy-dispersive spectra (EDS) were recorded on a JEOL JEM-2100F field-emission high-resolution transmission electron microscope. The surface chemical composition of the nanomaterial was analyzed using X-ray photoelectron spectrophotometry (XPS, Thermo Scientific K-Alpha). Thermal gravimetric analysis was performed using a thermogravimetric analyzer (TGA, TG 209 F3). A Nicolet Nexus 670 FTIR spectrometer was used to obtain Fourier transform infrared spectra. The fluorescence lifetime was obtained on an FLS1000 fluorescence spectrometer.

2.3. Synthesis of PEG–Tb–CaWO₄ NCs

In a typical synthesis procedure, aqueous solutions of CaCl₂ (1 mL, 0.173 mmol) and terbium nitrate (0.2 mL, 0.026 mmol) were mixed with 10 mL of polyethylene glycol and stirred for 5 min at room temperature. Then, an aqueous solution of sodium tungstate (1 mL, 0.199 mmol) was quickly injected into the above mixture and vigorously stirred for another 5 min. The mixture was then transferred into a 20 mL Teflon-lined autoclave and then heated in an oven for 5 h at 140 °C. After cooling to room temperature, the white-milky product was collected by centrifugation at 3500 rpm for 5 min. Then, through consecutive re-dispersion in water (via sonication for 5 min) and centrifugation, the as-obtained product was washed several times (at least 3 times) to remove unreacted substances and impurities. Finally, the purified product was re-dispersed in water via sonication and occasional shaking for 10 minutes and stored as a 5 mg mL⁻¹ stock solution.

2.4. Detection of ALP

In a typical procedure, 54 μL of AAP (10 mM) and various concentrations of ALP were mixed with 100 μL of Tris–HCl buffer solution (10 mM, pH 8.5) and incubated for 60 min at 37 °C. Then, 88 μL of aqueous solution of Ce⁴⁺ ions (10 mM) was added into the mixture and stored for another 5 min. Subsequently, 60 μL of PEG–Tb–CaWO₄ NCs (5 mg mL⁻¹) was added and the final volume was made to 3 mL with ultrapure water. After storing the mixture for another 5 min at room temperature, the fluorescence spectra were measured at excitation and emission wavelengths of 262 nm and 546 nm, respectively. The same procedure was applied to detect ALP in 10-fold diluted serum samples spiked with various amounts of ALP.

2.5. In vitro cytotoxicity assay

The cytotoxicity of PEG–Tb–CaWO₄ NCs was evaluated by the methyl thiazolyl tetrazolium (MTT) reduction assay using HeLa cells cultured in Dulbecco's modified Eagle's medium (DMEM) supplemented with 10% FBS at 37 °C and 5% CO₂ in a humidified atmosphere. In a typical procedure, HeLa cells were cultured at 5000 cells per well in a 96-well plate for 12 h. Subsequently, cells were incubated with various concentrations of Tb–CaWO₄ NCs in a 5% CO₂ atmosphere at a constant temperature of 37 °C for 24 h. Finally, cell viabilities were determined by CCK-8 according to the manufacturer's protocol.

2.6. Biological imaging of HeLa cells

HeLa cells were cultured in humidified 5% CO₂ for 24 h with DMEM supplemented with 10% FBS. Then, the cultured HeLa cells were transferred to a four-well chambered slide and further incubated for 24 h. Next, the adherent cells were incubated with 100 μg mL⁻¹ PEG–Tb–CaWO₄ NCs for 24 h. After washing the adherent cells (3 times) with fresh PBS solution, the cells were observed under a confocal laser scanning microscope (CLSM).

3. Results and discussion

3.1. Synthesis and characterization of Tb–CaWO₄ NCs

In this study, a solvothermal synthesis approach was employed to prepare PEG coated Tb–CaWO₄ NCs. During the synthesis approach, the effect of some of the experimental factors was optimized to obtain the desired product. For instance, Fig. S1A (ESI†) presents the effect of synthesis temperature on the fluorescence intensity of PEG–Tb–CaWO₄ NCs. The PEG–Tb–CaWO₄ NCs with higher fluorescence intensity were obtained at a heating temperature of 140 °C. Beyond this temperature, the fluorescence intensity started declining, which could be due to cross-relaxation among Tb³⁺ ions resulting from the agglomeration of particles at higher temperature.^21,22 Also, the PEG–Tb–CaWO₄ NCs synthesized at lower temperature appeared as a milky white dispersion, while those prepared at higher temperature appeared dark brown (Fig. S1A, inset, ESI†). This could be due to decomposition of surface PEG molecules at higher temperature, which could also result in a decrease in the fluorescence emission intensity of the as-synthesized PEG–Tb–CaWO₄ NCs.^23,24 Hence, a synthesis temperature of 140 °C was used to prepare the NCs as it enabled the highest fluorescence emission intensity. Next, the effect of synthesis time was optimized. As presented in Fig. S1B (ESI†), a synthesis time of 5 h gave the highest florescence intensity and it was taken as an optimum value. The effect of %Tb³⁺ ion doping on photoluminescence response was studied and the results are presented in Fig. S2A (ESI†). Based on the fluorescence intensity response, 15% doping showed the highest fluorescence response and it was taken as an optimum. Moreover, the effect of various amounts of PEG in the reaction mixture on the colloidal stability of Tb–CaWO₄ NCs was studied. As presented in Fig. S2B (ESI†), Tb–CaWO₄ NCs showed higher colloidal stability when the amount of PEG was higher than 50%.

Next, the morphology and particle size of the as-synthesized PEG–Tb–CaWO₄ NCs were characterized by HR-TEM. The PEG–Tb–CaWO₄ NCs showed well-dispersed quasi-spherical particles (Fig. 1A) and size distribution in the range of 6 to 22 nm with an average size of 13 nm (Fig. 1B). Also, the HR-TEM image in Fig. 1C illustrates the lattice fringes with a characteristic spacing of the lattice surface of 0.307 nm corresponding to the (103) crystallographic plane of PEG–Tb–CaWO₄ NCs (JCPDS Card No. 41-1431). Fig. 1D presents the selected area electron diffraction (SAED) image of PEG–Tb–CaWO₄ NCs. The SAED image reveals dense diffraction patterns arranged into rings forming continuous concentric circles. This indicates the polycrystalline nature of the as-synthesized PEG–Tb–CaWO₄ NCs, which is also in good agreement with HR-TEM results (Fig. S3, ESI†). Moreover, the elemental mapping results (Fig. 1E–H) show the presence of calcium, terbium, tungsten and oxygen. More importantly, the results confirmed the successful doping of terbium ions into the crystal structure of CaWO₄ NCs. The successful doping of Tb³⁺ ions was further proved by the presence of a Tb-peak in the EDS spectra (Fig. S4, ESI†).

Fig. 1 (A) HR-TEM image of PEG–Tb–CaWO₄ NCs. (B) Corresponding size distribution. (C) HR-TEM image showing lattice spacing. (D) SAED image. (E)–(H) Elemental mapping of W, O, Ca, and Tb, respectively.

To understand the crystal structure of the as-synthesized PEG–Tb–CaWO₄ NCs, X-ray diffraction patterns were recorded. As presented in Fig. S5 (ESI†), PEG–Tb–CaWO₄ NCs exhibit diffraction patterns similar to the standard data of CaWO₄ NCs (JCPDS Card No. 41-1431).²⁵ Both uncoated and PEG coated Tb–CaWO₄ NCs (Fig. 2A) showed identical diffraction patterns with four intense diffraction peaks at 2θ values of 28.7, 47.1, 57.8, and 76.1°, which, respectively, belong to the (112), (204), (312) and (208) planes (JCPDS no. 41-1431).²⁵ From the results, it can be concluded that both the doping of Tb³⁺ ions and surface coating with PEG molecules had no or little effect on the crystal nature of CaWO₄ NCs.

Fig. 2 (A) XRD patterns and (B) FT-IR spectra of uncoated and PEG coated Tb–CaWO₄ NCs.

Furthermore, the FT-IR, XPS, and TGA measurements were performed to understand the surface chemical composition of Tb–CaWO₄ NCs. In the FT-IR spectra (Fig. 2B), both uncoated and PEG coated Tb–CaWO₄ NCs showed strong absorption bands at about 800 cm⁻¹, characteristic of the anti-symmetric stretching vibrations of O–W–O within the WO₄ tetrahedron.²⁶ The strong absorption bands at about 3600–3000 cm⁻¹ could be assigned to the O–H stretching vibration from PEG molecules in coated Tb–CaWO₄ NCs or physically adsorbed water molecules in uncoated Tb–CaWO₄ NCs. The coating of PEG molecules on the surface of Tb–CaWO₄ NCs was confirmed by the appearance of additional peaks of CH₂ stretching (2910 cm⁻¹), CH₂ bending/scissoring (1595 cm⁻¹), and C–OH stretching (1016 cm⁻¹) vibrations.²⁷Fig. 3A–F shows the XPS spectra of the as-synthesized PEG–Tb–CaWO₄ NCs. The survey XPS results confirmed the presence of calcium (Ca), tungsten (W), terbium (Tb), carbon (C), and oxygen (O) elements. More importantly, the appearance of the carbon peak proved the PEG surface coating on PEG–Tb–CaWO₄ NCs. Also, the two peaks at 287.68 and 284.73 eV in the deconvoluted C 1s spectrum (Fig. 3E) can be assigned to the C–O and C–C/C–H of surface PEG molecules,^28,29 respectively. Moreover, the presence of a C–O peak (531.6 eV) in the high resolution XPS spectrum of O 1s (Fig. 3F) confirmed the presence of PEG molecules on the surface of PEG coated Tb–CaWO₄ NCs. Finally, TGA analysis was performed to further confirm the coating of PEG molecules on the surface of PEG–Tb–CaWO₄ NCs. As presented in Fig. S6 (ESI†), due to the decomposition of surface PEG molecules, the PEG coated Tb–CaWO₄ NCs exhibited a higher decomposition rate than that of uncoated Tb–CaWO₄ NCs. This result, along with FT-IR and XPS results, strongly confirmed the coating of PEG molecules on PEG–Tb–CaWO₄ NCs. The presence of PEG molecules on the surface of PEG–Tb–CaWO₄ NCs significantly improved the colloidal stability, while uncoated PEG–Tb–CaWO₄ NCs aggregated within a few minutes of dispersion (Fig. S7, ESI†).

Fig. 3 (A) XPS survey spectrum of PEG–Tb–CaWO₄ NCs. High resolution spectra of (B) Ca 2p, (C) W 4f, (D) Tb 3d, (E) C 1s, and (F) O 1s.

3.2. Optical properties of PEG–Tb–CaWO₄ NCs

The UV-visible absorption spectra of CaWO₄ and PEG–Tb–CaWO₄ NCs are presented in Fig. 4A. The CaWO₄ NCs showed a maximum absorption peak at 246 nm, which is attributed to the O-to-W charge transfer transition within the [WO₄]²⁻ group.^30,31 PEG–Tb–CaWO₄ NCs showed a maximum absorbance at 254 nm with an additional broad absorption peak in the wavelength range of 260–370 nm, which could be attributed to the f–f electronic transitions of Tb³⁺ ions.³² It can be noted that the absorption edge of PEG–Tb–CaWO₄ NCs red-shifted by about 8 nm in comparison to CaWO₄ NCs. This could be due to the occurrence of energy transfer from the host [WO₄]²⁻ group to Tb³⁺ ions as has been reported elsewhere.^32,33 The band gaps of CaWO₄ and PEG–Tb–CaWO₄ NCs computed according to the Tauc model³⁴ were 4.5 and 4.2 eV (Fig. 4B), respectively.

Fig. 4 (A) UV-visible absorption spectra of CaWO₄ and PEG–Tb–CaWO₄ NCs. (B) Corresponding spectra showing the Tauc plot curve for calculating the energy band gap.

Fig. 5A depicts the possible excitation, emission, and energy transfer processes in CaWO₄ and PEG–Tb–CaWO₄ NCs. The photoluminescence emission spectrum of undoped CaWO₄ consists of a strong and broad band from 300 to 600 nm, with the maximum emission at 415 nm (Fig. S8, ESI†). This is due to O-to-W charge transfer transitions within the [WO₄]²⁻ group.^35,36 The blue luminescence of solid CaWO₄ NCs was also demonstrated using UV-light at 254 nm (Fig. S8, inset, ESI†) and the chromaticity coordinates of x = 0.1472 and y = 0.0413 (Fig. S9A, ESI†). Upon the introduction of Tb³⁺ ions, the luminescence properties of CaWO₄ NCs changed due to the energy transfer from the [WO₄]²⁻ group to luminescent Tb³⁺ ions as schematically shown in Fig. 5A and B. This was proved by the presence of a strong O-to-W charge transfer transition band in the excitation spectrum of PEG–Tb–CaWO₄ NCs at an emission wavelength of 547 nm (Fig. 5C).³⁷ In the photoluminescence emission spectrum of PEG–Tb–CaWO₄ NCs, the wide and broad band of the host CaWO₄ almost disappeared with the appearance of sharp and narrow bands and characteristic green luminescence in both solid powder and aqueous dispersion (Fig. 5D and insets). The emission spectrum of PEG–Tb–CaWO₄ NCs consists of four distinct bands at wavelengths of 470 (⁵D₄ → ⁷F₆), 546 (⁵D₄ → ⁷F₅), 588 (⁵D₄ → ⁷F₄), and 622 nm (⁵D₄ → ⁷F₃),³⁸ among which the ⁵D₄ → ⁷F₅ transition dominated and resulted in a characteristic vibrant green luminescence. Also, the calculated chromaticity coordinates (x = 0.3192, y = 0.5908) were in the green region of the CIE diagram (Fig. S9B, ESI†).

Fig. 5 (A) and (B) Schematic representation of possible excitation, emission, and energy transfer within CaWO₄ and PEG–Tb–CaWO₄ NCs. (C) Excitation spectra at an emission wavelength of 547 nm. (D) Fluorescence emission spectra at an excitation wavelength of 262 nm of PEG–Tb–CaWO₄ NCs. Insets show the corresponding luminescence of solid powder and aqueous dispersion under a UV-lamp.

3.3. Stability of PEG–Tb–CaWO₄ NCs

The stability of a nanomaterial is a key determining factor for its practical sensing applications. In this regard, the luminescence stability of PEG–Tb–CaWO₄ NCs against ionic concentration, storage and pH was examined. From the fluorescence intensity response (Fig. S10A, ESI†), it can be observed that PEG–Tb–CaWO₄ NCs demonstrated excellent stability against various ionic concentrations. Also, the fluorescence intensity of PEG–Tb–CaWO₄ NCs showed excellent storage stability (Fig. S10B, ESI†). Moreover, as we were intended to employ PEG–Tb–CaWO₄ NCs as a fluorescence probe for the detection of ALP enzyme, which requires alkaline conditions, its fluorescence emission stability in alkaline medium was also investigated. Interestingly, the excellent fluorescence intensity of PEG–Tb–CaWO₄ NCs in the pH range from 7.0 to 9.5 proved its potential for application in alkaline medium (Fig. S10C, ESI†).

3.4. Designing a PEG–Tb–CaWO₄ NC based sensor for ALP detection

To test the fluorescence sensing potential of PEG–Tb–CaWO₄ NCs, a “signal-off–on” sensing strategy was proposed to detect ALP (Scheme 2A). In the detection system, Ce⁴⁺ ions were employed as an effective fluorescence signal quencher. From Fig. S11 (ESI†), the fluorescence emission of the PEG–Tb–CaWO₄ probe significantly decreased with increasing concentrations of Ce⁴⁺ ions, demonstrating their effectiveness as a fluorescence quencher. However, as presented in Scheme 2B, ALP can dephosphorylate its substrate AAP into AA and phosphate ions. The in situ generated AA undergoes a redox reaction with Ce⁴⁺ to produce Ce³⁺ ions (eqn (S1), ESI†),³⁸ which then combine with phosphate ions to form a non-quenching CePO₄ precipitate, hence resulting in fluorescence recovery.

Scheme 2 Schematic diagram showing (A) fluorescence responses with and without target ALP and (B) possible reactions in the detection system.

Fig. 6A and the inset present the quenched and recovered fluorescence emission of the detection system. To understand the fluorescence quenching process in the PEG–Tb–CaWO₄/Ce⁴⁺ system, the involvement of various possible mechanisms was examined. First, the involvement of static quenching was investigated. One of the rapid identification techniques for static quenching is a change in the absorption spectrum of the fluorophore due to the formation of a fluorophore–quencher complex.^39,40 However, there is no noticeable change in the absorption of PEG–Tb–CaWO₄ after the addition of Ce⁴⁺ ions (Fig. S12, ESI†) and hence the involvement of static quenching can be ruled out. But it should be noted that the UV-visible absorption spectrum of Ce⁴⁺ ions significantly overlapped with the excitation spectrum of PEG–Tb–CaWO₄ NCs (Fig. S13, ESI†). This suggests the involvement of energy transfer (FRET) or inner filter effect (IFE) quenching, which leads to disruption of energy transfer from [WO₄]²⁻ to Tb³⁺ ions and hence results in fluorescence quenching. To identify which one occurred, we measured the lifetimes of the PEG–Tb–CaWO₄ NCs with and without the addition of Ce⁴⁺ ions (Fig. 6B). For the IFE, the quencher did not cause a change in average lifetime (τ₀/τ = 1), while the lifetime was expected to change in the resonance energy transfer process (τ₀/τ ≠ 1).⁴¹ From the lifetime measurement results, the average lifetimes of PEG–Tb–CaWO₄ and PEG–Tb–CaWO₄/Ce⁴⁺ were found to be 1.14 and 0.98 ms, respectively. The result indicated that the addition of Ce⁴⁺ did not lead to a significant change in the lifetime of PEG–Tb–CaWO₄ NCs, and hence the quenching mechanism could be the IFE rather than FRET.

Fig. 6 (A) Fluorescence responses of the detection system with and without target ALP. (B) Fluorescence decay lifetime curves of PEG–Tb–CaWO₄ and Tb–CaWO₄/Ce⁴⁺ (τ_av = 1.14 and 0.93 ms, respectively).

3.5. Analytical performance of the PEG–Tb–CaWO₄ NC based sensor

To obtain the best analytical performance, the effects of various experimental conditions such as pH, the concentration of AAP, enzymatic reaction time, and the concentration of Ce⁴⁺ ions were optimized. As alkaline phosphatase enzyme performs well in alkaline conditions,⁴² a pH higher than 7.0 was adopted. Also, a pH > 9.0 was not considered since Ce⁴⁺ displayed low quenching efficiency beyond this pH (Fig. S14A, ESI†). Moreover, ALP exhibited higher activity at a pH of 8.5 (Fig. S14B, ESI†) and thus this pH was used for the following experiments. In addition, the detection system showed better fluorescence recovery at an AAP concentration of 180 μM and an enzymatic reaction time of 60 min (Fig. S14 C and D, ESI†). Moreover, the amounts of PEG–Tb–CaWO₄ NCs were optimized and 100 μg mL⁻¹ was taken as an optimum (Fig. S14E, ESI†). Lastly, as 260 μM of Ce⁴⁺ ions (1.625 times higher) were required to completely oxidize 160 μM of AA (Fig. S15, ESI†), and assuming complete conversion of 180 μM of AAP into AA, 292 μM of Ce⁴⁺ ions (180 × 1.625) was considered as an optimum concentration.

Next, the analytical performance of the proposed sensor for the detection of ALP was evaluated. As presented in Fig. 7A, the fluorescence intensity of the detection system gradually increased with an increase in ALP concentration in the range from 6 to 70 U L⁻¹. By plotting the fluorescence intensity ratio (F/F₀) versus the concentration of ALP, the linear response range from 6 to 40 U L⁻¹ with a linear fitting equation of F/F₀ = 0.27 + 0.13[ALP] (U L⁻¹) could be obtained (Fig. 7B). The LOD (3σ rule) was calculated to be 0.5 U L⁻¹. Considering the normal range of ALP (40–120 U L⁻¹) in adults,⁴³ the proposed detection procedure is sensitive enough to detect low concentrations of ALP. Moreover, the analytical performance of the proposed detection system was compared with previous approaches (Table S1, ESI†). It can be seen that the proposed detection system exhibited a lower or comparable LOD with these methods. This proves the capability of the proposed fluorescence method for the detection of ALP.

Fig. 7 (A) Fluorescence response of the detection system with the addition of various concentrations of ALP. (B) Corresponding linear range.

3.6. Selectivity and anti-interference test

The selectivity of the detection system was evaluated by recording its fluorescence response toward various potentially interfering biomolecules and commonly coexisting cations and anions. The tested biomolecules include amylase, bovine serum albumin (BSA), glucose oxidase (GOx), glutathione (GSH), human serum albumin (HAS), cysteine (Cyst), histidine (His), sulfatase, trypsin (Try), Na⁺, K⁺, Ca²⁺, Fe³⁺, Cl⁻, HCO₃⁻, CO₃²⁻, SO₄²⁻, and NO₃⁻. As presented in Fig. 8A, only the introduction of ALP caused significant fluorescence recovery, while other biomolecules and enzymes hardly caused changes in the fluorescence of the detection system. Also, the anti-interference performance of the detection system (Fig. 8B) was proved as the coexistence of these biomolecules and enzymes did not significantly affect the fluorescence response of the detection method. In conclusion, the detection platform exhibits excellent selectivity and anti-interference performance.

Fig. 8 (A) Fluorescence response of the detection system for ALP and interfering biomolecules and ions. (B) The anti-interference response of the detection system with the coexistence of biomolecules and ions with ALP. Concentrations: ALP = 15 U L⁻¹ and biomolecules = 10 mg mL⁻¹, ions = 50 μM.

3.7. Detection of ALP in serum samples

The applicability of the proposed method for the detection of ALP in a real biological matrix was tested by using serum samples. Considering the similarity in the components of mammals (including humans) and easy availability,⁴⁴ fetal bovine serum (FBS) was used to test the practical applicability of the as-proposed method for ALP detection. As presented in Table S2 (ESI†), the recoveries of the spiked ALP ranged from 83.9% to 107.7% with a RSD of 1.5% to 2.1%. The results demonstrate the potential applicability of the proposed method for the detection of ALP in real and complex biological samples.

3.8. Cytotoxicity and cell imaging applications

We assessed the cytotoxicity of the PEG–Tb–CaWO₄ NCs by examining their effect on the cell viability of HeLa cells using the MTT assay. It can be seen from Fig. S16 (ESI†) that increasing the concentration of PEG–Tb–CaWO₄ NCs did not affect the cell viability of HeLa cells. Even at a relatively high dose of PEG–Tb–CaWO₄ NCs (400 μg mL⁻¹), the cell viability still remained above 90%, demonstrating the very low cytotoxicity of the NCs. The observed non-toxicity of PEG–Tb–CaWO₄ NCs could be attributed to the good biocompatibility of PEG to the biological system⁴⁵ and the minimal probability of ion leakage from the PEG–Tb–CaWO₄ NCs in the cells.^18,46 Based on these results, it can be inferred that PEG–Tb–CaWO₄ NCs are nontoxic to live cells and can be used for in vivo biological applications.

Furthermore, HeLa cells were employed to investigate the biological imaging potential of PEG–Tb–CaWO₄ NCs (Fig. 9). From the green channel of CLSM images, no fluorescence signal can be detected from the control group (Fig. 9B), while a bright green fluorescence can be observed from the PEG–Tb–CaWO₄ NC treated cells (Fig. 9D). This indicates effective cellular uptake of PEG–Tb–CaWO₄ NCs by the HeLa cells. Also, the green fluorescence emission was observed in the cytoplasmic area, while no luminescence was observed at the nucleus, suggesting that PEG–Tb–CaWO₄ NCs were internalized through endocytosis and diffused into the cytoplasm.^47,48 Hence, based on the viability test and cell imaging results, it can be concluded that PEG–Tb–CaWO₄ NCs exhibited low cytotoxicity and could be considered as a potential probe for in vivo biological applications.

Fig. 9 CLSM images of (A) and (B) untreated and (C) and (D) PEG–Tb–CaWO₄ NC treated HeLa cells upon 488 nm laser irradiation.

4. Conclusions

Unlike conventional solid-state methods, which require annealing at extremely high temperatures and result in micron-sized NCs, we successfully synthesized nano-sized PEG–Tb–CaWO₄ NCs with excellent luminescence properties at a mild temperature (140 °C). In addition, coating with the PEG ligand significantly improved the colloidal stability of Tb–CaWO₄ NCs in aqueous and biological solutions. Furthermore, for the first time, we demonstrated the sensing and biological imaging applications of PEG–Tb–CaWO₄ NCs. To this end, a fluorescence “signal-off–on” sensing strategy was developed to detect a useful disease biomarker, ALP. The as-constructed sensor exhibited excellent sensitivity for the detection of ALP in both buffer solution as well as serum samples. Moreover, the cell imaging potential of PEG–Tb–CaWO₄ NCs was demonstrated by the bright green luminescence observed in HeLa cells incubated with the NCs. The viability study also confirmed the very low cell cytotoxicity of PEG–Tb–CaWO₄ NCs to HeLa cells. Overall, in this study, we demonstrated and highlighted the potential of PEG–Tb–CaWO₄ NCs as a promising probe for sensing and biological applications.

Author contributions

Terefe Tafese Bezuneh contributed to the investigation involving synthesis, characterization, visualization, and writing original draft, review, and editing of the manuscript. Xin Li, Tongtong Kou, Fuchun Nan, Zilong Wu, and Lanbo Shen supported various characterization studies and investigation. Xinyuan Xia provided funding acquisition and resources. Bin Li was responsible for funding acquisition, supervision and resources. Liang Wang was responsible for project administration, conceptualization, and supervision. William W. Yu conceptualized this work and was responsible for supervision, project administration, funding acquisition, and review and editing of the manuscript.

Data availability

All experimental details and data supporting the findings of this article are available within the paper and also included in the ESI.†

Conflicts of interest

The authors declare no competing financial interests.

Acknowledgements

This work was financially supported by the Natural Science Foundation of China (62374103), the Jinan Central Hospital (1190022050), the Taishan Scholar Foundation of Shandong Province (tsqn2023120051105), the Shandong Postdoctoral Innovation Project (SDCX-ZG-202400274), and Shandong Provincial Natural Science Foundation (ZR2022QH069, ZR2020MB083, and ZR2023QE322).

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Footnote

† Electronic supplementary information (ESI) available. See DOI: https://doi.org/10.1039/d5qm00122f

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