Synthesis of Up-Conversion CaTiO₃: Er³⁺ Films on Titanium by Anodization and Hydrothermal Method for Biomedical Applications

Abstract

The present study investigates the effects of Er³⁺ doping content on the microstructure and up-conversion emission properties of CaTiO₃: Er³⁺ phosphors as a potential material in biomedical applications. The CaTiO₃: x%Er³⁺ (x = 0.5, 1.0, 1.5, 2.0, 2.5, and 3.0%) films were synthesized on Ti substrates by a hydrothermal reaction at 200 °C for 24 h. The SEM image showed the formation of cubic nanorod CaTiO₃: Er³⁺ films with a mean edge size value of (1–5) μm. When excited with 980 nm light, the CaTiO₃: Er³⁺ films emitted a strong green band and a weak red band of Er³⁺ ions located at 543, 661, and 740 nm. The CaTiO₃: Er³⁺ film exhibited excellent surface hydrophilicity with a contact angle of ~zero and good biocompatibility against baby hamster kidney (BHK) cells. CaTiO₃: Er³⁺ films emerge as promising materials for different applications in the biomedical field.

1. Introduction

CaTiO₃ emerges as a promising host material for luminescence-based applications and biomedicine due to its exceptional combination of properties: high chemical durability, long luminescence lifetime, high color rendering index, low power consumption, and biocompatibility. Previous work also reported the biocompatibility of CaTiO₃ particles [1]. However, to our knowledge, the UC luminescence and cell compatibility of CaTiO₃: Er³⁺ films on Ti substrate synthesized by a hydrothermal method have yet to be well documented.

Semiconductors doped with rare earth (RE) ions are potential materials for a variety of applications, such as antimicrobial activities, photocatalytic treatment, and environmental purification [2,3]. Perovskite and oxide-based materials are effective hosts for the up-luminescence of RE ions [4,5] because of their low phonon energy, low cost, and easy synthesis [6,7]. Recent research of luminescent RE ions has focused on the Er³⁺ ion thanks to its unique electronic and optical properties and various applications, such as three-dimensional displays [8], solar cells [9,10], temperature sensors [11], photocatalysts [12,13], and biomedical field applications [14,15]. The UC luminescence of CaTiO₃: Er³⁺ particles is highly attractive for various applications, including drug delivery, tumor therapy, and cell imaging. Er³⁺ ions can be substitutionally doped into the CaTiO₃ lattice, where they occupy well-defined positions because the material’s crystal structure and the close correspondence between the ionic radii of Er³⁺ and Ca²⁺ ions contribute to the prevention of lanthanide element release [16,17]. The characteristics of Er³⁺ emissions from ⁴F_9/2→⁴I_15/2 (red) and ⁴S_3/2, ²H_11/2→⁴I_15/2 (green) of the material displayed various transitions under room temperature conditions. The UC luminescence properties of the material can be tailored by adjusting the Er³⁺ doping concentration and the UC photon excitation mechanism [1,18,19].

The hydrophilic property plays a crucial role in numerous biomedical applications. For instance, it serves several purposes, including tissue integration, enhanced cell interactions, and drug delivery. In the context of tissue integration, hydrophilic materials possess the ability to absorb water-based substances from the surrounding environment, enabling them to integrate better with body tissues. This can potentially reduce the risk of implant rejection and improve the long-term functionality of implants [20]. Additionally, the hydrophilic property can foster a favorable environment for cell adhesion and growth, promoting wound healing and tissue regeneration around the implant [21]. Furthermore, hydrophilic materials can be utilized to deliver drugs or growth factors to specific locations within the body, enhancing treatment efficacy and minimizing side effects [22].

In this study, we reported the hydrothermal synthesis of up-conversion (UC) emission of CaTiO₃: Er³⁺ film on Ti and discussed the influence of Er³⁺ concentrations on the UC luminescence of CaTiO₃: Er³⁺ film. We also investigated the hydrophilic properties and cell compatibility of the CaTiO₃: Er³⁺ film for biomedical applications.

2. Experimental

2.1. Synthesis of CaTiO₃: x%Er³⁺

CaTiO₃: Er³⁺ films were synthesized using the hydrothermal method. Calcium hydroxide [Ca(OH)_2, Merck (Rahway, NJ, USA), 99.9%], sodium hydroxide (NaOH, Merck, ≥97.0%), TiO₂ nanotubes template, and erbium chloride hexahydrate (ErCl₃·6H₂O, Merck, 99.9%) were used as starting materials. TiO₂ nanotubes templates were synthesized using the anodization method as reported in previous work [18]. A total of 7.4 mg of Calcium hydroxide (Ca(OH)₂) was dissolved in 40 mL of H₂O, and 3.2 mg of NaOH was dissolved in 10 mL of H₂O. After stirring the two mixtures for 15 min at room temperature, the mixtures were combined under continued magnetic stirring. Next, 7.8 mg of ErCl₃·6H₂O was dissolved in H₂O and slowly added to the beaker with the previous solution. Finally, the mixture solution was used for the hydrothermal synthesis of CaTiO₃ at 200 °C for 24 h. The synthesized CaTiO₃: Er³⁺ powder was subjected to a high-temperature treatment (annealing) at 800 °C for 2 h to obtain the final samples.

2.2. Characterization

2.2.1. Physicochemical Analysis Methods

The crystallographic properties of the obtained samples were investigated using X-ray techniques powder diffractometer (XRD, Siemens D500, Munich, Germany). Field emission scanning electron microscopy (FESEM, FE-SEM, JEOL JSM-7600F) was employed to investigate the morphology of CaTiO₃: Er³⁺. FESEM equipped with EDS (Gatan, UK) was used for the elemental analysis of the samples. A special instrument (NANO LOG spectrophotometer) was used to measure the light emission properties (luminescence) of CaTiO₃: Er³⁺. The instrument used two light sources: a high-power xenon lamp and a specific wavelength laser 980 nm. All the analyses were performed at room temperature. Water contact angle measurements were performed to assess the surface properties of the films by using a digital camera at room temperature. Close-up pictures were taken of tiny drops (about 1 microliter) of water placed on the top surface of each film.

2.2.2. Biocompatibility Assessment Methods

To assess cell compatibility in vitro, both the titanium substrate and CaTiO₃: Er³⁺ films were sterilized through autoclaving. This sterilization process involved exposure to high pressure steam at 121 °C for 60 min. Meanwhile, baby hamster kidney cells (BHK cells) were cultured in a growth medium called DMEM at 27 °C. The culture environment was maintained with humidified air and 5% CO₂. Cells at the same concentration and density were then applied to both the titanium and CaTiO₃: Er³⁺ films. A special microscope with lasers (confocal laser scanning microscopy, FV3000RS, Olympus, Tokyo, Japan) was used to see how well the cells attached to the surfaces. After culturing for 48 h, the BHK cells on the CaTiO₃: Er³⁺ films and the Ti substrates underwent fixation (4% paraformaldehyde/PBS, 10 min), washing, permeabilization (0.1% Triton X-100/PBS, 5 min), washing, and fluorescent phalloidin labeling (45 min). Cell nuclei were specifically stained with a fluorescent dye called DAPI for a duration of 5 min. The stained cells adhering to the samples were mounted onto glass cover slips for subsequent observation of cell attachment. Cell growth was evaluated by CellTiter 96^® AQ_ueous one solution compound, which contains a tetrazolium compound [3-(4,5-dimethylthiazol-2-yl)-5-(3-carboxymethoxyphenyl)-2-(4-sulfophenyl)-2H-tetrazolium, inner salt; MTS]. The quantity of the formazan product, which is measured by the absorbance at 490 nm using a microreader (Chromate 4300 Microplate Reader, Awareness Technology, Palm City, FL, USA), is directly proportional to the number of living cells on the sample.

3. Results and Discussion

3.1. Results of Material Synthesis

Following the fabrication of the material with varying Er dopant concentrations, the morphological and structural characterization of these samples was analyzed. The successful fabrication of CaTiO₃: Er³⁺ thin films was clearly demonstrated by using analytical techniques, including XRD, Raman, EDS, and SEM. Figure 1a shows the XRD patterns of the as-synthesized samples with different Er³⁺ doping content. Based on JCPDS No. 22–0153 reference database, all diffraction patterns of the samples correspond to pure orthorhombic phase CaTiO₃ with space group Pbnm. The orthorhombic structure of CaTiO₃ contains a Ca²⁺ ion with eight coordinates (CaO₈) and the Ti⁴⁺ ion with six coordinates in an octahedron (TiO₆) [5]. Ca²⁺ atoms at the dodecahedron (CaO₈) site are easily replaced by Er³⁺, while Ti⁴⁺ atoms continue to be unaltered at the TiO₆ site. Given the similarity of the ionic radii of Er³⁺ (1.19 Å, coordination number = 12) and Ca²⁺ (1.34 Å, coordination number = 12), RE³⁺ can replace Ca²⁺ in the CaTiO₃ structure [1]. To compensate for charge imbalances caused by lattice defects, a substitution process occurs, including Ca²⁺ (V’_Ca) and/or O²⁻ vacancies (V_O•) [19,23]. The diffraction profile of the Pbnm space exhibits typical (hkl) planes such as (111), (121), (031), (220), (040), (042), and (242) at 27.4°, 33.1°, 39.4°, 41.2°, 47.03°, 59.1°, and 69.8°, respectively. The most dominant diffraction peaks appeared and were centered at 2θ = 33.1°, corresponding to the (121) plane of the CaTiO₃ phase. As shown in Figure 1a, The XRD model of the CaTiO₃ film with optimized Er³⁺ concentration suggests the presence of a minor phase (peaks marked by symbol ▪) corresponding to Ti (JCPDS No. 44–1294), but this phenomenon did not affect much the phase of CaTiO₃ and optical properties. These results indicated the successful synthesis of the crystalline structure of CaTiO₃ films. Moreover, the XRD result revealed that the intensity of CaTiO₃: Er³⁺ diffraction peaks increases gradually with an increasing dopant concentration. When the concentration of Er³⁺ is increased to 2.5%, two characteristic peaks, 33.1$°$ and 47.03$°$, emerge with relatively high intensity and sharpness. This indicates that the crystal structure of CaTiO₃: Er³⁺ is well formed.

Raman spectroscopy serves as a valuable tool for studying symmetry changes in various compounds. In the case of the CaTiO₃ material, twenty-four Raman-active modes were identified within its orthorhombic Pbnm crystal structure (with Z^B = 4), featuring four molecular units within the primitive cell. The material’s irreducible representation is denoted as Γ_Raman,Pbnm = 7A_g + 5B_1g + 7B_2g + 5B_3g. Figure 1b depicts the Raman spectra of CaTiO₃: Er³⁺ films span the frequency scope of 100–900 cm⁻¹. Raman-active modes at 134 cm⁻¹ is attributed to the oscillation of Ca bonded to the TiO₃ (Ca–TiO₃) lattice. Modes at 226, 244, 281, and 362 cm⁻¹ are linked to O–Ti–O bending modes, while those at 464 and 495 cm⁻¹ correspond to Ti–O₆ twisted modes (bending or internal oscillation of the oxygen cage), with a second large band observed in the scope of 600–700 cm⁻¹. These observations agree with previous works [24,25,26,27].

Figure 2 shows the EDS spectra of the CaTiO₃: xEr³⁺ films (x = 0.5, 2.0, and 3.0 mol%). All samples show attendance of O, Ca, Ti, Er, and Na elements. The presence of Na could be due to the residual input solution. Analysis revealed no traces of other impurities, suggesting the high purity of the synthesized phosphors.

Figure 3 shows the FESEM picture of the CaTiO₃: Er³⁺ films. As shown in Figure 3, when the CaTiO₃ is doped at a concentration of 0.5% Er³⁺, the material begins to form fiber clumps. Increasing the doping concentration of (1–2)% Er³⁺ leads to the formation of increasingly homogeneous square-shaped fiber bundles. The CaTiO₃: Er³⁺ films consist of uniform cubic particles with a bar length of ~(1–5) μm and a width of ~(1–2) μm. The results of this SEM analysis are consistent with the previously analyzed XRD data.

3.2. Material Properties Evaluation Findings

3.2.1. UC Luminescence Properties

Following the successful fabrication of the material, we proceeded to investigate its optical properties when doped with rare earth erbium (Er³⁺) ions. Samples doped with Er³⁺ at varying concentrations, ranging from 0.5% to 3%, were employed to investigate their optical properties. Figure 4a,b depict the UC luminescence spectra for all samples with an excitation wavelength of 980 nm. Upon 980 nm excitation, all samples show intense green UC emission bands at 523/550 nm (²H_11/2→⁴I_15/2/⁴S_3/2→⁴I_15/2), a faint red emission band at 650/680 nm (⁴F_9/2→⁴I_15/2), and a far-red band at 740 nm (⁴I_9/2→⁴I_15/2). Across all samples with different Er³⁺ doping concentrations, a robust green emission band and a diminishing red emission band are consistently observed. The dominant emissions are located in the green luminescence range for CaTiO₃: Er³⁺ films. The comparative intensity of the three energy level transitions ⁴F_9/2→⁴I_15/2, ²H_11/2→⁴I_15/2, and ⁴S_3/2→⁴I_15/2 increases with increasing Er³⁺ concentration. The peak up-conversion (UC) emission intensity for the CaTiO₃: Er³⁺ films was identified by upholding the optimal convergence of the Er³⁺ activator. The discernible red and blue emission peaks suggest enhanced conversion of infrared light to visible light, attributed to the synergistic effect of Er³⁺ ion co-doping in CaTiO₃. This phenomenon notably broadens the emission spectrum in the visible region, expanding the range of electromagnetic waves emitted [28,29]. As illustrated in Figure 4a, the luminescence intensity of the material varies with the doping concentration. When the Er³⁺ dopant concentration is increased from 0.5% to 2%, the luminescence intensity also increases. However, further increasing the dopant concentration leads to a decrease in luminescence intensity. This phenomenon of intensity reduction is known as luminescence quenching.

The excitation spectrum of the 550 nm emission band (green) in 2.0 mol% Er³⁺–doped CaTiO₃ films is depicted in Figure 5a. The seven bands with centers at 357, 365, 379, 408, 445, 451, and 491 nm correspond to the excited levels, namely, ²G_7/2, ⁴G_9/2, ⁴G_11/2, ²G_9/2, ⁴F_3/2, ⁴F_5/2, and ⁴F_7/2, respectively. The most prominent peak at 379 nm aligns with the ⁴I_15/2→⁴G_11/2 transition. The excitation spectrum reveals various pathways to obtain green luminescence from the CaTiO₃ films. CaTiO₃: Er³⁺ emits bright green emission when the films are excited by 390 nm laser light. The visible emission spectra for samples doped with 0.5, 1.0, 1.5, 2.0, 2.5, and 3.0 mol% Er³⁺ are presented in Figure 5b. The two prominent green bands in the spectral ranges of 522–535 and 537–560 nm align with the ²H_11/2→⁴I_15/2 and ⁴S_3/2→⁴I_15/2 transitions, respectively. The fainter red band in the 650–675 nm region aligns with the ⁴F_9/2→⁴I_15/2 transition [30].

The rapport between up-conversion intensity (I_upc) bands and pump power excitation (P_pump) was examined to elucidate the up-conversion process in Er³⁺ co-doped films, as illustrated in Figure 6a,b. Understanding the power accessorial of up-conversion luminescence is crucial for unraveling the up-conversion convert mechanism. Typically, when up-conversion luminescence is generated at low pump intensity, the correlation between up-conversion emission intensity (I) and pump power (P) can be written as I_upc ∝ (P_pump)ⁿ or log (I_upc) ∝ n log (P_pump), where ‘n’ signifies the number of photons required to be excited to attain the up-conversion emitting level [31]. Figure 6a shows the power-dependent UC fluorescence spectra, and Figure 6b shows that the integrated intensity of the green and red emissions of CaTiO₃: Er³⁺ (2%) varies with pump power on the double logarithmic scale. Figure 6b shows that the log–log power accessorial slopes of 523, 550, and 670 nm are 3.05, 3.07, and 2.73, respectively. Our findings suggest that the green and red up-conversion (UC) luminescence of Er³⁺ stems from three-photon processes in the circumstances of the CaTiO₃: Er³⁺. Supported by the observed quadratic dependence on pump power and the alignment of energy levels, the most likely transitions responsible for the UC emissions are illustrated in Figure 7.

3.2.2. UC Mechanism of Er-Doped Systems

Er (⁴I_15/2) + hν_980nm→Er (⁴I_11/2)

(1)

Er (²h_11/2)→Er (⁴I_15/2) + hν_523nm

(2)

Er (⁴I_11/2) + Er (⁴I_11/2)→Er (⁴S_3/2) + Er (²I_15/2)→Er (⁴I_15/2) + hν_550nm

(3)

Er (⁴I_11/2) + Er (⁴I_13/2)→Er (⁴F_9/2) + Er (²I_15/2)→Er (⁴I_15/2) + hν_675nm

(4)

Figure 7 shows the system’s energy level diagram to understand the phosphor’s dual-mode green emission behavior. Several steps are proposed for selectively enhancing green UC emission of CaTiO₃: Er³⁺ phosphors [9,32].

Step 1: The process commences with the absorption of a photon by the electron at the ⁴I_15/2 state, elevating it to the ⁴I_11/2 level. This initial step, known as ground state absorption (GSA), is pivotal to the system’s behavior.

Step 2: The electron at the ⁴I_11/2 level absorbs a second photon and transitions to a higher energy excited state, the ²F_7/2 level, by excited condition absorption (ESA). It is notable owing to the unstable 2F_7/2 state that leads to no radiative relaxation of the electron to the ²H_11/2 and ⁴S_3/2 levels (two meta-stable levels).

Step 3: The electrons in ²H_11/2 and ⁴S_3/2 return to the ground condition ⁴I_15/2, producing intense green emission. Meanwhile, a few electrons underwent non-radiative relaxation from the ⁴S_3/2 level to the ⁴F_9/2 level and then moved to the initial condition (⁴I_15/2), forming a weak red UC emission.

The Er³⁺ ions initially undergo excitation from the ²F_7/2 to ²F_5/2 level by absorbing laser photons (ground/excited state absorption, GSA/ESA) due to the strong absorption at wavelength 980 nm. Under 980 nm excitation, the ⁴I_11/2 level of Er³⁺ becomes populated by absorbing a single infrared photon from the ⁴I_15/2 level (called ground state absorption, GSA), as depicted in Equation (1). Through the nonradiative relaxation (NR) progress from the ⁴F_7/2 level to the ²H_11/2/⁴S_3/2 (two meta-stable levels) lower energy level, practically all of the electrons in the ²H_11/2/⁴S_3/2 level progressed down to the first state ⁴I_15/2 of Er³⁺, which form intense green emissions (523–550 nm). The electrons in the Er³⁺ (⁴F_7/2) excited state could decay to the ²H_11/2 excited state through a process called thermal radiation. This process emits a 523 nm photon as the electrons return to their ground state, Er³⁺ (⁴I_15/2), as illustrated in Equation (2). Moreover, a part of electrons in the Er³⁺ (⁴I_11/2) stimulated state can transfer energy to an adjacent electron within a similar energy level. Consequently, an electron transition from Er³⁺ (⁴I_11/2) to Er³⁺ (⁴S_3/2) stimulated state ensues, triggering radiative relaxation to Er³⁺ (⁴I_15/2) along with the emission of a 550 nm green photon, as described by Equation (3). The electrons in the ⁴S_3/2 level undergo no radiative relaxation to the ⁴F_9/2 level; the next step is their return to the initial state of ⁴I_15/2, creating a red emission band, as indicated in Equation (4). The energy of the ⁴S_3/2 level surpasses that of the ⁴F_9/2 level, resulting in a subdued red emission band.

In the direct current (DC) emission, the strong green emission and weak red emission can be explained as follows: Initially, Er³⁺ ions in the basic state undergo excitation to the ⁴G_11/2 level (refer to Figure 7) through a xenon lamp with excitation wavelengths of 379 nm. Subsequently, a majority of the electrons in the ⁴G_11/2 level relax to the ⁴S_3/2 no radiative (NR) level with the swiftest multi-phonon relaxation rate, followed by their return to the radiative basic state, ⁴I_15/2, leading to the generation of two green emission bands. Notably, there is minimal excitation of electrons to the ⁴I_11/2 state, leading to a limited electron population in the ⁴F_9/2 level. Therefore, red emission occurs with weak intensity. Our findings indicate that the proposed mechanism effectively explains the dual-mode green emission observed in the CaTiO₃: Er³⁺ films from the perspective of the electronic structure scale.

3.3. Biocompatibility of CaTiO₃: Er³⁺

To confirm the biocompatibility of the material, the research team prepared a CaTiO₃: 2%Er³⁺ sample and conducted a study on its hydrophilicity. The hydrophilicity of a material is determined by using a contact angle measurement technique. In this work, we used the contact angle method to verify the hydrophilic properties of the Ti substrate, CaTiO₃ film, and CaTiO₃: Er³⁺ films. Interestingly, as shown in Figure 8, the result of all CaTiO₃: Er³⁺ films were considerably lower than that of the Ti substrate and CaTiO₃ films. Among the three kinds of films, the CaTiO₃: Er³⁺ films had the most evident decrease in contact angle, which indicated the best improvement in hydrophilic. Hence, surface transformation from the hydrophobic Ti to hydrophilic CaTiO₃: Er³⁺ films can be used to design water dispersible film phosphors in bio-medical fields. The findings demonstrate that the fabricated material possesses promising biomedical applications, particularly in the realm of implants. The material’s hydrophilic plays a crucial role in enhancing cell adhesion and growth, which can potentially promote wound healing and tissue regeneration in the surrounding area. Furthermore, highly hydrophilic materials can be utilized for delivering drugs or growth factors to specific locations within the body. This capability holds the potential to improve treatment efficacy and minimize side effects.

To confirm the crucial role of hydrophilicity in biomedical applications, such as enhancing cell adhesion and growth, cell culture experiments were conducted on the material’s surface to evaluate cell proliferation. Confocal laser scanning microscopy (CLSM) images in Figure 9a,b depict BHK cells adhering to the surfaces of Ti and CaTiO₃: Er³⁺ films, respectively. The uniform distribution and spread-out, fibrous morphology of the cells in both samples indicate good biocompatibility.

An MTS assay was employed to further assess the biocompatibility of the materials with the cell proliferation of the Ti and CaTiO₃: Er³⁺ films. The rate of proliferation was measured after culturing up to 72 h, using MTS for mitochondrial reduction. This assay is based on the ability of metabolically active cells to reduce a tetrazolium-based compound, MTS, to a purple formazan product. The quantity of formazan product is directly proportional to the number of living cells in the culture. This assay is a widely recognized tool for evaluating a material’s suitability for biological applications [33]. Figure 9c shows cell proliferation on the Ti and the CaTiO₃: Er³⁺ films. The rate of cell proliferation on the CaTiO₃: Er³⁺ films was significantly higher than that of the Ti substrate. This suggests that the CaTiO₃: Er³⁺ films supported the proliferation and growth of BHK cells without inducing any cytotoxic effects.

4. Conclusions

CaTiO₃: Er³⁺ films were successfully synthesized by a hydrothermal process on a Ti plate. The CaTiO₃: Er³⁺ films showed a cubic structure, with a bar length of ~5 μm and a width of ~(1–2) μm. The CaTiO₃: Er³⁺ films displayed highly intensive UC emission under 980 nm laser beam excitation. The emitted light was primarily composed of green and red bands originating from Er³⁺ ions, with maxima at 543, 661, and 740 nm. The CaTiO₃: Er³⁺ film had excellent surface hydrophilicity, with an almost zero contact angle, which can help increase the application of guide water and good substances in biomedicine. Furthermore, the CaTiO₃: Er³⁺ film significantly promoted the adhesion and growth of cells. Hence, CaTiO₃: Er³⁺ films are promising materials for future directions in luminescence and biomedical fields.