Polyacrylic Acid-Ca(Eu) Nanoclusters as a Luminescence Sensor of Phosphate Ion

Abstract

In this study, we synthesized polyacrylic acid (PAA)-Ca (Eu) nanoclusters as a luminescence sensor of phosphate ion by a complex method, and we aimed to achieve the quantitative detection of PO₄³⁻ based on the sensitivity of the charge transfer band of Eu³⁺ to anionic ligand. The resulting PAA-Ca(Eu) nanoclusters showed a well-dispersed and a dot-like morphology, with an ultra-small diameter (the average size of 2.17 nm) under high resolution transmission electron microscopy(HRTEM) observation. A dynamic light scattering particle size analyzer (DLS) showed a hydrodynamic size of 2.39 nm. The (PAA)-Ca (Eu) nanoclusters as a luminescence sensor showed a significantly higher sensitivity for PO₄³⁻ than other anions (CO₃²⁻, SiO₃²⁻, SO₄²⁻, SO₃²⁻, Br⁻, Cl⁻, F⁻). The luminescence intensity displayed a linear increase (y = 19.32x + 74.75, R² > 0.999) in a PO₄³ concentration range (0–10 mM) with the concentration of PO₄³⁻ increase, and the limit of detection was 0.023 mM. The results showed good recovery rates and low relative standard deviations. These (PAA)-Ca (Eu) nanoclusters are hopeful to become a luminescence sensor for quantitatively detecting PO₄³⁻.

1. Introduction

Europium element with a unique 4f electron layer structure is a commonly used luminescent probe [1,2,3] due to its good optical stability, high thermal and chemical stability, narrow emission band, high resistance to photobleaching, and light quenching [4,5,6]. The excitation wavelength of Europium mainly includes the 350–475 nm band of energy levels transition and the charge transfer band (CTB) in the ultraviolet region [5,7]. The energy level transition excitation can obtain better near-infrared emission luminescence, which is mainly used in the biomedical field [6,8,9,10,11,12]. The CTB has unique properties, Eu³⁺ binds to the anionic ligand to form a CTB. The position of the charge transfer transition band depends on the ligand [13,14,15,16,17,18]. Therefore, the CTB of Eu³⁺ can be used for qualitative and quantitative analysis of the types and contents of anionic ligands. For example, CTB formed with phosphate in hydroxyapatite is at 254 nm, while CTB formed with anionic ligand in LaOF is at 285 nm [7,19].

Phosphorus plays an important role in organisms and the environment [20,21]. Excessive phosphate content in water can cause water pollution [22,23]. Phosphate in organisms participates in a variety of metabolism processes. Phosphate content is one of the important indicators of human health, and its quantitative detection is of great significance [24,25]. In this study, inspired by the biomineralization process of calcium phosphate, we used polyacrylic acid (PAA) to complex Ca²⁺ and Eu³⁺ ions to obtain PAA-Ca (Eu) nanoclusters as a sensor for the quantitative detection of PO₄³⁻ based on the sensitivity of charge transfer band of Eu³⁺ to anionic ligand. The morphology, size, ion selectivity and luminescence of PAA-Ca (Eu) nanoclusters were characterized, and the mechanism of quantitative phosphate radical detection was analyzed and explained by luminescence spectra and molecular dynamics simulation (MDS).

2. Materials and Methods

2.1. Synthesis of PAA-Ca (Eu) Nanoclusters

The PAA-Ca(Eu) nanoclusters were prepared by a complex method. An aqueous Ca(Eu) solution (20 mL) was prepared using CaCl₂·2H₂O (99.42 mg, Sinopharm, Beijing, China) and Eu(NO₃)·6H₂O (33.52 mg, Aladdin, Shanghai, China) with a concentration of 37.575 mM in which the Eu³⁺/(Ca²⁺ + Eu³⁺) molar ratio was 10%. The solution was stirred vigorously to make it fully dissolved. An aqueous solution of PAA (average molecular weight of ~1800 g/mol, 216.43 mg, 20 mL, Sigma, St. Louis, USA) was quickly added to the aqueous Ca(Eu) solution, and the pH was adjusted to 7.5–8.0 using NH₃·H₂O (Sinopharm, Beijing, China) to yield the PAA-Ca(Eu) nanoclusters. The temperature of all the above solutions was room temperature (25 °C).

2.2. Characterization

High resolution transmission electron microscopy (HRTEM, Talos F200S, Waltham, MA, USA) was used to observe and to analyze the microstructure of the materials. Fourier transform infrared spectroscopy (FT-IR, Nicolet6700, Waltham, MA, USA) was used to record the spectra of the near infrared region (4000~400 cm⁻¹), analyze and study the vibration mode of the characteristic peak of the material, identify the substance, and determine the chemical composition or relative content of the substance. A dynamic light scattering particle size analyzer (DLS, Malvern, UK) was used to measure the particle size distribution and the dispersion coefficient of solution. Luminescence excitation and emission spectra of samples were measured by luminescence spectrophotometer (970CRT, Shanghai Sanco, Shanghai, China).

2.3. Detection of PO₄³⁻

An aqueous solution of phosphate ion was prepared by Na₂HPO₄·12H₂O and added to the PAA-Ca(Eu) nanoclusters solution. Finally, NH₃·H₂O was used to adjust the pH to 9.0–9.5 for luminescence detection.

2.4. Preparation of Buffer Solution

A total of 1.07 g of NH₄Cl (Sinopharm, Beijing, China) was added to 100 mL of deionized water. After it was fully dissolved, ammonia was added to adjust the pH of the aqueous solution to 8.0 to obtain the buffer solution.

2.5. Molecular Dynamics Simulation

All MDS employed the AMBER/general AMBER force field. In the cubic simulation unit with an initial size of 10 nm, the step change was set to 1 fs, and all simulations were run for 50 ns in real time using Gromacs 2018 software package [26,27].

3. Results and Discussion

3.1. Structure Characterization

First, the microstructure and the particle size of PAA-Ca (Eu) nanoclusters were characterized (Figure 1). Through HRTEM, it can be seen that the nanoclusters present dot-like particles, and the nanoclusters do not gather directly. The particle size also presents a relatively uniform distribution. Through the statistics of the nanoclusters in the HRTEM image, their particle size is concentrated in the range of 1.8–2.4 nm (this particle size range accounts for 88% of the total particle size), with an average particle size of 2.17 nm. DLS test results also showed a similar hydrodynamic size (2.39 nm).

In addition, FT-IR spectra of PAA-Ca (Eu) nanoclusters and samples with different PO₄³⁻ additions are shown in Figure 2. The absorption peak at 3478 cm⁻¹ is the O-H stretching vibration peak in PAA molecule [28]. The absorption peaks at 1556 cm⁻¹ and 1401 cm⁻¹ are the asymmetric stretching vibration peak (ν_as(COO⁻)) and the symmetric stretching vibration peak (ν_s(COO⁻)) of COO⁻ in the PAA molecule, respectively. Compared with pure PAA, the C=O absorption peak shifts to a low frequency and the C-O absorption peak shifts to a high frequency, which ν_as(COO⁻)–ν_s(COO⁻) is approximately 150 cm⁻¹, indicating that the coordination between carboxylic acid and the metal ions in PAA is a bridge coordination compound [29,30]. After adding PO₄³⁻, the absorption peak of the phosphate ion appeared obviously in the infrared spectrum, which was located at 1104 cm⁻¹, 1072 cm⁻¹ and 536 cm⁻¹, belonging to the asymmetric stretching (ν₃) and the asymmetric angle change (ν₄) of PO₄³⁻ [31,32].

3.2. Luminescent Characterization

3.2.1. Ion Selectivity

PAA-Ca(Eu) nanoclusters were used as sensors to detect common anions (the anion concentration was 10 mM). As shown in Figure 3a, PO₄³⁻ is the most sensitive to the sensor, and it has the highest luminescence intensity. The luminescence emission peak with the maximum luminescence intensity (617 nm) was selected for comparison, as shown in Figure 3b. It can be more intuitively observed that the sensor is sensitive to PO₄³⁻. Figure 3c shows that CTB positions and intensities are different for different anionic ligands. The CTB of PO₄³⁻ position is unique, and it is the strongest. All of the above indicated that PAA-Ca (Eu) nanoclusters could be used for the detection of PO₄³⁻ concentration.

3.2.2. Detection of PO₄³⁻ Concentration

In the emission spectrum excited at 254 nm, Eu³⁺ showed characteristic emission at 594 (⁵D₀ → ⁷F₁), 617 (⁵D₀ → ⁷F₂), 654 (⁵D₀ → ⁷F₃), and 699 nm (⁵D₀ → ⁷F₄) (Figure 4a). Figure 4b shows that with the increase of PO₄³⁻ concentration, the increase of luminescence first increased and then remained basically unchanged. The linear fitting of PO₄³⁻ concentration in the range of 0–10 mM showed that the linear equation was y = 19.32x + 74.75, and its R² was 0.999, indicating that PAA-Ca(Eu) nanoclusters can quantitatively detect PO₄³⁻ in this concentration range. In the excitation spectrum, Eu-O CTB gradually moved to the left from 273.7 nm to 258.6 nm with the increase of PO₄³⁻ concentration, indicating that the anion ligand connected to Eu³⁺ changed during this process.

LOD = 3σ/K

(1)

The detection limit of the fluorescent sensor is calculated using Formula (1), where LOD is limit of detection, σ is the standard deviation of the blank, and K is the slope of the linear relationship. We tested six groups of blank samples, obtained their standard deviation, and calculated that the detection limit of the luminescence sensor for PO₄³⁻ was 0.023 mM. It shows that the sensor can be used to detect PO₄³⁻ in serum and other samples [33]. We added a known concentration of PO₄³⁻ to the sample, which reacted with PAA-Ca(Eu) nanoclusters, and then tested its luminescence at 254 nm excitation wavelength. According to the emission peak intensity at 617 nm and the linear equation in Figure 4b, the spiked recovery rate of PO₄³⁻ in the sample was calculated. The results are shown in

Table 1. Results and recovery of samples (n = 3).

PO43− Spiked (mM)	PO43− Found (mM)	Recovery (%)	RSD (%)
1	1.060	106.0	4.2
4	4.200	105.0
5	4.793	95.9
8	7.951	99.4
10	9.914	99.1

. Overall, all samples showed good recovery rates and low relative standard deviations (RSD) within the linear range, making PAA-Ca(Eu) nanoclusters a sensor for PO₄³⁻ quantitative detection.

3.2.3. Buffer Solution

It can be seen from Figure 5 that in an aqueous solution and a buffer solution, the luminescence intensity of the PAA-Ca(Eu) nanoclusters is basically the same after reacting with PO₄³⁻ of the same concentration. It proved that the luminescence sensor also has a good sensing function in the buffer solution.

3.3. Mechanism of PO₄³⁻ Concentration Detection

After adding PO₄³⁻ to PAA-Ca(Eu) nanoclusters, the vibrational peak of PO₄³⁻ appeared in FT-IR, and the peak position and intensity of CTB changed in the excitation spectra (λem = 617 nm), indicating that the anions bonded with Eu changed in this process. In addition, MDS showed that Eu³⁺ combines with the oxygen anion of the PAA carboxyl group in PAA-Ca(Eu) nanoclusters, showing Eu–O₁ CTB (Figure 6a). When PO₄³⁻ was added to the PAA-Ca(Eu) nanoclusters, the COO^– bonded Eu³⁺ was bound by the oxygen anion of PO₄³⁻, displaying a new Eu–O₂ CTB (Figure 6b). This change in the bonding state of Eu³⁺ caused an increased energy state, corresponding to the shift to a low wavelength and an increased luminescence intensity. Based on this mechanism, the quantitative detection of PO₄³⁻ can be realized.

4. Conclusions

In conclusion, we synthesized ultra-small PAA-Ca(Eu) nanoclusters with an average particle size of 2.17 nm under HRTEM observation. The nanoclusters are sensitive to PO₄³⁻, and they can be used for quantitative detection of PO₄³⁻ in a certain concentration range (0–10 mM), with good linear correlation. The LOD is 0.023 mM. Based on the sensitivity of CTB of Eu³⁺ to anionic ligand, the quantitative detection of PO₄³⁻ can be carried out. In addition, the detected concentration range by the PAA-Ca(Eu) nanoclusters sensor covers the content of PO₄³⁻ in serum, urine, and sewage. So, it is hoped that it can detect PO₄³⁻ in physiological conditions and a natural environment.