Method for analyzing aggregation crystallization process of calcium phosphate pre-nucleation cluster
The aggregation and crystallization process of calcium phosphate pre-nuclear clusters is detected by the fluorescent double-probe method using aggregation-induced luminescent organic molecules and rare earth ions, which solves the problem of difficult characterization of calcium phosphate biomineralization process in the prior art, and achieves efficient and low-cost dynamic monitoring.
Patent Information
- Application Number
- CN202510290844.2
- Authority / Receiving Office
- CN · China
- Patent Type
- Applications(China)
- Current Assignee / Owner
- Filing Date
- 2025-03-12
- Publication Date
- 2025-06-13
AI Technical Summary
The prior art is difficult to accurately and reliably characterize the biomineralization process of calcium phosphate, especially when the sample crystallinity requirements are high, detection information is limited, or testing costs are high.
The fluorescent dual-probe method is used to signal the PNCs aggregation crystallization process through fluorescence signals by using aggregation-induced luminescent organic molecules and rare earth ions as detectors in response to the aggregation and crystallization process of calcium phosphate pre-nuclear clusters.
Continuous dynamic monitoring of the aggregation and crystallization process of calcium phosphate pre-nuclear clusters is realized, with the advantages of low cost and rich detection material characteristics, and overcomes the problems of limited information and high cost in the prior art.
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Abstract
Description
Technical Field
[0001] The present invention belongs to the technical field of material analysis, and particularly relates to a method for analyzing the aggregation and crystallization process of calcium phosphate prenucleation clusters. Background Art
[0002] The main inorganic phase in biological hard tissues (bones, teeth) is calcium phosphate, which is formed by biomineralization under the regulation of biomolecules in vivo. Exploring the main factors regulating calcium phosphate mineralization and elucidating the related mineralization mechanism have guiding significance for the treatment of clinical pathological mineralization diseases and the deepening of the understanding of biomineralization. Research shows that biomineralization belongs to a non-classical crystallization process, that is, calcium phosphate clusters are formed in solution, the clusters aggregate into an amorphous phase, and finally a phase transformation forms a crystalline phase. Currently, it is known that there are different phases of calcium phosphate in vivo, such as amorphous calcium phosphate and hydroxyapatite, but the mechanism of its molecular-regulated aggregation and crystallization still needs to be further studied.
[0003] Non-in-situ methods cannot achieve continuous in-situ signal acquisition because the samples need to be separated, and the information that can be obtained is limited. In-situ characterization methods such as high-energy X-ray diffraction (HE-XRD) can detect the phase transformation of perovskite nanocrystals (PNCs), but have high requirements for the crystallinity of the samples; atomic force microscopy (AFM) and Raman spectroscopy can reveal the dynamic changes in the morphology and composition of PNCs, but the detected information is limited; in-situ liquid transmission electron microscopy (In situ-TEM) can simultaneously study the morphology and phase transformation process of PNCs, but the test cost is high. Therefore, there is an urgent need to develop an accurate and reliable characterization technology applicable to the calcium phosphate biomineralization process. Summary of the Invention
[0004] In order to overcome the problems existing in the above-mentioned prior art, the purpose of the present invention is to provide a method for analyzing the aggregation and crystallization process of calcium phosphate prenucleation clusters.
[0005] Aggregation-induced emission (AIE) organic molecules can effectively restrict intramolecular motion and exhibit unique fluorescence properties of non-emission at low concentrations and strong emission in the condensed state, and their applications in fields such as solid-state lighting, specific detection, and near-infrared imaging have received extensive attention. Research has found that the macroscopic dispersion and spatial distribution of montmorillonite in a polyvinyl chloride matrix can be visualized using a tetraphenylethylene fluorescent probe. Rare earth ions have narrow emission bands, with definite magnetic dipole and electric dipole transitions, and can also be used as spectroscopic probes for the structural analysis of substances. Research shows that Eu 3+The probe can be used to identify the phase transitions among monoclinic, tetragonal, and cubic zirconia phases. The present invention utilizes the aggregation-induced emission (AIE) characteristics of organic molecules to respond to the "cluster aggregation process" and the hypersensitive transition characteristics of rare-earth ions to respond to the "asymmetry of coordination environment" to construct a fluorescence dual-probe, which respectively detects the aggregation and crystallization processes of calcium phosphate prenucleation clusters, and signals the fluorescence of the aggregation and crystallization processes of PNCs, providing a novel in-situ characterization method for studying calcium phosphate biomineralization.
[0006] To achieve the above object, the technical solution adopted by the present invention is as follows:
[0007] The present invention provides a method for analyzing the aggregation and crystallization processes of calcium phosphate prenucleation clusters, comprising the following steps:
[0008] Mix a polyanion, an aggregation-induced emission organic molecule, calcium ions, and rare-earth ions in water to obtain a prenucleation cluster precursor solution; mix the prenucleation cluster precursor solution with phosphate ions to obtain a prenucleation cluster solution; induce the prenucleation cluster solution to undergo aggregation and crystallization in sequence, perform fluorescence detection on the aggregation and crystallization product, and analyze the formation process of calcium phosphate prenucleation clusters using the fluorescence signal.
[0009] Preferably, the polyanion is selected from at least one of polyacrylic acid, polyaspartic acid, polyglutamic acid, and polyacrylamine hydrochloride.
[0010] Biomineralization is a non-classical crystallization process, that is, prenucleation clusters are first formed, then aggregated into an amorphous phase, and finally transformed into a crystal structure through a phase change. In this process, charged substances are often involved. For example, proteins are often involved in the regulation of the mineralization process of human bones and teeth, and organic matter is involved in the formation process of the shell of a shell. The present invention adds a polyanion as a process guiding agent to guide the formation of polymer-induced precursors (PILP), simulating the non-classical crystallization path of actual biomineralization; moreover, the entire mineralization process can be regulated by setting the parameters of the polymer guiding agent, providing convenience for studying the specific mechanism of mineralization.
[0011] Preferably, the aggregation-induced emission organic molecule includes at least one of tetrakis(4-carboxyphenyl)ethylene, 9,10-bis(4-carboxyphenyl)anthracene, 1,2,4,5-tetrakis(4-carboxyphenyl)benzene, and 2,3,5,6-tetrakis(4-carboxyphenyl)pyrazine.
[0012] Preferably, the rare-earth ion includes at least one of europium ion, terbium ion, cerium ion, dysprosium ion, thulium ion, and samarium ion.
[0013] Preferably, the molar ratio of the calcium ions to the rare-earth ions is 1:(0.005 - 0.05).
[0014] Preferably, the molar ratio of the calcium ions to the polyanion is 1: (1 to 3).
[0015] Preferably, the molar ratio of the calcium ions to the aggregation-induced emission organic molecule is 1: (0.005 to 0.05).
[0016] Preferably, the molar ratio of the calcium ions to the phosphate ions is 1: (0.1 to 1).
[0017] Preferably, the following steps are further included: mixing an aqueous solution containing a polyanion and calcium ions with an aqueous solution of an aggregation-induced emission organic molecule, and stirring at 25 to 80 °C for 2 to 12 h to obtain a prenucleation cluster precursor solution.
[0018] Preferably, the pH value of the prenucleation cluster precursor solution and the prenucleation cluster solution is 6 to 12.
[0019] More preferably, the pH value is adjusted with NaOH.
[0020] Preferably, the method for inducing aggregation of the prenucleation cluster solution includes the following steps: adding calcium ions to the prenucleation cluster solution to obtain a mixed solution, and evaporating the water in the mixed solution.
[0021] More preferably, the evaporation method includes the following steps: stirring the mixed solution at 25 to 50 °C for 1 to 3 h.
[0022] Further preferably, the mixed solution is stirred at 35 to 40 °C for 1.5 to 2.5 h.
[0023] More preferably, the molar ratio of the amount of calcium ions added to the prenucleation cluster solution to the calcium ions in the prenucleation cluster solution is (0.5 to 3): 1.
[0024] Preferably, the crystallization method includes the following steps: adding phosphate to the mixed solution after aggregation.
[0025] Preferably, the analysis method includes the following steps: fitting the Boltzmann model according to the fluorescence signal to analyze the aggregation behavior of calcium phosphate prenucleation clusters.
[0026] More preferably, the fluorescence signal includes the fluorescence emission characteristic peaks of organic molecules and rare earth ions; for the fluorescence characteristic peak of the organic molecule, the emission light intensity increase rate is calculated: ΔI = (I - I pre ) / I pre , where I and I pre respectively represent the fluorescence characteristic peak intensities of the PNCs solution and the PNCs aggregation crystallization product; the ΔI change curve is fitted with the Boltzmann model: where A 1 and A 2 are ΔIc at high and low calcium ion concentrations respectively 0 is the midpoint of the curve mutation and can be used as the critical micelle concentration (CMC) of the colloid; for the fluorescence characteristic peak of rare earth ions, calculate the ratio R of the fluorescence characteristic peak intensities corresponding to the electric dipole transition (hypersensitive transition) and the magnetic dipole transition I .
[0027] More preferably, the analysis method further includes the following steps: judging the agglomeration size of calcium phosphate according to the fluorescence signal amplification rate of organic molecules and the ratio of rare earth ion characteristic peaks
[0028] More preferably, the analysis method further includes the following steps: judging the crystal form of calcium phosphate according to the ratio of rare earth ion characteristic peaks
[0029] More preferably, the analysis method further includes the following steps: monitoring the crystal phase transformation from octacalcium phosphate to hydroxyapatite according to the ratio of rare earth ion characteristic peaks
[0030] The beneficial effects of the present invention are as follows
[0031] The present invention provides a method for analyzing the aggregation and crystallization process of calcium phosphate prenucleation clusters. A small amount of aggregation-induced emission organic molecules and rare earth ions are incorporated into the calcium phosphate prenucleation clusters, and the two are used as dual fluorescence probes to participate in the subsequent evolution process. The deprotonated organic molecules bind to calcium ions in the clusters. As the PNCs aggregate, the local molecular density of the organic molecules gradually increases, the restriction of intramolecular motion is further strengthened, the reduction of excited state energy loss leads to the opening of the electron transition channel, and the fluorescence signal enhancement phenomenon appears. The electric dipole transition (hypersensitive transition) of rare earth ions is sensitive to the asymmetry of the surrounding coordination environment, while the magnetic dipole transition is not sensitive. Therefore, the ratio of the two can be used to preliminarily judge the chemical structure of rare earth ions. In short, the present invention utilizes the aggregation-induced emission characteristics of organic molecules to respond to the "cluster aggregation process" and the hypersensitive transition characteristics of rare earth ions to respond to the "coordination environment asymmetry" to match the aggregation and crystallization process of PNCs and explain its biomineralization mechanism. Compared with the prior art, the present invention can realize the continuous dynamic monitoring of the aggregation and crystallization behavior of PNCs, and has the advantages of low cost and rich detection material characteristics Description of the Drawings
[0032] Figure 1 : Aggregation and crystallization of CaP PNCs detected by in-situ fluorescence probe method at different Ca 2+ addition amounts
[0033] Figure 2 : Aggregation and crystallization of CaP PNCs detected by in-situ fluorescence probe method at different temperatures
[0034] Figure 3: Detection of the aggregation and crystallization of CaP PNCs at different pH values by in-situ fluorescence probe method;
[0035] Figure 4 : I of HAP and OCP blends with different content ratios 613 / I 591 value relationship diagram.
[0036] Figure 5 : Luminescence fluorescence spectra of TPE-COOH solution at different Ca 2+ addition amounts.
[0037] Figure 6 : In-situ detection of dual fluorescence probes during calcium phosphate crystallization, where (a) is the XRD pattern of the product after triggering crystallization by adding different concentrations of phosphate, and (b) is the relationship diagram between different phosphate concentrations and the I 613 / I 591 value relationship diagram. Specific implementation manner
[0038] The content of the present invention will be further described in detail through specific examples below. The raw materials used in the following examples can be obtained from conventional commercial channels or prepared and separated by simple synthesis, unless otherwise specified; the processes used, unless otherwise specified, are conventional processes in the art.
[0039] Example 1
[0040] Example 1 provides a method for analyzing the aggregation and crystallization process of calcium phosphate prenucleation clusters. The specific analysis method is as follows:
[0041] Prepare a 1 g / L aqueous solution of TPE-COOH and adjust its pH value to 9-10 using NaOH. Stir 5 mL of the TPE-COOH aqueous solution, 2 mmol, 25 mL of the PAA aqueous solution, 1 mmol, 5 mL of the calcium ion aqueous solution, and 0.03 mmol, 5 mL of the europium ion aqueous solution to form a 40 mL mixed solution. Add an appropriate amount of NaOH aqueous solution to adjust the pH value of the mixed solution to 9, and stir at 37 °C for 30 min to obtain a calcium phosphate prenucleation cluster precursor solution. Add 0.6 mmol of phosphate aqueous solution to the precursor solution, set the final volume to 50 mL, and continue to stir at 37 °C for 2 h to obtain a calcium phosphate prenucleation cluster solution. Add 0, 0.2, 0.4, 0.6, 0.8, 1.0, 1.2, 1.5, 2.0, 2.5, 3.0, 4.0, 5.0 mmol of calcium ions to the prenucleation cluster solution to trigger cluster aggregation, and keep it at 37 °C for 2 h.
[0042] Transfer 2 mL of the aggregated product into a quartz cuvette of a fluorescence spectrophotometer, and record the emission light intensities of the sample at wavelengths of 467 nm, 591 nm, and 613 nm under the excitation light at wavelengths of 367 nm and 398 nm, 25 °C, and pH = 7.4, Figure 1 (a) in 2+ is the fluorescence emission spectrum of the product under the excitation of light with a wavelength of 367 nm at different Ca Figure 1 (b) in 2+ is the fluorescence emission spectrum of the product under the excitation of light with a wavelength of 398 nm at different Ca pre addition amounts. Calculate the growth rate of the fluorescence emission intensity of the organic molecule ΔI: ΔI = (I - I pre ) / I pre , where I and I Figure 1 respectively represent the fluorescence characteristic peak intensities of the PNCs solution and the PNCs aggregated crystallization product. Plot a relationship graph with the added calcium ion concentration as the abscissa and ΔI as the ordinate. See (c) in Figure 1 . As seen from (d) in 3+ , compared with the Eu Figure 1 rare earth fluorescence probe, the TPE-COOH fluorescence probe is more sensitive to the concentration of added calcium ions, and has higher sensitivity under the excitation light with a wavelength of 398 nm. It can be seen that with the addition of a small amount of calcium ions, the ΔI value increases slightly. When the added calcium ions exceed the critical amount, the ΔI value increases significantly and then levels off. This is similar to the colloidal property. After fitting this curve with Boltzmann, it is found that the critical calcium ion concentration is 2.16 mmol. See (e) in
[0043] Calculate the ratio R I of the fluorescence emission peaks of rare earth ions, I 613 / I 591 and the relationship with the added amount of Ca 2+ is specifically as shown in (f) in Figure 1 . With the addition of calcium ions, the R I value decreases slightly. When it exceeds the critical amount, the R I value decreases significantly and then levels off, which is consistent with the fluorescence change law of organic molecules, indicating that the asymmetry of the coordination environment around europium ions gradually decreases with the aggregation of clusters.
[0044] Appendix Figure 1 (g) in 2+ is the transmission electron microscope image of the CaP PNCs aggregates at different Ca
[0045] Example 2
[0046] Example 2 provides a method for analyzing the aggregation crystallization process of calcium phosphate prenucleation clusters. The specific analysis method is as follows:
[0047] Prepare a 1 g / L TPE-COOH solution and adjust its pH value to 9-10 using NaOH. Stir 5 mL of the TPE-COOH aqueous solution, 2 mmol, 25 mL of the PAA aqueous solution, 1 mmol, 5 mL of the calcium ion aqueous solution, and 0.03 mmol, 5 mL of the europium ion aqueous solution to form a 40 mL mixed solution. Add an appropriate amount of NaOH aqueous solution to adjust the pH value of the mixed solution to 9, and stir at 37 °C for 30 min to obtain a calcium phosphate prenucleation cluster precursor solution. Add 0.6 mmol of the phosphate aqueous solution to the precursor solution, set the final volume to 50 mL, and continue to stir at 37 °C for 2 h to obtain a calcium phosphate prenucleation cluster solution. Add 0.5 mmol, 1 mmol, 1.5 mmol, 2 mmol, 3 mmol of calcium ions to the prenucleation cluster solution to trigger cluster aggregation, and incubate at temperatures of 25 °C, 37 °C, and 50 °C in an oven for 2 h. Take 2 mL of the aggregated product and place it in a quartz cuvette of a fluorescence spectrophotometer, and record the emission light intensity of the sample at wavelengths of 467 nm, 591 nm, and 613 nm under the excitation light at a wavelength of 398 nm.
[0048] Figure 2 (a), (b), and (c) in are 1.5 mmol Ca 2+ Fluorescence emission spectra of triggering the aggregation of CaP PNCs for 2 h at 25 °C (a), 37 °C (b), 50 °C (c), and pH = 7.4. Calculate the growth rate of the fluorescence emission intensity of the organic molecule ΔI, and plot a relationship diagram under different temperature conditions with the calcium ion concentration added as the abscissa and ΔI as the ordinate. See Figure 2 (d) in. As the temperature increases, the value of ΔI gradually increases, and the overall change trend remains the same. Therefore, heating promotes cluster aggregation crystallization.
[0049] Example 3
[0050] Example 3 provides a method for analyzing the aggregation crystallization process of calcium phosphate prenucleation clusters. The specific analysis method is as follows:
[0051] Prepare a 1 g / L TPE-COOH solution and adjust its pH value to 9 - 10 using NaOH. Stir 5 mL of the TPE-COOH aqueous solution, 2 mmol (25 mL) of the PAA aqueous solution, 1 mmol (5 mL) of the calcium ion aqueous solution, and 0.03 mmol (5 mL) of the europium ion aqueous solution to form a 40 mL mixed solution. Add an appropriate amount of NaOH aqueous solution to adjust the pH value of the mixed solution to 6, 7, 8, 9, and stir at 37 °C for 30 min to obtain a calcium phosphate prenucleation cluster precursor solution. Add 0.6 mmol of the phosphate root aqueous solution to the precursor solution, set the final volume to 50 mL, and continue to stir at 37 °C for 2 h to obtain a calcium phosphate prenucleation cluster solution. Add 0.5 mmol, 1 mmol, 1.5 mmol, 2 mmol, and 3 mmol of calcium ions to the prenucleation cluster solution to trigger cluster aggregation and incubate at 37 °C for 2 h. Take 2 mL of the aggregated product and place it into a quartz cuvette of a fluorescence spectrophotometer, and record the emission light intensity of the sample at wavelengths of 467 nm, 591 nm, and 613 nm under the excitation light of 398 nm wavelength.
[0052] Figure 3 (a), (b), (c), (d) in are 1.5 mmol Ca 2+ Fluorescence emission spectra of triggering CaP PNCs aggregation at 25 °C, pH = 6 (a), 7 (b), 8 (c), 9 (d) for 2 h. Calculate the growth rate of the fluorescence emission intensity of the organic molecule ΔI, and plot a relationship graph with the calcium ion concentration added as the abscissa and ΔI as the ordinate under different pH value conditions. Figure 3 (e) in is the comparison of the ΔI value changes at different pH values. As the pH value increases, the ΔI value gradually increases, and the overall change trend remains the same. A high pH value promotes cluster aggregation and crystallization.
[0053] Calculate the fluorescence emission peak ratio R of rare earth ions I , Figure 3 (f) in is the comparison of the I 613 / I 591 value changes at different pH values. As the pH value increases, the R I value decreases with a larger amplitude, which is consistent with the fluorescence change law of organic molecules.
[0054] Example 4
[0055] Example 4 explored the fluorescence characteristics and crystal phase relationship, established a standard curve for the correlation between the two, and provided a reference standard library for judging the crystal phase through fluorescence signals in the next Example 5. The specific analysis method is as follows:
[0056] Mix 5 mmol of Na 2 HPO 4 ·12H 2O and 5 mmol NaH 2 PO 4 were dissolved in 750 ml of deionized water, and the pH value was adjusted to 3 to 6 with HCl to prepare a mixed solution A. Eu(NO 3 ) 3 ·6H 2 O and Ca(CH 3 COO) 2 were used to prepare a mixed solution B with a Ca 2+ / Eu 3+ concentration of 0.04 mol / L and a Eu / (Eu + Ca) molar ratio of 1% to 5%. Solution A was heated and maintained at 80 °C, and then solution B was added to solution A under stirring conditions. After reacting for 10 minutes, the precipitate was separated by centrifugation, washed four times, and freeze-dried to obtain Eu-OCP powder.
[0057] Eu(NO 3 ) 3 ·6H 2 O and CaCl 2 ·2H 2 O were used to prepare a mixed solution with a Ca 2+ / Eu 3+ concentration of 0.0668 mol / L and a Eu / (Eu + Ca) molar ratio of 1% to 5%. The mixed solution was heated and maintained at 37 °C. 0.04 mol / L Na 2 HPO 4 ·12H 2 O was quickly poured into the mixed solution while adjusting the pH value with NaOH. The reaction was maintained for 60 minutes. Finally, the precipitate was separated by centrifugation, washed four times, and freeze-dried to obtain Eu-HAP powder.
[0058] By comparing the fluorescence differences between Eu-OCP and Eu-HAP, the two showed different fluorescence characteristics in terms of fluorescence intensity and I 613 / I 591 ratio. Based on the Eu 3+ fluorescent probe, the relationship between the I 613 / I 591 ratio and the mass ratio of HAP to OCP in the HAP / OCP mixture was revealed. Figure 4 is a relationship diagram of the I 613 / I 591 values for HAP and OCP blends with different content ratios. According to the fluorescence signal, the Boltzmann model was fitted to analyze the aggregation behavior of calcium phosphate prenucleation clusters, as shown in Table 1 below:
[0059] Table 1
[0060] Model Boltzmann Equation y = A2+(A1 - A2) / (1 + exp((x - x0) / dx)) A1 1.39464 ± 0.02003 A2 2.0971 ± 0.03132 x0 0.68956 ± 0 dx 0.11274 ± 0 Reduced Chi-Sq 0.00233 COD(R^2) 0.96957
[0061] I 613 / I 591 The ratio is related to the content (x) of HAP in the HAP / OCP mixture as (I 613 / I 591 = 2.0971 - 0.70246 / (1 + exp((x - 0.68956) / 0.11274)), R 2 = 0.970). Meanwhile, the relationship between the I (1 1 2) / I (0 1 0) ratio and the mass ratio of HAP to OCP in the HAP / OCP mixture was revealed. I (1 1 2) / I (0 1 0) The relationship between the ratio and the content (x) of HAP in the HAP / OCP mixture is (I (112) / I (010) = 4.91063E-9exp(-x / -0.04601) + 0.76765exp(-x / 0.76765) + 0.52433exp(-x / -5.03863E89) - 1.28922, R 2 = 0.997). Through this fluorescence probe method, the phase transformation from OCP to HAP during the hydrolysis of OCP was quantitatively analyzed, which was consistent with the Figure 1 XRD analysis in
[0062] Comparative Example 1
[0063] Under the same experimental conditions, different amounts of Ca were added to the mixed solution of PAA and AIE molecules, 2+ and analyzed with a fluorescence spectrophotometer. The results are as Figure 5 shown. There was no obvious change in the fluorescence intensity, indicating that the change in the fluorescence intensity of the AIE characteristic peak in the examples was caused by the aggregation of calcium phosphate prenucleation clusters, and the AIE molecules themselves did not aggregate and crystallize.
[0064] The above is the preferred embodiment of the present invention. It should be noted that for those of ordinary skill in the art, without departing from the principle of the present invention, several improvements and refinements can still be made, and these improvements and refinements are also regarded as the protection scope of the present invention.
[0065] Example 5
[0066] Example 5 provides a method for analyzing the aggregation and crystallization process of calcium phosphate prenucleation clusters. The specific analysis method is as follows:
[0067] Prepare a 1 g / L TPE-COOH solution and adjust its pH value to 9 - 10 using NaOH. Mix 5 mL of the TPE-COOH aqueous solution, 2 mmol (25 mL) of PAA, 1 mmol (5 mL) of calcium ions, and 0.03 mmol (5 mL) of europium ions to form a 40 mL mixed solution. Add an appropriate amount of NaOH aqueous solution to adjust the pH value of the mixed solution to 7, and stir at 37 °C for 30 min to obtain a calcium phosphate prenucleation cluster precursor solution. Add 0.6 mmol of phosphate aqueous solution to the precursor solution, set the final volume to 50 mL, and continue to stir at 37 °C for 2 h to obtain a calcium phosphate prenucleation cluster solution. Add 1.5 mmol of calcium ions to the prenucleation cluster solution to trigger cluster aggregation, and keep it at 37 °C for 2 h. Add 0 mmol, 0.3 mmol, 0.6 mmol, 0.9 mmol, 1.2 mmol, 1.5 mmol, 1.8 mmol, and 2.1 mmol of phosphate solutions to the aggregated solution to trigger the crystallization process. Take 2 mL of the crystallized product and place it in a quartz cuvette of a fluorescence spectrophotometer, and record the emission light intensity of the sample at wavelengths of 591 nm and 613 nm under an excitation light of 398 nm wavelength.
[0068] Figure 6 In (a), it is the XRD pattern of the product after triggering the crystallization of calcium phosphate aggregates with different concentrations of added phosphate. As the concentration of added phosphate increases, the diffraction peaks of (002) and (211) representing the hydroxyapatite crystal phase gradually become obvious, indicating the gradual formation of the hydroxyapatite crystal phase. Figure 6 In (b), it is the fluorescence emission peak ratio of the above product at 613 nm and 591 nm. It can be judged that during the crystallization process, the asymmetry of the local chemical coordination environment experiences a process of first decreasing and then increasing.
Claims
1. A method for analyzing the crystallization process of calcium phosphate pre-nucleation clusters, characterized in that: The steps include: The polyanion, aggregation-induced luminescent organic molecule, calcium ion and rare earth ion are mixed in water to obtain a pre-nucleation cluster precursor solution; the pre-nucleation cluster precursor solution is mixed with phosphate ions to obtain a pre-nucleation cluster solution; the pre-nucleation cluster solution is induced to aggregate and crystallize in sequence, the aggregated crystal product is fluorescently detected, and the formation process of the calcium phosphate pre-nucleation cluster is analyzed using the fluorescent signal.
2. The method for analyzing the calcium phosphate pre-nucleation cluster aggregation crystallization process according to claim 1, wherein The polyanion is selected from at least one of polyacrylic acid, polyaspartic acid, polyglutamic acid and polyacrylamine hydrochloride.
3. The method for analyzing the calcium phosphate pre-nucleation cluster aggregation crystallization process according to claim 1, wherein The aggregation-induced emission organic molecules include at least one of tetrakis(4-carboxyphenyl)ethylene, 9,10-di(4-carboxyphenyl)anthracene, 1,2,4,5-tetrakis(4-carboxyphenyl)benzene, and 2,3,5,6-tetrakis(4-carboxyphenyl)pyrazine.
4. The method for analyzing the calcium phosphate pre-nucleation cluster aggregation crystallization process according to claim 1, wherein The rare earth ions include at least one of europium ions, terbium ions, cerium ions, dysprosium ions, thulium ions, and samarium ions.
5. The method for analyzing the calcium phosphate pre-nucleation cluster aggregation crystallization process according to claim 1, characterized in that, The molar ratio of the calcium ion to the rare earth ion is 1:(0.005-0.05); and / or, the molar ratio of the calcium ion to the polyanion is 1:(1-3); And / or, the molar ratio of the calcium ions to the aggregation-induced emission organic molecules is 1:(0.005-0.05).
6. The method for analyzing the calcium phosphate pre-nucleation cluster aggregation crystallization process according to claim 1, characterized in that, The pH value of the pre-nucleation cluster precursor solution and the pre-nucleation cluster solution is 6-12.
7. The method for analyzing the calcium phosphate pre-nucleation cluster aggregation crystallization process according to claim 1, characterized in that, The method of inducing the pre-nucleation cluster solution to aggregate comprises the following steps: adding calcium ions to the pre-nucleation cluster solution to obtain a mixed solution, and evaporating water in the mixed solution; And / or, the crystallization method comprises the following steps: adding phosphate to the mixed solution after aggregation.
8. The method for analyzing the calcium phosphate pre-nucleation cluster aggregation crystallization process according to claim 7, characterized in that, The evaporation method comprises the following steps: the mixed solution is stirred at 25-50° C. for 1-3 hours.
9. The method for analyzing the calcium phosphate pre-nucleation cluster aggregation crystallization process according to claim 1, characterized in that, The analysis method comprises the following steps: fitting a Boltzmann model according to the fluorescence signal to analyze the aggregation behavior of calcium phosphate pre-nucleation clusters.
10. The method for analyzing the crystallization process of calcium phosphate pre-nucleation clusters according to claim 9, characterized in that: The analysis method further comprises the following steps: judging the size of calcium phosphate aggregation according to the amplification rate of the organic molecule fluorescence signal and the ratio of the rare earth ion characteristic peak; And / or, the analysis method further comprises the following steps: determining the crystal form of calcium phosphate according to the ratio of the characteristic peaks of rare earth ions and monitoring the crystal phase transformation of octacalcium phosphate to hydroxyapatite.