Transformation Process Analysis of Amorphous Calcium Phosphate to Hydroxyapatite by In Situ Fluorescence Dual Probe

To characterize the transformation process of ACP to HAP (ACP-HAP) regulated by poly(acrylic acid) (PAA), an in situ fluorescence dualprobe method was established using Eu³⁺ and tetracarboxylic acid tetraphenylethylene (TCPE). Based on the principles of the Eu³⁺ hypersensitive transition matching the asymmetry of the crystal field environment and the aggregation-induced emission (AIE) of TCPE, the characteristics of the ACP-HAP process were successfully visualized through the Eu³⁺ fluorescence ratio (I₆₁₄/I₅₉₁) for the crystallization feature and the TCPE fluorescence intensity (I₄₆₈) for the state of PAA. During the crystallization process in the cycling system, the I₆₁₄/I₅₉₁ ratio of Eu³⁺ first decreased and then increased, and I₄₆₈ of TCPE continued to decrease. This indicated that the Ca²⁺ ions bonded to the carboxyl groups of PAA were transferred to the phosphate group, and PAA was gradually separated during the ACP-HAP process. Based on the Eu³⁺ fluorescence probe, the relationship between the normalized I₆₁₄/I₅₉₁ (y) and the mass ratio (x) of Eu-HAP/Eu-PAA-ACP was established (y = 0.347 + 0.673 × 0.874^x, R² = 0.993). The HAP content was quantitatively analyzed during the early stage of the ACP-HAP process in a cycling system simulating the blood flow. On the second, third, and fourth days, the content of HAP was approximately 0.900, 1.732, and 3.288%, respectively, which could not be detected by XRD. It can be speculated that this in situ fluorescence probe method is helpful to investigate the early stages of biomineralization.