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 (<i>I</i>₆₁₄/<i>I</i>₅₉₁) for the crystallization feature and the TCPE fluorescence intensity (<i>I</i>₄₆₈) for the state of PAA. During the crystallization process in the cycling system, the <i>I</i>₆₁₄/<i>I</i>₅₉₁ ratio of Eu³⁺ first decreased and then increased, and <i>I</i>₄₆₈ 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 <i>I</i>₆₁₄/<i>I</i>₅₉₁ (<i>y</i>) and the mass ratio (<i>x</i>) of Eu-HAP/Eu-PAA-ACP was established (<i>y</i> = 0.347 + 0.673 × 0.874<i>^x</i>, <i>R</i>² = 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.