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 Eu 3+ and tetracarboxylic acid tetraphenylethylene (TCPE). Based on the principles of the Eu 3+ 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 Eu 3+ fluorescence ratio ( I 614 / I 591 ) for the crystallization feature and the TCPE fluorescence intensity ( I 468 ) for the state of PAA. During the crystallization process in the cycling system, the I 614 / I 591 ratio of Eu 3+ first decreased and then increased, and I 468 of TCPE continued to decrease. This indicated that the Ca 2+ 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 Eu 3+ fluorescence probe, the relationship between the normalized I 614 / I 591 ( y ) and the mass ratio ( x ) of Eu-HAP/Eu-PAA-ACP was established ( y = 0.347 + 0.673 × 0.874 x, R 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.