In situ fluorescence dual probe for detecting evolution of calcium phosphate prenucleation clusters in
Jia Chen1, Ziyou Ding1, Heng Zhou1
1State Key Laboratory of Advanced Technology for Materials Synthesis and Processing, Biomedical Materials and Engineering Research Center of Hubei Province, Wuhan University of Technology, Wuhan, 430070, PR China.
Background:
Biomineralization, regulated by biomolecules, produces hard tissues with diverse structures and physiological functions. Calcium phosphate (CaP) prenucleation clusters (PNCs) serve as the initial components in this process. However, the mechanisms underlying the evolution of CaP PNCs have not yet been elucidated. Due to the molecular and atomic-scale nature of these processes, the sensitivity and accuracy of in situ characterization methods are crucial for analysis. Current in situ methods are limited by high costs, high radiation emissions, and demanding experimental conditions.
Results:
This study establishes an in situ fluorescence dual probe method using Eu3+ and tetracarboxylic acid tetraphenylethylene (TCPE) to characterize the formation, aggregation, and crystallization of CaP PNCs. The method leverages the following properties: (1) the charge transfer transition of Eu3+ matches the Ca2+-PO43- bonding process, (2) TCPE fluorescence emission enhancement correlates with CaP PNC aggregation, and (3) the hypersensitive transition of Eu3+ reflects the asymmetry of the crystal field environment. The accuracy of this method was validated through ex situ characterization and analog calculations. Results reveal competitive bonding between citrate/DNA and inorganic phosphorus with PNC precursors (pre-PNCs). Citrate-containing CaP PNCs exhibit inhibited aggregation, while DNA-containing CaP aggregates display a "contacting but not fusing" behavior. Additionally, the asymmetric variation trend of the amorphous phase during crystallization was investigated.
Significance:
This study introduces a novel optical strategy for in situ monitoring of CaP PNC formation and aggregation involving biomolecules. By combining molecular dynamics simulations and extended DLVO theory, the mechanisms underlying biomineralization are elucidated. The findings provide insights into early-stage biomineralization and pathological mineralization, offering potential applications in disease treatment and biomaterial design.
