Study On Calcium Phosphate Prenucleation Cluster Formation By Eu³⁺ In-Situ Fluorescence Probe

Whether it is normal mineralization of hard tissues such as mammalian bones and teeth,or pathological mineralization such as ectopic osteogenesis and atherosclerosis,the common key scientific problem is the mineralization process and mechanism of calcium phosphate（Ca P）biominerals.Among,prenucleation clusters（PNCs）formed in the early stage play a key role in the subsequent nucleation and crystallization of Ca P.Therefore,the study of the formation and evolution behavior of PNCs is very important for the understanding of mineralization process.Because of the unique properties of PNCs:ultra-small size（～2 nm）,liquid-like and extremely fast formation,the research on its formation process has always been a difficult problem to be solved.The structure,stoichiometry,kinetics and thermodynamics of PNCs have not been clear.The traditional ex-situ methods can not realize continuous monitoring and may destroy its structure.The in-situ methods are effective means to study the formation process of PNCs.In this paper,referring to the process of Polymer induced liquid precursor（PILP）,polyacrylic acid（PAA）was used as the process guide to construct prenucleation cluster precursors（pre-PNCs）to simulate the formation process of PNCs.On this basis,an in-situ fluorescence probe method was developed to study the formation of PNCs by partially replacing Ca²⁺in pre-PNCs with Eu³⁺.Combined with HRTEM,DLS,XPS,FT-IR and other characterization,Ca²⁺—PO₄^3-bonding was observed during the formation of PNCs,and the change of Eu-O charge transfer band（CTB）fluorescence was observed:with the increase of PO₄^3-concentration,the peak position of Eu-O charge transfer band（CTB）of Eu³⁺moved to low wavelength,and the peak intensity gradually increased,indicating that Ca（Eu）-O bonding changed from PAA carboxyl oxygen to phosphorus oxygen.In other words,Eu-O CTB fluorescence is responsive to the Ca²⁺—PO₄^3-bonding process,that is,the formation of PNCs.The formation of PNCs can be detected in situ through the change of fluorescence intensity increase rate.The thermodynamic and kinetic behavior of PNC formation was investigated by Eu³⁺in situ fluorescence probe.Firstly,the pseudo second-order kinetic model is used to describe the kinetic process of PNCs.The formation of PNCs can be attributed to chemical adsorption;Further combined with DLS and zeta potential characterization,it shows that PNCs have the characteristics of ionic liquid and tend to be thermodynamically stable.Secondly,the thermodynamic characteristics of the formation of PNCs are revealed and supplemented.The formation of PNCs conforms to the Langmuir isothermal adsorption model and is endothermic and hydroxyl driven.Third,we infer that PNCs are composed of Ca²⁺and PO₄^3-binding region and PO₄^3-adsorption region.Because of the existence of polyanions,the bonding region is PO₄^3-deficient,while the PO₄^3-adsorption region is formed through the hydrogen bond between HPO₄^2-and polyanions.PNCs have relatively fixed and high Ca/P ratio in the bonding region（depending on the polyanion）,and the orthophosphate ion adsorption region can be regarded as the phosphorus pool of PNCs.It is this structural feature that gives PNCs strong flexibility.As a precursor,PNCs can start a non-classical crystallization pathway and regulate the biomineralization of Ca P.Finally,consistent experimental conclusions were obtained by molecular dynamics simulation,which verified the effectiveness of Eu³⁺in-situ fluorescence probe method.