In Situ Detection of Calcium Phosphate Clusters in Solution and Wet
Amorphous Phase by Synchrotron X‑ray Absorption Near-Edge
Spectroscopy at Calcium K‑Edge
Qun Zhang,
#,†
Yun Jiang,
#,†
Bao-Di Gou,
†
Jian Huang,
†
Yu-Xi Gao,
‡
Jia-Ting Zhao,
‡
Lei Zheng,
‡
Yi-Dong Zhao,
‡
Tian-Lan Zhang,*
,†
and Kui Wang
†
†
Department of Chemical Biology, Peking University School of Pharmaceutical Sciences, 38 Xueyuan Road, Beijing 100191, P.R.
China
‡
Beijing Synchrotron Radiation Facility and Key Laboratory of Nuclear Analytical Techniques, Institute of High Energy Physics,
Chinese Academy of Sciences, 19B Yuquan Road, Beijing 100049, P.R. China
ABSTRACT: Calcium phosphate clusters are present in both
aqueous solutions and an amorphous phase during crystallization. It
is a challenging task to acquire the structural information on such
clusters, owing to their small size, chemical lability, and
inaccessibility to most detection techniques. Here, we demonstrate
the feasibility of detecting such clusters in situ by synchrotron X-ray
absorption near-edge spectroscopy at calcium K-edge, a technique
that is sensitive to the short-range order in calcium coordination
sphere. At the initial stage of crystallization, the most abundant
clusters are detected to be Ca(η
2
-PO
4
3-
)
2
L
2
(L = H
2
O or η
1
-PO
4
3-
)
in solution. More reactive clusters engage in the development of an amorphous phase via growing and fusing. The amorphous
phase exhibits a dual character in its short-range order: Some of its clusters are similar to hydrated calcium ions, and some others
to those in crystalline hydroxyapatite. When the amorphous dissolves, the detected unit is mainly Ca(η
2
-PO
4
3-
)(H
2
O)
4
in the
released solution clusters. While these findings provide a basis for a better understanding and rational control of calcium
phosphate crystallization at molecular level, the experimental technique in assessing wet samples adopted in this work might be
applicable to the crystallization studies of other materials as well.
■
INTRODUCTION
Calcium phosphates comprise the major inorganic components
in human bone and teeth, and find wide applications in
materials science and industries. Among them the least stable
solid phase is amorphous calcium phosphate (ACP), a
metastable precursor that can convert to crystalline hydrox-
yapatite [HA, Ca
10
(OH)
2
(PO
4
)
6
] through either structural
adjustment or dissolutionrecrystallization in aqueous sol-
ution.
1,2
The structure and property of ACP are closely
associated with its composing clusters. In 1974, by comparing
the X-ray radial distribution functions of ACP and HA, Betts
and Posner proposed a cluster model with the composition of
Ca
9
(PO
4
)
6
and an atomic arrangement similar to a fragment of
HA unit cell.
3,4
The Ca
9
(PO
4
)
6
cluster, also known as “Posner’s
cluster”, was assumed to be present in solution and to aggregate
into ACP particles, with water molecules being interstitial to
the domains. To make provision for the existence of protonated
phosphates
5-7
and tightly bound water molecules in ACP,
8
Eanes suggested that the cluster have a spatial domain carved
from the lattice of an acidic calcium phosphate, such as
octacalcium phosphate.
9
Studies with cryogenic transmission
electron microscopy
10
and atomic force microscopy
11
provided
morphological evidence for the existence of clusters in ACP.
Onuma and Ito even suggested that the Ca
9
(PO
4
)
6
clusters be
the growth unit of HA.
12
Recently, Habraken et al. reported a
calcium triphosphate structure [Ca(HPO
4
)
3
]
4-
that initially
emerged in solution and, afterward, developed into ACP via
binding additional calcium ions and aggregation.
13
Ionic
clusters of calcium carbonate, another kind of ubiquitously
existing biomineral, have also been reported.
14-16
The structural information on the clusters involved in ACP
formation and dissolution is essential for a better understanding
of biomineralization (such as bone metabolism) mechanisms
and for the rational control in developing the relevant materials.
However, progress has been slow in the study of solution
clusters and wet ACP, owing to their small size, chemical
lability, and inaccessibility to most detection techniques.
Thanks to the application of synchrotron radiation source to
X-ray absorption near-edge structure (XANES) spectroscopy,
17
we were able to make measurements on the suspension samples
of calcium phosphate with a qualified signal-to-noise ratio and
proposed a model of “idealized cluster” with the formula
Ca
9
(PO
4
)
6
(H
2
O)
30
.
18
The “idealized cluster” could serve as a
working model for the clusters presented at the late stage of
Received: December 21, 2014
Revised: March 9, 2015
Published: March 17, 2015
Article
pubs.acs.org/crystal
© 2015 American Chemical Society 2204 DOI: 10.1021/cg5018505
Cryst. Growth Des. 2015, 15, 2204-2210