7. Controlled formation of calcium-phosphate-based hybrid mesocrystals by
organic–inorganic co-assembly
Halei Zhai,a
Xiaobin Chu,a
Li Li,a
Xurong Xuab
and Ruikang Tang*ab
Received 28th July 2010, Accepted 27th August 2010
DOI: 10.1039/c0nr00542h
An understanding of controlled formation of biomimetic mesocrystals is of great importance in
materials chemistry and engineering. Here we report that organic–inorganic hybrid plates and even
mesocrystals can be conveniently synthesized using a one-pot reaction in a mixed system of protein
(bovine serum albumin (BSA)), surfactant (sodium bis(2-ethylhexyl) sulfosuccinate (AOT)) and
supersaturated calcium phosphate solution. The morphologies of calcium-phosphate-based products
are analogous to the general inorganic crystals but they have abnormal and interesting substructures.
The hybrids are constructed by the alternate stacking of organic layer (thickness of 1.31 nm) and
well-crystallized inorganic mineral layer (thickness of 2.13 nm) at the nanoscale. Their morphologies
(spindle, rhomboid and round) and sizes (200 nm–2 mm) can be tuned gradually by changing BSA,
AOT and calcium phosphate concentrations. This modulation effect can be explained by a competition
between the anisotropic and isotropic assembly of the ultrathin plate-like units. The anisotropic
assembly confers mesocrystal characteristics on the hybrids while the round ones are the results of
isotropic assembly. However, the basic lamellar organic–inorganic substructure remains unchanged
during the hybrid formation, which is a key factor to ensure the self-assembly from molecule to
micrometre scale. A morphological ternary diagram of BSA–AOT–calcium phosphate is used to
describe this controlled formation process, providing a feasible strategy to prepare the required
materials. This study highlights the cooperative effect of macromolecule (frame structure), small
biomolecule (binding sites) and mineral phase (main component) on the generation and regulation of
biomimetic hybrid mesocrystals.
Introduction
Scientists are eager to mimic nature’s ability to design functional
materials whose properties are often superior to the synthetic
ones. In nature, biominerals are widely produced by bacteria,
protists, plants, invertebrates and vertebrates, including
humankind.1
These biological materials are featured by a smart
combination of multi-components especially in the form of
integrated organic–inorganic hybrid materials, in which the
organic parts are often proteins and low-molecular-mass mole-
cules.2
They are constructed by using organic components to
control the nucleation, growth, organization and transformation
of inorganic phases. Interactions between organic and inorganic
phases at the molecular level, although complex, are common
occurrences to determine the size, shape, and properties of the
resulting products.1,3
Different from the synthesized ones, the
functions of biominerals depend to a large extent on the ordered
association of biomolecules with mineral phases. The organized
hybrid materials, unlike the single components, can be tailored
into different compositions and morphologies, e.g. bone,4
tooth5
and mollusc shells6
etc., to ensure the optimal mechanical and
physicochemical characteristics.
The controls that determine the sizes, shapes, and properties of
crystals are a key to addressing numerous challenges in material
designs and applications. It has been revealed that organic
molecules can influence the shape and properties of inorganic
crystals.7
However, it is difficult for the two distinct organic and
inorganic phases to spontaneously assemble into highly ordered
structures. In living organisms, biological mineralization is able
to combine particular building blocks or entities into functional
hybrid composites. An understanding of these biochemical
controls is essential and important, not only to study
biomineralization mechanisms further, but also to design novel
hybrid materials and processing technologies. Despite the
complicated hierarchical structures of biominerals, their basic
building blocks are frequently the nano-sized organic–inorganic
composites.8
Therefore, an ordered and periodic assembly of
organic and inorganic nanophases at the nanoscale is crucial to
biomimetically synthesize hybrid materials. But, how can we
design ordered hybrid composites and how can we conveniently
control their structures, sizes and morphologies under mild
conditions?
Although organic–inorganic hybrid materials have been
approached by various methods such as layer-by-layer (LbL)9
and template-directed crystallization,10
the bottom-up fabrica-
tion from ions or molecules is still a great challenge in the
laboratory since the control of periodic deposition is difficult to
achieve at hierarchical scales. In conventional biomimetic crys-
tallization studies, organic molecules, which act as structure-
directing agents, modulate the crystal morphology by their
a
Centre for Biomaterials and Biopathways, Zhejiang University,
Hangzhou, Zhejiang, 310027, China. E-mail: rtang@zju.edu.cn; Fax:
+86-571-87953736; Tel: +86-571-87953736
b
State Key Laboratory of Silicon Materials, Zhejiang University,
Hangzhou, Zhejiang, 310027, China
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8. selective absorption onto crystal faces, altering crystal facet
stability and growth kinetics.7,11
Recently, a non-classical crystal
growth pathway based upon nano assembly has received
considerable attention.12
The nanoparticles, which are directed
by specific organic additives, can act as the basic building units to
assemble into superstructures or mesocrystals. During such
a process, the organic molecules (especially macromolecules)
selectively absorb and interact with primary nanocrystals. The
assembly process follows programmed arrangement into high
order hybrid structures.13
The morphology can be tuned by
varying the interactions between different organic and inorganic
phases. However, the one step bottom-up process, which starts
from the molecular level rather than from preformed nano-
particle precursors, may be readily able to control the orientation
and order of assembly processes to form integrated hybrid
nanocomposite. But this strategy requires a precise and sponta-
neous co-assembly of both organic and inorganic phases
alternately at both the molecular level and the nanoscale.14
In this paper, we reported an easy but effective method for
direct synthesis of organic–inorganic hybrid mesocrystals by
a emergent co-assembly process of protein (bovine serum
albumin (BSA)) and surfactant (sodium bis(2-ethylhexyl) sulfo-
succinate (AOT)) in a supersaturated calcium phosphate solu-
tion. The calcium-phosphate-based hybrid crystals with lamellar
structure have different properties from conventional ones. Here
we emphasize that the size and morphology of the resulting
hybrids could be regulated readily by varying BSA, AOT and
calcium phosphate concentrations according to a suggested
morphological ternary diagram. This study provided a novel
pathway to one-pot preparation of functional hybrid crystal
materials with tuneable size and morphologies by organic–inor-
ganic co-assembly.
Results and discussion
It is believed that functional organic molecules can interact with
calcium species at the organic–inorganic interfaces to modulate
the growth and assemble of inorganic crystals. BSA is one of the
most studied proteins but this biological macromolecule is not an
effective modifier in calcium phosphate crystallization.15
It has
been previously confirmed that the interaction between BSA and
calcium or phosphate ions in aqueous solutions is poor.16
BSA
itself is inert in mineral deposition. In contrast, many surfactant
molecules are widely used as effective promoters and templates in
biomimetic calcium mineralization since their hydrophilic groups
(especially the sulfonate and carboxylate groups) provide active
binding sites to calcium ions. AOT is one among typical agents
that can modulate calcium phosphate precipitation significantly.
AOT molecules have a strong binding effect with calcium ions
due to their highly charged -SO3
2À
groups.16,17
However, hierar-
chical or complicated biomineral-like structures cannot be
achieved by using this small molecule due to the lack of higher-
order structures. In our control experiments, only poor crystal-
line HAP was obtained if BSA was added into the supersaturated
calcium phosphate solutions; AOT alone produced the conven-
tional rod-like HAP crystals without any organized hybrid
structure. These results matched the previous studies and
understandings well. However, the cooperative effect of BSA and
AOT in the calcium phosphate solution could lead to the
formation of unique hybrids in a one-pot reaction.
Under an experimental condition of 2 mM AOT, 1 mg mlÀ1
BSA and 1.25 mM calcium ions (the molar ratio of calcium to
phosphate was fixed at 1.67 in all experiments), the uniform
rhombic plates precipitated spontaneously as shown by scanning
electron microscopy (SEM, Fig. 1(A)). Their size distribution
was homogeneous. The typical rhombic plates were 1.23 Æ 0.21
and 0.91 Æ 0.18 mm along their long and short axes, respectively
(statistical results from $100 plates); the aspect ratio was about
1.4. The thickness of the plates was 130 Æ 20 nm. These rhombic
plates had exactly same morphology (Fig. 1(B)) and this char-
acteristic was similar to the general inorganic crystals. However,
the chemical compositions of the obtained plates were relatively
complicated. Besides the elements of calcium and phosphorus,
the element of sulfur was detected in the solids by using energy-
dispersive X-ray spectroscopy (EDS). This result indicated the
presence of AOT (-SO3
2À
) in the hybrid plates. It was also
revealed that inorganic part in the plates was a kind of calcium
phosphate minerals with Ca : P molar ratio of 1.5–1.6. The
coexistence of organic–inorganic components was also
confirmed by Fourier transform infrared spectroscopy (FT-IR,
Fig. 1(C)). The peaks at 1737, 1459 and 1419 cmÀ1
were the
characteristic signals of AOT, while the bands at 1656 (amide I)
and 1555 cmÀ1
(amide II) showed the involvement of BSA in the
solids.18
The broad peaks at 1022 and 564 cmÀ1
were assigned to
the inorganic phosphate groups.19
Thermogravimetric analysis
(TGA) showed that the mineral phase was the main composition
in the solids. The weight loss of 38% between 100 and 500
C was
corresponded predominantly to removal of the organic phase,
while the weight contents of the inorganic phases were 62%. In
addition, the plates became ‘crimped-paper’-like after calcina-
tions at 500
C in air for 2 h. Without the organic frame, the
solids became brittle and the structures were collapsed readily
into small pieces under an ultrasonic condition. Many previous
studies suggested that the organic compounds play a regulation
role in inorganic mineralization rather than being involved in
Fig. 1 (A) SEM image of the rhombic plates. (B) Enlarged image of the
rhombic plate in the white circle; the double-headed arrow shows the
extended orientation. (C) FT-IR pattern of the products. (D) The
rhombic plates after calcination at 500
C in air.
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9. structural recombination. However, the current results implied
that BSA and AOT were the key components in the hybrid
construction. Thus, these solids were different from the other
precipitated inorganic crystals in the presence of organic addi-
tives.
The resulting rhombic plates shared the same size and aniso-
tropic morphology similar as general inorganic crystals.
However, in-depth examination revealed that they were distinct
from the conventional calcium phosphate crystals.20
The
rhombic plates were examined by wide angle X-ray diffraction
(WAXD, Fig. 2(A)) and as expectated, the crystalline HAP-like
calcium phosphate phase was detected. The WAXD pattern was
very similar to that of pure HAP but small peak shifts were also
observed. We suggested that the binding effect between the
organic component and calcium ions would cause the lattice
distortion. The lattice structure of the inorganic phase could be
revealed at the atomic scale by using high resolution transmission
electron microscopy from a top view of the plates (HRTEM,
Fig. 2(B)). This image represents a typical ultrathin inorganic
crystal layer embedded in the rhombic plates. However, another
independent set of diffraction peaks was found in the X-ray
diffraction (XRD) pattern, which revealed that a superstructure
was present in the hybrids. The characteristic peaks of lamellar
structure (interspacing distance, d ¼ 3.43 nm) could be found
from both small angle X-ray diffraction (SAXD) and WAXD
(Fig. 2(A)), indicating an ordered arrangement of subunits along
a crystallographic direction rather than a simple mixture of the
organic and inorganic phases. A side view of the ultra-thin
sectioned samples under transmission electron microscopy
(TEM, Fig. 2(C)) confirmed the internal structure: the organic
layers (light, 1.31 nm) and the inorganic layers (dark, 2.13 nm)
alternately stacked at the nanoscale to form the compact hybrid
structure. Thus, the organic molecules (BSA and AOT) were well
organized to form the layered organic phase. Each organic–
inorganic ultra-thin unit had a thickness of 3.44 nm, which
agreed with the XRD data, 3.43 nm, and the individual inorganic
layer was a calcium phosphate crystal plate with a thickness of
only 2.13 nm. These nanoplates acted as the building blocks that
could self-assemble together with the organic layers to generate
the lamellar complex. Additionally, a wave-like superficial
texture of the hybrids could be observed (Fig. 1(B)) and the
profiles were similar to the hybrid crystal morphology. This
phenomenon indicated that the assembly might be an anisotropic
process.
In order to understand the orientation of each inorganic layer,
selected area electron diffraction study (SAED, Fig. 2(D)) was
applied. It was noted that the anisotropic diffraction dots rather
than the isotropic diffraction rings were obtained during the
examination of a whole rhombic crystal, which represented
a similar characteristic of single crystal. It was interesting that the
orientation reflected by these dots (arrow in the insert image) was
exactly same as the long axis of the examined rhombic crystal.
Such a coincidence implied that all the ultrathin inorganic crystal
layers within the hybrid plates should share the same crystallo-
graphic orientation. Additionally, the experimental diffractions
dots of the whole crystal were almost same as the fast Fourier
transform (FFT) result (Fig. 2(B)) of an individual crystal layer.
Therefore, the formed hybrid crystal exhibited similar features to
a single crystal; however, it had additional superlattice structure.
Since the rhombic plates had a specific morphology while they
were not constructed as the conventional single crystals, these
hybrids could be considered as a kind of artificial meso-
crystal.12,21
However, the imperfect dots on Fig. 2 (D) might
indicate that the misaligned orientation still occurred during
nano assembly. Since the material was constructed by ultrathin
calcium phosphate units, it was interesting that flexible and
elastic features were conferred onto the mesocrystal along the
lamellar packing direction in spite of that; its main composition
was a brittle ceramic phase. These mechanical properties of the
hybrids had been characterized by our previous study,16
demonstrating the advantages of organized assembly for
formation of mesocrystals in material functionalization.
The convenient control of the size and morphology of the
organic–inorganic hybrids and mesocrystals is a challenge,
although those for single hybrid crystals are nowadays sophis-
ticated. In our experiments, the calcium phosphate–BSA–AOT
hybrid mesocrystals with different size and morphology could be
feasibly regulated within a simple reaction system by changing
the reactant concentrations. We fixed BSA and calcium
concentration at 0.5 mg mlÀ1
and 1.25 mM, respectively. When
the AOT concentration was 1.00 mM, the obtained hybrid plates
were not rhombic plates any more. Their shapes became spindle-
like. The hybrid plates changed into a round shape when the
AOT concentration was increased to 4.00 mM. However, the
further decreasing or increasing of AOT concentration result into
the disappearance of the co-assembly or hybrid in the system. In
this experiment, their morphologies were gradually adjustable
from spindle, to rhombus to round by increasing the AOT
concentration from 1.00 to 4.00 mM (Fig. 3). During the
evolution process, the length along the short axis of the formed
Fig. 2 (A) WAXD and SAXD (insert) patterns of the rhombi;
(B) HRTEM of a rhombus (top view). Insert: FFT simulation result;
(C) TEM image of ultra-thin sectioned rhomb from side view. The values
of 2.13, 1.31 and 3.44 nm corresponded to the thicknesses of inorganic
(dark), organic (light) and organic–inorganic complex layers, respec-
tively. Insert: TEM image of the side view of the ultra-thin sections of the
plates, bar is 0.5mm. (C) is the enlargement of the region within the white
circle; (D) TEM image of the hybrids. Insert was the SEAD pattern
(white circle area). The HRTEM image in (B) was also obtained on the
same area by the in situ technique. Arrows showed that each individual
inorganic plate in the hybrid shared the same crystallographic orienta-
tion, which was the long axis of the rhombus.
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10. hybrid plates did not change significantly, it was maintained at
300–400 nm. However, the long axis kept on decreasing from
1.50 mm to 300–400 nm with increasing AOT concentration.
Accordingly, the hybrid morphology became isotropic. This
phenomenon implied that AOT component was an important
factor to control the a degree of anisotropic co-assembly of the
hybrids.
Although the morphologies and sizes of the resulted hybrid
plates were influenced remarkably by the changing of AOT
concentrations in the reaction solutions, the internal organic–
inorganic subunit remained. The WAXD and SAXD patterns of
the spindles, rhombi and rounds were exactly same without any
change. But the misalignments of each individual inorganic layer
in the hybrid increased with the increasing of AOT concentra-
tion. The crystallographic mismatch of the inorganic layers could
be examined by using SEAD. During the evolution from the
regular rhombi to round shapes, the diffraction dots disappeared
gradually while the diffraction rings existed (Fig. 4). This
tendency indicated that the preferred orientation of the thin
calcium phosphate planes in the hybrid was weakened. Although
AOT itself could result in aggregates in solution to induce
calcium mineralization, the aggregation was simple and isotropic
due to the lack of complicated configuration. Therefore, it was
reasonable that the excessive AOT could destroy the anisotropic
assembly of the ultrathin mineral plates in the hybrid rhombi.
Although the inorganic and organic layers were still packed
layer-by-layer strictly along the thickness direction, the crystal-
lographic directions of the inorganic crystal planes in the hybrids
became disordered. The anisotropic assembly transformed the
orientation of the long axes into the isotropic mode with
increasing AOT concentration; thus, the round plates were
finally yielded at 4.00 mM AOT and the hybrid was not meso-
crystalline any more. Besides, it should be mentioned that the
percentages of organic and inorganic contents in the hybrid
solids was not changed significantly during the morphology
modulation; in which the inorganic content was kept within
a range of 69–72% from the spindles to the rounds.
Besides the AOT concentrations, the formation of hybrid
crystals could be also adjustable by BSA concentration. In this
examination, the concentrations of AOT and calcium were
maintained at 2 mM and 1.25 mM, respectively, and the BSA
concentrations were increased from 0.25 to 2.00 mg mlÀ1
. It was
noted that the morphologies of hybrid plates underwent another
gradual evolution from the irregular quadrilaterals to rhombi
and then to plump spindles (Fig. 5). The sizes and aspect ratios of
the hybrids increased from 200 nm to 2 mm and 1.1 to 2.0,
respectively, during the modulation. Although the hybrid width
increased along the short axis, the more extended length along
the long axis indicated that the anisotropy assembly process was
affected significantly by the protein concentration. It was noticed
that in biomineralization, the complicated hierarchical building
structures of biominerals are frequently contributed by the
ordered aggregates of proteins. Again, the basic organic–inor-
ganic units and their ordered packing behaviours were not
changed during the morphology and size regulations. It was
mentioned that, when the BSA concentration increased, the role
of AOT in the synthesis decreased. Therefore, the ratio or the
Fig. 3 SEM images of the hybrids synthesised at AOT concentrations of
1.00 (A), 2.00 (B) and 4.00 mM (C). (D)–(F) are the corresponding XRD
patterns of (A)–(C), respectively.
Fig. 4 During the morphology change from rhombus (A) to round (B),
anisotropic diffraction dots became isotropic rings in the corresponding
SEAD pattern.
Fig. 5 SEM images of the hybrids at BSA concentration of 0.25 (A),
1.13 (B) and 2.00 mg mlÀ1
(C).
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11. cooperative effect of BSA and AOT was another key factor in
mesocrystal formation and regulation.
It was known that the co-assembly could not occur in the
absence of the inorganic phase. Thereby, it was reasonable that
the concentrations of calcium and phosphate could control the
mesocrystal formation too (Fig. 6). Under BSA and AOT
concentrations of 1.00 mg mlÀ1
and 2.00 mM, respectively, the
resulting rhombi shared the same intermediate state with
increasing calcium and phosphate concentration in the reaction
solution. If calcium concentration was decreased to 0.63 mM, the
poly-dispersed quadrilaterals-like plates (size of 400–800 nm)
formed with the small aspect ratio of 1.1. If the concentration
was increased to 2.50 mM, the slender spindle-like plates were
obtained and their size distribution was 1.8–2.3 mm with an
aspect ratio was 2.5. From the evolution from quadrilaterals,
rhombi to slender spindles, it could be seen that the anisotropic
co-assembly process was enhanced.
The previous studies of biomimetic fabrication of hybrid
materials with artificial molecules such as peptide-amphiphile,22
block copolymer,23
and amphiphilic dendro-calixarene,24
sug-
gested that the specific sites and sterically constrained effect may
control the assembly of the organic template and then the size
and morphology of the final hybrid materials. Different from the
above-mentioned understanding, under our experimental
conditions, the change of BSA and AOT concentrations were
directly related to the different modification state of BSA. The
BSA protein, which was constituted by a single chain of
583 amino acid residues, acted as a stable and relatively rigid
fragment connected with the special motif (AOT aggregates).25
The hydrophilic groups of aggregates exposed to aqueous solu-
tions and their configuration can be adjusted. The highly charged
group (–SO3
2À
) in AOT could greatly interact with calcium ions
and then modulated calcium phosphate precipitation
significantly, which had been demonstrated experimentally in
many works and in our previous paper.16,26
However, the binding
ability of BSA with calcium ions is weak and the controlling
effect on the mineral formation is relatively poor. As a result,
BSA acted as structural frame while the AOT aggregates
provided the nucleation sites of mineral during the co-assembly
process. In the current study, BSA macromolcules combined
with smaller AOT molecules to form a BSA–AOT complex and
such a modified protein could effective control the crysallization
and assembly of the calcium phosphate mineral. To some extent,
this method provides an efficient way to turn a non-mineraliza-
tion protein into a mineralization protein by using surfactants.
The conformation of the macromolecules restricts the assembly
only along certain specific directions. However, the larger
concentration of AOT is accompanied by an increase in the
amount and size of AOT aggregates, offering more sites for
the assembly process.27
As a result, the controlling effect from the
protein was counteracted and the assembly process could happen
at more directions to form the isotropic rounds. Furthermore,
increasing the amount or the relative amount of BSA concen-
trations partly restricted the assembly process in specific prefer-
ential orientations by spatial configuration to form the
anisotropic hybrids or mesocrystals.28
Thus, the co-assembly
process preferred to occur in certain directions, especially along
the long axis of the hybrid plates rather than the short axis.
Although the short axis partly extended under some experi-
mental cases, the greatly increase along the long axis resulted into
the spindles-like mesocrystal formation. The competitive
controlling effect of BSA and AOT led to the transformation of
an isotropic and anisotropic assembly process during hybrid
crystal construction. Thus, the formation of different hybrids
and mesocrystals with tuneable size and morphologies could be
achieved.
An anisotropic co-assembly process could also be promoted by
increasing the mineral ion concentrations. In the formation
process of mesocrystals, the inorganic precursor controlled the
size and morphology of the final product by tuning the amounts,
size and shapes of the nano-sized building blocks.29
Under our
experimental conditions, the controlling role of mesocrystal
growth became dominant in greater saturation to decide the
product size and structure. As the preferred orientation of
the calcium phosphate crystal plates is parallel to the long axis of
the rhombic plates, the fast growth of the calcium phosphate
plate crystals along this preferred orientation promoted the
formation of the slender spindle-like plates with larger aspect
ratios during the co-assembly process. However, the interaction
between BSA-AOT complex and calcium phosphate crystal was
also responsible for the co-assembly of the organic and inorganic
phase to form highly ordered hybrid materials and maintain their
internal structure.
Actually, the generation of hybrid material via the cooperative
effect of macromolecules (mainly proteins), small biomolecules
and the mineral phase is a common strategy in natural bio-
mineralization.30
In the biological construction, high-molecular-
weight macromolecules, such as collagen, act as support matrix
to provide a structural frame for the mineralization, the
biomineralization proteins themselves have nucleation sites
but most matrices receive mineralization function by binding
and stabilizing functional motifs that are carboxylate- or
Fig. 6 SEM images of the hybrids at calcium concentration of 0.63 (A),
1.56 (B) and 2.50 mM. In all experiments, the ratio of calcium to phos-
phate in the reaction solution was maintained at 1.67.
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12. sulfonate-rich. Thus, the combination of organic–inorganic
mineralization interfaces and the organized organic matrices can
concentrate the mineral ions to induce the deposition as well as to
regulate the size, morphology and orientation of the inorganic
building blocks to form integrated organic–inorganic hybrid
composites with complicated structure. We suggest that in this
system, BSA is the structural frame to control the anisotropic
assembly; the adsorption of AOT onto BSA enhances the
mineralization ability of the protein; and the mineral acts as an
inorganic conjunction phase to solidify the organic–inorganic
hybrid structure. In the experiments, the increase of BSA
promoted the formation of larger hybrid plates with increased
aspect ratio, while AOT exhibited the opposite controlling effect.
The increasing of inorganic concentrations preferred the
formation of slender hybrid plates with a larger size. In order to
show the controlling effect of the reactant concentrations, the
simplified morphological maps in the form of solution ternary
diagrams was proposed (Fig. 7). The biomimetic formation
hybrid and mesocrystals could be yielded in the grey region. In
the specific regions, the formed hybrid plates had a similar size
and morphology. From points A to B, the increase of aspect ratio
was preferred as the hybrid rounds transformed into the spindle
ones. Since the anisotropic assembly behaviour was enhanced,
this evolution implied that the resulting mesocrystals became
more organized and the mismatch degrees of the inorganic layers
in the hybrids could be reduced. From points A to C, both the
size and aspect ratio of the resulted hybrids were increased and
their morphologies were changed from rounds to spindles. From
points B to C, the hybrids turned from wide spindles to slender
spindles with increased size and aspect ratio too. By using this
morphological ternary diagram, we could design readily hybrids
and mesocrystals with the required size and morphology.
Conclusions
We demonstrate that the ordered and uniform hybrids or mes-
ocrystals can be biomimetically synthesized by the co-assembly
of proteins, small functional molecules and minerals using
a simple one-pot reaction. Their size distributions and
morphologies can be adjusted by varying the component
concentration in reaction solutions. The anisotropic co-assembly
of the BSA–AOT complex and ultrathin calcium phosphate
crystal plates is a key to the control of mesocrystal formation.
A morphological ternary diagram can be used to design different
hybrid materials as requireed. This work may give another
inspiration to the assembly of multi components into one inte-
grated hybrid material with a highly ordered structure.
Furthermore, the bottom-up pathway of controlled fabrication
may be developed as a simple and effective strategy to prepare
feasibly functional hybrid and mesocrystal materials.
Experimental
Materials
Triply distilled water was used in all the experiments. Ca(NO3)2
and (NH4)2HPO4 were of analytical and their solution were
filtered twice using 0.22mm Millipore films prior to use. BSA
(Albumin Bovine fraction V, BR, purity 98%, LABMAX) and
AOT (Aldrich) were used without any further purification.
Hybrid plate preparation
Using a typical experiment as an example, 100 ml aqueous
solution containing 4 mM AOT and 0.20 g BSA was mixed with
50 ml Ca(NO3)2 solution (5mM). The solution pH was adjusted
to 10.0 Æ 0.5 at room temperature by 3 M ammonia solution.
Then 50 ml (NH4)2HPO4 solution (3 mM, pH ¼ 10.0 Æ 0.5) was
added dropwise at a rate of 1.5 ml minÀ1
. The reaction solution
contained 2.00 mM AOT, 1.00 mg mlÀ1
BSA, 1.25 mM Ca(NO3)2
and 0.75 mM (NH4)2HPO4. The mixture was gently stirred at
30 Æ 1
C for 24 h. The precipitated solids were collected by
centrifugation at 6000 rpm. The solid were washed by water for
three times and were vacuum-dried at 35 Æ 1
C. In order to
examine the controlling effect of reactant concentrations on
hybrid formation, different concentrations of AOT, BSA and
calcium phosphate ions were used and all the experimental
processes were the same.
Fig. 7 Controlled synthesis of hybrids by a morphological ternary diagram. The co-assembly occurred within the grey area and the formation of
mesocrystals was preferred in its left and bottom sections. The typical morphology of the final products were also demonstrated. Bar ¼ 1mm.
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13. Characterizations
SEM was performed by using a HITACHI S-4800 at a typical
acceleration voltage of 5 kV. FT-IR spectra (Nicolet Nexus 670)
were applied to analysis the hybrid compositions. WAXD and
SAXD were characterized by a Rigaku D/max-2550pc with
monochromatized Cu-Ka radiation; the scanning step was 0.02
.
TGA was performed by a TA Instrument SDT Q600. The
experiment was measured in a temperature range from room
temperature to 1000
C under nitrogen atmosphere. TEM
observations were performed by a CM200UT TEM (Philips) at
an acceleration voltage of 160 kV. During the ultra-thin
sectioned TEM examination, rhombi were embedded in epoxy.
The mixture was solidified at 80
C for 12 h and then carefully
microtomed by a Reichert-Jung Ultracut E using a diamond
knife.
Acknowledgements
We thank Jieru Wang, Xinting Cong, Xiaomin Tang, Yin Xu
and Linshen Chen for their help with characterization, Haihua
Pan and Yuan Su for discussions. This work was supported by
the Fundamental Research Funds for the Central Universities,
National Natural Science Foundation of China (20871102),
Zhejiang Provincial Natural Science Foundation (R407087) and
Daming Biomineralization Foundation.
Notes and references
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14. Spontaneously amplified homochiral organic–inorganic
nano-helix complexes via self-proliferation†
Halei Zhai,a
Yan Quan,a
Li Li,a
Xiang-Yang Liu,b
Xurong Xuc
and Ruikang Tang*ac
Most spiral coiled biomaterials in nature, such as gastropod shells, are homochiral, and the favoured chiral
feature can be precisely inherited. This inspired us that selected material structures, including chirality,
could be specifically replicated into the self-similar populations; however, a physicochemical
understanding of the material-based heritage is unknown. We study the homochirality by using calcium
phosphate mineralization in the presence of racemic amphiphilic molecules and biological protein. The
organic–inorganic hybrid materials with spiral coiling characteristics are produced at the nanoscale. The
resulted helixes are chiral with the left- and right-handed characteristics, which are agglomerated
hierarchically to from clusters and networks. It is interesting that each cluster or network is homochiral
so that the enantiomorphs can be separated readily. Actually, each homochiral architecture is evolved
from an original chiral helix, demonstrating the heritage of the matrix chirality during the material
proliferation under a racemic condition. By using the Ginzburg–Landaue expression we find that the
chiral recognition in the organic–inorganic hybrid formation may be determined by a spontaneous
chiral separation and immobilization of asymmetric amphiphilic molecules on the mineral surface, which
transferred the structural information from the mother matrix to the descendants by an energetic
control. This study shows how biomolecules guide the selective amplification of chiral materials via
spontaneous self-replication. Such a strategy can be applied generally in the design and production of
artificial materials with self-similar structure characteristics.
1 Introduction
Through long time periods of evolution, most spiral coiled bio-
materials in nature, like gastropod shells, adopt a specic
homochirality.1,2
For example, the majority of current gastropod
shells have a right-handed (R-) coiling pattern (Fig. 1a).3
The
chiral minority was eliminated eventually by a frequency-
dependent selection and the dominant one proliferated.4,5
This
inspires us that materials with specic structural properties,
like chirality, can spontaneously develop into a large self-similar
community.6
Biologically, it is accepted that regularly expressed
biomolecules, together with inorganic minerals, constitute the
physical chirality of gastropod offsprings under the guidance of
a controlling gene.7
For instance, at the growth front of shells,
the tiny chitin nanocrystals behave as the amphiphilic mole-
cules and self-assemble into the liquid crystal layers (Fig. 1b).8
Fig. 1 The chirality of gastropod shells and a schematic drawing of the shell
mineralization front. (a) General gastropod species have right-handed shells. (b)
During the natural generation of shell structure b-chitin molecules assemble into
supermolecules (chitin crystallites) and their liquid-crystal layers induce the spiral
mineralization of calcium carbonate (this scheme is prepared based upon a
mechanism proposed by Cartwright et al.).8,9
a
Centre for Biomaterials and Biopathways and Department of Chemistry, Zhejiang
University, Hangzhou, 310027, China. E-mail: rtang@zju.edu.cn; Fax: +86 571-
8795-3736
b
Department of Physics and Department of Chemistry, National University of
Singapore, Singapore 117542, Singapore
c
Qiushi Academy for Advanced Studies, Zhejiang University, Hangzhou, Zhejiang
310027, China
† Electronic supplementary information (ESI) available: Supporting gures and
tables. See DOI: 10.1039/c3nr33782k
Cite this: Nanoscale, 2013, 5, 3006
Received 23rd November 2012
Accepted 29th January 2013
DOI: 10.1039/c3nr33782k
www.rsc.org/nanoscale
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15. These chitin layers provide growing sites for the inorganic
phase and modulate the mineralization together with related
proteins. Thus, it follows that the spiral micro-pattern consti-
tuted by a chitin–calcium carbonate lamellar structure is grad-
ually constructed (Fig. 1b).9
In this sense, an understanding on
the physicochemical regulations of the organic–inorganic bio-
inspired materials with selective chirality will advance our
knowledge in chemistry and materials sciences. A challenging
question to be addressed is whether we can mimic the self-
evolution (symmetry breaking) process of shells in our labora-
tories so that the chiral materials can be separated and propa-
gated to generate self-similar articial production.
It has been demonstrated that some organic molecules can
control the morphology of biominerals, like calcium phosphate
and calcium carbonate crystals.10
The chiral organic molecules
usually act as templates to control the crystal morphology,
rather than incorporated organic composition to constitute the
chiral hybrid materials.11,12
In the articial design of chiral
nanomaterials, a variety of dispersed chiral superstructures,
such as nano-helixes and nano-tubes, can be generated with the
twisted assembly of chiral molecules, or even nano-sized crys-
talline units.13
However, each nano-helix or nano-tube is con-
structed by independent assembly, rather than a successive
proliferation procedure to pass down the chirality and nal
formation of the homochiral complex. As a result, the archi-
tecture of a homochiral material complex is rarely achieved.14
Herein, by employing a racemic mixture of a chiral amphiphile
(bis-(2-ethylhexyl) sulfosuccinate sodium salt, AOT) and bovine
serum albumin (BSA) in supersaturated calcium phosphate
solution, two kinds of chiral organic–inorganic hybrid nano-
helixes (L- and R-enantiomers) can spontaneously form and
each kind of chiral helix eventually proliferates into a larger
homochiral helix complex. We feel that such an experimental
phenomenon may be relevant to the proliferation of chiral
materials.
2 Experimental section
2.1 Materials
Triply distilled CO2-free water was used in the experiment.
Ca(NO3)2 and (NH4)2HPO4 were of analytical grade and their
solutions were ltered twice through 0.22 mm Millipore lms
prior to use. BSA (Albumin Bovine fraction V, BR, purity 98%)
and AOT (Aldrich, racemic mixture) were directly used without
further purication.
2.2 Preparation of the homochiral nano-helix complex
The temperature during all the synthesis processes was main-
tained at 30 Æ 1
C. Briey, a 100 ml aqueous solution con-
taining 1 mM AOT and 1 mg mlÀ1
BSA was prepared. The
solution pH was adjusted to 10.0 Æ 0.5 by 3 M ammonia solu-
tion. 50 ml Ca(NO3)2 solution (5 mM, pH ¼ 10.0 Æ 0.5) was
added. Then 50 ml (NH4)2HPO4 solution (3 mM, pH ¼ 10.0 Æ
0.5) was added dropwise at a rate of 1.5 ml minÀ1
. The solution
was gently stirred for 10 h and the formed solids were
collected by centrifugation at 3600 rpm. All the solid samples
were washed by water three times and were vacuum-dried at
35 Æ 1
C. Freshly prepared samples were dispersed in ethanol
($0.5 mg mlÀ1
) and collected on carbon-coated copper grids for
TEM examinations. In the seed growth experiment, 1/20 percent
of the obtained product underwent intense ultrasonic treat-
ment (KUDOS, 35 kHz, 20 min) and the helix clusters or
networks were collapsed into dispersed helixes. Then the
dispersed helixes were added as seeds into freshly prepared
reaction solutions and the reaction solutions were collected by
centrifugation and observed with Transmission Electron
Microscopy (TEM). For ultrathin sectioned TEM examination,
dried samples were embedded in 0.5 ml epoxy. The mixture was
solidied at 80
C for 12 h and then carefully microtomed by a
Reichert-Jung Ultracut E ultramicrotome using a diamond
knife.
2.3 Au-labelled BSA absorptions
BSA-Au nanoparticles were synthesized according to the work by
J. Xie et al.15
Briey, 5 ml 10 mM HAuCl4 solution was added into
5 ml 50 mg mlÀ1
BSA solutions and stirred for 5 min. Then,
0.5 ml 1 M NaOH was added and the solution was kept at 37
C
for 24 h. The product was dialyzed with 1000 ml distilled water
for 24 h. The BSA-Au was used instead of pure BSA in order to
probe the location of BSA on the surface of the helix.
2.4 Examination of calcium concentrations
The concentration of free calcium ions in BSA, AOT, BSA + AOT
solutions were measured by a PCa-1 calcium ion selective
electrode with a saturated calomel electrode as the reference
electrode. The electrode was calibrated according to the
instructions before use.
2.5 Characterizations
Scanning electron microscopy (SEM) was performed by using a
HITACHI S-4800 eld-emission scanning electron microscope
at an acceleration voltage of 5 kV. Fourier-transform infrared
spectroscopy (FT-IR, Nicolet Nexus 670) was used to determine
the composition of the products. Thermogravimetric analysis
(TGA) was carried out by a TA Instrument SDT Q600. The
experiments were measured over a temperature range of 22–
800
C at a rate of 10
C minÀ1
under air atmosphere. TEM
observations were performed by a JEM-1200EX at a typical
acceleration voltage of 80 kV. Small angle X-ray diffraction
(SAXRD) and Wide angle X-ray diffraction (WAXRD) were char-
acterized by a Rigaku D/max-2550pc with monochromatized Cu
Ka radiation and the scanning step was 0.02
. Solid state
nuclear magnetic resonance (ssNMR) was kindly performed by
Prof. Jarry Chan's group at the National Taiwan University on a
Bruker DSX300 NMR spectrometer.
3 Results and discussion
3.1 Structure and composition of the nano-helix
In our biomimetic case, AOT and BSA were adopted as the
models for biological amphiphilic and proteins, respectively.
AOT is of asymmetric double-chain amphiphile (Fig. S1†). It can
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16. assemble into various mesomorphous phases, which have been
widely used in biomimetic crystallization.16
BSA is one of the
common proteins in biomineralization studies.17
The syner-
gistic effect of AOT and BSA on calcium phosphate minerali-
zation gave rise to the formation of nano-helix (Fig. 2 and S2†).
In the control experiments, the use of AOT and BSA alone only
generated the calcium phosphate nanorods and nanospheres,
respectively (Fig. S3†). Clearly, the helix formation was attrib-
uted to the coexistence of AOT, BSA and calcium phosphate.
It followed that the individual helixes could further develop
into micron-sized aggregated clusters (Fig. 2a and b). In an
individual cluster, the nano helixes extended radially outward
from a dense core, indicating the successive proliferation
procedure. Furthermore, some clusters connected with each
other to form a larger network (Fig. 2 and S4†). As the basic
building blocks of the clusters, the helixes were chiral and they
had two kinds of spiral enantiomers, L- and R-forms. Although
the overall amounts of the L- and R-helixes in the reaction
system were equal (Fig. S5†), only one helix enantiomer could be
identied within a cluster or connected network (Fig. 2a–c, see
more in ESI†). This suggested that the spontaneous chiral
recognition and chiral separation occurred during the cluster
and network generation. Concerning the composition and
structure of the helixes, they were constituted by organic and
mineral phases, which accounted for 20.5 wt% and 58.8 wt%,
respectively (the rest 20.7% was attributed to absorbed and
crystal water, Fig. S6†). FT-IR (Fig. 2d) and energy-dispersive
X-ray spectroscopy (EDS, Fig. S7†) revealed that the main
components in the helixes were AOT and a calcium phosphate
phase. X-ray diffraction (Fig. 2e) showed that the mineral phase
was close to brushite. Moreover, the mineral phase in the helix
was conrmed by Multiple Pulse Sequence Nuclear Magnetic
Resonance spectroscopy (CRAMPS-NMR) and a Heteronuclear
Correlation (HETCOR) spectrum between the 31
P and 1
H nuclei
31
P{1
H} combined rotation, indicating that the phosphate
groups were protonated (HPO4
2À
) in the calcium mineral
(Fig. 2f, S8 and Table S1†). The NMR data indicated the absence
of PO4
3À
in the complex. As a result, the calcium phosphate
species containing PO4
3À
groups such as hydroxyapatite, octa-
calcium phosphate and tri-calcium phosphate could be
excluded in the phase analysis. Additionally, Ca(H2PO4)2 could
not be precipitated under our experimental conditions due to
its high solubility. Both examinations shows that the signals of
the helix were close to those of brushite. Therefore, the brush-
ite-like mineral was considered as the primary inorganic
component in the helixes.
The internal structure of the nano-helixes could be consid-
ered as the alternative and spiral stacking of thin calcium
phosphate phase and AOT bilayers. The cross-section images of
the nano-helix showed that the thickness of the wall of the
nano-helix was about 2.1–2.2 nm (Fig. 3a–c). Furthermore,
SAXRD and WAXRD results also showed the alternative lamellar
superstructure in the helixes with a constant interspacing
distance (d ¼ 3.34 nm). The lamellar structure was also
demonstrated by TEM (Fig. 2e): the dark lines (1.7 nm) and light
lines (1.6 nm) correspond to the inorganic calcium phosphate
and organic AOT ultrathin layers, respectively. The thickness of
each organic–inorganic hybrid unit was about 3.3 Æ 0.2 nm,
which is in agreement with the d value calculated from the
SAXRD data. In the spiral helix, there existed a pitch angle of
about 43
between the strip edge and the long axis. It was noted
that the AOT molecules preferred to assemble into a bilayer
structure. Concisely, the organic bilayer could have a thickness
of about 1.6 nm if the molecules tilted by 43
. There were two
mirror forms for both the helix pitch angle and the AOT tilt
angle, +43
or À43
, as the denitions (Fig. 3d and e). The
mirror packing of AOT corresponded to the formation of R- and
L-enantiomers of the helixes. Apart from AOT, a small amount
of BSA was detected in the helix by FT-IR and 13
C{1
H} NMR
(Fig. S9†). Using nano Au particle labelled BSA as the imaging
agent, we found that the protein did not incorporate into the
hybrid inner structure, but absorbed onto the helix wall
surfaces (Fig. S10†), which might be due to its relatively large
dimension.19
We suggested that BSA served as a surface or
Fig. 2 Characterizations of nanohelixes. (a and b) Homochiral clusters consisting
of R-helixes and L-helixes, respectively. (c) A homochiral helix network; circles
indicate the cluster centres; inset is a magnification of the rectangular region. (d)
FT-IR of the helix and pure AOT. The typical and undisturbed peaks of AOT
(1750 cmÀ1
), BSA (1540 cmÀ1
, amino) and phosphate ions could be noted. The
peak located at 2342 cmÀ1
was generally attributed to CO2 from the air during the
FT-IR determination. (e) SAXRD and WAXRD patterns of the helixes. d ¼ 3.34 nm
and d ¼ 1.65 nm represent the first and the second diffractions of the lamellar
structure in the helixes, respectively. WAXRD also showed that the mineral phase
was similar to brushite. The XRD peaks of 11.3
and 31.0
were close to those of
brushite (020) and (121), respectively. The case of the small left-shift of charac-
teristic peaks could be found in small nanocrystals.18
The inset TEM image shows
each organic (light line)–inorganic (dark line) unit in the helix. (f) 31
P{1
H}HETCOR
spectra between the 31
P and 1
H nuclei measured in the helixes. The spectra was
acquired at a spinning frequency of 10 kHz and the contact time was set to 2.5 ms.
A total of 64 transients with an increment of 100 ms was accumulated.
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17. structure stabilizer for the hybrid spiral strips. An experimental
fact was that no chiral product formed by using BSA alone.
During biomineralization, some biomacromolecules can adopt
an extended conformation when they interact with the inor-
ganic phase surface.20
Therefore, there was a possibility that the
BSA molecules might incorporate into the helix using their
extended forms. However, we could conrm that the AOT
molecules with highly charged sulphuric groups, rather than
BSA, were the primary organic composition in the nano-helix.
For example, the FT-IR study showed a very weak amino peak
(Fig. 2d) in the composites, implying the ignorable contents of
BSA in comparison with the strong AOT bands. Therefore, it was
suggested that the hybrid helixes were formed by the ordered
assembly of the calcium phosphate mineralized layer and the
AOT bilayer, and were stabilized by BSA absorption on the
hybrid surfaces. In this architecture, the assembly behaviour of
chiral AOT molecules in the hybrid helixes determined the
material’s chirality.
3.2 Proliferation of the nano-helixes
Originally, the homochiral clusters and networks evolved from a
single nano-helix (mother matrix). The preformed chiral nano-
helix spontaneously passed down the structure information
(chirality) from one generation to the next and then generated
the homochiral complexes (Fig. 4). Firstly, tiny hybrid buds
sprouted from the surface of the matrix (Fig. 4a). Both organic
and inorganic parts of the buds directly integrated with the
corresponding parts of mother matrix. This could be considered
as a kind of matrix outgrowth. Secondly, these hybrid buds grew
longer and twisted into the helical ribbons. At this stage, the
organic and inorganic parts at the growing front of hybrid buds
did not integrate with mother matrix any more. At this time,
there should be a choice in the twist direction (L- or R-).
Nevertheless, we noted that the newly formed helical ribbons
replicated precisely the twist direction (chirality) of the mother
matrix. This meant that the mother matrix induced the later
AOT molecules to assemble into a coherent packing direction,
even the AOT bilayers at the budding region and growth front
were separated by calcium phosphate layers. Thus, the original
structure was inherited through the budding and proliferation
process (Fig. 4b). Thirdly, the homochiral proliferation process
of the helixes continued by generating more “daughters” and
“grand-daughters” based upon the matrix. Due to the space
limitation, the newly formed helixes tended to stretch outward,
which generated radial homochiral clusters (Fig. 4c). Finally, a
few of the helixes at the cluster edge acted as “bridges” to
provide additional growing sites for new buds and initiated
another proliferation process (Fig. 4e). This new proliferation
Fig. 3 (a and b) Cross-section images of nano-helixes under TEM. (c) Schematic
structure of the nano helixes (dark grey: inorganic phase; light grey: organic
phase). (d and e) TEM and schemes of the R- and L-helix. The width of the AOT
bilayer is 1.6 nm from TEM observation. As AOT molecules have a length of 1.1
nm, AOT molecules in a bilayer should arrange with a tilt angle of about 43
.
Note: the AOT molecules in the same bilayer are simply treated as direct contact
and this small variation of tilt angle doesn't affect our qualitative analysis.
Fig. 4 TEM images of the evolution from a single helix to a homochiral cluster
and then a homochiral network (community). (a) A sprouting bud from R-helix
matrix for the new “daughter” helix generation. (b) Growth and twist of the
“daughter” helix, which duplicated the chiral feature to be R-form; insets show
the details of the growth front on the matrix. (c) More buds formed and they
replicated the structure of matrix precisely. (d) Rudiment of the homochiral helix
cluster; insets: magnification of the branching sites. (e) Homochiral helix cluster
(R-form); arrows indicate the proliferation directions of the cluster; inset shows
the new buds formed at an extended helix. (f) Homochiral helix networks (R-
form); arrows show the proliferation directions.
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18. could happen at multiple directions (Fig. 4f). Through the self-
repeating processes, a single nano-sized helix eventually evolved
into a large homochiral complex (network) at the micrometer
scale (Fig. 4f). In each network, the chirality of the newly born
helixes was precisely “inherited” from the original mother
matrix from generation to generation, which can be considered
as a spontaneous process of material-based self-proliferation.
We note that it is impossible for the dispersed helixes aer
intense ultrasonic treatment to aggregate into homochiral
clusters again. However, aer the dispersed helixes are re-
dispersed into the freshly prepared reaction solutions as seeds,
the time to induce the formation of helix clusters can be rela-
tively reduced according to the seed amount, indicating that the
mother helix acts as the seed to induce the proliferation of new
helixes to form helix clusters (Fig. S11†). As a result, in each
network, the chirality of the newly born helixes was precisely
“inherited” from the original mother matrix from generation to
generation, which can be considered as a spontaneous process
of material-based self-proliferation.
3.3 Model for the homochiral material
Analogous to the shell formation, the co-assembly of organic and
inorganic phases is restricted at the local domains of the growth
front (Fig. 4a and b). AOT molecules are more able of binding
calcium ions than BSA (Table S2†) and these amphiphilic
molecules greatly modulate the growth of calcium phosphate
species.21
In this case, the helix formation and replication are
controlled dominantly by the assembly behaviours of AOT at the
growth front. The AOT molecules can tightly absorb onto the
surface of calcium phosphate species with a strong binding
effect between calcium ions and sulphuric groups, which facili-
tates the assembly of AOT bilayers.22
Unlike the free AOT mole-
cules in aqueous solution, the relatively rigid CaP crystal, rather
than the mobile water layer, can imobilize the adjacent AOT
bilayers. Thus, the hybrid structure could be ‘solidied’ and
stabilized with a decreasing disordered uctuation of AOT
molecules comparing the free state, which then induces the next
layer mineralization.23
This alternative and cooperated deposi-
tion of the AOT bilayer and calcium phosphate phase layer
gradually constitutes a thin AOT–calcium phosphate hybrid
strip, which is similar to the associated assembly of lipids and
inorganic phase reported by Seddon, et.al.13b
In our work, the chiral AOT molecules are responsible for the
twist of the hybrid strip to form the L- and R-nano-helixes.
Although the BSA used here is constituted with chiral L-amino
acids, the chirality of nano-helixes are unlikely to be controlled
by BSA. Due to its large size, it is difficult to incorporate into the
ordered structure with ultra-small units of 1.7 nm (calcium
phosphate layer) and 1.6 nm (organic layer), while the twisted
arrangement of these units forms the chirality at the nanoscale.
In addition, the equal number of L- and R-nano-helixes also
indicates that the BSA with single chiral units (L-amino acids)
has little contribution to the chirality of the nano-helixes. Many
works have been reported that strips constituted with chiral
molecules tend to twist into nano-helixes to reduce the elastic
energy.24
Similarly, the chirality of the nano-helixes in our
system is determined by the assembly behaviour of chiral AOT
molecules.
However, the racemic mixture generally dilutes the chiral
interaction between the chiral molecules, so that the chiral
superstructure might fail to form.25
Nevertheless, some studies
have shown that both R- and L-enantiomers can emerge in
racemic systems if an energy favoured chiral phase separation
occurs, especially for the lipids with chiral headgroups and
inexible double chains structures.26
Interestingly, AOT owns a
similar structure and phase behaviour to these lipids.27
More-
over, chiral molecules can also undergo a phase separation
when they are restricted at interfaces.28
Therefore, in our
system, it follows that a spontaneous chiral phase separation of
amphiphilic AOT may occur on the calcium phosphate mineral
substrate, resulting in the bilayers with exactly the same
molecular packing behaviour.
Due to the complicated structure, the conformation infor-
mation (chirality) of AOT in each nano-sized helix is difficult to
identify. Besides, methods of the synthesis or separation of AOT
diastereoisomers is rarely reported.29
Based upon the mirror
arrangement of AOT in L- and R-helixes, we divide the AOT
molecules into two types with different tilt directions of +43
or À43
. Aer this simplication, only the tilt angle needs to be
taken into account in the qualitative analysis of the energy
during the formation process. The AOT molecules in the bila-
yers have two different tilt angles, +43
or À43
, which can be
considered as the enantiomers to induce R- and L-chiral helix
formations, respectively (Fig. 3d and e). The favoured tilt angle
should maintain the same value during the alternative dispo-
sition. Thus, the energy favoured recognition is a key to main-
tain the molecular assembly according to the chiral breaking
model supposed by Selinger et al.30
The model suggests that the
elastic energy of the strip can be reduced by a chiral separation
even under racemic conditions. An order parameter, j, is
introduced, which is treated as the local net amount of right-
handed minus le-handed molecular packing here. The elastic
free energy, F, of the thin chiral bilayer strip can be written as
eqn (1)
F ¼
ð
dS
1
2
k
1
r
2
þ
1
2
k0
1
r
2
cos2
f À lHPj
1
r
sin f cos f
þ
1
2
KðVjÞ2
þ
1
2
tj2
þ
1
4
uj4
þ Eedge (1)
where, S is the area, the rst term is the standard Helfrich
bending energy of the hybrid membrane and the coefficient k is
the isotropic rigidity. In the right side of eqn (1), the second
term represents the anisotropy of the rigidity and the coefficient
k0
is the anisotropic term and f is the title angle of the chiral
molecules (Fig. 5a); the third term is a chiral term that favours
twisting in a tilt angle f; the coefficient lHP, is the chirality
parameter, which exists only in chiral membranes and depends
on the chiral order. The sign of lHP can be changed when the
membrane transforms into its mirror image. lHPj increases
with the greater chiral phase separation degree of j. The last
three terms in the bracket are the Ginzburg–Landau expres-
sions in powers of j, which represent the free energy change
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19. during the ordering transition. The values of K and u are
temperature independent constants while the coefficient of t
relates to temperature and t 0 for chiral phase separation.30,31
Because AOT molecules in the helix have the xed tilt angle of
43
, the domain wall energy on the edge is a constant.
When simultaneously minimizing the free energy over tilt
angle and radius, r, in eqn (1), the following is obtained,
f0 ¼ arctan
k þ k0
k
1=
4
(2)
r0 ¼
k
1=
4
ðk þ k0
Þ
1=
4
h
k
1=
2
þ ðk þ k0
Þ
1=
2
i
lHPj
(3)
in which, (k + k0
)/k represents the energy cost for the ratio of the
bend parallel to the tilt direction to bend perpendicular to
the tilt direction. In our system, its value is about 0.76 because
fAOT ¼ 43
, which indicates that the hybrid strip favours
twisting parallel to the AOT tilt direction. Besides, the radius of
the nano-helix equals 1.33k/lHPj. Usually, the lipid amphi-
philes form helical tubes or a helix with a larger diameter of
hundreds nanometres or even a few micrometers. Since the
organic–inorganic hybrid structure exists in our helixes, it is
reasonable that the rigidity coefficient k should be greater than
single component chiral lipid membranes. As a result, lHPj
must be signicantly greater to produce such a slender helix
with a very small radius ($10 to 25 nm).
We note that the favoured energy barrier DF plays an
essential role to control the twist direction and chiral prolifer-
ation of nano-helixes (eqn (1)). Here, the qualitative description
energy barrier DF between the racemic state (j ¼ 0) and sepa-
ration state (jmÆ) can be described by eqn (4),30
DF ¼
ð
dS
lHPjmÆ
1
r
sin f cos f þ
1
2
KðVjmÆÞ2
þ
1
2
tjmÆ
2
þ
1
4
ujmÆ
4
(4)
As the radius of the nano-helix is r f 1/lHPj, the relatively
small radius of the helix (10–25 nm) implies that lHPj is of great
value in our case, which facilitates the chiral separation. The
equation shows that the free energy of the strip has two local
minima representing the two types of energy favoured AOT
packing with the mirror symmetry (Fig. 5b) if chiral phase
separation occurs. Without chiral phase separation, the strip
cannot twist into a helix because the radius becomes innite
when j ¼ 0 (r / N).
Fig. 5b shows the constant arrangement of AOT molecules
(constant tilt angle of +43
or À43
) within a strip is energeti-
cally preferred due to an energy barrier. First, the energy barrier
DF can promote the formation of energy favoured and stable
nano-helixes, rather than unstable hybrid strips. If the different
arrangements of AOT molecules (L- and R-) coexist in the same
strip, the elastic energy increases so that the resulting strip
becomes unstable (Fig. 5b, middle). Therefore, the elastic
energy cannot be reduced and generate twisted nano-helixes. By
contrast, the same arrangements of AOT molecules (L- or R-) can
successfully reduce the unfavoured elastic energy and twist to
form L- or R-nano-helixes, respectively (Fig. 5b, le and right).
Second, the energy barrier of DF is also responsible for the
homochiral proliferation. In our system, the preformed helix
matrix has an inductive effect on the sequent proliferation
because the emerging organic and inorganic parts in the new
buds directly extend from their mother matrix. Thus, new buds
share the same AOT packing form with the mother matrix. The
same AOT packing can be replicated under the guidance of the
mother matrix due to the favoured energy reduction, which
means that L- to L- or R- to R-proliferation is a preferential way.
Subsequently, the buds grow following the determined AOT
packing to form a new chiral helix with the same chirality. For
example, the new buds generated from the R-nano-helixes in
Fig. 4 faithfully adopt the R-twist direction and keep the selected
form during the growth process. The mutated proliferation of
L- to R- or R- to L- also require extra energy to overcome DF in
comparison with the matched L- to L- or R- to R-. Accordingly,
the chiral structure proliferation always initiates at the pre-
formed helixes and amplies the chiral structure from the
mother matrix to subsequent generations. Finally, large
homochiral complexes (helix clusters and networks) can be
generated under the guidance of the energy controlled recog-
nition of AOT packing.
4 Conclusions
This study reveals that the homochiral complex of the organic–
inorganic hybrid helix can form via a self-proliferation process.
The energy controlled chiral recognitions and separations of
asymmetric chiral AOT molecules are essential in both helix
formation and homochiral proliferation. The nding is of
importance to approach homochiral biomimetic materials in
the laboratory. We expect this strategy of bio-inspired chiral
structure proliferation can be developed into a convenient
pathway for the articial synthesis of self-similar functional
materials.
Acknowledgements
We thank Prof. Jerry Chen for the ssNMR studies, Dr Jinhui Tao,
Dr Haihua Pan and Yuan Su for discussions, Hua Wang, Jieru
Fig. 5 (a) The geometry of AOT molecules in the helix discussed in eqn (1) for helix
formation. (b) Two local minima of the elastic free energy (F) with symmetry
packing (jm+ or jmÀ) lead to an energy barrier of DF, which ensures the oriented
packing vector of AOT bilayers to produce chiral helix and homochiral proliferation.
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![© 2010 WILEY-VCH Verlag GmbH & Co. KGaA, WeinheimAdv. Mater. 2010, 22, 3729–3734 3729
www.advmat.de
www.MaterialsViews.com
COMMUNICATION
By Halei Zhai, Wenge Jiang, Jinhui Tao, Siyi Lin, Xiaobin Chu, Xurong Xu,
and Ruikang Tang*
Self-Assembled Organic–Inorganic Hybrid Elastic Crystal
via Biomimetic Mineralization
[*] H. Zhai, W. Jiang, J. Tao, S. Lin, X. Chu, Dr. X. Xu, Prof. R. Tang
Center for Biomaterials and Biopathways
and Department of Chemistry
Zhejiang University
Hangzhou, 310027 (P.R. China)
E-mail: rtang@zju.edu.cn
Dr. X. Xu, Prof. R. Tang
State Key Laboratory of Silicon Materials
Zhejiang University
Hangzhou, 310027 (P.R. China)
DOI: 10.1002/adma.201000941
It is generally accepted that biomaterials have unique physi-
cochemical properties.[1] Inspired by biological systems, sci-
entists have been studying biomimetic methods to fabricate
functional materials.[2] Almost all biomaterials possess a
common multi-component feature.[1,3] These composites fre-
quently have ordered organic–inorganic hybrid structures and
their properties are distinct from the individual components.
For example, in a multilayered complex of inorganic aragonite
tablets and organic substrate, the fracture toughness of nacre is
significantly improved to three thousand times greater than that
of synthetic aragonite.[4] Another striking example is biological
bone. In bone, the hydroxyapatite (HAP) phase crystallizes in
the nanoscaled channels formed by the staggered alignment
of the protein matrix. The typical HAP crystals in bone are
plate-shaped with extremely thin thickness (1.5–2 nm), which
is the smallest known dimension of the biologically formed
crystals.[5] In nature the organic and inorganic components inti-
mately associate into well-organized hybrid structures to ensure
optimal strength and flexural stress.[6,7] Therefore, in biomi-
metic designs and fabrications the formation of such ordered
nanostructures is a key challenge.
The formation of inorganic crystals in living organisms is
regulated by the organic matrix. Generally, different organic
templates and additives lead to variety in the morphology, size,
orientation, and assembly of the inorganic crystal by medi-
ating its nucleation and growth.[8,9] Although many organic–
inorganic nanocomposites have been reported,[10] the self-
formation of ultrathin organic–inorganic substructures is still
difficult to achieve by using a simple bottom-up approach. But
the self-formed ordered and intimate combination of organic
additives and inorganic crystals at the nanoscale is a crucial
requirement for bioactive composites.[11] Here we prepare an
organic–inorganic hybrid crystal by the self-assembly of cal-
cium phosphate, surfactant, and protein. This hybrid crystal is
composed of uniform and alternate organic–inorganic layers
at the nanoscale. Both the inorganic crystalline phase and
organic phases in the hybrid crystals have an ultrathin thick-
ness of 1–2 nm. The two ordered components form simultane-
ously during the crystal generation so that they integrate well
with each other to form a superstructure. It is of great impor-
tance that such biomimetic crystals are considerably flexible
and elastic.
It is believed that functional organic molecules can interact
with calcium species at the organic–inorganic interfaces to
modulate the growth and assembly process of the inorganic
crystals. The globular protein bovine serum albumin (BSA),
which comprises a single chain of 583 amino acid residues,
is one of the most studied proteins. It is widely used as a
model protein in many fields including biomimetic miner-
alization.[12] Surfactants are widely applied as the crystalliza-
tion templates in many biomimetic studies.[13] However, the
cooperation of different organic additives has been frequently
overlooked in previous works because of the complicated inter-
actions in the system.[14] Actually, the interactions of a sur-
factant molecule and protein are widely found in biological
systems, for example, the interaction of protein with cell mem-
brane surfactants. The selected two compounds can represent
the protein matrix and special small functional molecules in
biomimetic mineralization studies. Usually, proteins and sur-
factants can form complexes in solution, which are frequently
described by a “necklace bead model”. The micelle-like clus-
ters of surfactants scatter along the polypeptide chains like the
pearls in a necklace.[15] The hydrophilic groups of micelles
are exposed to aqueous solutions and their configuration can
be adjusted. In such protein–surfactant complexes, the protein
is functionalized by the surfactant; meanwhile the aggrega-
tion behavior of the surfactant is also affected by the protein
structure. Here we find that the complex of BSA and an ani-
onic surfactant (sodium bis(2-ethylhexyl) sulfosuccinate, AOT)
could self-assemble into regular rhombus plates with a spe-
cific organic–inorganic substructure in a calcium phosphate
solution.
Scanning electron microscopy (SEM) shows the uniform
rhombic plates formed by the collaboration of calcium phos-
phate, BSA, and AOT (Figure 1a).The typical rhombs are
300–400 nm in the long axis and 200–300 nm in the short axis.
Their typical thickness is 80–100 nm. These rhombs are stable
and their structures can endure in solution or in air for months.
The energy-dispersive X-ray spectroscopy (EDS) reveals the pres-
ence of calcium and phosphate ions in the rhombs; the atomic
ratio of Ca:P is around 1.5. In addition to the elements of C
and O, S was also detected (Figure S1 and Table S1 of the Sup-
porting Information), indicating the presence of AOT (–SO3
2−).
The organic–inorganic hybrid composite was also confirmed by](https://image.slidesharecdn.com/413b0de5-a21b-4f75-a139-1fcf46b410b6-160908204742/85/5-all-published-paper-1-320.jpg)
