ARTICLE pubs.acs.org/crystal
Control over Different Crystallization Stages of CaCO3-Mediated by Silk Fibroin Ting Wang,†,‡ Renchao Che,‡ Wentao Li,† Ruixin Mi,† and Zhengzhong Shao*,†,‡ †
Key Laboratory of Molecular Engineering of Polymers of Ministry of Education, Department of Macromolecular Science, Fudan University, Shanghai, 200433, P. R. China ‡ Advanced Materials Laboratory, Fudan University, Shanghai, 200433, P. R. China
bS Supporting Information ABSTRACT: The crystallization process of CaCO3-mediated by the addition of silk fibroin at different crystalline stages was examined. During earlier stages of crystallization, time-resolved transmission electron microscopy (TEM) was applied to demonstrate that the crystallization of an amorphous precursor was based on randomly oriented domains. Different addition times of silk fibroin primarily led to two kinds of morphology of CaCO3, that is, lens-like and multilayered vaterite. Additionally, the thickness or number of layers of such vaterite would increase with the delay of silk fibroin addition, ascribing to the control of silk fibroin over different basic units during the aggregation and reorientation process. It was found that those squeezedout silk fibroins, which probably resulted from the relatively weak interaction between silk fibroin chains and (001) plane of vaterite phase during the crystallization process could lead to the formation of oblate aggregates via vectorial assembly of units with consistent orientation (nanoparticle for lens-like vaterite or flake for layered vaterite) and inhibition to the growth of (001) faces of fused intermediates. For comparison, the crystallization process of CaCO3 regulated by poly (acrylic acid) (PAA) was observed by cryoSEM, presenting a “stepwise aggregation” pathway to form spherical polycrystals which may be attributed to strong electrostatic interaction between carboxyl groups in PAA chains and nanoparticles. Therefore, the extent of binding affinity between organic and inorganic substances was proposed to be relevant to the reconstructuring process and the morphologies of final product.
’ INTRODUCTION Biominerals, such as bones, teeth, and shells, exhibit elaborate architectures and remarkable physical properties because of their complex hierarchical designs optimized over hundreds of millions of years.1 These hybrid materials are combinations of organic and inorganic components and serve a variety of purposes, including excellent mechanical and optical properties. This is based on the extraordinary control the organic constituents have over the size, morphology, and polymorph of inorganic components.1 However, whether the organic substances regulate the nucleation and growth of biominerals is unkown. Practically, the complexity of biological systems leads to difficulties in investigation of mineralization in vivo, yet there is a clear need to create in vitro systems that permit simplification and convenience to address this key gap in our knowledge. One of the most striking biomaterials, nacre has inspired researchers to investigate the formation mechanisms involved that have been found to be highly regular “brick-and-mortar” arrangement of aragonite tablets.24 Because nacre is comprised of a small percentage of β-chitin, silk-fibroin-like proteins, and acidic biopolymers, many approaches have been carried out to mimic the in vivo regulation action of these biopolymers in the growth process, including Langmuir monolayer,5 self-assembly monolayer,6,7 r 2011 American Chemical Society
micelle,8 and microemulsion.911 Additionally various additives such as proteins,1216 peptide,17 polysaccharides,1823 double hydrophilic block copolymers (DHBCs),2432 low molecule weight compounds,33 and simple ions34 have been adopted to direct the growth of CaCO3. Much effort has focused on exploring the interaction between additives and certain crystal faces or relating primary and secondary structure of organic additives to the crystal morphology.20,22,35,36 Recently, a series of quantitative analysis have been carried out during the early stages of CaCO3 precipitation in the absence and presence of such additives regarding the existence of prenucleation clusters, and the concept of “short-range order” has been placed in much earlier period.37,38 Therefore, it is necessary to investigate if such order structure in prenucleation clusters stage or the decorations derived from additives to different mineral phases could influence final morphology and polymorph. Generally, the crystallization process can be divided into several continuous periods, including nucleation, crystallization, growth, and reorientation. Therefore the interaction between Received: October 21, 2010 Revised: April 13, 2011 Published: April 15, 2011 2164
dx.doi.org/10.1021/cg101410u | Cryst. Growth Des. 2011, 11, 2164–2171
Crystal Growth & Design additives and a mineral form (ions, clusters, amorphous precursor, and crystalline phases) should be taken into consideration to better understand the crystallization process of CaCO3. Naka et al.39 has reported a concept for controlling polymorph of CaCO3 by adding poly (acrylic acid) (PAA) at different stages, and obtained stable vaterite particles. On the other hand, in terms of the importance of silk-fibroin-like protein (or its analogues such as silk fibroin derived from silkworm silk) in the formation process of nacreous platelets,2,40 it is important to investigate which steps greatly depended on the presence of silk fibroin in the mineralization process, while the relationship between the conformation of silk fibroin and morphology of CaCO3 was understand in a certain extent.35 Herein, we presented a detailed examination of the role of silk fibroin over different crystalline stages throughout the growth process of CaCO3 by altering the timing at which silk fibroin was present as an additive. Moreover, in situ observation on the growth process of CaCO3 regulated by PAA was used to make a comparison with that induced by silk fibroin, to understand the importance of the binding affinity of additive to crystallites or certain crystal faces.
’ EXPERIMENTAL SECTION Materials and Preparation of Regenerated Silk Fibroin (RSF). All chemicals (AR) as received without further purification and deionized water (DIW) were used. PAA (Mw = 2000) was purchased from Aldrich. Calcium chloride and sodium bicarbonate were purchased from Meixing Chemical Engineering Co., Ltd. (Shanghai). The cocoon silk of Bombyx mori constitutes of two core fibroin fibers coated by the water-soluble protein, that is, sericin. The fibroin contains up to 90% of amino acids of glycine (G), alanine (A), and serine (S) primarily in the repeated motifs of GAGAGS or GAGAGAGS, which leads to the domination of antiparallel β-sheet structure in silk fiber.41 The degumming (removing the sericin) and dissolving process of Bombyx mori silk followed established procedures. A semipermeable membrane (MEMBRA-CEL, 1200014000 MWCO) was used for dialysis. The dialyzed silk fibroin solution was centrifuged at 6000 rpm for about 5 min and the supernatant, which was about 4% (w/w) of aqueous regenerated silk fibroin (RSF), was collected at room temperature and stored at 4 °C. Crystallization of CaCO3. Small pieces of glass substrates were cleaned and sonicated in a bath containing ethanol for 10 min, then further soaked with a H2OHNO3(65 wt %)H2O2 (1:1:1, v/v/v) solution, rinsed with DIW and acetone, and finally dried in air. The precipitation of CaCO3 was carried by rapid mixing equal volume of CaCl2 (20 mmol/L) and NaHCO3 (20 mmol/L) with gentle stirring for 10 s. RSF solution was then added into above reaction mixture after a setting interval of reaction (0, 5, 10, 20, 30, 60, 90, 120, 240, and 360 min, respectively). A piece of cleaned glass slide was carefully put at the bottom of the beaker to collect any deposits. Mineralization was performed for 24 h at room temperature (around 25 °C). The glass slide with deposits was gently rinsed by DIW for several times and dried in vacuum overnight for further characterization. The crystallization of CaCO3 without additives was conducted under the same experimental conditions. Similarly, reversed addition procedure by mixing NaHCO3 (20 mmol/L) and RSF solution first was performed as well. Equal volumes of CaCl2 (20 mmol/L) were added after several minutes after the above solution was mixed for 0, 30, 60, 120, and 240 min, later the operation was the same as mentioned above. In Situ Observation. Mixed solution was dropped onto a copper grid with and additional solvent removed. Afterward, the grid was inserted into the TEM (JEM-2100F) chamber immediately. In situ experiments were carried out by continually keeping the sample under
ARTICLE
electron beam irradiation, and electron diffraction patterns and TEM images were collected at an interval of 5 min. CryoSEM is a suitable high-resolution observation method for hydrated samples.42 The crystallization was carried out at room temperature by mixing CaCl2 (20 mmol/L), NaHCO3 (20 mmol/L) in equal volume and PAA (4 wt %) at pH of 8 adjusted by NaOH solution (25 mmol/L). After set intervals, a glass slide with deposits was directly placed onto a stub, and then frozen in a Gatan-Alto 2500 high pressure freezer under liquid nitrogen. After transferring into the preparation chamber, the specimen was very slowly heated to 178 K to remove ice, and then scrape and incision steps were applied to further get rid of ice crystals and impurities. Samples were then imaged using a Hitachi-S4800 FE-SEM. Characterization. Scanning electron microscopy (SEM) was performed on a TS 5136MM microscope at a 20 kV acceleration voltage and Hitachi-S-4800 FE-SEM (Pt-coated prior to examination). Transmission electron microscope (TEM) observation and selected area electron diffraction (SAED) experiments were carried out using a JEM-2100F equipped with Gatan Image Filter system (Tridium). EDX was employed to determine elementary composition. The polymorphs of CaCO3 were examined by a Renishaw inVia Reflex Raman spectrometer equipped with 633 nm Helium/Neon laser, CCD detector and Leica 2500 optical microscopy. X-ray powder diffraction (XRD) data were recorded on an X'pert Pro with Cu Ka radiation. Sections of particles were prepared by Gatan Model 691 precision ion polishing system. Thermogravimetric analysis (TGA) was performed at 10 K 3 min1 on DTG-60H under nitrogen gas with flow rate of 40 cm3 3 min1, and the samples were strictly collected according to the individual fusion period before their dissolution.
’ RESULTS AND DISCUSSION Time-Resolved TEM Observation on Early Crystallization Process of CaCO3. Before the effects of silk fibroin addition on
CaCO3 crystallization were studied, it was necessary to establish the transformation period between each crystalline phase, especially the initial most transient and unstable phase. Therefore, in situ TEM observation and electronic diffraction (ED) detection was employed to investigate the evolution process of CaCO3. This was achieved initially by using a small amount of CaCl2 and NaHCO3 solution immediately dropped onto a copper grid. At the onset, the image of the original sample, shown in Figure 1a, indicated that there was liquid-like substance, and corresponding EDX spectrum demonstrated that the selected area was mainly composed of Ca, O, and C. Obvious dispersion ring of ED reflected that CaCO3 was in the amorphous phase (Figure 1c). With the TEM beam exposure time increasing, the gradual crystallization process was observed. The diffraction ring appeared to show that the crystallization of amorphous calcium carbonate (ACC) had started at 5 min (Figure 1d) and also showed the transformation period from single ion into amorphous precursor was very short. Over time a few disordered diffraction points appeared (Figure 1e) and became centrosymmetric after 20 min (Figure 1f), which may be assigned to vaterite with multiple orientations. At 30 min spherical nanoparticles (around 30 nm in size) with a rough profile and no obvious boundaries formed (Figure 1b), and the ED patterns indicated the nanoparticles having a 6-fold symmetric axis (Figure 1g), suggesting that it trended to be vaterite phase. In accordance with above development of ED patterns, control sample (i.e., the nanoparticles directly collected in the solution after 30 min reaction, Figure S1a, Supporting Information) possessed randomly oriented vaterite domains with a size of about 5 nm (Figure S1b, Supporting Information), indicating that the crystallization 2165
dx.doi.org/10.1021/cg101410u |Cryst. Growth Des. 2011, 11, 2164–2171
Crystal Growth & Design
Figure 1. TEM images of the early (0 to 1 min, a) and later (30 min, b) stage of CaCO3 crystallization period in the absence of additives. Inset in (a) indicates EDX spectrum of early substances. Scale bars, 100 nm. During the mineralization, ED patterns of CaCO3 in the area of (a) are showed as c (0 min), d (5 min), e (10 min), f (20 min), and g (30 min). Scale bar, 5 nm1. The spots pointed out by blue and orange arrow show that the d-spacing values corresponding to vaterite but the pattern is not indexed. The red, yellow, and green arrow indicate vaterite polycrystal, (002), (102), and (202). Parameters of the crystal are found in JCPDF (24-0030).
pathway of amorphous precursor is hardly affected by electronic beam. In addition, the vaterite intermediate would completely transform into thermodynamically stable rhombohedral calcite in the absence of silk fibroin after 4 h. For comparison, the earlier crystallization process of CaCO3 in the presence of silk fibroin was observed in the same way (Figure S2, Supporting Information). Gradual and stepwise phase transformation from initial liquid-like amorphous precursor to spherical vaterite crystalline phase was also detected. However, the crystallization of CaCO3 was postponed and retarded, probably owing to the inhibiting effect of silk fibroin in the nucleation and growth of CaCO3. Noticeably, when exposed to the electron beam, the crystallization process occurred more rapidly. We suggested that exposure to the electron beam provides additional energy, accelerating the dehydration and transformation of the amorphous precursor.43 Upon inspection of the distinctive features at each phase during crystallization process without additives, different addition timings of silk fibroin were used to investigate the relationship between additives and each stage. Specifically these were prenuleation, nucleation, formation of the amorphous precursor, and the later assembly process. Crystallization of CaCO3 with Different Addition Timings of Silk Fibroin. The effect of silk fibroin on CaCO3 crystallization with different addition timings was analyzed in supersaturated solution experiments, which facilitated the comparison among CaCO3 particles formed with addition of silk fibroin at different crystalline stages. The crystallization process of CaCO3 was performed at an almost constant pH value (pH = 8), and the conformation of silk fibroin was mainly random coil in an ambient environment.44 In the control experiment (without silk fibroin additive), plate-like vaterite aggregates (Figure S3, Supporting Information) mixed with rhombohedral calcite were produced,35 with vaterite completely transforming into calcite after 4 h, as mentioned above. In contrast, silk fibroin showed clearly influenced the different crystalline stages of CaCO3, as illustrated by three representative morphologies of CaCO3 particle in Figure 2. When silk fibroin was added 30 min into the reaction, lens-like CaCO3 particles with 4 μm diameter and 2 μm thicknesses were obtained (Figure 2a), and the surfaces of these particles were granular and rugged with densely packed
ARTICLE
Figure 2. FE-SEM images of vaterite particles obtained after 24 h with differing addition times of silk fibroin. (a, b 30 min; d, e 60 min; g, h 120 min). (b, e, h) show the detailed image for each deposit. Red lines outline the feature of hexagon-like shape. Insets indicate the side view and panoramic image of each sample, respectively. (c, f, i) are FE-TEM images and corresponding ED patterns of above particles. All the samples are assigned to vaterite (001) and SAED patterns are same as follows: 1 = (100), 2 = (110), 3 = (200). For parameters of the crystal please refer to JCPDF (24-0030).
30 nm nanoparticles (Figure 2b). When silk fibroin was added at 60 min, hexagonal lens-like particles with relatively uniform size were produced (Figure 2d). These were distinct from the lens-like ones produced at 30 min and exhibited a rudimentary multilayered structure, the edge of which consisted of hexagonal flakes with average size around 150 nm and the center of the surface was irregular and fused (Figure 2e). Adding silk fibroin at 120 min produced CaCO3 particles with integrated multilayer superstructures (Figure 2g), and both of their profile and basic units (a tablet with 1 μm in size) retained hexagonal features, implying a continual assembly process of hexagonal units (Figure 2h). Slightly different sizes of basic unit indicated different extents of fusion. All three kinds of particle displayed obvious single crystal features and were indexed as (001) oriented vaterite (Figure 2c, f, i), while the particles obtained at 12 h (Figure S4a, b, Supporting Information) presented the intermediates between a polycrystal and single crystal phase. The crystal phase of CaCO3 obtained through different timings of silk fibroin addition (30, 60, and 120 min) was further analyzed by XRD and Raman spectroscopy. The XRD patterns indicated that the CaCO3 products were a mixture of vaterite and calcite (Figure 3a and Table S1, Supporting Information). Calcite was formed due to unavoidable homogeneous nucleation and can be found in the inset images of Figure 2a, d, and g. However, the Raman spectra confirmed that the lens-like, hexagonal lens-like and multilayered particles were vaterite (Figure 3b). The shape, size, and polymorphs of CaCO3 particles formed in various conditions are summarized in Table 1. We found that the different addition timings of silk fibroin would primarily lead to two kinds of morphology of CaCO3 crystals, that is, lens-like and multilayered (the hexagonal lens-like particle were classified as multilayered particles as shown in Figure 2d). It should be noted 2166
dx.doi.org/10.1021/cg101410u |Cryst. Growth Des. 2011, 11, 2164–2171
Crystal Growth & Design
ARTICLE
Figure 3. XRD patterns (all deposits) and Raman spectra (specific lens-like and layered particles) of crystalline CaCO3 obtained with different addition time of silk fibroin: (i) 30 min; (ii) 60 min; (iii) 120 min. The parameters of the crystal are referenced to JCPDF (24-0030) and (72-1937).
Table 1. Shape and Polymorph of CaCO3 Particles a with Different Addition Time of Silk Fibroin moment of SF addition (min)
widthb ( μm) (n = 50)
shape and polymorph
thicknessb ( μm) (n = 50)
0
lens-like vaterite
3.8 ( 0.2
1.0 ( 0.1
5
lens-like vaterite and rhombohedral calcite
4.0 ( 0.3
1.0 ( 0.1
10
4.0 ( 0.5
1.0 ( 0.4
20
4.0 ( 0.6
1.5 ( 0.5
30
4.0 ( 0.6
2.0 ( 0.5
3555
multilayered hexagonal vaterite and rhombohedral calcite
c
3.0 ( 0.5 and 0.6 ( 0.05 6.0 ( 0.8
60 90
0.5 ( 0.01c 2.0 ( 0.2
120
10.0 ( 0.8
4.1 ( 0.3
240
10.0 ( 1.0
5.0 ( 0.4
360
rhombohedral calcite
a
Note: All particles were the products after 24 h reaction. b Only the vaterite particles were counted. c The size distribution of particles was too large to be statistical analyzed.
Figure 4. CryoSEM images of crystallization process of CaCO3 at different interval: (a) 2, (b) 4, (c) 8, (d) 10, and (e) 12 h. (f) ED patterns of final spherical vaterite polycrystal. The parameters of the crystal are referenced to JCPDF (24-0030).
that the silk fibroin introduced at earlier crystalline stages (within 30 min, including prenucleation, nucleation as well as the formation of amorphous precursor and primary nanoparticle), CaCO3 tended to precipitate as lens-like particles with the same width and slightly increased thickness. However, when the addition time of silk fibroin was delayed to around in 35 min until in 240 min after the mixing of CaCl2 and NaHCO3, the precipitation of CaCO3 was in the form of layered hexagons with increased size and number of layers. All of above
observations indicated that the silk fibroin was able to regulate the morphology to produce vaterite through its effect at the different crystalline stages of CaCO3. Formation of Lens-like Vaterite. As we have shown, adding silk fibroin into reaction system during either prenucleation or formation of ACC or stage of primary nanoparticle (i.e., during 030 min of the reaction) all led to the production of lens-like vaterite particles with the same orientation. The aggregation process of nanoparticles suggested an optimal stability derived 2167
dx.doi.org/10.1021/cg101410u |Cryst. Growth Des. 2011, 11, 2164–2171
Crystal Growth & Design
ARTICLE
Table 2. Changes of Morphology and Organic Content of CaCO3/Additive Hybrid Particles during the Fusion Process sample
a
reaction time
crystalline stagea
shape
units arrangement
organic content
CaCO3/SF
12 h
early
lens-like
loosely packed
5.2%
CaCO3/SF
18 h
middle
lens-like
densely fused
3.7%
CaCO3/SF
24 h
later
lens-like
solid
2.3%
CaCO3/PAA
4h
early
spherical
loosely packed
5.4%
CaCO3/PAA
10 h
middle
spherical
densely fused
4.6%
CaCO3/PAA
12 h
later
spherical
solid
4.1%
Note: All particles were collected to make comparison according to their individual fusion periods before dissolution.
from silk fibroin, similar to previous reports in the studies of growth based on nanoparticles.45,46 It is well-known that the carboxyl groups in macromolecular chains can generate nucleation points enabling CaCO3 nucleation in a specific position to form ACC nanoparticles, resulting in versatile morphology and controlled crystal phase of final products.26 Considering the extremely low content of carboxyl groups in silk fibroin chains (Glu þ Asp
