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SCIENCE CHINA Physics, Mechanics & Astronomy •

February 2010

Research Paper •

Vol. 53 No.2: 380−388

doi: 10.1007/s11433-010-0134-x

Chemical-mechanical stability of the hierarchical structure of shell nacre SUN JinMei & GUO WanLin* Institute of Nanoscience, Nanjing University of Aeronautics and Astronautics, Nanjing 210016, China Received June 24, 2009; accepted September 20, 2009

The hierarchical structure and mechanical property of shell nacre are experimentally investigated from the new aspects of chemical stability and chemistry-mechanics coupling. Through chemical deproteinization or demineralization methods together with characterization techniques at micro/nano scales, it is found that the nacre of abalone, haliotis discus hannai, contains a hierarchical structure stacked with irregular aragonite platelets and interplatelet organic matrix thin layers. Yet the aragonite platelet itself is a nanocomposite consisting of nanoparticles and intraplatelet organic matrix framework. The mean diameter of the nanoparticles and the distribution of framework are quite different for different platelets. Though the interplatelet and intraplatelet organic matrix can be both decomposed by sodium hydroxide solution, the chemical stability of individual aragonite platelets is much higher than that of the microstructure stacked with them. Further, macroscopic bending test or nanoindentation experiment is performed on the micro/nanostructure of nacre after sodium hydroxide treatment. It is found that the Young’s modulus of both the stacked microstructure and nanocomposite platelet reduced. The reduction of the microstructure is more remark than that of the platelet. Therefore the chemical-mechanical stability of the nanocomposite platelet itself is much higher than that of the stacked microstructure of nacre. nacre, aragonite, organic matrix, hierarchical structure, chemical-mechanical stability

Nacre (mother of pearl) from the molluscan shells is a typical self-assembled complex by the bottom-up nanofabrication method and it is composed of about 95 wt% ‘hard’ calcium carbonate crystal (aragonite) and only a few percent of ‘soft’ organic matrix, which is a mixture of proteins, glycoproteins, and polysaccharides [1–6]. The natural hierarchical structure and the excellent mechanical behavior of nacre have got extensive attention for several decades. Most researches of nacre have concentrated on its brick-and-mortar like multilayer microstructure with aragonite platelets and thin organic interlayers [7] and the submicro/nano structures between platelets such as aspertities [8], microwaviness [9] and mineral bridges [10]. Much work has been done about the toughness mechanisms of nacre: crossed lamellar microstructure design and crack deflection [11–13], plastic stretch of organic interlayers and aroused slip of adjacent *Corresponding author (email: [email protected]) © Science China Press and Springer-Verlag Berlin Heidelberg 2010

interfaces of platelets [4,11,14], shear resistance provided by inner submicro/nano structures [8,9,15–17]. Recently nanoparticles have been observed in individual aragonite platelets and it is suggested that the plastic deformation from the aragonite nanoparticles may be a key issue to the fracture toughness of nacre [18–21]. However much need to be explored about nacre on micro and nanometer scales. For example nanoindentation can show us the elastic modulus of an individual aragonite platelet, but its stability and strength remain unexplored yet. Therefore to unveil thoroughly the secret how nacre achieves the notable high ductility, it is necessary to probe the chemical stabilities and mechanical properties of nacre at different length scales. Here we investigated the nacre of shell was studied from a new perspective of chemical stability and chemical-mechanical coupling by chemical deproteinization and demineralization methods combined with macro/nano mechanical tests and AFM and SEM imaging. phys.scichina.com

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1 Materials and methods 1.1 Materials All specimens were cut from living abalone shells (Haliotis discus hannai，which belongs to the class of gastropod) from in-shore fisheries of north coast of China. The specimens were cut from the ellipse area marked by dashed line in Figure 1. The square specimens were for AFM or nanoindentation experiments and the rectangle ones were for three-point bending tests. The specimens for three-point bending tests were cut from abalone shell along certain orientation (Figure 1). The periostracum and the prismatic part of the specimens were mechanically removed along the direction more or less perpendicular to the c-axis and only the nacreous part was for investigation. The specimens of 2.5 mm×2.5 mm for AFM and nanoindentation experiments (Figure 2(a)) were prepared with the nacre surfaces being mechanically ground and polished by using W1.5 diamond paste. All surfaces of the nacre specimens for three-point bending tests (Figure 2(a)) were mechanically ground and the loading surfaces were polished using W1.5 diamond paste. The sizes of individual bending specimens have average depth, width and length about 0.5, 2.4 and 14 mm, respectively. All specimens were washed successively with 0.5% NaOH solution and the distilled water for 20 minutes in an ultrasonic cleaner and air-dried. Such specimens were called as fresh ones. The NaOH-etched nacre specimens for AFM imaging were prepared by soaking in 5% NaOH solution for 3, 6 and 19 h respectively. During the soaking process, the NaOH solution was replaced once per hour. Before AFM imaging, each of

Figure 1 Optical picture of Haliotis discus hannai shell and sampling location and orientation.

Figure 2 Schematic illustrations of AFM/nanoindenation and three-point bending tests on nacre.

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the NaOH-etched specimens was washed ultrasonically with distilled water for 10 min and air-dried. The NaOHetched nacre specimens for bending or nanoindentation tests were prepared in the same way. A second group of fresh specimens used in comparison tests was prepared by soaking in 1% HCl solution for 30, 60 and 90 s respectively, and then washing gently by small drops of the distilled water. Finally the 90 s-HCl-etched sample was washed ultrasonically with distilled water. The EDTA- etched nacre samples were prepared by soaking in 0.5 mol/L EDTA-2Na solution (disodium salt ethylenediaminetetraacetic acid, pH 8.0) for 5, 10, 15 and 20 min respectively and the washing process was the same as that for NaOH-etched specimens. 1.2 Experimental methods The hierarchical structure of nacre was characterized by using a scanning probe microscope (SPM) (SPI3800N-HV300) with the tapping mode. The tip was in intermittent-contact with the surface of sample to reduce the potentially destructive lateral forces. By mapping the phase lag of the cantilever oscillation, phase imaging was used to detect variations in composition, adhesion, friction, viscoelasticity and perhaps other properties such as different components in composite materials [22,23]. A commercial silicon probe with a cantilever of 1.4 N/m force constant and 27 kHz work frequency was used for AFM imaging. All data were collected under ambient conditions at ~60% relative humidity and ~25°C. The images were taken at resolution of 256 pixels×256 pixels. Scanning electron microscopy (SEM) (LEO1530VP) was used to image the nacreous structure as well. The SEM samples were coated with 10 nm Au. The working distance is 10 mm and the electron acceleration is 15 kV. Three-point bending tests were carried out on a universal testing machine (Instron 4466) with a span of 10 mm. Externally applied load was along the c-axis direction with loading speed of 0.5 mm/min. At least five specimens were repeatedly tested in each set. Nanoindentation experiments were operated on a nanoindenter (MTS-SA2) with a Berkovich diamond probe tip under displacement-controlled condition. Dynamic contact module and continuous stiffness measurements were used. The loading speed was 10 nm/s and along the c-axis direction. Thermal drift rate was not more than 0.15 nm/s. There is few research about the Poisson’s ratio of individual platelets in nacre. For most materials, the Poisson’s ratio is between 0.2 and 0.3, which has slight effect on the measured results of nanoindentation [24–26]. Therefore here the Poisson’s ratio was set as 0.25. The experiment temperature was about 20°C and relative humidity was about 60%. At least five indents were performed for one specimen and the space between indents was 10 μm.

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2 Results and discussion 2.1 Hierarchical structure of nacre The nacre in Haliotis discus hannai shells was stacked with pseudo-polygonal aragonite platelets of approximately 450 nm in thickness and 8 μm in size and organic matrix thin interlayers (Figure 3). Fresh nacreous microlayers are shown distinctly and this imaged surface was denoted as face-A (Figure 4(a)). The average distance between the edges of aragonite-microlayers, l, is 2.35 µm. However according to the SEM image of Figure 3(b), the average thickness of the nacreous microlayer (i.e. the thickness of aragonite platelet), d, is approximately 450 nm. So face-A is not perpendicular to the c-axis. The interfaces of individual aragonite-microlayers denoted as face-B are presented with the thicker diagonal pattern in Figure 4(b). Therefore the angle


θ between face-A and face-B is about 11°. By further imaging, the nanocomposite structure of hard aragonite nanoparticles (dark color) and surrounding soft intraplatelet organic matrix (light color) was found in aragonite platelets (Figure 5(a)). The similar nanocomposite structure can also be observed from face-C (perpendicular to face-A, Figure 4(b)). Coupling with the SEM image of the cross section of a platelet shown in Figure 6, it shows that the aragonite platelets consist of masses of nanoparticles among the organic network. Grain size analysis by the software SPIPTM [27] indicates that the mean diameter of the nanoparticles is (50±19) nm

Figure 3 SEM images of the microscale multilayer structure of nacre. (a) Fracture section; (b) the growth surface. Scale bars: 6 μm.

Figure 5 Nanocomposite structure of individual aragonite platelets from fresh nacre by AFM phase imaging. (a), (b) Nanocomposite structure imaged from face-A and face-C orientation; (c) nanocomposite structure from face-A orientation for another platelet; (a′)–(c′) frequency percentage and Gauss distribution of nanoparticles diameter of (a)–(c). Number values of insets indicate mean ± standard deviation, scale bars: 200 nm.

Figure 4 Multilayer stacked structure of fresh nacre and the imaging orientation. (a) AFM topography image of the prepared surface of nacre (face-A); (b) schematic illustration of the location relationship with face-A and face-B (interfaces of individual nacreous microlayers).

Figure 6 SEM image of an individual aragonite platelet.

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for a platelet from face-A (Figure 5(a)) and (51±16) nm from face-C (Figure 5(b)). There is one peak in nanoparticle diameter distribution on face-A, which is in normal distribution. Yet there are two peaks in the diameter distribution on face-C around 36 nm and 55 nm. The mean diameter and distribution of the nanoparticles change from platelet to platelet. For example, the mean diameter of nanoparticles on face-A from another platelet is only (42±13) nm (Figure 5(c)). On the other hand, the distribution of the intraplatelet organic matrix is also quite different among platelets. For the platelet shown in Figure 5(a), the organic matrix forms a continuous network, the same as mentioned by Rousseau et al. [20]. The ratio of the organic matrix’s area is about 7.13% calculated by the software SPIWin attached to the SPM. However, the organic matrix is much less and not continuous for the platelet shown in Figure 3(c) and the area ratio is only about 2.83%. The large scatter in nanoparticle size and in the distribution of organic matrix may due to differences in growth season and environment [28–30]. 2.2 Chemical stability of nacre 2.2.1 NaOH NaOH solution is usually used to dissolve the organic components in natural biomineral materials [31]. Figure 7 shows the AFM images of NaOH-etched nacre for 3, 6 and 19 h, respectively. With the increasing of the etched time the shedding fracture starts from the edges of the multilayer structure of nacre and spreads into the interior (Figures 7(a)–(c)). After 19 h, the interfaces of the microlayers (i.e. face-B) in nacre were exposed finally. The orientation relationship between face-A and face-B can be observed directly, coinciding with Figure 4(b). It is deduced that the


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behavior of shedding fracture of nacre is mainly attributed to chemical decomposition of the organic interlayers. Figure 8 shows that the organic matrix within the aragonite composite platelet itself can also be decomposed by NaOH solution. After 6 hours soaking in the NaOH solution, the area ratio of the intraplatelet organic matrix is decreased to 3.65% from original 7.13%. After 19 h the organic matrix in the imaging face is nearly completely decomposed. Grain size analysis shows that the mineral nanoparticles are not affected by NaOH solution and their mean diameter has little change. 2.2.2 HCl As shown in Figure 9, the intensive decomposition of calcium carbonate by the HCl solution makes the imaging surface of nacre covered by the chemical residues (white in Figures 9(b′) and 9(b″)), which accumulate gradually with increasing etched duration. Ultrasonic washing can remove the accumulated residues and expose the underlying multilayer microstructure of nacre and inner nanocomposite structure (Figures 9(c), (c′) and (c″)). 2.2.3 EDTA As a Chelating agent, EDTA molecule can bind to calcium ions in 1:1 to gain chelate complex as: edta4−+Ca2+→[Ca edta]2−. Thereby the nacre is decalcified by EDTA [32]. Here we observe the detailed decalcification process of nacre. The fresh nacre in Figure 10(a) was soaked in EDTA solution for 5, 10, 15 and 20 min respectively. For face-A of nacre, the outermost part of the sample is decomposed to exhibit big grains after 5 min EDTA-etched (Figures 10(b) and (b′). Then the underlying structure is exposed gradually. After 20 min-etched, the microlayers made up of aragonite platelets are exposed perfectly (Figure 10(e)). For face-C of nacre, shedding by layers can be found easily with its typical stacked way in columnar (Figure 11). 2.3 Mechanical properties of NaOH-etched nacre To characterize chemical-mechanical coupling stability of the hierarchical structure of nacre, we applied macroscopic three-point bending tests and nanoindentation experiments to study the stacked microstructure and the nanocomposite structure of aragonite platelets of the NaOH-etched nacre.

Figure 7 Stacked microstructure of NaOH-etched nacre by AFM topography imaging. (a)–(c) NaOH-etched for 3, 6 and 19 h, respectively; (c′) a three-dimensional picture of (c). Scale bars: 2 μm.

2.3.1 Mechanical properties of the stacked microstructure of NaOH-etched nacre Three-point bending tests were performed on the stacked nacre with loading direction along c-axis. Figure 12(a) shows the typical load-displacement curves of the fresh nacre and the NaOH-etched nacre after 1 to 19 h soaking in the NaOH solution. The elastic modulus and the flexural strength of the fresh nacre are about 51 GPa and 278 MPa, respectively. And these values both present falling with the decomposition of the organic interlayers by the NaOH solu-

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Figure 8 Nanocomposition structure of NaOH-etched nacre by AFM phase imaging. (a) Fresh nacre; (b), (c) NaOH-etched nacre for 6 and 19 h; (a′)–(c′) the three-dimensional pictures of (a)–(c); (d) Gauss distribution of the mean diameter of the nanoparticle of (a)–(c). Scale bars: 200 nm.

Figure 9 Images of HCI-etched nacre by AFM. (a), (a′) Stacked microstructure of fresh nacre and inner nanocomposite structure; (b), (b′) hierarchical structure of 60 s-HCI-etched nacre (washing gently); (c), (c′) hierarchical structure of 90 s-HCI-etched nacre (washing ultrasonically); (a″)–(c″) The three-dimensional pictures of (a′)–(c′).

tion (Figure 12(b)). Therefore the decomposition of organic interlayers can decrease the stiffness and strength of the stacked microstructure of nacre greatly. It is also found that the flexural strength of the stacked nacre keeps decreasing step by step, however its modulus keeps at 50 GPa around till 6 hours then decreases remarkablely. It shows that at the

early etched process, the gradual decomposition of the organic interlayers has less effect on the stiffness of the whole stacked nacre. Therefore it indicates that besides the organic interlayers, the microstructure design mechanism of layerby-layer cross stacked form is also significant to the dominated factor of the stiffness of nacre.

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Figure 10 Images of EDTA-etched nacre (face-A) by AFM. (a) Fresh nacre; (b), (b′) 5 min-EDTA etched nacre; (c)–(e) EDTA-etched nacre for 10, 15 and 30 min. Scale bar: 2 μm.

Figure 11 30 min.

Images of EDTA-etched nacre (face-C) by AFM. (a) Fresh nacre; (b), (b′) 10 min-EDTA-etched nacre; (c), (d) EDTA-etched nacre for 20 and

Figure 13(a) shows that by the three-point bending test, the outmost nacreous microlayers curls up into the large roll on the loading surface nearby the fracture section of 19

h-NaOH-etched nacre. This phenomenon is never found in fresh nacre specimens. It is indicated that the organic interlayers between the aragonite platelets were decom posed to

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Figure 12 Results of three-point bending tests upon stacked microstructure of nacre. (a) Typical load-displacement curves for fresh or NaOH-etched nacre; (b) mechanical properties of fresh and NaOH-etched nacre.

Figure 13 SEM images of the fracture section of the 19 h-NaOH- etched nacre by three-point bending tests. (a) Large crimps of the outmost nacreous microlayers presented on the loading surface; (b) fracture section near the loading surface.

lose adhesiveness. But the rolling nacreous microlayers made up of aragonite platelets is still in a integral state. Therefore the individual aragonite platelet has higher chemical stability than the stacked microstructure of nacre. Many pot-holes can be observed in the fracture section of 19 h-NaOH-etched nacre (Figure 13(b)), which is rarely found in that of fresh nacre. It shows that the stacked microstructure of nacre is not stable at all, due to the chemical decomposition. 2.3.2 Mechanical properties of nanocomposite aragonite platelets of NaOH-etched nacre Nanoindentation experiments were performed upon the

fresh as well as 19 h-NaOH-etched individual aragonite platelets and typical results are presented in Figure 14. Sudden displacement jumps are found on the load-depth curves of the 19 h-NaOH-etched aragonite platelets (Figure 14(b)). These jumps mainly occur at the depth ranging from 350 to 450 nm. This phenomenon is thought to ascribe to the decomposition of the organic interlayer between upper and lower platelets due to the NaOH solution. The DCM·CSM technique makes the continuous measurement of mechanical properties of individual aragonite platelets possible. It is found that both the hardness and Young’s modulus show noticeable change with depth of indentation. Take the average thickness of 450 nm of aragonite platelets into account and neglecting the very low values caused by surface effect in the beginning, there is a valid and reliable hardness/Young’s modulus data segment up to 200 nm depth, which should reflect the mechanical properties of aragonite platelets themselves. With the deepening of indentation, the hardness and modulus values decrease gradually, because of the influence of the underlying organic interlayer and other platelets. Beyond 400 nm of depth, the acquired hardness and modulus trend to stable values, which reflect the mechanical properties of the stacked microstructure with platelet-organic interlayer-platelet. Therefore, the Young’s modulus and hardness of a single aragonite platelet are 57.43 ± 6.8 GPa and 3.96 ± 0.9 GPa, respectively. 2.3.3 Discussion Figure 15 shows the results of the mechanical properties from the macroscopic three-point bending tests and nanoindentation. After 19 h-NaOH-etched, the modulus is reduced by about 27% to 41.98 GPa and the hardness is reduced by about 54% to 1.81 GPa (Figure 15(a)). However, according to the three-point bending tests, the modulus of the 19 h-etched stacked nacre decreases to 27 GPa, nearly half of the fresh one. And the decrease of the flexural strength is about 78% (Figure 15(b)). Therefore, for the chemical decomposition of NaOH, the nanocomposite structure of

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Figure 14 Nanoindentation experiments upon individual aragonite platelets in nacre. (a) Load-depth curves of the fresh and 19 hour-NaOH-etched aragonite platelets; (b) the magnified image in the rectangle frame in (a), displacement jumps are marked by arrows.

Figure 15 Comparison of mechanical properties between the aragonite platelet and the stacked nacre. (a) Modulus and hardness from nanoindentation experiments upon fresh and 19 h-NaOH-etched aragonite platelets; (b) modulus from three-point bending tests upon fresh and 6 and 19 h-NaOH- etched aragonite platelets.

individual aragonite platelets has much higher mechanical stability than the stacked structure of nacre.

3 Conclusion (1) By chemical demineralization or deproteinization techniques together with nanostructure analyses, it is found that the size and uniformity of the nanoparticles show large variation among individual aragonite platelets, with the mean diameter ranging from (50±19) nm to (42±13) nm. And the distribution of the intraplatelet organic matrix for different platelets is also different and exhibits as continuous or non-continuous network. (2) The chemical stability of nacre was investigated through the deproteinization method with NaOH solution. Both organic interlayer between adjacent aragonite platelets and organic matrix within a single platelet can be decomposed by the NaOH solution. Yet for 19 h-etched nacre, shedding-off found from the edges of the stacked multilayer and large crimps of the outmost microlayer found on the loading surface near the fracture section indicate that the organic interlayer is decomposed more easily than the intraplatelet organic matrix. Therefore the nanocomposite struc-

ture of aragonite platelets has higher chemical stability than the stacked microstructure of nacre. (3) Through three-point bending and nanoindentation experiments respectively, it is found that the mechanical properties of both stacked microstructure of nacre and the interior single aragonite platelet can be significantly reduced by NaOH-deproteinization treatment. But the modulus of the former decreases much more rapidly. Therefore, the chemical-mechanical stability of nanocomposite platelet itself is much higher than the stacked microstructure of nacre. This work is supported by the National Basic Research Program of China (Grant No. 2007CB936204), the Program for Changjiang Scholars and Innovative Research Team in University of China (Grant Nos. 705021 and IRT0534), the National Natural Science Foundation of China (Grant No. 10732040) and the Natural Science Foundation of Jiangsu Province (Grant No. BK2008042). 1 2 3

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