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Langmuir 2007, 23, 5014-5021
AFM Nanoindentations of Diatom Biosilica Surfaces Dusan. Losic,*,† Ken Short,§ James G. Mitchell,‡ Ratnesh Lal,| and Nicolas H. Voelcker*,† School of Chemistry, Physics, and Earth Sciences, and School of Biological Sciences, Flinders UniVersity, Bedford Park 5042, Australia, Materials and Engineering Science, Australian Nuclear Science Technology Organisation, Menai 2234, Australia, and Center for Nanomedicine and Department of Medicine, UniVersity of Chicago, Chicago, Illinois 60637 ReceiVed September 12, 2006. In Final Form: February 1, 2007 Diatoms have intricately and uniquely nanopatterned silica exoskeletons (frustules) and are a common target of biomimetic investigations. A better understanding of the diatom frustule structure and function at the nanoscale could provide new insights for the biomimetic fabrication of nanostructured ceramic materials and lightweight, yet strong, scaffold architectures. Here, we have mapped the nanoscale mechanical properties of Coscinodiscus sp. diatoms using atomic force microscopy (AFM)-based nanoindentation. Mechanical properties were correlated with the frustule structures obtained from high-resolution AFM and scanning electron microscopy (SEM). Significant differences in the micromechanical properties for the different frustule layers were observed. A comparative study of other related inorganic material including porous silicon films and free-standing membranes as well as porous alumina was also undertaken.
* Corresponding authors. E-mail:
[email protected], phone: +61 8 8201 5339, fax: +61 8 8201 2905. E-mail: dusan.losic@ flinders.edu.au, phone: +61 8 8201 2465, fax: +61 8 8201 2905. † School of Chemistry, Physics, and Earth Sciences, Flinders University. ‡ School of Biological Sciences, Flinders University. § Australian Nuclear Science Technology Organisation. | University of Chicago.
separation.18-21 The fine structures of both pennate and centric diatom frustules have been examined extensively.6 Their mechanical properties are less well understood.22-24 Hamm et al. showed an inverse relationship between frustule size and mechanical strength.22 Using AFM nanoindentation on the separated valves of pennate diatoms, Almqvist et al. reported high hardnesses and elastic moduli, in some cases approaching values obtained for pure silicon.23 Subhash et al. used a nanoindenter to investigate the fracture mode of the cleaned valve of centric diatoms at mN loads.24 In their study, however, the position of the indentations could not be located with precision due to the nature of the indenter used; SEM images had to be acquired before and after indentations. Both of these studies reported considerable variation in mechanical properties in different regions of the frustule. This variation was attributed to the general nonuniformity and porosity of the material but in part also to possible differences in silica content and phase. These studies did not compare the nanoscale mechanical properties of the intact diatom with those of the individual frustule components. Here, we have mapped the nanoscale mechanical properties of Coscinodiscus sp. diatoms using an AFM nanoindenter and have compared them with the mechanical properties of synthetic inorganic porous films and membranes. A series of AFM nanoindentation tests were performed at different locations along the diatom frustule. Micromechanical properties such as hardness
(1) Stupp, S. I.; Braun, P. V. Science 1997, 277, 1242-1248. (2) Sarikaya, M.; Tamerler, C.; Jen, A. K. Y.; Schulten, K.; Baneyx, F. Nat. Mater. 2003, 2, 577-585. (3) Vrieling, E. G.; Beelen, T. P. M.; van Santen, R. A.; Gieskes, W. W. C. J. Biotechnol. 1999, 70, 39-51. (4) Hasle, G. R.; Syversten, E. E. In Marine Diatoms. Identifying Marine Diatoms and Dinoflagellates; Tomas, K., Ed.; Academic Press: San Diego, 1996. (5) Noll, F.; Sumper, M.; Hampp, N. Nano Lett. 2002, 2, 91-95. (6) Round, F. E.; Craford, R. M.; Mann, D. G. Diatoms: Biology and Morphology of the Genera; Cambridge University Press: Cambridge, 1990. (7) Sumper, M. Science 2002, 295, 2430-2433. (8) Wetherbee, R.; Crawford, S.; Mulvaney, P. In Biomineralisation: From Biology to Biotechnology and Medical Application, Beurlein, E., Ed.; WileyVCH: Weinheim, 2000; p 189. (9) Parkinson, J.; Gordon, R. Trends Biotechnol. 1999, 17, 190-196. (10) Drum, R. W; Gordon. R. Trends Biotechnol. 2003, 21, 325-331. (11) Fuhrmann, T.; Landwehr, S.; El Rharbi-Kucki, M.; Sumper, M. Appl. Phys. B. 2004, 78, 257-260. (12) Rosi, N. L.; Thaxton, C. S.; Mirkin, C. A. Angew. Chem., Int. Ed. 2004, 43, 5500-5503. (13) Coradin, T.; Lopez, P. J. ChemBioChem 2003, 4, 251-259.
(14) Sandhage, K. H.; Dickerson, M. B.; Huseman, P. M.; Caranna, M. A.; Clifton, J. D.; Bull, T. A.; Heibel, T. J.; Overton, W. R.; Schoenwaelder, M. E. A. AdV. Mater. 2002, 14, 429-433. (15) Anderson, M. W.; Holmes, S. M.; Hanif, N.; Cundy, C. S. Angew. Chem. 2000, 112, 2819-2822. (16) Losic, D.; Mitchell, J. G.; Voelcker, N. H. Chem. Commun. 2005, 49054907. (17) Losic, D.; Mitchell, J. G.; Voelcker, N. H. New J. Chem. 2006, 30, 908919. (18) Hale, M. S.; Mitchell, J. G. Nano Lett. 2001, 1, 617-623. (19) Hale, M. S.; Mitchell, J. G. Nano Lett. 2002, 2, 657-663. (20) Hale, M. S.; Mitchell, J. G. Aquat. Microb. Ecol. 2001, 24, 287-295. (21) Losic, D.; Rosengarten, G.; Mitchell, J. G.; Voelcker, N. H. J. Nanosci. Nanotechnol. 2006, 6, 982-989. (22) Hamm, C. E.; Merkel, R.; Springer, O.; Jurkojc, P.; Maier, C.; Prechtel, K.; Smetacek, V. Nature (London) 2003, 421, 841-843. (23) Almqvist, N.; Delamo, Y.; Smith, B. L.; Thomson, N. H.; Bartholdson, A.; Lal, R.; Brzezinski, M.; Hansma, P. K. J. Microscopy 2001, 202, 518-532. (24) Subhash, G.; Yao, S.; Bellinger, B.; Gretz, M. R. J. Nanosci. Nanotechnol. 2005, 5, 50-56.
Introduction Diatoms are unicellular algae which have an intricately and uniquely nanopatterned silica exoskeleton.1-4 These exoskeletons, the so-called frustules, consist of fused silica nanoparticles assembled in stacks of membranes with highly organized and self-consistent pore arrangements and multiple pore sizes.5 These intricate structural features provide them an appropriate mechanical advantage when exposed to mechanical and biotic stress factors at the water/solid or water/air interface. Diatoms are a common target of biomimetic investigations. A better understanding of the diatom frustule structure and function at the nanoscale could provide new insights for the biomimetic fabrication of nanostructured ceramic materials and lightweight yet strong scaffold architectures for a variety of technological applications, including the design of nanodevices with relevance to biophotonics, immunoisolation, controlledrelease systems, bioencapsulation, biosensing,8-15 and nanofabrication,16,17 as well as for molecular or particle sorting and
10.1021/la062666y CCC: $37.00 © 2007 American Chemical Society Published on Web 03/31/2007
AFM Nanoindentation of Diatom Biosilica Surfaces
and elastic modulus were calculated from force penetration curves. Mechanical properties were correlated with AFM and scanning electron microscopy (SEM) images. AFM was chosen rather than the nanoindenter because the correlation of high-resolution structural and micromechanical properties was deemed more important than ultrahigh-precision hardness measurements at low spatial resolution. Coscinodiscus sp. was selected for this study because it is one of the largest genera of marine planktonic diatoms with diameters up to a few hundreds of micrometers and because it could be reproducibly grown in our laboratory. Experimental Details Coscinodiscus sp. was obtained from the CSIRO Marine Culture Collection, Australia, and cultured in GSE medium at 20 °C using a 12 h light/12 h dark cycle as described.25 Diatoms were harvested after 2-3 weeks of culturing and cleaned as described previously.26 For full removal of the organic matrix covering the siliceous frustules, concentrated sulfuric acid was used with small quantities of saturated potassium permanganate/oxalic acid, followed by centrifugation (3000 rpm for 5 min), and washing 5-10 times with distilled water followed by ethanol. These frustules are referred to as AC for acidcleaned. This procedure yields clean individual valves and girdle bands. To preserve the diatom whole frustule while removing most of the organic coating, a more gentle cleaning procedure was performed using 2% SDS in 100 mM EDTA solution followed by filtration and subsequent washing with distilled water.5 These frustules are referred to as SC for surfactant-cleaned. Cleaned diatoms were stored in a 100% ethanol solution. One drop of diatom suspension was deposited on a poly(lysine)-coated silicon wafer or glass slide and dried with nitrogen. The deposition and orientation of diatoms on the surface was monitored by light microscopy. AC diatom frustules adsorb on the surface either in concave orientation (exposing their internal surface) or in convex orientation (exposing their external surface). SC diatoms adsorb on the substrate surface in two orientations, either along their long axis (side-on) or along their short axis (end-on). The topography of the frustules was investigated by scanning electron microscopy (SEM) and AFM. For SEM imaging, the deposited diatoms were coated with a thin platinum film and mounted on a microscopy stub with carbon sticky tabs. The images were acquired using a Philips XL 30 field-emission SEM operated at 5-10 kV. To image internal structures of diatom valves, profile images were obtained on large cracks at a tilt angle greater than 30° normal to the surface. AFM imaging of diatom frustules was performed using a Nanoscope IV controller connected to a MultiMode or a Dimension 3000 AFM (Digital Instruments, Veeco Meteorology Group, Santa Barbara, U.S.A.). The topography images were obtained before indentation, using tapping mode in air and standard silicon tips (TESP, Digital Instruments). Nanoindentation was performed exclusively using the Dimension 3000 AFM with a special diamond tip, mounted at the end of a stainless steel cantilever (PDNSP, Digital Instruments). The cantilever specifications were as follows: length 350 µm, width 100 µm, thickness 13 µm, resonant frequency 50 kHz, and a spring constant of 132 N/m. The diamond tip was a three-sided pyramid with an ∼25 nm radius of curvature and apex angle of ca. 60° specified by the manufacturer. The shape and angle of the diamond tip were verified using sharp silica calibration gratings TGT1 (NTMDT, Russia).27 From these images, the real tip angle was derived (59.8 ( 1.5°) and was used in the equation for defining hardness. After obtaining an initial large scale image, the desired area for indentation was selected and an indentation was performed by selecting parameters such as the initial force (trigger threshold), (25) Guillard, R. R. L.; Ryther, J. H. Can. J. Microbiol. 1962, 8, 229-239. (26) Hasle, R.; Fryxell, G. A. Trans. Am. Microsc. Soc. 1970, 89, 469-475. (27) VanLandingham, M. R. J. Res. Natl. Inst. Stand. Technol. 2003, 108, 249-265. (28) VanLandingham, M. R.; McKnight, S. H.; Palmese, G. R.; Eduljee, R. F.; Gillepsie, J. W.; McCulough, R. L. J. Mater. Sci. Lett. 1997, 16, 117-119.
Langmuir, Vol. 23, No. 9, 2007 5015 force increment (threshold steps, if required), number of indents, and distance between indents. Both single indentations and a series of automated indentations (up to 10 indents in 1-3 rows) were performed on different frustule layers at different locations. An indent was made by forcing the tip into the surface until the required cantilever deflection was reached. For each indent, a plot of the cantilever deflection versus displacement in the z direction was recorded. These graphs were transformed into force (nN) versus tip penetration curves using DI (Veeco Corp., U.S.A.) off-line software and further used for the calculation of hardness and elastic modulus.27,28 Topographic AFM images were also obtained after an indentation experiment by scanning the surface using the diamond tip at low applied force. Due to the roughness of the diatom surfaces, image analysis of indents was not an appropriate method to establish the indentation contact area. Therefore, the force/penetration curves were generally used to calculate hardness and elastic modulus (see below). The applied load Fmax was determined as Fmax )
k‚∆V C
(1)
where k is the spring constant, ∆V the photodetector voltage change, and C the cantilever sensitivity. The cantilever sensitivity was determined by taking force measurements using hard surfaces such as sapphire and crystalline silicon. Loads from 1 to 15 µN were used. The hardness (H) was calculated using the Oliver and Pharr method 29-36 from the maximum load and the projected contact area (A) as H)
Fmax A
(2)
The contact area A was obtained from the following formula (eq 3) A ) 3 x3 hp2 tan2 θ
(3)
with hp the plastic depth of penetration and the face angle θ (59.8°). The depth of penetration (hp) was obtained from the force penetration curves as the x-axis intersect (zero load) of a line tangent to the initial part of the unloading curve. The hardness was calculated at a narrow range of maximum applied load of 2-4 µN to minimize the influence of the applied load.29-30 Hardness was averaged for each location on the diatom frustule from a minimum of 5 indents. The shape of the unloading force curves also allowed the derivation of qualitative information of the elastic properties of the frustule.28,31 The elastic modulus (indentation modulus) was calculated from the slope of the linear portion of the unloading force curve at near maximum load using eq 4 where E is the indentation modulus and dP/dh the slope of the unloading curve at maximum load (contact stiffness).32-36 This modulus in reality corresponds to a reduced E)
1 xπ dP 2 xA dh
(4)
modulus because it includes the deformation of sample and tip, but, given that the elastic modulus of the tip is much larger than that of the sample, no further correction was applied. Nanoindentations were performed on both the external and internal surfaces (convex and concave orientation) of the AC diatom frustule. A similar series of tests were performed on SC diatoms immobilized on the substrate surface in end-on orientation. Finally, indentations of side-on immobilized SC diatoms were performed at several (29) Oliver, W. C.; Pharr, G. M. J. Mater. Res. 1992, 7, 1564-1583. (30) Oliver, W. C.; Pharr, G. M. J. Mater. Res. 2004, 19, 3-20. (31) Fischer-Cripps, A. C. Nanoindentation, 2nd ed.; Springer-Verlag: New York, 2004. (32) Go¨ken, M.; Kempf, M.; Bordenet, M.; Vehoff, H. Surf. Interface Anal. 1999, 27, 302-308. (33) Kracke, B.; Damaschke, B. Appl. Phys. Lett. 2000, 77, 361-363. (34) Bhushan, B.; Koinkar, V. N. Appl. Phys. Lett. 1994, 64, 1653-1655. (35) Doerner, M. F.; Nix, W. D. J. Mater. Res. 1986, 1, 601-609. (36) Sneddon, I. N. Int. J. Eng. Sci. 1965, 3, 47-57.
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Figure 1. SEM images of diatom (Coscinodiscus sp.) identifying the locations where nanoindentations were performed. (a) The external surface (cribrum) of an acid-cleaned diatom frustule (central part and areas between central part and edges). (b) The internal surface (areola) of an acid-cleaned diatom frustule (central part and areas between central part and edges). (c) Top surface (cribellum) of a surfactant-cleaned diatom in end-on position. (d) Surfactantcleaned diatoms in side-on position with girdle bands The white dashed lines are marking the areas where indentations were performed. locations along the porous part of girdle bands. Figure 1 shows the locations of indentation points for both series. To determine the lateral distribution of the mechanical properties, hardness and elastic modulus were calculated from the indentation data and averaged over an area of approximately 5 × 5 µm2 across the marked radial lines (from the center to the edge). Indentations on porous silicon films and free-standing membranes as well as porous alumina membranes were performed as described above at random locations. A minimum of 5 series with 10 indents per series were acquired. Porous silicon was prepared by anodic oxidation of p-type silicon (resistivity 0.005-0.001 mΩ cm, Virginia Semiconductor, Fredericksburg, U.S.A.) in HF/ethanol solution as described in ref 37. A porous silicon membrane (65% porosity, 150 µm thickness, pores size 60 nm, pore-to-pore distance 50-70 nm) was obtained from pSimedica, Ltd. (Malvern, U.K.). Porous alumina membranes (with 20 nm pores) were obtained from Whatman (Brentford, U.K.). Also, control nanoindentation experiments were carried out on gold calibration grids (Micromasch, Estonia), crystalline (100) p-type silicon (Virginia Semiconductor, U.S.A.), and polycarbonate sheets (thickness 2 mm, Goodfellow, U.K.).
Results and Discussion Structural Characterization of Diatom Frustules. Figure 2 shows a series of SEM and AFM images which summarize the structure and organization of the porous layers of the silica frustule of Coscinodiscus sp. Three different porous layers of the frustule (Figure 2a-g) and the porous girdle bands (Figure 2i-k) of Coscinodiscus sp. are depicted here. A thin external silica layer (cribellum) (1) is shown in Figure 2a,b. This layer was only observed on surfactant-cleaned (SC) diatoms, since harsh acid treatment generally removed or dissolved the thin silica plate. An array of five to seven pores of 40-50 nm diameter with hexagonal arrangement decorates shallow, dome-shaped elevations (observed only in AFM image, Figure 2b) with an average size of about 1500 nm.38 Figure 2c,d shows SEM and AFM images of the second layer2 (cribrum) of the acid-cleaned (AC) diatom frustule, an ap(37) Steinem, C.; Janshoff, A.; Lin, V. S. L.; Voelcker, N. H.; Ghadiri, M. R. Tetrahedron 2004, 60, 11259-11267. (38) Losic, D.; Pillar, R. J.; Dilger, T.; Mitchell, J. G.; Voelcker, N. H. J. Porous Mat. DOI: 10.1007/s10934-006-9009-y, 2006.
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proximately 200 nm thick silica membrane consisting of porous dome structures that are organized in a hexagonal honeycomb pattern and match the cribellum domes in size. These domes are shallow with an elevation of about 60-80 nm over 1.5 µm diameter and therefore are only observed in AFM images (Figure 2d) but not in SEM images (Figure 2c). The predominant cribrum perforations are around 200 nm in diameter, but larger holes are also seen at the intersections of neighboring domes. These are likely to be the result of the corrosive cleaning procedure. Highresolution images indicate that the biosilica of this membrane is composed of fused particles of 50-80 nm diameter.21,38 The internal plate3 of the Coscinodiscus sp. frustule, on the other hand, shows marked differences to the external side (Figure 2e-g). Here, large, micron-sized holes (foramen) are organized in radial hexagonal rows. The foramen openings are approximately 1200 nm in diameter and are around 600 nm apart. The electron beam and the AFM tip also probe the underlying cribrum membrane at a depth of 350-450 nm (Figure 2e,f). These chambers formed by vertical walls (areolae) connecting the foramen and the cribrum plates are likely to mechanically strengthen the frustules. High-resolution AFM images of the surface of a silica rib between two foramen openings show a nodular topography, consistent with fused silica nanoparticles21,38 In between the holes, long meandering channels running along the silica ribs are observed (Figure 2g, inset). These channels, the function of which is unknown to date, extend radially from the nonporous thickened central plate.38 The cell nucleus is usually located underneath this central region of the frustule, and it is believed that the thickened silica plate on the central region can better withstand elastic deformations.38 So far, we have described three layers of biosilica membranes in Coscinodiscus sp. with pores increasing in size from the external to the internal side. Smaller pores form arrays that are centripetal in relation to the underlying large perforations. An SEM cross section obtained from the fractured frustule is shown in Figure 2h, confirming that the porous layers are integrated into a hierarchically organized structure. The function of this complex organization of the frustule valve is still not understood, but it is species-specific and likely to be the result of evolutionary pressure acting on diatoms. However, certain common design principles exist, such as the areolae chambers being arranged in hexagons and a size limit of 40 nm for the smallest perforations.21,38 Figure 2h also elegantly highlights the fact that a large percentage (∼60-70%) of the diatom’s frustule is essentially void space. This fact is exploited in materials such as kieselguhr and diatomite that have become common popular lightweight materials for a variety of support, filter, and insulation applications. The two halves of the diatom frustule are joined by a series of girdle bands (collectively know as cincture). The girdle band structure must withstand high mechanical stress in vivo to provide an intact seal between the two frustule halves. On SC diatoms, the girdle bands were conserved and could be characterized by SEM and AFM (Figure 2i-k). The girdle pores are of 120 nm diameter on average and are separated by 150-200 nm. Pores exhibit a perfect square lattice arrangement. Imperfections, as observed on the cribrum/cribellum, were absent. Where girdle bands overlap each other, a nonporous thin annular region can be seen (Figure 2j). Although we could only image the external side of the girdle band membrane, we obtained a profile structure of this part (Figure 2k) which shows vase-shaped chambers with a length of about 500 nm. This data suggests that both faces of the girdle band membrane are identical. Yet, the structure and morphology of this part is remarkably different to the frustule valves. While the function of the symmetrical pores on the girdle
AFM Nanoindentation of Diatom Biosilica Surfaces
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Figure 2. SEM and AFM structural characterization of porous layers of the silica frustule of Coscinodiscus sp. (a) SEM and (b) AFM images of the cribellum surface (z scale 500 nm). (c) SEM and (d) AFM images of the cribrum surface (z scale 500 nm). (e) SEM and (f) AFM images of the internal plate (z scale 700 nm). (g) AFM image of the central part of internal surface (z scale 700 nm) with inset showing channels and ribs. (h) SEM image of a cross section of the diatom frustule showing the internal shape of pores connecting cribellum and cribrum. (i) SEM and (j) AFM images of girdle band area of surfactant-cleaned diatom in side-on position (z scale 200 nm) and (k) SEM image of a cross section of the girdle band. Numbers within white circles denote the three porous layers of the diatom frustule: (1) cribellum, (2) cribrum, and (3) internal plate. Cross section image (h) is shown before images of internal layer (e,f,g).
band is unknown, a recent report speculates that this membrane might have photonic properties involved in maximizing the diatom’s light uptake.11 Micromechanical Properties of the External Frustule Membrane. Nanoindentation experiments on the external surface of a diatom frustule were performed first on the cribellum layer of SC diatoms. In order to perform these experiments, the diatom needed to be immobilized firmly on a substratum in an end-on position (Figure 1c). A typical AFM image of the cribellum after performing a series of indentations is shown in Figure 3a. The inset shows that cracks were induced at relatively small loads (1.5-3 µN), suggesting that this layer was mechanically weak. This is not a surprising result, considering the 12 µN), an indentation performed on the silica rib close to a foramen opening produced a crack which cut through the lip surrounding the opening but did not propagate along the silica rib. Mechanical Properties of the Girdle Bands. A fourth series of indentations was performed on the porous part of the girdle bands of SC diatoms adsorbed on the substratum in side-on position (Figure 1d). The surface was imaged before starting the indentation experiment to ensure that no sliding or rolling of the
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Figure 8. (a) AFM image of series of indentations on the girdle band surface of surfactant-cleaned diatom in side-on position obtained using force 1.5-3 µN (solid arrows) and 5-10 µN (dashed arrows). Cracks were observed when distance between indents was short (marked by dashed rectangular) and (b) typical force penetration curve showing loading and unloading parts (dashed arrows).
diatom shell occurred during the experiment. To further avoid movement of the diatom, indentations were limited to a small region on the porous part of the girdle band. An AFM image obtained after the indentation experiment is shown in Figure 8a and a typical force penetration curve in Figure 8b. Cracks are occasionally seen (dashed rectangle, Figure 8a), consistent with the fragility of this surface. The force curves consistently have more noise in them than those obtained on the other membranes signals. The average hardness is 0.059 ( 0.043 (n ) 50) GPa, and the elastic modulus is 4.0 ( 1.90 (n ) 50) GPa (Table 1). The average indentation depth for an applied force between 1.5 µN and 3 µN is ∼69.4 nm. These results show that the girdle band silica is one of softest parts of the valve silica. This could be a consequence of totally different morphological structure and higher porosity of this surface. Porosity has a large influence on the mechanical properties of silicon based film.38 The observed differences in the micromechanical properties between the valve and girdle band might also be related to a different silica biomineralization process. In comparison to the top part of the diatom frustule, which is formed from the SDV during cell division, the girdle bands are formed by the growing diatom when required. The girdle bands connecting the two frustule valves might have evolved to provide sufficient mechanical strength to diatoms without requiring multilayered patterned silica plates formed during the vesicular biomineralization. Similar Ceramic Materials of Synthetic Origin. A last series of indentations were performed on related manufactured membranes to compare the mechanical properties with those of the biosilica membranes. We used a p-type supported porous silicon
Figure 9. A series of AFM images of indentation points obtained on (a) porous silicon film and (b) a free-standing porous silicon membrane obtained with force between 1.5 and 5 µN. (c) Typical force penetration curve taken from a porous silicon film showing loading and unloading parts (dashed arrows) and several kinks in the loading curve (full arrows).
film of about 3 µm thickness, a free-standing porous silicon membrane, and a porous alumina membrane. A series of AFM images of indents on a porous silicon film and a free-standing porous silicon membrane are shown in Figure 9a,b. Highermagnification AFM images show irregular pore shape (data not shown) and a high dispersity in pore diameter (Table 1). A typical force penetration curve obtained from the porous silicon film is shown in Figure 9c. The loading curve shows several kinks (Figure 9, full arrows), which can be explained in terms of a collapsing porous silicon structure. The unloading curve is linear. The hardness and elasticity data for inorganic membranes are presented in Table 1. Results are in good agreement with previously published data for these materials.39-44 The mechanical properties of the porous silicon film (hardness 0.043 ( 0.016 GPa, elastic (39) Drory, M. D.; Searson, P. C.; Liu, L. J. Mater. Sci. Let. 1991, 10, 81-82 (40) Populaire, Ch.; Remaki, B.; Lysenko, V.; Barbier, D.; Artmann, H.; Pannek, T. Appl. Phys. Lett. 2003, 83, 1370-1372. (41) Bellet, D.; Lamagnere, P.; Vincent, A.; Brechet, Y. J. Appl. Phys. 1996, 80, 3772. (42) Xia, Z.; Riester, L.; Sheldon, B. W.; Curtin, W. A.; Liang, J.; Yin, A.; Xu, J. M. ReV. AdV. Mater. Sci. 2004, 6, 131-139.
AFM Nanoindentation of Diatom Biosilica Surfaces
modulus 8.11 ( 2.54 GPa) are comparable to those of the cribellum silica, while the porous silicon membranes (hardness 0.62 ( 0.15 GPa, elastic modulus 22.33 ( 3.78 GPa) show better mechanical properties, similar to those of the internal plate. On the other hand, the mechanical properties of porous alumina are remarkably different (hardness 1.72 ( 0.81 GPa, elastic modulus 58.3 ( 20.1 GPa), suggesting that crystal structure and material composition is more important than porosity for the mechanical properties for these biomaterials.
Conclusions The micromechanical properties of a centric diatom frustule were investigated using AFM-based nanoindentation and compared to the respective properties of porous silicon and alumina. Despite existing uncertainties, AFM nanoindentation provides useful information about the nanoscale mechanical properties of biological materials such as diatoms. AFM enabled us to correlate (43) Yan, J.; Takahashi, H.; Tamaki, J.; Gai X.; Harada, H.; Patten, J. Appl. Phys. Lett. 2005, 86, 181913-1-181913-3. (44) Drechsler, D.; Karbach, K.; Fuchs, H. Appl. Phys. A: Mater. Sci. Process. 1998, 66, S825-S829.
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hardness and elasticity with morphological features on the frustule valve with high spatial resolution. The results show that hardness and elastic modulus varied at different parts of the diatom frustule. The lowest values were found on the cribellum and the girdle band. The observed variation of mechanical properties could be attributed to difference in pore size, pore distances, and porosity, but also to a different biomineralization process. Many other parameters can also influence micromechanical properties of diatoms such as the size of the diatom frustule and the quality of nutrition (Si concentration) during culturing, and further studies are required to address these issues. Porous silicon fabricated by anodization shows much greater dispersity in pore size and shape, but shows similar mechanical properties to those of the diatom biosilica. Acknowledgment. The authors gratefully acknowledge support from the Australian Research Council (ARC) and the Australian Institute of Nuclear Science and Engineering (AINSE). LA062666Y
