NANO LETTERS
Nanostructure of Diatom Silica Surfaces and of Biomimetic Analogues
2002 Vol. 2, No. 2 91-95
Frank Noll,*,† Manfred Sumper,‡ and Norbert Hampp† Institute of Physical Chemistry and Materials Science Center, Philipps UniVersity, Hans-Meerwein-Str., D-35043 Marburg, Germany, and Department of Biology, UniVersity of Regensburg, UniVersita¨ tsstr. 1, D-93053 Regensburg, Germany Received July 13, 2001; Revised Manuscript Received October 11, 2001
ABSTRACT Diatoms generate their cell walls by silica biomineralization. The cell walls are composed of silica and organic macromolecules and show a complex microscopic structure. Analysis of this structure by different atomic force microscopy (AFM) techniques revealed an unexpected nanostructured granular surface. Silaffins, proteins that are posttranslationally modified with long-chain polyamines, and oligo-N-methylpropyleneamines were identified as the main organic constituents of diatom biosilica.1 Silaffins as well as free propyleneamines of different chain lengths induce rapid precipitation of nanosized particles from silicic acid solutions in vitro. In a biomimetic approach, we reacted aqueous silicic acid solution with tripropylenetetramine in CHCl3 in a biphasic system. As a result, thin nanostructured silica layers that show a granular nanostructure very similar to that of the diatom cell walls were obtained. This finding may serve as a good model to study the mechanisms that lead to the nanostructure of the diatom cell walls.
Diatoms are eukaryotic, unicellular algae that are ubiquitously present in almost every water habitat on earth. Apart from their ecological significance, they are well known for the spectacular structures of their silica-based cell walls. The shape of the cell walls is characteristic for the different species. The cell walls are structured on a nanometer to micrometer scale,2-5 and they are reproduced precisely during each cell division cycle. Investigation of the mechanisms that control nanostructured biosilica generation is not only of biochemical interest but also of interest in materials sciences. As far as biomineralization leads to reproducible nanostructures, it is believed that biomimicking this process may allow the production of advanced materials at ambient conditions. It is expected that these structures exhibit superior properties in a wide range of applications,6,7 and perhaps one will be able to produce custom-tailored nanostructures easily. Diatom cell walls are composed of inorganic and organic components. About 97% of the diatom cell wall consists of inorganic compounds, in particular almost pure hydrated silica doped with trace amounts of aluminum and iron. There is evidence from biochemical studies that the organic compounds are (glyco)proteins,8-11 and in the past few years a number of proteins associated with diatom biosilica have been purified and structually characterized. Recently, a set of cationic polypeptides (named silaffins, Figure 1) isolated form purified cell walls of the diatom * Corresponding author. E-mail:
[email protected] † Philipps University. ‡ University of Regensburg. 10.1021/nl015581k CCC: $22.00 Published on Web 01/11/2002
© 2002 American Chemical Society
Figure 1. Chemical structure of a part of a silaffin extracted from C. fusiformis posttranslationally modified with oligo-N-methylpropyleneamine at one lysine residue and methylated twice at a second lysine.
Cylindrotheca fusiformis were shown to generate networks of silica nanospheres within seconds when added to a solution of silicic acid.1 Furthermore, high amounts of long-chain polyamines have been found in diatom cell walls. Upon addition to monosilicic acid solution, the polyamines induce rapid precipitation of silica spheres with characteristic diameters in vitro.12 In this study, we present results of AFM investigations of diatom cell walls (valves) showing a distinguished nanostructured surface, which was not observed earlier with SEM.
Figure 2. SEM images of the inner (a) and outer (b) surface of a valve of C. granii. The valves had been cleaned intensely and were not coated prior to SEM investigation.
A biomimetic model for the cell wall formation was developed where newly synthesized oligopropyleneamines and silicic acid solution react at the interface between two immiscible liquid phases and form thin silica sheets. Diatoms Coscinodiscus granii were isolated from the North Sea and cultivated in an artificial seawater medium. To isolate diatom valves, cells were harvested and boiled in 2% SDS, 100 mM EDTA solutions in order to remove intracellular components. Details of the isolation are described elsewhere.12,13 As a result of the purification process, single diatom valves suspended in water were obtained. AFM images have been collected using a TopoMetrix Explorer AFM equipped with a 150 µm and a 2.5 µm scanner (Veeco Instruments, Santa Barbara, CA). Contact and noncontact mode imaging has been used. In contact mode, different AFM tips (standard, twin, oxide-sharpened) were used and different load forces were applied. All images presented are raw data except for first- or second-order twodimensional flattening. Details of the scanning parameters are given in the figure captions. Approximately 10 µL of a diatom cell wall suspension was deposited on freshly cleaved mica muscovit (Plano, Wetzlar, Germany). This droplet was allowed to dry at ambient conditions prior to AFM investigations. Typically, up to 10 diatom valves were deposited on each mica sheet. A single valve was then removed from the mica surface by 92
Figure 3. AFM images of the inner surface of a valve of C. granii at two different magnifications. The images were taken in contact mode with a Digital Instruments oxide sharpened tip.
a pair of tweezers under an optical microscope and deposited on a second, freshly cleaved mica substrate. This procedure allows to easily determine whether the inner or the outer surface of the valve is oriented toward the AFM tip. SEM imaging has been performed on a LEO 1530 SEM (LEO Elektronenmikroskope, Oberkochen, Germany) equipped with a Gemini field emission column, which offers a resolution of 3 nm at an acceleration voltage of 1 kV. The diatom valves under investigation were not coated. It may be assumed that the “true” geometry of the surface is observed. SEM images of silica nanoparticles have been taken with a Hitachi S-4100-SEM (Hitachi Europe Nissei Sangyo, Ratingen, Germany) equipped with a field emission cathode. Microscopic Analysis of Diatom Surfaces. Two different sides of a valve of C. granii imaged with the LEO SEM are shown in Figure 2. At the inner surface (Figure 2a) small crater-like structures with a diameter of about 400 nm are observed. The area between these structures appears to be fairly smooth. At the right side of the image the structure is broken. The outer surface (Figure 2b) shows a different Nano Lett., Vol. 2, No. 2, 2002
Figure 4. (a) AFM image of the outer surface of a valve of C. granii. This image was taken in contact mode with a Digital Instruments oxide sharpened tip. A granular nanostructure of the surface is observed. (b) Height profile of a line scan taken through the part of (a) marked by the green bar. The profile indicates that the nanostructure consists of fused silica grains with a size on the order of 100 nm. This size is of a similar order of magnitude as the silica spheres precipitated from silicic acid solution induced by silaffins1 or polyamines.12
topography. Holes with diameters of 200-300 nm, with very small “teeth” pointing to the center characterize this side of the valve. The area between the holes shows only very few porous structures. In Figure 3 two AFM images of the inner surface of C. granii at different magnifications are presented. The craterlike structure (Figure 3a) is in good agreement with the SEM result (Figure 1a). A closer look to the surface structure (Figure 3b) yields a quite different result compared to the SEM analysis (Figure 2a). Here a distinct surface roughness on the order of tens of nanometers is observed. The granular nanostructure is observed on the rim of the “craters”, as well as in the flat areas between craters. The same results were obtained for the outer surface of C. granii (Figure 4a). The AFM image shows the larger holes as seen in the SEM image (Figure 2b), as well as a nanostructured surface similar to the inner surface (Figure 3). The granular nanostructure appears to be composed of 100-200 nm spherically shaped particles (Figure 4b). This Nano Lett., Vol. 2, No. 2, 2002
Figure 5. AFM images of the outer surface of a valve of C. granii taken in noncontact mode. (a) Larger scan area, nanostructure to be seen. (b) Very high magnification, nanostructured surface and substructure of the holes are visible.
line scan shows a height profile of the line marked by a green bar in Figure 4a. The small “teeth” within the holes observed with SEM are not visible in contact mode AFM. To check whether the nanostructured surface observed in AFM but not in SEM might be an artifact, experimental parameters such as load force of the AFM tip, AFM tip type, scan mode (contact mode, noncontact mode), scratching along the surface with high load force, and purification procedure of the diatoms were varied. As an example in Figure 5, two AFM images of the outer surface taken in noncontact mode are shown. In this case even the small teeth-like structure can be seen, particularly in the highly resolved Figure 5b. Because of the large tipsample distance in noncontact mode AFM, the resolution of Figure 5 is not as good as in the case of SEM, but the nanostructure of the surface is observed. To check whether soft materials, such as organic molecules, cover the surface, high load forces during scanning were applied. For example, it had been demonstrated by several authors that DNA strands and chromosomes can be manipulated by AFM tips.14-19 We 93
Figure 6. Chemical structure of tripropylenetetramine (I) and pentapropylenehexamine (II).
Figure 7. SEM image of gold-coated silica nanoparticles on mica precipitated from silicic acid solution by pentapropylenehexamine. The thickness of the gold layer is approximately 30 nm.
performed horizontal line scans at high load forces in the center of an image, but no significant difference in the images taken before and after the line scans was observed. This indicates that there are either no removable molecules on top of the valve surface, or these molecules are bound very tightly to the surface and appear to be as solid as the silica surface of the valve. In both images the nanostructure was observed. Also, diatom valves cleaned more intensely, i.e., valves that were treated more often with boiling SDS and EDTA solution, show the nanostructure to be independent from the load force of the AFM tip. Even the tip shape shows only a qualitative effect on the AFM results. This was checked by using TopoMetrix standard tips (tip radius ∼50 nm), Digital Instruments oxide sharpened twin tips (Veeco Instruments, Santa Barbara, CA, tip radius 5-20 nm), and UltraSharp silicon tips (NT-MDT, Moscow, Russia, tip radius
