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A natural source of porous biosilica for nanotech applications: the diatoms microalgae

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A natural source of porous biosilica for nanotech applications: the diatoms microalgae
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     © 2010 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim pss current topics in solidstatephysics c      s      t     a      t     u     s     s     o      l      i      d      i   www.pss-c.com      p      h     y     s      i     c     a Early View publication on onlinelibrary.wiley.com (issue and page numbers not yet assigned; citable using Digital Object Identifier – DOI )   Phys. Status Solidi C, 1–6 (2010) / DOI   10.1002/pssc.201000328   A natural source of porous biosilica for nanotech applications: the diatoms microalgae Luca De Stefano *1 , Mario De Stefano 2 , Edoardo De Tommasi 1 , Ilaria Rea 1 , and Ivo Rendina 1   1  National Council of Research Institute for Microelectronics and Microsystems, Unit of Naples, Via P. Castellino 111, 80131 Naples, Italy 2  Environmental Science Department, Second University of Naples, 81100 Caserta, Italy Received 12 May 2010, revised 18 June 2010, accepted 21 June 2010 Published online 25 November 2010 Keywords  porous materials, diatoms, optical devices, nanotechnology   * Corresponding author: e-mail luca.destefano@na.imm.cnr.it , Phone: +39 081 6132375, Fax: +39 081 6132598 Several biological organisms, from some sea shells to  butterflies, exhibit beautiful and sophisticated organs, developed during the evolution of each species, which  properties are defined by their nanostructures. The ma-rine diatoms are microscopic algae enclosed between two valves of hydrated amorphous silica. These intricate structures, called frustules, show quite symmetric pat-terns of micrometric and nanometric pores. Their strong similarity with man-made materials, such as synthetic zeolites, or porous silicon and alumina, suggests to ex- ploit the physical properties of the frustules in nanotech applications. In this paper, we review the most relevant results achieved in our laboratory, and all over the world, about the discovery of surprising features that can be found in the characterization of these natural porous  biosilica materials.  © 2010 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim 1 Introduction  Porous materials represent a very ex-iting field of investigation in the field of material science due to the astonishing variety of morphology and physical-chemical properties which can be strongly modulated start-ing from those of the bulk materials. Depending on poros-ity and on pores sizes, the applications of porous materials range from those depending on their large surface area, such as sensing, catalysis, adsorption and chromatography, to that favored by the presence of voids in the bulk, like fil-tration and lightweight structures. Sometimes the porosity also adds new features to the material, as in the case of po-rous silicon, which is photoluminescent when irradiated by a laser beam of proper wavelength, while crystalline sili-con is not. For all these reasons there is a strong effort in fabrication procedures both in academic and in industrial laboratories. Porous metal and semiconductors are gener-ally fabricated by electrochemical methods, like dealloying (Cu, Pt, Pd, Au, and Ag) [1-3] and selective etching (Si, SiC, TiO 2 , InP, GaP, GaAs) [4, 5], but also exploiting the  possibilities of classic micromachining techniques, such as chemical vapors deposition or plasma enhanced chemical vapors deposition, and electro-spinning [6]. Depending on the parameters of he fabrication processes, but also on the characteristics of the bulk materials (doping level, orienta-tion, purity, and so on), a very broad range of porosity and  pore size dimensions can be achieved: again, in the case of the porous silicon, porosities between 30% and 90%, and  pores size ranging in the nanometer to micrometer interval, can be easily obtained. Man-made materials are in strong competition with natural porous materials: zeolites, for example, are well-known microporous crystalline solids with structures con-taining silicon, aluminium and oxygen in their framework. Many of them occur naturally as minerals, and, due to their impressive features, are widely used at industrial level in catalysis, adsorption, separation, and in general as molecu-lar sieves [7]. There is another important class of natural porous ma-terials: the diatoms microalgae. The diatoms are aquatic unicellular organisms, and they represent a good example of naturally evolved complex nanostructures. In these mi-cro algae, the organic matter is enclosed in a hydrated  2   L. De Stefano et al.: Porous biosilica for nanotech applications  © 2010 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim www.pss-c.com        p       h     y     s       i     c     a p s s      s      t     a      t     u     s     s     o       l       i       d       i c amorphous silica cell wall, called the frustule, consisting of two valves joined together by a series of silica bands linked together along the margins [8]. Diatoms are usually grouped on the basis of the frustule symmetry: centric dia-toms, which have a circular symmetry, and pennate dia-toms, which are bilaterally symmetrical. Anyway, as a matter of fact, the shape and the size of frustules are ex-tremely different among the 100000 and more species of existing diatoms: they can be circular, oval, stick-shaped, star like, and so on, and range in dimensions from micro-metres up to one millimetre. Valve surfaces exhibit specie-specific patterns of regular arrays of chambers, called areo-lae, developed into the frustule depth. Areolae range in di-ameter from few hundreds of nanometers up to few mi-crons, and can be circular, polygonal or elongate. In addi-tion, they are internally or externally occluded by cribra, which are thin, silica laminas pierced by pores ranging in diameter few nanometers. From this point of view, diatoms frustules are really micro and nanoporous materials, natu-rally available in almost every water pond. Moreover, dia-toms show other peculiar properties: they can cast into shaped colonies, and during the asexual reproduction cycle, the average dimensions of progenies are smaller then those of parents preserving all the aspects ratios; when the dia-tom size reaches a critical value, a sexual cycle starts and srcinal dimensions are recovered. Due to their peculiar morphologies, diatoms have been recently proposed to be employed in nanotechnology as natural made nano-devices [9]. A synopsis of diatoms in biotechnology can be also found in a very recent paper of Bozart et al. [10], where applications in nutritional field, fuel synthesis, and phy-toremediation are reported. In this review, we report the principal applications re-cently published in literature of diatom based devices in nanotechnological field: corresponding to the astonishing variety of species, optical, chemical, electrical, and also  biomedical applications have been exploited in different laboratories, all over the world. We also summarize our al-ready published results in this research field, but using ex- perimental data and images never appeared on other scien-tific journals. 2 Experimental section   In order to analyze the fine details of diatoms microshells, a cleaning procedure that destroys the organic matrix of the cell which covers the si-liceous frustule is mandatory. There are at least two proce-dures for removing the organic content from the frustules: one is based on acid treatment, the other on hydrogen per-oxide dipping [17, 35]. A mixture of acids of different strengths is generally used for this purpose. It is very im- portant to accurately calibrate the strength of the mixture to avoid silica skeleton damages and acid treatment should be used only when the diatom species possess highly silicified frustules. The items of this procedure are: (i) 50 ml of a highly concentrated, diatom strain sample was centrifuged at 3000 rpm for 10 min; (ii) the pellet was washed in dis-tilled water five times to remove excess of culturing mate-rial; (iii) 2 ml of the pellet was mixed with a similar vol-ume of 97% sulphuric acid for 5 min at 60 °C; (iv) the acid was removed and the pellet was washed again in distilled water five times. Some micro- and nanostructures and less silicified frustules may dissolve in strong acids, in this case several washing in H 2 O 2  can be used to remove the organic matter. Cleaned material was mounted on aluminium stubs and sputter coated with gold or platinum for scanning elec-tron microscopy (Philips 505, Philips Electron Optics BV, Eindhoven, The Netherlands) or mounted on formvar-coated grids for transmission electron microscopy (Philips 400, Philips 505, Philips Electron Optics BV, Eindhoven, The Netherlands). Cleaned frustules for experimental measurements were wet deposited on a single-polished, intrinsic silicon wafer, which gives a negligible contribution to photolumines-cence (PL) signal at the wavelength considered. The PL was induced by c.w. solid state laser light at the wave-length 406 nm and measured at room temperature in a test chamber with quartz windows for optical access through a monochromator (Oxford Instruments) equipped by 1200 grooves/mm grating blazed for 500 nm and thermo-cooled charge-coupled device camera. The spectra were recorded within an accumulation time of 3 s. 3 Results and discussion 3.1 Diatoms as 3D templates: bioclastic and biological approaches The first applications fore-sighted for diatoms were directly related to their symmetric shapes and typical dimensions at the micro and nano scale: Sandhage et al. [11 ] proposed in 2002 the use of diatom frustules as lithographic mask, instead of the layer by layer common approach used in microelectronics for lithography. Many details of diatoms frustules have a regular, repeat-able and well defined geometry which can be easily ex- ploited in realizing complex structures: in Fig. 1 some de-tails of frustules are reported just as examples of micro and nano ordered arrays which could be used as templates in  photolithographic processes. Figure 1  (Left) A template for a multi-analyte microarray. Scale  bar: 0.8  μ m. (Centre) A template for microchambers array with in/out channel already built in. Scale bar: 0.4 μ m. (Right)   A tem- plate of equally spaced microchannels. Scale bar: 1.5 μ m. Figure 1(left) shows a detail of the diatom Cocconeis scutellum  var. sullivanensis valve external surface: this areolae arrangement could be used as a template for a three analyte microarray; the central image of the Cocconeis  Phys. Status Solidi C   (2010) 3 www.pss-c.com    © 2010 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim   ContributedArticle scutellum  var. posidoniae shows the regular disposition of microchambers (estimated volume of few picoliters) with already in/out channels built in could be envisaged; finally, the photo of Cocconeis pseudomarginata  internal valve could be viewed as a template for an array of micro-channels. Of course, these are only examples but between an idea and its practical application there is a lot of effort, so that at the moment the utilization of diatoms frustules as masks templates has not been exploited on large scale. The Sandhage’s group transformed the diatom valves  by carbothermal reduction in other materials for specific applications preserving all the morphological aspects of the srcinal specie. This was really very important, since it makes possible to extend the range of applications for dia-tom frustules, limited by the properties of silica, substitut-ing the silicon atoms with other atoms without losses of the tridimensional structure: the exact chemical reactions and all the details can be found in Ref. [11, 12]. More recently other methods have been proposed for frustules modifica-tions: Losic et al. used atomic layer deposition to modify the pore structure in a controlled way [13]. Other scientists groups have altered frustules properties by proper biologi-cal procedures, obtaining very interesting results in terms of new or improved material performances. For example, the group of Parker has changed frustules characteristics  by growing the diatoms in a culturing medium containing a metal pollutant at sub-lethal concentration [14, 15]. The Rorrer group also modified the diatom frustules by metab-olically inserted titanium, and used dose-dependent meta- bolic incorporation of germanium to induce nanocomb structures [16, 17]. The titanium deposition has also be im- proved by this group using a peptide-mediated deposition method [18]. 3.2 Diatoms optical properties Despite the beauty of the pattern ornamentations, which was well known since the adoption of the microscope by the marine biologists, their extremely regular geometries suggested the investiga-tion of the optical properties exploited by the porous biosi-lica of the diatom frustules. Due to the most diffused hy- pothesis on frustules function, i.e. the nanopores could be a natural barrier against virus infections and the whole mi-cro-shell could be a protection against predators, only the mechanical properties of the frustules were investigated [19] revealing unsuspected mechanical strength. The group of Fuhrmann first studied the optical characteristics of a diatom as a natural photonic crystals, founding that light frequencies were differently focused in the protoplasm of the cell with some important possible biological conse-quences [20]. In the same period our group published an optical characterization of the frustules determining by spectroscopic reflectometry the effective refractive index of a frustule in an effective medium approximation [21]. The amorphous hydrated silica of the diatoms frustules is also photoluminescent when irradiated by a laser light, as it was pointed out by the experiments of Butcher et al. and, approximately at the same time, by our group [21, 22]. In Fig. 2 the photoluminescence (PL) of the Thalassiosira rotula  diatom under 406 nm laser light exposure is reported: this is also an important discovery since PL of manmade micro and nano silica beads is often used in monitoring and diagnostics. Figure 2  Photoluminescence normalized spectrum of Thalassi-osira rotula diatom. Dashed lines are the Gaussian profiles corre-sponding to different bands used to fit the experimental data. The srcin of this multiband PL in the energy range (1.8-2.76 eV), is attributed to bulk and surface non bridg-ing oxygen absorptions and the radiative decay of self-trapped excitons [23, 24], but also the role of OH groups and nanostructures has been pointed out by Rorrer et al. [25]. The group of Rorrer also obtained a blue PL emission from diatom frustules by cultivation of cells to silicon star-vation [25]. Figure 3  Left: Light intensity in the diatom focus plane regis-tered by a charged coupled device camera. Right: Light intensity recorded at 104 μ m from the focus plane: the transmitted laser  beam is narrowed by a factor of 12. Few years later, we investigated the transmission of light through the frustule of a centric marine diatom, the Coscinodiscus wailesii , and we found that this radial sym-metric frustule has unsuspected optical properties: a 100 μ m spot size of a red laser beam was narrowed up to less than 10 μ m at a distance of 104 μ m after the transmission through the regular geometry of the diatom structure, which thus acts as a microlens [26, 27]. Our experimental 4204504805105405706006306606900.00.51.01.52.02.5 533.4609.3661.5     P .   L .   (  a .  u .   ) Wavelength (nm)  4   L. De Stefano et al.: Porous biosilica for nanotech applications  © 2010 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim www.pss-c.com        p       h     y     s       i     c     a p s s      s      t     a      t     u     s     s     o       l       i       d       i c data, an example is reported in Fig. 3, and the numerical calculations indicated that this phenomenon is due to the  porous surface of the diatom: the coherent superposition of the light scattered by the areolae focus the light into a nar-row beam. This was the first proposal of diatoms frustules utilization as an optical device itself [26]. Even if the key features of diatoms, from the optical  point of view, are the intricate morphologies and the sym-metry of pores patterns, some teams have focused their re-searches on improving the diatoms photonic properties for optoelectronic device applications: in particular, the group of Rorrer has published some relevant results in this field which have bee also cited in a Nature Research Highlight (Nature, 453, 1146, 2008) [29]. From these papers, it is clear that there nanofabrication procedures for finely tun-ing the emission properties of diatom frustules: modifying the diatom surface chemistry by a simple solution deposi-tion method, it is possible to obtain a green photolumines-cence from the biogenic biosilica of diatoms [30-32]. 3.3 Diatoms as biochemical optical sensors When we discovered the PL of the diatom frustules, we were working on porous silicon based optical biosensors: due to the impressive morphological similitude between the porous silicon chip and the diatom surface, we tried to expose this material to different vapors atmospheres in or-der to verify if diatoms could be used as optical transduc-ers just as it happens with porous silicon [33, 34]. In pres-ence of gaseous species, both the PL optical intensity and the peak position shift. The effect is specific against the substances: the PL is quenched on exposure to those com- pounds, such as NO 2 , Acetone or Ethanol, which can at-tract electrons from the silica skeleton of diatoms thus de- pressing the photoluminescence [35-38]. On the contrary, other substances like Xylene and Pyridine, strongly en-hance the PL signal by giving free electrons to the diatom surface. Moreover the principal peak of the PL bands tends to red-shift towards longer wavelengths, due to two physi-cal phenomena: the capillary condensation of the gases in a liquid phase into the nanometric pores and the adsorption of the volatile substances on the larger pores surface. Both mechanisms increase the average refractive index of the structures. The transduction mechanism is also highly sen-sitive, since we have measured large variation of the PL light on exposure to sub-ppm levels of gases [36]. The very large specific surface area of the diatoms is a direct conse-quence of their regular porous structures. Beside the high sensitivity, the other    key feature for an optical sensor is the selectivity against a target analyte pre-sent in a complex mixture. One way to make a transducer selective is to chemical modify its surface in order to link a  proper bioprobe which is naturally selective against its ligand, such as an antibody or a protein. Since the diatoms frustule surface is covered by the reactive and unstable -OH groups, one possible strategy is the substitution of the hydroxyl groups by other organic linkers bringing specific functional groups. We have   demonstrated that the porous  biosilica of diatoms is compatible with the organic matter  by covalently bind an antibody on its surface (see for ex-ample Fig. 4) and studying the interaction between the an-tibody and its antigen by PL variations [39]. Figure 4  Fluorescence image of a single diatom frustule after the  binding with Rhodamine labeled antibodies. Scale bar: 50 μ m.   Once the antibody has been linked to the diatoms sur-face, we have checked its ability to recognize specifically and selectively a known antigen by recording the PL varia-tions on exposure of the diatoms to solutions with different concentrations of antigen. In Figure 5 we report the wave-length peak shift (a) and PL intensity changes (b) dose-response curves for antibody-antigen recognition. All ex- perimental details can be found in the reference [40]. In this case the diatoms used was a mixed strain of pennate diatoms. Figure 5  Wavelength peak shift (up) and PL intensity changes (down) dose-response curves. 0510152025300102030 Antigen concentration ( μ M)       Δ      λ    (  n  m   ) 051015202530024681012       Δ    I    P   L    (  a .  u .   ) Antigen concentration ( μ M)  Phys. Status Solidi C   (2010) 5 www.pss-c.com    © 2010 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim   ContributedArticle From these curves a dissociation constant of the order of μ M -1  can be estimated in quite good agreement with lit-erature data. Diatoms as biochemical optical sensors have  been reported in a recent paper of Gale et al. which also  provided a comprehensive analysis of enhanced photolu-minescence resulting from immunocomplex formation on antibody functionalized diatom frustules [41]. 3.4 Next future: diatoms in drugs delivery The micrometer size and the presence of a porous network in the whole frustule makes diatoms a perfect candidate for drug loading and delivery. Being a residue of a natural or-ganism, the cytotoxicity of diatoms frustules is very low, so that is not so strange to think about diatoms as an ideal vehicle for drug dispensing. The surface area of diatoms  pellets is of about hundred of m 2 /gr when estimated by BET measurements and the presence of hydroxyl groups assists the uploading of hydrophilic drugs [42]. In Fig. 6 two particular of two centric diatoms are reported, just as an examples of structures that can be found already built in for this specific application: in the left, some microcham- bers, where drugs can be loaded by capillarity and stored up to the release, are shown, while in the right, some glassy microtubules connecting the inner of the diatom to the outer. These tubes could act as micro-needles: the di-mensions of the tubes diameters are of the order of few mi-crons so that derma or other tissues could be holed without damage. Then, the drugs could be slowly released in a par-ticular point. These observations are just hints and sugges-tions for a new class of experiments. Figure 6  Diatom structures which can be used for drugs delivery. (Left) Internal microchambers for drugs loading. Scale bar: 10 μ m. (Right) A particular of microtubules connecting the chamber to the outer of the diatoms. Scale bar: 5 μ m. 4 Conclusions Top-down approaches have been until now the most spread fabrication methods for micro and nanostructured  porous materials, even if they necessitate of big invest-ments, continuous practice and dedicated laboratories and  personnel. A completely different approach to engineering systems at the nanoscale consists in recognizing the nano-structures and morphologies that nature has optimized dur-ing life’s history on earth: biologically-inspired devices and materials are often superior to human-made articles. Different evolutionary constraints lead to a variety of func-tional morphologies in diatoms, most of them are still of unknown srcin. We believe that the experimental work of several groups in different laboratories all around the world have clearly demonstrated the huge possibilities of diatoms frustules, as a low-cost, readily available source of  porous biosilica in practical technological applications ranging from photolithographic templates [9] to patterned arrays [43]. Acknowledgements The authors gratefully acknowledge the group of Prof. P. Maddalena of University of Naples “Fede-rico II”, Italy, for PL measurements. References [1] A.J. Smith, T. Tran, and M.S. Wainwright, J. Appl. Electro-chem. 29 , 1085-1091 (1999). [2] U.S. Min and J.C.M. Li, J. Mater. Res. 9 , 2878-2881 (1994). [3] S. Koh, N. Hahn, C.F. Yu, and P. Strasser, J. Electrochem. Soc. 155 , B1281-3 (2008). [4] L. Canham (Ed.), Properties of Porous Silicon (DERA, Mal-vern, UK, 1999). [5] M. Tiginyanu, V.V. Ursaki, L. Sirbu, M. Enaki, and E. Monaico, Phys. Status Solidi C 6 (7), 1587-1591 (2009). [6] A.M. Rossi, F. Giorgis, V. Ballarini, and S. Borini, Phys. Sta-tus Solidi A 202 , 8-10 (2005). [7] F.A. Mumpton, Proc. Natl. Acad. Sci. USA 96 (7), 3463-3470 (1999). [8] F.E. Round, R.M. Crawford, and D.G. Mann, The Diatoms: Biology and Morphology of the Genera (Cambridge Uni-versity Press, Cambridge, 1990). [9] R.W. Drum and R. Gordon, Trends Biotechnol. 21 , 325-328 (2003). [10] A. Bozarth, U.G. Maier, and S. Zauner, Appl. Microbiol. Biotechnol. 82 , 195-201 (2009). [11] K.H. Sandhage, M.B. Dickerson, P.M. Huseman, M.A. Ca-ranna, J.D. Clifton, T.A. Bull, T.J. Heibel, W.R. Overton, and M.E.A. Schoenwaelder, Adv. Mater. 14 , 429-433 (2002). [12] Z. Bao, M.R. Weatherspoon, S. Shian, Y. Cai, P.D. Graham, S.M. Allan, G. Ahmad, M.B. Dickerson, B.C. Church, Z. Kang, H.W Abernathy, C.J. Summers, M. Liu, and K.H. Sandhage, Nature 446 , 172-175 (2007). [13] D. Losic, G. Triani, P.J. Evans, A. Atanacio, J.G. Mitchell, and N.H. Voelcker, J. Mater. Chem. 16 , 4029-4034 (2006). [14] H.E. Townley, K.L. Woon, F.P. Payne, A.R. Parker, and H. White-Cooper, Nanotechnology 18 , 295101-5 (2007). [15] H.E. Townley, A.R. Parker, and H. White-Cooper, Adv. Func. Mater. 18 , 369-374 (2008). [16] T. Qin, T. Gutu, J. Jiao, C.-H. Chang, and G.L. Rorrer, ACS  Nano 2 , 1296-1304 (2008). [17] C. Jeffryes, T. Gutu, J. Jiao, and G.L. Rorrer, ACS Nano 2 , 2103-2112 (2008). [18] C. Jeffryes, T. Gutu, J. Jiao, and G.L. Rorrer, J. Mater. Res. 23 , 3255-3262 (2008). [19] C.E. Hamm, J. Nanosci. Nanotechnol. 5 , 108-112 (2005). [20] T. Fuhrmann, S. Landwehr, M.E. Rharbi-Kucki, and M. Sumper, Appl. Phys. B 78 , 257-261 (2004). [21] L. De Stefano, M. De Stefano, I. Rea, L. Moretti, A. Bismu-to, P. Maddalena, and I. Rendina, Proc. SPIE 5925 , 59250S-5925310S (2005).
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