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Simple method for coating Si (1 0 0) surfaces with ferritin monolayers—Iron oxide quantum dots
Materials Science and Engineering B 176 (2011) 500–503
Contents lists available at ScienceDirect
Materials Science and Engineering B journal homepage: www.elsevier.com/locate/mseb
Simple method for coating Si (1 0 0) surfaces with ferritin monolayers—Iron oxide quantum dots Georgios Papadopoulos a,∗ , Constantine Anetakis b , Christoforos Gravalidis b , Spiros Kassavetis b , Nikolaos Vouroutzis b , Nikolaos Frangis b , Stergios Logothetidis b a
University of Thessaly, Department of Biochemistry & Biotechnology, Ploutonos 26 & Aeolou, 41221 Larisa, Greece Aristotle University of Thessaloniki, Department of Physics, Laboratory for Thin Films – Nanosystems and Nanometrology and Laboratory of Electronic Microscopy, 54124 Thessaloniki, Greece b
a r t i c l e
i n f o
Article history: Received 15 September 2009 Received in revised form 17 April 2010 Accepted 17 May 2010 Keywords: Ferritin Iron oxide Quantum dots Atomic force microscopy Transmission electron microscopy Spectroscopic ellipsometry
a b s t r a c t With the goal to develop iron oxide quantum dots we developed a simple method to spread horse spleen ferritin monolayers on a Si (1 0 0) surface. Application of atomic force microscopy and spectroscopic ellipsometry showed the existence of regions with dense ferritin monolayers. Application of transmission electron microscopy identified the core of the spread ferritin as FeO nanocrystals. © 2010 Elsevier B.V. All rights reserved.
1. Introduction Iron oxides are interesting due to their catalytic, magnetic and semiconducting properties. Their applications include usage as pigments, catalysts in styrene synthesis and as a material for high density magnetic storage as well as possible applications in optical devices [1]. Nanosized iron oxide particles can be expected to show size-dependent optical, magnetic, and chemical properties. Moreover, the optical properties of semiconducting nanoparticles, such as quantum dots (QDs), are different from those of the corresponding bulk material. The size dependence of their properties can be associated with an influence of the size on the electronic structure of the material [2]. The most important feature of these materials is the increase in optical band gap due to quantum confinement effect. This increase is observed as a blueshift in photoluminescence emission or absorption spectra [3,4]. This can be explained as combination of the confinement-induced blueshift of the electronic levels and the redshift caused by the increased Coulomb interaction due to the compression of the exciton radii [5]. Besides, the
∗ Corresponding author. Tel.: +30 2410 565249; fax: +30 2410 565290. E-mail addresses: [email protected] (G. Papadopoulos), [email protected] (C. Anetakis), [email protected] (C. Gravalidis), [email protected] (S. Kassavetis), [email protected] (N. Vouroutzis), [email protected] (N. Frangis), [email protected] (S. Logothetidis). 0921-5107/$ – see front matter © 2010 Elsevier B.V. All rights reserved. doi:10.1016/j.mseb.2010.05.014
aforementioned influence can be due to the surface tension of the particle, resulting in an effective pressure or to changes in the stoichiometry (and resulting changes of the state of oxidation of Fe) of such particles, driven by the large surface to-volume ratio [2]. In the present study, in order to produce nanosized iron oxide particles, we utilized the ability of ferritin to self-assemble and construct a core of iron oxide. The major function of ferritin in animals, plants and bacteria, is to store and detoxify intracellular iron [6,7]. Crystallographic studies have revealed that its structure is uniquely well designed to contain large amounts of iron in a soluble, nontoxic, and available form [8]. Ferritins in vertebrates are assembled from 24 subunits into a spherical shell (Ø = 10–12 nm) from two genetically distinct subunit types, designated H and L [9,10]. The cage formed by the subunits encloses up to 4500 ferrihydrite molecules (approximate formula: 5Fe2 O3 ·9H2 O) in nanocrystalline form [6]. This protein shell functions in several manners: it acquires iron (II), catalyses its oxidation, and induces mineralization within its cavity [2,6,9]. Several procedures have been reported for the development of iron oxide thin films utilizing ferritin. Manning and Yau [11] have demonstrated immobilization of ferritin onto a Si/SiO2 surface, while Yamashita [12] and Matsui et al. [13] applied the interesting “Bio Nano Process”, which exploits the self-assembly properties of ferritin on an air–water surface to transfer a layer of ferritin onto a Si surface. Okuda et al. [14] reported the fabrication of nanometric iron and indium particles using ferritin. In a step forward Ichikawa et al. [15] developed LTPS-TFT flash memory
G. Papadopoulos et al. / Materials Science and Engineering B 176 (2011) 500–503
501
applying the “Bio Nano Process”. The above-mentioned methods differ in complexity, as well as in required time and expenses. Here we introduce a simple and cost-effective technique (paintbrush like), described in Section 2, to coat silicon (1 0 0) surfaces with ferritin monolayers as the first part of an effort to produce arrays of iron oxide quantum dots. The derived surfaces have been characterized with atomic force microscopy (AFM), spectroscopic ellipsometry (SE) and transmission electron microscopy (TEM). 2. Experimental As a substrate for the formation of the ferritin monolayer, silicon (1 0 0) surfaces were used. In order to remove possible organic contamination, the surfaces were treated successively with tetrachloroethylene (99%), acetone (99.5%), and methanol (99.9%), at 60 ◦ C in a supersonic bath, for 15 min for each step. Then, they were treated with hydrogen peroxide, at 85 ◦ C for 4 h. The surfaces were further cleansed mechanically. For this purpose, a poly vinyl chloride (PVdc) membrane, wrapped around a cotton stick, was used. In particular, 40 l of ddH2 O was posed on the surface, which was thoroughly passed with the stick for 5 min. In the present study, a horse spleen ferritin solution 1 mg/ml, in HEPES 0.1 M, pH 7, was used for the coating of the surface. Generally, the horse derived ferritins, have an isoelectric point (pI) extending from pH 4.1 to 5.1 [16], so that in neutral pH they have a net positive charge. For this reason, the silicon surfaces were treated overnight with HCl 10N, pH 1.2, to obtain a net negative charge [17] which will facilitate the deposition of the protein. The surfaces were then neatly dried, with a N2 draught in a clean chamber and 40 l of the ferritin solution were deposited on the surface. Again a cotton stick, wrapped with a PVdc membrane, was used, to cover the surface, with movements to a single direction. This step was repeated with 40 l of ddH2 O. Finally, the samples were dried thoroughly with a N2 draught. For the above treatment a clean chamber has been used. All samples were studied with AFM (NT-MDT) in semicontact mode, SE (Uvisel p/n 23 301 909, Jobin-Yvon) and TEM (JEOL 2011). AFM measurements of the silicon surfaces, after the two steps of cleansing, as well as SE measurements of the Si substrate, before the protein coverage, were also performed.
Fig. 1. Ellipsometric model for the sample of Figs. 2 and 4. Red lines and data points refer to the imaginary part of the dielectric function ε(ω), whereas black ones refer to the real part. (—) Si substrate, (•••) SiO2 /Si layer, (***) protein/SiO2 /Si layers. The effective thickness of the protein sublayer has been measured to 4.99 ± 0.50 nm. The refractive index associated with the model is 1.451. (For interpretation of the references to color in this figure legend, the reader is referred to the web version of the article.)
3. Results and discussion The aim of this work was to spread a monolayer of ferritin molecules with iron oxide core on a Si (1 0 0) surface as a first step for the development of iron oxide nanoparticles. Very clean Si surfaces are required. Chemical cleansing of the Si substrate left some contamination observed with AFM with average roughness (Ra) ∼1.52 nm, and RMS ∼2.55 nm. After the mechanical treatment, the surfaces were clean at the nanoscale level with Ra ∼0.47 nm and RMS ∼0.42 nm. Ra and RMS are defined as usually. Our sample is a clean surface, which has been coated with ferritin following the simple procedure described in Section 2. The SE measurements of the dielectric function ε(ω) shown in Fig. 1 were fitted using a simple Tauc–Lorenz model [19]. Analysis of the data in terms of a four phase model provided a profile consisted of the Si substrate, a SiO2 sublayer (due to the treatment of the surface with H2 O2 ) with a thickness of 0.60 ± 0.03 nm and a protein sublayer, with voids extending to about 50% of the covered surface. The effective thickness of the protein sublayer was calculated to be 4.99 ± 0.50 nm, so that the real thickness of the protein coverage corresponds, as expected, to about 10 nm. The AFM image of the sample shows coverage of the Si surface with molecular units (Fig. 2a) including some voids and aggregates, with Ra ∼2.37 nm and RMS ∼3.27 nm. A comparison of the heights
Fig. 2. (a) AFM image of a protein coated Si surface. The image shows a total coverage of the substrate, with some voids. (b) Sections through the AFM topography images of a clean Si surface (black line) and of a Si surface coated with a ferritin monolayer (blue and red line). The blue line shows an arbitrary section, whereas the red one is chosen to show a protein aggregate (highest peak) with a height corresponding to about two or three ferritin units. (For interpretation of the references to color in this figure legend, the reader is referred to the web version of the article.)
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G. Papadopoulos et al. / Materials Science and Engineering B 176 (2011) 500–503
Fig. 3. Height histogram of the sample depicted in Fig. 2, showing an average height of the coverage, 10–12 nm.
of a clean Si surface and of a Si surface coated with a ferritin monolayer is presented in Fig. 2b. The low height black line represents a section through the AFM image of the clean surface. The blue line represents an arbitrary section through the ferritin monolayer coating, whereas the red one represents a similar section chosen to show a protein aggregate (highest peak) with a height corresponding to about two or three ferritin molecules. Fig. 3 shows a histogram of the height distribution of Fig. 2a with a mode of ∼11 nm corresponding to the diameter of ferritin in agreement with our SE results. The presence of a smaller peak in the histogram at about 4.5 nm can be considered as an indication for the existence of bare nanocrystals from protein core and (or) to protein subunits. A clearer picture of the protein distribution is obtained from the AFM phase image (Fig. 4). Regular arrays of ferritin units are visible
Fig. 4. AFM phase image. A total coverage is visible. The region indicated by a white bar on the left (146 nm) covers 10 molecular units, including spacing between them.
Fig. 5. (a) An electron diffraction pattern from a plane view specimen. The (1 1 1) Sireflections are indicated by circles. The four polycrystalline rings correspond to the cubic FeO structure. (b) A HRTEM image revealing the presence of FeO nanocrystals (QD) with diameters between 2 and 8 nm.
as well as some larger objects, probably aggregates. We measured the length of 10 molecular units to be 146 nm (Fig. 4), which gives an average distance between the centers of the molecular units ∼14.6 nm. This value is consistent with the dimensions of ferritin units calculated from the topography (∼11 nm) and to those previously published [2,6,18], if we consider intermolecular water and a ferritin organization slightly adapted to the SiO2 background. Electron diffraction and high-resolution transmission electron microscopy (HRTEM) were used in order to elucidate the crystallinity of the quantum dots. Plane view specimens were prepared by ion thinning, bombarding only the substrate. A representative electron diffraction pattern is shown in Fig. 5a. Apart of some reflections of the Si substrate, rings containing a large number of spots are observed. Using the Si spots as an internal standard, the d-spacings of the rings are found to be: 0.248 ± 0.002 nm, 0.216 ± 0.002 nm, 0.153 ± 0.002 nm, and 0.128 ± 0.002 nm. These values are in very good agreement with the main d-spacings of the cubic FeO: d1 1 1 = 0.248 nm, d2 0 0 = 0.215 nm, d2 2 0 = 0.152 nm, d3 1 1 = 0.129 nm. This is quite surprising, since according to the literature [2,6,8,20], a structure similar to that of the mineral ferrihydrite (5Fe2 O3 ·9H2 O) would be expected. Exceptionally,
G. Papadopoulos et al. / Materials Science and Engineering B 176 (2011) 500–503
Preisinger et al. [2] needed to anneal the sample in air atmosphere at a temperature range of 575–675 K in order to obtain the Fe2 O3 form. Although ferrihydrite has some reflections close to those of FeO (0.247 nm, 0.234 nm and 0.148 nm according to [21]), the absence of its reflections corresponding to 0.198 and 0.173 nm, does not support the existence of ferrihydrite, at least as the main observed phase. Some isolated spots can also be seen in the pattern of Fig. 5a, denoting the possible existence as minority of other Fe phases, including ferrihydrite. A HRTEM image is shown in Fig. 5b. Almost spherical nanocrystals are seen, with diameters ranging from 3 to 8 nm with a mean size of ∼5 nm, close to the values observed by others [12,21] as well as that found from the histogram of Fig. 3. The lattice fringes observed in the nanocrystals are compatible with the d-spacings determined from the electron diffraction pattern. 4. Conclusions The future use of QDs in electronic devices will require large arrays of them in a known order. The assembly of these nanostructures on planar substrates is crucial, since it is very important to control collective behavior [22]. In the present work we have shown by a combination of characterization techniques that it is possible to construct arrays of ferritin molecular units on a Si substrate with a very simple and cost-effective method. Moreover, we have shown that the ferritin core contains mainly FeO nanocrystals. In order to produce bare FeO nanocrystals arrays on the Si substrate the protein cage must be removed. Acknowledgement The authors are thankful to Dr. Sylvie Lousinian for her invaluable assistance.
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References [1] J.D. Desai, H.M. Pathan, S.K. Min, K.D. Jung, O.S. Joo, Appl. Surf. Sci. 252 (2006) 8039. [2] M. Preisinger, M. Krispin, T. Rudolf, S. Horn, D.R. Strongin, Phys. Rev. B 71 (2005) 165409. [3] A.A. Malik, P.O. Brain, N. Revaprasadu, Adv. Mater. 11 (1999) 1441. [4] P. Ramvall, S. Tanaka, S. Nomura, P. Riblet, Y. Aoyagi, Appl. Phys. Lett. 73 (8) (1998) 1104–1106. [5] A. Zora, C. Simserides, G.P. Triberis, Phys. Stat. Sol. (c) 5 (12) (2008) 3806–3808. [6] N.D. Chasteen, P.M. Harrison, J. Struct. Biol. 126 (1999) 182. [7] S. Levi, A. Luzzago, G. Cesareni, A. Cozzi, F. Franceschinelli, A. Albertini, P. Arosio, J. Biol. Chem. 263 (34) (1988) 18086. [8] G.C. Ford, P.M. Harrison, D.W. Rice, J.M.A. Smith, A. Treffry, J.L. White, J. Yariv, Philos. Trans. R. Soc. Lond. B: Biol. Sci. 304 (1984) 551. [9] C.M. Barnes, S. Petoud, S.M. Cohen, K.N. Raymond, J. Biol. Inorg. Chem. 8 (2003) 195. [10] P. Santambrogio, S. Levi, A. Cozi, E. Rovida, A. Albertini, P. Arosio, J. Biol. Chem. 268 (17) (1993) 12744. [11] E. Manning, S.-T. Yau, J. Vac. Sci. Technol. B: Microelectron. Nanometer Struct. 23 (2005) 2309. [12] I. Yamashita, Thin Solid Films 393 (2001) 12–18. [13] T. Matsui, N. Matsukawa, K. Iwahori, K.-I. Sano, K. Shiba, I. Yamashita, Jpn. J. Appl. Phys. 46 (2007) L713–L715. [14] M. Okuda, Y. Kobayashi, K. Suzuki, K. Sonoda, T. Kondoh, A. Wagawa, A. Kondo, H. Yoshimura, Nano Lett. 5 (2005) 991. [15] K. Ichikawa, Y. Uraoka, P. Punchaipetch, H. Yano, T. Hatayama, T. Fuyuki, I. Yamashita, Jpn. J. Appl. Phys. Part 2: Lett. 46 (2007) L804. [16] S.M. Russell, P.M. Harrison, Biochem. J. 175 (1978) 91. [17] M.M. Heynes, T. Bearda, I. Cornelissen, S. De Gendt, R. Degraeve, G. Groeseneken, C. Kenens, D.M. Knotter, L.M. Loewenstein, P.W. Mertens, S. Mertens, M. Meuris, T. Nigam, M. Schaekers, I. Teerlinck, W. Vandervorst, R. Vos, K. Wolke, IBM J. Res. Dev. 43 (3) (1999) 339. [18] D.M. Lawson, P.J. Artymiuk, S.J. Yewdall, J.M.A. Smith, J.C. Livingstone, A. Treffry, A. Luzzago, S. Levi, P. Arosio, G. Cesareni, C.D. Thomas, W.V. Shaw, P.M. Harrison, Nature 349 (1991) 541. [19] G.E. Jellison, F.A. Modine, Appl. Phys. Lett. 69 (1996) 371. [20] P. Santabrogio, P. Pinto, S. Levi, A. Cozzi, E. Rovida, A. Albertini, P. Artymiuk, P.M. Harrison, P. Arosio, Biochem. J. 322 (1997) 461. [21] S.-W. Kim, H.-Y. Seo, Y.-B. Lee, Young, S. Park, K.-S. Kim, Bull. Korean Chem. Soc. 29 (10) (2008) 1969–1972. [22] P. Zhang, J. Fluor. 16 (3) (2006) 349.
Contents lists available at ScienceDirect
Materials Science and Engineering B journal homepage: www.elsevier.com/locate/mseb
Simple method for coating Si (1 0 0) surfaces with ferritin monolayers—Iron oxide quantum dots Georgios Papadopoulos a,∗ , Constantine Anetakis b , Christoforos Gravalidis b , Spiros Kassavetis b , Nikolaos Vouroutzis b , Nikolaos Frangis b , Stergios Logothetidis b a
University of Thessaly, Department of Biochemistry & Biotechnology, Ploutonos 26 & Aeolou, 41221 Larisa, Greece Aristotle University of Thessaloniki, Department of Physics, Laboratory for Thin Films – Nanosystems and Nanometrology and Laboratory of Electronic Microscopy, 54124 Thessaloniki, Greece b
a r t i c l e
i n f o
Article history: Received 15 September 2009 Received in revised form 17 April 2010 Accepted 17 May 2010 Keywords: Ferritin Iron oxide Quantum dots Atomic force microscopy Transmission electron microscopy Spectroscopic ellipsometry
a b s t r a c t With the goal to develop iron oxide quantum dots we developed a simple method to spread horse spleen ferritin monolayers on a Si (1 0 0) surface. Application of atomic force microscopy and spectroscopic ellipsometry showed the existence of regions with dense ferritin monolayers. Application of transmission electron microscopy identified the core of the spread ferritin as FeO nanocrystals. © 2010 Elsevier B.V. All rights reserved.
1. Introduction Iron oxides are interesting due to their catalytic, magnetic and semiconducting properties. Their applications include usage as pigments, catalysts in styrene synthesis and as a material for high density magnetic storage as well as possible applications in optical devices [1]. Nanosized iron oxide particles can be expected to show size-dependent optical, magnetic, and chemical properties. Moreover, the optical properties of semiconducting nanoparticles, such as quantum dots (QDs), are different from those of the corresponding bulk material. The size dependence of their properties can be associated with an influence of the size on the electronic structure of the material [2]. The most important feature of these materials is the increase in optical band gap due to quantum confinement effect. This increase is observed as a blueshift in photoluminescence emission or absorption spectra [3,4]. This can be explained as combination of the confinement-induced blueshift of the electronic levels and the redshift caused by the increased Coulomb interaction due to the compression of the exciton radii [5]. Besides, the
∗ Corresponding author. Tel.: +30 2410 565249; fax: +30 2410 565290. E-mail addresses: [email protected] (G. Papadopoulos), [email protected] (C. Anetakis), [email protected] (C. Gravalidis), [email protected] (S. Kassavetis), [email protected] (N. Vouroutzis), [email protected] (N. Frangis), [email protected] (S. Logothetidis). 0921-5107/$ – see front matter © 2010 Elsevier B.V. All rights reserved. doi:10.1016/j.mseb.2010.05.014
aforementioned influence can be due to the surface tension of the particle, resulting in an effective pressure or to changes in the stoichiometry (and resulting changes of the state of oxidation of Fe) of such particles, driven by the large surface to-volume ratio [2]. In the present study, in order to produce nanosized iron oxide particles, we utilized the ability of ferritin to self-assemble and construct a core of iron oxide. The major function of ferritin in animals, plants and bacteria, is to store and detoxify intracellular iron [6,7]. Crystallographic studies have revealed that its structure is uniquely well designed to contain large amounts of iron in a soluble, nontoxic, and available form [8]. Ferritins in vertebrates are assembled from 24 subunits into a spherical shell (Ø = 10–12 nm) from two genetically distinct subunit types, designated H and L [9,10]. The cage formed by the subunits encloses up to 4500 ferrihydrite molecules (approximate formula: 5Fe2 O3 ·9H2 O) in nanocrystalline form [6]. This protein shell functions in several manners: it acquires iron (II), catalyses its oxidation, and induces mineralization within its cavity [2,6,9]. Several procedures have been reported for the development of iron oxide thin films utilizing ferritin. Manning and Yau [11] have demonstrated immobilization of ferritin onto a Si/SiO2 surface, while Yamashita [12] and Matsui et al. [13] applied the interesting “Bio Nano Process”, which exploits the self-assembly properties of ferritin on an air–water surface to transfer a layer of ferritin onto a Si surface. Okuda et al. [14] reported the fabrication of nanometric iron and indium particles using ferritin. In a step forward Ichikawa et al. [15] developed LTPS-TFT flash memory
G. Papadopoulos et al. / Materials Science and Engineering B 176 (2011) 500–503
501
applying the “Bio Nano Process”. The above-mentioned methods differ in complexity, as well as in required time and expenses. Here we introduce a simple and cost-effective technique (paintbrush like), described in Section 2, to coat silicon (1 0 0) surfaces with ferritin monolayers as the first part of an effort to produce arrays of iron oxide quantum dots. The derived surfaces have been characterized with atomic force microscopy (AFM), spectroscopic ellipsometry (SE) and transmission electron microscopy (TEM). 2. Experimental As a substrate for the formation of the ferritin monolayer, silicon (1 0 0) surfaces were used. In order to remove possible organic contamination, the surfaces were treated successively with tetrachloroethylene (99%), acetone (99.5%), and methanol (99.9%), at 60 ◦ C in a supersonic bath, for 15 min for each step. Then, they were treated with hydrogen peroxide, at 85 ◦ C for 4 h. The surfaces were further cleansed mechanically. For this purpose, a poly vinyl chloride (PVdc) membrane, wrapped around a cotton stick, was used. In particular, 40 l of ddH2 O was posed on the surface, which was thoroughly passed with the stick for 5 min. In the present study, a horse spleen ferritin solution 1 mg/ml, in HEPES 0.1 M, pH 7, was used for the coating of the surface. Generally, the horse derived ferritins, have an isoelectric point (pI) extending from pH 4.1 to 5.1 [16], so that in neutral pH they have a net positive charge. For this reason, the silicon surfaces were treated overnight with HCl 10N, pH 1.2, to obtain a net negative charge [17] which will facilitate the deposition of the protein. The surfaces were then neatly dried, with a N2 draught in a clean chamber and 40 l of the ferritin solution were deposited on the surface. Again a cotton stick, wrapped with a PVdc membrane, was used, to cover the surface, with movements to a single direction. This step was repeated with 40 l of ddH2 O. Finally, the samples were dried thoroughly with a N2 draught. For the above treatment a clean chamber has been used. All samples were studied with AFM (NT-MDT) in semicontact mode, SE (Uvisel p/n 23 301 909, Jobin-Yvon) and TEM (JEOL 2011). AFM measurements of the silicon surfaces, after the two steps of cleansing, as well as SE measurements of the Si substrate, before the protein coverage, were also performed.
Fig. 1. Ellipsometric model for the sample of Figs. 2 and 4. Red lines and data points refer to the imaginary part of the dielectric function ε(ω), whereas black ones refer to the real part. (—) Si substrate, (•••) SiO2 /Si layer, (***) protein/SiO2 /Si layers. The effective thickness of the protein sublayer has been measured to 4.99 ± 0.50 nm. The refractive index associated with the model is 1.451. (For interpretation of the references to color in this figure legend, the reader is referred to the web version of the article.)
3. Results and discussion The aim of this work was to spread a monolayer of ferritin molecules with iron oxide core on a Si (1 0 0) surface as a first step for the development of iron oxide nanoparticles. Very clean Si surfaces are required. Chemical cleansing of the Si substrate left some contamination observed with AFM with average roughness (Ra) ∼1.52 nm, and RMS ∼2.55 nm. After the mechanical treatment, the surfaces were clean at the nanoscale level with Ra ∼0.47 nm and RMS ∼0.42 nm. Ra and RMS are defined as usually. Our sample is a clean surface, which has been coated with ferritin following the simple procedure described in Section 2. The SE measurements of the dielectric function ε(ω) shown in Fig. 1 were fitted using a simple Tauc–Lorenz model [19]. Analysis of the data in terms of a four phase model provided a profile consisted of the Si substrate, a SiO2 sublayer (due to the treatment of the surface with H2 O2 ) with a thickness of 0.60 ± 0.03 nm and a protein sublayer, with voids extending to about 50% of the covered surface. The effective thickness of the protein sublayer was calculated to be 4.99 ± 0.50 nm, so that the real thickness of the protein coverage corresponds, as expected, to about 10 nm. The AFM image of the sample shows coverage of the Si surface with molecular units (Fig. 2a) including some voids and aggregates, with Ra ∼2.37 nm and RMS ∼3.27 nm. A comparison of the heights
Fig. 2. (a) AFM image of a protein coated Si surface. The image shows a total coverage of the substrate, with some voids. (b) Sections through the AFM topography images of a clean Si surface (black line) and of a Si surface coated with a ferritin monolayer (blue and red line). The blue line shows an arbitrary section, whereas the red one is chosen to show a protein aggregate (highest peak) with a height corresponding to about two or three ferritin units. (For interpretation of the references to color in this figure legend, the reader is referred to the web version of the article.)
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G. Papadopoulos et al. / Materials Science and Engineering B 176 (2011) 500–503
Fig. 3. Height histogram of the sample depicted in Fig. 2, showing an average height of the coverage, 10–12 nm.
of a clean Si surface and of a Si surface coated with a ferritin monolayer is presented in Fig. 2b. The low height black line represents a section through the AFM image of the clean surface. The blue line represents an arbitrary section through the ferritin monolayer coating, whereas the red one represents a similar section chosen to show a protein aggregate (highest peak) with a height corresponding to about two or three ferritin molecules. Fig. 3 shows a histogram of the height distribution of Fig. 2a with a mode of ∼11 nm corresponding to the diameter of ferritin in agreement with our SE results. The presence of a smaller peak in the histogram at about 4.5 nm can be considered as an indication for the existence of bare nanocrystals from protein core and (or) to protein subunits. A clearer picture of the protein distribution is obtained from the AFM phase image (Fig. 4). Regular arrays of ferritin units are visible
Fig. 4. AFM phase image. A total coverage is visible. The region indicated by a white bar on the left (146 nm) covers 10 molecular units, including spacing between them.
Fig. 5. (a) An electron diffraction pattern from a plane view specimen. The (1 1 1) Sireflections are indicated by circles. The four polycrystalline rings correspond to the cubic FeO structure. (b) A HRTEM image revealing the presence of FeO nanocrystals (QD) with diameters between 2 and 8 nm.
as well as some larger objects, probably aggregates. We measured the length of 10 molecular units to be 146 nm (Fig. 4), which gives an average distance between the centers of the molecular units ∼14.6 nm. This value is consistent with the dimensions of ferritin units calculated from the topography (∼11 nm) and to those previously published [2,6,18], if we consider intermolecular water and a ferritin organization slightly adapted to the SiO2 background. Electron diffraction and high-resolution transmission electron microscopy (HRTEM) were used in order to elucidate the crystallinity of the quantum dots. Plane view specimens were prepared by ion thinning, bombarding only the substrate. A representative electron diffraction pattern is shown in Fig. 5a. Apart of some reflections of the Si substrate, rings containing a large number of spots are observed. Using the Si spots as an internal standard, the d-spacings of the rings are found to be: 0.248 ± 0.002 nm, 0.216 ± 0.002 nm, 0.153 ± 0.002 nm, and 0.128 ± 0.002 nm. These values are in very good agreement with the main d-spacings of the cubic FeO: d1 1 1 = 0.248 nm, d2 0 0 = 0.215 nm, d2 2 0 = 0.152 nm, d3 1 1 = 0.129 nm. This is quite surprising, since according to the literature [2,6,8,20], a structure similar to that of the mineral ferrihydrite (5Fe2 O3 ·9H2 O) would be expected. Exceptionally,
G. Papadopoulos et al. / Materials Science and Engineering B 176 (2011) 500–503
Preisinger et al. [2] needed to anneal the sample in air atmosphere at a temperature range of 575–675 K in order to obtain the Fe2 O3 form. Although ferrihydrite has some reflections close to those of FeO (0.247 nm, 0.234 nm and 0.148 nm according to [21]), the absence of its reflections corresponding to 0.198 and 0.173 nm, does not support the existence of ferrihydrite, at least as the main observed phase. Some isolated spots can also be seen in the pattern of Fig. 5a, denoting the possible existence as minority of other Fe phases, including ferrihydrite. A HRTEM image is shown in Fig. 5b. Almost spherical nanocrystals are seen, with diameters ranging from 3 to 8 nm with a mean size of ∼5 nm, close to the values observed by others [12,21] as well as that found from the histogram of Fig. 3. The lattice fringes observed in the nanocrystals are compatible with the d-spacings determined from the electron diffraction pattern. 4. Conclusions The future use of QDs in electronic devices will require large arrays of them in a known order. The assembly of these nanostructures on planar substrates is crucial, since it is very important to control collective behavior [22]. In the present work we have shown by a combination of characterization techniques that it is possible to construct arrays of ferritin molecular units on a Si substrate with a very simple and cost-effective method. Moreover, we have shown that the ferritin core contains mainly FeO nanocrystals. In order to produce bare FeO nanocrystals arrays on the Si substrate the protein cage must be removed. Acknowledgement The authors are thankful to Dr. Sylvie Lousinian for her invaluable assistance.
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