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Surface modification of core–shell nanowire with protein adsorption
Materials Chemistry and Physics 129 (2011) 256–260
Contents lists available at ScienceDirect
Materials Chemistry and Physics journal homepage: www.elsevier.com/locate/matchemphys
Surface modification of core–shell nanowire with protein adsorption B. Kalska-Szostko ∗ , E. Orzechowska Institute of Chemistry, University of Białystok, Hurtowa 1, 15-399 Białystok, Poland
a r t i c l e
i n f o
Article history: Received 1 September 2010 Received in revised form 18 March 2011 Accepted 2 April 2011 Keywords: Nanostructures Electrochemical techniques Composite materials Magnetic materials
a b s t r a c t Silver and ferrite nanotubes were obtained in nanoporous alumina templates (AAO) of 200 nm diameter by wetting chemical deposition followed by thermal crystallization. Iron filling in the nanotubes was fabricated by DC electrodeposition. Step by step fabrication in AAO was followed by scanning electron microscopy (SEM) and transmission electron microscopy (TEM) techniques. The core–shell nanowires were removed from the templates and verified by TEM and energy dispersive spectroscopy (EDS) techniques. Metallic (Ag) surfaces of nanowires were functionalized with alkenothiols terminated by methyl groups. Ferrite nanotubes were functionalized to –NH2 groups on the surface. In addition, these groups were bonded to protein–trypsin in two ways: covalently bonded to amine groups by glutaraldehyde and noncovalent absorption to alkenothiols. All the modifications were observed by infra-red spectroscopy (IR). © 2011 Elsevier B.V. All rights reserved.
1. Introduction In recent years nanotechnology became very important in many fields of our life. Minimalization of the scale achieved to develop new properties of the materials and create innovative applications. Nanowires are very important building blocks in nanotechnology. They have become the subject of intensive research in view of their potential applications as nanosensors, nanoactuators and nanocarriers [1]. The ability to prepare nanowires with different aspect ratios allows fine tuning of their electronic and optical properties [2]. Otherwise, integration of the nanowires and biomolecules leads to a novel hybrid system which couples recognition or catalytic properties of biomaterials with the attractive electronic, magnetic and structural characteristics of nanostructures [3]. Therefore, nanowires can be functionalized with various biomolecules through different linkage chemistries [4]. Metallic nanostructures can be bonded directly with organic molecules to the surface forming self-assembled monolayers [5]. These monolayers can be further modified either by the formation of a new covalent bond with, e.g., a receptor by the connector or directly through noncovalent interactions [6]. Noncovalent bonds are the most important in biological systems especially in hydrogen bonding network [7]. A large library of studies on chemical modification of planar surfaces has been prepared. It is well known that thiols have a huge affinity to gold surface, which often leads to form selfassembled monolayers. Otherwise, carboxylic acids will readily bond to a metal oxide surface as well as siloxanes and phospho-
∗ Corresponding author. Tel.: +48 85 745 7814; fax: +48 85 747 0113. E-mail address: [email protected] (B. Kalska-Szostko). 0254-0584/$ – see front matter © 2011 Elsevier B.V. All rights reserved. doi:10.1016/j.matchemphys.2011.04.014
nates. There exist many other ligands with different affinity to modified surfaces, which is reported in Meyer and co-workers’ publication [8]. In this work, two types of functionalized core–shell nanowire surfaces were used in this study: alkyl-terminated monolayers on silver and amine groups linked with glutaraldehyde on ferrite surface. In addition, covalent bonding protein to glutaraldehyde and noncovalent adsorption of the same protein to alkyl groups by hydrophobic interactions are present here. 2. Experimental The crystalline nanotubes were fabricated by wetting chemical deposition followed by thermal crystallization in nanoporous alumina templates (AAO) with pore diameter of around 200 nm. Magnetic material (Fe) as a filling of the nanotubes was electrodeposited from aqueous solution. The templates were afterwards dissolved in 1 mol l−1 NaOH and free nanowires were cleaned and isolated for further characterization. Next, silver nanotubes were functionalized with thiols and ferrite surfaces were modified by amine groups. Finally, a protein–trypsin was added to both nanotubes showing two methods of bonding. The obtained material was characterized by transmission electron microscopy (JEOL) and scanning electron microscopy (TM1000). Results of functionalization were tested by infra-red spectroscopy (NICOLET). 2.1. Preparation of nanotubes The AAO templates were used to fabricate metallic and ferrite nanotubes with the diameter of 200 nm [9]. Ni(NO3 )2 ·6H2 O or Co(NO3 )·6H2 O or Mn(NO3 )2 ·6H2 O and Fe(NO3 )2 ·6H2 O were mixed with a molar ratio 1:2 respectively, to form nitrate aqueous solutions. To fabricate metallic nanotubes 1 mol l−1 AgNO3 and 0.007 mol l−1 AuCl3 ·HCl aqueous solutions were prepared. All nanotubes were obtained following the same deposition method [10]. The AAO templates were put into a vessel containing the appropriate amount of each above-mentioned solution for 3 h at room temperature. Then, the templates were placed into a sampler holder upright and dried in an oven at 60 ◦ C for 20 h. During this process a thin film covering the pore wall was obtained. At last, the templates were removed to another oven for 3 h with temperature of 550 ◦ C to formed crystalline nanotubes. Fig. 1 presents the schematic sketch of the preparation process of nanotubes.
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Fig. 1. Steps of nanotubes preparation: (a) AAO template, (b) template immersed in the solution, (c) a thin film adsorbed on the pore wall, (d) crystalline nanotube, (e) DC deposition.
Fig. 2. Schematic presentation of the modification steps: (a) ferrite nanotube, (b) nanotube filled with Fe, (c) attachment of NH2 groups, (d) modification with glutaraldehyde, (e) final attachment of trypsin.
2.2. Synthesis of core–shell nanowires To fill the nanotubes with magnetic material the two-electrode electrodeposition process was used (Fig. 1). In all types of nanotubes Fe was deposited at constant current (DC) mode of I = 0.01 A for 10 min. The used electrolyte was composed from the aqueous solution of 0.43 mol l−1 FeSO4 ·7H2 O, 0.73 mol l−1 H3 BO3 , 0.0043 mol l−1 FeCl3 and 1 g ascorbic acid [11]. After deposition all samples were dissolved in 1 mol l−1 NaOH to remove nanowires from the template. Next, the obtained material was isolated using a permanent magnet and washed in pure water. Cleaned nanowires were removed to a beaker and left to evaporate the water. 2.3. Surface functionalization with protein 2.3.1. Ferrite nanotubes Clean and dry nanowires covered by ferrites were suspended in water. Then, a mixture of toluene, oleic acid and oleyamine in a molar ratio 1:2:2 was added to the sample and shaken a while to replace nanowires from the aqueous phase to the organic solvent. The sample prepared in this way was left for 2 days. After that time, the mixture of the solutions was removed. The solid sample was washed in toluene and left to evaporate the residual solvent for a night. Afterwards, nanowires were bathed in buffer Tris–HCl, then isolated using a permanent magnet and mixed with glutaraldehyde meanwhile. The sample was shaken from time to time for 2 h. After that time, the rest of glutaraldehyde was removed and the nanowires were left for a night to evaporate the solvent – to dry. Finally, a mixture of buffer Tris–HCl and trypsin in a molar ratio 1:3 was added to the solid sample, shaken from time to time for 1.5 h at room temperature and then for 30 min at 4 ◦ C [12]. In the end, all residual solvent was removed and the nanowires were left to get dry. Fig. 2 shows schematic steps of the nanowires surface modification. 2.4. Metallic nanotubes (Ag) To metallic nanotubes (Ag) filled with Fe the appropriate amount (∼1 ml) of 1hexadecanethiol was added. The sample was shaken from time to time during 1.5 h at room temperature. After that time, the sample was washed from thiol in ethanol four times to remove the rest of the reagents [13]. Nanowires treated in such a way were left to evaporate the solvent for a night. Afterwards, a mixture of buffer Tris–HCl and trypsin in a molar ratio 1:3 was added to the solid sample, shaken
from time to time for 1.5 h at room temperature and then 30 min at 4 ◦ C. All residual solvent was removed and the nanowires left to get dry. Fig. 3 shows schematically the steps of the surface modification.
3. Results and discussion Metallic and ferrite nanotubes filled with magnetic material were prepared as previously described. The morphology of obtained structures was verified by transmission electron microscopy (TEM) (Fig. 4). SEM image presents empty Ag nanotube (AgNT) in AAO matrix (Fig. 4b). TEM image presents: empty ferrite nanotube (Fig. 4a) and silver nanotube filled with Fe (Fig. 4c). To indicate if it presents expected nanostructures, the EDS analysis was done and the result is depicted in Fig. 5. As TEM image shows (Fig. 4a), the nanotubes have a granual crystalline structure. The walls of the tubes are rather tiny, which results in brittleness of the tubes. The walls, however, are very even, which suggests that the deposition of the material is rather even along the wall. In Fig. 4c, the wire is presented in STEM mode by which a fine regular structure is proven. EDS analysis (Fig. 5) confirmed the inert morphology of Ag nanotube filled with Fe, which should be expected to be formed after the deposition. Comparing the two data sets from (Fig. 5), it can be seen that for the analysis of the position assigned as 0 0 3 (Fig. 4c) more wall of the wire was analyzed and it pronounced the relative ratio between Ag and Fe peaks rather well. For 0 0 4 position the amount of Fe is so high that the Ag intensity almost disappears (for the same time of deposition), which is rather reasonable and proves the core–shell wire structure. In addition, the magnetic properties of the prepared nanotubes filled with Fe were verified by external magnetic field. Nanowires
Fig. 3. Schematic presentation of the modification steps: (a) metallic nanotube, (b) nanotube filled with Fe, (c) modification with thiol, (d) final attachment of trypsin.
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Fig. 4. Images of nanotubes: (a) TEM – empty CoFe2 O4 , (b) SEM – empty Ag in AAO, (c) STEM – Ag filled with Fe.
Fig. 5. EDS spectrum of AgNT filled with Fe: (a) point 0 0 3 which is chosen to the edge of the structure, (b) point 0 0 4 in the middle of nanotube on STEM image (Fig. 4c).
not influenced by external magnetic field form dispersed powder or they tend to agglomerate due to strong magnetic interaction between each other (Fig. 6a). In Fig. 6b, nanowires standing perpendicular to the bottom of the flask to the magnet which was placed under the flask with the powder sample are presented. IR spectroscopy was used to verify the result of executed functionalization. Fig. 7 presents the spectra obtained before and after modification with amine groups and with thiols (Fig. 8). In both cases modification includes the attachment of the trypsin as a final step.
The analysis of the spectrum (Fig. 7a and b) shows that they are very different. In the basic spectrum (a) it is possible to indicate two signals: 3417 cm−1 and 1611 cm−1 , which can be identified probably to –OH bonds from adsorbed water, from the air, during the drying process [14]. The third significant signal around 500 cm−1 comes from Fe–O bonds which confirm the surface of ferrite nanotubes [15]. In the spectrum received after modification some of the signals described above become more pronounced but also another strong signal at about 1025 cm−1 and 1636 cm−1 clearly appear. These wave numbers may be identified to covalently
Fig. 6. Visualization of magnetic properties: (a) nanowires without influence of external magnetic field, (b) nanowires standing perpendicular to the magnet under the flask.
B. Kalska-Szostko, E. Orzechowska / Materials Chemistry and Physics 129 (2011) 256–260
Fig. 7. IR spectrum of MnFe2 O4 nanotubes filled with Fe: (a) before, (b) after modification (trypsin attachment).
bonded trypsin and show amide bond which should be expected from the position of this signal. In addition, we can identify small but sharp signals at 2850–2920 cm−1 which belong to C–H bond in –CH2 – group in oleylamine chain and trypsin [16,17]. In all types of the tested ferrite nanotubes the same changes were observed in spectrum after modification, which gives the evidence about trypsin bonded to the nanowires. The comparison of spectra collected in Fig. 8 confirms that they are different in every step of modification. In the basic spectrum (a) there are two signals at 3450 cm−1 and 1650 cm−1 , which belong to –OH bonds from adsorbed water from the air. The rest of the signals around 2900 cm−1 and 1120 cm−1 can be identified to –NO3 − groups from other solvents participating in nanowire synthesis [18]. In the second spectrum (b) after modification with thiols we can observe characteristic sharp signals appearing originally in thiols bonds around 1470 cm−1 and 719 cm−1 [19]. Finally in the last spectrum (c) every earlier signal is present in it with a bit smaller intensity. Similar to the modification with amine groups, here we
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can observe a new signal, not so well defined as the previous one at 1025 cm−1 , which means that trypsin has adsorbed on the surface. There is also a large signal below 750 cm−1 [20]. In this case trypsin does not covalently bond with methyl groups but it appeared on the wire surface via hydrophobic interactions which give that signal. The composition of metallic (Ag) and ferrite (NiFe2 O4 , CoFe2 O4 and MnFe2 O4 ) nanotubes filled with magnetic material – iron inside allowed to create core–shell nanowires. Protein immobilization on the nanowires forced to produce two types of nanotubes modified in two different ways. Firstly, ferrite tubes have an oxidant structure, which is convenient to form monolayers with amine groups. These structures can also be modified with carboxylic groups, which can be investigated in future research. Because amine groups are quite short, there is no need to use any extreme conditions of experiment to attach them on the surface. Additionally, amine groups cause the presence of hydrophilic properties in the structure. Following the amination process, ferrite tubes were connected with trypsin using glurataldehyde as homo-bifunctional linker which is covalently bonded to both sides. A strong signal on IR spectrum confirmed trypsin attachment and the presence of amide bond. This procedure presented a simple method to form amine groups on the surface of the nanowires including protein immobilization which allows direct biofunctionalization without using thin films such as silanes [21]. The usage of linker (glutaraldehyde) allows to attach many others biomolecules which possess functional groups such as primary amines and sulfhydryls. For the metallic nanotubes, a sixteen-carbon thiol chain was used to modified homogeneous Ag surface. Thiols cause hydrophobic properties to the structure. In this method it can be suggested that the reaction time between Ag surface and thiol chains is very important because the longer the interaction lasts, the more thiol chains are standing perpendicular to the surface and the protein may easier adsorb to them. Moreover, the attachment of trypsin to methyl groups of thiol was observed without including linker. Moreover, functionalization proved that protein had influenced thiol, which is confirmed by a small signal on IR spectrum. The signal is not so well pronounced because in this case trypsin did not covalently bond to the methyl groups but just adsorbed on them. Adsorption is possible through hydrophobic interactions between methyl groups and carbon chains included in the trypsin structure. The comparison of two signals after modifications with amine and thiol groups including trypsin attachment indicates that covalently bonded structures show strongest signals in IR spectrum than those which interact through noncovalent adsorption. The usage of a functional group like thiol means that these groups may be used to other modified materials such as Au, Pd, CdS, CdSe, ZnS. Both experiments can be used to produce nanocapsules for antibodies or transporting medicines into sick areas by magnetic means.
4. Conclusion
Fig. 8. Spectrum of AgNT filled with Fe: (1) basic, (2) with thiols, (3) after trypsin attachment.
Preparation of metallic and ferrite nanotubes by wetting chemical deposition followed by thermal crystallization filled with magnetic material forming core–shell nanowires has been obtained. Two methods of functionalization: with amine and thiol groups, were successfully achieved. In addition, trypsin has shown two ways of bonding – covalently with amine groups through crosslinker – glutaraldehyde, and also noncovalent adsorption to methyl groups which ended a long thiol chain. Every step of the nanowire synthesis was verified by SEM, TEM and EDS techniques. The results of the surface modification were observed by IR spectroscopy. All procedures give clear step by step view of the execute process.
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Acknowledgments The authors acknowledge equipment support from JEOL and COMEF. We are very grateful for Dr. A. Dubis for IR measurements. The work was financed by Polish National Funding under contract number N N204246435. References [1] (a) J. Wang, Chem. Phys. Chem. 10 (2009) 1748; (b) A.K. Wanekaya, W. Chen, N.V. Myung, A. Mulchandani, Electroanalysis 18 (2006) 533. [2] R.L. Zong, J. Zhou, Q. Li, B. Du, B. Li, M. Fu, X.W. Qi, L.T. Li, J. Phys. Chem. B 108 (2004) 16713. [3] (a) R. Contreras, H. Sahlin, J.A. Frangos, J. Biomed. Mater. Res. A 80 (2007) 480; (b) C.R. Martin, Science 266 (1994) 1961. [4] (a) Q. Ren, Y.P. Zhao, J.C. Yue, Y.B. Cui, Biomed. Microdevices 8 (2006) 201; (b) K. Skinner, C. Dwyer, S. Washburn, Nano Lett. 6 (2006) 2758; (c) L.A. Bauer, D.H. Reich, G.J. Meyer, Langmuir 19 (2003) 7043. [5] K.L. Prime, G.M. Whitesides, Science 252 (1991) 1164. [6] (a) N.S. Birenbaum, B.T. Lai, C.S. Chen, D.H. Reich, G.J. Meyer, Langmuir 19 (2003) 9580; (b) T.P. Sullivan, W.T.S. Huck, Eur. J. Org. Chem. 2003 (2003) 17. [7] R.M. Crooks, L.J. Kepley, L. Sun, Langmuir 8 (1992) 2101. [8] L.A. Bauer, N.S. Birenbaum, G.J. Meyer, J. Mater. Chem. 14 (2004) 517.
[9] (a) B. Kalska-Szostko, E. Brancewicz, P. Mazalski, J. Sveklo, W. Olszewski, K. ´ SidorF A., Acta Phys. Pol. A 115 (2009) 542; Szymanski, (b) B. Kalska-Szostko, E. Brancewicz, P. Mazalski, J. Sveklo, W. Olszewski, K. ´ Szymanski, A. Sidor, Solid State Phen. 151 (2009) 190. [10] F. Li, L. Song, D. Zhou, T. Wang, Y. Wang, H. Wang, J. Mater. Sci. 42 (2007) 7214. ˜ M. Muhammed, Chem. [11] J. Qin, J. Noguès, M. Mikhaylova, A. Roig, J.S. Munoz, Mater. 17 (2005) 1829. [12] K. Kluchova, R. Zboril, J. Tucek, M. Pecova, L. Zajoncova, I. Safarik, M. Mashlan, I. Markova, D. Jancik, M. Sebela, H. Bartonkova, V. Bellesi, P. Novak, D. Petridis, Biomaterials 30 (2009) 2855. [13] Z. Gu, H. Ye, D.H. Gracias, J. Miner. Met. Mater. Soc. 57 (2005) 60. [14] J. Soria, J. Sanz, I. Sobrados, J.M. Coronado, A.J. Maira, M.D. Hernandez-Alonso, F. Fresno, J. Phys. Chem. C 111 (2007) 10590. [15] (a) G. Verlaj, K. Janaki, A. Mohamed Musthafa, R. Palanivel, Appl. Clay Sci. 43 (2009) 303; (b) D. Barilaro, G. Barone, V. Crupi, M.G. Donato, D. Majolino, G. Messina, R. Poterino, J. Mol. Struct. 744–747 (2005) 827; (c) V.S. Zaitser, D.S. Filimonov, I.A. Presnyakov, R.J. Gambino, B. Chu, J. Colloid Interface Sci. 212 (1999) 49. [16] L. Li, J. Weng, Nanotechnology 21 (2010) 305603 (supporting information). [17] T. Sainsbury, T. Ikuno, D. Okawa, D. Pacile, J.M.J. Frechet, A. Zettl, J. Phys. Chem. C 111 (2007) 12992. [18] J.T. Kloprogge, D. Wharton, L. Hickey, R.L. Frost, Am. Mineral. 87 (2002) 623. [19] M. Brust, M. Walker, D. Bethell, D.J. Schriffin, R. Whyman, J. Chem. Soc., Chem. Commun. 7 (1994) 801. [20] L. Li, J. Weng, Nanotechnology 21 (2010) 305603. [21] R. Stine, B.S. Simpkins, S.P. Mulvaney, L.J. Witchman, C.R. Tamanaha, Appl. Surf. Sci. 256 (2010) 4171.
Contents lists available at ScienceDirect
Materials Chemistry and Physics journal homepage: www.elsevier.com/locate/matchemphys
Surface modification of core–shell nanowire with protein adsorption B. Kalska-Szostko ∗ , E. Orzechowska Institute of Chemistry, University of Białystok, Hurtowa 1, 15-399 Białystok, Poland
a r t i c l e
i n f o
Article history: Received 1 September 2010 Received in revised form 18 March 2011 Accepted 2 April 2011 Keywords: Nanostructures Electrochemical techniques Composite materials Magnetic materials
a b s t r a c t Silver and ferrite nanotubes were obtained in nanoporous alumina templates (AAO) of 200 nm diameter by wetting chemical deposition followed by thermal crystallization. Iron filling in the nanotubes was fabricated by DC electrodeposition. Step by step fabrication in AAO was followed by scanning electron microscopy (SEM) and transmission electron microscopy (TEM) techniques. The core–shell nanowires were removed from the templates and verified by TEM and energy dispersive spectroscopy (EDS) techniques. Metallic (Ag) surfaces of nanowires were functionalized with alkenothiols terminated by methyl groups. Ferrite nanotubes were functionalized to –NH2 groups on the surface. In addition, these groups were bonded to protein–trypsin in two ways: covalently bonded to amine groups by glutaraldehyde and noncovalent absorption to alkenothiols. All the modifications were observed by infra-red spectroscopy (IR). © 2011 Elsevier B.V. All rights reserved.
1. Introduction In recent years nanotechnology became very important in many fields of our life. Minimalization of the scale achieved to develop new properties of the materials and create innovative applications. Nanowires are very important building blocks in nanotechnology. They have become the subject of intensive research in view of their potential applications as nanosensors, nanoactuators and nanocarriers [1]. The ability to prepare nanowires with different aspect ratios allows fine tuning of their electronic and optical properties [2]. Otherwise, integration of the nanowires and biomolecules leads to a novel hybrid system which couples recognition or catalytic properties of biomaterials with the attractive electronic, magnetic and structural characteristics of nanostructures [3]. Therefore, nanowires can be functionalized with various biomolecules through different linkage chemistries [4]. Metallic nanostructures can be bonded directly with organic molecules to the surface forming self-assembled monolayers [5]. These monolayers can be further modified either by the formation of a new covalent bond with, e.g., a receptor by the connector or directly through noncovalent interactions [6]. Noncovalent bonds are the most important in biological systems especially in hydrogen bonding network [7]. A large library of studies on chemical modification of planar surfaces has been prepared. It is well known that thiols have a huge affinity to gold surface, which often leads to form selfassembled monolayers. Otherwise, carboxylic acids will readily bond to a metal oxide surface as well as siloxanes and phospho-
∗ Corresponding author. Tel.: +48 85 745 7814; fax: +48 85 747 0113. E-mail address: [email protected] (B. Kalska-Szostko). 0254-0584/$ – see front matter © 2011 Elsevier B.V. All rights reserved. doi:10.1016/j.matchemphys.2011.04.014
nates. There exist many other ligands with different affinity to modified surfaces, which is reported in Meyer and co-workers’ publication [8]. In this work, two types of functionalized core–shell nanowire surfaces were used in this study: alkyl-terminated monolayers on silver and amine groups linked with glutaraldehyde on ferrite surface. In addition, covalent bonding protein to glutaraldehyde and noncovalent adsorption of the same protein to alkyl groups by hydrophobic interactions are present here. 2. Experimental The crystalline nanotubes were fabricated by wetting chemical deposition followed by thermal crystallization in nanoporous alumina templates (AAO) with pore diameter of around 200 nm. Magnetic material (Fe) as a filling of the nanotubes was electrodeposited from aqueous solution. The templates were afterwards dissolved in 1 mol l−1 NaOH and free nanowires were cleaned and isolated for further characterization. Next, silver nanotubes were functionalized with thiols and ferrite surfaces were modified by amine groups. Finally, a protein–trypsin was added to both nanotubes showing two methods of bonding. The obtained material was characterized by transmission electron microscopy (JEOL) and scanning electron microscopy (TM1000). Results of functionalization were tested by infra-red spectroscopy (NICOLET). 2.1. Preparation of nanotubes The AAO templates were used to fabricate metallic and ferrite nanotubes with the diameter of 200 nm [9]. Ni(NO3 )2 ·6H2 O or Co(NO3 )·6H2 O or Mn(NO3 )2 ·6H2 O and Fe(NO3 )2 ·6H2 O were mixed with a molar ratio 1:2 respectively, to form nitrate aqueous solutions. To fabricate metallic nanotubes 1 mol l−1 AgNO3 and 0.007 mol l−1 AuCl3 ·HCl aqueous solutions were prepared. All nanotubes were obtained following the same deposition method [10]. The AAO templates were put into a vessel containing the appropriate amount of each above-mentioned solution for 3 h at room temperature. Then, the templates were placed into a sampler holder upright and dried in an oven at 60 ◦ C for 20 h. During this process a thin film covering the pore wall was obtained. At last, the templates were removed to another oven for 3 h with temperature of 550 ◦ C to formed crystalline nanotubes. Fig. 1 presents the schematic sketch of the preparation process of nanotubes.
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Fig. 1. Steps of nanotubes preparation: (a) AAO template, (b) template immersed in the solution, (c) a thin film adsorbed on the pore wall, (d) crystalline nanotube, (e) DC deposition.
Fig. 2. Schematic presentation of the modification steps: (a) ferrite nanotube, (b) nanotube filled with Fe, (c) attachment of NH2 groups, (d) modification with glutaraldehyde, (e) final attachment of trypsin.
2.2. Synthesis of core–shell nanowires To fill the nanotubes with magnetic material the two-electrode electrodeposition process was used (Fig. 1). In all types of nanotubes Fe was deposited at constant current (DC) mode of I = 0.01 A for 10 min. The used electrolyte was composed from the aqueous solution of 0.43 mol l−1 FeSO4 ·7H2 O, 0.73 mol l−1 H3 BO3 , 0.0043 mol l−1 FeCl3 and 1 g ascorbic acid [11]. After deposition all samples were dissolved in 1 mol l−1 NaOH to remove nanowires from the template. Next, the obtained material was isolated using a permanent magnet and washed in pure water. Cleaned nanowires were removed to a beaker and left to evaporate the water. 2.3. Surface functionalization with protein 2.3.1. Ferrite nanotubes Clean and dry nanowires covered by ferrites were suspended in water. Then, a mixture of toluene, oleic acid and oleyamine in a molar ratio 1:2:2 was added to the sample and shaken a while to replace nanowires from the aqueous phase to the organic solvent. The sample prepared in this way was left for 2 days. After that time, the mixture of the solutions was removed. The solid sample was washed in toluene and left to evaporate the residual solvent for a night. Afterwards, nanowires were bathed in buffer Tris–HCl, then isolated using a permanent magnet and mixed with glutaraldehyde meanwhile. The sample was shaken from time to time for 2 h. After that time, the rest of glutaraldehyde was removed and the nanowires were left for a night to evaporate the solvent – to dry. Finally, a mixture of buffer Tris–HCl and trypsin in a molar ratio 1:3 was added to the solid sample, shaken from time to time for 1.5 h at room temperature and then for 30 min at 4 ◦ C [12]. In the end, all residual solvent was removed and the nanowires were left to get dry. Fig. 2 shows schematic steps of the nanowires surface modification. 2.4. Metallic nanotubes (Ag) To metallic nanotubes (Ag) filled with Fe the appropriate amount (∼1 ml) of 1hexadecanethiol was added. The sample was shaken from time to time during 1.5 h at room temperature. After that time, the sample was washed from thiol in ethanol four times to remove the rest of the reagents [13]. Nanowires treated in such a way were left to evaporate the solvent for a night. Afterwards, a mixture of buffer Tris–HCl and trypsin in a molar ratio 1:3 was added to the solid sample, shaken
from time to time for 1.5 h at room temperature and then 30 min at 4 ◦ C. All residual solvent was removed and the nanowires left to get dry. Fig. 3 shows schematically the steps of the surface modification.
3. Results and discussion Metallic and ferrite nanotubes filled with magnetic material were prepared as previously described. The morphology of obtained structures was verified by transmission electron microscopy (TEM) (Fig. 4). SEM image presents empty Ag nanotube (AgNT) in AAO matrix (Fig. 4b). TEM image presents: empty ferrite nanotube (Fig. 4a) and silver nanotube filled with Fe (Fig. 4c). To indicate if it presents expected nanostructures, the EDS analysis was done and the result is depicted in Fig. 5. As TEM image shows (Fig. 4a), the nanotubes have a granual crystalline structure. The walls of the tubes are rather tiny, which results in brittleness of the tubes. The walls, however, are very even, which suggests that the deposition of the material is rather even along the wall. In Fig. 4c, the wire is presented in STEM mode by which a fine regular structure is proven. EDS analysis (Fig. 5) confirmed the inert morphology of Ag nanotube filled with Fe, which should be expected to be formed after the deposition. Comparing the two data sets from (Fig. 5), it can be seen that for the analysis of the position assigned as 0 0 3 (Fig. 4c) more wall of the wire was analyzed and it pronounced the relative ratio between Ag and Fe peaks rather well. For 0 0 4 position the amount of Fe is so high that the Ag intensity almost disappears (for the same time of deposition), which is rather reasonable and proves the core–shell wire structure. In addition, the magnetic properties of the prepared nanotubes filled with Fe were verified by external magnetic field. Nanowires
Fig. 3. Schematic presentation of the modification steps: (a) metallic nanotube, (b) nanotube filled with Fe, (c) modification with thiol, (d) final attachment of trypsin.
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Fig. 4. Images of nanotubes: (a) TEM – empty CoFe2 O4 , (b) SEM – empty Ag in AAO, (c) STEM – Ag filled with Fe.
Fig. 5. EDS spectrum of AgNT filled with Fe: (a) point 0 0 3 which is chosen to the edge of the structure, (b) point 0 0 4 in the middle of nanotube on STEM image (Fig. 4c).
not influenced by external magnetic field form dispersed powder or they tend to agglomerate due to strong magnetic interaction between each other (Fig. 6a). In Fig. 6b, nanowires standing perpendicular to the bottom of the flask to the magnet which was placed under the flask with the powder sample are presented. IR spectroscopy was used to verify the result of executed functionalization. Fig. 7 presents the spectra obtained before and after modification with amine groups and with thiols (Fig. 8). In both cases modification includes the attachment of the trypsin as a final step.
The analysis of the spectrum (Fig. 7a and b) shows that they are very different. In the basic spectrum (a) it is possible to indicate two signals: 3417 cm−1 and 1611 cm−1 , which can be identified probably to –OH bonds from adsorbed water, from the air, during the drying process [14]. The third significant signal around 500 cm−1 comes from Fe–O bonds which confirm the surface of ferrite nanotubes [15]. In the spectrum received after modification some of the signals described above become more pronounced but also another strong signal at about 1025 cm−1 and 1636 cm−1 clearly appear. These wave numbers may be identified to covalently
Fig. 6. Visualization of magnetic properties: (a) nanowires without influence of external magnetic field, (b) nanowires standing perpendicular to the magnet under the flask.
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Fig. 7. IR spectrum of MnFe2 O4 nanotubes filled with Fe: (a) before, (b) after modification (trypsin attachment).
bonded trypsin and show amide bond which should be expected from the position of this signal. In addition, we can identify small but sharp signals at 2850–2920 cm−1 which belong to C–H bond in –CH2 – group in oleylamine chain and trypsin [16,17]. In all types of the tested ferrite nanotubes the same changes were observed in spectrum after modification, which gives the evidence about trypsin bonded to the nanowires. The comparison of spectra collected in Fig. 8 confirms that they are different in every step of modification. In the basic spectrum (a) there are two signals at 3450 cm−1 and 1650 cm−1 , which belong to –OH bonds from adsorbed water from the air. The rest of the signals around 2900 cm−1 and 1120 cm−1 can be identified to –NO3 − groups from other solvents participating in nanowire synthesis [18]. In the second spectrum (b) after modification with thiols we can observe characteristic sharp signals appearing originally in thiols bonds around 1470 cm−1 and 719 cm−1 [19]. Finally in the last spectrum (c) every earlier signal is present in it with a bit smaller intensity. Similar to the modification with amine groups, here we
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can observe a new signal, not so well defined as the previous one at 1025 cm−1 , which means that trypsin has adsorbed on the surface. There is also a large signal below 750 cm−1 [20]. In this case trypsin does not covalently bond with methyl groups but it appeared on the wire surface via hydrophobic interactions which give that signal. The composition of metallic (Ag) and ferrite (NiFe2 O4 , CoFe2 O4 and MnFe2 O4 ) nanotubes filled with magnetic material – iron inside allowed to create core–shell nanowires. Protein immobilization on the nanowires forced to produce two types of nanotubes modified in two different ways. Firstly, ferrite tubes have an oxidant structure, which is convenient to form monolayers with amine groups. These structures can also be modified with carboxylic groups, which can be investigated in future research. Because amine groups are quite short, there is no need to use any extreme conditions of experiment to attach them on the surface. Additionally, amine groups cause the presence of hydrophilic properties in the structure. Following the amination process, ferrite tubes were connected with trypsin using glurataldehyde as homo-bifunctional linker which is covalently bonded to both sides. A strong signal on IR spectrum confirmed trypsin attachment and the presence of amide bond. This procedure presented a simple method to form amine groups on the surface of the nanowires including protein immobilization which allows direct biofunctionalization without using thin films such as silanes [21]. The usage of linker (glutaraldehyde) allows to attach many others biomolecules which possess functional groups such as primary amines and sulfhydryls. For the metallic nanotubes, a sixteen-carbon thiol chain was used to modified homogeneous Ag surface. Thiols cause hydrophobic properties to the structure. In this method it can be suggested that the reaction time between Ag surface and thiol chains is very important because the longer the interaction lasts, the more thiol chains are standing perpendicular to the surface and the protein may easier adsorb to them. Moreover, the attachment of trypsin to methyl groups of thiol was observed without including linker. Moreover, functionalization proved that protein had influenced thiol, which is confirmed by a small signal on IR spectrum. The signal is not so well pronounced because in this case trypsin did not covalently bond to the methyl groups but just adsorbed on them. Adsorption is possible through hydrophobic interactions between methyl groups and carbon chains included in the trypsin structure. The comparison of two signals after modifications with amine and thiol groups including trypsin attachment indicates that covalently bonded structures show strongest signals in IR spectrum than those which interact through noncovalent adsorption. The usage of a functional group like thiol means that these groups may be used to other modified materials such as Au, Pd, CdS, CdSe, ZnS. Both experiments can be used to produce nanocapsules for antibodies or transporting medicines into sick areas by magnetic means.
4. Conclusion
Fig. 8. Spectrum of AgNT filled with Fe: (1) basic, (2) with thiols, (3) after trypsin attachment.
Preparation of metallic and ferrite nanotubes by wetting chemical deposition followed by thermal crystallization filled with magnetic material forming core–shell nanowires has been obtained. Two methods of functionalization: with amine and thiol groups, were successfully achieved. In addition, trypsin has shown two ways of bonding – covalently with amine groups through crosslinker – glutaraldehyde, and also noncovalent adsorption to methyl groups which ended a long thiol chain. Every step of the nanowire synthesis was verified by SEM, TEM and EDS techniques. The results of the surface modification were observed by IR spectroscopy. All procedures give clear step by step view of the execute process.
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