Biomineralization of Fine Selenium Crystalline ... - ACS Publications

Jul 1, 2009 - Gurinder Kaur, Mohammad Iqbal and Mandeep Singh Bakshi*. Nanotechnology Research Laboratory, College of North Atlantic, Labrador City, ...
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Biomineralization of Fine Selenium Crystalline Rods and Amorphous Spheres Gurinder Kaur,† Mohammad Iqbal,‡ and Mandeep Singh Bakshi*,§ Nanotechnology Research Laboratory, College of North Atlantic, Labrador City, A2 V 2Y1 Newfoundland, Canada, College of North Atlantic, Prince Philip DriVe Campus, St. John’s, A1C 5P7 Newfoundland, Canada, and Department of Chemistry, Acadia UniVersity, Elliot Hall, WolfVille, B4P 2R6, NoVa Scotia Canada ReceiVed: April 21, 2009; ReVised Manuscript ReceiVed: June 12, 2009

A simple aqueous phase method containing a water-soluble carrier protein, bovine serum albumin (BSA), has been presented for the synthesis of well-defined morphologies of nanobiomaterials. BSA has been used as a shape-directing agent to synthesize crystalline Se nanobars (NBs) and amorphous nanospheres in aqueous phase at a relatively low temperature of 85 °C. Na2SeO3 is used as the Se source to achieve nanoselenium following hydrazine reduction. Well-defined multifacet NBs are produced when the amount of Na2SeO3 is at least 6 times greater than that of BSA (on the basis of per residue), while amorphous spheres are formed with nearly a 1:1 ratio. Both morphologies have been fully characterized by field emission scanning electron microscopy (FESEM), high-resolution transmission electron microscopy (HRTEM), energy dispersive X-ray spectroscopy (EDX), X-ray diffraction (XRD), and X-ray photoelectron spectroscopic (XPS) analysis. Results have shown that the shape-directing ability of unfolded BSA helped to achieve the formation of crystalline NBs, while its soft template effect directed the nanosphere formation. 1. Introduction Bionanomaterials are highly important constituents of biocompatible devices with many applications in bioengineering, biomedical imaging, molecular diagnostics, and most importantly a new class of hybrid materials.1 Material properties affect biological outcomes including the half-life of drugs, biocompatibility of implanted devices, and release rates and toxicity of drug carriers.1g Similarly, physical and chemical properties of biomaterials can have a profound impact on cell proliferation and remodeling of tissues.1f A precise shape-controlled synthesis of a biomaterial is possible only if capping biomolecules could selectively control the crystal growth. Anionic phospholipids (PLs) have been found to be excellent capping/stabilizing agents for gold nanoparticles (Au NPs).2 Surprisingly, their zwitterionic homologues (phosphocholines) showed the least shape controlled effects.2b Fine PL-capped Au NPs were then used as model air pollutants to study their effect on the surface activity of semisynthetic pulmonary surfactants.3 More recently, bovine serum albumen (BSA), a water-soluble and highly important carrier protein, showed remarkable shape-controlled effects on PbS nanocrystals with respect to a temperature variation within 40-80 °C.4 The unfolded form of BSA worked effectively in controlling the crystal structure and led to well-defined cubic nanomorphologies in comparison to its native folded state. The exposed hydrophobic domains of unfolded form provided desired surface activity to control the crystal growth. Use of a carrier protein like BSA in a shape-controlled synthesis of bionanomaterials provides a direct opportunity to produce desired biomaterials for devices with applications in bioengineering. Although BSA has been used as a capping/stabilizing agent for different materials,5 precise shape-controlled morphologies are still elusive. We herein report the synthesis of fine crystalline nanobars (NBs) and amorphous spheres of * Corresponding author. E-mail: [email protected]. † College of North Atlantic, Labrador City. ‡ College of North Atlantic, St. John’s. § Acadia University.

selenium (Se) under different experimental conditions using BSA as a shape-directing agent. Selenium is an important inorganic semiconducting material with a large Bohr radius. In addition to its interesting physical properties such as thermoelectric and nonlinear optical responses, high conductivity, and piezoelectric effects,6 Se in appropriate amounts is an essential element for living organisms.7 Se has been shown to prevent cancer in numerous animal model systems when fed at levels exceeding the nutritional requirement.8 Clarke et al. showed cancer chemopreventive efficacy using a Se supplement in humans.9 Protein-selenium bioconjugate nanomaterials are reported to be cytotoxic for tumor cells.10 Se NPs have also been studied for their antioxidant activity.11a Apart from its many useful applications, excess of Se intake causes selenosis in animals and humans.11b Use of BSA in synthesizing the shape-controlled BSA-Se bioconjugate semiconducting nanomaterials are expected to have several advantages oversimple Se nanomaterials as far as their biomedical applications are concerned.11c 2. Experimental Section 2.1. Synthesis of Se NPs. Sodium selenite, hydrazine, and BSA, all 99% pure, were purchased from Aldrich. Double distilled water was used for all preparations. Se nanocrystals were synthesized by a chemical reduction of sodium selenite by hydrazine. In a typical procedure, 10 mL of aqueous BSA (1-10 × 10-4 g/mL) along with 2.5% hydrazine was taken in a round-bottom glass flask. Under constant stirring, sodium selenite (6-25 mM) was added in it. After mixing all the components at room temperature, the reaction mixture was kept in a water thermostat bath (Julabo F 25) at 85 °C for 48 h. The color of the solution changed from colorless to deep orange within 2 h and remained the same for 48 h. Initial pH of the BSA+water solution was 6.7, which increased to 7.8 upon addition of hydrazine. Addition of Na2SeO3 further increased the pH to 9 as a result of the following reaction:

10.1021/jp903685g CCC: $40.75  2009 American Chemical Society Published on Web 07/01/2009

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N2H4(aq) + SeO32-(aq) f Se(s) + N2(g) + 2OH-(aq) + H2O

(1) Within 2 h, pH further rose to 11 due to the denaturation of BSA (at 85 °C) and remained fairly constant for 48 h. The N (normal) to B (basic) transitions, which take place around pH ) 7-9, affect Cys-Cys as well as C-S bonds and cause unfolding at alkaline pH. The samples were purified by spinning the reaction product at 10 000 rpm for 10 min with repeated washing with distilled water. 2.2. Methods. Field Emission Scanning Electron Microscopy (FESEM), Transmission Electron Microscopy (TEM), X-ray Diffraction (XRD), and X-ray Photoelectron Spectroscopy (XPS) Measurements. FESEM analysis was carried out on a Zeiss NVision 40 Dual Beam FIB/SEM instrument. TEM

analysis was done on a JEOL 2010F at an operating voltage of 200 kV. Photomicrographs were obtained in bright field scanning/imaging mode, using a spot size of ∼1 nm and a camera length of 12 cm. Energy dispersive X-ray (EDX) microanalysis was carried out using an Oxford-INCA Atmospheric Ultrathin Window (UTW), and the data was processed using the Oxford INCA Microanalysis Suite, version 4.04. XRD patterns were recorded by using Bruker-AXS D8-GADDS with Tsec ) 480. Samples were prepared on glass slides by putting a concentrated drop of aqueous sample and then dried in vacuum desiccator. The chemical composition was confirmed with the help of XPS measurements. A portion of an aqueous NP solution was placed onto a clean silicon wafer and then it was put into the introduction chamber of the XPS instrument. The liquid was

Figure 1. (a) Low-resolution FESEM image of several bundles of NBs. (b) Magnified image of several multifaceted NBs. (c) Close-up image showing surface-adsorbed BSA and the presence of BSA in between the NBs. Inset, dark-field image showing the BSA coating. (d) HAADF image showing bright patches of adsorbed BSA and a few dark patches were created by the exposure to electron beam. (e) TEM images of a single NB with a selected area electron diffraction (SAED) image (inset) and (f) its EDX spectrum. (g) Line EDX line spectrum across two fused NBs showing emission due to Se.

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Figure 2. (a) Dark-field TEM image of a single bar showing the area in the box used for HRTEM analysis (b). (c) The same NB sensitive to electron beam splits into two pieces and the boxed area used for HRTEM analysis (d). (e) XRD patterns showing only one prominent peak due to predominant growth at {100} planes.

then pumped away. The XPS analyses were carried out with a Kratos Axis Ultra spectrometer using a monochromatic Al KR source (15 mA, 14 kV). Survey and high-resolution analyses were carried out with an analysis area of ∼300 × 700 µm using pass energies of 160 and 20 eV, respectively. Special care was taken to completely remove the uncapped BSA before XPS measurements. 2.3. Protein Assay. Bradford method12 was used to determine the total protein contents in the BSA-NP conjugate suspension. For this purpose, standard BSA (reference) solutions of concentrations 0, 2, 4, 6, 8, and 10 µg/µL were prepared in 100 µL distilled water. 10 µL of each of these solutions was taken in triplicate in different wells of the UV-plate. 1 mg of the dried Se samples (purified and dried at 40 °C) was taken in doublet in the UV-plate. 20 µL of pure water was added to the wells containing the reference (BSA) and 30 µL was added to the

wells with dried samples. After this, 170 µL of the Bradford reagent were mixed in all the wells to get total volume of 200 µL. The absorbance of each solution was measured and from the absorbance values the amount of BSA conjugated to Se NCs in both samples was calculated. 3. Results and Discussion Figure 1a shows the FESEM image of Se NBs synthesized with [Na2SeO3] ) 6 mM in the presence of BSA ) 1 × 10-4 g/mL. A BSA macromolecule contains 607 residues with average molecular weight of 66432 Da.13 Assuming all residues of the same nature, each one will contribute about 109.4 mols, and thus [Na2SeO3/BSA] mole ratio ) 6.7/residue. Bundles of fine multifaceted bars are evident (Figure 1b) with an average aspect ratio of 4.7 ( 1.8 (Supporting Information, Figure S1). Figure 1c indicates (block arrows) the presence of conjugated

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Figure 3. Low- (a) and high-resolution XPS spectra of (b) Se 3d, (c) C 1s, and (d) N 1s (see details in text).

BSA (BSAc) on the surface of NBs. A close up bright-field TEM image (inset) further confirms this. It appears that most of the surface of each NB is covered with BSAc. A high angle annular dark field (HAADF) image (Figure 1d) clearly shows bright surface patches on each NB indicated by block arrows. A dark dotted circle encloses a few spots of BSA-covered areas of the sample that were damaged by the electron beam. A TEM image of a single bar is shown in Figure 1e, which is positioned along the [110] zone as evident from the diffraction image (inset). EDX analysis of this particle further confirms the presence of Se (Figure 1f). A few weak Cu emissions are due to the Cu grid. Se EDX line spectrum is performed across the short axes of two NBs (Figure 1g). A simultaneous emission even from the attached portions of both NBs indicates a slight degree of fusion facilitated by the BSAc. Se has a trigonal arrangement due to spiral chains of Se atoms associated with each other through van der Waals interactions in a hexagonal lattice.14 Such an arrangement provides a unidirectional growth tendency. A thermodynamically stable trigonal (t) Se is expected to favor growth along the 〈100〉 direction and eventually lead to the formation of onedimensional (1D) nanostructures such as NBs. Figure 2 demonstrates the high-resolution transmission electron microscopy (HRTEM) characterization of a single NB. A lattice resolved image of a magnified part of a single NB (Figure 2a) is shown in Figure 2b with lattice spacing of 0.38 nm, which refers to {100} crystal planes of trigonal geometry. Interestingly, the HAADF image is highly sensitive to the electron beam, and it breaks the NB into two pieces, as shown in Figure 2c. A lattice resolved image of a broken part (see the scan area in Figure 2c) gives a lattice spacing of 0.30 nm, which corresponds to {101} crystal planes. Powder pattern XRD (Figure 2e) gives

only one sharp peak with prominent growth along the 〈100〉 crystal planes of trigonal hexagonal geometry of Se. The effective capping ability of BSA helps to attain well-defined rod shape geometries. BSA binds endogenous as well as exogenous substrates in its hydrophobic pockets.15 The overall shape of a BSA macromolecule is oblate ellipsoid in its native state, but denaturation is often followed by a massive “unfolding” of the protein. The secondary structure consists of hydrogen-bonded R-helices and β-sheets, and is called the largescale structure. At 85 °C, BSA is considered to be in its unfolded form because the overall denaturation temperature is usually reported to be close to 60 °C depending on different methodologies and detection techniques.16 Although the exact mechanism is still unclear, the unfolded BSA with predominantly hydrophobic domains might be adsorbed favorably on freshly cleaved Se nucleating centers.10,11,17 Thus, a selective adsorption of the unfolded form of BSA on low atomic density {100} crystal planes will direct the crystal growth on {111} planes and will result in the rod shape formation. The adsorption of BSA on Se NBs is further confirmed from XPS studies. Figure 3a presents a low-resolution spectrum of this sample, while highresolution spectra for Se 3d, C 1s, and N 1s are shown in Figure 3b, c, and d, respectively. Elemental selenium is generally observed between 54.9 and 56.3 eV.18 In our case, Se 3d peak exists in a weak doublet. Deconvolution of it gives two prominent peaks of Se 3d5/2 at 55.8 and 55.0 eV. The later peak refers to the elemental Se, while the former can be due to oxidation. The XPS peak for C 1s at 284.8 eV refers to C-C and C-H functional groups19 of BSA macromolecules. Similarly, N 1s produces a peak at 400.1 eV due to associated amine groups.20 Finally, the amount of BSAc on NBs was determined by following the Bradford method. In order to determine the

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Figure 4. (a) Low-resolution FESEM image of several groups of spheres. (b) Magnified image of a single group containing several spheres. (c) Close-up image showing fused spheres. (d) HAADF image showing a pair of fused spheres along with (e) a line spectrum across them indicating the emission due to Se. (f) TEM image of a single sphere with SAED image (inset) and (g) its EDX spectrum.

mode of association of BSA with growing NBs, samples at regular intervals were drawn from the reaction mixture. The particles of each sample were thoroughly washed with pure water to remove unassociated BSA. Then each sample was carefully dried and used to estimate BSAc (see Experimental Section). The amount of BSAc increases as the reaction proceeds (Figure S2) and then becomes constant within 18 h. It means that NBs continuously grow for 18 h before attaining a limiting growth. Se 1D nanostructures usually take 24 h to grow into well-defined geometries21a which is due to the fact that the least stable allotropes precipitate first, and then slowly develop into a thermodynamically more stable phase.21 Increase in the amount of BSA from 1 to 10 × 10-4 g/mL does not help to further improve the morphology of NBs; instead the shape of the bars becomes deformed (see Figure S3). But a [Na2SeO3/BSA] mole ratio of 2.6 per residue, produced large

spheres along with fine long needles (see Figure S4). We were interested in the nature of large spheres. Interestingly, further decrease in [Na2SeO3/BSA] mole ratio to 1.3 per residue suddenly eliminates most of the NBs or needles leaving behind only large groups of spheres. A low magnification FESEM image of such several groups of spheres is shown in Figure 4a. Size distribution histogram computes an average size of a sphere equal to 346 ( 110 nm (Figure S5). A high-magnification image (Figure 4b) shows several interconnected spheres in a single group. The spheres are in fact fused together sidewise (Figure 4c). In order to further evaluate the mode of fusion, we carefully selected a pair of spheres and got the HAADF image. This image (Figure 4d) fully confirms the fact that the spheres are indeed fused with each other. An EDX line spectrum (Figure 4e) running across the two balls further confirms that the fused part does not contain BSA because full Se emission is visible

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Figure 5. (a,b) HRTEM images of a nanosphere surface to show the absence of crystalline morphology and BSA coating. (c) XRD patterns showing no peaks indicating the amorphous nature.

from the total surface area. Figure 4f shows a TEM image of a single ball. Surprisingly, the diffraction image (inset) of this ball gives no diffraction rings, suggesting the amorphous nature of spheres. EDX spectrum (Figure 4g) further proves that this ball is entirely made up of Se. Hence, the interparticle fusion of such spheres is predominantly caused by their amorphous nature and not because of BSA capping. Figure 5a,b demonstrates two HRTEM images from different angles. No lattice planes were observed in both cases, and there was no sign of associated/capped BSA. Likewise, no diffraction peak were observed in the XRD patterns (Figure 5c), further confirming the presence of amorphous nature. Amorphous Se (a-Se) spheres have already been reported by other groups22 and are believed to be formed as a result of an insufficient amount of capping/ stabilizing agent. The above results clearly indicate that the NB and sphere formation is related to the [Na2SeO3/BSA] mole ratio. The fine NBs shown in Figure 1 are obtained at a mole ratio of 6.7 per residue, or in other words, when the amount of Na2SeO3 is much higher than that of BSA. Large spheres (Figure 5) on the other hand are synthesized with a ratio of 1.3 per residue. At an intermediate ratio of 2.6, both rods and spheres are observed (Figure S4). However, a large increase in the ratio even up to 28 does not help to achieve any more precise shape control; instead long needles of several micrometers become the major reaction product, as observed by other researchers.23 Thus, an appropriate balance between the amounts of both Na2SeO3 and BSA is essential to achieve shape-controlled morphologies. The mechanism of the reaction is considered to follow the following steps: In the first step, SeO3- ions adsorb electrostatically on the surface of unfolded BSA. Hydrazine reduces SeO3- ions into Se nucleating centers, which are simultaneously adsorbed

at the BSA-water interface.11,24 We do not see any significant effect of hydrazine on BSA. UV-visible spectrum shows that the intensity of the tryptophan absorbance around 278 nm slightly increases in the presence of hydrazine (Figure S6), which might be due to some conformational changes in view of an increased pH. At high temperature (85 °C), more SeO3ions will help to grow such nucleating centers by following an autocatalytic process. When the [Na2SeO3/BSA] mole ratio is 6.7, there are more than 6 times SeO3- ions in comparison to electropositive sites per residue (due to ammonium groups) available on BSA. The actual number of electropositive sites must be even far less than this because the unfolded state is considered to be predominantly hydrophobic in nature. Thus, an unfolded state not only helps in achieving the capping/ stabilizing effects but also acts as a soft template where an excess of SeO3- ions will direct the crystal growth in the 〈100〉 direction. Such a mechanism would obviously lead to the NB formation. On the contrary, when the [Na2SeO3/BSA] mole ratio is 1.3, all SeO3- ions may find their way into small Se nucleating centers adsorbed on the unfolded BSA with little probability of further autocatalytic process. Intramolecular dynamics influenced by the conformation changes and changing topographic effects24 can induce Ostwald repining among the nucleating centers and may lead to the formation of large spheres of amorphous nature. Protein adsorption characteristics are generally governed by the surface topography, i.e., curvature,25 and albumen is usually less ordered on large substrates.24 This effect should be more pronounced on a curved surface of a sphere rather than on a NB, hence it will reduce the capping ability of BSA to control the crystal growth in an ordered manner.

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4. Conclusions This study addresses a very significant and important problem of shape-controlled synthesis of ordered bionanomaterials in the presence of proteins. Ordered morphologies are highly important to build biochip devices. The results conclude that BSA works well in controlling the overall geometry of NPs when the [Na2SeO3/BSA] mole ratio is ∼6. At this mole ratio, denatured BSA becomes even more hydrophobic as a result of the neutralization of oppositely charged sites by SeO3- ions. A predominantly hydrophobic BSA is a better shape-directing agent. However, at a too high mole ratio of 28, BSA proves to be a poor capping agent because of the presence of too many nucleating centers whose growth cannot be simultaneously controlled by BSA macromolecules. On the other hand, when the mole ratio is ∼1, then unfolded BSA macromolecule works as a soft template by accommodating maximum nucleating centers on it and thereby facilitating the Ostwald ripening. Therefore, in order to observe the best shape directing effect of BSA, the following features have to be taken into consideration: (a) BSA should be in the unfolded and predominantly hydrophobic state. (b) The precursor concentration should be greater than that of BSA so that the growing nucleating centers can be properly stabilized. (c) Too many nucleating centers cannot be simultaneously stabilized by BSA because of its time-dependent surface adsorption. Acknowledgment. The authors would like to extend sincere thanks to Dr. Richard Sawyer at CNA, Lab West, for arranging financial assistance for the work. We thankfully acknowledge the help rendered by Julia Huang and Carmen Andrei at the Canadian Centre for Electron Microscopy, McMaster University. Supporting Information Available: Size distribution histograms, SEM images, other information is available free of charge via the Internet at http://pubs.acs.org. References and Notes (1) (a) Taft, B. J.; Lazareck, A. D.; Withey, G. D.; Yin, A.; Xu, J. M.; Kelley, S. O. J. Am. Chem. Soc. 2004, 126, 12750. (b) Li, W.; Gao, C. Langmuir 2007, 23, 4575. (c) Gao, C.; Muthukrishnan, S.; Li, W.; Yuan, J.; Xu, Y.; Muller, A. H. E. Macromolecules 2007, 40, 1803. (d) Eggenberger, K.; Merkulov, A.; Darbandi, M.; Nann, T.; Nick, P. Bioconjugate Chem. 2007, 18, 1879. (e) Wang, Y.; Tang, Z.; Tan, S.; Kotov, N. A. Nano Lett. 2005, 5, 243. (f) Langer, R.; Tirrel, D. A. Nature 2004, 428, 487. (g) Mooney, D. J.; Mikos, A. G. Sci. Am. 1995, 280, 60. (2) (a) Bakshi, M. S.; Possmayer, F.; Petersen, N. O. Chem. Mater. 2007, 19, 1257. (b) Bakshi, M. S.; Possmayer, F.; Petersen, N. O. J. Phys. Chem. C 2007, 111, 14113. (c) Bakshi, M. S.; Kaur, G.; Thakur, P.; Banipal, T. S.; Possmayer, F.; Petersen, N. O. J. Phys. Chem. C 2007, 111, 5932. (d) Meister, A.; Drescher, S.; Mey, I.; Wahab, M.; Graf, G.; Garamus, V. M.; Hause, G.; Mogel, H. J.; Janshoff, A.; Dobner, B.; Blume, A. J. Phys. Chem. B 2008, 112, 4506. (3) (a) Bakshi, M. S.; Zhao, L.; Smith, R.; Possmayer, F.; Petersen, N. O. Biophys. J. 2008, 94, 855. (b) Nakahara, H.; Lee, S.; Sugihara, G.; Chang, C.-H.; Shibata, O. Langmuir 2008, 24, 3370. (4) Bakshi, M. S.; Thakur, P.; Kaur, G.; Kaur, H.; Banipal, T. S.; Possmayer, F.; Petersen, N. O. AdV. Funct. Mater. 2009, 19, 1451.

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