selinium nanoparticles

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, andDepartment 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 usedas a shape-directing agent to synthesize crystalline Se nanobars (NBs) and amorphous nanospheres in aqueousphase at a relatively low temperature of 85 °C. Na2SeO3 is used as the Se source to achieve nanoseleniumfollowing hydrazine reduction. Well-defined multifacet NBs are produced when the amount of Na2SeO3 is atleast 6 times greater than that of BSA (on the basis of per residue), while amorphous spheres are formed withnearly a 1:1 ratio. Both morphologies have been fully characterized by field emission scanning electronmicroscopy (FESEM), high-resolution transmission electron microscopy (HRTEM), energy dispersive X-rayspectroscopy (EDX), X-ray diffraction (XRD), and X-ray photoelectron spectroscopic (XPS) analysis. Resultshave shown that the shape-directing ability of unfolded BSA helped to achieve the formation of crystallineNBs, while its soft template effect directed the nanosphere formation.

1. Introduction

Bionanomaterials are highly important constituents of bio-compatible devices with many applications in bioengineering,biomedical imaging, molecular diagnostics, and most impor-tantly a new class of hybrid materials.1 Material properties affectbiological outcomes including the half-life of drugs, biocom-patibility of implanted devices, and release rates and toxicityof drug carriers.1g Similarly, physical and chemical propertiesof biomaterials can have a profound impact on cell proliferationand remodeling of tissues.1f A precise shape-controlled synthesisof a biomaterial is possible only if capping biomolecules couldselectively control the crystal growth. Anionic phospholipids(PLs) have been found to be excellent capping/stabilizing agentsfor gold nanoparticles (Au NPs).2 Surprisingly, their zwitterionichomologues (phosphocholines) showed the least shape con-trolled effects.2b Fine PL-capped Au NPs were then used asmodel air pollutants to study their effect on the surface activityof semisynthetic pulmonary surfactants.3 More recently, bovineserum albumen (BSA), a water-soluble and highly importantcarrier protein, showed remarkable shape-controlled effects onPbS nanocrystals with respect to a temperature variation within40-80 °C.4 The unfolded form of BSA worked effectively incontrolling the crystal structure and led to well-defined cubicnanomorphologies in comparison to its native folded state. Theexposed hydrophobic domains of unfolded form provideddesired surface activity to control the crystal growth. Use of acarrier protein like BSA in a shape-controlled synthesis ofbionanomaterials provides a direct opportunity to producedesired biomaterials for devices with applications in bioengi-neering. Although BSA has been used as a capping/stabilizingagent for different materials,5 precise shape-controlled mor-phologies are still elusive. We herein report the synthesis offine crystalline nanobars (NBs) and amorphous spheres of

selenium (Se) under different experimental conditions usingBSA as a shape-directing agent.

Selenium is an important inorganic semiconducting materialwith a large Bohr radius. In addition to its interesting physicalproperties such as thermoelectric and nonlinear optical re-sponses, high conductivity, and piezoelectric effects,6 Se inappropriate amounts is an essential element for living organ-isms.7 Se has been shown to prevent cancer in numerous animalmodel systems when fed at levels exceeding the nutritionalrequirement.8 Clarke et al. showed cancer chemopreventiveefficacy using a Se supplement in humans.9 Protein-seleniumbioconjugate nanomaterials are reported to be cytotoxic fortumor cells.10 Se NPs have also been studied for their antioxidantactivity.11a Apart from its many useful applications, excess ofSe intake causes selenosis in animals and humans.11b Use ofBSA in synthesizing the shape-controlled BSA-Se bioconjugatesemiconducting nanomaterials are expected to have severaladvantages oversimple Se nanomaterials as far as their biomedi-cal applications are concerned.11c

2. Experimental Section

2.1. Synthesis of Se NPs. Sodium selenite, hydrazine, andBSA, all 99% pure, were purchased from Aldrich. Doubledistilled water was used for all preparations.

Se nanocrystals were synthesized by a chemical reductionof sodium selenite by hydrazine. In a typical procedure, 10 mLof aqueous BSA (1-10 × 10-4 g/mL) along with 2.5%hydrazine was taken in a round-bottom glass flask. Underconstant stirring, sodium selenite (6-25 mM) was added in it.After mixing all the components at room temperature, thereaction mixture was kept in a water thermostat bath (Julabo F25) at 85 °C for 48 h. The color of the solution changed fromcolorless to deep orange within 2 h and remained the same for48 h. Initial pH of the BSA+water solution was 6.7, whichincreased to 7.8 upon addition of hydrazine. Addition ofNa2SeO3 further increased the pH to 9 as a result of thefollowing reaction:

* Corresponding author. E-mail: [email protected].† College of North Atlantic, Labrador City.‡ College of North Atlantic, St. John’s.§ Acadia University.

J. Phys. Chem. C 2009, 113, 13670–1367613670

10.1021/jp903685g CCC: $40.75 2009 American Chemical SocietyPublished on Web 07/01/2009

Within 2 h, pH further rose to 11 due to the denaturation ofBSA (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 causeunfolding at alkaline pH. The samples were purified by spinningthe reaction product at 10 000 rpm for 10 min with repeatedwashing with distilled water.

2.2. Methods. Field Emission Scanning Electron Micros-copy (FESEM), Transmission Electron Microscopy (TEM),X-ray Diffraction (XRD), and X-ray Photoelectron Spectros-copy (XPS) Measurements. FESEM analysis was carried outon a Zeiss NVision 40 Dual Beam FIB/SEM instrument. TEM

analysis was done on a JEOL 2010F at an operating voltage of200 kV. Photomicrographs were obtained in bright fieldscanning/imaging mode, using a spot size of ∼1 nm and acamera length of 12 cm. Energy dispersive X-ray (EDX)microanalysis was carried out using an Oxford-INCA Atmo-spheric Ultrathin Window (UTW), and the data was processedusing the Oxford INCA Microanalysis Suite, version 4.04. XRDpatterns were recorded by using Bruker-AXS D8-GADDS withTsec ) 480. Samples were prepared on glass slides by putting aconcentrated drop of aqueous sample and then dried in vacuumdesiccator. The chemical composition was confirmed with thehelp of XPS measurements. A portion of an aqueous NP solutionwas placed onto a clean silicon wafer and then it was put intothe 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 imageshowing surface-adsorbed BSA and the presence of BSA in between the NBs. Inset, dark-field image showing the BSA coating. (d) HAADF imageshowing bright patches of adsorbed BSA and a few dark patches were created by the exposure to electron beam. (e) TEM images of a single NBwith a selected area electron diffraction (SAED) image (inset) and (f) its EDX spectrum. (g) Line EDX line spectrum across two fused NBsshowing emission due to Se.

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

(1)

Biomineralization of Se NBs and Amorphous Spheres J. Phys. Chem. C, Vol. 113, No. 31, 2009 13671

then pumped away. The XPS analyses were carried out with aKratos Axis Ultra spectrometer using a monochromatic Al KRsource (15 mA, 14 kV). Survey and high-resolution analyseswere carried out with an analysis area of ∼300 × 700 µm usingpass energies of 160 and 20 eV, respectively. Special care wastaken to completely remove the uncapped BSA before XPSmeasurements.

2.3. Protein Assay. Bradford method12 was used to determinethe total protein contents in the BSA-NP conjugate suspension.For this purpose, standard BSA (reference) solutions of con-centrations 0, 2, 4, 6, 8, and 10 µg/µL were prepared in 100 µLdistilled water. 10 µL of each of these solutions was taken intriplicate in different wells of the UV-plate. 1 mg of the driedSe samples (purified and dried at 40 °C) was taken in doubletin the UV-plate. 20 µL of pure water was added to the wellscontaining the reference (BSA) and 30 µL was added to the

wells with dried samples. After this, 170 µL of the Bradfordreagent were mixed in all the wells to get total volume of 200µL. The absorbance of each solution was measured and fromthe absorbance values the amount of BSA conjugated to Se NCsin both samples was calculated.

3. Results and Discussion

Figure 1a shows the FESEM image of Se NBs synthesizedwith [Na2SeO3] ) 6 mM in the presence of BSA ) 1 × 10-4

g/mL. A BSA macromolecule contains 607 residues withaverage molecular weight of 66432 Da.13 Assuming all residuesof the same nature, each one will contribute about 109.4 mols,and thus [Na2SeO3/BSA] mole ratio ) 6.7/residue. Bundles offine multifaceted bars are evident (Figure 1b) with an averageaspect ratio of 4.7 ( 1.8 (Supporting Information, Figure S1).Figure 1c indicates (block arrows) the presence of conjugated

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 toelectron beam splits into two pieces and the boxed area used for HRTEM analysis (d). (e) XRD patterns showing only one prominent peak due topredominant growth at {100} planes.

13672 J. Phys. Chem. C, Vol. 113, No. 31, 2009 Kaur et al.

BSA (BSAc) on the surface of NBs. A close up bright-fieldTEM image (inset) further confirms this. It appears that mostof the surface of each NB is covered with BSAc. A high angleannular dark field (HAADF) image (Figure 1d) clearly showsbright surface patches on each NB indicated by block arrows.A dark dotted circle encloses a few spots of BSA-covered areasof the sample that were damaged by the electron beam. A TEMimage of a single bar is shown in Figure 1e, which is positionedalong the [110] zone as evident from the diffraction image(inset). EDX analysis of this particle further confirms thepresence of Se (Figure 1f). A few weak Cu emissions are due tothe Cu grid. Se EDX line spectrum is performed across the shortaxes of two NBs (Figure 1g). A simultaneous emission evenfrom the attached portions of both NBs indicates a slight degreeof fusion facilitated by the BSAc.

Se has a trigonal arrangement due to spiral chains of Se atomsassociated with each other through van der Waals interactionsin a hexagonal lattice.14 Such an arrangement provides aunidirectional growth tendency. A thermodynamically stabletrigonal (t) Se is expected to favor growth along the ⟨100⟩direction and eventually lead to the formation of one-dimensional (1D) nanostructures such as NBs. Figure 2 dem-onstrates the high-resolution transmission electron microscopy(HRTEM) characterization of a single NB. A lattice resolvedimage of a magnified part of a single NB (Figure 2a) is shownin Figure 2b with lattice spacing of 0.38 nm, which refers to{100} crystal planes of trigonal geometry. Interestingly, theHAADF image is highly sensitive to the electron beam, and itbreaks the NB into two pieces, as shown in Figure 2c. A latticeresolved image of a broken part (see the scan area in Figure2c) 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. Theeffective capping ability of BSA helps to attain well-definedrod shape geometries. BSA binds endogenous as well asexogenous substrates in its hydrophobic pockets.15 The overallshape of a BSA macromolecule is oblate ellipsoid in its nativestate, but denaturation is often followed by a massive “unfold-ing” of the protein. The secondary structure consists ofhydrogen-bonded R-helices and �-sheets, and is called the large-scale structure. At 85 °C, BSA is considered to be in its unfoldedform because the overall denaturation temperature is usuallyreported to be close to 60 °C depending on different methodolo-gies and detection techniques.16 Although the exact mechanismis still unclear, the unfolded BSA with predominantly hydro-phobic domains might be adsorbed favorably on freshly cleavedSe nucleating centers.10,11,17 Thus, a selective adsorption of theunfolded form of BSA on low atomic density {100} crystalplanes will direct the crystal growth on {111} planes and willresult in the rod shape formation. The adsorption of BSA onSe NBs is further confirmed from XPS studies. Figure 3apresents a low-resolution spectrum of this sample, while high-resolution spectra for Se 3d, C 1s, and N 1s are shown in Figure3b, c, and d, respectively. Elemental selenium is generallyobserved between 54.9 and 56.3 eV.18 In our case, Se 3d peakexists in a weak doublet. Deconvolution of it gives twoprominent peaks of Se 3d5/2 at 55.8 and 55.0 eV. The laterpeak refers to the elemental Se, while the former can be due tooxidation. The XPS peak for C 1s at 284.8 eV refers to C-Cand C-H functional groups19 of BSA macromolecules. Simi-larly, N 1s produces a peak at 400.1 eV due to associated aminegroups.20 Finally, the amount of BSAc on NBs was determinedby following the Bradford method. In order to determine the

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


mode of association of BSA with growing NBs, samples atregular intervals were drawn from the reaction mixture. Theparticles of each sample were thoroughly washed with purewater to remove unassociated BSA. Then each sample wascarefully dried and used to estimate BSAc (see ExperimentalSection). The amount of BSAc increases as the reaction proceeds(Figure S2) and then becomes constant within 18 h. It meansthat NBs continuously grow for 18 h before attaining a limitinggrowth. Se 1D nanostructures usually take 24 h to grow intowell-defined geometries21a which is due to the fact that the leaststable allotropes precipitate first, and then slowly develop intoa thermodynamically more stable phase.21

Increase in the amount of BSA from 1 to 10 × 10-4 g/mLdoes not help to further improve the morphology of NBs; insteadthe 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 wereinterested in the nature of large spheres. Interestingly, furtherdecrease in [Na2SeO3/BSA] mole ratio to 1.3 per residuesuddenly eliminates most of the NBs or needles leaving behindonly large groups of spheres. A low magnification FESEMimage of such several groups of spheres is shown in Figure 4a.Size distribution histogram computes an average size of a sphereequal to 346 ( 110 nm (Figure S5). A high-magnificationimage (Figure 4b) shows several interconnected spheres in asingle group. The spheres are in fact fused together sidewise(Figure 4c). In order to further evaluate the mode of fusion, wecarefully selected a pair of spheres and got the HAADF image.This image (Figure 4d) fully confirms the fact that the spheresare indeed fused with each other. An EDX line spectrum (Figure4e) running across the two balls further confirms that the fusedpart does not contain BSA because full Se emission is visible

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 indicatingthe emission due to Se. (f) TEM image of a single sphere with SAED image (inset) and (g) its EDX spectrum.


from the total surface area. Figure 4f shows a TEM image of asingle ball. Surprisingly, the diffraction image (inset) of thisball gives no diffraction rings, suggesting the amorphous natureof spheres. EDX spectrum (Figure 4g) further proves that thisball is entirely made up of Se. Hence, the interparticle fusionof such spheres is predominantly caused by their amorphousnature and not because of BSA capping. Figure 5a,b demon-strates two HRTEM images from different angles. No latticeplanes were observed in both cases, and there was no sign ofassociated/capped BSA. Likewise, no diffraction peak wereobserved in the XRD patterns (Figure 5c), further confirmingthe presence of amorphous nature. Amorphous Se (a-Se) sphereshave already been reported by other groups22 and are believedto be formed as a result of an insufficient amount of capping/stabilizing agent.

The above results clearly indicate that the NB and sphereformation is related to the [Na2SeO3/BSA] mole ratio. The fineNBs shown in Figure 1 are obtained at a mole ratio of 6.7 perresidue, or in other words, when the amount of Na2SeO3 is muchhigher than that of BSA. Large spheres (Figure 5) on the otherhand are synthesized with a ratio of 1.3 per residue. At anintermediate ratio of 2.6, both rods and spheres are observed(Figure S4). However, a large increase in the ratio even up to28 does not help to achieve any more precise shape control;instead long needles of several micrometers become the majorreaction product, as observed by other researchers.23 Thus, anappropriate balance between the amounts of both Na2SeO3 andBSA is essential to achieve shape-controlled morphologies. Themechanism of the reaction is considered to follow the followingsteps: In the first step, SeO3

- ions adsorb electrostatically onthe surface of unfolded BSA. Hydrazine reduces SeO3

- ionsinto Se nucleating centers, which are simultaneously adsorbed

at the BSA-water interface.11,24 We do not see any significanteffect of hydrazine on BSA. UV-visible spectrum shows thatthe intensity of the tryptophan absorbance around 278 nmslightly increases in the presence of hydrazine (Figure S6),which might be due to some conformational changes in viewof an increased pH. At high temperature (85 °C), more SeO3

-

ions will help to grow such nucleating centers by following anautocatalytic process. When the [Na2SeO3/BSA] mole ratio is6.7, there are more than 6 times SeO3

- ions in comparison toelectropositive sites per residue (due to ammonium groups)available on BSA. The actual number of electropositive sitesmust be even far less than this because the unfolded state isconsidered 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 anexcess of SeO3

- ions will direct the crystal growth in the ⟨100⟩direction. Such a mechanism would obviously lead to the NBformation. On the contrary, when the [Na2SeO3/BSA] mole ratiois 1.3, all SeO3

- ions may find their way into small Se nucleatingcenters adsorbed on the unfolded BSA with little probability offurther autocatalytic process. Intramolecular dynamics influencedby the conformation changes and changing topographic effects24

can induce Ostwald repining among the nucleating centers andmay lead to the formation of large spheres of amorphous nature.Protein adsorption characteristics are generally governed by thesurface topography, i.e., curvature,25 and albumen is usually lessordered on large substrates.24 This effect should be morepronounced on a curved surface of a sphere rather than on aNB, hence it will reduce the capping ability of BSA to controlthe crystal growth in an ordered manner.

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


4. Conclusions

This study addresses a very significant and important problemof shape-controlled synthesis of ordered bionanomaterials in thepresence of proteins. Ordered morphologies are highly importantto build biochip devices. The results conclude that BSA workswell in controlling the overall geometry of NPs when the[Na2SeO3/BSA] mole ratio is ∼6. At this mole ratio, denaturedBSA becomes even more hydrophobic as a result of theneutralization of oppositely charged sites by SeO3

- ions. Apredominantly hydrophobic BSA is a better shape-directingagent. However, at a too high mole ratio of 28, BSA proves tobe a poor capping agent because of the presence of too manynucleating centers whose growth cannot be simultaneouslycontrolled by BSA macromolecules. On the other hand, whenthe mole ratio is ∼1, then unfolded BSA macromolecule worksas a soft template by accommodating maximum nucleatingcenters on it and thereby facilitating the Ostwald ripening.Therefore, in order to observe the best shape directing effect ofBSA, the following features have to be taken into consideration:(a) BSA should be in the unfolded and predominantly hydro-phobic state. (b) The precursor concentration should be greaterthan that of BSA so that the growing nucleating centers can beproperly stabilized. (c) Too many nucleating centers cannot besimultaneously stabilized by BSA because of its time-dependentsurface adsorption.

Acknowledgment. The authors would like to extend sincerethanks to Dr. Richard Sawyer at CNA, Lab West, for arrangingfinancial assistance for the work. We thankfully acknowledgethe help rendered by Julia Huang and Carmen Andrei at theCanadian Centre for Electron Microscopy, McMaster University.

Supporting Information Available: Size distribution his-tograms, SEM images, other information is available free ofcharge via the Internet at http://pubs.acs.org.

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selinium nanoparticles

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