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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: ms_bakshi@yahoo.com.
Acadia University.
(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
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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Kaur et al.
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 m 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 g/L were prepared in 100 L
distilled water. 10 L 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 L of pure water was added to the wells
containing the reference (BSA) and 30 L was added to 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).
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 -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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Kaur et al.
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.
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
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.
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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.
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