Electronic Supplementary Information
Bismuth Ferrite Nanoparticles Induced Hydrogel Formation in Human Serum Albumin
GarimaThakur, a* Prashanthi Kovur,a Roger M. Leblancb and Thomas Thundatb
a Department
of Chemical and Materials Engineering, University of Alberta, Edmonton,
Canada
b Department
of Chemistry, 1301 Memorial Drive, University of Miami, Florida, USA
Materials: Human serum albumin (30%, Sigma Aldrich), sodium chloride, sodium phosphate,
2-methoxyethanol,Thioflavin T, bismuth nitrate, iron nitrate and hydrochloric acid were
purchased from Sigma Aldrich, Oaksville, ON, Canada . All chemicals were analytical grade,
and used without further purification. Deionized water with resistivity of 18 MΩ∙cm was used for
preparation of solutions. BiFeO3 nanoparticles - with size 24-30 nm and 99.9% phase purity were
purchased from nanograde LIc, Switzerland.
Methods:
Transmission Electron Microscopy (TEM): Briefly, copper grids (carbon coated 400 mesh:
TED Pella Inc., Redding, CA) were inverted on an 5 μL aliquot of sample for 2 min. Excess
sample was removed and the grids immediately placed briefly on a droplet of distilled water.
Grids requiring the negative stain were then placed on droplets of 2% uranyl acetate solution for
2 min. Excess stain was removed and the grid was allowed to dry thoroughly. For unstained
grids, the excess water was removed and the dried grids examined on a Philips electron
microscope Morgagni 268 (FEI, Hillsbrough, OR) operating at 80 kV and images were collected
using a GatanOrius CCD camera at Biology Department, University of Alberta .
Scanning Electron Microscopy (SEM): Vega-3 Scanning Electron Microscope for SEM
(Tescan, USA) was used in High vacuum mode (pressure< 3 x 10-3 Pa) with accelerating voltage
between 0.2 to 30 keV for SEM imaging at the Alberta Centre for Surface Engineering and
Science (ACSES), University of Alberta. Gel was rinsed with deionized water and mounted on
SEM Stubs before imaging.
Dynamic Light scattering (DLS): DLS experiments were conducted on commercial apparatus
ALV/CGS-3 compact Goniometer system (ALV, GmbH, Germany) at an angle of 900. JDS
Uniphase 22mW He–Ne laser, operating at wavelength 632.8 nm was interfaced with a ALV5000/EPP multi-tau digital correlator with 288 channels and a ALV/LSE-5003 light scattering
electronics unit for stepper motor drive and limit switch control. Autocorrelation functions were
collected 3 times for each solution and they were analyzed by the cumulants method and the
CONTIN routine using the software provided by the manufacturer.
Infrared Spectroscopy (IR): NEXUS 670 FTIR (Thermo Nicolet, Madison, WI, USA) was
used for ATR-IR analysis of HSA samples equipped with ZnSe crystal. ATR-IR for the gel was
taken after putting the piece of hydrogel on the ZnSe crystal and dried under nitrogen until the
gel was dried without visible water on the crystal.
UV-vis and Fluorescence Spectroscopy: Fluorescence measurements were carried out on Cary
Eclipse- Varian Fluorescence Spectrometer (Agilent Technologies, Santa Clara, CA, USA) with
Xenon flash lamp. The emission and excitation slit width was set at 5 nm each. Cary 50 scan
UV-vis spectrophotometer was used for measuring UV-vis spectra (Agilent Technologies, Santa
Clara, CA, USA). Quartz cuvette with pathlength of 1 cm was used for all measurements.
Circular Dichroism (CD) Spectropolarimetery: The CD spectra were measured on an OLIS
DSM 17 Circular Dichroism instrument (OLIS Inc. Bogart, Georgia, USA). Quartz cell of 0.02
cm path length was used to contain sample, and the spectra were recorded in the far-UV region
with wavelength between 190 and 260 nm. The spectrum was recorded with five scan
accumulations. The solution for CD analysis was prepared by taking 20 µL of the incubated
sample diluted in 800 uL of PBS buffer (pH 7.4). For the preparation of the gel sample a piece of
gel was sonicated in 800 uL of PBS buffer for 10 min.
X-Ray Photoelectron Spectroscopy (XPS) : The XPS measurements were performed on AXIS
165 spectrometer (Kratos Analytical) at the Alberta Centre for Surface Engineering and Science
(ACSES), University of Alberta. The base pressure in the analytical chamber was lower than 4 x
10-8 Pa. Monochromatic Al Kα source (hν = 1486.6 eV) was used at a power of 210 W. The
analysis spot was 300 x700 µm. The resolution of the instrument is 0.55 eV for Ag 3d and 0.70
eV for Au 4f peaks. The survey scans were collected for binding energy spanning from 1100 eV
to 0 with analyzer pass energy of 160 eV and a step of 0.4 eV. For the high-resolution spectra the
pass-energy was 20 eV with a step of 0.1 eV. Electron flood neutralizer was applied to
compensate sample charging.
Presence of Fe3+ state in BFO NPs was revealed by XPS spectra (Fig. S3). Due to the very low
concentration of NPs (1mg/mL) in the hydrogel sample, NPs were not observed using XPS for
the hydrogel sample (Fig. S4). Fe3+ ions in aqueous solutions are acidic and hydrolysis can lead
to relatively insoluble polymeric hydroxo/oxo Fe3+ precipitates.1-2 It is known that the pI of HSA
is 4.7 and at pH 7.4 HSA carries negative charge.3 There is a possibility of controlling
mineralization or periodic arrangement of NPs by protein resulting in formation of HSA complex
with Fe3+ or Bi3+.1,2,4 However, structure of NPs is a key factor that is playing a role in the
formation of hydrogel. NPs suspended in an organic solvent are present as cluster of inorganic
solid approaching towards the property of bulk material and behave as nucleation centres
creating local oversaturation and crystallization occurring in non-specific manner.5 Moreover, it
has been reported by Gosh et al. that simple biological systems are able to crystallize inorganic
NPs in defined form and distances.6 Furthermore, in present study it was observed that NPs
might be oriented in a specific fashion in the fibrillar structures. It is worthwhile to mention that
organic solvent was essential for inducing gel formation. It was observed that NPs with similar
size in absence of organic solvent did not show any gel formation (sample 3 and 4). However,
the role of organic solvent has been clearly demonstrated in gel formation.
Preparation of solutions: Phosphate buffer saline was prepared in deionized water having 20
mM sodium phosphate and 0.15 M NaCl (pH 7.4) and pH was adjusted using HCl (0.1M).
Sample 1, 2, 3 and 4 were prepared in PBS buffer as described in manuscript.
Synthesis of Nanoparticles: Bismuth iron oxide nanoparticles were prepared by sol-gel route.
Bismuth nitrate and iron nitrate in stoichiometric proportions were dissolved in 2methoxyethanol. Ethanolamine was added to control the pH value of the solution to be around 4.
The solution was under constant magnetic stirring for 2 h at room temperature. All the chemicals
were of analytical grade and used as received without further purification. TEM analysis and
UV-vis spectroscopy (Fig. S10) was used to characterize the particles.
Particles were
precipitated out using toluene by centrifugation and were dried to get powdered form. BiFeO3
nanoparticles - with size 24-30 nm and 99.9% phase purity were purchased from Nanograde LIC,
Switzerland. Nanoparticles were annealed at 600 0C for 1h. Freshly prepared nanoparticles and
commercially purchased nanoparticles gave the same results.
Thioflavin T assay: 1 mM Th T stock solution was prepared in PBS buffer (pH 7.4) and diluted
to 10 μM. This solution was used for analyzing the fibrillation process before and after
incubation of HSA solutions for 23 h. It is worthwhile to mention that the intensity of emission
band at approximately 482 nm increases with increase in fibrillation process. Intensity of
fluorescence for the initial samples without incubation was little. However, there was increase in
intensity of fluorescence for sample 1, 3 and 4 as seen in Figure S8.
diameter of dispersed nanoparticles
b
700
C
average diameter( 9 nm)
600
500
400
Counts
a
300
200
100
0
5
10
15
20
25
Diameter, nm
Fig. S1 TEM image of dispersed (a) and suspended (b) nanoparticles. (c) Histogram of size of
nanoparticles when dispersed in 2-methoxyethanol.
1700
Fe 2p3/2
Intensity (cps)
75000
Binding Energy
710 eV
806 eV
532 eV
1500
1400
satellite peaks
1300
158 eV
Bi 4f
Fe 2p1/2
1600
Intensity (Cps)
Element
Fe 2p
100000 Bi 4p
O 1s
1200
740
730
720
710
Binding Energy (eV)
50000
O 1s
OKLL
Fe 2p
25000
Bi 4p
Bi 4d
0
1200
1000
800
600
400
Bi4f
200
Binding energy (eV)
Fig. S2 XPS spectra for the pure nanoparticle sample.
0
700
Intensity (Cps)
Element
120000 C 1s
O 1s
N 1s
100000
Na 1s
Cl 2s
Binding energy
284 eV
532 eV
399 eV O1s
1070 eV
270 eV
80000
60000
40000
Na 1s
Na KLL C1s
O KLL
N1s
20000
0
1200
1000
800
600
400
200
Binding energy (eV)
Fig. S3 XPS spectra for the hydrogel sample.
0
Size distribution of nanoparticles (sample 1)
8
nanoparticles inside the fibrils
7
6
counts
5
4
3
2
1
0
6
8
10
12
14
16
18
20
22
24
Size, nm
Fig.S4 Size distribution of naoparticles embedded inside the fibrils in sample 1 after incubation
for 23 h.
Fig. S5 SEM images of the gel rinsed in deionized water.
Size of oligomers
Nanoparticles dispersed in water ( sample 3)
7
6
Counts
5
4
3
2
1
0
50
100
150
200
250
Size, nm
Fig. S6 Size distribution of spherical structures formed in sample 3 after incubation for 23 h.
Size of oligomers
Nanoparticles suspended in water (sample 4)
16
14
12
Counts
10
8
6
4
2
0
20
25
30
35
40
45
50
size, nm
Fig. S7 Size distribution of spherical structures formed in sample 4 after incubation for 23 h.
ThT
ThT + pure HSA solution (initial)
ThT + sample 4 (initial)
ThT + sample 3 (initial)
ThT + sample 4 (incubated)
ThT + sample 3 (incubated)
ThT + sample 1 (initial)
ThT +sample1 (incubated)
35
30
Intensity (a.u.)
25
20
15
10
5
0
450
500
550
600
650
Wavelength, nm
Fig. S8 Comparison of ThT fluorescence before and after incubating different samples for 23 h.
Excitation wavelength was set at 440 nm with excitation and emission slit width of 5 nm each.
Bi FeO3 nanoparticles in suspended in 2-methoxyethanol
-Nanoparticles dispersed in 2-methoxyethanol
A
B
0.8
% of particles
0.8
% of particles
380 nm
1.0
22 nm
1.0
0.6
0.4
0.6
0.4
0.2
0.2
0.0
0.0
0
50
0
100
200
400
Radius, nm
nanoparticles dispersed in water
D
90 nm
1000
1003 nm
1.0
0.8
0.8
% of particles
% of nanoparticles
800
nanoparticles suspended in water
C
1.0
600
Radius, nm
0.6
0.4
0.6
0.4
0.2
0.2
0.0
0.0
0
100
200
300
Radius, nm
400
500
0
2000
4000
6000
8000
10000
Radius, nm
Fig. S9 Hydrodynamic radius of different set of BiFeO3 nanoparticles: A) dispersed in 2-methoxyethanol
(sample 1); B) suspended in 2-methoxyethanol (sample 2); C) dispersed in water (sample 3); D)
suspended in water (sample 4).
1.0
D
Absorbance
0.8
0.6
480
0.4
0.2
0.0
400
500
600
wavelength, nm
Fig. S10 Absorption spectrum of bismuth ferrite nanoparticles.
Table S1 Gel formation related to hydrodynamic radius of nanoparticles
BiFeO3 Nanoparticles
Dispersed in 2-methoxyethanol
(sample 1)
Suspended in 2-methoxyethanol
(sample 2)
Dispersed in water (sample 3)
Suspended in water (sample 4)
Radius (DLS
measurements)
22 or 40 nm
Gel formation
No
250 or 400 nm
Yes
90 nm
1031 nm
No
No
Table S2 ATR-IR of various HSA samples in presence of nanoparticles
sample
Initial HSA samples in
presence of nanoparticles
(without incubation) -sample
1, 2, 3 and 4
Initial HSA samples in absence
of nanoparticles (without
incubation)
HSA sample in presence of
nanoparticles dispersed in 2methoxyethanol (incubated for
23 h) -sample 1
HSA sample in presence of
nanoparticles dispersed in
water (incubated for 23 h) sample 3
HSA sample in presence of
nanoparticles suspended in
water (incubated for 23 h)sample 4
HSA sample in presence of
nanoparticles suspended in 2methoxyethanol (incubated for
23 h) (gel)-sample 2
Amide I (cm-1) (major
structure)
1652, 1630 (α-helix, random
coil)
Amide II (cm-1)
1655, 1632 (α-helix, random
coil)
1556, 1521
1650, 1624 (α-helix, random
coil )
1548, 1528, 1511
1655, 1630 (α-helix, random
coil)
1547, 1528, 1515
1645, 1622 (denatured protein)
1547, 1527, 1513
1657, 1650, 1640, 1630 (αhelix, β-sheet, denatured
protein)
1545, 1519
1540, 1517
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