Electronic Supplementary Material
Novel blue-emitting gold nanoclusters confined in human hemoglobin, and their use as
fluorescent probes for copper(II) and histidine
Mojtaba Shamsipur · Fatemeh Molaabasi · Maryam Shanehsaz · Ali Akbar MoosaviMovahedi
Preparation of hemoglobin
The experimental procedures and protocols for preparation of hemoglobin were carried out
according to the method of William and Tsay [1]. Briefly, blood (20 mL) was obtained by vein
puncture from a healthy person, and collected into a sterile bottle containing 2 mL of 4 % sodium
citrate and kept in a fridge at 4 ºC prior to use. The heparinized bloods were centrifuged to
remove plasma components. The packed red cells were washed three times in an isotonic saline
solution (0.9 % NaCl) and were lysed with cold double-distilled water. Membrane components
were removed by centrifugation at 3000 rpm for 15 min. The soluble Hb was centrifuged at least
two additional times at 10000 rpm for 15 min to remove residual insoluble materials. The Hb
solution was then brought to 20 % saturation with ammonium sulfate, left to stand for about 15
min, and centrifuged at 14000 rpm for 1 h at 2 ºC. The supernatants containing the Hb were
dialyzed at least three times in 50 mM phosphate buffer of pH 7.5 for 24 h [2]. The concentration
of the protein was determined by the method of Antonini and Brunori (λ415nm = 125 mM-1 cm-1 or
λ541nm = 13.8 mM-1 cm-1 per heme). The concentrations were high enough (i.e., ≥ 100 µM) to
avoid the formation of a considerable amount of dimers, which should always be ≤ 5 % [3].
In order to clarify the purity of prepared Hb, it was inserted in the Native-Page jel and
the result was compared with that of a Hb of more than 95% purity obtained from Sigma
Aldrich. The results shown in Fig S1-A clearly revealed the high purity of the Hb prepared
(>95%). Meanwhile, according SDS–Page result shown in Fig S1-B, hemoglobin chains are not
separated in highly basic pH, but as CD spectra confirmed the unfolding has occurred.
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A
B
Figure S1. (A) Native –Page (10%) of Hb; lane 1 standard Hb, lane 2 prepared Hb. (B) SDS–
Page (15%) of Hb: lane1, Hb in highly basic pH with high concentration, lane 2 Hb in highly
basic pH with low concentration, and lane 3 prepared Hb.
Optimization of experimental conditions for synthesis of Hb-AuNCs
a. Effect of NaOH concentration.
b. Effect of relative amount of Hb-to-HAuCl4.
c. Effect of reaction time.
Figure S2. Effect of NaOH concentration (A) and relative amount of Hb-to-HAuCl4 (B) on the
fluorescence spectra in the course of synthesis of Hb-AuNCs. Conditions: (A) solution pHs are:
(a) 8.0, (b) 10.0, (c) 12.2, (d) 12.4, (e). 12.6; (B) constant concentration of HAuCl4 (2.8 mM) and
varying amount of Hb (mg mL-1): (a) 3.5, (b) 7.0, (c) 21, (d) 28 and (e) 35.
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Time dependent studies of fluorescence and UV-Vis spectra
The time dependent fluorescence spectra of a solution at pH 12.4 containing 28 mg mL-1 of Hb
and 2.8 mM of HAuCl4 at 37 °C were recorded at varying reaction time periods from 1 to 12
days and the results are shown in Fig. S3-A. The corresponding UV-Vis spectra are also included
in Fig. S3-B for further information. As is obvious from Fig. S3-A, solutions of pure Hb (a) and
Hb/HAuCl4 in the absence of NaOH show no fluorescence emission at 450 nm. While, under
optimized pH of 12.4 and relative Hb/HAuCl4 concentration of 28 mg mL-1/2.8 mM, the reaction
for formation Hb-AuNCs begins as confirmed by observation of a weak emission at 450 nm,
while the intensity was significantly increased with increasing reaction time up to 12 days. It
should be noted that at longer time periods no measureable increase in fluorescence intensity was
observed. Thus, a time period of 12 days was selected for further studies.
Meanwhile, from the time-dependent UV-Vis spectra shown in Fig. S3-B, it is clear that
the as-prepared Hb-AuNCs exhibited absorption bands centered at a wavelength of 365 nm with
a blackish green color, whereas this peak cannot be seen before adding NaOH (pH 12.4). As time
progressed, the absorption band at 365 nm gradually increased and shifted from 360 nm (0 days)
to 325 nm (15 days) without any changes over time. However, no obvious shift was seen in
maximum emission at 450 nm (Fig. S3-A). The evolution of the excitation peak at 365 nm shows
the same trend as the emission spectra with time, confirming the presence of a single fluorescent
species in solution. Moreover, the sharp visible peak at 403 nm corresponds to the characteristic
absorption of the porphyrin–Soret band of hemoglobin that is decreased in intensity with a shift
toward the UV region (blue-shift) from the control absorption (412 nm) [4].
It has been reported that the shape and position of the Soret band are associated with the
porphyrin ring and therefore can provide insight into the environmental effect on the native
structural configuration of Hb, especially around the heme group, embedded in a hydrophobic
pocket formed by the protein’s backbone through appropriate folding. In this regard, synthesis of
AuNCs can affect the hydrophobic environment of the heme group. The strong absorption peak
at 215 nm that appears due to the α-helix structure of the protein increases with slight red shift,
indicating that the formation AuNCs in Hb leads to the loosening and unfolding of the protein
skeleton. Similarly, the absorption spectra of native Hb show a number of bands located at 280
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(due to the phenyl group of Trp and tyrosine residues), 349 (ε band), 540, and 575 nm (oxy-band
or Q-band. The position of the Q-band reflects the changes of heme microenvironment in Hb.
The features of aromatic amino acids in protein retained at 280 nm, while the Q-bands disappear
after formation of AuNCs, reflecting the fact that the microenvironment surrounding heme in
Hb-AuNCs is different from that of native Hb. The change in intensity of the Trp/tyrosine band,
ε band, Soret band, as well as the Q-band with AuNCs implies that nanoclusters can access both
the heme and the Trp residues.
Figure S3. Effect of time on the fluorescence (A) and UV-Vis (B) spectra in the course of
synthesis of Hb-AuNCs. (A, B) (a) and (b) are spectra of Hb (28 mg mL-1) and HAuCl4 (2.8
mM), respectively, in the absence of NaOH. The other spectra are belong to 28 mg mL-1 Hb at
2.8 mM HAuCl4 at pH 12.4 at varying reaction times (days): (c) immediately, (d) 1, (e) 2, (f) 4,
(g) 7, (h) 9, (i) 10, (j) 11, (k) 12, (l) 15 days.
These results indicate that the formation of AuNCs also affects the conformation of the
Hb as capping agent in the Hb-AuNCs system. At the optimized conditions (HAuCl4:Hb = 6.5:1;
37 °C; 30 days), Hb-AuNCs exhibit a blue fluorescence with a maximum excitation/emission
wavelength located at 365/450 nm (Figure S3-A). Reactions at the physiological temperature (37
°C) showed 0.5 % quantum yield using quinine sulfate as the standard (QY = 54 %, in 0.1 M
H2SO4) [5, 6]. Considering the weak autofluorescence of Hb at excitation at 365 nm, the increase
of the blue fluorescence after adding NaOH, and the blue shift in visible region from 412 to 403
nm (Figs. S3-Ba and S3-Bc, respectively) could be related to the formation of small AuNCs.
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Figure S4. (A) FTIR spectra of Hb (a) and Hb–AuNCs (b). (B) Far UV-CD spectra of Hb (a)
and Hb–AuNCs (b). (C) X-ray photoelectron spectroscopy (XPS) spectrum of Au(4f) for HbAuNCs: (a) Hb-AuNCs, (b) fitting line, (c) Au (0) line, (d) Au (I) line. (D) Emission spectra of
2.2 µM Hb-AuNCs taken at different excitation wavelengths: (a) 350, (b) 355, (c) 360, (d) 365,
(e) 370, (f) 375, (g) 380, (h) 385, (i) 390, (j) 395, (k) 400 nm. (F) pH dependence fluorescence of
Hb-AuNCs (2.2 µM) in 10 mM of buffer solution of varying pH.
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XPS study of Hb-AuNCs
To clarify chemical states of the AuNCs, XPS was utilized after drying the solution on a
silicon wafer (Fig. S4-C). In the Au 4f high resolution region (a), two binding energies were
detected (84.2 and 87.9 eV) which are assigned to 4f5/2 and 4f7/2, similar to Au (0) 4f7/2 (84.0 ev)
and Au (0) 4f5/2 (87.7 ev), with a spin-orbit splitting of 3.7 eV [7, 8], confirming reduction of Au
(III) to Au (0) valance state during the formation of stable Au nanoclusters. The Au 4f7/2 binding
energy spectrum (a, b) could be deconvoluted into two components centered at Au (0) at 83.7 eV
(c) and Au (I) centered at 84.8 eV (d). The atomic ratio between Au(0) and Au(I) was evaluated
as 1.5:1.0.
Stability of the Hb-AuNCs probe
The stability of the Hb-AuNCs was then evaluated by measuring their zeta potential. The
zeta potential of the Hb-AuNCs was found to be -30.4 ± 4.0 mV, reflecting a great electrostatic
repulsion between particles that, consequently, will result in well dispersion of the Hb-AuNCs in
aqueous solution [9]. It should be noted that the measured zeta potential of Hb-AuNCs is about
20 mV more negative than that of Hb alone (i.e., -10 ± 5.52 mV) at a pH of 7.4 confirming the
formation of Hb-AuNCs [9, 10].
To further confirm the stability of the Hb-AuNCs, their fluorescence was measured in a
solution of 10 mM phosphate (pH 7.4) containing high salt concentration (1 M NaCl) [11, 12]. A
slight decrease of <5 % in the fluorescence intensity revealed that Hb-AuNCs is quite stable
under conditions of high ionic strength.
The photostability of the prepared Hb-AuNCs was studied under ultraviolet (UV)
irradiation [13]. Here, the Hb-AuNCs were irradiated at 365 nm for different excitation times
from 5 to 120 min and found that the resulting fluorescence intensity did not change significantly
after irradiation for 30 min, while it showed only 6 % change after irradiation for 2 h (Fig. S5).
The great UV stability of AuNCs may be resulted from the large size of the protein template
(574 aa) and the completely coverage of surface atoms [14-16]. In addition, when Hb-AuNCs
were kept at ambient conditions for 3 months, no flocculation or precipitation was observed, and
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they still preserved their fluorescence. These results indicated that the prepared quantum clusters
possess favorable monodispersity, good stability and exhibit bright and large Stokes shift. These
findings suggested that Hb-AuNCs have great potential for sensing of the materials of interest
under physiological conditions and they can be used as cell markers for long-term studies such as
cell-cell interactions [13].
Figure S5. Effect of irradiation time on the fluorescence signal λem of 450 nm (λex = 365 nm) of
Hb-AuNCs.
Determination of spectral overlap integral (J)
The spectral overlap integral (J) between the donor emission spectrum and the acceptor
absorbance spectrum was also approximated by the following summation:
J=
where F(λ) and ɛ(λ) represent the fluorescence intensity of the donor and the molar extinction
coefficient of the acceptor, respectively.
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Figure S6. (C) Quenching effect of 5 µM Cu2+ on fluorescence intensity of 2.2 µM Hb-AuNCs
at different pH in 10 mM PBS of varying pH. The observed decreased fluorescence intensity at
pH> 7.4 is due to formation of some Cu2+-OH- species in solution.
Figure S7. Selectivity of Cu2+ (1) in the presence of Ni2+ (2) and Co2+ (3) at different pHs.
Conditions: 10 mM PBS, [M2+] = 5 µM, [Hb-Au NCs] = 2.2 µM. As seen, a more selective and
sensitive response of Cu2+ relative to its competing Ni2+ and Co2+ ions was observed under acidic
conditions of pH 5 that may be due to the lesser availability of the third binding site of histidine.
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Figure S8. (A) Time dependence fluorescence of (a) Hb-AuNCs; b, Hb-NCs/Cu2+; (c) HbAuNCs/Cu2+/His. Conditions: 2.2 µM Hb-AuNCs; 20 µM Cu2+; 20 µM histidine in 10 mM PBS
of pH 7.4.
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