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Unraveling the Stoichiometric Interactions and Synergism between
Ligand-Protected Gold Nanoparticles and Proteins
Bihan Zhang,◆ María Francisca Matus,◆ Qiaofeng Yao,* Xiaorong Song, Zhennan Wu, Wenping Hu,*
Hannu Häkkinen,* and Jianping Xie*
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ABSTRACT: Nanomaterials that engage in well-defined and tunable interactions
with proteins are pivotal for the development of advanced applications. Achieving
a precise molecular-level understanding of nano-bio interactions is essential for
establishing these interactions. However, such an understanding remains
challenging and elusive. Here, we identified stoichiometric interactions of
water-soluble gold nanoparticles (Au NPs) with bovine serum albumin (BSA),
unraveling their synergism in manipulating emission of nano-bio conjugates in the
second near-infrared (NIR-II) regime. Using Au25(p-MBS)18 (p-MBS = paramercaptobenzenesulfonic acid) as paradigm particles, we achieved precise binding
of Au NPs to BSA with definitive molar ratios of 1:1 and 2:1, which is
unambiguously evidenced by high-resolution mass spectrometry and transmission
electron microscopy. Molecular dynamics simulations identified well-defined
binding sites, mediated by electrostatic interactions and hydrogen bonds between the p-MBS moieties on the Au25(p-MBS)18 surface
and BSA. Particularly, positively charged residues on BSA were found to be pivotal. By careful control of the molar ratio of Au25(pMBS)18 to BSA, atomically precise [Au25(p-MBS)18]x−BSA conjugates (x = 1 or 2) could be formed. Through a comprehensive
spectroscopy study, an electron transfer process and synergistic effect were manifested in the Au25(p-MBS)18−BSA conjugates,
leading to drastically enhanced emission in the NIR-II window. This work offers insights into the precise engineering of
nanomaterial−protein interactions and opens new avenues for the development of next-generation nano-bio conjugates for
nanotheranostics.
■
INTRODUCTION
Synthesizing nanomaterials with tunable interactions with
biological matter has emerged as one of the exciting frontiers in
nanoscience with several potential applications in biosensing,1,2
therapeutics,3−6 diagnostics,7−9 and targeted drug delivery.10−13 On the surface of nanomaterials, adsorption of
proteins is a common phenomenon determining the
therapeutic effect, targeting effect, biocompatibility and cellular
uptake of nanomaterials in vivo.14−18 Documented effort has
been made to reveal the interactions between nanomaterials
and proteins.19−21 However, the lack of clear molecular-level
details of nanomaterial surfaces and the random adsorption of
proteins on these surfaces make it challenging to achieve a
molecular-level understanding of nanomaterial-protein interactions. This ambiguity hinders the development of precise
nano-bio conjugates for advanced applications.
Ligand-stabilized, atomically precise gold nanoclusters (Au
NCs) are an emerging subclass of metal nanoparticles.22,23
These clusters can, in most cases, be synthesized with a precise
chemical composition, e.g., Aum(SR)n where m and n denote
the number of Au atoms and thiolate ligands (SR),
respectively, and the crystal structure of a large number of
these precise compounds is also known. Stabilizing the clusters
© XXXX American Chemical Society
with hydrophilic ligands creates charging patterns and potential
sites for hydrogen bonding on the nanocluster surface,
increasing the diversity of favorable noncovalent interactions
with proteins. The size of the metal core of Au NCs is
generally below 2 nm, creating strong quantum confinement
effects and molecular-like properties such as enhanced
photoluminescence24,25 due to discrete electronic structures26−28 and good biocompatibilities,29 facilitating a series
of successful implementations in bioimaging9,30 and biomedicine9,29−33 It has also been shown that the electronic structure
of Au NCs is sensitive to their atomic structure and
environment, which can help to unveil the mechanism of the
interactions between proteins and Au NCs.34−36
Herein, we employ atomically precise Au25(p-MBS)18 (pMBS = para-mercaptobenzenesulfonic acid) as a model
nanoparticle to unravel the stoichiometric interactions between
Received: July 20, 2024
Revised: October 9, 2024
Accepted: December 17, 2024
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Figure 1. Formation of Au NCs−BSA conjugates. (a) Model structure of Au25(p-MBS)18 (top) and crystal structure of BSA (bottom, PDB ID:
4F5S) with the domains (I, II, and III) and subdomains (A and B) depicted as ribbons in different colors. UV−vis absorption (black line),
photoluminescence emission (red line), and excitation (blue dotted line) spectra of (b) Au25(p-MBS)18 (1100 nm emission under 470 nm
excitation) and (c) BSA (350 nm emission under 290 nm excitation). (d) Photoluminescence emission spectra of 0.01 mM Au25(p-MBS)18 in the
presence of different molar ratios of BSA (from 0 to 10 molar ratios of BSA:Au25(p-MBS)18) with 470 nm excitation. (e) BSA:Au25(p-MBS)18
molar ratio-dependent photoluminescence intensity of Au25(p-MBS)18. (f) DLS graph of Au25(p-MBS)18 at different BSA:Au25(p-MBS)18 molar
ratios. Error bars represent the standard deviation of triplicate independent measurements. (g) PAGE result of Au25(p-MBS)18 + BSA at different
BSA:Au25(p-MBS)18 molar ratios. Coomassie blue was used to stain the gel for the visualization of BSA at the bottom panel. All of the experiments
were conducted in 0.01 M PBS solution at pH 7.4 and room temperature.
naturally occurring proteins and water-soluble nanoparticles.
The selected Au25(p-MBS)18 NCs exhibit a second nearinfrared window (NIR-II) photoluminescence, high negative
surface charge at neutral pH conditions in water, as well as
distinct stabilities.37 Bovine serum albumin (BSA)38 is used as
the protein model, given its abundance in blood plasma39
(Figure 1A). Because of their high surface charge density and
well-organized SR ligands on the surface, Au25(p-MBS)18 NCs
can be preferentially bonded to the specific sites of BSA.
Comprehensive matrix-assisted laser desorption ionizationtime-of-flight (MALDI-TOF) and transmission electron
microscopy (TEM) analyses imply that Au25(p-MBS)18 NCs
can react with BSA with a definitive molar ratio of 1:1 or 2:1 to
form atomically precise [Au25(p-MBS)18]x−BSA conjugates (x
= 1 or 2), suggesting two distinct binding sites in BSA. The
nature of such two distinct binding sites is resolved by
performing large-scale molecular dynamics (MD) simulations
of nanocluster−protein conjugates in water up to 0.2microsecond time scale. The Au NCs bind to the protein via
a combination of electrostatic interactions and hydrogen bonds
between the p-MBS ligands on the cluster surface and residues
from BSA domains I and II. More importantly, detailed steadystate photoluminescence and femtosecond transient absorption
(TA) spectroscopy measurements manifest an electron transfer
mechanism via such stoichiometric interactions, giving rise to
significantly enhanced emission in the NIR-II regime. Besides
the electronic and optical property manipulation of Au25(pMBS)18 by BSA, the stability of BSA can be reinforced by the
anchoring effects of Au25(p-MBS)18 in the [Au25(p-MBS)18]x−
BSA conjugates (x = 1 or 2). Such identified stoichiometric
and synergistic Au NCs-proteins interaction paves the way for
the creation of advanced nano-bio conjugates with broad
applicability in various fields.
■
RESULTS
Formation of Au NCs-BSA Conjugates. The discrete
electronic structure of Au25(p-MBS)18 enables the NIR-II
fluorescence emission40 at 1100 nm under a broad excitation
region (300−900 nm) (Figure 1b). We chose 470 nm as the
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Table 1. Lifetimes of Au25(p-MBS)18 and [Au25(p-MBS)18]1−BSA Conjugates
Au25(p-MBS)18
[Au25(p-MBS)18]1−BSA
[Au25(p-MBS)18]1−BSA 80 °C
[Au25(p-MBS)18]1−BSA 8 M Urea
τ1 (ns)
res%
τ2 (ns)
res%
τave (ns)
32.6
34.2
48.7
35.3
77.7
13.8
7.6
4.5
78.2
124.1
159.7
125.3
22.3
86.2
92.4
95.5
42.8
111.7
151.3
121.3
excitation wavelength that can excite the strongest fluorescence
emission at 1100 nm. According to the relationship between
the optical properties and electronic structures of Au25 NCs,26
470 nm excitation includes both the intraband and interband
transitions related with both Au atoms in Au13 core and S
atoms in ligands, which correlate with the ligand-to-metal
charge-transfer (LMCT) process in the NIR-II emission of
Au25 NCs.41 Although it was widely accepted that the NIR-II
emission of Au25 NCs originates from the relaxation of core
excited states,40 two lifetimes, 32.6 and 78.2 ns (Table 1), were
simulated from the time-resolved spectroscopy of Au25(pMBS)18 (Figure S1). These two lifetimes may represent the
singlet and triplet excited states due to the heavy atoms
facilitating the intersystem crossing (ISC) process.42 The
relatively shorter lifetime of the triplet excited states here may
be due to the heavy NIR-II quenching effect of H2O and O2 in
aqueous solution.43 This conclusion is further supported by the
observed quenching of Au25(p-MBS)18 emission in the
presence of O2 (Figure S2). The pristine fluorescence of
BSA at 350 nm shows simpler mechanisms compared to
Au25(p-MBS)18 and originates from tryptophan residues
(Trp134 and Trp213)44 upon excited at 290 nm (Figure 1c).
Upon mixing Au25(p-MBS)18 and BSA in phosphatebuffered saline (PBS) solution at room temperature (see
supplemental experimental procedures for details), the visible
fluorescence of BSA was quenched (Figure S3). In contrast,
the NIR-II fluorescence of Au25(p-MBS)18 was enhanced
saliently (up to 4.5 times) and remained nearly unchanged for
two hours (Figures 1d and Figure S4), indicating the energy or
electron transfer between Au25(p-MBS)18 and BSA. The BSA
concentration-dependent emission enhancement of Au25(pMBS)18 (Figure 1e) is reminiscent of the Langmuir adsorption
curve. It is worthy pointing out that the emission enhancement
saturates after the feeding BSA:Au25(p-MBS)18 molar ratio of
∼2:1 and the curve fits well with the Hill equation for a
multibinding model34 (Figure S5), implying the formation of
stable and stoichiometric Au NCs−BSA conjugates. Further
substantiation of such conjugate formation across varying
BSA:Au25(p-MBS)18 ratios is provided by dynamic light
scattering (DLS) analysis (Figure 1f). It reveals that the
emergence of Au25 NCs−BSA conjugates with a hydrophilic
diameter of ∼7 nm occurs at a feeding BSA:Au25 NCs ratio
between 1:2 and 2:1. Beyond this ratio, further increasing the
BSA dosage would result in excess BSA and lower the mean
collective sizes measured by DLS. The formation of Au25
NCs−BSA conjugates at different molar ratios was also
examined through polyacrylamide gel electrophoresis
(PAGE). As shown in Figure 1g, two distinct bands were
observed in the PAGE gels, assignable to free Au25(p-MBS)18
and Au25 NCs−BSA conjugates, respectively. With the
increasing molar ratio of BSA:Au25(p-MBS)18, the band
corresponding to free Au25(p-MBS)18 fades while the band of
Au25 NCs−BSA conjugates intensifies. Notably, the free
Au25(p-MBS)18 band dominates in the PAGE gels until a
feeding BSA:Au25(p-MBS)18 ratio of 1:1, which disappears
entirely at a feeding BSA:Au25(p-MBS)18 ratio of 2:1. The trace
amount of free Au25(p-MBS)18 observed at the 1:1 ratio may
be attributed to the partial denaturation of BSA in the PAGE
separation process. This, in conjunction with the DLS results,
strongly supports the conclusion that the enhancement of
nanocluster emission in the NIR-II regime stems from the
formation of stoichiometric Au NCs−BSA conjugates.
BSA Possesses Two Binding Sites for Au NCs. In order
to shed fundamental light on the binding sites in the formed
Au NCs−BSA conjugates, we performed MALDI-TOF mass
spectrometry and negative-stain TEM analysis (Figure 2).
Figure 2. BSA possesses two binding sites for Au 25 (pMBS)18nanoclusters. (a) MALDI-TOF mass spectra of Au25(pMBS)18 + BSA at different BSA:Au25(p-MBS)18 molar ratios. (b)
Representative negative-stain TEM micrographs of Au NCs−BSA
conjugates with 1:1 binding stoichiometry. (c) Representative
negative-stain TEM micrograph of Au NCs−BSA conjugates with
2:1 binding stoichiometry.
There are only three sets of peaks discernible in the MALDITOF spectra (Figure 2a), where the peaks centered at m/z =
67,000, 75,256, and 83,434 correspond to free BSA, BSA
bound with one Au25(p-MBS)18 and BSA bound with two
Au25(p-MBS)18 (i.e., [Au25(p-MBS)18]1−BSA, and [Au25(pMBS)18]2−BSA, respectively). Moreover, with the concentration increase of Au25(p-MBS)18, the peaks of [Au25(pMBS)18]x−BSA with higher x value becomes dominant. When
the concentration of Au25(p-MBS)18 is less than that of BSA,
the spectra displayed peaks only for free BSA and [Au25(pMBS)18]1−BSA. Peaks indicative of [Au25(p-MBS)18]2−BSA
emerge only when the concentration of Au25(p-MBS)18
exceeds that of BSA, which aligns with the negative
cooperativity of Au25(p-MBS)18−BSA calculated from the
Hill equation in the BSA concentration-dependent PL spectra
(Figure S5). Notably, no peaks were detected beyond a massto-charge ratio (m/z) of 90,000 (Figure S6), suggesting the
absence of additional binding sites on BSA or the formation of
the “protein corona”, i.e., a layer of BSA proteins around
nanoclusters.15 The above mass spectrometry analysis strongly
indicates up to two binding sites existing in BSA for cluster
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Figure 3. Computational modeling of the specific binding sites for Au25(p-MBS)18on BSA using 1:1 Au NC−BSA conjugates. (a and b)
Representative snapshots from the 200 ns MD trajectory of [Au25(p-MBS)18]1−BSA conjugate showing the identified binding sites on BSA in blue
(BSite1) and green (BSite2) surfaces. (c and d) Representative zoom-in snapshots from the 200 ns MD trajectory showing the H-bonds (black
dashed lines) formed between the ligand layer of Au25(p-MBS)18 and residues from (c) BSite1 and (d) BSite2 on BSA. (e) Number of H-bonds
formed between Au25(p-MBS)18 and any region of BSA (cyan line) or specific residues that compose BSite1 (blue line) as a function of the
simulation time. (f) Number of H-bonds formed between Au25(p-MBS)18 and any region of BSA (cyan line) or specific residues that compose
BSite2 (green line) as a function of the simulation time. Color code for Au25(p-MBS)18: gold atoms are shown in large mustard spheres, sulfur
atoms at the metal−ligand interface in small yellow spheres, p-MBS ligands in sticks with carbon atoms in gray, sulfur atoms in yellow, oxygen
atoms in red, and hydrogen atoms in white. Color code for BSA: Full structure of BSA is shown in pastel cyan ribbons and binding sites residues in
sticks, with carbon atoms in pastel cyan, oxygen atoms in red, nitrogen atoms in blue, and hydrogen atoms in white.
anchoring, which is in good agreement with the aforementioned emission enhancement and DLS result. The PAGE
results, which clearly show free Au 25 (p-MBS) 18 at a
BSA:Au25(p-MBS)18 ratio of 1:2, may be attributed to the
biased affinities of these two binding sites (vide infra). The
Au25 NCs binding to the relatively weaker site becomes less
durable in the PAGE conditions, rendering their desorption
from the [Au25(p-MBS)18]2−BSA conjugates. This hypothesis
is also corroborated by the detection of [Au25(p-MBS)18]1−
BSA in MALDI-TOF spectra at a feeding BSA:Au25(p-MBS)18
ratio of 1:2 (Figure 2a).
Negative-stain TEM imaging (using uranyl acetate dihydrate
as the contrast agent) was performed to visualize the Au25 NCs
within individual protein. In the TEM micrographs (Figures
2b-c), BSA appears bright and white, while Au25(p-MBS)18 and
uranyl acetate appear dark due to the heavy metal atoms. A
distinct dark spot is evident within the BSA structure at a 1:1
molar ratio of Au25(p-MBS)18 to BSA (Figures 2b). Upon
elevating the molar ratio to 2:1, dual dark spots are observed
(Figure 2c), suggesting that precise control over the binding
sites and stoichiometry in Au NCs−BSA conjugates can be
achieved by modulating the relative molar ratios of Au25 NCs
and BSA. We extracted images of representative conjugates
from a larger TEM image to highlight the binding in Figure 2.
More comprehensive views of the Au NCs distribution in BSA
can be found in Figures S7 and S8. To gain a better
understanding of this nano-bio conjugate formation, we used a
computational approach based on MD simulations to
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Figure 4. Computational modeling of the specific binding sites for Au25(p-MBS)18on BSA using 2:1 Au NCs−BSA conjugates. (a) Snapshots from
the 200 ns MD trajectory of [Au25(p-MBS)18]2−BSA showing the proximity of Au25(p-MBS)18 nanoclusters (Au25 NC1 and Au25 NC2) with
BSite1 (blue surface) and BSite2 (green surface) at different points of the simulation. (b) Distances between the center of mass (COM) of Au25
NC1 and COM of BSite1 during the simulation time. (c) Distances between COM of Au25 NC2 and COM of BSite2 during the simulation time.
(d) Number of H-bonds formed between Au25 NC1 and any region of BSA (cyan line) or specific residues that compose BSite1 (blue line) as a
function of simulation time. (e) Number of H-bonds formed between Au25 NC2 and any region of BSA (cyan line) or specific residues that
compose BSite2 (green line) as a function of simulation time. (f and g) Zoom-in snapshots at different points of the 200 ns MD trajectory of
[Au25(p-MBS)18]2−BSA showing the H-bonds (black dashed lines) formed between the ligand layer of (f) Au25 NC1 and residues from BSite1, and
(g) Au25 NC2 and residues from BSite2. Color code for Au25(p-MBS)18: gold atoms are shown in large mustard spheres, sulfur atoms at the metal−
ligand interface in small yellow spheres, p-MBS ligands in sticks with carbon atoms in gray, sulfur atoms in yellow, oxygen atoms in red, and
hydrogen atoms in white. Color code for BSA: Full structure of BSA is shown in pastel cyan ribbons and binding site residues in sticks, with carbon
atoms in pastel cyan, oxygen atoms in red, nitrogen atoms in blue, and hydrogen atoms in white.
MBS)18]1−BSA (see Figures S9−10, and supplemental
computational procedures for details). By using these initial
configurations, two binding sites for Au25(p-MBS)18 (BSite1
and BSite2) were successfully identified (Figures 3a and 3b)
and interestingly, featured different affinities (Figures 3c-3f).
investigate the specific interactions between Au25(p-MBS)18
and BSA.
Computational Modeling of the Specific Binding
Sites for Au NCs on BSA. Atomistic MD simulations of 200
ns were conducted for three initial models of [Au25(pE
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Figure 5. Electron transfer from BSA to Au NCs. (a and b) Ultrafast TA spectra of (a) Au25(p-MBS)18 and (b) [Au25(p-MBS)18]1−BSA conjugate.
(c and d), Global fitting of the TA maps of (c) Au25(p-MBS)18 and (d) [Au25(p-MBS)18]1−BSA conjugate. (e) XPS spectra of Au25(p-MBS)18 and
[Au25(p-MBS)18]1−BSA conjugate. (f) Schematic diagram of the excited state dynamics of [Au25(p-MBS)18]1−BSA conjugate.
BSite1 includes residues from BSA domains I and II, including
Asp1, Thr2, Lys4, His9, Lys12, and Lys261 and it roughly
follows the curvature of the nanocluster’s ligand surface
(Figure 3c). BSite2 is located at a small loop in domain I
containing the residues Lys116, Lys173, Gly174, and Ala175
(Figure 3d). In both cases, we observed that hydrogen bonds
(H-bonds) and electrostatic interactions are the main
facilitators of Au25(p-MBS)18−BSA binding. Lysine residues,
which are positively charged basic amino acids, greatly benefit
the stable interactions with the negatively charged ligand layer
(sulfonate groups) of Au25(p-MBS)18. However, the number of
H-bonds between Au25(p-MBS)18 and BSA is higher in BSite1
(Figure 3e) than in BSite2 (Figure 3f), and this attraction
occurs faster in BSite1 (H-bond formation at 0.4 and 26 ns for
BSite1 and BSite2, respectively). In addition, energy
decomposition also shows that the contribution of the
Coulombic terms (electrostatic interactions) to the total
interaction energy is higher in BSite1 (Figures S11−12).
We further built a model for [Au25(p-MBS)18]2−BSA based
on the identified binding sites BSite1 and BSite2, and analyzed
its stability through 200 ns MD simulations (Figure 4).
Interestingly, when both binding sites are occupied simultaneously, they exhibit similar affinity for Au25(p-MBS)18, as the
initial location remained almost intact during the simulation
(Figure 4a). The short distances (<2 nm) between the center
of mass (COM) of each binding site and Au25 NCs indicate
that both Au25(p-MBS)18 are tightly bound to the protein and
stayed inside BSite1 (Figure 4b) and BSite2 (Figure 4c). When
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comparing the formation of H-bonds between the Au25 NCs
and BSA, a nearly identical number of interactions is observed
inside the two binding sites (Figures 4d and 4e). However, no
contribution of electrostatic interactions was observed for
BSite2 as it was seen for BSite1 (Figure S13), suggesting that
BSite1 has a higher affinity for Au25(p-MBS)18. Overall, the
same residues previously described were observed to be the
main contributors to the Au25(p-MBS)18−BSA interactions
(Figures 4f and 4g). Nevertheless, the flexibility of the loops
and the subtle movements of Au25 NCs inside the binding sites
promote the formation of new interactions with charged
residues neighboring the defined binding sites (for example,
with Lys239 in BSite1) and, more interestingly, special
arrangements of their side chains inside the nanocluster’s
ligand layer. For instance, in BSite1, the side chain of Lys4 can
be found between three p-MBS groups, very close to the
metal−ligand interface (Figure 4f). The mutual attraction
between the Lys4 positively charged amino group and the
partially negative sulfur atoms at the gold−sulfur interface
induces a more open conformation of the p-MBS groups,
which eventually could rigidify the metal−ligand interface and
thus cause the photoluminescence enhancement effect of
Au25(p-MBS)18 when conjugated with BSA, as reported before
for similar thiolate-protected Au NCs.45
Thus, despite the high stability of the [Au25(p-MBS)18]2−
BSA observed in the MD simulations, the potential different
affinity of the binding sites can account for the detection of
free Au25(p-MBS)18 in PAGE analysis and [Au25(p-MBS)18]1−
BSA presented in MALDI-TOF spectra at a BSA:Au25(pMBS)18 ratio of 1:2 (Figures 1g and 2a). Specifically, BSite2
exhibits a lower affinity, which cannot hold the bound Au25(pMBS)18 throughout the assay conditions used.
Electron Transfer from BSA to Au NCs. In our previous
discussion, the strong effect of BSA on photoluminescence
enhancement of Au25(p-MBS)18 is clear when the conjugate is
observed at room temperature (Figure 1d), and without
structural disruption of any individual component (Figures
S14, S15), which implies the energy or electron transfer
between Au25(p-MBS)18 and BSA. Given that the illumination
of 470 nm exceeds the excitation wavelength of BSA (Figure
1c), Förster energy transfer between Au25(p-MBS)18 and BSA
can be ruled out. As a sufficient electron-rich environment
would accelerate the electron transfer process,44,46 we suspect
an electron transfer process from BSA to Au25(p-MBS)18.
Moreover, BSA contains multiple electron-rich amino acids,
and their reduction potential is typically low enough to
facilitate the electron transfer process to other materials.47 To
shed light on the underlying electron transfer mechanism, a
more detailed spectroscopy study was conducted. We chose
the [Au25(p-MBS)18]1−BSA conjugates as the model system
due to its higher stability and monodispersity, which can
provide understanding of this nano-bio interactions at the
molecular level. As shown in Figures 5a and 5b, the TA maps
of the Au25(p-MBS)18 and [Au25(p-MBS)18]1−BSA conjugates
were taken under the excitation of 470 nm, and both showed
similar excited-state absorption (ESA) overlapped with
ground-state bleaching (GSB). Singular value decomposition
(SVD) and global fitting were carried out to extract the time
constants of these two TA maps (Figures S16 and S17). Two
components, 1 ps and >1 ns, were identified in the Au25(pMBS)18 TA map (Figure 5c). The 1 ps process is attributed to
the internal conversion (IC) and ISC as no additional
relaxation is observed and the longer lifetime (>1 ns)
Article
corresponds to the photoluminescence emission decay. Upon
the formation of the [Au25(p-MBS)18]1−BSA conjugate, a new
electron dynamic process was identified with a decay of 479 fs
(Figure 5d). This superfast decay shows enhanced adsorption
around 630 and 680 nm. As BSA showed no absorption at 470
nm (Figure 1c), this new component originates from Au25(pMBS)18. Increased absorption implies increased electron
density; according to the electronic structure of Au25 NCs,
the 680 nm absorption belongs to the Au (sp-sp) transition.
Both the slightly increased steady absorptions of [Au25(pMBS)18]1−BSA at around 470 nm in UV−vis spectra (Figure
S14) and the decreased content of Au(I) in the shell of Au25(pMBS)18 in X-ray photoelectron spectroscopy (XPS) (Figure
5e) confirmed the electron transfer from BSA to Au25(pMBS)18. Thus, this 479 fs decay was assigned to electron
transfer from BSA to Au25(p-MBS)18. Another two components of [Au25(p-MBS)18]1−BSA conjugate, 992 fs and >1 ns,
are IC and ISC relaxation and photoluminescence emission
decay, respectively. Slightly decreased IC and ISC relaxation of
the Au25(p-MBS)18−BSA conjugate compared with that of
Au25(p-MBS)18 may result from the rigidified metal−ligand
interface after the binding with BSA, which facilitates the ISC
process. This also agrees with the enhanced triplet excited state
emission after the [Au25(p-MBS)18]1−BSA conjugate formation (Table 1).
To figure out how this electron transfer occurs and
considering that ligands play an important role in the
interactions between Au NCs and BSA, we further constructed
three Au25 NCs protected by para-mercaptobenzoic acid (pMBA), 6-mercaptohexanoic acid (MHA), and 3-mercapto-1propanesulfonic acid (MPS). These ligands were chosen to
represent different structural motifs, including aromatic rings
(p-MBA), alkyl chains of varying lengths (MHA and MPS),
and terminal functional groups with different acidities
(carboxylic groups and sulfonic groups). We hypothesized
that the electronic structures and molecular characteristics of
these ligands would influence the electron transfer process
between BSA and Au25 NCs. All three Au25 NCs, namely,
Au25(p-MBA)18, Au25(MHA)18 and Au25(MPS)18, exhibit NIR
photoluminescence under 470 nm excitation (Figure S18).
Following incubation with BSA, these Au25 NCs all showed
enhanced NIR-II fluorescence. However, only Au25(p-MBA)18
showed comparable BSA photoluminescence enhancement
effects and PAGE results (Figure S19) with Au25(p-MBS)18.
The weak fluorescence enhancement of alkyl-thiolate-protected Au25 NCs unambiguously indicates that the delocalized
electrons on benzene ring is crucial for promoting the electron
process between BSA and Au25 NCs. Delocalized orbitals are
more likely to be the electron acceptor in the XH-π (X = C or
N),48 facilitating the electron transfer from BSA to Au25 NCs.
The S XPS spectra of Au25(p-MBS)18 confirm this hypothesis,
where increased electron density is observed on S after binding
with BSA (Figure S20). Additionally, we observed that
Au25(MPS)18, which is protected by a shorter alkyl chain,
exhibited a stronger emission enhancement compared to
Au25(MHA)18. This can be explained by the shorter alkyl chain
facilitating faster electron transfer due to the reduced distance
between the interacting surfaces of BSA and Au25 NCs.
However, the influence of the terminal functional groups is less
significant. Thus, we can conclude that the electron transfer
from BSA to Au25 NCs is significantly influenced by the ligand
structure. In the case of Au25(p-MBS)18, the electron transfer is
from BSA to p-MBS on the surface of Au25(p-MBS)18 and then
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Figure 6. Synergistic effect of the Au NCs−BSA conjugate. (a) Temperature-dependent photoluminescence spectra of the [Au25(p-MBS)18]1−BSA
conjugate. (b) Temperature-dependent CD spectra of the [Au25(p-MBS)18]1−BSA conjugate. (c) Urea concentration-dependent photoluminescence spectra of the [Au25(p-MBS)18]1−BSA conjugate. All the experiments were conducted in 0.01 M PBS solution at pH 7.4 and room
temperature unless otherwise indicated.
affects the photoluminescence of Au25(p-MBS)18 through
LMCT. This highlights the importance of ligand selection in
tuning the electron transfer and optical properties of Au NCs
in protein-conjugated systems.
As the electron transfer process highly depends on the
proximity between the electron donor and acceptor,49,50 and
the LMCT process relies on the ligands and motif
conformation, we hypothesized that the electronic structure
of Au25(p-MBS)18 can be further tuned by manipulating the
structure of BSA. Thus, we subsequently explored the
synergistic effect in [Au25(p-MBS)18]1−BSA conjugates.
Synergistic Effect of Au NCs−BSA Conjugate. The
configuration of BSA is irreversibly/reversibly alterable by
diverse stimuli, including temperature and H-bond disruptor.
First, we used high temperatures to denature the protein, i.e.,
loosen the structural configuration of BSA. Incubated under
high temperatures (>60 °C) for 30 min, the structure of BSA
suffers from a severe distortion (Figure S21), which has been
identified mainly as the loosening of α helix and turn
structure.51 Just as we suspected, regulating the structure of
BSA in this way can further regulate the electronic structure
and thus the photoluminescence of the Au25(p-MBS)18. NIR-II
photoluminescence of the [Au25(p-MBS)18]1−BSA conjugate
intensified with increasing temperature (Figure 6a), which is
not observed for free Au25(p-MBS)18 (Figure S22). However,
temperature-varied UV−vis absorption and CD spectra
showed that the [Au25(p-MBS)18]1−BSA conjugate can also
maintain its stability at 80 °C for at least 30 min without
compromising the structures of either Au25(p-MBS)18 (Figure
S23) or BSA (Figure 6b). Considering the already proved
distinct stability of Au25(p-MBS)18 under high temperatures,37
these results suggest that the enhanced nano-bio conjugate
stability is greatly attributed to the structural templating effect
of Au25(p-MBS)18, which also induces thermal stability on BSA
by increasing its denaturation temperature (Figure 6b),
consistent with a previously reported effect of sulfonatecontaining ligands on the stability and conformation of BSA.52
Although the structure of Au25(p-MBS)18 and BSA in
conjugates remains unchanged under high temperatures (<80
°C), the fact that the protein structure tends to become loose
due to the destruction of hydrogen bonds at high temperatures
cannot be ignored. Under high temperatures, the loosening
tendency of BSA stretches the shell of Au25(p-MBS)18 through
its interactions with p-MBS, which can increase the Au(I)−
Au(I) distance in the protecting shell of NCs and thus lead to
the enhanced emission with blue-shifted emission maximum. It
is documented in the literature that increasing Au(I)−Au(I)
distance in the frame of aurophilic interactions is capable of
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inducing blue-shift of cluster emission, in good agreement with
our observation.53,54 This is further supported by the
photoluminescence lifetime of [Au25(p-MBS)18]1−BSA conjugate at 80 °C, where the ratio of triplet excited states
emission drastically enhanced (Table 1). Under extreme
conditions, such as 100 °C for 30 min, the conjugate is
disrupted, where Au25(p-MBS)18 decomposition (Figure S23)
and BSA denaturation (Figure 6a) are observed. Consequently,
the photoluminescence starts to decay (Figure 6b). Thus, the
optical properties of Au NCs can reflect the structure of the
protein in the formed conjugates. By monitoring the UV−vis
spectra of Au25(p-MBS)18 (Figure S24), it is evident that the
[Au25(p-MBS)18]1−BSA conjugate shows distinct stability,
remaining structurally stable in PBS solution for at least 6 days.
In addition to temperature, the electronic structure of
Au25(p-MBS)18 can be tuned by adding urea, which can disrupt
the H-bonds within BSA and thus alter the structure of BSA.55
By cyclic adding and removing (by dialysis) of aliquots amount
of urea into the solution of [Au25(p-MBS)18]1−BSA conjugate,
the photoluminescence intensity of Au25(p-MBS)18 exhibits a
cyclic ascending and descending pattern (Figure 6c). This
reversible photoluminescence enhancement effect corroborates
that the stress exerted by BSA on the shell of Au25(p-MBS)18
induces minor and reversible structure distortion. In this vein,
the BSA can act as tweezers in the [Au25(p-MBS)18]1−BSA
conjugate, finely and controllably tailoring the photoluminescence of Au25 NCs. This observation further demonstrates that
the optical properties of Au NCs can reflect the state of the
protein in nano-bio conjugates, implying the potential of Au
NCs to serve as protein structure probes in bioapplications.
These findings suggest a synergistic effect in the nanocluster−
protein conjugates, where the nanocluster provides thermal
stability to the protein, and simultaneously, the structure of
protein manipulate the electronic structure of nanoclusters.
Article
ASSOCIATED CONTENT
sı Supporting Information
*
The Supporting Information is available free of charge at
https://pubs.acs.org/doi/10.1021/jacs.4c09879.
Detailed experimental procedures, descriptions of
characterization methods, supplemental computational
methodologies, time-resolved PL spectroscopy, PL
spectra, fitted Hill equation plot, MALDI-TOF mass
spectra, negative-stained TEM micrographs, UV−vis
spectra, CD spectra, XPS spectra, MD models of BSite1
and BSite2, global fitting of TA spectra (PDF)
■
AUTHOR INFORMATION
Corresponding Authors
Qiaofeng Yao − Key Laboratory of Organic Integrated
Circuits, Ministry of Education & Tianjin Key Laboratory of
Molecular Optoelectronic Sciences, Department of Chemistry,
School of Science, Tianjin University, Tianjin 300072, China;
Collaborative Innovation Center of Chemical Science and
Engineering (Tianjin), Tianjin 300072, China; orcid.org/
0000-0002-5129-9343; Email: qfyao@tju.edu.cn
Wenping Hu − Key Laboratory of Organic Integrated Circuits,
Ministry of Education & Tianjin Key Laboratory of
Molecular Optoelectronic Sciences, Department of Chemistry,
School of Science, Tianjin University, Tianjin 300072, China;
Collaborative Innovation Center of Chemical Science and
Engineering (Tianjin), Tianjin 300072, China; orcid.org/
0000-0001-5686-2740; Email: huwp@tju.edu.cn
Hannu Häkkinen − Departments of Physics and Chemistry,
Nanoscience Center, University of Jyväskylä, FI-40014
Jyväskylä, Finland; orcid.org/0000-0002-8558-5436;
Email: hannu.j.hakkinen@jyu.fi
Jianping Xie − Department of Chemical and Biomolecular
Engineering, National University of Singapore, Singapore
117585, Singapore; Joint School of National University of
Singapore and Tianjin University, International Campus of
Tianjin University, Binhai New City, Fuzhou 350207,
China; orcid.org/0000-0002-3254-5799;
Email: chexiej@nus.edu.sg
■
CONCLUSION
In summary, our study has revealed the stoichiometry and
structural basis for the nanoclusters−protein interactions at the
atomic and quantitative levels, presenting the specific and
selective noncovalent interactions between Au25(p-MBS)18 and
BSA, in which the MD simulations highlighted the crucial role
of charged residues within specific domains (such as lysine and
histidine) in forming the stable interactions. By finely tuning
the molar ratio of Au25(p-MBS)18 to BSA, we achieved
molecularly precise [Au25(p-MBS)18]x−BSA conjugates (x = 1
or 2) without structural disruption of any individual
component. Spectroscopic analyses further revealed that BSA
enhances the NIR-II emission of Au25(p-MBS)18 through
electron transfer between BSA residues and ligands on the
surface of the Au NCs. The [Au25(p-MBS)18]1−BSA conjugate
exhibits a synergistic effect, in which Au25(p-MBS)18 enhances
the stability of BSA and the structural dynamics of BSA can be
employed to delicately manipulate the electronic and physical
structure of Au25(p-MBS)18, while keeping the nanocluster
structure largely unaltered. This study not only elucidates the
fundamental interactions between NCs and proteins but also
illuminates the potential for strategic manipulation of the
interactions between nanomaterials and proteins through
ligand engineering, which opens promising avenues for the
development of advanced nanomedicine.
Authors
Bihan Zhang − Department of Chemical and Biomolecular
Engineering, National University of Singapore, Singapore
117585, Singapore; Joint School of National University of
Singapore and Tianjin University, International Campus of
Tianjin University, Binhai New City, Fuzhou 350207, China
María Francisca Matus − Departments of Physics and
Chemistry, Nanoscience Center, University of Jyväskylä, FI40014 Jyväskylä, Finland; orcid.org/0000-0002-4816531X
Xiaorong Song − MOE Key Laboratory for Analytical Science
of Food Safety and Biology & State Key Laboratory of
Photocatalysis on Energy and Environment, College of
Chemistry, Fuzhou University, Fuzhou 350116, China;
orcid.org/0000-0001-5484-0978
Zhennan Wu − State Key Laboratory of Integrated
Optoelectronics, College of Electronic Science and Engineering,
Jilin University, Changchun 130012, China; orcid.org/
0000-0001-5887-7129
Complete contact information is available at:
https://pubs.acs.org/10.1021/jacs.4c09879
I
https://doi.org/10.1021/jacs.4c09879
J. Am. Chem. Soc. XXXX, XXX, XXX−XXX
Journal of the American Chemical Society
pubs.acs.org/JACS
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◆
B.Z. and M.F.M. contributed equally to this work.
Notes
The authors declare no competing financial interest.
■
ACKNOWLEDGMENTS
The experimental work is financially supported by the Ministry
of Education, Singapore (A-8000054-01-00), National Natural
Science Foundation of China (22071174, 22371204), and the
Fundamental Research Funds for the Central Universities. The
computational work at the University of Jyväskylä is supported
by the Academy of Finland. The computations were performed
in the LUMI supercomputer, owned by the EuroHPC Joint
Undertaking and hosted by CSC (Finland), through the
Finnish Grand Challenge Project BIOINT. We thank Sami
Malola for help in building the initial Au NC models for
molecular dynamics simulations.
■
Article
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