Plasmon-Enhanced Fluorescent Sensor based on Aggregation-Induced Emission for the Study of Protein Conformational Transformation

Article abstract of DOI:10.1002/adfm.201807211

The alteration in protein conformation not only affects the performance of its biological functions, but also leads to a variety of protein-mediated diseases. Developing a sensitive strategy for protein detection and monitoring its conformation changes is of great significance for the diagnosis and treatment of protein conformation diseases. Herein, a plasmon-enhanced fluorescence (PEF) sensor is developed, based on an aggregation-induced emission (AIE) molecule to monitor conformational changes in protein, using prion protein as a model. Three anthracene derivatives with AIE characteristics are synthesized and a water-miscible sulfonate salt of 9,10-bis(2-(6-sulfonaphthalen-2-yl)vinyl)anthracene (BSNVA) is selected to construct the PEF–AIE sensor. The sensor is nearly non-emissive when it is mixed with cellular prion protein while emits fluorescence when mixed with disease-associated prion protein (PrP^Sc). The kinetic process of conformational conversion can be monitored through the fluorescence changes of the PEF–AIE sensor. By right of the amplified fluorescence signal, this PEF–AIE sensor can achieve a detection limit 10 pM lower than the traditional AIE probe and exhibit a good performance in human serum sample. Furthermore, molecular docking simulations suggest that BSNVA tends to dock in the β-sheet structure of PrP by hydrophobic interaction between BSNVA and the exposed hydrophobic residues.

Full text of DOI:10.1002/adfm.201807211

FULL PAPER
Protein Conformational Transformation Monitoring
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Plasmon-Enhanced Fluorescent Sensor based on
Aggregation-Induced Emission for the Study of Protein
Conformational Transformation
Yanyun Cui, Chang Yuan, Hongwei Tan, Zhanbin Zhang, Yijing Jia, Na Na,
and Jin Ouyang*
in monitoring protein conformational
The alteration in protein conformation not only affects the performance of its
biological functions, but also leads to a variety of protein-mediated diseases.
Developing a sensitive strategy for protein detection and monitoring its
conformation changes is of great significance for the diagnosis and treatment
of protein conformation diseases. Herein, a plasmon-enhanced fluorescence
(PEF) sensor is developed, based on an aggregation-induced emission (AIE)
molecule to monitor conformational changes in protein, using prion protein
as a model. Three anthracene derivatives with AIE characteristics are synthe-
sized and a water-miscible sulfonate salt of 9,10-bis(2-(6-sulfonaphthalen-2-yl)
vinyl)anthracene (BSNVA) is selected to construct the PEF–AIE sensor. The
sensor is nearly non-emissive when it is mixed with cellular prion protein
while emits fluorescence when mixed with disease-associated prion protein
(PrPSc). The kinetic process of conformational conversion can be monitored
through the fluorescence changes of the PEF–AIE sensor. By right of the
amplified fluorescence signal, this PEF–AIE sensor can achieve a detection
limit 10 pM lower than the traditional AIE probe and exhibit a good perfor-
mance in human serum sample. Furthermore, molecular docking simulations
suggest that BSNVA tends to dock in the β-sheet structure of PrP by hydro-
phobic interaction between BSNVA and the exposed hydrophobic residues.
changes, such as nuclear magnetic reso-
nance (NMR), circular dichroism (CD),
Fourier transform infrared spectroscopy,
atomic force microscopy, scanning elec-
tron microscopy (SEM), fluorescence
spectroscopy, positron emission tomog-
raphy, and surface-sensitive measurement
techniques.[2] Among them, fluorescence
methods have received increasing atten-
tion owing to the advantages of rapidity,
simplicity, low cost, and high sensitivity.[3]
Some types of fluorophores have been
reported to be able to bind to oligomers
or fibrils to present different fluorescence
emissions, such as congo red, thioflavin T,
1-anilino-naphthalene-8-sulfonate, rhoda-
mine derivatives, and quantum dots.[2a,d,4]
However, these conventional dyes suffer
from several drawbacks including low
specificity, insufficient sensitivity, and
false-positive response.[5] In addition, pro-
tein transformation involves multiple pro-
cesses, and most fluorescent molecules
are not able to detect intermediates, hence
being unsuitable for kinetic studies.[6] Moreover, these fluoro-
phore molecules usually go through serious self-quenching
when they are concentrated or bounded to biomacromolecules
due to the aggregation caused quenching (ACQ) effect.[7]
1. Introduction
The alteration in protein conformation not only affects the per-
formance of its biological functions, but also leads to a large
variety of protein-mediated diseases including Alzheimer’s
disease (AD), Parkinson’s disease (PD), type II diabetes, prion
disease, etc.[1] Developing an effective method for monitoring
the changes of protein conformation is very important for
the diagnosis and treatment of protein conformation disease.
At present, several techniques have been successfully used
Aggregation-induced emission (AIE) molecules are a class
of fluorophores that are non-emissive at dissolved state, but
become highly fluorescent emission at aggregated state.[8] They
can effectively conquer the ACQ effect of conventional dyes.[9]
In addition, they have several advantages, including the large
Stokes’ shift, strong photostability, and high signal-to-noise
ratio.[7a,10] Based on the unique properties of AIE molecules,
they are widely used in the detection of metal ions and small
molecules, stimuli response, fluorescence imaging, etc.[11]
More importantly, the AIE molecules are very sensitive to the
changes of microenvironment, so they are especially suitable
for monitoring the change progress of protein conformational.
Tang et al. synthesized 1,2-bis[4-(3-sulfonatopropoxyl)phenyl]-
1,2-diphenylethene, which has good AIE properties and has
been successfully implemented in monitoring the unfolding
process of human serum albumin.[12] Since then, AIE mole-
cules have stepped onto the stage of protein conformation
monitoring. Subsequently, a large number of AIE molecules
Y. Cui, C. Yuan, Prof. H. Tan, Prof. Z. Zhang, Y. Jia, Prof. N. Na,
Prof. J. Ouyang
Key Laboratory of Theoretical and Computational Photochemistry
Ministry of Education
College of Chemistry
Beijing Normal University
Beijing 100875, China
E-mail: jinoyang@bnu.edu.cn
The ORCID identification number(s) for the author(s) of this article
can be found under https://doi.org/10.1002/adfm.201807211.
DOI: 10.1002/adfm.201807211
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are synthesized and used to detect the fibrillation or unfolding
process of insulin, erythropoietin, α-synuclein, etc.[5,6,13] How-
ever, most AIE molecules have poor water solubility which
limits their practical applications. In addition, the hydrophobic
interaction between AIE molecules and proteins is the main
detection mechanism, which leads to poor binding selectivity.
Furthermore, the low binding efficiency of recognition assay
limits the sensitivity. Therefore, it is necessary to develop new
AIE sensors with high sensitivity and specificity in aqueous
solution for biological macromolecules detection.
Signal amplification is a promising approach to obtain high
sensitivity sensors.[14] The localized surface plasmon resonance
of metal nanoparticles (NPs) can greatly amplify the electro-
magnetic (EM) field around them, which in turn enhances
the surrounding fluorescence to achieve plasmon-enhanced
fluorescence (PEF).[15] PEF can improve the fluorescence per-
formance of fluorescent molecules and the detection sensi-
tivity of target molecules. At present, a variety of fluorescent
molecules have achieved fluorescence enhancement through
the PEF effect, including porphyrin dyes, cyanine dyes, nucleic
acid stain, and fluorescein.[16] However, these conventional dyes
usually undergo serious self-quenching when they are con-
centrated or bounded to biological macromolecules due to the
intermolecular π–π stacking interactions, which greatly limits
their applications in biological macromolecular detection.[11c,17]
In addition, they are not sensitive to the changes of microen-
vironment and cannot monitor biological processes such as
protein conformation changes. Inspired by the advantages of
PEF and AIE effects, we has designed to introduce the AIE
molecules into PEF system, enabling the PEF sensor to show
new potential in protein conformation monitoring and other
biological processes.
AIE characteristics and self-assembly effects, especially the sul-
fonate salt of 9,10-bis[2-(6-sulfonatopropoxyl)naphthylethenyl]
anthracene (BSNVA). Subsequently, BSNVA molecules were
introduced into the PEF system by taking advantages of the
specific recognition of protein by aptamer and the interaction
between protein and BSNVA molecules. The Au@SiO2 NPs
can significantly enhance the fluorescence intensity of BSNVA
molecules in solution. Although organic–inorganic hybrid
sensors have been reported, the combination of AIE and PEF
systems has been rarely reported.[11d,15a,18] By regulating the
thickness of silicon shell, we successfully achieved the fluo-
rescence enhancement of AIE molecules, which expands the
application scope of PEF. Water-soluble AIE molecules have a
lower background interference level in aqueous solutions than
traditional dyes, which makes them more suitable for PEF
system. This design can not only improve the performance of
fluorescent molecule, but also overcome the disadvantage of
self-quenching of traditional dyes. Compared with most pre-
vious methods, the new method in this paper constructed a
turn-on and label-free fluorescence sensor by combining PEF
and AIE. By means of the signal amplification, the PEF–AIE
sensor exhibits outstanding sensitivity and selectivity for target
protein detection. Furthermore, using prion protein (PrP) as
the model, the PEF–AIE sensor is further applied to monitor
the change of protein conformation. This strategy may serve as
a common method for protein conformational transformation,
which is of great importance to the diagnosis and treatment of
protein-conformational diseases.
2. Results and Discussion
In this work, a hybrid PEF–AIE sensor consisting of plas-
monic Au@SiO2 NPs coupled with AIE molecule was designed
for monitoring protein conformational changes. First, three
anthracene derivatives with different substituents were success-
fully designed and synthesized. All of them exhibited excellent
The preparation of the PEF–AIE sensor was illustrated in
Scheme 1. First, we successfully synthesized a anthracene
derivative of BSNVA. Good water solubility and low quantum
yield in aqueous solution make BSNVA suitable for PEF sensor.
Au nanocubes (Au NCs) coated with silica (Au@SiO2) was
Scheme 1. Schematic illustration of PEF sensor based on AIE molecules for the detections of A) PrPC and B) PrPSc.
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selected as the PEF matrix, and BSNVA as fluorescence emitter
was introduced into PEF system. The Au@SiO2 NPs were fur-
ther modified with aptamer to capture PrP in the solution. In
the presence of cellular prion protein (PrPC), BSNVA mole-
cules are dispersed in water, as BSNVA is difficult to bind to
PrPC (Scheme 1A). Instead, with the introduction of disease-
associated prion protein (PrPSc), the highly specific interactions
between aptamer and PrPSc can promote the formation of Au@
SiO2-Apt-PrPSc complexes. At the same time, AIE molecules
bind spontaneously to PrPSc through hydrophobic interactions,
leading to an obvious fluorescence enhancement (Scheme 1B).
Accordingly, changes in the conformation of PrP can be
obtained by monitoring the variation of fluorescence intensity.
fluorescence (FL) intensity gradually increased and reached the
maximum value when the volume fraction of water (fw) was 80%,
and BMNVA emitted strong green light with the FL emission
peak centered at 525 nm (Figure 2A,B). The absorption spectra
of BMNVA in THF-water mixture are shown in Figure S6A
in the Supporting Information, and the change trend is con-
sistent with the FL spectra. The maximum absorption of
BMNVA in THF is 418 nm. When the fw reaches 60%, the
absorption intensity at 418 nm disappears, while a weak band
appears at 476 nm. When fw is 80%, a remarkable absorption
band at 476 nm represents the formation of BMNVA aggregates
in suspensions. Similarly, the AIE performances of BHNVA
were evaluated in the same mixed solvent. BHNVA exhibited
weak fluorescence when completely dissolved in pure THF.
However, the FL intensity of BHNVA increased dramatically
upon increasing the fw and reached the highest level when the
volume of water was 70% (Figure 2C, D). As a sulfonate salt,
BSNVA can dissolve in water and exhibits weak fluorescence.
With the THF fraction (fT) increasing, the FL intensity go up
by 13.2-fold when the fT is 80% (Figure 2E,F). As mentioned
above, all three compounds have good AIE properties. In addi-
tion, BMNVA possesses solvatochromism characteristics, along
with enhancement of the solvent polarities, and the emission
bands redshift from 516 to 535 nm (Figure S6B, Supporting
Information). The mechanical grinding and thermal stimula-
tion make the luminous colors of the as-prepared powders
convert between orange and green emission under UV light,
which further enables BMNVA to be used on the eraser board
(Figure S6C,D, Supporting Information).
2.1. Synthesis and Characterization of Anthracene Derivatives
Anthracene and its derivatives are a typical class of AIE mole-
cules due to their excellent optical properties and chemical
stability.[19] Through the Witting–Horner reaction, three
anthracene derivatives were designed and synthesized, namely
BMNVA, BHNVA, and BSNVA (Scheme S1, Supporting Infor-
mation). The general synthetic route is shown in Figure 1. The
products and key intermediates are characterized by NMR and
mass spectrometry (Figures S1–S5, Supporting Information).
It is envisioned that BMNVA, BHNVA, and BSNVA will have
AIE performances benefiting from the 9,10-anthylene core.[20]
They have weak fluorescence in the dispersed state, because the
vibration of 9,10-anthylene and rotation of aryl peripheries in
the solution can dissipate excitation energy. However, an inten-
sive fluorescence was observed in the aggregated state owing
to the restriction of intramolecular motions (RIM), resulting in
the blocking of the non-radiative decay channel.[21]
Shape and size determine the physicochemical properties of
materials, turning the shape control into a hot topic in mate-
rial science.[22] Studies have shown that AIE molecules could
self-assemble into various shapes. In this work, three anthra-
cene derivatives with different substituents were synthesized
and the solution evaporation approach was employed to pre-
pare their self-assembled structures. The fluorescence micro-
scopic images demonstrated that solution evaporation process
led to the formation of aggregation into different shapes in the
Furthermore, poor–good solvent systems were chosen to
confirm their AIE characteristics. Tetrahydrofuran (THF)-H2O
mixed solvent was used to investigate the AIE characteristics
of BMNVA. BMNVA showed weak fluorescence when dis-
solved in pure THF. However, with the addition of water, the
Figure 1. Synthesis routes of AIE molecules.
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Figure 2. A) FL spectra and B) plot of relative FL intensity (I/I0) of BMNVA in THF/water mixtures with different water fractions ( fw), where I0 is the FL
intensity in THF. C) FL spectra and D) plot of relative FL intensity (I/I0) of BHNVA in THF/water mixtures with different fw, where I0 is the FL intensity
in THF. E) FL spectra and F) plot of relative FL intensity (I/I0) of BSNVA in THF/water mixtures with different THF fractions ( fT), where I0 is the FL
intensity in water. The inset photo shows the corresponding emission colors under UV illumination.
THF/water mixtures (Figure 3). BMNVA with methoxy group
could assemble into irregular flake-like, needle-shaped, leaf-
like, and xiphophyllous structures in the THF/water mixtures
with fw = 30%, 70%, 80%, and 90% (Figure 3A). In the same
proportion of solvents, the compound BHNVA with hydroxyl
appeared in needle-shaped, diamond-shaped, spherical, and
blend shapes, respectively (Figure 3B). Similarly, sulfonate
derivatives also exhibited excellent self-assembly performance.
As displayed in Figure 3C, BSNVA forms green emission nee-
dles, and the length and width are different along with the
increasing of fT. In addition, the fluorescence microscopic
images also describe the luminescence colors of different
aggregates, such as diamond BHNVA emitting green fluores-
cence, while spherical BHNVA showing yellow emission. By
comparing the fluorescence microscopy results of the three AIE
molecules, it can be concluded that the introduction of different
substituents to anthracene derivative can effectively change the
self-assembly behavior of AIE compounds. The above results
are consistent with the AIE properties of the three molecules,
indicating that the change of luminescence properties of three
AIE molecules are related to their self-assembly morphology
and aggregation degree.
2.2. Fabrication of PEF–AIE Sensor
The enhanced local EM fields around plasmonic nanostruc-
tures can dramatically modify the optical properties of adjacent
fluorescent molecules, known as PEF.[16a] PEF is considered
as an attractive biosensing technique as it can improve detec-
tion sensitivity of target molecules. The plasmon metallic NPs
are key factors in PEF because they can serve as antennas
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aptamer had been successfully conjugated
to the surface of Au NC@SiO2 (Figure S9,
Supporting Information). The conjugation
number of aptamer to Au NC@SiO2 was cal-
culated to be 409 24 aptamer per Au NC@
SiO2 (Supporting Information).
The selection of fluorescent molecule
is one of the factors to be considered in
designing a PEF sensor. The spectral overlap
between the plasmon resonance wavelength
and the absorption or emission of the fluo-
rophore influences the enhancement of the
emission intensity.[25] BSNVA is selected
as the signal molecule in this work, which
possesses two negative charges, exhibiting
good water solubility. More importantly, the
emission wavelength of BSNVA is 540 nm,
which completely overlaps with the plasma
absorption of Au NCs (Figure 4E). Unlike
traditional dyes, BSNVA shows the unique
AIE properties that dissolve in aqueous solu-
tion with almost no luminescence, greatly
reducing the interference of background flu-
orescence. Moreover, BSNVA is sensitive to
hydrophobic environments, and its aromatic
Figure 3. Fluorescence microscopy images of A) BMNVA, and B) BHNVA generated by evapo-
rating suspensions of THF/water mixtures with fw = 30%, 70%, 80%, and 90%, and C) BSNVA
in THF/water mixtures generated the same way with fT = 30%, 70%, 80%, and 90% .
providing enhanced EM field.[23] Au NCs, with multiple sharp
corners and edges of “hot spots”, are considered as suitable
materials for PEF.[16d] In this work, Au NCs with an average
diameter of about 50 nm were prepared by a seed-growth
method, which possessed a surface plasmon absorption at
540 nm (Figure 4A,E). When the distance between the dyes and
the metallic nanostructures is less than 10 nm, the fluorescence
of dyes is quenched due to fluorescence resonance energy
transfer.[24] Therefore, silica was chosen as the dielectric spacer
to control the spacing distance, and an average thickness of the
silica shell was about 15.7 nm (Figure 4B). Silica can easily be
modified, providing convenience for the aptamer conjugation.
Subsequently, Au NC@SiO2 NPs was modified by –NH2 groups
and further connected to the aptamer. Compared with the Au
NCs, there was a redshift in the plasmon absorption from 540 to
555 nm by a series of surface modifications, which was induced
by the change of dielectric environment. However, the shape
of the plasmon absorption of Au NC remained unchanged,
suggesting that the functionalization process did not affect the
morphology of the Au NCs (Figure 4C). Zeta potential meas-
urements revealed that the potential changed after the modifi-
cations of –NH2 and aptamer, and the corresponding potential
values were respectively 28.1 and −10.7 eV (Figure 4D). In addi-
tion, the stability of Au@SiO2-Apt NPs is estimated. Figures S7
and S8 in the Supporting Information show that the UV–vis
spectrum and the size remain unchanged within 3 d, which
confirms Au@SiO2-Apt NPs are stable in the short term. Being
a fluorescent nucleicacid stain for double-strand DNA, pico-
Green (PG) was employed to further confirm the connection
between the aptamer and Au NC@SiO2 NPs. After removing
unconnected aptamer by multiple washes, Au NC@SiO2-Apt
was incubated with the complementary DNA (cDNA) of Apt. As
a result, a fluorescence emission of PG that gradually increased
with the increase of cDNA was observed, indicating that the
core can spontaneously bind to the hydrophobic region of PrPSc
rich in β-sheet structure, hence switching on its light emission.
In the enhancement system, Au NC@SiO2-Apt can capture the
PrPSc molecules in the solution through the specific binding of
the Apt. At the same time, the BSNVA molecules are indirectly
connected to the surface of the core–shell structure by hydro-
phobic interactions with PrPSc. Therefore, an obvious fluores-
cence enhancement of BSNVA is observed. As reported, the
separation distance determined the overall PEF efficiency.[25]
In order to achieve the optimal fluorescence enhancement, six
thicknesses of the silica shell ranging from 8.1 to 41.6 nm were
prepared (Figure 5A–F). The highest fluorescence enhance-
ment for Au@SiO2-Apt-PrPSc-BSNVA NPs when the silica shell
thickness was 15.7 nm, which is 18.5 times higher than BSNVA
(Figure 4F and Figure S10, Supporting Information). SiO2-Apt-
PrPSc-BSNVA NPs are used as control sample because they do
not affect the fluorescence of dyes, hence serving as a common
method to observe PEF.[16c,26]
2.3. PEF–AIE Sensor for Discrimination of PrP
Prion disease, also known as transmissible spongiform enceph-
alopathy (TSE), is a fatal neurodegenerative disease caused by
the infection of prion.[27] PrPC and PrPSc are two isoforms of
PrP, with the same amino acids sequence but different in con-
formation. PrPSc is considered as an early biomarker for prion
disease, and the conversion of PrPC to PrPSc is the mechanisms
of TSE.[28] Therefore, the discrimination of two conformers
of PrP is the premise of studying its conformational changes.
PrPC has a mainly α-helical structure, while PrPSc has a rich
β-sheet structure.[29] It is hypothesized that the PEF–AIE sensor
can be used to distinguish PrPC and PrPSc, because BSNVA can
bind to the grooves of β-sheet structures. To prove its feasibility,
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Figure 4. A) TEM image of Au NCs. B) TEM image of core–shell Au NC@SiO2 NPs. C) UV–vis absorbance spectra of Au NCs, Au NC@SiO2, Au NC@
SiO2-NH2, and Au NC@SiO2-Apt. D) Zeta-potential values of Au NCs coated with CTAB, silica shell, functionalized with the amino, and connected
with Apt. E) The extinction spectrum of Au NCs, excitation, and emission spectra of BSNVA. F) FL spectra of BSNVA, SiO2-Apt-PrPSc-BSNVA, and Au
NC@SiO2-Apt-PrPSc-BSNVA.
the FL responses of the PEF–AIE sensor to PrPC and PrPSc were
carried out. As shown in Figure 6A, BSNVA solution is almost
non-emissive due to its excellent water solubility. After the addi-
tion of PrPC, the solution of Au NC@SiO2-Apt-PrPC-BSNVA
shows a weak fluorescence. PrPC is positively charged, while
BSNVA carries a negatively charged under neutral conditions,
resulting in weak fluorescence due to a small amount of electro-
static adsorption.[30] However, the sensor is switched on when
the PrPSc is added. At this point, PrPSc is negatively charged,
which prevents BSNVA molecules from binding to PrPSc due to
the electrostatic repulsion between the same charges.[31] How-
ever, the extended β-sheet structures of PrPSc allow BSNVA
molecules accumulate on its surface through hydrophobic
interaction, which limits the intramolecular motion and thus
lits up the fluorescent signal of BSNVA. The Au NC@SiO2-Apt-
PrPSc-BSNVA and Au NC@SiO2-Apt-PrPC-BSNVA solutions
in sunlight are transparent (Figure 6B). Under the ultraviolet
irradiation, Au NC@SiO2-Apt-PrPC-BSNVA solution is almost
non-emissive, while Au NC@SiO2-Apt-PrPSc-BSNVA solution
switches on green emission (Figure 6C). The large FL intensity
differences between Au NC@SiO2-Apt-PrPC-BSNVA and Au
NC@SiO2-Apt-PrPSc-BSNVA, which indicates that the sensor
can effectively distinguish PrPSc from PrPC.
2.4. PEF–AIE Sensor for Monitoring the Conformational Change
of PrP
The distinct fluorescence responses of AIE–PEF sensor in the
presence of PrPC and PrPSc prompted us to try to use it to monitor
the conformational change process of PrP. PrPC is stable under
neutral pH and changes in conformational at low pH, resulting in
the formation of PrPSc.[32] We dissolved PrPC in a sodium acetate–
acetic acid buffer (pH 4.0) and incubated it at 65 °C to achieve
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The FL signals of Au NC@SiO2-Apt-PrPSc-
BSNVA varying with PrPSc concentrations
are recorded at an excitation wavelength of
410 nm. The emission of Au NC@SiO2-
Apt-PrPSc-BSNVA increases gradually fol-
lowing the increase of PrPSc concentration
(Figure 8A). However, the FL intensity of Au
NC@SiO2-Apt-PrPC-BSNVA is lower than
that of Au NC@SiO2-Apt-PrPSc-BSNVA and
remains almost unchanged with the change
of PrPC concentration (Figure 8B,C). A good
liner relationship with the PrPSc concen-
tration in the range from 0 to 15 × 10−9
m
with R2 of 0.999 is observed (Figure 8D),
and the limit of detection is calculated to be
10 pM (3σ). Compared with the traditional
fluorescence assay, which is direct detection
of PrPSc by BSNVA, the sensitivity of this
method is higher and improves by 100 times
(Figure S11, Supporting Information). In
addition, the selectivity of the present strategy
for PrPSc detection has been further con-
firmed by the comparison of signals obtained
Figure 5. TEM images of Au NC@SiO2 NPs with increasing silica shell thicknesses A) 8.1,
B) 11.3, C) 15.7, D) 23.1, E) 34.5, and F) 41.6 nm.
the transformation from PrPC to PrPSc.[30] At the specified interval,
aliquots of the incubation solution were taken out and added to
the detection system containing Au NC@SiO2-Apt. Subsequently,
BSNVA was added to the above mixture for the following fluores-
cence measurement. The FL of detection system emitted faintly
and remained almost unchanged when incubation time was less
than 20 min. After 30 min of incubation, the mixture became
emissive and the FL intensity increased rapidly as incubation time
went by. The FL intensity reached its maximum at 90 min, after
which the fluorescence no longer changed (Figure 7).
in the presence of other proteins, glucides, and some amino
acids (Figure S12, Supporting Information). As demonstrated,
these interferences have not significantly changed the signal,
and thus the PEF–AIE sensor could be further utilized for
detecting PrPSc in human serum sample (Table S1, Supporting
Information). The relative standard deviation for 5 nmol L−1
PrPSc was 2.28% (n = 5), and the corresponding recovery is
102.8%. This indicated that the method could be utilized for
detecting PrPSc in real sample.
The results indicate that the transformation for the PrPC
includes a three-step process. The first step represents a lag
phase of nucleation. Nucleation is a rate-limiting step in pro-
tein conformational changes, during which the β-sheet struc-
ture has not been produced due to the absence of seeds. Sub-
sequently, the PrPC is unstable under acidic conditions and
high temperature, which can trigger conformation changes to
form a β-sheet-rich form of the PrPSc. The formation of PrPSc
can in turn promote the conversion of PrPC
2.5. Possible Mechanism of the PEF–AIE Sensor
The mechanism of PEF–AIE sensor to monitor the conforma-
tional transformation is directly related to the properties of flu-
orescent molecule and protein structure. The molecule BSNVA
itself is non-emissive by annihilating its excited states through
phenyl of PEF–AIE sensor torsional motions. Our experimental
to PrPSc, which rapidly increases the fluo-
rescence of the detection system. This is the
second stage that represents the elongation
process. Finally, the transformation from
PrPC to PrPSc reaches an equilibrium stage,
and the fluorescence signal of the mixture
tends to be stable. The results are consistent
with
a
nucleation-dependent elongation
model, in which the three phases correspond
to nucleation, extension, and equilibrium
phases, respectively.[33] It is worth men-
tioning that, based on the hydrophobic effect
between AIE molecules and proteins, the
PEF–AIE sensor is also expected to monitor
the conformational changes of other proteins
by selecting appropriate aptamers and corre-
sponding proteins.
Figure 6. A) FL spectra of Au NC@SiO2-Apt-PrPC-BSNVA (red line), Au NC@SiO2-Apt-PrPSc-
BSNVA (blue line) and BSNVA (black line). Photographs of mixtures of Au NC@SiO2-Apt-PrPC-
BSNVA and Au NC@SiO2-Apt-PrPSc-BSNVA mixtures taken under B) normal laboratory lighting
and C) illumination with a UV light of 365 nm.
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specifically recognizes and interacts with β-sheet structure in
PrPSc.
To gain deeper insights into the interactions between
BSNVA and PrP and to better understand the binding mecha-
nism, the docking simulations were performed. As shown in
Figure 9A, PrPC is stable under neutral pH, which contains
three well-resolved α-helices (α1, α2, and α3) and almost no
β-sheet structure. In contrary to PrPC, the PrPSc contains
two significant β-sheet structures (Figure 9B). The transfor-
mation from α-helices to β-sheets structure is a necessary
feature in the formation of PrPSc from PrPC. Our docking
results clearly show the preference of BSNVA’s affinity to the
β-sheet region of PrPSc. (Figure 9D). In the docked BSNVA-
PrPSc complex, except the hydrophobic interactions between
the aromatic rings of BSNVA and the exposed hydrophobic
residues (L130 and Y162) from PrPSc, there are also two
hydrogen bonds formed between the oxygen atoms of sul-
fonate groups of BSNVA and two residues, K194 and L125
from PrPSc. Whereas in PrPC, the hydrophobic residues that
interact with BSNVA in PrPSc are wrapped in inside into the
α-helices. Hence in the docked BSNVA-PrPC complex, only
two hydrogen bonds are formed between PrPC and BSNVA
molecule (Figure 9C). The loose binding to PrPC thus allows
the phenyl groups in BSNVA to be dynamic rotational and
renders fluorescence quenching of it. The looser binding
between PrPC and BSNVA could also be evidenced from
the binding energy. The obtained lowest docking energy
between BSNVA and PrPC (−9.40 kcal mol−1) from 500 runs
of docking is much higher than that between BSNVA and
Figure 7. The conformation change process of PrP monitored by Au
NC@SiO2-Apt-PrPSc-BSNVA sensor.
results clearly show that BSNVA molecule turns to be lumi-
nescent in complex with the PrPSc with obvious AIE char-
acter. The experimental results inferred that bound with PrPSc
effectly limits intramolecular motions (RIM) or rotations (RIR)
of BSNVA thus switches on the fluorescence emission. How-
ever, we also have an interesting observation that the BSNVA
remains nearly non-emissive in complex with PrPC, which con-
tains less β-sheet contents in its effector binding site compared
with PrPSc, which prompts us to speculate that the BSNVA
Figure 8. A) FL spectra of Au NC@SiO2-Apt-PrPSc-BSNVA complexes upon addition of different concentrations of PrPSc. B) FL spectra of Au NC@
SiO2-Apt-PrPC-BSNVA complexes upon addition of different concentrations of PrPC. C) FL intensity of PEF–AIE sensor varies with the concentration of
PrPSc (red line) or PrPC (black line). D) The linear relationship between the PrPSc concentrations and the FL intensity of Au NC@SiO2-Apt-PrPSc-BSNVA.
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the secondary structure changes of PrP
during the conformational transformation
(Figure 11). At first, the secondary struc-
ture of PrPC was α-helices, with two nega-
tive bands at 207 and 222 nm. There was
no significant change in the spectral pro-
file as the incubation time has increased
from 0 to 30 min, which corresponded to
the initial lag phase for nucleation. Subse-
quently, an obvious decrease in intensity is
observed, indicating that PrP has changed
from α-helices to β-sheets. After incubation
for 90 min, the CD spectra had changed to
a broad negative peak at 218 nm, indicating
that the secondary structure was dominated
by β-sheet. Since then, the β-sheet-rich
structure has remained almost unchanged
with the increase of incubation time. The
extended β-sheet structure leads the con-
formers to bind to BSNVA. The changes of
secondary structure with incubation time are
consistent with the change of FL intensity.
The morphology of the PrP was further
observed by SEM. At the first stage, the mor-
phology of PrP molecules was small spher-
ical particles with no apparent aggregation.
Then, several PrP molecules were integrated
into one oligomer with large size. After 2 h of
incubation, a number of fibrils were formed
(Figure S13, Supporting Information). The
CD and SEM results reveal that the struc-
tural change of PrP is the important factor
leading to the fluorescence change of PEF–
AIE sensor.
Figure 9. Crystal structures of A) PrPC and B) PrPSc. C) The best docked complex between
BSNVA and PrPC with the lowest binding free energies (E; kcal mol−1). D) The best docked
complex between BSNVA and PrPSc with the lowest binding free energies (E; kcal mol−1). The
ligand BSNVA is represented as in ball and stick mode, with carbon, oxygen, and sulfonate
atoms colored in green, red, and orange, respectively. The crystal structure of PrPC and PrPSc
are represented as cartoon, with α-helices, β-sheets, and fibril colored in red, yellow, and green,
respectively. The key residues in the crystal structures of PrPC and PrPSc are represented with
sticks, with carbon, oxygen, and nitrogen atoms colored in yellow, red, and blue, respectively.
PrPSc (−11.36 kcal mol−1). To further identify the relationship
between the binding strength of BSNVA to PrPs and their
3. Conclusion
β-sheet content degree, we choose the other three repre-
sentative PrP structures (PDB ID: 2K1D, 3HAK, and 1QLX),
with different degrees of β-sheets content from low to high,
to examine their binding behavior with BSNVA molecule
(Figure 10). The resulted binding energy of BSNVA to them
calculated from docking results are listed in the Table S2 in
the Supporting Information. The binding energy of BSNVA to
PrP listed in the table displays obvious positive corrleation
to the degree of β-sheet contents in PrPs. The preference
of the BSNVA to β-sheet structure could also observed from
the docked complex structures. In short, the docking simula-
tions confirmed the experiment observation that the BSNVA
specifically recognizes and binds to the β-sheet structure in
PrPs.
In conclusion, a label-free AIE-based PEF sensor was success-
fully constructed to monitor the conformational changes in pro-
tein, using PrP as a model. A series of anthracene derivatives
were synthesized and a water-soluble sulfonate BSNVA with
excellent AIE properties was selected to prepare the PEF–AIE
sensor. Using an aptamer of PrP as the bridge, we success-
fully introduced BSNVA into PEF system. To the best of our
knowledge, this is the first PEF sensor to realize the fluores-
cence enhancement of AIE molecule, which expands the scope
of application of PEF. The PEF–AIE sensor emits weakly in
the presence of PrPC and strongly in the presence of PrPSc,
which enables the sensor to distinguish the PrPSc from PrPC
effectively. Furthermore, the sensor has been successfully
applied to quantify PrPSc and monitor conformational changes
of PrP. Compared with immunological methods and direct
fluorescence assay, such strategy is more sensitive, selective,
and economic because it is signal amplification and does not
require fluorescent labeling. The strategy provides a new sight
for protein detection and conformational monitoring, which
is expected to promote the search of protein-conformational
diseases.
2.6. Method Validation by CD and SEM
CD is a common method for the analysis of protein confor-
mation.[34] To further verify the validity of the above-men-
tioned results, CD spectroscopy is performed to monitor
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Figure 10. A–C) The docked complexes of BSNVA and PrPs with the lowest binding free energies (E; kcal mol−1). D–F) The detailed docked complexes
of BSNVA and PrPs with different degrees of the β-sheet content. The ligand BSNVA is represented in ball and stick mode, with carbon, oxygen, and
sulfonate atoms colored in green, red, and orange, respectively. The crystal structures of PrPC and PrPSc are represented as cartoon, with α-helices,
β-sheets, and fibril colored in red, yellow, and green, respectively. The key resudies in crystal structures of PrPC and PrPSc are represented as sticks,
with carbon, oxygen, and nitrogen atoms colored in yellow, red, and blue, respectively.
Information S1. PrPSc aptamer was synthesized by Sangon Tech. Ltd.
(Shanghai, China), and the sequence is shown in Supporting Information
4. Experimental Section
Chemicals and Materials: All reagents were purchased from suppliers
without further purification. Anthracene, paraformaldehyde, potassium
tert-butoxide (t-BuOK), and (3-Aminopropyl) triethoxysilane (APTES,
98%) were obtained from Alfa Aesar (Shanghai, China). 6-Methoxy-
2-naphthaldehyde was purchased from Tokyo Chemical Industry
(Shanghai, China). Chloroauric acid hydrated (HAuCl4·4H2O) was
obtained from Sinopharm Chemical Reagent Co., Ltd (China). Boron
tribromide, THF, dry dichloromethane (DCM), sodium borohydride,
hexadecyltrimethylammonium bromide (CTAB), L-Ascorbic acid,
tetraethyl orthosilicate, and 2,4,6-trichloro-1,3,5-triazine were purchased
from Sigma–Aldrich (Shanghai, China). Triethyl phosphite was get from
InnoChem (Beijing, China). Hydrochloric acid, methanol, petroleum ether
(PE), sodium chloride, concentrated sulfuric acid were purchased from
Beijing Chemical Works. PrP was obtained from Calbiochem (Germany).
The amino acid sequence of human PrP is shown in Supporting
S2. Human serum was purchased from Biodee (Beijing, China). Deionized
water (18.2 MΩ cm) was purified with a Milli-Q water purification.
Instrumentation: 1H NMR spectra were measured on a JEOL 400 MHz
spectrometer using chloroform-d (CDCl3) or dimethyl sulfoxide (DMSO)
as solvent. Mass spectral measurements were taken by a Thermo
Fisher Scientific LTQ-XL. UV–vis spectra were recorded on UV-2450
spectrometer (Shimadzu, Japan). Fluorescence spectra were measured
using a RF-6000 (Shimadzu, Japan). Transmission electron microscopy
(TEM) images were measured on a JEOL 2010 transmission electron
microscope. The morphology of the products were obtained on an
SEM (FESEM, S-8010, Hitachi). The zeta potential measurements were
obtained on a Nano-ZS Zetasizer ZEN3600 (Malvern Instruments Ltd,
UK). Fluorescence imaging was performed on a confocal laser scanning
microscope (Nikon ALR, Japan). CD spectra were recorded on a J-810
spectropolarimeter (JASCO) from 180 to 260 nm.
Figure 11. A) CD spectra of PrP in a pH 4 buffer at 65 °C for different periods of time (0–600 min). B) Change of CD spectra intensity at 208 nm with
different incubation time.
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Synthesis of 9,10-bis[2-(6-methoxy)naphthylethenyl]anthracene (BMNVA):
Compounds 1 and 2 were synthesized according to the literature, and the
synthetic details are shown in the Supporting Information.[35] Compound
2 (1 g, 2.08 mmol) and t-BuOK (1.2 g, 10.69 mmol) was dissolved in
THF (40 mL) under nitrogen. The solution was stirred for 1 h at 0 °C.
6-methoxy-2-naphthaldehyde (1.4 g, 7.52 mmol) in THF (40 mL) was
added to the mixture. Then, the mixture was kept at room temperature
and stirred for 12 h. The resultant precipitate was washed successively
with MeOH and filtered off to give compound as yellow powder (56.68%
yield). 1H NMR (400 MHz, CDCl3) δ 8.47 (dd, J = 6.8, 3.3 Hz, 4H),
8.03 (d, J = 16.4 Hz, 2H), 7.96–7.78 (m, 8H), 7.50 (dd, J = 6.9, 3.2 Hz,
4H), 7.19 (d, J = 8.2 Hz, 4H), 7.09 (d, J = 16.5 Hz, 2H), 3.97 (s, 6H); m/z
543.23 [M + H]+; and elemental analysis calcd (%) for C40H30O2: C 88.53,
H 5.57,O 5.90; found: C 88.51, H 5.58, and O 6.92.
Synthesis of 9,10-bis[2-(6-hydroxy)naphthylethenyl]anthracene (BHNVA):
Compound 3 (0.4066 g, 1 mmol) and dry DCM (10 mL) were added into
a 50 mL three-necked bottle. The mixture was kept in liquid nitrogen-
ethanol at −78 °C. Boron tribromide (3 mL, 1 m) in CH2Cl2 solution
was added carefully to the mixture while stirring. Then, the mixture was
stirred for 12 h and allowed to gradually return to room temperature.
Subsequently, 10 mL water was added carefully to the mixture and
stirred for 30 min to make the product and unreacted boron tribromide
hydrolyze. After that, the mixture was extracted with DCM, the organic
phase was washed with brine, dried with MgSO4 and concentrated
with to dryness under vacuum. Finally, the crude product was
chromatographed over silica gel. The column was eluted with a mixture
(3:2) of PE and acetone to yield 0.52 g (69%) of a yellow powder. 1H
NMR (400 MHz, DMSO) δ 9.84 (s, 2H), 8.44 (dd, J = 6.8, 3.3 Hz, 4H),
8.18 (d, J = 16.5 Hz, 2H), 8.09–7.99 (m, 4H), 7.79 (dd, J = 8.7, 4.2 Hz,
4H), 7.57 (dd, J = 6.9, 3.2 Hz, 4H), 7.18 (d, J = 2.2 Hz, 2H), 7.11 (dd,
J = 8.7, 2.3 Hz, 2H), 7.04 (d, J = 16.4 Hz, 2H). m/z = 515.23 [M + H]+;
and elemental analysis calcd (%) for C38H26O2: C 88.69, H 5.09, O 6.22;
found: C 90.01, H 5.58, and O 6.92.
PEF–AIE Sensor for Monitoring Conformational Changes of PrP: PrPC
was dissolved in a sodium acetate-acetic acid buffer (pH 4.0) and
incubated at 65 °C to achieve the transformation from PrPC to PrPSc.
An aliquot of the PrPC solution taken out from the incubation mixture at
a defined time and added to the detection system containing Au NC@
SiO2-Apt. Subsequently, BSNVA was added to the above mixture for the
following fluorescence measurement. The final concentration of PrP and
BSPOTPE were both 5 × 10−6 m.
Calculation Details: The geometry structure of the ligand BSNVA
molecule was optimized by density functional theory (DFT) method
B3LYP functional with 6–31G(d) basis set. Five representative
conformations of human PrP which present different degrees of β-sheet
content(PDB ID: 2K1D, 1HJM, 3HAK, 1QLX, and 1HJN) were chosen
to investigate their interaction with BSNVA. In order to establish the
complex model, BSNVA was first docked into the human PrP structures
by using AutoDock 4.2 software package with the AutoDock Lamarckian
Genetic algorithm.[36] The proteins were all kept to be rigid in the docking
process, while the ligand BSNVA was set to be rotatable bonds to allow
it to better fit in the pocket. A default docking parameters were applied.
All the docking simulations were performed for 500 runs.
Supporting Information
Supporting Information is available from the Wiley Online Library or
from the author.
Acknowledgements
J.O. gratefully acknowledges the support from the National Natural
Science Foundation of China (21675014 and 21475011) and the
Fundamental Research Funds for the Central Universities. H.T. gratefully
acknowledges the support from the National Natural Science Foundation
of China (21571019). N.N. gratefully acknowledges the support from the
National Natural Science Foundation of China (21675015).
Synthesis of 9,10-bis[2-(6-sulfonatopropoxyl)naphthylethenyl]anthracene
(BSNVA): Compound 4 (0.515 g, 1 mmol) and 20 mL of anhydrous
ethanol were added into 100 mL three-necked bottle under nitrogen.
Then a solution of NaOEt (0.1565 g, 2.3 mmol) in anhydrous ethanol
(20 mL) was added by dropwise to the above mixture and stirred until the
solution color turned to orange–red. Subsequently, 1,3-propanesultone
(200 µL) in ethanol (20 mL) was added to the reaction mixture. The
mixture was vigorously stirred overnight and a yellow product was
precipitated out from the solution. Finally, the product was filtrated and
then washed with ethanol and acetone two times to give g of a yellow
powder. 1H NMR (400 MHz, DMSO) δ 8.46 (dd, J = 6.8, 3.3 Hz, 4H),
8.19 (d, J = 16.5 Hz, 2H), 8.10–8.02 (m, 4H), 7.81 (dd, J = 8.7, 5.0 Hz,
4H), 7.58 (dd, J = 6.9, 3.2 Hz, 4H), 7.21–7.10 (m, 4H), 7.06 (d, J =
16.5 Hz, 2H), 4.07 (t, J = 6.5 Hz, 4H), 2.63 (t, J = 7.4 Hz, 4H), 2.02–1.97
(m, 4H). 13C NMR (126 MHz, DMSO) δ 156.25, 138.02, 134.95, 132.93,
131.94, 130.14, 129.52, 128.30, 127.31, 127.09, 126.78, 126.04, 124.50,
Conflict of Interest
The authors declare no conflict of interest.
Keywords
aggregation-induced emission, fluorescence sensor, plasmonic
enhancement, prion protein, protein conformation
124.02, 119.51, 109.43, 67.24, 49.11, and 25.31. m/z = 378.07 [M-2Na]2−
,
and elemental analysis calcd (%) for C44H36Na2O8S2: C 65.82, H 4.52,
Na 5.73, O 15.94, S 7.99; found: C 65.84, H 4.49, Na 5.73, O 15.96, and
S 7.98.
Received: October 12, 2018
Revised: December 21, 2018
Published online:
Fabrication of PEF Sensor: According to the literature and the previous
work, core–shell Au NC@SiO2 NPs were prepared.[14d] The detailed
information about the immobilization of aptamer onto Au NC@SiO2
NPs (Au NC@SiO2-Apt, ASA) is located in the Supporting Information.
It is worth noting that, in order to ensure the stability of ASA, the
prepared samples need to be used as soon as possible. The formation
of Au NC@SiO2-Apt-PrPSc–BSNVA NPs followed the schematic diagram
in Scheme 1. In short, 1 mL of ASA NPs solution with a certain
concentration was first mixed with the PrPSc in varied concentrations.
Then, the mixtures were incubated at 4 °C for 30 min. During this
procedure, PrPSc was specifically captured by ASA NPs. After that, the
suspensions were collected via centrifugation. The concentrated Au
NC@SiO2-Apt-PrPSc conjugates were redispersed in the phosphate
buffer saline (PBS) buffer via gentle shaking. Finally, BSNVA was added
to the dispersed Au NC@SiO2-Apt-PrPSc solution.
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