Functionalization of Silk by AIEgens through Facile Bioconjugation: Full-Color Fluorescence and Long-Term Bioimaging

Article abstract of DOI:10.1002/anie.202015592

Silkworm silk is a promising natural biopolymer for textile and biomedical applications for its remarkable flexibility, excellent biocompatibility and controllable biodegradability. The functionalization of silks makes them more versatile for flexible displays and visible bioscaffolds. However, fluorescent silks are normally fabricated through unstable physical absorption or complicated chemical reactions under harsh conditions. Herein, we developed a simple strategy for preparing fluorescent silks. Five aggregation-induced emission luminogens (AIEgens) with activated alkynes were synthesized by rational molecular design, and then reacted with silk fibers through facile metal-free click bioconjugation. The resulting conjugates show bright full-color emissions and high stability. A white light-emitting silk was fabricated by simultaneous bioconjugation with red-, green- and blue-emissive AIEgens. The red-emissive AIEgen-functionalized silks were successfully applied for long-term cell tracking and two-photon bioimaging, demonstrating great potential for tissue engineering and bioscaffold monitoring.

Full text of DOI:10.1002/anie.202015592

Angewandte
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How to cite:
International Edition: doi.org/10.1002/anie.202015592
German Edition: doi.org/10.1002/ange.202015592
Silk Functionalization
Functionalization of Silk by AIEgens through Facile Bioconjugation:
Full-Color Fluorescence and Long-Term Bioimaging
Chenchen Liu, Haotian Bai,* Benzhao He, Xuewen He, Jianyu Zhang, Chao Chen,
Yanping Qiu, Rong Hu, Fangxin Zhao, Yunxiao Zhang, Wei He, Joe H. C. Chau, Sijie Chen,
Jacky W. Y. Lam, and Ben Zhong Tang*
Abstract: Silkworm silk is a promising natural biopolymer for
textile and biomedical applications for its remarkable flexi-
bility, excellent biocompatibility and controllable biodegrada-
bility. The functionalization of silks makes them more versatile
for flexible displays and visible bioscaffolds. However, fluo-
rescent silks are normally fabricated through unstable physical
absorption or complicated chemical reactions under harsh
conditions. Herein, we developed a simple strategy for prepar-
ing fluorescent silks. Five aggregation-induced emission lumi-
nogens (AIEgens) with activated alkynes were synthesized by
rational molecular design, and then reacted with silk fibers
through facile metal-free click bioconjugation. The resulting
conjugates show bright full-color emissions and high stability.
A white light-emitting silk was fabricated by simultaneous
bioconjugation with red-, green- and blue-emissive AIEgens.
The red-emissive AIEgen-functionalized silks were success-
fully applied for long-term cell tracking and two-photon
bioimaging, demonstrating great potential for tissue engineer-
ing and bioscaffold monitoring.
flexibility, luster smoothness, light weight and high mechan-
ical strength. Recently, the remarkable biocompatibility and
controllable biodegradability of silk make it favorable as the
structural material in tissue engineering.[2] Additionally, silk
can be functionalized to show advanced properties such as
fluorescence, stimulus and conductivity, making them more
attractive for flexible display, electronic skin and bioimaging
applications.[3]
Fluorescent silk materials exhibit great advantages in
functional bio-optical devices[4] and bio-scaffolds.[5] Thus, it is
urgent to explore high performance fluorescent dyes and
efficient dyeing procedures for silk functionalization. How-
ever, conventional dyeing procedures require harsh condi-
tions like high temperature and low pH,[6] which consume
much energy and may damage the protein structures of silk.
In addition, many fluorescent silk materials were fabricated
by physical absorption through hydrogen bonding or electro-
static attraction. For example, fluorescent silk proteins can be
modified with inorganic quantum dots (QDs) through hydro-
gen bond.[7] However, such non-covalent interaction is
unstable in physiological environment to result in easy
leakage of inorganic QDs that may cause harm to health.[8]
In addition, many organic fluorescent dyes suffer from the
aggregation-caused quenching (ACQ) effect in the aggregate
state due to the strong p-p stacking, they have to be
molecularly dissolved to avoid the ACQ effect for silk
functionalization.[9] Therefore, chemical modification of silk
with solid-state emissive fluorophores seems to be the best
approach to fabricate bright and stable fluorescent silk.
However, there still remains many challenges and difficulties
which restrict the development of silk functionalization.
Introduction
Natural biopolymers have been widely utilized in bio-
medical areas with good biocompatibility and biodegradabil-
ity, and are accessible from abundant natural sources. For
example, polysaccharides like cellulose and chitosan could be
obtained directly from trees and crab shells, but need
complicated preparation procedures in the presence of harsh
chemical treatment for their synthesis in the laboratory.[1] As
a unique natural fibrous protein, silkworm silk can be easily
prepared from cocoon and has been used for textile applica-
tions historically with excellent physical properties, such as
[*] C. Liu, Dr. H. Bai, Dr. B. He, Dr. X. He, J. Zhang, Dr. C. Chen, W. He,
J. H. C. Chau, Dr. J. W. Y. Lam, Prof. B. Z. Tang
F. Zhao
Department of Ocean Science, The Hong Kong University of Science
and Technology
Clear Water Bay, Kowloon, Hong Kong (China)
Department of Chemistry, Hong Kong Branch of Chinese National
Engineering Research Center for Tissue Restoration and Recon-
struction, Division of Life Science and State Key Laboratory of
Molecular Neuroscience, Institute for Advanced Study and Depart-
ment of Chemical and Biomedical Engineering, The Hong Kong
University of Science and Technology
Y. Zhang
Department of Mechanical and Aerospace Engineering, The Hong
Kong University of Science and Technology
Clear Water Bay, Kowloon, Hong Kong (China)
Clear Water Bay, Kowloon, Hong Kong (China)
E-mail: tangbenz@ust.hk
Prof. S. Chen, Prof. B. Z. Tang
Ming Wai Lau Centre for Reparative Medicine, Karolinska Institute
Sha Tin, Hong Kong (China)
Y. Qiu, Dr. R. Hu, Prof. B. Z. Tang
Center for Aggregation-Induced Emission, SCUT-HKUST Joint
Research Institute, State Key Laboratory of Luminescent Materials
and Devices, South China University of Technology
Guangzhou 510640 (China)
Supporting information and the ORCID identification number(s) for
the author(s) of this article can be found under:
https://doi.org/10.1002/anie.202015592.
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In contrast to traditional ACQ dyes, aggregation-induced Results and Discussion
emission luminogens (AIEgens) exhibit intense emission in
the solid state due to the restriction of molecular motion. For
example, tetraphenylethylene (TPE) is a typical AIE mole-
cule that is not emissive in the solution state, because the
energy is dissipated from non-radiative excited-state decay.
However, it can exhibit intense emission in the aggregation
state due to the restriction of molecular motion. In addition,
the modification of traditional ACQ dyes with AIE units is an
efficient method to design new AIE molecules, which are
inherently suitable for fabricating solid-state luminescent
devices like organic light-emitting diodes.[10] Actually, AIE-
gens have already been successfully applied for the function-
alization of silk materials for flexible display and biomedical
application.[11] Undoubtedly, AIEgens are remarkable candi-
dates for fabricating bright and stable fluorescent silks
through bioconjugation. However, the well-known azide-
alkyne cycloaddition or the so called “click” reaction requires
the use of copper catalyst which may quench the fluorescence
and cause damage to health. And prefunctionalization of the
silk proteins makes the dyeing process more complicated.[12]
Recently, our group developed a simple strategy for metal-
free “click” bioconjugation based on activated alkynes for
protein modification, which is promising for silk functional-
ization.[13] Inspired by the inherent bright emission of
AIEgens in the solid state and the facile bioconjugation
process, in this work we synthesized full-color emissive
AIEgens with activated alkynes by rational molecular design,
for the purpose of fabricating chemical conjugated fluores-
cent silk with bright emission and great stability. As a proof of
concept, a flexible white light-emitting silk was fabricated by
simultaneous bioconjugation of red-, green- and blue- emis-
sive AIEgens. In addition, AIEgen-functionalized silk with
red emission manifested great potential for long-term cell
tracking and two-photon bioimaging.
Five AIE molecules with propynone groups (AIE-pyo)
and emission color covering the whole visible region (blue,
green, yellow, orange and red) were designed and synthesized
(Figure 1a). TPE is a typical AIEgen that emits blue
fluorescence under UV irradiation, so TPE was functional-
ized with propynone group to design blue-emissive AIE-pyo
luminogen according to the reported method (Scheme S1).[13a]
Then, methoxy and benzothiadiazole groups were selected as
the electron-donating and electron-withdrawing groups, re-
spectively. They were introduced to synthesize MTPEP-pyo,
TPEBP-pyo and MTPEBP-pyo with D-A structures and
longer-wavelength emissions (Scheme S2–S4). Triphenyla-
mine (TPA) was widely utilized as the electron-donating
group and molecular rotor to build new AIEgens, therefore
stronger electron-donating methoxytriphenylamine (MTPA)
group was chosen to synthesize MTPABP-pyo with red
emission (Scheme S5). The intermediates and products were
carefully characterized by 1H NMR, 13C NMR and MS
spectra with satisfactory results (Supporting Information).
For example, the FT-IR spectra of AIE-pyo definitely
ꢀ
confirmed the existence of alkyne groups (C C stretching at
ꢁ 2160 cmÀ1) and ketone groups (C O stretching at
=
ꢁ 1680 cmÀ1) (Figure S1).
The absorption spectra of AIE-pyo luminogens were first
measured and the absorption maximum red-shifted from
ultraviolet to visible spectral region with increasing D-A
interaction (Figure 1b). Then we evaluated the AIE property
of TPE-pyo. TPE-pyo showed weak emission in DMSO
solution due to the free rotation of the phenol rings that
facilitates the non-radiative excited-state decay.[10a] After
addition of a poor solvent of water into its DMSO solution,
the molecular motion could be largely restricted to result in
remarkable enhancement of fluorescent emission (Figure 1c).
Other AIE-pyo luminogens also manifested similar AIE
Figure 1. a) Molecular structures of AIE-pyo luminogens. b) Normalized absorption spectra of AIE-pyo luminogens in DMSO solutions.
c) Fluorescence spectra of TPE-pyo (10À5 M) in DMSO/H2O mixtures with different fractions of water (fw). d) Plots of relative fluorescence
intensity (I/I0) of AIE-pyo luminogens versus the composition of their DMSO/H2O mixtures. e) Normalized fluorescence spectra of AIE-pyo
luminogens in the solid state.
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behaviors (Figure 1d and Figure S2). Among them, TPEBP-
pyo exhibited obvious twisted intramolecular charge transfer
(TICT) effect and its emission was weakened and red-shifted
gradually in solvents with increasing polarity. However, when
aggregates formed, the emission became stronger again with
a slight blue-shift owing to the comparatively lower polar
environment inside the nanoparticles (Figure S2d). Full-color
emissions were realized by rationally tuning the D-A
structures of the AIE-pyo luminogens (Figure 1e). Theoret-
ical calculation confirmed the efficient separation of HOMO
and LUMO in molecules with strong D-A interaction, which
resulted in decreased energy gaps and red-shifted absorption
and emissions. (Figure S3)
The efficient metal-free “click” bioconjugation of acti-
vated alkynes with amine groups was previously reported, and
showed great potential for facile functionalization of bioma-
terials.[13] As a proof of concept, the model metal-free “click”
reaction was evaluated between n-butylamine and five AIE-
pyo luminogens, the spontaneous reaction finished within 30
minutes at room temperature and molecular structures of the
Figure 3. a) AIEgen-silk bioconjugates with full-color emissions in the
visible spectral region. b) Fluorescent photos of (upper) threads and
(lower) fabrics of AIEgen-silks taken under 365 nm UV light illumina-
tion. c) Normalized fluorescence spectra of AIEgen-silk fabrics.
1
products were successfully characterized by H-NMR, 13C-
NMR and HRMS analysis (Scheme S6). In addition, native
bovine serum albumin (BSA) and MTPABP-pyo were taken
as examples to evaluate the feasibility of amino-yne click
bioconjugation (Figure 2). BSA consists of 59 lysine residues
and 30–35 of them have primary amines that are capable of
reacting with activated alkynes.[13a,14] As shown in Figure 2a,
one primary amine group of BSA was taken to demonstrate
the metal-free “click” bioconjugation process, which could be
readily occurred by stirring a mixture of BSA and MTPABP-
pyo in aqueous solution at room temperature. The formation
of fluorescent MTPABP-BSA bioconjugates was successfully
verified by sodium dodecyl sulfate polyacrylamide gel elec-
trophoresis, where the MTPABP-labelled BSA exhibited
bright fluorescence under UV irradiation (Figure 2b). There-
fore, AIE-pyo luminogens manifested great potential for silk
functionalization.
mechanical properties of the AIEgen-silk threads were
compared with the untreated ones and no obvious difference
was observed (Figure S5). This meant that the bioconjugation
process did not affect the mechanical properties of natural
silk.
To demonstrate the advantages of AIE-pyo over tradi-
tional fluorescent dyes in silk functionalization, fluorescein
was chosen for comparison. First, as a typical ACQ fluoro-
phore, fluorescein showed strong fluorescence in solution, but
much weakened emission in the solid state due to strong p-p
interaction (Figure 4c and Table S2). Actually, the ACQ
effect has limited the solid-state applications of many
fluorophores including silk functionalization.[9] Thus, ACQ
As there are many primary amine groups in the lysine
residues of silk proteins, they can be employed for facile
bioconjugation with activated alkyne groups.[15] Thus, AIE-
pyo functionalized silks (AIEgen-silks) were conveniently
fabricated by immersing silk fibers into AIE-pyo solutions at
room temperature overnight, and AIEgen-silk threads and
fabrics with uniform fluorescence covering the entire visible
light region were obtained (Figure 3 and Table S1). Next, the
Figure 4. Photos of a) TPE-pyo, b) MTPABP-pyo and c) fluorescein in
solution and powdery states, and dye-modified silk fabrics before and
after washing (with soapy water for 10 times) taken under (upper)
room lighting and (lower) UV illumination. d) Variation of dyes
retained on the silk fabrics after repeated washing.
Figure 2. a) Metal-free “click” reaction of MTPABP-pyo with native BSA
without prefunctionalization at room temperature. b) Verification of
bioconjugation between MTPABP-pyo and BSA by SDS-PAGE separa-
tion: (left) bright field and (right) fluorescent image.
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dyes like fluorescein should be dispersed molecularly in silk
Due to the remarkable biocombability and controllable
fibers, which is hard to control and unstable for long-term
usage. Consequently, AIE luminogens with intense solid-state
emissions manifested inherent advantages in fabricating
fluorescent silk. Next, fluorescein-silk was fabricated through
hydrogen bond interaction, and its stability was evaluated and
compared with chemically conjugated AIEgen-silks. The blue
AIEgen-silk (AIE-silk-b) and red AIEgen-silk (AIE-silk-r)
fabrics exhibited intense emission even after ultrasonic
cleaning with soapy water for ten times (Figure 4a,b). In
contrast, although fluorescein modified-silk exhibited yellow
fluorescence due to the partial segregation of the fluorescein
molecules by silk polymeric chains, its fluorescence was
reduced dramatically after soap washing for ten times due to
the unstable physical interaction (Figure 4c). Obviously, the
retention rates of AIEgens on silk fabrics were much higher
than that of fluorescein, in corresponding to higher stability
contributed by chemical bonding than physical absorption
(Figure 4d and Figure S6). Owing to inherent solid-state
emission and stable covalent bonding, AIE-pyo luminogens
exhibited great advantages in fabricating fluorescent silks
with high brightness and high stability.
White light-emitting (WLE) materials and devices have
attracted considerable attention in lighting and displays. In
principle WLE could be achieved by mixing three primary
RGB colors (red, green and blue).[7,16] Thus, WLE silk was
fabricated through simultaneous bioconjugation with blue-,
green- and red-emissive AIE-pyo luminogens at a precisely
controlled molar ratio of 88:6:6, with a CIE coordinate of
(0.33, 0.36) (Figure 5a,b). The fluorescence resonance energy
transfer occurred due to the overlapping of absorption of red
AIEgen with the fluorescence of blue and green AIEgens
(Figure S7). Notably, WLE silk fibers of different shapes were
easily fabricated with excellent flexibility, demonstrating
potential applications in flexible displays and wearable
electronic devices (Figure 5c).
degradability, functionalized silks have been successfully
utilized for tissue engineering scaffolds.[5] In addition, red
and near infrared-emissive AIEgens play important roles in
bioimaging with high penetration depth and low tissue
autofluorescence.[17] Therefore, we attempted to fabricate
red-emissive silk materials through bioconjugation of hydro-
lyzed silk protein with red-emissive MTPABP-pyo. The
hydrodynamic size distribution of MTPABP-hydrolyzed silk
in 1% DMSO aqueous solution was evaluated and the
average diameter is 26.8 nm (Figure S8). Both hydrolyzed silk
and MTPABP-functionalized hydrolyzed silk showed excel-
lent biocombability on A549 cells even at a high concen-
tration of 125 mgmLÀ1 (Figure 6a,b). Next, one-photon cell
imaging study was evaluated by staining the A549 cells with
red-emissive MTPABP-functionalized hydrolyzed silk. As
indicated in Figure 6c, the plasma membrane could be
visualized clearly with excellent image contrast within
2 min, attributed to the remarkable cell attachment ability
of the silk protein.[18] Then, the dye gradually entered the cells
with increased emission intensity within 2 h, showing an
entire cellular uptake process. Moreover, we continued to
monitor the cell growth for different passages by confocal
imaging (Figure 7). Although the concentration of MTPABP-
functionalized hydrolyzed silk in cells gradually decreased
during the process of cell division, red emission could still be
clearly observed for up to 11 days, which demonstrated the
great potential of AIE-silk bioconjugates for long-term cell
tracking.
Recently, two-photon fluorescence (2PF) imaging has
attracted much attention in deep-tissue imaging due to its
great advantages such as low tissue autofluorescence, long-
wavelength excitation and high spatiotemporal resolution.[19]
Materials with large two-photon absorption (2PA) cross-
section (s2PA) are found to show enhanced 2PF imaging
performance, and the introduction of strong electron donor
and acceptor groups is an efficient strategy to increase the
s2PA value.[20] The s2PA values of the AIE-pyo luminogens were
measured by two-photon-induced fluorescence experiments.
As shown in Figure 8a, the s2PA values increased in accord-
ance with the extent of D-A interaction and among them, the
red-emissive MTPABP-pyo showed the highest s2PA value of
up to 190 GM under 920 nm excitation. Therefore, the
MTPABP-functionalized silk fabrics were chosen for further
two-photon imaging experiments. First, the fine morphology
of MTPABP-functionalized silk fabric was clearly visualized
under two-photon excitation, manifesting much better spa-
tiotemporal resolution than one-photon excitation (Fig-
ure S9a). In addition, the three-dimensional (3D) structure
of the silk fabric with a thickness of 160 mm could be precisely
reconstructed from top to bottom by 2PF imaging (Fig-
ure S9b). On the other hand, the noninvasive real-time
monitoring of bioscaffold is of great importance in drug
delivery and tissue engineering. Encouraged by the high
penetration depth of 2PF imaging, we further evaluated the
MTPABP-functionalized silk fabric for deep-tissue imaging
and bioscaffold monitoring. Chicken breast tissues were
selected to mimic the optical diffusion in biological systems.
As shown in Figure 8b, the structure of the silk fabrics was
Figure 5. a) Preparation of white light-emitting (WLE) silk through
bioconjugation with TPE-pyo, MTPEP-pyo and MTPABP-pyo at a molar
ratio of 88:6:6. b) Fluorescence spectrum of the WLE silk fabric and
(insert) its CIE coordinate. c) Fluorescence photos of flexible WLE
fabrics fabricated from the AIEgen-silk fibers.
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Figure 6. a) Fluorescence spectrum of hydrolyzed silk conjugated with MTPABP-pyo. b) Biocompatibility of the hydrolyzed silk and its AIEgen-
conjugate. c) Staining of A549 cells with MTPABP-functionalized hydrolyzed silk (100 mgmLÀ1); fluorescent images taken at lex =488 nm and
lem =565–700 nm.
Figure 7. Long-term tracking of A549 cells by MTPABP-functionalized hydrolyzed silk; fluorescent images taken at lex =488 nm and lem =565–
700 nm.
clearly visualized even the fabrics were covered by chicken
tissues. The red emission could penetrate through a thickness
of up to 1200 mm. Such a result demonstrated the great
possibility of the present bioconjugate for monitoring the silk
bioscaffold in real physiological systems.
exhibited great stability and realized full-color emissions by
rational molecular design. A white light-emitting silk was
achieved by simultaneous bioconjugation with red-, green-
and blue-emissive AIEgens. Additionally, red emissive
MTPABP-functionalized hydrolyzed silk was successfully
applied for real-time and long-term cell tracking. MTPABP-
functionalized silk fabrics showed large two-photon absorp-
tion and demonstrated great potential for deep-tissue imaging
and bioscaffold monitoring. It is believed that the facile
bioconjugation strategy developed in the present work will
inspire the development of functionalized silk materials with
Conclusion
In summary, we developed a simple strategy for silk
functionalization by AIEgens through facile metal-free click
bioconjugation. The chemically conjugated fluorescent silks
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Acknowledgements
We are grateful for financial support from the National
Science Foundation of China (21788102), the Research
Grants Council of Hong Kong (16305618, 16304819,
N_HKUT609/19 and C6009-17G), the Innovation and Tech-
nology Commission (ITC-CNERC14SC01 and ITCPD/17-9),
the Science and Technology Plan of Shenzhen
(JCYJ20170818113602462 and JCYJ20180507183832744),
and the Ming Wai Lau Centre for Reparative Medicine
Associate Member Programme (MWLC11SC2021).
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The authors declare no conflict of interest.
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Keywords: aggregation-induced emission · flexible display ·
long-term cell tracking · silk functionalization ·
two-photon emission
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ꢀ 2021 Wiley-VCH GmbH
www.angewandte.org
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Research Articles
Chemie
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Manuscript received: November 23, 2020
Revised manuscript received: March 18, 2021
Accepted manuscript online: March 24, 2021
Version of record online: && &&
,
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www.angewandte.org
ꢀ 2021 Wiley-VCH GmbH
Angew. Chem. Int. Ed. 2021, 60, 2 – 9
These are not the final page numbers!

Angewandte
Research Articles
Chemie
Research Articles
Silk Functionalization
Fluorescent silks with full-color emis-
sions were fabricated through facile
metal-free click bioconjugation with five
aggregation-induced emission lumi-
nogens (AIEgens). White light-emitting
silk was prepared and demonstrated
potential applications in flexible displays.
Moreover, red-emissive AIEgen-function-
alized silk materials were successfully
applied for long-term cell tracking and
two-photon bioimaging.
C. Liu, H. Bai,* B. He, X. He, J. Zhang,
C. Chen, Y. Qiu, R. Hu, F. Zhao, Y. Zhang,
W. He, J. H. C. Chau, S. Chen,
J. W. Y. Lam, B. Z. Tang*
&&&& — &&&&
Functionalization of Silk by AIEgens
through Facile Bioconjugation: Full-Color
Fluorescence and Long-Term Bioimaging
Angew. Chem. Int. Ed. 2021, 60, 2 – 9
ꢀ 2021 Wiley-VCH GmbH
www.angewandte.org
&&&&
These are not the final page numbers!