A FRET probe with AIEgen as the energy quencher: Dual signal turn-on for self-validated caspase detection

Article abstract of DOI:10.1039/c6sc00055j

The accurate detection of biological substances is highly desirable to study various biological processes and evaluate disease progression. Herein, we report a self-validated fluorescent probe which is composed of a coumarin fluorophore as the energy dono

Full text of DOI:10.1039/c6sc00055j

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DOI: 10.1039/C6SC00055J
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FRET Probe with AIEgen as the Energy Quencher: Dual Signal Turn-
On for Self-Validated Caspase Detection
Received 00th January 20xx,
Accepted 00th January 20xx
Youyong Yuan,a,† Ruoyu Zhang,a,† Xiamin Cheng,a,† Shidang Xu,a and Bin Liua,b,
*
Accurate detection of biological substance is highly desirable to study various biological processes and evaluate disease
progression. Herein, we report a self-validated fluorescent probe which is composed of a coumarin fluorophore as energy
donor and a fluorogen with aggregation-induced emission characteristics (AIEgen) as energy quencher linked through a
caspase-3 specific peptide substrate. Unlike the traditionally widely studied fluorescence resonance energy transfer (FRET)
probes, our new generation of FRET probe itself is non-fluorescent due to energy transfer as well as the dissipation of the
acceptor energy through free molecular motion of the AIEgen. Upon interaction with caspase-3, the probe displays strong
green and red fluorescent signals synchronously due to the separation of donor-quencher and aggregation of the released
AIEgen. The fluorescence turn-on with dual signal amplification allows real-time and self-validated enzyme detection with
high signal-to-background ratio, providing a good opportunity to accurately monitor various biological processes in real-
time manner.
DOI: 10.1039/x0xx00000x
www.rsc.org/
different image modalities have been developed for self-
validated bioimaging in order to provide more accurate
results.[5] Up to now, a variety of biological probes based on
Introduction
Fluorescent probes have attracted increasing attention in
biomedical research due to their high sensitivity, good
selectivity, non-invasiveness and the capability of real-time
detection.[1] Fluorescence turn-on probes are superior to turn-
off probes due to their lower background signal and higher
signal output.[2] Fluorescence resonance energy transfer (FRET)
is one of the most widely exploited mechanisms for the design
of fluorescence turn-on probes, which have been successfully
utilized in sensing, imaging, environmental monitoring, and
medical diagnosis.[3] Two strategies have been generally
utilized to design FRET probes. One is to conjugate a
fluorescent dye with a quencher (donor-quencher model) and
the other is to link two fluorescent dyes which can form the
donor-acceptor pair (donor-acceptor model).[4] Once these
FRET probes are exposed to analytes, the separation between
the donor and quencher/acceptor leads to readable signals.
While the donor-quencher model exhibits single fluorescent
signal output, the donor-acceptor model shows donor
fluorescence turn-on at the expense of the acceptor emission.
The single fluorescent signal turn-on can only provide limited
information, which is sometimes insufficient for accurate
detection.[5] Recently, several “always on” probes with
fluorescence imaging, magnetic resonance imaging (MRI),
positron emission tomography (PET) and photoacoustic
imaging have been actively explored to offer dual signal output
in one study.[6] However, single fluorescence turn-on probe
with dual signal output upon encountering specific analyte
have not been reported yet. It is thus of great interest to
develop single fluorescent probes with dual signal turn-on at
different emission wavelengths for self-validated sensing and
imaging, which remains challenging.
In Recent years, fluorogens with aggregation-induced
emission characteristics (AIEgens) have attracted considerable
attention in biosensing and bioimaging.[7] Opposite to
traditional fluorophores that show a notorious phenomenon
known as aggregation-caused quenching (ACQ),[8] AIEgens are
non-emissive in molecularly dissolved state but can be induced
to emit strongly in aggregates due to the restriction of
intramolecular motion (RIM) and prohibition of energy
dissipation via non-radiative channels.[9] Based on this unique
property, several fluorescence turn-on probes have been
developed for detection and imaging of various analytes.[7c]
These probes are generally based on AIEgens conjugated to
hydrophilic recognition elements. When the AIE probes are
well-dissolved in aqueous media, the background fluorescence
is very low. Upon specific analyte recognition, the probes are
cleaved to release the hydrophobic AIEgens and yield bright
fluorescence. As compared to traditional FRET probes which
require dual labelling to realize fluorescence turn-on, these
singly labelled AIE probes show high simplicity, which makes
them very useful in the development of multifunctional probes
a. Department of Chemical and Biomolecular Engineering, National University of
Singapore, 4 Engineering Drive 4, Singapore 117585, E-mail: cheliub@nus.edu.sg
b. Institute of Materials Research and Engineering, Agency for Science, Technology
and Research (A*STAR), 3 Research Link, Singapore, 117602
† These authors contributed equally to this work.
Electronic Supplementary Information (ESI) available: [details of any
supplementary information available should be included here]. See
DOI: 10.1039/x0xx00000x
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for monitoring of multiple processes in one go.[7c] These
successful examples have motivated us to explore whether the
energy dissipation of AIEgens could affect other fluorophores
conjugated to the probe. In this case, it would offer a new
generation of energy quenchers, which is able to fluoresce
once it is separated from the energy donor and offers for the
first time a dual signal turn-on probe for self-validated
biosensing and bioimaging.
O
O
O
HBTU, TEA
O
View Article Online
S
O
S
N
HO
DOI: 10.1039/C6SC00055J
NMe(OMe).HCl
O
N
OH
2
O
O
O
O
O
O
O
O
O
S
N
O
N
O
N
O
TiCl4, Zn
THF
2
O
+
O
BuLi
S
Br
1
3
Br
O
O
1. NaOH in MeOH-
water (3:1); 2. HCl
O
O
O
As a proof of concept, in this contribution, we report a
fluorescent probe based on coumarin (Cou) as energy donor
and an AIEgen as the energy quencher conjugated via a Asp-
Glu-Val-Asp (DEVD) substrate for caspase-3 detection (Scheme
1).10 The probe itself is non-fluorescent due to the energy
transfer and dissipation of the acceptor energy through free
motion of AIEgens. Upon addition of caspase-3, it displays
strong green fluorescence of Cou due to the separation of
donor-acceptor and intense red fluorescence of AIEgen due to
the aggregation of the released AIEgen residue. This dual
fluorescent signal turn-on can be used for caspase-3 detection
both in solution and in cells, which opens new avenues for the
development of next generation self-validated FRET probes
with high signal-to-background ratio and fluorescence
amplification.
O
H2N
N3
OH
S
HBTU, TEA, DMF
H
N
N3
4
S
5
O
O
O
NC
CN
TiCl4/Pyridine
O
O
COOH
COOH
H
N
CuSO4,
sodium ascorbate
O
O
O
H
N
+
H2N
N
N
NH2
DMSO/H2O
(10:1, v/v)
H
H
H
N
O
O
N3
COOH
S
O
Alkyne-DEVD
NC
CN
TPETP-N3
O
O
COOH
N
O
COOH
H
N
NC
S
CN
O
O
O
H
N
O
H2N
N
O
O
N
N
NH2
N
H
H
O
O
Cou-NHS
H
N
COOH
N
DIPEA
O
N
TPETP-DEVD
O
O
COOH
O
COOH
H
N
NC
CN
O
O
O
H
NH
N
N
H
N
NH2
N
H
N
O
O
O
O
H
S
COOH
N
N
O
Results and discussion
N
Cou-DEVD-TPETP
O
O
Scheme 2. Synthetic route to the probe of Cou-DEVD-TPETP.
HBTU: O-(benzotriazol-1-yl)-N,N,N′,N′-tetramethyluronium
hexafluorophosphate; TEA: triethylamine; TiCl4: titanium(IV)
chloride; Zn: zinc; THF: tetrahydrofuran; BuLi: butyllithium;
DMF:
N,
N-dimethylformamide;
DIPEA:
N,
N-
diisopropylethylamine; Cou-NHS: 7-(diethylamino)coumarin-3-
carboxylic acid N-succinimidyl ester.
support to produce azide-functionalized TPETP (TPETP-N3). The
“click” reaction between TPETP-N3 and alkyne-functionalized
DEVD catalyzed by copper(II) sulfate (CuSO4) and sodium
ascorbate (NaAsc) afforded amine terminated TPETP-DEVD
which was further reacted with 7-(diethylamino)coumarin-3-
carboxylic acid N-succinimidyl ester (Cou-NHS) in the presence
of N, N-diisopropylethylamine (DIPEA) to afford Cou-DEVD-
TPETP in 52% yield after HPLC purification. Detailed
characterization data are shown in Figures S1-S8. The AIE
property of TPETP-N3 was confirmed by studying its PL spectra
in DMSO or DMSO/water mixtures (v/v = 1/99). As shown in
Figure S9A, TPETP-N3 is almost non-fluorescent in DMSO which
should be due to the easy intramolecular motions of the TPE
phenyl rings in benign solvents.[7a] However, TPETP-N3 in
DMSO/water mixtures (v/v = 1/99) is highly fluorescent, which
shows 110-fold brighter fluorescence than that in DMSO. The
increase in fluorescence intensity is attributed to the
aggregation of TPETP-N3, which restricts the intramolecular
motion when the water fraction is increased. The formation of
TPETP-N3 aggregates in aqueous media was also confirmed by
laser light scattering (LLS) measurements and transmission
electron microscopy (TEM) image (Figure S9), while there is no
Scheme 1. Schematic illustration of the FRET probe using
AIEgen as energy quencher with dual signal output for self-
validated caspase-3 detection.
Recently, some AIEgens with red emission has been
developed for bioimaging and tetraphenylethenethiophene
(TPETP) was selected for this study.11 The synthetic route to
the azide-functionalized TPETP (TPETP-N3) and the probe Cou-
DEVD-TPETP are shown in Scheme 2. Compound
synthesized by McMurry reaction between bis(4-
methoxyphenyl)methanone and (4-
bromophenyl)(phenyl)methanone in the presence of TiCl4 and
1 was
Zn. Subsequently, compound
in the presence of butyllithium to yield compound
carboxyl group of compound
hydroxide in methanol-water mixture, which was further
reacted with 3-azidopropan-1-amine to yield compound
Compound was further reacted with malononitrile on SiO2
1
was reacted with compound
2
3. The
3
was deprotected by sodium
5.
5
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LLS signal detected in DMSO solution. The absorption and 3 at different concentrations for 1 h at 37 °C, theVeiemw AisrtsiciloenOsnlionef
emission spectra of Cou and TPETP-N3 are shown in Figure S10. Cou-DEVD and TPETP residue exhibit conDceOnI:t1r0a.t1i0o3n9-/dCe6SpCe0n0d0e5n5Jt
The emission of Cou and the absorption of TPETP-N3 shows a turn-on at 465 nm and 665 nm, respectively. When the probe
perfect spectral overlap, indicating that they could form an was treated with 200 pM caspase-3 for 1 h, the fluorescence of
energy transfer pair.
Cou-DEVD and TPETP residue showed 55 and 37 fold increase
compared to their intrinsic emission in the probe, respectively.
In addition, the peak PL intensities of Cou and TPETP are
shown in Figure S12, which correlate linearly to the
concentration of caspase-3 with R2 = 0.97, indicating that the
enzyme concentration can be quantified and self-validated
through monitoring the PL intensity changes of Cou and TPETP.
In addition, in the presence of caspase-3 inhibitor 5-[(S)-(+)-2-
(methoxymethyl)pyrrolidino] sulfonylisatin, the probe remains
weakly emissive even after the treatment with caspase-3. The
kinetic analysis of the enzymatic reaction was also studied by
incubating casp-3 with different concentrations of Cou-DEVD-
TPETP at 37 °C. As shown in Figure S13, the Michaelis
constants (KM) and the kinetic constants (kcat) were calculated
to be 7.70 μM and 2.69 s-1, which are comparable to the
previous FRET probe.[10a] The specific cleavage of DEVD by
caspase-3 was further confirmed by reverse phase HPLC and
mass spectrometry study through the formation of Cou-DEVD
and TPETP residue (Figure S14).
300
250
200
150
100
50
150
120
90
60
30
0
200
150
100
50
B
A
PL @ 465 nm
PL @ 665 nm
PL @ 465 nm
0
0
20 40 60 80 100 120
100
75
50
25
0
Figure 1. (A) Photoluminescence (PL) spectra of Cou, TPETP-N3
and Cou-DEVD-TPETP (10 μM) in DMSO/PBS buffer (v/v = 1/99).
(B) PL spectra of Cou-DEVD-TPETP (10 μM) upon incubation
with different concentrations of caspase-3 (λex: 405 nm,
PL @ 665 nm
0
0
20 40 60 80 100 120
-3
Casp
-7
Casp
sin
Time (min)
emission collected from 430
 550 nm is from the Cou-DEVD
BSA
Pep
Trypsin
Lysozyme
and that at 600 – 780 nm is from the TPETP residue).
Figure 2. (A) PL intensities monitored at both 465 and 665 nm
for Cou-DEVD-TPETP (10 μM) upon treatment with various
proteins; (B) Time-dependent PL intensities for Cou-DEVD-
TPETP (10 μM) upon addition of caspase-3 (λex: 405 nm).
To validate the energy transfer between Cou and TPETP-N3,
the optical properties of Cou, TPETP-N3 and Cou-DEVD-TPETP
were studied in DMSO/PBS buffer (v/v = 1/99). Cou and TPETP-
N3 have absorption maxima at 445 and 470 nm, with emission
maxima at 465 and 665 nm, respectively (Figure S10). As both
Cou and TPETP-N3 show obvious absorption at 405 nm, it
offers the possibility of dual signal collection upon a single
wavelength excitation. As shown in Figure 1A, upon excitation
at 405 nm, both Cou and TPETP-N3 are highly fluorescent while
Cou-DEVD-TPETP emits very weakly, which indicates that the
probe has extremely low background signal as designed. The
rationale behind the fluorescence quenching of Cou and TPETP
parts in the probe is due to energy transfer from the former to
the latter, and the dissipation of the AIEgen energy through
free motion of the molecules. The fluorescence of the probe
remains weak in aqueous media with different ionic strength
or in cell culture medium (Figure S11). Upon addition of
caspase-3, the cleavage of DEVD separates Cou-DEVD away
from the proximity of TPETP. This leads to the recovery of the
green fluorescence of Cou and synchronously allows the
formation of TPETP residue aggregates with red fluorescence
turn-on. As shown in Figure 1B, upon treatment with caspase-
The selectivity of the probe was studied by incubating the
probe with different caspase enzymes as well as several
proteins including lysozyme, pepsin, bovine serum albumin
and trypsin. As shown in Figure 2A and Figure S15A, only the
probe treated with caspase-3/-7 displays significant
fluorescence increase, confirming the specificity of the probe
for caspase-3/-7. The time-dependent fluorescence change of
the probe after incubation with caspase-3/-7 and cell lysate of
normal and apoptotic cells were also studied. As shown in
Figure 2B, a quick and steady fluorescence increase at both
465 and 665 nm is observed when caspase-3 is added. On the
other hand, the HeLa cells were induced to undergo apoptosis
by the treatment of staurosporine (STS), a commonly used
apoptosis inducer,[12] and the cell lysates were incubated with
the probe. As shown in Figure S15, the fluorescent signals at
both wavelengths increase along with incubation time which
are similar to the solution study shown in Figure 2B. In
contrast, no fluorescence change is observed when the probe
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is incubated with HeLa cell lysate without STS treatment, Figure 4. Confocal images of apoptotic HeLa cellsVtierweaArtteicdle Ownilitnhe
DOI: 10.1039/C6SC00055J
indicating that the probe is stable with cellular proteins and it Cou-DEVD-TPETP (10 μM) in the absence (A) and (B) presence
can be specifically recognized by caspase enzyme with self- of caspase-3 inhibitor and stained with anti-caspase-3 primary
validation.
antibody and a Texas Red-labeled secondary antibody. Green
fluorescence (Cou-DEVD, A1, B1, Ex: 405 nm; Em: 505-525 nm);
orange fluorescence (Texas Red, A2, B2, Ex: 543 nm, Em:
610−640 nm); red fluorescence (TPETP residue, A3, B3, Ex: 405
nm, Em: > 650 nm); A4, B4 are the overlay images of A1-A3 and
B1-B3, respectively. Due to the low absorbance of TPETP at
543 nm, its emission spectral overlap with Texas Red is
negligible.
To further confirm that the probe can image cell apoptosis,
HeLa cells were co-stained with anti-caspase-3 primary
antibody and a Texas Red-labeled secondary antibody. As
shown in Figure 4A, the fluorescence of Cou-DEVD and TPETP
residue in HeLa cells with STS treatment overlaps well with the
immunofluorescence signals from Texas Red. However, when
the cells are pretreated with caspase-3/-7 inhibitor, both
intensities are greatly reduced while the fluorescence of Texas
Red remains (Figure 4B). This is because the activity of
caspase-3/-7 enzyme is prohibited upon inhibitor treatment,
but the apoptosis process is not affected. Similar results were
also observed in MDA-MB-231 cells (Figure S17). Overall, these
results further confirm the caspase-3/-7 specific turn-on of the
probe fluorescence in cells.
It is known that most drugs induce cell death through
apoptosis pathway.[13] To verify the potential of this probe for
self-validated drug screening, the probe incubated cells were
treated with several cell apoptosis inducing drugs and the
apoptosis-inducing capabilities were evaluated by monitoring
the fluorescent signal changes. As shown in Figure 5, the
strongest fluorescence of Cou-DEVD and TPETP residue is
observed when the cells are treated with STS, while moderate
fluorescence is observed when the cells are treated with the
anticancer drug of doxorubicin (DOX) or cisplatin. The cells
treated with Na Asc show the lowest fluorescence, indicating
Na Asc is not a good apoptosis inducer. Similar results were
also observed in MDA-MB-231 cells (Figure S18), revealing the
generality of the probe. To test whether the probe could be
used to quantify the capability of different drugs in inducing
cell apoptosis, the cells were further treated with different
amount of Na Asc, cisplatin, DOX and STS and the fluorescent
signals were studied. As shown in Figure 5C, the fluorescent
signals of Cou-DEVD and TPETP residue intensify
synchronously with the drug concentration for all the four
drugs. It should be noted that the probe (up to 25 μM) do not
show any obvious cytotoxicity to both cells after 48 h
incubation (Figure S19). Collectively, these results indicate that
Cou-DEVD-TPETP can quantitatively analyze the capability of
different drugs to induce cell apoptosis in living cells with self-
validation, which offers a new opportunity to accurately
evaluate the efficiency of new anticancer drugs.
Figure 3. Confocal images of Cou-DEVD-TPETP (10 μM)
incubated HeLa cells upon treatment with STS (1 μM) for
different time. Green fluorescence (Cou-DEVD, Ex: 405 nm; Em:
505-525 nm); orange fluorescence (nucleus dyed with SYTO®
orange, Ex: 543 nm, Em: 610-640 nm); A1-D1 are the overlay
images of the fluorescence of Cou and SYTO® orange; red
fluorescence (TPETP residue, A2-D2, Ex: 405 nm, Em: > 650 nm).
(E, F) Flow cytometric analysis of Cou-DEVD (E) and TPETP
residue (F) fluorescence in HeLa cells after treatment with STS
(1 μM).
To explore the potential of using Cou-DEVD-TPETP for
caspase imaging in live cells, the probe was incubated with
HeLa and MDA-MB-231 cells and subsequently treated with
STS. The fluorescence changes at both wavelgnths were
monitored by confocal laser scanning microscopy. As shown in
Figure 3 and Figure S16, the fluorescent signals of Cou-DEVD
and TPETP residue increase gradually and synchronously with
the cellular apoptotic progress upon addition of STS. The
fluorescence changes after STS treatment were also confirmed
by flow cytometric analysis (Figures 3E and 3F). These results
clearly support that the probe with dual signal turn-on can be
used for self-validation of caspase-3 activation and for real-
time monitoring of apoptosis process in live cells.
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Acknowledgements
View Article Online
We thank the Ministry of Defence (R279-D0O0I0: -1304.1003-29/3C26)S,CS0M00A5R5TJ
(R279-000-378-592), the Ministry of Education (R279-000-391-
112), Singapore NRF Investigatorship (R279-000-444-281) and
the Institute of Materials Research and Engineering of
Singapore (IMRE/14-8P1110) for financial support.
References
1
(a) M. I. J. Stich, L. H. Fischer, O. S. Wolfbeis, Chem. Soc. Rev.
2010, 39, 3102-3114; (b) Z. G. Yang, J. F. Cao, Y. X. He, J. H.
Yang, T. Kim, X. J. Peng, J. S. Kim, Chem. Soc. Rev. 2014, 43
,
4563-4601; (c) J. Zheng, R. H. Yang, M. L. Shi, C. C. Wu, X. H.
Fang, Y. H. Li, J. H. Li, W. H. Tan, Chem. Soc. Rev. 2015, 44
,
3036-3055.
2
3
4
5
6
J. Chan, S. C. Dodani, C. J. Chang, Nat. Chem. 2012, 4, 973-
984.
J. J. Li, F. F. Cheng, H. P. Huang, L. L. Li, J. J. Zhu, Chem. Soc.
Rev. 2015, 44, 7855-7880.
K. E. Sapsford, L. Berti, I. L. Medintz, Angew. Chem. Int. Ed.
2006, 45, 4562-4588.
D. E. Lee, H. Koo, I. C. Sun, J. H. Ryu, K. Kim, I. C. Kwon, Chem.
Soc. Rev. 2012, 41, 2656-2672.
Figure 5. Confocal images of Cou-DEVD-TPETP (10 μM)
incubated HeLa cells upon treatment with (A) sodium
ascorbate (Na Asc), (B) cisplatin, (C) DOX and (D) STS. Green
fluorescence (Cou-DEVD, Ex: 405 nm; Em: 505-525 nm); orange
fluorescence (nucleus dyed with SYTO® orange, Ex: 543 nm, Em:
610-640 nm); A1-D1 are the overlay images of the
fluorescence of Cou and SYTO® orange; red fluorescence
(TPETP residue, A2-D2, Ex: 405 nm, Em: > 650 nm). (E) PL
intensities of Cou-DEVD and TPETP residue in HeLa cells
treated with of Na Asc, cisplatin, DOX and STS at different
concentrations.
(a) B. E. Rolfe, I. Blakey, O. Squires, H. Peng, N. R. B. Boase, C.
Alexander, P. G. Parsons, G. M. Boyle, A. K. Whittaker, K. J.
Thurecht, J. Am. Chem. Soc. 2014, 136, 2413−2419; (b) V. S. R.
Harrison, C. E. Carney, K. W. MacRenaris, E. A. Waters, T. J.
Meade, J. Am. Chem. Soc., 2015, 137, 9108–9116; (c) Y. Sun,
X. W. Ma, K. Cheng, B. Y. Wu, J. L. Duan, H. Chen, L. H. Bu, R.
P. Zhang, X. M. Hu, Z. X. Deng, L. Xing, X. C. Hong, Z. Cheng,
Angew. Chem. Int. Ed. 2015, 54, 5981–5984 (d) Y. H. Wang, S.
Y. Song, J, H. Liu, D. P. Liu, H. J. Zhang, Angew. Chem. Int. Ed.
2015, 54, 536–540; (e) X. J. Wu, B. J. Lin, M. Z. Yu, L. Yang, J.
H. Han, S. F. Han, Chem. Sci. 2015,
Q. Cheng, S. Yuan, J. C. Qian, K. Zhong, Y. F. Qian, Y. Z. Liu,
Chem. Sci. 2015, , 6607-6613.
6, 2002-2009; (f) Q. Wu,
6
Conclusions
7
(a) J. Mei, N. L. C. Leung, R. T. K. Kwok, J. W. Y. Lam, B. Z.
Tang, Chem. Rev. 2015, 115, 11718-11940; (b) W. J. Guan, W.
J. Zhou, C. Lu, B. Z. Tang Angew. Chem. Int.
Ed. 2015, 54, 15160-15164; (c) J. Liang, B. Tang, B. Liu, Chem.
Soc. Rev. 2015, 44, 2798-2811; (d) K. Li, B. Liu, Chem. Soc.
Rev. 2014, 43, 6570-6597; (e) X. Zhang, X. Zhang, L. Tao, Z.
In summary, we developed a simple but unique fluorescent
probe with dual signal turn-on for accurate caspase-3
detection with self-validation. Thanks to the unique property
of the AIEgen, the fluorescence of the probe is initially
quenched, but two-signal turn-on is produced upon interaction
with caspase-3 enzyme. The dual-signal turn-on enables real-
time monitoring of caspase-3 activity in solution and in live
cells with high efficiency, which has been utilized for self-
validated enzyme detection and drug screening. Compared to
traditional FRET probes that show single fluorescence turn-on
upon interaction with the analytes, the probe developed in
this work using AIEgen as the energy quencher does not
complicate the probe design, but offers for the first time two-
signal turn-on upon analyte recognition. In addition, this is the
first time that the energy quencher could change its role to a
signal reporter upon analyte recognition. Our design strategy
of AIEgen based FRET probe can be generalized to other
probes simply by changing DEVD to other cleavable substrate,
which will open new avenues for self-validated diagnosis,
imaging and drug screening applications. Potential translation
would still be needed to evaluate the in vivo cytotocity and
design probes with longer absorption wavelength.
Chi, J. Xu, Y. Wei, J. Mater. Chem. B 2014, 2, 4398-4414; (f) Q.
L. Hu, M. Gao, G. X. Feng, B. Liu, Angew. Chem. Int. Ed. 2014,
53, 14225-14229; (g) Y. Yuan, R. T. Kwok, B. Z. Tang, B. Liu, J.
Am. Chem. Soc. 2014, 136, 2546-2554; (h) S. Xu, Y. Yuan, X.
Cai, C. Zhang, F. Hu, J. Liang, G. Zhang, D. Zhang, B. Liu. Chem.
Sci. 2015, 6, 5824-5830; (i) X. D. Xue, Y. Y. Zhao, L. R. Dai, X.
Zhang, X. H. Hao, C. Q. Zhang, S. D. Huo, J. Liu, C. Liu, A.
Kumar, W. Q. Chen, G. Z. Zou, X. J. Liang, Adv. Mater. 2014,
26, 712-717; (j) Y. Y. Yuan, C. J. Zhang, M. Gao, R. Y. Zhang, B.
Z. Tang, B. Liu, Angew. Chem. Int. Ed. 2015, 54, 1780-1786; (k)
A. D. Shao, Y. S. Xie, S. J. Zhu, Z. Q. Guo, S. Q. Zhu, J. Guo, P.
Shi, T. D. James, H. Tian, W. H. Zhu, Angew. Chem. Int. Ed.
2015, 54, 7275-7280; (l) Z. Xie, C. Chen, S. Xu, J. Li, Y. Zhang, S.
Liu, J. Xu, Z. Chi, Angew. Chem. Int. Ed. 2015, 54, 7181-7184.
J. B. Birks, Photophysics of Aromatic Molecules; Wiley:
London, 1970.
8
9
Y. N. Hong, J. W. Y. Lam, B. Z. Tang, Chem. Soc. Rev. 2011, 40
,
5361-5388.
10 (a) K. Boeneman, B. C. Mei, A. M. Dennis, G. Bao, J. R.
Deschamps, H. Mattoussi, I. L. Medintz, J. Am. Chem. Soc.
2009, 131, 3828-3829; (b) J. F. Lovell, M. W. Chan, Q. C. Qi, J.
Chen, G. Zheng, J. Am. Chem. Soc. 2011, 133, 18580–18582;
(c) L. Zhang, J. P. Lei, J. T. Liu, F. J. Ma, H. X. Ju, Chem. Sci.
This journal is © The Royal Society of Chemistry 20xx
J. Name., 2013, 00, 1-3 | 5
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Chemical Science
Page 6 of 6
ARTICLE
Journal Name
2015, 6, 3365-3372; (d) D. Ye, A. J. Shuhendler, P. Pandit, K.
View Article Online
DOI: 10.1039/C6SC00055J
D. Brewer, S. S. Tee, L. Cui, G. Tikhomirov, B. Rutt, J. H. Rao,
Chem. Sci. 2014, 5, 3845–3852; (e) Z. Gu, A. Biswas, K. Joo, B.
L. Hu, P. Wang, Y. Tang, Chem. Commun. 2010, 46, 6467-
6469.
11 (a) Y. Y. Huang, F. Hu, R. Zhao, G. X. Zhang, H. Yang, D. Q.
Zhang, Chem. Eur. J. 2014, 20, 158-164; (b) F. Hu, Y. Y. Huang,
G. X. Zhang, R. Zhao, H. Yang, D. Q. Zhang, Anal. Chem. 2014,
86, 7987–7995; (c) Y. Y. Yuan, C. Zhang, R. Kwok, S. Xu, R.
Zhang, J. Wu, B. Z. Tang, B. Liu, Adv. Funct. Mater. 2015, 25
,
6586-6595; (d) Y. Y. Yuan, C. J. Zhang, B. Liu, Angew. Chem.
Int. Ed. 2015, 54, 11419-11423.
12 X. D. Zhang, S. K. Gillespie, P. Hersey, Mol. Cancer Ther. 2004,
3
, 187-197.
13 H. Okada, T. W. Mak, Nat. Rev. Cancer 2004,
4
, 592-603.
6 | J. Name., 2012, 00, 1-3
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