Water-soluble near-infrared fluorescent probes enhanced by ionic co-assembly of a four-armed amphiphile with SDBS: Toward application in cell imaging

Article abstract of DOI:10.1016/j.dyepig.2020.108541

Near-infrared (NIR) organic fluorescent probes have attracted great deal of attention for application in cell imaging, where the probes demand good water solubility, strong emission and good cytocompatibility. In this study, a conjugate of anthraquinone a

Full text of DOI:10.1016/j.dyepig.2020.108541

Journal Pre-proof
Water-soluble near-infrared fluorescent probes enhanced by ionic co-assembly of a
four-armed amphiphile with SDBS: Toward application in cell imaging
Lina Zhang, Shixin Zhou, Shenxi Min, Xiaobo Yu, Guangliang Hou, Qin Tong, Bin
Dong, Huifang Zhu, Bo Song
PII:
S0143-7208(19)33001-3
DOI:
https://doi.org/10.1016/j.dyepig.2020.108541
DYPI 108541
Reference:
To appear in: Dyes and Pigments
Received Date: 22 December 2019
Revised Date: 2 May 2020
Accepted Date: 10 May 2020
Please cite this article as: Zhang L, Zhou S, Min S, Yu X, Hou G, Tong Q, Dong B, Zhu H, Song
B, Water-soluble near-infrared fluorescent probes enhanced by ionic co-assembly of a four-armed
amphiphile with SDBS: Toward application in cell imaging, Dyes and Pigments (2020), doi: https://
doi.org/10.1016/j.dyepig.2020.108541.
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2020 Published by Elsevier Ltd.

Lina Zhang: Writing - original draft, Data curation,
Investigation. Shixin Zhou: Formal analysis. Shenxi Min: Formal
analysis. Xiaobo Yu: Resources. Guangliang Hou: Formal
analysis. Qin Tong: Data curation. Bin Dong: Resources. Huifang
Zhu: Resources. Bo Song: Conceptualization, Supervision, Project
administration, Writing - review & editing.

Water-soluble near-infrared fluorescent probes enhanced by ionic co-assembly of
a four-armed amphiphile with SDBS: toward application in cell imaging
a
b
a
a
a
a
Lina Zhang, Shixin Zhou, Shenxi Min, Xiaobo Yu, Guangliang Hou, Qin Tong,
c
d
a
Bin Dong, Huifang Zhu* , Bo Song*
a
College of Chemistry, Chemical Engineering and Materials Science, Soochow
University, Suzhou 215123, P. R. China. E-mail: songbo@suda.edu.cn
b
Department of Cell Biology and Stem Cell Research Center, School of Basic Medical
Sciences, Peking University Health Science Center, Beijing 100191, P. R. China
c
Jiangsu Key Laboratory for Carbon-Based Functional Materials & Devices, Soochow
University, Suzhou, 215123, P. R. China.
d
Analysis and Testing Center, Soochow University, Suzhou, 215123, P. R. China.
E-mail: hfzhu@suda.edu.cn
Keywords
Near-infrared fluorescence
Ionic co-assembly
Emission enhancement
Aggregation-induced emission
Cell imaging

Abstract
Near-infrared (NIR) organic fluorescent probes have attracted great deal of attention
for application in cell imaging, where the probes demand good water solubility, strong
emission and good cytocompatibility. In this study, a conjugate of anthraquinone and
tetraphenylethylene is introduced into an amphiphile (denoted by BTPEAQ-10) with
four pyridinium terminated aliphatic chains. The assemblies of BTPEAQ-10 show an
NIR emission peaked at 670 nm. Ionic co-assembly with sodium dodecyl benzene
sulfonate leads to drastic enhancement of the fluorescent emission to 30-fold.
Controlling experiments on different surfactants and anionic groups show that both
the hydrophilic group and the alkyl chain play critical role to the emission
enhancement. Both the assemblies and co-assemblies have good water solubility and
cytocompatibility, and were applied in cell imaging. The co-assemblies showed much
better contrast of cell imaging due to the higher emission property.
1
. Introduction
Supramolecular assembly presents a versatile platform for tuning the
emission of fluorescent dyes, and thus owns great potential in preparation of
fluorescent probes applicable in the field of cell imaging.[1-4] Among them,
fluorophores with near-infrared (NIR) emission are developing rapidly owing to
their advantages, such as overcoming the auto-fluorescence of the
bio-systems,[5-7] reducing photo-damage,[8, 9] and providing deeper tissue
penetration[10, 11] relative to fluorophores in the short-wavelength
region.[12-17] Cells contain a large quantity of water as well as organelles and
lipid membranes. Thus, the organic fluorescent dyes, as being applied as probes
in the cells, inevitably aggregate due to their inherent hydrophobic nature.[18,
1
9] Therefore, good water-solubility and strong emissive property are requisite

in their aggregated state for labelling the cells.[20, 21] On this regard, marriage
of supramolecular assembly and fluorescent dyes with aggregation-induced
emission (AIE) properties would produce new fluorescent probes that meet the
requirement of applications in cell imaging.[22-24]
To obtain NIR emission, narrow band gap dyes are classically realized by
extending π-conjugation system,[25, 26] or introducing strong donor-acceptor
groups to the aromatics.[27, 28] For the traditional fluorescent dyes, a big
π-system often suffers from the aggregation-caused quenching of fluorescence
emission.[29-32] On this regard, the AIE dyes show great advantages, and such
kinds of dyes usually own big Stokes shift, which are favoured for application
as probes in biosystems.[33-35] Recently, Bin Liu et al. reported a NIR AIE
dots, which is composed of fluorescent dyes by conjugating one anthraquinone
and two tetraphenylethylene (TPE) groups. This molecule shows rather weak
fluorescent emission. However, encapsulation by DSPE-PEG block copolymer
or silica colloids led to strong emission and efficient singlet oxygen.[28]
Ionic co-assembly has been demonstrated an effective method to tune the
emission in terms of intensity and wavelength,[36-42] which was usually
realized by changing the packing mode or the distance of π-plane.[43-47] For
instance, Arun K. Nandi et al. found that the controllable co-assembly of ionic
surfactants with opposite charges could be used to turn on/off reversible
fluorescence of ionic agglomerates.[48] Subodh Kumar et al. showed the
possibility of enhancing the emission intensity by ionic assembly far below the
critical micelle concentration (CMC) of the surfactants.[49]
In this study, we introduced the AIE dyes designed by Bin Liu et al.[28] as
a core to an amphiphile with four pyridinium terminated alkyl chains, and
attempted to tune the emission by ionic co-assembly through adding a second
amphiphile. It turns out that both the assemblies and co-assemblies have good
water solubility, and the latter showed 30-fold higher intensity of NIR emission
compared to the former. Controlling experiments on different amphiphiles and
salts show that both the hydrophilic group and the alkyl chain play critical role

to the emission enhancement. The co-assemblies have excellent
cytocompatibility and were successfully applied on HeLa cells imaging.
Scheme 1. The synthetic route of BTPEAQ-10.
2
. Results and discussion
The synthetic route of BTPEAQ-10 is illustrated in Scheme 1. The details
1
of synthesis are shown in the experimental section, and the corresponding H
NMR and mass spectra (MS) are shown in electronic supplementary
information (ESI). BTPEAQ-10 has good solubility in methanol, and moderate
solubility in water. Being dissolved in methanol, BTPEAQ-10 showed an
emission peak at around 425 nm, excited by 340 nm light. Keeping the
-
5
-1
concentration of BTPEAQ-10 (CB) at 1 × 10 mol L , and adding water into
the solution resulted in the decrease of the emission intensity. At the same time,
a new peak appeared at around 670 nm, which covered from 520 to 840 nm.
The intensity of the new peak increases with the water fraction (fw, by volume)
in methanol. The new emission peak should be attributed to the aggregation of
BTPEAQ-10. The enhanced emission accords well with the AIE property of
BTPEAQ,[28] reported by Liu et al. The peak intensity at 670 nm is plotted
against fw, as shown in Fig. 1. The emission did not show observable increase

until fw was approximately 60%, and thereafter increased drastically, and then
increased slowly as fw was more than 90%. It is worth to note that the
aggregation of BTPEAQ-10 led to the Stokes shift of 240 nm, and the whole
emission peak is far away from the excitation light (340 nm). The absorption
band has a little overlap with the emission band (Fig. S1). Therefore, the
auto-absorption is negligible. These features make the aggregated BTPEAQ-10
suitable for application in cell imaging or other bio-related labelling.
(
a)
(b)6
fw (%)
50
60
70
80
90
100
6
0
1
0
2
0
4
2
0
4
2
0
3
0
40
400
500
600
700
800
0
20
40
60
80
100
Wavelength (nm)
fw (%)
Fig. 1. (a) Fluorescence spectra of BTPEAQ-10 with different f . (b) Plot of
w
-5
-1
fluorescence intensity at 670 nm versus f . C = 1 × 10 mol L , λ = 340 nm.
w
B
ex
Utilizing the AIE property, we are able to determine the CMC of
BTPEAQ-10. The fluorescent intensity was plotted versus the corresponding
concentration, as shown in Fig. 2. Linear fitting of the data showed an
-7
-1
intersection at around 6.6 ×10 mol L , which should correspond to the CMC.
This result is understandable. Below the CMC, BTPEAQ-10 does not form
micelles (small bundles of molecules may happen), and hence the fluorescence
at 670 nm is very weak and increases slowly with concentration; above the
CMC, the fluorescence increases drastically due to the formation of micelles.

4
3
2
1
-
7
-1
6
.6 ×10 mol L
0
1
2-
3
6
-1
Concentration (×10 mol L )
Fig. 2. Plot of fluorescence intensity versus the corresponding concentration. λ =
ex
3
40 nm.
During the study, we found an interesting phenomenon. The fluorescence
was significantly enhanced upon addition of sodium dodecyl benzene sulfonate
SDBS), as shown in Fig. 3a (where the molar concentration of SDBS (CS) was
-fold of CB). The emission difference can also be observed by the pictures of
the solutions excited by 365 nm UV light (inset of Fig. 3a). As shown in Fig. 3b,
plotting of the peak intensity versus the molar ratio of CS CB (denoted by
shows an intersection at 4. As ≤ 4, the emission intensity increases drastically
with ; as > 4, the increase slows down. We assume that the interaction
(
4
/
R)
R
R
R
between each cationic alkyl chain on BTPEAQ-10 and anionic SDBS has no
association effect, and hence the combination of BTPEAQ-10 with one SDBS
molecule will not make another combination of SDBS molecule easier or more
difficult. As
R > 4, most of the BTPEAQ-10 molecules are involved in the
complexation, and pushing the equilibrium to right side needs more SDBS
molecules. The fluorescence enhancement was also evaluated by
photoluminescence quantum yield (PLQY). The PLQY of neat BTPEAQ-10
solution (above CMC) is 0.3% and increases to 2.9% as adding 4-fold SDBS (i.e.
R = 4). The fluorescence enhancement also brings about the changes of average
lifetimes (τ s). The τ s are 1.12 and 4.16 ns for neat BTPEAQ-10 and
ave
ave
co-assemblies of BTPEAQ-10 & SDBS, respectively. The corresponding transient
fluorescence spectra are shown in Fig. S3. With these values, we are able to estimate

the radiative and non-radiative decay rates (the calculation is presented in the ESI).
-3
-3
The radiative decay rates increased from 2.68 × 10 to 6.98 × 10 , and the
non-radiative decay rates reduced from 0.89 to 0.23 (Table. S1) upon addition of
SDBS into BTPEAQ-10 aqueous solution.
Fig. 3. (a) Fluorescence spectra of BTPEAQ-10 in absence and presence of 4-fold
SDBS in aqueous solution. Inset: Photographs of the corresponding solution under
illumination of 365 nm light. (b) Plot of fluorescence intensity at 670 nm versus C .
S
-5
-1
C = 1 ×10 mol L , λ = 340 nm.
B
ex
The complexation between BTPEAQ-10 and SDBS was investigated by
MS. As shown in Fig. 4, the mass-to-charge ratios show peaks at 710.42,
228.51 and 2783.48, corresponding to the complexes of BTPEAQ-10@SDBS,
1
BTPEAQ-10@(SDBS)2 and BTPEAQ-10@(SDBS) . The experimental
3
isotopic peaks are consistent with the theoretical simulation. It is worth to note
that these three groups of peaks are all found for the solutions with different R.
We did not find the peaks of complexes of BTPEAQ-10@(SDBS) , but we
4
assume that this complex should also exist in the solution. The absence from
the spectra is because that the charge of the co-assembly is neutralized when
binding 4 molecules of SDBS on BTPEAQ-10. These results confirm that the
complexation between BTPEAQ-10 and SDBS has no association effect. Based
on the above results, we conclude that the addition of SDBS into the aqueous
solution of BTPEAQ-10 will produce 4 kinds of complexes, as shown in Fig.
4
d. The increase of the amount of SDBS will push the equilibrium to right side,
and thus the complexes with more SDBS are formed.

Fig. 4. Theoretical simulation and experimental MS of BTPEAQ-10 and SDBS
mixture: (a) BTPEAQ-10@SDBS, (b) BTPEAQ-10@(SDBS)2, (c)
BTPEAQ-10@(SDBS) , (d) Schematic illustration of the possible complexes formed
3
by BTPEAQ-10 and SDBS.
The ionic interaction between BTPEAQ-10 and SDBS was verified by
Fourier transform infrared (FTIR) spectra. As shown in Fig. 5, the two
-1
characteristic peaks of sulfonate group at around 1133 and 1045 cm are
assigned to the antisymmetric and symmetric stretching vibration, respectively.
-1
- 1
Upon complexation, the peak at around 1133 cm shifted to 1130 cm , and the
-1
-1
single peak at 1045 cm split to double peaks at 1045 and 1036 cm . The
spectral changes should be attributed to the electrostatic interaction between
sulfonate and pyridinium groups.
(a)
BTPEAQ-10
(b)
SDBS
1
133
1045
(c)
BTPEAQ-10 & SDBS
1
045 1036
1130
1200
1150
1100
1050-
1000
1
Wavenumber (cm )
Fig. 5. FTIR spectra of (a) BTPEAQ-10, (b) SDBS and (c) mixture of BTPEAQ-10 &

SDBS (R = 4).
We were wondering whether the complexation has effect on the assembly
of BTPEAQ-10, and thus we investigated the morphologies of the aggregates of
BTPEAQ-10 before and after addition of SDBS. Atomic force microscopy
(
AFM) images are shown in Fig. 6, and grain-like nanostructures are observed
on silicon substrate, which should stand for the micellar aggregates formed by
BTPEAQ-10 in the aqueous solution. The apparent sizes of the grains are
length of 50 - 100 nm, width of 30 - 60 nm and height of 5 - 25 nm. Upon
addition of SDBS, the grain-like structures are converted to spherical structures.
Particle analysis shows an average lateral diameter of 170 nm, in range of 40 -
3
60 nm. The height is in range of 20 - 45 nm. We speculate that the
co-assemblies of BTPEAQ-10 and SDBS formed spherical structures in the
solution, and these structures collapsed during the drying process. This makes a
reasonable explanation to the big difference of the diameter and height.
Transmission electron microscope (TEM) showed similar structures, as shown
in Fig. S5. The morphological changes can also be reflected by the increased
intensity in the whole spectra and stronger scattering of the co-assemblies
formed by BTPEAQ-10 and SDBS indicated by UV-vis absorption spectra and
Tyndall effect (Fig. S6), respectively. Both the assembly and the co-assembly
have good stability, and there is no collapse, cracking or other morphological
changes during storage (as shown in Fig. S7).

Fig. 6. AFM images of (a) the assemblies BTPEAQ-10 and (b) co-assemblies of
BTPEAQ-10 and SDBS. Section analysis of (c) the assemblies BTPEAQ-10 and (d)
co-assemblies of BTPEAQ-10 and SDBS (R = 4).
The photostability of the probes is one of the most important concerns. As
shown in Fig. S8, we investigated the photostability of BTPEAQ-10 and
co-assemblies of BTPEAQ-10 & SDBS (
R = 4). The results show that after
irradiating with a 365 nm ultraviolet (12 W) for 40 minutes, the emission of
BTPEAQ-10 hardly changes, which means that BTPEAQ-10 has excellent
photostability. For the co-assemblies of BTPEAQ-10 & SDBS, the emission
showed a little decrease after being continuously irradiation. Based on the
above two groups of experiment, we conclude that dissociation might happen
upon irradiation.
The co-assembly of BTPEAQ-10 and SDBS mainly relies on electron static
and hydrophobic interaction. Therefore, the formation of the co-assembly can
also be reflected by the changes in zeta potential (ζ). The ζs were 44.7 and -29.3
mV for the assemblies of BTPEAQ-10 and co-assemblies of BTPEAQ-10 &
SDBS (
R = 4), respectively. The ζ conversion further confirms the
complexation and co-assembly of BTPEAQ-10 and SDBS in the aqueous
solution.

Except for SDBS, we also testified the effect of other surfactants and anionic
groups on the emission of BTPEAQ-10. Herein, sodium dodecyl sulfonate (SDS),
sodium laurate (SL), sodium stearate (SS), sodium p-toluene sulfonate (SpTS),
sodium mesitylenesulfonate (SMeS), sodium 3-nitrobenzene sulfonate (S3-NBS),
sodium methanesulfonate (SMS), sodium trifluoromethanesulfonate (STFMS),
sodium acetate (NaAc) and sodium bromide (NaBr) were selected for investigation.
The corresponding chemical structures are shown in Table S2. The intensity of the
fluorescence at 670 nm is plotted versus the categories of anionic groups. As shown in
Fig. 7, F and F are the initial emission and the emission of BTPEAQ-10 after adding
0
different amounts of additives. Addition of SDBS (R = 4) resulted in approximately
3
0-fold enhancement of fluorescent emission, while addition of SDS, SL, SS caused
approximately 15-fold, 5.5-fold, 6.0-fold enhancement, respectively. In contrast, the
rest anionic groups showed a little effect on the fluorescence of BTPEAQ-10. These
results indicate that both the tosyl head and alkyl chain are indispensable for the
strong emission of BTPEAQ-10.
30
20
10
0
0
S
S
L
S
S
S
S
S
S
c
r
1
S
S
T
e
A
B
-
B
D
B
M
M
a
a
Q
D
S
+
+
p
M
-
N
S
F
N
S
S
T
N
A
S
+
3
+
E
+
+
S
+
+
+
S
P
+
+
T
B
Fig. 7. The fluorescence intensity of BTPEAQ-10 at 670 nm versus categories of
-5
-1
-5
anionic groups. C = 1 ×10 mol L , and concentration of the additives are 4 ×10
mol L .
B
-1
Cytocompatibility is a very important parameter for application in labelling the
cells. Herein, the cytocompatibility of BTPEAQ-10 and BTPEAQ-10 & SDBS was
testified by the cell viabilities. In doing so, cell counting kit-8 (CCK-8) assay was
employed to measure the cell viabilities of HeLa cells treated with BTPEAQ-10 and

BTPEAQ-10 & SDBS. Note that the cells were cultured in the mediums with the
fluorescent probes for 12 h before being fixed. As shown in Fig.8, the viabilities of
-6
-1
HeLa cells were close to 100% as C < 8×10 mol L , and start to drop afterward. In
B
case of BTPEAQ-10 & SDBS mixture, the viabilities are much better than the neat
-6
-1
BTPEAQ-10, and kept almost 100 % as C < 12×10 mol L . These results indicate
B
that both BTPEAQ-10 and BTPEAQ-10 & SDBS have good cytocompatibility.
BTPEAQ-10
1
20
00
BTPEAQ-10 & SDBS
1
80
60
40
20
0
0
2
4
8
12 16 -1 32
−
6
Concentration(×10 mol L )
Fig. 8. Viability of HeLa cells being cultured in mediums containing BTPEAQ-10 or
mixture of BTPEAQ-10 & SDBS.
BTPEAQ-10 and mixture of BTPEAQ-10 & SDBS were applied to label
HeLa cells. In the present study, the cells were cultured in the mediums
containing the fluorescent probes for 2 h, and then fixed with
polyoxymethylene (see the details in the experimental section). Note here, for
-6
-1
both probes, CB was controlled to be 5×10 mol L , far below the toxic dose.
As shown by the confocal laser scanning microscope (CLSM) images in Fig. 9,
both BTPEAQ-10 and BTPEAQ-10 & SDBS are successfully applied to label
the HeLa cells, and showed clear topological structure of the cells, and the
latter showed much stronger contrast. These results indicate that the mixture of
BTPEAQ-10 & SDBS presents a better NIR probe in cell imaging in
comparison of neat BTPEAQ-10.

Fig. 9. CLSM images of HeLa cells (excitation wavelength: 405 nm): (a-c) cells
treated with BTPEAQ-10, (d-f) cells treated with mixture of BTPEAQ-10 & SDBS.
From left to right columns are fluorescence, bright field, and merged images.
3
. Conclusions
In summary, we designed and synthesized an amphiphilic fluorescent probe (i.e.
BTPEAQ-10) containing a conjugated group of anthraquinone and TPE. BTPEAQ-10
shows AIE property in aqueous solution and NIR emission covers in range of 520-840
nm. However, the emission is rather weak and the PLQY is merely 0.3%.
Co-assembly of BTPEAQ-10 and SDBS can drastically improve the emission, and the
PLQY was promoted to 2.9% under existence of 4-fold SDBS. Both the ionic and
hydrophobic interactions play important roles in the fluorescent enhancement. The
co-assemblies of BTPEAQ-10 and SDBS show better cytocompatibility and high
contrast fluorescent images, presenting a good NIR probe in cell imaging.
4
. Experimental
4
.1 Materials
4
-Bromobenzophenone, CDCl , DMSO-d6 were purchased from J&K
3
Chemical Co., Ltd (Shanghai). 4,4'-Dihydroxybenzophenone and
,10-Dibromodecane were purchased from Energy Chemical Co., Ltd. Zinc
powder, TiCl , Tetrahydrofuran (THF), N, N-dimethylformamide (DMF),
1
4

methanol, pyridine, anhydrous Na SO , anhydrous NaCl, anhydrous K CO ,
2
4
2
3
HCl, KAc, NaAc, and NaBr were purchased from Sinopharm Chemical
Reagent Co., Ltd. Pd(PPh ) , SDBS were purchased from TCI Development
3
4
Co., Ltd (Shanghai). Ethyl acetate, petroleum ether, dichloromethane (DCM)
and chloroform were obtained from Yonghua Chemical Technology Co., Ltd
(
Jiangsu). 2,6-Dibromoanthracene-9,10-dione was purchased from Bide
Pharmaceutical Technology Co., Ltd (Shanghai). 1,4-Dioxane was purchased
from 3A Chemicals Co., Ltd. Pd(dppf)Cl ·CH Cl , SDS, SL, SS, SpTS, SMeS,
2
2
2
S3-NBS, SMS, STFMS were obtained from Macklin Biochemical Technology
Co., Ltd. Bis(pinacolato)diboron was obtained from Aladdin Biochemical
Technology Co., Ltd (Shanghai). Milli-Q water with resistivity of 18 MΩ cm
was produced by Direct-Q5uv manufactured by Merck Millipore.
4
.2 Instruments
1
H NMR spectra were recorded on Avance III 400 MHz (Bruker, USA) and
6
00 MHz NMR spectrometer (Agilent Technologies, USA). MS of the products
were obtained on micro Q-TOF III mass spectrometer (Bruker, USA) in the
electrospray ionization (ESI) mode and matrix-assisted laser
desorption/ionization time-off light mass spectroscopy (MALDI-TOF-MS).
The MS of complexation between BTPEAQ-10 and SDBS was investigated
with the solution of
R = 20. The fluorescence spectra were recorded on FLS980
(
Edinburgh Instrument, UK). The UV-vis absorption spectra were recorded on
Cary 60 (Agilent Technologies, USA). The PLQYs were obtained by
C9920-02G (HAMAMASTU Instrument, Japan) using the integration sphere
by absolute method. AFM images were taken on Multimode 8 microscope
(
Bruker, USA). The peak force quantitative nanomechanical mapping mode
-1
with a ScanAasyst-Air probe (nominal spring constants 0.4 N m , frequency 70
kHz, from Bruker) was adopted during the measurement. The samples were
cast on silicon substrates and dried in vacuum. TEM characterization was

performed by FEI TECNAI F20 operating at 200 kV. Approximately 6 μL of
sample solution was cast onto a carbon-coated copper grid, and then the grid
was dried in vacuum. FTIR spectra were recorded on Nicolet 6700 produced by
Thermo Scientific (USA). The cell images were obtained by STP6000 CLSM
(
LEICA, Germany). Transient fluorescence spectra measurements were carried
out by using time-correlated single-photon counting lifetime spectroscopy
system (Edinburgh FL920) with a semiconductor laser as the excitation source.
The ζ measurements were performed at 25 ± 0.1 °C using a Malvern Zetasizer
Nano-ZS instrument (Malvern, UK).
4
.3 Synthesis and characterization
4
.3.1
Synthesis
of
4,4'-(2-(4-bromophenyl)-2-phenylethene-1,1-diyl)diphenol
(compound A).
Compound A was synthesized following the procedures reported in the
literature[50] with some modifications. 4-Bromobenzophenone (3.90 g, 15
mmol), 4,4'-Dihydroxybenzophenone (1.60 g, 7.50 mmol) and zinc powder
(
4.50 g, 70 mmol) were added to a 250-mL three-necked round-bottom flask.
The flask was vacuumed and purged with nitrogen for 3 times. Afterward, 100
mL of anhydrous THF was injected. The reaction system was cooled down to 0
°
C, and TiCl (3.40 mL, 31 mmol) was added dropwise to the mixture. Then the
4
mixture was vigorously stirred in an ice bath for 2 h. Finally, the mixture was
refluxed at 78 °C for 24 h. After cooling to room temperature, hydrochloric
-1
acid (150 mL, 1 mol L ) was added. The mixture was stirred for 15 min and
then filtered. The filtrate was extracted with DCM for 3 times, and the organic
layer was dried with anhydrous sodium sulfate. Then the solvent was removed
on a rotavapor. The coarse product was separated by silica gel column
chromatography with hexane / ethyl acetate (5:2, v/v) as eluent. The white solid
1
was the final product (1.80 g, yield 54%). H NMR (400 MHz, DMSO-d6) δ
9
.35 (t, J = 15.2 Hz, 2H), 7.31 (d, J = 8.3 Hz, 2H), 7.12 (dd, J = 14.7, 7.3 Hz,

3
6
H), 6.93 (d,
J = 7.1 Hz, 2H), 6.85 (d, J = 8.3 Hz, 2H), 6.73 (t, J = 8.3 Hz, 4H),
.50 (dd, = 18.5, 8.4 Hz, 4H).
J
4
.3.2 Synthesis of 4,4'-(2-(4-bromophenyl)-2-phenylethene-1, 1-diyl)bis(((10
bromodecyl)oxy) Benzene (compound B).
Compound B was synthesized following the procedures reported in the
literature[51] with some modifications. 1, 10-Dibromodecane (2.40 g, 8 mmol)
and K CO (1.38 g, 10 mmol) were added to a 100-mL three-necked
2
3
round-bottom flask. The flask was vacuumed and purged with nitrogen for 3
times. Afterward, 10 mL of anhydrous DMF was injected. The mixture was
heated to 70 °C, then the anhydrous DMF (10 mL) containing compound A
(
0.44 g, 1 mmol) was added dropwise to the mixture. The mixture was refluxed
at 70 °C for 20 h. After cooling to room temperature, sodium chloride solution
40 mL) was added. Afterward, the mixture was extracted with DCM, washed
(
with distilled water for 3 times. Then the organic layer was dried with
anhydrous sodium sulfate, and the solvent was removed on a rotavapor. The
crude product was purified by silica column chromatography with hexane /
DCM (5:3, v/v) as eluent. The yellow oily droplet was the final product (0.27 g,
1
yield 31%). H NMR (400 MHz, CDCl ) δ 7.21 (d,
J
= 8.4 Hz, 2H), 7.09 (t,
= 8.4, 5.9 Hz, 6H), 6.63 (dd,
14.1, 8.7 Hz, 4H), 3.93 - 3.82 (m, 4H), 3.47 (dt,
.69 (m, 8H), 1.43 (s, 8H), 1.30 (s, 16H).
J
=
3
9
.3 Hz, 3H), 7.00 (d,
J
= 7.6 Hz, 2H), 6.89 (td,
J
J
=
J = 49.7, 6.8 Hz, 4H), 1.90 -
1
4
.3.3 Synthesis of
2
,6-bis(4,4,5,5-tetramethyl-1,3,2-dioxaborolan-2-yl)anthracene-9,10-dione
(
compound C).
Compound C was synthesized following the procedures reported in the
literature[52] with some modifications. 2,6-dibromoanthracene-9,10-dione
0.76 g, 2.06 mmol), Bis(pinacolato)diboron (1.32 g, 5.20 mmol),
Pd(dppf)Cl ·CH Cl (0.17 g, 0.21 mmol) and KAc (0.73 g, 7.43 mmol) were
(
2
2
2

added to a 100-mL two-necked round-bottom flask. The flask was vacuumed
and purged with nitrogen for 3 times. Then anhydrous 1, 4-dioxane (40 mL)
was added to the flask. The mixture was stirred at room temperature for 30 min
and refluxed at 84 °C for 40 h. The reactant was cooled down and quenched by
adding water (20 mL). Then the mixture was extracted with DCM for 3 times,
and the organic phase was dried with anhydrous sodium sulfate. Then the
solvent was removed on a rotavapor. The coarse product was separated by
silica gel column chromatography with DCM / methyl alcohol (20:1, v/v) as
1
eluent. The primrose powdery was the final product (0.35 g, yield 37%). H
NMR (400 MHz, CDCl ) δ 8.75 (s, 2H), 8.30 (d,
J = 7.7 Hz, 2H), 8.20 (d, J =
3
7
.7 Hz, 2H), 1.38 (s, 24H).
4
2
9
.3.4 Synthesis of
,6-bis(4-(2,2-bis(4-((10-bromodecyl)oxy)phenyl)-1-phenylvinyl)phenyl)ant-hracene-
,10-dione (compound D).
Compound B (0.88 g, 1 mmol), compound C (0.18 g, 0.40 mmol) and
K CO (0.55 g, 4 mmol) were added to a 250-mL two-necked round-bottom
2
3
flask. The flask was vacuumed and purged with nitrogen for 3 times, then
anhydrous THF (100 mL) and nitrogen-bubbled water (20 mL) were added to
the flask. After the mixture was purged with nitrogen for 30 min, Pd(PPh3)4
(
46.20 mg, 0.04 mmol) was added. Then the mixture was refluxed at 75 °C for
2 h. After cooling to room temperature, the mixture was extracted with DCM
7
for 3 times. The organic layer was dried with anhydrous sodium sulfate, and the
solvent was removed on a rotavapor. The crude product was separated by silica
gel column chromatography with hexane / ethyl acetate (15:1, v/v) as the
1
eluent. The orange solid was the final product (0.11 g, yield 15%). H NMR
(
400 MHz, CDCl ) δ 8.49 (s, 2H), 8.33 (d,
J
= 8.0 Hz, 2H), 7.96 (d,
= 8.4 Hz, 4H), 7.17 - 7.01 (m, 14H), 6.93 (dd, = 15.4, 8.7 Hz,
= 11.7, 8.7 Hz, 8H), 3.86 (d, = 3.3 Hz, 8H), 3.44 (ddd,
9.8, 15.7, 6.8 Hz, 8H), 1.87 - 1.67 (m, 16H), 1.40 (s, 16H), 1.27 (d, = 6.3 Hz,
J = 8.1 Hz,
3
2
8
4
H), 7.48 (d,
J
J
H), 6.62 (dd,
J
J
J =
J

+
3
2H). MALDI-TOF-MS (m/z): calcd for [C H Br O + H] :1810.5889,
106 121 4 6
found: 1810.831.
4
.3.5 Synthesis of BTPEAQ-10.
Compound D (0.12 g, 0.07 mmol) was dissolved in pyridine (80 mL). The
mixture was refluxed at 110 °C for 48 h. The solvent was removed on a
rotavapor. Then the product was washed for 5 times with ethyl ether. The
1
crimson solid was the final product (0.11 g, yield 76%). H NMR (600 MHz,
DMSO-d6) δ 9.09 (dd,
J
= 12.3, 5.7 Hz, 8H), 8.63 - 8.56 (m, 4H), 8.39 (s, 2H),
= 8.2 Hz, 2H), 8.21 (d, = 8.7 Hz, 2H), 8.19 - 8.10 (m, 8H), 7.69 (d,
= 8.3 Hz, 4H), 7.21 - 7.10 (m, 10H), 7.01 (d, = 7.4 Hz, 4H), 6.92 (d, = 8.6
= 8.3 Hz, 4H), 6.70 (dd, = 23.9, 8.7 Hz, 8H), 4.58 (dt,
5.0, 7.4 Hz, 8H), 3.86 (t, = 6.1 Hz, 8H), 1.90 (dd, = 18.3, 7.3 Hz, 8H), 1.64
d, = 6.5 Hz, 8H), 1.32 (d, = 39.6 Hz, 16H), 1.23 (s, 32H). ESI-MS (m/z):
calcd for [C H N O ] : 451.5202, found: 451.5199.
8
.27 (d,
J
J
J
J
J
Hz, 4H), 6.87 (d,
J
J
J =
1
J
J
(
J
J
4+
126 140
4
6
4
.4 Cell viability assay
The viability of HeLa cells was evaluated by CCK-8 method, where
BTPEAQ-10 and co-assemblies of BTPEAQ-10 and SDBS were separately
co-cultured with the HeLa cells at 37 °C and with existence of 5% CO . Note
2
that the HeLa cells were pre-cultured in the mediums without the fluorescent
probes for 12 h. In the following, the pre-cultured cells were cultured in the
mediums with the fluorescent probes for 12 h. Herein, CB was varied as 0, 2, 4,
-6
-1
8
, 12, 16, 32 × 10 mol L , and CS was 4-fold of CB. Then 10 μL of CCK-8
solution was added and continuously cultured for 2 h. The absorbance (i.e. OD
value) at 450 nm was measured by using a microplate reader (Spectramax i3,
Molecular Devices, USA). For each condition, three groups of cells were
parallel tested. The biocompatibility was evaluated by comparing the viability
of the cells treated with the fluorescent probes with the control.

4
.5 Cell culturing and imaging
HeLa cells were cultured in Dulbecco’s Modified Eagle Medium (DMEM,
ThermoFisher) and 10% fetal bovine serum for 12 h. The pre-cultured cells
-6
-1
were then cultured in the mediums with BTPEAQ-10 (CB = 5 × 10 mol L ) or
-6
-1
-6
-1
BTPEAQ-10 & SDBS (CB = 5 × 10 mol L , CS = 20 × 10 mol L ) for 2 h,
and then gently washed with phosphate buffered saline for 3 times and fixed
with 2% paraformaldehyde for 40 min. The cells were imaged with CLSM.
(
Excitation wavelength: 405 nm).
Acknowledgements
We thank the financial support from the National Natural Science
Foundation of China (21674075), the Natural Science Foundation of Jiangsu
Province (BK20161211), and the Key University Science Research Project of
Jiangsu Province (17KJA150007). This project is also supported by the Jiangsu
Key Laboratory for Carbon-Based Functional Materials & Devices, at Soochow
University.
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Highlights
1. A four-armed amphiphile with near-infrared emission is synthesized.
2. The fluorescence emission is greatly enhanced by ionic co-assembly.
3. The co-assemblies are successfully applied in fluorescent labeling of HeLa cells.

Declaration of interests
☐
The authors declare that they have no known competing financial interests or personal relationships
that could have appeared to influence the work reported in this paper.
☒
The authors declare the following financial interests/personal relationships which may be considered
as potential competing interests: