Near-infrared squaraine fluorescent probe for imaging adenosine 5′-triphosphate in live cells

Article abstract of DOI:10.1016/j.dyepig.2019.107698

Adenosine 5′-triphosphate (ATP), as a “molecular currency” for intracellular energy transfer, not only participates in various physiological activities, but also involves in various pathological processes of living cells. Therefore, the establishment of a simple, rapid, sensitive and accurate ATP analysis method is of great significance for the study of its physiological functions. In this work, we rationally design a near-infrared phenylboronic acid group-functionalized dicyanovinyl squaraine (SQ-PBA3), with which a supramolecular assembly was constructed for fluorescent imaging of ATP. In PBS buffer, SQ-PBA3 changes self-assembly when adding cetyltrimethyl ammonium bromide (CTAB) deriving fluorescence response of SQ-PBA3 to ATP with the maximum emission at 700 nm, due to the specificity of phenylboronic acid to diols and the multiple electrostatic interactions between SQ-PBA3, ATP and CTAB molecules. Meanwhile, the probe was successfully applied to monitor intracellular ATP level in MCF-7 cells with high sensitivity and selectivity.

Full text of DOI:10.1016/j.dyepig.2019.107698

Dyes and Pigments 171 (2019) 107698
Contents lists available at ScienceDirect
Dyes and Pigments
journal homepage: www.elsevier.com/locate/dyepig
Near-infrared squaraine fluorescent probe for imaging adenosine 5′-
triphosphate in live cells
Guimei Wanga,b, Xiaoxue Jianga,b, Nanyan Fua,b,∗
a
MOE Key Laboratory for Analytical Science of Food Safety and Biology & Fujian Provincial Key Laboratory of Analysis and Detection Technology for Food Safety, State
Key Laboratory of Photocatalysis on Energy and Environment, College of Chemistry, Fuzhou University, Fuzhou, 350116, PR China
b
Key Laboratory of Bioorganic Phosphorus Chemistry & Chemical Biology (Ministry of Education), Tsinghua University, Beijing, 100084, PR China
A R T I C L E I N F O
A B S T R A C T
Keywords:
Adenosine 5′-triphosphate (ATP), as a “molecular currency” for intracellular energy transfer, not only partici-
pates in various physiological activities, but also involves in various pathological processes of living cells.
Therefore, the establishment of a simple, rapid, sensitive and accurate ATP analysis method is of great sig-
nificance for the study of its physiological functions. In this work, we rationally design a near-infrared phe-
nylboronic acid group-functionalized dicyanovinyl squaraine (SQ-PBA3), with which a supramolecular assembly
was constructed for fluorescent imaging of ATP. In PBS buffer, SQ-PBA3 changes self-assembly when adding
cetyltrimethyl ammonium bromide (CTAB) deriving fluorescence response of SQ-PBA3 to ATP with the max-
imum emission at 700 nm, due to the specificity of phenylboronic acid to diols and the multiple electrostatic
interactions between SQ-PBA3, ATP and CTAB molecules. Meanwhile, the probe was successfully applied to
monitor intracellular ATP level in MCF-7 cells with high sensitivity and selectivity.
Phenylboronic acid functionalized squaraine
Cetyltrimethyl ammonium bromide (CTAB)
Adenosine 5′-triphosphate (ATP) detection
Supramolecular assembly
NIR fluorescent probe
Bioimaging
1. Introduction
fluorescence imaging offers many advantages like simple operation,
cost-effectiveness, high sensitivity to analytes, and good selectivity. On
Adenosine 5′-triphosphate (ATP) is not only a universal energy
top of it all, it can achieve in-situ visible imaging of ATP, which has
attracted more and more interest [14–19].
currency in various cellular activities, but also a signal of many biolo-
gical processes involved in cell division [1], ion channel [2], DNA
synthesis [3], etc. A recent study showed that at micromolar con-
centrations, ATP can act as a source of energy that drives metabolism in
the body, while reaching millimolar concentrations, it in turn serves as
a hydrotrope to help solubilize hydrophobic proteins in the cytoplasm
4]. On the other hand, extracellular ATP can function as mediators in
tumor metastasis, including cellular proliferation, invasion and death
5]. Therefore, the establishment of a detection method that can be
used to monitor changes in ATP level in real time is of great significance
for the study of its physiological functions.
Till now, many ATP detection methods have been developed in-
cluding electrophoresis [6], chemiluminescence [7], bioluminescence
NIR absorbing and emitting fluorescent probes have minimal light
damage to organisms, better deep tissue penetration and can effectively
reduce background fluorescence from living systems. Squaraine dyes
have a non-classical resonance system with a unique zwitterionic
structure. The distinctive push-pull electronic structure of the donor-
acceptor-donor (D-A-D) results in their strong absorption in the region
from red (620–670 nm) to near-infrared. Additionally, squaraine dyes
are prone to form diverse aggregates under certain conditions, and
these aggregates exhibit distinctly different optical properties from
monomers [20–22]. The diversity of dye aggregate forms makes it
possible to develop functional fluorescent probes for different target by
regulating of the aggregates [23–26].
[
[
[
8], and electrochemical analysis [9]. These methods face different
Boronic acid derivatives can rapidly and reversibly interact with
1,2- or 1,3-diols, which have been widely applied in the recognition of
1,2- or 1,3-diols structure [15,27–30]. In this work, we report a series of
dicyanovinyl squaraines (SQ-PBA1-3) with boronic acid group in dif-
ferent positions of phenyl ring, whose ability in fluorescent imaging of
challenges in the detection of intracellular ATP, such as the use of off-
line methods to obtain cell extracts for analysis by disrupting cells,
which will increase the measurement cost, and cannot realize real-time
and in-situ detection [10–13]. Compared to these methods, however,
∗
Corresponding author. MOE Key Laboratory for Analytical Science of Food Safety and Biology & Fujian Provincial Key Laboratory of Analysis and Detection
Technology for Food Safety, State Key Laboratory of Photocatalysis on Energy and Environment, College of Chemistry, Fuzhou University, Fuzhou, 350116, PR
China.
E-mail address: nanyan_fu@fzu.edu.cn (N. Fu).
https://doi.org/10.1016/j.dyepig.2019.107698
Received 25 May 2019; Received in revised form 30 June 2019; Accepted 2 July 2019
Available online 03 July 2019
0143-7208/ © 2019 Elsevier Ltd. All rights reserved.

G. Wang, et al.
Dyes and Pigments 171 (2019) 107698
+
ATP in the NIR region have been studied. The phenylboronic acid group
is designed as a recognition unit, and an electron withdrawing cyano
Calcd for C18
H
21NBO
2
([M] 1): 294.1665; found: 294.1657.
4c: 140 mg, yield 53%. H NMR (400 MHz, DMSO‑d
6
) δ 7.87 (d,
(
–CN) group was introduced in central four-membered ring of squaric
J = 7.0 Hz, 1H), 7.81 (d, J = 7.8 Hz, 2H), 7.78 (d, J = 7.7 Hz, 1H), 7.60
(t, J = 7.2 Hz, 1H), 7.56 (d, J = 7.2 Hz, 1H), 7.37 (d, J = 7.6 Hz, 2H),
acid to improve its photostability. To improve the responsibility of SQ-
PBA1-3 to ATP, supramolecular assemblies were constructed with them
and cetyltrimethyl ammonium bromide (CTAB). We speculated that the
fluorescence of SQ-PBA1-3 would be quenched due to the aggregation
caused quenching (ACQ) effect. Then in the presence of ATP, it will go
through a process of disaggregation and reassembly owing to the co-
operation of the specificity of phenylboronic acid to diols and the
multiple electrostatic interactions between SQ-PBA1-3, ATP and CTAB
molecules, along with the fluorescence “turn-on”, by which time
fluorescence imaging of intracellular ATP can be achieved, as well as
real-time monitoring of changes in intracellular ATP level.
5.82 (s, 2H), 2.98 (s, 3H), 1.60 (s, 6H); 13C NMR (100 MHz, DMSO‑d
6
):
δ 198.85, 142.43, 141.91, 136.45, 135.81, 130.89, 129.89, 129.40,
128.00, 125.89, 124.06, 116.32, 54.91, 51.74, 22.60, 15.01; HRMS
+
(ESI) m/z: Calcd for C18
H
21NBO
2
([M] ): 294.1665; found: 294.1657.
2.2.3. Synthesis of SQ-PBA1-3
Phenylboronic acid substituted indole 4 (99 mg, 0.265 mmol) and
dicyanovinyl substituted squaric acid derivative 2 (38 mg, 0.132 mmol)
were dissolved in 20 mL of n-butanol and toluene (1:1, v/v) in a 50 mL
two-necked flask equipped with a Dean-Stark trap and then the solution
was refluxed under nitrogen with stirring for 5 h. The reaction was
monitored by thin layer chromatography (TLC) until the completion of
the reaction. After that, the solution was cooled to room temperature,
washed with water, and dried the organic layer with anhydrous sodium
sulfate. After removing the solvent, the residue was purified by column
chromatography eluting with dichloromethane: methanol (15:1, v/v) to
afford a dark green solid.
2. Experimental
2.1. Materials and instruments
Adenosine-5′-triphosphate disodium salt hydrate (ATP), adenosine-
′-diphosphate sodium salt (ADP), adenosine-5′-monophosphate
5
monohydrate (AMP), cytidine-5′-diphosphate disodium salt (CDP), cy-
tidine-5′-triphosphate disodium salt (CTP), uridine-5′-monophosphate
disodium salt (UMP), uridine-5′-diphosphate sodium salt (UDP), ur-
idine-5′-triphosphate trisodium salt (UTP), guanosine-5′-monopho-
sphate disodium salt hydrate (GMP), guanosine-5′-diphosphate dis-
odium salt (GDP), and guanosine-5′-triphosphate trisodium salt (GTP)
were purchased from Aladdin Reagent Co., Ltd. (Shanghai, China).
Other reagents not mentioned above are at least analytically pure,
purchased from Sinopharm Chemical Reagent Co., Ltd. (Shanghai,
China), and used without further purification; the organic solvents were
SQ-PBA1: 53 mg, yield 56%. 1H NMR (400 MHz, DMSO‑d
6
): δ 7.75
(d, J = 7.2 Hz, 2H), 7.62 (d, J = 7.3 Hz, 2H), 7.33–7.21 (m, 8H), 7.15
(d, J = 6.9 Hz, 2H), 6.73 (d, J = 7.2Hz, 2H), 6.35 (s, 2H), 5.57 (s, 4H),
1.82 (s, 12H); 13C NMR (100 MHz, DMSO‑d
): δ 172.88, 172.53,
6
166.65, 165.48, 142.38, 142.27, 133.98, 132.49, 128.70, 128.40,
125.42, 122.85, 118.54, 111.92, 89.06, 79.04, 66.75, 49.81, 47.37,
33.46, 31.99, 31.62, 29.95, 29.47, 26.54, 20.95; HRMS (ESI) m/z: Calcd
for C43
H
39
B
2
N
4
O
5
([M+H]+): 713.3107; found: 713.3104.
1
SQ-PBA2: 33 mg, yield 35%. H NMR (400 MHz, DMSO‑d
7.72–7.63 (m, 8H), 7.37–7.32 (m, 8H), 6.37 (s, 2H), 5.34 (s, 4H), 1.76
6
): δ
dried according to standard procedures.
(s, 12H); 13C NMR (100 MHz, DMSO‑d
6
): δ 172.88, 172.53, 166.65,
1
H NMR (400 MHz) and 13C NMR (100 MHz) spectra were measured
165.48, 142.38, 142.27, 133.98, 132.49, 128.70, 128.42, 125.42,
122.85, 118.54, 111.91, 89.16, 78.98, 66.79, 49.81, 47.38, 33.42,
on
a Bruker AV-400 spectrometer (TMS as internal standard).
Absorption spectra were recorded on a PerkinElmer Lambda 750
UV–vis spectrophotometer. Fluorescent emission spectra were acquired
using a Cary Eclipse fluorescence spectrophotometer.
32.02, 29.92, 26.54, 20.95; HRMS (ESI) m/z: Calcd for C43
H
39
B
2
N
4
O
5
+
([M+H] ): 713.3107; found: 713.3101.
SQ-PBA3: 31 mg, yield 33%. 1H NMR (400 MHz, DMSO‑d
): δ 7.74
d, J = 7.6 Hz, 4H), 7.62 (d, J = 7.2 Hz, 2H), 7.44–7.37 (m, 4H), 7.28
(d, J = 7.3 Hz, 2H), 7.22 (d, J = 7.0 Hz, 4H), 6.34 (s, 2H), 5.33 (s, 4H),
.74 (s, 12H); 13C NMR (100 MHz, DMSO‑d
6
(
2
2
.2. Synthesis
1
6
): δ 172.76, 172.40,
.2.1. Synthesis of 2
172.39, 165.45, 142.44, 142.25, 137.00, 135.10, 128.76, 126.06,
125.47, 122.90, 118.56, 111.86, 89.20, 49.78, 47.30, 33.42, 32.07,
Dicyanovinyl substituted squaric acid derivative 2 was synthesized
according to the reported procedure [31].
39 2 4 5
29.47, 26.52, 20.95; HRMS (ESI) m/z: Calcd for C43H B N O ([M
+
+
H] ): 713.3107; found: 713.3140.
2.2.2. Synthesis of 4
2,3,3-Trimethyl-3H-indole (3) (120 mg, 0.75 mmol) was dissolved
2.3. General procedure for spectral measurement
in 4 mL of acetonitrile in a 25 mL round bottom flask, then bromo-
methyl phenylboronic acid (150 mg, 0.70 mmol) in 4 mL of acetonitrile
was injected and refluxed overnight at 80 °C. The solution was dark red
and the crude product was purified by column chromatography eluting
with dichloromethane: methanol (20:1, v/v) and a khaki solid was
obtained.
Stock solutions of SQ-PBA1-3 (10 mM) were prepared by dissolving
them in DMSO. A stock solution of ATP (10 mM) was prepared in twice
distilled water. Solutions of AMP, ADP, CDP, CTP, GMP, GDP, GTP,
UMP, UDP, UTP were prepared in twice distilled water (50 mM), re-
spectively. 0.8 μL of SQ-PBA1-3 stock solutions were added to 2 mL of
PBS buffer solutions (10 mM, pH 7.4) in quartz cuvettes of 1 cm path
length to acquire 4.0 μM dye solutions, and different volumes of the
ATP solutions were added.
4
a: 170 mg, yield 65%. 1H NMR (400 MHz, DMSO‑d
J = 7.2 Hz, 1H), 7.80 (t, J = 4.3 Hz, 1H), 7.58 (d, J = 10.2 Hz, 2H),
6
) δ 7.88 (d,
7
1
.51 (d, J = 7.6 Hz, 1H), 7.37 (t, J = 4.6 Hz, 2H), 6.93 (t, J = 4.9 Hz,
1
3
H), 6.03 (s, 2H), 2.89 (s, 3H), 1.63 (s, 6H); C NMR (100 MHz,
): δ 198.72, 142.43, 141.91, 136.43, 135.81, 130.89, 129.89,
29.40, 128.00, 125.89, 124.06, 116.32, 54.91, 51.74, 22.60, 15.01;
DMSO‑d
1
6
2.4. Dynamic light scattering measurement (DLS)
+
HRMS (ESI) m/z: Calcd for C18
94.1656.
b: 153 mg, yield 58%. 1H NMR (400 MHz, DMSO‑d
) δ 7.88 (d,
H
21NBO
2
([M] ): 294.1665; found:
Solutions of SQ-PBA3, CTAB and ATP were filtered through a filter
membrane with a pore size of 0.25 μm, and then transferred to clean
cuvettes for testing.
2
4
6
J = 6.4 Hz, 1H), 7.80 (d, J = 6.9 Hz, 1H), 7.67–7.57 (m, 3H), 7.46 (d,
J = 6.7 Hz, 1H), 7.25 (d, J = 6.7 Hz, 1H), 5.84 (s, 2H), 2.99 (s, 3H),
2.5. Field emission scanning electron microscope (FESEM)
1
3
1
1
1
6
.62 (s, 6H); C NMR (100 MHz, DMSO‑d ): δ 172.44, 161.24, 146.14,
37.36, 136.75, 134.84, 133.12, 128.51, 127.99, 124.08, 122.37,
Solutions of SQ-PBA3 alone in PBS solution, in the presence of
surfactant CTAB (0.25 mM) and subsequently addition of ATP were
18.92, 116.40, 105.95, 54.95, 51.33, 21.52, 15.01; HRMS (ESI) m/z:
2

G. Wang, et al.
Dyes and Pigments 171 (2019) 107698
prepared respectively. After standing at room temperature for 20 min, a
drop of the solution was added on the cleaned single crystal silicon
wafer, evaporated at room temperature, and tested after spraying gold.
background signal, the concentration of CTAB should be optimized. In
the presence of ATP, the molecules are reassembled to form a short and
small rod-like structure owing to the electrostatic interaction between
the SQ-PBA3, ATP and CTAB molecules, accompanied by the fluores-
cence “turn-on” of SQ-PBA3.
2.6. Cell incubation and fluorescence microscopy imaging
The MCF-7 cells were cultivated in a 35 mm confocal culture dish in
3.2. Construction of supramolecular assembly
an atmosphere of 5% CO
2
at 37°C in DEME culture medium containing
1
0% fetal bovine serum (FBS), 1.0% penicillins and treptomycin. After
In order to improve the response of compounds under physiological
conditions, we first studied the effect of different surfactants on the ATP
response ability of SQ-PBA3. The cationic surfactant CTAB and the
nonionic surfactant Tween 80 (TW80) were added into PBS buffer
(pH = 7.4, 10 mM), respectively, then the same amount of ATP was
added for UV–Vis spectral analysis. It was found from Fig. S19 (Sup-
plementary Data), that the absorption of the monomer around 680 nm
was significantly enhanced with a bathochromic shift only in the pre-
sence of CTAB, indicating that CTAB was helpful to improve the re-
sponse of SQ-PBA3 to ATP. This may be attributed to the electrostatic
interaction between SQ-PBA3 and CTAB, which can modulate the ag-
gregation state of dye.
incubation for 24 h, the MCF-7 cells were further treated with 4.0 μM
SQ-PBA3 at 37 °C for 1.5 h. Following the removal of the culture
medium, the cells were washed with PBS buffer three times and then
incubated with 10 nM paclitaxel in DEME culture medium for 24 h.
Fluorescence imaging was performed on a Nikon confocal microscope
C2/C2si.
3
. Results and discussion
3.1. Synthesis and sensing mechanism of supramolecular assembly
The concentration of CTAB was then optimized. Absorbance
changes of SQ-PBA3 solutions before and after the addition of ATP
were measured with different concentrations of CTAB. The results are
shown in Fig. 1. When the concentration of CTAB is 0.25 mM, the ab-
sorbance change of the solution is the largest, indicating the formation
of the supramolecular assembly. Thus it is determined that 0.25 mM is
the best test concentration.
The chemical structure and synthetic procedure of SQ-PBA1-3 are
summarized in Scheme 1. Substitution reaction between bromomethyl
phenylboronic acid and 2,3,3-trimethyl-3H-indole (3) gave compound
. SQ-PBA1-3 were prepared by condensation reaction between di-
4
cyanovinyl substituted squaric acid derivative 2 and phenylboronic
1
13
acid substituted indole 4, and characterized by H NMR, C NMR, and
high-resolution mass spectrometry (Figs. S1–S18, Supplementary Data).
As shown in Scheme 2, the proposed sensing supramolecular as-
sembly was composed of ATP probe and CTAB. When SQ-PBA3 alone is
dissolved in PBS solution, the molecules are self-aggregated due to
hydrophobic interaction and π-π interaction, leading to the fluores-
cence quenching. With further addition of CTAB, the electrostatic in-
teraction between SQ-PBA3 and CTAB would change the aggregates of
the probe, contributing to the sensing of ATP. In order to obtain a low
3.3. Spectral response of supramolecular assemblies to AMP, ADP and ATP
Firstly, we studied the fluorescence response of supramolecular as-
semblies to AMP, ADP and ATP in PBS buffer solution (pH = 7.4,
10 mM, 0.25 mM CTAB). As shown in Fig. 2, supramolecular assembly
containing SQ-PBA2 displayed no response to AMP, ADP and ATP,
Scheme 1. Synthesis of SQ-PBA1-3.
3

G. Wang, et al.
Dyes and Pigments 171 (2019) 107698
Scheme 2. Schematic illustration of self-assembly of squaraine dye SQ-PBA3 in aqueous solution and detection of ATP.
Fig. 3. Fluorescence spectra of SQ-PBA3 with the increasing addition of ATP in
Fig. 1. Absorption spectra of SQ-PBA3 (4.0 μM) in PBS solution (pH = 7.4,
PBS solution (pH = 7.4, 10 mM, 0.25 mM CTAB).
1
0 mM) in the presence of increasing concentration of CTAB from 0 to 0.3 mM.
space steric hindrance of the ortho position. Thus, SQ-PBA3 was chosen
to evaluate its response towards ATP.
Following, we performed ATP fluorescence titration experiments in
PBS solution (pH = 7.4, 10 mM, 0.25 mM CTAB). Fig. 3 shows the
fluorescence spectra of SQ-PBA3 with different amounts of ATP. It can
be seen that the fluorescence of SQ-PBA3 in PBS solution was weakly
fluorescent, with the addition of ATP, gradual increase in the fluores-
cence intensity at 700 nm was found, and then the saturated fluores-
cence intensity with a 15-fold enhancement was obtained upon the
addition of 40 μM ATP. As shown in Fig. S20 (Supplementary Data), a
good linear relationship was discovered between the fluorescence in-
tensity of the solution and the ATP concentration from
2 to
2
8
22 μM (R = 0.9918, k = 3.12 × 10 ). According to 3σ/k, the calcu-
lated detection limit is 28 nM.
The relative amount of ATP and its dephosphorylated products ADP
and AMP, control the metabolic activity of the cell. Thus, we performed
AMP, ADP and ATP fluorescence titration experiments in PBS solution
(
pH = 7.4, 10 mM, 0.25 mM CTAB). As can be seen in Fig. 4a, SQ-PBA3
Fig. 2. Fluorescence spectra of SQ-PBA1-3 responding to ATP. (λex = 670 nm,
slit: 5 nm/5 nm, PMT Volts: 650).
exhibited different response towards AMP, ADP and ATP, and the ad-
dition of ATP lead to the largest increases of fluorescence, which was
about 2 times of ADP and 3 times of AMP.
while SQ-PBA1 and SQ-PBA3 all exhibited different levels of fluores-
cence response to ATP, and the strongest fluorescence response of SQ-
PBA3 to ATP was observed. This result may be ascribed to the electron
effect on the benzene ring. Although the ortho-position substitution is
responsive to ATP, it is not conducive to the recognition due to the large
3
.4. The specificity of SQ-PBA3 to ATP
Meanwhile we examined the specificity of SQ-PBA3 to ATP over
some metal ions, amino acids and nucleotides present in biological
4

G. Wang, et al.
Dyes and Pigments 171 (2019) 107698
Fig. 4. (a) Fluorescence titration curve of SQ-PBA3 (4.0 μM) in PBS solution (pH = 7.4, 10 mM, 0.25 mM CTAB) upon the addition of ATP (blue), ADP (red) and AMP
(
black). (b) The fluorescence response of SQ-PBA3 (4.0 μM) in the PBS solution (pH = 7.2, 10 mM, 0.25 mM CTAB) in the presence of 5 mM proline (1), histidine (2),
2+ + + 2+ 2+
cysteine (3), arginine (4), GSH (5), lysine (6), glutamic acid (7), asparaginic acid (8), tyrosine (9), serine (10), Zn (11), Na (12), K (13), Mg (14), Ca (15),
CDP (16), CTP (17), UTP (18), GTP (19), UMP (20), AMP (21), ADP (22), ATP (23), GDP (24), UDP (25). (λex = 670 nm, slit: 5 nm/5 nm, PMT Volts: 650). (For
interpretation of the references to colour in this figure legend, the reader is referred to the Web version of this article.)
systems by recording fluorescence spectra of SQ-PBA3. Due to the si-
milarity of nucleotide structures, such as UTP, GTP and CTP, these
substances all induced increase of fluorescence (Fig. 4b), which may
indirectly indicate that the recognition of ATP is mainly due to the
specificity of phenylboronic acid to diols. ATP plays a major role in the
energy metabolism of the human body. The release and absorption of
energy in the body is mainly manifested by the production and con-
sumption of ATP. In addition, UTP, CTP and GTP are also sources of
energy in the anabolism of some substances. Thus SQ-PBA3 can be used
to monitor changes in the total intracellular nucleoside triphosphate
content caused by drugs or diseases.
3.5. Response mechanism
First, the binding ratio between the SQ-PBA3 and ATP was studied
by Job's Plot experiment. The change of the maximum absorbance of
solution increases linearly with the increase of the ATP concentration.
Then the inflection point occurs when [ATP]/[ATP]+[SQ-PBA3] is
0.67, so the binding ratio of ATP to SQ-PBA3 is 2:1 (Fig. S21, Sup-
plementary Data).
To further confirm that ATP induced reassembly of SQ-PBA3 in
aqueous solution, we conducted dynamic light scattering experiments
(
DLS) to study the particle size distribution of aggregates. As shown in
Fig. 5, the average hydrated particle size of SQ-PBA3 in PBS buffer
solution is 570 nm. When the CTAB was added to the solution, the
particle size decreased to 120 nm, indicating that CTAB has a good
solubilizing effect on the dye, which is consistent with the experimental
results of the absorbance spectrum. With the subsequent addition of
ATP, the particle size of the solution was further decreased to 70 nm. All
the results revealed the reassembly of SQ-PBA3 by the addition of ATP.
The aggregates morphology of SQ-PBA3 alone in PBS solution, in
the presence of surfactant CTAB and subsequently addition of ATP were
observed respectively by field emission scanning electron microscopy.
When SQ-PBA3 was alone in PBS solution, it self-aggregated and ag-
glomerated together in the form of small spheres (Fig. 6a). After adding
CTAB, the morphology of the aggregates changed greatly (Fig. 6b).
With further addition of ATP, the agglomerated aggregates in the so-
lution disappeared, exhibiting a short and small rod-like structure, and
the structures spread in a dendritic shape (Fig. 6c).
Fig. 5. DLS analysis of SQ-PBA3 (4.0 μM) alone, in the presence of CTAB, and
CTAB + ATP in PBS solution (pH = 7.4, 10 mM).
3.6. Intracellular imaging for endogenous ATP
We then examined the capacity of SQ-PBA3 to monitor ATP levels
in living cells. Paclitaxel was used for fluorescence imaging because it
interferes with the normal function of the cytoskeletal component mi-
crotubules, stopping cell division in mitosis, until death [32]. It can be
seen from Fig. 7 that MCF-7 cells incubated with 4.0 μM SQ-PBA3
displayed bright red fluorescence and have good dispersion in the cells,
indicating that the probe has good cell permeability. The cells con-
tinued to be incubated with paclitaxel, as expected, showed sig-
nificantly weakened fluorescence, suggesting that the decreased ATP
level in the cells and the abilities of SQ-PBA3 to sense the changes of
intracellular ATP level.
5

G. Wang, et al.
Dyes and Pigments 171 (2019) 107698
Fig. 6. FESEM images of SQ-PBA3 (4.0 μM) in PBS solution (pH = 7.4, 10 mM) (a), with 0.25 mM CTAB (b), with 0.25 mM CTAB and 40 μM ATP (c).
sensitivity and better selectivity.
Acknowledgements
This work was financially supported by the Program for Chang
Jiang Scholars and Innovative Research Team in University of Ministry
of Education of China (IRT15R11), the Fujian Provincial Natural
Science Foundation, P.R. China (No. 2017J01577, 2019J01208), the
Open Project Program of the State Key Laboratory of Photocatalysis on
Energy and Environment, Fuzhou University (No. SKLPEE-KF201724,
SKLPEEKF201727), and the Open Project Program of Key Laboratory of
Bioorganic Phosphorus Chemistry & Chemical Biology (Ministry of
Education), Tsinghua University.
Appendix A. Supplementary data
Supplementary data to this article can be found online at https://
doi.org/10.1016/j.dyepig.2019.107698.
References
[
[
[
[
[
1] Shen X, Mizuguchi G, Hamiche A, Wu C. A chromatin remodelling complex involved
in transcription and DNA processing. Nature 2000;406:541–4.
2] Ashcroft FM, Gribble FM. ATP-sensitive K+ channels and insulin secretion: their
role in health and disease. Diabetologia 1999;42:903–19.
3] Dennis PB, Jaeschke A, Saitoh M, Fowler B, Kozma SC, Thomas G. Mammalian TOR:
a homeostatic ATP sensor. Science 2001;294:1102–5.
4] Patel A, Malinovska L, Saha S, Wang J, Alberti S, Krishnan Y, Hyman AA.
Biochemistry: ATP as a biological hydrotrope. Science 2017;356:753–6.
5] Liu Y, Geng YH, Yang H, Yang H, Zhou YT, Zhang HQ, Tian XX, Fang WG.
Extracellular ATP drives breast cancer cell migration and metastasis via S100A4
production by cancer cells and fibroblasts. Cancer Lett 2018;430:1–10.
6] Yangyuoru PM, Dhakal S, Yu Z, Koirala D, Mwongela SM, Mao H. Single-molecule
measurements of the binding between small molecules and DNA aptamers. Anal
Chem 2012;84:5298–303.
[
[
[
[
7] Zhang S, Yan Y, Bi S. Design of molecular beacon as signaling probes for ATP de-
tection in cancer cells based on chemiluminescence resonance energy transfer. Anal
Chem 2009;81:8695–701.
8] Liu BF, Ozaki M, Hisamoto H, Luo Q, Utsumi Y, Hattori T, Terabe S. Microfluidic
chip toward cellular ATP and ATP-conjugated metabolic analysis with biolumi-
nescence detection. Anal Chem 2005;77:573–8.
9] Goyal RN, Oyama M, Singh SP. Simultaneous determination of adenosine and
adenosine-5’-triphosphate at nanogold modified indium tin oxide electrode by os-
teryoung square-wave voltammetry. Electroanalysis 2007;19:575–81.
Fig. 7. Fluorescent images of MCF-7 cells. (a) MCF-7 cells incubated with SQ-
PBA3 for 2 h, (b) pretreated with SQ-PBA3 for 2 h and further incubated with
paclitaxel (10 nM) for 24 h.
[
[
10] Manfredi G, Yang L, Gajewski CD, Mattiazzi M. Measurements of ATP in mamma-
lian cells. Methods 2002;26:317–26.
11] Li M, Zhang J, Suri S, Sooter LJ, Ma D, Wu N. Detection of adenosine triphosphate
with an aptamer biosensor based on surface-enhanced Raman scattering. Anal
4
. Conclusion
In conclusion, based on multisite combining and “aggregation-re-
Chem 2012;84:2837–42.
assembly” strategies, we have developed a near-infrared squaraine (SQ-
PBA3) attached with two phenylboronic acid groups for selectively and
sensitively detection of ATP in living cells. The dye can effectively
monitor changes in intracellular ATP level. We expect that our work
should provide new attempts for the design of near-infrared squaraine
based fluorescent probes for biosensing detection with higher
12] Gout E, Rebeille F, Douce R, Bligny R. Interplay of Mg2+, ADP, and ATP in the
[
[
2
+
cytosol and mitochondria: unravelling the role of Mg in cell respiration. Proc Natl
Acad Sci Unit States Am 2014;111:E4560–7.
13] Wang G, Su X, Xu Q, Xu G, Lin J, Luo X. Antifouling aptasensor for the detection of
adenosine triphosphate in biological media based on mixed self-assembled aptamer
and zwitterionic peptide. Biosens Bioelectron 2018;101:129–34.
[14] Xu Z, Singh NJ, Lim J, Pan J, Ha NK, Park S, Kim KS, Yoon J. Unique sandwich
6

G. Wang, et al.
Dyes and Pigments 171 (2019) 107698
stacking of pyrene-adenine-pyrene for selective and ratiometric fluorescent sensing
Org Lett 2011;13:1162–5.
of ATP at physiological pH. J Am Chem Soc 2009;131:15528–33.
15] Wang L, Yuan L, Zeng X, Peng J, Ni Y, Er JC, Xu W, Agrawalla BK, Su D, Kim B,
Chang YT. A multisite-binding switchable fluorescent probe for monitoring mi-
tochondrial ATP level fluctuation in live cells. Angew Chem Int Ed 2016;55:1773–6.
16] Tan KY, Li CY, Li YF, Fei J, Yang B, Fu YJ, Li F. Real-time monitoring ATP in
mitochondrion of living cells: a specific fluorescent probe for ATP by dual re-
cognition sites. Anal Chem 2017;89:1749–56.
[24] Anees P, Sreejith S, Ajayaghosh A. Self-assembled near-infrared dye nanoparticles
as a selective protein sensor by activation of a dormant fluorophore. J Am Chem Soc
2014;136:13233–9.
[25] Wang G, Xu W, Guo Y, Fu N. Near-infrared squaraine dye as a selective protein
sensor based on self-assembly. Sensor Actuator B Chem 2017;245:932–7.
[26] Sun P, Wu Q, Sun X, Miao H, Deng W, Zhang W, Fan Q, Huang W. J-Aggregate
squaraine nanoparticles with bright NIR-II fluorescence for imaging guided pho-
tothermal therapy. Chem Commun 2018;54:13395–8.
[27] Ma R, Yang H, Li Z, Liu G, Sun X, Liu X, An Y, Shi L. Phenylboronic acid-based
complex micelles with enhanced glucose-responsiveness at physiological pH by
complexation with glycopolymer. Biomacromolecules 2012;13:3409–17.
[28] Yang H, Sun X, Liu G, Ma R, Li Z, An Y, Shi L. Glucose-responsive complex micelles
for self-regulated release of insulin under physiological conditions. Soft Matter
2013;9:8589–99.
[29] Sun X, Xu Q, Kim G, Flower SE, Lowe JP, Yoon J, Fossey JS, Qian X, Bull SD, James
TD. A water-soluble boronate-based fluorescent probe for the selective detection of
peroxynitrite and imaging in living cells. Chem Sci 2014;5:3368–73.
[30] Zhang P, Zhu MS, Luo H, Zhang Q, Guo LE, Li Z, Jiang YB. Aggregation-switching
strategy for promoting fluorescent sensing of biologically relevant species: a simple
near-infrared cyanine dye highly sensitive and selective for ATP. Anal Chem
2017;89:6210–5.
[
[
[
[
[
[
[
[
[
17] Zhang M, Ma WJ, He CT, Jiang L, Lu TB. Highly selective recognition and fluor-
escence imaging of adenosine polyphosphates in aqueous solution. Inorg Chem
2
013;52:4873–9.
18] Liu Y, Lee D, Wu D, Swamy KMK, Yoon J. A new kind of rhodamine-based fluor-
escence turn-on probe for monitoring ATP in mitochondria. Sensor Actuator B
Chem 2018;265:429–34.
19] Liu JH, Li RS, Yuan B, Wang J, Li YF, Huang CZ. Mitochondria-targeting single-
layered graphene quantum dots with dual recognition sites for ATP imaging in
living cells. Nanoscale 2018;10:17402–8.
20] Chen H, Law KY, Perlstein J, Whitten DG. Amphiphilic squaraine dye aggregates:
evidence for a cyclic chiral structure as a general supramolecular structure for
aggregates of dyes and aromatic molecules. J Am Chem Soc 1995;117:7257–8.
21] De Miguel G, Ziółek M, Zitnan M, Organero JA, Pandey SS, Hayase S, Douhal A.
Photophysics of H- and J-aggregates of indole-based squaraines in solid state. J Phys
Chem C 2012;116:9379–89.
[31] Mayerhöffer U, Fimmel B, Würthner F. Bright near-infrared fluorophores based on
squaraines by unexpected halogen effects. Angew Chem Int Ed 2012;51:164–7.
[32] Alushin GM, Lander GC, Kellogg EH, Zhang R, Baker D, Nogales E. High-Resolution
microtubule structures reveal the structural transitions in α β-tubulin upon GTP
hydrolysis. Cell 2014;157:1117–29.
22] Xu Y, Li Z, Malkovskiy A, Sun S, Pang Y. Aggregation control of squaraines and their
use as near-infrared fluorescent sensors for protein. J Phys Chem B
2
010;114:8574–80.
23] Chen C, Wang R, Guo L, Fu N, Dong H, Yuan Y. A squaraine-based colorimetric and
2
+
“
turn on” fluorescent sensor for selective detection of Hg in an aqueous medium.
7