Dicyanoisophorone-Based Near-Infrared-Emission Fluorescent Probe for Detecting NAD(P)H in Living Cells and in Vivo

Article abstract of DOI:10.1021/acs.analchem.8b03563

NADH and NADPH are ubiquitous coenzymes in all living cells that play vital roles in numerous redox reactions in cellular energy metabolism. To accurately detect the distribution and dynamic changes of NAD(P)H under physiological conditions is essential f

Full text of DOI:10.1021/acs.analchem.8b03563

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Article
Dicyanoisophorone-Based Near-Infrared Emission Fluorescent
Probe for Detecting NAD(P)H in Living Cells and in Vivo
Yuehui Zhao, Keyan Wei, Fanpeng Kong, Xiaonan Gao, Kehua Xu, and Bo Tang
Anal. Chem., Just Accepted Manuscript • DOI: 10.1021/acs.analchem.8b03563 • Publication Date (Web): 11 Dec 2018
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Analytical Chemistry
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Dicyanoisophorone-Based Near-Infrared Emission Fluorescent Probe
for Detecting NAD(P)H in Living Cells and in Vivo
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Yuehui Zhao, Keyan Wei, Fanpeng Kong, Xiaonan Gao, Kehua Xu* and Bo Tang*
College of Chemistry, Chemical Engineering and Materials Science, Key Laboratory of Molecular and Nano Probes,
Ministry of Education, Collaborative Innovation Center of Functionalized Probes for Chemical Imaging in
Universities of Shandong, Shandong Normal University, Jinan 250014, P. R. China.
ABSTRACT: NADH and NADPH are ubiquitous coenzymes in all living cells and play vital roles in numerous redox
reactions in cellular energy metabolism. To accurately detect the distribution and dynamic changes of NAD(P)H under
physiological condition is essential for understanding its biological functions and pathological roles. In this work, we
developed a near-infrared (NIR) emission fluorescent small-molecule probe (DCI-MQ) composed of a dicyanoisophorone
chromophore conjugated with a quinolinium moiety for in vivo NAD(P)H detection. DCI-MQ owns the advantages of
high water solubility, rapid response, extraordinary selectivity, great sensitivity (detection limit of 12 nM), low cytotoxicity
and a NIR emission (660 nm) in response to NAD(P)H. Moreover, the probe DCI-MQ was successfully applied for the
detection and imaging of endogenous NAD(P)H in both living cells and tumor-bearing mice, which provides an effective
tool for the study of NAD(P)H-related physiological and pathological processes.
Nicotinamide adenine dinucleotide (NAD), reduced
nicotinamide adenine dinucleotide (NADH),
NAD(P)H have also been developed.22-26 Up to date,
several conveniently used small-molecule fluorescent
NAD(P)H probes have been reported. Komatsu et al.
developed a turn-off ubiquinone-rhodol fluorescent probe
UQ-Rh (λex/λem = 492/518 nm) for imaging intracellular
NAD(P)H with Ir complex as a promoter.27 Inspired by
the enzyme-catalyzed reaction, Chang’s group has
designed a boronic acid-containing fluorescent NADH
probe based on resazurin, in which boronic acid was
selected as the binding site of NADH and accelerate the
reduction process of weakly fluorescent resazurin to
strongly fluorescent resorufin.28 In addition, fluorescent
probes based on coumarin,29 fluorescein,30 cyanine,31
calix[4]arene dimer,32 and others,33,34 also have been
reported. However, most of them possess short emission
wavelengths that hinder their application in living
systems (Table S1). Up to now, organic small-molecule
fluorescent probes suitable for real-time detection and
imaging of NAD(P)H in vivo is still urgently needed. Near-
infrared (NIR) range from 650–900 nm becomes most
optical for in vivo imaging due to its less cell damage, low
background autofluorescence and high tissue penetration
depth.35,36 Therefore, designing NIR fluorescent probes for
imaging of NAD(P)H in living cells and in vivo is still
quite challenging but extremely significant.
nicotinamide adenine dinucleotide phosphate (NADP),
and reduced nicotinamide adenine dinucleotide
phosphate (NADPH) are ubiquitous coenzymes in all
living organisms. Electron carriers, NAD(P)H, are
involved in many biological processes, including energy
metabolism, mitochondrial function, gene expression,
calcium homeostasis, antioxidation, carcinogenesis, cell
death and aging.1-7 The level changes of NAD(P)H is also
associated with different pathological states, such as
diabetes, cancer and neurodegeneration.1,8-10 Therefore,
dynamic monitoring and imaging of intracellular
NAD(P)H are imperative for understanding the
contributions of NAD(P)H in physiological and
pathological processes.
Numerous classical methods for the detection of
NAD(P)H in vitro have been reported, including
electrochemical analysis,11,12 enzymatic cycling assay,13
high performance liquid chromatography,14 and capillary
electrophoresis.15 For imaging of the intracellular
NAD(P)H, single-photon or two-photon excitation
microscopy were used based on autofluorescence of
NAD(P)H.16,17 However, the above methods are still
limited by their low sensitivity, easily ultraviolet
irradiation photodamage, and biomaterials’ interference
in cells. Fluorescence bioimaging offers a powerful tool
for visualizing various biologically relevant species in
living cells and animals with high resolution.18-21 Recently,
genetically encoded fluorescent sensors and nanosensors
for dynamic monitoring and imaging of intracellular
In this regard, a turn-on NIR emission fluorescent
probe for the detection of NAD(P)H was developed (DCI-
MQ, Scheme 1). The probe contains dicyanoisophorone as
the
NIR
fluorescent
chromophore
and
1-
methylquinolinium cation as the NAD(P)H recognition
unit. Dicyanoisophorone-based fluorescent dyes have a
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typical push-pull structure, NIR emission wavelength and
Page 2 of 8
mL ，0.92 mmol) were added into 5 mL chloroform. The
mixture was stirred at room temperature under a nitrogen
atmosphere for 24 hours. Afterwards, the solvent was
removed under reduced pressure, and 10 mL ether was
added to precipitate the desired product DCI-MQ as a
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large Stokes shift, hence many NIR fluorescent probes
based on dicyanoisophorone have been reported for Au3+,
H2S, biothiols, leucine aminopeptidase and HOCl.37-41 The
DCI-MQ displays a rapid and specific response to
NAD(P)H: in the presence of NAD(P)H, the quinolinium
moiety was specially reduced, and the electron-rich amine
moiety was released, then a strong fluorescence was
emitted at 660 nm (Stocks shift 92 nm) with a low
detection limit of 12 nM. DCI-MQ was then successfully
applied in bioimaging of endogenous NAD(P)H in living
cells and in vivo. To the best of our knowledge, the probe
DCI-MQ is the first NIR emission fluorescent probe for
real-time imaging of endogenous NAD(P)H in tumor-
bearing mice, and the probe provides a contributing tool
for monitoring changes of NAD(P)H in biosystems.
1
yellow solid. Yield: 70 mg (70%). H NMR (DMSO-d6, 400
MHz): δ = 9.89 (s, 1H), 9.40 (s, 1H), 8.51 (d, J = 8.9 Hz, 1H),
8.39 (d, J = 8.3 Hz, 1H), 8.27 (t, J = 8.7 Hz, 1H), 8.07 (t, J =
7.6 Hz, 1H), 7.77 (d, J = 16.3 Hz, 1H), 7.52 (d, J = 16.3 Hz,
1H), 6.96 (s, 1H), 4.64 (s, 3H), 2.70 (s, 2H), 2.60 (s, 2H),
1.07 (s, 6H); 13C NMR (DMSO-d6, 101 MHz): δ = 170.36,
154.13, 150.08, 143.73, 137.90, 135.97, 134.84, 131.04, 130.97,
130.67, 129.45, 125.59, 119.77, 113.82, 113.17, 79.46, 46.20,
42.60, 38.47, 32.24, 27.88. HRMS: (ESI, m/z) calcd for
C24H22F3N3O3S, [M  TfO]: 340.1808, found: 340.1802.
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Scheme 2. Synthesis of DCI-MQ
Scheme 1. DCI-MQ for Fluorescent Detection of
NAD(P)H
NC
CN
NC
CN
ICT
NAD(P)H
N
N
OTf
DCI-MQ
Fluorescence off
DCI-MQH
Strong NIR Fluorescence
EXPERIMENTAL SECTION
In Vitro Detection of NADH. The stock solution of
DCI-MQ (2  10-3 M) was prepared in DMSO. Stock
solutions of NAD(P)H and various interferential reagents
(K+, Na+, Ca2+, Mg2+, Zn2+, Fe3+, Fe2+, glycine, lysine,
methionine, serine, cysteine, homocysteine, glutathione,
Vitamin C, dithiothreitol, H2O2, NaClO, t-BuOOH and
NO) were prepared in double-distilled water, while
superoxide (O2) was prepared from a solution of KO2 in
DMSO. For spectral measurements, the stock solution of
the probe was diluted with PBS buffer (10 mM, pH = 7.4)
to give a final concentration of 10 μM. NAD(P)H and
various analytes were added to the solution of DCI-MQ
(10 μM) in PBS buffer (10 mM, pH = 7.4, containing 0.5%
DMSO). The UV-Vis absorption spectra were collected
from 280–700 nm, and the fluorescence emission spectra
were recorded in a range from 592–800 nm (λex = 568 nm,
λem = 660 nm, slit widths: 10 nm/10 nm).
Synthesis of DCI-MQ. Synthesis of Compound 1. To a
solution of isophorone (2.1 mL, 13.8 mmol) and
malononitrile (0.91 g, 13.8 mmol) in 30 mL anhydrous
ethanol, catalytic amount of piperidine (14 μL, 0.14 mmol)
was added. The mixture was stirred at 60 oC for 8 h. After
cooling to room temperature, the mixture was slowly
transfered into 50 mL water and the precipitated solid
1
was filtered to give product 1 (1.2 g, yield 47%). H NMR
(Chloroform-d, 400 MHz): δ = 6.60 (s, 1H), 2.50 (s, 2H),
2.16 (s, 2H), 2.02 (s, 3H), 1.00 (s, 6H); 13C NMR
(Chloroform-d, 101 MHz): δ = 170.44, 159.87, 120.56, 113.19,
112.41, 78.17, 45.66, 42.63, 32.37, 27.81, 25.32. HRMS: (ESI,
m/z) calcd for C12H14N2, [M  H]: 185.1073, found: 185.1082.
Synthesis of Compound 2. Compound 1 (200 mg, 1.07
mmol), 3-quinolinecarboxaldehyde (202 mg, 1.28 mmol)
and catalytic amount of piperidine (11 μL, 0.11 mmol) were
dissolved in 8 mL dry acetonitrile. The mixture was
stirred at 50 oC for 5 h. After cooling to room temperature,
the precipitate was filtered to obtain the desired product
Cell Culture and Fluorescence Imaging. HepG2 and
HL-7702 cells were cultured in DMEM (Dulbecco’s
modified Eagle’s medium) supplemented with 1%
1
2 (150 mg, yield 43%). H NMR (DMSO-d6, 400 MHz): δ =
o
penicillin-streptomycin, 10% fetal bovine serum at 37 C
9.24 (s, 1H), 8.64 (s, 1H), 8.03 (d, J = 8.4 Hz, 1H), 7.97 (d, J
= 8.2 Hz, 1H), 7.78 (t, J = 7.7 Hz, 1H), 7.72 (d, J = 16.3 Hz,
1H), 7.65 (t, J = 7.6 Hz, 1H), 7.49 (d, J = 16.3 Hz, 1H), 6.96 (s,
1H), 2.64 (s, 2H), 2.60 (s, 2H), 1.04 (s, 6H); 13C NMR
(DMSO-d6, 101 MHz): δ = 170.72, 155.67, 150.49, 147.95,
134.61, 134.57, 131.89, 130.75, 129.70, 129.29, 129.00, 127.98,
127.81, 124.08, 113.40, 77.69, 42.72, 38.52, 32.18, 27.93.
HRMS: (ESI, m/z) calcd for C22H19N3, [M  H]: 326.1651,
found: 326.1657.
in an incubator of 5% CO2. The cells were incubated in
confocal dishes for 12 h to adhere, and then the solution
of DCI-MQ in DMSO was added to the adherent cells to
give a final concentration of 10 μM DCI-MQ. All
fluorescence images were acquired by a Leica TCS SP8
confocal laser scanning microscopy with a 63× oil
objective lens. Time-dependent images were acquired
every five minutes after addition of 10 μM DCI-MQ into
HepG2 cells. To compare the difference of NADH content
between tumor cells and normal cells, HepG2 cells and
HL-7702 cells were respectively treated with 10 μM DCI-
Synthesis of the probe DCI-MQ. Compound 2 (60 mg，
0.18 mmol) and methyl trifluoromethanesulphonate (0.1
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MQ for 15 min, washed three times with PBS buffer. For
Optical Properties. To testify the detection
sensitivity of the probe towards NADH, the optical
properties in the presence/absence of NADH were
measured in PBS buffer (10 mM, pH = 7.4, containing
0.5% DMSO, v/v). From the UV-Vis spectra, DCI-MQ
exhibits a maximum absorption wavelength at 380 nm in
the presence of NADH, and then redly shifted to 568 nm
after reaction with NADH (Figure 1A). Concurrently, the
fluorescence emission spectral of DCI-MQ treated with
NADH have also enhanced significantly at 660 nm
(Quantum Yield = 0.16) (Figure 1B). Both changes can be
attributed to the reduction of quinolinium moiety, which
transfers the probe to a typical donor-π-acceptor (D-π-A)
structure and for producing remarkable fluorescence
signal. The time-dependent (0–30 min) fluorescence
response of DCI-MQ with NADH (50 μM) was tested
(Figure 1C): the fluorescence intensity at 660 nm of DCI-
MQ (10 μM) exhibits negligible changes, whereas it
rapidly intensified to a plateau at 15 min after the
treatment of NADH. These results prove that DCI-MQ is
an ideal turn-on NIR emission fluorescent probe for
detecting NADH.
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glucose stimulation, HepG2 cells were pretreated with 20
mM glucose for 30 min, and then treated with 10 μM DCI-
MQ for 15 min. For colocalization assay, HepG2 cells or
HL-7702 cells were co-stained with 10 μM DCI-MQ and 1
μM Mito-Tracker Green for 15 min. DCI-MQ (red channel)
was excited at 561 nm and the fluorescence emission
range was set from 590 to 700 nm. Mito-Tracker Green
(green channel) was excited at 488 nm and the
fluorescence emission range was set from 500 to 550 nm.
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RESULTS AND DISCUSSION
Design and Synthesis of DCI-MQ. The reduction of 1-
methylquinolinium cations followed previously report,
which bearing an electron-withdrawing substituent in the
3-positon, with NADH analogues to yield the 1,4-
dihydroquinolines predominantly.42 In this regard, the
probe DCI-MQ was designed using quinolinium moiety
as the reduction site of NAD(P)H and the
dicyanoisophorone electron-withdrawing group as the
NIR chromophore. It is envisioned that, upon reaction
with NAD(P)H, quinolinium moiety could be reduced
into an electron-rich amine moiety, and the reaction
product possesses an intramolecular charge transfer (ICT)
process, thereby leading to a distinct NIR fluorescence
signal. Consequently, the probe was conveniently
prepared in three steps and purified by filtration (Scheme
2), and all the products were characterized by nuclear
magnetic resonance (1H NMR, 13C NMR) and high-
resolution mass spectrometry (HRMS), as shown in the
Experimental Section and Supporting Information.
Quantification of NADH and Detection Limit.
Fluorescent titration experiments of DCI-MQ to different
concentration of NADH (0–1 μM) were examined to
investigate the sensitivity of DCI-MQ toward NADH. The
fluorescence emission intensities at 660 nm enhanced
gradually, and exhibit excellent linearity when plotting
versus the concentration of NADH (Figure 1D). The
regression equation is F = 22.12 + 134.27 [NADH], with a
linear coefficient of 0.9915, and the limit of detection
(signal to noise ratio = 3) for NADH was further
calculated as low as 12 nM. The probe’s fluorescence
intensities after addition of different concentrations of
NADH (0−50 μM) was also observed (Figure S1), which
agrees with the performance of the previous study also.
Hence, DCI-MQ with excellent sensitivity to NADH
applies as an effective tool for imaging endogenous
NADH in biological samples.
1.0
A
B
600
450
300
150
0
DCI-MQ
DCI-MQ + NADH
DCI-MQ
DCI-MQ + NADH
0.8
0.6
0.4
0.2
0.0
300
400
500
600
700
600
650
700
750
800
Wavelength (nm)
Wavelength (nm)
C750
D
150
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120
80
600
450
300
150
0
1 μM
DCI-MQ + NADH
DCI-MQ
600
F=22.12+134.27[NADH]
40
0
2
R
=0.9915
0
0.0 0.2 0.4 0.6 0.8 1.0
NADH (μM)
1
450
300
150
0
0
0
5
10
15
20
25
30
600
650
700
750
800
Wavelength (nm)
Time (min)
Figure 1. (A) UV-Vis absorption and (B) fluorescence
emission spectra of DCI-MQ (10 μM) before and after
reaction with NADH (50 μM). (C) The time-dependent
fluorescence intensities at 660 nm of DCI-MQ (10 μM) with
NADH (50 μM) in PBS buffer (10 mM, pH 7.4, 0.5% DMSO).
(D) Fluorescence response of DCI-MQ (10 μM) after
incubated with different concentration of NADH (0–1 μM) in
PBS buffer (10 mM, pH 7.4, 0.5% DMSO) at 37 C for 15 min.
Inset: Linear fitting curve of the fluorescence intensity at 660
nm towards the concentration of NADH (0−1 μM). λex/λem
+
+
+
+
+
2
+
+
t
e
r
e
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3
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l
s
s
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c
V
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S
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H
P
H
D
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c
H
S
2
O
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T
B
a
S
e
G
K
G
a
g
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L
S
M
C
S
D
P
N
H
D
A
Z
F
F
C
M
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a
A
N
N
Figure 2. Fluorescence intensity of 10 μM DCI-MQ at 660
nm in the presence of various analytes: 1 mM of (K+, Na+,
Ca2+, Mg2+, Zn2+, Fe3+, Fe2+, GSH, Gly, Lys, Met, Ser); 100 μM
of (Cys, Hcy, VC, DTT); 5 μM of (H2S2, Na2SO3); and 50 μM
of (NADPH, NADH). Error bars represent standard deviation
(n = 3).
o
=
568/660 nm. Error bars represent standard deviation (n = 3).
Selectivity of DCI-MQ. Before applying the probe to
complicated biological sample, the selectivity of DCI-MQ
toward NAD(P)H was investigated by fluorescence
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measurements of the probe to other biological relevant
Page 4 of 8
influence on the fluorescence response of DCI-MQ with
NADH was also investigated in PBS buffer (pH = 5.8–8.0),
which indicated that the probe is capable of detecting
NAD(P)H under physiological pH conditions (Figure S6).
species in PBS buffer, including inorganic salts (K+, Na+,
Ca2+, Mg2+, Zn2+, Fe3+, Fe2+), common amino acids (glycine,
lysine, methionine, serine, cysteine, homocysteine), and
reducing agents (glutathione, Vitamin C, dithiothreitol,
H2S2 and SO32-). The fluorescence intensities at 660 nm
only increased dramatically when NAD(P)H was added,
but all the other analytes occupied quite low response
(Figure 2). Notably, sulfite has considerably higher
fluorescence intensity change than other materials,43 but
still negligible to NAD(P)H due to its low in vivo
concentration (0.2 and 4.87 µM).44-48 Meanwhile, after
DCI-MQ reacted with NADH, the fluorescence intensities
yielded a negligible change after various reactive oxygen
species (ROSs) addition, including O2, H2O2, NaClO, t-
BuOOH and NO (Figure S2), which represents the
reaction product also owns high stability. These data
further validates that DCI-MQ possesses a high selectivity
for NAD(P)H under physiological conditions and
therefore could be potentially used to specific NAD(P)H
detection in biological samples.
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Cytotoxicity Assays. Prior to analysis in living cells, we
tested the cytotoxicity of DCI-MQ using a conventional
MTT assay in HepG2 cells. In Figure S7, HepG2 cells kept
higher viability when incubated with 0–100 μM of DCI-
MQ for 12 h, thereby validating that DCI-MQ holds low
cytotoxicity to cells at the concentration of 10 μM for 12 h
under experimental conditions, and it is potentially
applicable for the biological application.
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A)
B)
D)
1 min C)
5 min
10 min
G)
E)
15 min F)
20 min
25 min H)
30 min
Scheme 3. Proposed Reaction Mechanism for the
Response of DCI-MQ to NADH
Figure 3. Time-dependent fluorescence imaging of
endogenous NADH in HepG2 cells incubated with DCI-MQ
(10 μM) for 30 min. (A) Bright filed and (B-H) fluorescence
images of HepG2 cells captured without washing. Cell images
were acquired with λex/λem of 561/590–700 nm. Scale bar: 50
μm.
Fluorescence imaging in living cells. Encouraged by
the above satisfactory outcome, we further investigated
the imaging ability of DCI-MQ for endogenous NAD(P)H.
HepG2 cells were treated with DCI-MQ (10 μM) and
recorded their fluorescence changes within 30 min using
confocal analysis. After addition of DCI-MQ, the NIR
fluorescence signal of HepG2 cells increased gradually in
30 min, and almost no fluorescence outside the cells was
observed even without washing (Figure 3). In addition, a
control experiment with H2O2 was carried out. The HepG2
cells were pretreated with 100 μM H2O2 for 30 min and
then treated with 10 μM probe for 30 min. The
fluorescence intensity of HepG2 cells decreased obviously
duo to the lower NADH level in the process of H2O2-
induced oxidative stress (Figure S8).15 The result indicated
that the probe has decent cell permeability and could
react with endogenous NAD(P)H in living cells.
Reaction Mechanism and pH Influence. The
proposed mechanism for the response of DCI-MQ to
NADH is that the quinolinium moiety can be reduced to
the 1, 4-dihydroquinoline by reacting with NADH
(Scheme 3). In order to explore the reductive sensing
mechanism, the probe and the product of DCI-MQ with
NADH were analyzed by ESI-HRMS and 1H NMR
spectroscopy. After completing reaction of the probe with
NADH in PBS buffer, the product was extracted with
dichloromethane and then purified by silica gel column
chromatography. The reaction product, DCI-MQH, was
observed by the [M  H] signal at m/z = 340.1855 (Figure
S3, calculated for [M  H] C23H23N3: 340.1808). To further
Recent studies have shown that the ratio of
NAD+/NADH is significantly lower in certain tumor cell
lines, representing that the level of NADH in tumor cells
is higher than normal cells.20 To compare the difference of
NADH content between tumor cells and normal cells, we
investigated the fluorescence signal intensity in tumor
cells (HepG2 cells) and normal cells (HL-7702 cells) by
DCI-MQ. The fluorescence intensity in HepG2 cells was
much stronger than in HL-7702 cells (Figure 4), verifying
the concentration level of NADH in HepG2 cells was
higher than in HL-7702 cells. The data suggests that the
probe is capable of tracking endogenous NADH in living
1
confirm the proposed mechanism, H NMR spectroscopy
of DCI-MQ and DCI-MQH was also carried out. The
reduction of quinolinium induced pronounced upfield
shift of N-CH3 from 4.64 to 3.27 ppm, and a new singlet at
3.72 ppm emerged clearly (Figure S4). In addition, the
HPLC analysis was also performed in order to explain the
mechanism. After the reaction of DCI-MQ with NADH, a
new peak at 7.87 min belonging to reaction product (DCI-
MQH) was observed, and the peak at 5.17 min
corresponding to DCI-MQ decreased remarkably (Figure
S5). Hereafter, these data confirmed the reductive sensing
mechanism of DCI-MQ with NADH. Besides, the pH
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cells and reveals the different NADH content between
tumor cells and normal cells.
and 0.91 for HL-7702 cells) of Mito-Tracker Green and
DCI-MQ merged well, which designated that
quinolinium moiety did bring DCI-MQ to the
mitochondria of living cells. The colocalization
experiments indicated that the probe was organelle-
specifically trapped in the mitochondria of living cells.
HepG2
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HepG2
HL-7702
A
B
30
25
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a)
b)
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A
HL-7702
f)
a)
b)
e)
c)
d)
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c)
d)
g)
h)
0
HepG2
HL-7702
Figure 4. (A) Fluorescence and bright field images of HepG2
cells (a, c) and HL-7702 cells (b, d) incubated with DCI-MQ
(10 μM) for 15 min, respectively. (B) Quantified fluorescence
intensities of (a) and (b). Cell images were acquired with
λex/λem of 561/590–700 nm. Scale bar: 50 μm.
1.2
1.0
0.8
0.6
0.4
0.2
0.0
C
D
Mito-tracker
DCI-MQ
Mito-tracker
DCI-MQ
1.0
0.8
0.6
0.4
0.2
0.0
In tumor cells, the intracellular NADH level is mainly
dependent on the glycolysis, which consumes glucose and
generates NADH. Therefor the addition of glucose can
elevate the intracellular NADH level. HepG2 cells were
firstly pretreated with 20 mM glucose for 30 min, and
then incubated with 10 μM DCI-MQ for another 15 min
before confocal imaging. As expected, the fluorescence
signal intensity of DCI-MQ stained cells was enhanced
after incubation with 20 mM glucose (Figure 5).
0
5
10 15 20 25 30 35
Distance (μM)
0
5
10
15
20
Distance (μM)
Figure 6. Intracellular localization of HepG2 and HL-7702
cells incubated with DCI-MQ. (A) & (B) CLSM images of
cells pretreated with 10 μM DCI-MQ and 1 μM Mito-Tracker
Green for 15 min. (a) (e) Bright filed; (b) (f) green channel,
Mito-Tracker Green; (c) (g) red channel, probe fluorescence;
(d) (h) the overlap (b) (f) and (c) (g) respectively. (C) & (D)
Intensity profile of ROIs across HepG2 and HL-7702 cells
respectively. Scale bar: 10 μm.
Control
+ 20 mM glucose
b)
A
B
50
40
30
20
10
0
a)
Imaging of NAD(P)H in Vivo. The current small-
molecule fluorescent NAD(P)H probes with emission
wavelengths located in the visible region are not suitable
for in vivo images. Therefore, to evaluate the suitability of
DCI-MQ to respond to NAD(P)H in vivo, Kunming mice
with homograft murine hepatoma cell line H22 tumor
were orthotopically injected with buffer solutions
containing DCI-MQ (10 μM) and then fluorescence
images were obtained at different times using a small
animal in vivo imaging system (IVIS). The fluorescence
signal was gradually intensified in the tumor region,
indicating that the probe can detect endogenous
NAD(P)H in H22 tumor-bearing mice (Figure 7). In this
regard, it can be concluded that DCI-MQ is an effective
probe for imaging endogenous NAD(P)H at the organism
level.
c)
d)
Control
20 mM glucose
Figure 5. (A) Fluorescence and bright field images of
endogenous NAD(P)H in HepG2 cells: (a, c) Cells stained
with DCI-MQ (10 μM, 15 min), (b, d) Cells pretreated with
glucose (20 mM, 30 min) and then stained with DCI-MQ (10
μM, 15 min), respectively. (B) Quantified fluorescence
intensities of (a) and (b). Cell images were acquired with
λex/λem of 561/590–700 nm. Scale bar: 50 μm.
A photobleaching test was performed for study the
photostability of the reaction product in living cells by
using confocal laser scanning microscopy. Within 600 s,
the fluorescence signal remains unchanged (Figure S9),
signifying that the reaction product with great
photostability. The subcellular distribution of DCI-MQ
was also completed by co-staining HepG2 cells or HL-
7702 cells with DCI-MQ and Mito-Tracker Green, a
typical commercially available mitochondrial tracker.
Figure 6 displays the overlapped fluorescence intensities
(Pearson’s colocalization coefficient 0.85 for HepG2 cells
5 min
10 min
30 min
15 min
25 min
20 min
Max
Min
Figure 7. In vivo fluorescence images of H22 tumor-bearing
mice injected with DCI-MQ (10 μM, 100 μL) via
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Analytical Chemistry
Page 6 of 8
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of clock and NPAS2 DNA binding by the redox state of NAD
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Tankyrase 1 as a target for telomere-directed molecular cancer
therapeutics. Cancer Cell 2005, 7, 25-37.
orthotopically injection for 30 min. λex/λem of 560/670 nm
was applied.
1
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3
CONCLUSIONS
In conclusion,
a
novel dicyanoisophorone-based
4
5
6
7
8
9
fluorescent NAD(P)H probe, DCI-MQ, with near-infrared
emission has been developed successfully on the basis of a
specific reduction. DCI-MQ exhibits high NAD(P)H
selectivity over other biologically relevant species, as well
as a significantly low detection limit of 12 nM. With the
low cytotoxicity and great cell membrane permeability,
DCI-MQ was successfully applied for bioimaging of
endogenous NAD(P)H in living cells and in vivo. As for its
easy preparation, DCI-MQ shows substantial potential for
applications in the study of the physiological and
pathological roles of NAD(P)H, and provides an effective
tool for monitoring changes of NAD(P)H level in
reductive stress in our future works.
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Harris, A. L.; Hawkes, D. J.; Hoekstra, O. S.; Huang, E. P.;
Hutton, B. F.; Jackson, E. F.; Jayson, G. C.; Jones, A.; Koh, D.-M.;
Lacombe, D.; Lambin, P.; Lassau, N.; Leach, M. O.; Lee, T.-Y.;
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ASSOCIATED CONTENT
Supporting Information
Materials
and
instruments,
experimental
details,
supplementary data and characterization figures of
compounds. The Supporting Information is available free of
charge via the Internet at http://pubs.acs.org.
AUTHOR INFORMATION
Corresponding Author
*Phone: 86-531-86180010
*E-mail: xukehua@sdnu.edu.cn; tangb@sdnu.edu.cn
Author Contributions
All authors have given approval to the final version of the
manuscript.
Notes
The authors declare no competing financial interests.
ACKNOWLEDGMENT
This work was supported by National Natural Science
Foundation of China (21575081, 21535004, 21775091, 91753111
and 21705098) and the Key Research and Development
Program of Shandong Province (2018YFJH0502).
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