Development of a Highly Selective Two-Photon Probe for Methylglyoxal and its Applications in Living Cells, Tissues, and Zebrafish

Article abstract of DOI:10.1007/s10895-018-2323-3

Methylglyoxal (MGO) is one of the most important active carbonyl compounds in living organisms. It is a metabolic by product of glycolysis. MGO participates in glycosylation of proteins and nucleic acids to trigger carbonyl stress, inducing pathological status and even exacerbateing the development of chronic degenerative diseases. In order to study the diseases caused by MGO, it is meaningful for us to develop methods that could efficiently detect MGO. In our work, a new two-photon fluorescent turn-on probe which named NP has been designed which was made up of naphthalimides dye as the two-photon fluorescent platform and the o-phenylenediamine as recognition site. When reacted with MGO, NP showed excellent sensitivity and selectivity. Based on the two-photon fluorescence imaging technology, NP has firstly successful application in living cells, tissues and zebrafish to detecting MGO.

Full text of DOI:10.1007/s10895-018-2323-3

Journal of Fluorescence
https://doi.org/10.1007/s10895-018-2323-3
ORIGINAL ARTICLE
Development of a Highly Selective Two-Photon Probe for Methylglyoxal
and its Applications in Living Cells, Tissues, and Zebrafish
Shiying Gao1 & Yonghe Tang1 & Weiying Lin1
Received: 16 August 2018 /Accepted: 5 November 2018
#
Springer Science+Business Media, LLC, part of Springer Nature 2018
Abstract
Methylglyoxal (MGO) is one of the most important active carbonyl compounds in living organisms. It is a metabolic by product of
glycolysis. MGO participates in glycosylation of proteins and nucleic acids to trigger carbonyl stress, inducing pathological status and
even exacerbateing the development of chronic degenerative diseases. In order to study the diseases caused by MGO, it is meaningful
for us to develop methods that could efficiently detect MGO. In our work, a new two-photon fluorescent turn-on probe which named
NP has been designed which was made up of naphthalimides dye as the two-photon fluorescent platform and the o-phenylenediamine
as recognition site. When reacted with MGO, NP showed excellent sensitivity and selectivity. Based on the two-photon fluorescence
imaging technology, NP has firstly successful application in living cells, tissues and zebrafish to detecting MGO.
.
.
.
.
Keywords Fluorescence probe Methylglyoxal Two-photon Turn-on Naphthalimides dye
Introduction
Medical research shows that MGO is directly related to diabe-
tes, alzheimer’s disease, cardiovascular disease and other dis-
eases [7]. Therefore, it is of great significance to developing
effective methods for the detection of MGO and it is the basis
of learning its toxicity to disease research.
Methylglyoxal (MGO), also known as pyruvaldehyde, is an
important bioactive carbonyl compound. MGO can be pro-
duced gradually in the process of food production and storage.
In addition, higher concentrations of MGO were detected in
milk, coffee, cola and other beverages [1]. The endogenous
MGO is mainly comes from the by-products of glycolysis
through oxidative metabolism [2–5]. For example, endogenous
MGO is produced by propionic phosphate, including dihy-
droxyacetone phosphate and glyceraldehyde triphosphate, cat-
alyzed by the propionic phosphatase and methyl ethylaldehyde
synthase [6, 7]. The processes of lipid and acetone peroxida-
tion, amino acid degradation, and Amadori rearrangement can
also produce endogenous MGO [8, 9]. MGO has high reactiv-
ity and is easy to react with the biomolecules containing amino
or mercapto groups to form glycosylated compounds [10].
Currently, the fluorescence imaging technology is a promis-
ing method to detect biologically active molecules in living
systems due to the non-destructive detection, high selectivity
and sensitivity [11–15]. To data, several MGO fluorescent
probes have been reported [16–18]. However, they generally
utilize one-photon (OP) light and provide short emission wave-
length fluorescent signal output (less than 600 nm). By con-
trast, the two-photon (TP) imaging microscopy technology
usually employed near-infrared excitation source (more than
600 nm), and exhibited some advantages, including the less
photo-damage, deep penetration, three-dimensional (3D) imag-
ing, and less auto-fluorescence interference [18]. Accordingly,
it’s urgent to develop a two-photon fluorescence probe which
can be utilized to detecting the MGO in biological systems.
Herein, we presented a new two-photon fluorescent probe
NP for detecting of MGO. The NP contained the two-photon
fluorescent platform of naphthalimides dye, and the recognition
site of o-phenylenediamine. With the introduction of MGO, NP
can give out significant fluorescence enhancement signal at
about 555 nm. NP showed excellent sensitivity and selectivity
to MGO. What’s more, NP has been propitious utilized in the
two-photon imaging of the MGO in cells, tissues and zebrafish.
Electronic supplementary material The online version of this article
(https://doi.org/10.1007/s10895-018-2323-3) contains supplementary
material, which is available to authorized users.
* Weiying Lin
weiyinglin2013@163.com
1
Institute of Fluorescent Probes for Biological Imaging, School of
Chemistry and Chemical Engineering, School of Materials Science
and Engineering, University of Jinan, Shandong 250022, People’s
Republic of China

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Experimental
overnight. Then removed solvent under reduced pressure. The
crude material was purified by flash chromatography (dichloro-
methane: methanol = 10:1) to give the compound 2 (382 mg,
Materials and Instruments
1
yield: 77%). H NMR (400 MHz, DMSO-d6) δ 8.76 (d, J =
All raw materials and reagents from supplier were directly used
without further purification. 3-Methoxyaniline was purchased
from Tianjin Heowns Biochemical Technology Co., Ltd., 1-
bromo-3-chloropropane was purchased from J&K Scientific
Ltd., hydriodic acid (HI) was purchased from Zhengzhou
Alfachem Co.,Ltd. The hydrazine was dissolved into ultrapure
water. Absorption spectra were tested by a UV-vis spectropho-
tometer (Shimadzu UV-2600, Japan). Fluorescent spectra were
tested by a fluorescence spectrophotometer (HITACHI F4600,
Japan). The pH measurements were performanced on a pH me-
ter (Mettler-Toledo Delta 320, Switzerland). High-resolution
mass spectra (HRMS) were recorded on a mass spectrometer
(Bruker Apex Ultra 7.0 T FTMS, Germany) in electrospray
ionization (ESI) mode. NMR spectra were recorded on a
400 MHz Digital NMR Spectrometer (Bruker AVANCE III,
Germany), using tetramethylsilane (TMS) as internal reference.
8.4 Hz, 1H), 8.43 (d, J = 7.6 Hz, 1H), 8.26 (d, J = 8.4 Hz, 1H),
7.95 (d, J = 2.4 Hz, 1H), 7.83 (t, J = 5.6 Hz, 1H), 7.71 (t, J =
8.0 Hz, 1H), 7.62 (s, 2H), 7.36 (dd, J1 = 8.8 Hz, J2 = 2.4 Hz, 1H),
7.10 (d, J = 8.8 Hz, 1H), 6.81 (d, J = 8.8 Hz, 1H), 3.40 (q, J =
6.8 Hz, 2H), 1.71 (m, 2H), 1.45 (m, 2H), 0.96 (t, J = 7.2 Hz, 3H);
13C NMR (100 MHz, DMSO-d6) δ 164.63, 163.70, 151.30,
146.24, 137.47, 134.84, 131.25, 130.37, 130.19, 129.25,
126.16, 124.62, 124.36, 122.68, 120.67, 119.67, 108.09,
104.23, 30.43, 26.80, 20.32, 14.23; HR-MS calculated for
C22H20N4O4 [M + H]+ m/z 405.1557, found [M + H]+ m/z
405.1554(Fig. S1-S3).
The Synthesis of NP
Compound 2 (404 mg, 1 mmol) in ethanol (20 mL) stirred
slowly with adding 1 mL containing 5 mmol SnCl2 concen-
trated hydrochloric acid solution, then refluxed for 3 h. Until
the mixture cooled, and the yellow solid would be settled.
After vacuum filtration, the cake obtained by ultra-pure water
washing to neutral. The crude product was purified by flash
chromatography (dichloromethane: methanol = 10:1) to get
The NP Synthesis Steps
The routes of compound 1, 2 and NP synthesis was shown in
Scheme 1.
1
the probe NP (248 mg, yield: 67%). H NMR (400 MHz,
The Synthesis of Compound 1
DMSO-d6) δ 8.75 (d, J = 8.4 Hz, 1H), 8.41 (d, J = 7.2 Hz,
1H), 8.24 (d, J = 8.4 Hz, 1H), 7.82 (t, J = 5.6 Hz, 1H), 7.70
(t, J = 7.6 Hz, 1H), 6.83 (m, 2H), 6.64 (s, 1H), 6.63 (s, 4H),
6.54 (d, J = 8.0 Hz, 1H), 3.39 (q, J = 6.8 Hz, 4H), 1.71 (m,
2H), 1.44 (m, 2H), 0.96 (t, J = 7.2 Hz); 13C NMR (100 MHz,
DMSO-d6) δ 164.60, 163.75, 151.26, 134.86, 134.85, 131.28,
130.28, 129.19, 124.75, 122.63, 122.61, 121.77, 121.63,
120.62, 119.24, 119.10, 108.04, 104.29, 43.02, 30.38, 20.27,
14.22; HR-MS calculated for C22H22N4O2 [M + H]+ m/z
375.1816, found [M + H]+ m/z 375.1813(Fig. S4-S6).
4-bromo-1,8-naphthalic anhydride (554 mg, 2 mmol) and 2-
nitrobenzene-1,4- diamine (306 mg, 2 mmol) were dissolved in
20 mL ethanol, refluxed for 5–6 h. After the solution gradually
deepened from the pale yellow to dark brown, the whole reac-
tion system was cooled down and gradually deposited with
yellow solid. After vacuum filtration, the cake was washed with
ice ethanol for 2–3 times. After drying, the compound 1
(774 mg, yield: 91%) was obtained without purification.
The Synthesis of Compound 2
Preparation for the Spectroscopic Test
Compound 1 (441 mg, 1 mmol) in 2-methoxyethanol (30 mL)
All the spectrum measurements were carried out as following.
added with n- butylamine (1 mL, 1 mmol) dropwise, refluxed
The stock solution of NP was prepared in DMSO and the
Scheme 1 Synthesis route of the
NP

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concentration was 1.0 mM. MGO was dissolved in ultrapure
water to get the test storage solution with a concentration of
10 mM. Other analytes (cationic/anionic) were also prepared
with ultrapure water as test storage solutions with a concentration
of 10 mM.
Before the spectrum test, 25 μL probe NP test storage
solution and 250 μL DMSO were placed in the 5 mL volu-
metric flask, then used 10 mM PBS buffer to adjust the final
volume to 5 mL to get test probe solution.
For the fluorescent titration test, different concentra-
tions of MGO test storage solution were added to the
test probe solution and recorded after 60 min of
incubation.
For the pH study, 25 μL NP test storage solution and
250 μL DMSO were placed in the 5 mL volumetric
flask, then used 10 mM PBS buffer (different pH value)
to adjust the final volume to 5 mL to get pH test probe
solution. Then added 10 μL MGO test storage solution
to the above pH test probe solution, then recorded after
60 min of incubation.
Cell Culture and Bio-Imaging
HeLa cells were cultured in Dulbecco’s modified eagle medi-
um (DMEM) supplemented with 10% FBS (fetal bovine se-
rum) and incubated at 37 °C in air atmosphere (5% CO2).
t?>Before the imaging experiments, Hela cells were seeded
in confocal plates (2 × 104 cells/mL). After 24 h, in the control
group, the cells were pretreated with 5 μM NP for 60 min at
37 °C. Cell imaging was performed after the cells were washed
twice with PBS. In the imaging of exogenous MGO, Hela cells
were pretreated with MGO (100 μM) for 60 min and washed
three times with PBS. Then Hela cells were treated with NP
(5 μM) for 60 min, washed three times with PBS. The one-
photon fluorescence excited at 488 nm for the green channel
(500–550 nm). The two-photon fluorescence excited at 760 nm.
Liver Tissue Bio-Imaging Using MGO
Rat brain slices were prepared from the liver of 4-weeks-old
living Balb/c mice, which was approved by the Animal Ethical
Experimentation Committee of Shandong University. The
fresh brain tissues were incubated with 5 μM probe NP for
60 min, and then, 100 μM MGO was added for another
60 min. Before imaging, the tissues were washed with PBS
three times. A Nikon A1R confocal microscope with a 40 ×
objective lens was used for fluorescence images. All of these
experiments meet the relevant laws and institutional guidelines.
For the selective spectrum test, requisite amount of
analytes test storage solutions was added to the above
test probe solutions, and then incubated for 60 min at
room temperature.
DFT Calculations
The theoretical calculations were performed by the soft-
ware of Gaussian 09. The orbital energy of excited dye
and recognition sites (before and after reacted with
MGO) were performed by using DFT with B3LYP func-
tional and 6-31G basis set. The initial geometric shapes
of excited dye and recognition sites (before and after
reacted with MGO) were generated by the software of
GaussView.
Zebrafish Bio-Imaging Using MGO
Wild type zebrafishes were obtained from the Nanjing Eze-
Rinka Biotechnology Co., Ltd. 3-day-old zebrafishes were trans-
ferred into confocal plates, and 5 μM probe NP was fed for
60 min; then, 100 μM MGO was added and further incubated
for another 60 min. After that, the zebrafishes were transferred
Scheme 2 Reaction mechanism
of the NP

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Fig. 1 Molecular orbital plots
(LUMO and HOMO) and
HOMO/LUMO energy gaps of
NP. In the ball-and-stick
representation, hydrogen, carbon,
nitrogen and oxygen are colored
in light gray, gray, blue, red, and
pink, respectively
into new glass bottom dishes for imaging. Fluorescence images
were acquired with a Nikon A1R confocal microscope with a 4×
objective lens. The excitation and emission wavelengths were
the same as for fluorescence imaging in living cells.
fluorescent. With the introduction of MGO, there is no
existence of the PET effect because of the condensation-
cyclization reaction between o-phenylenediamine group
and MGO. At the same time, the fluorescent intensity of
NP showed significant enhancement (Scheme 2). This
hypothesis was supported by the theoretical calculations
(Fig. 1). The structure of NP was confirmed by 1H
NMR, 13C NMR, and HR-MS.
Results and Discussion
Design and Synthesis of the NP
Spectral Response of Probe towards MGO
Naphthalimides dyes have many significant features, in-
cluding excellent two-photon properties, excellent opti-
cal stability, and high fluorescence quantum yield, so
the naphthalimides dyes are widely used in the fluores-
cence probe design [19–28]. Because of the PET
(Photoinduced Electron Transfer) effect caused by the
o-phenylenediamine group, the probe showed non-
The fluorescent spectra of NP for MGO were investi-
gated. As shown in Fig. 2a, only NP which was excited
with 440 nm showed weak fluorescence in 555 nm be-
cause of the effect of PET. After the introduction of
MGO, a distinct fluorescence peak was observed with
the equivalent concentration increased of MGO, and the
Fig. 2 a The fluorescence titration curve of the interaction between 5 μM
NP and different concentrations of MGO (0–200 μM solution); b The
change curve of the fluorescence intensity enhancement with equivalent
change of the MGO and NP in the DMSO/PBS buffer (pH = 7.4, v/v =
1:19) λex = 440 nm

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Fig. 3 Normalized variation of
fluorescence signal with the
changing concentration of MGO
intensity of fluorescence at 555 nm reached the maxi-
mum in the presence of 150 μM MGO (Fig. 2b). At the
same time, an effective linear relation between the in-
tensity of fluorescence at 555 nm and MGO level rang-
ing from 0 μM to 6 μM could be obtained, and the low
detection limit was calculated to 1.47 μM referred to
Fig. 3. It is noteworthy that the low detection limit of
NP may make it very desirable to observe the level of
MGO in vivo.
(150 μM) about 40 min. Under the pseudo-first-order
conditions, the rate constant for the probe was deter-
mined to be k = 0.07 min−1 (Fig. 4b). Therefore, NP
can be served as a sensitive probe for MGO and may
help to detect the changes of MGO in bio-systems.
Recognition Mechanism of Probe towards MGO
Subsequently, dynamic tests of NP towards MGO
were evaluated. After the treatment of 5 μM NP with
0 μM, 100 μM and 150 μM MGO, the fluorescence
intensity at 555 nm grew with time (Fig. 4). The equi-
librium state achieved of NP (5 μM) treated with MGO
The recognition mechanism of NP to MGO was investigated
by the MS assay. After the treatment of NP with MGO in
10 mM PBS (pH 7.4), the MS data (Fig. S8) showed that a
peak at 411.2 corresponding to compound 3 (calculated for
+
[M + H] m/z 411.2) could be observed clearly. It confirms
Fig. 4 a Kinetic studies curve of NP (5 μM) treated with MGO (0,100 and 150 μM) in the DMSO/ PBS buffer (pH = 7.4, v/v = 1:19). λex = 440 nm; b
Pseudo-first-order kinetic plot of the reaction of NP (5 μM) with MGO (150 μM) in the DMSO/ PBS buffer (pH = 7.4, v/v = 1:19). Slope = 0.07 min−1

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Fig. 5 a Fluorescence intensity of free sensor NP (5 μM) [ ] and in the presence of 150 μM MGO [ ] with different pH; b Photostability profiles of NP
(5 μM) in the absence or presence of UV-irradiated (365 nm). The fluorescence intensities were continuously monitored at time [ ]light; [ ] unlight
that NP undergoes the condensation-cyclization reaction with
MGO to provide the compound 3.
Although the o-phenylenediamine group is also the recognition
site for nitric oxide (NO), with the introduction of NO only
slightly fluorescence changes (about 1.6-fold) was obtained
during the same incubating time. These results indicated that
NP is highly sensitive to MGO over other analytes.
The Effect of pH, Selectivity and Photo-Stability
Studies
The pH of the test solution (or cellular microenvironment) may
affected the ability of the probe to recognize MGO. Then we
carried out the pH effect tests about NP in the absence or
presence of MGO. In the pH range of the test, the fluorescence
intensity of NP was almost invariable. At the same time when
MGO was added, the fluorescence signal fluctuated slightly in
the range of 4.5–10.0 (Fig. 5a). The experimental data indicated
the potential applications of the probe in biological samples at
physiological pH environment. After that, the stability of NP
was detected, and the results indicated that NP has excellent
photo-stability (Fig. 5b). The selectivity of NP to MGO was
evaluated by the determination of its fluorescence spectra treat-
ed with a variety of biologically relevant species. As shown in
the Fig. 6, only MGO could make significant change in fluo-
rescence intensity at 555 nm, whereas the other relevant
analytes showed almost no change in fluorescence intensity.
Cell Imaging of NP towards MGO
We had taken MTT assay to determined cell viability before
the imaging experiment. The viability was more than 80%
when the HeLa cells were incubated with NP (0–40 μM) for
24 h, meaning that NP has low cytotoxicity and can be used
for cell imaging (Fig. S7).
The utility of probe NP for imaging of MGO in living cells
was investigated under OP and TP modes. As shown in
Fig.7e-h, the HeLa cells incubated with only the probe NP
displayed almost no fluorescence. In contract, when incubated
with 5 μM probe NP for 60 min and then treated with 100 μM
MGO for another 60 min, the HeLa cells exhibited enhanced
fluorescence in the green channel at the OP mode. (Fig.7i-k).
Meanwhile, the obvious (Fig.7l) green fluorescence increase
can also be noted at the TP mode. These studies showed that
Fig. 6 The fluorescence intensity
of NP (5 μM) in the presence of
150 μM MGO in the DMSO/PBS
buffer (pH = 7.4, v/v = 1:19) and
200 μM ions, anions and ROS;
The analytes were in turn: 1: only
the probe; 2: Cu2+; 3: Mg2+; 4:
NO3−; 5: NO2−; 6: HSO3−; 7: S2−
;
8:SO42−; 9: Fe2+; 10: I−; 11: Br−;
12: ClO−; 13: H2O2; 14: Thr; 15:
Hcy; 16: Cys; 17: GSH; 18: NO;
19: Glu; 20: ONBD; 21: Pyr; 22:
FA; 23: Alk; 24: Tri; 25: Gly; 26:
MGO

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Fig. 7 The fluorescence imaging
of NP in Hela cells. a-d) The
imaging for the HeLa cells; e-h)
The imaging for the HeLa cells
treated with NP (5 μM). i-l) The
imaging for the HeLa cells treated
with MGO (100 μM) and NP
(5 μM). The cells were excited at
488 nm, and the emission
collection was from 500 to
550 nm. Scale bar: 20 μm
Fig. 8 The fluorescence imaging of NP in liver tissues. a Tissue slices only treated with NP (30 μM);(b) tissue slices added NP (30 μM) in present of
MGO (200 μM). The liver slides were excited at 760 nm. And the emission collection was from 500 to 550 nm

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Fig. 9 Fluorescence images of
zebrafish at one-photon and two-
photon modes. a-d) are the blank
group. e − h) are the
fluorescence images of zebrafish
treated with 10 μM NP. i − l) are
the fluorescence images of
zebrafish treated with 100 μM
MGO and then treated with
10 μM NP. Scale bar: 500 μm
NP has a good membrane permeability and can be applied for
monitoring exogenous MGO in living cells.
Conclusion
In conclusion, a new two-photon fluorescent turn-on probe
NP which was combined with naphthalimides dye as
fluorophore platform and o-phenylenediamine as the MGO
recognition site. NP had almost no fluorescence due to the
PET effect from the o-phenylenediamine group to the
naphthalimides dye. With the intervention of MGO, the PET
effect was broken because of the condensation-cyclization
reaction between the group of o-phenylenediamine and
MGO, showed great enhancement of fluorescence signal.
The probe showed the characteristics of highly sensitive and
selective responded to MGO. Significantly, NP has been pro-
pitious applied to the two-photon imaging of MGO in cells,
tissues and zebrafish for the first time. All these features
showed that the probe NP has the potential to practical appli-
cations in biological systems.
Liver Tissue Imaging of NP towards MGO
Encouraged by cells imaging applications of NP with
MGO, then we applied NP to image MGO in living
liver tissue slices. As a control group, the living tissues
only loaded NP displayed weak fluorescence (Fig. 8a).
After the further incubation with MGO for another
60 min, the living tissues exhibited strong green fluo-
rescence (Fig. 8b). It can be observed that the penetra-
tion depth could approximate 110 μm. These data indi-
cated that the probe NP can be applied for the visuali-
zation of MGO in living tissues.
Acknowledgements This work was financially supported by NSFC
(21472067, 21672083, 21877048), Taishan Scholar Foundation (TS
201511041), and the startup fund of the University of Jinan (309-10004).
Zebrafish Imaging of NP towards MGO
Finally, we proceeded to test the ability of NP to visu-
alize MGO in living zebrafish. As shown in Fig.9e-h,
the the living zebrafish incubated with only the probe
NP exhibited weak fluorescence. In comparison, when
incubated with the probe NP and MGO, the zebrafish
displayed enhanced fluorescence under OP excitation.
(Fig.7i-l). Notably, the same fluorescence changes were
also obtained (Fig.7l) under TP excitation. These studies
showed that NP could enter zebrafish and detect MGO
in living zebrafish under OP and TP modes.
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