Detection sensitivity enhancement of naphthalimide PET fluorescent probes by 4-methoxy-substitution

Article abstract of DOI:10.3390/molecules25194465

Naphthalimide photoinduced electron transfer (PET) fluorescent probes are widely used in fluorescence imaging. Thereinto, detection sensitivity is the vital parameter of PET probes. However, the modulation of detection sensitivity is yet to be reported for naphthalimide PET probes. Herein, the detection sensitivity enhancement of naphthalimide PET fluorescent probes through 4-methoxy-substitution is proposed in this work. Taking Zn2+ detection an example, 4-methoxy-naphthalimide PET probe 2-(2-(bis(pyridin-2-ylmethyl)amino)ethyl)-6-methoxy-1H-ben zo[de]isoquinoline-1,3(2H)-dione (BPNM) and control PET probe 2-(2-(bis(pyridin-2-ylmethyl)amino) ethyl)-1H-benzo[de]isoquinoline-1,3(2H)-dione (BPN) are separately synthesized. The addition of 4-methoxy group with ability of strong electron donating to naphthalimide facilitates the construction of electronic push-pull system in the fluorophore resulting in the bathochromic shift of absorption and fluorescence emission spectra ofBPNMand is further conducive to the enhancement of molar extinction coefficient and fluorescence quantum yield f of BPNM. Compared with BPN, BPNM shows lower Zn2+ detection limit in titration assays. Meanwhile, the fluorescence signal change (off-on) before and after Zn2+ addition of intracellular BPNM is more obvious and easier to control in confocal laser scanning imaging. Therefore, 4-methoxy-substitution improves the detection sensitivity of naphthalimide PET probe, which is favorable for the precise sensing of analyte, and further lays a good foundation for the synthesis of PET probe with high sensitivity.

Full text of DOI:10.3390/molecules25194465

molecules
Article
Detection Sensitivity Enhancement of Naphthalimide
PET Fluorescent Probes by 4-Methoxy-Substitution
Ye Tian 1,2,
*
, Miao Li 3,* and Ying Liu 1,2
1
College of Marine Technology and Environment, Dalian Ocean University, Dalian 116023, China;
yingliu@dlou.edu.cn
Key Laboratory of Environment Controlled Aquaculture, Ministry of Education, Dalian 116023, China
School of Biological Engineering, Dalian Polytechnic University, Dalian 116023, China
Correspondence: tianye@dlou.edu.cn (Y.T.); lim@mail.dlut.edu.cn (M.L.); Tel.: +86-0411-8476-3255 (Y.T.);
+86-0411-8632-2228 (M.L.)
2
3
*
ꢀꢁꢂꢀꢃꢄꢅꢆꢇ
Received: 2 September 2020; Accepted: 27 September 2020; Published: 29 September 2020
ꢀꢁꢂꢃꢄꢅꢆ
Abstract: Naphthalimide photoinduced electron transfer (PET) fluorescent probes are widely
used in fluorescence imaging. Thereinto, detection sensitivity is the vital parameter of PET
probes. However, the modulation of detection sensitivity is yet to be reported for naphthalimide
PET probes. Herein, the detection sensitivity enhancement of naphthalimide PET fluorescent probes
through 4-methoxy-substitution is proposed in this work. Taking Zn2+ detection an example,
4-methoxy-naphthalimide PET probe 2-(2-(bis(pyridin-2-ylmethyl)amino)ethyl)-6-methoxy-1H-ben
zo[de]isoquinoline-1,3(2H)-dione (BPNM) and control PET probe 2-(2-(bis(pyridin-2-ylmethyl)amino)
ethyl)-1H-benzo[de]isoquinoline-1,3(2H)-dione (BPN) are separately synthesized. The addition of
4-methoxy group with ability of strong electron donating to naphthalimide facilitates the construction
of electronic push-pull system in the fluorophore resulting in the bathochromic shift of absorption and
fluorescence emission spectra of BPNM and is further conducive to the enhancement of molar extinction
coefficient ε and fluorescence quantum yield Φf of BPNM. Compared with BPN, BPNM shows lower
Zn2+ detection limit in titration assays. Meanwhile, the fluorescence signal change (off-on) before
and after Zn2+ addition of intracellular BPNM is more obvious and easier to control in confocal
laser scanning imaging. Therefore, 4-methoxy-substitution improves the detection sensitivity of
naphthalimide PET probe, which is favorable for the precise sensing of analyte, and further lays
a good foundation for the synthesis of PET probe with high sensitivity.
Keywords: 1,8-naphthalimide; detection sensitivity; fluorescence quantum yield; molar extinction
coefficient; photoinduced electron transfer
1. Introduction
Fluorescence imaging is a sensor technique with non-invasive characteristics, high sensitivity,
and excellent spatial and temporal resolution [1,2]. Fluorescence probe molecular devices, which are
specially designed for analysis and detection purposes, have been widely used in the fields of biological,
environmental, and medical testing, such as the detection of active small molecules, over-expressed
enzymes and proteins in certain diseases, and intracellular micro-environment parameters [3,4].
The design of fluorescence probes can be realized through the utilization of different fluorescence
detection mechanisms based on various detection environments and purposes. The development
and understanding of fluorescence mechanisms promote a continuous, wider, and more flexible
application of the fluorescence probe in the microscopic area. Fluorescence probes with large Stokes
shift designed according to fluorescence resonance energy transfer (FRET) mechanism can generate
accurate fluorescence ratiometric detection without the need for calibration [5–7]. Probes designed
Molecules 2020, 25, 4465; doi:10.3390/molecules25194465
www.mdpi.com/journal/molecules

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based on intra-molecular charge transfer (ICT) can realize small molecule long wavelength fluorescence
emission, and detection of the micro-environment such as pH value and solvent polarity [ 12].
Meanwhile, the probes produced based on photo-induced electron transfer (PET) can realize remarkable
fluorescence off-on change before and after detection [13 17]. A typical PET fluorescence probe is
8
–
–
composed of a fluorophore conjugated with a recognition receptor via a short spacer. The PET process
can be divided into a-PET and d-PET process, which correspond to the different transition directions of
the electrons among the fluorophore and analyte recognition receptor. In the a-PET process, electron
transfers from the recognition receptor to the fluorophore, because the highest occupied molecular
orbital (HOMO) energy level (EHOMO) of the receptor is higher in magnitude than that of fluorophore.
During d-PET, the excited state of the fluorophore donates electron to the lowest unoccupied molecular
orbital (LUMO) of receptor and quenches fluorescence [18,19]. Thereinto, a-PET process is a more
commonly used PET process.
To date, several fluorophores, including naphthalimide, cyanine dyes, and fluoroborate pyrrole,
have been successfully applied to the design and preparation of fluorescence probes. 1,8-naphthalimide
fluorophore has attracted extensive attentions due to its ease of structural modification, low cytotoxicity,
high photo stability, pH stability, and two-photon absorption characteristics [20–23]. Specifically,
the photophysical properties and ease of structural modification provide desirable diversity for the
design and synthesis of naphthalimide fluorescence probes. Considering that the imine group of
naphthalimide can be used as the condensing site to connect the fluorescence probe and electron-rich
recognition group, naphthalimide is widely used as the fluorophore of a-PET fluorescence probe.
When the recognition group of the probe is in the free state, the electrons transit from the HOMO
of the recognition receptor to the HOMO of the naphthalimide fluorophore, quenching fluorescence
of naphthalimide. On the contrary, when the analyte is combined with the recognition receptor,
PET process is restrained and the fluorescence is recovered. Fluorescence probes require high sensitivity
for improving detection efficiency. However, the improvement of the naphthalimide PET fluorescence
probe sensitivity has yet to be reported.
The detection sensitivity of the fluorescence probe is closely related to the rate of change of
the fluorescence signal with concentration [17]. The improvement of the fluorescence intensity of
PET probe after PET is restrained can promote the detection sensitivity of probe under the constant
probe concentration. The fluorescence intensity of probe at the specific concentration corresponds
to two physical parameters; fluorescence quantum yield Φf and molar extinction coefficient
ε [24].
Therefore, fluorophore with larger Φf and has higher detection sensitivity. The C4-substitution of
ε
naphthalimide with electron donating group is favorable for the construction of fluorophore electronic
push-and-pull systems, which decreases the energy level difference between the fluorophore HOMO
and LUMO and increases the Φf and ε. Therefore, C4-substitution with electron donating group
can improve detection sensitivity of the naphthalimide fluorophore under the same experimental
conditions. Usually, C4-substitution of naphthalimide mainly derives from the nucleophilic reaction
of secondary or tertiary amine as the electron-donating group [21–23]. However, nitrogen atoms are
prone to protonation, which weakens the electron donating ability of the amine groups, disturbs the
original fluorophore electronic push-and-pull system, changes the fluorophore’s physical properties,
and influences the detection performance of PET fluorescence probe. In contrast, strong electron
donating groups, such as methoxy, are not easily influenced by the environment. Hence, C4-substitution
with methoxy is more suitable for improving the detection sensitivity of naphthalimide PET probe.
Zinc ions as the second most abundant transition metal ions in body and important metal ions
in living organism, participate in various biological processes, such as apoptosis, gene expression,
enzyme regulation, and neurotransmission [25]. Taking Zn2+ detection as an example, Zn2+ PET probe
BPNM and unsubstituted Zn2+ naphthalimide PET probe BPN as the control were simultaneously
synthesized to obtain the influence of C4-substitution with methoxy on detection sensitivity (Scheme 1).
After Zn2+ recognition by BPNM (or BPN), the enhancement of fluorescence quantum yields indicated
that the PET process is inhibited and BPNM (or BPN) can detect Zn2+ through monitoring fluorescence

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enhancement. Moreover, fluorometric titration testing showed that BPNM has lower detection limit
compared to BPN. C4-substitution of naphthalimide with methoxy improving the PET probe detection
sensitivity were further confirmed by confocal laser scanning microscope imaging; when BPNM was
used for Zn2+ detection in cells, the signal off-on change was more substantial compared with BPN
under the same detection conditions. Therefore, under the same detection conditions, C4-substitution
with methoxy significantly improves detection sensitivity of naphthalimide PET probe, promoting the
precise sensing of analyte and design of the PET fluorescence probe with high sensitivity.
Scheme 1. Structures of BPNM and BPN.
2. Results and Discussion
2.1. 4-Methoxy Substitution of Naphthalimide
The development of C4-substitution of naphthalimide with methoxy is lacking due to the
difficulty in modifying the C4 position. The only reported cases of such preparations are as follows:
4-methoxy-1,8-naphthalene dimethyl anhydride was synthesized via the reaction of 1,8-naphthalene
dimethyl anhydride and sodium methylate in pyridine under iodide copper catalyzed conditions [26].
However, the method mentioned above possess some shortcomings, including that pyridine as solution
is irritant and high toxicity; the iodide copper catalyst has a small particle size, making it difficult to
separate from the final product by filtration and column chromatography.
Preparation of 4-methoxy-substituted naphthalimide can also be achieved via the reaction of
4-bromo naphthalimide and sodium methylate in methanol using anhydrous cupric sulfate as the
catalyst [27]. The anhydrous cupric sulfate dissolved in methanol catalyzing the 4-Br-substituted
naphthalimide replaced the original synthetic method of iodine copper catalyzing 1,8-naphthalene
dimethyl anhydride in pyridine to undergo the reaction of C4-substitution of naphthalimide with
methoxy. This modified method has the advantages of good water solubility and easy separation of the
catalyst. Therefore, 4-methoxy-substituted Zn2+ PET probe BPNM using 2,2’-dipicolylamine (DPA) for
molecular recognition were synthesized under these conditions. The corresponding synthetic route and
compound characterization are shown in Scheme S1 and Figures S1–S10. Probes BPNM and BPN were
successfully synthesized for further Zn2+ detection in the solutions and biological micro-environment.
2.2. Zn2+ Titration Experiments of BPNM and BPN
In Figure S11, we can know that the HOMO energy levels of the BPNM and BPN fluorophore
is lower than that of free receptors (DPA), but higher than that of zinc ion-combined receptors
(DPA-Zn2+). Hence, in the free state of DPA, the PET process can originate in BPNM and BPN probes,
where fluorophore emission is quenched. On the contrary, the PET is restrained and fluorescence
light up after DPA coordination with zinc ions. The addition of BPNM and BPN to Tris-HCl solution
(
10 mM, pH = 7.4) and dimethyl sulfoxide (DMSO) (V:V = 9:1) solution generated varying degrees of
response to Zn2+ in certain range of concentration, which is evident from the comparison between the
absorbance (or emission spectra) of BPNM and BPN (Figure 1a,b). The comparison of BPNM and BPN
spectra showed that the maximum absorption peak of BPNM had an obvious red shift relative to BPN
(Figure 1a). Moreover, under the same reactant concentrations, the absorbance of BPNM showed a

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greater increase than that of BPN (Figure 1a). Similar results were observed in the fluorescence emission
spectra of BPNM, which possessed a red shift compared to BPN (Figure 1b). Additionally, the BPNM
displayed a 2-fold increased fluorescence intensity compared to BPN (Figure 1b). By comparing
chemical structure, it can be concluded that the differences between BPNM and BPN spectra mainly
originate from the 4-methoxy substitution of naphthalimide. The strong electron donating methoxy
group acting as the auxochromic group, which enhances the fluorophore electron push-and-pull,
connected to the naphthalimide fluorophore, reducing the energy level difference between the
fluorophore HOMO and LUMO (from 4.01 to 3.79 eV) with the red-shift of the maximum absorbance
peak and fluorescence emission peak (Figure 1 and Figure S11). The molar extinction coefficients of
BPNM and BPN for Zn2+ recognition are 1.617
×
105 and 1.407
×
105 L
·
Mol
−1 cm−1, where the value
·
of BPNM showed a significant increases compared to BPN. Furthermore, the Φf of BPNM and BPN
before and after Zn2+ recognition are shown in Table 1. Under constant probe concentration conditions,
after the addition of a specific amount of Zn2+, the Φf of BPNM (0.628) is considerably higher than that
of BPN (0.014). In summary, both BPNM and BPN can be used as PET probe for fluorescence detection
of Zn2+. Additionally, incorporation of the 4-methoxy group provides the naphthalimide PET probe
with higher molar extinction coefficient and fluorescence quantum yield.
Figure 1. BPNM and BPN response spectra to Zn2+. (
of BPN (10 M, = 345 nm) and BPNM (10 M,
a
) Absorption spectra and (
λ
= 379 nm) with different Zn2+ concentrations in
ex
b) fluorescence spectra
µ
λ
ex
µ
the mixture of Tris-HCl (10 mM, pH = 7.4) and DMSO (V:V = 9:1).
Table 1. Photophysical properties of BPNM and BPN.
3
4
λabs (nm) 1
λ
(nm) 2
Φf
Φf
em
379
345
2
457
396
0.058
0.003
0.628
0.014
BPNM
BPN
1
Wavelengths of absorption peaks and emission peaks excited by the corresponding excited wavelengths in
10 mM Tris-HCl buffer (pH = 7.4) and DMSO (V:V = 9:1) solution; fluorescence quantum yields of chemosensors
3
4
(0.1 mM
)
before and after the addition of Zn2+ (0.1 mM) to 10 mM Tris-HCl buffer (pH = 7.4) and DMSO
(V:V = 9:1) solution.
2.3. Determination and Comparison of Limit of Detection (LOD) of BPNM and BPN Probes
The detection sensitivity of the PET fluorescence probe was quantitatively analyzed by LOD.
Zn2+ concentration in the Tris-HCl (10 mM, pH = 7) and DMSO (V:V = 9:1) solutions was plotted as the
abscissa and the fluorescence intensity of the probe after Zn2+ was plotted as the ordinate. As shown
in Figure 2, within a certain Zn2+ concentration range, the fluorescence intensities of BPNM and BPN
are linearly correlated to the Zn2+ concentration, in which the slope fitted is k.
σ value is the standard
deviation value of the three measurements of the fluorescence intensity without the addition of Zn2+
The probe LOD can be calculated using Equation (1):
.
Detection limit = 3 σ/κ
(1)

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Figure 2. Determination of (a) BPN and (b) BPNM detection limit.
2.4. BPNM and BPN Ion Competitive Experiment
In the Tris-HCl solution (10 mM, pH = 7) and DMSO (V:V = 9:1) system, the fluorescence intensity
M BPNM (or BPN) was determined as I0. A variety of metal ions such as Na+, Fe2+, Fe3+, Mg2+
Mn2+, Cr3+, K+, or Ca2+ (30
M, nitrates), were added into the system, where no new fluorescence
peaks were observed for BPNM (or BPN), and the I/I0 value was approx 1 (Figure 3 and Figure S12).
However, when Zn2+ (30
M) was continuously added into the system, the fluorescence intensity of
of 10
µ
,
µ
µ
BPNM (or BPN) increased (Figure 3 and Figure S12). Therefore, in the presence of other metal ions,
BPNM (or BPN) can selectively recognize Zn2+ with the obvious increase of fluorescence intensity,
and the intensity change after Zn2+ recognition is simple to monitor.
Figure 3. Fluorescence intensity (I) responding to other metal ions of nitrates (30
(30 µM) existed or not. I0 is the fluorescence intensity of BPNM (30 µM).
µM) with zinc ions
2.5. BPNM and BPN Cytotoxicity Analysis
The biocompatibility of BPNM (or BPN) was evaluated using 3-(4,5-dimethylthiazol-2-yl)-2,5-dip
henyltetrazo-lium bromide (MTT) assay. The results showed that after incubation of BPNM (or BPN)
(0, 0.25, 0.5, 1.0, 2.5, 5.0, 8.0, and 10.0 µM) with human breast cancer cells MCF-7 for 12 h, the cells
displayed above 75% survival rate and high activity (Figure 4 and Figure S13). Hence, BPNM (or BPN)
probe has good biocompatibility and low cytotoxicity, which can be further used as a fluorescence
sensor for Zn2+ detection in biological micro-environment.

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Figure 4. BPNM cytotoxicity analyzed by MTT assays at various concentrations (0.25, 0.5, 1, 2.5, 5.0,
8.0, and 10.0 µM) in living MCF-7 cells for 12 h.
2.6. Confocal Laser Scanning Imaging of BPNM and BPN in Cells
The fluorescence signal can be detected by confocal laser scanning microscopy (CLSM). For cell
passage, MCF-7 cells were vaccinated on the confocal cell culture dish at density of 105/dish, followed
by the addition of 10% fetal Dulbecco’s modified Eagle’s (DMEM) with bovine serum. The cell
◦
dish was placed in an incubator filled with 5% CO2 and 95% air at 37 C and the adhesive cells
were cultured for 24 h. The experimental steps of the intracellular BPNM probe imaging are as
follows: BPNM probe (50 µM) was added into the confocal cell culture dish and incubated in the
incubator for 20 min. The dish was removed from the incubator, the culture medium was disposed,
the cells were rinsed 3 times (2 mL) with phosphate buffer (PBS, pH = 7.4, 0.01 µM), and new DMEM
culture medium was subjected to confocal laser scanning imaging. The BPNM probe fluorescence
was excited by a two-photon laser at 800 nm wavelength using a titanium-sapphire lock mode laser
(Maitai, Spectra-Physics) and collected by RXD3 channel at 380–560 nm wavelength. The inverted cell
fluorescence images were imaged by the laser confocal scanning microscope (Olympus FV1000) and
the results were further post-treated and analyzed by Olympus FV10-ASW 3.0 software. The regions of
interest (ROI) were selected on the cell fluorescence image, in which the average fluorescence intensity
of the cell was statistically analyzed and the average fluorescence intensity of the cell in the above
experiment was calculated as 473 in the ROI (Figure 5). After cell fluorescence imaging of BPNM,
Zn2+ (1 equiv.) was added to the above culture dish and mixed with the culture medium, the cells
were placed in the incubator for a further 20 min. Then, the original culture medium was disposed and
the new DMEM culture medium was imaged on the confocal microscope. After fluorescence imaging,
the average fluorescence intensity of the BPNM with Zn2+ addition in the same ROI was calculated
and the average fluorescence intensity of the cell after Zn2+ addition was calculated as 1150 (Figure 5).
Furthermore, the fluorescence imaging of the MCF-7 cell dyed by BPN (50 µM) was conducted in the
same manner. After imaging, the average fluorescence intensity of BPN before and after the addition
of Zn2+ (1 equiv.) calculated by the above calculating method of fluorescence intensity were 85 and
123, respectively (Figure 5).
The results show that the fluorescence intensity change of BPNM probe for Zn2+ recognition is
more substantial than that of BPN. As shown in Figure S14, we can know that the TPA cross section of
BPNM is about one time larger than TPA cross section of BPN. Moreover, the change rate of BPNM
fluorescence signal with Zn2+ concentration is much larger than that of BPN. Considering that the
change rate of probe fluorescence intensity with analyte concentration corresponds to TPA cross section,
which is equivalent to the role of molar extinction coefficient in one-photon fluorescence imaging,
and fluorescence quantum yield in two-photon fluorescence imaging. Hence, it can be concluded that
C4-substitution with methoxy group simultaneously increases TPA cross section and molar extinction
coefficient of naphthalimide PET probe, improving that the detection sensitivity of naphthalimide PET
probe BPNM.

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Figure 5. Two-photon confocal fluorescence images and average fluorescence intensity of MCF-7 cells
stained with probe (1) BPNM and (2) BPN. Before imaging, (a) cells and BPNM (or BPN) (50 µM),
(b) cells, BPNM (or BPN) (50 µM), and Zn2+ (50 µM) were co-incubated in culture medium.
3. Materials and Methods
3.1. Materials and Instrument
The reagent grade solvents and reagents used in the experiment were purchased from Shanghai
Sahn chemical technology Co., Ltd. The experimental water was taken from Milli-Q ultrapure water
system. When preparing the experiment, BPNM and BPN dissolved in DMSO (20 mM) was stored in
1
a refrigerator at
−
20 ◦C for use. H NMR and 13C NMR spectra of BPNM and BPN were measured by
Bruker Avance II 400 MHz nuclear magnetic resonance spectrometer (Madison, WI, USA). Meanwhile,
the mass spectra of BPNM and BPN are measured by Agilent LC/Q-TOF mass spectrometer (Palo Alto
,
CA, USA). The fluorescence emission and ultraviolet-visible absorbance spectra were separately
measured by Agilent Technologies Cary Eclipse fluorescence spectrophotometer (Palo Alto, CA, USA)
and Agilent Technologies Cary 60 UV-Vis spectrophotometer (Palo Alto, CA, USA) separately. In order
to adjust the fluorescence intensity within the appropriate range, the excitation and emission slit
width of the fluorescence spectrometer was 5/5 nm respectively. The silica gel used in the fast
column chromatography method was 200–300 mesh, purchased from Qingdao Ocean Chemical
Limited company.
3.2. Methods
3.2.1. BPNM and BPN Synthesis Method
The synthetic methods of BPN, BPNM, and its intermediate compounds 1–4 have been described
in reference [27]. The 1H NMR spectra of BPN, BPNM, and compounds
spectra of BPN and BPNM are shown in Supporting Information.
1–4,
13C NMR and HRMS
3.2.2. Fluorescence Quantum Yield Determination
The absolute fluorescence quantum yield was determined by the absolute quantum yield
instrument and the testing steps are as follows: Firstly, the absorbance wavelength of the probe
in testing solvent was inputted, the solvent background was corrected by running only the solvent.
Next, the sample transmittance was tested by loading the probe sample at 1
×
10−4 M concentration.

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The fluorescence quantum yield (Φf) was defined as the ratio between the emission photon number of
probe molecule (PNem) and the absorbance photo number of probe molecule (PNabs) (Equation (2)).
The absorbance photo number of probe molecule (PNabs) was obtained through subtracting the
transmittance photon number (PNtrans) from the total photon number (PNall).
Φf = PNen/PNabs = PNen/(PNall − PNtrans
3.2.3. Cell Culture and MTT Experiment
)
(2)
Human breast cancer MCF-7 cells were commercially supplied by KeyGEN BioTECH Co., Ltd.
(Nanjing, Jiangsu, China) The culture medium was the Dulbecco’s modified Eagle’s culture medium
containing 10% fetal bovine serum and 1% streptomycin-penicillin, and the culture conditions were
the adherent culture in an incubator filled with the mixture of 5% CO2 and 95% air. The cell activity
was determined and evaluated by MTT experiment.
Mitochondrial dehydrogenases in the mitochondria of the living cells has the ability to restore
3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide (MTT) in water-insoluble blue purple
formazan, while the dead cells do not have this ability. Within a certain cell counting range, the amount
of formazan crystalline formation amount was proportional to the living cell amount. The formazan
crystalline deposited in the cell was dissolved in DMSO and its absorbance was determined by
microplate reader instrument at specific wavelength. The cells not cultured by probe were used as the
negative control group, and by calculating the ratio between the absorbances of the probe cultured
group and negative control group, the percentage of cell activity under the culture conditions was
obtained (cell activity of the negative control group: 100%).
Microplate reader required the utilization of 96-hole plate for MTT assays. Therefore, after the cells
were digested, 100
to the 96-hole plate (Costar) for cell passage. In order to ensure data effectiveness, the cell inoculum
density in each hole should reach 1
105 /mL. After cell adhesion for 24 h, the original culture medium
was disposed and the 96-hole plate was rinsed with phosphate buffered saline (PBS, 0.01 M, pH = 7.4)
(100 L/hole). Then, every 6 multiple holes were divided as a group of parallel experiment, the cells in
each group were cultured by the complete culture medium containing 0, 0.25, 0.5, 1, 2, 5, 8 or 10
µL culture medium containing MCF-7 cells and 10% fetal bovine serum was added
×
µ
µM
BPMN. The results of the experimental and control groups were statistically obtained by each parallel
experiment containing 6 multiple pores. After being cell-cultured for 12 h, PBS solution of MTT was
added into the 96-pore plate (10
Then the culture medium was removed and the formazan deposited at the bottom of the cell was
dissolved in DMSO (100 L/pore). Finally, the microplate reader was used to measure the absorbances
at 490 and 570 nm wavelengths by calculating the cell activity according to Equation (3).
µL/pore, 5 mg/mL), which was placed in the cell incubator for 4 h.
µ
OD490
OD490nm Control
− OD570nm
nm
Probe
Probe − OD570nm Control
Cell activity(%) =
× 100%
(3)
4. Conclusions
Naphthalimide is commonly used as a PET fluorescence probe for biological micro-environment
detection, but requires high detection sensitivity to be effective. Therefore, the method for improving
the sensitivity of the naphthalimide PET fluorescence probe is worth exploring. In this work,
the 4-methoxy-naphthalimide PET probe BPNM and control naphthalimide PET probe BPN for Zn2+
detection were simultaneously synthesized to compare the influence of 4- methoxy substitution
on detection sensitivity of naphthalimide PET probe. 4-methoxy-naphthalimide PET probe BPNM
was prepared using anhydrous cupric sulfate as the catalysis in methanol, while PET probe BPN
without 4-Methoxy substitution were synthesized as the control molecular. By comparing the
absorbance and fluorescence spectra, and analysis of HOMO-LUMO orbit energy between BPNM and
BPN probes, it revealed that C4-substitution with methoxy of naphthalimide enhanced the electron

Molecules 2020, 25, 4465
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push-and-pull of the fluorophore system, reduced the energy level difference between the HOMO and
LUMO orbits of the fluorophore, promoted a red shift of the maximum absorbance and fluorescence
emission peaks, and increased the molar extinction coefficient and fluorescence quantum yield of the
probe. Furthermore, compared to BPN (3.04
detection limit. Meanwhile, BPNM, which showed increased detection sensitivity, compared to BPN,
as well as low cytotoxicity and specific Zn2+ recognition, can be used to recognize intracellular Zn2+
×
10−8 M), BPNM (4.29
×
10−9 M) showed lower Zn2+
.
In intracellular microenvironment, BPNM exhibited more substantial fluorescence intensity change for
Zn2+ recognition, compared to BPN. Hence, C4-substitution with methoxy provides a novel approach
to improve the naphthalimide PET probe, which is favorable for precise study of intended analyte and
allows for further design and synthesis of PET probes with high sensitivity.
Supplementary Materials: Scheme S1: Synthesis routes of probe BPN and BPNM, Figure S1: 1H NMR spectrum
1
of compound
1
recorded in CDCl3, Figure S2: H NMR spectrum of compound
2
recorded in CDCl3, Figure S3:
recorded
1
1H NMR spectrum of compound
3
recorded in CDCl3, Figure S4: H NMR spectrum of compound
4
in CDCl3, Figure S5: 1H NMR spectrum of compound
2
recorded in CDCl3, Figure S6: 1H NMR spectrum of
recorded in CDCl3, Figure S7: 1H NMR spectrum of compound BPNM recorded in CDCl3, Figure S8:
compound
6
13C NMR spectrum of compound BPNM recorded in CDCl3, Figure S9: Q-TOF mass spectrum of compound BPN,
Figure S10: Q-TOF mass spectrum of compound BPNM, Figure S11: (a) Molecular structures and illustrations of
frontier molecular orbital energy and PET process between different states of receptor (DPA) and (b) BPNM or (c)
BPN fluorophore moieties by B3LYP/ 6–31 g (d, p) Gaussian calculation, Figure S12: Fluorescence intensity (I)
responding to other metal ions of nitrates (30
intensity of BPN (30 M), Figure S13: BPN cytotoxicity analyzed by MTT assays at various concentrations (0.25, 0.5
1, 2.5, 5.0, 8.0, and 10.0 M) in living MCF-7 cells for 12 h, Figure S14: TPA spectra of BPNM (0.1 mM) and BPN
(0.1 mM Tris-HCl 10 mM, 60 mM KCl, pH = 7.4).
µM) with zinc ions (30 µM) existed or not. I0 is the fluorescence
µ
,
µ
Author Contributions: Conceptualization, M.L.; data curation, Y.T. and M.L.; formal analysis, Y.T. and M.L.;
investigation, Y.T. and M.L.; methodology, Y.T. and M.L.; resources, Y.L.; funding acquisition, Y.T. and Y.L.;
validation, Y.T. and M.L.; writing—original draft, Y.T.; writing—review and editing, Y.T., M.L. and Y.L. All authors
have read and agreed to the published version of the manuscript.
Funding: The research was funded by [National key R&D Program of China] grant number [2017YFD0701700],
[National Natural Science Foundation of China] grant number [31901775 and 31672673], [Key R&D Program of
Guangdong Province] grant number [2019B020215001], and [Liaoning S&T Project] grant number [2017203002].
Conflicts of Interest: The authors declare no conflict of interest.
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Sample Availability: Samples of the compounds are not available from the authors.
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