A new aggregation-induced emission fluorescent probe for rapid detection of nitroreductase and its application in living cells

Article abstract of DOI:10.1016/j.saa.2017.06.065

The biological activity of nitroreductase (NTR) is closely related to biological hypoxia status in organisms. The development of effective methods for monitoring the activity of NTR is of great significance for medical diagnosis and tumor research. Toward this goal, we have developed a new aggregation-induced emission (AIE) fluorescence NTR probe TPE-HY used the tetraphenylethene as the fluorophore, and used the nitro group as the NTR recognition site. The probe TPE-HY has many excellent properties, including rapid response, AIE characteristics, high sensitivity and selectivity, and low cytotoxicity. Importantly, the probe TPE-HY is successfully applied to monitor endogenous NTR in living HeLa cells.

Full text of DOI:10.1016/j.saa.2017.06.065

Accepted Manuscript
A new aggregation-induced emission fluorescent probe for rapid
detection of nitroreductase and its application in living cells
Gaoping Xu, Yonghe Tang, Yanyan Ma, An Xu, Weiying Lin
PII:
DOI:
Reference:
S1386-1425(17)30540-1
doi: 10.1016/j.saa.2017.06.065
SAA 15281
Spectrochimica Acta Part A: Molecular and Biomolecular
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30 June 2017
Please cite this article as: Gaoping Xu, Yonghe Tang, Yanyan Ma, An Xu, Weiying Lin , A
new aggregation-induced emission fluorescent probe for rapid detection of nitroreductase
and its application in living cells, Spectrochimica Acta Part A: Molecular and
Biomolecular Spectroscopy (2017), doi: 10.1016/j.saa.2017.06.065
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ACCEPTED MANUSCRIPT
A new aggregation-induced emission fluorescent probe for rapid
detection of nitroreductase and its application in living cells
Gaoping Xu, Yonghe Tang, Yanyan Ma, An Xu, and Weiying Lin*
Institute of Fluorescent Probes for Biological Imaging, School of Chemistry and Chemical Engineering, School of
Materials Science and Engineering, University of Jinan, Jinan, Shandong 250022, P.R. China.
*Corresponding Author.
Tel.: +86 53182769108.
E-mail address: weiyinglin2013@163.com.
Abstract
The biological activity of nitroreductase (NTR) is closely related to biological hypoxia status in
organisms. The development of effective methods for monitoring the activity of NTR is of great
significance for medical diagnosis and tumor research. Toward this goal, we have developed a
new aggregation-induced emission (AIE) fluorescence NTR probe TPE-HY used the
tetraphenylethene as the fluorophore, and used the nitro group as the NTR recognition site. The
probe TPE-HY has many excellent properties, including rapid response, AIE characteristics, high
sensitivity and selectivity, and low cytotoxicity. Importantly, the probe TPE-HY is successfully
applied to monitor endogenous NTR in living HeLa cells.
Keywords
Fluorescent Probe; Nitroreductase; Aggregation-Induced Emission; Tetraphenylethene; Hypoxia
1. Introduction
Hypoxia, caused by the lack of oxygen supply [1], is associated with a variety of diseases, such
as, cardiac ischemia [2-4], stroke [5], inflammatory diseases [6, 7], and solid tumors [8, 9].
Especially, inadequate supply of oxygen (≤ 4.4 %) is an important characteristic of solid tumors.
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Medical research showed that the hypoxic status of solid tumors is directly related to tumor
progression, metastasis, drug resistance, and resistance to treatment [10, 11]. Therefore, the
development of new methods for monitoring the hypoxic conditions in cancer cells and body is of
great significance for medical diagnosis and tumor research.
At present, there are many detection methods for oxygen level reported, such as nuclear
magnetic resonance (NMR) [12, 13], clark electrode [14], immunostaining [15, 16], positron
emission tomography imaging [17, 18], most of which offer high sensitivity and selectivity.
However, those detection methods can’t guarantee the integrity of the sample. In the past decade,
fluorescence imaging as a powerful tool to monitor biomolecules in biological systems has been
widely concerned. Fluorescent probes have many obvious advantages, including good selectivity,
high sensitivity, as well as real-time imaging [19-22]. The recent studies indicated that the
biological activity of nitroreductase (NTR) is directly related to the hypoxic status in the living
cells [23-26]. Therefore, to monitor the activity of the NTR can reflect the oxygen content of the
organism. Under hypoxic condition, in the presence of NTR, the nitro group is reduced to amino
moiety through three steps. The nitro group first can be reduced to a nitroso group, then to a
hydroxylamine moiety, finally to an amino group. So far, there is a large number of fluorescent
NTR probes have been reported [23-31]. However, the response time of most fluorescent probes
for NTR more than 20 min, it is more desirable to develop the fluorescent NTR probes with rapid
response for NTR.
The aggregation-induced emission (AIE) fluorophores have been exploited to serve as novel
optical materials and probes for biosensing and imaging applications [32]. AIE fluorophores have
high emission efficiency when their molecules are aggregated. However, conventional organic
fluorophores often give strong emission in dilute solution, but their emission is quenched in the
aggregated state or in concentrated solution. A number of AIE-based probes have been reported
[33], but AIE fluorescent probe for NTR detection has not been reported so far.
In this work, we have designed and synthesized a new AIE fluorescent probe TPE-HY for
detecting NTR. The fluorescent probe has many obvious advantages, such as, fast response (about
5 min), high selectivity, and high sensitivity to NTR. In addition, we successfully apply the new
probe TPE-HY for monitoring NTR in living cells.
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2. Experimental
2.1. Materials and instruments
Unless otherwise specified, all reagents were mentioned in this work of analytical grade and
directly used without further purification. The Escherichia coli nitroreductase (NTR) was
purchased from Sigma-Aldrich. To keep the enzyme activity, NTR was dissolved into ultrapure
water and stored immediately at -80 °C [34]. The β-Nicotinamide adenine dinucleotide disodium
salt (NADH) was purchased from J&K Scientific Ltd.
The 1H NMR and 13C NMR spectra were collected on an AVANCE III 400 MHz Digital NMR
spectrometer, and using tetramethylsilane (TMS) as internal; High resolution mass spectrometric
(HRMS) analyses were measured by using a Finnigan MAT 95 XP spectrometer; UV-vis and
fluorescence spectra were acquired on a Shimadzu UV-2700 Power spectrometer (with a 1 cm
standard quartz cell), HITACHI F4600 fluorescence spectrophotometer (with a 1 cm standard
quartz cell); The pH measurements were carried out with a Mettler-Toledo Delta 320 pH meter;
The fluorescence imaging of living cells was performed through Nikon A1MP confocal
microscopy.
2.2. Synthesis
The synthetic routes of compound 1, TPE-HY, and TPE-NH2 were shown in Scheme 1. The
detailed synthetic procedures were given below.
Scheme 1 Design strategy of the fluorescent probe TPE-HY and TPE-NH2.
2.2.1. Synthesis of 1,1,2,2-tetraphenylethene (1).
Zinc powder (7.20 g, 0.11mol) was added in dry THF (80 mL) with rigorous magnetic stirring,
and after the suspension was stirred at 0 °C for 15 min. TiCl4 (6 mL, 0.055 mol) was injected the
suspension slowly over a period of 30 min. Then the reaction system was refluxed for about 2 h.
And a solution of benzophenone (5.00 g, 0.028 mol) in dry THF (20 ml) was injected slowly using
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a syringe, and the reaction system was refluxed for 12 h. Then the reaction was quenched with 5 %
aqueous solution of ammonium chloride ((w/v), 100mL), and extracted with ethylacetate. The
organic extracts were combined, washed with water, and dried with MgSO4. After filtration and
the solvent was removed using a rotary evaporator. The crude product was re-crystallized from
EtOH to afford a white solid compound 1 with a yield of 90%. 1H NMR (400 MHz, CDCl3) δ 7.12
13
(m, 12H), 7.05 (m, 8H); C NMR (100 MHz, CDCl3) δ 143.75, 140.99, 131.35, 127.67, 126.43;
HR-MS calculated for C26H20 [M+H]+ m/z 333.1638, found [M+H]+ m/z 333.1651.
2.2.2. Synthesis of (2-(4-nitrophenyl)ethene-1,1,2-triyl)tribenzene (TPE-HY).
Compound 1 (3.32 mg, 10 mmol) was added in CH2Cl2 (30 mL). Then glacial acetic acid (1 mL)
was slowly dropped into the solution at 0 °C with rigorous magnetic stirring. Maintaining the
reaction temperature, concentrated HNO3 (65 wt %, 0.72 mL) was slowly dropped into the
solution within 30 min. After stirred at 0 °C for 30 min, the reaction was stirred at room
temperature for another 6 h, and then poured into water. The formed precipitate was collected, and
washed with water. The crude product was purified by flash chromatography on silica gel with
petroleum ether/ethyl acetate (50:1, v/v) as the eluent to afford a yellow solid TPE-HY with a
1
yield of 80%. H NMR (400 MHz, CDCl3) δ 7.98 (d, J = 8.8 Hz, 2H), 7.20 (d, J = 8.8 Hz, 2H),
7.16 (m, 9H), 7.03 (m, 6H); 13C NMR (100 MHz, CDCl3) δ 151.05, 145.98, 143.89, 142.74,
142.67, 142.54, 138.83, 132.09, 131.27, 131.26, 131.19, 128.12, 128.11, 127.82, 127.37, 127.13,
127.11, 123.04; HR-MS calculated for C26H19NO2 [M+H]+ m/z 378.1489, found [M+H]+ m/z
378.1494.
2.2.3. Synthesis of (2-(4- aminophenyl)ethene-1,1,2-triyl)tribenzene (TPE-NH2).
Tin(II) chloride (4.015 g, 21.2 mmol) was dissolved in concentrated HCl (36%, 3.5 mL), then
the solution was added dropwise to a suspension of compound TPE-HY (1.55 g, 4.1 mmol) in
ethanol (3 mL) with rigorous magnetic stirring. After reflux for 3 h, the reaction system was
cooled to room temperature. The formed precipitate was collected, and washed with water. The
crude product was purified by flash chromatography on silica gel with petroleum ether/ethyl
acetate (50:1, v/v) as the eluent and re-crystallized from EtOH to afford a white solid TPE-NH2
with a yield of 51%. 1H NMR (400 MHz, CDCl3) δ 7.08 (m, 13H), 7.01 (m, 2H), 6.81 (d, J = 8.4
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Hz, 2H), 6.46 (d, J = 8.4 Hz, 2H), 4.09 (s, 2H); 13C NMR (100 MHz, CDCl3) δ 144.69, 144.36,
144.23, 144.19, 140.95, 139.34, 134.11, 132.52, 131.50, 131.44, 131.39, 127.69, 127.57, 127.54,
126.27, 126.09, 114.39; HR-MS calculated for C26H21N [M+H]+ m/z 348.1747, found [M+H]+
m/z 348.1744.
2.3. Preparation of Solutions of probe TPE-HY
The concentration of the probe stock solution was 1.0 mM in DMSO. The concentration of the
NTR stock solution was 10.0 μg/mL in ultrapure water. The concentration of the NADH stock
solution was 100 mM in ultrapure water. The concentration of analytes stock solutions were 100
mM prepared in the ultrapure water or DMSO.
For the test solution, the concentration of the probe was 5.0 μM and the concentration of the
NADH was 300 μM in 10 mM PBS buffer (pH=7.4, 10 % DMSO). The concentration of analytes
were different: biothiols, cations, anions (2.5 mM); reactive oxygen/nitrogen species (ROS/RNS)
(100 μM). The resulting solution was incubated at 37 °C for 20 min before recording the spectra.
Unless otherwise noted, for all the measurements, the excitation wavelength was 405 nm, the
excitation slit widths were 5 nm, and the emission slit widths were 5 nm.
2.4. HeLa cells culture and hypoxic imaging
The requisite density HeLa cells were seeded into the imaging dish (35 mm glass-bottom
culture dishes). In the next 12 hours, the cells were incubated under different oxygen level
environments (the normoxic condition: 20 % O2; and the hypoxic condition: 1 % O2). Then the
probe was added to the HeLa cells, further incubated for another 20 min under respective
conditions. After washed with PBS buffer (pH=7.4) for three times, and the fluorescence images
were acquired through a Nikon A1MP confocal microscopy inverted fluorescence microscopy
equipped with a cooled CCD camera. The fluorescence emission was collected at 500-550 nm
upon excitation at 405 nm with a femtosecond pulse.
3. Results and discussion
3.1. Design and synthesis of the probe TPE-HY
The tetraphenylethene fluorophore has many advantages, including aggregation-induced
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emission, high photochemical stability, and high emission efficiency [32-33], and has been
reported as intermediate product in several research areas [35-39]. We designed a new NTR
fluorescent probe TPE-HY employed nitro group as the responding site for NTR as it could be
easily reduced by NTR to the amine moiety. With the introduction of nitro group (electron
donating group), the electronic structure of the probe became “D-π-A”. The probe emitted strong
fluorescence emission because of the intramolecular charge transfer (ICT). When treated with
NTR, via a series of one-electron reduction processes, the nitro group of the probe would be
reduced to the amino moiety, and the electronic structure of the probe became “D-π-D”, results in
the excitation wavelength of the reduction product (TPE-NH2) becoming shorter than that of
probe TPE-HY. The fluorescence signal couldn’t be observed (Scheme 2). The new probe
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TPE-HY was fully characterized by 1H NMR, C NMR and HRMS and used as an AIE organic
fluorescent probe for NTR detection for the first time.
Scheme 2. Proposed reaction mechanism of TPE-HY with NTR.
3.2. Photophysical properties of the probe CoNO2-HY
Due to the tetraphenylethene fluorophore was the typical representative aggregation induced
emission dye, we first tested the fluorescence intensity changes of TPE-HY and TPE-NH2 in
DMSO/water mixtures with different fractions of water (fw). As shown in Fig. S1, the solution of
TPE-HY in DMSO (contain 1 vol % water) were almost non-fluorescent. With the increase of
water content, the fluorescence intensity increased gradually. However, the fluorescence intensity
still weak until 80 vol % water added. At the 90 vol % water content, a large fluorescence signal
observed. The compound TPE-NH2 showed the same fluorescence intensity changes. As shown in
Fig. S2, with the increase of water content, the fluorescence intensity increased gradually, but the
fluorescence intensity of the compound TPE-NH2 was excitated by the 405 nm excitation
light was very low. By contrast, the compound TPE-NH2 was excitated by the 340 nm
excitation light exhibited strong fluorescent changes. These studies demonstrated that TPE-HY
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and TPE-NH2 have the aggregation induced emission (AIE) characteristics, and slso confirmed
that the reaction mechanism of the probe TPE-HY with NTR as proposed we expected in Scheme
2.
In the titration experiments, as expected, the free probe TPE-HY displayed a strong fluorescent
emission at 525 nm. However, when incubated with NTR, the fluorescence intensities of the probe
TPE-HY significantly decline. As shown in the Fig. 1, with the increase of concentration of NTR,
the fluorescence intensities of the probe TPE-HY gradually reduced. When 5 μM probe TPE-HY
incubated with 10.0 μg/mL NTR for 20 min, it was about 4.7-fold decrease in fluorescence is
detected. Based on the titration experiments, the detection limit of the probe for NTR was
calculated to be 11.5 ng/mL with the assay conditions as shown in Fig. S3. Similar to the emission
spectrum, the absorption spectrum of the probe also changed significantly. As shown in the Fig. S4,
with the increase of the introduction of the concentration of NTR (0-10 μg/mL), the absorption
value of the probe at about 340 nm gradually reduced. These studies demonstrated that the probe
TPE-HY could detect the basal level of NTR in the living samples.
Fig. 1. The fluorescence response of the probe TPE-HY to NTR at the varied concentrations in
PBS buffer (pH 7.4, 5 % DMSO). λex = 405 nm. The spectra were recorded upon treatment of the
probe (5.0 μM) with NTR (0- 10.0μg/mL) in the presence of NADH (300 μM) for 20 min.
The kinetic profiles of the probe TPE-HY (10 µM) treated with the varied concentrations of
NTR (0-10.0 μg/mL) were shown in Fig 2. The probe TPE-HY untreated with NTR, the
fluorescence intensity almost had no change at 525 nm. However, a fast fluorescence reduced
phenomenon was noted when the incubated with NTR in PBS buffer, and the signal reached an
equilibrium state after about 5 min. These results suggested that the rapid fluorescence response of
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the probe may make it suitable for detection of NTR.
Fig. 2. Reaction-time profiles of the probe TPE-HY (5.0 μM) treated with the varied
concentrations of NTR in the presence of NADH (300 μM). The fluorescence intensities at 525
nm are continuously monitored at time intervals for 60 min in PBS buffer (pH 7.4, 5 % DMSO).
In order to confirm the reaction mechanism, the reaction system was characterized by the
HR-MS analyses. 50 μM probe TPE-HY was reacted with 10.0 μg/mL NTR in the presence of
300 μM NADH at 37 °C for 20 min, and then the reaction solution was investigated and
determined by HR-MS analysis. The HRMS showed a peak at m/z= 348.1750 [M+H]+, which
attributing to compound TPE-NH2 (calcd. for C26H21N 348.1747 [M+H]+) (Fig. S5). The result
was in agreement with the proposed mechanism in Scheme 2.
The high selectivity for target molecules is one of the important properties of fluorescent probes
for the complex biological application. In order to better illustrate the selectivity of the probe, the
probe TPE-HY was treated with various biologically relevant species, including cations, anions,
reactive oxygen/nitrogen species (ROS/RNS), and biothiols, to investigate the selectivity. As
shown in the Fig. 3, the probe TPE-HY incubated with the other analytes triggered almost
non-fluorescence change. By contrast, when the probe treated with NTR for 20 min, there was a
decrease of approximately 4.7-fold as compared with that of the probe TPE-HY. Similar to the
emission spectrum, the absorption spectrum of the probe showed the same phenomenon. The
probe treated with the other analytes, the absorption spectrum of the probe barely changed. By
contrast, when NTR was introduced, the absorption value of the probe at 340 nm decreased
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significantly (Fig. S6). These results showed that the probe TPE-HY was highly selective for
NTR over other tested analytes, suggesting that the probe is favourable for investigation of NTR
in biological systems.
Fig. 3. Fluorescence changes (F0/F) of the probe TPE-HY (5 μM) treated with various relevant
analytes. The data are obtained after the incubation of the probe TPE-HY with the analytes for 20
min in the presence of NADH (300 μΜ). Legend: 1) free probe; 2) NaNO2; 3) CaCl2; 4) FeSO4; 5)
KI; 6) KNO3; 7) MgCl2; 8) NaBr; 9) ZnCl2; 10) NaHSO3; 11) Na2S; 12) Cys; 13) GSH; 14) Hcy;
15) H2O2; 16) NaClO; 17) tert-butyl hydroperoxide; 18) di-tert-butylperoxide; 19) NTR.
The solution of pH and temperature have great influence on the enzyme activity. We also
investigated the fluorescent changes of the probe TPE-HY with or without NTR in different pH
PBS buffer and at different temperature. As shown in Fig. S7 and S8, the fluorescence intensities
of the free probe TPE-HY have produced subtle change over a wide pH range of 5.0-8.0 and a
wide temperature range of 20-40 °C, suggesting that the probe TPE-HY was stable in this pH
range and this temperature range. Obviously, with the introduction of NTR, a significant
fluorescence signal decrease was observed, especially at physiological pH 7.4 and physiological
temperature 37 °C, suggesting that the probe has the potential value for biological applications.
These results reveal that the probe TPE-HY functions well under physiological conditions (about
pH 7.4 and 37 °C).
Furthermore, the photo-stability of probe TPE-HY also was surveyed. The probe was excited at
365 nm using a UV lamp, the fluorescent intensity displayed almost no change (Fig. S9),
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suggesting that the probe is highly stable and may have potential usefulness in living system.
3.3. Fluorescence imaging of NTR in the living cells
Encouraged by the excellent properties of the probe, including fast response, working
appropriately at physiological pH and temperature, high sensitivity and selectivity for NTR, and
excellent photo-stability, the fluorescence imaging experiments were carried out in living cells.
As imaging reagents, the probe TPE-HY should have low biological toxicity. The cytotoxicity
of the probe TPE-HY for HeLa cells was carried out. As shown in the Fig. S10, the standard MTT
assay indicated that the probe TPE-HY (0-20 µM) has no remarkable cytotoxicity to the HeLa
cells, meaning that the probe can be used for the bio-imaging.
For the endogenous NTR imaging experiments, as the anoxic condition would improve the
NTR biological activity, the HeLa cells were incubated under different oxygen contents (the
normoxic condition: 20 % O2; the hypoxic condition: 1 % O2) for 12 h. Then the probe TPE-HY
(5 μM) was added to the HeLa cells under the original condition for another 20 min, respectively.
As shown in the Fig. 4, only the living cells displayed non-fluorescence (Fig. 4e, 4f). At the
normoxic condition, the HeLa cells incubated with probe showed strong fluorescence (Fig. 4g).
And as we expected, at hypoxic condition, weak fluorescence signal was detected in the HeLa
cells treated with the probe TPE-HY (Fig. 4h). These results demonstrated that the probe
TPE-HY has good cell-membrane permeability and is responsive to intracellular NTR. The probe
TPE-HY is the first AIE NTR fluorescent probe and can be used to the activity of NTR in living
cells.
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Fig. 4. Fluorescence imaging of NTR in the living HeLa cells under normoxic (20% O2) and
hypoxic (1% O2) conditions incubated with the probe TPE-HY for 20 min. a-d) Bright-field
image of the HeLa cells under normoxic and hypoxic conditions; e-h) Fluorescence image of a)-d);
i-l) The merge of the bright-field image of and a)-d) and e-h).
Scale bar: 20 μm.
4. Conclusion
In summary, we have designed and synthesized a new aggregation induced emission (AIE)
fluorescent probe TPE-HY for detecting NTR for the first time. The probe used tetraphenylethene,
the classic AIE dye, as the fluorophore, and used the nitro group as the NTR recognition site. With
the introduction of NTR, the nitro group of the probe would be reduced to the amino moiety, the
electronic structure of the probe had been changed, and then the ICT process was suppressed. The
probe TPE-HY had many excellent properties, such as, aggregation induced emission (AIE)
characteristics, rapid response, high sensitivity and selectivity to NTR. More importantly, the
probe was successfully applied to monitor NTR in living HeLa cells. Further applications of the
probe for the biological functions, pathological roles and disorders of NTR were under progress.
Acknowledgements
This work was financially supported by NSFC (21472067, 21672083), Taishan Scholar
Foundation (TS 201511041), and the startup fund of the University of Jinan (309-10004).
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Graphical abstract
A new fluorescent quenched probe for nitroreductase (TPE-HY) was engineered for monitoring
nitroreductase in the living cells.
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Highlights
1) A new aggregation induced emission (AIE) fluorescent probe TPE-HY
of detecting nitroreductase has designed and synthesized for the first
time.
2) The probe TPE-HY is a new fluorescent nitroreductase probe, with
high selectivity and sensitivity for nitroreductase.
3) When the probe TPE-HY treated with NTR in PBS solution, a fast
fluorescence reduce phenomenon is noted, and the signal reached
equilibrium state at about 5 min.
4) Fluorescence imaging shows that the probe TPE-HY is
membrane-permeable and suitable for detecting endogenous NTR in
living cells.
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