English

• 0.   Introduction

  Hypoxia^[1-4] usually happens in many solid tumors because the abnormal metabolism and proliferation of tumor cells will increase the oxygen consumption, in addition to the reason that the insufficient blood vessels in tumors block the transportation of oxygen toward tumor cells^[5-6]. Besides, recent studies have found that the dense extracellular matrix^[7], solid stress^[8], and abnormal vascular structures^[9-11] in the tumor microenvironment^[12] are also critical factors that may affect the oxygen content in tumor cells. Hypoxia has a great effect on the biological behavior and malignant phenotype of tumor cells, and it mediates the effects of tumor therapy through some complex mechanisms, which are closely related to the poor prognosis of tumors in many patients^[13-14]. All these make hypoxia atmosphere become one of the most attractive research hotspots in tumor microenvironment (TME), of which the exact sensing offers a vital guidance for the enhanced molecular diagnostics and image-guided therapeutic applications^[15-17].

  Of the reported hypoxia detection approaches, functional molecule probes^[18-19] allow the covalent connection of fluorophores and hypoxia-responsive moieties together for the selective imaging and therapy of tumors^[20-21]. Usually, an azo bond is a well-known hypoxia-triggering group, and the introduction of the azo moiety via a covalent bond into a probe would significantly quench its fluorescence emission^[22-25]. When the hypoxia-responsive probe is selectively activated by sodium dithionite (SDT, a hypoxia simulator used to generate and simulate hypoxic conditions in solution tests) under hypoxic conditions, it releases the fluorophore and shows an obvious fluorescence emission^[26-30]. This hypoxia-responsive fluorescent detection method makes the imaging and diagnosis of tumors a simple operation, with low cost, good sensitivity, specific selectivity, real-time detection, etc. Although recent advances in probes with azo structures are widely applied for detecting hypoxic tumor cells, which are mainly based on different fluorophores, such as rhodamine^[31-32], boron dipyrromethene^[33-34], and cyanine^[35]. Developing other azo-structure-based probes for the imaging of hypoxic tumor cells is fairly essential to expand the hypoxia probe library, which provides researchers more ways for tumor detection and diagnosis.

  Quinoxaline derivative is a heterocyclic compound with a large conjugated planar^[36-37], which often has a stable fluorescence property. Moreover, the synthesis procedures of quinoxaline-based fluorophore derivatives often have cheap raw materials, a high reaction efficiency, and a simple post-reaction processing. Quinoxaline fluorophore derivatives possess suitable fluorescence emissions that can be easily distinguished from the environment of cells^[38], which shows a relatively low background fluorescent signal in cell imaging tests. Additionally, quinoxaline fluorophore derivatives also exhibit good biological activity and biocompatibility, which have been widely used in tumor cell imaging^[39-41].

  In this work, a fluorescent probe AQD, based on a quinoxaline fluorophore derivative, was designed to achieve hypoxia detection in tumor cells, which was synthesized by the covalent connection of a quinoxaline fluorophore derivative and a N, N-dimethylaniline moiety with an azo bond. Fluorescent probe AQD showed good selectivity toward hypoxic conditions in solution tests, and its emission intensity gradually enhanced with the increase of hypoxia degree. In addition, AQD was successfully used to conduct the hypoxia A549 cells imaging tests, which implied the potential of this probe for cell imaging applications. When the hypoxia-responsive AQD was activated under hypoxic conditions, its azo linker broke and the probe released the fluorophore^[42-43], resulting in an obvious fluorescence emission (Scheme 1).

  Scheme 1

  

  Scheme 1.  Fluorescence turn-on mechanism of AQD toward hypoxia reduction

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  1.   Experimental

  1.1   Materials and methods

  All chemicals were obtained from commercial suppliers and used without further purification. NMR spectra were measured on a Bruker Avance Ⅱ 400 NMR spectrometer and a Bruker AVANCE Ⅲ 500 NMR spectrometer (in DMSO-d₆, CF₃COOD or CDCl₃, TMS as internal standard). ESI-MS was carried out on a Thermo Scientific Q Exactive Plus LC-MS. Fluorescent spectra were performed on an Edinburgh FLS1000 steady-state fluorescence spectrophotometer. High- performance liquid chromatography (HPLC) analysis tests were conducted in an Agilent 1100 reversed- phase system. Ultrapure water purified by the Milli-Q reference system (Millipore) was used throughout. All compounds were dissolved in DMSO to prepare a stock solution (1, 5, 10 mmol·L^-1). Fluorescence cell imaging was performed on an Olympus FV1000 fluorescence microscope with a laser source at a wavelength of 405 nm.

  1.2   Synthesis of probe AQD

  The synthetic route of probe AQD is shown in Scheme 2.

  Scheme 2

  

  Scheme 2.  Synthetic route of probe AQD

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  1.2.1   Synthesis of compound 1

  Acenaphthylene-1, 2-dione (1.82 g, 10.0 mmol) and 4-nitrobenzene-1, 2-diamine (1.53 g, 10.0 mmol) were mixed in a round-bottomed flask, and then acetic acid solution (25 mL) was added to the reaction flask. The mixture solution was stirred and refluxed at 110 ℃ for about 12 h, and the reaction solution turned into a brown, turbid liquid. After the reaction was complete, the mixture was filtered while it was hot. The light- yellow solid powder obtained by the filtration was washed with MeOH and CH₂Cl₂ solution three times, and dried in a vacuum, resulting in a light-yellow powder product compound 1^[37]. Yield: 2.78 g, 93%. ¹H NMR (500 MHz, CF₃COOD): δ 8.92 (s, 1H), 8.42-8.32 (m, 1H), 8.19 (d, J=9.0 Hz, 1H), 8.12 (d, J=8.0 Hz, 1H), 8.04 (d, J=8.0 Hz, 1H), 7.66 (dd, J=15.8, 7.7 Hz, 1H). ¹³C NMR (101 MHz, CF₃COOD): δ 150.04, 146.72, 144.13, 134.81, 131.86, 131.69, 130.07, 129.91, 125.67, 125.40, 124.13, 122.98, 121.58, 121.07, 120.38, 118.60, 118.48. Atmospheric pressure chemical ionization MS (APCI-MS, m/z): [M+H]⁺ Calcd. for [C₁₈H₁₀N₃O₂]⁺ 300.076 8, found 300.076 9.

  1.2.2   Synthesis of compound 2

  Compound 1 (1.50 g, 5.0 mmol) and concentrated hydrochloric acid (30 mL) were added to a round- bottom flask and stirred for 10 min. Then, a white powder SnCl₂·H₂O (4.51 g, 20 mmol) was slowly added into the mixture at room temperature, and the reaction was stirred at 80 ℃ for over 12 h. After the reaction was complete, the reaction solution changed to a black, turbid solution. The crude product was collected by filtration, and then it was washed with hydrochloric acid (1 mol·L^-1, 10 mL), NaOH solution (1 mol·L^-1, 10 mL), and water (5 mL×3) orderly. Finally, it was dried under vacuum to afford the crude product, which was further recrystallized in CH₂Cl₂ and methanol solution to obtain the brown yellow solid compound 2. Yield: 1.23 g, 91%. ¹H NMR (500 MHz, DMSO-d₆): δ 8.28 (d, J=6.9 Hz, 1H), 8.18 (d, J=6.9 Hz, 1H), 8.14 (d, J=8.2 Hz, 1H), 8.09 (d, J=8.2 Hz, 1H), 7.86 (d, J=8.9 Hz, 1H), 7.84-7.78 (m, 2H), 7.25 (dd, J=8.9, 1.9 Hz, 1H), 7.14 (s, 1H), 6.13 (s, 2H). ¹³C NMR (126 MHz, DMSO-d₆): δ 153.20, 150.50, 148.19, 143.20, 134.36, 134.32, 132.18, 131.89, 130.00, 129.51, 129.29, 128.78, 128.65, 128.20, 121.35, 120.96, 120.25, 106.88. APCI-MS (m/z): [M+H]⁺ Calcd. for [C₁₈H₁₂N₃]⁺ 270.102 6, found 270.102 6.

  1.2.3   Synthesis of probe AQD

  Compound 2 (269 mg, 1.0 mmol) was dissolved in a mixture of hydrochloric acid (12 mol·L^-1, 0.45 mL) and water (15 mL) at 0 ℃. Then, NaNO₂ (228 mg, 3.3 mmol) dissolved in 2 mL of water was slowly dripped into the acidified mixture solution. The mixture solution was stirred in an ice bath for 3 h to obtain the reddish brown, clear diazonium salt solution. After that, urea (120 mg, 2.0 mmol) was added to the reaction solution and stirred for 30 min. N, N-dimethylaniline (785 mg, 3.6 mmol) was dissolved in 5 mL EtOH, and the solution was stirred and cooled down to 0-5 ℃^[44]. The diazonium salt solution was added dropwise into the above EtOH solution within 30 min. After the mixture had reacted in an ice bath for 12 h, the mixture was poured into 100 mL of saturated brine. The precipitate was collected by filtration and washed with excess water several times (10 mL×6). The residue was purified by column chromatography on silica gel (V_CH2Cl2∶V_methanol=200∶1) to give the probe AQD as a red powder solid. Yield: 315 mg, 78%. ¹H NMR (500 MHz, CDCl₃): δ 8.58 (d, J=2.0 Hz, 1H), 8.40 (d, J=6.9 Hz, 1H), 8.36 (d, J=6.9 Hz, 1H), 8.26 (dd, J=8.9, 2.1 Hz, 1H), 8.19 (d, J=8.9 Hz, 1H), 8.04 (dd, J=8.2, 2.9 Hz, 1H), 7.93 (d, J=9.0 Hz, 1H), 7.80 (dd, J=14.4, 6.8 Hz, 1H), 6.74 (d, J=9.0 Hz, 1H), 3.07 (s, 1H). ¹³C NMR (126 MHz, CDCl₃): δ 153.44, 152.82, 152.01, 151.70, 142.76, 140.97, 140.83, 135.40, 130.83, 130.77, 129.01, 128.93, 128.43, 127.61, 124.47, 124.17, 120.98, 120.82, 120.78, 110.47, 39.23. ESI-MS (m/z): [M+H]⁺ Calcd. for [C₂₆H₂₀N₅]⁺ 402.171 3, found 402.171 8.

  1.3   Optical properties tests

  The probe AQD and the fluorophore (compound 2) stock solution were prepared with dimethyl sulfoxide (DMSO) to a concentration of 10, 5, and 1 mmol·L^-1, respectively. AQD stock solution was diluted to obtain 10 μmol·L^-1 with phosphate-buffered saline (PBS, 10 mmol·L^-1, pH=7.4, 10% DMSO), and the fluorescent tests were performed in that concentration. The emission stability tests of the fluorophore under different pH values (from 5.0 to 8.0) and at different times were conducted to investigate the imaging application potential in solution, which was the basis of the further cell imaging. The excitation wavelength of the fluorophore and probe AQD was set at 400 nm in PBS (10 mmol·L^-1, pH=7.4, 10% DMSO), and the maximum emission wavelength of AQD and its hypoxia reduction product showed around 550 nm.

  1.4   Anti-interference ability test among various biomolecules

  It's necessary to evaluate whether the interference of other ions and biomolecules will affect the accurate imaging and detection results of the probe toward hypoxic tumor cells, which is important for their application potential in the complex life system. With these in mind, a broader range of relevant ions and biomolecules were selected to conducted the tests, including metal ions (Na⁺, K⁺, Ca²⁺, Cu²⁺, Mg²⁺, and Zn²⁺, 20 mmol·L^-1), glucose (50 mmol·L^-1), adenosine triphosphate (ATP, 10 mmol·L^-1), glutathione (GSH, 10 mmol·L^-1), H₂O₂ (10 mmol·L^-1), amino acids [L-Glutamic acid (L-Glu), L-Tyrosine (L-Tyr), L-Arginine (L-Arg), L-Aspartic acid (L-Asp), L-Proline (L-Pro), L-Homocysteine (L-Hcy), L-Cysteine (L-Cys), 10 mmol·L^-1], bovine serum albumin (BSA, 10 μg·L^-1), glucose oxidase (Glu_ox, 10 μg·L^-1), and cytochrome C (Cyt C, 10 μg·L^-1). These distractors were mixed with AQD (10 μmol·L^-1) for 3 h in PBS (10 mmol·L^-1, pH=7.4, 10% DMSO), and the results were compared with those of SDT (5 mmol·L^-1) and AQD (10 μmol·L^-1). The excitation wavelength of all solutions was set at 400 nm, and the relative intensity at 550 nm was used to evaluate the selectivity of AQD.

  1.5   HPLC test conditions

  HPLC tests were used to evaluate the hypoxia- responsive mechanism of probe AQD. Firstly, fluorophore and AQD solutions (10 mmol·L^-1) were configured in DMSO, respectively, and then they were diluted to a concentration of 100 μmol·L^-1 in methanol. The initial mobile phase conditions of HPLC were set as 70% methanol and 30% pure water. After 5 min of gradient elution, the mobile phase was changed to 100% methanol and 0% pure water. The subsequent mobile phase was still 100% methanol for 15 min elution. Then, three experimental groups, 100 μmol·L^-1 fluorophore, 100 μmol·L^-1 AQD, and 100 μmol·L^-1 AQD with 100 mmol·L^-1 SDT reacting for 12 h, were analyzed for 10 μL under these conditions. The hypoxia-responsive mechanism of AQD can be explained by the peak retention time of the three groups of experiments.

  1.6   Cell culture and confocal imaging

  Cell culture and foetal bovine serum were purchased from Gibco (Thermo Fisher Scientific). A549 cells (human alveolar basal epithelial cells derived from lung tissue) were cultured in 1640 medium with 10% foetal bovine serum and % penicillin-streptomycin under 5.0% (volume fraction) CO₂ at 37 ℃. All cells were maintained at 37 ℃ under 5% CO₂ in air (the standard conditions). Before the experiment, cells were treated under different hypoxic conditions (O₂ concentration of 20%, 8%, or 0.1%) for another 8 h. The hypoxic condition of 0.1% O₂ was generated with an Anaero Pack TM-Anaero (Mitsubishi Gas Chemical Company, Inc.) and a 2.5 L rectangular jar (Mitsubishi Gas Chemical Company, Inc.). The hypoxic condition of 8% O₂ was generated with an Anaero Pack TM-Micro Aero (Mitsubishi Gas Chemical Company, Inc.) and a 2.5 L rectangular jar.

  To generate different hypoxia, cells were incubated for 8 h at 37 ℃ under different hypoxic conditions (8% or 0.1% O₂). The cells were further incubated with probe AQD (10 μmol·L^-1) for another 120 min under different hypoxia levels for cell imaging. Fluorescence imaging of cells was performed by incubation with probe AQD (10 μmol·L^-1) for 0, 30, 60, 120, and 180 min. After being washed with PBS three times, the dishes were used for imaging. Fluorescence cell imaging was performed on an Olympus FV1000 fluorescence microscope with a laser source at a wavelength of 405 nm, and fluorescence signals of the cells were collected at 510-610 nm.

  2.   Results and discussion

  2.1   Design and synthesis of AQD

  The large conjugated planar probe AQD was designed by covalent connection of acenaphtho[1, 2-b]quinoxaline moiety and N, N-dimethylaniline moiety with an azo bond. As shown in Scheme 2, probe AQD was synthesized by a diazotization coupling reaction between fluorophore and N, N-dimethylaniline, resulting in a brick red powder solid product. Structures of intermediates and AQD were confirmed by ¹H NMR, ¹³C NMR, and MS spectra (Fig.S1-S9, Supporting information).

  2.2   Spectral characterizations of AQD toward hypoxia reduction conditions

  In consideration of the cells′ imaging application of AQD, the stability of the fluorophore (compound 2) needed to be investigated. The fluorophore showed a higher emission intensity with the increase of DMSO content in PBS (Fig.S10), and the solution of fluorescence tests was chosen as PBS (10 mmol·L^-1, pH=7.4, 10% DMSO). Time-dependent UV-Vis spectra tests of compound 2 were performed in PBS (Fig.S11), and the UV-Vis absorption showed little changes with time, indicating the good stability of the fluorophore. Similarly, time-dependent fluorescence tests of compound 2 (10 μmol·L^-1) were conducted in PBS (Fig. 1a). The results showed that the emission of fluorophore around 550 nm had little change within 3 h, showing its relatively good fluorescence stability (Fig. 1b). This could guarantee a certain stability of the fluorescence signal generated from probe AQD in solution tests or cell imaging tests. In addition, we also conducted the fluorescence tests of the fluorophore (10 μmol·L^-1) in various pH values (Fig. 1c and 1d). The data showed that the emission intensity of the fluorophore at 550 nm had little change around physiological pH conditions (from 6.5 to 7.4). All these indicated our probe AQD had a relatively good stability in cell imaging.

  Figure 1

  

  Figure 1.  (a) Time-dependent fluorescence tests of compound 2 (10 μmol•L^-1) in PBS (10 mmol•L^-1, pH 7.4, 10% DMSO); (b) Relative fluorescence intensity changes against time at 550 nm; (c) Fluorescence tests of compound 2 (10 μmol•L^-1) in various pH values (pH=5.0, 5.5, 6.0, 6.5, 7.0, 7.4, 8.0) of PBS (10 mmol•L^-1, 10% DMSO); (d) Relative fluorescence intensity changes against various pH values at 550 nm

  λ_ex=400 nm.

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  Because of the hypoxia-triggering ability of the azo bond, probe AQD may have a fluorescence response to the hypoxia-reducing atmosphere. To begin with, the fluorescence emission spectra of AQD and compound 2 were conducted in PBS, respectively, to evaluate the potential emission intensity of AQD. Probe AQD in the test had no emission, and compound 2 showed over 200-fold fluorescent emission in the same testing condition (Fig. 2a), which indicated the reduction product of probe AQD (fluorophore compound 2) had a good fluorescence response signal and the test results were not interfered by other compounds without fluorescent emission. In this work, sodium hyposulfite was selected to simulate a hypoxic environment in solution tests, and the added concentration of SDT was positively correlated with the degree of hypoxia. Fluorescence spectra changes of AQD (10 μmol·L^-1) with time were recorded to investigate the hypoxia-responsive reaction (Fig. 2b). The relative fluorescence intensity changes of AQD (10 μmol·L^-1) at 550 nm with or without the addition of SDT were recorded for 3 h (Fig. 2c). The test results indicated that the fluorescence intensity of the AQD solution increased with time going on after the addition of SDT, and the initial reaction rate was the fastest upon the addition of SDT. The fluorescence spectra of AQD with the addition of SDT were similar to that of fluorophore compound 2, which implied hypoxia reduction of AQD would release the fluorophore and result in a fluorescence emission recovery. All these tests exhibited the good fluorophore response of AQD toward hypoxic conditions.

  Figure 2

  

  Figure 2.  (a) Fluorescence tests of AQD (10 μmol•L^-1) and compound 2 (10 μmol•L^-1) in PBS (10 mmol•L^-1, pH=7.4, 10% DMSO)under the same excitation light at 400 nm; (b) Fluorescence spectra changes of AQD (10 μmol•L^-1) with time after the addition of SDT (20 mmol•L^-1); (c) Relative emission intensity changes of AQD at 550 nm as a function of time following the addition of SDT (20 mmol•L^-1)

  λ_ex=400 nm, λ_em=550 nm.

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  2.3   Response mechanism

  As is known to all, azo compound shows sensitivity toward hypoxia-reducing atmosphere, which will be reduced into amino compounds eventually. Besides, azo compound usually has little fluorescence emission due to their quenching effect on fluorophores. While the hypoxia reduction of AQD would release the fluorophore and show the fluorescence response signal. To investigate this fluorescence response mechanism of AQD, the HPLC tests^[45] were performed to analyze its reduction product (Fig. 3). AQD (100 μmol·L^-1) reacted with SDT (100 mmol·L^-1) for 4 h, and then the reaction mixture was analyzed by HPLC. AQD (100 μmol·L^-1) and fluorophore compound 2 (100 μmol·L^-1) were also analyzed by HPLC as the control group experiments. According to the HPLC results, the addition of SDT into AQD solution resulted in a peak that had the same retention time as that of fluorophore compound 2, which indicates the break of the azo bond and the release of fluorophore. Therefore, the azo-bond- breaking mechanism is the reason for the fluorescence response of AQD.

  Figure 3

  

  Figure 3.  HPLC analysis of AQD (100 μmol•L^-1), fluorophore compound 2 (100 μmol•L^-1), and AQD (100 μmol•L^-1) treated withSDT (100 mmol•L^-1) for 10 h

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  2.4   Selectivity tests and cytotoxicity assay

  Selective response ability and low cytotoxicity are essential for a fluorescent probe to be used in biological applications. Before the application of probe AQD in cell imaging, the interference effect of other ions and biomolecules in living organisms was investigated to determine whether they would affect the detection accuracy of hypoxic conditions^[46]. Various ions and biomolecules had little effect on the fluorescence response of AQD, and the results implied that AQD showed good selectivity toward hypoxia among these interfering substances. Additionally, MTT assay was also conducted to evaluate the cytotoxicity of AQD toward A549 cells^[47]. The results showed that the cell viability was over 80% with the incubation of different concentrations of AQD, indicating the low cytotoxicity of AQD. These tests prove the selective response ability toward hypoxia and the low cytotoxicity of AQD, and it has potential applications in cell imaging.

  Figure 4

  

  Figure 4.  (a) Selectivity of AQD (10 μmol•L^-1) in the presence of various ions, biomolecules, or SDT in PBS (10 mmol•L^-1, pH=7.4, 10% DMSO); (b) Relative cell viability of A549 cells with different concentrations of AQD for 24 h estimated by MTT assay in normoxia

  1: Na⁺, 2: K⁺, 3: Ca²⁺, 4: Cu²⁺, 5: Mg²⁺, 6: Zn²⁺, 7: L-Glu, 8: L-Tyr, 9: L-Arg, 10: L-Asp, 11: L-Pro, 12: L-Hcy, 13: L-Cys, 14: GSH, 15: H₂O₂, 16: ATP, 17: BSA, 18: Glu_ox, 19: Cyt C, 20: SDT; Data were presented as the mean value±SD (n=3).

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  2.5   Confocal imaging

  Hypoxia is a general characteristic in many tumors, where the abnormal glycolytic activity results in a lower O₂ level than that in normal tissue cells^[48-50]. To investigate the imaging performance toward the hypoxic tumor cells^[51], AQD was used to conduct the A549 cell imaging tests in different O₂ concentrations (20%, 8%, or 0.1%). A549 cells were pretreated with different O₂ concentrations for 8 h, and then A549 cells were incubated with AQD for another 3 h to perform the confocal imaging tests (Fig. 5a). The cell imaging effect of AQD became better with the increase of hypoxia degree, after being incubated for the same time. The fluorescence imaging intensity of cells was measured to evaluate the imaging effect in different hypoxic conditions (Fig. 5b). The data indicated that the imaging effect of AQD toward A549 cells in 0.1% O₂ was better than that in 8% or 20% O₂ condition, showing its selectively response toward hypoxic tumor cells. Additionally, A549 cells were pretreated in 0.1% O₂ concentration for 8 h, and then they were incubated with AQD for different time to evaluate the confocal imaging effect with time (Fig. 6a). The fluorescence intensity of cells was measured to investigate the imaging effect with different incubation time in the same hypoxic condition (Fig. 6b). The cell imaging effect of AQD became better with the increase of incubation time in the same hypoxic condition. All these results implied the good hypoxia-responsive imaging ability of AQD in tumor cells.

  Figure 5

  

  Figure 5.  (a) Fluorescence confocal microscopy images of A549 cells incubated with probe AQD (10 μmol•L^-1) for 3 h at different O₂ levels (20%, 8%, and 0.1%); (b) Cells imaging intensity changes of A549 cells incubated with probe AQD (10 μmol•L^-1) for 3 h at different O₂ levels (20%, 8%, and 0.1%)

  Scale bar=40 μm; Data were presented as the mean value±SD (n=3, ^*p < 0.05, ^**p < 0.01, ^***p < 0.001).

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  Figure 6

  

  Figure 6.  (a) Fluorescence confocal microscopy images of A549 cells incubated with probe AQD (10 μmol•L^-1) at 0.1% O₂ level for different times (0, 30, 60, 120, and 180 min); (b) Cells imaging intensity changes of A549 cells incubated with probe AQD (10 μmol•L^-1) at 0.1% O₂ level for different times

  Scale bar=40 μm; Data were presented as the mean value±SD (n=3, ^*p < 0.05, ^**p < 0.01, ^***p < 0.001).

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  3.   Conclusions

  In summary, a novel hypoxia-responsive fluorescent probe AQD was synthesized for the detection and imaging of hypoxic tumor cells, which was obtained by the covalent connection of a large planar conjugated fluorophore and a N, N-dimethylaniline moiety via the azo bond. AQD was used to detect hypoxic conditions by fluorescence enhancement in PBS solution, which showed a significantly fluorescent emission at 550 nm after reduction. AQD exhibited excellent biocompatibility and a good selectivity toward hypoxia, and other ions and biomolecules hardly interfered with the detection results. Additionally, it had been successfully applied for the imaging of hypoxia in A549 cells, providing a way for the detection of hypoxic tumors.

  
  Supporting information is available at http://www.wjhxxb.cn
  
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  Scheme 1  Fluorescence turn-on mechanism of AQD toward hypoxia reduction

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  Scheme 2  Synthetic route of probe AQD

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  Figure 1  (a) Time-dependent fluorescence tests of compound 2 (10 μmol•L^-1) in PBS (10 mmol•L^-1, pH 7.4, 10% DMSO); (b) Relative fluorescence intensity changes against time at 550 nm; (c) Fluorescence tests of compound 2 (10 μmol•L^-1) in various pH values (pH=5.0, 5.5, 6.0, 6.5, 7.0, 7.4, 8.0) of PBS (10 mmol•L^-1, 10% DMSO); (d) Relative fluorescence intensity changes against various pH values at 550 nm

  λ_ex=400 nm.

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  Figure 2  (a) Fluorescence tests of AQD (10 μmol•L^-1) and compound 2 (10 μmol•L^-1) in PBS (10 mmol•L^-1, pH=7.4, 10% DMSO)under the same excitation light at 400 nm; (b) Fluorescence spectra changes of AQD (10 μmol•L^-1) with time after the addition of SDT (20 mmol•L^-1); (c) Relative emission intensity changes of AQD at 550 nm as a function of time following the addition of SDT (20 mmol•L^-1)

  λ_ex=400 nm, λ_em=550 nm.

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  Figure 3  HPLC analysis of AQD (100 μmol•L^-1), fluorophore compound 2 (100 μmol•L^-1), and AQD (100 μmol•L^-1) treated withSDT (100 mmol•L^-1) for 10 h

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  Figure 4  (a) Selectivity of AQD (10 μmol•L^-1) in the presence of various ions, biomolecules, or SDT in PBS (10 mmol•L^-1, pH=7.4, 10% DMSO); (b) Relative cell viability of A549 cells with different concentrations of AQD for 24 h estimated by MTT assay in normoxia

  1: Na⁺, 2: K⁺, 3: Ca²⁺, 4: Cu²⁺, 5: Mg²⁺, 6: Zn²⁺, 7: L-Glu, 8: L-Tyr, 9: L-Arg, 10: L-Asp, 11: L-Pro, 12: L-Hcy, 13: L-Cys, 14: GSH, 15: H₂O₂, 16: ATP, 17: BSA, 18: Glu_ox, 19: Cyt C, 20: SDT; Data were presented as the mean value±SD (n=3).

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  Figure 5  (a) Fluorescence confocal microscopy images of A549 cells incubated with probe AQD (10 μmol•L^-1) for 3 h at different O₂ levels (20%, 8%, and 0.1%); (b) Cells imaging intensity changes of A549 cells incubated with probe AQD (10 μmol•L^-1) for 3 h at different O₂ levels (20%, 8%, and 0.1%)

  Scale bar=40 μm; Data were presented as the mean value±SD (n=3, ^*p < 0.05, ^**p < 0.01, ^***p < 0.001).

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  Figure 6  (a) Fluorescence confocal microscopy images of A549 cells incubated with probe AQD (10 μmol•L^-1) at 0.1% O₂ level for different times (0, 30, 60, 120, and 180 min); (b) Cells imaging intensity changes of A549 cells incubated with probe AQD (10 μmol•L^-1) at 0.1% O₂ level for different times

  Scale bar=40 μm; Data were presented as the mean value±SD (n=3, ^*p < 0.05, ^**p < 0.01, ^***p < 0.001).

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