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Self-Assembled Micellar Nanosensor toward pH with high photo-stability and its application in living cells
Accepted Manuscript Title: Self-Assembled Micellar Nanosensor toward pH with high photo-stability and its application in living cells Authors: Huanhuan Song, Weiwei Du, Chunxia Liu, Zhanxian Li, Hongyan Zhang, Liuhe Wei, Mingming Yu PII: DOI: Reference:
S0925-4005(18)31246-2 https://doi.org/10.1016/j.snb.2018.07.009 SNB 24978
To appear in:
Sensors and Actuators B
Received date: Revised date: Accepted date:
14-3-2018 1-7-2018 2-7-2018
Please cite this article as: Song H, Du W, Liu C, Li Z, Zhang H, Wei L, Yu M, Self-Assembled Micellar Nanosensor toward pH with high photo-stability and its application in living cells, Sensors and Actuators: B. Chemical (2018), https://doi.org/10.1016/j.snb.2018.07.009 This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.
Self-Assembled Micellar Nanosensor toward pH with high photo-stability and its application in living cells Huanhuan Songa, Weiwei Dua, Chunxia Liua, Zhanxian Lia,b*, Hongyan Zhangc*, Liuhe Weia, Mingming Yua,b* College of Chemistry and Molecular Engineering, Zhengzhou University, Zhengzhou,
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a.
450001, China.
Department of Chemical, Biological and Pharmaceutical Engineering, New Jersey
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b.
Institute of Technology, Newark, NJ 07102, USA
Key Laboratory of Photochemical Conversion and Optoelectronic Materials, Technical
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c.
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Institute of Physics and Chemistry, Chinese Academy of Sciences, Beijing, 100190,
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China
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* Corresponding author.
Tel: +(86)371-67781205
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Fax: +(86)371-67781205
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E-mail address: [email protected] (Z. Li), [email protected] (M. Yu).
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Graphical Abstract
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Based on photo-induced electron transfer, 1,8-naphthalic anhydride-based organic molecular fluorescent pH-sensor (1) was designed and synthesized. Hydrophobic, fluorescent hybrid nanosensor (1-PS35-b-PAA30) encapsulated with the hydrophobic pH
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responsive fluorophore for sensing intracellular pH has been fabricated based on the
1-PS35-b-PAA30
sensor
exhibits
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as-synthesized
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self-assembly of amphiphilic diblock copolymer PS35-b-PAA30 and with 1. The excellent
photo-stability,
good
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water-solubility and anti-disturbance ability, fast real time pH response, and enhanced
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fluorescence intensity under acidic environment with respect to the corresponding free dye in highly polar aqueous system because of the encapsulation of 1 inside nanoparticle
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cores with weak polarity environment. The fluorescence intensity of 1-PS35-b-PAA30 is enhanced by 7.3-fold upon changing from base (pH = 9.0) to acid (pH = 4) in aqueous
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system, which can exactly meet the physiological pH range in cells. Moreover, its linear fluorescent response from pH 5.2 to 7.4 makes this sensor suitable for the practical
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tracking of pH fluctuation in live cells. With this sensor, intracellular stimulated pH fluctuation has been studied via fluorescence imaging and TEM experiments.
Highlights
Based on 1,8-naphthalimide unit, a fluorescent sensor for pH was synthesized.
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Amphiphilic block copolymer PS-b-PAA was prepared.
Micellar pH-nanosensor was fabricated by self-assembly method.
The as-prepared nanosensor exhibits a linear response from pH 5.2 to 7.4.
Intracellular stimulated pH fluctuation has been racked via fluorescence imaging.
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Abstract
Based on photo-induced electron transfer, 1,8-naphthalic anhydride-based organic molecular fluorescent pH-sensor (1) was designed and synthesized. Hydrophobic,
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fluorescent hybrid nanosensor (1-PS35-b-PAA30) encapsulated with the hydrophobic pH
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responsive fluorophore for sensing intracellular pH has been fabricated based on the
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self-assembly of amphiphilic diblock copolymer PS35-b-PAA30 and 1. The as-synthesized 1-PS35-b-PAA30 sensor exhibits excellent photo-stability, good anti-disturbance ability,
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and enhanced fluorescence intensity under acidic environment with respect to the
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corresponding free dye in highly polar aqueous system because of the encapsulation of 1 inside nanoparticle cores with weak polarity environment. The fluorescence intensity of
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1-PS35-b-PAA30 is enhanced by 7.3-fold upon changing from base (pH = 9.0) to acid (pH = 4) in aqueous system, which can exactly meet the physiological pH range in cells.
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Moreover, its linear fluorescent response from pH 5.2 to 7.4 makes this sensor suitable for the practical tracking of pH fluctuation in live cells. The fluorescence imaging and TEM experiments indicated that the sensor permeated into cells and could not be observed because of aggregation in acidic condition.
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Keywords: Self-Assembled Micellar Nanosensor; pH fluctuation; Cell imaging. 1. Introduction As a crucial physiological parameter, pH plays a critical role in both intracellular (pHi) and extracellular (pHe) milieu, which is influenced by diverse physiological and
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pathological processes. Hence quantitatively measuring pH is useful for cellular analysis or diagnosis [1,2]. Fluorescence-based method has been widely used to
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detect various analytes due to its simplicity, high sensitivity, rapid response, and
capacity of real-time and in situ monitoring of the dynamic biological processes in
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living cells [3–23].
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So far, many organic molecular fluorescent pH sensors have been reported and
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some of them exhibit excellent properties [24–34]. Considering the drawbacks of
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molecular sensors such as the photobleaching and solvent-dependent luminescence
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properties, some hybrid pH-nanosensors have been developed and shown expansive potential of development [35–46]. Compared with small molecular probes, polymer
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composite fluorescent probes are more highly stable, easy to operate and can amplify
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signals, polymer fluorescent probes are widely used for detection of metal ions, biological small molecules, pH and temperature, and as a result, the design and development of polymer fluorescent probes have become the focus of researchers
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[47–49].
As a very important kind of organic fluorescent dyes, 1,8-naphthalimide derivatives have many merits such as high fluorescent quantum yields, large Stoke’s shift, and their absorption and emission spectra being in the visible region. Further, their photophysical
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properties can be easily tuned through judicious structural modifications. Based on the 1,8-naphthalimide unit, many fluorescent probes/sensors toward metal ions, anions, biological molecules, and environmental pollutants have been widely and deeply studied [50–53]. In our laboratory, we have synthesized some 1,8-naphthyridine derivatives and
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studied their sensing properties [54–57]. In this paper, based on 1,8-naphthalimide unit, a fluorescent molecule 1, as the
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pH-responsive fluorophore, was synthesized (Scheme 1) [58,59]. In addition, amphiphilic block copolymer PS-b-PAA (Scheme 2) [60] was synthesized. micellar pH-nanosensor was fabricated by self-assembly method. The as-prepared nanosensor exhibits excellent
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photo-stability, good anti-disturbance ability, and a linear response from pH 5.2 to 7.4.
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With this sensor, intracellular stimulated pH fluctuation has been studied via fluorescence
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imaging.
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2. Experimental
2.1. Methods and materials
H and 13C NMR spectra were recorded on a Bruker 400 NMR spectrometer. Mass
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1
spectra were obtained on a IonSpec4.7 Tesla FTMS-MALDI/DHB high resolution mass spectrometer. Fourier transform infrared spectroscopy (FTIR) was performed on a
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NEXUS-470 spectrometer at frequencies ranging from 400 to 4,000 cm-1. Samples for transmission electron microscopy (TEM) analysis were prepared by drying a colloidal solution of nanoparticles on amorphous carbon-coated copper grids. Low-resolution TEM were operated on a JEOL-JEM 2100 transmission electron microscopy operated at 200 kV. High resolution TEM (HRTEM) were carried out on a JEOL-JEM 2100F field emission
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transmission electron microscope operated at 200 kV. All of the chemicals were purchased from commercial suppliers and used without further purification. All of the reactions were performed under an argon atmosphere using solvents purified by standard methods. Compound 1 and amphiphilic block copolymer PS-b-PAA were synthesized according to Schemes 1 and 2.
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2.2. Spectral characterizations
All spectral characterizations were carried out in HPLC-grade solvents at 20 °C within
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a 10 mm quartz cell. UV-vis absorption spectra were measured with a TU-1901
double-beam UV-vis spectrophotometer. Fluorescence spectroscopy was determined on a
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Hitachi F-4600 spectrometer.
The excitation wavelength was 400 nm. The concentration of the nanosensor was 0.1
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mg/mL (2× 10−3 mg/mL of compound 1, 9.8× 10−2 mg/mL of PS35-b-PAA30). The sensing
2.3. Cell incubation and imaging
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solution was the mixture of DMSO−HEPES buffer solutions (1:9, v/v).
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HeLa cells were seeded in a 12-well plate in Dulbecco’s modified Eagle’s medium (DMEM) supplemented with 10% fetal bovine serum and 1% penicillin. The cells were
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incubated under an atmosphere of 5% CO2 and 95% air at 37 °C for 24 h before the cell
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imaging experiments. The cells were washed three times with PBS buffer before used. Fluorescence imaging experiments in Living Hela cells were operated with Nikon A1plus. 405 nm semiconductor laser was used for fluorescence imaging experiments.
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2.4. Synthesis of compound 3 4-Bromo-1,8-naphthalic
anhydride
(0.554
g,
2
mmol)
and
4-(2-aminoethyl)morpholine (525 μL, 4 mmol) were dissolved in 10 mL ethanol. The mixture was heated to 78℃ and refluxed for 1.5 h. After cooled to room temperature, the final product 3 (0.663 g, yield was 85%) was obtained after filtration and vacuum drying.
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Characterization of 3: 1H NMR (CDCl3, 400 MHz): δH 2.62 (s, 4H), 2.73 (t, J = 6.0 Hz, 2H), 3.70 (t, J = 6.0 Hz, 4H), 4.36 (t, J = 6.0 Hz, 2H), 7.86 (t, J = 4.0 Hz, 1H), 8.05 (d, J = 8.0 Hz, 1H), 8.41 (d, J = 8.0 Hz, 1H), 8.57 (d, J = 8.0 Hz, 1H), 8.85 (t, J = 4.0 Hz, 1H). C NMR (100 MHz, CDCl3): δC 163.62, 163.60, 133.31, 132.04, 131.23, 131.12, 130.64,
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130.32, 129.03, 128.10, 123.05, 122.18, 67.02, 56.09, 53.81, 37.30.
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2.5. Synthesis of compound 2
Compound 3 (298.7 mg, 0.75 mmol) and piperazidine (129.2 mg, 1.5 mmol) were
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dissolved in 10 mL 2-methoxyethanol. The mixture was cooled to room temperature and heated to 124℃ and refluxed for 4 h. The reaction liquid was filtered and the filter cake
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was obtained as the crude product. The final product 2 (231.2 mg, 78.2%) was obtained
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by column chromatography over silica gel column using dichloromethane/ethanol (25 : 1) as eluent.
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Characterization of 2: 1H NMR (400 MHz, DMSO-d6, Me4Si): δH 1.19 (m, 4H), 2.50 (m, 10H), 2.56 (t, J = 6.0 Hz, 2H), 3.08 (m, 4H), 4.17 (t, J = 6.0 Hz, 2H), 7.43 (d, J = 8.0
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Hz, 1H), 7.85 (t, J = 4.0 Hz,1H), 8.43 (d, J = 8.0 Hz, 1H), 8.52 (d, J = 8.0 Hz, 2H). 2.6. Synthesis of compound 1
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Compound 2 (100 mg, 0.25 mmol), K2CO3 (138.2 mg, 1 mmol) and n-C12H25Br (56 μL, 0.25 mmol) were dissolved in 15 mL acetonitrile. After cooled to room temperature, the
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mixture was filtered and the filter cake was obtained as the crude product. The final product 1 (101.9 mg, 72.4%) was obtained by column chromatography over silica gel
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column using dichloromethane/ethanol (20:1) as eluent. Characterization of 1: HRMS (EI) m/z: calcd for C34H52N4O3 [M], 564.4039; found,
564.3981. 1H NMR (CDCl3, 400 MHz): δH 0.92 (t, J = 8.0 Hz, 3H), 1.32 (m, 20H), 1.59 (s, 2H), 2.51 (t, J = 8.0 Hz, 2H), 2.61 (s, 4H), 2.71 (t, J = 8.0 Hz, 2H), 2.80 (s, 4H), 3.33 (s, 3H). 3.7 (t, J = 4.0 Hz, 3H), 4.35 (t, J = 4.0 Hz, 2H), 7.25 (d, J = 8.0 Hz, 1H), 7.7 (t, J =
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4.0 Hz, 1H), 8.43 (d, J = 8.0 Hz, 1H), 8.52 (d, J = 8.0 Hz, 1H), 8.58 (d, J = 8.0 Hz, 1H). C NMR (100 MHz, CDCl3): δC 164.51, 164.01, 158.08, 132.62, 131.09, 130.39, 129.94,
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126.17, 125.60, 123.20, 116.57, 114.91, 67.08, 58.80, 56.22, 53.83, 53.30, 53.04, 37.07, 31.93, 29.69 , 29.66, 29.61, 29.37, 27.58, 22.70, 14.14.
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2.7. Synthesis of PS-Br CuBr (0.05 g), toluene (4 mL), Methyl 2-Bromopropionate (MBP, 39 mL),
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pentamethyldiethylenetriamine (PMDETA, 146 μL), styrene (3.96 g, 4 mL), were added
in a 50 mL Schlenk bottle and mixed. The reaction was carried out at 90℃ in neither oxygen nor water condition and reacted for 21 h. after cooling to room temperature, THF
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was added in the reaction mixture and green solution was obtained. The colorless liquid
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was obtained by column chromatography over neutral alumina using toluene as eluent.
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With the rotary evaporator, the colorless clarified liquid was concentrated to about 20 mL and added in dropwise to 200 mL cooled methanol. The final product PS-Br (4.6 g) was
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obtained as white solid by filtration and drying of the filter cake from the mixture. The average number of molecular weight is 3540 and the distribution coefficient is 1.34.
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2.8. Synthesis of PS-b-PtBA
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In nitrogen atmosphere, 2.5 g PS-Br, 0.122 g CuBr, 355 μL PMDETA, 4.1 mL tert-butyl acrylate (t-BA) and 15 mL toluene were added in 50 mL Schlenk and stirred. In no oxygen or water, the reaction was carried out at 100℃ and reacted for 4 h. After
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cooling to room temperature, THF was added and green solution was obtained. The pale yellow liquid was obtained by column chromatography over neutral alumina using toluene as eluent. With the rotary evaporator, the pale yellow clarified liquid was concentrated to about 20 mL and added in dropwise to 200 mL cooled methanol. The final product PS-b-PtBA (2.0 g) was obtained as white solid by filtration and drying of
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the filter cake from the mixture. The average number of molecular weight is 7300 and the distribution coefficient is 1.45. 2.9. Synthesis of PS35-b-PAA30 1.93 g PS-b-PtBA and 2.96 mL trifluoroacetic acid were added in 20 mL dichloromethane and the reaction was carried out at room temperature for 24 h. With the
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rotary evaporator, the reaction liquid was concentrated to 15 mL and added in dropwise to the mixture of cooled methanol and water. The final product PS35-b-PAA30 (1.5 g) was
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collected with centrifugation (9000 rpm). The average number of molecular weight is 7300 and the distribution coefficient is 1.45.
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2.10. Self-assembly of compound 1 and PS35-b-PAA30
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In this part, we have obtained three examples which contain 1%, 2% and 4% of
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compound 1 respectively. Herein, 2% was selected as the example. The preparation
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process was as bellow: PS35-b-PAA30 (100 mg) and compound 2 (2 mg) were dissolved
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in 10 mL DMSO, and then, 30 mL ultra pure water was quickly added into the above mixture. After intense stirring, and dialysis with dichloromethane as solvent, the hybrid
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pH-nanosensor (1-PS35-b-PAA30) was obtained by vacuum drying.
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3. Results and Discussion
3.1. Best doping content of compound 1 in 1-PS35-b-PAA30
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As shown in Fig. 1a, upon excitation at 400 nm and the doping content of compound 1
is 1%, no obvious fluorescence change was observed for the hybrid nanomaterial 1-PS35-b-PAA30 in pH = 4 and 9. When the doping content was increased to 2% (Fig. 1b) and 4% (Fig. 1c), great fluorescence change can be observed for 1-PS35-b-PAA30 in the
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same condition as that when the doping content is 1%, and no difference was observed for the doping content of 2% and 4%. Therefore, 2% was selected as the best doping content and 1-PS35-b-PAA30 which contains 2% of compound 1 was used in all the following experiments.
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3.2. FT-IR Spectral and Morphology Characterization
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Fig. 1d indicates the FT-IR spectra of PS35-b-PAA30 (black line), compound 1 (green
line) and 1-PS35-b-PAA30 (red line). In the FT-IR spectrum of PS35-b-PAA30, the peaks at
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3000 cm-1, 1700 cm-1, 1500 and 1450 cm-1, 690 cm-1 are ascribed to the O-H, C=O, C-H
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of benzene ring, and C-Br stretching vibration respectively. In the FT-IR spectrum of
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compound 1, the peaks at 3000 cm-1, 1700 cm-1, 1400 cm-1, 1010 cm-1 can be ascribed to
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the C-H of benzene ring, C=O, C-O stretching vibration respectively. Combined FT-IR
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spectra of PS35-b-PAA30, compound 1 and 1-PS35-b-PAA30, it is concluded that the successful self-assembly of PS35-b-PAA30 and compound 1 was finished.
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Fig. 2 indicates the TEM images of PS35-b-PAA30 and 1-PS35-b-PAA30. As shown in
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Fig. 2a and Fig. 2b, PS35-b-PAA30 exists as 50 nm Vesicles and these vesicles form Micelle group by intermolecular interaction. From Fig. 2c and Fig. 2d, it is concluded that after self-assembly, the hybrid nanosensor 1-PS35-b-PAA30 still exists as vesicles.
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Further studies indicate that membranes are formed on the edge of the vesicles and the particles in the vesicles may be compound 2.
3.3. pH sensing properties of 1-PS35-b-PAA30
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The fluorescence spectra of 1-PS35-b-PAA30 at different apparent pH (pHapp) were examined in a HEPES buffer containing 10% DMSO. As shown in Fig. 3a, when pHapp increased from 4.0 to 9.0, the fluorescence peak at 507 nm of 1-PS35-b-PAA30 decreased. The changes in emission spectrum are ascribed to the structural transformation from
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compound 4 to compound 1 (Scheme 1). In acidic condition, the nitrogen atom can be protonated, which inhibits the photo-induced electron transfer from piperazine unit to the
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1,8-naphthalic anhydride group [50, 58, 59]. As a result, in acidic condition, fluorescence of compound 1 is much stronger than that in neutral and basic condition. The linear range
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for the response from emission spectra of 1-PS35-b-PAA30 are pHapp of 5.2 to 7.4 (Fig.
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2b).
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3.4. The anti-disturbance effect study of 1-PS35-b-PAA30 for pH sensing
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To further assess its utility as a pH fluorescent sensor, its emission spectral response to pH 7.4 in the presence of biological species including biological molecules, metal ions,
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and anions (figures 4a, 5a and 5b) was also tested. The emission intensity at 507 nm (I507)
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exhibits a negligible change in the presence of these species. All these results suggest that 1-PS35-b-PAA30 might be a suitable candidate for intracellular pH imaging. 3.5. Photostability study of sensor 1-PS35-b-PAA30
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An ideal sensor ought to have good photostability. As shown in Fig. 4b, with
irradiation time extending, the emission intensity at 507 nm of 1-PS35-b-PAA30 in HEPES solutions hardly changes, indicating excellent photostability.
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3.6. Application of 1-PS35-b-PAA30 in cellular imaging The hybrid nanosensor 1-PS35-b-PAA30 was further studied for its pHi imagining ability. In this study, the intracellular pH calibration was carried out in live HeLa cells using a standard procedure followed by a further incubation with high K+ buffers of different pH,
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also containing 1 mM nigericin. As shown in the first and second rows of Fig. 6, upon excitation at 405 nm laser, the emission intensities at green channel with 525/50 filter
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(figures 6a, 6b, 6c, 6d) gradually increase with pH changing from 5.0 to 8.0. The result obtained from fluorescence imaging of pHi in live HeLa cells was quite different from
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those in solution. To further study such difference, the TEM images of 1-PS35-b-PAA30
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(Fig. 7) revealed that the protonation of the hybrid nanosensor resulted in the block
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network formation among the amphiphilic block polymers in acidic solution (Fig. 7a and
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Fig. 7b). Poor dispersity of nanosensor in live cells results in the weak fluorescence in
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acidic condition (figures 6a and 6b). Upon pH increasing, the weakened protonation makes good dispersity of the hybrid nanosensor (Fig. 7c and Fig. 7d), which results in the
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strong fluorescence in neutral and basic intracellular condition (figures 6c and 6d). Such
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results mean that this nanosensor can be as an extracellular pH-sensor.
3. Conclusion In summary, a hydrophilic fluorescent hybrid nanosensor (1-PS35-b-PAA30) was fabricated via self-assembly between a 1,8-naphthalic anhydride-based organic molecular fluorescent pH-sensor and an amphiphilic diblock copolymer PS35-b-PAA30. The
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nanosensor 1-PS35-b-PAA30 exhibits excellent photo-stability, good anti-disturbance ability, and a linear fluorescent response from pH 5.2 to 7.4. Moreover, TEM experiments and fluorescence imaging results indicated that the sensor permeated into cells and could not be observed because of aggregation in acidic condition. Acknowledgements
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We are grateful for the financial supports from National Natural Science Foundation of China (U1704161, U1504203, 21601158), and Zhengzhou University (32210431).
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Biographies Huanhuan Song received her B.S. degree in 2016. She is currently a M.S. candidate at
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College of Chemistry and Molecular Engineering, Zhengzhou University. Her research interests focus on developing fluorescent chemosensors. Weiwei Du received her B.S. degree in 2014. She is currently a M.S. candidate at College of Chemistry and Molecular Engineering, Zhengzhou University. Her research interests
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focus on developing fluorescent chemosensors. Chunxia Liu received her B.S. degree in 2012. She is currently a M.S. candidate at
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College of Chemistry and Molecular Engineering, Zhengzhou University. Her research interests focus on developing fluorescent chemosensors.
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Zhanxian Li received her Ph.D. degree in 2006 from Chinese Academy of Sciences. She
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is an associate professor of College of Chemistry and Molecular Engineering, Zhengzhou
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University. Her current research interests are mainly in fluorescent chemosensors and
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molecular recognition.
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Hongyan Zhang received her Ph.D. in 2005 from Changchun Institute of Optics and Fine Mechanics and Physics, Chinese Academy of Sciences. She joined Technical Institute of
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Physics and Chemistry, Chinese Academy of Sciences in 2007. Her main research
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interests include design and develop optical detection system for bimolecular and sensor molecules.
Liuhe Wei is a professor of College of Chemistry and Molecular Engineering, Zhengzhou
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University. Her current research interests focus on functional polymers and supermolecular chemistry. Mingming Yu is an associate professor of College of Chemistry and Molecular Engineering, Zhengzhou University. His current research interests are mainly in
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fluorescent chemosensors and functional polymers.
Figure Captions Fig. 1. Emission spectra of 1-PS35-b-PAA30 with different doping content of
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compound 1 1% (a), 2% (b), 4 % (c) in different pH condition with excitation at 400 nm.
(d) FT-IR spectra of PS35-b-PAA30 (black line of Fig. 1d), compound 1 (green line of Fig.
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1d), and 1-PS35-b-PAA30 (red line of Fig. 1d).
Fig. 2. TEM images of PS-b-PAA (scale bar is 0.5 μm (a), scale bar is 200 nm (b)),
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and TEM images of 1-PS35-b-PAA30 (scale bar is 0.5 μm (c), scale bar is 100 nm (d)).
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Fig. 3. Emission spectra of 1-PS35-b-PAA30 (2 × 10−3 mg/mL of compound 1, 9.8 ×
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10−2 mg/mL of PS35-b-PAA30) in DMSO−HEPES buffer solutions (1:9, v/v) of different
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pHapp values under excitation at 400 nm (a). The pH titration curve was plotted by the
wavelength was 400 nm.
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emission spectra of 1-PS35-b-PAA30 as linear function of pHapp (b). The excitation
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Fig. 4. Emission spectra response of 1-PS35-b-PAA30 (2 × 10−3 mg/mL of compound 1, 9.8 × 10−2 mg/mL of PS35-b-PAA30, VDMSO:VHEPES=1:9, pH=7.4) upon addition of
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different metal ions (9.5×10–5 mol/L) (a), the excitation wavelength was 400 nm. I507 represents the emission intensity at 507 nm. Emission intensity at 507 nm of
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1-PS35-b-PAA30 (0.1 mg/mL, VDMSO:VHEPES=1:9, pH=7.4) with irradiation (λex = 470 nm) time change (b). Fig. 5. Emission spectral response of 1-PS35-b-PAA30 (2 × 10−3 mg/mL of compound 1, 9.8 × 10−2 mg/mL of PS35-b-PAA30, VDMSO:VHEPES=1:9, pH=7.4) upon addition of different anions (a) and biological molecules (b) (9.5×10–5 mol/L), the excitation
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wavelength was 400 nm. I507 represents the emission intensity at 507 nm. The anions used were ClO4–, HCO3–, CO32–, SO42–, NO3–, C2O42–, HSO3–, HSO4–, S2O32–, SH–, I–, N3–, PO43–, P2O74–, H2PO4–, SO32–, HPO42–, SiO32–, ClO3–, CH3COO–, F–, Br–, and Cl–. The biological molecules used were Gly, lle, Ala, Cys, GSH, Hcy, Arg, Met, Phe, Thr, Gln, Lys, Asn, Val, Trp, Ser, Pro, Leu, and Tyr.
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Fig. 6. Confocal microscopy images of HeLa cells incubated with 1-PS35-b-PAA30 (2
× 10−3 mg/mL of compound 1, 9.8 × 10−2 mg/mL of PS35-b-PAA30) at different pH (pH=5,
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a,e), (pH=6, b, f), (pH=7, c, g), and (pH=8, d, h). The excitation wavelength was 405 nm. The images were collected at green channel with 525/50 filter (a, b, c, d).
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Fig. 7. TEM images of 1-PS35-b-PAA30 in different pH conditions.
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Scheme 2. Synthesis route of PS-b-PAA.
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Scheme 1. Synthesis route and sensing mechanism of compound 1.
Fig. 1. Emission spectra of 1-PS35-b-PAA30 with different doping content of compound 1 1% (a), 2% (b), 4 % (c) in different pH condition with excitation at 400 nm. (d) FT-IR spectra of PS35-b-PAA30 (black line of Fig. 1d), compound 1 (green line of Fig. 1d), and 1-PS35-b-PAA30 (red line of Fig. 1d).
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Fig. 2. TEM images of PS-b-PAA (scale bar is 0.5 μm (a), scale bar is 200 nm (b)), and
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TEM images of 1-PS35-b-PAA30 (scale bar is 0.5 μm (c), scale bar is 100 nm (d)).
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Fig. 3. Emission spectra of 1-PS35-b-PAA30 (2 × 10−3 mg/mL of compound 1, 9.8 × 10−2
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mg/mL of PS35-b-PAA30) in DMSO−HEPES buffer solutions (1:9, v/v) of different pHapp values under excitation at 400 nm (a). The pH titration curve was plotted by the emission
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spectra of 1-PS35-b-PAA30 as linear function of pHapp (b). The excitation wavelength was
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400 nm.
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Fig. 4. Emission spectra response of 1-PS35-b-PAA30 (2 × 10−3 mg/mL of compound 1,
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9.8 × 10−2 mg/mL of PS35-b-PAA30, VDMSO:VHEPES=1:9, pH=7.4) upon addition of different metal ions (9.5×10–5 mol/L) (a), the excitation wavelength was 400 nm. I507
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represents the emission intensity at 507 nm. Emission intensity at 507 nm of 1-PS35-b-PAA30 (0.1 mg/mL, VDMSO:VHEPES=1:9, pH=7.4) with irradiation (λex = 470 nm)
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time change (b).
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Fig. 5. Emission spectral response of 1-PS35-b-PAA30 (2 × 10−3 mg/mL of compound 1, 9.8 × 10−2 mg/mL of PS35-b-PAA30, VDMSO:VHEPES=1:9, pH=7.4) upon addition of
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different anions (a) and biological molecules (b) (9.5×10–5 mol/L), the excitation wavelength was 400 nm. I507 represents the emission intensity at 507 nm. The anions used were ClO4–, HCO3–, CO32–, SO42–, NO3–, C2O42–, HSO3–, HSO4–, S2O32–, SH–, I–, N3–, PO43–, P2O74–, H2PO4–, SO32–, HPO42–, SiO32–, ClO3–, CH3COO–, F–, Br–, and Cl–. The biological molecules used were Gly, lle, Ala, Cys, GSH, Hcy, Arg, Met, Phe, Thr, Gln,
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Lys, Asn, Val, Trp, Ser, Pro, Leu, and Tyr.
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Fig. 6. Confocal microscopy images of HeLa cells incubated with 1-PS35-b-PAA30 (2 × 10−3 mg/mL of compound 1, 9.8 × 10−2 mg/mL of PS35-b-PAA30) at different pH (pH=5, a,e), (pH=6, b, f), (pH=7, c, g), and (pH=8, d, h). The excitation wavelength was 405 nm. The images were collected at green channel with 525/50 filter (a, b, c, d).
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Fig. 7. TEM images of 1-PS35-b-PAA30 in different pH condition.
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Scheme 1. Synthesis route and sensing mechanism of compound 1.
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Scheme 2. Synthesis route of PS-b-PAA.
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S0925-4005(18)31246-2 https://doi.org/10.1016/j.snb.2018.07.009 SNB 24978
To appear in:
Sensors and Actuators B
Received date: Revised date: Accepted date:
14-3-2018 1-7-2018 2-7-2018
Please cite this article as: Song H, Du W, Liu C, Li Z, Zhang H, Wei L, Yu M, Self-Assembled Micellar Nanosensor toward pH with high photo-stability and its application in living cells, Sensors and Actuators: B. Chemical (2018), https://doi.org/10.1016/j.snb.2018.07.009 This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.
Self-Assembled Micellar Nanosensor toward pH with high photo-stability and its application in living cells Huanhuan Songa, Weiwei Dua, Chunxia Liua, Zhanxian Lia,b*, Hongyan Zhangc*, Liuhe Weia, Mingming Yua,b* College of Chemistry and Molecular Engineering, Zhengzhou University, Zhengzhou,
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a.
450001, China.
Department of Chemical, Biological and Pharmaceutical Engineering, New Jersey
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b.
Institute of Technology, Newark, NJ 07102, USA
Key Laboratory of Photochemical Conversion and Optoelectronic Materials, Technical
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c.
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Institute of Physics and Chemistry, Chinese Academy of Sciences, Beijing, 100190,
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China
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* Corresponding author.
Tel: +(86)371-67781205
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Fax: +(86)371-67781205
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E-mail address: [email protected] (Z. Li), [email protected] (M. Yu).
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Graphical Abstract
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2
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Based on photo-induced electron transfer, 1,8-naphthalic anhydride-based organic molecular fluorescent pH-sensor (1) was designed and synthesized. Hydrophobic, fluorescent hybrid nanosensor (1-PS35-b-PAA30) encapsulated with the hydrophobic pH
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responsive fluorophore for sensing intracellular pH has been fabricated based on the
1-PS35-b-PAA30
sensor
exhibits
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as-synthesized
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self-assembly of amphiphilic diblock copolymer PS35-b-PAA30 and with 1. The excellent
photo-stability,
good
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water-solubility and anti-disturbance ability, fast real time pH response, and enhanced
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fluorescence intensity under acidic environment with respect to the corresponding free dye in highly polar aqueous system because of the encapsulation of 1 inside nanoparticle
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cores with weak polarity environment. The fluorescence intensity of 1-PS35-b-PAA30 is enhanced by 7.3-fold upon changing from base (pH = 9.0) to acid (pH = 4) in aqueous
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system, which can exactly meet the physiological pH range in cells. Moreover, its linear fluorescent response from pH 5.2 to 7.4 makes this sensor suitable for the practical
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tracking of pH fluctuation in live cells. With this sensor, intracellular stimulated pH fluctuation has been studied via fluorescence imaging and TEM experiments.
Highlights
Based on 1,8-naphthalimide unit, a fluorescent sensor for pH was synthesized.
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Amphiphilic block copolymer PS-b-PAA was prepared.
Micellar pH-nanosensor was fabricated by self-assembly method.
The as-prepared nanosensor exhibits a linear response from pH 5.2 to 7.4.
Intracellular stimulated pH fluctuation has been racked via fluorescence imaging.
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Abstract
Based on photo-induced electron transfer, 1,8-naphthalic anhydride-based organic molecular fluorescent pH-sensor (1) was designed and synthesized. Hydrophobic,
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fluorescent hybrid nanosensor (1-PS35-b-PAA30) encapsulated with the hydrophobic pH
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responsive fluorophore for sensing intracellular pH has been fabricated based on the
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self-assembly of amphiphilic diblock copolymer PS35-b-PAA30 and 1. The as-synthesized 1-PS35-b-PAA30 sensor exhibits excellent photo-stability, good anti-disturbance ability,
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and enhanced fluorescence intensity under acidic environment with respect to the
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corresponding free dye in highly polar aqueous system because of the encapsulation of 1 inside nanoparticle cores with weak polarity environment. The fluorescence intensity of
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1-PS35-b-PAA30 is enhanced by 7.3-fold upon changing from base (pH = 9.0) to acid (pH = 4) in aqueous system, which can exactly meet the physiological pH range in cells.
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Moreover, its linear fluorescent response from pH 5.2 to 7.4 makes this sensor suitable for the practical tracking of pH fluctuation in live cells. The fluorescence imaging and TEM experiments indicated that the sensor permeated into cells and could not be observed because of aggregation in acidic condition.
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Keywords: Self-Assembled Micellar Nanosensor; pH fluctuation; Cell imaging. 1. Introduction As a crucial physiological parameter, pH plays a critical role in both intracellular (pHi) and extracellular (pHe) milieu, which is influenced by diverse physiological and
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pathological processes. Hence quantitatively measuring pH is useful for cellular analysis or diagnosis [1,2]. Fluorescence-based method has been widely used to
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detect various analytes due to its simplicity, high sensitivity, rapid response, and
capacity of real-time and in situ monitoring of the dynamic biological processes in
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living cells [3–23].
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So far, many organic molecular fluorescent pH sensors have been reported and
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some of them exhibit excellent properties [24–34]. Considering the drawbacks of
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molecular sensors such as the photobleaching and solvent-dependent luminescence
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properties, some hybrid pH-nanosensors have been developed and shown expansive potential of development [35–46]. Compared with small molecular probes, polymer
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composite fluorescent probes are more highly stable, easy to operate and can amplify
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signals, polymer fluorescent probes are widely used for detection of metal ions, biological small molecules, pH and temperature, and as a result, the design and development of polymer fluorescent probes have become the focus of researchers
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[47–49].
As a very important kind of organic fluorescent dyes, 1,8-naphthalimide derivatives have many merits such as high fluorescent quantum yields, large Stoke’s shift, and their absorption and emission spectra being in the visible region. Further, their photophysical
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properties can be easily tuned through judicious structural modifications. Based on the 1,8-naphthalimide unit, many fluorescent probes/sensors toward metal ions, anions, biological molecules, and environmental pollutants have been widely and deeply studied [50–53]. In our laboratory, we have synthesized some 1,8-naphthyridine derivatives and
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studied their sensing properties [54–57]. In this paper, based on 1,8-naphthalimide unit, a fluorescent molecule 1, as the
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pH-responsive fluorophore, was synthesized (Scheme 1) [58,59]. In addition, amphiphilic block copolymer PS-b-PAA (Scheme 2) [60] was synthesized. micellar pH-nanosensor was fabricated by self-assembly method. The as-prepared nanosensor exhibits excellent
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photo-stability, good anti-disturbance ability, and a linear response from pH 5.2 to 7.4.
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With this sensor, intracellular stimulated pH fluctuation has been studied via fluorescence
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imaging.
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2. Experimental
2.1. Methods and materials
H and 13C NMR spectra were recorded on a Bruker 400 NMR spectrometer. Mass
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1
spectra were obtained on a IonSpec4.7 Tesla FTMS-MALDI/DHB high resolution mass spectrometer. Fourier transform infrared spectroscopy (FTIR) was performed on a
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NEXUS-470 spectrometer at frequencies ranging from 400 to 4,000 cm-1. Samples for transmission electron microscopy (TEM) analysis were prepared by drying a colloidal solution of nanoparticles on amorphous carbon-coated copper grids. Low-resolution TEM were operated on a JEOL-JEM 2100 transmission electron microscopy operated at 200 kV. High resolution TEM (HRTEM) were carried out on a JEOL-JEM 2100F field emission
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transmission electron microscope operated at 200 kV. All of the chemicals were purchased from commercial suppliers and used without further purification. All of the reactions were performed under an argon atmosphere using solvents purified by standard methods. Compound 1 and amphiphilic block copolymer PS-b-PAA were synthesized according to Schemes 1 and 2.
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2.2. Spectral characterizations
All spectral characterizations were carried out in HPLC-grade solvents at 20 °C within
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a 10 mm quartz cell. UV-vis absorption spectra were measured with a TU-1901
double-beam UV-vis spectrophotometer. Fluorescence spectroscopy was determined on a
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Hitachi F-4600 spectrometer.
The excitation wavelength was 400 nm. The concentration of the nanosensor was 0.1
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mg/mL (2× 10−3 mg/mL of compound 1, 9.8× 10−2 mg/mL of PS35-b-PAA30). The sensing
2.3. Cell incubation and imaging
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solution was the mixture of DMSO−HEPES buffer solutions (1:9, v/v).
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HeLa cells were seeded in a 12-well plate in Dulbecco’s modified Eagle’s medium (DMEM) supplemented with 10% fetal bovine serum and 1% penicillin. The cells were
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incubated under an atmosphere of 5% CO2 and 95% air at 37 °C for 24 h before the cell
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imaging experiments. The cells were washed three times with PBS buffer before used. Fluorescence imaging experiments in Living Hela cells were operated with Nikon A1plus. 405 nm semiconductor laser was used for fluorescence imaging experiments.
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2.4. Synthesis of compound 3 4-Bromo-1,8-naphthalic
anhydride
(0.554
g,
2
mmol)
and
4-(2-aminoethyl)morpholine (525 μL, 4 mmol) were dissolved in 10 mL ethanol. The mixture was heated to 78℃ and refluxed for 1.5 h. After cooled to room temperature, the final product 3 (0.663 g, yield was 85%) was obtained after filtration and vacuum drying.
7
Characterization of 3: 1H NMR (CDCl3, 400 MHz): δH 2.62 (s, 4H), 2.73 (t, J = 6.0 Hz, 2H), 3.70 (t, J = 6.0 Hz, 4H), 4.36 (t, J = 6.0 Hz, 2H), 7.86 (t, J = 4.0 Hz, 1H), 8.05 (d, J = 8.0 Hz, 1H), 8.41 (d, J = 8.0 Hz, 1H), 8.57 (d, J = 8.0 Hz, 1H), 8.85 (t, J = 4.0 Hz, 1H). C NMR (100 MHz, CDCl3): δC 163.62, 163.60, 133.31, 132.04, 131.23, 131.12, 130.64,
13
130.32, 129.03, 128.10, 123.05, 122.18, 67.02, 56.09, 53.81, 37.30.
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2.5. Synthesis of compound 2
Compound 3 (298.7 mg, 0.75 mmol) and piperazidine (129.2 mg, 1.5 mmol) were
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dissolved in 10 mL 2-methoxyethanol. The mixture was cooled to room temperature and heated to 124℃ and refluxed for 4 h. The reaction liquid was filtered and the filter cake
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was obtained as the crude product. The final product 2 (231.2 mg, 78.2%) was obtained
N
by column chromatography over silica gel column using dichloromethane/ethanol (25 : 1) as eluent.
M
A
Characterization of 2: 1H NMR (400 MHz, DMSO-d6, Me4Si): δH 1.19 (m, 4H), 2.50 (m, 10H), 2.56 (t, J = 6.0 Hz, 2H), 3.08 (m, 4H), 4.17 (t, J = 6.0 Hz, 2H), 7.43 (d, J = 8.0
ED
Hz, 1H), 7.85 (t, J = 4.0 Hz,1H), 8.43 (d, J = 8.0 Hz, 1H), 8.52 (d, J = 8.0 Hz, 2H). 2.6. Synthesis of compound 1
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Compound 2 (100 mg, 0.25 mmol), K2CO3 (138.2 mg, 1 mmol) and n-C12H25Br (56 μL, 0.25 mmol) were dissolved in 15 mL acetonitrile. After cooled to room temperature, the
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mixture was filtered and the filter cake was obtained as the crude product. The final product 1 (101.9 mg, 72.4%) was obtained by column chromatography over silica gel
A
column using dichloromethane/ethanol (20:1) as eluent. Characterization of 1: HRMS (EI) m/z: calcd for C34H52N4O3 [M], 564.4039; found,
564.3981. 1H NMR (CDCl3, 400 MHz): δH 0.92 (t, J = 8.0 Hz, 3H), 1.32 (m, 20H), 1.59 (s, 2H), 2.51 (t, J = 8.0 Hz, 2H), 2.61 (s, 4H), 2.71 (t, J = 8.0 Hz, 2H), 2.80 (s, 4H), 3.33 (s, 3H). 3.7 (t, J = 4.0 Hz, 3H), 4.35 (t, J = 4.0 Hz, 2H), 7.25 (d, J = 8.0 Hz, 1H), 7.7 (t, J =
8
4.0 Hz, 1H), 8.43 (d, J = 8.0 Hz, 1H), 8.52 (d, J = 8.0 Hz, 1H), 8.58 (d, J = 8.0 Hz, 1H). C NMR (100 MHz, CDCl3): δC 164.51, 164.01, 158.08, 132.62, 131.09, 130.39, 129.94,
13
126.17, 125.60, 123.20, 116.57, 114.91, 67.08, 58.80, 56.22, 53.83, 53.30, 53.04, 37.07, 31.93, 29.69 , 29.66, 29.61, 29.37, 27.58, 22.70, 14.14.
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2.7. Synthesis of PS-Br CuBr (0.05 g), toluene (4 mL), Methyl 2-Bromopropionate (MBP, 39 mL),
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pentamethyldiethylenetriamine (PMDETA, 146 μL), styrene (3.96 g, 4 mL), were added
in a 50 mL Schlenk bottle and mixed. The reaction was carried out at 90℃ in neither oxygen nor water condition and reacted for 21 h. after cooling to room temperature, THF
N
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was added in the reaction mixture and green solution was obtained. The colorless liquid
A
was obtained by column chromatography over neutral alumina using toluene as eluent.
M
With the rotary evaporator, the colorless clarified liquid was concentrated to about 20 mL and added in dropwise to 200 mL cooled methanol. The final product PS-Br (4.6 g) was
ED
obtained as white solid by filtration and drying of the filter cake from the mixture. The average number of molecular weight is 3540 and the distribution coefficient is 1.34.
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2.8. Synthesis of PS-b-PtBA
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In nitrogen atmosphere, 2.5 g PS-Br, 0.122 g CuBr, 355 μL PMDETA, 4.1 mL tert-butyl acrylate (t-BA) and 15 mL toluene were added in 50 mL Schlenk and stirred. In no oxygen or water, the reaction was carried out at 100℃ and reacted for 4 h. After
A
cooling to room temperature, THF was added and green solution was obtained. The pale yellow liquid was obtained by column chromatography over neutral alumina using toluene as eluent. With the rotary evaporator, the pale yellow clarified liquid was concentrated to about 20 mL and added in dropwise to 200 mL cooled methanol. The final product PS-b-PtBA (2.0 g) was obtained as white solid by filtration and drying of
9
the filter cake from the mixture. The average number of molecular weight is 7300 and the distribution coefficient is 1.45. 2.9. Synthesis of PS35-b-PAA30 1.93 g PS-b-PtBA and 2.96 mL trifluoroacetic acid were added in 20 mL dichloromethane and the reaction was carried out at room temperature for 24 h. With the
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rotary evaporator, the reaction liquid was concentrated to 15 mL and added in dropwise to the mixture of cooled methanol and water. The final product PS35-b-PAA30 (1.5 g) was
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collected with centrifugation (9000 rpm). The average number of molecular weight is 7300 and the distribution coefficient is 1.45.
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2.10. Self-assembly of compound 1 and PS35-b-PAA30
N
In this part, we have obtained three examples which contain 1%, 2% and 4% of
A
compound 1 respectively. Herein, 2% was selected as the example. The preparation
M
process was as bellow: PS35-b-PAA30 (100 mg) and compound 2 (2 mg) were dissolved
ED
in 10 mL DMSO, and then, 30 mL ultra pure water was quickly added into the above mixture. After intense stirring, and dialysis with dichloromethane as solvent, the hybrid
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pH-nanosensor (1-PS35-b-PAA30) was obtained by vacuum drying.
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3. Results and Discussion
3.1. Best doping content of compound 1 in 1-PS35-b-PAA30
A
As shown in Fig. 1a, upon excitation at 400 nm and the doping content of compound 1
is 1%, no obvious fluorescence change was observed for the hybrid nanomaterial 1-PS35-b-PAA30 in pH = 4 and 9. When the doping content was increased to 2% (Fig. 1b) and 4% (Fig. 1c), great fluorescence change can be observed for 1-PS35-b-PAA30 in the
10
same condition as that when the doping content is 1%, and no difference was observed for the doping content of 2% and 4%. Therefore, 2% was selected as the best doping content and 1-PS35-b-PAA30 which contains 2% of compound 1 was used in all the following experiments.
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3.2. FT-IR Spectral and Morphology Characterization
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Fig. 1d indicates the FT-IR spectra of PS35-b-PAA30 (black line), compound 1 (green
line) and 1-PS35-b-PAA30 (red line). In the FT-IR spectrum of PS35-b-PAA30, the peaks at
U
3000 cm-1, 1700 cm-1, 1500 and 1450 cm-1, 690 cm-1 are ascribed to the O-H, C=O, C-H
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of benzene ring, and C-Br stretching vibration respectively. In the FT-IR spectrum of
A
compound 1, the peaks at 3000 cm-1, 1700 cm-1, 1400 cm-1, 1010 cm-1 can be ascribed to
M
the C-H of benzene ring, C=O, C-O stretching vibration respectively. Combined FT-IR
ED
spectra of PS35-b-PAA30, compound 1 and 1-PS35-b-PAA30, it is concluded that the successful self-assembly of PS35-b-PAA30 and compound 1 was finished.
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Fig. 2 indicates the TEM images of PS35-b-PAA30 and 1-PS35-b-PAA30. As shown in
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Fig. 2a and Fig. 2b, PS35-b-PAA30 exists as 50 nm Vesicles and these vesicles form Micelle group by intermolecular interaction. From Fig. 2c and Fig. 2d, it is concluded that after self-assembly, the hybrid nanosensor 1-PS35-b-PAA30 still exists as vesicles.
A
Further studies indicate that membranes are formed on the edge of the vesicles and the particles in the vesicles may be compound 2.
3.3. pH sensing properties of 1-PS35-b-PAA30
11
The fluorescence spectra of 1-PS35-b-PAA30 at different apparent pH (pHapp) were examined in a HEPES buffer containing 10% DMSO. As shown in Fig. 3a, when pHapp increased from 4.0 to 9.0, the fluorescence peak at 507 nm of 1-PS35-b-PAA30 decreased. The changes in emission spectrum are ascribed to the structural transformation from
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compound 4 to compound 1 (Scheme 1). In acidic condition, the nitrogen atom can be protonated, which inhibits the photo-induced electron transfer from piperazine unit to the
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1,8-naphthalic anhydride group [50, 58, 59]. As a result, in acidic condition, fluorescence of compound 1 is much stronger than that in neutral and basic condition. The linear range
U
for the response from emission spectra of 1-PS35-b-PAA30 are pHapp of 5.2 to 7.4 (Fig.
N
2b).
A
M
3.4. The anti-disturbance effect study of 1-PS35-b-PAA30 for pH sensing
ED
To further assess its utility as a pH fluorescent sensor, its emission spectral response to pH 7.4 in the presence of biological species including biological molecules, metal ions,
PT
and anions (figures 4a, 5a and 5b) was also tested. The emission intensity at 507 nm (I507)
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exhibits a negligible change in the presence of these species. All these results suggest that 1-PS35-b-PAA30 might be a suitable candidate for intracellular pH imaging. 3.5. Photostability study of sensor 1-PS35-b-PAA30
A
An ideal sensor ought to have good photostability. As shown in Fig. 4b, with
irradiation time extending, the emission intensity at 507 nm of 1-PS35-b-PAA30 in HEPES solutions hardly changes, indicating excellent photostability.
12
3.6. Application of 1-PS35-b-PAA30 in cellular imaging The hybrid nanosensor 1-PS35-b-PAA30 was further studied for its pHi imagining ability. In this study, the intracellular pH calibration was carried out in live HeLa cells using a standard procedure followed by a further incubation with high K+ buffers of different pH,
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also containing 1 mM nigericin. As shown in the first and second rows of Fig. 6, upon excitation at 405 nm laser, the emission intensities at green channel with 525/50 filter
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(figures 6a, 6b, 6c, 6d) gradually increase with pH changing from 5.0 to 8.0. The result obtained from fluorescence imaging of pHi in live HeLa cells was quite different from
U
those in solution. To further study such difference, the TEM images of 1-PS35-b-PAA30
N
(Fig. 7) revealed that the protonation of the hybrid nanosensor resulted in the block
A
network formation among the amphiphilic block polymers in acidic solution (Fig. 7a and
M
Fig. 7b). Poor dispersity of nanosensor in live cells results in the weak fluorescence in
ED
acidic condition (figures 6a and 6b). Upon pH increasing, the weakened protonation makes good dispersity of the hybrid nanosensor (Fig. 7c and Fig. 7d), which results in the
PT
strong fluorescence in neutral and basic intracellular condition (figures 6c and 6d). Such
A
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results mean that this nanosensor can be as an extracellular pH-sensor.
3. Conclusion In summary, a hydrophilic fluorescent hybrid nanosensor (1-PS35-b-PAA30) was fabricated via self-assembly between a 1,8-naphthalic anhydride-based organic molecular fluorescent pH-sensor and an amphiphilic diblock copolymer PS35-b-PAA30. The
13
nanosensor 1-PS35-b-PAA30 exhibits excellent photo-stability, good anti-disturbance ability, and a linear fluorescent response from pH 5.2 to 7.4. Moreover, TEM experiments and fluorescence imaging results indicated that the sensor permeated into cells and could not be observed because of aggregation in acidic condition. Acknowledgements
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We are grateful for the financial supports from National Natural Science Foundation of China (U1704161, U1504203, 21601158), and Zhengzhou University (32210431).
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Actuators B: Chem. 212 (2015) 364–370.
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naked-eye detection with a 1, 8-naphthalimide-based colorimetric probe, Sens.
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detection of acidic pH in lysosome with carbon nanodots, Chin. Chem. Lett. 28 (2017) 1969–1974.
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[59] L. Yin, C. He, C. Huang, W. Zhu, X. Wang, Y. Xu, X. Qian, A dual pH and
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temperature responsive polymeric fluorescent sensor and its imaging application in living cells, Chem. Commun. 48(2012) 4486–4488.
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Biographies Huanhuan Song received her B.S. degree in 2016. She is currently a M.S. candidate at
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College of Chemistry and Molecular Engineering, Zhengzhou University. Her research interests focus on developing fluorescent chemosensors. Weiwei Du received her B.S. degree in 2014. She is currently a M.S. candidate at College of Chemistry and Molecular Engineering, Zhengzhou University. Her research interests
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focus on developing fluorescent chemosensors. Chunxia Liu received her B.S. degree in 2012. She is currently a M.S. candidate at
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College of Chemistry and Molecular Engineering, Zhengzhou University. Her research interests focus on developing fluorescent chemosensors.
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Zhanxian Li received her Ph.D. degree in 2006 from Chinese Academy of Sciences. She
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is an associate professor of College of Chemistry and Molecular Engineering, Zhengzhou
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University. Her current research interests are mainly in fluorescent chemosensors and
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molecular recognition.
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Hongyan Zhang received her Ph.D. in 2005 from Changchun Institute of Optics and Fine Mechanics and Physics, Chinese Academy of Sciences. She joined Technical Institute of
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Physics and Chemistry, Chinese Academy of Sciences in 2007. Her main research
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interests include design and develop optical detection system for bimolecular and sensor molecules.
Liuhe Wei is a professor of College of Chemistry and Molecular Engineering, Zhengzhou
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University. Her current research interests focus on functional polymers and supermolecular chemistry. Mingming Yu is an associate professor of College of Chemistry and Molecular Engineering, Zhengzhou University. His current research interests are mainly in
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fluorescent chemosensors and functional polymers.
Figure Captions Fig. 1. Emission spectra of 1-PS35-b-PAA30 with different doping content of
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compound 1 1% (a), 2% (b), 4 % (c) in different pH condition with excitation at 400 nm.
(d) FT-IR spectra of PS35-b-PAA30 (black line of Fig. 1d), compound 1 (green line of Fig.
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1d), and 1-PS35-b-PAA30 (red line of Fig. 1d).
Fig. 2. TEM images of PS-b-PAA (scale bar is 0.5 μm (a), scale bar is 200 nm (b)),
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and TEM images of 1-PS35-b-PAA30 (scale bar is 0.5 μm (c), scale bar is 100 nm (d)).
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Fig. 3. Emission spectra of 1-PS35-b-PAA30 (2 × 10−3 mg/mL of compound 1, 9.8 ×
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10−2 mg/mL of PS35-b-PAA30) in DMSO−HEPES buffer solutions (1:9, v/v) of different
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pHapp values under excitation at 400 nm (a). The pH titration curve was plotted by the
wavelength was 400 nm.
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emission spectra of 1-PS35-b-PAA30 as linear function of pHapp (b). The excitation
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Fig. 4. Emission spectra response of 1-PS35-b-PAA30 (2 × 10−3 mg/mL of compound 1, 9.8 × 10−2 mg/mL of PS35-b-PAA30, VDMSO:VHEPES=1:9, pH=7.4) upon addition of
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different metal ions (9.5×10–5 mol/L) (a), the excitation wavelength was 400 nm. I507 represents the emission intensity at 507 nm. Emission intensity at 507 nm of
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1-PS35-b-PAA30 (0.1 mg/mL, VDMSO:VHEPES=1:9, pH=7.4) with irradiation (λex = 470 nm) time change (b). Fig. 5. Emission spectral response of 1-PS35-b-PAA30 (2 × 10−3 mg/mL of compound 1, 9.8 × 10−2 mg/mL of PS35-b-PAA30, VDMSO:VHEPES=1:9, pH=7.4) upon addition of different anions (a) and biological molecules (b) (9.5×10–5 mol/L), the excitation
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wavelength was 400 nm. I507 represents the emission intensity at 507 nm. The anions used were ClO4–, HCO3–, CO32–, SO42–, NO3–, C2O42–, HSO3–, HSO4–, S2O32–, SH–, I–, N3–, PO43–, P2O74–, H2PO4–, SO32–, HPO42–, SiO32–, ClO3–, CH3COO–, F–, Br–, and Cl–. The biological molecules used were Gly, lle, Ala, Cys, GSH, Hcy, Arg, Met, Phe, Thr, Gln, Lys, Asn, Val, Trp, Ser, Pro, Leu, and Tyr.
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Fig. 6. Confocal microscopy images of HeLa cells incubated with 1-PS35-b-PAA30 (2
× 10−3 mg/mL of compound 1, 9.8 × 10−2 mg/mL of PS35-b-PAA30) at different pH (pH=5,
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a,e), (pH=6, b, f), (pH=7, c, g), and (pH=8, d, h). The excitation wavelength was 405 nm. The images were collected at green channel with 525/50 filter (a, b, c, d).
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Fig. 7. TEM images of 1-PS35-b-PAA30 in different pH conditions.
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Scheme 2. Synthesis route of PS-b-PAA.
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Scheme 1. Synthesis route and sensing mechanism of compound 1.
Fig. 1. Emission spectra of 1-PS35-b-PAA30 with different doping content of compound 1 1% (a), 2% (b), 4 % (c) in different pH condition with excitation at 400 nm. (d) FT-IR spectra of PS35-b-PAA30 (black line of Fig. 1d), compound 1 (green line of Fig. 1d), and 1-PS35-b-PAA30 (red line of Fig. 1d).
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Fig. 2. TEM images of PS-b-PAA (scale bar is 0.5 μm (a), scale bar is 200 nm (b)), and
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TEM images of 1-PS35-b-PAA30 (scale bar is 0.5 μm (c), scale bar is 100 nm (d)).
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Fig. 3. Emission spectra of 1-PS35-b-PAA30 (2 × 10−3 mg/mL of compound 1, 9.8 × 10−2
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mg/mL of PS35-b-PAA30) in DMSO−HEPES buffer solutions (1:9, v/v) of different pHapp values under excitation at 400 nm (a). The pH titration curve was plotted by the emission
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spectra of 1-PS35-b-PAA30 as linear function of pHapp (b). The excitation wavelength was
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400 nm.
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Fig. 4. Emission spectra response of 1-PS35-b-PAA30 (2 × 10−3 mg/mL of compound 1,
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9.8 × 10−2 mg/mL of PS35-b-PAA30, VDMSO:VHEPES=1:9, pH=7.4) upon addition of different metal ions (9.5×10–5 mol/L) (a), the excitation wavelength was 400 nm. I507
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represents the emission intensity at 507 nm. Emission intensity at 507 nm of 1-PS35-b-PAA30 (0.1 mg/mL, VDMSO:VHEPES=1:9, pH=7.4) with irradiation (λex = 470 nm)
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time change (b).
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Fig. 5. Emission spectral response of 1-PS35-b-PAA30 (2 × 10−3 mg/mL of compound 1, 9.8 × 10−2 mg/mL of PS35-b-PAA30, VDMSO:VHEPES=1:9, pH=7.4) upon addition of
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different anions (a) and biological molecules (b) (9.5×10–5 mol/L), the excitation wavelength was 400 nm. I507 represents the emission intensity at 507 nm. The anions used were ClO4–, HCO3–, CO32–, SO42–, NO3–, C2O42–, HSO3–, HSO4–, S2O32–, SH–, I–, N3–, PO43–, P2O74–, H2PO4–, SO32–, HPO42–, SiO32–, ClO3–, CH3COO–, F–, Br–, and Cl–. The biological molecules used were Gly, lle, Ala, Cys, GSH, Hcy, Arg, Met, Phe, Thr, Gln,
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Lys, Asn, Val, Trp, Ser, Pro, Leu, and Tyr.
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Fig. 6. Confocal microscopy images of HeLa cells incubated with 1-PS35-b-PAA30 (2 × 10−3 mg/mL of compound 1, 9.8 × 10−2 mg/mL of PS35-b-PAA30) at different pH (pH=5, a,e), (pH=6, b, f), (pH=7, c, g), and (pH=8, d, h). The excitation wavelength was 405 nm. The images were collected at green channel with 525/50 filter (a, b, c, d).
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Fig. 7. TEM images of 1-PS35-b-PAA30 in different pH condition.
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Scheme 1. Synthesis route and sensing mechanism of compound 1.
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Scheme 2. Synthesis route of PS-b-PAA.
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