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Preparation of gold-carbon dots and ratiometric fluorescence cellular imaging Lingyang Zhang, Donghui Wang, Haowen Huang, Lanfang Liu, Yuan Zhou, Xiaodong Xia, Keqin Deng, and Xuanyong Liu ACS Appl. Mater. Interfaces, Just Accepted Manuscript • DOI: 10.1021/acsami.5b12084 • Publication Date (Web): 23 Feb 2016 Downloaded from http://pubs.acs.org on February 24, 2016
Just Accepted “Just Accepted” manuscripts have been peer-reviewed and accepted for publication. They are posted online prior to technical editing, formatting for publication and author proofing. The American Chemical Society provides “Just Accepted” as a free service to the research community to expedite the dissemination of scientific material as soon as possible after acceptance. “Just Accepted” manuscripts appear in full in PDF format accompanied by an HTML abstract. “Just Accepted” manuscripts have been fully peer reviewed, but should not be considered the official version of record. They are accessible to all readers and citable by the Digital Object Identifier (DOI®). “Just Accepted” is an optional service offered to authors. Therefore, the “Just Accepted” Web site may not include all articles that will be published in the journal. After a manuscript is technically edited and formatted, it will be removed from the “Just Accepted” Web site and published as an ASAP article. Note that technical editing may introduce minor changes to the manuscript text and/or graphics which could affect content, and all legal disclaimers and ethical guidelines that apply to the journal pertain. ACS cannot be held responsible for errors or consequences arising from the use of information contained in these “Just Accepted” manuscripts.
ACS Applied Materials & Interfaces is published by the American Chemical Society. 1155 Sixteenth Street N.W., Washington, DC 20036 Published by American Chemical Society. Copyright © American Chemical Society. However, no copyright claim is made to original U.S. Government works, or works produced by employees of any Commonwealth realm Crown government in the course of their duties.
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ACS Applied Materials & Interfaces
Preparation of gold-carbon dots and ratiometric fluorescence cellular imaging Lingyang Zhang, † Donghui Wang,
‡
Haowen Huang,* † Lanfang Liu, † Yuan
Zhou,† Xiaodong Xia,† Keqin Deng,† Xuanyong Liu*‡
†
Key Laboratory of Theoretical Organic Chemistry and Function Molecule, Ministry
of Education, Hunan Provincial University Key Laboratory of QSAR/QSPR, School of Chemistry and Chemical Engineering, Hunan University of Science and Technology, Xiangtan, China. ‡
State Key Laboratory of High Performance Ceramics and Superfine Microstructure,
Shanghai Institute of Ceramics, Chinese Academy of Sciences, Shanghai 200050, China
Corresponding authors Haowen Huang: [email protected], Tel:+86-731-58290045, Fax: +86-731-58290509 Xuanyong Liu:
[email protected], Tel:+86-21-52412409, Fax: +86-21-52412409
1
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ACS Applied Materials & Interfaces
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ABSTRACT
In this study, we synthesized novel gold-carbon dots (GCDs) with unique properties by microwave-assisted method. The characterization of high-resolution transmission electron microscope (HRTEM), XRD, high-angle annular dark field scanning transmission
electron
microscope
(HAADF-STEM),
and
energy
dispersive
spectrometer demonstrates the GCDs are composed of carbon and Au. Tiny Au clusters are dispersed in 2-nm-size carbon skeleton, which integrate the properties of typical CDs and gold nanoclusters (AuNCs), displaying fascinating peroxidase-like activity and single excitation/dual emission. Dual emission of the GCDs exhibits different fluorescent response to the target species enables the GCDs to be exploited for sensing and bioimaging. The highly photostable and biocompatible GCDs were applied to dual fluorescent imaging for breast cancer cells and normal rat osteoblast cells under a single excitation. Moreover, ratiometric fluorescence imaging was used to monitor Fe3+ level in normal rat osteoblast cells.
Keywords: gold-carbon dots, carbon dots, Au nanoclusters, dual fluorescent imaging, sensor
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ACS Applied Materials & Interfaces
INTRODUCTION Multiplex fluorescence determination allows for the detection of multiple emissions in a single fluorescent matter, which increase the detection specificity, and enable more quantitative measurements.1-5 Fluorescent ratio imaging is an effective approach for the quantitative detection of biologically active molecules in cells.6,7 However, the existing multiplex fluorescent probes are mostly based on two-wavelength excitation, which require more complicated instrumental settings. To accurately measure the levels of intracellular active molecules varying continuously with time, the development of fluorescent ratio imaging with a single excitation wavelength has attracted interest.8-11 New strategies for bioanalysis and bioimaging techniques have been recently developed based on the integration of highly specific organic molecules or biomolecules into inorganic nanomaterials of unique electronic and optical characteristics,
such
as
gold
nanoparticles,12,13
carbon
nanotubes,14,15
or
semiconductor nanomaterials.16,17 Nevertheless, seeking single excitation/dual maximum emission of the inorganic nanoparticles which possesses water-soluble, biocompatible, photostable and multicolor properties remains a big challenge. Carbon-based materials like carbon dots (CDs) exhibit unique properties, such as photostability, low cytotoxicity and high biocompatibility.18-20 As an alternative to traditional semiconducting quantum dots usually containing heavy metal, the fascinating properties have enabled CDs to be a great tool for biological labeling.21-23 Although the excitation-dependent photoluminescence (PL) of CDs can provide various fluorescence emissions at different excited wavelengths, it seems difficult to 3
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directly obtain single excitation/dual maximum emission from the CDs. Therefore, various approaches have been designed to synthesize CD-based nanoparticles allowing development of multifunctional materials with dual fluorescence.24-27 Most of the composite nanoparticles are hybrid from different particles, which may lead to the change of their intrinsic properties. In addition, the preparation of these composites was a bit of troublesome. Accordingly, it is necessary to develop a simple method to prepare multifunctional nanoparticles. Herein, we design and prepared innovative fluorescent gold-CDs (GCDs) possessing two fluorophores with identical excitation wavelengths, which can be facilely synthesized by microwave-assisted method. Tiny Au clusters are dispersed in the fluorescent 2-nm-size GCDs, which effectively integrate the properties of typical CDs and gold nanoclusters (AuNCs). Compared with the organic fluorophores, dual-emission GCDs do not need elaborate molecular design and complicated synthesis. The highly photostable and biocompatible GCDs was applied to dual fluorescent imaging for breast cancer cells and normal rat osteoblast cells. Moreover, ratiometric fluorescence imaging was used to monitor Fe3+ level in cells.
EXPERIMENT SECTION Synthesis
of
CDs
microwave-assisted method.
and 28
AuNCs.
The
CDs
were
synthesized
by
Freshly prepared aqueous solution of glucose was
heated in a domestic microwave for 20 min. The obtained product was centrifuged and then the resultant supernate containing luminescent CDs was dialyzed with a 4
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ACS Applied Materials & Interfaces
dialysis tube (molecular weight 1000) against doubly distilled water for 24 h, and finally the product was obtained. Meanwhile, fluorescent gold nanoclusters (AuNCs) were prepared by HAuCl4 and glutathione (GSH) using microwave-assisted method for 20 min. 29 Synthesis of GCDs. The GCDs were synthesized based on the modification of preparing CDs and AuNCs. Typically, 5 mL of 20 mM freshly prepared HAuCl4 aqueous solution and 1.5 mL of 100 mM GSH were mixed, and then the mixture solution was diluted by distilled water up to 50 mL in a round bottomed flask at room temperature. The mixture was heated to 80 ℃ under gentle stirring for 2 h. Then the mixture was transferred into a conical flask and dissolved 2 g glucose. The resulting solution was heated in a domestic microwave for 20 min. The obtained product was dissolved again and centrifuged (16,000 ×g, 30 min). After that, the resultant supernatant containing luminescent GCDs were dialyzed with a dialysis tube (molecular weight 8000~14000) against doubly distilled water for 24 h, and finally the product was obtained. Terephthalic Acid Photoluminescence Probing Assay (TA−PL) To Detect the HO·. Terephthalic acid (5 mM) was prepared by adding THA into phosphate buffered saline (PBS) containing 10 mM NaOH at pH 7.4. After adding a suitable amount of catalyst into the buffer, the mixture was irradiated by visible light or no light. Product fluorescence (THA−OH) was measured by a RF-5301PC (Shimadzu, Japan) fluorescence spectrometer) with 350 nm excitation and 429 nm emission. Cell Culture. The cancer cell line human breast cancer cells (MCF-7) and 5
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normal cell line rat osteoblasts (MC3T3-E1) were obtained from Cell Bank of Chinese Academy of Science, and cultured in the RPMI-1640 medium (Gibco, Life Technologies) supplemented with 10% fetal calf serum (FCS) (Exell Biology, Inc, South America), and 1% antimicrobial of penicillin and streptomycin (HyClone Laboratories, Inc, Utah, USA). The cells were cultured in 75 cm2 flasks (Thermo Fisher Scientific, USA) at 37 ℃ in humidified atmosphere with 5% CO2. Based on the cell condition, cells were passaged at ratio of 1:2-1:3 every 2-4 days. Assessment of Cellular Viability. The cell viability was determined by the alarmarBlue assay (AbD Serotec Ltd, UK). Briefly 5 ×103 cells in 100 µL medium were seeded in each well of a 96-multiwell plate (Thermo Fisher Scientific, USA) for overnight growth. Then the culture medium was removed and cells were treated with 100 µL of a series of GCDs suspensions with final concentrations of 0, 3, 10, 31, 93, 278, 833, 2500 µg/mL respectively. After culturing 24 h, the suspensions were removed and the cells were rinsed with PBS for three times. Then 100 µL fresh medium containing 10% alarmarBlue was added into the 96-well plate. After incubation for 4 h, the 96-well plate was taken out and measured by an enzyme labeling instrument at excitation wavelengths of 570 nm and 600 nm. The cell viability was calculated according to the instruction of the alamarBlue assay, and expressed as the reduction ratio of alarmarBlue. Cell Imaging. Cells were seeded onto the carry sheet glass with a density of 5 × 104 cell/well for overnight growth. Then, the culture medium was replaced by 1 mL fresh medium containing 833 µg GCDs. After incubation for 10 hours, the carry sheet 6
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glass was rinsed by PBS for three times, placed into a flask containing 1 mL PBS, and observed under CLSM (Ex = 405 nm, Em = 470 ± 30 nm and 570 ± 30 nm). Then ferric chloride solution (30 µM) was added into the observed sample. After 30 minutes’ standing, another CLSM image was taken in the same area without changing any condition. The taken pictures were analyzed by Image Pro-Plus 6 software package. Data Analysis. GraphPad Prism 5 statistical software package was used to perform statistical analysis. All of the data were expressed as means ± standard deviation (SD). Statistically significant differences (P) between different groups were got by one-way analysis of variance and Turkey’s multiple comparison tests. P
Article
Preparation of gold-carbon dots and ratiometric fluorescence cellular imaging Lingyang Zhang, Donghui Wang, Haowen Huang, Lanfang Liu, Yuan Zhou, Xiaodong Xia, Keqin Deng, and Xuanyong Liu ACS Appl. Mater. Interfaces, Just Accepted Manuscript • DOI: 10.1021/acsami.5b12084 • Publication Date (Web): 23 Feb 2016 Downloaded from http://pubs.acs.org on February 24, 2016
Just Accepted “Just Accepted” manuscripts have been peer-reviewed and accepted for publication. They are posted online prior to technical editing, formatting for publication and author proofing. The American Chemical Society provides “Just Accepted” as a free service to the research community to expedite the dissemination of scientific material as soon as possible after acceptance. “Just Accepted” manuscripts appear in full in PDF format accompanied by an HTML abstract. “Just Accepted” manuscripts have been fully peer reviewed, but should not be considered the official version of record. They are accessible to all readers and citable by the Digital Object Identifier (DOI®). “Just Accepted” is an optional service offered to authors. Therefore, the “Just Accepted” Web site may not include all articles that will be published in the journal. After a manuscript is technically edited and formatted, it will be removed from the “Just Accepted” Web site and published as an ASAP article. Note that technical editing may introduce minor changes to the manuscript text and/or graphics which could affect content, and all legal disclaimers and ethical guidelines that apply to the journal pertain. ACS cannot be held responsible for errors or consequences arising from the use of information contained in these “Just Accepted” manuscripts.
ACS Applied Materials & Interfaces is published by the American Chemical Society. 1155 Sixteenth Street N.W., Washington, DC 20036 Published by American Chemical Society. Copyright © American Chemical Society. However, no copyright claim is made to original U.S. Government works, or works produced by employees of any Commonwealth realm Crown government in the course of their duties.
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ACS Applied Materials & Interfaces
Preparation of gold-carbon dots and ratiometric fluorescence cellular imaging Lingyang Zhang, † Donghui Wang,
‡
Haowen Huang,* † Lanfang Liu, † Yuan
Zhou,† Xiaodong Xia,† Keqin Deng,† Xuanyong Liu*‡
†
Key Laboratory of Theoretical Organic Chemistry and Function Molecule, Ministry
of Education, Hunan Provincial University Key Laboratory of QSAR/QSPR, School of Chemistry and Chemical Engineering, Hunan University of Science and Technology, Xiangtan, China. ‡
State Key Laboratory of High Performance Ceramics and Superfine Microstructure,
Shanghai Institute of Ceramics, Chinese Academy of Sciences, Shanghai 200050, China
Corresponding authors Haowen Huang: [email protected], Tel:+86-731-58290045, Fax: +86-731-58290509 Xuanyong Liu:
[email protected], Tel:+86-21-52412409, Fax: +86-21-52412409
1
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ACS Applied Materials & Interfaces
1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60
Page 2 of 31
ABSTRACT
In this study, we synthesized novel gold-carbon dots (GCDs) with unique properties by microwave-assisted method. The characterization of high-resolution transmission electron microscope (HRTEM), XRD, high-angle annular dark field scanning transmission
electron
microscope
(HAADF-STEM),
and
energy
dispersive
spectrometer demonstrates the GCDs are composed of carbon and Au. Tiny Au clusters are dispersed in 2-nm-size carbon skeleton, which integrate the properties of typical CDs and gold nanoclusters (AuNCs), displaying fascinating peroxidase-like activity and single excitation/dual emission. Dual emission of the GCDs exhibits different fluorescent response to the target species enables the GCDs to be exploited for sensing and bioimaging. The highly photostable and biocompatible GCDs were applied to dual fluorescent imaging for breast cancer cells and normal rat osteoblast cells under a single excitation. Moreover, ratiometric fluorescence imaging was used to monitor Fe3+ level in normal rat osteoblast cells.
Keywords: gold-carbon dots, carbon dots, Au nanoclusters, dual fluorescent imaging, sensor
2
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ACS Applied Materials & Interfaces
INTRODUCTION Multiplex fluorescence determination allows for the detection of multiple emissions in a single fluorescent matter, which increase the detection specificity, and enable more quantitative measurements.1-5 Fluorescent ratio imaging is an effective approach for the quantitative detection of biologically active molecules in cells.6,7 However, the existing multiplex fluorescent probes are mostly based on two-wavelength excitation, which require more complicated instrumental settings. To accurately measure the levels of intracellular active molecules varying continuously with time, the development of fluorescent ratio imaging with a single excitation wavelength has attracted interest.8-11 New strategies for bioanalysis and bioimaging techniques have been recently developed based on the integration of highly specific organic molecules or biomolecules into inorganic nanomaterials of unique electronic and optical characteristics,
such
as
gold
nanoparticles,12,13
carbon
nanotubes,14,15
or
semiconductor nanomaterials.16,17 Nevertheless, seeking single excitation/dual maximum emission of the inorganic nanoparticles which possesses water-soluble, biocompatible, photostable and multicolor properties remains a big challenge. Carbon-based materials like carbon dots (CDs) exhibit unique properties, such as photostability, low cytotoxicity and high biocompatibility.18-20 As an alternative to traditional semiconducting quantum dots usually containing heavy metal, the fascinating properties have enabled CDs to be a great tool for biological labeling.21-23 Although the excitation-dependent photoluminescence (PL) of CDs can provide various fluorescence emissions at different excited wavelengths, it seems difficult to 3
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1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60
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directly obtain single excitation/dual maximum emission from the CDs. Therefore, various approaches have been designed to synthesize CD-based nanoparticles allowing development of multifunctional materials with dual fluorescence.24-27 Most of the composite nanoparticles are hybrid from different particles, which may lead to the change of their intrinsic properties. In addition, the preparation of these composites was a bit of troublesome. Accordingly, it is necessary to develop a simple method to prepare multifunctional nanoparticles. Herein, we design and prepared innovative fluorescent gold-CDs (GCDs) possessing two fluorophores with identical excitation wavelengths, which can be facilely synthesized by microwave-assisted method. Tiny Au clusters are dispersed in the fluorescent 2-nm-size GCDs, which effectively integrate the properties of typical CDs and gold nanoclusters (AuNCs). Compared with the organic fluorophores, dual-emission GCDs do not need elaborate molecular design and complicated synthesis. The highly photostable and biocompatible GCDs was applied to dual fluorescent imaging for breast cancer cells and normal rat osteoblast cells. Moreover, ratiometric fluorescence imaging was used to monitor Fe3+ level in cells.
EXPERIMENT SECTION Synthesis
of
CDs
microwave-assisted method.
and 28
AuNCs.
The
CDs
were
synthesized
by
Freshly prepared aqueous solution of glucose was
heated in a domestic microwave for 20 min. The obtained product was centrifuged and then the resultant supernate containing luminescent CDs was dialyzed with a 4
ACS Paragon Plus Environment
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ACS Applied Materials & Interfaces
dialysis tube (molecular weight 1000) against doubly distilled water for 24 h, and finally the product was obtained. Meanwhile, fluorescent gold nanoclusters (AuNCs) were prepared by HAuCl4 and glutathione (GSH) using microwave-assisted method for 20 min. 29 Synthesis of GCDs. The GCDs were synthesized based on the modification of preparing CDs and AuNCs. Typically, 5 mL of 20 mM freshly prepared HAuCl4 aqueous solution and 1.5 mL of 100 mM GSH were mixed, and then the mixture solution was diluted by distilled water up to 50 mL in a round bottomed flask at room temperature. The mixture was heated to 80 ℃ under gentle stirring for 2 h. Then the mixture was transferred into a conical flask and dissolved 2 g glucose. The resulting solution was heated in a domestic microwave for 20 min. The obtained product was dissolved again and centrifuged (16,000 ×g, 30 min). After that, the resultant supernatant containing luminescent GCDs were dialyzed with a dialysis tube (molecular weight 8000~14000) against doubly distilled water for 24 h, and finally the product was obtained. Terephthalic Acid Photoluminescence Probing Assay (TA−PL) To Detect the HO·. Terephthalic acid (5 mM) was prepared by adding THA into phosphate buffered saline (PBS) containing 10 mM NaOH at pH 7.4. After adding a suitable amount of catalyst into the buffer, the mixture was irradiated by visible light or no light. Product fluorescence (THA−OH) was measured by a RF-5301PC (Shimadzu, Japan) fluorescence spectrometer) with 350 nm excitation and 429 nm emission. Cell Culture. The cancer cell line human breast cancer cells (MCF-7) and 5
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1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60
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normal cell line rat osteoblasts (MC3T3-E1) were obtained from Cell Bank of Chinese Academy of Science, and cultured in the RPMI-1640 medium (Gibco, Life Technologies) supplemented with 10% fetal calf serum (FCS) (Exell Biology, Inc, South America), and 1% antimicrobial of penicillin and streptomycin (HyClone Laboratories, Inc, Utah, USA). The cells were cultured in 75 cm2 flasks (Thermo Fisher Scientific, USA) at 37 ℃ in humidified atmosphere with 5% CO2. Based on the cell condition, cells were passaged at ratio of 1:2-1:3 every 2-4 days. Assessment of Cellular Viability. The cell viability was determined by the alarmarBlue assay (AbD Serotec Ltd, UK). Briefly 5 ×103 cells in 100 µL medium were seeded in each well of a 96-multiwell plate (Thermo Fisher Scientific, USA) for overnight growth. Then the culture medium was removed and cells were treated with 100 µL of a series of GCDs suspensions with final concentrations of 0, 3, 10, 31, 93, 278, 833, 2500 µg/mL respectively. After culturing 24 h, the suspensions were removed and the cells were rinsed with PBS for three times. Then 100 µL fresh medium containing 10% alarmarBlue was added into the 96-well plate. After incubation for 4 h, the 96-well plate was taken out and measured by an enzyme labeling instrument at excitation wavelengths of 570 nm and 600 nm. The cell viability was calculated according to the instruction of the alamarBlue assay, and expressed as the reduction ratio of alarmarBlue. Cell Imaging. Cells were seeded onto the carry sheet glass with a density of 5 × 104 cell/well for overnight growth. Then, the culture medium was replaced by 1 mL fresh medium containing 833 µg GCDs. After incubation for 10 hours, the carry sheet 6
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glass was rinsed by PBS for three times, placed into a flask containing 1 mL PBS, and observed under CLSM (Ex = 405 nm, Em = 470 ± 30 nm and 570 ± 30 nm). Then ferric chloride solution (30 µM) was added into the observed sample. After 30 minutes’ standing, another CLSM image was taken in the same area without changing any condition. The taken pictures were analyzed by Image Pro-Plus 6 software package. Data Analysis. GraphPad Prism 5 statistical software package was used to perform statistical analysis. All of the data were expressed as means ± standard deviation (SD). Statistically significant differences (P) between different groups were got by one-way analysis of variance and Turkey’s multiple comparison tests. P
