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Jun 23, 2016 - acid and basic fuchsin, the carbon dots showing dual emission bands at 475 and 545 nm under single-wavelength excitation were synthesiz...
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Label-Free Carbon-Dots-Based Ratiometric Fluorescence pH Nanoprobes for Intracellular pH Sensing Jingfang Shangguan, Dinggeng He, Xiaoxiao He,* Kemin Wang,* Fengzhou Xu, Jinquan Liu, Jinlu Tang, Xue Yang, and Jin Huang State Key Laboratory of Chemo/Biosensing and Chemometrics, College of Biology, College of Chemistry and Chemical Engineering, Hunan University, Key Laboratory for Bio-Nanotechnology and Molecule Engineering of Hunan Province, Changsha 410082, China S Supporting Information *

ABSTRACT: Measuring pH in living cells is of great importance for better understanding cellular functions as well as providing pivotal assistance for early diagnosis of diseases. In this work, we report the first use of a novel kind of label-free carbon dots for intracellular ratiometric fluorescence pH sensing. By simple one-pot hydrothermal treatment of citric acid and basic fuchsin, the carbon dots showing dual emission bands at 475 and 545 nm under single-wavelength excitation were synthesized. It is demonstrated that the fluorescence intensities of the as-synthesized carbon dots at the two emissions are pH-sensitive simultaneously. The intensity ratio (I475 nm/ I545 nm) is linear against pH values from 5.2 to 8.8 in buffer solution, affording the capability as ratiometric probes for intracellular pH sensing. It also displays that the carbon dots show excellent reversibility and photostability in pH measurements. With this nanoprobe, quantitative fluorescence imaging using the ratio of two emissions (I475 nm/I545 nm) for the detection of intracellular pH were successfully applied in HeLa cells. In contrast to most of the reported nanomaterials-based ratiometric pH sensors which rely on the attachment of additional dyes, these carbon-dots-based ratiometric probes are low in toxicity, easy to synthesize, and free from labels.

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instead of measuring single emission intensity changes. Using the ratio of two different fluorescent emission intensities to sense the pH-induced fluctuation, the ratiometric measurements can offer self-calibration for environmental interference and effectively overcome the limitations in the single emission intensity measurements. To date, a great number of ratiometric fluorescence pH sensors have been developed by various groups.13−17 Up to now, the strategies for constructing of ratiometric fluorescence pH sensors can be mainly divided three types according to the carrier of the pH-sensitive indicators, including organic small molecules, fluorescent proteins, and nanomaterials. The organic small molecules strategy was based on the different pH-response fluorophores existing in the molecular structures, which resulted in ratiometric fluorescence emission during protonated and deprotonated procedure. By rational design of the pH-sensitive fluorophores, they can have a wide linear range of measurement pH values.13,14 With respect to the fluorescent proteins strategy, the fluorescent proteins were encoded genetically or combined with another fluorescent protein to achieve ratiometric fluorescence pH sensing, allowing for the specific targeting of intracellular organelles.15,16 However, there were several disadvantages that could not be ignored in the above

ntracellular pH plays vital roles in various physiological and pathological processes such as cell cycle1 and apoptosis,2 receptor-mediated signal transduction,3 ion transport,4 muscle contraction,5 inflammation,6 and tumor growth.7 It modulates the structure and function of all biologically active macromolecules. Measurement of pH distribution and variation in living cells has great significance in better understanding cellular functions as well as providing pivotal assistance for early diagnosis of diseases.8,9 For this reason, various methods and techniques for intracellular pH sensing have been developed. In particular, optical methods based on pH-induced fluctuation in fluorescence intensity are attracting attention in pH measurements because of the advantages of excellent spatial and temporal resolution, rapid response, high signal-to-noise ratio, noninvasiveness, and good sensitivity. Many optical probes such as organic dyes and fluorescent proteins have been widely used to monitor local pH values inside cells by measuring the increase or decrease in fluorescence intensity.10−12 However, the single emission intensity change based on intracellular pH sensing might be influenced by many factors, such as the stability of the light source, the concentration of fluorescence probes, the bleaching of dyes, the binding of macromolecules, among others, which may result in degradation of the reliability of the measured results. With this in mind, much effort has been made to develop ratiometric pH measurements, which can permit simultaneous recording of the relative changes of two separated wavelengths © 2016 American Chemical Society

Received: May 18, 2016 Accepted: June 23, 2016 Published: June 23, 2016 7837

DOI: 10.1021/acs.analchem.6b01932 Anal. Chem. 2016, 88, 7837−7843

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carbon dots were carried out, and the proposed drawbacks mentioned above were still unavoidable. Hence, it is of great importance to explore carbon dots with intrinsic pH-sensitive ratiometric fluorescence emission and further construct carbondots-based ratiometric fluorescence nanoprobes for pH measurement in living cells. Toward this end, we develop a label-free carbon dots as ratiometric fluorescence pH nanoprobes and then apply them for intracellular pH sensing (Scheme 1). In this work, the

two strategies. For example, some organic small-molecule-based pH sensors, such as cyanine, may not an appropriate candidate for pH quantification because of their rapid photobleaching. The fluorescent-protein-based pH sensors needed a complicated extraction and purification steps, which could not meet the demand for rapid pH sensing. Apart from these strategies, nanomaterials-based ratiometric fluorescence pH sensors possessed great potential for measuring pH in living cells due to their good solubility, colloidal stability, and ease of cellular uptake. By using a central, nonfluorescent or fluorescent nanoparticle as matrixes, the ratiometric fluorescence signals could be obtained through covalent cross-linking or embedding dyes to the nanomaterials.17 So far, a number of ratiometric fluorescence pH probes, based on silica nanoparticle,18 semiconductor quantum dots,19 gold nanoparticle,20 fluorescence metal nanoclusters,21 and carbon nanomaterials22 have been built to quantify intracellular pH. Owning to the necessity of combining additional dyes to the nanoparticles, there are some factors that have to be considered in building nanomaterials-based ratiometric fluorescence pH sensors. First, these sensors usually utilize the dual excitation mode or fluorescence resonance energy transfer with single excitation, which lead to the limitations for dyes selection. Second, the leaching of the embedded dyes from the matrixes may influence the accuracy of the method. Finally, complex separation and purification steps are necessary in preparation process, resulting in timeconsuming and tedious procedures. These limitations may impede their applications in pH sensing. Therefore, it is important to develop a pH-sensitive nanomaterial which can be free from labeling and exhibit intrinsic pH-sensitive ratiometric fluorescence emission. Carbon dots, an emerging star in the nanocarbon family, have attracted tremendous attention due to their strong fluorescence, nonblinking property, excellent solubility, and nontoxicity. There has been ongoing interest in exploring carbon dots as a new nanocarrier in various applications, such as photocatalysis,23,24 sensors,25−28 bioimaging,29,30 and drug delivery.31,32 Among these works, one of the studied properties is pH-dependent features of emission for carbon dots. With different precursors, the pH-sensitive carbon dots have been prepared.33−35 Wang et al. developed a simple method to prepare pH-sensitive fluorescence carbon dots by hydrothermal treatment of threonine. With the increase of pH values, the fluorescence intensity of carbon dots were decreased.33 Kang et al. reported a green method to synthesize pH-sensitive fluorescent carbon dots by a one-step sodium hydroxideassisted electrochemical treatment of ethanol.34 However, the pH-induced fluorescence intensity change property in these reported carbon dots were the fluctuation of a single fluorescence emission, which may be subjected to many factors. To overcome these shortages, carbon-dots-based ratiometric fluorescence sensors for intracellular pH measuring have been investigated by some groups in recent years.36,37 For instance, Ma et al. developed a ratiometric pH sensor based on two-dyedecorated carbon dots. Combining different molar ratios of pHsensitive fluoresceinisothiocyanate (FITC) and pH-insensitive rhodamine B isothiocyanate (RBITC) with carbon dots, a tunable ratiometric pH sensor was established.36 Li et al. have built a ratiometric pH sensor using two emission wavelengths of FITC-labeled carbon dots as independent references to improve the reliability and accuracy for the measurement of intracellular pH.37 However, in these ratiometric pH-sensing systems, the cross attachment or embedding of dyes to the

Scheme 1. Schematic Diagram for the Preparation of LabelFree Carbon Dots and Their Application for Intracellular pH Sensing

carbon dots exhibiting dual emissions at 475 and 545 nm were obtained through one-pot hydrothermal treatment of citric acid and basic fuchsin, in which the citric acid is a commonly used carbon source and the basic fuchsin is a small molecule with pH-sensitive property. The fluorescence intensity of the asprepared carbon dots at the two emissions were pH-dependent simultaneously. Without the need of dyes labeling, the one-pot synthesized carbon dots displayed intrinsic ratiometric fluorescence emission (I475 nm/I545 nm) against pH variation and have a good linear relationship with a pH response range of 5.2−8.8 in buffer solution. Taking advantage of their unique emission properties, the carbon dots were applied as a kind of ratiometric fluorescence pH nanoprobe to measure the cellular pH and the stimulus-responsive changes of intracellular pH values in HeLa cells.

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EXPERIMENTAL SECTION Chemicals and Reagents. Citric acid monohydrate (98%), 3-(4,5-dimethyl-2-thiazoyl)-2,5-diphenyl tetrazolium bromide (MTT, 98%), chloroquine (CQ), dexamethasone (DEX), and nigericin sodium salt were purchased from Sigma-Aldrich (St. Louis, MO, U.S.A.). Basic fuchsin was obtained from Xiangzhong Reagent Company (Hunan, China). Dimethyl sulfoxide (DMSO) was obtained from Xilong Reagents Company (Guangdong, China). Quinine sulfate (98%, suitable for fluorescence) was purchased from Sangon Biotechnology Inc. (Shanghai, China). Nanopure water (18.2 MΩ, Millpore Co., U.S.A.) was used in all experiments. All chemicals used were of analytical grade without any further purification. Measurements. The high-resolution transmission electron microscopy (HRTEM) images were obtained on a Hitachi- F20 microscope and an acceleration voltage of 200 kV. Malvern Zetasizer Nano ZS was used to perform the mean diameter of resultant carbon dots. X-ray photoelectron spectra (XPS) measurement was performed on an ESCALAB 250Xi spectrometer using a monochromatic Al Kα excitation. The X-ray diffraction (XRD) analysis was carried out using a scintag XDS-2000 powder diffractometer with Cu Kα irradiation (λ = 0.154 nm). UV−vis absorption spectra were recorded on a DU 800 UV−vis spectrophotometer. All the fluorescence spectroscopy was obtained on a Hitachi F-7000 fluorescence 7838

DOI: 10.1021/acs.analchem.6b01932 Anal. Chem. 2016, 88, 7837−7843

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spectrophotometer. Fourier transform infrared spectroscopy (FT-IR) was conducted on a Tensor 27 FT-IR spectrometer, and the confocal laser scanning microscopy (CLSM) images were acquired on a Fluoview FV500. Preparation of Carbon Dots. Briefly, 4.8 mg of basic fuchsin and 3 g of citric acid monohydrate were dissolved in 30 mL of deionized water with a molar ratio of 1:1000, and then the mixture was transferred into a 50 mL Telfon equipped stainless steel autoclave and heated at 200 °C for 8 h. The resultant transparent yellow solution was filtered with a 0.22 μm filter membrane and further dialyzed against deionized water for 24 h to remove the excess reactants. The pH value of the carbon dots solution was adjusted to neutral and stored at 4 °C for further use. Simultaneously, several parallel experiments were also carried out through hydrothermal treatment of basic fuchsin and citric acid with different molar ratios of 0:1000, 0.5:1000, 5:1000, and 1000:0, respectively. The same preparation and purification processes were applied to the control groups. Cytotoxicity Assay. HeLa cells were cultured in medium of RPMI 1640 (1640, HyClone) containing 15% fetal bovine serum (FBS, heat inactivated) and 100 IU mL−1 penicillinstreptomycin at 37 °C in 5% CO2 humidified atmosphere. For the cytotoxicity assay, HeLa cells were seeded in a 96-well plate (about 7 × 103 cells per well) and incubated for 24 h. Then 200 μL of medium containing different concentrations of carbon dots were added to every well and incubated for another 24 h. Afterward, cells were incubated in medium containing 0.5 mg mL−1 of MTT for 4 h. Finally, the medium was replaced by 150 μL of dimethyl sulfoxide (DMSO) solution, and the absorbance of MTT at 492 nm was measured by an automatic ELISA analyzer (Benchmark plus, Biorad, Japan). Intracellular pH Calibration. HeLa cells were seeded in a 35 mm × 12 mm style cell culture dish and cultured for 24 h prior to treatment. Then, the cells were incubated with 1 mL of fresh medium containing 0.5 mg mL−1 carbon dots for 2 h at 37 °C. Prior to imaging, cells were washed with 10 mM PBS (137 mM NaCl, 2.7 mM KCl, 10 mM Na2HPO4, 2 mM K2HPO4, pH 7.4) for three times and treated with 1 mL of high-K+ HEPES-buffered solution (125 mM KCl, 20 mM NaCl, 0.5 mM CaCl2, 0.5 mM MgCl2, 5 mM glucose, and 20 mM HEPES) at various pH values (5.01, 5.64, 6.46, 7.21, and 8.01) in the presence of 5 μg mL−1 of nigericin. The cells were incubated for 10 min, and the intracellular ratiometric pH calibration experiment was then carried out on a laser confocal fluorescence microscope. The images were acquired by sequential line scanning model with excitation at 405 nm. The emission signals were collected from 465 to 495 nm of channel 1 and 535−565 nm of channel 2, respectively. All the images were obtained under constant settings. The image processing was conducted using software Image Proplus 6.0. Determination of pH in Living Cells. HeLa cells were seeded in the culture dish and grown overnight before use. For the drug-stimuli experiment, HeLa cells were cultured in the medium supplemented with 200 μM Chloroquine (CQ) for 40 min and 1 μM dexamethasone (DEX) for 6 h, respectively. After removal of the medium, the cells were washed with PBS buffer for three times and incubated with 0.5 mg mL−1 carbon dots in 1 mL of fresh culture medium for 2 h at 37 °C. Then, the cells were washed with 10 mM PBS to remove the remaining carbon dots and imaged on a laser-scanning confocal microscope. The image processing was conducted using software Image Proplus 6.0.

Article

RESULTS AND DISCUSSION

Synthesis of Carbon Dots. We synthesized the carbon dots by using different molar ratio of basic fuchsin and citric acid as reactants. The fluorescence emission spectra of the products with a molar ratio of 0:1000, 1:1000 and 1000:0, were investigated first. As shown in Figure S1A, the fluorescence of the product with a molar ratio of 0:1000 (see line a) exhibited a 450 nm emission under 380 nm excitation, which was attributed to the synthesized fluorescence carbon dots from citric acid. With hydrothermal treatment of the reactants with a molar ratio of 1000:0 (see line b), the fluorescence spectra showed two emission peaks at 530 and 590 nm from basic fuchsin, which were extremely weak so that the fluorescence intensity could be considered negligible. However, two emission peaks at 475 and 545 nm with high fluorescence intensity could be observed from the synthesized carbon dots using basic fuchsin and citric acid as coreaction precursors (see line c), indicating the fabrication of dual-emission carbon dots during the hydrothermal treatment of citric acid in the presence of basic fuchsin. In addition, the fluorescence spectra of the mixed solution containing two products with a molar ratio of 0:1000 and 1000:0 was also investigated. As shown in line d, only one emission peak centered at 455 nm was observed in the mixture solution, which has no difference with their own fluorescence spectra before mixing. The result demonstrated that the dual-emission carbon dots could only be fabricated using basic fuchsin and citric acid as the starting materials. To obtain the carbon dots with highest possible fluorescence intensity, the fluorescence spectra of different types of carbon dots were investigated by changing the molar ratio to 0.5:1000, 1:1000, and 5:1000 in the process of reaction (Figure S1B, a− c). The result revealed that the fluorescence intensity at 475 and 545 nm increased with the increase of the molar ratio from 0.5:1000 to 5:1000. It was also demonstrated that all three types of carbon dots prepared from different molar ratios displayed very similar fluorescence properties (Figure S1C−E). With a broad absorption under 500 nm in the UV−vis spectrum, they exhibited the same maximum excitation at 380 nm and the dual emission peak position at 475 and 545 nm. However, the carbon dots synthesized with molar ratio of 5:1000 has a tendency to aggregated after settled for 7 days (Figure S1F, insert), and the fluorescence intensity was slightly decreased (Figure S1F)). Therefore, we decided to use basic fuchsin and citric acid as reactants in a molar ratio of 1:1000 to synthesize carbon dots with high and stable fluorescence for further characterize. Characterization. The morphology, structure, and optical properties of the synthesized carbon dots with the molar ratio of 1:1000 were characterized. The size and dispersion of the carbon dots were characterized using high-resolution transmission electron microscopy (HRTEM), atomic force microscopy (AFM), and ZetaSizer Nano, respectively. As illustrated in Figure 1A,B, the carbon dots were mostly spherical in shape and well-dispersed in aqueous solution. The size distribution, as measured by dynamic light scattering (DLS) analysis, displayed a narrow size distribution in the range of 5−12 nm and exhibited a maximum population at 7.9 nm (Figure 1 (C)). For the structure measurement, the as-prepared carbon dots were measured by means of X-ray diffraction (XRD). The surface functional groups were identified by Fourier transform infrared spectroscopy (FT-IR) spectra. As shown in Figure 2A, two broad peaks in XRD spectra centered at 20° and 31° were 7839

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was also carried out to explore the elemental composition and chemical bonds of carbon dots. Figure 2C depicted the element identification of carbon dots analyzed by XPS, which illustrated that there are three elements existed, 54.82% of carbon, 44.84% of oxygen, and 0.34% of nitrogen. It is worthwhile to note that with very small amounts of basic fuchsin added in the reaction system, a nitrogen peak was not apparent in the XPS spectra until the high-resolution survey scan of N 1s was performed (Figure 2F). As shown in Figure 2D, the high-resolution C 1s spectra peaks at 284.3, 284.9, 285.7, 286.2, 288.2, and 289.2 eV demonstrate the existence of CC, C−C, C−N, C−O, CO, and O−CO bond, respectively. The high-resolution O 1s spectra (Figure 2E) peaks at 531.0 and 533.6 eV indicate the formation of C−O/C−O−C, whereas the 531.8 eV was attributed to oxygenated carbon atoms (CO, OC−O). The high-resolution N 1s spectra peaks at 400.2, 401.7 eV in Figure 2F showed that the nitrogen primarily existed in the form of C−N and N−H bonds. The XPS and FT-IR analysis have provided convincing evidence for the surface states and composition of the carbon dots, indicating the successful introduction of nitrogen to the as-prepared carbon dots. We then explored the optical properties of the synthesized carbon dots. The absorption and fluorescence spectra were depicted in Figure 3A. It showed a broad absorption under 500

Figure 1. Size and morphology characterization of carbon dots. (A) HRTEM image, (B) AFM image, and (C) particle size distribution analysis of carbon dots. Scale bar: 20 nm.

Figure 3. Optical properties of carbon dots. (A) UV−vis absorption spectrum and fluorescence spectra of carbon dots. Inset is a photo of the carbon dots irradiated by 365 nm UV light. (B) fluorescence spectroscopy of carbon dots with excitation wavelength changing from 360 to 420 nm with interval of 20 nm.

nm, which is possibly due to the relatively broad size distribution of the resultant carbon dots. With irradiation by a 365 nm UV light, a bright fluorescence could be seen with the naked eye (inset). Further, a detailed study on fluorescence properties were carried out by changing the excitation wavelength ranging from 360 to 420 nm with interval of 20 nm. As shown in Figure 3B, the fluorescence intensity of carbon dots at 475 and 545 nm were decreased with the increase of excitation wavelength. Accompanied with the remarkable gradual decrease of the fluorescence intensity at 475 nm, that at 545 nm displayed irregular behavior with the change of excitation wavelength. Moreover, the maximum emission peak position of the two emissions were fixed during excitation variation and exhibited λex-independent behavior. In addition, the quantum yield (QY) of the carbon dots was found using Quinine sulfate as a standard (measured at 370 nm excitation wavelength, QY = 54%). The average quantum yield (QY) of carbon dots in aqueous solution at room temperature was about 4.7%. Investigation of pH-Response Property. After the carbon dots with dual emissions were successfully synthesized, the pH-sensitive behavior of the carbon dots was investigated in 10 mM PBS buffer with different pH values. As illustrated in Figure 4A, with pH values changing from 4.0 to 11.0, a gradient

Figure 2. Characterization of structure and functional groups of carbon dots. (A) XRD pattern; (B) FT-IR characterization ((a) basic fuchsin, (b) citric acid treated by hydrothermal, (c) pH-sensitive carbon dots); (C) XPS survey scan of carbon dots; and the XPS highresolution survey scan of (D) C 1s, (E) O 1s, and (F) N 1s region of carbon dots.

observed, demonstrating that the carbon dots were highly disordered carbon atoms. Figure 2B depicted the FT-IR spectrum of basic fuchsin (line a), hydrothermal-treated citric acid (line b), and the prepared fluorescence carbon dots with basic fuchsin and citric acid in the molar ratio of 1:1000 (line c), respectively. The result revealed that the carbon dots synthesized from basic fuchsin and citric acid showed typical signals compared with a and b, which were arising from C−N/ CN stretching vibration at 1240 cm−1, N−H deformation vibration at 830 cm−1, and the CO stretching in amine bond at 1640 cm−1. The X-ray photoelectron spectroscopy (XPS) 7840

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usually sensitive to the existed various ions, such as Fe3+, Cu2+, Hg2+, and so on, which may limit their application in biosensing and bioimaging. For pH sensing in living cells, the potential interference from metal ions should be ruled out. Additionally, the effect of high concentrations of biothiol and reactive oxygen species (ROS) were also investigated. We explored the fluorescence stability of the prepared pH-sensitive carbon dots by measuring the ratiometric fluorescence intensity (I475 nm/I545 nm) in the presence of ROS (H2O2, HClO), biothiol (GSH, Cys) as well as various metal ions (Na+, K+, Ca2+, Mg2+, Cu2+, Ni2+, Mn2+, Pb2+, Hg2+, and Fe3+) in 10 mM PBS buffer with different pH values. As shown in Figure 5, no

Figure 4. pH-response of as-prepared carbon dots. (A) Fluorescence spectrum of carbon dots in PBS buffer with different pH values range from 4.0 to 11.0 under 380 nm excitation wavelength. Inset is a photo of color change of the carbon dots irradiated by 365 nm UV light. (B) Linear relationship of the ratiometric fluorescence intensity (I475 nm/ I545 nm) versus pH values. (C) Time scan of the ratiometric fluorescence intensity (I475 nm/I545 nm) of carbon dots for 1 h, and the ratiometric fluorescence intensity was normalized. (D) Fluorescence reversibility against pH change between 4.0 and 11.0, repeatedly.

color change could be observed under UV light (365 nm, insert) along with the pH changes and the fluorescence intensity of carbon dots emission at 475 nm increasing continuously; however, that at 545 nm exhibited a slight increase from acid to neutral and then decreased in alkaline solutions. More importantly, it was confirmed that the fluorescence intensity ratios (I475 nm/I545 nm) is linear against pH variation. A pH calibration curve in the PBS buffer solution was plotted in Figure 4B, which displayed a good linear relationship in the range of 5.2−8.8. The fluorescence intensity ratios (I475 nm/I545 nm) of the carbon dots excited under different excitation with different pH values in PBS buffer were also investigated (Figure S2). The above results demonstrate the fluorescence intensity of carbon dots at 475 and 545 nm are pH-sensitive simultaneously, and with a good linear relationship of fluorescence intensity ratios (I475 nm/I545 nm) against pH under different excitation wavelength, they could applied as a new kind of nanoprobes for pH sensing. To investigate their photostability, time scans of the fluorescence intensity in 10 mM PBS buffer with different pH values (5.0, 7.0 and 9.0) were performed for 1 h. It was demonstrated that the ratiometric fluorescence intensity (I475 nm/I545 nm) of carbon dots remained stable during the scanning process (Figure 4C), indicating their stability to light and air at the certain pH value. Then, the fluorescence reversibility against pH was carried out by adjusting the pH value between 4.0 and 11.0 repeatedly. As shown in Figure 4D, the ratiometric fluorescence intensity (I475 nm/I545 nm) of carbon dots presented a good reproducibility between the strong acidic solution (pH 4.0) and the strong alkaline solution (pH 11.0). The synthesized pH-sensitive carbon dots exhibited good photostability and fluorescence reversibility in different pH solutions. Thus, they can be used as excellent nanoprobes for intracellular pH measurement. Effect of Potential Interfering Substances in Different pH Solutions. The fluorescence intensity of carbon dots are

Figure 5. Ratiometric fluorescence intensity (I475 nm/I545 nm) of carbon dots in 10 mM PBS buffer at different pH in the presence of 1 mM for metal ions, biothiol (5 mM for GSH, 1 mM for Cys) and 500 μM for ROS (H2O2, HClO). (A) pH at 5.0, (B) pH at 6.0, (C) pH at 7.0, and (D) 8.0.

marked changes on the ratiometric fluorescence intensity (I475 nm/I545 nm) at pH 5.0, 6.0, 7.0, and 8.0 were observed for high concentrations of biothiol, ROS, and metal ions, indicating the excellent stability of carbon dots against these additives. MTT Assay. The spectroscopy data has confirmed that the fluorescence intensity of carbon dots at 475 and 545 nm were pH-sensitive, and they exhibited ratiometric fluorescence intensity (I475 nm/I545 nm) against pH variation. We then studied the application of carbon dots for intracellular pH imaging. Prior to cellular imaging, MTT assay was performed to investigate the toxicity of carbon dots. As shown in Figure 6, no significant drop of cell viability was observed for cells treated with carbon dots at a high concentration (up to 0.5 mg mL−1), demonstrating that they have very low toxicity in vitro. The result suggested that they have satisfactory biocompatibility and were suitable for fluorescence detection of pH in living cells. Ratiometric Calibration of pH in Living Cells. Before using the pH-sensitive carbon dots for pH measurement in living cells, the intracellular calibration experiment was performed in high-K+ HEPES-buffered solution containing 5 μg mL−1 of nigericin. Nigericin is a kind of polycyclic ether carboxylic acid compound that enables exchanges of H+ /K+ and rapid equilibration of pH across the cell membrane. For the ratiometric calibration of intracellular pH experiments, HeLa cells were first incubated with 0.5 mg mL−1 of carbon dots for 2 h at 37 °C in PBS buffer (pH 7.4). After removing the medium, the cells were washed with PBS buffer for three times and 7841

DOI: 10.1021/acs.analchem.6b01932 Anal. Chem. 2016, 88, 7837−7843

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pH was shown in Figure 7B. The intensity ratio (I475 nm/I545 nm) was linear against pH values from 5.0 to 8.0 in living cells, which covers the physiological pH level inside biological cells. Intracellular pH Imaging. To further demonstrate the quantitative detection in living cells, we then performed the measurements of pH in HeLa cells treated by different stimuli, such as chloroquine (CQ) and dexamethasone (DEX). CQ is an antimalarial drug which has been shown to induce a lysosomal pH increase in a living cell, and DEX is an antiinflammatory drug which could induce cell apoptosis accompanied by a decrease of intracellular pH. Figure 8

Figure 6. Cell viability for HeLa cells in the presence of different concentrations of carbon dots.

further treated with high-K+ HEPES-buffered solution in the presence of nigericin with different pH (5.01, 5.64, 6.46, 7.21, and 8.01) for 15 min, respectively. As shown in Figure 7A, with

Figure 8. Confocal microscope analysis of HeLa cells treated with chloroquine (CQ) and dexamethasone (DEX).

showed the fluorescence imaging of HeLa cells treated with CQ and DEX, respectively. Compared with the fluorescence intensity of control cells, the fluorescence intensities of channel 1 in CQ treated cells (Figure 8, second row) and channel 2 in DEX treated cells (Figure 8, third row) were increased. The addition of CQ causes an increase in cellular pH, while the DEX causes a decrease, which were reflected by the change in the ratiometric fluorescence intensity (I475 nm/I545 nm), suggesting that the pH-sensitive fluorescence carbon dots could sense the pH change in living cells.

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CONCLUSION In conclusion, we presented a novel ratiometric fluorescence pH sensor with label-free carbon dots as nanoprobes. Using citric acid and basic fuchsin as reaction source, the carbon dots with dual emissions at 475 and 545 nm were simply synthesized through a one-pot hydrothermal procedure. Carbon dots show pH-dependent emission features at both 475 and 545 nm; the fluorescence intensity ratio (I475 nm/I545 nm) of the carbon dots was linear against pH variation and has a good linear relationship against pH values from 5.2 to 8.8 in buffer solution. It also exhibited excellent fluorescence reversibility and photostability against potential interfering substances. Meanwhile, it has been successfully used in measurement of pH in HeLa cells. In comparison with other pH-sensitive nanocomposites-based sensors, the advantages of our nanoprobes include the following: (1) Noninvasive and fast measurement of intracellular pH can be achieved due to the ease of cellular uptake, strong fluorescence, and low toxicity of the carbon dots. (2) It can overcome the drawbacks of dye leaching in the most of the reported nanomaterials-based ratiometric pH sensors. (3) Complex separation and

Figure 7. Ratiometric calibration of pH in living cells. (A) Confocal fluorescence images of 0.5 mg mL−1 carbon dots incubating with HeLa cells in high-K+ HEPES-buffered solution in the presence of nigericin at different pH. (B) Calibration curve of intracellular pH sensing.

the increase of pH from 5.0 to 8.0, the fluorescence intensity in channel 1 (blue pseudocolor) was gradually enhanced while that of channel 2 (yellow pseudocolor) was slightly decreased when observed with the naked eye. The merge graph of channel 1 and channel 2 showed an obvious color change along with the pH variation. The fluorescence ratio image (Iblue/ Iyellow) was obtained using Image-Pro Plus software (fourth row in Figure 7A). By calculating the average fluorescence intensity of channel 1 and channel 2, the calibration curve of intracellular 7842

DOI: 10.1021/acs.analchem.6b01932 Anal. Chem. 2016, 88, 7837−7843

Article

Analytical Chemistry

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purification steps can be avoided because it does not require the attachment of additional dyes to the nanoparticles. Thus, we believe that these label-free carbon-dots-based nanoporobes can be employed as a new promising candidate for intracellular pH sensing.

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ASSOCIATED CONTENT

* Supporting Information S

The Supporting Information is available free of charge on the ACS Publications website at DOI: 10.1021/acs.analchem.6b01932. PL properties of carbon dots synthesized with different ratio of reactants, fluorescence intensity ratios (I475 nm/ I545 nm) of the carbon dots under 405 nm excitation (PDF)

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AUTHOR INFORMATION

Corresponding Authors

*E-mail: [email protected]. Phone: +86-731-8882-3073; Fax: +86-731-8882-1566. *E-mail: [email protected]. Phone: +86-731-8882-1566; Fax: +86-731-8882-1566. Author Contributions

The manuscript was written through contributions of all authors. All authors have given approval to the final version of the manuscript. Notes

The authors declare no competing financial interest.

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ACKNOWLEDGMENTS This work was supported in part by the Key Project of Natural Science Foundation of China (Grants 21190044, 21322509, 21305035, 21305038, and 21221003).

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REFERENCES

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DOI: 10.1021/acs.analchem.6b01932 Anal. Chem. 2016, 88, 7837−7843