Design of a New Near-Infrared Ratiometric Fluorescent Nanoprobe for

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Design of a new Near-Infrared Ratiometric Fluorescent Nanoprobe for Real-Time Imaging Superoxide Anion and Hydroxyl Radical in Live Cells and in Situ Tracing Inflammation Process in Vivo Rongjun Liu, Liangliang Zhang, Yunyun Chen, Zirong Huang, Yong Huang, and Shulin Zhao Anal. Chem., Just Accepted Manuscript • DOI: 10.1021/acs.analchem.7b04488 • Publication Date (Web): 07 Mar 2018 Downloaded from http://pubs.acs.org on March 7, 2018

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Analytical Chemistry

Design of a new Near-Infrared Ratiometric Fluorescent Nanoprobe for Real-Time Imaging Superoxide Anion and Hydroxyl Radical in Live Cells and in Situ Tracing Inflammation Process in Vivo

Rongjun Liu ,†,‡ Liangliang Zhang,*,† Yunyun Chen,† Zirong Huang,† Yong Huang,† Shulin Zhao*,†

†

State Key Laboratory for the Chemistry and Molecular Engineering of Medicinal

Resources, College of Chemistry and Pharmacy, Guangxi Normal University, Guilin, 541004, China ‡

Guangxi Key Laboratory of Agricultural Resources Chemistry and Biotechnology,

College of Chemistry and Food Science, Yulin Normal University, Yulin, 537000, China

Corresponding author:

Professor Shulin Zhao and Liangliang Zhang E-mail: [email protected] [email protected]

1

ACS Paragon Plus Environment

Analytical Chemistry 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

ABSTRACT: Superoxide anion (O2•−) and hydroxyl radical (•OH) are important reactive oxygen species (ROS) used as biomarkers in physiological and pathological processes. The ROS generation is closely related to the development of a variety of inflammatory diseases. However, in situ tracing inflammation process by real time monitoring the changes of ROS is difficult owing to short half-lives of ROS and high tissue autofluorescence in vivo. Here we developed a new near-infrared (NIR) ratiometric fluorescence imaging approach by using a förster resonance energy transfer (FRET)-base ratiometric fluorescent nanoprobe for real-time monitoring O2•− and •OH generation, and in situ tracing inflammation process in vivo. Proposed nanoprobe was composed of PEG functionalized GQDs as the energy donor connecting to hydroIR783 as both the O2•−/•OH recognizing ligand and the energy acceptor. The nanoprobe not only exhibited fast response to O2•− and •OH, but also presented good biocomapatibility, high photostability and signal-to-noise ratio. We have demonstrated that proposed NIR ratiometric fluorescent nanoprobe can monitor the changes of O2•− and •OH in living RAW 264.7 cells via drug mediating inflammation model, and further realized visual monitoring the change of O2•− and •OH in mice for in situ tracing the inflammation process. Our design may provide a new paradigm for long-term and real-time imaging applications on tracing the pathological process related to the inflammatory diseases in vivo.

Reactive oxygen species (ROS) are small bio-active molecules that contain oxygen, and play vital roles in a wide range of physiological and pathological 2


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processes.1 Superoxide anion (O2•−) and hydroxyl radical (•OH) are two primary ROS, and used as biomarkers for disease diagnosis. Although O2•− and •OH in normal cells with low concentration are essential for the life, their overaccumulation also can leads to oxidative stress and oxidative damage, and bringing many pathological conditions, such as inflammation, cellular damage and cancer.2-4 On the other hand, the inflammation plays a key role in pathogen invasion, tissue repair, adjustment of stress response, and the pathogenesis of various diseases. In situ tracing the inflammation process is crucial for the pathogenesis

study

and

therapies.5

individual

Inflammation

provide

a

microenvironment, where can produce more O2•− and •OH, and the ROS produced is closely related to the development of a variety of inflammatory diseases.6-8 Thus, real-time monitoring O2•− and •OH levels can in situ trace the inflammation process.9 Therefore, developing the methods for real-time monitoring O2•− and •OH, and tracing inflammation process are highly desirable. In the past decade, a number of methods were reported for the detection of O2•− and •OH levels by using spectrophotometry, chemiluminescence, electron spin resonance (ESR) spectroscopy, and electrochemical sensing.10-14 However, these methods are difficult to use for the detection of ROS in vivo. The fluorescence and bioluminogenic imaging has been established as one of the most useful techniques for monitoring ROS in live cells and in vivo.6,15-18 Compared with other techniques for monitoring ROS, the fluorescence imaging feature more advantages such as high sensitivity and visualization. However, conventional 3



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fluorescence imaging methods are single-signal channel imaging that is difficult to show the change of inflammation, and fail to accurately monitor O2•− and •OH dynamic changes in vivo. The ratiometric fluorescence approach allows analyte-induced fluorescence intensity changes in two signal channels, which provides a self-calibration for eliminating interferences, and improves the sensitivity and reliability.19-22 Therefore, several ratiometric fluorescence imaging methods has been applied for monitoring ROS in live cells.23-26 Although these ratiometric fluorescence imaging methods has high sensitivity and selectivity, these methods are still difficult to use for O2•− and •OH imaging in vivo. The imaging applications in vivo require that the fluorescenc probe provides NIR signals emission because the light in NIR region (650–900 nm) can improve the tissue penetration, and minimize the effect of light scattering and tissue autofluorescence.27-30 In addition, the ratiometric fluorescent probe for real time monitoring O2•− and •OH in vivo need meeting at least three special features including two NIR signal channels, fast response to target, and good photostability. To date, only a few NIR ratiometric fluorescent probes were reported.31-33 However, most reported NIR fluorescence probes are synthetical fluorescence small-molecule, while organic

small-molecule

probe

exhibits

small

Stokes

shifts

and

poor

photostability.34 Therefore, developing the NIR ratiometric fluorescent nanoprobe with low cytotoxicity and high photostability for in situ real-time monitoring O2•− and •OH in vivo retains still a challenge. 4


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Luminescent graphene quantum dots (GQDs), new fluorescence materials, are superior in terms of photostability, negligible cytotoxicity and biocompatibility, which make GQDs promising in sensors and bioimaging. 35,36 In this study, we developed a new NIR ratiometric fluorescent nanoprobe that allows imaging O2•− and •OH in vivo. This especial nanoprobe was fabricated by connecting yellow fluorescent GQDs to hydroIR783. HydroIR783 respond specifically to O2•− and •OH, and was converted to NIR fluorescent IR783 molecule, which turns on the förster resonance energy transfer (FRET) from GQDs to IR783. We have demonstrated that the GQDs-HydroIR783 nanocomposites can be applied as a noninvasive bioimaging nanoprobe for real-time monitoring the change of O2•− and •OH levels, and in situ tracing inflammation process in living mice.

EXPERIMENTAL SECTION Materials and Reagents. Polyacrylonitrile (PAN)-based carbon fibers were acquired from SGL Group-The Carbon Company. 1-Ethyl-3-(3-dimethylaminopropyl)carbodiimide (EDC), polyoxyethylenebis (amine) (NH2-PEG-NH2 Mw=2,000 Da), and N-hydroxy-succinimide (NHS) were purchased from Aladdin Reagent Co., Ltd. IR783, lipopolysaccharide (LPS), and phorbol-12-myristate-13-acetate (PMA) were obtained from Sigma-Aldrich. All animals were supplied by Hunan SJA Laboratory Animal Co., Ltd. Animal handing procedures were approved by Animal Ethics Committee of Guangxi Normal University (No. 20150325-XC). Preparation of HydroIR783. HydroIR783 were synthesized based on the method 5



reported by Kundu et al.18 Briefly, IR783 (6 mg) was dissolved in water (6 mL); 5 mg/mL sodium borohydride (NaBH4) solution was added dropwise to the IR783 solution with a stir, till its color changed to yellow. The solution was then placed in a dialysis bag (0.5 kDa) for 10 h to remove impurities and finally freeze-dried. Synthesis of GQDs, GQDs-PEG and GQDs-HydroIR783. Short carbon fibers (100 mg), sulfuric acid (80 mL, 98%), and nitric acid (20 mL, 60%) were placed in a three-neck round-bottom flask. The mixed solution was heated with continuous stir to 90 °C and refluxed for 10 h. After cooling to room temperature, 500 mL water was added the dark brown solution, and then part of dilute solution was neutralized by adding NaOH solid with a stir. The obtained GQDs solution was preserved at 4°C for a certain period to remove the precipitated salts. GQDs-PEG was prepared by a covalent connection between the GQDs and NH2-PEG-NH2. Firstly, GQDs with carboxyl groups were activated by adding EDC and NHS. Then, 2 mg/mL NH2-PEG-NH2 was added to the activated GQDs solution. After reaction at 0–4 °C overnight, GQDs-PEG was separated from free EDC/NHS and unreacted NH2-PEG-NH2 using a dialysis bag (3 kDa), and then lyophilized. For fabrication of the FRET-based nanoprobe, hydroIR783 was conjugated with GQDs-PEG through π-π stacking. 0.5 mg/mL GQDs-PEG in PBS buffer (10 µM, pH=7.4) was added to a hydroIR783 (5 mg/mL) solution. After stirring at room temperature for about 2 h, the mixed solution was transferred to a dialysis bag (1 kDa), then dialyzed for 8 h to obtain uniform GQDs -HydroIR783 nanoprobe. Measurements of Quantum Yield. Photoluminescence quantum yields of GQDs and GQDs-PEG were estimated by comparing with that of quinine sulfate with 45% 6


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quantum yield in 0.05 M H2SO4,37 using the following equation: φx=φs(Bx/Bs)(ηx/ηs)2. The subscripts “x” and “s” refer to the test samples and quinine sulfate, respectively. φ is the quantum yield, η is the refractive index of the solvent, and B is the gradient from the plot of integrated fluorescence intensity vs absorbance. Fluorescence Responses of the GQDs-HydroIR783 Nanoprobe to ROS. The fluorescence intensities were detected at room temperature. Various concentrations of O2•− were directly added to the solution containing GQDs-HydroIR783 at pH=7.4, and then the fluorescence spectra of solution were obtained on a Cary Eclipse spectrofluorophotometer. The O2•− solution was prepared by dissolving KO2 in DMSO solution. The absorption intensity was recorded to quantify the concentration of O2•− using a Cary 60 UV-vis spectrophotometer. The •OH solution was prepared from the Fenton reaction (Fe2+/H2O2). Singlet oxygen (1O2) was generated by adding NaClO into H2O2. Peroxynitrite (ONOO-) was chemically generated by H2O2 and NaNO2. Hypochlorite ion (ClO-) was provided by NaClO. Cytotoxicity Testing. The cytotoxicity was investigated using an MTT assay. RAW 264.7 cells were put into a 96-well plate and maintained at 37 °C for 24 h. The cells were incubated with GQDs, GQDs-PEG, and GQDs-HydroIR783 with different concentrations of 5, 10, 50, 90, 120 and 200 µg/mL, respectively. Then, 20 µL of 0.5 mg/mL MTT was added to each cell well. After incubation for 4 h, 80 µL DMSO was added. An enzyme linked immunosorbent assay reader was employed to measure the absorbance of samples.

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Ratioimaging of ROS in Live Cells. RAW 264.7 cells were put into 35-mm glass culture dishes in suited culture medium in 5% CO2 at 37 °C. When the cells grew to ~80% confluence, GQDs-HydroIR783 was added to the RAW 264.7 cells and incubated for 10 h. For imaging ROS in living cells, the RAW 264.7 cells are stimulated with PMA for different periods (0-4 h). The ratiometric fluorescence imaging of live cells were obtained using a laser scanning confocal microscopy (LSM710, Zeiss). In Vivo Ratioimaging. Female BALB/C-NU mice (8 weeks, 20 g) were anesthetized via inhalation of isoflurane and injected intraperitoneally with the nanoprobe (3 mg/mL, 100 µL). Fluorescence signal photographs (700±20 nm from GQDs and GQDs-PEG, 790±20 nm from IR783) were taken after injection at different time points. For in situ real-time monitoring of ROS in living animals, mice were given an intraperitoneal injection of LPS and GQDs-HydroIR783. In vivo ratioimaging was performed on a Kodak in-vivo FX Pro imaging system (Bruker). Fluorescence images were captured using a fixed exposure time (40 s) at different time points. Fluorescence images and their corresponding X-ray images were merged to clearly indicate the signal site of the mice. The fluorescence images were analyzed using the Bruker Molecular Imaging (BMI) Software.

RESULTS AND DISCUSSION Design and Principle of GQDs-HydroIR783 Nanoprobe. Design and principle of GQDs-HydroIR783 nanoprobe for O2•− and •OH detection and tracing inflammation process in vivo is illustrated in Figure 1A. The hydroIR783 with reducibility can 8


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respond specifically to O2•− and •OH, and are converted to fluorescent IR783 molecule which has been characterized by MS (Figures S1, S2) due to regenerating their extended π conjugation (Figure 1B).9,18,26 The IR783 molecule with NIR fluorescence is suitable for detecting O2•− and •OH in vivo. Therefore, hydroIR783 was chosen as a recognition molecule to capture O2•− and •OH. Furthermore, it was noted an overlap between the emission spectrum of GQDs and the absorption spectrum of IR783 (Figure 1C), which suggests that they were likely a donor-acceptor pair of FRET.38 Thus, GQDs was selected as the FRET donor, and connected with hydroIR783 to forming a FRET system. In the presence of O2•− and •OH, hydroIR783 was oxidized by O2•− and •OH, and converted to IR783, which turns on the FRET from GQDs to IR783, and lead to the fluorescence intensity of GQDs decrease, and fluorescence intensity of IR783 increase. Preparation and Characterization of GQDs-HydroIR783 Nanoprobe. To construct the GQDs-HydroIR783 nanoprobe, we first synthesized the GQDs with yellow emission using a top-down method that was based on the oxidation of polyacrylonitrile (PAN)-based carbon fibers in sulfuric acid and nitric acid under heating condition (Figure 2). Transmission electron microscopy (TEM) and X-ray photoelectron spectroscopy (XPS) images showed respectively that the as-prepared GQDs were 5±2.5 nm in diameter, dispersed uniformly in water, and made up of carbon, oxygen, and nitrogen (Figures S3, S4). The GQDs exhibited yellow fluorescence under irradiation with a 420 nm light (Figure 1C, inset b), and an excitation-dependent mulriple-color emission behavior (Figure S5). The photoluminescence quantum yields (PLQY) of the GQDs was found to be 8.2% 9



when using quinine sulfate as a reference. To increase the stability of the GQDs, PEG-modified GQDs (GQDs-PEG) were obtained by covalently coupling NH2-PEG-NH2 to the surface of the GQDs with coupling reagents (EDC/NHS). The FT-IR spectrum data suggested a successful conjugation of NH2-PEG-NH2 with GQDs (Figure S6).39 The GQDs-HydroIR783 nanoprobe was then fabricated by trapping the hydroIR783 molecules within the NH2-PEG-NH2 network, and immobilizing on the GQDs surface via π–π stacking.40 The GQDs-HydroIR783 nanoprobe was very stable in both PBS and serum (Figure S7). Feasibility Study of Nanoprobe for O2•− and •OH Detection. To verify the feasibility of GQDs-hydroIR783 as a FRET-based ratiometric fluorescent nanoprobe for O2•− and •OH detection, the FRET between GQDs and IR783 was investigated by measuring the fluorescence change of GQDs in the presence of different concentrations IR783. The results indicate that with increasing IR783 concentration from 0.2% to 1.4%, the fluorescence intensity of IR783 at 800 nm (emission peak) increase gradually, whereas the fluorescence intensity of GQDs at 520 nm (emission peak) decrease gradually. Further increasing the concentration of the IR783 to 2.0%, the fluorescence intensity of IR783 is slightly lower, this is due to lower overlap between fluorescence spectrum of GQDs and the absorption spectrum of IR783, resulting in limited FRET efficiency (Figure S8). Besides, the influence of IR783 (acceptor) on the fluorescence lifetime of GQDs (donor) was also investigated. It was found that the GQD-PEG had an initial lifetime of 4.27 ns in the absence of IR783. However, its fluorescence lifetime decreased to 2.18 ns in the presence of IR783 (Figure S9). The difference of 10


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fluorescence lifetime and the fluorescence spectra demonstrated also the FRET between GQDs and IR7833.41 Detection of O2•− and •OH. As a proof-of-concept, the fluorescence response of the as-prepared GQDs-hydroIR783 nanoprobe to O2•− was tested to verify the ratiometric fluorescence detection method. Firstly, the influence of pH on the detection of O2•− and •OH was investigated, it is found that the acidity of the solution in the range of pH 6-8 have no effect on the detection of O2•− and •OH (Figure S10). Then, the O2•− with a concentration range of 0.5–30 µM were added into the nanoprobe solution (0.35 mg/mL) at pH 7.4, and the fluorescence spectra of GQDs-hydroIR783 nanoprobe solutions were recorded. It is found that the fluorescence intensity of IR783 at 800 nm increase, and that of GQDs at 520 nm decrease with the increase of O2•− concentration (Figure 3A). The relationship between the fluorescence intensity ratio (I800/I520) and O2•− concentration in the range of 0 to 20 µM showed a good linearity (I800/I520=0.041 [O2•−] µM+0.028, R2=0.9910; inset of Figure 3A). A detection limit of 0.2 µM was achieved (S/N=3), which is comparable to previously reported O2 • − ratiometric fluorescent probe.23,24 In addition, the response time of GQDs-hydroIR783 nanoprobe toward O2•− was also investigated via a kinetics method. After adding O2•− to the GQDs-hydroIR783 nanoprobe solution 0.1 min, the fluorescence intensity of IR783 increase to its maximum and that of GQDs decrease to its minimum (Figure 3B). As expected, GQDs-hydroIR783 nanoprobe exhibited fast response and high selectivity towards O2•− and •OH (Figure S11). The influence of O2•− and •OH on the fluorescence intensity of GQDs was very small (Figure S12), implying the decrease of fluorescence intensity at 11



520 nm in GQDs-hydroIR783 nanoprobe solution was due to the activation of FRET. Moreover, we also evaluated the cytotoxicity of the GQDs, GQDs-PEG, and GQDs-hydroIR783 nanoprobe in RAW 264.7 cells via an MTT assay. The results indicate that the GQDs, GQDs-PEG, and GQDs-hydroIR783 nanoprobe presented negligible biotoxicity (Figure S13). These experimental results suggest that the developed GQDs-based ratiometric fluorescent nanoprobe possesses low biotoxicity, high selectivity and fast response, and may be applied for the real-time detection of O2•− and •OH levels in biological samples. Intracellular Real-Time Ratioimaging of O2•− and •OH. We evaluated the ability of GQDs-hydroIR783 nanoprobe for ratiometric fluorescence imaging of O2•− and •OH in live macrophages after continuously stimulating by PMA.42 The activation of macrophages by PMA is a common model to increase the concentration of O2•− and •OH in cells. Taking the GQDs-hydroIR783 nanoprobe into cells was achieved after incubation the cells for 10 h. Then, nanoprobe-loaded cells were incubated with 0.4 mg/mL PMA. Time-sequential imaging showed that the yellow fluorescence channel from GQDs became gradually darker within 3 h, and finally tended to stabilize at 4 h. In contrast, the red fluorescence channel from IR783 became gradually brighter within 3 h, reaching its brightest at 4 h (Figures 4A, 4B). Besides, we can also see that the color of ratio channel (color generated by image analysis software Image Pro-plus) changed clearly from purple to red when the cells were induced with PMA form 0 to 4 h, respectively. From Figure 4C, the mean fluorescence intensity ratios of red channel and yellow channel (Fred/Fyellow) increase to 0.73±0.05 after in situ stimulation for 4 h. Our 12


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results show that the amounts of O2•− and •OH is gradually enhanced with prolonged time by the stimulation of PMA, which is consistent with the finding from Tang’s group.42-44 To confirm that these changes were caused by the generated O2•− and •OH, the cells were pretreated with 1.0 mM GSH (an O2•− and •OH scavenger)6 for 30 min, and then exposed to PMA stimulation. Comparison the fluorescence images of two channels (Figure 5), it is clear that only the fluorescence of IR783 was strengthened with the reaction of O2•− and •OH from the PMA stimulation (Figure 5B), while negligible fluorescence changes were observated in Probe+GSH+PMA group (Figure 5C). Pretreatment with GSH inhibited obviously the red fluorescence, where the GQDs-based FRET turned off. This result not only confirms that the signal changes of two channel are related to intracellular O2•− and •OH levels, but also implied that the developed GQDs-hydroIR783 ratioimaging nanoprobe is a reliable sensing platform for biomarkers research. In Situ Real-Time Ratioimaging of O2•− and •OH in Vivo for Tracing Inflammation Process. Before real-time ratioimaging of O2•− and •OH in vivo, the excitation and emission wavelengths of GQDs-PEG were investigated. A mouse was treated with GQDs-PEG by intraperitoneal injection; a control mouse was injected with saline. Three lights with different wavelength (470 nm, 510 nm and 540 nm) were used as exciting light, and three different emission filters (535±20 nm, 600±20 nm, and 700±20 nm) were used to collect the fluorescence signal of GQDs-PEG. Surprisingly, the fluorescence signal of GQDs-PEG could only be observed when using 540 nm exciting light and 700 nm emission filter (Figure S14). Thus, 540 nm exciting light and 13



700 nm emission filter were selected for collecting the fluorescence signal from GQDs in subsequent in vivo ratioimaging. Undoubtedly, the photostability is another important factor for long-term in vivo fluorescence imaging.25,40 Next, we compare the fluorescence stability of GQDs, GQDs-PEG, and IR783 in the peritoneal cavity of mice via long-term fluorescence imaging. The GQDs-PEG remained stable and bright fluorescence after 6 h. IR783 remained stable and bright fluorescence within 3 h，and the fluorescence signals of IR783 decrease gradually after 3 h (Figure 6). The fluorescence signals of GQDs decrease gradually with time increasing owing to biodegradation in the peritoneal cavity of mice (Figure S15). Finally, we explored whether GQDs-hydroIR783 nanoprobe could be utilized to monitor O2•− and •OH levels in vivo for in situ tracing inflammation process using a LPS-induced inflammation model. A female BALB/C-NU mouse was successively given an intraperitoneal injection of LPS and GQDs-hydroIR783. The O2•− and •OH levels in vivo increase gradually with the onset of inflammation. As the inflammation time go up, the fluorescence intensity of 790 nm channel from IR783 rise gradually, while the fluorescence intensity of 700 nm channel from GQDs declined gradually (Figure 7A, 7B and 7C). The ratio images (F790/F700) were obtained from the above two channels, and a distinguishable color change was observed with the increase of inflammation time. Furthermore, the fluorescence intensity ratio of two channels (F790/F700) rise gradually with the increase of inflammation time, and reached maximum within 4 h. Then, the fluorescence intensity ratio remains fixedness with the inflammation time further increased (Figure 7D). The ratioimaging results demonstrate that proposed 14


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GQDs-hydroIR783 nanoprobe can be applied for in situ real-time monitoring O2•− and •

OH, and tracing inflammation process in vivo. Although the as-prepared GQDs show intense emission in the yellow light region,

in vivo imaging results show the GQDs have still high signal-to-noise ratio in NIR region due to its excitation-dependent mulriple-color emission behavior. NIR fluorescence of GQDs-PEG can efficiently penetrate the mouse tissues and reduce the interferences of light scattering and tissue autofluorescence.45 Based on this finding, we investigated ratiometric imaging capability of the GQDs-HydroIR783 nanoprobe in vivo. The results shows that the in situ ratiometric imaging allow real-time monitoring of O2•− and •OH levels in vivo. In addition, the overaccumulation of O2•− and •OH would generate oxidative stress. Meanwhile continued oxidative stress can cause inflammation. Thus, long-time and real-time monitoring O2•− and •OH levels in vivo can trace inflammation process. While the inflammation is early features of tumour progression, a lot of cancers are caused by the sites of infection and inflammation.46 Therefore, the localization tracing the inflammation process is crucial for the pathogenesis study and individual therapies of cancers.

CONCLUSIONS In summary, we have developed a novel method to prepare NIR ratiometric fluorescence nanoprobe, which is composed of GQDs-PEG connecting to hydroIR783. The GQDs-HydroIR783 nanoprobe can specifically and quickly respond to O2•− and •

OH with a low detection limit down to 0.2 µM. Taking the advantages of good 15



selectivity, desirable biocomapatibility, high sensitivity and excellent photostability, the GQDs-HydroIR783 nanoprobe could serve as a robust NIR ratiometric fluorescence image platform for visual real-time monitoring O2•− and •OH in vivo, and in situ tracing inflammation process. We believe that the NIR ratiometric fluorescent nanoprobe proposed here may be a promising tool for the pathogenesis study of many diseases, and further clinical diagnosis.

ASSOCIATED CONTENT Supporting Information The Supporting Information is available free of charge on the ACS Publications website at DOI: 10.1021/acs.analchem.XXXXXXX HRMS spectrum of HydroIR783, HRMS spectrum of IR783, TEM image of GQDs from carbon fiber, XPS spectrum of the GQDs, emission spectra of GQDs at different excitation wavelengths, effect of pH on the fluorescence intensity ratios (I800/I520) of GQDs-HydroIR783 in the presence of O2•−, FT-IR spectra of GQDs, NH2-PEG-NH2 and GQDs-PEG, pictures of GQDs-HydroIR783 probe in PBS and serum after standing for different time, fluorescence spectra of GQDs-PEG in different concentrations of IR783, fluorescence decays of GQDs-PEG and GQDs-PEG-IR783, selectivity and specificity assays of GQDs-HydroIR783 probe in the presence of various analytes, fluorescence spectra of GQDs in the presence of O2•− and •OH, respectively, viability of RAW 264.7 cells under different concentrations of GQDs, GQDs-PEG, and GQDs-HydroIR783, in vivo fluorescence 16


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imaging of GQDs-PEG in BALB/C-NU mice, with different emission wavelengths, and photostability of GQDs in mice (PDF).

AUTHOR INFORMATION Corresponding Authors *E-mail: [email protected] (S. Zhao), [email protected] (L. Zhang). Notes The authors declare no competing financial interest.

ACKNOWLEDGMENTS This work was supported by the National Natural Science Foundations of China (Grant Nos. 21327007 and 21575031), Natural Science Foundations of Guangxi Province (Grant No. 2015GXNSFDA139006) and BAGUI Scholar Program. We thank Dr Ying Jiang for her suggestions on the manuscript.

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FIGURE CAPTIONS Figure 1.

(A) Rational design of GQDs-hydroIR783 nanoprobe for in situ, real-time, ratiometric fluorescence monitoring O2•− and •OH in vivo. (B) The reaction between O2•− or •OH and the hydroIR783. (C) Normalized absorption spectra (Abs) and fluorescence spectra (FL) of GQDs and IR783 in PBS (pH=7.4). The purple area indicates the spectral overlap between the FL of GQDs and the Abs of IR783. Inset: bright-field photographs of GQDs (a) and fluorescence photographs of GQDs (b).

Figure 2. Schematic diagram for synthesis of GQDs and GQDs-HydroIR783 nanoprobe. Figure 3.

(A) Fluorescence spectra of the FRET-based GQDs-hydroIR783 nanoprobe in the presence of different concentrations O2•− (0, 0.5, 1.0, 1.5, 2.5, 3.5, 5.0, 7.0, 10.0, 20.0, 30.0 µM). pH=7.4, Ex=440 nm. Inset: plot of the fluorescence intensity ratios (I800/I520) to O2•− concentrations. (B) Kinetics curve for the change of fluorescence intensity of 0.3 mg/mL GQDs-hydroIR783 solution with 4 µM O2•−.

Figure 4.

(A) Real-time fluorescence image of stimulation-produced ROS in RAW 264.7 cells. Yellow channel and red channel ware respectively collected at 510-570 nm and 750-790 nm, under excitation of 488 nm. Cells were treated with 0.15 mg/mL of the nanoprobe for 10 h, followed by adding 0.4 mg/mL PMA and incubated for 0, 1, 2, 3 and 4 h. (B) The mean fluorescence intensity of yellow channel and red channel with time increase. 22


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(C) The mean fluorescence intensity ratios (Fred/Fyellow) in different time points. Figure 5.

Confocal ratiometric fluorescence image of RAW 264.7 cells incubated with GQDs-HydroIR783 nanoprobe (A), probe-loaded cells after stimulating by 0.5 mg/mL PMA for 4 h (B), and probe-loaded cells pretreated with GSH (1 mM) for 30 min and then stimulating by 0.5 mg/mL PMA for 4 h (C). Yellow and red channels ware respectively collected at 510-570 nm and 750-790 nm under excitation of 488 nm light.

Figure 6. Photostability of GQDs-PEG and IR783 in mice. (A) In vivo fluorescence images of mice taken at different times after intraperitoneal injection with GQDs-PEG (3 mg/mL, 100 µL). (B) Net fluorescence intensity obtained from GQDs-PEG at different times (Ex: 540 nm; Em: 700±20 nm).

(C) In

vivo fluorescence images of mice taken at different times after intraperitoneal injection with IR783 (0.05 mg/mL, 100 µL). (D) Net fluorescence intensity obtained from IR783 at different times (Ex: 700 nm; Em: 790±20 nm). Figure 7.

(A) In situ real time and in vivo ratiometric fluorescence imaging of O2•− and •OH from peritoneal cavity of mice using GQDs-HydroIR783 nanoprobe with stimulation of LPS (0.8 mg/mL, 100 µL). The mean fluorescence intensity of 700 nm channel (B) and 790 nm channel in different time points (C). (D) The mean fluorescence intensity ratios (F790/F700) in different time points. 23



Figure 1

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Figure 2

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Figure 3

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Figure 4

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Figure 5

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Figure 6

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Figure 7

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For TOC only

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Design of a New Near-Infrared Ratiometric Fluorescent Nanoprobe for

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