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Dec 19, 2016 - Mukesh Kumar Kumawat , Mukeshchand Thakur, Raju B. Gurung, and .... The cells were stained with DAPI (2 μg/mL) for 5 min and washed ...
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Graphene Quantum Dots from Mangifera indica: Application in NearInfrared Bioimaging and Intracellular Nanothermometry Mukesh Kumar Kumawat, Mukeshchand Thakur, Raju B. Gurung, and Rohit Srivastava* Department of Biosciences and Bioengineering, Indian Institute of Technology Bombay, Mumbai, India

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S Supporting Information *

ABSTRACT: We report a simple one-pot microwave-assisted greensynthesis route for the fabrication of bright red-luminescent graphene quantum dots (GQDs) using ethanolic extracts of Mangifera indica (mango) leaves, hence addressing them as mGQDs. The mGQDs were quantum-sized ranging from 2 to 8 nm and exhibited excitationindependent fluorescence emission in the near-infrared (NIR) region between 650 and 750 nm. The mGQDs showed defects in their structure and were highly crystalline in nature as confirmed by Raman spectroscopy and powdered X-ray diffraction analysis, respectively. These mGQDs showed 100% cellular uptake and excellent biocompatibility on L929 cells even at high concentration (0.1 mg/ mL) 24 h post-treatment. Cell cycle analysis showed increased proliferation in L929 cells upon mGQDs treatment. Furthermore, the mGQDs were demonstrated as NIR-responsive fluorescent bioimaging probes, self-localizing themselves selectively in the cell cytoplasm. Also, the temperature-dependent fluorescence intensity of these GQDs proved them as a very competent temperature sensing probe (at 10−80 °C). The temperature sensing stability analysis showed that the temperature signal remains stable even after multiple cycles of temperature switching between 30−80 °C. Furthermore, we analyzed intracellular temperature (25−45 °C) of live L929 cells based on the fluorescence intensity of the mGQDs. It was observed that with an increasing temperature there was a decrease in the fluorescence intensity of the mGQDs making it a suitable probe for temperature sensing. In sum, a biocompatible, scalable, photostable, green synthesis based mGQDs were prepared for NIR imaging and nanothermometry applications which can play a pivotal role in biomedical nanotechnology. KEYWORDS: Mangifera indica, Graphene quantum dots, Near-infrared, Bioimaging, Intracellular, Temperature sensors



INTRODUCTION Graphene quantum dots (GQDs) are zero-dimensional carbon nanomaterials which have been explored extensively since their serendipitous discovery during purification of carbon nanotubes.1,2 When two-dimensional graphene or graphene oxide sheets are cut down to zero dimension, they form GQDs,3,4 sometimes also referred to as carbon quantum dots (CQDs) or C-dots. They possess unique and exciting properties such as excitation-dependent/-independent fluorescence,5−7 multicolor emission,8−10 excellent dispersibility and solubility in aqueous and organic solvents11,12 resistant to photobleaching, competitive quantum yields,3,13 and long fluorescence lifetimes.14 The GQDs are eco-friendly, relatively nontoxic, and photostable as compared to semiconductor QDs and organic dyes.7,15,16 Since their discovery in 2004,2 various syntheses approaches have been employed for fabrication of GQDs which can be divided into two categories: top-down and bottom-up.7,17,18 The top-down approach, as the term implies, involves breakdown of bulk material into smaller nanomaterials and consists of methods like electrochemical cutting,19 nanolithography,20 solvothermal synthesis,21 and chemical exfoliation,22 whereas the bottom-up approach involves smaller units © 2016 American Chemical Society

forming bigger entities and consists of methods like thermal combustion,23 microwave irradiation,10,24 hydrothermal heating,25 or cage-opening method of fullerene.26 However, these syntheses approaches demand high-quality carbon precursors, concentrated acid/alkali treatments, high temperature, toxic organic solvents, and complicated purification methods. Thus, green-chemistry approaches have been employed which has an added advantages of the myriad of carbon sources available, economical and eco-friendly synthesis methods, biocompatibility, large-scale production, and recycling of waste products into value added products. For instance, GQDs fabricated from green synthesis routes using different carbon sources like fruit extracts,27 peels,28 food-wastes,29 algal blooms,30 bacteria,31 milk,10 cabbage,32 and human urine.33 The advantages of using these precursors for GQDs synthesis involve large-scale availability, easy handling, and nontoxicity. Some enzymes, vitamins, polysaccharides, proteins, and other biomolecules present in the plants have a natural capacity to perform reduction and capping of nonbiocompatible materials.34 Plant Received: August 8, 2016 Revised: December 2, 2016 Published: December 19, 2016 1382

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2.2.2. Biocompatibility Study. For biocompatibility study, mouse fibroblast L929 cells were procured from National Centre for Cell Sciences (NCCS), Pune. Briefly, 8000 cells/well were seeded in a 96well plate and incubated for 24 h under humidified condition at 5% CO2 and 37 °C. The actively growing cells were then gently washed with PBS (1×, pH 7.0) and seeded with different concentrations of mGQDs (5 μg/mL to 0.1 mg/mL) for 24 h. Percentage cell viability was calculated using an MTT assay49 by recording the absorbance at 570 nm against the reference wavelength 620 nm using Tecan Infinite M200 Pro Plate reader. The cell viability was calculated using following eq 1, where Atest is an absorbance value of test sample, Ablank is an absorbance value for blank and Acontrol is an absorbance value for untreated control cells:

materials prove to be an excellent source for biosynthesis of carbon nanomaterials as they are high in carbon content for the production of the carbon nanomaterial. 35 Herein, we demonstrate a simple one-step microwave-assisted method for synthesis of GQD using an ethanolic extracts of mango leaves. Mango leaves extracts are valued for antioxidant properties effective against reactive oxygen species.36,37 This property enables them to be active against some diseases acting as an antiallergy,38 antiviral,39 and anticancer agent.40 Mangiferin, a polyphenolic antioxidant, is a major constituent of mango leaf extracts present in various capacities.41 Since these GQDs are synthesized from metal-free precursors, they have the least possibility of cellular toxicity and do not require additional capping or passivation to make them biocompatible and stable. Thus, mango leaves would serve as an excellent carbon precursor for fabrication of GQDs. In this report, we focus on mango leaves assisted fabrication of GQDs (mGQDs) for near-infrared (NIR) imaging of mammalian cells and photoluminescence-based intracellular nanothermometry applications. Lately, GQDs have been widely used for biomedical applications like drug delivery, biosensing, and mainly bioimaging purposes.17,18,42 The multifluorescence property of GQDs extends from ultraviolet to infrared region of the light spectrum.43 However, very few GQDs show NIR imaging capability for bioimaging applications. For an in vivo imaging application, NIR fluorescing nanoprobes are preferred due to their least absorbance along with deep penetration within the tissues.44 Probing temperature variation at the nanoscale is a challenge. The latest technology for nanothermometry envisages the concept of optical feature size method and techniques like scanning thermal microscopy,45 Raman spectroscopy,46 and fluorescence-based microscopy.47 Thermal sensing fluorescent materials include organic dyes, metal clusters, and quantum dots; however, their use for sensing applications has limitations in biological medium because of their toxic nature, photoinstability, and low sensitivity.48 The mGQDs served as a potential candidate for NIR imaging and thermal sensing of live cells in vitro due to their excellent biocompatibility and photostability.

Viability (%) =

A test − A blank × 100 Acontrol − A blank

(1)

2.2.3. Confocal Laser Scanning Microscopy (CLSM). Cell bioimaging was performed on L929 cell line using CLSM. Briefly, 6000 cells/well were seeded in a 12-well plate on a sterile and acidetched glass coverslips. Cells were allowed to adhere for 24 h under humidified condition at 5% CO2 and 37 °C. The cells were then treated with mGQDs until 0.1 mg/mL concentration and kept for incubation. After incubation for 24 h, cells were washed carefully using PBS (pH 7.0) and fixed using 2.7% formaldehyde (15 min), followed by washing thrice with PBS (pH 7.0). The cells were stained with DAPI (2 μg/mL) for 5 min and washed thrice with PBS (pH 7.0), then imaged at excitation/emission laser wavelengths 750/780 nm and 561/637 nm. 2.2.4. Cell Cycle Analysis Using Flow Cytometry. For cell cycle analysis, 2 × 106 cells were seeded in a T-25 flask and allowed to incubate for 24 h at 5% CO2 and 37 °C under humidified condition. The cells were then treated with mGQDs (50 μg/mL) for 24 h. The cells were then washed, trypsinized, fixed, and dispersed in 500 μL of PBS (pH 7) with PI (50 μg/mL) and RNase A (100 μg/mL) and kept in the dark for 15 min before flow cytometry analysis. A minimum of 10 000 events were recorded for each study and data were analyzed using FlowJo V10.1R7 software package. 2.2.5. Temperature Sensing Study. Thermal sensing study was performed by heating and cooling the dispersion of mGQDs (1.0 mg/ mL) in IKA HB10 water bath at different temperatures. The dispersion was set at various temperature ranges from 30 to 80 °C. The corresponding fluorescence intensity of the dispersion was measured by fluorescence excitation at 400 nm and emission at 690 nm. 2.2.6. Intracellular Temperature Sensing. To measure the intracellular temperature in live cells, L929 cells were seeded in a 35 mm glass-bottomed dish and kept under humidified incubation condition at 37 °C and 5% CO2. After 24 h, cells were treated with mGQDs (0.1 mg/mL) and kept under similar incubation condition. The cells were washed gently with PBS before imaging under CLSM. The temperature was varied from 25 to 45 °C with 5 °C interval in CLSM, and Z-stack imaging was carried out at every 5 °C temperature increment. Laser parameters and area of interest were kept unchanged for imaging at every temperature increment. Few representative cells were selected to assess the change in fluorescence intensity over temperature change. 2.2.7. Instrumentation. The size and morphology of mGQDs were investigated using field emission gun transmission electron microscopy (FEG-TEM, JEOL, Japan). Photoluminescence behavior of mGQDs was studied using fluorescence spectrophotometer (Hitachi F-2500 FL Spectrophotometer), and absorption spectra were recorded in a UV− vis spectrophotometer (Lambda25, PerkinElmer). The infrared (IR) spectra were recorded using a Bruker spectrophotometer, the highresolution X-ray diffraction (XRD) pattern was obtained from Smartlab Rigaku diffractometer, and Raman spectra were recorded using a Jobin-Yvon Labram spectrometer. Dynamic light scattering (DLS) was performed using DLS−BI200SM (Brookhaven Instruments Corporation, USA). X-ray photoelectron spectroscopy (XPS) was performed using a monochromatic Al Kα source (225 W) with a

2. EXPERIMENTAL SECTION 2.1. Materials. M. indica leaves were obtained from mango tree in the local campus of IIT Bombay, India. Ethanol was purchased from Hayman, UK. Dulbecco’s modified Eagle’s media (DMEM), minimum essential medium (MEM), fetal bovine serum (FBS), phosphatebuffered saline (PBS, pH 7.0), trypsin-EDTA solution (0.25% trypsin and 0.02% EDTA in Dulbecco’s PBS without phenol red) and antibiotic antimycotic solution (10 000 units of penicillin, 10.0 mg of streptomycin, and 25 μg of amphoterin B per mL in 0.9% normal saline) were purchased from Himedia, India. 3-(4,5-Dimethylthiazol-2yl)-2,5-diphenyltetrazolium bromide (MTT) dye was procured from Sigma, USA. 2.2. Methods. 2.2.1. Extract Preparation and Synthesis of mGQDs. Mango leaves were cut down into tiny pieces (1−2 cm) and dipped in absolute ethanol. The mixture was kept under constant stirring for 4 h, and the resultant extract was centrifuged at 8000 rpm for 10 min to achieve a clear supernatant. The extract was further filtered and concentrated by evaporating the ethanol in a rotary evaporator until residual slurry was obtained. The slurry was mixed with a small amount of milli-Q water and heated under 900 W domestic microwave oven for 5 min, and the residue dispersed in absolute ethanol for proper dispersion of mGQDs. The dispersion was further filtered through the syringe filter (0.22 μm) to obtain pure mGQDs; mGQDs were then allowed to dry at 65 °C for 24 h to obtain dried powder. 1383

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Figure 1. (a) UV−vis Sspectroscopy of mGQDs and MIE. (b) Comparative FTIR analysis of MIE and mGQDs, and (c) change in FL intensity of mGQDs at excitation range of 300−500 nm.

Figure 2. (a) FEG-TEM micrograph of mGQDs at 2 nm scale (inset showing size distribution characterization using DLS). (b) Micrograph showing mGQDs at 50 nm scale with an inset showing a frame with dimension of 12.2 × 12.2 nm displaying high-resolution image of mGQDs with fringe lattice width of 0.32 nm. Kratos Analytical instrument (Model AXIS Supra), and CLSM was performed using Olympus, FlowView.

3. RESULTS AND DISCUSSION 3.1. Optical and Surface Characterization of mGQDs. The absorption spectra of both M. indica leaf extract (MIE) and mGQDs are given in Figure 1a. The absorption spectrum of MIE (50 μg/mL) showed distinct peaks at 260, 317, and 360 nm corresponding to leaves extract components such as gallic acid, maclurin 3-(2-gallolyl)-β-D-glucoside, iriflophenone3-C-β50 D-glucoside, and mangiferin, while chlorophyll-a shows absorption at 420 and 670 nm, respectively. The synthesized mGQDs showed an absorption peak at 280 nm (Figure 1a) corresponding to the n−π* electronic transitions within the carbon structure. The FTIR spectrum is depicted in Figure 1b; the mGQDs display broadening and intensification of the vibration peaks at 3396, 1029, and 1072 cm−1 in contrast to those of MIE. These bands correspond to stretching and bending vibrations of the hydroxyl (−OH) groups. Also, vibration peaks at 1739, 1224, and 1371 cm−1 are ascribed to the carbonyl functional groups of the ester family present in the structure. The peaks at 1460, 1623, 2922, and 2854 cm−1 correspond to stretching vibrations of C−C, −CC−, and

Figure 3. (a) Raman spectrum of the mGQDs showing G band at 1574 cm−1 and D band at 1324 cm−1. (b) XPS survey spectrum. (c and d) C 1s and O 1s XPS spectrum, respectively.

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Figure 4. (a) Cell cycle analysis of L929 cells was performed using flow cytometry. The cells treated with mGQDs (50 μg/mL) in comparison to control (untreated) show an increase in G2-phase after 24 h, indicating the increase in cell proliferation rate. (b) Histogram showing biocompatibility of mGQDs estimated to be more than 95% after incubation for 24 h in L929 cells. PC = positive control, NC = negative control.

−C−H (both 2 and 1° carbon) bonds, respectively, present in the hydrocarbon skeleton of the mGQDs. An investigation of PL spectra of the mGQDs at different excitation wavelength from 300 to 500 nm showed a rise in the emission intensity until highest emission at 400 nm and then decreased further (Figure 1c). The increase in the excitation wavelength causes the corresponding decrease in the emission intensity. The mGQDs exhibited excitation-independent fixed emission at 680 nm. This is probably due to the energy released by the smaller graphitic domains of mGQDs being absorbed by the larger counterparts which release the energy altogether toward longer wavelength.51,52 Furthermore, the GQDs also showed excellent photostability and lifetime (Figures S1 and S2). The possibility of red emission from the mGQDs caused by residual MIE

could be ruled out by the fact that at the high temperature inside microwave oven (>150 °C, 5 min) nearly all organic molecules undergo oxidation process during carbonization and were decomposed or modified. This is evident by the difference in the absorption spectrum of MIE and mGQDs where many significant peaks disappeared in the case of the mGQDs. The origin of red fluorescence could be attributed to the introduction of oxygen-containing functional groups which relates to the tuning of oxidation states and reducing the band gap.43 This is evident by FTIR analysis and further confirmed by deconvoluted C 1s and O 1s XPS spectral analysis of mGQDs showing significant peaks of CO and C−O, indicative of oxidation during carbonization thereby giving red-emission. 1385

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Figure 5. Bioimaging and cellular uptake studies of L929 cells treated with mGQDs after 24 h. (a−d) Control (untreated) cells show only DAPIstained nuclei with no emission in NIR region. However, cells labeled with both DAPI and mGQDs (e−h) show clear NIR signal in the cell cytoplasm. The excitation/emission wavelengths used for DAPI and mGQDs were 750/780 nm and 561/637 nm, respectively. (i−j) Cellular uptake was performed using flow cytometry showing 100% cell uptake as compared to untreated control cells.

3.2. Morphological Characteristics of mGQDs. The morphology of mGQDs investigated by FEG-TEM showed that mGQDs (Figure 2a) were found to be sized in the range of 2−8 nm which is in agreement with results obtained by the DLS (inset Figure 2a), calculated to be the average size of 4−7 nm. The formation of these nanostructures may be attributed to carbonization of the MIE during microwave synthesis.53 The degree of carbonization of the material helps in controlling the size of the carbon nanomaterial. The lattice structure of mGQDs clearly indicates about its crystalline nature with lattice parameter 0.32 nm measured among its zigzag structure (Figure 2b). 3.3. Composition of mGQDs. Raman spectroscopy is an excellent method to determine the defect or impurity in graphitic carbon which influences the structure of native carbon matrix. Defects in the carbon structure lead to rising in the defect- or D-band in the Raman spectrum which appeared in mGQDs at ∼1324 cm−1 is indicative of disorder caused in the crystalline sp2 carbon skeleton and a graphitic G-band at ∼1574 cm−1 corresponding to the graphitic domains in the structure.54

Thus, the Raman spectrum reveals that the structure of the mGQDs is similar to graphene oxide with a clear demonstration of the presence of sp2 carbon as well as sp3 hybridized carbon atoms which are responsible for the defects and disorder within the carbon skeleton.55 The intensity ratio ID/IG for the mGQDs was calculated to be 0.84.7 Results from XRD analysis are displayed in Figure S3. The figure depicts a [002] diffraction peak centered at 24° for mGQDs corresponding to the graphitic structure with an interlayer spacing of 0.32 nm (due to oxygen-containing species at the edges of mGQDs).56 The occurrence of oxygen-containing functionalities in the structure has been already evident by FTIR spectrum and also supported by an XPS spectrum of mGQDs showing presence of oxygencontaining functional groups in the mGQDs skeleton (Figure 3b−d). The survey spectrum (Figure 3b) shows a presence of C 1s and O 1s peaks, and its deconvoluted forms depict these peaks in the Figure 3c,d, respectively. The C 1s spectrum reveals the presence of C−C, C−O, and CO binding energy at 284.5, 286.0, and 288.0 eV, respectively. The presence of 1386

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Figure 6. Intracellular localization of mGQDs in L929 cells stained with DAPI and mGQDs. (a) The optical section with x-, y- axes and corresponding projections of x-, z- and y-, z- axes of L929 cells; (b) 2.5D imaging and (c, d) 3D intracellular imaging showing an obvious localization signals from nuclei stained with DAPI and cytoplasm stained with mGQDs.

Figure 7. Temperature sensing study: (a) Fluorescence spectra of mGQDs in the temperature range of 10−80 °C, (b) fluorescence intensity histogram of mGQDs at different temperatures plotted at 680 nm, and (c) temperature sensing stability of mGQDs obtained by plotting the fluorescence intensities versus number of cycles of temperature switching at 30−80 °C.

mGQDs treatment indicating increased cell proliferation. Also, mGQDs treated cells did not show any effect on G0-phase (SubG1 phase) of cells (0% as compared to 0.27% in control cells) indicative of excellent biocompatibility. Biocompatibility is an important parameter that dictates the possibility for successful administration of nanomaterials for the biological applications like bioimaging and theranostics.57,49 Biocompatibility analysis using MTT assay revealed that more than 95% L929 cells were viable even after 24 h of incubation of mGQDs

CO and C−O binding energy peaks in the O 1s spectrum were observed at 532.2 and 533.3 eV, respectively. 3.4. Cell Cycle Analysis and Biocompatibility of mGQDs. Cell cycle analysis was performed to check the effect of mGQDs on cell cycle of L929 cells. The G2-phase of the cell cycle is characterized by protein synthesis and cell growth of the cell. Cells showed distinct G1, S, and G2-phases in control as well as mGQDs treated cells (Figure 4a). In contrast to control, the G2-phase increased from 21.6 to 45.4% after 1387

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Figure 8. Live intracellular temperature sensing using mGQDs (0.1 mg/mL) in L929 cells. A representative area was selected, and the temperature was varied from 25 to 45 °C with the aid of temperature controller in CLSM. The fluorescent intensity of the area was scanned under similar laser conditions at different temperatures. Each row in the figure shows an image of mGQDs internalized cells excited with a red laser, its overlay with DIC image, and the fluorescence intensity profile arose from the selected cells from the same area.

at a concentration ranging from 5 μg/mL to as high as 0.1 mg/ mL (Figure 4b). Cell cycle and MTT assay therefore ascertain that mGQDs are biocompatible could be used for in vitro and in vivo applications. 3.5. Bioimaging and Cytoplasm Labeling Using mGQDs. Cellular internalization of mGQDs was confirmed by confocal microscopic images shown in Figure 5a−d show untreated control L929 cells labeled with DAPI only whether Figure 5e−i show the images of cells treated with mGQDs and further stained with DAPI. The cells show no NIR emission (637 nm) upon laser excitation at 561 nm. Cell nucleus was stained with DAPI and mGQDs stained cytoplasm thus not colocalizing with each other (Figure S4). Cellular uptake study done by flow cytometry of mGQDs demonstrated the 100% internalization of mGQDs (Figure 5i).

Intracellular localization was further ascertained by analysis of Z-stacked images captured in CLSM using orthogonal imaging, 2.5D imaging, and 3D imaging modes (Figure 6). Orthogonal imaging mode shows intracellular localization of DAPI and mGQDs in x-, z- and y-, z- axes (Figure 6a). In Figure 6b, 2.5D imaging mode shows the distribution of cells along x-, y- axes projecting separate signals along z-axis from DAPI and mGQDs localized in different cellular compartments. Last, 3D imaging (Figure 6c,d) shows a different 3D view of the cells confirming complete intracellular localization of DAPI in the nucleus and mGQDs in the cytosol. Thus, mGQDs serve as a biocompatible and economical probe for cell cytoplasm labeling applications. 3.6. Temperature-Sensing Application of mGQDs. The temperature variation in the cell is an important phenomenon as it governs the intricate cellular functioning.58 Menter et al. showed fluorescence of cells changes with the denaturation of 1388

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ACS Sustainable Chemistry & Engineering proteins brought about by the temperature.59 Chang et al. reported a temperature study performed using organosilanedoped C-dots that displayed a decrease in fluorescence with increase in temperature.60 Thus, a green-synthesized sensing probe would be a very economical alternative that can be used as a sensor for evaluating the changes in the temperature. Figure 7a,b shows an inverse relationship between fluorescence intensity of mGQDs with the temperature change; the intensity of fluorescence decreased with the increase in the temperature. The decrease in the fluorescence intensity was calculated up to 58% with the increase in temperature from 10 to 80 °C. The temperature sensing efficiency and stability of mGQDs was further confirmed by changing the temperature of mGQDs dispersion from room temperature (30 °C) to 80 °C and subsequently recording the respective fluorescence intensity (Figure 7c); the fluorescence intensity was largely constant even after 5 cycles of temperature sensing experiment showing the stability in the mGQDs as a sensing probe. The increase in temperature of the environment causes the increase in the energy transfer efficiency which subsequently decreases the collision time of the molecules leading to a dynamic collision in the solution quenching the fluorescence of the particle.61,62 Studies on the effect of variations in temperature on the fluorescence of tissue have shown higher fluorescence intensity at the lower temperature.63,64 Figure 8 shows intracellular temperature sensing of L929 cells preincubated with mGQDs. The mGQDs internalized in L929 cells showed intense fluorescence at 25 °C which gradually decreased over temperature increment up to 45 °C. The fluorescence intensity decreases up to 95% within the 20 °C temperature change which indicates that it can be used to detect the minute temperature variation in the cellular environment. Changes in the intracellular cytosolic environment have been detected recently using C-dots and gold nanoclusters in 293T cells after fixation.65 We successfully carried out live intracellular temperature sensing of L929 cells using mGQDs as an NIR imaging probe. This will help to develop new biocompatible tools with inherent NIR emission capability to image and sense intracellular perturbations like temperatures in a noninvasive manner.



AUTHOR INFORMATION

Corresponding Author

*E-mail: [email protected]. Phone: +91-022-25764761. Tel.: +91 22 25767746. ORCID

Mukesh Kumar Kumawat: 0000-0002-8847-3691 Rohit Srivastava: 0000-0002-3937-5139 Author Contributions

M.T. and R.B.G. contributed equally to the creation of this work. Notes

The authors declare no competing financial interest.



ACKNOWLEDGMENTS M.K.K. acknowledges Ministry of Human Resources and Development (MHRD), India for providing financial support. We also acknowledge Sophisticated Analytical Instrumentation Facility (SAIF), Industrial Research Consultancy Centre (IRCC), Indian Institute of Technology Bombay (IITB), for providing central instrumentation facility.



REFERENCES

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4. CONCLUSIONS In conclusion, we demonstrate scalable, rapid, and economically viable green synthesis of mGQDs using mango leaves as a carbon source that showed bright red fluorescence. The mGQDs were highly biocompatible and photostable, demonstrated excellent cellular uptake, and showed selective emission in NIR region. Surface modification of mGQDs can tune the fluorescence emission realizing its potential toward in vivo bioimaging. Furthermore, mGQDs showed good resolution over the wide temperature range in vitro. We, therefore, showed the intracellular temperature sensing capability of mGQDs under live cellular conditions. This will pave the way in devising novel green-synthesis-based miniaturized temperature-sensing probes such as mGQDs with multifunctionalities in biomedical nanotechnology in future.



Data on photostability analysis, lifetime study, XRD spectrum of mGQDs, and CLSM imaging (PDF)

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S Supporting Information *

The Supporting Information is available free of charge on the ACS Publications website at DOI: 10.1021/acssuschemeng.6b01893. 1389

DOI: 10.1021/acssuschemeng.6b01893 ACS Sustainable Chem. Eng. 2017, 5, 1382−1391

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DOI: 10.1021/acssuschemeng.6b01893 ACS Sustainable Chem. Eng. 2017, 5, 1382−1391