Green preparation of versatile nitrogen-doped carbon quantum dots from watermelon juice for cell imaging, detection of Fe3+ ions and cysteine, and optical thermometry

Journal of Molecular Liquids 269 (2018) 766–774

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Journal of Molecular Liquids journal homepage: www.elsevier.com/locate/molliq

Green preparation of versatile nitrogen-doped carbon quantum dots from watermelon juice for cell imaging, detection of Fe3+ ions and cysteine, and optical thermometry Meice Lu, Yixing Duan, Yiheng Song, Jisuan Tan, Li Zhou ⁎ Key Laboratory of New Processing Technology for Nonferrous Metal and Materials (Ministry of Education), and College of Materials Science and Engineering, Guilin University of Technology, Guilin 541004, PR China

a r t i c l e

i n f o

Article history: Received 25 June 2018 Received in revised form 1 August 2018 Accepted 18 August 2018 Available online 20 August 2018 Keywords: Nitrogen-doping Carbon quantum dots Fluorescence Cell imaging Detection of Fe3+ and cysteine Optical thermometry

a b s t r a c t Photoluminescent nitrogen-doped carbon quantum dots (N-CQDs) hold great promise for numerous applications because of their attractive attributes. However, the development of N-CQDs with bright fluorescence and unique optical responsibility is still in its infancy. Herein we present a facile and green approach to prepare versatile N-CQDs via hydrothermal carbonization of watermelon juice. The resulted N-CQDs with high fluorescence quantum yield (10.6%) showed superior water dispersibility, stable photoluminescence, and low cytotoxicity. In addition, the N-CQDs exhibited selective and sensitive fluorescence quenching behavior towards Fe3+ ions and the limit of detection (LOD) can reach 0.16 μM. It was also demonstrated that the N-CQDs/Fe3+ system can be employed to selectively sense cysteine (LOD = 0.27 μM) based on the fluorescence turn on effect. Moreover, the N-CQDs also showed interesting temperature-dependent fluorescence behavior and can be utilized as a nanothermometer, as demonstrated by determining temperature in cells in this study. © 2018 Elsevier B.V. All rights reserved.

1. Introduction Carbon quantum dots (CQDs), with the merits of favorable biocompatibility, superior water dispersibility, unique optical property and low cost, have captured increasing attention for promising applications in fields ranging from bioimaging and sensor to catalysis [1–7]. To date, a variety of precursors have been utilized to prepare CQDs through “bottom up” or “top down” strategy [8–10]. Among them, the use of natural biomass to prepare CQDs has received considerable attention due to its sustainable, easy, available, and low-cost characteristics [11–15]. On the other hand, most of the reported CQDs presented relatively low fluorescence quantum yield (QY) (b10%), and thus their down-toearth applications are heavily restricted. Recently, doping CQDs with N atom was demonstrated to be an effective method to enhance the QY of CQDs [16–20]. Although nitrogen-doped CQDs (N-CQDs) can be achieved by tailoring the surface of CQDs with nitrogen-containing functional molecules, the surface modification process is generally complex and time consuming. Alternatively, the N-CQDs can be directly prepared by hydrothermal treatment of nitrogen-containing raw materials. Several kinds of nitrogen-containing biomass including orange juice, lemon juice, garlic, cocoon silk and cabbage have been used to prepare N-CQDs [21–25]. Furthermore, the nitrogen-doping ⁎ Corresponding author. E-mail address: [email protected] (L. Zhou).

https://doi.org/10.1016/j.molliq.2018.08.101 0167-7322/© 2018 Elsevier B.V. All rights reserved.

process can impart unexpected surface properties such as optical responsibility and catalytic activity to the CQDs [26–28]. Despite the progress made so far, the use of easily available biomass to prepare N-CQDs with high QY and versatile surface properties is still in its infancy. Fe3+ ion is one of the most essential metal ions for living systems. It plays crucial roles in numerous biochemical processes, such as formation of red cells, oxygen transport and exchange, and enzyme catalysis, and thus the sensing of Fe3+ ion is of great importance. Recently, the utilization of fluorescent CQDs for detection of Fe3+ ions have been reported [17,18,29,30]. On the other hand, cysteine (Cys) as a kind of important amino acid plays important role in human body, such as protein synthesis and detoxification. The abnormal level of Cys may cause serious physiological problem such as liver damage, slow growth rate, lethargy, hair depigmentation, and so on [31]. Several kinds of fluorescent probes including fluorescent small molecules, DNA and CQDs have been utilized to detect Cys [32–35]. However, it remains a big challenge to sensitive and selective detections of Cys from various amino acids because of their analogues molecular structure and composition. To address this problem, exploring fluorescent probes that are able to selective sensing of Cys is highly desired. Optical thermometry, a newly developed non-contact technique for temperature measurement, has drawn tremendous attention because of its promising for determining temperature in many harsh situations, such as cells and tissues, where the conventional thermometer is not applicable [36,37]. Among various optical thermometry approaches,

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particular attention was paid on the luminescence thermometry technique, which depends on the temperature-dependent luminescence behavior. Various materials including semiconductor quantum dots, dyes, polymers, and lanthanide doped inorganic particles have been studied for luminescence thermometry [37–40]. However, the drawbacks of low sensitivity, high toxicity, and poor optical stability in the surrounding environment significantly restrict their real applications. Although the CQDs have been widely studied in recent year, however, very limited papers on utilizing fluorescent CQDs for optical thermometry [41–43]. In this contribution, we report a facile approach to synthesize highly water-dispersible N-CQDs by hydrothermal carbonization of watermelon juice at a mild condition (Scheme 1). The as-prepared N-CQDs with high fluorescence QY value (10.6%) displayed superior aqueous stability, stable fluorescence against changes of solution pH and ionic strength, low cytotoxicity, and intensive fluorescence in cancer cells. Moreover, the N-CQDs showed on-off-on fluorescence behavior after addition of Fe3+ ions and followed by Cys. The detection of Fe3+ ions is based on their quenching effect on the fluorescence of N-CQDs, while the sensing of Cys is ascribe to the binding preference of Cys towards Fe3+ ions. The fluorescence of N-CQDs also highly depends on external temperature and the utilization of N-CQDs as a luminescence nanothermometer to determine the temperature in cells was studied in this study.

Analytical Ltd) with mono Al Kα radiation (hν = 1487.71 eV). Fourier transform infrared (FTIR) spectra were collected on a FTIR 2500 spectrometer using KBr disk method in the range of 400–4000 cm−1. Raman measurements were performed on a LabRam-1B Raman spectroscope equipped with a 632.8 nm laser source. X-ray diffraction (XRD) pattern was recorded on a Holland PANalytical X-Pert PRO Xray diffractometer with Cu Ka radiation. Emission and excitation spectra were determined using a Varian Cary 100 spectrometer. Absorption spectra were collected using a UV-3600 UV-VIS-NIR spectrophotometer (Shimadzu). Photoluminescence QY value of the N-CQDs were measured on a Hamamatsu absolute photoluminescence quantum yield spectrometer C11347 Quantaurus-QY. Confocal laser-scanning microscopy (CLSM) measurements were conducted on a Zeiss LSM 510 CLSM.

2. Experimental

Typically, 2.5 mL of aqueous N-CQDs dispersion (2.5 μg/mL) was added to a cuvette (3.5 mL). Then 0.5 mL of Fe3+ ions (or other metal ions) solution with known concentration was added. The mixture was shaken for 30 s before measuring the emission spectrum of mixture at room temperature (λex = 355 nm).

2.1. Materials NaCl (99.5%), CaCl 2 (99.99%), AgNO 3 (99.9%), CdCl 2 (99.99%), Co(NO 3 ) 2 ·6H 2 O (99.99%), MnCl 2 ·4H 2 O (99.99%), CuCl 2 ·2H 2 O (99.99%), Pb(NO3)2 (99.99%), Ni(NO3)2·6H2O (99.0%), FeSO4·7H2O (99.5%), Zn(NO3)2·6H2O (99.99%), FeCl3·6H2O (98.0%), dimethyl sulphoxide (DMSO, 99.9%), cysteine (Cys, 97.0%), arginine (Arg, 98.0%), Lysine (Lys, 98.0%), alanine (Ala, 99.0%), threonine (Thr, 98.0%), glycine (Gly, 99.0%), and methylthiazolyldiphenyl-tetrazolium bromide (MTT, 98%) were purchased from Innochem Co. Ltd. (Beijing, China) and used as received. Deionized water was employed throughout all experiments.

2.3. Synthesis of N-CQDs In a typical procedure, 50 mL of fresh-squeezed watermelon juice and 5 mL of ethanol were sealed into a Teflon equipped stainless steel autoclave (100 mL) and heated at 180 °C for 3 h. After the reaction, the resulted solution was centrifuged at 5000 rpm for 10 min to remove big particles. The obtained supernatant was then dialyzed against water (MW cutoff 500 Da) for 24 h to afford aqueous solution of N-CQDs. 2.4. Detection of Fe3+ ions

2.5. Detection of cysteine Typically, 3 mL of aqueous mixture of N-CQDs (2.5 μg/mL) and Fe3+ ions (300 μM) was added to a cuvette (3.5 mL). Subsequently, aqueous solution of cysteine with known concentration was added to the mixture of N-CQDs/Fe3+. After shaking for 30 s, the emission spectrum of mixture was determined at room temperature (λex = 355 nm).

2.2. Characterizations

2.6. Cytotoxicity evaluation

Transmission electron microscope (TEM) images were determined on a JEOL-2010 TEM at 160 kV. X-ray photoelectron spectroscopy (XPS) spectra were determined on Kratos AXIS ULTRA DLD (Kratos

MTT viability assay was employed to evaluate the cytotoxicity of NCQDs [44,45]. Typically, human hepatoblastoma (HepG2) cells were seeded in 96-well plates at a density of 2 × 104 cells/mL before adding

Scheme 1. Schematic illustration of the synthesis of N-CQDs from watermelon juice by hydrothermal treatment for sensing of Fe3+ ions, cysteine and temperature.

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M. Lu et al. / Journal of Molecular Liquids 269 (2018) 766–774

Fig. 1. (a) TEM image of N-CQDs. Raman (b), FTIR (c), XPS survey (d), XPS C1s (e) and XPS N1 s (f) spectra of N-CQDs.

N-CQDs solutions to replace the culture medium. After incubation for 24 h, the wells were washed with PBS buffer and then freshly prepared MTT medium solution (100 μL, 0.5 mg/mL) was added to each well.

After incubation for another 3 h, the MTT medium was removed before adding DMSO (100 μL) to each well. Finally, the viability of HepG2 cells was calculated by the absorbance ratio (λ = 570 nm) of the HepG2 cells

Fig. 2. (a) UV–vis absorption (Abs), photoluminescence excitation (Ex) (λem = 439 nm), and emission (Em) (λex = 355 nm) spectra of aqueous N-CQDs dispersion (inset: photographs of N-CQDs in water under daylight and 365 nm UV light illumination). (b) Emission spectra of aqueous N-CQDs dispersion with increasing excitation wavelength from 315 nm to 455 nm. (c) pH and (d) NaCl concentration dependent fluorescent intensity ratio (I/I0) of the N-CQDs. I0 is the PL intensity of N-CQDs dispersion in the absence of NaCl and at pH 7. I is the PL intensity of N-CQDs dispersion at diverse pH values (c) or diverse concentrations of NaCl (d).

M. Lu et al. / Journal of Molecular Liquids 269 (2018) 766–774

incubated with N-CQDs to that of the HepG2 cells incubated with culture medium only. 2.7. Cell imaging In a typical procedure, the aqueous N-CQDs dispersion (20 μg/mL, 0.5 mL) was incubated with HepG2 cancer cells in culture medium for 4 h at 37 °C. After that, the cells were washed three times with PBS buffer to remove the excess N-CQDs and fixed by 75% ethanol solution for 30 min. After washing with PBS buffer for two times, imaging of HepG2 cancer cells was conducted on a CLSM (Zeiss LSM 510). The temperature of sample could be adjusted using a heater during the CLSM observation.

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centered at around 439 nm. Because high fluorescent stability at harsh environment is of great importance for the practical application of NCQDs, the effect of solution pH and electrolyte on the fluorescent performance of N-CQDs dispersion was examined. As depicted in Fig. 2c, the N-CQDs solution showed relatively stable emission intensity with increasing solution pH from 3 to 9. The emission intensity decreases no N20% even at acidic or basic solution. We also studied the influence of electrolyte (NaCl) on the fluorescence property of N-CQDs. As presented in Fig. 2d, no obvious intensity decline is observed at low concentration of NaCl (0.1 M) and lower than 15% decline was detected even at high concentration of NaCl (1.0 M), demonstrating favorable fluorescence stability of the N-CQDs in the existence of electrolyte. Therefore, the N-CQDs possess stable fluorescence property, posing a favorable platform for sensing and imaging applications.

3. Results and discussion 3.2. Cytotoxicity evaluation, cellular imaging and photostability evaluation 3.1. Synthesis and characterization of N-CQDs Through facile hydrothermal treatment of watermelon juice at 180 °C for 3 h, highly fluorescent N-CQDs were synthesized (Scheme 1). The N-CQDs show excellent dispersibility and stability in water and no precipitates were observed even after storage in water for four months. Transmission electron microscopy (TEM) image in Fig. 1a reveals that the N-CQDs possess uniform spherical shape and narrow size distribution (3–7 nm). As can be observed in the high-resolution TEM image, most of the N-CQDs are crystalline with an interplanar spacing of 0.25 nm, which is in accordance with (020) lattice fringes of graphitic carbon [46]. In addition, some of amorphous N-CQDs could also be seen. The Raman spectrum of N-CQDs in Fig. 1b shows two obvious bands at 1365 and 1588 cm−1, corresponding to the D and G bands of graphitic structure, respectively. The intensity ratio of the D band to the G band (ID/IG) is calculated to be 0.89, further supporting the partial amorphous characteristic of the N-CQDs. The XRD pattern of N-CQDs was determined. As depicted in Fig. S1, a broad diffraction band cantered at around 25 o is observed, which is assigned to the (002) plane of graphitic carbon [18,47]. From the FTIR spectrum in Fig. 1c, the strong band at 3401 cm−1 assigned to OH group, two bands at 2854 and 2945 cm−1 associated with C\\H stretching, and four obvious bands at 1056, 1217, 1648 and1756 cm−1 respectively correspondence to the C\\O, C\\N, C_C and C_O groups can be clearly seen. These oxygen-containing functional groups can impart superior water dispersibility to the N-CQDs with even without further surface tailoring. To gain more insight on structure information, the N-CQDs was characterized by X-ray photoelectron spectroscopy (XPS). The XPS survey spectrum (Fig. 1d) shows a strong N1 s band centered at 400.3 eV and the content of N is 8.7 wt%, demonstrated the successful formation of N-CQDs by the hydrothermal treatment of watermelon juice. Similar to the FTIR results, the C1s spectrum in Fig. 1e indicates that the NCQDs contain C\\O, C\\N, C_C, C_N and C_O groups. The corresponding high-resolution N1 s spectrum (Fig. 1f) further confirmed the existence of pyrrolic and pyridinic N atoms. The absorption spectrum of N-CQDs exhibits a strong band centered at 282 nm and a relatively weak shoulder band centered at around 355 nm, which are attributed to the π-π* transition of C_C or C_N bonds and the n–π* transition of C_O bonds, respectively [15,48]. Owing to their narrow size distribution, the N-CQDs solution presented symmetrical emission spectrum and intense fluorescence under illumination of 365 nm UV light (Fig. 2a). The fluorescence QY of N-CQDs was determined to be 10.6%, which is much higher than many reported biomass-derived CQDs [49–51]. In addition, the obtained N-CQDs also showed excitation-dependent emission spectrum like other reported N-CQDs [15–22]. With the increase of excitation wavelength from 315 nm to 455 nm, the corresponding emission peak of N-CQDs dispersion gradually shifted from 424 nm to 512 nm (Fig. 2b). In agreement with the excitation spectrum, the optimal excitation wavelength occurred at 355 nm, corresponding to the maximum emission band

The cytotoxicity of N-CQDs was examined for human hepatoblastoma (HepG2) cells using standard methylthiazolyldiphenyl-tetrazolium bromide (MTT) assay. As presented in Fig. 3a, the cell viabilities within the tested period are close to 100% at low concentration of N-CQDs (≤100 μg/mL) and remain higher than 90% even at high concentration of N-CQDs (300 μg/mL), demonstrated the low cytotoxicity of N-CQDs. Moreover, the utilization of N-CQDs as a fluorescent probe for cellular imaging was also studied. Fig. 3b and c show the confocal laser scanning

Fig. 3. Cell viability of HepG2 cells after incubation with N-CQDs at varying concentrations for 4 h and 24 h, respectively. CLSM fluorescence (b) and merged (c) images of HepG2 cells upon incubation with N-CQDs (20 μg/mL) for 2 h. CLSM fluorescence (d) and merged (e) images of HepG2 cells without incubation with N-CQDs. The scale bar is 20 μm.

M. Lu et al. / Journal of Molecular Liquids 269 (2018) 766–774

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0 µM 10 µM 30 µM 50 µM 80 µM 100 µM 150 µM 200 µM 250 µM 300 µM

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B la nk N + a C 2 a + M n 2+ N 2 i + C d 2+ C o 2+

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N + a C 2 a + M n 2+

0.2 0.4

Fig. 4. (a) Emission spectra of aqueous N-CQDs dispersion upon addition of various concentrations of Fe3+ ions (λex = 355 nm). (b) The dependence of I/I0 value on the concentration of Fe3+ ions. (c) Emission spectra of N-CQDs after incubation with 300 μM of Fe3+ ions for different time (λex = 355 nm). (d) Photographs of aqueous N-CQDs dispersions (5 μg/mL) under 365 nm UV light with 200 μM of diverse metal ions. (e) I/I0 value of N-CQDs dispersions with diverse metal ions at 200 μM each. (f) Selective fluorescence response of N-CQDs dispersions towards 200 μM of Fe3+ ions (blue bar), and interference of 200 μM of other metal ions with 200 μM of Fe3+ ions (red bars). I0 is the emission intensity of N-CQDs dispersion in the absence of metal ions. I is the emission intensity of N-CQDs dispersion with various metal ions. (For interpretation of the references to colour in this figure legend, the reader is referred to the web version of this article.)

microscopy (CLSM) images of HepG2 cancer cells after incubation with NCQDs (20 μg/mL). In agreement with low cytotoxicity of the N-CQDs, all the HepG2 cancer cells maintained their normal morphology. Furthermore, the cellular cytoplasm emitted strong blue fluorescence, indicating that N-CQDs are efficiently internalized by HepG2 cells and accumulated in the cytoplasm. On the contrast, no fluorescence is seen for the HepG2 cells without incubation with N-CQDs (Fig. 3d and e). In addition, the photostability of N-CQDs was examined by treating the N-CQDs with continuous CLSM scanning for 10 min. For comparison, fluorescein was also studied under the same condition. As can be seen in Fig. S2, no obvious decline of emission intensity is observed for N-CQDs, while the emission intensity of fluorescein decreased by almost 90% after 10 min. Therefore, the obtained N-CQDs are promising for cell imaging because of their low toxicity, high fluorescence contrast, and favorable optical stability. 3.3. Detection of Fe3+ ions Upon addition of Fe3+ ions into the aqueous N-CQDs dispersion, the emission intensity of N-CQDs dispersion decreases gradually (Fig. 4a), implying that the introduction of Fe3+ ions can efficiently quench the

fluorescence of N-CQDs. The quenching efficiency (I/I0) presented good linear relationship (R2 = 0.9811) versus the concentration of Fe3+ ions in the range of 0–300 μM (Fig. 4b). The detection of limit was determined to be 0.16 μM at a signal-to-noise of 3. Such high sensitivity compares favorably to many reported CQDs (see Table 1) [52–55]. Moreover, the fluorescence quenching rate is very fast and the quenching interaction between the N-CQDs and Fe3+ ions could be completed within 0.5 min (Fig. 4c). To determine the selectivity of N-CQDs towards Fe3+ ions, various environmentally relevant metal ions, including Ag+, Na+, K+, Co2+, Ca2+, Mn2+, Cd2+, Zn2+, Ni2+, Cu2+, Fe2+, Pb2+ and Fe3+ ions were respectively added into the N-CQDs dispersions with the concentration of 200 μM. As depicted in Fig. 4d, the fluorescence of N-CQDs dispersion was significantly quenched by Fe3+ ions, while other metal ions exhibited negligible or slight fluorescence quenching effect, indicating that the N-CQDs have high selectivity towards Fe3+ ions. Consistent with the photographs shown in Fig. 4d, the I/I0 results (Fig. 4e) confirm that the fluorescence quenching capability of Fe3+ ions is much higher than other metal ions. Furthermore, the effect of coexisting metal ions on the selective sensing of Fe3+ ions was also investigated. The fluorescent intensity of N-CQDs dispersion

Table 1 CQDs-based sensors for detection of Fe3+ ions or Cys. Detection 3+

Fe Fe3+ Fe3+ Fe3+ + Fe3+ Fe3+ Fe3+ Fe3+ Fe3+ Cys Cys Cys Cys

Sensors

Linear range

LOD

Reference

N-CQDs from m-aminobenzoic acid N-CQDs from alginic acid N-CQDs from Chionanthus retusus N-CQDs from Phyllanthus acidus N-CQDs from Magnolia liliiflora N-CQDs from Prunus avium N-CQDs from rose-heart radish CQDs from blueberry N-CQDs from watermelon juice CQDs/Au nanocluster S, N-CQDs from citric acid and VB1 S, N-CQDs from citric acid cysteine N-CQDs from watermelon juice

0–1.6 μM 0–0.05 mM 0–20 μM 2–25 μM 1–1000 μM 0–100 μM 0.02–40 μM 12.5–100 μM 0–300 μM 0–60 μM 0–120 μM 10–200 μM 0–250 μM

0.05 μM 10.98 μM 70 μM 0.9 μM 1.2 μM 0.96 μM 0.13 μM 9.97 μM 0.16 μM 0.10 μM 0.35 μM 0.54 μM 0.27 μM

17 18 27 29 30 47 48 52 This study 33 61 62 This study

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blank 3+ Fe 3+ Fe +Cys 3+ Fe +Lys 3+ Fe +Arg 3+ Fe +Ala 3+ Fe +Thr 3+ Fe +Gly

200

100

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0.8 0.6 0.4 0.2

Th r G ly

la A

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0.0

rg

650

A

600

Ly s

550

ys

500

k

450

C

400

la n

0 350

B

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Cys

I/I0

PL Intensity (a.u.)

300

771

Fig. 5. (a) Emission spectra of the mixture of N-CQDs/Fe3+ ions (300 μM) after adding diverse concentrations of Cys. (b) Photographs of aqueous N-CQDs dispersion upon addition of Fe3+ ions (300 μM) and followed by adding Cys (0.65 mM) under 365 nm UV light. (c) Emission spectra of aqueous N-CQDs dispersions before and after adding Fe3+ ions (300 μM) and various amino acid (0.65 mM). (d) I/I0 value of N-CQDs dispersions with Fe3+ ions (300 μM) and various amino acid (0.65 mM). I0 is the emission intensity of N-CQDs dispersion without adding Fe3+ ions and amino acid. I is the emission intensity of N-CQDs dispersion with Fe3+ ions (300 μM) and various amino acid (0.65 mM).

at 439 nm after adding 200 μM of Fe3+ ions alone (blue bar) and the mixtures of 200 μM of Fe3+ ions and 200 μM of other metal ions (red bars) were determined. As shown in Fig. 3f, other coexisting metal ions exhibit slight quenching effect on the fluorescence of N-CQDs as compared with the Fe3+ ions. The high detection sensitivity and selectivity of N-CQDs towards Fe3+ ion is possibly ascribed to the strong coordinate interactions between the Fe3+ ions and the surface functional groups (e.g. hydroxyl groups, amine groups and carboxylic groups) of N-CQDs [56–59]. The strong interactions can disrupt the radiative transition, leading to the fluorescence quenching of N-CQDs [29,30]. All the above results suggest that the N-CQDs could be utilized as a highly efficient fluorescent probe for detection of Fe3+ ions in water. 3.4. Detection of cysteine (Cys) Since the N-CQDs can selectively sense Fe3+ ions based on the fluorescence tune-off behavior, it was expected that the N-CQDs/Fe3+ system can be utilized to detect specific amino acid that can recover the fluorescence of N-CQDs. To our delight, the fluorescence of N-CQDs/ Fe3+ system was gradually recovered after addition of Cys (Fig. 5a). The plot of I/I0 against 0–250 μM of Cys presents a good linear relationship with the R2 value of 0.9869 (Fig. S3). As depicted in Fig. 5b, the strong blue fluorescence of aqueous N-CQDs dispersion was firstly quenched by adding Fe3+ ions and then recovered followed by addition of Cys, implying that the fluorescence of N-CQDs/Fe3+ system was sensitive to Cys. We think that the competitive interaction between the Cys and N-CQDs towards Fe3+ ions lead to remove Fe3+ ions from the surface of N-CQDs, and thus the fluorescence could be turned on. The limit of detection of N-CQDs/Fe3+ system for Cys was measured to be 0.27 μM, which compares favorably to many reported fluorescent probes (see Table 1) [60–62]. To examine the selectivity of N-CQDs/Fe3+ system for Cys, changes in the fluorescence intensity of N-CQDs/Fe3+

system caused by addition of other amino acids, including Lys, Arg, Ala, Thy and Gly were also determined. Fig. 5c shows the photoluminescent spectra of the aqueous dispersion of N-CQDs/Fe3+ system after addition of diverse amino acids. It was observed that other amino acids showed negligible effect on the fluorescence of N-CQDs/Fe3+ system (Fig. 5d), indicating the high selectivity of N-CQDs/Fe3+ system for Cys over other amino acids. The high selectivity is possibly attributed to the presence of thiol group in Cys since the interaction between thiol group and Fe3+ ion is very strong. 3.5. Optical thermometry Interestingly, the obtained N-CQDs also exhibited interesting temperature-dependent fluorescence behavior. As shown in Fig. 6a, the fluorescent intensity of aqueous N-CQDs dispersion declined significantly with increasing external temperature. Correspondingly, the photographs of aqueous N-CQDs dispersion at 10 °C and 50 °C under 365 nm UV light are presented in Fig. 6b. As can be seen, the N-CQDs dispersion exhibited strong blue fluorescence at 10 °C, while only weak fluorescence was observed at 50 °C. To further probe the relationship between the temperature and fluorescent intensity of N-CQDs dispersion, the curve of fluorescent intensity against temperature variation was plotted as shown in Fig. 6c. Clearly, linear relationship with high coefficient value (R2 = 0.9874) was obtained between the fluorescent intensity and temperature. The fluorescent intensity of N-CQDs declined by 51.0% within 97 °C, indicating a sensitivity of 0.52%/°C [42,43,63]. Accordingly, the N-CQDs hold great promise as a temperature sensor based on their high fluorescence sensitivity towards the variation of temperature. To further determine the reproducibility of N-CQDs with temperature change, five consecutive heating-cooling cycles between 20 °C and 40 °C were performed. As presented in Fig. 6d, the N-CQDs show stable fluorescence at the corresponding temperature. Therefore,

M. Lu et al. / Journal of Molecular Liquids 269 (2018) 766–774

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Cycle number

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Fig. 6. (a) Emission spectra of aqueous N-CQDs dispersion at different temperature. (b) Infrared thermal and fluorescent images of aqueous N-CQDs dispersion at 10 °C and 50 °C. (c) Emission intensity of N-CQDs dispersion against temperature variation. (d) Emission intensity of the N-CQDs dispersion as a function of heating-cooling cycles between 20 °C and 40 °C.

the N-CQDs can serve as an ideal temperature nanosensor with high accuracy and reproducibility. Considering that the obtained N-CQDs possess temperature-dependent fluorescence and high fluorescence stability at harsh environment, the utilization of N-CQDs as a nanothermometer for monitoring intracellular temperature was studied. HepG2 cancer cells were selected as a model system to incubate with N-CQDs and then the fluorescence of HepG2 cells at different temperatures were determined by CLSM (Fig. 7). Obviously, the cellular cytoplasm emitted strong blue fluorescence at 20 °C (Fig. 7a). Upon increasing temperature to 40 °C, the blue fluorescence in cell declined significantly (Fig. 7b), consistent with the changes seen from the photographs and fluorescent spectra. These results suggest the potential of N-CQDs in detecting temperature at the cellular level and, highlight their potential for applications in optical thermometry.

showed superior water dispersibility, favorable fluorescence stability against variations of pH and ionic strength, low cytotoxicity, and high fluorescent contrast in cells. The fluorescence of N-CQDs can be quenched by Fe3+ ions with high sensitivity and selectivity. In addition, the N-CQDs/Fe3+ system exhibited sensitive and selective fluorescence turn on behavior towards Cys. Moreover, the N-CQDs also presented interesting temperature-dependent fluorescence behavior and hold great promise as a nanothermometer to monitor intracellular temperature.

4. Conclusions

This work was supported by National Natural Science Foundation of China (No. 51663007 and No. 21364003), Natural Science Foundation of Guangxi Province (No. 2017GXNSFFA198002), and the Project of Thousand Outstanding Young Teachers' Training in Higher Education Institutions of Guangxi.

In summary, we have demonstrated a facile and viable approach to synthesize versatile N-CQDs by hydrothermal treatment of watermelon juice. The obtained N-CQDs with high fluorescence QY simultaneously

Conflict of interest The authors declare no competing financial interest. Acknowledgments

Fig. 7. CLSM fluorescence images of HepG2 cells at 20 °C (a) and 40 °C (b) and bight field image (c) after incubation with N-CQDs (20 μg/mL) for 2 h. The scale bar is 20 μm.

M. Lu et al. / Journal of Molecular Liquids 269 (2018) 766–774

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