Lysosome targeting carbon dots-based fluorescent probe for monitoring pH changes in vitro and in vivo

Chemical Engineering Journal 381 (2020) 122665

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Lysosome targeting carbon dots-based fluorescent probe for monitoring pH changes in vitro and in vivo ⁎

Pengli Gaoa, Jingwen Wanga, Min Zhenga, , Zhigang Xieb,

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a School of Chemistry and Life Science, Advanced Institute of Materials Science, Changchun University of Technology, 2055 Yanan Street, Changchun, Jilin 130022, PR China b State Key Laboratory of Polymer Physics and Chemistry, Changchun Institute of Applied Chemistry, Chinese Academy of Sciences, 5625 Renmin Street, Changchun, Jilin 130022, PR China

H I GH L IG H T S

G R A P H I C A L A B S T R A C T

with green emission are success• CDs fully synthesized. have high pH sensitivity and se• CDs lectivity with pKa of 6.0 ± 0.72. colocalization demonstrates • Lysosome that CDs can target lysosomes. can monitor pH changes in vitro • CDs and vivo.

A R T I C LE I N FO

A B S T R A C T

Keywords: Carbon dots Fluorescent probe Lysosome targeting pH sensitive Imaging

The anomalous fluctuations of pH in lysosome will cause the disorder of normal activity of cells and even many diseases. Thus, developing a convenient strategy for tracking pH changes of lysosome is highly desired. Herein, carbon dots (CDs)-based fluorescent probe with lysosome-targeting function was fabricated. CDs can monitor pH changes in live cells and organisms, with the aid of confocal laser scanning microscope and noninvasive optical imaging system. The advantages of CDs including robust photostability, high pH sensitivity and selectivity, good reversibility and low toxicity render them a prominent candidate to investigate pH-associated physiological and pathological processes.

1. Introduction Intracellular pH is one of the most considerable parameters, of which acid-base dynamic balance can be maintained by the three regulating systems of organisms [1–5]. In normal organisms, the acid-base balance of their physiological environment is always maintained in a certain stabilizing range and cellular dysfunction is often associated with abnormal pH values in organelles. In terms of lysosome, pH is preserved in the acidic environment of 4.5–6.5 [6–9], which plays an unquestionable pivotal function that accelerates the metabolism of

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macromolecular in cells and is closely related to cells aging and human diseases. Consequently, the anomalous fluctuations of the pH in lysosome will cause the disorder of normal activity of cells and even many diseases such as gout, silicosis, cancer and Alzheimer's disease Etc. [10–14]. Therefore, it is highly desirable to develop a convenient strategy for tracking and monitoring pH changes in lysosome [15–18]. Although various techniques such as voltammetry [19], nuclear magnetic resonance [20] and spectrophotometry [21] etc. have been developed for sensing pH, fluorescence spectroscopy is recognized as a more convenient way than other methods due to its low cost, high

Corresponding authors. E-mail addresses: [email protected] (M. Zheng), [email protected] (Z. Xie).

https://doi.org/10.1016/j.cej.2019.122665 Received 14 June 2019; Received in revised form 21 August 2019; Accepted 29 August 2019 Available online 30 August 2019 1385-8947/ © 2019 Elsevier B.V. All rights reserved.

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Chemical Reagent Co., China; Acros; Fluka) and used without further purification. 3-[4,5-dimethylthiazol-2-yl]-2,5-Diphenyltetra-zolium bromide (MTT) was purchased from Beyotime Biotechnology Co., Ltd. (China). Fetal Bovine Serum (FBS) and LysoTracker Red DND-99 were purchased from Dalian Meilun Biotechnology Co., Ltd. Ultrapure water was prepared from a Milli-Q system (Millipore, USA). All other chemicals were of analytical grade or above.

sensitivity, rapid response and operational simplicity. To this end, numerous pH responsive fluorescent probes based on organic dyes [22–32] had been exploited, however, the disadvantages of low water solubility, poor biocompatibility and photostability are a stumbling block for their potential applications in biomedical fields. Therefore, it is imperative to develop a highly efficient probe with high solubility, good biocompatibility and excellent photostability. As a new type of nanomaterial, carbon dots (CDs) exhibit variety of advantages such as small sizes, outstanding photoluminescence, excellent biocompatibility, high water-solubility, good photochemical stability [33–38,39–42] as well as simple synthesis, which have wide applications for sensing [42–43], bioimaging [44–49], nanomedicine [50] and other fields [51–55]. The development of CDs-based fluorescent nanosensors for the detection of ions [43–44,56–57,58–60] (such as H+, Hg2+, Cr (VI), Ag+, Fe3+ and Cu2+) and organic molecules [61–63] (for example volatile organic compounds, glucose and ascorbic acid etc.) have attracted increasing interest. However, according to our understanding, the relatively few reports about CDs-based materials for the detection organelles pH changes have been developed [15,17]. Herein, a kind of CDs-based fluorescent probe with lysosome-targeting ability was synthesized and its reversibility and selectivity were systemically investigated. As illustrated in Scheme 1, with the help of confocal laser scanning microscope (CLSM) and in vivo optical imaging system, the real-time imaging of cellular pH in HeLa cells and tracking pH fluctuations in live mouse were successfully achieved. These results demonstrate that CDs possess excellent photostability, high pH sensitivity and selectivity, good reversibility and low toxicity, which enable them to act as a promising candidate for high resolution pH imaging in cells and organisms. This work highlights the potential application of CDs as a high-performance platform for sensing and bioimaging.

2.2. Characterization Transmission electron microscope (TEM) images were carried out on a JEOL JEM-1011 (Japan) at the accelerating voltage of 100 kV. Fourier transform infrared (FT-IR) spectrum of CDs were recorded on a Bruker Vertex 70 spectrometer from 4000 to 500 cm−1. High-resolution transmission electron microscopy (HR-TEM) image was recorded with FEI-TECNAI G2 transmission electron microscope operating at 200 kV. X-Ray photoelectron spectra (XPS) were obtained on a Thermo Scientific ESCALAB 250 Multitechnique Surface Analysis. Fluorescence emission spectra were carried out on a LS-55 fluorophotometer. UV–vis absorption spectra were conducted on a Shimadzu UV-2450 spectrophotometer. X-ray photoelectron spectra were obtained on a Thermo Scientific ESCALAB 250 Multitechnique Surface Analysis. The Edinburgh FLS 920 spectrometer with a calibrated integrating sphere was used to measure absolute Quantum Yield.

2.3. Synthesis of CDs p-Phenylenediamine (0.5 mmol) and thiourea (1 mmol) were dissolved in water (25 mL), then transferred into a Teflon-lined autoclave. The mixture was heated up to 160 °C and maintained for 10 h. Finally, the crude product was naturally cooled to room temperature and purified via silica column chromatography using a mixture of ethyl acetate and dichloromethane as the eluent. CDs were obtained with a yield of 12.8%.

2. Materials and methods 2.1. Materials All the reagents were purchased from commercial suppliers (Beijing

Scheme 1. Schematic illustrating the fabrication of CDs, their lysosome targeting ability and applications for probing pH changes in vitro and in vivo. 2

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Fig. 1. (A) Transmission electron microscopy (TEM) images of CDs. The inset image is the diameter distribution of CDs. (B) High-resolution TEM image of CDs. (C) The highlighted area of B. (D) FT-IR spectrum of CDs. (E) Full XPS of CDs. (F) High-resolution XPS of N1s. (G) High-resolution XPS of C1s. (H) High-resolution XPS of S2p. (I) XRD patterns of CDs. (J) Raman spectrum of CDs. (K) UV–vis absorption spectra of CDs. (L) Fluorescence spectra of CDs under different excitation wavelengths. (M) Fluorescence lifetime of CDs. (N) Changes of fluorescence intensity of CDs at 532 nm vs irradiation time. (O) Effect of ionic strength on the fluorescence intensity of CDs.

DHP-CA buffers (pH 4.0–8.0) were prepared by mixing DHP (0.2 mol L−1) and CA (0.1 mol L−1) in different proportions (Table S1).

diluting the stock solution to (15 µg L−1) in different pH DHP-CA buffers. The pH-dependent fluorescence behaviours of CDs were measured. And the excitation wavelength was 380 nm. All the spectroscopic experiments were carried out at room temperature.

2.5. CDs fluorescence pH titrations

2.6. Cell culture and (3-(4,5-dmethylthiazol-2-yl)-2,5-diphenyltetrazolium

Stock solutions of CDs (30 µg L−1) were prepared in deionized (DI) water. The solution for spectroscopic determination was obtained by

HeLa cells were purchased from the Institute of Biochemistry and Cell Biology, Chinese Academy of Sciences, Shanghai, China. The cells

2.4. Disodium hydrogen phosphate (DHP)-citric acid (CA) buffer

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3. Results and discussion

were incubated in Dulbecco's modified Eagle's medium (DMEM, GIBCO) supplemented with 10% heat inactivated fetal bovine serum (FBS, GIBCO), 100 U mL−1 penicillin and 100 µg mL−1 streptomycin (Sigma), and the culture medium was replaced once every day.

3.1. Characterization of CDs CDs were synthesized from p-Phenylenediamine and thiourea by hydrothermal method. The transmission electron microscopy (TEM) image (Fig. 1A) shows that the as-prepared CDs possess a uniform diameter of 7.8 ± 0.65 nm with mono-dispersion. High resolution TEM (HR-TEM) image (Fig. 1B) illustrates that CDs have well-resolved lattice structures with lattice spacing d of 0.21 nm (Fig. 1C), in agreement with the basal spacing of graphite. Fourier transform infrared (FT-IR) spectroscopy (Fig. 1D) confirms the presence of numerous functional groups including –CONH2 (1625 cm−1), C]C (1510 cm−1), –N]C]S (2068 cm−1), C-N (1445 cm−1), C–C/C–O/C–S (1251 cm−1) and N–H, O–H (3451 cm−1). X-ray photoelectron spectroscopy (XPS) was used to further investigate the surfaces of CDs. Fig. 1E shows that the full-scan of CDs are mainly composed of carbon (C 1s, 285.1 eV), nitrogen (N 1s, 399.7 eV), sulfur (S 2s, 226.6 eV), sulfur (S 2p, 162.6 eV), and oxygen (O 1s, 533.2 eV). The high-resolution spectrum of N 1s (Fig. 1F) exhibits two main peaks at 399.1 and 400.6 eV, which reveal the presence of both pyridinic type and pyrrolic type N atoms, respectively. The high-resolution XPS spectrum of C 1s (Fig. 1G) can be well deconvoluted into three characteristic peaks at 284.6, 285.7 and 288.7 eV, which belong to C–C/C]C, the presence of C–O/C–S/C–N, and C]N/C]S/C]O, respectively. Fig. 1H displays the high-resolution spectrum of S 2p which reveals the presence of S 2p3/2C-S-C (161.9 eV) and S 2p1/2C-S-C (163.1 eV). The surface components of CDs determined by XPS are in good agreement with FT-IR results. As shown in Fig. 1I, the XRD pattern of CDs, displays two peaks centered at 21° and 41.3°, which are contributed to the graphitic structure with interlayer spacing (0 0 2) of 0.41 nm and interlayer spacing (1 0 0) of 0.21 nm, respectively, verifying the high-crystalline graphitic structure of CDs. The Raman spectrum of CDs (Fig. 1J) exhibits two peaks at 1366 and 1553 cm−1, the former corresponds to the sp3 domains of graphene edges (D band), while the latter is related to the in plane stretching vibration of sp2 carbon atoms (G band). The ratio of ID to IG is 0.97, which indicates that CDs possess a high degree of graphitization. As shown in Fig. 1K, the UV–vis spectrum of CDs exhibits three bands at 290 nm, 360 nm and 515 nm. The former one may be attributed to the formation of multiple polyaromatic chromophores and correspond to the π-π* transition of the aromatic sp2 domains, the latter two belong to the n-π* transition of CDs [44]. Fig. 1L displays the fluorescence spectra of CDs under different excitation wavelengths. Unlike most of CDs reported previously, the maximum emission wavelength at 532 nm does not shift obviously with increasing the excitation wavelength from 350 nm to 420 nm. Furthermore, the fluorescence lifetime also exhibits excitation-independent behaviour and remains constant under excitation of 350–400 nm, which is determined to be 2.45 ns (Fig. 2M) with a mono-exponential decay function. The above two characteristics further confirm that CDs have relatively uniform sizes and surface states. The absolute fluorescence quantum yield (QY) of CDs is 13.0%. The photostability of CDs was evaluated by monitoring changes of fluorescence intensity under green light irradiation. As shown in Fig. 1N, the fluorescence intensity at 532 nm is almost constant under the irradiation for 48 h, demonstrating the high photostability of CDs. The ionic-strength stability of CDs was studied in NaCl aqueous and shown in Fig. 1O. The fluorescence intensity of CDs has no significant change even the concentration of NaCl reaches up to 0.3 M, indicating that CDs have excellent ionic-strength stability.

2.7. Biocompatibility test HeLa cell lines harvested in a logarithmic growth phase were seeded in 96-well plates at a density of 1 × 104 cells per well and incubated in DMEM for 24 h, respectively. The medium was replaced by CDs at concentration from 10 to 200 µg mL−1 for 24 h. Then, 20 µL of MTT solution in PBS with the concentration of 5 mg mL−1 was added and cells were incubated for another 4 h at 37 °C. Followed by removal of the culture medium containing MTT and addition of 150 µL of DMSO to each well to dissolve the formed formazan crystals. Finally, the plate was shaken for 5 min, and the absorbance of formazan product was measured at 490 nm by a microplate reader.

2.8. Cell imaging Lysosome colocalization experiment. HeLa Cells were incubated in DMEM at a density of 5 × 104 per well for 24 h. Then CDs (100 µg mL−1) were incubated with the cells for 1 h. After CDs were removed, LysoTracker Red DND-99 (LTR, 75 nM) were added and incubated with the cells for 30 min at 37 °C. Finally, the cells were washed three times with preheated PBS and fixed with 4% formaldehyde. The slides were mounted and observed with a confocal laser scanning microscope imaging system. Cells inflammation experiment. HeLa cells were treated with different concentrations of LPS for 12 h at 37 °C, after LPS was removed, CDs (100 µg mL−1) was added and incubated with the cells for 1 h. Finally, the cells were washed three times with PBS and fixed with 4% formaldehyde.

2.9. Calculation of pKa values pKa values of as-prepared CDs and CDs in cells were calculated by the following formula:

log(Imax − I∣I − Imin ) = pH ± pKa Imin is minimum fluorescence intensity limiting values in base, Imax is maximum fluorescence intensity limiting values in acid. 2.10. In vivo fluorescence imaging Kunming mouse were obtained from the Hospital of Jilin University. A week later, the back area of Kun Ming (KM) mouse surrounding the injection area was shaved to avoid autofluorescence. The mice were subcutaneously injected with LPS (position 1), LPS + CDs (position 2) and PBS + CDs (position 3) at three different places on its back. The in vivo fluorescence imaging was successfully performed on a Maestro 500FL in vivo imaging system after injection for 0, 15, 30, 60 and 120 min, with a 503–555 nm excitation filter and a 580 nm emission filter

2.11. Statistical analysis 3.2. pH sensitivity and selectivity of CDs The data were expressed as mean ± standard deviation (SD). Student's t-test was used to determine the statistical difference between various experimental and control groups. Differences were considered statistically significant at a level of p < 0.05.

Standard pH titrations of fluorescence spectra were performed in DHP-CA buffers to study the optical responses of CDs toward pH. The fluorescence of CDs fades away as pH increases from 4.0 to 8.0 4

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Fig. 2. (A) Fluorescence spectra of CDs in Na2HPO4-citrate buffer solution with distinct pH. (B) The pH titration curve of fluorescence intensity of CDs as a function of pH. (C) Stern–Volmer plot of CDs in different pH of buffer solution. (D) Normalize fluorescence intensity of CDs between pH 4.0 and 7.4. (E) The anti-interference performance of CDs in the presence of various ions (Na+, Mg2+, Al3+, K+, Cr3+, Fe3+, Co3+, Ni2+, Cu2+, Zn2+, Ag+, Cd2+, Pb2+) and bioactive molecules (Glucose, GSH, H2O2, Cys, His, Glu, Asp) in the buffer solutions of pH 4.0 and 7.4. (F) Cell viability of Hela cells treated with CDs under various concentrations (0–200 µg mL−1) for 24 h. (G) CLSM images of HeLa cells incubated with different concentrations of CDs for 6 h at 37 °C. (H) CLSM images of HeLa cells treated with CDs (100 µg mL−1) at various time points at 37 °C. The first column: bright field images, the second column: fluorescence images at green channel (488 nm), the third column: overlay images of the first and second columns.

plot of the relative fluorescence intensity (I0/I-1) vs pH is plotted (y = 0.009 + 1.185 × 10−5 exp1.677×, I0 is the fluorescence intensity at pH 4.0), which further certificates that CDs have an excellent fitting effect in the range of pH 4.0–7.4. To determine the pH-dependent

(Fig. 2A). The pH titration curve of fluorescence intensity has a jump range from pH 5.4 to 7.4 (Fig. 2B), based on which the pKa of CDs is calculated to be 6.0 ± 0.72 [34,64], and this is comparable with the pH range of lysosomal (4.5–6.5). As shown in Fig. 2C, Stern–Volmer 5

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Fig. 3. CLSM images of HeLa cells stained with CDs (100 µg mL−1) and LTR (75 nM) for 1 h. (A) Bright-field image. (B) CLSM image of CDs in green channel (488 nm). (C) CLSM image of LTR in the red channel. (D) Overlay image of A, B and C. (E) Intensity profile (white line arrow) within the regions of interest (white line in A–D) of CDs and LTR across HeLa cells. (F) The fluorescence intensity correlation plot of CDs (green channel) and LTR (red channel). Time-dependent CLSM images of LTR (G) and CDs (H) with increasing of the laser exposure time. (I) Corresponding quantitative analysis of the cellular fluorescence intensity in (G) (the black column) and (H) (the red column).

living cells by MTT (3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide) assay in HeLa cell lines. As shown in Fig. 2F, the cell viability of HeLa cells exceeds 89% after incubating with different concentrations (0–200 µg mL−1) of CDs for 24 h. The result indicates that CDs have no obvious inhibitory effect on cells proliferation and can be applied to biomedical applications. We further employed CDs for in vitro imaging in HeLa cells by confocal laser scanning microscopy (CLSM). Firstly, HeLa cells were incubated with different concentrations of CDs for 6 h at 37 °C. As shown in Fig. 2G, the fluorescence intensity of HeLa cells gradually enhanced with the concentration of CDs increasing from 5 to 100 µg mL−1. However, no obvious fluorescence enhancement was observed when the concentration of CDs was up to 200 µg mL−1. Hence, 100 µg mL−1 is the optimal concentration of CDs. Secondly, HeLa cells were treated with 100 µg mL−1 of CDs at different time intervals (10 min, 0.5 h, 1 h, 2 h, 4 h). As displayed in Fig. 2H, the fluorescence intensity gradually enhanced with prolonging the incubation time from 10 min to 1 h. When the incubation time was further increased to 2 h or 4 h, the change of fluorescence intensity was negligible, indicating that the endocytosis of CDs reached saturation at 1 h. Thus, the optimal incubation time is 1 h. In order to study the lysosome-targeting ability of CDs,

mechanism of CDs, we investigated the Zeta potential of CDs in different pH buffer solutions. As shown in Fig. S1, CDs are positively charged in acidic environment and negatively charged in an alkaline environment. The protonation and deprotonation [4,17] of the amino groups on the surface of CDs may cause the fluorescence changes of CDs in different pH buffer solutions. We also studied the pH reversibility of CDs, the results (Fig. 2D) prove that CDs have a brilliant reversibility between pH 4.0 and 7.4. According to the above results, we conclude that CDs are expected to be an excellent fluorescence pH probe. The selectivity of CDs for preferential binding to H+ over other potential interfering agents under physiological environment was investigated by fluorescence spectra in DHP-CA buffer solutions of pH 4.0 and 7.4. As shown in Fig. 2E, the fluorescence intensity of CDs has no obvious change in the presence of various ions (Na+, Mg2+, Al3+, K+, Cr3+, Fe3+, Co3+, Ni2+, Cu2+, Zn2+, Ag+, Cd2+, Pb2+) and bioactive molecules (amino acids, glucose, and H2O2), further proving that CDs have admirable anti-interference capability and high proton selectivity. 3.3. Biocompatibility and in vitro fluorescence imaging of CDs. Before CDs are used as a fluorescent pH probe for intracellular imaging, it is imperative to evaluate that the cytotoxicity of CDs to 6

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Fig. 4. (A) CLSM images of HeLa cells incubated with CDs (100 µg mL−1) in DMEM with different pH. (B) Corresponding quantitative analysis of the cellular fluorescence intensity in (A). (C) Intracellular pH calibration curve constructed by average fluorescence intensity of images with pH. (D) CLSM images of HeLa cells treated with a series of concentrations LPS (0, 0.2, 0.5, 1, 2 µg mL−1) for 12 h at 37 °C, and then incubated with CDs (100 µg mL−1) for 1 h. (E) Corresponding quantitative analysis of the cellular average fluorescence intensity.

of pH 4.0–7.0 (Fig. 4C). The pKa of CDs in cells is calculated to be 6.0 ± 0.79 [28,63]. The above results certify that CDs can monitor pH changes in live cells. In addition, the relationship between pH and inflammatory process in cells was explored by treating HeLa cells with lipopolysaccharide (LPS), and characterized by CLSM [30]. HeLa cells were incubated with various concentrations of LPS (0, 0.2, 0.5, 1, 2 µg mL−1) for 12 h, and then stained with CDs (100 µg mL−1) for 1 h. The results display that fluorescent intensity of CDs associated with the increase of LPS concentration, the more severe the inflammation, the stronger the fluorescence intensity (Fig. 4D, E), revealing that pH in HeLa cells decreases during inflammation. These results verify that CDs can act as an ideal candidate for monitoring pH changes in inflammatory cells.

colocalization experiments with the commercial lysosome dye LysoTracker Red DND-99 (LTR) were conducted. As shown in Fig. 3A–F, CDs have terrific overlap with LTR probe, the corresponding overlap coefficient is calculated to be 0.88 and correlation R is determined to be 0.44 by ZEN 2011 software. The results demonstrate that CDs mainly localize into lysosome during the process of endocytosis in live HeLa cells. As the pH range is 4.5–6.5 in lysosome, and lysosome can provide CDs with protons, the protonation of amino groups of CDs lead to the enhanced fluorescence of CDs in lysosome after internalization [15]. Meanwhile, the photostabilities of LTR and CDs were evaluated by CLSM. As shown in Fig. 3G and Fig. 3I, the fluorescence intensity of LTR gradually weakens with prolonging the laser exposure time from 0 min to 10 min. When time of the exposure was increased to 20 min, the fluorescence of LTR is completely quenched. By contrast, the fluorescence intensity of CDs slightly weakens with increasing the laser exposure time from 0 min to 20 min (Fig. 3H and I). Therefore, CDs have higher photostability than the commercial lysosome probe (LTR). The ability of CDs to estimate cellular pH changes was analyzed by CLSM [65]. As shown in Fig. 4A, the fluorescence of CDs gradually increased with pH decreasing from 7.0 to 4.0. CDs exhibit pH-responsive optical behavior (Fig. 4B) in live cells with a good linear calibration curve of FL = −5.12 pH + 43.42 (R2 = 0.99192) in the range

3.4. In vivo fluorescence imaging Encouraged by the achieved successful in vitro imaging with CDs, we made a further exploratory to determine whether CDs would be qualified for in vivo imaging and reflect occurrence of biological processes induced by exogenous stimulation. Thus, we explored the application of CDs to visualize pH changes in an inflammation model induced by LPS. Before the subcutaneous injection, the back area of Kun Ming (KM) mouse surrounding the injection area was shaved to avoid 7

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Fig. 5. In vivo fluorescence images (A, true-color) and (B, pseudo-color) of a KM mouse injected with LPS (position 1), LPS + CDs (position 2) and PBS + CDs (position 3), respectively.

autofluorescence. The mouse was subcutaneously injected with LPS (position 1), LPS + CDs (position 2) and PBS + CDs (position 3) at three different places on its back. And then it was imaged by a Maestro 500FL in vivo optical imaging system under the excitation of green light. As displayed in Fig. 5, at the initial injection time (0 min), the fluorescence signals can be detected at position 2 and 3, on the contrary, no fluorescence is observed at position 1 which was treated with no CDs but LPS. Thus, CDs are competent for in vivo bioimaging. Along with the extension of time (15 min), LPS initiated a more severe inflammatory response and thereby lowered the local pH, leading to the signal at position 2 significantly enhanced. However, with the time prolonged (30 and 60 min), some CDs were metabolized by the organisms of mouse, fluorescence signal gradually decreased and very weak signal was observed at 120 min. Therefore, these experimental results fully prove that CDs are a prominent fluorescent probe for tracking pH changes in vivo.

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4. Conclusions In summary, a kind of CDs-based fluorescent probe with lysosometargeting function was synthesized. With the merits of excellent photostability, high pH sensitivity and selectivity, favorable water solubility and nice biocompatibility, CDs exhibit good performance in quantitatively measuring pH changes in live cells and monitoring pH fluctuations in organisms. The results demonstrate that CDs are promising candidates for the study of pH-associated physiological and pathological processes. Acknowledgements The financial support from the National Natural Science Foundation of China (Nos. 51873023 and 51522307) and Talent Development Fund of Jilin Province. Appendix A. Supplementary data Supplementary data to this article can be found online at https:// doi.org/10.1016/j.cej.2019.122665. References [1] H. Peretz-Soroka, A. Pevzner, G. Davidi, V. Naddaka, M. Kwiat, D. Huppert, F. Patolsky, Manipulating and monitoring on-surface biological reactions by lighttriggered local pH alterations, Nano Lett. 15 (2015) 4758–4768.

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