Folic acid encapsulated graphene quantum dots for ratiometric pH sensing and specific multicolor imaging in living cells

Sensors and Actuators B 268 (2018) 61–69

Contents lists available at ScienceDirect

Sensors and Actuators B: Chemical journal homepage: www.elsevier.com/locate/snb

Folic acid encapsulated graphene quantum dots for ratiometric pH sensing and specific multicolor imaging in living cells Xin Hai, Yiting Wang, Xiaoyao Hao, Xuwei Chen ∗ , Jianhua Wang ∗ Research Center for Analytical Sciences, Northeastern University, Box 332, Shenyang, 110819, China

a r t i c l e

i n f o

Article history: Received 16 January 2018 Received in revised form 18 April 2018 Accepted 19 April 2018 Available online 22 April 2018 Keywords: Graphene quantum dots Folic acid Encapsulation Ratiometric fluorimetry pH sensing Multicolor pH imaging

a b s t r a c t Folic acid (FA) encapsulated graphene quantum dots (FA-GQDs) were prepared via a one-pot microwaved alkali cutting method, using graphene oxide (GO) and FA as raw material. TEM and AFM images revealed that FA was successfully cross-linked on the surface of GQDs under alkali circumstance, giving rising to stable and homogeneous FA-GQDs with size of about 33.6 nm. XPS and FT-IR confirmed that there are abundant hydroxyl, carboxyl and amino groups on the encapsulated FA-GQDs. The as-prepared FA-GQDs possessed the fluorescence behaviors of multi-excitation and emission sites, and exhibited sensitive response to the pH variation within the pH range of 3–9, making it excellent fluorescence probe for pH sensing in cell lysate and environmental water samples. The broad emission with pH-sensitive characteristic and low toxicity of FA-GQDs also provided an alternative for the specific multicolor imaging and sensing of pH in living cells. © 2018 Elsevier B.V. All rights reserved.

1. Introduction pH plays a pivotal role in environmental monitoring as well as the regulation of physiological and pathological processes [1]. For instance, the acidification of pH in environment may lead to the pollution of water seriously, which bring health dangers in turn. In normal biological processes, intracellular pH modulates many cellular behaviors and is homeostatic under physiological levels (pH 7.36–7.44) [2]. However, a lower extracellular pH value is observed in tumour cells rather than normal cells [3]. Moreover, irreversible pH variation will cause dysfunction of cells, and the abnormal pH is usually associated with apoptosis, external stimulus or harmful diseases such as stroke or cancer [4,5]. Therefore, the accurate monitoring of pH value of environment and biosome is of vital importance. As a non-invasive measurement, fluorometry possesses high sensitivity, favorable selectivity and fast response [6]. What’s more, fluorescent imaging technology offers the feasibility for real-time monitoring and in situ observation of pH variation in living cells [7]. Up to now, various pH-sensitive fluorescent probes have been developed such as organic dyes [8] and semiconductor quantum dots [9], while the high toxicity limits their practical application in

∗ Corresponding author. E-mail addresses: [email protected] (X. Chen), [email protected] (J. Wang). https://doi.org/10.1016/j.snb.2018.04.090 0925-4005/© 2018 Elsevier B.V. All rights reserved.

bio-systems. Recently biocompatible nanomaterials such as metal nanoclusters and carbon dots have been devoted to monitor pH changes in living cells [10–12]. Nevertheless, these fluorescent nanomaterials usually exhibit single-wavelength excitation and emission behaviors, and their fluorescence are easily affected by complicated circumstance. The dual emission fluorescent probes have been demonstrated to be useful to above problem, while their preparation should be further improved as sophisticated procedures are usually needed to decorate the pH-sensitive groups onto the basic frameworks [13,14]. Graphene quantum dots (GQDs) are small graphene sheet of less than 100 nm in lateral size [15], exhibiting plenty of merits such as chemical inertness, photostability, favorable biocompatibility and low cytotoxicity [16]. In particular, GQDs emerge as unique and excellent fluorophores with tunable photoluminescence via varying the size, edge-state, doping, layer and surface chemistry of the GQDs [17], thereby GQDs are endowed with versatile functionalities for biosensing, bioimaging, drug delivery and therapy [18–20]. The functionalization of GQDs can be easily achieved due to the rich oxygen-containing groups such as hydroxyl and carboxyl groups on the surface [21]. Folic acid (FA) is a typical ligand for targeted identification and labelling of folate receptor (FR) overexpressed cancer cells (such as HeLa cells) [22,23] on account of the high affinity between FA and FR, and it can be transported into cell via receptor-mediated endocytosis [24]. In particular, FA is rich in a variety of functional moieties, making it easy to conjugate with GO or GQDs via amidation [25,26]. In present study,

62

X. Hai et al. / Sensors and Actuators B 268 (2018) 61–69

we propose a one-pot microwave-assisted alkali cutting strategy for the fabrication of FA encapsulated graphene quantum dots (FA-GQDs) using graphene oxide (GO) and FA as source materials. The encapsulation of GQDs with FA provides colloidal and homogeneous FA-GQDs along with a pH-sensitive characteristic. The as-prepared FA-GQDs possess multi-emission sites, and a ratiometric fluorescence method is established for pH sensing based on the fluorescence response of 360 nm/450 nm with pH under the excitation of 280 nm. The practicability of this ratiometric fluorescence method is demonstrated by the accurate sensing pH value of cell lysate and environmental water samples. Furthermore, the broad emission with pH-sensitive characteristic of FA-GQDs containing targeting ligand FA provide an alternative for the specific multicolor imaging and sensing of pH in living cells.

2. Experimental 2.1. Materials Graphite powder, potassium hydroxide (KOH) and other common solvents and reagents were purchased from Sinopharm Chemical Reagent Co., Ltd. (Shanghai, China). Hydrogen peroxide (H2 O2 ) and folic acid (FA) were purchased from Aladdin Chem. Co., Ltd (Shanghai, China). The chemical reagents used in this work were all analytical grade without any further purification. Deionized water of 18 M·cm was used throughout.

path. Raman spectra were collected on an XploRA Raman spectrometer (Horiba Co., France) with excitation wavelength at 532 nm. 2.4. Ratiometric fluorescent pH sensing Britton-Robinson (B-R) buffer solution (0.04 mol L−1 , pH 3–9) was prepared by adding different amounts of 0.2 mol L−1 NaOH aqueous solution into the blended solution of 0.04 mol L−1 acetic acid, boric acid, and phosphoric acid. The ratiometric pH sensing was developed using FA-GQDs as a probe to record the fluorescent emissions (360 nm and 450 nm) under the excitation of 280 nm within pH range of 3–9. We also investigated the pH-response of FA-GQDs under longer excitation wavelengths (405 nm and 488 nm) using various pH (5–8) of PBS buffer solution (10 mmol L−1 ). The pH-reversibility was realized through the treating of FA-GQDs with acid (at pH 3.0) and base (at pH 9.0) for 7 times. The selectivity for pH sensing was carried out in the presence of various interferences under pH 7 at identical conditions. 2.5. Multicolor pH imaging and intracellular pH sensing

GO was firstly synthesized based on the modified Hummers and Offeman’s method. 2 mL GO suspension (10 mg mL−1 ) with 20 mg FA in the presence of 1 mol L−1 KOH solution was achieved by ultrasonic dispersion. 500 ␮L fresh H2 O2 (30%) and 2.5 mL deionized water were added into the above system. Subsequently, the mixture was transferred into a 100 mL Teflon digestion jar and heated at 200 ◦ C for 40 min in a COOLPEX microwave chemical reaction apparatus (PreeKem Scientific Instruments, Co., Ltd., China). After cooled to room temperature, the products were taken out from the container and filtered through a 0.1 ␮m microporous membrane to discard the sediment. After the removal of residual H2 O2 via rotary evaporation, the pH of the filtrate was regulated to 7 by hydrochloric acid (HCl). Thereafter, the neutral solution was dialyzed over deionized water in a dialysis bag (MWCO membranes 100 ∼ 500 Da) for 2 days to remove the residue. Finally, solid FAGQDs were acquired via freeze-drying. GQDs were prepared using the same procedure without FA in the system.

The cytotoxicity of FA-GQDs was carried out using a standard MTT assay. HeLa cells were cultured for 12 h and treated with various concentrations of FA-GQDs (0, 0.05, 0.10, 0.20, 0.40, 1.00, 2.00, 4.00 mg mL−1 ) for 20 h. Then, 20 ␮L MTT solutions were injected into the plate to co-incubate for another 4 h. Afterwards, 150 ␮L DMSO was added to dissolve the precipitate after supernate was removed from the system. The cell viability was measured in a microplate reader (BioTek, USA). Multicolor imaging in living cells was executed by following steps. Firstly, HeLa cells were treated with FA-GQDs (1 mg mL−1 ) for 6 h and rinsed for three times with PBS (10 mmol L−1 , pH 7.4) to discard extra FA-GQDs. Then, HeLa cells were incubated in different pH of PBS buffers (10 mmol L−1 , pH 5, 6, 7, 8) with 10 ␮mol L−1 nigericin for another 30 min. Finally, the buffers were removed and the cells were immobilized by paraformaldehyde. The cell imaging under different pH condition was taken on a confocal laser scanning microscope (CLSM, Olympus, Japan) using two excitations of (␭ex 405 nm/␭em 425–490 nm, ␭ex 488 nm/␭em 500–570 nm). The imaging of other type of cells (293T cell) was conducted using the same procedure as HeLa cell under the same condition. The fluorescence intensity of images in the region of interest (ROI) was measured as optical density performed by Image J software. Finally, pH determination of cells under various circumstances (normal cells, treated with 100 ␮mol L−1 dexamethasone and 100 ␮mol L−1 chloroquine) were thoroughly investigated under the same conditions.

2.3. Characterizations

3. Results and discussion

Transmission electron microscopy (TEM) images of GQDs and FA-GQDs were recorded on a JEM-2100F field-emission electron microscope with an accelerating voltage of 200 kV (JEOL, Japan). Atomic force microscopy (AFM) images were taken on a Bruker Dimension icon atomic force microscopy operating under ScanAsyst in air mode using a tip of ScanAsyst-air (Bruker, Germany). X-ray photoelectron spectroscopy (XPS) was obtained via an ESCALAB 250 surface analysis system (Thermo, USA), which set Al K␣ 280 eV as excitation source. Fourier transform infrared (FTIR) spectra were conducted on a Nicolet-6700 FT-IR spectrophotometer (Thermo, USA) in a range of 400–4000 cm−1 . Fluorescence spectra were executed using an F-7000 fluorescence spectrophotometer (Hitachi High Technologies, Japan) with a 0.5 cm quartz cuvette. UV–vis spectra were received on a U-3900 spectrophotometer (Hitachi High Technologies, Japan) using a 1.0 cm optical

3.1. Preparation and characterizations of FA-GQDs

2.2. Preparation of FA-GQDs

In present study, pH-sensitive probe FA-GQDs were fabricated by a one-pot microwave-assisted alkali cutting strategy, as illustrated in Scheme 1. With the assistance of microwave radiation, GO sheets were cut into small GQDs with average sized of 5.5 nm (Fig. 1A, B) and topographic height of 0.88 nm (Fig. S1A, B) by KOH. FA is a longish small molecule containing an amino group at one side and a carboxyl group at the other side, which tends to cross-linked together to form a reticular structure under high temperature [27]. Therefore, the encapsulation of GQDs into the cross-linked FA under microwave radiation gives rising to stable and homogeneous FA-GQDs with average size of 33.6 nm (Fig. 1C, D). AFM results presented an average topographic height of 3.59 nm for FA-GQDs (Fig. S1C, D), which was higher than that of GQDs. In

X. Hai et al. / Sensors and Actuators B 268 (2018) 61–69

63

Scheme 1. Preparation scheme for FA-GQDs.

particular, the encapsulation of GQDs by FA was clearly observed, further confirming the structure of FA-GQDs. X-ray photoelectron spectroscopy (XPS) was conducted to confirm the structure and composition of GQDs and FA-GQDs. The high-resolution C1 s spectra of GQDs contained four typical peaks at 284.3 eV, 284.8 eV, 286 eV and 288.1 eV, attributing to C C, C C, C O and COOH, respectively (Fig. 2A) [28]. Besides the peaks in the spectra of GQDs, a new peak of C N (285.1 eV) appeared in the C1 s spectra of FA-GQDs, suggesting the existence of N content (Fig. 2B), and the N1 s spectra of FA-GQDs split into three peaks of pyridinic N (398.6 eV), pyrrolic N (399.6 eV) and graphitic N (401.2 eV) (Fig. 2C) [29,30]. Moreover, O1s XPS spectra including C O (531 eV), C OH (532.1 eV) and COOH (533.3 eV) displayed in GQDs as well as FA-GQDs, and these peaks were consistent with that in C1s spectra (Fig. S2). The functional groups of GQDs and FAGQDs are further identified by FT-IR spectra (Fig. 2D). The stretching vibration of C OH (3424 cm−1 ), C C of benzene ring (1596 cm−1 , 1424 cm−1 ) and −OH (1120 cm−1 ) appeared in both spectra of

GQDs and FA-GQDs, while only C O adsorption peak (1681 cm−1 ) was exhibited in GQDs, indicating the existence of hydroxyl and carboxyl groups [27,31]. After the encapsulation of GQDs by FA, the stretching vibrations of C N (1309 cm−1 ) and wagging vibrations of N H (770 cm−1 ) emerged, suggesting the presence of amino groups on the surface of FA-GQDs [26]. Moreover, the fingerprint peaks in FA also appear in the spectra of FA-GQDs, which further confirm the successful encapsulation GQDs by FA. Raman spectra demonstrated a typical D band (1350 cm−1 ) attributed to disordered sp3 defects and a G band (1590 cm−1 ) related to ordered sp2 C C bond in GQDs as well as FA-GQDs (Fig. 2E). The weak peak located between the D and G bands at ∼1460 cm−1 was ascribed to the asymmetric bending of the methyl group [32]. The intensity ratio of the disordered D band to the crystalline G band (ID /IG ) well illustrated the disorder degree of GQDs. The ID /IG of FA-GQDs was calculated to be 0.72, which was lower than that of GQDs (0.82), indicating the defect of GQDs was restored after the FA encapsulation of GQDs.

Fig. 1. TEM images of GQDs (A); FA-GQDs (C); Size distributions of GQDs (B) and FA-GQDs (D).

64

X. Hai et al. / Sensors and Actuators B 268 (2018) 61–69

Fig. 2. High-resolution C1 s XPS spectra of GQDs (A); FA-GQDs (B); N1 s XPS spectra of FA-GQDs (C); FT-IR spectra (D) of GQDs, FA and FA-GQDs. Raman spectra (E) of GQDs and FA-GQDs.

Fig. 3. (A) UV–vis spectra of GQDs and FA-GQDs; (B) 2D-fluorescence topographical map of FA-GQDs.

3.2. Ratiometric fluorescent sensing of pH The most exciting characteristic of GQDs is their charming optical behavior. GQDs possessed a UV absorption peak in 220 nm assigned to ␲–␲* transition of aromatic sp2 domains. In the spectra of FA-GQDs, except the ␲–␲* transition in 210 nm, a new visible n␲* transition of C O appeared in 260 nm (Fig. 3A), suggesting the presence of FA [26]. GQDs synthesized by alkali cutting and the as-prepared FAGQDs both exhibited a strong excitation-dependent PL behavior (Fig. S3A and B), which not only is associated with the quantum confinement effect, but also contributed by the abundant functional groups with various energy levels on the surface of GQDs/FA-GQDs [33,34]. The GQDs possess two-emission maximum centering at 280 nm/440 nm and 350 nm/440 nm (Fig. S3C). Compared to GQDs, FA-GQDs prepared in alkali condition presented fluorescence behaviors of multi-excitation and emission sites, with a dual emission (␭ex/␭em = 280 nm/360 nm, 450 nm, P1, bluish vio-

let emission) and two single emissions (␭ex/␭em = 360 nm/450 nm P2, blue emission; and ␭ex/␭em = 440 nm/510 nm, P3, green emission) (Fig. 3B). The multicolor emission might be ascribed to the distribution of different emissive sites and the different sizes of FA-GQDs [35]. Moreover, when FA was put into the synthesized system under the same microwave irradiation, only a single emission center was shown in P1 region (␭ex/␭em = 280 nm/360 nm) (Fig. S3D). This further indicates that one of the dual emission (␭ex/␭em = 280 nm/360 nm) is contributed by the encapsulation of FA. In comparison with FA-GQDs synthesized in alkali condition, weak and narrow PL behavior was observed in FA-GQDs prepared under neutral or acidic media (Fig. S3E, F). The PL diversity among different pH medium originates from the intrinsic structure of GQDs. The PL properties of GQDs derive from free zigzag sites with a carbene-like triplet ground state [36]. In alkali condition, FA-GQDs are prone to form more free edge-enriched zigzag sites [37], which emit strong PL intensity. Whereas zigzag sites are pro-

X. Hai et al. / Sensors and Actuators B 268 (2018) 61–69

65

Fig. 4. Fluorescence spectra (A); the linear fluorescence response of relative intensity (F360 /F450 ) (B) of FA-GQDs under different pH conditions (pH 3–9) at the excitation wavelength of 280 nm.

tonated and combine with H+ to form a reversible complex, leading to a break of emissive triple carbene state with inactive PL. In addition, the encapsulation of GQDs with FA provides rich functional groups such as hydroxyl, carboxyl and amino groups. Therefore, the edge-enriched FA-GQDs with functional groups present pHsensitive characteristic. It is reported that the blue emission of GQDs is ascribed to intrinsic state emission (such as electron hole recombination or quantum size effect/zig-zag effect) and the hydroxyl and amino group decorated at the lateral edge or in plane [31,38,39], while the green emission is derived from surface defects (defect state emission) including oxygen containing functional groups such as carboxyl group [40,41]. Then the fluorescence responses of FA-GQDs in three regions (P1, P2 and P3) under different pH circumstance have been investigated thoroughly. The variations of pH value nearly posed effect on the fluorescence emission at 360 nm in P1 center (excitation at 280 nm) originating from FA (Fig. S3D), while the fluorescence emission situated in 450 nm decreased with the increase of pH value (Fig. 4A). Hence, a ratiometric fluorescent strategy was proposed for pH sensing by utilizing the relationship between pH and the relative fluorescence ratio of FA-GQDs (F360 /F450 ) with regression equations of F360 /F450 = 0.1415 pH + 0.9481 (pH 3–9, R2 = 0.9928) (Fig. 4B). We speculate that the functionalization of amino groups dominate the blue emission in P1 region. The protonation of amino groups induced by the decrease of pH offer them improved ability to donate electrons to GQDs, thus resulting in the increasing blue emission at 450 nm. 3.3. Selectivity In order to evaluate the selectivity of this fluorescent probe towards pH, FA-GQDs were treated with foreign species (common metal ions, anions and biomolecules, final concentration: 100 ␮mol L−1 ) under pH 7. The experimental results showed that 100 ␮mol L−1 of the coexisting foreign species posed no obvious interference on pH sensing (Fig. S4). 3.4. Photostability and reversibility of FA-GQDs The as-prepared FA-GQDs showed excellent anti-salt ability and the fluorescence of FA-GQDs kept stable even under the condition of KCl concentration high up to 2.0 mol L−1 (Fig. S5A). At the same time, no obvious change on the fluorescence of FA-GQDs was observed after 2 h continuous irradiation (Fig. S5B). Furthermore, the reversible pH response of FA-GQDs has been evaluated (Fig. S6). The pH was switched back and forth between 3 and 9 by using concentrated NaOH and HCl for 7 consecutive cycles. The relative intensity of FA-GQDs increased as the pH changes from 3 to

Table 1 Determination results of pH in real samples. a

Samples

pH1

Nanhu lake water Rain water Tap water Yangtze River water Guanmenshan water Cell suspension

7.39 ± 0.02 7.68 ± 0.01 7.57 ± 0.01 7.51 ± 0.02 7.43 ± 0.01 6.42 ± 0.01

a b

pH2

b

7.96 7.72 7.53 7.73 7.68 6.14

R.S.D% (n = 6) 1.44 1.08 1.21 1.97 1.23 1.12

obtained from the ratiometric fluorescence method. measured by a pH meter.

9. Subsequently, when adjusting the pH from 9 to 3, the relative intensity downed to that as before. The relative intensity of FAGQDs remained virtually stable even after 7 cycles of change. The results declare that FA-GQDs possess an excellent reversibility to pH, which further indicate the FA-GQDs possess basic and acidic sites relevant to the PL emission [42]. 3.5. Sensing pH values in real samples To demonstrate the accuracy and feasibility of this FA-GQDs probe, the pH of Yangtze River water, Nahu lake water, GuanMenShan water, snow water, rain water and tap water sample was measured by using the proposed ratiometric pH sensing procedure. HeLa cells were collected after digesting by trypsin and washing with PBS (pH 7.4, 10 mmol L−1 ) for 2 times. The sesning results of pH values in real samples are shown in Table 1. The results obtained by the FA-GQDs probe were consistent with that of pH meter, indicating the high accuracy of this proposed ratiometric pH sensing procedure. 3.6. Multicolor pH imaging and intracellular pH sensing On account of the multicolor emission of FA-GQDs, the pHresponse in another two emission regions (P2 and P3) have been explored. A pH-independent behavior was observed in P2 regions (405 nm/500 nm) with blue emission (Fig. 5A, B), which came from the synergy effect of intrinsic state emissions (zigzag sites) and amino groups. As zigzag sites contribute to the decreased emission and amino groups facilitate the enhanced PL with the decrease of pH, the two totally contrast pH-response caused PL equilibration under various pH media. However, a pH-dependent performance (a bright PL under basic condition) with green emission attributed to carboxyl groups was appeared in P3 region (488 nm/550 nm) (Fig. 5C). This might lie in the fact that the protonation of carboxyl groups enables their electron-accepting capacity, leading to the quenching of green fluorescence at 550 nm. Thus, the PL inten-

66

X. Hai et al. / Sensors and Actuators B 268 (2018) 61–69

Fig. 5. Fluorescence spectra (A); fluorescence response (B) of FA-GQDs under different pH conditions (pH 5–8) at the excitation wavelength of 405 nm; Fluorescence spectra (C); the linear fluorescence response (D) of FA-GQDs under different pH conditions (pH 5–8) at the excitation wavelength of 488 nm.

Fig. 6. The confocal fluorescence images of HeLa cells (A); 293T cells (B); HeLa cells saturated with excess FA (C) incubated with 1 mg mL−1 FA-GQDs for 6 h. The fluorescence images of (1), (2) and (3) are collected in blue channel (425–490 nm, ␭ex 405 nm), green channel (500–570 nm, ␭ex 488 nm) and bright field. The scale bar stands for 50 ␮m. (For interpretation of the references to colour in this figure legend, the reader is referred to the web version of this article.)

X. Hai et al. / Sensors and Actuators B 268 (2018) 61–69

67

Fig. 7. (A-D) The confocal fluorescence images of HeLa cells incubated with 1 mg mL−1 FA-GQDs for 6 h and then treated with various pH (5, 6, 7, 8) for 30 min; The fluorescence images of (1), (2) and (3) are collected in blue channel (425–490 nm, ␭ex 405 nm), green channel (500–570 nm, ␭ex 488 nm) and bright field. The scale bar stands for 50 ␮m. (For interpretation of the references to colour in this figure legend, the reader is referred to the web version of this article.)

Fig. 8. The confocal fluorescence images of HeLa cells incubated with 1 mg mL−1 FA-GQDs for 6 h without any treatment (A); by the treatment of 100 ␮mol L−1 of dexamethasone (B) and 100 ␮mol L−1 of chloroquine (C). The fluorescence images of (1), (2) and (3) are collected in blue channel (425–490 nm, ␭ex 405 nm), green channel (500–570 nm, ␭ex 488 nm) and bright field. The scale bar stands for 50 ␮m. (For interpretation of the references to colour in this figure legend, the reader is referred to the web version of this article.)

68

X. Hai et al. / Sensors and Actuators B 268 (2018) 61–69

Table 2 Measurement of pH in HeLa cells under various stimulations. (n = 3, at 95% confidence level). Sample pH (HeLa cells) normal 100 ␮mol L−1 dexamethasone 100 ␮mol L−1 chloroquine √ n = 3, means ± t s/ n, f = 95%, t = 4.303.

Found pH 7.01 ± 0.19 5.57 ± 0.20 7.74 ± 0.08

sity maintained stable under 405 nm excitation (pH 5–8), while a linear relationship F550 = 664.76 pH–585.78 (pH 5–8, R2 = 0.9874) was observed with another 488 nm excitation (Fig. 5D). Therefore, a ratiometric pH sensor was developed by using the different pHresponse in blue and green region (P2 and P3). To assess the viability of pH imaging and intracellular pH sensing of FA-GQDs in live cells, the cytotoxicity of FA-GQDs (0–4 mg mL−1 ) was investigated by standard MTT assay (Fig. S7). The cell viability remained virtual unchanged when FA-GQDs concentration changed from 0 to 0.4 mg mL−1 , and a slight decrease was observed when the concentration of FA-GQDs was higher than 0.8 mg mL−1 . What’s more, a cell viability of ca. 80% was achieved even FA-GQDs concentration was high up to 2.0 mg mL−1 . The results suggest that the as-prepared FA-GQDs possess low cytotoxicity and good biocompatibility, which could be utilized as excellent fluorescence probe under high working concentration. The overexpression of FR on the surface of some mammalian cancer cells (such as HeLa cell) could serve as a recognition part for the specific binding with FA functionalized nanomaterials and trigger receptor-mediated endocytosis [43]. In order to verify the specific targeting imaging of FR-positive cancer cells with FA-GQDs as fluorescent probe, the uptake and imaging of FR overexpressed cancer cells (HeLa cells) and non-cancerous cells (human renal epithelial cells, 293T) has been investigated. A bright fluorescence image was observed in HeLa cells, while weak fluorescence displayed in 293T cells, suggesting that the FA-GQDs were suited for the discrimination of FR-positive cancer cells from other cells (Fig. 6A, B). To further confirm the entrance and target recognition of FA-GQDs into cells, the HeLa cells were saturated with excess FA for 2 h, and then treated with FA-GQDs for another 6 h. The cells exhibited weak fluorescence compared to the untreated cells (Fig. 6C), suggesting that FA-GQDs were internalized by HeLa cells via receptor-mediated delivery [22]. These results well reveal the cancer cell targeting and imaging capability of FA-GQDs. FA-GQDs were further employed as a probe to monitor pH value in HeLa cells by using confocal fluorescence imaging. First, the intracellular pH calibration experiment was carried out in FA-GQDs-loaded HeLa cells under various pH with nigericin to homogenize the pH between cells and the culture medium [44]. The blue channel (␭ex 405 nm/␭em 425–490 nm) of fluorescence intensity in HeLa cells treated with various pH was almost identical, while the green channel (␭ex 488 nm/␭em 500–570 nm) was getting brighter as the pH increased (pH 5–8) (Fig. 7). Then, a linear relationship was developed between pH and optical density of green fluorescence images (Fig. S8). This is in accordant with the fluorescence pH response of FA-GQDs. Hence, it was demonstrated that FA-GQDs could serve as an indicator to monitor the variation of pH in cells by the observation of fluorescence brightness. It is reported that dexamethasone could induce intracellular acidification of HeLa cells [45], while exposure of chloroquine brings basification effect on the cell cytoplasm [46]. Then, the pH value of normal HeLa cells, dexamethasone and chloroquine stimulated cells was measured under the same conditions, and was calculated according to the intracellular pH calibration curve (Fig. S8). The results are shown in Fig. 8 and Table 2. It was clear that HeLa cells without any treatment displayed nor-

mal shape with pH of 7.01 ± 0.19. The pH increased to 7.74 under the stimulation of chloroquine (100 ␮mol L−1 ), whereas the pH was down to 5.57 on the exposure of 100 ␮mol L−1 dexamethasone, which were all consistent with the reported results. All of these results indicate that the FA-GQDs could act as desirable pH sensor for monitoring the pH in live cells. 4. Conclusion In summary, a pH sensing and imaging system has been established by using FA-GQDs as a probe. Characterizations results revealed that FA was cross-linked on the surface of GQDs, forming stable and homogeneous encapsulated FA-GQDs. The as-prepared FA-GQDs exhibit multicolor emission properties with pH-sensitive characteristic. A ratiometric fluorescence pH sensor has been proposed based on the pH response under 280 nm excitations, and it has been successfully applied in the detection of pH values of water samples and cell suspensions. Furthermore, the broad emission with pH-sensitive characteristic of FA-GQDs provide an alternative for specific multicolor imaging and sensing of pH in living cells. Acknowledgements The authors appreciate financial support from National Natural Science Foundation of China (21475017, 21275027, 21235001), and Fundamental Research Funds for the Central Universities (N150502001). Appendix A. Supplementary data Supplementary data associated with this article can be found, in the online version, at https://doi.org/10.1016/j.snb.2018.04.090. References [1] Z.Y. Yang, W. Qin, J.W.Y. Lam, S.J. Chen, H.H.Y. Sung, I.D. Williams, B.Z. Tang, Fluorescent pH sensor constructed from a heteroatom-containing luminogen with tunable AIE and ICT characteristics, Chem. Sci. 4 (2013) 3725–3730. [2] Y.Y. Liu, M. Wu, L.N. Zhu, X.Z. Feng, D.M. Kong, Colorimetric and fluorescent bimodal ratiometric probes for pH sensing of living cells, Chem.-Asian J. 10 (2015) 1304–1310. [3] M. Stubbs, P.M.J. McSheehy, J.R. Griffiths, C.L. Bashford, Causes and consequences of tumour acidity and implications for treatment, Mol. Med. Today 6 (2000) 15–19. [4] S.J. Chen, Y.N. Hong, Y. Liu, J.Z. Liu, C.W.T. Leung, M. Li, R.T.K. Kwok, E. Zhao, J.W.Y. Lam, Y. Yu, B.Z. Tang, Full-range intracellular pH sensing by an aggregation-induced emission-active two-channel ratiometric fluorogen, J. Am. Chem. Soc. 135 (2013) 4926–4929. [5] W.F. Niu, L. Fan, M. Nan, Z.B. Li, D.T. Lu, M.S. Wong, S.M. Shuang, C. Dong, Ratiometric emission fluorescent pH probe for imaging of living cells in extreme acidity, Anal. Chem. 87 (2015) 2788–2793. [6] R. Wang, C.W. Yu, F.B. Yu, L.X. Chen, Molecular fluorescent probes for monitoring pH changes in living cells, TrAC Trends Anal. Chem. 29 (2010) 1004–1013. ´ M. Heidkamp, Y. Ming, B. Johansson, L. Terenius, R. Rigler, [7] V. Vukojevic, Quantitative single-molecule imaging by confocal laser scanning microscopy, Proc. Natl. Acad. Sci. U. S. A 105 (2008) 18176–18181. [8] R. Gotor, P. Ashokkumar, M. Hecht, K. Keil, K. Rurack, Optical pH sensor covering the range from pH 0–14 compatible with mobile-device readout and based on a set of rationally designed indicator dyes, Anal. Chem. 89 (2017) 8437–8444. [9] Y.S. Liu, Y.H. Sun, P.T. Vernier, C.H. Liang, S.Y.C. Chong, M.A. Gundersen, pH-sensitive photoluminescence of CdSe/ZnSe/ZnS quantum dots in human ovarian cancer cells, J. Phys. Chem. C 111 (2007) 2872–2878. [10] C.Q. Ding, Y. Tian, Gold nanocluster-based fluorescence biosensor for targeted imaging in cancer cells and ratiometric determination of intracellular pH, Biosens. Bioelectron. 65 (2015) 183–190. [11] H. Nie, M.J. Li, Q.S. Li, S.J. Liang, Y.Y. Tan, L. Sheng, W. Shi, S.X.A. Zhang, Carbon dots with continuously tunable full-color emission and their application in ratiometric pH sensing, Chem. Mater. 26 (2014) 3104–3112. [12] Y.H. Chan, C.F. Wu, F.M. Ye, Y.H. Jin, P.B. Smith, D.T. Chiu, Development of ultrabright semiconducting polymer dots for ratiometric pH sensing, Anal. Chem. 83 (2011) 1448–1455.

X. Hai et al. / Sensors and Actuators B 268 (2018) 61–69 [13] J. Zhou, C.L. Fang, T.J. Chang, X.J. Liu, D.H. Shangguan, A pH sensitive ratiometric fluorophore and its application for monitoring the intracellular and extracellular pHs simultaneously, J. Mater. Chem. B 1 (2013) 661–667. [14] J. Huang, L. Ying, X.L. Yang, Y.J. Yang, K. Quan, H. Wang, N.L. Xie, M. Ou, Q.F. Zhou, K.M. Wang, Ratiometric fluorescent sensing of pH values in living cells by dual-fluorophore-labeled i-motif nanoprobes, Anal. Chem. 87 (2015) 8724–8731. [15] F. Liu, M.H. Jang, H.D. Ha, J.H. Kim, Y.H. Cho, T.S. Seo, Facile synthetic method for pristine graphene quantum dots and graphene oxide quantum dots: origin of blue and green luminescence, Adv. Mater. 25 (2013) 3657–3662. [16] D. Qu, M. Zheng, L.G. Zhang, H.F. Zhao, Z.G. Xie, X.B. Jing, R.E. Haddad, H.Y. Fan, Z.C. Sun, Formation mechanism and optimization of highly luminescent N-doped graphene quantum dots, Sci. Rep. 4 (2014) 5294. [17] S. Kim, S.W. Hwang, M.K. Kim, D.Y. Shin, D.H. Shin, C.O. Kim, S.B. Yang, J.H. Park, E. Hwang, S.H. Choi, G. Ko, S. Sim, C. Sone, H.J. Choi, S. Bae, B.H. Hong, Anomalous behaviors of visible luminescence from graphene quantum dots: interplay between size and shape, ACS Nano 6 (2012) 8203–8208. [18] H.J. Sun, L. Wu, W.L. Wei, X.G. Qu, Recent advances in graphene quantum dots for sensing, Mater. Today 16 (2013) 433–442. [19] X.T. Zheng, A. Ananthanarayanan, K.Q. Luo, P. Chen, Glowing graphene quantum dots and carbon dots: properties, syntheses, and biological applications, Small 11 (2015) 1620–1636. [20] K.L. Schroeder, R.V. Goreham, T. Nann, Graphene quantum dots for theranostics and bioimaging, Pharm. Res. 33 (2016) 2337–2357. [21] S.J. Zhu, J.R. Shao, Y.B. Song, X.H. Zhao, J.L. Du, L. Wang, H.Y. Wang, K. Zhang, J.H. Zhang, B. Yang, Investigating the surface state of graphene quantum dots, Nanoscale 7 (2015) 7927–7933. [22] J. Qiao, X.Y. Mu, L. Qi, J.J. Deng, L.Q. Mao, Folic acid-functionalized fluorescent gold nanoclusters with polymers as linkers for cancer cell imaging, Chem. Commun. 49 (2013) 8030–8032. [23] D. Lei, W. Yang, Y.Q. Gong, J. Jing, H.L. Nie, B. Yu, X.L. Zhang, Non-covalent decoration of carbon dots with folic acid via a polymer-assisted strategy for fast and targeted cancer cell fluorescence imaging, Sens. Actuators B 230 (2016) 714–720. [24] D. Feng, Y.C. Song, W. Shi, X.H. Li, H.M. Ma, Distinguishing folate-receptor-positive cells from folate-receptor-negative cells using a fluorescence off-on nanoprobe, Anal. Chem. 85 (2013) 6530–6535. [25] L.M. Zhang, J.G. Xia, Q.H. Zhao, L.W. Liu, Z.J. Zhang, Functional graphene oxide as a nanocarrier for controlled loading and targeted delivery of mixed anticancer drugs, Small 6 (2010) 537–544. [26] X.J. Wang, X. Sun, J. Lao, H. He, T.T. Cheng, M.Q. Wang, S.J. Wang, F. Huang, Multifunctional graphene quantum dots for simultaneous targeted cellular imaging and drug delivery, Colloids Surf. B 122 (2014) 638–644. [27] C. Shen, Y.P. Sun, J. Wang, Y. Lu, Facile route to highly photoluminescent carbon nanodots for ion detection, pH sensors and bioimaging, Nanoscale 6 (2014) 9139–9147. [28] R.Z. Zhang, W. Chen, Nitrogen-doped carbon quantum dots: facile synthesis and application as a turn-off fluorescent probe for detection of Hg2+ ions, Biosens. Bioelectron. 55 (2014) 83–90. [29] D. Qu, M. Zheng, P. Du, Y. Zhou, L.G. Zhang, D. Li, H.Q. Tan, Z. Zhao, Z.G. Xie, Z.C. Sun, Highly luminescent S, N co-doped graphene quantum dots with broad visible absorption bands for visible light photocatalysts, Nanoscale 5 (2013) 12272–12277. [30] Z.H. Sheng, L. Shao, J.J. Chen, W.J. Bao, F.B. Wang, X.H. Xia, Catalyst-free synthesis of nitrogen-doped graphene via thermal annealing graphite oxide with melamine and its excellent electrocatalysis, ACS Nano 5 (2011) 4350–4358. [31] H.J. Sun, N. Gao, L. Wu, J.S. Ren, W.L. Wei, X.G. Qu, Highly photoluminescent amino-functionalized graphene quantum dots used for sensing copper ions, Chem. − Eur. J. 19 (2013) 13362–13368. [32] Z. Guo, Z.Q. Zhang, W. Zhang, L.Q. Zhou, H.W. Li, H.M. Wang, C. Andreazza-Vignolle, P. Andreazza, D.X. Zhao, Y.H. Wu, Q.L. Wang, T. Zhang, K.M. Jiang, Color-switchable, emission-enhanced fluorescence realized by engineering C-dot@C-dot nanoparticles, ACS Appl. Mater. Interfaces 6 (2014) 20700–20708. [33] Z.X. Gan, H. Xu, Y.L. Hao, Mechanism for excitation-dependent photoluminescence from graphene quantum dots and other graphene oxide derivates: consensus, debates and challenges, Nanoscale 8 (2016) 7794–7807. [34] A. Barati, M. Shamsipur, H. Abdollahi, Carbon dots with strong excitation-dependent fluorescence changes towards pH. Application as nanosensors for a broad range of pH, Anal. Chim. Acta 931 (2016) 25–33. [35] M.Y. Xue, Z.H. Zhan, M.B. Zou, L.L. Zhang, S.L. Zhao, Green synthesis of stable and biocompatible fluorescent carbon dots from peanut shells for multicolor living cell imaging, New J. Chem. 40 (2016) 1698–1703.

69

[36] D.Y. Pan, J.C. Zhang, Z. Li, M.H. Wu, Hydrothermal route for cutting graphene sheets into blue-luminescent graphene quantum dots, Adv. Mater. 22 (2010) 734–738. [37] M. Hassan, E. Haque, K.R. Reddy, A.I. Minett, J. Chen, V.G. Gomes, Edge-enriched graphene quantum dots for enhanced photo-luminescence and supercapacitance, Nanoscale 6 (2014) 11988–11994. [38] J.L. Du, H.Y. Wang, L. Wang, S.J. Zhu, Y.B. Song, B. Yang, H.B. Sun, Insight into the effect of functional groups on visible-fluorescence emissions of graphene quantum dots, J. Mater. Chem. C 4 (2016) 2235–2242. [39] L. Wang, S.J. Zhu, H.Y. Wang, Y.F. Wang, Y.W. Hao, J.H. Zhang, Q.D. Chen, Y.L. Zhang, W. Han, B. Yang, H.B. Sun, Unraveling bright molecule-like state and dark intrinsic state in green-fluorescence graphene quantum dots via ultrafast spectroscopy, Adv. Opt. Mater. 1 (2013) 264–271. [40] S.J. Zhu, J.H. Zhang, S.J. Tang, C.Y. Qiao, L. Wang, H.Y. Wang, X. Liu, B. Li, Y.F. Li, W.L. Yu, X.F. Wang, H.C. Sun, B. Yang, Surface chemistry routes to modulate the photoluminescence of graphene quantum dots: from fluorescence mechanism to up-conversion bioimaging applications, Adv. Funct. Mater. 22 (2012) 4732–4740. [41] H. Yoon, Y.H. Chang, S.H. Song, E.S. Lee, S.H. Jin, C. Park, J. Lee, B.H. Kim, H.J. Kang, Y.H. Kim, S. Jeon, Intrinsic photoluminescence emission from subdomained graphene quantum dots, Adv. Mater. 28 (2016) 5255–5261. [42] D.Y. Pan, J.C. Zhang, Z. Li, C. Wu, X.M. Yan, M.H. Wu, Observation of pHsolvent-, spin-, and excitation-dependent blue photoluminescence from carbon nanoparticles, Chem. Commun. 46 (2010) 3681–3683. [43] S. Dhar, Z. Liu, J. Thomale, H.J. Dai, S.J. Lippard, Targeted single-wall carbon nanotube-mediated Pt (IV) prodrug delivery using folate as a homing device, J. Am. Chem. Soc. 130 (2008) 11467–11476. [44] W. Shi, X.H. Li, H.M. Ma, A tunable ratiometric pH sensor based on carbon nanodots for the quantitative measurement of the intracellular pH of whole cells, Angew. Chem. Int. Ed. 51 (2012) 6432–6435. [45] W.J. Wang, J.M. Xia, J. Feng, M.Q. He, M.L. Chen, J.H. Wang, Green preparation of carbon dots for intracellular pH sensing and multicolor live cell imaging, J. Mater. Chem. B 4 (2016) 7130–7137. [46] F. Bruni, J. Pedrini, C. Bossio, B. Santiago-Gonzalez, F. Meinardi, W.K. Bae, V.I. Klimov, G. Lanzani, S. Brovelli, Two-color emitting colloidal nanocrystals as single-particle ratiometric probes of intracellular pH, Adv. Funct. Mater. 7 (2017) 1605533.

Biographies Xin Hai received her bachelor degree (2013) and Master degree (2015) in Analytical Chemistry from Northeastern University (China). She now is a Ph.D student at the Research Centre for Analytical Sciences in Northeastern University, China. Her work is devoted to the functionalization of graphene quantum dots and its applications in biological assays. Yi-ting Wang received Master degree (2016) in Analytical Chemistry from Northeastern University (China). She is currently a Ph.D student in the Department of Chemistry at Northeastern University. Her work is focus on the biosensing and bioimaging based on carbon nitride. Xiao-yao Hao received her bachelor degree (2017) in Applied Chemistry from Northeastern University (China). She now is a master student at the Research Centre for Analytical Sciences in Northeastern University, China. Her work is devoted to the functionalization of graphene-based materials for biosensing. Xu-wei Chen is a professor at the Research Centre for Analytical Sciences in Northeastern University, China. Prof. Chen received his PhD degree in Analytical Chemistry from Northeastern University(China) in July 2007. His current research interests focus on the development of new techniques and materials for the isolation of trace biomacromolecules from complex biological samples. Jian-hua Wang is currently a professor at the Research Centre for Analytical Sciences in Northeastern University, China. He obtained his B.Sc from the Department of Chemistry, Nankai University, and M.Sc from Jilin University, China. He was awarded a PhD degree by Technical University of Denmark in 2002. He has been serving as an Associate Editor for TALANTA since Dec. 2004. His current research interest is focused on the exploitation of flow analysis protocols and hyphenation techniques, isolation of trace level of analytes of biologically significant (metals and biomacromolecules) from complex sample matrices and life science analysis.