Rhodamine-Functionalized Graphene Quantum Dots for Detection of

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Rhodamine-functionalized graphene quantum dots for detection of Fe3+ in cancer stem cells Ruihua Guo, Shixin Zhou, Yunchao Li, Xiaohong Li, Louzhen Fan, and Nicolas H. Voelcker ACS Appl. Mater. Interfaces, Just Accepted Manuscript • DOI: 10.1021/acsami.5b06523 • Publication Date (Web): 28 Aug 2015 Downloaded from http://pubs.acs.org on August 31, 2015

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ACS Applied Materials & Interfaces

Rhodamine-functionalized graphene quantum dots for detection of Fe3+ in cancer stem cells Ruihua Guo,a Shixin Zhou, b Yunchao Li,a Xiaohong Li,a Louzhen Fan,*a and Nicolas H. Voelcker*c a

b

Department of Chemistry, Beijing Normal University, Beijing 100875, P. R. China

Department of Cell Biology, School of Basic Medicine, Peking University Health Science Center, Beijing 100191, P. R. China

c

ARC Centre of Excellence in Convergent Bio-Nano Science and Technology, University of South Australia, Mawson Lakes, SA 5095, Australia *Corresponding Author: [email protected]; [email protected]

ACS Paragon Plus Environment

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KEYWORDS: graphene quantum dots, rhodamine B derivative, detection, Fe3+, cancer stem cells

ABSTRACT

A turn-on orange-red fluorescent nanosensor based on rhodamine B derivative-functionalized graphene quantum dots (RBD-GQDs) has been successfully synthesized for Fe3+ detection with high sensitivity and selectivity. By connected with GQDs, the water solubility, sensitivity, photostability and biocompatibility of RBD are drastically improved. The most distinctive feature of the RBD-GQDs, which sets them apart from other previously reported fluorophores or GQDs is that they with the detection limits as low as 0.02 µM are demonstrated as a Fe3+ turn-on fluorescent nanosensor in cancer stem cells. Fe3+ binding to such GQDs (RBD-GQDs-Fe3+) with orange-red fluerescence of 43% quantum yield were demonstrated to be the biomarkers for cancer stem cell imaging.

1. INTRODUCTION Fe3+, as one of the most important physiologically relevant metal ions, performs indispensable and versatile roles in many physiological and pathological processes.1-4 Both deficiency and excess accumulation in the human body can induce various health problems related to anemia, cancers and dysfunction of organs.5,6 Thus, new theranostic methods are needed to visualize the concentration of Fe3+ in cells. Among various strategies,7,8 fluorescent Fe3+ sensors are particularly attractive due to their high sensitivity,9-14 in which rhodamine derivatives are extensively employed in turn-on fluorescent chemosensors for the selective recognition of Fe3+ due to the switching between the spirocyclic form (colorless and non-fluorescent) and the ringopened amide form (pink and strongly fluorescent).15-19 However, they intrinsically suffer from


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poor solubility and photostability, which hinders their long-term biological imaging because of fast photobleaching. Yang and Sun’s group has coupled these organic molecules to PEG-Fe3O4 nanoparticles to resolve the low solubility and improve the detection sensitivity.20,21 However, it has remained a challenge to creat a sensor that possesses the required photostability, biocompatibility and sensitivity for Fe3+ levels encountered inside of cells. Graphene quantum dots (GQDs) have ignited tremendous research interest for their outstanding properties in terms of good photostability, excellent biocompatibility and cell membrane permeability due to their small size, tunable surface functionalities and long term resistance of photobleaching,22-26 making them perfect alternatives to organic dyes and semiconductor quantum dots for fluorescent bioimaging and biosensing.27-29 Despite the highly anticipated potential of these nanostructures, there is a need to develop GQDs with longerwavelength emission and higher quantum yield (Ф), which are critically needed in biological imaging owing to the reduced background autofluorescence and improved signal-to-noise ratio. On the other hand, various carbon nanomaterials-based fluorescent probes30-35 for the detection of metal ions have been explored,36-42 whereas in most of reports on graphitic carbon quantum dots43,44 and fluorescent graphene oxide45-47 as fluorescent Fe3+ sensors, ion binding is signaled by fluorescence quenching through an electron transfer due to the special coordinate interaction between Fe3+ and phenolic hydroxyl on the surface. However, turn-on fluorescent sensors are usually preferred over turn-off sensors due to the enhanced sensing sensitivity.48 Herein, we report a N-(rhodamine B) lactam-ethylenediamine (RBD)-functionalized GQDs (RBD-GQDs) as a Fe3+ turn-on fluorescent nanosensor in living cells with improved water solubility, sensitivity, photostability and biocompatibility, and the detection limits as low as 0.02 µM. Significantly, RBD-GQDs can easily penetrate into cancer stem cells (CSCs) and be responsive to Fe3+ there.


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The strong orange-red fluorescence (Ф = 43%) of Fe3+ binding to RBD-GQDs (RBD-GQDs-Fe3+) are also demonstrated to be the biomarkers for CSCs imaging.

2. EXPERIMENTAL SECTION 2.1 Materials and reagents. High purity graphite rods were purchased from Shanghai Carbon Co., Ltd (China). Other chemical solvents and reagents were of analytical reagent grade and were used without further purification. Deionized water was used to prepare all aqueous solutions. The metal salts were Fe(NO3)3, AlCl3·6H2O, CaCl2, CdCl2·H2O, CoCl2·6H2O, FeSO4·7H2O, Hg(ClO4)2, KCl, MgSO4, NaCl, NH4Cl, NiCl2·6H2O, Zn(NO3)2·6H2O, CuCl2·2H2O, Pb(ClO4)2. The salts solutions of anions were the sodium salts of the respective anions (F-, Cl-, Br-, I-, CO32-, CH3COO-, C2O42-, ClO-, ClO4-, SO42-, S2-, NO3-, NO2-, CNO-, N3-, HPO42-, Cr2O72-, H2AsO4-). In the fluorescence measurements, the RBD-GQDs solution (5 mg/L) was prepared with 20 mM Tris-HCl buffer (ethanol-H2O, 1:1 v/v; pH 7.0). In the cellular experiments, the RBD-GQDs solution (25 mg/L) was prepared with PBS buffer (pH 7.4). 2.2 Synthesis of RBD. RBD was synthesized according to previous literature reports.49 Briefly, rhodamine B (0.9580 g, 2 mmol) was dissolved in hot ethanol (30 mL) with addition dropwise of ethylenediamine (0.67 mL, 10 mmol). The reaction mixture was refluxed for 24 h until the fluorescence of the solution disappeared. The reaction was cooled to room temperature and the solvent was removed by evaporation. Then, 20 mL of water was added to the resultant and extracted with CH2Cl2 (20 ml × 2). The organic phase was collected and washed several times with saturated NaCl and dried over anhydrous Na2SO4. The solvent was removed by evaporation and dried under vacuum. Yield: 0.59 g, 61.9%.


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Single

crystal

of

RBD

was

obtained

by

slow

evaporation

of

acetonitrile/hexane/dichloromethane solution (2.5 : 2.5 : 1, v/v/v). The single-crystal X-ray diffraction study showed that the molecular structure of RBD is of spirocyclic form in the crystal (Figure S1). ESI-MS m/z : 485.4 (M+H)+ (Figure S2). 1H NMR (400 MHz, CDCl3) δ 7.90 (dd, J = 5.5, 3.1 Hz, 1H), 7.56-7.33 (m, 2H), 7.09 (dd, J = 5.4, 3.0 Hz, 1H), 6.43 (d, J = 8.8 Hz, 2H), 6.37 (d, J = 2.4 Hz, 2H), 6.27 (dd, J = 8.9, 2.4 Hz, 2H), 3.53-3.23 (m, 8H), 3.19 (t, J = 6.5 Hz, 2H), 2.42 (t, J = 6.5 Hz, 2H), 1.56 (s, 3H), 1.16 (t, J = 7.0 Hz, 12H) (Figure S3). 2.3 Preparation of GQDs. GQDs were prepared by electrochemical exfoliation of graphite rod and acidic oxidation of exfoliation using concentrated mineral acid. The electrolysis of the graphite rod was performed on a HDV-7C transistor potentiostat with cathode current about 10 mA. The graphite rod was inserted as a working electrode into 5 mL DMSO solution which contained 0.01 M TBAP as electrolyte, paralleled to a Pt foil used as counter electrode. After electrolysis for 3 h, the resulting black solution was washed with ethanol and then centrifuged for several times to remove DMSO and TBAP. Finally, it was dried at 75 °C in a drying oven to a black powder. 0.1 g of the prepared black powder was added into a mixture of concentrated H2SO4 (15 mL) and HNO3 (5 mL). After refluxing at 100 °C for 24 h, the mixture was cooled to room temperature and diluted with deionized (DI) water. The excessive acid was neutralized by Na2CO3. The obtained solution was further dialyzed over DI water in a dialysis bag (molecular weight cutoff: 3500 Da) for one week. 2.4 Synthesis of RBD-GQDs. The synthetic route of RBD-GQDs is shown in Scheme 1. A mixture of as-prepared GQDs (30 mg) and SOCl2 (30 mL) was reacted in the presence of anhydrous DMF (0.5 mL) at 75 °C for 24 h under nitrogen atmosphere. And then excess SOCl2 was removed by distillation. Finally, in the presence of triethylamine (TEA, 0.5 mL), RBD (30


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mg) was added to the above product and heated in anhydrous DMF (30 mL) at 85 °C for 48 h. The resulting mixture was cooled to room temperature, transferred into dichloromethane and then washed three times with saturated NaHCO3. The organic phase was collected. The product was precipitated by adding excess n-hexane and collected by centrifugation at 12000 rpm, and washed several times with n-hexane/dichloromethane (5:1, v/v) and dried under vacuum. 2.5 Characterization. A JEOL JEM 2100 transmission electron microscope (TEM) was used to investigate the morphologies of the GQDs and RBD-GQDs. The FT-IR spectra were measured using a Nicolet 380 spectrograph. X-ray diffraction (XRD) patterns were obtained by using CuKα radiation (XRD, PANalytical X’Pert Pro MPD). X-ray photoelectron spectroscopy (XPS) were carried out by an ESCALab 250Xi electron spectrometer from Thermo Scientific using 300 W Al Kα radiation. The base pressure was about 3 × 10-9 mbar. The binding energies were referenced to the C1s line at 284.8 eV from adventitious carbon. The PL spectra were recorded on a Cary Eclipse fluorimeter. The 1H NMR spectra were recorded at 400 MHz on a Bruker Advance spectrometer in CDCl3, with chemical shift values in parts per million. An LEICA STP6000 (Germany) confocal fluorescence microcope was used to obtain fluorescence microscopy images, with excitation wavelengths at 530 nm. Crystal structure of RBD was collected on a Bruker Smart Apex П CCD diffractometer with graphite–monochromated Mo–K radiation (0.71073Å) at 110 K. The structure was solved by direct methods and refined with the full-matrix least-squares technique based on F2 using the SHELXL program with anisotropic thermal parameters for non-hydrogen atoms. 2.6 Detection of Fe3+ using RBD-GQDs. The detection of Fe3+ was performed at room temperature in Tris-HCl buffer (20 mM, pH 7.0). In a typical run, the RBD-GQDs solution (5 mg/L) was prepared with 20 mM Tris-HCl buffer (ethanol-H2O, 1:1 v/v; pH 7.0), followed by


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the addition of a calculated amount of Fe3+. The resulting solution was shaked well before recording the fluorescence emission spectra. The sensitivity and selectivity measurements were conducted in triplicate. 2.7 Cell fluorescence. HeLa cells were incubated in RPMI1640 medium supplemented with 10% fetal bovine serum (FBS) at 37 °C in a humidified atmosphere of 5% CO2 and 95% air, which were seeded in a flat-bottom 6-well plate (1.5 × 104 cells/well) with glass coverslips in 2 mL culture medium. After overnight incubation, the cells were treated with or without Fe3+ solutions for 5 h and washed three times with PBS buffer (pH 7.4) to remove any excess Fe3+. The treated HeLa cells were then incubated with RBD-GQDs (25 mg/L) in medium for 0.5 h. Then the HeLa cells on the plate were washed with PBS buffer and fixed by 4 % paraformaldehyde in PBS buffer for 15 min at room temperature. After being washed three times with PBS buffer, the cells were imaged by a confocal fluorescence microscope. Pancreatic cancer stem cells were identified and sorted by surface marker, CD44, from pancreatic cancer cell lines,50 which were incubated in DMEM medium supplemented with 10% fetal bovine serum (FBS) at 37 °C in a humidified atmosphere of 5% CO2 and 95% air. The cancer stem cells were treated with the same procedure as above HeLa cells.

3. RESULTS AND DISCUSSION An acylation reaction was used to synthesize RBD-GQDs, as illustrated in Scheme 1. GQDs (Figure S4), prepared from electrochemical exfoliation of graphite rods followed by refluxing with concentrated acid, were activated first to transform carboxylic groups into acyl chloride, followed by a nucleophilic substitution reaction with RBD between the acyl chloride and amine groups to give RBD-GQDs.


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Scheme 1. Synthetic route of RBD-GQDs. TEM images (Figure 1c, f) showed that the obtained GQDs and RBD-GQDs had the average diameter of 4.6 and 5.0 nm, respectively. The high-resolution TEM (HRTEM) images (Figure 1b, e) confirmed that the GQDs and RBD-GQDs exhibited high crystallinity with the lattice spacing of 0.21 nm, corresponding to (1120) lattice fringes of graphene, indicating that there were no significant changes in size and morphology of GQDs after modification with RBD.

Figure 1. TEM images of (a) GQDs and (d) RBD-GQDs. High-resolution TEM images of (b) GQDs and (e) RBD-GQDs. Size distribution of (c) GQDs and (f) RBD-GQDs. Figure 2a showed the XRD profiles for RBD, GQDs and RBD-GQDs. Both the GQDs and RBD-GQDs had a broad peak. The XRD peak for RBD-GQDs shifted to a lower degree (22.3


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degrees) compared with the GQDs (23.6 degrees), suggesting that RBD-GQDs had a larger interlayer spacing after introducing RBD into the GQDs sheets. The successful functionalization of GQDs was also corroborated by XPS analysis (Figure 2b). RBD-GQDs had a higher N content (7.08 %) and higher C:O atomic ratios (5.72) than that of GQDs (1.92). FTIR spectrum further revealed changes in the chemical functional groups on the GQDs (Figure 2c). The asprepared GQDs showed peaks at 1728 cm-1 (C=O stretching), 1623 cm-1 (C=C stretching), and a broad peak at 3434 cm-1 that corresponded to carboxylic acid and hydroxyl groups. Upon surface functionalization with RBD, the intensity of the carbonyl group peak diminished, while two new peaks appeared at 1769 and 1717 cm-1 corresponding to carbonyl bands of five-membered cyclic imides, indicating that the periphery of RBD-GQDs was principally functionalized with the phthalimide structures.51

Figure 2. (a) Powder X-ray diffraction (XRD) patterns of RBD, GQDs and RBD-GQDs. (b) XPS of GQDs and RBD-GQDs. (c) FTIR spectra of RBD, GQDs and RBD-GQDs. The UV-vis absorption spectra of GQDs, RBD, RBD-GQDs and RBD-GQDs-Fe3+ were shown in Figure 3a. The spectrum of GQDs showed the typical absorption peaks at 230 and 270 nm due to π-π* transition of aromatic sp2 domains and another weak peak at 360 nm assigned to a n-π* transition, while RBD exhibited absorption at 240, 275 and 318 nm. In comparison, the


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RBD-GQDs had absorption peaks at 223, 238, 272 and 314 nm, verifying the successful conjugation of RBD with the GQDs. Immediately after the introduction of Fe3+ into the RBDGQDs solution, a new peak appeared at 559 nm, indicating that the spirolactam ring of RBD was opened by Fe3+.16 The Fe3+ triggered absorption caused a conspicuous color change from light brown to pink (inset of Figure 3a). As shown in Figure 3b, the photoluminescence (PL) emission of the GQDs (excited at 440 nm) appeared at 520 nm, corresponding to greenish-yellow luminescence (inset of Figure 3b), while for RBD, no PL was detected in the visible-light region. The PL emission of RBD-GQDs was red-shifted by about 30 nm to 550 nm compared with that of GQDs, giving yellow luminescence (inset of Figure 3b). Upon addition of Fe3+ in the solution of RBD-GQDs, a new strong peak accompanied by orange-red emission (centered at 580 nm, inset of Figure 3b) appeared, which was the result of the on-switch of the spirocyclic moiety, mediated by the Fe3+ as illustratrated in Figure 3c.17,20 The binding of RBD-GQDs with Fe3+ was demonstrated to be reversible (Figure S5). The pink color and orange-red fluorescence of RBDGQDs-Fe3+ disappeared immediately when excess EDTA was added into the solution of RBDGQDs-Fe3+. If Fe3+ was further added into this solution, the pink color and orange-red fluorescence could recover, indicating that Fe3+ is reversibly binding to RBD-GQDs. The quantum yield (Ф) of RBD-GQDs-Fe3+ was as high as 43%, which was calculated using Rhodamine B as the reference. It was estimated that there was about 553 mg RBDs on 1 g GQDs (see Supporting information).


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Figure 3. (a) UV-vis absorption spectra of GQDs, RBD, RBD-GQDs, and RBD-GQDs-Fe3+. (b) Fluorescence emission spectra of GQDs, RBD-GQDs, and RBD-GQDs-Fe3+. The insets show photographs of GQDs, RBD-GQDs and RBD-GQDs-Fe3+ (from left to right) under day light (a) and under UV light (365 nm) (b). (c) The illustration for RBD-GQDs binding with Fe3+. A fluorescence titration of Fe3+ was carried out using 5 mg/L RBD-GQDs in 20 mM Tris-HCl buffer-ethanol (1/1, v/v) at pH 7.0. Without Fe3+, the solution was almost colorless and showed a rather weak fluorescence signal over the whole wavelength range. However, upon the addition of Fe3+ from 0 to 65 µM, the fluorescence intensity of RBD-GQDs at 580 nm increased gradually (Figure 4a). Figure 4b presented the relationship of the changes of fluorescence intensity F - F0 (F and F0 were the fluorescence intensities of RBD-GQDs at 580 nm in the presence and absence of Fe3+, respectively) with the concentration of Fe3+. Saturation was observed after addition of 22.5 µM of Fe3+ (Figure 4b). The luminescence enhancement factor for titration equilibrium was around 28.1-fold. The inset of Figure 4b showed that the F - F0 had a good linear dependence on the Fe3+ concentration in the range of 0 to 1 µM, and the linear regression equation is F - F0 = 16.335c - 0.161 with a correlation coefficient of 0.9975, where c is the concentration of Fe3+


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(µM). The detection limit of 0.02 µM (1.12 ppb) was obtained based on a 3σ/m (σ was the standard deviation of the blank signal and m was the slope of the linear calibration plot), which was much lower than most of the previous reported assays for Fe3+ detection,52-54 indicating that the RBD-GQDs possessed excellent sensitivity in the detection of trace Fe3+ than RBD and other Fe3+-sensing materials (Table S1).45,55,56

Figure 4. (a) Fluorescence response of RBD-GQDs (5 mg/L) in 20 mM Tris-HCl buffer (ethanol-H2O, 1:1 v/v; pH 7.0) solution upon addition of varying concentrations of Fe3+ with an excitation at 540 nm. (b) Plot of the changes of fluorescence intensity F-F0 at 580 nm versus Fe3+ concentrations ranging from 0 to 65 µM. The inset in (b) is the linear calibration plot for Fe3+ detection with the concentration ranging from 0 to 1 µM. The fluorescence responses of RBD-GQDs as a result of the addition of other cations, including Al3+, Ca2+, Cd2+, Co2+, Cu2+, Hg2+, K+, Mg2+, Mn2+, Na+, NH4+, Ni2+, Pb2+, Zn2+ and Fe2+ were also investigated. Fluorescence intensity changes of solutions of RBD-GQDs (5 mg/L), recorded in the presence of 20 µM Fe3+ and 1 mM each of these cations under identical condition, were displayed in Figure 5a. Of all the ions tested, only the addition of Al3+ resulted in a very small increase in fluorescence intensity. The enhancement in fluorescence intensity resulting from the addition of Fe3+ was not influenced by the presence of other cations (Figure


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5b). Moreover, the addition of various anions did not cause any discernible changes in fluorescence intensity and the wavelength of emission (Figure S6). The stability of RBD-GQDs in acidic or basic pH solutions was also investigated (Figure S7). When pH value was higher than 5.0, no any change was observed. But when pH < 5.0, the intensity of fluorescence increased gradually with the pH value decreased, indicating that spirolactam ring of RBD could be opened by H+ at very strong acidic solution, which clearly explained that the RBD-GQDs could be used in a broad range of pH (5 - 13). Based on these above results, it was very encouraging that the RBD-GQDs could detect Fe3+ in the presence of all possible interference ions, underlining the high selectivity of RBD-GQDs for detecting Fe3+.

Figure 5. (a) Fluorescence responses of RBD-GQDs (5 mg/L) in the presence of various metal ions (20 µM Fe3+, 1 mM other metal ions) in Tris-HCl buffer (20 mM; ethanol-H2O, 1:1 v/v; pH 7.0) solution with an excitation at 540 nm. (b) Fluorescence response of RBD-GQDs (5 mg/L) to various cations and selective of RBD-GQDs for Fe3+ in the presence of other metal ions in TrisHCl buffered (20 mM; ethanol-H2O, 1:1 v/v; pH 7.0) solution. The white bars represent the emission of RBD-GQDs in the presence of 1 mM of cations. The black bars represent the change of the emission that occurs upon the subsequent addition of 20 µM Fe3+ to a solution containing of RBD-GQDs and 1 mM cations. The emission intensities were recorded at 580 nm.


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The sensitive and selective binding of Fe3+ to RBD-GQDs was then first explored for the detection of Fe3+ in HeLa cells. HeLa cells were incubated with and without Fe3+ solutions for 5 h in RPMI1640 medium supplemented with 10% FBS at 37 °C, then washed with PBS buffer (pH 7.4) to remove any excess Fe3+. The treated HeLa cells were then incubated with RBDGQDs (25 mg/L) in medium for 0.5 h. After being washed with PBS buffer, the cells were imaged by a confocal fluorescence microscope. It can be seen that the cells incubated without Fe3+ exhibited weak yellow fluorescence (Figure 6a), whereas those with Fe3+ displayed strong orange-red fluorescence (Figure 6b). The confocal section images of HeLa cells (Figure S8) showed that the vision was dimmer both above and below the cells than that within the cells, connoting an efficient internalization of RBD-GQDs to the HeLa cells in the cytoplasmic areas, not just adsorbed on the outer membrane surfaces. This result testified that the RBD-GQDs easily penetrated into the cells but did not enter the nuclei. Furthermore, the cells exposed to 25 µM Fe3+ showed brighter orange-red fluorescence (Figure 6c) than those exposed to 10 µM Fe3+ (Figure 6b), indicating that the RBD-GQDs could be responsive to Fe3+ in HeLa cells.


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Figure 6. Confocal fluorescence microscopy images of HeLa cells incubated with 0 µM (a), 10 µM (b) and 25 µM (c) Fe3+ for 5 h, followed by further incubation with RBD-GQDs (25 mg/L) for 0.5 h. Cancer stem cells (CSCs), as a subpopulation of stem-like cells within tumors, exhibit characteristic of both stem cells and cancer cells.57 They have the capacity to self-renew inside a tumor and to generate heterogeneous lineages of cancer cells that drive cancer progression, metastasis and drug resistance.50 Therefore, they have been proposed as the cause of tumor relapse and are the relatively new target of cancer therapies.58 According to our previous report,


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the labelling of the stem cells poses a considerable challenge because of their particularity.59 Although almost all nanomaterials can penetrate the cancer cells effortlessly for imaging, only unique GQDs with specific structure can enter the stem cells as labeling agents.22,28 The exceptional binding of Fe3+ to RBD-GQDs (Figure S9) coupling with the durability and biological compatibility of GQDs are compelling and have prompted us to explore their potential use in CSCs. Pancreatic CSCs were cultured with the same procedure as above HeLa cells. The CSCs incubated with only RBD-GQDs exhibited weak yellow fluorescence (Figure S10), whereas those incubated with Fe3+ then washed with PBS buffer (pH 7.4) to remove any excess Fe3+ and further with RBD-GQDs displayed strong orange-red intracellular fluorescence (Figure 7), demonstrating that the RBD-GQDs could penetrate the Pancreatic CSCs membrane and bind to intracellular Fe3+. The intracellular fluorescence image grew brighter as the concentration of Fe3+ increased (Figure 7). The flow cytometry analysis of Pancreatic CSCs quantitatively showed that the intensity of cellular fluorescence increased gradually with the concentration of Fe3+ increased (Figure S11), which was consistent with the results of confocal fluorescence microscopy images, sufficiently indicating that the RBD-GQDs could be responsive to Fe3+ in the Pancreatic CSCs.


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Figure 7. Confocal fluorescence microscopy images of Pancreatic CSCs incubated with (a) 10 µM, (b) 25 µM and (c) 50 µM Fe3+ for 5 h, followed by further incubation with RBD-GQDs (25 mg/L) for 0.5 h. To further demonstrate the potential applications of strong orange-red fluorescence of RBDGQDs-Fe3+, Pancreatic CSCs were cultured with RBD-GQDs-Fe3+ directly in DMEM medium (10 % FBS) for 5 h at 37 °C then washed with PBS buffer (pH 7.4) to remove any excess RBDGQDs-Fe3+. From Figure S12, the morphology of Pancreatic CSCs can be clearly discerned with


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the internalized RBD-GQDs-Fe3+. The orange-red spots could only be observed in the cytoplasmic area, and the intensity at the central region of the nucleus was relatively weak. The result testified that the RBD-GQDs-Fe3+ easily penetrated into the CSCs but did not enter the nucleis, suggesting that genetic disruptions of the living CSCs by the RBD-GQDs-Fe3+ didn’t happen. In addition, after several runs of repeated excitation, no fluorescence intensity decreasing in the microscopy images could be detected, demonstrating high photostability of RBD-GQDs-Fe3+ in living cells. Furthermore, the better photostability of RBD-GQDs-Fe3+ than that of RBD-Fe3+ was also demonstrated. As shown in Figure S13, the fluorescence intensity of RBD-GQDs-Fe3+ did not change even after continuous excitation with a Xe lamp for 12 h, while that of RBD-Fe3+ was decreased by 25 %. The standard Cell Counting Kit-8 (CCK-8) assays were performed to evaluate the cytotoxicity of RBD-GQDs-Fe3+ and RBD-Fe3+ in living cells to compare the biocompatibility of RBDGQDs-Fe3+ with that of RBD-Fe3+. As shown in Figure 8, average cell viability of HeLa cells was nearly 50% after 24 h culturing with RBD-GQDs-Fe3+ at concentration of 100 mg/L (Figure 8a), while only 20% was for RBD-Fe3+ (Figure 8b), indicating the better biocompatibility of RBD-GQDs-Fe3+ than that of RBD-Fe3+. This result testified that the fluorescent RBD-GQDsFe3+ could serve as an effective biological imaging platform with little cytotoxicity.


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Figure 8. Cell viability of HeLa cells incubated with various concentrations of (a) RBD-GQDsFe3+ and (b) RBD-Fe3+. 4. CONCLUSIONS We have successfully synthesized a rhodamine B derivative functionalized GQDs (RBDGQDs) as a Fe3+ turn-on fluorescent nanosensor in living cells, especially in CSCs, with the detection limits as low as 0.02 µM. Fe3+ binding to such GQDs (RBD-GQDs-Fe3+) with orangered fluerence of 43% quantum yield were demonstrated to be the biomarkers for CSCs imaging. To the best of our knowledge, this is the first time that GQDs are used to detect Fe3+ and as fluorescent labeling agents in CSCs. Compared with other competing fluorescent nanomaterials, these GQDs are expected to be more practicable in biomedical applications, such as in tracking the proliferation, apoptosis, and differentiation of various cell lineages and in drug delivery, due to the scope for further straightforward functionalization. Detailed follow-up work is underway in our laboratory and will be reported in due course.

ASSOCIATED CONTENT Supporting Information. The characterization of RBD with single-crystal X-ray diffraction study, ESI-MS and

1

H NMR, the reversible binding nature of RBD-GQDs with Fe3+,

fluorescence responses of RBD-GQDs to various anions and pH, confocal fluorescence microscopy section images of HeLa cells incubated with Fe3+ and followed with RBD-GQDs, confocal fluorescence microscopy images of Pancreatic CSCs incubated with pure RBD-GQDs, flow cytometry analysis of Pancreatic CSCs incubated with different concentration of Fe3+ and followed with RBD-GQDs, confocal fluorescence microscopy images of Pancreatic CSCs


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incubated with RBD-GQDs-Fe3+, photostability test of RBD-GQDs-Fe3+ and RBD-Fe3+. This material is available free of charge via the Internet at http://pubs.acs.org. AUTHOR INFORMATION Corresponding Author ∗

E-mail: [email protected]; [email protected]

ACKNOWLEDGMENT This work is supported by the Major Research Plan of NSFC (21233003), the Fundamental Research Funds for the Central Universities, Key Laboratory of Theoretical and Computational Photochemistry, and the Australian Research Council Centre of Excellence in Convergent BioNano Science and Technology (CE140100036). REFERENCES (1) Wu, J. S.; Liu, W. M.; Ge, J. C.; Zhang, H. Y.; Wang, P. F. New Sensing Mechanisms for Design of Fluorescent Chemosensors Emerging in Recent Years. Chem. Soc. Rev. 2011, 40, 3483-3495. (2) Eisenstein, R S. Iron Regulatory Proteins and the Molecular Control of Mammalian Iron Metabolism. Annu. Rev. Nutr. 2000, 20, 627-662. (3) Autreaux, B. D.; Tucker, N. P.; Dixon, R.; Spiro, S. A Non-Haem Iron Centre in the Transcription Factor NorR Senses Nitric Oxide. Nature 2005, 437, 769-772. (4) Rouault, T. A. The Role of Iron Regulatory Proteins in Mammalian Iron Homeostasis and Disease. Nat. Chem. Biol. 2006, 2, 406-414. (5) Narayanaswamy, N.; Govindaraju, T. Aldazine-Based Colorimetric Sensors for Cu2+ and Fe3+. Sens. Actuators, B 2012, 161, 304-310.


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Graphic for manuscript


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