Author’s Accepted Manuscript A FRET chemsensor based on graphene quantum dots for detecting and intracellular imaging of Hg2+ Maoping Liu, Tao Liu, Yang Li, Hui Xu, Baozhan Zheng, Dongmei Wang, Juan Du, Dan Xiao www.elsevier.com/locate/talanta

PII: DOI: Reference:

S0039-9140(15)00376-8 http://dx.doi.org/10.1016/j.talanta.2015.05.023 TAL15615

To appear in: Talanta Received date: 3 March 2015 Revised date: 5 May 2015 Accepted date: 11 May 2015 Cite this article as: Maoping Liu, Tao Liu, Yang Li, Hui Xu, Baozhan Zheng, Dongmei Wang, Juan Du and Dan Xiao, A FRET chemsensor based on graphene quantum dots for detecting and intracellular imaging of Hg2+, Talanta, http://dx.doi.org/10.1016/j.talanta.2015.05.023 This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting galley proof before it is published in its final citable form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.

A FRET chemsensor based on graphene quantum dots for detecting and intracellular imaging of Hg2+

Maoping Liua, Tao Liuc, Yang Lia, Hui Xua, Baozhan Zhenga, Dongmei Wanga, Juan Dua*, and Dan Xiaoa,b* a

College of Chemistry, Sichuan University, NO.29 Wangjiang Rode, Chengdu,

610064, PR China. E-mail: [email protected]; fax: +86-28-85416029; tel: +86-28-85415029 b

College of Chemical Engineering , Sichuan University, NO. 29 Wangjiang Rode,

Chengdu, PR China c

College of Life Sciences, Sichuan University, NO. 29 Wangjiang Rode, Chengdu, PR

China

1

Abstract The detection of Hg2+ has attracted considerable attention because of the serious health and environmental problems caused by it. Herein, a novel ratiometric fluorescent chemsensor (GQDs-SR) based on fluorescence resonance energy transfer (FRET) process for detecting of Hg2+ was designed and synthesized with rhodamine derivative covalently linked onto graphene quantum dots. In this sensor, the graphene quantum dots (GQDs) served as energy donor and the rhodamine derivative turned into an energy acceptor when encountered Hg2+. The chemsensor exhibited high selectivity, low cytotoxicity, biocompatibility and good water solubility. The results of intracellular imaging experiment demonstrated that GQDs-SR was cell permeable and could be used for monitoring Hg2+ in living cells, and it was also successfully applied to the detection of Hg2+ in practical water samples. Keywords: Ratiometric fluorescent chemsensor; Graphene quantum dots; Hg2+; Living cell.

1. Introduction Mercury ion, one of the most significant cations among various heavy cations, is harmful for both environmental and biological systems [1]. It exists in a variety of different forms and can be transformed into methylmercury by microbial biomethylation in the aquatic environment, which then bioaccumulates through the food chain [2]. The exposure to mercury, even at very low concentration, leads to digestive, kidney and especially neurological diseases [3] as mercury can easily pass 2

through the biological membranes. Thus, simple, sensitive and rapid analytical methods for detection of Hg2+ in environmental and biological samples have been paid more attention to. A variety of approaches such as atomic absorption spectroscopy (AAS) [4], inductively coupled plasma mass spectrometry (ICP-MS) [5], cold-vapor atomic fluorescence

spectrometry

(CV-AFS)

[6]

and

high

performance

liquid

chromatography with detection by ICP-MS or AFS [7,8] have been developed. These techniques provide low detection limit and thus can be adapted to monitor ultratrace mercury, however, their running requires precise and expensive instruments operated by professionals [9]. Moreover, they are not suitable for in vivo monitoring of intracellular mercury. In order to overcome these drawbacks, many attempts have been made to develop portable sensors for facile and rapid detection. Thus, detection schemes based on fluorescence spectroscopy are prevalent due to its high sensitivity, fast analysis with high spatiotemporal resolution for providing in situ and real-time information, and nondestructive sample preparation when combined with microscopy. A number of fluorescent Hg2+ probes have been developed to date based on the quenching

mechanism

(“turn-off”)

[10-19]

or

target-triggered

fluorescent

enhancement (“turn-on”) [20-28]. For the “turn-off” probes, the complexation of mercury ion can induce a strong fluorescence quenching due to the spin-orbit coupling effect [29-31]. But they may report false positive results caused by other quenchers in practical samples and are undesirable for practical analytical applications. Although the “turn-on” fluorescent probes are more sensitive and favorable for 3

bioimaging applications due to their off-on response feature [32-34], the single-emission detection is readily perturbed by instrumental or environmental factors and the concentration of probe molecule. By contrast, ratiometric measurements, which involve the simultaneous measurement of two fluorescence signals at different wavelengths and use the ratio of the two fluorescence intensities to quantitatively detect the analytes, can alleviate most of these interferences and give greater precision to the data analysis [35-38]. In particular, fluorescence resonance energy transfer (FRET) [39], which is a nonradiative process that an excited state donor transfers energy to a proximal ground state acceptor through long-range dipole-dipole interactions, has been widely utilized in ratiometric detection due to its facile control as a sensing mode [40-44]. Conventionally, the FRET-based sensing systems constitute of a small-molecular dyad which contains two fluorophores connected by covalent links [45,46]. However, such fluorescent molecular sensors are difficult to be prepared and purified. On the other hand, building the FRET systems within the particles, such as quantum dots [47,48], silica [49,50] and polymer particles [51-53] can not only overcome these shortcomings but also enhance water-dispensability of the systems. Even though, their applications in the field of biological systems are still restricted due to the effect of particle size or toxicity. In the past few years, graphene and its derivatives have received a lot of attention for their super performance such as chemical stability, water dispensibility and biocompatibility [54,55]. The zero-dimensional graphene quantum dots (GQDs), which consist of small graphene nanosheets, possess strong 4

quantum confinement and edge effects [56]. However, most works on GQDs are focused on theoretical prediction and experiment synthesis [57,58], they rarely explored in ratiometric sensing applications [59,60]. In consideration of these excellent features, we report a novel ratiometric fluorescent chemsensor (GQDs-SR) comprised of graphene quantum dots and a Hg2+ recognition probe, and try to use this chemsensor for intracellular Hg2+ imaging. In this sensor, the GQDs not only serve as the energy donor, but also as the anchoring site for the probe, a rhodamine derivative. For the sensor, the presence of Hg2+ can induce an effective ring-opening reaction of the spirolactam rhodamine, affording the system an efficient FRET-based ratiometric detection for mercury ions. GQDs-SR exhibits good water solubility, little cytotoxicity, high selectivity and sensitivity, suggesting its potential and significance in bioanalysis and detection in the future. The schematic illustration for the selective detection of Hg2+ by GQDs-SR is shown in Scheme 1. Scheme 1.

2. Experimental section 2.1 Apparatus 1

H NMR measurements were performed with Bruker AV II-400 MHz

spectrometer. Mass spectra were obtained from a commercial ion trap mass spectrometer (Thermo Fisher Scientific, San Jose, CA). FT-IR was recorded on Thermo Scientific Nicolet 6700 FT-IR spectrometer (Sugar Land, TX, USA). Fluorescence spectra were acquired on F-7000 spectrophotometer equipped with a 1 5

cm quartz cell (HITACHI, Japan). UV-visible spectra were measured on U-2900 spectrophotometer (HITACHI, Japan). The transmission electron microscopy (TEM) and high resolution TEM (HRTEM) images were obtained by an FEI Tecnai F-2050 field emission HRTEM (Hills-boro, OR, USA) operating at 200 kV. X-ray diffraction (XRD) measurement was acquired on a Tongda TD-3500 X-ray powder diffractometer (Liaoning, China) with Cu Kα radiation (λ = 0.154 nm). The XRD pattern was recorded from 10°-60° at a scan rate of 0.06°/s. The lifetime measurements were performed on a HORIBA TemPro 01 fluorescence lifetime system (Glasgow, UK). A 400 nm NanoLED pulsed diode excitation source was used to excite the samples. Fluorescence images were obtained by using an Olympus IX71 with a DP70 color CCD. 2.2 Reagents Graphite Powder (spectral pure) was provided by Shanghai Huayi Company; Rhodamine B was purchased from Beijing Chemical Co., China; Lawesson’s reagent (2,4-bis(4-methoxyphenyl)-1,3-dithia-2,4-diphosphetane-2,4-disulfide) was purchased from Alfa Aesar; Anhydrous ethylenediamine was provided by Kemiou (Tianjin); Sodium salt of 4-(2-hydroxylethyl)-1-perazineethane sulfonic acid (HEPES), EDC (1-ethyl-3-(3-dimethylaminopropyl)carbodiimide) and NHS (N-hydroxy-succinimide) were

purchased

from

Sigma-Aldrich.

All

the

other

chemicals

were

of

analytical-reagent grade and used without further purification. The purified water used in this study was the double-distilled water. 2.3 Synthesis of GQDs -SR 6

2.3.1 Preparation of Graphene quantum dots (GQDs) GO was prepared from graphite powder by a modified Hummers' method [61]. The prepared GO (0.5 mg mL-1) was re-oxidized employed hydrogen peroxide (0.05 mL) and then treated with ozone for 1 h in ice-bath to form ozonized graphene oxide (O-GO) [62]. Subsequently, 3 mL O-GO and 10 mL H2SO4 were mixed and heated in a domestic microwave oven (maximum power 800 W, 2450 MHz) for 5 min, the black suspension turned into claybank solution with a few of suspended particles. After addition of 100 mL deionized water and cooled to room temperature, the mixture was filtered through 0.22 μm microporous membrane and collected the filter solution. The filter solution was further dialyzed in dialysis bag (retained molecular weight: 1000 Da) for 48 h and GQDs were obtained. 2.3.2 Synthesis of SR SR was facilely synthesized from the simple reaction of Rhodamine B derivative (R1) and Lawesson’s reagent. The synthesis and structure of SR were depicted in Scheme S1†. R1 was synthesized according to the literature [63]. R1 (0.97 g, 2 mmol) was dissolved in 20 mL dry toluene under N2, and then Lawesson’s reagent (0.4 g, 1 mmol) was slowly added. The mixture was refluxed for 12 h. After removal of the solvent, the residue was purified by silica gel column chromatography with ethyl acetate/petroleum ether (1/4, v/v) to provide the desired SR as a white solid (yield: 21%). 1H NMR spectrum of SR (CDCl3, 400 MHz) δ: 8.14 (d, 1H), 7.45 (m, 2H), 7.09 (d, 1H), 6.39 (d, 2H), 6.21 (m, 2H), 5.99 (m, 2H), 3. 58 (d, 2H), 3.20 (t, 8H), 2.57 (t, 2H), 1.10 (t, 12H). ESI m/z [M+H]+ = 501.73. 7

2.3.3 Preparation of GQDs-SR GQDs-SR was prepared according to the literature [64,65] with a little modification as follows: the carboxylcoated GQDs (50 mg) were dissolved in water (10 mL) and added EDC (76.8 mg, 0.4 mmol) in it, followed by slow addition of 0.01 M HCl to adjust the pH value of the reaction solution to 5. After stirring for 30 min at room temperature, NHS (23.0 mg, 0.2 mmol) was added into the GQDs solution and then the SR (50.1 mg, 0.1 mmol) in the solution of DMSO (3 mL) was added dropwise to the already activated GQDs solution, followed by adjusted the pH of mixture to 9.0 with 0.01 M NaOH. The reaction solution was stirred gently for 48 h under dark. Finally, the resulting solution was dialyzed (retained molecular weight: 1000 Da) against water overnight, and then GQDs-SR was dried under vacuum. 2.4 Cell incubating Hela cells (human cervical cancer cell) were provided by the College of Life Sciences at Sichuan University (Sichuan, China). The Hela cells were incubated with high glucose medium (H-DMEM) (Gibco, USA) supplemented with 10% fetal calf serum (FBS) (Gibco, USA) for 48 h at 37 oC. Subsequently, the GQDs-SR (final concentration 0.1 mg mL-1) introduced to the cell culture medium and incubated at 37 o

C under 5% CO2 for 1 hour. After renew the medium, the Hela cells were incubated

with 20 μM Hg(NO3)2 for another 30 min at 37 oC. Then, after washing with PBS three times, fluorescence images were taken by using an inverted fluorescence microscope. 2.5 MTT assay 8

Cytotoxicity was evaluated by MTT assay as previously described. Hela cells were seeded onto 96-well plates. After a 24 h incubation in serum containing media at 37 oC with 5% CO2, cells were treated with 200 μL indicated concentrations of GQDs-SR or plain GQDs for 24 h in serum containing media. Then, 20 μL of 5 mg mL-1 MTT solution (Sigma, St. Louis, MO) was added to each well and plates were incubated for 2 h at 37 oC. After carefully aspirating the media and MTT from each well, the formazan crystals were dissolved with 150 μL of DMSO (Sigma, St. Louis, MO), and absorbance was read at 570 nm with a Multiscan MK3 microplate reader (Thermofisher Inc.). Each assay was replicated 3 times. Cells without treatment with GQDs or GQDs-SR were taken as control.

3. Results and Discussion 3.1 Preparation and characterization of GQDs-SR The synthesis procedure for GQDs-SR was shown in Scheme S1†. The Hg2+ recognition element was characterized by 1H NMR and mass spectroscopy (Fig. S1-S2†). The GQDs were prepared by a microwave heating reaction. The transmission electron microscope (TEM) image was shown in Fig. 1A, revealing that the particles were uniformly dispersed and possessed a nearly spherical shape with diameters of 3.2-6.4 nm (Fig. 1B), and the average size was 4.38 nm. The high resolution TEM (HRTEM) image (inset of Fig. 1A) clearly unveiled the crystal lattice of GQDs, the 60 spacing of which were calculated to be 0.24 nm, which was comparable to the (1120) lattice fringes of graphene [66]. The corresponding X-ray 9

diffraction (XRD) pattern, shown in Fig. 1C, displayed a broad (002) peak centered at ca. 22°. Fig. 1. The surface functionalization was also characterized through NMR spectroscopy, UV-visible absorption spectra and Fourier transform infrared (FTIR) spectroscopy. As shown in Fig. S3†, the peaks of as-prepared GQDs mainly appeared at 6.2~8.0 ppm. However, typical resonance peaks of 1.1 ppm originated from the methyl of SR were displayed in the 1H NMR spectrum of GQDs-SR, indicating the successful surface functionalization of GQDs with the rhodamine derivatives. The UV-visible absorption spectra of GQDs, GQDs-SR and GQDs-SR + Hg2+ were shown in Fig. S4†. The absorption peak of GQDs at ca. 230 nm was due to π-π* of C=C, and the absorption at 280 nm corresponded to n-π* transition of the C=O bond [67]. GQDs-SR showed a new absorption at 310 nm, moreover, the typical characteristic absorption peak of Rhodamine derivatives appearing at 563 nm after addtion of Hg2+ confirmed that SR was conjugated to GQDs. The successful connection of SR onto GQDs was further verified by the FTIR. As shown in Fig. 1D, the as-prepared GQDs exhibited characteristic absorption bands of stretching vibration of O-H at 3415 cm-1, stretching vibration of C=O at 1712 cm-1, skeletal vibration of aromatic ring (νC=C ) at 1600 cm-1 and stretching vibration of C-O at 1120 cm-1 [68], confirming the existence of carboxyl groups on the surface of as-prepared GQDs, which was beneficial for further chemical modification on the surface of GQDs. The conjugation of SR onto the surface of the GQDs was achieved through the reaction between the carboxyl groups 10

on the surfaces of GQDs and the amino groups in SR. Compared to the as-prepared GQDs, the presence of the peaks at 2925 cm-1 and 2854 cm-1 can be observed in the FTIR spectra of GQDs-SR, which were assigned to the symmetric and asymmetric C-H stretches of SR, and the peak at 1673 cm-1 should be attributed to the carbonyl stretching vibration of the amides. Taken together, the results confirmed the successful synthesis of the GQDs-SR. The amount of the probe moieties conjugated onto the GQDs was calculated to be about 82.26 mg g

-1

(Fig. S5†) through the

standard addition experiments [69]. 3.2 Fluorescent ratiometric sensing for Hg2+ To investigate the optical properties of the GQDs, a detailed photoluminescence (PL) study was carried out by using different excitation wavelengths, and the results were shown in Fig. 2A. The as-prepared GQDs exhibited an excitation-dependent PL emission, the emission peak shifted from ca. 440 nm to ca. 550 nm while excitation wavelength changed from 280 to 460 nm. For the FRET process, a suitable emission wavelength of the donor for the acceptor was necessary. In the sensing system here, the SR exhibited no fluorescent emission due to the ring-closed form; but in the presence of Hg2+, the SR transformed into the ring-opened form that turned into an energy acceptor and accordingly exhibited maximum absorption peak at around 563 nm (Fig. 2B). Due to the property of λex-dependent PL emission of the GQDs, a suitable excitation wavelength can be selected to match a specific energy acceptor. From Fig. 2A, we found that the maximum emission peak of GQDs was 450 nm while the 11

excitation was 360 nm, but the overlap between the maximum emission of GQDs and the absorption of acceptor was relatively few under this condition that would result in poor efficiency of FRET. With the increasing of excitation, the emission peak appeared redshift and fluorescence intensity decreased. In order to adopt a suitable fluorescence intensity and emission wavelength of GQDs, we selected 400 nm as the suitable excitation wavelength of the GQDs. Hence, the SR could function as the energy acceptor, thus the FRET process could occur from the GQDs to the SR, shown in Fig. 2B. Fig. 2 The results of fluorescent response of the GQDs-SR toward Hg2+ were shown in Fig. 3A. The free chemsensor displayed a single emission band centered at 500 nm. Upon addition of Hg2+, the fluorescence intensity of GQDs at 500 nm gradually decreased, while a new emission band at 585 appeared. The changes in fluorescence occured with an isoemissive point at 546 nm, indicating the occurrence of FRET process. Under the ultraviolet lamp, the chemsensor solution changed continuously from light-green to orange as presented in Fig. S6†, enabling the detection of Hg2+ by the naked eye possible. We also established a working curve by plotting the emission intensity ratio (I585\I500) vs. Hg2+ concentration. As shown in Fig. 3B, the best linear response concentration range of Hg2+ was from 0.6 to 12 μM with a correlation coefficient of 0.992, and the detection limit was estimated to be 0.23 μM (3σ/slope). Fig. 3 3.3 Selectivity of GQDs-SR toward Hg2+ 12

Selectivity is a very important parameter to evaluate the performance of a fluorescence chemosensor. To examine the selectivity of GQDs-SR for Hg2+, the fluorescence intensity ratios (I585/I500) of GQDs-SR were recorded in the presence of Hg2+ (20 μM) and other ions (50 μM), including K+, Na+, Ag+, Ca2+, Zn2+, Ba2+, Co2+, Pb2+, Ni2+, Mg2+, Cu2+, Cd2+, Mn2+,Al3+, Fe3+ ,Cr3+, SO42-,PO43-,NO3-,CO32-. Fig. 4A showed that no obvious fluorescence intensity ratios changes were observed with the presence of the above mentioned ions except Hg2+. These results indicated that GQDs-SR had high selectivity for identification of Hg2+ over other metal ions or anions. Another important feature of GQDs-SR is its high selectivity toward Hg2+ in a competitive environment. The competitive experiments were conducted in the presence of Hg2+ and various other metal ions and anions respectively. The results were shown in Fig. 4B, there were no distinct variations of the fluorescence signal caused by co-existence of these species, therefore, they can not interfere with the subsequent FRET process. Fig. 4 3.4 Suitable pH range To study the practical applicability, the effects of pH on the fluorescence response of GQDs-SR to Hg2+ were investigated. The experiments were carried out at a pH range from 3.0 to 11.0, with a concentration of GQDs-SR fixed at 0.1 mg mL-1, and Hg2+ at 20 μM, respectively (Fig. 5). The results showed that for both GQDs-SR systems with and without Hg2+, the fluorescence intensity ratio increased with the 13

decreasing of pH value at acidic conditions ( pH < 5), which might be attributed to the formation of the open-ring state because of the strong protonation. In the absence of Hg2+, no obvious variations of the fluorescence emission have been observed in the pH range 5.0-11.0, suggesting that the spirocyclic form was still preferred in this pH range. Upon addition of Hg2+, a remarkable increase in fluorescence intensity ratio was observed between pH 5-8.5, which was attributed to the Hg2+-induced opening of the rhodamine ring. And the fluorescent emission remained fairly stable from pH 6.5 to 8.5. Thus the GQDs-SR could serve as a fluorescent ratiometric sensor for Hg2+ over a pH span of 6.5-8.5. Fig. 5 3.5 Fluorescence lifetime study We know that the excition lifetime properties of the FRET donor would alter due to nonradiative excition transfer [70,71]. To further demonstrate the occurrence of energy transfer from GQDs (donor) to SR (acceptor), the decay curves for the donor before and after adding Hg2+ had been recorded as shown in Fig. 6. In addition, the average lifetimes for the donor with or without Hg2+ were calculated by fitting the data to a three exponential decay curve, from which the average lifetime decreased from 4.80 ns to 4.02 ns after added 20 μM of mercury ions into the system (As seen in Table S1†). The decrease in fluorescence emission lifetime provided additional evidence that the FRET process was turned on by mercury ions. Fig. 6 3.6 Reversibility and reproducibility of GQDs-SR 14

Reversibility is another important aspect for a chemical sensor. Fig. 7 showed the fluorescence intensity ratio for the sensor when the Na2S (10μM) or Hg2+ (10 μM) was added into the solution of GQDs-SR (0.1mg mL-1) for five times. The ratio decreased after adding Na2S into the GQDs-SR + Hg2+ due to the decomplexation of Hg2+ by S2- and a subsequent spirolactam ring closure reaction. After five cycles, the ratio (I585/ I500) remained relatively stable. The reproducibility of the ratio of the emission intensity of the rhodamine derivative to that of GQDs clearly demonstrated the excellent reversibility and the robustness of GQDs-SR. We also tested the reproducibility of the sensor by determining of 10 μM Hg2+ (n=6), the resluts were shown in Table S3†. From the experiments, average value, standard deviation (SD) and Relative Standard Deviation (RSD) were calculated to be 10.16, 0.27, 2.66%. Fig. 7 3.7 Fluoresence imaging of GQDs-SR in living cells For further biological applications, MTT assays were carried out to evaluate the cytotoxicity of blank GQDs and GQDs-SR by using the Hela cell line. As shown in Fig. 8, no significant reduction in cell viability was observed for cells treated with GQDs or GQDs-SR. The viability of Hela cells retained more than 90 % after treated with GQDs-SR even at a high concentration of 0.1 mg mL-1 for 24 h. It’s probably due to the intrinsic biocompatibility and low toxicity of GQDs. This result demonstrated the outstanding biocompatibility of GQDs-SR and emphasized its potential application in detection of Hg2+ in live cells. Fig. 8 15

Taking the results above, the advantages of small size, high selectivity and outstanding biocompatibility of GQDs-SR made it superior to other fluorescence semiconductor quantum dots-based probes in the potential bioimaging application. Herein, in order to investigate the capability of biological application of GQDs-SR, intracellular Hg2+ imaging assays were carried out by using an inverted fluorescence microscope. Fig. 8 showed the double-channel fluorescence images for Hg2+ in living cells at 515 ± 10 nm (green channel) and 590 ± 10 nm (red channel). HeLa cells incubated with 0.1 mg mL-1 GQDs-SR for 1 h at 37 oC showed a green fluorescence signal in the green channel (Fig. 9B) and no fluorescence in the red channel (Fig. 9C). In the contrary, the control experiment for the Hela cells without being treated with GQDs-SR exhibited no green fluorescence under the same conditions (Fig. S7†). In contrast, the live cells treated with GQDs-SR and further incubated with 20 μM of Hg2+ for another 30 min at 37 oC, eliciting an obvious fluorescence decrease in the green channel (Fig. 9F) and a simultaneously remarkable fluorescence increase in the red channel (Fig. 9G). Bright-field microscopic image revealed that the cells were viable throughout the imaging experiments (Fig. 9A, 9E). The overlay of fluorescence and bright field images clearly showed that GQDs-SR can cross the cell membrane and label the cytoplasm of HeLa cells without reaching the nucleus (Fig. 9D, 9H). These results demonstrated that the FRET process occurred in the presence of Hg2+ inside live cells. Thus, the results indicated that GQDs-SR could be employed for ratiometric fluorescence imaging of Hg2+ with excellent cell-permeability and biocompatibility in the living cells. 16

Fig. 9 3.8 Detection of Hg2+ in water simples Moreover, to illustrate the practical applicability of this sensing system, the GQDs-SR was applied in the determination of Hg2+ in both tap and pond water samples respectively. The samples collected were simply pretreated with filtration before further determination. Standard addition method was adopted to analyze each sample in triplicate and the analytical results were shown in Table S2†. The recovery of Hg2+ was between 101.3 % and 104.7 % for pond water and between 102.1% and 104.6 % for tap water, respectively, which suggested that the proposed GQDs-SR was suited to detect Hg2+ in real water samples.

4. Conclusion In summary, we designed and synthesized a fluorescence ratiometric chemsensor GQDs-SR based on fluorescence resonance energy transfer mechanism for detection of Hg2+ by facilely conjugating rhodamine derivative onto graphene quantum dots. GQDs-SR exhibited excellent good water-solubility, sensitivity and selectivity towards Hg2+. What’s more, the biocompatible nature and small size of the graphene quantum dots made GQDs-SR easily permeate through the cell membrane to monitor Hg2+ in living cells with very low cytotoxicity. Furthermore, we have demonstrated that GQDs-SR was applicable for Hg2+ detection in practical water samples.

Acknowledgments 17

The work described in this paper was supported by the National Natural Science Foundation of China (21377089, 21407109) and Doctoral Program Foundation of Institutions of Higher Education of China (20120181120075). We would like to express our sincere thanks to Analytical & Testing Centre of Sichuan University for the MS measurements.

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Figure Captions

Scheme 1. The proposed response mechanism of Hg2+ detection using GQDs-SR. The inset photographs were taken under a hand-held UV lamp (365 nm).

Fig. 1. (A) The TEM images and HRTEM images (inset); (B) size histogram; (C) XRD of GQDs; (D) the FTIR spectra of SR (a), the as-prepared GQDs (b) and GQDs-SR (c).

Fig. 2. (A) Fluorescence emission spectra of GQDs in HEPES buffered (pH = 7.4) water-ethanol (8:1, v/v) with different excitation wavelengths; (B) The spectral overlap between the absorption of SR-Hg2+ (acceptor) and the fluorescence emission spectra of GQDs (donor, λex = 400 nm).

Fig. 3. (A) Fluorescence responses of GQDs-SR (0.1 mg mL-1) in the presence of different amounts of Hg2+; (B) Fluorescence intensity ratio (I585/I500) of GQDs-SR as a function of Hg2+ concentration in HEPES buffered ( pH = 7.4) water–ethanol (8 : 1, v/v) (λex = 400 nm).

Fig. 4. (A) Fluorescence intensity ratio (I585/I500) of GQDs-SR (0.1 mg mL-1) in the presence of 20 μM of Hg2+ or 50 μM of other different analytes respectively (λex = 400 nm). (B) Fluorescence emission intensity ratio (I585/I500) of GQDs-SR (0.1 mg 23

mL-1) in the presence of the mixture of 20 μM of Hg2+ and 50 μM of other different analytes respectively (λex = 400 nm). All spectra were measured in HEPES buffered (pH = 7.4) water–ethanol (8 : 1, v/v).

Fig. 5. Fluorescence intensity ratio of GQDs-SR (0.1 mg mL-1) as a function of pH in the absence and presence of Hg2+ (20 μM).

Fig. 6. Fluorescence decay curves for GQDs of the chemsensor with or without Hg2+. Black lines: in the absence of Hg2+. Red lines: in the presence of Hg2+ (20 μM).

Fig. 7 Reversibility of GQDs-SR for Hg2+.

Fig. 8. Cellular toxicity determined by MTT assay against Hela cells upon 24 hours of incubation concentrations of GQDs or GQDs-SR (a: 100 μg mL-1 of GQDs, b-e: 25.0, 50.0, 75.0, 100.0 μg mL-1 of GQDs-SR).

Fig. 9. Images of Hela cells treated with GQDs-SR: (A) bright field image of Hela cells incubated with GQDs-SR (100 μg mL-1); (B) fluorescence image (A) from green channel; (C) fluorescence image (A) from red channel; (D) overlay image of (B) and (C); (E) bright field image of Hela cells incubated with GQDs-SR (100 μg mL-1) and Hg2+ (20 μM); (F) fluorescence image (E) from green channel; (G) fluorescence image (E) from red channel; (H) overlay image of (F) and (G). 24

Scheme 1.

Fig. 1.

Fig. 2. 25

Fig. 3.

Fig. 4.

Fig. 5.

26

Fig. 6.

Fig. 7.

27

Fig. 8.

Fig. 9.

Highlights A chemsensor (GQDs-SR) is developed based on FRET process for detecting Hg2+. The sensor exhibites low cytotoxicity and good biocompatibility. The sensor has been used for monitoring Hg2+ in living cells and water samples.

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