Selenium Doped Graphene Quantum Dots as an Ultrasensitive Redox

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Selenium Doped Graphene Quantum Dots as an Ultrasensitive Redox Fluorescent Switch Siwei Yang, Jing Sun, Peng He, Xinxia Deng, Zhongyang Wang, Chenyao Hu, Guqiao Ding, and Xiaoming Xie Chem. Mater., Just Accepted Manuscript • DOI: 10.1021/acs.chemmater.5b00112 • Publication Date (Web): 23 Feb 2015 Downloaded from http://pubs.acs.org on March 1, 2015

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Chemistry of Materials

Selenium doped graphene quantum dots as an ultrasensitive redox fluorescent switch Siwei Yang,† Jing Sun,† Peng He,† Xinxia Deng,‡ Zhongyang Wang,‡ Chenyao Hu,† Guqiao Ding,†,* and Xiaoming Xie† †

State Key Laboratory of Functional Materials for Informatics, Shanghai Institute of microsystem and Information Technology, Chinese Academy of Sciences, Shanghai 200050, P. R. China. ‡

Shanghai Advanced Research Institute, Chinese Academy of Sciences, Shanghai 200050, P. R. China. KEYWORDS: graphene quantum dot, hydroxyl radical, fluorescent switch, Bio-imaging.

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Table of Contents (TOC) Graphic

ABSTRACT: A new reversible fluorescent switch for the detection of oxidative hydroxyl radical (·OH) and reductive glutathione (GSH) was designed based on the use of selenium doped graphene quantum dots (Se-GQDs). The Se-GQDs have a thickness of 1-3 atomic layers, a lateral size of 1-5 nm, a quantum yield of 0.29 and a photoluminescence lifetime of 3.44 ns, which ensured a high selectivity and stability for the fluorescent switch. The fluorescence of SeGQDs was reversibly quenched and recovered by ·OH and GSH, respectively, because of the reversible oxidation of C-Se groups and reduction of Se-Se groups. This brand-new GQD-based fluorescent switch gave a rapid response when tested in both aqueous solutions and living HeLa cells. In particular, the detection limit for ·OH was only 0.3 nM, which was much lower than that in switches made from organic dyes.

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INTRODUCTION Cellular redox homeostasis is essential to life. 1-4 It is believed that the in vivo damage of biomolecules is initiated by reactive oxygen species in a process known as oxidative stress.

1-7

The

hydroxyl radical (·OH), one of the most important reactive oxygen species in vivo, has important functions in bio-systems.

8-14

On the other hand, intracellular thiols provide abundant reducing

sources that are central to the antioxidant defense systems.

15, 16

Among these biothiols,

glutathione (GSH) is the most abundant endogenous thiol with a concentration from 1 to 15 mM depending on cell types.

17-20

GSH is the major non-protein thiol in most organisms, and it is a

multifunctional metabolite in various defense reactions and in ensuring appropriate intracellular redox homeostasis. 21-24 Since ·OH and GSH are the most important in vivo oxidiser and reducer, it is very important to develop a probe that can respond reversibly to ·OH/GSH changes for visualizing states of the redox cycles in living cells. Nowadays, most rapid detection methods for ·OH or GSH are based on organic dyes or scavengers. complex.

25-32

33, 34

However, the design, synthesis and preparation of most organic dyes are In addition, their low stability, weak anti-jamming ability, and potential

cytotoxicity to organisms retard their widespread application.

33-36

Graphene quantum dots

(GQDs) are a rising star in the family of fluorescent materials, and has attracted intensive attention.

37-43

Compared with organic dyes and semiconductor quantum dots, GQDs have

superior properties, such as high photo-stability against photo-bleaching and photo-blinking, biocompatibility and low toxicity.

44-49

GQDs can be easily taken up by living cells, making it

possible to detect ions or other molecules in living cells. 44-46 On the other hand, there are several reports about pH controlled fluorescent switch based on GQDs which has shown the wide application of GQDs of the fluorescent switch.

47, 50

But, the GQD-based redox fluorescent

switch has not been realized or reported to date. 3



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Heteroatom doping will not only affect the PL intensity and wavelength, 51-53 but also change the reactivity or sensitivity of GQDs to the environment.

54, 55

This indicates the feasibility of

preparing fluorescent probes and building fluorescent switch systems based on GQDs and their controllable doping.56, 57 Chen et al. reported N-GQDs with oxygen-rich functional groups which can be used in sensitive and selective detection of Fe(III) ions.

58

Hence, we designed and

synthesized a new reversible fluorescent switch for the detection of ·OH and GSH in living cells based on selenium doped GQDs (Se-GQDs). The C-Se group in Se-GQDs can be oxidised by ·OH to non-fluorescent Se-Se group and it can be reduced to C-Se again by GSH, which constitutes a reversible on-off fluorescent switch. The switch mechanism was experimentally confirmed through XPS and fluorescent lifetime measurements. Moreover, the fluorescence of Se-GQDs is ultra-sensitive and highly-selective to the presence of ·OH, and can be used as an ultrasensitive ·OH sensor in both aqueous solution and living cells.

EXPERIMENTAL SECTION Chemicals Sulfuric acid (H2SO4, 95.0-98.9 %), nitric acid (HNO3, 65.0-68.0 %), sodium hydroxide (NaOH, ≥ 96.0 %), hydrochloric acid (HCl, 36.0-38.0 %), and ammonium hydroxide (NH3·H2O, 25-28 %) was purchased from Shanghai Lingfeng chemical reagent. Co., Ltd. Sodium chlorate (NaClO3, 99.0 %) was supplied by Aladdin Reagents (Shanghai, China) Co., Ltd. The NaHSe aqueous solution was prepared via a reduction reaction of Se and NaBH4 in water under Ar. All chemicals are analytically pure and used as received without further purification. Deionized water (resistivity ~18.2 MΩ cm, 25 °C) obtained through a Milli-Q system was used throughout all experiments. Graphene powders (G-100) were purchased from SIBAT (Shanghai, China). Synthesis of graphene oxide quantum dots (GOQDs)

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GOQDs were prepared from graphene powder (SIMBATT, Shanghai, China) by a modified Staudenmaier method. Graphene powder (4 g) was put into H2SO4 (150 mL) and HNO3 (80 mL) with stirring at 15 °C, and was kept for 2 h. Then NaClO3 (40 g) was added gradually and the temperature was kept below 5 °C. The mixture was then stirred at 15 °C for 5 h. After that, the reaction was terminated by adding distilled water (80 mL). The pH value was neutralised to 7 by NaOH, before the mixture was filtered using an alumina inorganic membrane with 20 nm pores. The resulting light yellow filtrate was dialysed in a 3500 Da dialysis bag against deionised water for a week to remove excess salt. The resultant solution was freeze-dried to obtain the GOQD powder. The GOQD yield from graphene is approximately 45%. Synthesis of Se-GQDs The Se-GQDs were prepared as follows: typically, 1.0 mL 150 mM NaHSe aqueous solution was added to 9.0 mL GOQDs (1 mg mL-1) aqueous solution, and then the mixture was transferred into a 15 mL Teflon-lined autoclave and heated and kept at 250 °C for 24 h. After that, the mixture was filtered using an alumina inorganic membrane with 20 nm pores. The resulting orange filtrate was dialysed in a 500 Da dialysis bag against deionised water for a week to remove excess salt. The Se-GQD yield from GOQDs is approximately 86%. Characterization methods Transmission electron microscopy (TEM) measurements were carried out on a Hitachi H8100 EM (Hitachi, Tokyo, Japan) with an accelerating voltage of 200 kV. Atomic force microscopy (AFM) measurements were carried out on Bruker Dimension Icon AFM microscope. X-ray photoelectron spectra (XPS) were carried out on a PHI Quantera II system (Ulvac-PHI, INC, Japan). Fluorescent emission and excitation spectra were recorded on a PerkinElmer LS55 luminescence spectrometer (PerkinElmer Instruments, U.K.) at room temperature (25 °C) in aqueous solution. The stability of these products was determined via contrast the fluorescent emission intensity of products aqueous solution under different conservative time at room 5



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temperature (25 °C). Quantum yield (Φf) was measured according to established procedure (Lakowicz, J. R. Principles of Fluorescence Spectroscopy, 2nd Ed., 1999, Kluwer Academic/Plenum Publishers, New York). The UV-vis spectra were obtained on a UV5800 Spectrophotometer. Rhodamine 6G solution (quantum yield 0.98 in EtOH) was chosen as standards. Absolute values are calculated using the standard reference sample that has a fixed and known fluorescence quantum yield value. In order to minimize re-absorption effects, absorbencies in the 10 mm fluorescence cuvette were kept under 0.1 at the excitation wavelength. Time-resolved fluorescence behavior was measured via the time-correlated singlephoton counting (TCSPC) technique (Hydra Harp 400, Pico Quant). The samples were excited by a frequency-doubled titanium: sapphire oscillator laser with approximately a pulse duration of 150 fs, and a repetition rate of 80 MHz (Chameleon, Coherent). Fluorescence emission was sent to a spectrometer (iHR550, Horiba Jobin Yvon) with 300/mm grating and then detected by a photomultiplier tube.

RESULTS AND DISCUSSION Characterization of Se-GQDs Fig. 1a shows the low magnification TEM image and corresponding size distribution histogram of Se-GQDs thus formed. Homogeneous dots with a lateral size distribution of 1 to 5 nm and an average diameter of 3.08 nm were found. Fig. 1b shows a representative highresolution TEM image of an individual Se-GQD. The distinct crystal lattice indicates the crystallinity of the Se-GQD, and the lattice parameter of 0.242 nm represents the (1120) lattice fringe of graphene.59 AFM observations (Fig. 1c) reveal highly dispersed Se-GQDs on the mica substrate with a typical topographic height of 0.5 to 1.5 nm, indicating that most Se-GQDs consist of 1-3 graphene layers. To probe Se atoms in the Se-GQDs, XPS measurements were undertaken. As seen in Fig. S1, the XPS survey spectrum for Se-GQDs shows a predominant narrow graphitic C 1s peak at ca. 6


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284.2 eV, along with an O 1s peak at ca. 532 eV and a Se 3d peak at ca. 56 eV. The elemental composition of Se-GQDs and precursor (GOQDs) are shown in Table S1. The O/C ratio of precursor is 0.49, which is much higher than Se-GQDs (0.19). This indicates the removal of oxygen-containing groups in doping progress. Fig. 1d shows the well-fitted C1s XPS spectrum of Se-GQDs. The C1s XPS spectrum can be divided into five different peaks, which correspond to the signals of C-C/C=C (284.6 eV, 39.5 %), C-OH (285.2 eV, 44.5 %), C-O-C (286.1 eV, 8.4 %), C=O (289.2 eV, 4.9 %), and C-Se (292.2 eV, 2.7 %).60-64 The O 1s XPS spectrum can be divided into two different peaks (Fig. S2), which correspond to the signals of C-OH or C-O-C (533.4 eV, 64.1 %) and C=O (532.4 eV, 35.9 %).

60, 61

It is interesting that the contents of

oxygen-containing groups (C-C/C=C, C-OH, C-O-C, C=O, C-OH/C-O-C and C=O) have no significant change (Fig. S3-5) compared with the precursor. The possible mechanism is that the contents of oxygen-containing groups are in dynamic equilibrium in doping progress. Moreover, the Se 3d XPS spectrum of Se-GQDs can also be divided into two different peaks (Fig. 1e), which correspond to the signals of C-Se (56.1 eV) and Se-Se (57.0 eV). The C-Se content is 57 % and that of Se-Se forms the remaining 43 %.65, 66

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Figure 1. Characterization of Se-GQDs. (a) TEM image and corresponding size distribution histogram of Se-GQDs, (b) HRTEM image of a single Se-GQD particle, (c) AFM topography image of Se-GQDs on a mica substrate in tapping mode (Bruker Dimension Icon AFM microscope), inset: height profile analysis along the line shown in the image. High-resolution XPS spectra of Se-GQDs: (d) C 1s and (e) Se 3d spectra of Se-GQDs. Optical properties of Se-GQDs The optical properties of Se-GQDs were studied by UV-vis absorption and PL spectroscopy. As shown in Fig. 2a, it is noteworthy that the UV-vis absorption spectrum of Se-GQDs shows a typical absorption peak around 308 nm due to the π-π* transition of aromatic sp2 domains, and a long tail extending into the visible range.60 The absorption peak was red shifted compared with precursor (240 nm, Fig. S6) which can be attributed to the electron-donating capacity of Se in aromatic sp2 domains.31 Moreover, the Se-GQDs have another new noteworthy absorption peak centered at a wavelength of 450 nm, which can be attributed to the n-π* transition of C-Se.65-67 The optimum excitation condition of Se-GQDs is at 467 nm, which corresponds to the UV-vis peak at 450 nm. For the PL spectroscopy, the maximum excitation and emission wavelengths of the precursor is 375 nm and 460 nm, respectively (Fig. S7). However, the Se-GQDs exhibited red shifted maximum excitation (λex=467 nm) and emission wavelengths (λem=563 nm) compared with precursor. The spectral shift also can be attributed to the electron-donating capacity of Se in aromatic sp2 domains.31 The Se-GQDs exhibit a maximum emission wavelength shift of 110 nm when the excitation wavelength increases from 340 to 530 nm (Fig. S8), which can be attributed to other active groups, such as -OH, -COOH, and -C=O. This indicates that the n-π* transition can effectively increase the PL efficiency of Se-GQDs since it appears frequently in fluorescent dyes.58 The Stokes shift (∆νSt) and full width half maximum (FWHM) of Se-GQDs are 0.45 eV and 0.36 eV (at 80 nm), respectively. This indicates that the Se-GQDs have a weak self-absorption effect and low energy loss.68

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The Commission International d’Eclairage (CIE) chromaticity coordinates for Se-GQDs are shown in Fig. 2b. The CIE chromaticity coordinates of the Se-GQDs are (0.42, 0.49). The SeGQDs aqueous solution excited by a 365 nm lamp (6 W) emits an intense orange luminescence (Fig. 2c), which is attributable to the surface functional groups. Thus, this orange-light-emitting probe effectively avoids the influence of auto fluorescence in biological systems. The quantum yield (φ) of Se-GQDs is 0.29 which is higher than some previous reports (Table S2).

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Figure 2. Optical properties of Se-GQDs. (a) Normalized UV-vis absorption spectrum (red curve), Photoluminescence excitation (black curve) and PL (blue curve) spectra of Se-GQDs aqueous solution (b) The CIE chromaticity coordinates for Se-GQDs in aqueous solution. (c) digital image of Se-GQDs in aqueous solution under UV-light (centre wavelength: 365 nm). (d) PL decay curves of Se-GQDs measured at room temperature with excitation at 467 nm. (e) Stability of Se-GQDs under visible light (black curve) at room temperature. Photo-stability (red 9



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curve)of Se-GQDs under UV light (150 W Xe lamp with centre wavelength at 320 nm). F0 and F are PL intensities at the outset and specified times, respectively. The PL decay of Se-GQDs is measured by a time-correlated single photon counting technique, and fitted well with a bi-exponential decay as shown in Fig. 2d. The lifetime (τ) is dominated by a long decay component of 3.5 ns (97%) plus a small contribution from the short decay of 1.5 ns (3%), the weighted-average lifetime is approximately 3.44 ns. To combine the φ and τ by κr=φ/τ we can obtain the radiative rates κr.70, 71 The fluorescence radiative rate κr of SeGQDs is 8.43×107 s-1. The high radiative rate implies a high electronic transition probability in the Se-GQDs, which confirms the aforementioned n-π* transition model therein. For practical applications, the stability of Se-GQDs is very important, although with many active groups, the resulting Se-GQDs showed excellent photo-stability under adverse conditions (Fig. 2e). The Se-GQDs showed excellent stability after continuous excitation under visible light for more than 60 days (see black curve in Fig. 2e), or ultraviolet radiation (150 W Xe lamp with centre wavelength at 320nm, 48 h, see red curve in Fig. 2e). The PL intensity of Se-GQDs is also stable when the pH value was changed from 1 to 14 (Fig. S9). Moreover, Fig. S10 shows the PL stability of Se-GQDs against their ionic strength. There is no obvious change in PL intensity even with a concentrated phosphate buffer solution (PBS, pH=5.6, which is the most commonly used buffer system in organisms) at 1.0 M. Since our Se-GQDs have excellent PL properties and good stability, we further investigated such Se-GQDs for their potential use as a redox switch. A dramatic change in the PL properties of the Se-GQDs was observed (Fig. 3a) when Se-GQDs were introduced to Fenton’s reaction system (a well-known pathway to produce ·OH, experimental details are shown in ESI). 72 The PL intensity of Se-GQDs is 5.98 × 106 in the absence of ·OH. However, the PL intensity decreased gradually with the increased presence of ·OH. When the concentration of ·OH is 300 nM, the PL intensity of Se-GQDs is close to zero (Fig. 3b). The quenching progress can be due 10


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to the high oxidation capacities of ·OH (redox potential, 2.8 V). The fluorescence quenching data follows Eq. (1): F0 / F = 1+ K[Q]

(1)

Where [Q] is the analyte (·OH) concentration, and F0 and F, are PL intensities of the SeGQDs at 563 nm in the absence and presence of ·OH, respectively. The plot shown in Fig. 3b (insert) fits a linear equation over the concentration range 2.0 × 10-9 to 1.8× 10-8 M. The correlation coefficients (r2) and K are 0.9983 and 0.4731 determining ·OH over the linear concentration range of 3.0 × 10-10 to 1.8× 10-8 M, respectively. The detection limit is estimated to be 3.0 × 10-10 M at a signal-to-noise ratio of 3. This detection limit is remarkable and much lower than offered by other detection methods (Table S3). These results imply that the Se-GQDs are likely to be capable of practical ·OH detection with further development.

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Figure 3. Changes in the PL properties of Se-GQDs when Se-GQDs were introduced to Fenton’s reaction system. (a) Fluorescence emission spectra of Se-GQD for different 11



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concentrations of ·OH with an emission wavelength of 563 nm at 25 °C and pH = 7. The concentration of Se-GQD is 1.0 mg mL-1. (b) PL intensity of Se-GQD for different concentrations of ·OH. (c) The difference in PL intensity of Se-GQDs between the blank and samples in H2O2, FeSO4, and Fenton’s reaction system, respectively. F0 and F are PL intensities of the blank and samples in H2O2, FeSO4, and Fenton’s reaction system, respectively. (d) Fluorescence emission spectra of oxidised Se-GQD (the concentration of Se-GQD is 1.0 mg mL1 , the concentration of ·OH is 300 nM) for different concentrations of GSH at an excitation wavelength of 563 nm at 25 °C and pH = 7. The inset shows the PL intensity of oxidised SeGQD for different concentrations of GSH. Further experiments proved the role of the quencher in the quenching progress in Fenton’s reaction system. Fig. 3c shows the PL intensity of Se-GQDs in H2O2, FeSO4, and Fenton’s reaction system, respectively. It is clear that the PL intensity remains unchanged when FeSO4 was added. The PL intensity decreased slightly when H2O2 was added. The weak quenching function of H2O2 may be due to the low concentration of ·OH obtained from the decomposition process of H2O2 in H2O2 aqueous solution. Moreover we also compared the change of PL intensity of Se-GQDs with the presence of other oxidising agents (such as: KMnO4, NaClO, (NH4)2S2O8, K2Cr2O7, and HNO3). There are no obvious changes in PL intensity in the presence of these oxidising agents (Fig. S11) and this demonstrates that the Se-GQDs show high selectivity for ·OH. The high selectivity of Se-GQDs may be due to the high oxidation capacity of ·OH. On the other hand, the PL intensity of Se-GQDs can be recovered easily when GSH was introduced to this system (Fig. 3d). The PL intensity of Se-GQDs gradually recovered with elevated GSH concentration. The PL intensity recovered to 6.02 × 106 when the concentration of GSH is 2.5 × 10-7 M. Thus, an oxidation-reduction fluorescent switch system is prepared based on Se-GQDs. The operating state of this fluorescent switch depends on the redox state of the system. When this switch system is oxidised by ·OH, the fluorescence is quenched and the switch is “off”. On the contrary, this switch is "on". Fig. 4a shows the response surfaces for the interaction between GSH and ·OH concentrations and the PL intensity of Se-GQDs. The surface can be divided into the two regions: one 12


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corresponding to the “on” state, the other to the “off” state. The threshold value for the “on” state can be set to 1.2 × 107, and the threshold value for the “off” state can be set to 0.6 × 107. Fig. 4b shows a colour-coded contour plot of the slope values for this switch system. The added proportions of GSH and ·OH influence the switch state. The results indicate a high signal-noise ratio.

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Figure 4. Performances PL switch system. (a) Response surfaces for interaction between GSH and ·OH concentrations with PL intensity of Se-GQDs. The concentration of Se-GQDs is 1.0 mg mL-1. (b) Colour-coded contour plot of PL intensity for the switch system as a function of GSH and ·OH concentration (c) PL intensity of this fluorescent switch system in “on” or “off” states over 15 redox cycles. F0 and F are PL intensities in the absence and presence of ·OH, respectively. The concentrations of Se-GQDs, ·OH and GSH are 1.0 mg mL-1, 100 nM, 200 nM, respectively. (d) The difference in PL intensity of Se-GQDs in “off” (black) and “on” (red) states

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between the blank and solutions containing different metal ions (F0 and F are PL intensities in the absence and presence of ions, respectively). Fig. 4c shows that the reversible oxidation/reduction cycle could be repeated at least 15 times with no obvious loss of PL intensity. The selectivity and specificity of this switch system in both “on” and “off” states are also demonstrated (Fig. 4d). The PL intensity of this switch system does not give any observable change in its “off” state when various kinds of disturbing substances were added, such as: Pb2+, Ag+, Zn2+, Fe3+, Ni2+, Cd2+, Cu2+, Hg2+, Cl-, Mg2+, Al3+, Ca2+, Na+, K+, and SO42-, indicating good selectivity in the “off” state. This good selectivity may be due to the weak coordination ability of Se-Se groups in oxidised Se-GQD. However, the presence of heavy metal ions (Hg2+, Pb2+, and Ag+) results in a decrease in PL intensity in the “on” state. This interference may be attributed to a coordination effect between these ions and Se-H. The binding ways between heavy metal ions and Se-GQDs may be the coordination interactions between heavy metal ions and C-Se groups. Indeed, when the strong complexing agent (such as EDTA) is added, heavy metal ions are removed from the surface of Se-GQDs by forming a new complex, the PL of Se-GQDs recovered (Fig. S12-13). This efficient recovery of PL strongly confirms the above-mentioned coordination process. 31 The behavior mechanism underpinning of the switching process was investigated through control experiments, XPS, PL lifetime and UV-vis absorption measurements. To verify the effect of doping concentration on the performance of this switch system, the Se-GQDs with different doping concentrations and same oxygen content were prepared. The control experimental details of preparation progress were showed in supplementary information. As shown in Fig. 5a, SeGQDs with different doping concentrations show different performance in oxidation/reduction cycle. The Se-GQDs with lower doping concentration (Se-GQDs-2, doping concentration: 3.24 at. %) showed lower quenching efficiency and signal-noise ratio than Se-GQDs with higher doping concentration. What's more, Se-GQDs-3 with the lowest doping concentration (1.03 14


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at. % ) showed the lowest quenching efficiency and signal-noise ratio. Finally, the GQDs which were undoped showed no response of the redox progress. This indicates the Se is the key factor for the performance of this switch system. Fig. 5b and c show the high-resolution XPS spectra of Se 3d from the Se-GQDs when the switch is in “off” and “on” states, respectively. Both these spectra can be divided into two different peaks, corresponding to the signals of C-Se (56.1 eV) and Se-Se (57.0 eV). When the switch is in its “off” state (oxidised by ·OH), The C-Se content is 19.8 % and the C-Se-Se content is 80.2 %. Otherwise, the C-Se content is 64.7 % with an Se-Se content is 35.3 %. These results confirm that the C-Se can be oxidised by ·OH and be reduced by GSH. The quenching mechanism (static quenching or dynamic/collision quenching) can be directly verified through PL lifetime measurements. In static quenching, the fluorescence lifetime does not change (τ0/τ = 1) where τ0 and τ are the fluorescence lifetimes in the absence and presence of quencher, respectively. This does not make a complex with the quencher in the ground state having the original lifetime (τ0). In dynamic quenching, F0/F = τ0/τ, and the lifetime decreases on addition of the quencher. Fig. 5d shows the typical effects of ·OH concentration on the lifetime of the Se-GQDs. The lifetime of the Se-GQDs did not change (τ0/τ ≈ 1) upon Se-GQD addition, verifying the likelihood of a static quenching process over the concentration range 3.0 × 10-10 to 2 × 10-8 M. The static quenching process may be caused by chemical reactions between SeGQDs and ·OH. The UV-vis absorption spectra of the switch system in "on" and "off" state are shown in Fig. 5e. When the switch system is in "on" state, a noteworthy absorption peak centered at a wavelength of 450 nm, which can be attributed to the n-π* transition of C-Se.59, 61 However, when the switch system is in "off" state, the UV-vis absorption spectra shows no absorption peak at 450 nm. This can be due to the formation of Se-Se bond in "off" state which conforms to the static quenching process.

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The switch process therefore comes down to the redox progress in the Se-Se bonds (Fig. 5f). When ·OH was added, the C-Se bonds were oxidised. The Se-Se linkage is flexible and the energy of electrons in an excited state is associated with the vibration of Se-Se groups. So the fluorescence is quenched and the switch is “off”: when the Se-Se groups were reduced by GSH, the switch is “on”. We tested the in vitro cytotoxicity of Se-GQDs using the HeLa cell line. The activity of HeLa cells treated with different concentrations of Se-GQDs (Fig. S25) was observed. Different concentrations of Se-GQDs (0 to 500 µg/mL) were added to the cells cultured in 96 well-plates and incubated for 24 h. Subsequently, a standard assay was performed to assess the cell viabilities after the Se-GQDs treatments. No significant reduction in cell viability was observed for cells treated with Se-GQDs even at concentrations of up to 500 µg/mL. These results indicated that Se-GQDs can be used for intracellular imaging. Next, we used Se-GQDs for fluorescence sensing of ·OH in living cells. HeLa cells treated with Se-GQDs emitted strong orange fluorescence (Fig. 6b and 6c) around their nuclei. However, when the ·OH was introduced to these cells, the resulting quenched fluorescence (Fig. 6d and 6e) demonstrated that Se-GQDs were able to display a fluorescence turn-off response to ·OH in the living cells. Moreover, the quenched fluorescence was recovered easily when GSH was introduced to above cells (Fig. 6f and 6g). This result confirms that Se-GQDs are capable of sensing redox cycles through reversible fluorescence responses in living cells.

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a

b GOQDs

Se-GQD-3

Se-GQD-2

Se-GQDs

c

d

e

f

n-π*

Figure 5. Working mechanism of the switch system. (a) PL intensity of Se-GQD, Se-GQD-2, Se-GQD-3 and GOQDs over 15 redox cycles. F0 and F are PL intensities in the absence and presence of ·OH and GSH, respectively. The concentrations of Se-GQD, Se-GQD-2, Se-GQD-3 and GOQDs are 1.0 mg mL-1. The concentrations of ·OH and GSH are 100 nM, 200 nM, respectively. Se 3d spectra of Se-GQDs in: (b) “off” and (c) “on” states. (d) Lifetime of Se-GQD 17



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plotted against the concentration of the quencher, where τ0 and τ are the fluorescence lifetimes in the absence and presence of ·OH. (e) Normalized UV-vis absorption spectra of the switch system in "on" and "off" states. (f) Schematic diagram illustrating of the under pinning mechanism of the switching.

a

b

c

d

e

f

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Figure 6. Practical applications of switch system in vivo. HeLa cells loaded with 1 mL, 250.0 mg mL-1 Se-GQDs for 10 min. (a) Bright-field image of Se-GQD-loaded cells, (b) corresponding confocal fluorescence microphotograph of (a). (c) Bright-field image of Se-GQD-loaded cells treated with 1.0 µM H2O2 and 0.5 µM FeSO4 for 10 min. (d) corresponding confocal fluorescence microphotograph of (c). (e) Bright-field image of Se-GQD-loaded cells exposed to a second dose of 5.0 µM GSH for an additional 10 min. (f) corresponding confocal fluorescence microphotograph of (e). Scale bar: 20 µm.

CONCLUSION In summary, we have developed a reversible fluorescent probe that exhibits high sensitivity and selectivity in monitoring ·OH and reduction events in aqueous solution and living cells. The fluorescence of Se-GQDs is statically quenched by ·OH, and the Se-Se groups cause the fluorescence switch to be “off”. The Se-Se groups can be effectively and rapidly reduced to C-Se groups through GSH addition, and the fluorescence is recovered as the “on” state of the switch. This fluorescence switch provides a new detection approach for redox events involved in cellular redox regulation.

ASSOCIATED CONTENT Supporting Information Available Experimental details, XPS survey spectrum, elemental composition, O1s spectra and stability of Se-GQDs. PL emission spectra of Se-GQDs with different excitation wavelength. Table summarizing the quantum yield of GQDs. Table summarizing the detection limit for ·OH of different measurgin method This material is available free of charge via the Internet at http://pubs.acs.org.

AUTHOR INFORMATION Corresponding Author **Corresponding Author: [email protected] 19



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Notes The authors declare no competing ﬁnancial interest.

ACKNOWLEDGMENTS This work was supported by projects from the National Science and Technology Major Project (Grant no. 2011ZX02707), the National Natural Science Foundation of China (Grant no. 11104303), the Chinese Academy of Sciences (Grant no. KGZD-EW-303 and XDA02040000). The authors would like to acknowledge the guidance from Prof. Mianheng Jiang.

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Selenium Doped Graphene Quantum Dots as an Ultrasensitive Redox

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