Fabrication of Excitation-Independent Green Emissive Graphene Quantum Dots for Selective Detection of Hg2+and Fluorescence Imaging of Biothiols in Living Cells

MA Qifeng()， WANG Lei( )， CHEN Yansong()， LUO Xiaosong()， ZHU Zhijia()， ZHANG Xuan( )

College of Chemistry，Chemical Engineering&Biotechnology，Donghua University，Shanghai201620，China

Abstract：Nitrogen-doped graphene quantum dots(N-GQDs)exhibiting excitation-independent green fluorescence emission(536 nm)was facilely synthesized.The as-prepared N-GQDs showed a highly selective fluorescence quenching response toward Hg 2+ with a linear range of 0.1-30.0 μmol/L and detection limit of 50 nmol/L.Based on the high affinity of biothiols(such as cysteine)toward Hg 2+，the quenched fluorescence of N-GQDs could be recovered upon addition of biothiols，and thereby a new fluorescence turn-on probe for cysteine detection was further developed.The linear range and detection limit for cysteine were found to be 0.1-12.5 μmol/L and 46 nmol/L，respectively.The present fluorescent probe worked well in a physiological pure water medium，allowing a fluorescence imaging of cysteine in living cells.

Key words：fluorescent probe；graphene quantum dots(GQDs)；mercury ion；cysteine bioimaging

Introduction

Graphene quantum dots(GQDs) have attracted increasing attention in fluorescence sensing and bioimaging， due to their high fluorescence quantum yield， low toxicity， excellent water-solubility， chemical stability and biocompatibility[1-6]. Generally， the fluorescent GQDs could be fabricated by top-down and bottom-up approaches. The top-down method is operated by cutting the large sp2-hybridized carbon materials such as graphene oxide， carbon fiber， and carbon nanotube， into small pieces， whereas the bottom-up method is based on carbonization of small organic molecules such as citric acid， into nanoparticles[7-10].

The GQDs fabricated from these two approaches usually showed an excitation-dependent fluorescence emission， due to the mixture products have both various particle sizes distribution and complicated surface states[4-5]. Furthermore， most of GQDs reported previously showed blue fluorescence emissions(<500 nm)[11-17]， and only a few GQDs could emit long-wavelength green， yellow or red fluorescence[18-20]. For a practical application， the facile fabrication of more pure GQDs exhibiting excitation-independent and long-wavelength fluorescence emissive characters is highly desired.

Additionally，because the environmental toxicity of Hg2+and biological importance of biothiols such as cysteine(Cys)， glutathione(GSH)， and homocysteine(Hcy)， the detection of both Hg2+and biothiols has gathered much current interest[21-26]. Recently， several GQDs-based fluorescent probes have been developed for Hg2+and biothiols[27-30]. For example， the GQDs derived from citric acid have been reported to emit a fluorescence emission at 450 nm that could be quenched by Hg2+and further recovered by Cys， and thus a GQDs-based fluorescent probe for Hg2+and biothiols was constructed[29]. Yanetal. prepared blue emissive GQDs that showed an excitation-dependent fluorescence at 450 nm and applied to detect the Hg2+in water samples and biothiols in serum samples[30]. However， these previously reported probes exhibited short-wavelength(<500 nm) and excitation-dependent fluorescence emissions， largely limited their promising application in fluorescence bioimaging.

In this work， nitrogen-doped graphene quantum dots(N-GQDs) that showed excitation-independent long-wavelength fluorescence at 536 nm were facilely fabricated by a simple solvothermal method and combination of column chromatography purification. The as-prepared N-GQDs showed a highly selective fluorescence quenching response upon addition of Hg2+， allowing the selective detection of Hg2+available. Further introduction of biothiols such as Cys， the quenched fluorescence of N-GQDs was recovered， and thereby a new fluorescence turn-on probe for Cys imaging in living cells was developed.

1 Experimental

1.1 Materials and methods

Citricacid， urea， amino acids such as Cys， Hcy， GSH， glutamic acid(Glu)， asparagine(Asp)， lysine(Lys)， proline(Pro)， methionine(Met)， tryptophane(Trp)， tyrosine(Tyr)， histidine(His)， threonine(Thr)， glycine(Gly)， aspartic acid(Asp)， valine(Val)， leucine(Leu), argnine(Arg)， serine(Ser)， phenylalanine(Phe)， isoleucine(Ile)， and dichloroethane were all purchased from Sinopharm Chemical Reagent Co. (Shanghai， China). Mercury perchlorate trihydrate(HgClO4·3H2O) was obtained from Strem Chemicals Inc. (Newburyport， US). Phosphate buffered saline(PBS， pH=7.4) was prepared from disodium hydrogen phosphate(Na2HPO4)(0.1 mol/L) and sodium dihydrogen phosphate(KH2PO4)(0.1 mol/L) in deionized water.

Fluorescence spectra were measured on Edinburgh FS5 spectro fluorometer(Edinburgh，England) with Ex/Em slit widths of 5 nm. The absolute fluorescence quantum yields measurements were performed on EI-FS5-SC-30 integrating sphere( Edinburgh，England). Absorption spectra were performed with a PerkinElmer Lambda 35 spectrometer(PerkinElmer， USA). Fourier transform infrared spectra(FT-IR) were recorded on a Varian 640-IR FT-IR spectrometer(Varian， USA). Electron microscopy analysis and X-ray photoelectron spectroscopy(XPS) were carried out on JEM-2100F transmission electron microscope(TEM，JEOL， Japan) and PHI 5400 X-ray photoelectron spectrometer(ULVAC-PHI， Japan)， respectively. Fluorescence imaging in living A549 cells were conducted with Zeiss Axiovert(Zeiss， Germany).

1.2 Synthesis of N-GQDs

N-GQDs were similarly synthesized by the solvothermal route from citric acid and urea[31]， but using dichloroethane(EDC) as solvent. In a typical synthesis 1 g citric acid and 2 g urea were dispersed in 10 mL EDC， transferred into a 50 mL Teflon lined autoclave and heated at 160 ℃ for 6 h. The solvent was removed with the rotary evaporation and the dark solid was collected. The solid was dissolved in water and purified by dialysis(molecular weight cut off 500 Da) against water for 2 d to remove un-reacted small molecules. The obtained crude N-GQDs product were further purified by silica gel(400 mesh) column chromatography using water as the eluent and the pure N-GQDs powder was then obtained after removing the solvent under reduced pressure and freeze drying.

1.3 Detection of Hg 2+ and Cys

The detection of Hg2+was performed in PBS buffer solution(10 mmol/L， pH=7.4) at room temperature. The N-GQDs was dispersed in the PBS(0.05 g/L) and mixed with different amount of Hg2+(0-100 μmol/L). After standing 2 h at room temperature， the fluorescence intensity at 536 nm was recorded under the excitation wavelength of 411 nm， respectively. For a Cys detection， various Cys solutions(0-30 μmol/L) were sequentially added into the solution of N-GQDs(0.05 g/L) containing 30 μmol/L Hg2+and the fluorescence intensity at 536 nm was recorded immediately under the excitation wavelength of 411 nm， respectively.

1.4 Cell culture and imaging

Human lung adenocarcinoma A549 cells were cultured in Dulbecco’s modified Eagle medium(DMEM) supplemented with 10% fetal bovine serum at 37 ℃ in an atmosphere of 5% CO2environment. The cells were seeded in 24-wells glass-bottomed dishes at a density of 5 × 104cells per dish in DMEM for 24 h. For control experiments， the cells were pretreated with N-ethylmaleiminde(NEM， 1 mmol/L) for 30 min at 37 ℃ in an atmosphere of 5% CO2environment， followed by washing with PBS(10 mmol/L) three times before cells incubated with N-GQDs. The cells were cultivated with N-GQDs(100 μg/mL) for 1 h at 37 ℃， washed with PBS three times to remove free N-GQDs， and observed under Zeiss Axiovert. The NEM-treated cells were firstly incubated with 100 μmol/L Hg2+for 1 h at 37 ℃， washed with PBS three times to remove free Hg2+and observed with fluorescence microscopy. Subsequently， the cells were further incubated with Cys(50 μmol/L) for 5 min and observed with fluorescence microscopy.

2 Results and Discussion

2.1 Optical spectral character of N-GQDs

As shown in Fig. 1(a)， the crude N-GQD that obtained just after dialysis treatment， exhibited an excitation-dependent fluorescence， where the emission wavelength shifted from 465 nm to 552 nm with changing the excitation wavelengths from 300 nm to 500 nm. However， after a simple purification by silica gel column chromatography， the obtained N-GQDs showed an excitation-independent fluorescence at 536 nm(Fig. 1(b))， with the absolute fluorescence quantum yield of 8.6%. The fluorescence color obtained under UV-lamp irradiation(365 nm) was also clearly changed into green from blue after purification(insets in Figs. 1(a) and(b)). These results indicate that the more pure N-GQDs exhibiting excitation-independent and long-wavelength fluorescence emissive characters were successfully created in this work. It has been known that the photoluminescence properties of GQDs strongly depended on their surface states[32-34]. Thus the present purification treatment via silica gel column chromatography is effective on separation of GQDs based on controlling surface states. This is further confirmed by absorption spectra(Fig. 1(c)). While a broad spectrum was observed for as-prepared N-GQD， the pure N-GQD showed two typical absorption bands characterized at 272 nm and 411 nm， respectively(Fig. 1(c)). These two absorption bands could be readily assigned to the π-π*transitions of the aromatic sp2domains of N-GQD and the n-π*transitions related to the surface states， respectively[6， 33-34].

(a) Fluorescence emission spectra of as-prepared N-GQDs

(b) Fluorescence emission spectra of purified N-GQDs under various excitation wavelengths of 300-500 nm

(c) Corresponding absorption spectra

2.2 Characterization of N-GQDs

(a) TEM images

(b) Size histogram

(c) FT-IR spectrum of N-GQDs

(a) Survey spectrum of N-GQDs

(b) Spectrum of C 1s

(c) Spectrum of O 1s

(d) Spectrum of N 1s

2.3 Selective detection of Hg 2+ by using N-GQDs

The selectivity of N-GQDs towards Hg2+was conducted by examining the fluorescence change induced by various metal cations. As shown in Fig. 4(a)， the number 1 represents N-GQDs only and numbers 2-16 represent Hg2+， Cd2+， Ni2+， Na+， Pd2+， Mg2+， Mn2+， Co2+， Cu2+， Ag+， Zn2+， Ca2+， Fe3+， K+and Fe2+， respectively. Clearly， a significant fluorescence quenching of N-GQDs(0.05 g/L) was only observed with the addition of 100 μmol/L Hg2+， suggesting that the present N-GQDs were specific to Hg2+. Such fluorescence quenching effect could be similarly attributed to that the coordination interactions between the functional groups on surface of N-GQDs and Hg2+could drag quantum dots particles close to each other， thus effectively facilitate non-radiative transitions and lead to a decline of fluorescence emission[14]. Furthermore， it was noted that the co-existed other metal cations had negligible influence on fluorescence quenching effect of Hg2+toward N-GQDs(Fig. 4(a)). These results revealed that the present N-GQDs could serve as a highly selective fluorescent probe for Hg2+detection. Figure 4(b) showed the pH effect on the fluorescence of the N-GQDs in the absence and presence of Hg2+. Obviously， the fluorescence of free N-GQDs remained almost constant at pH 4-9， but a drastic decrease observed in the presence of Hg2+at pH>6. The weak quenching effect at pH < 5 could result from the weak coordination interaction between functional groups of N-GQDs and Hg2+. This indicated that the present probe could work well in neutral and weak alkaline conditions， and the pH 7.4 was selected in this work to match with the requirement in physiological environment. Figure 4(c) showed the fluorescence titration of the N-GQDs with Hg2+in PBS solution(10 mmol/L， pH=7.4). It can be noted that the fluorescence at 536 nm was continuously declined with increasing Hg2+amounts up to 100 μmol/L. A good linear relationship(R2=0.990 7) between the relative fluorescence intensity(F/F0) of N-GQDs at 536 nm and concentration of Hg2+was obtained over the range of 0.1-30.0 μmol/L(Fig. 4(d)). The detection limit for Hg2+was determined to be 50 nmol/L.

(a) Fluorescence intensity change of N-GQDs upon addition of various metal cations(black bars) and subsequent addition of(white bars)

(b) pH effect on fluorescence intensity change of the N-GQDs and N-GQDs+Hg2+ensemble， respectively

(c) Fluorescence titration spectra of N-GQDs with Hg2+

(d) Corresponding linear relationship between theF/F 0and the concentration of Hg2+

2.4 Detection of biothiols by using N-GQDs-Hg 2+ ensemble

Based on the high affinity of biothiols(such as cysteine) toward Hg2+， the fluorescence of N-GQDs quenched by Hg2+could be recovered upon addition of biothiols， and thereby a new fluorescence turn-on probe for biothiols detection in aqueous solution was readily developed. Figures 5(a) and 5(b) showed the fluorescence intensity(F/F0) change upon addition of 100 μmol/L of various amino acids， where the number 1 represented N-GQDs only in Fig. 5(a) and N-GQDs-Hg2+ensemble only in Fig. 5(b)， and the numbers 2-21 represented Cys， Hcy， GSH， Glu， Asp， Lys， Pro， Met， Trp， Tyr， His， Thr， Gly， Asp， Val， Leu, Arg， Ser， Phe， Ile， respectively. Notably， all of amino acids had no obvious effect on fluorescence of N-GQDs(Fig. 5(a)， but the quenched fluorescence of N-GQDs-Hg2+ensemble can be selectively restored by biothiols such as Cys， Hcy and GSH(Fig. 5(b))， where a green fluorescence was clearly enhanced(inset in Fig. 5(b)). It was believed that the high affinity of biothiols towards Hg2+could take out Hg2+from the N-GQDs-Hg2+ensemble and release free N-GQDs and thus restore the original green fluorescence. This demonstrated that the present N-GQDs-Hg2+ensemble can be used as a fluorescent probe for biothiols detection. As a proof of concept， theuorescence titration of N-GQDs-Hg2+ensemble with Cys was performed in PBS solution(10 mmol/L， pH=7.4). As shown in Fig. 5(c)， theuorescence intensity at 536 nm was continuously increased with increasing Cys from 0 to 30 μmol/L. A linear relationship(R2=0.995 3) between the relative fluorescence intensity(F/F0) and concentration of Cys was obtained over the range of 0.1-12.5 μmol/L(Fig. 5(d)) with a detection limit of 46 nmol/L.

(a) Fluorescence intensity change of the N-GQDs

(b) N-GQDs-Hg2+ensemble upon addition of various compounds

(c) Fluorescence change of the N-GQDs-Hg2+ensemble upon addition of Cys

(d) Corresponding linear relationship between theF/F 0and the concentration of Cys

2.5 Fluorescence imaging of Cys in living cells

To demonstrate the potential application for Cys imaging in living cells， the A549 cells were sequentially incubated with the N-GQDs， Hg2+， and Cys at 37 ℃ and observed by confocal fluorescence microscope(Fig. 6). It was observed that the cells incubated with N-GQDs(100 μg/mL) alone displayed a bright green fluorescence from the intracellular area(Fig. 6(b)). To remove the effect of intracellular biothiols， the cells were pretreated with N-ethylmaleimides(NEM， 1 mmol/L)， a thiol-blocking agent， prior to addition of N-GQDs， and further incubated with the addition of Hg2+(100 μmol/L) for another 1 h. The fluorescence of N-GQDs was clearly quenched upon addition of Hg2+(Fig. 6(d)). Further incubation with 50 μmol/L Cys led to a recovery of green fluorescence(Fig. 6(f))， indicating that the present fluorescent probe can be applied for Cys imaging in living cells. In Fig. 6， the cells incubated with the N-GQDs for 2 h(a， b)， the N-GQDs stained cells pretreated with NEM and incubated with addition of Hg2+for 1 h(c， d)，and the N-GQDs-Hg2+ensemble stained cells incubated with addition of Cys(e， f) for 5 min.

Fig. 6 Bright-eld images anduorescence images of living A549 cells

3 Conclusions

In conclusion， N-GQDs that exhibited excitation-independent and long-wavelength green fluorescence emission(536 nm) have been successfully synthesized. The present green emissive N-GQDs showed a highly selective fluorescence quenching response toward Hg2+and a fluorescent probe for Hg2+detection was developed with the detection limit of 50 nmol/L. Based on the high affinity of biothiols(such as Cys) toward Hg2+， the fluorescence of N-GQDs-Hg2+ensemble could be recovered upon addition of biothiols， and thereby a new fluorescence turn-on probe for Cys detection in aqueous solution was further developed with the detection limit of 46 nmol/L. Furthermore， the present fluorescent probe was successfully used for fluorescence imaging of Cys in living cells.