Integration detection of mercury(ii) and GSH with a fluorescent “on-off-on” switch sensor based on nitrogen, sulfur co-doped carbon dots

Using aurine and citric acid as precursors, we have synthesized stable blue-fluorescent nitrogen and sulfur co-doped carbon dots (NS-CDs), with a high quantum yield of up to 68.94% via a thermal lysis method. The fluorescent NS-CDs were employed as a sensitive sensor for the integration detection of Hg2+ and glutathione (GSH). This was attributed to Hg2+ effectively quenching the fluorescence of the NS-CDs by static quenching, and then GSH was able to recover the fluorescence owing to the stronger binding between Hg2+ and the sulfhydryl of GSH. Based on the “on-off-on” tactic, the detection limits of Hg2+ ions and GSH were 50 nM and 67 nM respectively. The fluorescence sensor was successfully applied to detect Hg2+ ions and GSH in actual samples (tap water and fetal bovine serum). Furthermore, we have proved that the sensor had good reversibility. Overall, our NS-CDs can serve as effective sensors for environmental and biological analysis in the future.


Introduction
As an important zero-dimensional carbon-based nanometer material, carbon dots (CDs) have been studied since 2004 (ref. [1][2][3][4] and widely used in biological analysis, 5,6 sensing, 7-9 catalysis, 10,11 bioimaging 12 and photothermal therapy [13][14][15] and biomedicine. 16 They have various properties, including stable uorescence photoluminescence, 17,18 low toxicity, 19 excellent biocompatibility, 20 good water solubility 21,22 and low-cost synthesis. 23 However, the applications of CDs are limited by their low quantum yield (QY) in conventional carbon sources. To increase QY, a hetero atom doping method has been utilized, in which nitrogen (N) and sulfur (S) are the commonest doped elements used to improve the uorescence performance of CDs and provide targeting properties for special functional groups. 24,25 In recent years, heavy metals have been considered as hazardous chemicals in the environment. Hg 2+ ions, as one of the most stable inorganic forms of mercury, are known to be highly toxic because of their easy bioaccumulation and inactive sulydryl group. Glutathione (GSH) is one of the most ubiquitous lowmolecular-weight (LMW) thiol in living cells 26,27 and can cause various physiological dysregulations with an anomalous concentration. Therefore, GSH 28 has been commonly used as a diagnostic biomarker for multiple diseases. Especially, it is very important to monitor the level of GSH in living cells considering the high GSH concentration in cancer cells. 29,30 To monitor Hg 2+ ions 31,32 and GSH, 33,34 numerous analytical methods have been developed, including AAS, 35 electrochemical, 36 electrophoresis, 37 HPLC, 38 mass spectrometry, 39 and etc. 40 However, most of them are laborious, intricate, and in need of professional operating and expensive instruments. Fluorescence probes providing a better choice of Hg 2+ ions and GSH detection in living cells, tissues, even animals due to the exclusive advantages of high sensitivity, 41,42 simple controls, convenient testing/monitoring and quick response, etc. However, most of the conventional uorescent probes such as small organic uorescent molecule, and semiconductor quantum dots are relatively toxic and poorly watersoluble, and greatly limit the detection of Hg 2+ ions and GSH in bioassay. Accordingly, CDs as a novel uorescent probe with excellent biocompatibility has attracted increasing attention.
In this study, we have successfully prepared CDs with citric acid and taurine, with its QY up to 68.94%. With the addition of Hg 2+ ions, the uorescence of NS-CDs can be quenched efficiently, and the uorescence was recovered while GSH was added. Moreover, NS-CDs have been tested in actual sample (tap water and fetal bovine serum) to detect Hg 2+ ions and GSH, showing their good selectivity and sensitivity.

Characterization of NS-CDs
As shown in Fig. 1a, NS-CDs are well dispersed and have nearly spherical shape. The particle sizes of NS-CDs are mainly distributed in a narrow range from 1.2 nm to 2.8 nm, with an average particle diameter of 1.7 nm (Fig. 1b). The XRD pattern result indicates a broad peak at 2q ¼ 23 , suggesting its attribution to the amorphous structure. Then, we have examined the surface groups on the NS-CDs. The FTIR results shows several strong absorption peaks at 2500-3440 cm À1 , 1710 cm À1 and 1210 cm À1 , 1349 cm À1 , 1410 cm À1 , 1052 cm À1 , and 741 cm À1 , which are attributed to O-H groups, C]O and C-O, S]O bonds, C-N bond, O]S]O bonds, and NH bonds, respectively (Fig. 1d). Overall, this suggests NS-CDs have high solubility in water because of their carboxyl, hydroxyl, and amino functional groups on surface.
To further analyze the composed elements of NS-CDs, we have performed XPS analysis, and the full XPS survey spectrum shows the elementary analysis of sulfur (S2p, 169.08 eV), carbon (C1s, 286.08 eV), nitrogen (N1s, 401.08 eV), and oxygen (O1s, 532.08 eV) (Fig. 2a). This indicated NS-CDs includes four elements, C, O, N, and S, with a content of 47.7 wt%, 32.03 wt%, 8.6 wt% and 8.06 wt%, respectively. To reveal the detailed bonds, we have conducts XPS spectrum experiments for each element. C1s spectrum displays four distinct peaks at 288. ). Hence, we concluded that N and S elements are on the surface of NS-CDs, which is consistent with our FTIR measurements data (Fig. 1d). Both XPS spectra and FTIR spectra suggests the hydrophilicity and stability of NS-CDs due to their functional hydroxyl, amide, amino, and carboxyl/carbonyl groups.

Optical properties of NS-CDs
Next, we determined the optical properties of NS-CDs. NS-CDs display an absorption peak at 332 nm because of the absorption of connected carbonyl/amine group 43 with its maximum excitation wavelength and emission at 345 nm and 443 nm, respectively (Fig. 3a). The aqueous solution of NS-CDs displays colorless under sunlight but emits blue uorescence while under UV lamp (Fig. 3a inset). The CIE color coordinate of NS-CDs is (0.1545, 0.1551), further indicating NS-CDs show blue uorescence with UV radiation (Fig. 3c). With the excitation wavelength of NS-CDs ranging from 325 nm to 385 nm, their emission peak shis from 439 nm to 446 nm, showing redshi (Fig. 3b). This suggests that N and S doping elements contribute to a surface condition and uniform size distribution of NS- CDs. 44 2D-emission plots show that a photoluminescence center could be observed at the emission wavelength of 443 nm and excitation wavelength of 345 nm (Fig. 3d). In addition, the uorescence QY of NS-CDs was calculated to be 68.94%.
Furthermore, ionic strength, illumination, and pH value were examined to assess the uorescence stability of NS-CDs. With increasing the concentration of sodium chloride (from 0 to 4 M), there was no obviously change on the uorescence intensity of NS-CDs, suggesting their excellent tolerance in ion solutions (Fig. S1 †). In addition, the uorescence intensity of NS-CDs remains unaltered under the UV light irradiation for at least 90 min, showing their stability under UV light (Fig. S2 †). The uorescence intensity of NS-CDs gradually increases when pH value increases from 1 to 7, and decreases when pH value raised from 7 to 13, with the maximum intensity at pH value of 7 (Fig. S3 †). In general, we have conrmed that NS-CDs have a stable and excellent optical performance even under extreme environmental conditions.

Detection of Hg 2+ and GSH
To evaluate their selectivity of specic metal ions, we investigated the uorescence response of NS-CDs to common metal ions. Compared with other ions, Hg 2+ ions have a stronger interaction with NS-CDs, with outstanding uorescence quenching (Fig. 4a). 45,46 When possible interference ions coexists, the addition of coexisted ions exert no effect on the uorescence quenching caused by the Hg 2+ ions, further indicating a high selectivity of NS-CDs for Hg 2+ ions detection even in the presence of possible interference ions (Fig. 4b).
The uorescence spectra were recorded with adding Hg 2+ ions of various concentrations into the NS-CDs solution. The uorescence intensity drops quickly when the concentration of Hg 2+ increases from 0 mM to 100 mM (Fig. 5a). The relationship between the relative uorescence intensity ((F 0 À F)/F 0 ) of NS-CDs and the concentration of Hg 2+ ions is shown in Fig. 5b. When the concentrations of Hg 2+ ions range from 0-10 mM, there is a strong linear correlation, with the relative coefficient (R 2 ) of 0.9932. The tted linear equation is (F 0 À F)/F 0 ¼ 0.0776c (Hg 2+ ) À 0.4405, where F and F 0 are the uorescence intensities of NS-CDs with or without adding Hg 2+ ions, respectively.  According to 3d/k, we have determined the detection limit of Hg 2+ ions at 50 nM. Table 1 shows the linear range and LOD of Hg 2+ are compared with previously literature values.
When we examined the selectivity of the NS-CDs-Hg 2+ system to different amino acids, other small biomolecules cannot or little alter the uorescence, except for GSH (Fig. 6a). The quenched uorescence is only recovered when GSH is   added into the NS-CDs-Hg 2+ system. Consequently, the NS-CDs-Hg 2+ system could be served as a turn-on sensor to detect GSH specically. When other biomolecules were mixed with GSH, the effect of coexistent biomolecules can be neglected, suggesting the specicity of the NS-CDs-Hg 2+ system to GSH detection (Fig. 6b).
With GSH concentration increased, the uorescence intensity of NS-CDs-Hg 2+ is gradually returned (Fig. 7a). Then, we calculated the relation between the relative uorescence intensity ((F À F 0 )/F 0 ) of NS-CDs-Hg 2+ and the concentration of GSH. The relative intensity of NS-CDs-Hg 2+ shows a strong linear correlation to the concentration of GSH in the range of 0-10 mM, with the related coefficient (R 2 ) of 0.9932 (Fig. 7b). The linear tting equation is (F À F 0 )/F 0 ¼ 0.05792c (GSH) + 0.01393, where F and F 0 are the uorescence intensities of NS-CDs-Hg 2+ with or without adding GSH, respectively. The detection limit of GSH is calculated at 67 nM. The NS-CDs-Hg 2+ as the uorescence sensor to test GSH is also compared with the other works in Table 2. In conclusion, these results demonstrate the NS-CDs can detect Hg 2+ and GSH at "on-off-on" mode in a sensitive and specic manner.

Mechanism of the "on-off-on" mode
In this work, the NS-CDs displays a fascinating "on-off-on" experimental phenomena with the gradual addition of Hg 2+ and GSH. Scheme 1 illustrates a proposed sensing mechanism of   (Fig. S4 †). On the other hand, since the sulydryl of GSH has a stronger binding interaction with Hg 2+ , it can take away Hg 2+ from the surface of NS-CDs and then recover the uorescence of NS-CDs.
The uorescence sensing procedure was demonstrated by uorescence lifetime and UV-vis experiments. In Fig. 8a, the absorption peak of NS-CDs (345 nm) is weakened aer the addition of Hg 2+ , which proves Hg 2+ compounds with NS-CDs. However, the absorption peaks intensity is strengthened with the addition of GSH, proving that Hg 2+ is removed from the surface of NS-CDs. 61 The measured FL decay curves exhibit a double-exponential function (Fig. 8b). The average excition lifetime of NS-CDs is 9.89 ns, containing long lifetime of 11.39 ns (50.15%) and short lifetime of 2.25 ns (49.85%). With the addition of Hg 2+ and GSH, there is no change in the average uorescence lifetime of NS-CDs, indicating the uorescence quenching mechanism is static quenching. 62,63

Detection of Hg 2+ and GSH in actual samples
To investigate the feasibility and sensitivity of the method, the N-CDs was applied to determine Hg 2+ in tap water. The tap water samples obtained from the laboratory and was not any pretreatment. The recoveries of Hg 2+ are 94.0-105.2 (RSD < 3.4%) in Table S1. † Tap water contains some other elements, we further study the interferences experiment of ions commonly present in tap water (Fig. S5 †), the result shows the addition of metal ions did not interfere with the uorescence intensity of NS-CDs/tap water system, further illustrating the metals does not affect the test results of actual samples.
Future, practical applications of N-CDs-Hg 2+ sensor for GSH determination in fetal bovine serum sample were executed. The measurement of GSH in fetal bovine serum is 0.35 mM, and the recoveries of GSH are 98.4-102.5% (RSD < 2.4%) (Table S2 †). All the results indicate that the N-CDs sensor have high precision and accuracy for determining Hg 2+ and GSH in the practical detection.
Reversibility (also known as fatigue resistance) is one of the important indicators for evaluating "on-off-on" uorescent sensors. In order to study the reversibility of NS-CDs for Hg 2+ and GSH detection, Hg 2+ and GSH were added alternately to the NS-CDs solution (Fig. 9). The results reects the uorescent intensity shows an alternating change, and retains good fatigue resistance aer 10 cycles, indicating that the uorescent sensor has good reversibility.

Conclusions
In summary, NS-CDs based uorescent "on-off-on" was prepared for detecting Hg 2+ and GSH. The blue emission NS- Fig. 8 (a) UV-vis spectra of NS-CDs, Hg 2+ , and GSH system. (b) Fluorescence decay curves of the NS-CDs, NS-CDs-Hg 2+ , and NS-CDs-Hg 2+ + GSH. Fig. 9 The reversibility of the NS-CDs by alternately adding Hg 2+ and GSH.
CDs showed excellent optical performance, good uorescent stability, with the uorescence quantum yield up to 68.94%. The uorescence of NS-CDs solution was quenched (namely turn-off) when Hg 2+ ions were added and subsequently recovered (turn-on) when GSH was added. These results illustrate that the NS-CDs can be used as uorescence sensor to detect Hg 2+ ions and GSH through uorescent "on-off-on" detection mode, with the detection limits as low as 50 nM and 67 nM, respectively. Furthermore, the uorescent sensor had good reversibility. Consequently, the NS-CDs offered an effective platform for biological analysis and environmental analysis.

Conflicts of interest
The researcher claims no conicts of interests.