The on–off-on Fluorescence Sensor of Hollow Carbon Dots for Detecting Hg2+ and Ascorbic Acid

Carbon dots (CDs) have excellent fluorescence properties and can be used in many research fields. In this paper, carbon dots were prepared by microwave-assisted pyrolysis of citric acid and urea, characterized by transmission electron microscope (TEM), X-ray diffractometer (XRD), 13C-NMR spectrum, zeta potential, Fourier transform infrared spectroscopy (FT-IR), ultraviolet–visible (UV–vis) absorption and fluorescence spectra, and detected the Hg2+ and ascorbic acid (AA) sequentially. It showed that carbon dots were hollow, spherical particles and less than 10 nm, photoluminescence quantum yield of carbon dots was about 15%. The CDs were selective and sensitive to Hg2+ and AA based on the “on–off-on” fluorescence behavior. The detection limits of CDs for Hg2+ and AA were 0.138 μM and 0.212 μM, respectively. Fluorescence response mechanism of CDs was also discussed.


Introduction
People pay more and more attention to health with the improvement of living standard. The detection of toxic substances and nutrients becomes very important. Ascorbic acid (AA) known as vitamin C is vital and plays a crucial role in biological processes. The recommend amount of the average daily AA intake for male and female adults is 90 mg and 70 mg, respectively [1]. The insufficient or excessive intake of AA may be harmful to the human body; lack of AA can induce to scurvy and the excess to gastritis. Therefore, the quantitative detection of AA is important in pharmaceutical and food industries.
In addition, we all know that mercury ion (Hg 2+ ) is toxic and accumulates in the ecosystem through the food chain. Hg 2+ can cause damage to the nervous system, immune system, kidney, and heart of humans or animals. Therefore, the detection of Hg 2+ is essential to human health. There are many methods have been reported for measuring Hg 2+ , such as, atomic absorption spectroscopy [2], surface-enhanced Raman scattering spectroscopy [3], inductively coupled plasma mass spectrometry [4], and fluorescence spectroscopy [5]. Among them, fluorescence spectroscopy has received more and more attention due to its low background noise, simple operation, and high sensitivity.
Carbon dots (CDs) are a novel type of fluorescent nanomaterial [6] with excellent water solubility, chemical stability and excellent biocompatibility [7][8][9][10][11], and so on. CDs have been used widely in cell and tissue imaging [12,13], ions and biomolecule detection [14], drug-loading [15], optoelectronics [16], photocatalytic [17], and especially in fluorescence sensing [9,18]. As fluorescent probes, CDs have the attractive advantages of high selectivity, sensitivity, and low limit of detection (LOD). Meanwhile, the multicomponent detection by CDs has been attracted extensive attention in recent years. The basic principle is that the fluorescence intensity of CDs is weakened or quenched by some metal ions (such as Cu 2+ [19], Hg 2+ [20], Fe 3+ [18,21]), and then can be restored with the addition of other substances (glutathione [22], AA [23], I − [24], ciprofloxacin [25], and endotoxin [26]). This "on-off-on" type of sensor could be used to detect two or more components. At present, many reports were about the sequential detection of Cu 2+ / Fe 3+ and AA [27][28][29][30][31]. The multiplex fluorescent "on-off-on" sensor based on CDs was time-saving, sensitive, simple, and high selectivity. To our knowledge, the "on-off-on" CDs sensor for detecting Hg 2+ and AA has not been reported. In our previous work, our group reported the application of CDs used as drug carrier [32,33]. In this work, we focus on the application of CDs in fluorescence sensing. CDs were prepared by microwave method using citric acid and urea as raw materials and characterized by transmission electron microscope (TEM), X-ray diffractometer (XRD), 13 C-NMR spectrum, zeta potential, Fourier transform infrared spectroscopy (FT-IR), ultraviolet-visible (UV-vis) absorption and fluorescence spectra. The fluorescence sensitivity of CDs to various substances was tested. Hg 2+ could decrease the fluorescence of CDs and AA could recover the fluorescence of CDs, and the response mechanism was discussed. And the detection of actual sample was also carried out.

Materials
The citric acid (Tianjin North Tianyi Reagent Company) and urea (Tianjin Bodi Chemical Co., Ltd.) were used to prepare CDs. The quantum yield was calculated referring to quinine sulfate (Sinopharm Group). Hg(NO 3 ) 2 (Jiangyan Huanqiu Reagent Factory), ascorbic acid(Tianjin Yongda Chemical Reagent Co., Ltd.) and Vitamin C (CSPC Ouyi Pharmaceutical Co., Ltd.) were used for fluorescence detection. All reagents used in the experiment were of analytical grade.

Preparation of CDs
The CDs were prepared using the microwave method. The citric acid and urea (molar ratio of citric acid to urea was 1:2) were dissolved in a beaker containing 10 mL of deionized water. And then, the beaker was put into the microwave oven and heated for 2 min. After the product cooled down naturally to room temperature, 10 mL water was added to beaker. And then the solution was centrifuged at 4000 rpm for 15 min to get rid of the precipitation, the supernatant was dialyzed and dried at 80 ℃. The black product was obtained and used to prepare CDs solution with 10 mg/mL stored at 4℃.

Characterization of CDs
Transmission electron microscope (TEM) was performed on JEM 100SX (Japan) with an acceleration voltage of 200 kV. The X-ray diffractometer (XRD) data was collected in the range of 10-70° using Bruker D8 Advance X-ray diffractometer (Cu Ka radiation, λ = 1.54178 Å) (Germany). 13 C-NMR (500.13 MHz) was characterized by Bruker DMX-500 spectrometer (Germany) in a D 2 O environment. XPS measurements were undertaken on an ESCALAB 250Xi X-ray Photoelectron Spectrometer microprobe using a monochromatic Al-K X-ray source (h = 1486.68 eV) (USA). Fourier Transform Infrared Spectroscopy (FT-IR) spectroscopy was accomplished on Thermo SCIENTIFIC equipment (USA) to complete at 4000-500 cm −1 using KBr pressed-disk technique. Fluorescence spectrum (FL) was obtained by F-4600 fluorescence spectrophotometer (Japan) in the field of 200-700 nm. Cary60 ultraviolet spectrophotometer (Agilent Technologies, USA) was used to record the ultraviolet-visible (UV-vis) absorption spectrum in the range of 200-700 nm. The Zeta potential results were obtained by using Nano-ZS90 (Malvern Instruments Ltd, Malvern, UK).

Quantum Yield (QY) Measurement
The QY of CDs was calculated according to the literature method [34] (quinine sulfate solution was selected as reference substance, and its QY was 0.55): In the above formula, Y represents QY, F represents the integrated peak area of the fluorescence emission spectra, A means the absorbance at the excitation wavelength, and the subscripts s and u represent the standard substance and the test substance, respectively.
Hg 2+ could reduce the fluorescence of CDs. To investigate the fluorescence recovery of CDs, some different substances (Try, Leu, Pro, Glu, Gly, Arg, Lys, Met, Gln, or AA) were added respectively to the CDs-Hg 2+ solution.
The limit of detection (LOD) of Hg 2+ /AA was calculated according to the formula LOD = 3σ/S [35] (the standard deviation of the blank signal was σ (n = 5) and the slope of the standard curve was S).

Sample Analysis of CDs Sensor
To test the applicability of CDs sensor, the vitamin C tablets was purchased from the pharmacy and dissolved in redistilled water with a certain concentration. The supernatant was taken out and calculated the AA amount by the working curve of AA. The supernatant was added CDs and Hg 2+ , and subsequently, different concentrations of AA (0, 30, 60, or 90 μM) were added respectively. And the recovery rate of AA was calculated.

Characterization of CDs
The size and morphology of CDs were observed by TEM (Fig. 1A). The prepared CDs were approximately spherical particles smaller than 10 nm, uniformly distributed in the water phase without apparent aggregation [36]. In addition, the TEM result indicated that the structure of CDs was hollow, which may be due to the production of ammonia and carbon dioxide during the reaction process.
The XRD (Fig. 1B) pattern of the CDs showed that there was a broad peak of about 20°-22°, which indicated that the CDs were amorphous and highly disordered [33]. The 13 C-NMR spectrum (Fig. 1C) of CDs showed three signals in the range of 30-45 ppm, 70-80 ppm, and 170-185 ppm. The signals at 30-45 ppm and 70-80 ppm belonged to the sp 3 hybrid aliphatic carbon atoms, and the signal at 170-185 ppm indicated the presence of sp 2 -hybridized carbon atoms, which corresponded to carboxyl/amide groups [33,37]. The functional groups of CDs can be deduced from the FT-IR as shown in Fig. 1D. The broad absorption peaks at 3442 and 1450 cm −1 were attributed to the O-H vibration, suggesting abundant hydroxyl groups on the CDs' surface [38]. The N-H stretching vibration [39]  To further explore the elemental composition and chemical bond of the CDs, XPS measurements were performed. The CDs contain three elements, C, N and O. As shown in Fig. 2A, three dominant peaks at 284 eV, 399 eV, 530 eV were attributed to C 1 s, N 1 s, and O 1 s, respectively. The high-resolution XPS spectrum of C 1 s (Fig. 2B) were subdivided into three peaks at 284.9 eV, 283.55 eV and 286.25 eV, which were attributed to the C-C/C = C, C = O/C = N and C-N/C-O groups [42], respectively. The N 1 s (Fig. 2C) spectrum showed a peak at 399.05 eV, which attributed to amino nitrogen [43]. XPS analysis of the O 1 s spectrum (Fig. 2D) showed two peaks at 529.65 and 530.75 eV, corresponding to C-O and C = O groups [44], respectively. The results show that the CDs surface contains functional groups such as − COOH, − OH and − NH 2 , which was consistent with the FT-IR results.
The ultraviolet-visible absorption and photoluminescence spectra of CDs were showed in Fig. 3A. In the UV-vis spectra, two distinct absorption peaks at 250 nm and 330 nm were observed, and the peaks were ascribed to the n → π* transition of C = O and the π → π* transition of conjugated C = C [45] respectively. The emission peak of CDs redshifted with the excitation wavelength increased gradually from 300 to 400 nm [46], and the fluorescence intensity increased firstly and then decreased. This phenomenon may be the effect of surface states on the band gap of the hollow CDs [33].The excitation and emission spectrum exhibited good symmetry (Fig. 3B) and the CDs emitted blue light (Fig. 3C).The quantum yield (QY) of CDs was 15% according to the reference method.

The "on-off" Signal of CDs for Detection of Hg 2+
The influence of metal ion to the fluorescence intensity of CDs was showed in Fig. 4A. It showed that Hg 2+ decreased the fluorescence intensity of CDs significantly and other ions nearly had no effects on the CDs fluorescence. The degree of fluorescence quenching (1-F/F 0 ) of CDs had a linear relationship with the concentration of Hg 2+ (0.5-40 μM) (Fig. 4C). Furthermore, the limit of detection (LOD) of CDs to Hg 2+ was  Table 1. By contrast, the hollow CDs had a lower LOD and wider detection range for Hg 2+ . It indicated that CDs had good selectivity and specificity with Hg 2+ and could be used to detecting Hg 2+ .

The"off-on" Signal of CDs for Detection of AA
In the above experiment, it showed that Hg 2+ could decrease the fluorescence of CDs. To observe the fluorescence recovery of CDs-Hg 2+ system, different substances (Try, Leu, Pro, Glu, Gly, Arg, Lys, Met, Gln, or AA) were added respectively into the CDs-Hg 2+ solution (Fig. 5A). It showed that only AA could obviously recover the fluorescence of CDs-Hg 2+ solution, and the fluorescence intensity was increased with the increasing concentration of AA (Fig. 5B). The degree of fluorescence recovery (F/F 0 -1) of CDs had a linear relationship with the concentration of AA (0-500 μM) (Fig. 5C). The LOD of CDs-Hg 2+ system to detect AA was calculated and was 0.212 μM. Different substances (Try, Leu, Pro, Glu, Gly, Arg, Lys, Met, Gln, and AA) were added to the previously obtained CDs-Hg 2+ system. The effects of other substances to the CDs-Hg 2+ system could be ignored or had no effect except for AA (Fig. 5A). The fluorescence intensity increases significantly as AA addition. Figure 5B shows the fluorescence intensity of CDs-Hg 2+ with increasing AA concentrations from 0 to 500 μM. The fluorescence recovery rate could reach up to 90% until the AA concentration had risen to 500 μM. Figure 5C shows the linear relationship between the AA concentration in the range of 10-100 μM and the fluorescence intensity (F/F 0 -1) of the CDs-Hg 2+ system, using 3σ/S to calculate the LOD as 0.212 μM. The experimental results indicated that this sensing assay for the detection of AA based on the principle of fluorescence "on-off-on" had satisfactory sensitivity and selectivity, which was highly superior to most reported fluorescence methods for AA detection (Table 2).
To demonstrate the practicability of this strategy, various concentrations of standard AA were added to the sample solution of vitamin C tablets. Table 3 showed that 0, 30, 60, and 90 μM standard AA was added to the CDs-Hg 2+ system, and the calculated concentration responses were 242.02, 274.29, 299.90, and 330.25 μM, respectively. The experiment results showed that the range of AA recovery was 96.4-107.5%, and the relative standard deviation for the detection of AA was within the scope of 0.31-1.04%. This detection assay based on the fluorescent "off-on" principle could detect the AA amount, which fit well with the expected results.

The "on-off-on" Mechanism of CDs Sensor
For the investigation of sensing mechanism, FRET and inner filter effect were first ruled out, because there was no obvious absorption of Hg 2+ in the range of 300-500 nm [56,57]. The response mechanism of CDs to Hg 2+ and AA may have relationship with coordination reaction: the "on-off" signal was the coordination of Hg 2+ with the hydroxyl groups on the surface of CDs. This interaction between Hg 2+ and CDs  affected the electron distribution, and accelerated the effective electron transfer in the non-radioactive recombination process, resulting in a significant decrease in fluorescence intensity of CDs [38,58]. The fluorescence recovery of CDs-Hg 2+ system by AA could be attributed to the competitive coordination mechanism [59,60], and the "off-on" signal was that the hydroxyl groups of AA reacted with Hg 2+ , which was deprived from CDs-Hg 2+ system by AA, so the fluorescence of CDs was recovered (Fig. 6A). Furthermore, the speculation was demonstrated by the changes of fluorescence intensity and zeta potential of CDs (Fig. 6B, C). When Hg 2+ and AA were added into the CDs solution subsequently, the fluorescence intensity of CDs decreased firstly and then increased, and the zeta potential   of CDs was changed from -24.3 mV to 21.8 mV and then to -30.8 mV. When the -OH was coordinated with Hg 2+ ion to form ground state complex, the oxygen obtained a positive charge with increasing zeta potential [61]. The AA has a stronger binding force with Hg 2+ because each AA molecular contains four -OH. When AA was added, Hg 2+ was taken away from the surface of CDs, thereby the fluorescence of CDs was recovery. The whole process responded quickly.

Conclusions
In this work, hollow CDs were prepared by microwaveassisted pyrolysis of citric acid and urea, characterized by TEM, XRD, 13 C-NMR, FT-IR, UV-vis and fluorescence spectra, and zeta potential. The obtained CDs could detect Hg 2+ and AA sequentially based on the "on-offon" fluorescence principle. The LOD of CDs for Hg 2+ and AA was 0.138 μM and 0.212 μM, respectively, which were high sensitivity and selectivity compared with the literature. This method could be used to detect AA in the actual samples. The response mechanism of CDs detecting Hg 2+ and AA was explained by coordination chemistry principle.

Authors' Contributions
All authors contributed to the study conception and design. Material preparation and characterization were carried out by Yunping Hao, the fluorescence sensitivity of the production was tested by Ronghui Li. data collection and analyses were performed by Yanxu Liu and Xuhong Zhang. The first draft of the manuscript was written by Lina Geng, and the check of the manuscript was done by Shenna Chen. All authors read and approved the final manuscript.