Preparation of Highly Catalytic N-Doped Carbon Dots and Their Application in SERS Sulfate Sensing.

Carbon dots (CD) have excellent stability and fluorescence activity, and have been widely used in fluorescence methods. However, there are no reports about using CD as catalysts to amplify SERS signals to detect trace sulfate. Thus, preparing CD catalysts and their application in SERS sulfate-sensing are significant. In this article, highly catalytic N-doped carbon dots (CDN) were prepared by a hydrothermal procedure. CDN exhibited strong catalysis of the gold nanoparticle (AuNP) reaction between HAuCl₄ and H₂O₂. Vitoria blue 4R (VB4R) has a strong SERS peak at 1614 cm-1 in the formed AuNP sol substrate. When Ba2+ ions were added, they were adsorbed on a CDN surface to inhibit the CDN catalytic activity that caused the SERS peak decreasing. Upon addition of analyte SO₄2-, a reaction with Ba2+ produced stable BaSO₄ precipitate and CDN, and its catalysis recovered to cause SERS intensity increasing linearly. Thus, an SERS method was developed for the detection of 0.02⁻1.7 μmol/L SO₄2-, with a detection limit of 0.007 μmol/L.

1. Introduction

Because carbon dots (CD) have excellent stability, excellent chemical properties, high fluorescence activity, anti-photobleaching abilities and low cell toxicity [1,2,3,4,5], they are of interest to scientists. Based on the redox, complex, enzyme and immune reactions, CD have been used to determine chlorine ion, phosphate, ATP, ferric ion, hydrogen peroxide, glucose, immunoglobulin G, biological thiols, deoxyribonucleic acid, trypsin and so on [6,7,8,9,10,11]. Freire et al. [12] used polyvinyleneimine to prepare carbon quantum dots (CQDs/BPEI) to detect proteins. The nitrogen-doped carbon dots with high fluorescence efficiency have attracted much attention. Liu et al. [13] prepared nitrogen-doped graphene quantum dots and a photoelectrochemical aptasensor for chloramphenicol determination. Gu et al. [14] used 2-azidoimidazole and ammonia as reactants to prepare a fluorescent quantum dots by a thermal procedure, and to determine cysteine (Cys) by the reaction of CD-Cu2+-Cys. An aptamer has good electivity and has been combined with CD. Feng et al. [15] reported a graphene quantum dots-aptamer fluorescent probe to detect lead (II) ions (as low as 0.6 nmol/L). However, there are no reports about preparation of highly catalytic N-doped carbon dots and their application to SERS quantitative analysis.

SERS is a highly sensitive and selective molecular spectral technique; it has been used in biomedical, environmental monitoring, and analytical chemistry [16,17,18]. Liang et al. [19] prepared silver nanorods/reduced graphene oxide (AgNR/rGO) nanosol as SERS substrate to determine 8–1500 nmol/L iodide. Yang et al. [20] prepared silver nanosol SERS substrate to determine 2–191.0 mg/L thiocyanate. Luo et al. [21] prepared triangular nanosilver based on graphene oxide catalysis, and the nanosilver was used to analysis of 0.7–72 nmol/L nitrite by SERS. Jiang et al. [22] examined the catalytic reduction of HAuCl4 by cysteine with AuNP nanoenzyme to prepare gold nanosol substrate with high SERS activity to determine surfactants. Zhang et al. [23] developed a SERS method for detection of SO2, with a detection limit of 1 mg/L, based on the Raman peak at 630 cm−1 of S atom. Shang et al. [24] prepared silver nanochain (AgNC) sol substrate to analyze 0.00725–0.3 μmol/L hexametaphosphate. Sulfate is one of the important anions in water science, food science, soil chemistry, biology, mineralogy and related disciplines. For analysis of trace SO42−, there are visible–ultraviolet spectrophotometry, turbidimetry, fluorescence spectrophotometry, electrochemical analysis, radiochemical analysis, resonance Rayleigh scattering, ion chromatography, and so on [25,26,27,28,29,30]. In this experiment, highly catalytic N-doped carbon dots were prepared for the HAuCl4-H2O2 reaction, and a new and sensitive SERS quantitative analysis method was proposed for the determination of sulfate in water and beer samples, based on the CD catalysis.

2. Materials and Methods

2.1. Apparatus and Reagents

The SERS spectra were recorded by a model of DXR smart Raman spectrometer (Thermo, Waltham, MA, USA) with laser wavelength of 633 nm, power of 3.5 mW, slit of 50 μm and acquisition time of 5 s. A model of 3K-15 high-speed refrigerated centrifuge (Sigma Co., Darmstadt, Germany) and a model of 79-1 magnetic stirrer with heating (Zhongda Instrumental Plant, Jiangsu, China) were used. A model of S-4800 field emission scanning electron microscope (Hitachi High-Technologies Corporation, Japan/Oxford Company, Oxford, UK) was used to record the graphs.

A 2.9 mmol/L HAuCl4 (National Pharmaceutical Group Chemical Reagents Company, Shanghai, China), 10 μmol/L VB4R (Shanghai Reagent Three Factory, Shanghai, China) stock solution, 1 mmol/L BaCl2 (Hunan Reagent Factory, Changsha, China), 1.00 mmol/L Na2SO4 (Xilong Science Co., Ltd., Shantou, China) and 3.4 mmoL/L trisodium citrate (Xilong Chemical Plant, Shantou, China) were prepared.

Preparation of N-doped carbon dot solution (CDN): A 1 g of citric acid and 0, 0.5, 1.0 and 2.0 g urea were dissolved respectively in 30 mL water, and the brown yellow transparent solution was transferred to a polytetrafluoroethylene autoclave. After sealing, the autoclave was heated at 180 °C for 5 h. It was cooled to room temperature with tap water and was dialysis a night with dialysis bag of 3500 Da, and neutralized with NaOH solution to pH 7.0 to get a 0.021 g/mL CDN that was named as CD0N, CD0.5N, CD1N and CD2N respectively.

2.2. Procedure

In a 5 mL graduated test tube, an appropriate amount of Na2SO4, 80 μL 1 mmol/L BaCl2 and 75 μL 100 μg/mL CD were added and mixed well. Then 100 μL 0.1% HAuCl4 and 50 μL 0.10 mol/L H2O2 solution were added and diluted to 1.5 mL. The tube was heated at 50 °C water bath for 20 min, cooled with ice-water, and 50 μL10 μmol/L VB4R molecular probe was added. The SERS spectrum was recorded by the spectrometer. The SERS peak intensity at 1614 cm−1 (I1614cm) and a blank (I1614cm)0 without SO42− were recorded. The value of ΔI = I1614cm − (I1614cm)0 was obtained.

3. Results and Discussions

3.1. Principle

The AuNP reaction was very slow, and the CD1N surface contained more surface electrons that enhanced the electron transfer of the HAuCl4-H2O2 redox reaction, and displayed strong catalytic activity on the AuNP reaction. The Ba2+ ions adsorb on the CD1N surface and repress the catalysis. When SO42− was added, stable BaSO4 formed and CD1N was released which caused the SERS peak to increase due to formation of more SERS active gold nanoparticles. The more SO42− was added, the more CD was released, the more Au nanoparticles formed, and the SERS signal enhanced greatly after addition of probe VB4R. Accordingly, a new SERS quantitative analysis method was proposed for trace sulfate, based on the regulation of CD1N catalysis (Figure 1).

Figure 1

Surface enhanced Raman scattering (SERS) determination of sulfate by BaSO4 regulation of CDN catalysis of the gold nanoreaction between HAuCl4 and H2O2.

3.2. SERS Spectra

Compared to common carbon nanomaterials such as graphene and C60, CD are very stable and dissolved in water, and were chosen for use. The CD0N, CD0.5N, CD1N and CD2N analytical systems were studied by an SERS technique with VB4R molecular probes. There are nine SERS peaks at 240, 432, 675, 800, 1175, 1202, 1290, 1394 and 1614 cm−1 (Figure 2). With the SO42− concentration increasing, the SERS signal increased greatly. Among the four systems, the CD1N analytical system at 1614 cm−1 SERS peak is the most sensitive. Thus, it was chosen to detect SO42−.

Figure 2

SERS spectrum of HAuCl4-H2O2-CD1N-Na2SO4-BaCl2-VB4R system. (a): 4.2 μmol/L HAuCl4 + 2.5 mol/L H2O2 + 5 μg/mL CD1N + 53 μmol/L BaCl2 + 0.33 μmol/L VB4R; (b): a + 0.05 μmol/L Na2SO4; (c): a + 0.10 μmol/L Na2SO4; (d): a + 0.2 μmol/L Na2SO4; (e): a + 0.7 μmol/L Na2SO4; (f): a + 1.0 μmol/L Na2SO4; (g): a + 1.7 μmol/L Na2SO4.

3.3. Scanning Electron Microscopy

Scanning electron microscopy (SEM, Hitachi High-Technologies Corporation, Japan/Oxford Company, Oxford, UK) and energy spectra of CD1N show that the small CD particles are spherical with an average size of 20 nm (Figure 3a) and the large aggregate may be the salt crystallization on the silicon wafer of SEM. There is a spectral peak at 0.25 keV for C, N and O elements. The SEM of HAuCl4-H2O2-CD1N-Na2SO4-BaCl2-VB4R was recorded. When there is no Na2SO4, the HAuCl4-H2O2 reaction is very slow to produce few quasi spherical AuNPs with an average size of 50 nm (Figure 3b); the morphology is not like the CD1N, and there is a spectral peak at 1.7 keV for Au. When Na2SO4 was added (Figure 3c), there were more AuNPs with an average size of 40 nm owing to CD1N catalysis recovering and enhancing the SERS peak. This also indicated that the particles are AuNPs in the analytical system.

Figure 3

Scanning electron microscopy of the CD1N analytical system. (a): 50 μg/mL CD1N; (b): 4.2 μmol/L HAuCl4 + 0.33 μmol/L VB4R + 5 μg/mL CD1N + 2.5 mmol/L H2O2 + 53 mol/L BaCl2; (c): b + 1.67 μmol/L Na2SO4.

3.4. Catalysis and Inhibition

Under the conditions as in the procedure, the AuNP reaction of H2O2-HAuCl4 is slow. The CDN exhibited catalysis of the AuNP reaction, and the SERS intensity increased with increasing CD concentration (Table 1, Figure 4). The CD without N element exhibited weak catalysis of the AuNP reaction of H2O2-HAuCl4, with a slope of 55.8. After doping N element such as CD1N with a slope of 249, the CD1N surface electrons were enhanced; the CD1N surface electrons accelerated the redox electron transfer so that the gold nanoreaction was greatly enhanced to produce more AuNPs which caused the SERS intensity to increase (Figure 5).

Table 1

Comparing of the catalysis by SERS method .

System	Linear Range	Regress Equation	Coefficient
CD0N	1.0–60 μg/mL	ΔI = 55.8x + 30	0.9898
CD0.5N	6.0–20 μg/mL	ΔI = 89.2x + 130	0.9869
CD1N	0.79–8 μg/mL	ΔI = 249.0x − 8.6	0.993
CD2N	0.79–10 μg/mL	ΔI = 197.4x + 13	0.9633

Figure 4

Relationship between the SERS intensity and CD catalyst concentration. 4.2 μmol/L HAuCl4 + 2.5 mmol/L H2O2 + CD0N–2N + 0.33 μmol/L VB4R. (a) CD0N; (b) CD0.5N; (c) CD1N; (d) CD2N.

Figure 5

Enhancement of the doped N element.

3.5. Optimization of Analytical Conditions

The effect of reagent concentration such as HAuCl4, H2O2, CD1N, BaCl2 and VB4R, reaction temperature and time were optimized, respectively (Figure 6). When HAuCl4 is 4.2 μmol/L, most AuNPs formed in analytical systems with large ΔI. With increasing H2O2, the ΔI increased due to the formed AuNPs increasing, and a 2.5 mmol/L H2O2 gives the largest ΔI. CD1N is the catalyst of the AuNP reaction, when the catalyst concentration increased, the ΔI enhanced, a 5 μg/mL CD1N gives the largest ΔI. BaCl2 can inhibit the CD catalysis, when its concentration increased the ΔI was enhanced due to the blank decreasing, a 53 μmol/L BaCl2 gives the largest ΔI. VB4R is a sensitive molecular probe; when the concentration increased the ΔI enhanced due to more VB4R adsorption on the AuNP surface, a 0.33 μmoL/L VB4R gives biggest ΔI. Reaction temperature and time were considered; 50 °C for 20 min gives biggest ΔI. Thus, a 4.2 μmol/L HAuCl4, 2.5 mmol/L H2O2, 5 μg/mL CD1N, 53 μmol/L BaCl2 and 0.33 μmoL/L VB4R, and a reaction temperature of 50 °C for 20 min was selected in this SERS method.

Figure 6

Effect of reagent concentration, reaction temperature and time. (a): HAuCl4 + 2.5 mmol/L H2O2 + 5 μg/mL CD1N + 0.67 μmol/L Na2SO4 + 53 μmol/L BaCl2 + 0.33 μmol/L VB4R; (b): 4.2 μmol/L HAuCl4 + 0.33 μmol/L VB4R + 5 μg/mL CD1N + 0.67 μmol/L Na2SO4 + 53 μmol/LBaCl2; (c): 4.2 μmol/L HAuCl4 + 2.5 mmol/L H2O2 + CD1N + 0.67 μmol/L Na2SO4 + 53 μmol/L BaCl2 + 0.33 μmol/L VB4R; (d): 4.2 μmol/L HAuCl4 + 2.5 mmol/L H2O2 + 5 μg/mL CD1N + 0.67 μmol/L Na2SO4 + BaCl2 + 0.33 μmol/L VB4R; (e): 4.2 μmol/L HAuCl4 + 2.5 mmol/L H2O2 + 5 μg/mL CD1N + 0.67 μmol/L Na2SO4 + 53 μmol/L BaCl2 + VB4R; (f): Reaction temperature, 4.2 μmol/L HAuCl4 + 2.5 mmol/L H2O2 + 5 μg/mL CD1N + 0.67 μmol/L Na2SO4 + 53 μmol/L BaCl2 + 0.33 μmoL/L VB4R; (g): Reaction time, 4.2 μmol/L HAuCl4 + 2.5 mmol/L H2O2 + 5 μg/mL CD1N + 0.67 μmol/L Na2SO4 + 53 μmol/L BaCl2 + 0.33 μmoL/L VB4R.

3.6. Performance Curve

The working curve of the system was drawn according to the experimental method. In the four systems (Table 2, Figure 7), the CD1N was most sensitive, with a linear range (LR) of 0.02–1.7 μmol/L and a detection limit (DL) of 0.007 μmol/L, and was selected for detection of sulfate. Comparison of the reported methods for detection of sulfate [25,26,27,28,29,30] showed the SERS method was more sensitive. The C60 catalytic SERS method was used to detect sulfate, but the preparation of C60 is complex, the C60 nanosol is unstable [25], and the CD1N analytical system overcomes the disadvantages.

Table 2

Analytical features of CD-catalytic SERS determination of sulfate.

CD	Linear Equation	Coefficient	LR (μmol/L)	DL(μmol/L)
CD0N	ΔI = 66.9C + 20.4	0.9283	1.0–6.0	0.50
CD0.5N	ΔI = 166.4C + 48.8	0.9463	0.5–2.31	0.20
CD1N	ΔI = 348.8C + 18.0	0.9384	0.02–1.7	0.007
CD2N	ΔI = 268.6C−73.9	0.9403	0.06–2.66	0.02

Figure 7

Working curves for CD catalytic-SERS method. 4.2 μmol/L HAuCl4 + 2.5 mmol/L H2O2 + 5 μg/mL CD + 53 μmol/L BaCl2 + 0.33 μmol/L VB4R. (a): CD0N; (b): CD0.5N; (c): CD1N; (d): CD2N.

3.7. Influence of Foreign Substances

The influence of foreign substance on the determination of 0.66 µmol/L SO42− was investigated according to the experimental method. When the relative error is within 10%, results show that 33 µmol/L Na+, Zn2+, Ca2+, ethanol, Pb+2, NH4+, K+, SO32−, Bi3+ and Cu+2, 26.4 µmol/L HCO3−, Mg2+, 16.5 µmol/L ethylene glycol, 6.6 µmol/L Cr6+, Fe3+, NO2− and glycolic acid did not interfer with the SERS detection. Table 3 shows that the SERS quantitative analysis method has good selectivity.

Table 3

Effect of interfering substances on the SERS detection of 0.66 µmol/L SO42−.

Foreign Substance	Tolerance Concentration (µmol/L)	Relative Error (%)	Foreign Substance	Tolerance Concentration (µmol/L)	Relative Error (%)
Na+	33	5.0	Cu2+	33	7.6
Zn2+	33	6.4	HCO3−	26.4	8.6
Ca2+	33	−6.7	Mg2+	26.4	6.0
ethanol	33	−5.6	ethylene glycol	16.5	5.8
Pb2+	33	7.0	Cr6+	6.6	−6.0
NH4+	33	3.9	Fe3+	6.6	−4.5
K+	33	6.0	NO2−	6.6	6.2
SO32−	33	−7.9	glycolic acid	6.6	5.0
Bi3+	33	6.4

3.8. Analysis of Samples

The water samples including tap water, Rong lake water and Shan lake water were taken into sample bottles. The lake water was filtered with filter paper, and then a 2.0 mL water sample was removed in a centrifuge tube. Three beer samples were purchased supermarkets. Samples were centrifuged at 7000 r/min for 10 min, to obtain a sample solution. The sulfate content was determined according to the SERS detection procedure. The SERS results were in agreement with that of ion chromatography (IC), the relative standard deviation was in the range of 0.90–4.77% and the recovery was between 92.3% and 105% (Table 4).

Table 4

Analytical results of sulfate in water samples.

Sample	Single Value (μmol/L)	Average (μmol/L)	Added (μmol/)	Found (μmol/L)	Recovery (%)	RSD (%)	Content (μmol/L)	IC Results (μmol/L)
Running water	0.39, 0.41, 0.38, 0.40, 0.43	0.40	0.13	0.52	92.3	4.77	0.40	0.38
Ronng lake water	1.12, 1.17, 1.11, 1.17, 1.17	1.15	0.13	1.274	95	2.6	1.15	1.22
Shan lake water	0.70, 0.71, 0.71, 0.72, 0.71	0.71	0.13	0.839	99.2	0.90	0.71	0.68
Beer 1	1.22, 1.26, 1.30, 1.28, 1.32	1.28	0.20	1.47	95	3.0	1.28	1.20
Beer 2	1.30, 1.35, 1.38, 1.39, 1.33	1.35	0.20	1.56	105	2.7	1.35	1.38
Beer 3	1.39, 1.30, 1.39, 1.32, 1.25	1.33	0.20	1.52	95	4.3	1.33	1.28

4. Conclusions

Highly catalytic CDN was prepared by a hydrothermal procedure, and it was used to catalyze the reduction of chlorauric acid by H2O2 to produce AuNP sol substrate with high SERS activity. Ba(II) ions can combined with CDN to inhibit the catalysis of CDN. Upon addition of sulfate ions, stable barium sulfate precipitates formed, and CDN was released, which causes CDN catalysis to be activated and the SERS signal to be enhanced. Based on this principle, a new, simple and selective SERS quantitative analysis method was established for the detection of trace sulfate.