Nitrosamines are formed from nitrites and are known carcinogens that have toxicological effects on humans [1
]. Nitrosamines are found in the natural environment, and also in many food products [2
]. Hence, it of is of great significance and a necessity to determine the presence of nitrites and to provide sensitive and selective assays for their early detection. Numerous methods for detecting and determining nitrite have been reported using ultraviolet-visible (UV-vis) spectrophotometric [3
], chemiluminescent (CL) [4
], electrochemical [7
] and spectrofluorimetric methods [9
]. Ultraviolet spectrophotometry is the most commonly used method for the detection of nitrite. Diazonium compounds can be formed during the reaction of a primary aromatic amine with nitrous acid. The absorbance of the product after this reaction is proportional to nitrite concentration. However, this method is greatly limited because of its poor sensitivity and interferences from other participating ions [11
]. Although, nitrite is electroactive at platinum, gold, copper, diamond, and transition metal oxide electrodes [12
], electrochemical methods are not preferred for trace analysis due to their poor selectivity. Nitrites can be detected in gaseous or aqueous phase systems using CL methods. In the former case, nitrites are reduced to nitrogen oxide by a reductant and NO2
* is formed by the subsequent reaction of nitrogen oxide with ozone, which is electronically excited and falls to a ground state with light emission [15
]. For aqueous phase detection, nitrites react with H2
forming the intermediate peroxynitrous acid (ONOOH) which is further treated with alkali to form peroxynitrite. Since the decomposition of peroxynitrite is associated with CL emission, the flow-injection technique was developed for nitrite determination [16
]. The intensity of CL emission is related to environmental factors and any change in these factors can adversely impact the stability and reproducibility of CL results [17
The underlying principle on which spectrofluorimetric methods are based, utilizes the variations recorded in fluorescence intensity during the reaction between fluorescent probes and nitrite. This technique has been more commonly used to detect nitrite concentration because it provides high sensitivity, good selectivity, excellent limits of detection and comprehensive suitability. Several such probes have been developed by utilizing the chemical specificity of nitrite ion towards diazotization or nitrosation [17
]. One of the probes, resorcinol, can react with nitrite to give nitroso derivatives, which cause a fluorescence intensity variation [10
]. In these studies, the recorded changes in fluorescence intensity have been used for quantitative analysis of nitrite. Axelrod et al., have demonstrated an increase in fluorescence intensity during the reaction of 5-aminofluorescein with nitrite [18
], however, it was found that the stability of such probes was pretty weak. This highlights the need for developing a stable, selective and robust probe.
Carbon quantum dots (CQDs) are small nanoparticles (less than 10 nm diameter) [19
]. They were found to exhibit photoluminescent properties when first discovered accidentally by Xu et al. in 2004 [20
]. CQDs present high chemical stability, bio-compatibility, and excellent optical properties, as well as ease of surface modification [21
], and have already been widely applied in diverse fields, including cell imaging [23
], biochemical sensing [26
], and analysis [28
]. In solution, the fluorescence intensity of CQDs can be quenched by an electron donor or an electron acceptor molecule, indicating that CQDs themselves are good electron donors or acceptors. By using this property, CQDs can assist in identifying certain specific ions in solution [29
]. At present, very few published studies are available in the literature on the use of CQDs to detect nitrite. CQDs have chemiluminescent properties in the presence of ONOOH, formed by the reaction between H2
. Lin et al., developed an injection method for its detection, however, this requires special pumps and added hydrogen peroxide reagents [16
]. Nitrogen-doped carbon quantum dots (N-CQDs) were prepared via carbonization of citric acid in the presence of triethylenetetramine as a nitrogen source, and were introduced as a novel fluorescence probe to determine NO3−
via their quenching behavior [31
]. However, due to poor specificity, the method could not directly distinguish between NO2−
In the present work, N-CQDs have been applied for building a direct, fast and simpler nitrite detection method.
In this research, we present a fluorescent assay for nitrite detection by using N-CQDs as fluorescence probes. N-CQDs were prepared by hydrothermal treatment of citric acid as the carbon source and EDA as the nitrogen source. At a pH of 2, the fluorescence of the N-CQDs can be selectively quenched by nitrite. A possible mechanism has been put forward whereby N-nitroso compounds can be formed in the reaction of amide group with nitrous acid, which result in fluorescence static quenching. Experimental results demonstrate that this proposed assay has robustness for the quantitative analysis of nitrite with high sensitivity, low cost and good selectivity. Furthermore, this method can also be applied for measuring nitrite in tap water samples.
3. Materials and Methods
Citric acid, sodium nitrite, hydrochloric acid, sodium hydroxide and ethylenediamine were bought from Aladdin Chemical Reagent Co. Ltd. (Shanghai, China). Metal salts (Na2CO3, Na2SO3, PbSO4, CuCl2·2H2O, KCl, NaCl, BaCl2, HgCl2, NaNO3, Co(NO3)2·6H2O, Ni(NO3)2·6H2O, Cr(NO3)3·9H2O, FeCl3, ZnCl2, Na3PO4, Na2HPO4, NaH2PO4, NH4F, KBr and KI) were purchased from YongDa Chemical Reagent Co. Ltd. (Tianjin, China). Ultrapure water prepared from a Milli-Q water purification system (Millipore, Billerica, MA, USA) was used throughout the experiments.
A FEI TF-20 instrument operating at 200 kV (FEI, Hillsboro, TX, USA) was used to obtain high resolution transmission microscopy (HRTEM) information. FTIR spectra were collected from 20 scans with a resolution of 4 cm−1 by a Magna-IR560 unit (Nicolet Co., Madison, WI, USA). UV-vis. spectroscopy was performed on a UV-2550 spectrophotometer (Shimadzu, Kyoto, Japan) though a quartz cell with a 1 cm optical path. An LS-55 fluorescence spectrometer (PerkinElmer, Waltham, MA, USA) recorded the fluorescence. X-ray photoelectron spectroscopy (XPS) data for the N-CQDs powder deposited on copper substrates were measured by an AXIS Ultra DLD spectrometer (Kratos, Manchester, UK) with a monochromatized Al Kα X-ray source (1486.6 eV) for determining the composition and chemical bonding configurations.
3.3. Preparation of N-CQDs
N-CQDs were prepared by hydrothermal treatment of citric acid and EDA. Citric acid (3 g) and EDA (3 mL) were mixed in a tetrafluoroethylene-lined autoclave (50 mL), and water was added until a final volume of 30 mL was reached. The resulting solution was then kept at 180 °C for 5 h. After cooling at room temperature, the mixture was dialyzed using 300 Da cut-off bag with ultrapure water for one day to remove by-products.
3.4. NO2− Determination
NO2− detection is performed using N-CQDs (50 μL, 1.5 mg mL−1) with 8 mL HCl-KCl buffer solution and different volumes of NO2− stock solution (0.001 M) were added into a 10 mL volumetric flask, and finally diluted with HCl-KCl buffer solution to 10 mL. After thorough mixing, the fluorescence spectra were recorded (equilibrated time 15 min). The NO2− selectivity is determined using 100 µL of a single metal ion stock solution (0.001 M) instead of NO2− in a similar way.
All the fluorescence detections were under the same conditions: the slit widths of the excitation and emission were both 10 nm, and the fluorescence spectra were recorded at an excitation wavelength of 370 nm with the emission recorded over the wavelength range of 370–600 nm. The fluorescence intensity of the maximum emission peak was used for quantitative and qualitative analysis.