In order to determine the concentration of melamine, nitrogen-doped carbon dots (NCDs) were synthesized in one step as a fluorescent probe. Uric acid and diethylenetriamine were used as carbon source and nitrogen source, respectively. The experimental results showed that the fluorescence of NCDs can be quenched by mercury ions (Hg2+). Due to the strong coordination affinity between the carbon-nitrogen heterocyclic of melamine and Hg2+, part of Hg2+ coordinated with melamine when melamine was mixed with Hg2+. Then, the fluorescence of the added NCDs was quenched by the remaining Hg2+. Therefore, the concentration of melamine could be determined. The results show that the method has high sensitivity in wide measuring range that the linear ranges are 50–400
Melamine (C3H6N6), an organic compound with triazine nitrogen-containing heterocycle, has been widely used in plastics and coating industry [
At present, there are a number of detection methods for melamine, such as enzyme-linked immune sorbent assay (ELISA) [
Fluorescence spectroscopy (FS) can reflect the characteristics of the target molecule. It can be used for qualitative or quantitative detection of the target molecule. Fluorescence spectroscopy has been applied to the precise detection of many complex mixture systems because it has the advantages of good selectivity, high sensitivity, simple operation, and small sample volume. Up to now, in order to improve the sensitivity of detecting the concentration of melamine, some nanomaterials had been used as fluorescent probes for the quantitative detection of melamine. The utilization of gold nanoparticles and CdTe quantum dots to detect melamine had been reported [
Carbon dots were widely used in heavy metal ions, photocatalysis, LED, and sensing because of its simple synthesis, low cost, low toxicity, and high specificity [
In this study, a method for indirectly detecting the concentration of melamine using the fluorescence intensity of the system was established. The prepared NCDs are used as fluorescent probes. By the mechanism of the strong coordination affinity between melamine and Hg2+, the fluorescence of NCDs can be quenched by Hg2+. The melamine-Hg2+-NCDs system is established by researching the interaction between these three kinds of materials. The relationship between the fluorescence intensity of the system and the melamine concentration was studied. Additionally, the method has the advantages of simple operation, short detection time, and high sensitivity in comparison with the methods employing other fluorescent probes.
Melamine, uric acid (UA), diethylenetriamine (DETA), Hg (NO3)2·H2O, urea, biuret, tryptophan (Trp), phenylalanine (Phe), tyrosine (Tyr), valine (Val), alanine (Ala), and glycine (Gly) were obtained from Sinopharm Chemical Reagent Co., Ltd. (Shanghai, China).
The fluorescence measurements were conducted using the FLS920P fluorescence spectrometer produced by Edinburg, England. Absorption spectra were collected on the Shimadzu UV2600 absorption spectrometer.
The NCDs was prepared as previously reported papers [
NCDs (500
Aqueous solution of melamine with a concentration range of 0–10 mg/L was configured. 1.2 mM mercury nitrate (100
The absorption spectrum, fluorescence excitation spectrum, and emission spectrum of NCDs were measured, and the results are shown in Figure
The absorption (black line), fluorescence excitation spectrum (blue line), and emission spectrum (red line) of NCDs. Insets show photographs of aqueous solution of NCDs with the exposure of visible (left) and ultraviolet (right) light (365 nm).
The fluorescence emission spectra of the mixed solutions of NCDs and mercury nitrate with different concentrations were obtained under the excitation wavelength of 370 nm (Figure
(a) The fluorescence emission spectra of the mixed solutions of mercury nitrate and NCDs with different concentrations (
The absorption spectra of mixed solutions with different concentrations of mercury nitrate and NCDs were measured (Figure
The fluorescence emission spectra of NCDs, NCDs-melamine mixed solution, melamine-Hg2+-NCDs mixed solution, and NCDs-Hg2+ mixed solution were measured (
The fluorescence emission spectra of NCDs (blue line), NCDs-melamine mixed solution (gray line), melamine-Hg2+-NCDs mixed solution (red line), and NCDs-Hg2+ mixed solution (black line) (
The process of the melamine-Hg2+-NCDs system is shown in Scheme
The process of the melamine-Hg2+-NCDs system.
The order of addition of melamine, Hg2+, and NCDs had a greater impact on the experiment. The fluorescence emission spectra of NCDs-Hg2+ mixed solution, melamine-Hg2+-NCDs mixed solution, melamine-NCDs-Hg2+ mixed solution, and NCDs-Hg2+-melamine mixed solution are shown in Figure
The fluorescence emission spectra of NCDs-Hg2+ mixed solution (black line), melamine-Hg2+-NCDs mixed solution (red line), melamine-NCDs-Hg2+ mixed solution (blue line), and NCDs-Hg2+-melamine mixed solution (gray line) (
It can be seen in Figure
The concentration of NCDs had a greater impact on the detection of melamine. Comparison of the fluorescence peak intensity (normalized) of the melamine-Hg2+-NCDs system is shown in Figure
Comparison of the fluorescence peak intensity (normalized) of the system when different concentrations of Hg2+ were mixed with melamine and then added with different concentrations of NCDs.
Due to the combination of Hg2+ and melamine, the fluorescence intensity of the system increases with the increase of melamine concentration. However, Hg2+ is not completely bound by melamine when the concentration of melamine is 0–10 mg/L due to the high concentration of Hg2+ shown as the black line. There is a low sensitivity under this condition. As shown as the red line, the decrease of Hg2+ concentration makes it to only need a small amount of melamine to cooperate with it. When the melamine concentration is 0–4 mg/L, the fluorescence intensity of the system gradually increases and the sensitivity is higher when the melamine concentration increases. The fluorescence intensity of the system has become flat when the concentration is 4–10 mg/L. This condition is used in this article. As shown as the blue line and the gray line, the low concentration of melamine (0–1 mg/L) is detected with better sensitivity when the concentration of Hg2+ is lowered again. But, when the concentration of melamine is high (1–4 mg/L), the detection sensitivity is reduced because the amount of NCDs are also small at this time and the fluorescence intensity is weak. Therefore, in order to detect the high sensitivity of melamine, NCDs (diluted 5 times) and Hg2+ (1.2 mM) are selected as experimental conditions.
The concentration of melamine was detected by using the melamine-Hg2+-NCDs system. The fluorescence emission spectrum of the system is shown in Figure
(a) The fluorescence emission spectrum of the system (
The inset in Figure
It can be seen from Figures
To verify the selectivity of the melamine-Hg2+-NCDs system, we investigate the fluorescence response of the system in the presence of different interfering substances. We have selected several common amino group containing molecules which includes urea, biuret, tryptophan (Trp), phenylalanine (Phe), tyrosine (Tyr), valine (Val), alanine (Ala), and glycine (Gly). The effect of amino group-containing molecules on the system is shown in Figure
The interference experiment of amino group-containing molecules on the system, including urea, biuret, Trp, Phe, Tyr, Val, Ala, and Gly. The concentration of all samples was 0.4 mM.
For the convenience of comparison, we set the same concentration of these samples to 0.4 mM. The black bars show the fluorescence peak intensity (normalized) of these samples mixed with NCDs. It can be seen that there is no obvious difference in the fluorescence intensity of these samples mixed with NCDs, which means that these samples have no significant influence on the NCDs. The blue bars are the fluorescence peak intensities of Hg2+ mixed with NCDs. The fluorescence of NCDs can be quenched by Hg2+. Since melamine can be combined with Hg2+, the fluorescence of NCDs is only quenched by a small part of the Hg2+ at the first of red bar. The fluorescence intensity of the system is relatively high. Then, these amino group-containing molecules samples replace the melamine in the melamine-Hg2+-NCDs system. The results are shown in the other red bars. The fluorescence intensity of other samples is close to the blue bars. It suggests that these amino-containing samples will not have a significant impact on the system. It is also proved that the mechanism of the system is the combination of the triazine nitrogen-containing heterocycle of melamine and Hg2+.
In order to prove the repeatability and quasidetermination of this method, the standard addition experiment was carried out. Table
Analysis of deionized water spiked with different amounts of melamine (
Sample | Concentration of melamine ( | Recovery (%) | RSD (%) | |
---|---|---|---|---|
Spiked | Measured | |||
1 | 250 | 265.02 | 106.01 | 4.5 |
2 | 1250 | 1161.52 | 92.92 | 2.14 |
3 | 2000 | 1986.9 | 99.36 | 1.77 |
In addition, this study is compared with other melamine detection methods. Table
Comparison of linear range, LOD, and detection time of different fluorescent probes for melamine detection.
Fluorescent probe | Linear range | LOD | Detection time (min) | Ref. |
---|---|---|---|---|
FRET between CDs and Au NPs | 50–500 nM | 36 nM | 35 | [ |
CDs-Hg2+ | 1–20 | 0.3 | 13 | [ |
NCDs-Fe3+ | 2–290 | 0.66 | 30 | [ |
Aptamer-modified Au NPs | 1.2–2.4 | 793 nM | 30 | [ |
NCDs-Hg2+ | 50–400 | 21.76 | 10 | This work |
In summary, a method for detecting the concentration of melamine based on changes in the fluorescence intensity of NCDs was explored. By using the mechanism of quench and interaction inside the melamine-Hg2+-NCDs system, the detection method was established. Then, the relationship between the concentration of melamine and the fluorescence intensity of the system was obtained. The results showed that the manufacturing process of NCDs is straightforward, the detection is convenient and quick, and it has high sensitivity and large measurement range. The two linear ranges are 50–400
The data used to support the findings of this study are available from the corresponding author upon request.
The authors declare that they have no conflicts of interest.
The authors greatly appreciate the National Key R&D Project (2018YFC1604204-3), National Food Science and Engineering First-Class Discipline Construction Project (JUFSTR20180302), National Natural Science Foundation of China (61378037), and the Key Research and Development Program of Jiangsu Province (BE2020756) for financial support.