A fluorescence detection method for pentachlorophenol based on natural nitrogen and sulfur co-doped carbon quantum dots

Abstract

The present invention discloses a fluorescence detection method for pentachlorophenol based on natural nitrogen and sulfur co-doped carbon quantum dots, belonging to the technical field of food safety and rapid detection. The fluorescence detection method for pentachlorophenol based on natural nitrogen and sulfur co-doped carbon quantum dots of the present invention uses crayfish shells as raw materials to prepare natural nitrogen and sulfur co-doped carbon quantum dots, and then mixes them with H2O2 and horseradish peroxidase solution to form a quenched carbon quantum dot system; the test samples containing pentachlorophenol and a series of standard solutions of pentachlorophenol are respectively added to the quenched carbon quantum dot system, cultured for a period of time, fluorescence data is collected using a fluorescence spectrometer, the fluorescence intensity ratio and the standard solution concentration are used as the quantitative relationship, a standard linear relationship diagram of pentachlorophenol is drawn, and the content of pentachlorophenol in the test sample is calculated. This detection method has high sensitivity, a wide detection range, good selectivity for pentachlorophenol, and can be widely used for the rapid detection of pentachlorophenol in food.

Description

Technical Field

[0001] The invention relates to a pentachlorophenol fluorescence detection method based on natural nitrogen and sulfur co-doped carbon quantum dots, belonging to the technical field of food safety and rapid detection. Background Art

[0002] Pentachlorophenol is widely used in industry and agriculture as a pesticide, preservative and mildew preventer. It is one of the most toxic chlorophenols. Animal toxicity studies and human clinical data show that long-term exposure to it is seriously harmful to human health. Pentachlorophenol is highly stable in the environment and can accumulate in organisms through the food chain, posing a serious threat to human health and aquatic ecosystems.

[0003] At present, many standard test methods, such as high performance liquid chromatography, gas chromatography-mass spectrometry, inductively coupled plasma mass spectrometry and liquid chromatography-mass spectrometry, have relatively accurate results, but these methods have cumbersome pre-treatment operations, complex and expensive equipment, and are time-consuming. Therefore, it is necessary to develop a simple, economical and portable fluorescence detection method.

[0004] Carbon quantum dots (CQDs) are a type of zero-dimensional carbon nanomaterial with a carbon skeleton and a spherical or quasi-spherical structure less than 10 nm in size. CQDs have excellent optical properties such as fluorescence, ultraviolet absorption, and photoluminescence. They also have excellent water solubility, biocompatibility, and low toxicity, as well as the advantages of a wide range of synthetic raw materials, low cost, and easy surface functionalization. They are often used as fluorescent probes for analytical detection and bioimaging research.

[0005] Since CQDs have surface defects, it is beneficial to modify the surface. A large number of experimental results show that element doping can give CQDs new properties and adjust the structural defects of CQDs. These new surface states suppress or eliminate the original oxidation state, resulting in a higher fluorescence quantum yield of the doped CQDs. Doped heteroatoms can also be embedded in the structure of CQDs to form new energy levels or adjust the initial band gap. For example, doping with nitrogen and sulfur is the most common way to change the fluorescence properties of CQDs. The performance of CQDs can be greatly improved by modifying the surface of CQDs.

[0006] On this basis, the present invention has developed and prepared natural nitrogen-sulfur co-doped carbon quantum dots, and constructed a switch-type fluorescent probe for the rapid detection of pentachlorophenol in crayfish meat and aquaculture water. Summary of the invention

[0007] In view of the shortcomings of the prior art, the present invention uses lobster shells to construct natural nitrogen-sulfur co-doped carbon quantum dots as fluorescent probes, which are combined with horseradish peroxidase solution and H2O2 solution to form a switch-type fluorescent probe that can be used for the rapid detection of pentachlorophenol. The method is simple, convenient, stable and highly sensitive.

[0008] The object of the present invention is to provide a method for detecting pentachlorophenol fluorescence based on natural nitrogen and sulfur co-doped carbon quantum dots, the method comprising the following steps:

[0009] (1) Using crayfish shells as raw materials, natural nitrogen-sulfur co-doped carbon quantum dots were prepared by hydrothermal method;

[0010] (2) adding the natural nitrogen-sulfur co-doped carbon quantum dots prepared in step (1) into a buffer solution to prepare a carbon quantum dot dispersion; and then mixing the dispersion with H2O2 and horseradish peroxidase solution to form a quenched carbon quantum dot system;

[0011] (3) adding the sample to be tested and a series of standard solutions of pentachlorophenol to the quenched carbon quantum dot system obtained in the above step (2), respectively, incubating for a period of time, collecting the fluorescence intensity of the standard solution by a fluorescence spectrometer, recorded as F, and taking the fluorescence intensity of the blank solution without pentachlorophenol as F0; constructing a quantitative relationship model with the fluorescence intensity ratio F / F0 and the concentration of the standard solution of pentachlorophenol as the quantitative relationship; then calculating the content of pentachlorophenol in the sample to be tested based on the ratio of the fluorescence intensity of the sample to be tested to F0.

[0012] In one embodiment, the buffer solution in step (2) is a phosphate buffer solution; the pH value of the phosphate buffer solution is 5.0-6.0, and the concentration is 0.02-0.05 mol / L.

[0013] In one embodiment, the mass ratio of the natural nitrogen-sulfur co-doped carbon quantum dots, H2O2 and horseradish peroxidase in step (2) is 400:1-4:10000-150000.

[0014] In one embodiment, the molar ratio of the horseradish peroxidase to H2O2 in step (2) is 1-4:20000-40000.

[0015] In one embodiment, the concentration of the carbon quantum dot dispersion in step (2) is 10 to 15 μg / mL.

[0016] In one embodiment, the concentration of the horseradish peroxidase solution in step (2) is 10-40 nmol / L; the concentration of the H2O2 solution is 200-400 μmol / L.

[0017] In one embodiment, the culturing in step (3) is carried out in an incubator at a temperature of 30-37° C. for a period of 20-40 min.

[0018] In one embodiment, the concentration of pentachlorophenol in the pentachlorophenol-containing sample to be tested in step (3) is 0-300 μmol / L.

[0019] In one embodiment, the detection conditions of the fluorescence spectrometer in step (3) are as follows: the excitation wavelength is 360 nm, the starting wavelength is 380.0 nm, the ending wavelength is 650.0 nm, and the slit ratio is 5:5 nm.

[0020] In one embodiment, the regression equation of the standard linear relationship of pentachlorophenol is y=0.0028x+1.0121; wherein y is the ratio of fluorescence absorption values ​​at different pentachlorophenol concentrations, and x is the corresponding pentachlorophenol concentration.

[0021] In one embodiment, the preparation of natural nitrogen-sulfur co-doped carbon quantum dots in step (1) specifically comprises the following steps:

[0022] 1) Rinse the crayfish shells, dry them, crush them and sieve them;

[0023] 2) treating the sieved crayfish shell powder in step 1) with acid, then rinsing it to neutrality, and drying it;

[0024] 3) using the dried crayfish shell powder in step 2) as a raw material and mixing it with water in a certain proportion to obtain a precursor solution;

[0025] 4) transferring the precursor solution obtained in step 3) to a polytetrafluoroethylene high-pressure reactor for hydrothermal reaction;

[0026] 5) filtering the product after the hydrothermal reaction in step 4), centrifuging, taking the supernatant for dialysis to obtain a carbon quantum dot solution, freezing and drying to obtain natural nitrogen-sulfur co-doped carbon quantum dots.

[0027] In one embodiment, the drying temperature in step 1) is 60-80° C.; and the sieving is through an 80-mesh sieve.

[0028] In one embodiment, the acid treatment in step 2) is to treat the lobster shells with ultrasound assistance in a hydrochloric acid solution for 1.5-2.5 hours; the concentration of the hydrochloric acid solution is 0.2-1.0 mol / L.

[0029] In one embodiment, the drying temperature in step 2) is 60-80°C.

[0030] In one embodiment, the mass ratio of the raw material to water in step 3) is 1:20-30.

[0031] In one embodiment, the hydrothermal reaction temperature in step 4) is 180-220°C.

[0032] In one embodiment, the hydrothermal reaction time in step 4) is 4-8 hours.

[0033] In one embodiment, the filtration in step 5) is performed using a 0.22 μm hydrophilic filter membrane.

[0034] In one embodiment, the centrifugation condition in step 5) is 10000-15000 r / min for 10-20 min.

[0035] In one embodiment, the dialysis in step 5) is performed using a dialysis bag with a molecular weight cutoff of 500-1000 Da for 48-72 hours.

[0036] Another object of the present invention is to provide an application of the above-mentioned pentachlorophenol fluorescence detection method based on natural nitrogen and sulfur co-doped carbon quantum dots in the field of food safety and rapid detection technology.

[0037] In one embodiment, the application includes application in the detection of water and food containing pentachlorophenol.

[0038] Beneficial Effects of the Invention

[0039] (1) The present invention uses nitrogen-sulfur co-doped carbon quantum dots prepared from lobster shells as fluorescent probes. The preparation method is simple, the raw materials are widely available, the price is low, and large-scale production is possible. The prepared carbon quantum dots are luminescent and have good stability. At the same time, the quantum yield of the carbon quantum dots is significantly improved by doping with nitrogen and sulfur elements.

[0040] (2) The pentachlorophenol detection method established by the present invention avoids cumbersome and complicated pre-treatment steps and equipment, has a wide linear range (0-300 μmol / L), a low detection limit (0.23 μmol / L) and good selectivity, and has also achieved satisfactory results in the detection of actual samples. BRIEF DESCRIPTION OF THE DRAWINGS

[0041] Figure 1 This is an electron transmission electron micrograph of nitrogen-sulfur co-doped carbon quantum dots prepared in Example 1 of the present invention;

[0042] Figure 2 This is a fluorescence emission spectrum of nitrogen-sulfur co-doped carbon quantum dots prepared in Example 1 of the present invention;

[0043] Figure 3 The ultraviolet-visible absorption spectrum, excitation and emission spectrum of the nitrogen-sulfur co-doped carbon quantum dots prepared in Example 1 of the present invention;

[0044] Figure 4 This is an infrared spectrum of nitrogen-sulfur co-doped carbon quantum dots prepared in Example 1 of the present invention;

[0045] Figure 5 This is an X-ray photoelectron spectrum of nitrogen-sulfur co-doped carbon quantum dots prepared in Example 1 of the present invention;

[0046] Figure 6 The fluorescence spectra of the carbon quantum dot system quenched by H2O2 in Example 2 of the present invention after adding pentachlorophenol at different concentrations;

[0047] Figure 7 This is a linear relationship diagram of the carbon quantum dot system detecting pentachlorophenol in Example 2 of the present invention. DETAILED DESCRIPTION

[0048] The preferred embodiments of the present invention are described below. It should be understood that the embodiments are for better explaining the present invention and are not used to limit the present invention.

[0049] Example 1 Synthesis of Carbon Quantum Dots

[0050] A method for preparing natural nitrogen-sulfur co-doped carbon quantum dots comprises the following steps:

[0051] (1) The crayfish shells were first rinsed with tap water and then washed with deionized water; the cleaned crayfish shells were dried at 70°C overnight; the dried samples were ground into powder and passed through an 80-mesh sieve;

[0052] (2) treating the dried crayfish shell powder obtained in step (1) with 0.2 mol / L hydrochloric acid under ultrasound assistance for 2 h, washing with deionized water until neutral, and drying at 70° C.;

[0053] (3) using the lobster shell powder obtained in step (2) as a carbon source, preparing a solution with a carbon source to water mass ratio of 1:25, and mixing to obtain a precursor solution;

[0054] (4) transferring the precursor solution obtained in step (3) into a polytetrafluoroethylene autoclave and reacting at 200° C. for 6 h;

[0055] (5) filtering the reaction product obtained in step (4) with a 0.22 μm hydrophilic filter membrane, centrifuging at 10,000 r / min for 10 min, and then dialyzing with a dialysis bag with a molecular weight cutoff of 1,000 Da for 48 h to obtain a carbon quantum dot solution;

[0056] (6) freeze-drying the carbon quantum dot solution obtained in step (5) to obtain natural nitrogen-sulfur co-doped carbon quantum dots.

[0057] Performance Characterization of Carbon Quantum Dots

[0058] The performance characterization test was carried out on the natural nitrogen-sulfur co-doped carbon quantum dots prepared in Example 1.

[0059] The fluorescence quantum yield of the natural nitrogen-sulfur co-doped carbon quantum dots prepared in Example 1 is 18.6% (the quantum yield of quinine sulfate under the same conditions is used as a reference); the electron transmission electron microscopy image of the nitrogen-sulfur co-doped carbon quantum dots is as follows: Figure 1 .

[0060] The fluorescence spectrum of natural nitrogen and sulfur co-doped carbon quantum dots is shown in Figure 2 As shown in the figure, it can be seen that carbon quantum dots have emission characteristics that are dependent on the excitation wavelength.

[0061] The UV-visible absorption spectrum, excitation and emission spectra of natural nitrogen-sulfur co-doped carbon quantum dots are shown in the figure below. Figure 3 As shown in the figure, it can be seen that carbon quantum dots have strong absorption in the ultraviolet visible light region, the best excitation wavelength is 360nm, and the best emission wavelength is 440nm.

[0062] The infrared spectrum of natural nitrogen and sulfur co-doped carbon quantum dots is shown in Figure 2. Figure 4 As shown in the figure, it can be seen that there are functional groups such as hydroxyl, carboxyl, and amino groups on the surface of carbon quantum dots.

[0063] X-ray photoelectron spectra of natural nitrogen-sulfur co-doped carbon quantum dots Figure 5 As shown in the figure, it can be seen that carbon quantum dots are mainly composed of carbon, oxygen, nitrogen, sulfur and other elements, indicating that nitrogen and sulfur are successfully doped into carbon quantum dots.

[0064] Example 2

[0065] A method for constructing a switch-type fluorescent probe for detecting pentachlorophenol based on natural nitrogen-sulfur co-doped carbon quantum dots prepared in Example 1, specifically comprising the following steps:

[0066] (1) 10 mg of freeze-dried natural nitrogen-sulfur co-doped carbon quantum dots was added to 1000 mL of pH 5.0 phosphate buffer (0.02 mol / L) to form a 10 μg / L carbon quantum dot dispersion;

[0067] (2) 100 μL of horseradish peroxidase solution (20 nmol / L) and 100 μL of H2O2 solution (300 μmol / L) were mixed with 800 μL of the carbon quantum dot dispersion obtained in step (1), and the mixture was placed in a 30°C incubator for reaction for 30 min;

[0068] (3) mixing the system after the reaction in step (2) with 100 μL of a sample containing pentachlorophenol, and placing the mixture in a 30° C. incubator for 30 min;

[0069] (4) A quartz cuvette is filled with the mixed solution after the reaction in step (3) and placed in an RF-6000 fluorescence spectrometer to record its fluorescence spectrum.

[0070] Pentachlorophenol standard curve establishment

[0071] A series of pentachlorophenol standard sample solutions (0-300 μmol / L) were prepared, and the fluorescence intensity of each sample was measured according to the method of Example 2 to draw a standard curve.

[0072] The fluorescence intensity ratio F / F0 was used to evaluate the recovery efficiency of pentachlorophenol on the H2O2 / horseradish peroxidase quenched carbon quantum dot system, where F0 and F represent the fluorescence intensity of the H2O2 / horseradish peroxidase quenched carbon quantum dot system in the absence and presence of pentachlorophenol.

[0073] The fluorescence spectrum of the carbon quantum dot system quenched by H2O2 after adding pentachlorophenol is shown in the figure below. Figure 6 As shown, it can be seen from the figure that pentachlorophenol can gradually restore the fluorescence intensity of carbon quantum dots.

[0074] The linear relationship diagram of the carbon quantum dot system detecting pentachlorophenol is shown in Figure 7 As shown in Figure 2, the fluorescence changes showed a significant linear correlation (R 2 =0.9981); the regression equation is y=0.0028x+1.0121, and the detection limit (LOD) is 0.23 μmol / L (3S / K, S is the standard deviation of the blank sample, and K is the slope of the linear calibration curve).

[0075] Example 3 Interference effects of common ions and phenolic compounds on pentachlorophenol detection

[0076] 100 μL of pentachlorophenol standard solution (200 μmol / L) was added to the detection system of Example 2, and then 100 μL of different common ions and phenolic compounds were added respectively; then placed in RF-6000 fluorescence spectrometer to record its fluorescence spectrum. The relative fluorescence intensity ratio (I-I0) / I0 was used to evaluate the interference effect of coexisting substances on pentachlorophenol detection. Wherein I0 represents the fluorescence intensity of the detection system in the absence of coexisting substances (i.e., the fluorescence intensity of 100 μL of pentachlorophenol standard solution (200 μmol / L)); I represents the fluorescence intensity of the detection system in the presence of coexisting substances (i.e., the fluorescence intensity of 100 μL of pentachlorophenol standard solution (200 μmol / L) coexisting with the substances in Table 1). The test results are shown in Table 1:

[0077] Table 1

[0078]

[0079] As can be seen from the table, the relative error of the interference effect of common ions and phenolic compounds on the detection of pentachlorophenol is between 0.67% and 5.24%, which is considered acceptable. The results show that common metal ions and phenolic compounds have no obvious interference with the detection of pentachlorophenol.

[0080] Example 4 Determination of pentachlorophenol in crayfish meat samples and aquaculture water samples

[0081] 1. Preparation of crayfish meat sample solution:

[0082] Take 2g of crushed crayfish meat sample, add 6mL of 5% triethylamine acetonitrile aqueous solution, vortex mix for 1min, ultrasonic extract for 5min, and collect the supernatant by centrifugation; repeat 3 times and combine the supernatants; remove the organic solvent from the supernatant, and then pass it through a 0.22μm hydrophilic membrane to obtain the crayfish meat sample liquid to be tested.

[0083] 2. Preparation of crayfish culture water sample solution

[0084] Take 10 mL of water sample and centrifuge it at 10000 rpm for 10 min. Take the supernatant and pass it through a 0.22 μm hydrophilic membrane to obtain the water sample to be tested.

[0085] The crayfish meat samples and water samples prepared in 1 and 2 were mixed with standard pentachlorophenol solutions (0, 50, 100 and 150 μmol / L) to prepare spiked samples; then the content of pentachlorophenol in the water samples and crayfish meat samples was detected by the detection method of Example 2. All measurements were repeated three times. The test results are shown in Table 1. The recovery rate of pentachlorophenol in crayfish meat samples and aquaculture water samples was between 95.62% and 103.22%, indicating that the method has good accuracy and can be used for the detection of actual samples.

[0086] Table 2

[0087]

[0088] Example 5 Optimization of the mass ratio of carbon source to water

[0089] The mass ratios of the carbon source to water in step (3) of Example 1 were adjusted to 1:20, 1:25, and 1:30, respectively, and the rest were kept consistent with Example 1 to obtain natural nitrogen-sulfur co-doped carbon quantum dots.

[0090]

[0091] Example 6 Optimization of reaction temperature

[0092] The reaction temperatures in step (4) of Example 1 were adjusted to 180° C., 200° C., and 220° C., respectively, and the rest were kept consistent with Example 1 to obtain natural nitrogen-sulfur co-doped carbon quantum dots.

[0093]

[0094] Example 7 Optimization of reaction time

[0095] The reaction time in step (4) of Example 1 was adjusted to 4 h, 6 h, and 8 h, respectively, and the rest was kept consistent with Example 1 to obtain natural nitrogen-sulfur co-doped carbon quantum dots.

[0096]

[0097] Example 8 Optimization of horseradish peroxidase concentration

[0098] The degree of quenching of the carbon dot system by hydroxyl radicals is a very important prerequisite for constructing a fluorescent switch detection method for pentachlorophenol. The concentration of horseradish peroxidase is a key factor affecting the generation of hydroxyl radicals. Therefore, the peroxidase concentrations in Example 2 were adjusted to 5, 10, 20, 30, and 40 nmol / L, respectively, and the other steps (1) and (2) in Example 2 were consistent. The fluorescence intensity ratio F / F0 was used to evaluate the quenching efficiency of the carbon quantum dot system by horseradish peroxidase concentration, where F0 and F represent the fluorescence intensity of the carbon quantum dot system with and without the addition of horseradish peroxidase.

[0099]

[0100] From the data in the table, it can be seen that with the addition of horseradish peroxidase, the fluorescence intensity of the carbon dot system decreased significantly, and when the horseradish peroxidase concentration was greater than 20 nM, the fluorescence intensity remained unchanged. Therefore, in further experiments, a horseradish peroxidase concentration of 20-40 nM was selected.

[0101] Example 9 Optimization of hydrogen peroxide concentration

[0102] The concentration of hydrogen peroxide is another key factor affecting the generation of hydroxyl radicals. Therefore, the concentration of hydrogen peroxide in Example 2 was adjusted to 50, 100, 200, 300, and 400 μmol / L, respectively, and the other steps were consistent with steps (1) and (2) in Example 2. The fluorescence intensity ratio F / F0 was used to evaluate the quenching efficiency of the carbon quantum dot system by the concentration of hydrogen peroxide, where F0 and F represent the fluorescence intensity of the carbon quantum dot system with and without the addition of hydrogen peroxide.

[0103]

[0104] From the data in the table, it can be seen that with the addition of hydrogen peroxide, the fluorescence intensity of the carbon dot system decreases significantly. When the hydrogen peroxide concentration is greater than 300 μmol / L, the fluorescence intensity remains unchanged. Therefore, the optimal hydrogen peroxide concentration is 300 μmol / L.

Claims

1. A method for detecting pentachlorophenol fluorescence based on natural nitrogen and sulfur co-doped carbon quantum dots, characterized in that: The method comprises the following steps: (1) Using crayfish shells as raw materials, natural nitrogen-sulfur co-doped carbon quantum dots were prepared by hydrothermal method; The preparation of the natural nitrogen-sulfur co-doped carbon quantum dots specifically comprises the following steps: 1) Rinse, dry, crush and sieve the crayfish shells; 2) treating the sieved crayfish shell powder in step 1) with acid, then rinsing it to neutrality, and drying it; 3) Using the dried crayfish shell powder from step 2) as a raw material, mixing it with water in a certain proportion to obtain a precursor solution; the mass ratio of the raw material to water is 1:20-30; 4) transferring the precursor solution obtained in step 3) to a polytetrafluoroethylene high-pressure reactor for hydrothermal reaction; 5) filtering the product after the hydrothermal reaction in step 4), centrifuging, taking the supernatant for dialysis, obtaining a carbon quantum dot solution, freezing, and drying to obtain natural nitrogen-sulfur co-doped carbon quantum dots; the hydrothermal reaction temperature is 180-220°C and the time is 4-8 h; (2) adding the natural nitrogen-sulfur co-doped carbon quantum dots prepared in step (1) into a buffer solution to prepare a carbon quantum dot dispersion; then mixing the dispersion with H2O2 and horseradish peroxidase solution to form a quenched carbon quantum dot system; the mass ratio of the natural nitrogen-sulfur co-doped carbon quantum dots, H2O2 and horseradish peroxidase is 400:1-4:10000-150000; the molar ratio of horseradish peroxidase to H2O2 is 1-4:20000-40000; (3) The sample to be tested and a series of standard solutions of pentachlorophenol are added to the quenched carbon quantum dot system obtained in the above step (2) respectively, and incubated for a period of time. The fluorescence intensity of the standard solution is collected by a fluorescence spectrometer and recorded as F, and the fluorescence intensity of the blank solution without pentachlorophenol is F0; a quantitative relationship model is constructed based on the fluorescence intensity ratio F / F0 and the concentration of the standard solution of pentachlorophenol; then, the content of pentachlorophenol in the sample to be tested is calculated based on the ratio of the fluorescence intensity of the sample to be tested to F0.

2. The method according to claim 1, characterized in that The culture in step (3) is carried out in an incubator at a temperature of 30-37°C for 20-40 min.

3. The method according to claim 1, characterized in that The detection conditions of the fluorescence spectrometer described in step (3) are as follows: the excitation wavelength is 360 nm, the starting wavelength is 380.0 nm, the ending wavelength is 650.0 nm, and the slit ratio is 5:5 nm.

4. The method according to claim 1, characterized in that: The acid treatment in step 2) is specifically to treat the lobster shells in a hydrochloric acid solution with ultrasound assistance for 1.5-2.5 h; the concentration of the hydrochloric acid solution is 0.2-1.0 mol / L.

5. Application of the method according to any one of claims 1 to 4 in the field of food safety and rapid detection technology.

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