A method for detecting tetracycline antibiotics in milk based on deep ultraviolet emission carbon dots

Abstract

The present invention discloses a method for detecting tetracycline antibiotics in milk based on deep ultraviolet emission carbon dots, and belongs to the field of analysis and detection. The method for detecting tetracycline antibiotics in milk based on deep ultraviolet emission carbon dots comprises the following steps: (1) preparing a substance rich in hydroxyl, amino and carboxyl groups into a carbon dot solution; (2) performing a simple pretreatment after mixing milk with tetracycline, and then mixing with the carbon dots in (1), and obtaining the degree of fluorescence quenching of the mixed solution by fluorescence spectrum detection, and finally constructing a standard curve of tetracycline concentration and the degree of fluorescence intensity quenching of the mixed solution; (3) according to the standard curve of step (2), the purpose of detecting tetracycline antibiotics in milk samples is achieved. The present invention uses deep ultraviolet emission carbon dots as fluorescent probes for the first time to realize the quantitative detection of tetracycline antibiotics in milk, and has the advantages of high sensitivity, simple operation, safety, high energy utilization and low cost, and is suitable for routine analysis.

Description

Technical Field

[0001] The invention relates to a method for detecting tetracycline antibiotics in milk based on deep ultraviolet emission carbon dots, and belongs to the field of analysis and detection. Background Art

[0002] Antibiotics play an important role in the treatment and prevention of bacterial infections, among which tetracycline antibiotics (TCs) are one of the most commonly used antibiotics. Common tetracycline antibiotics include tetracycline (TC), oxytetracycline (OTC), chlortetracycline (CTC) and doxycycline (DOX). This type of antibiotic is widely used due to its broad-spectrum antibacterial effect. However, the abuse of tetracycline will cause tetracycline residues in dairy products and eventually accumulate in the human body, which will have a certain impact on human health.

[0003] Currently, there are many methods for detecting tetracycline in milk, such as high performance liquid chromatography (HPLC), immunoassays, capillary electrophoresis (CE), colorimetry and chemiluminescence method, etc. However, most of these methods require expensive instruments, complicated operations and long time consumption.

[0004] Fluorescence method has the advantages of high sensitivity, simple operation, low cost and time saving. Using fluorescent nanomaterials as fluorescent probes for detection greatly improves the sensitivity of detection, and the detection has the advantages of simple operation, short detection time and high sensitivity. At present, fluorescence spectroscopy technology has been widely used in the fields of environment, chemistry, medicine and food. Some scholars have also proposed the use of fluorescence spectroscopy technology to detect tetracycline antibiotics in milk. According to our investigation, the application of deep ultraviolet emission carbon dots in food detection has not been reported. Summary of the invention

[0005] [Technical issues]

[0006] At present, the main methods for detecting tetracycline antibiotics in milk are: high performance liquid chromatography, immunoassay, capillary electrophoresis, colorimetry and chemiluminescence. Most of these tests are time-consuming and involve complex sample processing, or require professional technicians and expensive costs. The use of deep ultraviolet emission carbon dots for food testing has not been reported.

[0007] [Technical solution]

[0008] In response to the above problems, the present invention provides a method for detecting tetracycline antibiotics in milk based on deep ultraviolet emission carbon dots. Based on the combined action of the inner filtration effect and hydrogen bonding, the present invention proposes a method for detecting tetracycline antibiotics in milk based on deep ultraviolet emission carbon dots, which can achieve convenient, safe and sensitive quantitative detection of tetracycline antibiotics in milk.

[0009] The present invention provides a method for detecting tetracycline antibiotics in milk based on deep ultraviolet emitting carbon dots as a novel fluorescent probe, comprising the following steps:

[0010] (1) Preparation of deep ultraviolet emitting carbon dots: 2,4-dihydroxybenzoic acid and lysine are added to water, mixed and subjected to hydrothermal reaction. After the reaction is completed, solid-liquid separation is performed and the filtrate is collected to obtain a deep ultraviolet emitting carbon dot solution (DUV-CDs);

[0011] (2) A series of tetracycline milk solutions with known concentrations were mixed with acetonitrile and filtered through a membrane to obtain corresponding tetracycline test samples;

[0012] (3) diluting the deep ultraviolet emitting carbon dot solution obtained in step (1), and then mixing it with the tetracycline test sample in step (2), collecting corresponding fluorescence spectrum information, and obtaining the fluorescence intensity peak value F0 of the sample when the tetracycline concentration is 0, and the fluorescence intensity peak value F of the sample at other concentrations; calculating the quenching degree C by (F0-F) / F0, and linearly correlating the quenching degree C with the tetracycline concentration of the corresponding sample to obtain a quantitative detection model.

[0013] In one embodiment of the present invention, in step (1), the mass ratio of 2,4-dihydroxybenzoic acid to lysine is (0.8-1.2):1. Specifically, it can be 1:1.

[0014] In one embodiment of the present invention, in step (1), the concentration of lysine relative to water is 6-8 mg / mL.

[0015] In one embodiment of the present invention, in step (1), the hydrothermal reaction is carried out at 180° C. for 10 hours.

[0016] In one embodiment of the present invention, in step (2), the tetracycline milk solution is prepared by the following process: dissolving tetracycline in water to obtain a tetracycline aqueous solution; and then mixing with milk to obtain a tetracycline milk solution.

[0017] In one embodiment of the present invention, in step (2), the concentration range of the tetracycline milk solution is 0-200 mg / L.

[0018] In one embodiment of the present invention, in step (2), the volume ratio of the tetracycline milk solution to acetonitrile is 2:3.

[0019] In one embodiment of the present invention, in step (2), during the mixing with acetonitrile, shaking, ultrasound, centrifugation are used to collect the clear liquid.

[0020] In one embodiment of the present invention, the ultrasonic time is 10 minutes; the centrifugal speed is 12000 rpm, and the centrifugal time is 15 minutes.

[0021] In one embodiment of the present invention, in step (3), the dilution factor of the deep ultraviolet emitting carbon dot solution obtained in step (1) is 9 times.

[0022] In one embodiment of the present invention, in step (3), the fluorescence intensity peak refers to the fluorescence intensity peak at 308 nm.

[0023] In one embodiment of the present invention, in step (4), the fluorescence emission spectrum is measured using an FLS980 fluorescence spectrometer produced in Edinburg, UK, with an excitation wavelength of 273 nm, a peak wavelength of 308 nm, and excitation and emission slit widths of 1 nm and 1 nm, respectively.

[0024] The present invention provides application of the above method in the field of food detection.

[0025] Beneficial effects:

[0026] 1. The fluorescence emission of DUV-CDs used in the present invention is in the deep ultraviolet band, with a small Stokes shift and high energy utilization. In addition, the fluorescence emission of DUV-CDs overlaps with the absorption of tetracycline to a large extent, providing a good basis for the detection of tetracycline.

[0027] 2. The concentration of DUV-CDs used in the present invention has been optimized and has a good effect when applied to milk, thereby increasing the sensitivity of detection.

[0028] 3. The method of the present invention uses deep ultraviolet emitting carbon dots for the first time to achieve quantitative detection of tetracycline antibiotics in complex systems such as milk. The detection operation of this method is simple. During the detection, it is only necessary to perform a simple protein pretreatment on the milk and then measure the fluorescence emission spectrum.

[0029] 4. The present invention has similar detection capabilities for tetracycline antibiotics. Since several tetracycline antibiotics have similar structures and similar physical and chemical properties, DUV-CDs can detect this type of antibiotics.

[0030] 5. The linear range of the linear model constructed by the present invention is 0.5-40 mg / L, the standard curve equation is C=0.00795I+0.01535 (C is the quenching degree of carbon dots and I is the tetracycline concentration), and the determination coefficient R 2=0.991, the detection limit was calculated to be as low as 70.61μg / L, and the recovery rate obtained by the method was 93.47% to 97%. The detection limit was lower than the national standard and had good detection accuracy, which was of great significance for supervising the residue of tetracycline antibiotics in milk. BRIEF DESCRIPTION OF THE DRAWINGS

[0031] Figure 1 Flow chart for the detection of tetracyclines in milk by DUV-CDs.

[0032] Figure 2 The absorption spectrum (black line), fluorescence excitation spectrum (red line) and emission spectrum (blue line) of the DUV-CDs aqueous solution in Example 1.

[0033] Figure 3 Emission spectra of DUV-CDs mixed with tetracycline, chlortetracycline, oxytetracycline and doxycycline, respectively (excitation wavelength is 273 nm).

[0034] Figure 4 (a) Fluorescence emission spectra of carbon dot-tetracycline milk solution when the tetracycline concentration was 0-200 mg / L (excitation wavelength was 273 nm); (a) Inset: line graph of fluorescence peak intensity and tetracycline concentration; (b) fitting graph of quenching degree ((F0-F) / F0) and tetracycline concentration (tetracycline concentration was 0.5-40 mg / L, F0 and F were the peak intensities of the solution in the absence and presence of tetracycline, respectively).

[0035] Figure 5 (a) Comparison of normalized spectra of carbon dots, carbon dots-milk pretreatment and milk pretreatment; (b) Histogram of the quenching degree of carbon dots at different concentrations after pretreatment of milk and standard milk with tetracycline added. DETAILED DESCRIPTION

[0036] The embodiments of the present invention will be described in detail below with reference to examples, but those skilled in the art will appreciate that the following examples are only used to illustrate the present invention and should not be construed as limiting the scope of the present invention.

[0037] Example 1 Preparation of Deep Ultraviolet Emitting Carbon Dots

[0038] The preparation of deep ultraviolet emitting carbon dots comprises the following steps:

[0039] (1) 0.257 g of 2,4-dihydroxybenzoic acid and 0.244 g of lysine were added to 40 ml of ultrapure water. After stirring, the mixture was transferred to a 100 ml tetrafluoroethylene-lined autoclave and heated from room temperature to 180° C. in an electric furnace and maintained for 10 hours.

[0040] (2) After heating, the reaction product was naturally cooled to room temperature, and the solid precipitate in the reaction product was filtered to remove the obtained DUV-CDs stock solution. The obtained DUV-CDs stock solution was diluted 9 times and stored in a refrigerator at 4°C for subsequent use.

[0041] (3) Absorption spectra (squares), fluorescence excitation spectra (circles), and emission spectra (triangles) of DUV-CDs. Figure 2 shown.

[0042] Example 2 Response of Deep Ultraviolet Emitting Carbon Dots to Tetracycline Antibiotics

[0043] Deep UV emission carbon dots response experiment to tetracycline antibiotics:

[0044] 0.4 mL of DUV-CDs diluted 9 times was mixed with 2 mL of water, 100 mg / L tetracycline, 100 mg / L chlortetracycline, 100 mg / L oxytetracycline and 100 mg / L doxycycline, and the samples were subjected to fluorescence spectrum detection. The scanning conditions were as follows: the excitation wavelength was 273 nm, the emission wavelength scanning range was 280-500 nm, the scanning was performed every 1 nm, and the slit width was set to 1 / 1 nm (excitation slit / emission slit). Figure 3 The same concentration of the four tetracycline antibiotics quenched the fluorescence of carbon dots, and the quenching degree was similar.

[0045] Example 3 Construction of standard curve

[0046] A method for constructing a standard curve in the method for detecting tetracycline antibiotics in milk based on deep ultraviolet emission described in Example 1, taking tetracycline, a common antibiotic in this class, as an example, a detection experiment was carried out, comprising the following steps:

[0047] (1) Prepare a 0-400 mg / L tetracycline aqueous solution, mix it with milk at a ratio of 1:1 (2 mL each) to obtain a 4 mL 0-200 mg / L tetracycline milk solution, add 6 mL acetonitrile, shake it and ultrasonicate it for 10 minutes, centrifuge it at 12000 rpm for 15 minutes, and then filter it with a 0.22 μm filter membrane to obtain a pretreated tetracycline milk solution. Take 0.4 mL of the 9-fold diluted DUV-CDs stock solution and mix it with 2 mL of the pretreated sample.

[0048] (2) The sample in (1) was subjected to fluorescence spectrum detection, and the scanning conditions were: the excitation wavelength was 273 nm, the emission wavelength scanning range was 280-500 nm, the scanning was performed every 1 nm, and the slit width was set to 1 / 1 nm (excitation slit / emission slit), and the fluorescence intensity peak F at 308 nm was obtained; the milk solution with a tetracycline concentration of 0 was subjected to fluorescence spectrum detection, and the fluorescence intensity peak F0 at 308 nm was obtained; the quenching degree C of different tetracycline concentrations was calculated by formula (1);

[0049] C=(F0-F) / F0 (1)

[0050] (4) The relationship curve between the fluorescence intensity quenching degree of tetracycline milk solution with different concentrations is shown in the figure. Figure 4 (a) Inset, the fitting curve of quenching degree C and tetracycline concentration I is shown in Figure 4 (b) from Figure 4 (a) (b) It can be seen that when the tetracycline concentration is in the range of 25-500 mg / L, the quenching degree C of the carbon dots is linearly related to the tetracycline concentration I. The linear regression equation is C=0.00795I+0.01535, and the determination coefficient R 2 =0.991, and the detection limit was 70.61 μg / L.

[0051] Example 4: Verification of spike recovery

[0052] Table 1 shows the spiked recoveries and RSDs of tetracycline in milk by DUV-CDs method

[0053]

[0054] Example 5 Optimization of carbon dot concentration

[0055] Due to the existence of the fluorescence spectrum of milk itself, the concentration of DUV-CDs needs to be optimized before detecting tetracycline in milk. The DUV-CDs stock solution was diluted 3 times, 6 times, 9 times, 15 times, 30 times, and 60 times, respectively, and 2 mL of milk was mixed with 2 mL of water and 2 mL of 100 mg / L tetracycline to obtain a 50 mg / L tetracycline milk solution. Then 6 mL of acetonitrile was added, and after shaking, it was ultrasonicated for 10 minutes, centrifuged at 12,000 rpm for 15 minutes, and then filtered with a 0.22 μm filter membrane to obtain clear and transparent tetracycline-free milk pretreatment samples and tetracycline-containing milk pretreatment samples. Take 0.4 mL of carbon dots of each concentration and mix them with 2 mL of tetracycline-free milk pretreatment samples and 2 mL of tetracycline-containing milk pretreatment samples, respectively, and measure the emission spectrum under 273 nm excitation to obtain. Figure 5 . Figure 5(a) is a comparison of the normalized spectra of carbon dots, carbon dots-milk pretreatment, and milk pretreatment. It can be seen that the addition of milk will have a certain impact on the spectrum of carbon dots, the most obvious change is between 325nm-400nm, and the peak wavelength of carbon dots has also shifted to a certain extent. By comparison, it can be determined that the spectrum is formed by the superposition of the fluorescence spectrum of milk itself and carbon dots. Therefore, when the concentration of carbon dots is low in the experiment, it will be greatly affected by the spectrum of milk itself, and the concentration of carbon dots needs to be optimized. Figure 5 (b) The quenching degree of carbon dots added to milk and standard milk with tetracycline added to different concentrations is shown in the bar graph. When the carbon dots are diluted 9-60 times, the fluorescence of the carbon dots gradually weakens, and the quenching degree gradually decreases due to the increasing spectrum of the milk itself. When the carbon dots are diluted 3-9 times, the carbon dots concentration is high, and the effect of tetracycline on it is reduced. After comparison, the carbon dots concentration is selected to be diluted 9 times of the original solution. At this time, the fluorescence of the carbon dots is basically not affected by the fluorescence of the milk itself.

Claims

1. A method for detecting tetracycline antibiotics in milk based on deep ultraviolet emitting carbon dots as fluorescent probes, characterized in that: The following steps are involved: (1) Preparation of deep ultraviolet emitting carbon dots: 2,4-dihydroxybenzoic acid and lysine are added to water, mixed and subjected to hydrothermal reaction. After the reaction is completed, solid-liquid separation is performed and the filtrate is collected to obtain a deep ultraviolet emitting carbon dot solution; (2) A series of tetracycline milk solutions with known concentrations were mixed with acetonitrile and filtered through a membrane to obtain the corresponding tetracycline test samples; (3) diluting the deep ultraviolet emitting carbon dot solution obtained in step (1), and then mixing it with the tetracycline test sample in step (2), collecting corresponding fluorescence spectrum information, and obtaining the fluorescence intensity peak value F0 of the sample when the tetracycline concentration is 0, and the fluorescence intensity peak value F of the sample at other concentrations; calculating the quenching degree C by (F0-F) / F0, and linearly correlating the quenching degree C with the tetracycline concentration of the corresponding sample to obtain a quantitative detection model; In step (1), the mass ratio of 2,4-dihydroxybenzoic acid to lysine is (0.8-1.2):1; In step (1), the hydrothermal reaction is carried out at 180°C for 10 h.

2. The method according to claim 1, characterized in that In step (1), the concentration of lysine relative to water is 6-8 mg / mL.

3. The method according to claim 1, characterized in that In step (2), the tetracycline milk solution is prepared by the following process: dissolving tetracycline in water to obtain a tetracycline aqueous solution; and then mixing with milk to obtain a tetracycline milk solution.

4. The method according to claim 1, characterized in that: In step (2), the concentration range of the tetracycline milk solution is 0-200 mg / L.

5. The method according to claim 1, characterized in that In step (2), the volume ratio of tetracycline milk solution to acetonitrile is 2:

3.

6. The method according to claim 1, characterized in that In step (3), the dilution ratio of the deep ultraviolet emitting carbon dot solution obtained in step (1) is 9 times.

7. The method according to claim 1, characterized in that In step (3), the fluorescence intensity peak refers to the fluorescence intensity peak at 308 nm.

8. Application of the method according to any one of claims 1 to 7 in the field of food testing.

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