Ratio-type detection method for tetracycline and oxytetracycline

Abstract

The invention relates to detection of compounds, in particular to a ratio type method for detecting tetracycline (TC) and oxytetracycline (OTC). The method is realized through the following steps: firstly, preparing an H-CDs solution from carrot powder; and then mixing the buffer solution, TC and OTC standard solutions with different concentrations and H-CDs solution for reaction, and measuring the fluorescence intensity. According to the detection method provided by the invention, on the basis of the H-CDs, along with the increase of the concentration of tetracycline and oxytetracycline, the fluorescence emission peak of the H-CDs at 540 nm shows an enhancement phenomenon, the fluorescence at 430 nm shows a quenching phenomenon, and the color of the solution is gradually changed from blue to green under the irradiation of an ultraviolet lamp. The ratio-type detection of tetracycline and oxytetracycline is realized by adopting a fluorogram mode, the anti-interference capability is strong, the detection accuracy is improved, the detection process is simple and rapid, the detection can be completed in a short time, and the method is favorably popularized and used in the field of detection of tetracycline antibiotics in a water body.

Description

Technical Field

[0001] The present invention relates to the detection of compounds, and in particular to a ratiometric method for detecting tetracycline and oxytetracycline. Background Art

[0002] Tetracycline antibiotics are a class of broad-spectrum antibiotics, with representative drugs including oxytetracycline (OTC), tetracycline (TC), chlortetracycline (CTC), etc. They are widely used to treat various bacterial infections and can be used to prevent common aquatic diseases caused by bacteria. Tetracycline antibiotics are widely used as feed additives to promote their growth and reduce morbidity. Since tetracycline antibiotics are difficult to digest and absorb in the body, they are usually excreted in the form of urine or feces, which makes tetracycline antibiotics and their by-products spread among microorganisms, ultimately threatening human health.

[0003] Since the three tetracycline antibiotics (tetracycline, oxytetracycline, and chlortetracycline) have similar structures, they are all composed of four six-membered rings with hydroxyl groups at the C5 and C6 positions, and chlortetracycline has a chlorine substituent. Due to their structural similarities, how to achieve specific detection of tetracycline and oxytetracycline is very important. So far, many methods have been applied to the detection of tetracycline antibiotics, including high performance liquid chromatography, electrochemical analysis, atomic absorption spectrometry, and fluorescence analysis, but most of the methods respond to tetracycline antibiotics approximately as single signal detection, lacking a certain degree of accuracy.

[0004] Carbon quantum dots were first discovered by Xu et al. in 2004. Compared with traditional silicon quantum dots, carbon quantum dots have the advantages of being green and environmentally friendly, simple synthesis methods, excellent fluorescence properties, and good biocompatibility. Surface modification and doping with heteroelements can change its disadvantages such as low quantum yield, short fluorescence lifetime, and few active sites. Surface modification includes chemical modification, physical treatment, and self-assembly. Among them, the chemical modification method has attracted widespread attention due to its simplicity and environmental protection. In the prior art, the methods for detecting related tetracycline antibiotics mostly show fluorescence quenching, but the carbon dots are combined with Eu 3+ The combined composite ratio probe detects tetracycline antibiotics as fluorescence enhancement.

[0005] This is because the β-diketonate configuration in tetracyclines easily reacts with Eu 3+ Violent coordination is formed to form stable tetracycline-Eu 3+ Complex. From tetracyclines to Eu 3+ The energy transfer that occurs can effectively realize Eu 3+The sensitization of characteristic emission, also known as the "antenna effect", can achieve tetracycline-based Eu 3+ The complex was used to detect tetracyclines, thus showing fluorescence enhancement. However, most detections of tetracycline antibiotics still show fluorescence quenching. There are relatively few reports on the use of carbon quantum dots to achieve ratiometric tetracycline and oxytetracycline detection and fluorescence enhancement. Summary of the invention

[0006] In view of the problems existing in the prior art, the present invention provides a ratiometric method for detecting tetracycline and oxytetracycline.

[0007] The technical solution adopted by the present invention to achieve the above-mentioned purpose is: The present invention provides a ratiometric method for detecting tetracycline and oxytetracycline, comprising the following steps: 1) Mix carrot powder and anhydrous ethanol to obtain a mixture; reflux the mixture under nitrogen protection, then cool it to room temperature, filter the supernatant to obtain a filtrate; 2) Mix the filtrate with an anhydrous ethanol solution of polyoxyethylene diamine (H2N-PEG-NH2), heat the mixture under reflux, cool to room temperature, and filter the mixture to obtain a H-CDs solution; 3) Add different concentrations of TC and OTC standard solutions and H-CDs solution to the buffer solution, perform a mixed reaction, and measure the fluorescence intensity to obtain a standard curve; 4) Add the sample to be tested and H-CDs solution into the buffer solution, mix them and measure their fluorescence intensity.

[0008] Preferably, in step 1), the carrot powder is prepared by the following method: washing the carrots, drying them at 60° C. overnight, crushing them and sieving them; the solid-liquid ratio of the carrot powder to anhydrous ethanol is 1 g:20 mL.

[0009] Preferably, in step 1), the reflux heating is reflux at 85° C. for 18-26 h; and the filtration uses a 0.22 μm filter membrane.

[0010] Preferably, in step 2), the volume ratio of the filtrate to the anhydrous ethanol solution of polyoxyethylene diamine is 4:1; and the concentration of the anhydrous ethanol solution of polyoxyethylene diamine is 1.2 mg / mL.

[0011] Preferably, in step 2), the heating reflux is heating reflux at 75° C. for 4 hours; and the filtration uses a 0.22 μm filter membrane.

[0012] Preferably, in steps 3) and 4), the buffer is 0.04 M, 3 mL Britton-Robinson buffer with a pH of 9.

[0013] Preferably, in step 3), the concentration of TC or OTC in the buffer is 0.0667-25 µM.

[0014] Preferably, in steps 3) and 4), the mixing reaction is carried out at room temperature for 10 minutes.

[0015] The detection method provided by the present invention, in the presence of carbon dots, increases the concentration of tetracycline and oxytetracycline, so that the fluorescence intensity at 540 nm increases, and the mechanism of fluorescence enhancement may be caused by aggregation induction. The fluorescence at 430 nm is quenched, and the mechanism of fluorescence quenching is inner filter effect and static quenching. In the presence of carbon dots, the ratio of the fluorescence intensity at 540 nm to that at 430 nm (F 540 / F 430 ) shows a linear relationship. Therefore, the ratiometric detection of tetracycline and oxytetracycline was achieved. Since the carbon dots themselves have two emission peaks (located at 430 nm and 540 nm), and with the increase of tetracycline and oxytetracycline concentrations, the fluorescence intensity at 540 nm gradually increases, and the fluorescence intensity at 430 nm gradually quenches. Under ultraviolet light, Figure 2 and 3 As shown, it is observed that the color of the solution gradually changes from blue to green. Therefore, the presence of tetracycline and oxytetracycline can be relatively intuitively judged based on the color change under ultraviolet light, making the detection process simple and fast, and can be completed in a short time.

[0016] The beneficial effects of the present invention are: (1) The present invention uses biomass as raw material and synthesizes H-CDs by a method of mixing carrots and ethanol and refluxing, and has good compatibility with the substance to be detected; the H-CDs synthesized by the present invention have a simple preparation method, and based on the H-CDs prepared by the present invention, a new fluorescence detection method for tetracycline and oxytetracycline with high sensitivity and strong selectivity can be established; (2) The detection method provided by the present invention is based on H-CDs. As the concentration of tetracycline and oxytetracycline increases, the fluorescence emission peak of H-CDs at 540 nm shows an enhancement phenomenon, while the fluorescence at 430 nm is quenched, and under ultraviolet light, the color of the solution gradually changes from blue to green. The present invention uses fluorescence spectra to achieve ratiometric detection of tetracycline and oxytetracycline, which increases the accuracy of the detection. The detection process is simple and fast and can be completed in a short time. BRIEF DESCRIPTION OF THE DRAWINGS

[0017] Figure 1Fluorescence spectra of tetracycline and oxytetracycline before and after addition; Figure 2 and Figure 3 The photos are of tetracycline and oxytetracycline added at concentrations of 0 μM, 1.66 μM, 3.33 μM, 6.67 μM, 11.76 μM, 16.6 μM, 21.05 μM, and 25 μM under ultraviolet light in the presence of carbon dots; Figure 4 1 is the fluorescence spectra of aqueous solutions of tetracycline (TC) with different concentrations in the examples; Figure 5 is the change in fluorescence intensity at the emission wavelength of tetracycline (TC) of different concentrations in the example; Figure 6 The fluorescence spectra of aqueous solutions of oxytetracycline (OTC) with different concentrations in the examples; Figure 7 is the change in fluorescence intensity at the emission wavelength of oxytetracycline (OTC) of different concentrations in the example; Figure 8 The fluorescence images are for the specific detection of tetracycline and oxytetracycline. DETAILED DESCRIPTION

[0018] The present invention is described below in conjunction with examples, which are only used to explain the present invention and are not used to limit the scope of the present invention.

[0019] The polyoxyethylene diamine (H2N-PEG-NH2) used in the present invention was purchased from Sigma-Aldrich, with a molecular weight of Mw = 4000. Example 1 The detection method of tetracycline and oxytetracycline comprises the following steps: 1) Wash the carrots, dry them at 60 °C overnight, crush them and sieve them. Subsequently, mix 2 g of carrot powder and 40 mL of anhydrous ethanol in a 100 mL round-bottom flask. Under nitrogen protection, reflux the mixture at 85 °C for 24 h. After cooling to room temperature, filter the supernatant with a 0.22 μm filter membrane. Then, mix 6 mL of the filtrate with 1.5 mL of anhydrous ethanol solution of 1.2 mg / mL polyoxyethylene diamine (H2N-PEG-NH2), place the mixture in a 25 mL round-bottom flask, and heat reflux at 75 °C for 4 h. Cool to room temperature, filter the mixture with a 0.22 μm filter membrane to obtain a light yellow H-CDs solution; 2) Add 3 mL of pH 9 Britton-Robinson buffer to the cuvette, add 0.0667-25 µM TC / OTC standard solutions and the H-CDs obtained in step 1) to the buffer, mix and react at room temperature for 10 min, and then measure the fluorescence intensity. Figure 4-Figure 7shown.

[0020] Example 2 Pattern detection method of tetracycline and oxytetracycline: replace tetracycline (TC) and oxytetracycline (OTC) at a concentration of 25 µM in the embodiment with doxycycline (DOX), enrofloxacin (ENR), ciprofloxacin (CIP), chloramphenicol (CPL), lomefloxacin (LOM), azithromycin (AZI), clarithromycin (CLR), amoxicillin (AMC), kanamycin (KAN), erythromycin (EM), streptomycin (SM), thiamphenicol (TAP), chlortetracycline (CTC), gentamicin (GM), streptomycin (SM), L-lysine (L-Lys), L-alanine (L-Ala), L-asparagine (L-Asn), L-cysteine ​​(L-Cys), Fe 3+ , Hg 2+ ,Mg 2 + , Ni 2+ , Ba 2+ , Cu 2+ ,Co 2+ The fluorescence intensity was determined by the ratio of the fluorescence intensity at 540 nm to that at 430 nm (F 540 / F 430 ) is used to determine the specificity of H-CDs in detecting tetracycline and oxytetracycline, such as Figure 8 shown.

[0021] Fluorescence spectra before and after the addition of tetracycline and oxytetracycline ( Figure 1 ) It can be seen that when there is no target antibiotic, the fluorescence intensities at the maximum emission wavelengths of 430 nm and 540 nm are 220 and 158. After adding tetracycline, the fluorescence intensity at the emission wavelength of 540 nm is enhanced to 505, and the fluorescence intensity at the emission wavelength of 430 nm is 130; after adding oxytetracycline, the fluorescence intensity at the emission wavelength of 430 nm is 194, and the fluorescence intensity at the emission wavelength of 540 nm is enhanced to 581.

[0022] Figure 5 and Figure 7 The invention can quantitatively detect tetracycline and oxytetracycline at different concentrations. The concentrations of tetracycline and oxytetracycline are in the range of 0.0667-25 μM and F 540 / F 430 The ratio of fluorescence intensity is linear. Figure 8 It can be observed that when different interfering substances are present, F 540 / F 430 The ratio of the fluorescence intensity of the two emission peaks is compared with the value in the absence of antibiotic F 0 (540) / F 0 (430)The ratio of the fluorescence intensity of the two emission peaks (Blank) was compared and it was found that when ciprofloxacin (CIP) and enrofloxacin (ENR) were present, F 540 / F 430 Compared with the absence of antibiotics 0 (540) / F 0（430） Compared with the above, it can be seen that there is an obvious change. This is because quinolone antibiotics have fluorescence themselves, which makes the fluorescence at 430nm appear enhanced. Other antibiotics and interfering substances almost have an effect on the ratio of the fluorescence intensity at 540 nm to that at 430nm (F 540 / F 430 ) has little effect, which shows that the carbon dots (H-CDs) prepared in this experiment have good selectivity for tetracycline and oxytetracycline.

[0023] Effect Example 1 The lake water from Deqing Lake of Huzhou Normal University and the largemouth bass breeding water from the Aquaculture Center of the School of Life Sciences were selected as actual samples. The two water samples were centrifuged and filtered with a 0.22μm filter membrane. Then the same volume of water sample was mixed with Britton-Robinson buffer at pH=9, and different concentrations of TC and OTC (5μМ, 25μМ) were added to 3mL of the mixed actual sample. 540 / F 430 =0.113C TC +0.671) and the regression equation of OTC (F 540 / F 430 =0.093C OTC +0.833) to test the concentration of TC and OTC. Tables 1 and 2 show the detection results of TC and OTC in actual water samples by H-CDs. The results show that the recovery rate is between 96.26-100.21%.

[0024] Table 1 Table 2 The above description is only a preferred embodiment of the present invention and is not intended to limit the present invention. Any modifications, equivalent substitutions, improvements, etc. made within the spirit and principle of the present invention should be included in the protection scope of the present invention.

Claims

1. A method for ratiometric detection of tetracycline and oxytetracycline, characterized in that: The following steps are involved: 1) Mix carrot powder and anhydrous ethanol to obtain a mixture; reflux the mixture under nitrogen protection, then cool it to room temperature, filter the supernatant to obtain a filtrate; 2) mixing the filtrate with an anhydrous ethanol solution of polyoxyethylene diamine, heating the mixture under reflux, cooling to room temperature, and filtering the mixture to obtain an H-CDs solution; 3) Add different concentrations of TC and OTC standard solutions and H-CDs solution to the buffer solution, perform a mixed reaction, and measure the fluorescence intensity to obtain a standard curve; 4) Add the sample to be tested and H-CDs solution into the buffer solution, mix them and measure their fluorescence intensity.

2. The method according to claim 1, characterized in that In step 1), the carrot powder is prepared by the following method: washing the carrots, drying them at 60° C. overnight, crushing them and sieving them; the solid-liquid ratio of the carrot powder to anhydrous ethanol is 1 g:20 mL.

3. The method according to claim 1 or 2, characterized in that: In step 1), the reflux heating is reflux at 85°C for 18-26 hours; and the filtration adopts a 0.22 μm filter membrane.

4. The method according to claim 1, characterized in that: In step 2), the volume ratio of the filtrate to the anhydrous ethanol solution of polyoxyethylene diamine is 4:1; the concentration of the anhydrous ethanol solution of polyoxyethylene diamine is 1.2 mg / mL.

5. The method according to claim 1 or 4, characterized in that: In step 2), the heating reflux is heating reflux at 75° C. for 4 hours; and the filtration uses a 0.22 μm filter membrane.

6. The method according to any one of claims 1 to 5, characterized in that: In step 3), the buffer is 0.04 M, 3 mL, pH=9 Britton-Robinson buffer.

7. The method according to claim 1 or 6, characterized in that: In step 3), the concentration of TC or OTC in the buffer solution is 0.0667-25 µM.

8. The method according to any one of claims 1 to 7, characterized in that: In steps 3) and 4), the mixed reaction is carried out at room temperature for 10 minutes.

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