Cordyceps militaris polysaccharide fluorescent carbon dots for detecting and degrading tetracycline antibiotics, preparation method and application thereof

Abstract

The present invention discloses a Cordyceps militaris polysaccharide fluorescent carbon dot that can be used to detect and degrade tetracycline antibiotics, a preparation method and application thereof, and belongs to the field of nanomaterials and environmental monitoring technology. The Cordyceps militaris polysaccharide fluorescent carbon dot is prepared by the following method: Cordyceps militaris polysaccharide and zinc chloride are dissolved in water, and then subjected to high-temperature reaction; after the reaction is completed, filtering, dialyzing, and drying to obtain Cordyceps militaris polysaccharide fluorescent carbon dots. The carbon quantum dots prepared by the present invention realize the integration of detection and degradation of tetracycline antibiotics, and have a series of advantages such as simple operation, high selectivity, low detection limit, and environmental friendliness, thus having important value in the field of nanomaterials and environmental monitoring technology.

Description

Technical Field

[0001] The present invention belongs to the technical field of nanomaterials and environmental monitoring, and particularly relates to Cordyceps militaris polysaccharide fluorescent carbon dots that can be used for detecting and degrading tetracycline antibiotics, as well as a preparation method and application thereof. Background Art

[0002] Tetracycline antibiotics (TCs) are a typical class of broad-spectrum antibiotics widely used to prevent and treat microbial diseases such as filariasis and giardiasis. They can also be used to increase livestock yields. However, the overuse of tetracyclines can have serious consequences. Studies have shown that tetracyclines are commonly used to treat a variety of mammalian diseases, but less than 50% of the dose is metabolized. A significant portion of tetracycline is excreted into water and soil through animal feces and urine, causing pollution. Tetracycline residues in animals can also harm human health through the food chain. Antibiotic residues in foods such as meat, milk, and honey are highly toxic and can cause gastrointestinal disturbances, allergies, and central nervous system reactions. To address these issues, numerous tetracycline detection technologies have been developed. Among traditional tetracycline detection methods, fluorescence detection is widely used due to its high sensitivity, low detection limit, and fast response time. However, TCs actually include a variety of different antibiotics, including minocycline (MC), tetracycline (TC), oxytetracycline (OTC), doxycycline (DOC) and chlortetracycline (CTC). These antibiotics have similar structures but are difficult to detect simultaneously. Current research often only detects one specific antibiotic, and the detection methods for similar antibiotics are often complicated. Therefore, it is very important to find a simple and effective method to achieve the simultaneous detection of multiple tetracycline antibiotics. In addition, in practical applications, the detection and degradation of tetracycline antibiotics should complement each other, but there are very few related studies. Therefore, the development of a material that can be used to simultaneously detect and degrade tetracycline antibiotics faces huge challenges. Summary of the Invention

[0003] The invention provides a Cordyceps militaris polysaccharide fluorescent carbon dot for detecting and degrading tetracycline antibiotics. The fluorescent carbon dot is a blue fluorescent quantum dot which uses Cordyceps militaris polysaccharide as a carbon source and is co-doped with zinc and chlorine.

[0004] The above-mentioned Cordyceps militaris polysaccharide fluorescent carbon dots are prepared by the following method:

[0005] Cordyceps militaris polysaccharide and zinc chloride are dissolved in water, and then subjected to high-temperature reaction; after the reaction is completed, the mixture is filtered, dialyzed, and dried to obtain Cordyceps militaris polysaccharide fluorescent carbon dots.

[0006] In the above preparation method, the raw materials are selected from the following parts by weight: 0.1-0.5 parts of Cordyceps militaris polysaccharide, 0.14-0.8 parts of zinc chloride, and 5-15 parts of water.

[0007] In a specific embodiment, the raw materials are selected from the following parts by mass: 0.12 parts of Cordyceps militaris polysaccharide, 0.6 parts of zinc chloride, and 10 parts of water.

[0008] In the above preparation method, the conditions of the high temperature reaction are selected from: hydrothermal reaction at 160-240° C. for 8-14 h; preferably: hydrothermal reaction at 180° C. for 10 h.

[0009] In the above preparation method, the filtration is performed using a 0.22 μm filter membrane; and the dialysis is performed using a dialysis bag with a molecular weight cut-off of 1000 Da.

[0010] The present invention provides the use of the Cordyceps militaris polysaccharide fluorescent carbon dots in detecting and / or degrading tetracycline antibiotics.

[0011] In the present invention, the tetracycline antibiotic is selected from one or more of minocycline (MC), tetracycline (TC), chlortetracycline (CTC), oxytetracycline (OTC), and doxycycline (DOC).

[0012] The present invention provides a method for detecting tetracycline antibiotics, comprising the following steps:

[0013] Cordyceps militaris polysaccharide fluorescent carbon dots were mixed with tetracycline antibiotic standard solutions of different concentration gradients, and then the emission intensity was measured at an excitation wavelength of 366 nm, and the fluorescence intensity at the peak was recorded. A graph was plotted with the fluorescence intensity at the peak as the ordinate and the tetracycline antibiotic concentration as the abscissa to obtain a linear equation.

[0014] The test sample solution is then mixed with the Cordyceps militaris polysaccharide fluorescent carbon dots, and the emission intensity is measured at an excitation wavelength of 366 nm. The fluorescence intensity at the peak of the test solution is recorded and substituted into the linear equation to obtain the concentration of tetracycline antibiotics in the test sample solution.

[0015] In the above detection method, the usage ratio of the Cordyceps militaris polysaccharide fluorescent carbon dots to the tetracycline antibiotic standard solution is 1 mg:1 mL; the usage ratio of the Cordyceps militaris polysaccharide fluorescent carbon dots to the sample solution to be tested is 1 mg:1 mL.

[0016] The present invention provides a detection method for distinguishing tetracycline antibiotics, comprising the following steps:

[0017] Cordyceps militaris polysaccharide fluorescent carbon dots were mixed with aqueous solutions of various tetracycline antibiotics at the same concentration. The color of the carbon dots in each solution was then photographed under ultraviolet light using the colorimeter app on a smartphone. The resulting fluorescent color was digitized and output to obtain RGB values. Principal component analysis (PCA) was performed on the resulting experimental data to obtain a PCA plot. PCA was then used to analyze the differences in fluorescent color.

[0018] The sample solution to be tested was mixed with fluorescent carbon dots from Cordyceps militaris polysaccharide. The color of the carbon dots in each solution was then photographed under ultraviolet light using the colorimeter app on a smartphone. The obtained fluorescent color was digitized and output to obtain RGB values. Principal component analysis was then performed to determine the type of antibiotic in the sample solution to be tested.

[0019] In the above detection method, the usage ratio of the Cordyceps militaris polysaccharide fluorescent carbon dots to the tetracycline antibiotic aqueous solution is 1 mg:1 mL; the usage ratio of the Cordyceps militaris polysaccharide fluorescent carbon dots to the sample solution to be tested is 1 mg:1 mL.

[0020] In the above detection method, the concentration of the tetracycline antibiotic aqueous solution is selected from 60 to 120 μmol / L.

[0021] The present invention provides a method for degrading tetracycline antibiotics, comprising the following steps:

[0022] The Cordyceps militaris polysaccharide fluorescent carbon dots were mixed with a sample solution containing tetracycline antibiotics and then treated with light to achieve the degradation of tetracycline antibiotics.

[0023] In the above degradation method, the usage ratio of the Cordyceps militaris polysaccharide fluorescent carbon dots to the sample solution is 1 mg:1 mL.

[0024] In the above degradation method, a xenon lamp is used for illumination.

[0025] The method for detecting and degrading tetracycline antibiotics of the present invention is applicable to matrix samples such as aqueous solutions and foods, such as water, milk, honey, etc.; solid samples can be prepared in the form of aqueous solutions, or detected after filtering to remove water-insoluble components.

[0026] The beneficial effects of the present invention are:

[0027] The present invention uses Cordyceps militaris polysaccharide as a carbon source and doped with zinc chloride to prepare a fluorescent carbon dot with a lattice spacing of about 0.33nm. The carbon dot produces a significant fluorescent signal change as the concentration of tetracycline antibiotics changes. Under ultraviolet light, abundant color changes can be observed with the naked eye, thereby enabling sensitive detection of tetracycline antibiotics. At the same time, the fluorescent carbon dot can also be used as a catalyst for photocatalytic degradation to achieve efficient degradation of tetracycline antibiotics. Thus, the fluorescent carbon dot can be used as a portable product to achieve real-time, on-site visualization, high sensitivity, and environmentally friendly detection of tetracycline antibiotics (TCs), and achieve rapid degradation of tetracycline antibiotics.

[0028] Furthermore, the fluorescent carbon dots prepared in this invention can be used to differentiate TCs of the same concentration using a smartphone with an easily accessible color scanning app. The Colorimeter, a smartphone-installed Color Picker app, converts the red, green, and blue (RGB) channels of a fluorescent image into digital values. By leveraging the rich color variations of the fluorescent carbon dots during TC detection, the on-site visual detection and differentiation of TCs can be quickly and conveniently achieved without the need for expensive equipment or skilled operators.

[0029] The main raw material used in this invention, Cordyceps militaris polysaccharide, is a natural product extracted from nature, eliminating the need for traditional chemical agents. This system design embodies the sustainable development concept of "from nature, for nature, and integrated with nature," providing technical support for tetracycline antibiotic pollution source control, environmental planning and management, and public health and safety protection.

[0030] In summary, the carbon quantum dots prepared in the present invention realize the integrated detection and degradation of TCs, and have a series of advantages such as simple operation, easy portability, high selectivity, low detection limit, and environmental friendliness, thus having important value in the field of nanomaterials and environmental monitoring technology. BRIEF DESCRIPTION OF THE DRAWINGS

[0031] Figure 1 Transmission electron microscopy images (including high-resolution images) (a) and particle size (b) of the Cordyceps militaris polysaccharide fluorescent carbon dots described in Example 2;

[0032] Figure 2 This is the fluorescence emission image of the Cordyceps militaris polysaccharide fluorescent carbon dots described in Example 2;

[0033] Figure 3 This is the X-ray photoelectron spectrum of the Cordyceps militaris polysaccharide fluorescent carbon dots described in Example 2;

[0034] Figure 4 The fluorescence spectra and linear relationship diagrams obtained by detecting different concentrations of antibiotics with Cordyceps militaris polysaccharide fluorescent carbon dots as described in Example 2;

[0035] Figure 5 The mechanism of TC quenching the fluorescence of Cordyceps militaris polysaccharide carbon dots;

[0036] Figure 6 PCA diagram for the detection of tetracycline antibiotics by Cordyceps militaris polysaccharide fluorescent carbon dots combined with smartphones;

[0037] Figure 7 The effects of different ions and amino acids on the mixed system of Cordyceps militaris polysaccharide fluorescent carbon dots;

[0038] Figure 8 This is the UV spectrum of the fluorescent carbon dots of Cordyceps militaris polysaccharide during the degradation of TCs;

[0039] Figure 9 The degradation kinetics of Cordyceps militaris polysaccharide fluorescent carbon dots in the process of degrading TCs;

[0040] Figure 10 This is the mechanism of TCs degradation by Cordyceps militaris polysaccharide fluorescent carbon dots. DETAILED DESCRIPTION

[0041] In the present invention, Cordyceps militaris polysaccharide is prepared by the following method:

[0042] The Cordyceps militaris extract was dissolved in an ethanol-water solution and subjected to graded alcohol precipitation, wherein the volume ratio of water to ethanol was successively reduced from 4:1 to 1:1, and Cordyceps militaris polysaccharide was obtained after four steps of graded alcohol precipitation.

[0043] In the present invention, the Cordyceps militaris extract was purchased from Xi'an Best Biotechnology Co., Ltd.

[0044] The other materials used in the present invention, unless otherwise stated, can be obtained through commercial channels. Unless otherwise specified, other terms used in the present invention generally have the meanings commonly understood by those of ordinary skill in the art. The present invention will be further described in detail below with reference to specific examples and data. The following examples are merely for illustration of the present invention and are not intended to limit the scope of the present invention in any way.

[0045] Example 1

[0046] Preparation of Cordyceps militaris polysaccharide fluorescent carbon dots, the steps are as follows:

[0047] 180 mg of Cordyceps militaris polysaccharide and 740 mg of zinc chloride were dissolved in 10 mL of ultrapure water and sonicated for 10 minutes. The solution was thoroughly mixed, transferred to a 25 mL polytetrafluoroethylene-lined stainless steel autoclave, and maintained at 180°C for 10 hours. After cooling to room temperature, the resulting dark yellow solution was filtered through a 0.22 μm filter membrane. The filtrate was further dialyzed in ultrapure water for 24 hours using a dialysis bag (molecular weight cutoff of 1000 Da), with the water being refreshed every 6 hours to remove small molecules. Subsequently, the solution was freeze-dried for 24 hours to obtain Cordyceps militaris polysaccharide fluorescent carbon dot powder (Zn,Cl-CDs).

[0048] Example 2

[0049] Preparation of Cordyceps militaris polysaccharide fluorescent carbon dots, the steps are as follows:

[0050] 120 mg of Cordyceps militaris polysaccharide and 600 mg of zinc chloride were dissolved in 10 mL of ultrapure water and sonicated for 10 minutes. The solution was thoroughly mixed, transferred to a 25 mL polytetrafluoroethylene-lined stainless steel autoclave, and maintained at 180°C for 10 hours. After cooling to room temperature, the resulting dark yellow solution was filtered through a 0.22 μm filter membrane. The filtrate was further dialyzed in ultrapure water for 24 hours using a dialysis bag (molecular weight cutoff of 1000 Da), with the water being refreshed every 6 hours to remove small molecules. Subsequently, the solution was freeze-dried for 24 hours to obtain Cordyceps militaris polysaccharide fluorescent carbon dot powder (Zn,Cl-CDs).

[0051] The transmission electron microscopy (TEM) and high-resolution images of the above-mentioned Cordyceps militaris polysaccharide fluorescent carbon dots are shown in Figure 1. Figure 1 The morphology and microstructure of the above-mentioned Cordyceps militaris polysaccharide fluorescent carbon dots were characterized by TEM. Figure 1 As can be seen in the figure, the Cordyceps militaris polysaccharide fluorescent carbon dots are uniformly distributed spherical nanoparticles with diameters ranging from 3.0 to 5.0 nm and an average particle size of 4.3 nm. Further careful observation of the HRTEM image shows that the Cordyceps militaris polysaccharide fluorescent carbon dots exhibit distinct lattice fringes with a lattice spacing of 0.33 nm.

[0052] In order to explore the fluorescence properties of Cordyceps militaris polysaccharide fluorescent carbon dots, the fluorescence emission spectra of Cordyceps militaris polysaccharide fluorescent carbon dots at different excitation wavelengths of 290-400 nm were tested by fluorescence spectroscopy. Figure 2 As shown, it can be observed that the Cordyceps militaris polysaccharide fluorescent carbon dots obtain the strongest fluorescence emission at 430 nm under 360 nm excitation.

[0053] In order to further confirm the elemental composition and chemical bonds of the C. militaris polysaccharide fluorescent carbon dots, XPS measurements were performed, such as Figure 3 As shown. In the full spectrum scan, five main peaks were found: C1s, O 1s, N 1s, Cl 2p, and Zn 2p ( Figure 3a). Cordyceps militaris polysaccharide fluorescent carbon dots are composed of C (71.6%), O (19.9%), N (3.9%), Zn (2.5%) and Cl (2.1%) ( Figure 3 a), indicating the successful synthesis of zinc and chlorine co-doped carbon quantum dots. Figure 3 As shown in b, the high-resolution Zn 2p peaks can be divided into two peaks, which correspond to Zn-O (1045.29eV, 1022.37eV). Figure 3 The three peaks in c) correspond to C=O (288.75eV), CO / CN (286.69eV), and CC (284.80eV). Figure 3 d) is divided into three peaks, corresponding to Cl 2p3 / 2 (201.55eV, 198.38eV) and Cl 2p1 / 2 (199.89eV). High resolution N1s ( Figure 3 The two peaks in e) correspond to NH (401.69eV) and NC (400.03eV). High resolution O1s ( Figure 3 The two peaks in f) correspond to HOC (533.60 eV) and CO (532.05 eV), respectively.

[0054] Example 3

[0055] Preparation of Cordyceps militaris polysaccharide fluorescent carbon dots, the steps are as follows:

[0056] 200 mg of Cordyceps militaris polysaccharide and 800 mg of zinc chloride were dissolved in 10 mL of ultrapure water and sonicated for 10 minutes. The solution was thoroughly mixed, transferred to a 25 mL polytetrafluoroethylene-lined stainless steel autoclave, and maintained at 180°C for 10 hours. After cooling to room temperature, the resulting dark yellow solution was filtered through a 0.22 μm filter membrane. The filtrate was further dialyzed in ultrapure water for 24 hours using a dialysis bag (molecular weight cutoff of 1000 Da), with the water being refreshed every 6 hours to remove small molecules. Subsequently, the solution was freeze-dried for 24 hours to obtain Cordyceps militaris polysaccharide fluorescent carbon dot powder (Zn,Cl-CDs).

[0057] 1. Design of Tetracycline Antibiotic Detection Test

[0058] 4.0 mg of the Cordyceps militaris polysaccharide fluorescent carbon dots prepared in Example 2 were added to 4 mL of an antibiotic aqueous solution of different concentrations (0-100 μM) and mixed evenly. Antibiotics include tetracycline antibiotics (MC, TC, CTC, OTC, DOC) and non-tetracycline antibiotics (penicillin (PNG), erythromycin (ETC), ceftriaxone sodium (CRO), kanamycin (KAN)). Then transfer it to a cuvette, measure its emission intensity at an excitation wavelength of 366 nm, and record the emission spectrum of the mixture. The slit widths for emission and excitation are both 10 nm. Plot the recorded fluorescence spectrum test data and obtain its linear relationship, as shown in FIG. Figure 4 shown.

[0059] Depend on Figure 4 It can be seen that with the increase of the concentration of tetracycline antibiotics, the fluorescence intensity of the Cordyceps militaris polysaccharide fluorescent carbon dot system at 434nm decreased significantly, and as the concentration continued to increase, it was accompanied by varying degrees of red shift. Figure 4 The small figures above in the middle bf figures show, from left to right, the fluorescence color changes of the same mass of Cordyceps militaris polysaccharide fluorescent carbon dots (4.0 mg) when TCs solutions with concentrations of 10 μM, 20 μM, 30 μM, 40 μM, 50 μM, 60 μM, 80 μM, and 100 μM were added respectively. It can be seen that with the increase of TCs concentration, the fluorescence of the Cordyceps militaris polysaccharide fluorescent carbon dots system changes from blue to blue-green and then to green.

[0060] With the fluorescence intensity at the peak as the ordinate and the TCs concentration as the abscissa, the linear equation is obtained as follows: Figure 4 As shown in the small and medium figures, they are as follows: F = -57.30C MC +3665.88, F=-54.71C TC +3561.24, F=-55.98C OTC +3849.01, F=-46.72C DOC +3916.90, F=-59.29C CTC +3991.36, linear correlation coefficient R 2 >0.976.

[0061] The test sample solution and the Cordyceps militaris polysaccharide fluorescent carbon dot solution synthesized in Example 2 were mixed in a ratio of 1:1 (mg:mL) and mixed evenly; fluorescence spectroscopy was used under excitation at 366 nm to obtain the fluorescence intensity at the peak in the test solution. The content of tetracycline antibiotics in the test sample solution could be calculated by comparing with the linear equation obtained in the above steps.

[0062] The limit of detection (LOD) of the Cordyceps militaris polysaccharide fluorescent carbon dots for TCs was calculated using the equation LOD = 3Sb / s, where Sb represents the standard error of 10 consecutive scans of the blank sample and s represents the slope of the calibration curve. The results showed that the limit of detection of this method was between 3.5 and 7.3 nM.

[0063] 2. Detection of Tetracycline Antibiotics in Milk

[0064] Before the experiment, milk samples were pretreated with a protein precipitation procedure: equal volumes (5 mL) of milk and 1% (v / v) trichloroacetic acid solution were thoroughly mixed. The mixture was then sonicated at 40°C for 20 minutes and transferred to a centrifuge tube. The milk extract supernatant was obtained after centrifugation at 12,000 rpm for 8 minutes. The pH was adjusted to neutral with aqueous sodium hydroxide.

[0065] Tetracycline antibiotics were added to the milk extraction supernatant, and a series of standard milk sample solutions containing different concentrations of TCs (0-50 μM) were prepared.

[0066] The above-mentioned standard milk sample solutions containing different concentrations of tetracycline antibiotics were taken separately, and the Cordyceps militaris polysaccharide fluorescent carbon dots prepared in Example 2 were added thereto at a ratio of 1:1 (mg:mL); after mixing evenly, the mixture was incubated at room temperature for 30 minutes, the emission spectrum of the carbon dots was measured with an excitation intensity of 366 nm, and the color change of the carbon dots was observed under ultraviolet light.

[0067] The fluorescence intensity at the peak is used as the ordinate and the antibiotic concentration is used as the abscissa to obtain the relationship between the fluorescence intensity at the peak and the antibiotic concentration in the milk sample.

[0068] After the milk sample solution to be tested was treated with the above sample pretreatment step, it was mixed with the Cordyceps militaris polysaccharide fluorescent carbon dot solution synthesized in Example 2 in a ratio of 1:1 (mg:mL) and mixed evenly; incubated at room temperature for 30 minutes, the color change of the carbon dots was observed under ultraviolet light, and the fluorescence spectrum was measured; and the tetracycline content in the milk sample solution to be tested was calculated by referring to the linear equation obtained in the above steps.

[0069] The test results are shown in Table 1:

[0070] Table 1 Recovery results of spiked milk samples

[0071]

[0072]

[0073] As shown in Table 1, the Cordyceps militaris polysaccharide fluorescent carbon dots prepared by the present invention are used to detect tetracycline antibiotics in milk, with good stability and high sensitivity.

[0074] 3. Detection mechanism of Cordyceps militaris polysaccharide fluorescent carbon dots

[0075] In order to clarify the mechanism of TC-induced quenching of the fluorescent carbon dots of Cordyceps militaris polysaccharide, the excitation spectrum (Ex of Zn, Cl-CDs), emission spectrum (Em of Zn, Cl-CDs) of the fluorescent carbon dots of Cordyceps militaris polysaccharide and the UV-visible absorption spectrum of TC were detected. Figure 5 As shown in the figure, the TC absorption band at 357 nm partially overlaps with the excitation spectrum of the Cordyceps militaris polysaccharide fluorescent carbon dots. The results show that the TC-induced fluorescence quenching of the Cordyceps militaris polysaccharide fluorescent carbon dots is a stable quenching mechanism based on the inner filter effect (IFE).

[0076] The present invention uses Cordyceps militaris polysaccharide as a carbon source, adds ZnCl2 and water, and uses a one-step hydrothermal method to prepare blue-luminescent zinc-chloride co-doped carbon quantum dots (Zn,Cl-CDs). After the carbon quantum dots interact with tetracycline antibiotics (TCs), the internal filter effect (IFE) gradually quenches the blue fluorescence of the carbon quantum dots, while the characteristic green fluorescence of the zinc- and chlorine-doped elements gradually increases, resulting in a fluorescence signal that varies based on the type and content of the tetracycline antibiotic. The fluorescent carbon dots have a recognition space that specifically matches the TCs molecule. After recognizing TCs, the blue fluorescence emission spectrum of the fluorescent carbon dots at 434nm is gradually quenched, and different degrees of red shift occur depending on the type of TC. This allows for highly selective, visual on-site detection and differentiation of TCs molecules.

[0077] 4. Detection of tetracycline antibiotics using Cordyceps militaris polysaccharide fluorescent carbon dots combined with smartphones

[0078] 4.0 mg of the Cordyceps militaris polysaccharide fluorescent carbon dots prepared in Example 2 were added to 4 mL of an aqueous solution of tetracycline antibiotics of the same concentration (100 μM) and mixed evenly.

[0079] The mixed solution was then transferred to a cuvette, and a color scanning application (APP: colorimeter) was downloaded from the app store on a smartphone. The color of the carbon dots in the standard solution with the same tetracycline antibiotic concentration under ultraviolet light was photographed, and the obtained fluorescence color was digitized and output to obtain RGB values. The obtained experimental data was used for principal component analysis to obtain a PCA graph, as shown in Figure 2. Figure 6 shown.

[0080] Figure 6 a shows that at this concentration, the fluorescence colors of tetracycline antibiotics are significantly different, and the type of tetracycline antibiotics can be determined by visually identifying the fluorescence color. Figure 6 b shows that the change of fluorescence color of tetracycline antibiotics with concentration is a gradual process, and the change is obvious within 0-50 μM.

[0081] The Cordyceps militaris polysaccharide fluorescent carbon dots prepared in Example 2 are mixed with the sample solution to be tested (containing TC, MC, OTC, at a concentration of 100 μM) in a 1: 1 (mg: mL) ratio, and mixed evenly; The fluorescent color of the gel balls under ultraviolet light in the solution to be tested is photographed using a color scanning application program of a smart phone, and digitized and output to obtain RGB values; PCA graphs are obtained after principal component analysis. Result was compared with the PCA graph of 100 μM tetracycline antibiotic standard solution, and the type of antibiotic was accurately determined according to the position where the data was located. Simultaneously, the fluorescent color of the solution to be tested under 365 nm ultraviolet irradiation was observed with the naked eye and the type of antibiotic was successfully determined.

[0082] 5. Effects of different ions and amino acids on the Cordyceps militaris polysaccharide fluorescent carbon dot mixed system

[0083] The Cordyceps militaris polysaccharide fluorescent carbon dot prepared by 4.0mg embodiment 2 is added in 4mL tetracycline aqueous solution (100 μM), and the different ions and amino acid aqueous solution (concentration 50 μM) of 2000 μL are added respectively, and with the Cordyceps militaris polysaccharide fluorescent carbon dot mixed system without adding any ion and amino acid as control (Blank).Then the mixed solution is transferred to a cuvette, and the emission intensity is measured with the excitation wavelength of 366nm, and the emission spectrum of the mixture is recorded. The slit width of emission and excitation is 10nm.Record experimental data calculates F / F0, and makes a bar graph.

[0084] The test results are as follows Figure 7 As shown:

[0085] Depend on Figure 7 It can be seen that ions and amino acids have little effect on the fluorescence intensity of the sample.

[0086] 6. Photocatalytic degradation of tetracycline antibiotics

[0087] 4.0 mg of the Cordyceps militaris polysaccharide fluorescent carbon dots prepared in Example 2 were added to 4 mL of an aqueous solution of tetracycline antibiotics of the same concentration (100 μM) and mixed well. The mixture was placed in a dark environment and kept away from light for 24 h. The mixed solution was then transferred to a watch glass and then placed under a xenon lamp for uniform illumination for 24 min. The ultraviolet spectrum of the solution was recorded in real time, as shown in FIG. Figure 8 shown.

[0088] Combined with the standard curve of tetracycline antibiotic concentration and absorbance, the absorbance at the peak around 370nm indicates the concentration of tetracycline antibiotics, and the concentration c of tetracycline antibiotics is obtained in real time. t , the initial concentration is set to c0;

[0089] The experimental data were sorted and plotted as ln c t / c0 as the ordinate and time as the abscissa, and obtain the time and ln c t / c0 linear equation, such as Figure 9 shown.

[0090] The results of photocatalytic degradation of tetracycline antibiotics are shown in Table 2:

[0091] Table 2 Degradation rate of tetracycline antibiotics

[0092]

[0093]

[0094] From the above table and picture results, we can see that Cordyceps militaris polysaccharide fluorescent carbon dots can achieve rapid degradation of tetracycline antibiotics within 24 minutes, and its degradation rate conforms to pseudo-first-order kinetics, with a correlation coefficient R 2 >0.986.

[0095] 7. Degradation of Tetracycline Antibiotics in Milk

[0096] Before the experiment, the milk was pretreated, i.e., the protein precipitation procedure: an equal volume (5 mL) of milk and a 1% (v / v) trichloroacetic acid solution were thoroughly mixed, and the mixed sample was then ultrasonically treated at 40°C for 20 min and transferred to a centrifuge tube. After centrifugation at 12,000 rpm for 8 min, the milk extract supernatant was obtained. The pH was adjusted to neutral with an aqueous sodium hydroxide solution. Tetracycline antibiotics were added to the above-mentioned milk extract supernatant, and a series of standard milk sample solutions containing different concentrations of TCs (0-100 μM) were prepared. The Cordyceps militaris polysaccharide fluorescent carbon dots prepared in Example 2 were then added thereto at a ratio of 1:1 (mg:mL); after mixing evenly; incubated at room temperature for 30 min, and the ultraviolet spectrum was measured. The concentration of tetracycline antibiotics was indicated by the absorbance at the peak around 370 nm, and the concentration c of tetracycline antibiotics was obtained in real time. t , the initial concentration is set to c0.

[0097] The obtained experimental data were arranged and plotted, with absorbance as the ordinate and antibiotic concentration as the abscissa, to obtain a linear equation of antibiotic concentration and absorbance.

[0098] The milk sample solution to be tested was then treated with the sample pretreatment steps described above and then mixed with the Cordyceps militaris polysaccharide fluorescent carbon dots prepared in Example 2 at a 1:1 (mg:mL) ratio. After uniform mixing, the solution was incubated at room temperature for 30 minutes. The mixed solution was transferred to a watch glass and then placed under a xenon lamp for uniform illumination for 24 minutes. The UV spectrum of the solution was recorded. The degradation rate of tetracycline antibiotics was calculated based on the standard curve described above.

[0099] The test results are shown in Table 3:

[0100] Table 3 Degradation rate of tetracycline antibiotics in milk samples

[0101]

[0102]

[0103] As shown in Table 3, the fluorescent carbon dots of Cordyceps militaris polysaccharide have an obvious degradation effect on TCs in milk samples.

[0104] 8. Degradation Mechanism of Cordyceps militaris Polysaccharide Fluorescent Carbon Dots

[0105] In order to study the degradation mechanism of tetracycline antibiotics by fluorescent carbon dots of Cordyceps militaris polysaccharide, tert-butyl alcohol (TBA), benzoquinone (BQ), furfuryl alcohol (FFA) and ammonium oxalate (AO) were added as ·OH, O 2- ·、 1 O2 and H + The effects of different free radicals on the degradation rate of tetracycline antibiotics were studied using a quencher. The specific steps are as follows:

[0106] Cordyceps militaris polysaccharide fluorescent carbon dot prepared by 4.0mg embodiment 2 is added in the tetracycline antibiotic aqueous solution of 4mL same concentrations (100 μM), mix homogeneously.It is identical to control the kind of tetracycline antibiotic, and tert-butyl alcohol (TBA), benzoquinone (BQ), ammonium oxalate (AO), furfuryl alcohol (FFA) are added respectively, and the reagent concentration added is 3.2 μM, with the solution without adding any quencher as control group.Gained solution is placed in dark environment dark treatment 24h.Solution is transferred in watch glass, is then placed in xenon lamp and carries out uniform illumination, and light application time is 24min, the ultraviolet spectrum of real-time recording solution.With reference to the standard curve of tetracycline antibiotic concentration and absorbance, with the absorbance indication tetracycline antibiotic concentration at 370nm left and right peak value, calculate the degradation rate after illumination.

[0107] The test results are as follows Figure 10 As shown:

[0108] Depend on Figure 10 It can be seen that the mechanism of degradation of TCs by Cordyceps militaris polysaccharide fluorescent carbon dots is ·OH, O 2-·、 1 O2 and H + The result of a combined effect, but the main effect is 1 The extra positive charge inside the carbon dots leads to the generation of more unpaired electrons, which promotes the formation of singlet oxygen ( 1 O2) formation. 1 The long lifetime of O2 is conducive to the degradation of dilute solutions, which explains the high degradation rate of Cordyceps militaris polysaccharide fluorescent carbon dots.

[0109] The above description is merely a preferred embodiment of the present invention and is not intended to limit the present invention in any other manner. Any person skilled in the art may utilize the above-disclosed technical content to modify or modify the present invention into equivalent embodiments. However, any simple modifications, equivalent variations, and modifications to the above embodiments that do not depart from the technical content of the present invention and are based on the technical essence of the present invention remain within the scope of protection of the present invention.

Claims

1. A Cordyceps militaris polysaccharide fluorescent carbon dot for detecting and degrading tetracycline antibiotics, characterized in that: The fluorescent carbon dots are blue fluorescent quantum dots that use Cordyceps militaris polysaccharide as a carbon source and are co-doped with zinc and chlorine.

2. The Cordyceps militaris polysaccharide fluorescent carbon dots according to claim 1, characterized in that: The Cordyceps militaris polysaccharide fluorescent carbon dots are prepared by the following method: Dissolving Cordyceps militaris polysaccharide and zinc chloride in water and then subjecting them to a high-temperature reaction; after the reaction is complete, filtering, dialysis, and drying to obtain Cordyceps militaris polysaccharide fluorescent carbon dots; The high temperature reaction conditions are selected from: hydrothermal reaction at 160-240° C. for 8-14 hours.

3. The Cordyceps militaris polysaccharide fluorescent carbon dots according to claim 2, characterized in that: The raw materials are selected from the following parts by weight: 0.1-0.5 parts of Cordyceps militaris polysaccharide, 0.14-0.8 parts of zinc chloride, and 5-15 parts of water.

4. Use of the Cordyceps militaris polysaccharide fluorescent carbon dots according to any one of claims 1 to 3 in the detection or degradation of tetracycline antibiotics.

5. The use according to claim 4, characterized in that The tetracycline antibiotic is selected from one or more of minocycline, tetracycline, chlortetracycline, oxytetracycline and doxycycline.

6. A method for detecting tetracycline antibiotics, characterized in that: The steps include: The Cordyceps militaris polysaccharide fluorescent carbon dots according to any one of claims 1 to 3 are mixed with tetracycline antibiotic standard solutions of different concentration gradients, and then the emission intensity is measured at an excitation wavelength of 366 nm, and the fluorescence intensity at the peak is recorded; a graph is drawn with the fluorescence intensity at the peak as the ordinate and the tetracycline antibiotic concentration as the abscissa to obtain a linear equation; The test sample solution was then mixed with the Cordyceps militaris polysaccharide fluorescent carbon dots, and the emission intensity was measured at an excitation wavelength of 366 nm. The fluorescence intensity at the peak of the test solution was recorded and substituted into the linear equation to obtain the concentration of tetracycline antibiotics in the test sample solution.

7. A detection method for distinguishing tetracycline antibiotics, characterized in that: The steps include: Mixing the Cordyceps militaris polysaccharide fluorescent carbon dots according to any one of claims 1 to 3 with aqueous solutions of various tetracycline antibiotics at the same concentration, then photographing the color of each carbon dot solution under ultraviolet light using a colorimeter app on a smartphone, digitizing and outputting the obtained fluorescent color to obtain RGB values; performing principal component analysis on the obtained experimental data to obtain a PCA graph; and analyzing differences in fluorescent color using PCA; The sample solution to be tested was mixed with the fluorescent carbon dots of Cordyceps militaris polysaccharide. The color of the carbon dots in each solution was then photographed under ultraviolet light using the colorimeter app on a smartphone. The obtained fluorescent color was digitized and output to obtain RGB values. The types of antibiotics in the sample solution were obtained after principal component analysis.

8. A method for degrading tetracycline antibiotics, characterized in that: The steps include: The Cordyceps militaris polysaccharide fluorescent carbon dots according to any one of claims 1 to 3 are mixed with a sample solution containing tetracycline antibiotics, and then subjected to light treatment to achieve degradation of the tetracycline antibiotics.

9. The degradation method according to claim 8, characterized in that Xenon lamp was used for illumination treatment.

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