Method and kit for cooperatively detecting tetracycline antibiotics in water body based on multi-factor microenvironment

Abstract

The invention relates to a method and kit for cooperatively detecting tetracycline antibiotics in a water body based on a multi-factor microenvironment, and belongs to the field of chemical substance detection.The method comprises the steps that a carbon quantum dot probe solution and a multi-factor microenvironment solution are mixed, and a detection solution is obtained; mixing the detection solution and a to-be-detected object in a buffer solution to obtain a mixed solution, and detecting the mixed solution through a fluorescence spectrophotometer; wherein microenvironment factors in the multi-factor microenvironment solution comprise one or a combination of more of metal ions, protein and a surfactant; the tetracycline antibiotics comprise one or a combination of more of tetracycline, oxytetracycline, aureomycin and doxycycline. The multi-factor microenvironment substance is introduced and combined with the carbon quantum dot probe to detect the tetracycline antibiotics, so that the response speed is high, and the detection sensitivity and the anti-interference capability are further improved.

Description

Technical Field

[0001] The present invention relates to the field of chemical substance detection, and in particular to a method and a kit for detecting tetracycline antibiotics in water based on multi-factor microenvironment synergy. Background Art

[0002] Tetracycline antibiotics include tetracycline, chlortetracycline, oxytetracycline, doxycycline, etc., which are widely used in modern medicine and livestock and poultry breeding. The problem of their residues in the water environment is becoming increasingly prominent, posing a huge threat to the ecosystem and public health. In view of this severe situation, it is urgent to accurately and efficiently detect antibiotic residues and establish sensitive, rapid and reliable analytical methods to study the concentration, distribution and migration of antibiotics, so as to provide a more scientific and effective basis for water pollution control and environmental remediation. However, traditional detection methods mainly rely on technologies such as chromatography and enzyme-linked immunosorbent assay (ELISA), which usually require large-scale analytical equipment, and the cost of instrument operation and maintenance is high, which makes it difficult to meet the needs of on-site screening or emergency monitoring.

[0003] The Chinese patent with publication number CN 115558494 A discloses a carbon quantum dot for detecting mercury ions and oxytetracycline, a preparation method and an application thereof, wherein the specific steps are to mix black fungus with thiourea, react to obtain carbon quantum dots, and cool, coarsely filter, centrifuge and dialyze the obtained carbon quantum dot probe solution before using it to detect oxytetracycline and mercury ions in a pure water medium. However, the carbon quantum dots prepared by this method require the addition of a chemical substance (thiourea) with a special odor to the precursor, and if the sample contains both mercury ions and oxytetracycline, the selective detection of tetracycline antibiotics cannot be achieved.

[0004] At present, the detection method of tetracycline antibiotics based on optical analysis mainly focuses on the interaction between the target pollutant and a single probe to change the physical and chemical properties of the probe, thereby affecting its luminescence intensity, luminescence lifetime and spectral position. However, due to the complex matrix of the water environment, the interaction between different substances may lead to problems such as poor detection selectivity, low sensitivity and low detection efficiency, which limits its application in rapid on-site detection. Summary of the invention

[0005] In order to efficiently, quickly and sensitively detect tetracycline antibiotics, the present invention proposes a method and a kit for detecting tetracycline antibiotics in water based on multi-factor microenvironment synergy, which improves the detection sensitivity and specificity simultaneously through the synergy brought by the multi-factor microenvironment and the probe. The method is not only simple to operate, but also has high detection efficiency and low cost, providing technical support for the rapid monitoring of tetracycline antibiotics in water.

[0006] The purpose of the present invention can be achieved by the following technical solutions:

[0007] One of the purposes of the present invention is to provide a method for detecting tetracycline antibiotics in water based on multi-factor microenvironment synergy, comprising: mixing a carbon quantum dot probe solution with a multi-factor microenvironment solution to obtain a detection solution; mixing the detection solution and a test object in a buffer solution to obtain a mixed solution, and detecting the mixed solution by a fluorescence spectrometer;

[0008] Among them, the microenvironment factors in the multi-factor microenvironment solution include a combination of one or more of metal ions, proteins, and surfactants; the tetracycline antibiotics include a combination of one or more of tetracycline, oxytetracycline, chlortetracycline, and doxycycline.

[0009] Furthermore, the preparation process of the carbon quantum dot probe solution is: using biomass material as a precursor, preparing carbon quantum dot probes by a one-step hydrothermal method, filtering with a filter membrane and dialysis purification to obtain a carbon quantum dot probe solution.

[0010] Specifically, the biomass material is ultrasonically cleaned in water and air-dried until completely dry, and then cut into 4-6 cm raw material segments. The raw material segments are weighed into deionized water and placed in a reactor, the temperature is maintained at 150-200° C., and the reaction time is 6-10 hours.

[0011] Furthermore, the biomass material is a leaf rich in nitrogen and oxygen-containing functional groups, such as Radix Ophiopogonis, P. aceriflora or other suitable plant leaves.

[0012] Furthermore, the metal ion is selected from one or more of magnesium ion, zinc ion, and yttrium ion; the protein is selected from one or more of bovine serum albumin, human serum albumin, and lactoferrin; and the surfactant is selected from one or more of dodecyltrimethylammonium bromide and hexadecyltrimethylammonium bromide.

[0013] Furthermore, the microenvironmental factors in the multi-factor microenvironmental solution are a mixture of human serum albumin and magnesium ions.

[0014] Furthermore, the concentration of each microenvironment factor in the multi-factor microenvironment solution is 20 μM-3 mM, and the concentration of the carbon quantum dot probe in the carbon quantum dot probe solution is 5-20 mg / L.

[0015] Furthermore, the concentration of the carbon quantum dot probe in the carbon quantum dot probe solution is 2-20 mg / L.

[0016] Furthermore, the buffer is any one of 4-hydroxyethylpiperazineethanesulfonic acid, tris(hydroxymethylaminomethane) or phosphate buffer, preferably phosphate buffer.

[0017] Furthermore, the pH of the mixed solution is 8.0-10.0, and the reaction time of the mixed solution is 1-3 min. The pH value of the solution is adjusted to 9.5, the reaction time is 1-3 min, and the fluorescence signal is selected as the ratio of the fluorescence intensity at a wavelength of 520 nm to that at 425 nm.

[0018] Furthermore, the analytes can be taken from rivers near industrial parks, rivers near aquaculture farms, and rivers near residential areas, respectively. The water samples are filtered through a 0.22 μm water filter membrane, and the pH value thereof is adjusted to 9.0.

[0019] Furthermore, the potential interfering substances in the analyte include inorganic ions (silver ions, nitrate ions, sulfate ions, etc.), biological molecules (glucose, glutathione, cysteine, etc.), and various antibiotics (kanamycin, azithromycin, ciprofloxacin, erythromycin, chloramphenicol, etc.), and their concentrations are 10-50 times that of tetracycline antibiotics, and the concentration of humic acid is 20 mg / L.

[0020] The second object of the present invention is a kit for detecting tetracycline antibiotics, the kit comprising a disposable sampling tube and filter paper arranged in the disposable sampling tube;

[0021] The filter paper is a filter paper soaked in ethylenediaminetetraacetic acid solution and dried; the kit is encapsulated in a disposable sampling tube by a hydrophobic sealing tape partition, wherein the mixture of the carbon quantum dot probe solution, the multi-factor microenvironment solution and the buffer solution is encapsulated in the disposable sampling tube;

[0022] Wherein, the mixture of the carbon quantum dot probe solution, the multi-factor microenvironment solution and the buffer solution is the carbon quantum dot probe solution, the multi-factor microenvironment solution and the buffer solution as described above;

[0023] The kit is used to detect tetracycline antibiotics by the detection method described above.

[0024] Specifically, the kit of the present invention comprises a sampler, a carbon quantum dot probe solution, a hydrophobic sealing tape and an auxiliary liquid reagent, wherein the auxiliary liquid reagent is a mixture of a multi-factor microenvironment solution and a buffer. The construction of the kit includes soaking the filter paper in an ethylenediaminetetraacetic acid solution overnight, drying it naturally at room temperature, and then plugging it into the sampler; the probe and the auxiliary liquid reagent are encapsulated inside a disposable sampling tube by using a hydrophobic sealing tape partition. The detection of tetracycline antibiotics in water includes the following steps: adding 200 μL of water sample to the scale line in the sampling tube, then inserting the auxiliary liquid (comprising a microenvironment factor solution and a buffer), oscillating and mixing for 1-3 minutes, and comparing the color interval with the standard colorimetric card, that is, realizing semi-quantitative rapid detection of tetracycline antibiotics.

[0025] Mechanism of action :

[0026] The biomass-based carbon quantum dots prepared by the present invention use natural plant leaves as raw materials. The inherent oxygen, nitrogen, hydrogen and other elements in the leaves make the synthesized biomass-based carbon quantum dots contain rich functional groups. In addition, the prepared biomass-based carbon quantum dots realize self-doping of elements such as nitrogen, sulfur or phosphorus, which is very important for introducing higher density structural defects into carbon quantum dot probes, optimizing the chemical structure of carbon quantum dots, providing more surface groups and effective binding sites for hydrogen bonding, electrostatics, π-π stacking and other interactions with tetracycline antibiotics.

[0027] As alkalinity increases, tetracycline antibiotics will be deprotonated, and the proton transfer process in the excited state molecule will be inhibited, causing the emission spectrum to blue-shift and the ultraviolet absorption spectrum to red-shift. The introduction of microenvironmental factors, including metal ions, proteins, surfactants, etc., will enhance the green fluorescence of tetracycline antibiotics through interaction forces such as coordination, sensitization, and hydrophobic effects. At the same time, the binding effect between microenvironmental factors and tetracycline antibiotics will change the ultraviolet-visible absorption spectrum of tetracycline antibiotics and promote the energy transfer between biomass-based carbon quantum dots and tetracycline antibiotic-microenvironmental factor complexes, thereby triggering a significant ratiometric fluorescence signal response.

[0028] Compared with the prior art, the present invention has the following advantages:

[0029] (1) The present invention introduces multi-factor microenvironment substances and combines them with carbon quantum dot probes to detect tetracycline antibiotics, which not only has a faster response speed but also further improves the detection sensitivity and anti-interference ability.

[0030] (2) The present invention uses cheap, readily available, and environmentally friendly biomass materials as precursors to prepare carbon quantum dot probes with high dispersion and good stability without adding external heteroatoms, which can achieve high-value utilization of natural resources and contribute to environmental protection.

[0031] (3) The kit provided by the present invention has high detection efficiency, low cost, and simple operation. It can be used for rapid semi-quantitative detection of tetracycline antibiotics (such as oxytetracycline, chlortetracycline, tetracycline, doxycycline, etc.) in water bodies on site, and has good promotion value and application prospects. BRIEF DESCRIPTION OF THE DRAWINGS

[0032] Figure 1 is a transmission electron microscope photograph of the biomass-based carbon quantum dots provided in Preparation Example 1 of the present invention;

[0033] Figure 2 is an infrared spectrum of the biomass-based carbon quantum dots provided in Preparation Example 1 of the present invention;

[0034] Figure 3 It is the fluorescence emission spectrum and fluorescence photograph of the biomass-based carbon quantum dots provided in Preparation Example 1 of the present invention;

[0035] Figure 4 is the oxytetracycline detection result provided in Example 1 of the present invention;

[0036] Figure 5 This is a graph showing the detection results of oxytetracycline at different concentrations provided in Example 1 of the present invention;

[0037] Figure 6 The kit component provided in Example 4 of the present invention;

[0038] Figure 7 This is the test result of the kit provided in Example 4 of the present invention. DETAILED DESCRIPTION

[0039] The present invention is described in detail below in conjunction with the accompanying drawings and specific embodiments. This embodiment is implemented based on the technical solution of the present invention, and provides a detailed implementation method and specific operation process, but the protection scope of the present invention is not limited to the following embodiments.

[0040] Preparation Example 1: Preparation of Biomass-based Carbon Quantum Dots

[0041] Step 1: Ultrasonic clean the Ophiopogon japonicus leaves in water and air-dry until completely dry, cut into 5 cm long raw material segments, weigh the raw material segments and add them to deionized water, wherein the added amount of the raw material segments is 0.5 g, and the added amount of deionized water is 12 mL. Place the above mixture in a reactor and react at 160°C for 8 hours.

[0042] Step 2: After the reaction in step 1 is completed, the mixture is cooled naturally to room temperature, the supernatant is collected, filtered using a 0.22 μm filter membrane, and dialyzed continuously in deionized water for 24 h, wherein the molecular weight cutoff of the dialysis bag is 500 Da. The solution in the dialysis bag is collected and stored in a refrigerator at 4°C.

[0043] The samples obtained in Preparation Example 1 were tested and characterized.

[0044] refer to Figure 1 The transmission electron microscope photos in the figure show that the biomass-based carbon quantum dots are uniform in size, well dispersed, and have a particle size of 2-3nm. Figure 2 This is the infrared spectrum of biomass-based carbon quantum dots. -1 The characteristic peak at 1650cm is the stretching vibration of OH / NH -1 The characteristic peak at 1074 cm corresponds to the stretching vibration of C=O. -1The characteristic peak at is the stretching vibration of CO, which proves that there are abundant functional groups such as hydroxyl and carboxyl on the surface of carbon quantum dots. Figure 3 The fluorescence spectrum and fluorescence photograph of the sample show that the biomass-based carbon quantum dots emit bright blue fluorescence under ultraviolet light.

[0045] Preparation Example 2: Preparation of Biomass-based Carbon Quantum Dots

[0046] Step 1: Ultrasonic clean the leaves of Platanus pyrifera in water and air-dry them until completely dry, cut them into 5 cm long raw material segments, weigh the raw material segments and add them to deionized water, wherein the added amount of the raw material segments is 1.0 g, and the added amount of deionized water is 15 mL. Place the above mixture in a reactor and react at 150° C. for 10 hours.

[0047] Step 2: After the reaction is completed, cool naturally to room temperature, collect the supernatant, filter using a 0.22 μm filter membrane and dialyze continuously in deionized water for 48 hours, wherein the molecular weight cutoff of the dialysis bag is 1000 Da. Collect the solution in the dialysis bag and store it in a refrigerator at 4°C.

[0048] Example 1: Biomass-based carbon quantum dots combined with multi-factor microenvironment detection of oxytetracycline

[0049] Step 1: Prepare a biomass-based carbon quantum dot probe solution according to the steps in Preparation Example 1, mix 50 μL of human serum albumin solution (200 μM), 50 μL of magnesium ion solution (3 mM) and 10 μL of carbon quantum dot probe solution, add the test solution, and then dilute to 1 mL with 4-hydroxyethylpiperazineethanesulfonic acid buffer, adjust the pH of the solution to 9.0, and the final concentration of oxytetracycline in the mixed solution is 5 μM. After the above solutions are evenly mixed, stand at room temperature for 1 min, measure the fluorescence spectrum of the solution, and quantify it using the change in fluorescence response, where the fluorescence response is the ratio of the fluorescence intensity at a wavelength of 520 nm to that at a wavelength of 425 nm.

[0050] Step 2: Working curve and anti-interference ability test of the determination method:

[0051] A linear equation between oxytetracycline concentration and fluorescence intensity ratio was established. Figure 5 It can be seen that the linear range of the detection method for oxytetracycline is 0.01-20μM, and the minimum detection limit is 3.3nM.

[0052] The detection method detects potential interfering substances in water bodies, among which potential interfering substances include inorganic ions (calcium ions, magnesium ions, copper ions, etc.), biological molecules (glucose, glutathione, cysteine, etc.), and various antibiotics (kanamycin, azithromycin, ciprofloxacin, erythromycin, chloramphenicol, etc.). Their concentrations are 10-50 times that of oxytetracycline, and the concentration of humic acid is 20 mg / L.

[0053] Example 2: Rapid detection of doxycycline in water by biomass-based carbon quantum dots combined with multi-factor microenvironment

[0054] Step 1: Prepare a biomass-based carbon quantum dot probe solution according to the steps in Preparation Example 1, mix 100 μL of dodecyl trimethyl ammonium bromide solution, 50 μL of yttrium ion solution and 10 μL of carbon quantum dot probe solution, add doxycycline test solution, and then dilute to 1 mL with phosphate buffer, and adjust the pH of the solution to 9.5. Among them, the final concentrations of dodecyl trimethyl ammonium bromide and yttrium ions are both 1 mM. After the above solutions are evenly mixed, stand at room temperature for 1 minute, measure the fluorescence spectrum of the solution, and use the change in fluorescence response for quantification, wherein the fluorescence response uses the ratio of the fluorescence intensity at a wavelength of 520 nm to that at a wavelength of 425 nm.

[0055] Step 2: Working curve and anti-interference ability test of the determination method:

[0056] A linear equation between oxytetracycline concentration and fluorescence intensity ratio was established, and the detection effect of the method on potential interfering substances in water bodies was determined.

[0057] Example 3: Detection of tetracycline in different water samples

[0058] Environmental water samples were taken from a river near an industrial park, a river near an aquaculture farm, and a river near a residential area. The water samples were filtered through a 0.22 μm water filter membrane, and the pH was adjusted to 9.0. Referring to step 1 in Example 2, the tetracycline concentration in the sample was measured and analyzed, and the precision of the detection method was measured by the recovery rate and relative standard deviation.

[0059] Example 4: Portable kit for detecting oxytetracycline in water

[0060] Step 1: Select tea leaves as the biomass raw material and prepare biomass-based carbon quantum dots according to the steps in Preparation Example 1.

[0061] Step 2: Construct a detection kit, which includes soaking filter paper in EDTA solution overnight and drying it naturally at room temperature, and then inserting it into the sampling tube; using hydrophobic sealing tape to separate the carbon quantum dot probe solution and auxiliary reagents in the sampling tube.

[0062] Step 3: Utilize the kit in this embodiment to analyze and detect oxytetracycline in water, and the kit mainly comprises the following steps during detection: add 200 μL water sample to the scale line in the sampling tube, then insert into the auxiliary liquid (comprising lactoferrin solution, zinc ion solution and buffer solution), after mixing by pipetting for 1-3 minutes, compare the color interval with the standard colorimetric card, and realize the semi-quantitative rapid detection of oxytetracycline.

[0063] The biomass-based carbon quantum dots prepared by the present invention use natural plant leaves as raw materials. The inherent oxygen, nitrogen, hydrogen and other elements in the leaves make the synthesized biomass-based carbon quantum dots contain rich functional groups. In addition, the prepared biomass-based carbon quantum dots realize self-doping of elements such as nitrogen, sulfur or phosphorus, which is very important for introducing higher density structural defects into carbon quantum dot probes, optimizing the chemical structure of carbon quantum dots, providing more surface groups and effective binding sites for hydrogen bonding, electrostatics, π-π stacking and other interactions with tetracycline antibiotics.

[0064] As alkalinity increases, tetracycline antibiotics will be deprotonated, and the proton transfer process in the excited state molecule will be inhibited, causing the emission spectrum to blue-shift and the ultraviolet absorption spectrum to red-shift. The introduction of microenvironmental factors, including metal ions, proteins, surfactants, etc., will enhance the green fluorescence of tetracycline antibiotics through interaction forces such as coordination, sensitization, and hydrophobic effects. At the same time, the binding effect between microenvironmental factors and tetracycline antibiotics will change the ultraviolet-visible absorption spectrum of tetracycline antibiotics and promote the energy transfer between biomass-based carbon quantum dots and tetracycline antibiotic-microenvironmental factor complexes, thereby triggering a significant ratiometric fluorescence signal response.

[0065] In summary, the method for detecting tetracycline antibiotics in water based on multi-factor microenvironment synergistically provided by the present invention can use natural green biomass as raw material to prepare carbon quantum dot probes, thereby realizing high-value utilization of natural resources; at the same time, it can give play to the synergistic sensing effect of multi-factor microenvironment to further improve detection sensitivity and anti-interference ability; the detection kit is easy to operate, low-cost, and portable, which is helpful to realize on-site rapid detection of antibiotic pollutants in resource-scarce areas, and has broad application prospects in real-time analysis and emergency monitoring of water environment.

[0066] The above is only a preferred embodiment of the present invention, and does not limit the present invention in other forms. Any technician familiar with the profession may use the above disclosed technical content to change or modify it into an equivalent embodiment with equivalent changes. However, any simple modification, equivalent change and modification made to the above embodiment according to the technical essence of the present invention without departing from the technical solution of the present invention still belongs to the protection scope of the technical solution of the present invention.

Claims

1. A method for detecting tetracycline antibiotics in water based on multi-factor microenvironment synergy, characterized in that: include: Mixing the carbon quantum dot probe solution with the multi-factor microenvironment solution to obtain a detection solution; The test solution and the test object are mixed in a buffer solution to obtain a mixed solution, and the mixed solution is tested by a fluorescence spectrometer; Among them, the microenvironment factors in the multi-factor microenvironment solution include a combination of one or more of metal ions, proteins, and surfactants; the tetracycline antibiotics include a combination of one or more of tetracycline, oxytetracycline, chlortetracycline, and doxycycline.

2. The method for detecting tetracycline antibiotics in water based on multi-factor microenvironment synergy according to claim 1, characterized in that: The preparation process of the carbon quantum dot probe solution is as follows: using biomass material as a precursor, preparing carbon quantum dot probes by a one-step hydrothermal method, filtering with a filter membrane and dialysis purification to obtain the carbon quantum dot probe solution.

3. The method for detecting tetracycline antibiotics in water based on multi-factor microenvironment synergy according to claim 2, characterized in that: The biomass material is leaves rich in nitrogen and oxygen-containing functional groups.

4. The method for detecting tetracycline antibiotics in water based on multi-factor microenvironment synergy according to claim 1, characterized in that: The metal ions are selected from one or more of magnesium ions, zinc ions, and yttrium ions; The protein is selected from one or more of bovine serum albumin, human serum albumin, and lactoferrin; The surfactant is selected from one or more of dodecyltrimethylammonium bromide and hexadecyltrimethylammonium bromide.

5. The method for detecting tetracycline antibiotics in water based on multi-factor microenvironment synergy according to claim 4, characterized in that: The microenvironmental factors in the multi-factor microenvironmental solution are a mixture of human serum albumin and magnesium ions.

6. The method for detecting tetracycline antibiotics in water based on multi-factor microenvironment synergy according to claim 1, characterized in that: The concentration of each microenvironment factor in the multi-factor microenvironment solution is 20 μM-3 mM, and the concentration of the carbon quantum dot probe in the carbon quantum dot probe solution is 5-20 mg / L.

7. The method for detecting tetracycline antibiotics in water based on multi-factor microenvironment synergy according to claim 1, characterized in that: The concentration of the carbon quantum dot probe in the carbon quantum dot probe solution is 2-20 mg / L.

8. The method for detecting tetracycline antibiotics in water based on multi-factor microenvironment synergy according to claim 1, characterized in that: The buffer is any one of 4-hydroxyethylpiperazineethanesulfonic acid, tris(hydroxymethyl)aminomethane or phosphate buffer.

9. The method for detecting tetracycline antibiotics in water based on multi-factor microenvironment synergy according to claim 1, characterized in that: The pH of the mixed solution is 8.0-10.0, and the reaction time of the mixed solution is 1-3 minutes.

10. A kit for detecting tetracycline antibiotics, characterized in that: The kit comprises a disposable sampling tube and filter paper arranged in the disposable sampling tube; The filter paper is a filter paper soaked in ethylenediaminetetraacetic acid solution and dried; the kit is encapsulated in a disposable sampling tube by a hydrophobic sealing tape partition, wherein the mixture of the carbon quantum dot probe solution, the multi-factor microenvironment solution and the buffer solution is encapsulated in the disposable sampling tube; The kit is used for detecting tetracycline antibiotics by the detection method described in any one of claims 1-9.

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