A method for detecting tetracycline hydrochloride concentration in water using microbially synthesized carbon dots

Abstract

The present invention provides a method for detecting the concentration of tetracycline hydrochloride in water by using microbially synthesized carbon dots, comprising the following steps: (1) culturing a strain (Meyerozyma guilliermondii PG-1) in LB medium to prepare biocarbon dots (CDs); (1) drawing a standard curve: taking 40 μL of a CDs solution with a mass concentration of 44.32 g / L in an EP tube, then adding 960 μL of tetracycline hydrochloride solutions of different known concentrations, measuring the fluorescence intensity under the conditions of Ex=354 nm and Em=432 nm using an enzyme marker, calculating the fluorescence intensity ratio F / F0, and obtaining a standard curve in which (F / F0) is linearly related to the tetracycline hydrochloride concentration; (3) determining the concentration of tetracycline hydrochloride in a water sample to be tested: taking 40 μL of a CDs solution with a mass concentration of 44.32 g / L in an EP tube, then adding 960 μL of a water sample to be tested, repeating the determination method in step (2), and obtaining the tetracycline hydrochloride concentration by comparing with the standard curve in step (2); calculating the average value, and obtaining the result. The invention can be used for rapid detection of tetracycline hydrochloride in various water samples, has strong anti-interference ability, and is not interfered by other antibiotics and metal ions.

Description

Technical Field

[0001] The present invention relates to the field of water quality detection, and in particular to a method for detecting the concentration of tetracycline hydrochloride in water using microbially synthesized carbon dots (CDs) as fluorescent probes. Background Art

[0002] Since the advent of antibiotics, their uncontrolled use has caused a series of serious problems. Antibiotic residues not only pollute aquatic environments but also pose a threat to human health, making detection of these contaminants in aquatic environments essential. Tetracycline (TC) is a widely used broad-spectrum antibiotic. Common detection methods for tetracycline include microbiological testing, immunological analysis, and high-performance liquid chromatography. However, microbiological testing is time-consuming and lacks sensitivity; immunological analysis requires complex sample pretreatment and expensive instrumentation; and high-performance liquid chromatography has high maintenance costs, poor reproducibility, and difficult instrument operation. These shortcomings limit its application in the environment.

[0003] Fluorescence spectroscopy, as a simple, highly sensitive, and selective detection method, offers an excellent option for detecting tetracycline (TC). Many fluorescent nanomaterials have been reported as fluorescent probes for fluorescence detection. However, traditional physical and chemical methods for producing nanomaterials are associated with high capital and labor costs, significant energy losses, and significant environmental pollution from the toxic chemicals used in the production process. The present invention aims to synthesize nanomaterials through a cost-effective, environmentally friendly, and green method to rapidly detect tetracycline in the environment. Summary of the Invention

[0004] The technical problem to be solved by the present invention is to overcome the deficiencies in the prior art and provide a method for detecting the concentration of tetracycline hydrochloride in water by using microbial synthesized CDs.

[0005] To solve the above problems, the solution of the present invention is:

[0006] A method for detecting the concentration of tetracycline hydrochloride in water by using microbial synthesis of CDs is provided, comprising the following steps:

[0007] (1) Preparation of biocarbon dots (CDs):

[0008] The strain ( Meyerozyma guilliermondii PG-1) was cultured in LB medium;

[0009] The Meyerozyma guilliermondii PG-1 bacterial suspension was washed and resuspended with UP water, and then centrifuged at high speed to obtain the bacterial cells;

[0010] The collected bacteria in the centrifuge tube were resuspended in UP water. The resuspended bacterial solution was transferred to the inner lining of the reactor, placed in the reactor, hydrothermally heated in an oven, and removed after cooling to room temperature. The resulting solution was centrifuged to remove the precipitate and then repeatedly filtered through a 0.22 μm filter membrane to obtain the initial CDs solution. The CDs solution was stored in a refrigerator at 4 ˚C until use. After freeze-drying, the CDs solid powder was obtained and stored at -20 ˚C until use.

[0011] Draw a standard curve:

[0012] 40 μL of a CDs solution with a mass concentration of 44.32 g / L was placed in an EP tube, and then 960 μL of tetracycline hydrochloride solutions of different known concentrations were added. After mixing evenly, the mixture was allowed to stand at room temperature for 1 min. 200 μL of each solution in the EP tube was added to a 96-well plate, and three groups of parallel plates were prepared. The fluorescence intensity was measured using a microplate reader under the conditions of Ex = 354 nm and Em = 432 nm, and the fluorescence intensity ratio F / F0 was calculated, where F0 refers to the fluorescence intensity of CDs and F refers to the fluorescence intensity of CDs after tetracycline hydrochloride was added. A standard curve showing a linear relationship between the fluorescence intensity ratio (F / F0) of CDs and the concentration of tetracycline hydrochloride was obtained.

[0013] Determine the concentration of tetracycline hydrochloride in the water sample to be tested:

[0014] Take 40 μL of CDs solution with a mass concentration of 44.32 g / L in an EP tube, then add 960 μL of the water sample to be tested, mix well, and let it stand at room temperature for 1 minute. Take 200 μL of the solution in the above EP tube and add it to a 96-well plate. Make three sets of parallels. Use an enzyme marker to measure the fluorescence intensity under the conditions of Ex=354nm and Em=432nm, and calculate the fluorescence intensity ratio F / F0, where F0 refers to the fluorescence intensity of CDs and F refers to the fluorescence intensity of CDs after tetracycline hydrochloride is added. Obtain the tetracycline hydrochloride concentration by comparing with the standard curve in step (2). Average the three sets of concentration data, and the result obtained is the tetracycline hydrochloride concentration in the water sample to be tested.

[0015] Furthermore, in step (1), the cultured strain is Meyerozyma guilliermondii PG-1.

[0016] Furthermore, in step (1), the strain is cultured at 30° C. and 150 rpm under shaking conditions.

[0017] Furthermore, in step (1), the hydrothermal heating temperature is 200° C. and the heating time is 12 h.

[0018] Furthermore, in step (1), the conditions for centrifugation to remove precipitation and filtration are centrifugation at 9500 rpm for 10 min to remove precipitation, and then repeated filtration using a 0.22 μm filter membrane.

[0019] Specifically, microbial cells are rich in organic molecules such as carbohydrates, peptidoglycans, and proteins. The heteroatoms (e.g., O, N, and P) contained in these molecules allow their derived carbon dots to possess well-functionalized surfaces without any additional modification. Therefore, CDs are often used in research related to pollutant detection, bioimaging, catalysis, and sensing.

[0020] In the present invention, CDs prepared from the unicellular microorganism Meyerozyma guilliermondii PG-1 by a hydrothermal method have good physical and chemical stability. The fluorescence intensity can be measured using a microplate reader under the conditions of Ex = 354 nm and Em = 432 nm. Under ultraviolet light (354 nm), they emit blue fluorescence. In addition, experiments have found that the fluorescence of CDs can be rapidly quenched by tetracycline hydrochloride. Therefore, CDs can be used as an effective fluorescent probe for detecting tetracycline hydrochloride in a solution, and the concentration of tetracycline hydrochloride in the solution can be determined by the fluorescence change of CDs.

[0021] The invention also discloses application of the method in the field of tetracycline hydrochloride concentration detection in water.

[0022] Compared with the prior art, the present invention has the following beneficial effects:

[0023] 1. The absorption spectrum of tetracycline hydrochloride in the present invention has a great overlap with the excitation spectrum and generation spectrum of CDs, which provides a good basis for the detection of tetracycline hydrochloride.

[0024] 2. The present invention uses microorganisms to prepare CDs, an effective fluorescent probe, and adopts fluorescence spectroscopy to detect tetracycline hydrochloride in water. Compared with existing detection technologies, it does not require expensive instruments and equipment or multiple reaction reagents, and has the advantages of simple operation, low cost, fast reaction, and green and non-toxic.

[0025] 3. The present invention can be used for the rapid detection of tetracycline hydrochloride in various water samples, has strong anti-interference ability, is not interfered by other antibiotics and metal ions in the water, and has low requirements on reaction conditions.

[0026] 4. The linear range of the linear model constructed in the present invention is 10-200 μg / mL, and the standard curve equation is F / F0=–3.539c+0.9592 (F0 refers to the fluorescence intensity of CDs, F refers to the fluorescence intensity of CDs after adding tetracycline hydrochloride. c is the concentration of tetracycline hydrochloride), and the determination coefficient R 2= 0.9786. The detection limit was calculated to be as low as 0.21 μg / mL. The method recoveries ranged from 95.44% to 103.65%. BRIEF DESCRIPTION OF THE DRAWINGS

[0027] The present invention will be further described below with reference to the accompanying drawings.

[0028] Figure 1 The UV absorption-visible spectra were obtained by adding 40 μL (44.32 g / L) of CDs solution to 960 μL (0.2 g / L) of tetracycline hydrochloride solution, mixing evenly, and then standing at room temperature for 1 minute. 1 mL of tetracycline hydrochloride solution and 1 mL of CDs solution were scanned respectively.

[0029] Figure 2 The fluorescence intensity of the mixture of tetracycline hydrochloride solution with gradient concentration and CDs solution was measured using a microplate reader under the conditions of Ex=354 nm and Em=432 nm. The obtained standard curve showed a linear relationship between the fluorescence intensity ratio of CDs (F / F0) and the concentration of tetracycline hydrochloride.

[0030] Figure 3 The fluorescence intensity ratio was measured using a microplate reader at Ex = 354 nm and Em = 432 nm after adding 40 μL (44.32 g / L) of CDs solution to 960 μL (0.2 g / L) of different antibiotic solutions and mixing them evenly and then standing at room temperature for 1 min.

[0031] Figure 4 The fluorescence intensity ratio was measured by adding 40 μL (44.32 g / L) of CDs solution to 960 μL (0.2 g / L) of different heavy metal solutions, mixing them evenly, and then standing at room temperature for 1 min. The fluorescence intensity ratio was measured using a microplate reader under the conditions of Ex=354 nm and Em=432 nm. Implementation Method

[0032] The following will clearly and completely describe the technical solutions in the embodiments of the present invention in conjunction with the embodiments of the present invention and the accompanying drawings. Obviously, the embodiments described are only part of the embodiments of the present invention, not all of the embodiments. Based on the embodiments of the present invention, all other embodiments obtained by ordinary technicians in this field without making creative efforts are within the scope of protection of the present invention.

[0033] Meyerozyma guilliermondii PG-1 is a widely used bacterial strain, and its commercial products are available through various channels. For example, the National Center for Type Culture Collection (NTCC)'s Plasmid Vector, Strain, Cell, and Gene Collection sells its strain products. The strain Meyerozyma guilliermondii PG-1 used in the present invention was isolated and screened from soil in the Daqing Oilfield in Heilongjiang Province.

[0034] The present invention provides a method for detecting the concentration of tetracycline hydrochloride in water using microbially synthesized carbon dots, comprising the following steps:

[0035] (1) Preparation of biocarbon dots (CDs):

[0036] The strain (Meyerozyma guilliermondii PG-1) was cultured in LB medium at 30°C and 150 rpm in a shaking incubator.

[0037] The Meyerozyma guilliermondii PG-1 bacterial suspension was washed and resuspended with UP water, and then centrifuged at high speed to obtain the bacterial cells;

[0038] Resuspend the collected cells in the centrifuge tube with UP water. Transfer the resuspended bacterial solution to the reactor liner, place it in the reactor, and hydrothermally heat it in an oven at 200°C for 12 hours. Cool to room temperature and remove from the oven. Centrifuge the resulting solution to remove the precipitate. Centrifuge and filter at 9500 rpm for 10 minutes to remove the precipitate, then filter repeatedly through a 0.22 μm filter membrane to obtain the initial CDs solution. Store the CDs solution in a refrigerator at 4°C until use. Freeze-dry the resulting CDs solid powder and store it at –20°C until use.

[0039] Draw a standard curve:

[0040] 40 μL of a CDs solution with a mass concentration of 44.32 g / L was placed in an EP tube, and then 960 μL of tetracycline hydrochloride solutions of different known concentrations were added. After mixing evenly, the mixture was allowed to stand at room temperature for 1 min. 200 μL of each solution in the EP tube was added to a 96-well plate, and three groups of parallel plates were prepared. The fluorescence intensity was measured using a microplate reader under the conditions of Ex = 354 nm and Em = 432 nm, and the fluorescence intensity ratio F / F0 was calculated, where F0 refers to the fluorescence intensity of CDs and F refers to the fluorescence intensity of CDs after tetracycline hydrochloride was added. A standard curve showing a linear relationship between the fluorescence intensity ratio (F / F0) of CDs and the concentration of tetracycline hydrochloride was obtained.

[0041] Determine the concentration of tetracycline hydrochloride in the water sample to be tested:

[0042] Take 40 μL of CDs solution with a mass concentration of 44.32 g / L in an EP tube, then add 960 μL of the water sample to be tested, mix well, and let it stand at room temperature for 1 minute. Take 200 μL of the solution in the above EP tube and add it to a 96-well plate. Make three sets of parallels. Use an enzyme marker to measure the fluorescence intensity under the conditions of Ex=354nm and Em=432nm, and calculate the fluorescence intensity ratio F / F0, where F0 refers to the fluorescence intensity of CDs and F refers to the fluorescence intensity of CDs after tetracycline hydrochloride is added. Obtain the tetracycline hydrochloride concentration by comparing with the standard curve in step (2). Average the three sets of concentration data, and the result obtained is the tetracycline hydrochloride concentration in the water sample to be tested. Example

[0043] The method for detecting the concentration of tetracycline hydrochloride in water using microbially synthesized carbon dots comprises the following steps:

[0044] (1) Preparation of Luria–Bertani (LB) medium: Dissolve trypsin (10 g / L), sodium chloride (10 g / L), and yeast extract (5 g / L) in an appropriate amount of ultrapure water in a conical flask. Adjust the solution to pH 7 with 0.1 M NaOH or HCl, then seal the solution. Sterilize the solution in an autoclave (121°C, 20 min) and cool it for later use.

[0045] (2) Cultivation of Meyerozyma guilliermondii PG-1: Inoculate an appropriate amount of Meyerozyma guilliermondii PG-1 monoclonal cells into a certain amount of liquid sterile LB medium and incubate at 30°C, 150 rpm, and a constant-temperature shaker for 24 h. Centrifuge (6000 rpm, 5 min, 4°C) to separate the cells. Wash off the residual LB medium with sterile water and resuspend in a small amount of sterile water for later use.

[0046] (3) Dilute the Meyerozyma guilliermondii PG-1 stock solution to 30 mL with sterile deionized water, transfer it to a high-temperature resistant reactor (polytetrafluoroethylene lining), and continue heating. After the reaction is completed, cool it naturally to room temperature. The product is first centrifuged at high speed (9500 rpm, time 10 min) to remove the precipitate and then filtered (0.22 μm) three times to obtain the CDs stock solution. The CDs solution is stored in a refrigerator at 4 ˚C until use. After freeze-drying, the CDs solid powder is obtained and stored at -20 ˚C until use.

[0047] (4) Take 40 μL (44.32 g / L) of CDs solution in an EP tube, then add 960 μL of tetracycline hydrochloride solution with different known gradient concentrations, mix well, and let it stand at room temperature for 1 min. Take 200 μL of the solution in the above EP tube and add it to a 96-well plate (do three sets of parallel). Use a microplate reader to measure the fluorescence intensity under the conditions of Ex = 354 nm and Em = 432 nm, and calculate the fluorescence intensity ratio F / F0 (where F refers to the fluorescence intensity of CDs and F0 refers to the fluorescence intensity of CDs after adding antibiotics.) A standard curve showing a linear relationship between the fluorescence intensity ratio (F / F0) of CDs and the concentration of tetracycline hydrochloride was obtained.

[0048] (5) Take 40 μL (44.32 g / L) of CDs solution in an EP tube, then add 960 μL of the water sample to be tested, mix well, and let it stand at room temperature for 1 min. Take 200 μL of the solution in the above EP tube and add it to a 96-well plate (do three sets of parallel). Use an enzyme marker to measure the fluorescence intensity under the conditions of Ex = 354 nm and Em = 432 nm, and calculate the fluorescence intensity ratio F / F0 (where F refers to the fluorescence intensity of CDs and F0 refers to the fluorescence intensity of CDs after adding antibiotics). Obtain the concentration of tetracycline hydrochloride by comparing with the standard curve; calculate the average of the three sets of concentration data, and the result obtained is the concentration of tetracycline hydrochloride in the water sample to be tested. Example

[0049] In this embodiment, the operation steps are the same as those in Example 1.

[0050] Except that the concentration of the CDs solution was 22.16 g / L, and the CDs solution was mixed evenly with 960 μL of the water sample and then allowed to stand for 2 minutes, other conditions were the same as those in Example 1.

[0051] Description of the accompanying drawings:

[0052] Figure 1 Ultraviolet-visible absorption spectra obtained by scanning a CDs solution containing tetracycline hydrochloride, as well as a tetracycline hydrochloride solution and a CDs solution separately, reveal that the absorption spectrum changes significantly after tetracycline hydrochloride is added to the CDs solution. This demonstrates that the CDs solution can detect the presence of tetracycline hydrochloride in the solution. The present invention involves adding CDs to water containing tetracycline hydrochloride and exposing it to ultraviolet light (354 nm). The blue fluorescence emitted by the mixed solution is clearly weakened, thereby determining the presence of tetracycline hydrochloride in the water.

[0053] Figure 2The fluorescence intensity of a mixture of a tetracycline hydrochloride solution and a CDs solution at gradient concentrations was measured using a microplate reader under the conditions of Ex = 354 nm and Em = 432 nm. The fluorescence intensity ratio (F / F0) was found to be inversely proportional to the tetracycline hydrochloride concentration, demonstrating that the tetracycline hydrochloride concentration is directly proportional to the fluorescence intensity ratio (F / F0). Therefore, in the present invention, the tetracycline hydrochloride concentration is indirectly measured by measuring the fluorescence intensity under the conditions of Ex = 354 nm and Em = 432 nm.

[0054] Figure 3 Five common antibiotics were selected to record the changes in CDs fluorescence intensity after their addition. Microplate reader analysis revealed that tetracycline hydrochloride had the most pronounced fluorescence quenching effect on CDs, with the fluorescence intensity ratio (F / F0) dropping to approximately 0.25. This contrasts sharply with the negligible decrease in fluorescence observed after the addition of other antibiotics. This demonstrates that CDs exhibit significantly higher selectivity for tetracycline hydrochloride than for other antibiotics.

[0055] Figure 4 By adding certain concentrations of iron (III), manganese (II), and copper (II) ions to the CDs solution and measuring the fluorescence intensity, it was found that under the conditions of Ex = 354 nm and Em = 432 nm, the fluorescence intensity of the CDs solution did not change significantly after the addition of various ions. Therefore, it was confirmed that ions such as iron (III), manganese (II), and copper (II) ions do not affect the determination of tetracycline hydrochloride.

[0056] The above content is merely an example of the structure of the present invention. Those skilled in the art may make various modifications or additions to the described specific embodiments or replace them in a similar manner. As long as they do not deviate from the structure of the present invention or exceed the scope defined by the claims, they should all fall within the scope of protection of the present invention.

Claims

1. A method for detecting the concentration of tetracycline hydrochloride in water using microbially synthesized carbon dots, characterized in that: The following steps are involved: (1) Preparation of bio-carbon dots CDs: The strain Meyerozyma guilliermondii PG-1 was cultured in LB medium; The Meyerozyma guilliermondii PG-1 bacterial suspension was washed and resuspended with UP water, and then centrifuged at high speed to obtain the bacterial cells; The collected bacteria in the centrifuge tube were resuspended in UP water. The resuspended bacterial solution was transferred to the inner lining of the reactor, placed in the reactor, hydrothermally heated in an oven, and removed after cooling to room temperature. The resulting solution was centrifuged to remove the precipitate and then repeatedly filtered with a filter membrane to obtain the initial CDs solution. The CDs solution was stored in a refrigerator at 4 ˚C until use. After freeze-drying, the CDs solid powder was obtained and stored at -20 ˚C until use. (2) Draw the standard curve: 40 μL of a CDs solution with a mass concentration of 44.32 g / L was placed in an EP tube, and then 960 μL of tetracycline hydrochloride solutions of different known concentrations were added. After mixing evenly, the mixture was allowed to stand at room temperature for 1 min. 200 μL of each solution in the EP tube was added to a 96-well plate, and three groups of parallel plates were prepared. The fluorescence intensity was measured using a microplate reader under the conditions of Ex = 354 nm and Em = 432 nm, and the fluorescence intensity ratio F / F0 was calculated, where F0 refers to the fluorescence intensity of CDs and F refers to the fluorescence intensity of CDs after tetracycline hydrochloride was added. A standard curve showing a linear relationship between the fluorescence intensity ratio F / F0 of CDs and the concentration of tetracycline hydrochloride was obtained. (3) Determine the concentration of tetracycline hydrochloride in the water sample to be tested: Take 40 μL of CDs solution with a mass concentration of 44.32 g / L in an EP tube, then add 960 μL of the water sample to be tested, mix well, and let it stand at room temperature for 1 minute. Take 200 μL of the solution in the above EP tube and add it to a 96-well plate. Make three sets of parallels. Use an enzyme marker to measure the fluorescence intensity under the conditions of Ex=354nm and Em=432nm, and calculate the fluorescence intensity ratio F / F0, where F0 refers to the fluorescence intensity of CDs and F refers to the fluorescence intensity of CDs after tetracycline hydrochloride is added. Obtain the concentration of tetracycline hydrochloride by comparing with the standard curve in step (2). Average the three sets of concentration data, and the result obtained is the concentration of tetracycline hydrochloride in the water sample to be tested. In the step (1), the strain is cultured at 30°C and 150 rpm on a shaker; In the step (1), the hydrothermal heating temperature is 200°C and the heating time is 12 hours; In the step (1), the conditions for centrifugation to remove precipitation and filtration are centrifugation at 9500 rpm for 10 min to remove precipitation, and then repeated filtration using a 0.22 μm filter membrane.

2. Application of the method according to claim 1 in the field of tetracycline hydrochloride concentration detection in water bodies.

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