Fluorescent hydrogel microspheres for rapidly detecting tetracycline and preparation and detection methods thereof

Abstract

The invention discloses fluorescent hydrogel microspheres for rapidly detecting tetracycline as well as a preparation method and a detection method of the fluorescent hydrogel microspheres. The fluorescent hydrogel microsphere is mainly prepared from sodium alginate and a fluorescent complex, wherein the sodium alginate is a carrier of the fluorescent complex; the fluorescent complex is mainly formed by self-assembling guanylic acid, a europium ion compound and luminol; the fluorescent hydrogel microspheres are formed by dropwise adding a mixed solution of sodium alginate and a fluorescent complex into a calcium salt solution and solidifying; the fluorescent hydrogel microspheres are added into a sample solution containing tetracycline, after a reaction is carried out for a period of time at room temperature in a dark place, quantitative detection of tetracycline is realized by utilizing the ratio fluorescence change of the fluorescent hydrogel microspheres, and the detection limit is 5 mu g / kg. The fluorescent hydrogel microsphere provided by the invention is simple to prepare and mild in synthesis condition, after the fluorescent hydrogel microsphere responds to tetracycline, the hue change is obvious, the change is visible to naked eyes, and the fluorescent hydrogel microsphere has the advantages of simplicity, convenience, rapidness, sensitivity and the like.

Description

Technical Field

[0001] The invention relates to the technical field of rapid detection of tetracycline, and in particular to fluorescent hydrogel microspheres for rapid detection of tetracycline and preparation and detection methods thereof. Background Art

[0002] Tetracycline (TC) is widely used in the treatment of bovine mastitis because of its low price and strong antibacterial ability. However, irrational use of tetracycline may lead to excessive tetracycline residues in milk. If humans drink milk with excessive tetracycline residues for a long time, the excessive tetracycline residues will enter the human body with the milk and accumulate, which will increase the resistance of the human flora to tetracycline and increase the risk of disease. After tetracycline accumulates to a certain threshold, it may cause direct toxicity in the human body, such as liver damage and impaired detoxification and metabolic functions. Therefore, the development of a simple, efficient and rapid tetracycline detection method is of great significance to ensure the quality and safety of milk.

[0003] Among the many methods for detecting tetracycline, the most commonly used method for detecting tetracycline is the instrumental detection method. However, the instrumental detection method has the disadvantages of complex sample pretreatment, long detection time, and the need for professional operation. In recent years, researchers have developed many tetracycline detection methods based on nanomaterials, including colorimetry, fluorescence, and electrochemical methods. Among these methods, the fluorescence method has attracted much attention due to its high sensitivity, significant response, and visual changes visible to the naked eye. The fluorescent materials currently used for tetracycline detection include quantum dots, carbon dots, metal organic frameworks, etc. However, the cumbersome preparation process and high synthesis cost limit the application of these methods, and complicated sample pretreatment steps are still required before detection to extract, separate and enrich tetracycline from milk, which cannot meet the needs of rapid on-site detection. Summary of the invention

[0004] In order to overcome the shortcomings of the prior art, the present invention proposes a fluorescent hydrogel microsphere for rapid detection of tetracycline and a preparation and detection method. The present invention uses sodium alginate microspheres as carriers, encapsulates a fluorescent complex with blue fluorescence inside the carrier, and prepares a fluorescent hydrogel microsphere. Tetracycline can react and bind with the fluorescent complex through the pore channels in the sodium alginate microspheres, opening the europium ion (Eu 3+ )’s characteristic red fluorescence quenches the blue fluorescence of the complex, achieving rapid detection without extraction and separation.

[0005] The technical solution adopted by the present invention is as follows:

[0006] 1. A fluorescent hydrogel microsphere for rapid detection of tetracycline

[0007] The fluorescent hydrogel microspheres are mainly prepared from sodium alginate and a fluorescent complex. The sodium alginate is used to form sodium alginate microspheres, and the sodium alginate microspheres are used as carriers of the fluorescent complex. The fluorescent complex is mainly composed of guanosine monophosphate (GMP), europium ions (Eu 3+ ) compound and luminol (Luminol, LML) self-assembled.

[0008] The sodium alginate is used to form sodium alginate microspheres, which are used to encapsulate fluorescent complexes and selectively adsorb tetracycline in milk samples. The fluorescent complex is used to quickly respond to tetracycline adsorbed by the sodium alginate microspheres and produce significant and visible ratio fluorescence changes. After tetracycline reacts and combines with the fluorescent complex, tetracycline transfers excitation energy to europium ions (Eu 3+ ), so that Eu 3+ The characteristic red fluorescence of the fluorescent complex is excited, and the blue fluorescence of the fluorescent complex is quenched by the inner filter effect.

[0009] The fluorescent hydrogel microspheres and the fluorescent complex both have blue fluorescence, and after reacting with tetracycline, the fluorescent hydrogel microspheres and the fluorescent complex both turn into purple-red.

[0010] The europium ion compound is used to provide europium ions, and the molar ratio of the guanylic acid, europium ions and luminol is 2-3:2:1.

[0011] The guanylic acid, europium ion compound and luminol are self-assembled under the condition of pH=7.0-8.0.

[0012] The molecular formula of the fluorescent complex is EuC 18 H 19 N8Na2O 10 P.

[0013] The europium ion compound is used to provide Eu 3+ , preferably Eu(NO3)3·6H2O.

[0014] The fluorescent hydrogel microspheres are prepared by dropping a mixed solution of sodium alginate and a fluorescent complex into a calcium salt solution. 2+ ) cross-linked.

[0015] The particle size of the fluorescent hydrogel microspheres ranges from 1 mm to 5 mm. When the particle size of the fluorescent hydrogel microspheres is within the range of 1 mm to 5 mm, the fluorescent hydrogel microspheres can be spherical and not easy to collapse, which is conducive to regular color change during final detection.

[0016] The fluorescence emission peak ratio of the fluorescent hydrogel microspheres after reacting with tetracycline is positively correlated with the concentration of tetracycline.

[0017] 2. A method for preparing fluorescent hydrogel microspheres for rapid detection of tetracycline

[0018] The preparation method comprises the following steps:

[0019] 1) mixing the guanylate solution, the europium ion compound solution, the luminol solution and the Hepes buffer solution, stirring and reacting at room temperature in the dark, and after the reaction is completed, filtering and collecting the precipitate, washing and obtaining the fluorescent complex;

[0020] The step 1) is specifically as follows: a guanylic acid solution with a concentration of 5 to 10 mmol / L, a europium ion compound solution with a concentration of 5 to 10 mmol / L, a luminol aqueous solution with a concentration of 50 to 100 mmol / L, and a Hepes buffer solution with a concentration of 1 mol / L and a pH of 7.0 to 8.0 are mixed at room temperature, vortexed vigorously, stirred in the dark for 0 to 2 hours, filtered and washed with water to obtain the fluorescent complex; the volume ratio of the guanylic acid solution, the europium ion compound solution, the luminol solution and the buffer solution is 10 to 30:10 to 30:1:10 to 30.

[0021] 2) dropping a mixed solution of a sodium alginate (SA) solution and a fluorescent complex solution into a crosslinker solution to obtain the fluorescent hydrogel microspheres;

[0022] The step 2) comprises the following steps:

[0023] 2.1) preparing a sodium alginate solution with a mass fraction of 1% to 2% and a fluorescent complex solution with a mass fraction of 1% to 2%, mixing the sodium alginate solution and the fluorescent complex solution in a volume ratio of 1:1 and stirring them evenly to obtain a fluorescent microsphere preparatory solution;

[0024] 2.2) preparing a cross-linking agent solution with a mass fraction of 1% to 2%, wherein the cross-linking agent is calcium chloride (CaCl2);

[0025] 2.3) Add the fluorescent microsphere preparatory solution to a syringe with a volume of 5 mL, connect the syringe containing the fluorescent microsphere preparatory solution to a peristaltic pump, and set the flow rate of the peristaltic pump to 1-2 mL / min; make the needle of the syringe point vertically downward, and drip the fluorescent microsphere preparatory solution in the syringe vertically from the needle into the cross-linker solution. After the addition is completed, let it stand for 25 minutes to obtain the fluorescent hydrogel microspheres.

[0026] In step 2.3), the fluorescent microsphere preparation solution is dropped into the calcium chloride aqueous solution, and the Ca 2+The fluorescent hydrogel microspheres are formed by cross-linking of ions.

[0027] 3. Tetracycline Detection Method Using Fluorescent Hydrogel Microspheres

[0028] The detection method comprises the following steps:

[0029] 1) Mix a citric acid aqueous solution with a concentration of 10 to 40 mmol / L, a Hepes buffer solution with a concentration of 1 mol / L and a pH of 7.0 to 8.0, and water, and then add a fluorescent hydrogel microsphere to obtain a detection solution;

[0030] The volume ratio of the citric acid aqueous solution, the Hepes buffer solution and water is 1:10:70-80;

[0031] 2) adding a milk sample containing tetracycline to the detection solution, oscillating and mixing, and reacting at room temperature in the dark for 2 to 20 minutes; wherein the volume ratio of the milk sample to the detection solution is preferably 1:9 to 10;

[0032] 3) Take out the fluorescent hydrogel microspheres from the reaction mixture, set the excitation wavelength to 365 nm, and detect and obtain the fluorescence emission peak value F of the fluorescent hydrogel microspheres at 424 nm after the reaction. 424 and the fluorescence emission peak F at 618 nm 618 , proceed to step 4);

[0033] Alternatively, under the irradiation of ultraviolet light with a wavelength of 365 nm, the color change of the fluorescent hydrogel microspheres after the reaction is observed, and the concentration of tetracycline in the milk sample is visualized and analyzed to obtain a visualized detection result of the tetracycline concentration;

[0034] 4) Calculate the fluorescence emission peak ratio F of the fluorescent hydrogel microspheres after the reaction 618 / F 424 , the fluorescence emission peak ratio F 618 / F 424 The fluorescence emission peak F at 618 nm 618 The fluorescence emission peak F is at 424 nm. 424 The ratio of

[0035] 5) The peak value of fluorescence emission of the fluorescent hydrogel microspheres after the reaction is F 618 / F 424 Substitute it into the pre-established linear regression curve to calculate the concentration of tetracycline in the sample solution.

[0036] The specific process of establishing the linear regression curve is: after the detection liquid is reacted with multiple tetracycline standard solutions of different concentrations and known concentrations, the fluorescence emission peak ratio of each fluorescent hydrogel microsphere after reacting with the corresponding tetracycline standard solution is obtained, and the fluorescence emission peak ratio of each fluorescent hydrogel microsphere is fitted with the corresponding tetracycline standard solution concentration value to obtain a linear regression curve.

[0037] Optionally, in step 2), the concentration of tetracycline in the milk sample ranges from 5 μg / kg to 10 mg / kg.

[0038] In the present invention, unless otherwise specified, room temperature refers to a temperature of 25±5°C.

[0039] In the present invention, unless otherwise specified, the solvent of all solutions is water.

[0040] The present invention combines sodium alginate with lanthanide coordination compounds to prepare a fluorescent hydrogel microsphere, wherein the fluorescent hydrogel microsphere is mainly formed by dropping a mixed solution of sodium alginate and a fluorescent complex into a calcium salt solution through an ionic crosslinking reaction promoted by calcium ions. The fluorescent complex loaded in the sodium alginate microsphere is mainly self-assembled by guanylic acid, europium ion compounds and luminol at room temperature and pH = 7.0 to 8.0. The present invention also develops a detection method for quickly detecting tetracycline in milk based on the fluorescent hydrogel microsphere without sample pretreatment such as extraction and separation steps, and the detection solution prepared based on the fluorescent hydrogel microsphere is mixed with a sample solution containing tetracycline, and after a period of reaction at room temperature in the dark, the ratio fluorescence change of the fluorescent hydrogel microsphere is used to realize the quantitative detection of tetracycline, and the detection limit is 5 μg / kg. The fluorescent hydrogel microspheres proposed by the present invention are simple to prepare and have mild synthesis conditions. The fluorescent hydrogel microspheres have a short response time to tetracycline, obvious hue changes and changes visible to the naked eye, have good sensitivity and specificity for detecting tetracycline, and have the advantages of simplicity, rapidity, sensitivity, etc.

[0041] Beneficial effects of the present invention:

[0042] 1. The present invention uses a fluorescent complex combined with sodium alginate to prepare fluorescent hydrogel microspheres. The fluorescent complex in the fluorescent hydrogel microspheres can not only react quickly with tetracycline, thereby realizing rapid detection of tetracycline, but also has a significant fluorescence response. The detection result can be judged by the naked eye, thereby realizing visual detection of tetracycline content.

[0043] 2. When the fluorescent hydrogel microspheres provided by the present invention are used to detect tetracycline in milk samples, the pore channels inside the microspheres can allow tetracycline in the milk to pass through, and isolate other substances in the milk from the outside of the fluorescent hydrogel microspheres. They have extremely strong anti-interference ability, and are therefore simple to operate and easy to use. There is no need to perform pre-treatment such as extraction, separation, and enrichment on the milk samples. The content of tetracycline antibiotics in the milk samples can be quickly detected with just a simple dilution.

[0044] 3. The preparation method of the fluorescent hydrogel microspheres provided by the present invention has simple synthesis steps and mild synthesis conditions.

[0045] 4. When the fluorescent hydrogel microspheres provided by the present invention detect tetracycline antibiotics, a good linear relationship is shown in the concentration range of 0 to 10 mg / kg, and the minimum detection limit is 5 μg / kg. BRIEF DESCRIPTION OF THE DRAWINGS

[0046] Figure 1 This is a schematic diagram of the principle of direct detection of tetracycline in milk samples by fluorescent hydrogel microspheres in the present invention;

[0047] Figure 2 The figures are the physical pictures and fluorescence spectra of the fluorescent hydrogel microspheres in the present invention; wherein, (a) is the physical picture of the fluorescent hydrogel microspheres under natural light; (b) is the physical picture of the fluorescent hydrogel microspheres under 365nm ultraviolet light; (c) is the physical picture of the fluorescent hydrogel microspheres under 365nm ultraviolet light after the reaction with tetracycline; (d) is the fluorescence spectra of the fluorescent hydrogel microspheres before (blue curve) and after (red curve) the reaction with tetracycline;

[0048] Figure 3 The figure is a schematic diagram of the results of the fluorescent hydrogel microspheres in the present invention detecting tetracycline standard solutions of different concentrations; wherein (a) is a fluorescence spectrum of the fluorescent hydrogel microspheres at an excitation wavelength of 365 nm; (b) is a linear regression curve when the fluorescent hydrogel microspheres detect tetracycline of different concentrations and a fluorescence physical image under a 365 nm ultraviolet lamp;

[0049] Figure 4 The present invention is a schematic diagram of the results of direct detection of fresh milk containing different concentrations of tetracycline by fluorescent hydrogel microspheres; wherein, (a) is a physical picture of fluorescent hydrogel microspheres detecting different concentrations of tetracycline under 365nm ultraviolet light; (b) is a physical picture of the milk sample solution after reaction under natural light; (c) is a physical picture of the milk sample solution after reaction under 365nm ultraviolet light. DETAILED DESCRIPTION

[0050] The present invention will be further described below in conjunction with the accompanying drawings and embodiments. The embodiments described herein are only used to illustrate and explain the present invention and are not used to limit the present invention.

[0051] The present invention provides a fluorescent hydrogel microsphere that can quickly respond to tetracycline. The principle of the fluorescent hydrogel microsphere responding to tetracycline is as follows: Figure 1 First, guanylate, luminol and europium ions (Eu 3+ ) compounds combined and self-assembled to form a fluorescent complex LML-Eu / GMP. 3+ ) is quenched by the high-frequency OH oscillation of the surrounding water molecules (H2O). The fluorescent complex only emits blue fluorescence mainly composed of luminol, and no europium ions (Eu 3+ ) characteristic fluorescence. Then, the fluorescent complex was combined with sodium alginate by ion crosslinking to form fluorescent hydrogel microspheres LML-Eu / GMP MSs with blue fluorescence. After the fluorescent hydrogel microspheres reacted with milk with residual TC, the tetracycline in the milk, as a small molecule, was reacted and combined with the fluorescent complex encapsulated inside the fluorescent hydrogel microspheres through the pore channels in the fluorescent hydrogel microspheres, and the europium ions (Eu 3+ )’s characteristic red fluorescence is turned on through the “antenna effect”, and the blue fluorescence of the fluorescent complex is greatly quenched through the inner filter effect, thereby achieving a ratiometric fluorescence response to tetracycline; other large molecular protein impurities in milk cannot pass through the surface of the microspheres and are isolated outside, which plays a role in separating tetracycline from other substances in milk, successfully realizing the rapid detection of TC in dairy cows without sample pretreatment.

[0052] The specific embodiments of the present invention are as follows:

[0053] Example

[0054] In this embodiment, a fluorescent hydrogel microsphere LML-Eu / GMP MSs is prepared. The preparation process of the fluorescent hydrogel microsphere includes the following steps:

[0055] 1) Mix 1 mL of 100 mmol / L Hepes buffer solution with a pH of 8.0, 1 mL of 10 mmol / L Eu(NO3)3·6H2O aqueous solution, 50 μL of 100 mmol / L luminol aqueous solution, and 1 mL of 10 mmol / L GMP aqueous solution at room temperature, vortex vigorously, and react for 2 h in a dark environment to obtain a fluorescent complex reaction solution;

[0056] 2) centrifuging the fluorescent complex reaction solution obtained in step 1) at 12000 rpm for 10 min, removing the supernatant to obtain a yellow precipitate;

[0057] 3) The obtained yellow precipitate was re-dissolved with ultrapure water, and then centrifuged at 10000 rpm for 10 min, and the supernatant was removed to obtain a yellow precipitate;

[0058] 4) Repeat step 3) three times to wash the yellow precipitate three times to obtain the washed yellow precipitate, which is the fluorescent complex LML-Eu / GMP; redisperse the obtained fluorescent complex in 5 mL of ultrapure water to obtain a fluorescent complex solution;

[0059] 5) preparing a sodium alginate aqueous solution with a mass fraction of 2%, and mixing the sodium alginate aqueous solution with the fluorescent complex solution obtained in step 4) at a volume ratio of 1:1 to obtain a fluorescent microsphere preparatory solution;

[0060] 6) Prepare a 1% calcium chloride aqueous solution, use a 5 mL syringe to absorb the fluorescent microsphere preparatory solution obtained in step 5), connect the syringe to a peristaltic pump, and place the syringe vertically downward. Set the peristaltic pump speed to 1.5 mL / min, drip the fluorescent microsphere preparatory solution into the calcium chloride aqueous solution, and let it stand in the calcium chloride aqueous solution for 25 minutes to obtain fluorescent hydrogel microspheres LML-Eu / GMP MSs. Finally, store the obtained fluorescent hydrogel microspheres in a 1% calcium chloride aqueous solution at 4°C.

[0061] In order to verify the fluorescence response effect of fluorescent hydrogel microspheres and tetracycline, the present invention tested the response effect of fluorescent hydrogel microspheres. The test process and results are as follows:

[0062] a. Physical picture and fluorescence emission spectrum of fluorescent hydrogel microspheres

[0063] The actual picture of fluorescent hydrogel microspheres is as follows Figure 2 (a) and Figure 2 As shown in (b). Among them, Figure 2 (a) is a real picture of fluorescent hydrogel microspheres. Under visible light, the fluorescent hydrogel microspheres appear as uniform transparent microspheres. Figure 2 (b) is a real picture of fluorescent hydrogel microspheres under 365nm ultraviolet light. Under the excitation of 365nm ultraviolet light, the fluorescent hydrogel microspheres emit strong blue fluorescence.

[0064] Next, the fluorescence emission spectrum of the fluorescent hydrogel microspheres was measured under an excitation light wavelength of 365nm. The specific process of measuring the fluorescence emission spectrum is as follows: the fluorescent hydrogel microspheres to be tested are placed in the sample wells of a 96-well ELISA plate suitable for measuring fluorescence spectra, the excitation light wavelength of the ELISA instrument is set to 365nm, the emission spectrum collection range is set to 390-750nm, the step size is set to 2nm, and the fluorescence emission spectrum of the fluorescent hydrogel microspheres is obtained by scanning. The fluorescence emission spectrum of the fluorescent hydrogel microspheres is shown in Figure 2. Figure 2As shown in the blue curve in (d), it can be seen that under the excitation wavelength of 365nm, the fluorescent hydrogel microspheres have characteristic peaks at 424nm, 590nm, 618nm, 652nm and 692nm, but the characteristic peaks at 590nm, 618nm, 652nm and 692nm are weak.

[0065] b. Physical picture and fluorescence emission spectrum of fluorescent hydrogel microspheres after reaction with tetracycline

[0066] The specific process of the reaction between fluorescent hydrogel microspheres and tetracycline is as follows:

[0067] b1) taking out the fluorescent hydrogel microspheres from the storage solution for use; preparing a citric acid aqueous solution with a concentration of 20 mmol / L for use;

[0068] b2) Prepare a total volume of 360 μL of detection solution, add 316 μL of water, 4 μL of 20 mmol / L citric acid aqueous solution, 40 μL of 1 mol / L Hepes buffer solution with a pH of 8.0 and a fluorescent hydrogel microsphere, mix with 40 μL of tetracycline solution and react at room temperature for 10 min;

[0069] b3) After the reaction is completed, the fluorescent hydrogel microspheres reacted with tetracycline are taken out from the detection solution and transferred to the sample wells of a 96-well ELISA plate. The fluorescence emission spectrum of the fluorescent hydrogel microspheres reacted with tetracycline is detected by an ELISA instrument, the excitation wavelength is set to 365 nm, the fluorescence emission spectrum collection range is set to 390-750 nm, the step size is set to 2 nm, and the fluorescence emission spectrum is scanned.

[0070] Figure 2 (c) is a real picture of the fluorescent hydrogel microspheres after reacting with tetracycline under 365nm ultraviolet light. It can be seen that under the excitation of 365nm ultraviolet light, the fluorescent hydrogel microspheres after reacting with tetracycline emit strong purple-red fluorescence, which is Figure 2 The blue fluorescence in (b) has a significant color change. This is due to the fact that Eu 3+ The characteristic red fluorescence is turned on through the "antenna effect", and the blue fluorescence of the fluorescent complex is greatly quenched through the inner filter effect, thereby achieving a ratiometric fluorescence response to TC.

[0071] Figure 2 (d) is the fluorescence emission spectrum of the fluorescent hydrogel microspheres after reacting with tetracycline. It can be seen that after the fluorescent hydrogel microspheres react with tetracycline, the fluorescence intensity at 424nm is significantly reduced, and the fluorescence intensity at 590nm, 618nm, 652nm and 692nm is significantly increased. Therefore, the fluorescent hydrogel microspheres can be used for the detection of tetracycline.

[0072] c. Rapid detection of tetracycline using fluorescent hydrogel microspheres

[0073] According to the same process as steps b1) to b3), the fluorescent hydrogel microspheres were reacted with multiple tetracycline standard samples of different concentrations, and the fluorescence emission spectra of the fluorescent hydrogel microspheres after reacting with the tetracycline standard samples were detected. Each concentration of tetracycline standard sample was measured three times in parallel. The results of rapid detection of tetracycline using fluorescent hydrogel microspheres are shown in Figure 1. Figure 3 shown.

[0074] like Figure 3 As shown in (a), as the detection concentration of tetracycline increases from 0 to 20 mg / kg (from low to high, 0, 5, 10, 25, 50, 100, 250, 500, 750, 1000, 2000, 3000, 4000, 5000, 6000, 7000, 8000, 9000, 10000, 20000 μg / kg), after reacting with the tetracycline standard sample, the fluorescence emission peak of the fluorescent hydrogel microspheres at 424 nm gradually decreases, and the fluorescence emission peaks at 590, 618, 652 and 692 nm gradually increase.

[0075] like Figure 3 As shown in (b), the fluorescence intensity ratio of the fluorescent hydrogel microspheres at 618 nm and 424 nm is F 618 / F 424 Linear fitting was performed with the concentration of tetracycline standard sample. When the concentration of tetracycline was in the range of 0 to 10 mg / kg, the fluorescence ratio F of the fluorescent hydrogel microspheres 618 / F 424 It is linearly related to the tetracycline concentration. The fitting results show that the tetracycline concentration of 0-10 mg / kg and the fluorescence ratio F 618 / F 424 The linear regression equation is y = 0.2927x + 0.03839, and the linear correlation coefficient R 2 It is 0.9948. Therefore, the linear range of the fluorescent hydrogel microspheres provided by the present invention is 0 mg / kg to 10 mg / kg, and the detection limit of tetracycline is 5 μg / kg.

[0076] d. Rapid detection of tetracycline in milk using fluorescent hydrogel microspheres

[0077] The specific steps include:

[0078] d1) adding different concentrations of tetracycline (0, 5, 10, 100, 250, 500, 1000, 2000 μg / kg, from low to high) to milk without tetracycline to obtain multiple milk samples containing different concentrations of tetracycline;

[0079] d2) Prepare multiple test solutions with a total volume of 360 μL. The preparation process is as follows: add 316 μL of water, 4 μL of 20 mmol / L citric acid aqueous solution, 40 μL of 1 mol / L Hepes buffer solution with a pH of 8.0, and a fluorescent hydrogel microsphere into the container, add 40 μL of milk sample containing tetracycline to each test solution, mix well, and react at room temperature for 10 minutes;

[0080] d3) taking out the fluorescent hydrogel microspheres from the reaction detection solution, transferring them to the sample wells of a 96-well ELISA plate, and detecting the fluorescence emission spectrum of the sample using an ELISA reader, with the excitation wavelength set to 365 nm, the fluorescence emission spectrum collection range set to 390-750 nm, the step size set to 2 nm, and scanning the fluorescence emission spectrum.

[0081] The actual picture of fluorescent hydrogel microspheres after reacting with fresh milk containing different concentrations of tetracycline under 365nm ultraviolet light is as follows Figure 4 (a)~ Figure 4 as shown in (c).

[0082] It can be seen that after reacting with milk samples containing different tetracycline concentrations, the fluorescence color of LML-Eu / GMP MSs changed from blue to pink, realizing the visual detection of tetracycline content. The milk sample only needs a simple dilution process, without other complicated pre-treatment steps, because the hydrogel microsphere structure of the fluorescent hydrogel microspheres isolates macromolecular substances such as protein in milk from entering the microspheres, reducing interference with detection. It can be seen that the fluorescent hydrogel microspheres provided by the present invention have good sensitivity, specificity and anti-interference ability when used to detect tetracycline.

[0083] e. Rapid detection of tetracycline in milk using fluorescent hydrogel microspheres

[0084] This addition recovery test is used to verify the reliability of this detection method. The specific process is as follows:

[0085] First, the tetracycline content in three unspiked samples was measured using fluorescent hydrogel microspheres. Then, tetracycline standard samples of known concentrations (0 mg / kg, 0.01 mg / kg, and 1 mg / kg) were added to the three unspiked samples, and three spiked samples were obtained and the tetracycline content was detected respectively. Finally, the spike recovery was calculated using the following formula:

[0086]

[0087] Where K0 is the blank measurement value of the unspiked sample, K1 is the tetracycline concentration in the standard sample, and K2 is the measurement value of the spiked sample.

[0088] The measurement results of the addition recovery experiment are as follows:

[0089]

[0090] It can be seen that the fluorescent hydrogel microspheres provided by the present invention have good reliability, sensitivity, specificity and anti-interference ability when used to detect tetracycline.

[0091] In summary, the fluorescent hydrogel microspheres provided by the present invention are loaded with fluorescent complexes, which can not only react quickly with tetracycline to achieve rapid detection of tetracycline, but also have a significant fluorescence response, and the test results can be judged by the naked eye, thereby realizing visual detection of tetracycline content. The preparation method of the fluorescent hydrogel microspheres provided by the present invention has simple synthesis steps and mild synthesis conditions. The tetracycline detection method provided by the present invention has extremely strong anti-interference ability, simple operation and easy application. The tetracycline detection method provided by the present invention can detect tetracycline in a concentration range of 0 to 10 mg / kg, and the minimum detection limit is 5 μg / kg.

[0092] The present invention has been described in detail above. For those skilled in the art, without departing from the purpose and scope of the present invention, and without the need to carry out unnecessary experimental conditions, the present invention can be implemented in a wide range under equivalent parameters, concentrations and conditions. Although the present invention provides specific embodiments, it should be understood that the present invention can be further improved. In a word, according to the principles of the present invention, the application is intended to include any changes, uses or improvements to the present invention, including departure from the disclosed scope in the application, and changes made with conventional techniques known in the art.

Claims

1. A fluorescent hydrogel microsphere for rapid detection of tetracycline, characterized in that: The fluorescent hydrogel microspheres are mainly prepared from sodium alginate and a fluorescent complex; the fluorescent complex is mainly formed by self-assembly of guanylic acid, europium ion compounds and luminol.

2. The fluorescent hydrogel microspheres for rapid detection of tetracycline according to claim 1, characterized in that: The europium ion compound is used to provide europium ions, and the molar ratio of the guanylic acid, europium ions and luminol is 2-3:2:

1.

3. The fluorescent hydrogel microspheres for rapid detection of tetracycline according to claim 1, characterized in that: The molecular formula of the fluorescent complex is EuC 18 H 19 N8Na2O 10 P.

4. The fluorescent hydrogel microspheres for rapid detection of tetracycline according to claim 1, characterized in that: The fluorescent hydrogel microspheres are mainly formed by dropping a mixed solution of sodium alginate and a fluorescent complex into a calcium salt solution for solidification; the particle size of the fluorescent hydrogel microspheres ranges from 1 mm to 5 mm.

5. A method for preparing fluorescent hydrogel microspheres as claimed in any one of claims 1 to 4, characterized in that: The preparation method comprises the following steps: 1) mixing a guanylate solution, a europium ion compound solution, a luminol solution and a buffer solution, stirring and reacting at room temperature in the dark, and after the reaction is completed, filtering and collecting the precipitate, washing and obtaining the fluorescent complex; 2) dripping a mixed solution of the sodium alginate solution and the fluorescent complex solution into the crosslinker solution to obtain the fluorescent hydrogel microspheres.

6. The preparation method according to claim 5, characterized in that: The step 1) is specifically as follows: a guanylic acid solution with a concentration of 5 to 10 mmol / L, a europium ion compound solution with a concentration of 5 to 10 mmol / L, a luminol aqueous solution with a concentration of 50 to 100 mmol / L, and a buffer solution with a pH of 7.0 to 8.0 are mixed at room temperature, vortexed, stirred to react in the dark, filtered, and the precipitate is washed with water to obtain the fluorescent complex; the volume ratio of the guanylic acid solution, the europium ion compound solution, the luminol solution, and the buffer solution is 10 to 30:10 to 30:1:10 to 30.

7. The preparation method according to claim 5, characterized in that: The step 2) comprises the following steps: 2.1) preparing a sodium alginate solution with a mass fraction of 1% to 2% and a fluorescent complex solution with a mass fraction of 1% to 2%, mixing the sodium alginate solution and the fluorescent complex solution in a volume ratio of 1:1 and stirring them evenly to obtain a fluorescent microsphere preparatory solution; 2.2) preparing a cross-linking agent solution with a mass fraction of 1% to 2%, wherein the cross-linking agent is calcium chloride; 2.3) Add fluorescent microsphere preparatory solution to the syringe, connect the syringe to a peristaltic pump, and set the flow rate of the peristaltic pump to 1-2 mL / min; vertically drip the fluorescent microsphere preparatory solution in the syringe from the needle tip into the crosslinker solution. After the dripping is completed, let it stand for 25 minutes to obtain the fluorescent hydrogel microspheres.

8. A method for rapid detection of tetracycline using the fluorescent hydrogel microspheres according to any one of claims 1 to 4 or the fluorescent hydrogel microspheres prepared by the preparation method according to any one of claims 5 to 7, characterized in that: The following steps are involved: 1) Mix a citric acid aqueous solution with a concentration of 10 to 40 mmol / L, a buffer solution with a pH of 7.0 to 8.0, and water, and then add a fluorescent hydrogel microsphere to obtain a detection solution; The volume ratio of the citric acid aqueous solution, the buffer solution and water is 1:10:70-80; 2) Add the sample solution containing tetracycline to the test solution, shake and mix, and react at room temperature in the dark for 2 to 20 minutes; 3) Take out the fluorescent hydrogel microspheres from the reaction mixture, set the excitation wavelength to 365 nm, and detect and obtain the fluorescence emission peak value F of the fluorescent hydrogel microspheres at 424 nm after the reaction. 424 and the fluorescence emission peak F at 618 nm 618 , proceed to step 4); Alternatively, under the irradiation of ultraviolet light with a wavelength of 365 nm, the color change of the fluorescent hydrogel microspheres after the reaction is observed to realize the visualization analysis of the tetracycline concentration in the sample solution, and obtain the visualization result of the tetracycline concentration; 4) Calculate the fluorescence emission peak ratio F of the fluorescent hydrogel microspheres after the reaction 618 / F 424 , the fluorescence emission peak ratio F 618 / F 424 The fluorescence emission peak F at 618 nm 618 The fluorescence emission peak F is at 424 nm. 424 The ratio of 5) The peak value of fluorescence emission of the fluorescent hydrogel microspheres after the reaction is F 618 / F 424 Substitute it into the pre-established linear regression curve to calculate the concentration of tetracycline in the sample solution.

9. The method for rapid detection of tetracycline according to claim 8, characterized in that: The specific process of establishing the linear regression curve is: after the detection liquid is reacted with multiple tetracycline standard solutions of different concentrations and known concentrations, the fluorescence emission peak ratio of each fluorescent hydrogel microsphere after reacting with the corresponding tetracycline standard solution is obtained, and the fluorescence emission peak ratio of each fluorescent hydrogel microsphere is fitted with the corresponding tetracycline standard solution concentration value to obtain a linear regression curve.

10. The method for rapid detection of tetracycline according to claim 8, characterized in that: In the step 2), the sample solution is milk; the concentration of tetracycline in the sample solution ranges from 5 μg / kg to 10 mg / kg.

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