A device and method for detecting antibiotics in aquaculture water

Abstract

The application provides a device and method for detecting antibiotics in aquaculture water, and belongs to the technical field of environmental pollutant detection.The device is characterized in that Fe3O4@GO magnetic nano adsorbent material is encapsulated in a microfluidic chip reaction chamber, and a magnetic sticker is used to realize the immobilization and replaceable design of the adsorbent; an electromagnetic oscillation module is integrated to drive the adsorbent to vibrate in the microchannel, so that the mass transfer and capture efficiency of antibiotics such as sulfonamides, quinolones and tetracyclines is significantly improved.The application innovatively provides an integrated process of "adsorption-elution-detection", the pretreatment time is shortened from hours to 10 minutes, the synchronous detection of ultra-trace multi-component antibiotics in 5-10 muL water samples at the level of 0.01-5 mu g / L is realized, the detection precision is significantly improved compared with traditional methods, the anti-matrix interference ability is strong, and the device is suitable for real-time online monitoring of multi-antibiotic residues in aquaculture environment.

Description

TECHNICAL FIELD

[0001] The present application belongs to the technical field of environmental pollutant detection, and particularly relates to a device and method for detecting antibiotics in aquaculture water. BACKGROUND

[0002] Aquaculture is one of the most important food production industries in the world, playing a key role in meeting the growing demand for protein by humans. However, in the process of aquaculture, antibiotics are widely used to prevent and control diseases in aquatic animals. Although the use of these antibiotics has ensured the yield and quality of aquaculture to some extent, it has also brought serious environmental and health problems.

[0003] With the extensive use of antibiotics, the problem of antibiotic residues in aquaculture water has become increasingly prominent. These residual antibiotics not only cause damage to the aquaculture ecosystem, affecting the growth, reproduction and survival of aquatic organisms, but also can be transmitted to humans through the food chain, posing a potential threat to human health. For example, long-term consumption of aquatic products containing antibiotic residues may lead to drug resistance in the human body, reducing the effectiveness of antibiotics in treating human diseases and increasing the difficulty and risk of treating infectious diseases. In addition, antibiotic residues can also cause allergic reactions, intestinal flora imbalance and other health problems.

[0004] Currently, the detection of antibiotic residues in aquaculture water mainly relies on traditional analysis methods such as high-performance liquid chromatography (HPLC), gas chromatography-mass spectrometry (GC-MS), etc. Although these methods have high accuracy and sensitivity, they have some obvious limitations. First, the pre-treatment process of traditional methods is complex and time-consuming, usually requiring multiple steps such as sample extraction, purification, concentration, etc. The entire pre-treatment process may take several hours or even days, which cannot meet the demand of real-time online monitoring. This makes it difficult to discover the changes of antibiotic residues in aquaculture water in a timely manner, and effective measures cannot be taken to control and manage in a timely manner. Second, the detection cost of traditional methods is high, requiring the use of expensive instruments and a large amount of chemical reagents, which is difficult for small-scale aquaculture farms to bear. Moreover, the operation requirements of traditional methods are high, requiring professional technical personnel to operate, which also limits its widespread application in primary aquaculture.

[0005] In addition, the traditional method has weak anti-interference ability when dealing with complex aquaculture water matrix. The aquaculture water contains a large amount of impurities such as organic matter, inorganic matter and microorganism, which will interfere with the detection result and cause inaccurate detection result. In order to eliminate these interferences, complex sample pretreatment and purification steps are often required, which further increases the difficulty and cost of detection. There are also some problems in the selection and use of adsorbents in the existing detection technology. The traditional adsorbent has low selectivity and adsorption efficiency for multiple types of antibiotics, and it is difficult to realize the simultaneous and efficient enrichment of multiple types of antibiotics. Moreover, the regeneration and reuse performance of the adsorbent is poor, which increases the detection cost and resource waste. Therefore, there is an urgent need to develop a rapid, accurate, sensitive and low-cost online detection technology for multiple types of antibiotics in aquaculture water. SUMMARY

[0006] In view of the defects and deficiencies of the existing detection technology for multiple types of antibiotics in aquaculture water, such as complex and time-consuming pretreatment, high detection cost and weak anti-interference ability, the present application provides a detection device and method for antibiotics in aquaculture water. The method can be used for real-time online monitoring of multiple types of antibiotic residues in aquaculture environment, can simultaneously detect multiple types of antibiotics such as sulfonamides, quinolones and tetracyclines, and has simple device structure, easy assembly and operation. According to the actual detection requirements, the method can realize accurate detection of specific types and contents of antibiotics, and provide strong technical support for protecting the safety of aquaculture environment and the quality of aquatic products.

[0007] To achieve the above-mentioned purpose, the present application provides the following technical solutions:

[0008] One of the technical solutions of the present application is:

[0009] A detection device for antibiotics in aquaculture water, comprising: a microfluidic chip module, an electromagnetic oscillation module, a microfluidic driving module and a Chip-HPLC-MS combined interface; the microfluidic driving module comprises a microfluidic injection pump and a two-way valve;

[0010] The electromagnetic oscillation module is physically fixed below the microfluidic chip module loaded with magnetic adsorbent material through a chip fixing component, and the fluid direction of the reaction chamber of the microfluidic chip module is perpendicular to the magnetic field direction of the electromagnetic oscillation module;

[0011] The microfluidic injection pump of the microfluidic driving module is connected to the channel inlet of the microfluidic chip module through the two-way valve; and the Chip-HPLC-MS combined interface is connected to the channel outlet of the microfluidic chip module through the two-way valve of the microfluidic driving module;

[0012] A magnetic sticker is affixed to the bottom of the outer chamber of the magnetic microfluidic chip module, which fixes the Fe3O4@GO magnetic adsorption material to the inner wall of the microfluidic channel.

[0013] Furthermore, the preparation method of the Fe3O4@GO magnetic adsorption material includes the following steps:

[0014] Graphene oxide was dispersed in 2-morpholine ethanesulfonic acid (MES) buffer, and ethylene glycol dichloro ether (EDC) and N-hydroxysuccinimide (NHS) were added. After magnetic stirring, solution A was obtained.

[0015] NH2-Fe3O4 magnetic beads were dispersed in 2-morpholine ethanesulfonic acid (MES) buffer to obtain solution B;

[0016] The solutions A and B are mixed, and the products are magnetically separated to obtain the Fe3O4@GO magnetic adsorption material.

[0017] Furthermore, in solution A, the ratio of the amount of graphene oxide to MES buffer, EDC, and NHS is 30 mg: 3 mL: 10 mg: 6 mg;

[0018] In solution B, the ratio of the NH2-Fe3O4 magnetic beads to the MES buffer is 1 mg: 1 mL.

[0019] Furthermore, in solutions A and B, the mass ratio of the NH2-Fe3O4 magnetic spheres to the graphene oxide is 2:3.

[0020] Furthermore, the step of fixing the Fe3O4@GO magnetic adsorption material to the inner wall of the microfluidic channel using the magnetic sticker includes:

[0021] The Fe3O4@GO magnetic adsorption material is injected into the reaction chamber of the magnetic microfluidic chip module via the microfluidic injection pump of the microfluidic drive module. A magnetic sticker is attached to the outer surface of the chip. The magnetic adsorption material flowing through this area is adsorbed onto the inner wall of the reaction chamber of the microfluidic chip module under the action of the magnetic field of the magnetic sticker.

[0022] Furthermore, the electromagnetic oscillation module also includes a current controller, a chip fixing component, and an electromagnetic coil, which can generate an alternating magnetic field to drive the magnetic adsorption material to vibrate, significantly improving the mass transfer and capture efficiency of antibiotics and ensuring that the adsorption effect of antibiotics on samples from the same batch remains consistent. Simultaneously, the magnetic field parameters can be set according to actual detection needs, thereby controlling the adsorption efficiency and effect.

[0023] Furthermore, the Chip-HPLC-MS interface includes a two-way injection valve connecting the inlet and outlet of the microfluidic chip and a high-pressure resistant fluid path connecting the liquid chromatography system, ensuring that the adsorbed antibiotics can be successfully eluted and introduced into the HPLC-MS (High Performance Liquid Chromatography-Mass Spectrometry) system for detection.

[0024] Furthermore, the microfluidic chip of the microfluidic drive module and the electromagnetic oscillation module are located between the mobile phase storage bottle and the liquid chromatograph. The chip inlet and outlet are connected, and the two channels are switched by a switching valve. Channel A is connected to the injection pump for injecting magnetic adsorption materials and sample solutions, while channel B is controlled by the high-performance liquid chromatography program, realizing an integrated process of "adsorption-elution-detection".

[0025] The second technical solution of the present invention:

[0026] A method for detecting antibiotics in aquaculture water, using a detection device for antibiotics in the aquaculture water, includes the following steps:

[0027] Remove the magnetic sticker on the bottom of the reaction chamber of the magnetic microfluidic chip module, start the electromagnetic oscillation module, and inject the aquaculture water sample into the reaction chamber of the magnetic microfluidic chip module through the microfluidic injection pump of the microfluidic drive module. The water sample is mixed with Fe3O4@GO magnetic adsorption material to enrich the antibiotics. After enrichment is completed, the rinsing waste liquid is discharged.

[0028] Switch the two-way valve to introduce the methanol-water mixture as the HPLC mobile phase into the reaction chamber of the magnetic microfluidic chip module, gradient eluent the enriched antibiotics, and eluent to the C18 column through the Chip-HPLC-MS coupling interface.

[0029] The antibiotics separated by the C18 column were introduced into a mass spectrometer and qualitative and quantitative analyses were performed using the mass spectrometry multiple reaction monitoring mode.

[0030] Furthermore, the injection flow rate of the aquaculture water sample to be tested is 0.2-0.5 mL / min.

[0031] Furthermore, the gradient elution procedure is as follows: 5 wt.% methanol concentration in the mixture for 0-2 min, and 95 wt.% methanol concentration in the mixture for 2-15 min.

[0032] Furthermore, the output frequency of the magnetic field of the electromagnetic oscillation module is 100-500Hz, and the magnetic field strength is 50-200mT; during the enrichment stage, the vibration frequency of the Fe3O4@GO magnetic adsorption material is 300Hz, and the enrichment time is 3-5min.

[0033] Furthermore, the antibiotics in the aquaculture water samples to be tested include sulfonamides, quinolones, tetracyclines, and sedative antibiotics, with a detection range of 0.01-5 μg / L.

[0034] Furthermore, the aquaculture water sample to be tested is injected via a microfluidic injection pump and fully mixed with the Fe3O4@GO magnetic adsorption material in the reaction chamber. After the reaction is complete, it is rinsed out. The introduction of the mobile phase can be precisely controlled by a switching valve to achieve efficient elution of antibiotics.

[0035] Furthermore, the method for detecting multiple types of antibiotics in aquaculture water also includes a Fe3O4@GO magnetic adsorption material regeneration step: injecting 0.1 mol / L NaOH solution into the microfluidic chip for 3 minutes to clean it, and the Fe3O4@GO magnetic adsorption material can be reused ≥5 times.

[0036] In the device for online high-sensitivity detection of multiple types of antibiotics in aquaculture water based on Chip-HPLC-MS of this invention, after the aquaculture water sample is taken, the Fe3O4@GO magnetic adsorption material forms a dynamic adsorption interface in the microfluidic channel, which can fully contact the water sample and achieve efficient adsorption of various antibiotics such as sulfonamides, quinolones, and tetracyclines. This avoids the interference of complex matrices on detection, and the adsorbed antibiotic content is linearly correlated with the initial antibiotic concentration in the water sample.

[0037] Compared with the prior art, the present invention has the following advantages and technical effects:

[0038] (1) The method for detecting multiple types of antibiotics in aquaculture water in this invention innovatively employs surface-modified Fe3O4@GO magnetic nano-adsorption material and magnetic sticker immobilization technology, combined with an electromagnetic oscillation module to improve mass transfer capture efficiency, realizing an integrated "adsorption-elution-detection" process. This reduces pretreatment time from hours to within 10 minutes, enabling simultaneous detection of ultra-trace amounts of multiple antibiotics (0.01-5 μg / L) in 5-10 μL water samples, significantly improving detection accuracy and resistance to matrix interference. This invention is of great significance for real-time online monitoring of multiple antibiotic residues in aquaculture environments, enabling timely detection of antibiotic residue problems, providing strong support for the scientific management of aquaculture and the safety of aquatic product quality, and promoting the sustainable development of the aquaculture industry. Simultaneously, this invention also provides a new approach and method in the field of environmental pollutant detection technology, and is expected to be widely applied in other fields.

[0039] (2) The device of the present invention includes a magnetic microfluidic chip module, an electromagnetic oscillation module, a microfluidic drive module and a Chip-HPLC-MS interface. The first three parts are integrated and linked together to ensure efficient enrichment, elution and detection of a variety of antibiotics in aquaculture water.

[0040] (3) This invention solves the problems of complex and time-consuming pretreatment, low detection accuracy and weak anti-interference ability of traditional detection methods. It can simultaneously improve and accurately control the detection capability of multiple types of antibiotics. The device has a high degree of integration and intelligence, but the assembly module is relatively simple, easy to operate and maintain, and the material cost is low. The invention has a very high universality and can be applied to the detection of antibiotic residues in different aquaculture environments and different types of antibiotics. Attached Figure Description

[0041] The accompanying drawings, which form part of this invention, are used to provide a further understanding of the invention. The illustrative embodiments of the invention and their descriptions are used to explain the invention and do not constitute an undue limitation of the invention. In the drawings:

[0042] Figure 1 This is a schematic diagram illustrating the principle of detecting multiple types of antibiotics in aquaculture water in an embodiment of the present invention;

[0043] Figure 2 The schematic diagram of the detection device for antibiotics in aquaculture water in Example 1 shows that: 1-microfluidic injection pump, 2-mobile phase reservoir, 3-two-way valve, 4-magnetic microfluidic chip, 5-electromagnetic oscillation module, 6-waste tank, and 7-Chip-HPLC-MS coupling interface.

[0044] Figure 3 The device described in Example 2 of this invention was used to repeatedly test the removal efficiency of antibiotics in aquaculture water;

[0045] Figure 4 The results of the capture efficiency experiment of 26 commonly used fish antibiotics using the device in Embodiment 2 of the present invention;

[0046] Figure 5 The results of the capture efficiency of 18 antibiotics using the device in Embodiment 2 of the present invention under different concentrations of magnetic adsorption material are presented. Detailed Implementation

[0047] Various exemplary embodiments of the present invention will now be described in detail. This detailed description should not be considered as a limitation of the present invention, but rather as a more detailed description of certain aspects, features, and embodiments of the present invention.

[0048] It should be understood that the terminology used in this invention is merely for describing particular embodiments and is not intended to limit the invention. Furthermore, with respect to numerical ranges in this invention, it should be understood that each intermediate value between the upper and lower limits of the range is also specifically disclosed. Every smaller range between any stated value or intermediate value within a stated range, and any other stated value or intermediate value within said range, is also included in this invention. The upper and lower limits of these smaller ranges may be independently included or excluded from the range.

[0049] Unless otherwise stated, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art. While only preferred methods and materials have been described herein, any methods and materials similar or equivalent to those described herein may be used in the implementation or testing of this invention. All references to this specification are incorporated by way of citation to disclose and describe methods and / or materials associated with those references. In the event of any conflict with any incorporated reference, the content of this specification shall prevail.

[0050] Various modifications and variations can be made to the specific embodiments described in this specification without departing from the scope or spirit of the invention, as will be apparent to those skilled in the art. Other embodiments derived from this specification will also be obvious to those skilled in the art. This specification and embodiments are merely exemplary.

[0051] The terms “include,” “including,” “have,” “contain,” etc., used in this article are all open-ended terms, meaning that they include but are not limited to.

[0052] This invention provides a device for detecting antibiotics in aquaculture water, comprising: a microfluidic chip module, an electromagnetic oscillation module, a microfluidic drive module, and a Chip-HPLC-MS interface; the microfluidic drive module includes a microfluidic injection pump and a two-way valve;

[0053] The electromagnetic oscillation module is physically fixed below the microfluidic chip module carrying magnetic adsorption material by a chip fixing component, and the fluid direction of the reaction chamber of the microfluidic chip module is perpendicular to the magnetic field direction of the electromagnetic oscillation module.

[0054] The microfluidic injection pump of the microfluidic drive module is connected to the channel inlet of the microfluidic chip module through a two-way valve; the Chip-HPLC-MS coupling interface is connected to the channel outlet of the microfluidic chip module through the two-way valve of the microfluidic drive module.

[0055] A magnetic sticker is affixed to the bottom of the outer chamber of the magnetic microfluidic chip module, which fixes the Fe3O4@GO magnetic adsorption material to the inner wall of the microfluidic channel.

[0056] In a preferred embodiment of the present invention, the microfluidic chip of the magnetic microfluidic chip module is a PMMA (acrylic) material chip prepared by laser engraving, with a channel cross-sectional size of 200×200μm, a pressure resistance limit of 10MPa, and an operating temperature range of 4-60℃.

[0057] In a preferred embodiment of the present invention, the preparation method of Fe3O4@GO magnetic adsorption material includes the following steps:

[0058] (1) Synthesis of NH2-Fe3O4 magnetic spheres

[0059] FeCl3·6H2O was dissolved in ethylene glycol under ultrasonic assistance to form an orange-yellow solution. Then, anhydrous sodium acetate and 1,6-hexanediamine were added to the solution and the mixture was stirred vigorously at room temperature for 30 minutes. The mixture was then transferred to a high-pressure reactor and heated at 198°C for 6 hours. After the high-pressure reactor cooled naturally, the product was collected with the aid of an external magnetic field. The product was washed three times alternately with ultrapure water and ethanol and then freeze-dried for 24 hours to obtain NH2-Fe3O4 magnetic spheres.

[0060] (2) Synthesis of graphene oxide (GO)

[0061] Add 1g of graphite powder and 1g of NaNO3 to a 500mL beaker, then add 46mL of sulfuric acid. Stir in an ice bath for 10 minutes, then add 6g of potassium permanganate. Within 10 minutes, add the potassium permanganate to the solution. Observe that the potassium permanganate is reduced and turns dark green. Stir continuously for 2 hours to allow the redox reaction to proceed fully. Slowly add distilled water; the solution turns dark brown. Place the mixture in a 98℃ water bath and stir for 15 minutes, adding a large amount of distilled water while stirring. Then add an appropriate amount of 30% hydrogen peroxide to terminate the reaction for 1 hour. The solution will initially appear yellow, gradually turning brown. After centrifugation, remove sulfate ions with 5wt.% HCl, wash three times with pure water until neutral, and vacuum dry overnight to obtain graphene oxide.

[0062] (3) Synthesis of Fe3O4@GO magnetic adsorption material

[0063] First, disperse 300 mg GO in 30 mL of MES buffer (pH=5.6), add 100 mg EDC, sonicate and stir for 30 minutes, then add 60 mg NHS and continue magnetic stirring for 15 minutes to obtain solution A; weigh 200 mg NH2-Fe3O4 magnetic beads and disperse them in 200 mL of MES buffer, sonicate for 30 minutes to obtain solution B;

[0064] Solution B was mixed dropwise with solution A and placed in a magnetic stirrer. The mixture was extracted, and the product was obtained by magnetic separation. The product was washed three times alternately with ultrapure water and ethanol, and then freeze-dried for 24 hours to obtain Fe3O4@GO magnetic adsorption material.

[0065] In a preferred embodiment of the present invention, the step of fixing the Fe3O4@GO magnetic adsorption material to the inner wall of the microfluidic channel with a magnetic sticker includes: injecting the Fe3O4@GO magnetic adsorption material into the reaction chamber of the magnetic microfluidic chip module via a microfluidic injection pump of the microfluidic drive module; attaching a magnetic sticker to the outer surface of the chip; and adsorbing the magnetic adsorption material flowing through the area onto the inner wall of the reaction chamber of the microfluidic chip module under the action of the magnetic field of the magnetic sticker.

[0066] In a preferred embodiment of the present invention, the electromagnetic oscillation module further includes a current controller, a chip fixing component, an electromagnetic coil, and other components, which can generate an alternating magnetic field to drive the magnetic adsorption material to vibrate, significantly improving the mass transfer and capture efficiency of antibiotics and ensuring that the adsorption effect of antibiotics on samples from the same batch remains consistent. Simultaneously, the magnetic field parameters can be set according to actual detection needs, thereby controlling the adsorption efficiency and effect.

[0067] In a preferred embodiment of the present invention, the Chip-HPLC-MS coupling interface includes a two-way injection valve connecting the inlet and outlet of the microfluidic chip and a high-pressure resistant fluid path connecting the liquid chromatography system, ensuring that the adsorbed antibiotics can be successfully eluted and introduced into the HPLC-MS (High Performance Liquid Chromatography-Mass Spectrometry) system for detection.

[0068] In a preferred embodiment of the present invention, the microfluidic chip of the microfluidic drive module and the electromagnetic oscillation module are located between the mobile phase storage bottle and the liquid chromatograph. The chip inlet and outlet are connected, and the two channels are switched by a switching valve. Channel A is connected to an injection pump for injecting magnetic adsorption materials and sample solutions, while channel B is controlled by a high-performance liquid chromatography program, realizing an integrated process of "adsorption-elution-detection".

[0069] This invention also proposes a method for detecting antibiotics in aquaculture water, using the aforementioned antibiotic detection device for aquaculture water, comprising the following steps:

[0070] Remove the magnetic sticker on the bottom of the reaction chamber of the magnetic microfluidic chip module, start the electromagnetic oscillation module, and inject the aquaculture water sample into the reaction chamber of the magnetic microfluidic chip module through the microfluidic injection pump of the microfluidic drive module. The water sample is mixed with Fe3O4@GO magnetic adsorption material to enrich the antibiotics. After enrichment, the rinsing waste liquid is discharged into the waste liquid pool.

[0071] Switch the two-way valve to introduce the methanol-water mixture as the HPLC mobile phase into the reaction chamber of the magnetic microfluidic chip module, gradient eluent the enriched antibiotics, and eluent to the C18 column through the Chip-HPLC-MS coupling interface.

[0072] The antibiotics separated by the C18 column were introduced into a mass spectrometer and qualitative and quantitative analyses were performed using the mass spectrometry multiple reaction monitoring mode.

[0073] In a preferred embodiment of the present invention, the injection flow rate of the aquaculture water sample to be tested is 0.2-0.5 mL / min.

[0074] In a preferred embodiment of the present invention, the gradient elution program is as follows: 0-2 min, methanol concentration in the mixture is 5 wt.%, and 2-15 min, methanol concentration in the mixture is 95 wt.%.

[0075] In a preferred embodiment of the present invention, the output frequency of the magnetic field of the electromagnetic oscillation module is 100-500Hz, and the magnetic field strength is 50-200mT; during the enrichment stage, the vibration frequency of the Fe3O4@GO magnetic adsorption material is 300Hz, and the enrichment time is 3-5min.

[0076] In a preferred embodiment of the present invention, the antibiotics in the aquaculture water sample to be tested include sulfonamides, quinolones, tetracyclines, and sedative antibiotics, with a detection range of 0.01-5 μg / L.

[0077] In a preferred embodiment of the present invention, the aquaculture water sample to be tested is injected through a microfluidic injection pump and fully mixed with the Fe3O4@GO magnetic adsorption material in the reaction chamber. After the reaction is complete, it is rinsed out. The introduction of the mobile phase can be precisely controlled by a switching valve to achieve efficient elution of antibiotics.

[0078] In a preferred embodiment of the present invention, the method for detecting multiple types of antibiotics in aquaculture water further includes a Fe3O4@GO magnetic adsorption material regeneration step: injecting 0.1 mol / L NaOH solution into the microfluidic chip for 3 minutes to clean it, and the Fe3O4@GO magnetic adsorption material can be reused ≥5 times.

[0079] The schematic diagram of the principle for detecting multiple types of antibiotics in aquaculture water in this embodiment of the invention is shown below. Figure 1Specifically, a sample solution containing antibiotics from aquaculture water is injected into a microfluidic chip using a syringe pump. This chip is placed in a magnetic field environment constructed by an electromagnetic oscillation module (the "S" and "N" poles in the diagram indicate the magnetic field distribution). The Fe3O4@GO magnetic nanomaterial loaded within the chip vibrates under the influence of the magnetic field, significantly improving the mass transfer efficiency with antibiotic molecules in the sample solution. The target antibiotics are efficiently adsorbed and captured through the interaction of functional groups such as hydroxyl and carboxyl groups on the material surface, while impurities are discharged with the waste liquid. Subsequently, a mobile phase (methanol / water mixture) is introduced into the chip, eluting the antibiotics adsorbed on the magnetic material under the influence of the magnetic field. The eluted target antibiotic molecules are then carried into an HPLC-MS system. Gradient elution using an HPLC column (e.g., a C18 column) separates different types of antibiotics. Mass spectrometry, employing multiple reaction monitoring (MRM) mode, performs ultra-trace (0.01-5 μg / L) qualitative and quantitative detection of various antibiotics, including sulfonamides and quinolones. After detection, the results can also be obtained by applying an alkaline solution (e.g., 0.1 mol / L). NaOH solution is used to regenerate the magnetic adsorption material, enabling the material to be reused.

[0080] In this embodiment of the invention, room temperature refers to "25±2℃".

[0081] The technical solution of the present invention will be further illustrated by the following embodiments.

[0082] Example 1

[0083] A device for detecting antibiotics in aquaculture water, structural schematic diagram shown below. Figure 2 Among them, 1-microfluidic injection pump, 2-mobile phase reservoir, 3-two-way valve, 4-magnetic microfluidic chip, 5-electromagnetic oscillation module, 6-waste liquid reservoir, 7-Chip-HPLC-MS coupling interface, specifically including: magnetic microfluidic chip module (including magnetic microfluidic chip 4), electromagnetic oscillation module 5, microfluidic drive module and Chip-HPLC-MS coupling interface 7; the microfluidic drive module includes microfluidic injection pump 1 and two-way valve 3;

[0084] The electromagnetic oscillation module is physically fixed below the magnetic microfluidic chip module by a chip fixing component, so that the fluid direction in the reaction chamber of the magnetic microfluidic chip module is perpendicular to the magnetic field direction of the electromagnetic oscillation module.

[0085] The microfluidic injection pump of the microfluidic drive module is connected to the channel inlet of the magnetic microfluidic chip module through a two-way valve; the Chip-HPLC-MS coupling interface is connected to the channel outlet of the magnetic microfluidic chip module through the two-way valve of the microfluidic drive module.

[0086] The outer wall of the reaction chamber of the magnetic microfluidic chip module is covered with magnetic stickers, which are used to fix Fe3O4@GO magnetic adsorption material.

[0087] Among them, the microfluidic chip of the magnetic microfluidic chip module of the device is a PMMA (acrylic) material chip prepared by laser engraving, with a channel cross-sectional size of 200×200μm, a pressure resistance limit of 10MPa, and an operating temperature range of 4-60℃;

[0088] The steps of immobilizing Fe3O4@GO magnetic adsorption material onto the surface of a magnetic sticker include:

[0089] The Fe3O4@GO magnetic adsorbent material is injected into the reaction chamber of the magnetic microfluidic chip module via a microfluidic injection pump driven by the microfluidic driving module. A magnetic sticker is affixed to the outer surface of the microfluidic reaction chamber. Under the influence of an external magnetic field, the magnetic adsorbent material flowing through this area is fixed onto the inner wall of the reaction chamber. The output frequency of the electromagnetic oscillation module is 100-500Hz, and the magnetic field strength is 50-200mT. The preparation method of the Fe3O4@GO magnetic adsorbent material includes the following steps:

[0090] (1) Synthesis of NH2-Fe3O4 magnetic spheres

[0091] FeCl3·6H2O was dissolved in ethylene glycol under ultrasonic assistance to form an orange-yellow solution. Then, anhydrous sodium acetate and 1,6-hexanediamine were added to the solution and the mixture was stirred vigorously at room temperature for 30 minutes. The mixture was then transferred to a high-pressure reactor and heated at 198°C for 6 hours. After the high-pressure reactor cooled naturally, the product was collected with the aid of an external magnetic field. The product was washed three times alternately with ultrapure water and ethanol and then freeze-dried for 24 hours to obtain NH2-Fe3O4 magnetic spheres.

[0092] (2) Synthesis of graphene oxide (GO)

[0093] Add 1g of graphite powder and 1g of NaNO3 to a 500mL beaker, then add 46mL of sulfuric acid. Stir in an ice bath for 10 minutes, then add 6g of potassium permanganate. Within 10 minutes, add the potassium permanganate to the solution. Observe that the potassium permanganate is reduced and turns dark green. Stir continuously for 2 hours to allow the redox reaction to proceed fully. Slowly add distilled water; the solution turns dark brown. Place the mixture in a 98℃ water bath and stir for 15 minutes, adding a large amount of distilled water while stirring. Then add an appropriate amount of 30% hydrogen peroxide to terminate the reaction for 1 hour. The solution will initially appear yellow, gradually turning brown. After centrifugation, remove sulfate ions with 5wt.% HCl, wash three times with pure water until neutral, and vacuum dry overnight to obtain graphene oxide.

[0094] (3) Synthesis of Fe3O4@GO magnetic adsorption material

[0095] First, disperse 300 mg GO in 30 mL of MES buffer (pH=5.6), add 100 mg EDC, sonicate and stir for 30 minutes, then add 60 mg N-hydroxysuccinimide and continue magnetic stirring for 15 minutes to obtain solution A; weigh 200 mg NH2-Fe3O4 magnetic beads and disperse them in 200 mL of MES buffer, sonicate for 30 minutes to obtain solution B;

[0096] Solution B was mixed dropwise with solution A and placed in a magnetic stirrer. The mixture was extracted, and the product was obtained by magnetic separation. The product was washed three times alternately with ultrapure water and ethanol, and then freeze-dried for 24 hours to obtain Fe3O4@GO magnetic adsorption material.

[0097] Example 2

[0098] The method for detecting multiple types of antibiotics in aquaculture water using the detection device for antibiotics in aquaculture water described in Example 1 specifically includes the following steps:

[0099] Remove the magnetic sticker on the bottom of the reaction chamber of the magnetic microfluidic chip module, start the electromagnetic oscillation module (the output frequency of the magnetic field is 100-500Hz, and the magnetic field strength is 50-200mT), and pump the aquaculture water sample to be tested into the reaction chamber of the magnetic microfluidic chip module at a flow rate of 0.5-5 mL / min through the microfluidic injection pump of the microfluidic drive module. Set the adsorbent vibration frequency to 300 Hz, the enrichment time to 3 minutes, and the water sample treatment volume to 5μL. The Fe3O4@GO magnetic adsorbent material vibrates under the drive of the alternating magnetic field, and fully contacts the antibiotics in the water sample to achieve efficient enrichment. After enrichment, discharge the rinsing waste liquid into the waste liquid tank.

[0100] Switch the two-way valve to introduce a methanol-water mixture as the HPLC mobile phase into the reaction chamber of the magnetic microfluidic chip module. Elute the enriched antibiotics with a gradient elution using 500 μL of eluent (i.e., mobile phase) (program: 5 wt.% methanol concentration in the mixture for 0-2 min, 95 wt.% methanol concentration in the mixture for 2-15 min), achieving an elution efficiency ≥98.5%. Elute the solution to a C18 column via the Chip-HPLC-MS interface.

[0101] The antibiotics separated by the C18 column were introduced into a mass spectrometer and qualitative and quantitative analyses were performed using the mass spectrometry multiple reaction monitoring mode.

[0102] The results of antibiotic detection in aquaculture water were obtained according to the method in Example 2:

[0103] 5 μL of a mixed solution containing 18 kinds of fishery antibiotics (2 mg / L, types and...) Figure 5(The types of antibiotics used are consistent throughout the process.) The adsorption is directly injected into the composite chip using a syringe pump, achieving high efficiency within 5 minutes. The adsorbed solution is then analyzed by mass spectrometry with an accuracy exceeding 90%. After detection, the microfluidic chip is cleaned with 0.1 mol / L NaOH solution for 3 minutes, and the above detection process is repeated. The results are shown below. Figure 3 As can be seen, the Fe3O4@GO magnetic adsorption material of the present invention can be reused ≥5 times.

[0104] Water from the pond was collected and spiked using the isotope spiking method. The true concentration of antibiotics in the pond water was calculated based on the yield of the isotopically labeled antibiotics. The detection method includes the following steps:

[0105] Twenty-six commonly used aquatic antibiotics were selected and mixed solutions were prepared at concentration gradients of 1 mg / L, 2 mg / L, 5 mg / L, 8 mg / L, and 10 mg / L. 5 μL of each mixed solution was used as a sample solution, and online detection and analysis were performed using this method. The results are as follows: Figure 4 As shown in the figure (1-1 and 2-1 represent different concentration gradients, for example, 1-1 represents an antibiotic with a concentration of 1 mg / L), this method was used to perform online detection of mixed solutions of 26 commonly used veterinary antibiotics. Pefloxacin and malachite green had relatively low capture efficiencies, ranging from 69.7% to 83.2% and 81.3% to 88.6% at five different concentrations, respectively, which may be related to their molecular structures. The capture efficiencies of the other 24 antibiotics were all greater than 90%, which fully demonstrates that this method is universally applicable to conventional veterinary antibiotics and can perform efficient analysis and detection.

[0106] Eighteen other commonly used fish antibiotics were spiked for testing. The concentration of the mixed solution was 2 mg / L. Magnetic adsorption materials at concentrations of 1 mg / L, 2 mg / L, 5 mg / L, 8 mg / L, and 10 mg / L were injected into the microfluidic chip channels, respectively. The experimental results are shown below. Figure 5 (In the figure, 1-1, 2-1, etc. represent the concentrations of different magnetic adsorption materials. For example, 1-1 indicates that the concentration of magnetic adsorption material is 1 mg / L, while 8-(1) and 8-(2) represent two experiments with 8 mg / L magnetic adsorption material.) The results show that as the concentration of adsorption material increases, the capture efficiency of 18 antibiotics all show a gradually increasing trend. When the concentration of magnetic adsorption material is ≥8 mg / L, the capture efficiency of 18 antibiotics can reach more than 98%, which fully demonstrates that by adjusting the concentration of magnetic adsorption material, efficient capture of different samples can be achieved, thereby further ensuring the efficient and sensitive detection of multiple types of antibiotics in aquaculture water.

[0107] The results show that the method in Example 2 can accurately detect more than 50 antibiotics of 8 types, including quinolones, sulfonamides, and sedatives, with a detection limit of 0.1 μg / L. In summary, this invention successfully solves the problems of low adsorption efficiency and cumbersome pretreatment in the detection of multiple antibiotic residues in aquatic environments by combining the structural design of magnetic adsorption materials with microfluidic preparation processes. The data from the examples show that the material has a recovery rate of over 90% for multiple antibiotics and can meet the needs of both qualitative and quantitative screening in laboratories. Those skilled in the art can adjust parameters such as the amount of magnetic adsorption material and the fluid flow rate according to actual needs; all such improvements fall within the scope of protection of this invention.

[0108] The above are merely preferred embodiments of the present invention, but the scope of protection of the present invention is not limited thereto. Any variations or substitutions that can be easily conceived by those skilled in the art within the scope of the technology disclosed in the present invention should be included within the scope of protection of the present invention.

Claims

1. A method for detecting antibiotics in aquaculture water, characterized in that, The detection of antibiotics in aquaculture water is performed using a detection device, which includes: a microfluidic chip module, an electromagnetic oscillation module, a microfluidic drive module, and a Chip-HPLC-MS interface; the microfluidic drive module includes a microfluidic injection pump and a two-way valve. The electromagnetic oscillation module is physically fixed below the microfluidic chip module carrying magnetic adsorption material by a chip fixing component, and the fluid direction of the reaction chamber of the microfluidic chip module is perpendicular to the magnetic field direction of the electromagnetic oscillation module. The microfluidic injection pump of the microfluidic drive module is connected to the channel inlet of the microfluidic chip module through a two-way valve; the Chip-HPLC-MS coupling interface is connected to the channel outlet of the microfluidic chip module through the two-way valve of the microfluidic drive module. A magnetic sticker is affixed to the bottom of the outer chamber of the microfluidic chip module, which fixes the Fe3O4@GO magnetic adsorption material to the inner wall of the microfluidic channel. The preparation method of the Fe3O4@GO magnetic adsorption material includes the following steps: Graphene oxide was dispersed in 2-morpholine ethanesulfonic acid buffer, and ethylene glycol dichloro ether and N-hydroxysuccinimide were added. After magnetic stirring, solution A was obtained. NH2-Fe3O4 magnetic beads were dispersed in 2-morpholine ethanesulfonic acid buffer solution to obtain solution B; The solutions A and B were mixed, and the products were magnetically separated to obtain the Fe3O4@GO magnetic adsorption material. The method for detecting antibiotics in aquaculture water includes the following steps: Remove the magnetic sticker from the bottom of the reaction chamber of the magnetic microfluidic chip module. Start the electromagnetic oscillation module and inject the aquaculture water sample into the reaction chamber of the magnetic microfluidic chip module via the microfluidic injection pump of the microfluidic drive module. This allows the water sample to mix with the Fe3O4@GO magnetic adsorption material to enrich antibiotics. After enrichment, the rinsing waste liquid is discharged. Switch the two-way valve to introduce a mixture of methanol and water as the HPLC mobile phase into the reaction chamber of the magnetic microfluidic chip module. The enriched antibiotics are then eluted using a gradient elution method and eluted to a C18 column via the Chip-HPLC-MS interface. The antibiotics separated by the C18 column are then introduced into a mass spectrometer for qualitative and quantitative analysis using multiple reaction monitoring (MRM) mode.

2. The method for detecting antibiotics in aquaculture water according to claim 1, characterized in that, In solution A, the ratio of the amount of graphene oxide to 2-morpholine ethanesulfonic acid buffer, ethylene glycol dichloro ether, and N-hydroxysuccinimide is 30 mg: 3 mL: 10 mg: 6 mg. In solution B, the ratio of the NH2-Fe3O4 magnetic beads to the 2-morpholine ethanesulfonic acid buffer is 1 mg: 1 mL.

3. The method for detecting antibiotics in aquaculture water according to claim 1, characterized in that, In solutions A and B, the mass ratio of the NH2-Fe3O4 magnetic spheres to the graphene oxide is 2:

3.

4. The method for detecting antibiotics in aquaculture water according to claim 1, characterized in that, The step of fixing the Fe3O4@GO magnetic adsorption material to the inner wall of the microfluidic channel using the magnetic sticker includes: The Fe3O4@GO magnetic adsorption material is injected into the reaction chamber of the magnetic microfluidic chip module via the microfluidic injection pump of the microfluidic drive module. A magnetic sticker is attached to the outer surface of the chip. The magnetic adsorption material flowing through this area is adsorbed onto the inner wall of the reaction chamber of the microfluidic chip module under the action of the magnetic field of the magnetic sticker.

5. The method for detecting antibiotics in aquaculture water according to claim 1, characterized in that, The injection flow rate of the aquaculture water sample to be tested was 0.2-0.5 mL / min.

6. The method for detecting antibiotics in aquaculture water according to claim 1, characterized in that, The gradient elution program is as follows: 5 wt.% methanol in the mixture for 0-2 min, and 95 wt.% methanol in the mixture for 2-15 min.

7. The method for detecting antibiotics in aquaculture water according to claim 1, characterized in that, The output frequency of the magnetic field of the electromagnetic oscillation module is 100-500Hz, and the magnetic field strength is 50-200mT; during the enrichment stage, the vibration frequency of the Fe3O4@GO magnetic adsorption material is 300Hz, and the enrichment time is 3-5min.

8. The method for detecting antibiotics in aquaculture water according to claim 1, characterized in that, The antibiotics in the aquaculture water samples to be tested included sulfonamides, quinolones, tetracyclines, and sedative antibiotics, with a detection range of 0.01-5 μg / L.

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