Device and method for detecting antibiotics in aquaculture water

Abstract

The invention provides a device and a method for detecting antibiotics in aquaculture water, and belongs to the technical field of environmental pollutant detection. According to the device disclosed by the invention, a Fe3O4 (at) GO magnetic nano adsorption material is packaged in a micro-fluidic chip reaction chamber, and a magnetic sticker is innovatively adopted to realize immobilization and replaceable design of an adsorbent; the integrated electromagnetic oscillation module drives the adsorbent to vibrate in the microchannel, and the mass transfer capture efficiency of antibiotics such as sulfonamides, quinolones and tetracyclines is remarkably improved. According to the method, an adsorption-elution-detection integrated process is initiated for the first time, the pretreatment time is shortened to be within 10 minutes from the hour level, synchronous detection of 0.01-5 [mu] g / L ultra-trace multi-component antibiotics in 5-10 [mu] L of a water sample is achieved, compared with a traditional method, the detection precision is remarkably improved, the matrix interference resistance is high, and the method is suitable for large-scale popularization and application. The method is suitable for real-time online monitoring of multiple antibiotic residues in the 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] The reaction chamber outer bottom of the magnetic microfluidic chip module is attached with a magnetic sticker, and the Fe3O4@GO magnetic adsorption material is fixed on the inner wall of the microfluidic channel.

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

[0014] The graphene oxide is dispersed in 2-morpholinoethanesulfonic acid (MES) buffer solution, and ethylene glycol dichloride (EDC) and N-hydroxysuccinimide (NHS) are added, and after magnetic stirring, solution A is obtained;

[0015] The NH2-Fe3O4 magnetic ball is dispersed in 2-morpholinoethanesulfonic acid (MES) buffer solution to obtain solution B;

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

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

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

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

[0020] Further, the step of fixing the Fe3O4@GO magnetic adsorption material on the inner wall of the microfluidic channel by the magnetic sticker comprises:

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

[0022] Further, the electromagnetic oscillation module further comprises a current controller, a chip fixing component, an electromagnetic coil and the like, can generate an alternating magnetic field to drive the magnetic adsorption material to vibrate, significantly improve the mass transfer and capture efficiency of the antibiotic, and ensure that the adsorption effect of the same batch of samples on the antibiotic remains consistent. At the same time, the magnetic field parameters can be set according to the actual detection requirements, so as to control the adsorption efficiency and effect.

[0023] Further, the Chip-HPLC-MS interface comprises a two-way injection valve connected with the inlet and outlet of the microfluidic chip and a high-pressure fluid passage connected with the liquid chromatograph, so that the adsorbed antibiotics can be eluted and introduced into the HPLC-MS (high performance liquid chromatography-mass spectrometry) system for detection.

[0024] Further, the microfluidic chip of the microfluidic driving module is located between the flow phase storage bottle and the liquid chromatograph, the chip inlet and outlet are connected, and the two channels are switched by the switching valve, the A channel is connected with the injection pump for injecting the magnetic adsorption material and the sample solution, and the B channel is controlled by the high performance liquid chromatography program, so that the integrated process of "adsorption-elution-detection" is realized.

[0025] The second technical scheme of the application:

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

[0027] 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 to be tested into the reaction chamber of the magnetic microfluidic chip module through the microfluidic injection pump of the microfluidic driving module, so that the water sample is mixed with the Fe3O4@GO magnetic adsorption material to enrich the antibiotics, and then the flushing waste liquid is discharged after the enrichment is completed;

[0028] Switch the two-way valve, introduce the mixed liquid containing methanol and water into the reaction chamber of the magnetic microfluidic chip module as the HPLC flow phase, gradient elute the enriched antibiotics, and elute to the C18 chromatographic column through the Chip-HPLC-MS interface;

[0029] Introduce the antibiotics separated by the C18 chromatographic column into the mass spectrometer, and perform qualitative and quantitative analysis by mass spectrometry multi-reaction monitoring mode.

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

[0031] Further, the gradient elution program is that the methanol concentration in the mixed liquid is 5wt.% at 0-2min, and the methanol concentration in the mixed liquid is 95wt.% at 2-15min.

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

[0033] Further, the antibiotics in the water sample to be detected of the aquaculture water include sulfonamides, quinolones, tetracyclines and tranquilizer antibiotics, and the detection range is 0.01-5 μg / L.

[0034] Further, the water sample to be detected of the aquaculture water is injected by a micro-fluid injection pump and mixed with the Fe3O4@GO magnetic adsorption material in the reaction chamber, and after the reaction is completed, the reaction product is flushed out, and the introduction of the mobile phase can be accurately controlled by switching the valve, so that the antibiotics can be efficiently eluted.

[0035] Further, the method for detecting multiple types of antibiotics in the aquaculture water further comprises a regeneration step of the Fe3O4@GO magnetic adsorption material: injecting 0.1 mol / L NaOH solution into the micro-fluidic chip for cleaning for 3 minutes, and the reuse number of the Fe3O4@GO magnetic adsorption material is ≥5 times.

[0036] After the water sample to be detected of the aquaculture water in the device for online high-sensitivity detection of multiple types of antibiotics in the aquaculture water based on Chip-HPLC-MS, the Fe3O4@GO magnetic adsorption material forms a dynamic adsorption interface in the micro-fluidic channel, can be in full contact with the water sample, realizes efficient adsorption of multiple types of antibiotics such as sulfonamides, quinolones and tetracyclines, avoids the interference of complex matrix on detection, and the content of the adsorbed antibiotics is linearly related to the initial concentration of the antibiotics in the water sample.

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

[0038] (1) The method for detecting multiple types of antibiotics in the aquaculture water of the present application realizes the integration of "adsorption-elution-detection" process by innovatively using the surface-modified Fe3O4@GO magnetic nano-adsorption material and the magnetic sticker immobilization technology, combining the electromagnetic oscillation module to improve the mass transfer and capture efficiency, shortening the pretreatment time from hours to 10 minutes, realizing the synchronous detection of 0.01-5 μg / L ultra-trace multi-component antibiotics in 5-10 μL water sample, and significantly improving the detection accuracy and anti-matrix interference ability. The technical scheme of the present application has important significance for real-time online monitoring of multiple antibiotic residues in aquaculture environment, can timely find the antibiotic residue problem, provides strong guarantee for scientific management of aquaculture and quality and safety of aquatic products, and promotes the sustainable development of aquaculture industry. At the same time, the present application also provides a new idea and method for the field of environmental pollutant detection technology, and is expected to be widely applied in other fields.

[0039] (2) The device of the present application comprises a magnetic micro-fluidic chip module, an electromagnetic oscillation module, a micro-fluid driving module and a Chip-HPLC-MS combined interface, the first three parts are integrated and designed, and are linked to each other to ensure efficient enrichment, elution and detection of multiple antibiotics in the aquaculture water.

[0040] (3) The application solves the problems of traditional detection methods, such as complex pretreatment, time-consuming, low detection precision, weak anti-interference ability, etc., and can improve and accurately control the detection ability of various types of antibiotics at the same time; the device has high integration and intelligence, but the assembly module is relatively simple, easy to operate and maintain, and has low material cost; the application has very high universality and can be applied to the detection of residues of different types of antibiotics in different aquaculture environments. BRIEF DESCRIPTION OF DRAWINGS

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

[0042] Figure 1 The principle diagram for detecting multiple types of antibiotics in aquaculture water in the embodiments of the application;

[0043] Figure 2 The detection device for antibiotics in aquaculture water in embodiment 1, the structure schematic diagram, wherein, 1-microfluidic injection pump, 2-mobile phase storage pool, 3-two-way valve, 4-magnetic microfluidic chip, 5-electromagnetic oscillation module, 6-waste liquid pool, 7-Chip-HPLC-MS combined interface;

[0044] Figure 3 The removal efficiency result of the device in embodiment 2 of the application for repeated detection of antibiotics in aquaculture water;

[0045] Figure 4 The capture efficiency experimental result of the device in embodiment 2 of the application for 26 commonly used fish antibiotics;

[0046] Figure 5 The capture efficiency experimental result of the device in embodiment 2 of the application for 18 antibiotics under different concentrations of magnetic adsorption materials. DETAILED DESCRIPTION

[0047] The detailed description of the various exemplary embodiments of the application should not be considered as limiting the application, but should be understood as a more detailed description of certain aspects, characteristics and embodiments of the application.

[0048] It should be understood that the terms described in the application are only for the description of the specific embodiments, and are not used to limit the application. In addition, for the numerical range in the application, it should be understood that each intermediate value between the upper limit and the lower limit of the range is also specifically disclosed. Each smaller range between any stated value or intermediate value in the stated range and any other stated value or intermediate value in the stated range is also included in the application. The upper limit and the lower limit of these smaller ranges can be independently included or excluded from the range.

[0049] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this application belongs. Although methods and materials similar or equivalent to those described herein can be used in the practice or testing of the present application, the preferred methods and materials are described. All documents mentioned herein are incorporated by reference to disclose and describe in full the methods and / or materials which are described therein. In case of conflict, the content of the present specification will control.

[0050] Many modifications and variations of the present application described in the specific embodiments of the application can be made by those skilled in the art without departing from the scope or spirit of the application. Other implementations of the application will be apparent to those skilled in the art from consideration of the specification and practice of the application. The specification and examples of the application are exemplary only.

[0051] As used herein, the terms "comprises", "comprising", "includes", "including", "has", "having" or variants thereof are open-ended, meaning they do not exclude additional elements or steps.

[0052] The embodiment of the present application provides a detection device for antibiotics in aquaculture water, which comprises a microfluidic chip module, an electromagnetic oscillation module, a microfluidic driving module and a Chip-HPLC-MS interface; the microfluidic driving module comprises a microfluidic injection pump and a two-way valve;

[0053] The electromagnetic oscillation module is physically fixed below the microfluidic chip module loaded with the magnetic adsorption 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;

[0054] 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 interface is connected to the channel outlet of the microfluidic chip module through the two-way valve of the microfluidic driving module.

[0055] The reaction chamber of the magnetic microfluidic chip module is attached with a magnetic sticker on the outer bottom, and the magnetic sticker fixes the Fe3O4@GO magnetic adsorption material on the inner wall of the microfluidic channel.

[0056] In the preferred embodiment of the present application, the microfluidic chip of the magnetic microfluidic chip module is a PMMA (acrylic) material chip prepared by laser engraving method, the channel cross-sectional size is 200x200μm, the upper limit of pressure resistance is 10MPa, and the working temperature range is 4-60℃.

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

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

[0059] FeCl3·6H2O was dissolved in ethylene glycol under the assistance of ultrasonic 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, then the mixture was transferred to a high-pressure reaction kettle, heated at 198℃ for 6 hours, after the high-pressure reaction kettle was naturally cooled, the product was collected by an external magnetic field, washed with ultrapure water and ethanol alternately for three times, and then freeze-dried for 24 hours to obtain NH2-Fe3O4 magnetic balls;

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

[0061] In a 500mL beaker, 1g of graphite powder and 1g of NaNO3 were added, followed by the addition of 46mL of sulfuric acid, and the mixture was stirred in an ice bath for 10 minutes, then 6g of potassium permanganate was added, and the addition was completed within 10 minutes, and it was observed that the potassium permanganate was reduced to a dark green color, and the stirring was continued for 2 hours to ensure that the redox reaction was complete, then distilled water was slowly added, and the solution turned dark brown, the mixture was placed in a 98℃ water bath and stirred for 15 minutes, and a large amount of distilled water was added during stirring, and an appropriate amount of 30% hydrogen peroxide was added to terminate the reaction for 1 hour, and the solution first turned yellow and then brown. After centrifugal separation, 5wt.% HCl was used to remove sulfate ions, and then the mixture was washed with pure water three times until it was neutral, and then vacuum dried overnight to obtain graphene oxide;

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

[0063] First, 300mg of GO was dispersed in 30mL of MES buffer solution (pH=5.6), 100mg of EDC was added, and the mixture was ultrasonically dispersed and stirred for 30 minutes, then 60mg of NHS was added, and the stirring was continued for 15 minutes to obtain solution A; 200mg of NH2-Fe3O4 magnetic balls were weighed and dispersed in 200mL of MES buffer solution, and ultrasonic dispersion was performed for 30 minutes to obtain solution B;

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

[0065] In the preferred embodiment of the present application, the step of fixing the Fe3O4@GO magnetic adsorption material on the inner wall of the microfluidic channel by the magnetic sticker includes: injecting the Fe3O4@GO magnetic adsorption material into the reaction chamber of the magnetic microfluidic chip module through the microfluidic injection pump of the microfluidic driving module, attaching a magnetic sticker to the outer surface of the chip, and adsorbing the magnetic adsorption material flowing through the region on 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 the preferred embodiment of the present application, the electromagnetic oscillation module further includes a current controller, a chip fixing component, an electromagnetic coil and the like, can generate an alternating magnetic field to drive the magnetic adsorption material to vibrate, significantly improve the mass transfer and capture efficiency of the antibiotic, and ensure that the adsorption effect of the same batch of samples on the antibiotic remains consistent. At the same time, the magnetic field parameters can be set according to the actual detection requirements, so as to control the adsorption efficiency and effect.

[0067] In the preferred embodiment of the present application, the Chip-HPLC-MS combined interface includes a two-way injection valve connected to the inlet and outlet of the microfluidic chip and a high-pressure fluid passage connected to the liquid chromatograph, so as to ensure that the adsorbed antibiotic can be smoothly eluted and introduced into the HPLC-MS (high performance liquid chromatography-mass spectrometry) system for detection.

[0068] In the preferred embodiment of the present application, the microfluidic chip of the microfluidic driving 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, the two channels are switched by a switching valve, the A channel is connected to the injection pump for injecting the magnetic adsorption material and the sample solution, and the B channel is controlled by the high performance liquid chromatography program to realize the integrated process of "adsorption-elution-detection".

[0069] The embodiment of the present application also provides a method for detecting antibiotics in aquaculture water, using the detection device for antibiotics in aquaculture water, and the method comprises the following steps:

[0070] The magnetic sticker on the outer bottom of the reaction chamber of the magnetic microfluidic chip module is removed, the electromagnetic oscillation module is started, and the aquaculture water sample to be detected is injected into the reaction chamber of the magnetic microfluidic chip module through the microfluidic injection pump of the microfluidic driving module, so that the water sample and the Fe3O4@GO magnetic adsorption material are mixed to enrich the antibiotic, and after the enrichment is completed, the flushing waste liquid is discharged to the waste liquid pool;

[0071] The two-way valve is switched, the mixed liquid containing methanol and water is introduced into the reaction chamber of the magnetic microfluidic chip module as the HPLC mobile phase, the enriched antibiotic is gradient eluted, and is eluted to the C18 chromatographic column through the Chip-HPLC-MS combined interface;

[0072] The antibiotic separated by the C18 chromatographic column is introduced into the mass spectrometer, and qualitative and quantitative analysis is performed by using the mass spectrometry multiple reaction monitoring mode.

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

[0074] In the preferred embodiment of the present application, the gradient elution procedure is 5wt.% of methanol in the mixture for 0-2 min and 95wt.% of methanol in the mixture for 2-15 min.

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

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

[0077] In the preferred embodiment of the present application, the water sample to be tested of the aquaculture water is injected by a micro-fluid injection pump and mixed with the Fe3O4@GO magnetic adsorption material in the reaction chamber, and after the reaction is completed, it is flushed out, and the introduction of the mobile phase can be accurately controlled by switching the valve to realize efficient elution of the antibiotics.

[0078] In the preferred embodiment of the present application, the method for detecting multiple types of antibiotics in aquaculture water further comprises a regeneration step of the Fe3O4@GO magnetic adsorption material: injecting 0.1 mol / L NaOH solution into the micro-fluidic chip for cleaning for 3 min, and the repeated use number of the Fe3O4@GO magnetic adsorption material is ≥5 times.

[0079] The principle diagram of detecting multiple types of antibiotics in aquaculture water in the embodiment of the present application is shown in Figure 1Specifically, a sample solution containing antibiotics in aquaculture water is injected into a microfluidic chip by using a syringe pump, and the chip is in a magnetic field environment constructed by an electromagnetic oscillation module and the like (the magnetic field distribution is shown by the "S, N" poles in the figure); the Fe3O4@GO magnetic nanoadsorbent material loaded in the chip is driven to vibrate by the magnetic field, which significantly improves the mass transfer efficiency of the antibiotic molecules in the sample solution, and the target antibiotics are efficiently adsorbed and captured by the interaction between the functional groups such as hydroxyl and carboxyl groups on the surface of the material and the antibiotics, and the impurities are discharged with the waste liquid; then, the mobile phase (methanol / water mixture) is introduced into the chip, and the antibiotics adsorbed on the magnetic material are eluted under the action of the magnetic field, and the target antibiotic molecules eluted are introduced into the HPLC-MS system with the mobile phase, and different types of antibiotics are separated by gradient elution on the HPLC chromatographic column (such as a C18 column), and then the mass spectrometer is used in the multiple reaction monitoring mode to complete the qualitative and quantitative detection of multiple types of antibiotics such as sulfonamides and quinolones at ultra-trace levels (0.01-5 μg / L level); after the detection is completed, the magnetic adsorbent material can also be regenerated by an alkaline solution (such as 0.1 mol / L NaOH solution) to realize the reuse of the material.

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

[0081] The technical solutions of the present application are further illustrated by the following examples.

[0082] Example 1

[0083] A device for detecting antibiotics in aquaculture water, a structural schematic diagram is shown in Figure 2 , wherein 1-microfluidic syringe pump, 2-mobile phase reservoir, 3-two-way valve, 4-magnetic microfluidic chip, 5-electromagnetic oscillation module, 6-waste liquid pool, 7-Chip-HPLC-MS combined interface, specifically comprising: a magnetic microfluidic chip module (including a magnetic microfluidic chip 4), an electromagnetic oscillation module 5, a microfluidic driving module and a Chip-HPLC-MS combined interface 7; the microfluidic driving module includes a microfluidic syringe pump 1 and a 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 of the reaction chamber of the magnetic microfluidic chip module is perpendicular to the magnetic field direction of the electromagnetic oscillation module;

[0085] The microfluidic syringe pump of the microfluidic driving module is connected to the channel inlet of the magnetic microfluidic chip module through the two-way valve; the Chip-HPLC-MS combined interface is connected to the channel outlet of the magnetic microfluidic chip module through the two-way valve of the microfluidic driving module;

[0086] The reaction chamber outer wall of the magnetic microfluidic chip module is loaded with a magnetic sticker, and the magnetic sticker is used to fix the Fe3O4@GO magnetic adsorbent material;

[0087] The microfluidic chip of the magnetic microfluidic chip module of the device is a PMMA chip prepared by laser engraving, the channel cross-sectional size is 200x200 μm, the upper limit of pressure resistance is 10 MPa, and the working temperature range is 4-60℃;

[0088] The step of immobilizing the Fe3O4@GO magnetic adsorbent material on the surface of the magnetic sticker includes:

[0089] The Fe3O4@GO magnetic adsorbent material is injected into the reaction chamber of the magnetic microfluidic chip module through the microfluidic injection pump of the microfluidic driving module, the magnetic sticker is attached to the outer surface of the microfluidic reaction chamber, and under the action of the external magnetic field, the magnetic adsorbent material flowing through the region is fixed on the inner wall of the reaction chamber; the output frequency of the electromagnetic oscillation module magnetic field is 100-500 Hz, and the magnetic field strength is 50-200 mT; the preparation method of the Fe3O4@GO magnetic adsorbent material includes the following steps:

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

[0091] FeCl3·6H2O is dissolved in ethylene glycol under the assistance of ultrasonic waves to form an orange yellow solution, then anhydrous sodium acetate and 1,6-hexanediamine are added to the solution, and the mixture is stirred vigorously at room temperature for 30 minutes, then the mixture is transferred to a high-pressure reaction kettle, heated at 198℃ for 6 hours, after the high-pressure reaction kettle is naturally cooled, the product is collected by an external magnetic field, washed with ultrapure water and ethanol alternately for three times, and then freeze-dried for 24 hours to obtain NH2-Fe3O4 magnetic balls;

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

[0093] In a 500 mL beaker, 1 g of graphite powder and 1 g of NaNO3 are added, 46 mL of sulfuric acid is added, and the mixture is stirred in an ice bath for 10 minutes, then 6 g of potassium permanganate is added, and the potassium permanganate is added to the above solution within 10 minutes, and the potassium permanganate is reduced to present dark green, and the stirring is continued for 2 hours to make the redox reaction proceed fully, distilled water is slowly added, and the solution presents dark brown, the mixture is placed in a 98℃ water bath and stirred for 15 minutes, a large amount of distilled water is added during stirring, and an appropriate amount of 30% hydrogen peroxide is added to terminate the reaction for 1 hour, and the solution will first present yellow and then brown. After centrifugal separation, 5wt.% HCl is used to remove sulfate ions, and then the mixture is washed with pure water for three times until neutral, and then vacuum dried overnight to obtain graphene oxide;

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

[0095] First, 300 mg of GO was dispersed in 30 mL of MES buffer (pH = 5.6), 100 mg of EDC was added, and after ultrasonic dispersion, it was stirred for 30 minutes, then 60 mg of N-hydroxysuccinimide was added, and magnetic stirring was continued for 15 minutes to obtain solution A; 200 mg of NH2-Fe3O4 magnetic balls were weighed and dispersed in 200 mL of MES buffer, and ultrasonic dispersion was performed for 30 minutes to obtain solution B;

[0096] Solution B was added dropwise to solution A and placed in a magnetic stirrer, and the mixture was extracted and magnetically separated to obtain the product, which was washed with ultrapure water and ethanol three times alternately, and freeze-dried for 24 hours to obtain Fe3O4@GO magnetic adsorbent 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 in Example 1, specifically comprising the following steps:

[0099] Remove the magnetic sticker from 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 100-500 Hz, and the magnetic field strength is 50-200 mT), and pump the 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 driving module. The vibration frequency of the adsorbent is set to 300 Hz, the enrichment time is 3 minutes, the water sample treatment volume is 5 μL, and the Fe3O4@GO magnetic adsorbent material is driven to vibrate under the alternating magnetic field to achieve efficient enrichment with the antibiotics in the water sample. After enrichment, the flushing waste liquid is discharged to the waste liquid pool;

[0100] Switch the two-way valve, and introduce the mixture containing methanol and water as the HPLC mobile phase into the reaction chamber of the magnetic microfluidic chip module, and elute the enriched antibiotics with 500 μL of eluent (i.e. mobile phase) volume elution gradient elution (the program is 0-2 min, the methanol concentration in the mixture is 5 wt.%, and 2-15 min, the methanol concentration in the mixture is 95 wt.%), and the elution efficiency is ≥98.5%, and the elution is carried out to the C18 chromatographic column through the Chip-HPLC-MS combined interface;

[0101] The antibiotics separated by the C18 chromatographic column are introduced into the mass spectrometer, and qualitative and quantitative analysis is performed by mass spectrometry in multiple reaction monitoring mode.

[0102] The detection results of antibiotics in aquaculture water according to the method of Example 2 are as follows:

[0103] 5 μL of a mixed solution containing 18 types of fish antibiotics (2 mg / L, types and Figure 5The injection pump is directly used to inject into the composite chip, high-efficiency adsorption can be completed in 5 minutes, the solution after adsorption is detected by mass spectrometry, and the accuracy is as high as 90% or more. After detection, 0.1 mol / L NaOH solution is injected into the microfluidic chip for 3 minutes, and the above detection process is repeated, and the detection results are shown in Figure 3 It can be seen that the Fe3O4@GO magnetic adsorption material of the application can be reused for more than 5 times.

[0104] Take pond water, and use isotopic labeling method to add labels, calculate the real concentration of antibiotics in pond water according to the yield of isotopically labeled antibiotics. The detection method includes the following steps:

[0105] 26 kinds of commonly used fish antibiotics are selected, and a mixed solution is prepared according to the gradient concentration of 1 mg / L, 2 mg / L, 5 mg / L, 8 mg / L and 10 mg / L. 5 μL of the mixed solution with different concentrations is taken as a sample solution, and the method is used for online detection and analysis. The results are shown in Figure 4 The method is used for online detection of 26 kinds of commonly used fish antibiotic mixed solutions, and the capture efficiency of Pefloxacin (Pefloxacin) and Malachite Green is relatively low, and the capture efficiency at five different concentrations is in the range of 69.7%-83.2% and 81.3%-88.6% respectively, which may be related to the molecular structure. The capture efficiency of the other 24 antibiotics is more than 90%, which fully shows that the method has universality for conventional fish antibiotics and can be used for efficient analysis and detection.

[0106] Take another 18 kinds of commonly used fish antibiotics for labeling detection, and the concentration of the mixed solution is 2 mg / L, and 1 mg / L, 2 mg / L, 5 mg / L, 8 mg / L and 10 mg / L of magnetic adsorption material are injected into the microfluidic chip channel. The experimental results are shown in Figure 5 (1-1, 2-1, etc. represent different concentrations of magnetic adsorption materials, such as 1-1 represents the concentration of magnetic adsorption material is 1 mg / L, and 8- (1), 8- (2) represent two experiments under 8 mg / L of magnetic adsorption material), the results show that with the increase of the concentration of adsorption material, the capture efficiency of 18 kinds of antibiotics shows a gradually increasing trend, when the concentration of magnetic adsorption material is greater than or equal to 8 mg / L, the capture efficiency of 18 kinds of antibiotics can reach more than 98%, which fully shows that by adjusting the concentration of magnetic adsorption material, high-efficiency capture of different samples can be realized, so as to further guarantee the high-efficiency and sensitive detection of the method for detecting multiple types of antibiotics in aquaculture water.

[0107] The results show that the method of example 2 can accurately detect more than 50 kinds of antibiotics of 8 types such as quinolones, sulfonamides, tranquilizers and the like, and the minimum detection limit is 0.1 μg / L. In summary, by combining the structural design of the magnetic adsorption material and the microfluidic preparation process, the problems of low adsorption efficiency and complicated pretreatment in the detection of various types of antibiotic residues in the water environment are successfully solved. The example data show that the recovery rate of the material to various antibiotics is more than 90%, and it can adapt to the dual screening requirements of laboratory qualitative and quantitative. The person skilled in the art can adjust the parameters such as the amount of magnetic adsorption material and fluid flow rate according to the actual needs, and the improved scheme all belongs to the protection scope of the present application.

[0108] The above is only a preferred specific embodiment of the present application, but the protection scope of the present application is not limited thereto, any person skilled in the art can easily think of changes or replacements within the technical range disclosed by the present application, which should be covered within the protection scope of the present application.

Claims

1. A device for detecting antibiotics in aquaculture water, characterized in that, include: The system 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 magnetic microfluidic chip module, which fixes the Fe3O4@GO magnetic adsorption material to the inner wall of the microfluidic channel.

2. The detection device for antibiotics in aquaculture water according to claim 1, characterized in that, 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 are mixed, and the products are magnetically separated to obtain the Fe3O4@GO magnetic adsorption material.

3. The detection device for antibiotics in aquaculture water according to claim 2, 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.

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

3.

5. The detection device for 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.

6. A method for detecting antibiotics in aquaculture water, characterized in that, The following steps are performed using the detection device for antibiotics in aquaculture water according to any one of claims 1-5: 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 antibiotics. After enrichment, the rinsing waste liquid is discharged. Switch the two-way valve to introduce the mixture of methanol and water as the HPLC mobile phase into the reaction chamber of the magnetic microfluidic chip module. The enriched antibiotics are eluted by gradient elution and eluted to the C18 column through the Chip-HPLC-MS coupling interface. 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.

7. The method for detecting antibiotics in aquaculture water according to claim 6, characterized in that, The injection flow rate of the aquaculture water sample to be tested was 0.2-0.5 mL / min.

8. The method for detecting antibiotics in aquaculture water according to claim 6, 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.

9. The method for detecting antibiotics in aquaculture water according to claim 6, 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.

10. The method for detecting antibiotics in aquaculture water according to claim 6, 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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