Micro-fluidic chip and detection method for content of sulfonamides in dairy products

Abstract

The invention provides a micro-fluidic chip for detecting the content of sulfonamides in dairy products and a method for detecting the content of sulfonamides in dairy products, and relates to the technical field of food detection. The micro-fluidic chip comprises a chip body, a sample inlet and a sample outlet are formed in the chip body, and a sample pretreatment channel communicated with the sample inlet, a filtering, buffering and mixing channel communicated with the sample pretreatment channel and a filtering and buffering channel communicated with the filtering, buffering and mixing channel are formed in the chip body. The reaction tank and the waste liquid tank are communicated with the filtering buffer channel, and the sample outlet is communicated with the reaction tank and the waste liquid tank; wherein the sample pretreatment channel is filled with a filter filler, the filter buffer mixing channel is used for full pretreatment reaction, a filter membrane is arranged between the filter buffer mixing channel and the filter buffer channel, and the reaction tank is filled with a biological composite material; the content of the sulfonamides in the dairy product can be effectively detected.

Description

Technical Field

[0001] The present disclosure relates to the technical field of food detection, and in particular, to a microfluidic chip for detecting the content of sulfonamide drugs in dairy products and a method for detecting the content of sulfonamide drugs in dairy products. Background Art

[0002] Sulfonamides are a class of broad-spectrum antibiotics that are commonly used to prevent and treat bacterial and parasitic diseases in dairy cows to reduce the impact of diseases on the health and production efficiency of the herd and to increase the milk production and feed conversion rate of dairy cows. Although sulfonamides play an important role in animal husbandry.

[0003] However, its residues in dairy products may pose a threat to human health, such as causing hematopoietic system disorders, allergic reactions and potential carcinogenic risks. The maximum limit standards for sulfonamide residues in dairy products vary from country to country. For example, the standards of the European Union and Canada are 100μg / kg, Japan is 20μg / kg, and China has also set a maximum limit of 100μg / kg. Despite the differences in standards, all countries have strengthened the control of its residues, showing that they attach great importance to dairy product safety testing.

[0004] It should be noted that the information disclosed in the above background technology section is only used to enhance the understanding of the background of the present disclosure, and therefore may include information that does not constitute the prior art known to ordinary technicians in the field. Summary of the invention

[0005] The purpose of the present disclosure is to provide a microfluidic chip for detecting the content of sulfonamide drugs in dairy products and a method for detecting the content of sulfonamide drugs in dairy products, which can effectively detect the content of sulfonamide drugs in dairy products.

[0006] According to one aspect of the present disclosure, a microfluidic chip for controlling the content of sulfonamide drugs in dairy products is provided, the microfluidic chip comprising a chip body, a sample inlet and a sample outlet formed on the chip body, a sample pretreatment channel connected to the sample inlet, a filtration buffer mixing channel connected to the sample pretreatment channel, a filtration buffer channel connected to the filtration buffer mixing channel, a reaction pool and a waste liquid pool connected to the filtration buffer channel, and the sample outlet is connected to the reaction pool and the waste liquid pool;

[0007] The sample pretreatment channel is filled with filter fillers, the filter buffer mixing channel is used for pretreatment and sufficient reaction, a filter membrane is provided between the filter buffer mixing channel and the filter buffer channel, and the reaction pool is filled with biocomposite materials.

[0008] In an exemplary embodiment of the present disclosure, the chip body includes:

[0009] A first chip layer and a second chip layer, wherein the first chip layer and the second chip layer are sealed and connected to form the chip body.

[0010] In an exemplary embodiment of the present disclosure, the filter membrane is a PVDF filter membrane, and the pore size of the filter membrane is 0.22 μm to 5 μm.

[0011] In an exemplary embodiment of the present disclosure, the filter filler is a composite filler of phosphate and ethylenediamine-N-propyl.

[0012] In an exemplary embodiment of the present disclosure, the biocomposite material is a carbonic anhydrase freeze-dried hydrogel.

[0013] In an exemplary embodiment of the present disclosure, the carbonic anhydrase freeze-dried hydrogel includes carbonic anhydrase, silk fibroin, PEG and gelatin composite embedding.

[0014] In an exemplary embodiment of the present disclosure, a plurality of the reaction pools are formed in the chip body.

[0015] According to another aspect of the present disclosure, a method for detecting the content of sulfonamide drugs in dairy products is provided, the method comprising:

[0016] Providing a microfluidic chip as described above, and placing the microfluidic chip vertically so that the waste liquid pool is located above the reaction pool;

[0017] Injecting the dairy product sample to be tested into the sample pretreatment channel through the sample injection port, and spontaneously reacting or adsorbing with the filter filler in the sample pretreatment channel during the injection process;

[0018] The sample enters the filtering buffer mixing channel from the sample pretreatment channel under the driving of the injection pressure, and the sample and the filtering filler fully react with each other in the filtering buffer mixing channel;

[0019] The mixed solution after the reaction is driven by the injection pressure from the filtering buffer mixing channel through the filtering membrane into the filtering buffer channel;

[0020] Driven by the injection pressure, the treatment solution in the filtration buffer channel fills the reaction tank and the waste liquid tank from bottom to top, and reacts with the biocomposite material in the reaction tank;

[0021] Inverting the microfluidic chip so that the injection port is located below the microfluidic chip;

[0022] Applying a first preset temperature to the microfluidic chip, and incubating the dairy product sample to be tested in the reaction pool for a first preset time;

[0023] Blow air through the injection port to discharge excess liquid injected into the reaction pool, so that the waste liquid is concentrated in the waste liquid pool or discharged through the outlet port;

[0024] Slowly injecting the color developing solution into the microfluidic chip through the injection port, so that the color developing solution fills the reaction pool;

[0025] Applying a second preset temperature to the microfluidic chip, incubating the color developing solution in the reaction pool for a second preset time, and then optically detecting the reaction pool to obtain an absorbance value of the reaction pool;

[0026] The obtained absorbance value is compared with the database to obtain the detection result of the sulfonamide content in the dairy product.

[0027] In an exemplary embodiment of the present disclosure, the first preset temperature is 30° C. to 40° C., and the first preset time is 8 min to 12 min.

[0028] In an exemplary embodiment of the present disclosure, the second preset temperature is 20° C. to 30° C., and the second preset time is 1 min to 5 min.

[0029] The microfluidic chip provided by the present disclosure can realize the detection of sulfonamide drugs in dairy products through simple operation steps, without the need for additional sample pre-treatment process, which greatly improves the operability of non-professionals. The interference of the sample itself to optical detection is eliminated by reasonable reaction area design, the sensitivity of detection is improved, and the chip can be applied to different batches or different types of dairy products. The design of the microfluidic chip is upper and lower layers, which saves the production cost of the chip. Carbonic anhydrase freeze-dried hydrogel is easy to store and has a good shelf life, and has good application prospects in the detection of sulfonamide drugs in dairy products.

[0030] It is to be understood that the foregoing general description and the following detailed description are exemplary and explanatory only and are not restrictive of the present disclosure. BRIEF DESCRIPTION OF THE DRAWINGS

[0031] The accompanying drawings herein are incorporated into the specification and constitute a part of the specification, illustrate embodiments consistent with the present disclosure, and together with the specification are used to explain the principles of the present disclosure. Obviously, the accompanying drawings described below are only some embodiments of the present disclosure, and for ordinary technicians in this field, other accompanying drawings can be obtained based on these accompanying drawings without creative work.

[0032] Figure 1 A schematic diagram of a microfluidic chip provided for one embodiment of the present disclosure.

[0033] Figure 2A schematic diagram of a first chip layer provided for an embodiment of the present disclosure.

[0034] Figure 3 A schematic diagram of a second chip layer provided for an embodiment of the present disclosure.

[0035] Figure 4 A flow chart of a method for detecting the content of sulfonamide drugs provided in one embodiment of the present disclosure.

[0036] Figure 5 An absorbance fitting curve diagram for detecting sulfonamide drugs of different concentrations provided in one embodiment of the present disclosure.

[0037] Figure 6 A storage life curve diagram of a microfluidic chip provided in one embodiment of the present disclosure.

[0038] Description of reference numerals:

[0039] 10. Microfluidic chip;

[0040] 100, first chip layer; 110, injection port; 120, sample pretreatment channel; 130, filtration buffer channel; 140, reaction pool; 150, waste liquid pool; 160, sample outlet;

[0041] 200, second chip layer; 210, filtering buffer mixing channel. DETAILED DESCRIPTION

[0042] Example embodiments will now be described more fully with reference to the accompanying drawings. However, example embodiments can be implemented in a variety of forms and should not be construed as limited to the embodiments set forth herein; rather, these embodiments are provided so that this disclosure will be comprehensive and complete and will fully convey the concepts of the example embodiments to those skilled in the art. The same reference numerals in the figures represent the same or similar structures, and thus their detailed description will be omitted.

[0043] Although relative terms such as "upper" and "lower" are used in this specification to describe the relative relationship of one component of the illustration to another component, these terms are used in this specification only for convenience, such as according to the orientation of the examples described in the drawings. It is understood that if the device of the illustration is turned upside down, the component described as "upper" will become the component "lower". When a structure is "on" other structures, it may mean that the structure is formed integrally on the other structure, or that the structure is "directly" disposed on the other structure, or that the structure is "indirectly" disposed on the other structure through another structure.

[0044] The terms "a", "an", "the", "said" and "at least one" are used to indicate the presence of one or more elements / components / etc.; the terms "including" and "having" are used to express an open-ended inclusive meaning and mean that additional elements / components / etc. may exist in addition to the listed elements / components / etc.; the terms "first", "second" and "third" etc. are used merely as labels and are not intended to limit the quantity of their objects.

[0045] Traditional detection of sulfonamides in dairy products mainly includes chromatography and colloidal gold methods, which have the characteristics of high sensitivity, high specificity and high accuracy, and can effectively detect sulfonamide residues in dairy products.

[0046] However, these methods require expensive instruments and equipment, professional technicians to operate, long detection time and complex sample pretreatment processes, which limits their application in large-scale rapid detection. In contrast, microfluidics can integrate multiple detection steps, including sample pretreatment, reaction and signal detection, to achieve fully automated operation. This integrated and automated design reduces manual operation errors and improves the repeatability and reliability of sulfonamide drug detection in dairy products.

[0047] The present disclosure provides a microfluidic chip 10 for measuring the content of sulfonamide drugs in dairy products, characterized in that it includes a chip body, a sample inlet 110 and a sample outlet 160 are formed on the chip body, a sample pretreatment channel 120 connected to the sample inlet 110, a filter buffer mixing channel 210 connected to the sample pretreatment channel 120, a filter buffer channel 130 connected to the filter buffer mixing channel 210, a reaction pool 140 and a waste liquid pool 150 connected to the filter buffer channel 130, and a sample outlet 160 connected to the reaction pool 140 and the waste liquid pool 150;

[0048] The sample pretreatment channel 120 is filled with filter fillers, the filter buffer mixing channel 210 is used for pretreatment and sufficient reaction, a filter membrane is provided between the filter buffer mixing channel 210 and the filter buffer channel 130, and the reaction pool 140 is filled with biocomposite materials.

[0049] The microfluidic chip 10 provided by the present disclosure can realize the detection of sulfonamide drugs in dairy products through simple operation steps, without the need for additional sample pre-treatment processes, which greatly improves the operability of non-professionals. The interference of the sample itself to optical detection is eliminated by reasonable reaction area design, the sensitivity of detection is improved, and the chip can be applied to different batches or different types of dairy products. The design of the microfluidic chip is upper and lower layers, which saves the production cost of the chip. Carbonic anhydrase freeze-dried hydrogel is easy to store and has a good shelf life, and has a good application prospect in the detection of sulfonamide drugs in dairy products.

[0050] like Figure 2 and Figure 3 As shown, the chip body includes: a first chip layer 100 and a second chip layer 200, and the first chip layer 100 and the second chip layer 200 are sealed and connected to form the chip body.

[0051] Among them, Figure 2 As shown, the first chip layer 100 is formed with an injection port 110 , a sample outlet 160 , a sample pre-treatment channel 120 connected to the injection port 110 , a filtering buffer mixing channel 210 , a reaction pool 140 and a waste liquid pool 150 .

[0052] Among them, Figure 3 As shown, a filtration buffer channel 130 is formed on the second chip layer 200; after the first chip layer 100 and the second chip layer 200 are stacked and connected together, the inlet of the filtration buffer mixing channel 210 is aligned and connected with the outlet of the sample pretreatment channel 120, and the outlet of the filtration buffer mixing channel 210 is aligned and connected with the inlet of the filtration buffer channel 130.

[0053] Among them, the sample pretreatment channel 120, the filtration buffer mixing channel 210, the reaction pool 140 and the waste liquid pool 150 can be structures directly formed on the first chip layer 100, or can be structures formed by the first chip layer 100 and the second chip layer 200 being stacked together and connected; the filtration buffer channel 130 can be a structure directly formed on the second chip layer 200, or can be a structure formed by the second chip layer 200 and the first chip layer 100 being stacked together and connected.

[0054] The width of the sample pretreatment channel 120, the filter buffer mixing channel 210 and the filter buffer channel 130 is 2 mm to 3 mm, and the depth is 0.5 mm to 1 mm; preferably, the width is 2.5 mm, and the depth of the microchannel is 1 mm. The width and / or depth of the sample pretreatment channel 120, the filter buffer mixing channel 210 and the filter buffer channel 130 may be the same or different.

[0055] The reaction pool 140 may have a length of 8 mm to 12 mm, a width of 6 mm to 10 mm, and a depth of 1 mm to 3 mm; preferably, the reaction pool 140 may have a length of 10 mm, a width of 8 mm, and a depth of 2 mm.

[0056] The waste liquid pool 150 may have a length of 8 mm to 12 mm, a width of 6 mm to 10 mm, and a depth of 1 mm to 3 mm; preferably, the waste liquid pool 150 may have a length of 10 mm, a width of 8 mm, and a depth of 2 mm. The size of the waste liquid pool 150 may be the same as or different from that of the reaction pool 140.

[0057] The filter filler can be a phosphate and ethylenediamine-N-propyl composite filler, which is used for pre-treatment purification of dairy products and adsorption of small molecule proteins, and optimizes the detection precision caused by liquid chromaticity on optical signals. Preferably, the filter filler can form a filter column with a structure that is not easy to loose in the sample pre-treatment channel 120 when tightly filled, so as to improve the filtering effect.

[0058] Among them, the mass ratio of phosphate to ethylenediamino-N-propyl in the filter filler can be (0.8-1.2):(0.8-1.2); preferably, the mass ratio of phosphate to ethylenediamino-N-propyl is 1:1.

[0059] Among them, the reaction biocomposite material can be a carbonic anhydrase freeze-dried hydrogel; further, the components of the carbonic anhydrase freeze-dried hydrogel can be a composite embedding of carbonic anhydrase, silk fibroin, PEG and gelatin, which effectively prolongs the shelf life of the immobilized enzyme.

[0060] The material of the first chip layer 100 and the second chip layer 200 is PMMA (polymethyl methacrylate) or PDMS (polydimethylsiloxane), preferably PMMA.

[0061] The first chip layer 100 and the second chip layer 200 may be formed into corresponding structures by laser printing.

[0062] The microfluidic detection chip is arranged in a sheet shape, that is, the first chip layer 100 and the second chip layer 200 can be in the shape of a circular sheet, a rectangular sheet or a polygonal sheet, preferably a rectangular sheet.

[0063] The first chip layer 100 and the second chip layer 200 can be sealed by bonding, clamping, screwing or welding. Preferably, PMMA special glue is used to seal the entire microfluidic chip 10.

[0064] Specifically, a filter membrane is provided between the first chip layer 100 and the second chip layer 200 , and the filter membrane covers the outlet of the filter buffer mixing channel 210 and the inlet of the filter buffer channel 130 .

[0065] Among them, the filter membrane is a PVDF filter membrane (polyvinylidene fluoride membrane), and the pore size on the filter membrane is 0.22μm to 5μm, for example, 0.22μm, 0.25μm, 0.3μm, 0.5μm, 1μm, 1.5μm, 2μm, 2.5μm, 3μm, 3.5μm, 4μm, 4.5μm, 5μm, etc., which are not listed one by one in the present disclosure; preferably, the pore size of the filter membrane is 0.22μm.

[0066] The filter membrane may be clamped and fixed between the first chip layer 100 and the second chip layer 200 .

[0067] Specifically, a plurality of reaction pools 140 are formed in the chip body, and the plurality of reaction pools 140 are used to eliminate the influence of solution matrices brought by different batches or different types of dairy products on the detection system.

[0068] Among them, Figure 1 As shown, two reaction pools 140 are formed in the chip body; of course, three, four or more reaction pools 140 may also be provided, and the present disclosure does not impose any limitation on this.

[0069] The embodiments of the present disclosure also provide a method for detecting the content of sulfonamide drugs in dairy products, such as Figure 4 As shown, the detection method includes:

[0070] S100, providing the microfluidic chip 10 described in the above embodiment, and placing the microfluidic chip 10 vertically so that the waste liquid pool 150 is located above the reaction pool 140;

[0071] S200, injecting the dairy product sample to be tested into the sample pre-treatment channel 120 through the injection port 110, and spontaneously reacting or adsorbing with the filter filler in the sample pre-treatment channel 120 during the injection process;

[0072] S300, the sample enters the filter buffer mixing channel 210 from the sample pre-treatment channel 120 under the driving of the injection pressure, and the sample and the filter filler react fully with each other in the filter buffer mixing channel 210;

[0073] S400, the mixed solution after the reaction is driven by the injection pressure from the filter buffer mixing channel 210, through the filter membrane into the filter buffer channel 130;

[0074] S500, driven by the injection pressure, the treatment solution in the filtration buffer channel 130 fills the reaction tank 140 and the waste liquid tank 150 from bottom to top, and reacts with the biocomposite material in the reaction tank 140;

[0075] S600, invert the microfluidic chip 10 so that the injection port 110 is located below the microfluidic chip 10;

[0076] S700, applying a first preset temperature to the microfluidic chip 10, and incubating the dairy product sample to be tested in the reaction pool 140 for a first preset time;

[0077] S800, blowing air through the injection port 110 to discharge the excess liquid injected into the reaction pool 140, so that the waste liquid is concentrated in the waste liquid pool 150 or discharged through the sample outlet 160;

[0078] S900, slowly injecting the color developing solution into the microfluidic chip 10 through the injection port 110, so that the color developing solution fills the reaction pool 140;

[0079] S1000, applying a second preset temperature to the microfluidic chip 10, incubating the color developing solution in the reaction pool 140 for a second preset time, and then optically detecting the reaction pool 140 to obtain an absorbance value of the reaction pool 140;

[0080] S1100, comparing the obtained absorbance value with the database to obtain the detection result of the sulfonamide drug content in the dairy product.

[0081] The monitoring method provided by the present disclosure can realize the detection of sulfonamide drugs in dairy products through simple operation steps, without the need for additional sample pre-treatment process, which greatly improves the operability of non-professionals. The interference of the sample itself to optical detection is eliminated by reasonable reaction area design, the sensitivity of detection is improved, and the chip can be applied to different batches or different types of dairy products. The design of the microfluidic chip is upper and lower layers, which saves the production cost of the chip. Carbonic anhydrase freeze-dried hydrogel is easy to store and has a good shelf life, and has good application prospects in the detection of sulfonamide drugs in dairy products.

[0082] Below, each step in the method for detecting the content of sulfonamide drugs in dairy products provided by the present disclosure will be described in detail.

[0083] S100 , providing the microfluidic chip 10 described in the above embodiment, and placing the microfluidic chip 10 vertically so that the waste liquid pool 150 is located above the reaction pool 140 .

[0084] Specifically, Figure 2 As shown, the microfluidic chip 10 is placed vertically so that the waste liquid pool 150 is located above the reaction pool 140, so that the dairy product sample to be tested can completely fill the reaction pool 140 later.

[0085] S200 , injecting the dairy product sample to be tested into the sample pre-treatment channel 120 through the sample injection port 110 , and spontaneously reacting or adsorbing with the filter filler in the sample pre-treatment channel 120 during the injection process.

[0086] Specifically, the dairy product sample to be tested is slowly injected into the sample pretreatment channel 120 through the injection port 110 of the first chip layer 100. The pretreatment channel is filled with phosphate and ethylenediamine-N-propyl composite filler, which reacts or adsorbs spontaneously during the slow injection process.

[0087] Among them, the injection volume of the dairy sample to be tested can be about 2 mL.

[0088] S300 , the sample enters the filtering buffer mixing channel 210 from the sample pre-treatment channel 120 driven by the injection pressure, and the sample and the filtering filler fully react with each other in the filtering buffer mixing channel 210 .

[0089] Specifically, after the dairy sample to be tested reacts or adsorbs spontaneously in the pretreatment channel, the dairy sample to be tested enters the second chip layer 200 from the pretreatment channel driven by the injection pressure, passes through the filtration buffer channel 130, and at this time, the filtration buffer mixing channel 210 enables the dairy sample to be tested and phosphate to react fully with each other.

[0090] S400 , the mixed solution after the reaction is driven by the injection pressure from the filtering buffer mixing channel 210 , through the filtering membrane and into the filtering buffer channel 130 .

[0091] Specifically, after the dairy product sample to be tested and the phosphate in the filter buffer mixing channel 210 react fully with each other, driven by the injection pressure, the sample enters the filter buffer channel 130 through the filter membrane from the filter buffer mixing channel 210 and re-enters the first chip layer 100. Impurities can be trapped by the filter membrane to obtain a relatively pure processing solution, thereby improving the detection sensitivity of the sample under the optical method.

[0092] S500 , driven by the injection pressure, the treatment solution in the filtration buffer channel 130 fills the reaction tank 140 and the waste liquid tank 150 from bottom to top, and reacts with the biocomposite material in the reaction tank 140 .

[0093] Specifically, driven by the injection pressure, the treatment solution in the filtration buffer channel 130 fills the reaction tank 140 and the waste liquid tank 150 from bottom to top, and reacts with the biocomposite material in the reaction tank 140 .

[0094] The carbonic anhydrase freeze-dried hydrogel disposed in the reaction tank 140 absorbs water and swells to perform a reaction.

[0095] S600 , invert the microfluidic chip 10 so that the sample inlet 110 is located below the microfluidic chip 10 .

[0096] Specifically, the microfluidic chip 10 is inverted so that the sample inlet 110 is placed below the microfluidic chip 10 , and the excess reaction liquid enters the waste liquid pool 150 or is discharged through the sample outlet 160 .

[0097] S700 , applying a first preset temperature to the microfluidic chip 10 , and incubating the dairy product sample to be tested in the reaction pool 140 for a first preset time.

[0098] Specifically, a first preset temperature is applied to the microfluidic chip 10 , and the dairy product sample to be tested is incubated in the reaction tank 140 for a first preset time, so as to incubate the dairy product sample to be tested in the reaction tank 140 .

[0099] The first preset temperature is 30°C to 40°C, such as 30°C, 32°C, 35°C, 37°C, and 40°C; the first preset time is 8min to 12min, such as 8min, 9min, 10min, 11min, and 12min. Preferably, the first preset temperature is 35°C, and the first preset time is 10min.

[0100] S800 , blowing air through the sample inlet 110 to discharge the excess liquid injected into the reaction pool 140 , so that the waste liquid is concentrated in the waste liquid pool 150 or discharged through the sample outlet 160 .

[0101] Specifically, air is blown in through the sample addition port, so that the waste liquid is concentrated in the waste liquid pool 150 or flows out through the outflow sample port 160 .

[0102] S900 , slowly inject the color developing solution into the microfluidic chip 10 through the injection port 110 , so that the color developing solution fills the reaction pool 140 .

[0103] Specifically, the color developing solution is slowly injected into the microfluidic chip 10 through the sample injection port and effectively fills the sample reaction pool 140 , and the excess color developing solution enters the waste liquid pool 150 .

[0104] S1000 , applying a second preset temperature to the microfluidic chip 10 , incubating the color developing solution in the reaction pool 140 for a second preset time, and then optically detecting the reaction pool 140 to obtain an absorbance value of the reaction pool 140 .

[0105] Specifically, a second preset temperature is applied to the microfluidic chip 10 to be tested, and after the color developing solution is incubated in the reaction pool 140 for a second preset time, the reaction pool 140 is optically detected to obtain the absorbance value of the reaction pool 140 .

[0106] The second preset temperature is 20°C to 30°C, such as 20°C, 22°C, 25°C, 27°C, and 30°C; the second preset time is 1min to 5min, such as 1min, 2min, 3min, 4min, and 5min. Preferably, the second preset temperature is 25°C, and the second preset time is 3min.

[0107] S1100, comparing the obtained absorbance value with the database to obtain the detection result of the sulfonamide drug content in the dairy product.

[0108] Specifically, Figure 5 As shown, the multiple absorbance values ​​obtained are fitted or compared with the database to obtain the detection results of the sulfonamide content in the dairy product.

[0109] Specifically, the preparation process of carbonic anhydrase freeze-dried hydrogel is as follows:

[0110] Take 5g PEG-2000 and dissolve it in 15mL of dichloromethane solvent, add 0.3g dimethylacrylic anhydride and 300μL triethylamine, fully react for 48h, then add 100μL SOCl2, fully react for 2 hours, vacuum evaporation of the solution after reaction, add ultrapure water to dissolve, remove excess impurities with a dialysis bag, and then freeze-dry at low temperature to obtain a relatively pure product. Add water to prepare a PEG chloride solution, preferably with a solution mass fraction of 15%. Take 0.5g soluble silk protein and 0.3g gelatin dissolved in 20mL PEG chloride solution, react at 40℃ for 6 hours, add carbonic anhydrase to the liquid after reaction and mix and stir. Take out the mixed solution and add it dropwise to the two sample reaction areas 105, freeze-dry at low temperature and store at -30℃ away from light.

[0111] Specifically, the detection of sulfadimethoxine and the drawing of the standard curve:

[0112] like Figure 5 As shown, a standard solution of sulfadimethoxine phosphate was prepared with a concentration gradient of 10 ng / mL, 50 ng / mL, 100 ng / mL, 200 ng / mL, and 300 ng / mL, and the solution was added to the reaction pool 140 of the microfluidic chip 10 to react at 35° C. for 10 minutes. After removing the excess reaction solution with water, a DMSO aqueous solution of p-nitrophenyl acetate was added to the reaction pool 140 of the microfluidic chip 10. After incubation at 25° C. for 3 minutes, the absorbance of the corresponding concentration was tested at a wavelength of 405 nm.

[0113] Specifically, the freeze-dried carbonic anhydrase hydrogel effectively prolongs the shelf life of the immobilized enzyme. The storage activity of the prepared hydrogel is described below using sulfadimethoxine as a detection object.

[0114] After the preparation of the carbonic anhydrase freeze-dried hydrogel, the absorbance test of the standard solution was performed on the 1st day, 5th day, 10th day, 15th day, 20th day, 25th day and 30th day after the preparation.

[0115] For example, Figure 6 As shown, a 100 ng / mL concentration gradient of sulfadimethoxine phosphate standard solution was prepared, the solution was added to the reaction pool 140 of the microfluidic chip 10, and the reaction was carried out at 35° C. for 10 minutes. After removing the excess reaction solution with water, a DMSO aqueous solution of p-nitrophenyl acetate was added to the reaction pool 140 of the microfluidic chip 10. After incubation at 25° C. for 3 minutes, the absorbance of the corresponding concentration was tested at a wavelength of 405 nm.

[0116] Those skilled in the art will readily appreciate other embodiments of the present disclosure after considering the specification and practicing the invention disclosed herein. This application is intended to cover any modification, use or adaptation of the present disclosure, which follows the general principles of the present disclosure and includes common knowledge or customary techniques in the art that are not disclosed in the present disclosure. The specification and examples are intended to be exemplary only, and the true scope and spirit of the present disclosure are indicated by the appended claims.

Claims

1. A microfluidic chip for measuring the content of sulfonamides in dairy products, characterized in that: The chip body comprises a sample inlet and a sample outlet, a sample pretreatment channel connected to the sample inlet, a filter buffer mixing channel connected to the sample pretreatment channel, a filter buffer channel connected to the filter buffer mixing channel, a reaction pool and a waste liquid pool connected to the filter buffer channel, and the sample outlet is connected to the reaction pool and the waste liquid pool; The sample pretreatment channel is filled with filter fillers, the filter buffer mixing channel is used for pretreatment and sufficient reaction, a filter membrane is provided between the filter buffer mixing channel and the filter buffer channel, and the reaction pool is filled with biocomposite materials.

2. The microfluidic chip according to claim 1, characterized in that: The chip body comprises: A first chip layer and a second chip layer, wherein the first chip layer and the second chip layer are sealed and connected to form the chip body.

3. The microfluidic chip according to claim 1, characterized in that: The filter membrane is a PVDF filter membrane, and the pore size of the filter membrane is 0.22 μm to 5 μm.

4. The microfluidic chip according to claim 1, characterized in that: The filtering filler is a composite filler of phosphate and ethylenediamine-N-propyl.

5. The microfluidic chip according to claim 1, characterized in that: The biocomposite material is a freeze-dried carbonic anhydrase hydrogel.

6. The microfluidic chip according to claim 5, characterized in that: The carbonic anhydrase freeze-dried hydrogel comprises carbonic anhydrase, silk fibroin, PEG and gelatin composite embedding.

7. The microfluidic chip according to claim 1, characterized in that: A plurality of reaction pools are formed in the chip body.

8. A method for detecting the content of sulfonamide drugs in dairy products, characterized in that: include: Providing the microfluidic chip according to any one of claims 1 to 7, and placing the microfluidic chip vertically so that the waste liquid pool is located above the reaction pool; Injecting the dairy product sample to be tested into the sample pretreatment channel through the sample injection port, and spontaneously reacting or adsorbing with the filter filler in the sample pretreatment channel during the injection process; The sample enters the filtering buffer mixing channel from the sample pretreatment channel under the driving of the injection pressure, and the sample and the filtering filler fully react with each other in the filtering buffer mixing channel; The mixed solution after the reaction is driven by the injection pressure from the filtering buffer mixing channel through the filtering membrane into the filtering buffer channel; Driven by the injection pressure, the treatment solution in the filtration buffer channel fills the reaction tank and the waste liquid tank from bottom to top, and reacts with the biocomposite material in the reaction tank; Inverting the microfluidic chip so that the injection port is located below the microfluidic chip; Applying a first preset temperature to the microfluidic chip, and incubating the dairy product sample to be tested in the reaction pool for a first preset time; Blow air through the injection port to discharge excess liquid injected into the reaction pool, so that the waste liquid is concentrated in the waste liquid pool or discharged through the outlet port; Slowly injecting the color developing solution into the microfluidic chip through the injection port, so that the color developing solution fills the reaction pool; Applying a second preset temperature to the microfluidic chip, incubating the color developing solution in the reaction pool for a second preset time, and then optically detecting the reaction pool to obtain an absorbance value of the reaction pool; The obtained absorbance value is compared with the database to obtain the detection result of the sulfonamide content in the dairy product.

9. The detection method according to claim 8, characterized in that: The first preset temperature is 30° C. to 40° C., and the first preset time is 8 min to 12 min.

10. The detection method according to claim 8, characterized in that: The second preset temperature is 20° C. to 30° C., and the second preset time is 1 min to 5 min.

Images