Micro-fluidic chip for real-time high-flux pretreatment of various fungaltoxins

Abstract

The utility model provides a micro-fluidic chip for real-time high-flux pretreatment of various mycotoxins, and belongs to the field of micro-fluidic chip manufacturing technology and rapid detection. The micro-fluidic chip is a centrifugal micro-fluidic chip, the micro-fluidic chip comprises a lower-layer substrate provided with a micro-fluidic channel and an upper-layer substrate bonded with the lower-layer substrate, and the lower-layer substrate is provided with a sample mixing area, a centrifugal area, a diluent chamber, a siphon micro-valve and a dilution area which are communicated in sequence; the micro-fluidic chip is rapid and portable, does not need complex instruments, can be used for rapid in-situ pretreatment of various mycotoxins, and can be applied to instant pretreatment of various mycotoxins in grains.

Description

Technical Field

[0001] The utility model belongs to the field of microfluidic chip manufacturing technology and rapid detection, and specifically relates to a microfluidic chip for real-time high-throughput pretreatment of multiple fungal toxins. Background Art

[0002] Wheat is one of the three major grains in the world and is extremely susceptible to contamination by mycotoxins. Mycotoxins not only cause serious economic losses, but also have carcinogenic, teratogenic, and mutagenic hazards, which have become an important issue related to the food safety of wheat and its products. Therefore, it is necessary to strictly control the quality and safety of various agricultural products such as wheat, standardize and restrict mycotoxin prevention and control behaviors, and prevent and control mycotoxins in wheat through early detection of mycotoxins in wheat.

[0003] Common and harmful mycotoxins in wheat mainly include vomitoxin (DON), aflatoxin B1 (AFB1) and ochratoxin A (OTA). The contamination of the above-mentioned mycotoxins in wheat and its products is characterized by wide distribution and great harm. Microfluidic technology, as an integrated micro-laboratory platform, has the advantages of integration, compactness and portability, and can be used for the detection of mycotoxins. The research of the prior art discloses a convenient, automated and parallelized microfluidic chip, which can simultaneously detect 104 endotoxin samples on the chip; it also discloses a portable automated microfluidic chip, which can realize the full automation of injecting the specified solution into the centrifugal device on demand, program-controlled chip rotation and multiplexed gold nanoparticle synthesis on the chip. However, the above-mentioned microfluidic chips are all used for the separation and enrichment of spores, viruses, etc., and there are no reports on the pretreatment of small molecules such as mycotoxins.

[0004] Pretreatment for mycotoxin detection mostly relies on traditional centrifuges or blenders, but traditional pretreatment methods are time-consuming and labor-intensive, require a large amount of reagents, and are inconvenient to carry, making them unsuitable for on-site testing. Therefore, designing a microfluidic chip that can simultaneously pretreat multiple mycotoxins in wheat is very necessary to improve the automation and convenience of mycotoxin detection. Utility Model Content

[0005] In view of some deficiencies in the prior art, the utility model provides a microfluidic chip for real-time high-throughput pretreatment of multiple fungal toxins and its application; the microfluidic chip described in the utility model is a centrifugal microfluidic chip, the microfluidic chip comprises a lower film provided with a microfluidic channel and an upper substrate bonded to the lower film, the lower film is provided with a sample mixing area, a centrifugal area, a dilution chamber, a siphon valve area and a dilution area which are connected in sequence; the microfluidic chip is fast, portable, and does not require complex instruments, the microfluidic chip can quickly pretreat multiple fungal toxins in situ, and can be applied to the real-time pretreatment of fungal toxins in multiple grains.

[0006] In order to achieve the above technical purpose, the utility model adopts the following technical means:

[0007] The utility model first provides a microfluidic chip for real-time high-throughput pretreatment of multiple fungal toxins, wherein the microfluidic chip comprises a lower substrate having at least one microfluidic channel and an upper substrate bonded to the lower substrate;

[0008] The microfluidic channel includes a sample mixing area, a centrifugal area, a diluent chamber, a siphon valve area, and a dilution area;

[0009] The sample mixing area is connected to one side of the centrifugal area; the other side of the centrifugal area is connected to one side of the dilution area through the siphon microvalve A of the siphon valve area, and the other side of the dilution area is connected to the diluent chamber through the siphon microvalve B of the siphon valve area; the dilution area is provided with a sample outlet.

[0010] Preferably, the number of the microfluidic channels is 1 to 6.

[0011] Preferably, the sample mixing zone includes a sample inlet and a mixing zone connected thereto, and the mixing zone is connected to one side of the centrifugal zone.

[0012] Preferably, the mixing zone is in the shape of a serpentine curved channel, and the centrifugal zone is an elliptical chamber.

[0013] Preferably, the siphon microvalve A is provided with a siphon channel A, and the siphon microvalve B is provided with a siphon channel B; the siphon channel A and the siphon channel B are arranged symmetrically.

[0014] Preferably, the siphon channel A and the siphon channel B are both curved channels pointing to the center of the microfluidic chip; the top of the curved channel is semicircular.

[0015] Preferably, the siphon channel A is connected to the centrifugal zone and the dilution zone, and the siphon channel B is connected to the dilution liquid chamber and the dilution zone.

[0016] Preferably, the sample inlet is provided with a blocking membrane; the blocking membrane is preferably a PVDC (polyvinylidene chloride) membrane.

[0017] Preferably, the dilution zone is an irregular pattern channel; the bottom of the dilution zone is an arc-shaped structure, which is convenient for sample dilution and mixing.

[0018] The utility model also provides the above-mentioned microfluidic chip for real-time high-throughput pretreatment of multiple mycotoxins, specifically comprising:

[0019] (1) designing a chip pattern according to the above-mentioned microfluidic chip, engraving a lower film using a laser engraving machine, and cleaning the debris remaining on the surface of the lower film and in the channel to obtain a lower film;

[0020] The upper substrate is engraved by a laser engraving machine so that a circular hole for the DC motor shaft to pass through is engraved at the center of the circle;

[0021] (2) The microfluidic channel on the lower substrate is subjected to hydrophilic treatment, and then the lower substrate and the upper substrate are bonded to obtain the microfluidic chip.

[0022] The utility model also provides the use of the microfluidic chip in pre-treating mycotoxins; the mycotoxins include mycotoxins in grains.

[0023] The utility model also provides a method for pretreating fungal toxins in food, the method comprising:

[0024] (1) Grinding the grain to be tested into powder, adding the grain powder and the methanol solution to the sample inlet of the above-mentioned microfluidic chip respectively, and mixing them evenly at a certain speed to obtain a sample solution;

[0025] (2) After the sample liquid flows into the centrifugal zone, the rotation speed is further increased for centrifugal treatment;

[0026] (3) After the centrifugation is completed, the rotation speed is reduced, and the sample liquid is sucked into the dilution zone through the siphon channel A. At the same time, the Tris buffer in the dilution liquid chamber is sucked into the dilution zone through the siphon channel B. The sample liquid is mixed with the Tris buffer. After the dilution is completed, the rotation is stopped to obtain the pretreated sample to be tested, which is taken out from the sample outlet.

[0027] Preferably, in step (1), the dosage ratio of the grain powder to the methanol solution is 1 mg:5 μL; the methanol solution is 80% (v / v) methanol solution;

[0028] The rotation speed is 500 rpm / min.

[0029] Preferably, in step (2), the centrifugal treatment is performed at 2000 rpm / min for 10 min.

[0030] Preferably, in step (3), the rotation speed is reduced to 500 rpm / min after the centrifugation is completed.

[0031] Compared with the prior art, the beneficial effects of the utility model are:

[0032] The microfluidic chip of the utility model integrates sample mixing, centrifugation and dilution, and can realize rapid and instant high-throughput pretreatment of multiple mycotoxins in grain samples. The microfluidic chip can quickly pretreat grain samples in situ, simplifying the separation and enrichment process of mycotoxins. The microfluidic chip of the utility model can greatly save the amount of sample and reagent required for pretreatment, reduce pollution while improving portability, and can complete the separation and enrichment of multiple mycotoxins in wheat within 20 minutes without any large instruments.

[0033] The microfluidic chip described in the utility model adopts a siphon microvalve. Under high-speed rotation, the centrifugal force is greater than the capillary force, and the sample will be blocked at the front end of the microvalve. After switching to low-speed rotation, the capillary force dominates and the sample will be sucked into the siphon channel. The fluid in the controlled area can be adjusted by the rotation speed, and the operation is more convenient.

[0034] The utility model measured the reliability of the pretreatment results of the microfluidic chip and used it to evaluate the accuracy of quantitative detection through ORIGIN 2018. The test found that there was no significant difference in the detection results obtained by the microfluidic pretreatment method described in the utility model and the traditional pretreatment method, and it had good reliability and repeatability. BRIEF DESCRIPTION OF THE DRAWINGS

[0035] Figure 1 The schematic diagram is a microfluidic channel structure of the lower substrate of the microfluidic chip of the utility model.

[0036] Figure 2 It is a schematic diagram of the structure of the microfluidic chip of the utility model.

[0037] Figure 3 Figure 1 is a schematic diagram of the pretreatment process of the microfluidic chip; in the figure, a is the mixing process of the wheat sample and the methanol solution; b is the connection between the sample mixing area and the centrifugal area; c is the centrifugal process of the sample in the centrifugal area; d is the process of the sample being sucked into the siphon microvalve A after the rotation speed is reduced; f is a schematic diagram of the dilution liquid chamber, and e is the mixing and dilution process of the sample and Tris buffer.

[0038] Figure 4 This is the pre-processing flow chart of the microfluidic chip.

[0039] In the figure, 1-upper substrate; 2-lower film; 3-sample inlet; 4-mixing zone; 5-centrifugal zone; 6-siphon microvalve A; 6-1-siphon channel A; 7-siphon microvalve B; 7-1-siphon channel B; 8-dilution chamber; 9-dilution zone; 10-sample outlet; 11-microfluidic channel. DETAILED DESCRIPTION

[0040] The present invention will be further described below in conjunction with the accompanying drawings and specific embodiments, but the protection scope of the present invention is not limited thereto.

[0041] Embodiment 1:

[0042] like Figures 1-2 The microfluidic chip of the utility model comprises a lower film 2 provided with at least one microfluidic channel 11 and an upper substrate 1 bonded to the lower film 2, and the upper substrate 1 and the lower film 2 are preferably circular in shape. The microfluidic channel 11 comprises a sample mixing area, and the sample mixing area is connected to one side of the centrifugal area 5; the other side of the centrifugal area 5 is connected to one side of the dilution area 9 through a siphon microvalve A 6, and the other side of the dilution area 9 is connected to the dilution liquid chamber 8 through a siphon microvalve B7; the dilution area 9 is provided with a sample outlet 10.

[0043] like Figures 1-2 As shown, the sample mixing area includes a sample injection port 3 and a mixing area 4 connected thereto. The mixing area 4 is connected to one side of the centrifugal area 5; the centrifugal area 5 is an elliptical channel area, which is connected to the sample mixing area 4 and the siphon valve area A6. The siphon microvalve A6 is provided with a siphon channel A6-1, and the siphon microvalve B7 is provided with a siphon channel B7-1; the siphon channel A6-1 and the siphon channel B7-1 are arranged symmetrically. The siphon channel A6-1 is connected to the centrifugal area 5 and the dilution area 9, and the siphon channel B7-1 is connected to the dilution liquid chamber 8 and the dilution area 9. In actual use, in order to speed up the pretreatment speed of the sample, the various areas in the chip are optimized. In order to mix the sample evenly in the mixing area 4, the mixing area 4 is set to a serpentine curved channel shape, and the width of the curved channel of the mixing area 4 in the serpentine curved channel shape is 0.3mm, the diameter of the curved part of the channel is 1.2mm, and the height is 0.1mm. In addition, the centrifugal zone 5, the diluent chamber 8 and the dilution zone 9 are set to be 0.1 mm high to facilitate the storage of the sample liquid and the diluent. The width of the siphon microvalve A 6 and the siphon microvalve B 7 are set to 0.1 mm and the height is set to 0.1 mm. In addition, the siphon channel A 6-1 and the siphon channel B 7-1 are set to be curved channels, pointing to the center of the microfluidic chip, and the top of the curved channel is semicircular.

[0044] In actual use, the sample inlet 3 is provided with a blocking film, and the blocking film is a PVDC (polyvinylidene chloride) film.

[0045] Embodiment 2:

[0046] This embodiment is a method for preparing the microfluidic chip described in Example 1, which comprises the following steps:

[0047] (1) Chip cutting:

[0048] Import the designed chip pattern into the laser engraving machine, place PMMA horizontally in the processing room of the laser engraving machine, adjust the distance from the laser gun head to the PMMA surface to 0.5cm, start the machine to process the operating unit on the chip, and obtain the lower film; similarly, adjust the laser gun head to penetrate the center of the upper cover film, leaving a 6mm circular hole. Since laser cutting will produce some MMA monomers and PMMA debris, the MMA monomers and PMMA debris remaining on the surface of the lower film and inside the channel surface need to be cleaned.

[0049] The cleaning process is as follows: wash each layer of PMMA with detergent to remove the MMA monomer and PMMA debris on the surface, then place it in a 95% v / v ethanol solution for ultrasonic cleaning for 15 minutes, then place it in triple distilled water for ultrasonic cleaning for 15 minutes to remove residual detergent and alcohol, and blow it dry with nitrogen until there are no obvious droplets and water stains on the surface.

[0050] (2) Hydrophilic treatment of channel layer:

[0051] Dilute TWEEN-20 with 95% ethanol to a concentration of 2.0% v / v, and ultrasonically clean the bottom film in 95% ethanol and distilled water for 15 minutes to remove dust and impurities on the chip. After cleaning, dry it with nitrogen, dry it in a 40°C drying oven for 30 minutes, and then soak it directly in 2.0% v / v TWEEN-20 for 24 hours. After soaking, take it out and place it in a dry, dark environment.

[0052] (3) Chip bonding

[0053] The upper substrate and the lower film after laser engraving are bonded with acrylic glue. The specific operation is as follows: the upper substrate and the lower film are bonded together, and the acrylic glue is sucked from the bottle with a syringe with a capacity of 1 mL. The needle is inserted into the gap between the layers and the acrylic glue is injected until the layers of PMMA are completely bonded together without obvious gaps, thereby obtaining the microfluidic chip. The bonded microfluidic chip still needs to be ultrasonically cleaned in alcohol and triple distilled water for 10 minutes each to remove the glue remaining in the chip during the bonding process. After drying with nitrogen, the microfluidic chip for real-time high-throughput pretreatment of multiple fungal toxins is obtained.

[0054] Embodiment 3:

[0055] This embodiment takes wheat as an example, and uses the microfluidic chip described in Example 1 to pre-treat fungal toxins. However, the pre-treatment scope of the microfluidic chip described in the utility model is not limited to wheat. The specific treatment method is as follows: Figure 4 As shown, the following steps are included:

[0056] (1) The wheat to be tested (provided by the School of Food and Bioengineering, Jiangsu University) was crushed by a pulverizer and passed through a 165 μm (100 mesh) pore size test sieve to obtain wheat powder. 1 mg of wheat powder and 5 μL of 80% methanol solution were added to the sample injection port of the microfluidic chip described in Example 1 through a syringe, and the rotation speed was increased to 500 rpm. The mixture was fully mixed through the serpentine curved channel in the mixing zone to obtain a sample solution.

[0057] (2) After the sample solution in step (1) flows into the centrifugal chamber, the speed is increased to 2000 rpm and centrifuged for 10 min.

[0058] (3) After the centrifugation is completed, the rotation speed is reduced to 500 rpm, and the sample liquid is sucked into the dilution zone through the siphon channel A. At the same time, the Tris buffer in the dilution liquid chamber is sucked into the dilution zone through the siphon channel B; the sample liquid is mixed with the Tris buffer, and the rotation is stopped after the dilution is completed to obtain the pretreated sample to be tested, which is taken out from the sample outlet.

[0059] The schematic diagram of the preprocessing steps is as follows Figure 3 As shown, part a shows the pretreatment results of wheat samples with 80% methanol solution, part b shows the sample mixture flowing from the mixing area into the centrifugal area, part c shows the sample being centrifuged in the centrifugal chamber, part d shows the sample being sucked into the siphon valve after the rotation speed is reduced, part f shows the Tris buffer pre-existing in the diluent chamber, and part e shows the dilution of the sample with the Tris buffer.

[0060] In order to verify the reliability of this method, this embodiment also carried out a reliability test on the pretreatment results of the microfluidic chip. The specific method of the reliability test is: using a variety of wheat samples sold on the market and samples collected from farmland, the various samples were pretreated by traditional methods and centrifugal microfluidic chip methods respectively. The traditional pretreatment method refers to the "Determination of 16 Fungal Toxins in Major Cereals in Grain and Oil Inspection by Liquid Chromatography-Tandem Mass Spectrometry" in the "Grain Industry Standards of the People's Republic of China". The standard curves were drawn by liquid chromatography-tandem mass spectrometry for the samples pretreated by the traditional method and the microfluidic chip method, and the quantitative results of various fungal toxins under the two methods were compared, and the accuracy of quantitative detection was evaluated by ORIGIN 2018.

[0061] Table 1. Quantitative detection results of mycotoxins under different pretreatment methods

[0062]

[0063] Table 1 shows the quantitative detection results of mycotoxins under different pretreatment methods. It can be seen from the table that there is no significant difference in the detection results of the two methods, indicating that this method has good reliability and repeatability.

[0064] In summary, the microfluidic chip of the utility model integrates sample mixing, centrifugation and dilution, and can realize rapid and instant high-throughput pretreatment of various mycotoxins in grain samples. The microfluidic chip can quickly pretreat grain samples in situ, simplifying the separation and enrichment process of mycotoxins. The microfluidic chip of the utility model can greatly save the amount of sample and reagent required for pretreatment, reduce pollution while improving portability, and can complete the separation and enrichment of various mycotoxins in wheat within 20 minutes without any large instruments.

[0065] The embodiments are preferred implementations of the present invention, but the present invention is not limited to the above-mentioned implementations. Any obvious improvements, substitutions or modifications that can be made by those skilled in the art without departing from the essential content of the present invention belong to the protection scope of the present invention.

Claims

1. A microfluidic chip for real-time high-throughput pretreatment of multiple mycotoxins, characterized in that: The microfluidic chip comprises a lower substrate (2) having at least one microfluidic channel (11) and an upper substrate (1) bonded to the lower substrate (2); The microfluidic channel (11) comprises a sample mixing area, a centrifugal area (5), a diluent chamber (8), a siphon valve area and a dilution area; The sample mixing zone is connected to one side of the centrifugal zone (5); the other side of the centrifugal zone (5) is connected to one side of the dilution zone (9) through a siphon microvalve A (6) of the siphon valve zone, and the other side of the dilution zone (9) is connected to the diluent chamber (8) through a siphon microvalve B (7) of the siphon valve zone; the dilution zone (9) is provided with a sample outlet (10).

2. The microfluidic chip for real-time high-throughput pretreatment of multiple mycotoxins according to claim 1, characterized in that: The number of the microfluidic channels (11) is 1 to 6.

3. The microfluidic chip for real-time high-throughput pretreatment of multiple mycotoxins according to claim 1, characterized in that: The sample mixing area comprises a sample injection port (3) and a mixing area (4) connected thereto, and the mixing area (4) is connected to one side of the centrifugal area (5).

4. The microfluidic chip for real-time high-throughput pretreatment of multiple mycotoxins according to claim 1, characterized in that: The mixing zone (4) is in the shape of a serpentine curved channel, and the centrifugal zone (5) is an elliptical chamber.

5. The microfluidic chip for real-time high-throughput pretreatment of multiple mycotoxins according to claim 1, characterized in that: The siphon microvalve A (6) is provided with a siphon channel A (6-1), and the siphon microvalve B (7) is provided with a siphon channel B (7-1); the siphon channel A (6-1) and the siphon channel B (7-1) are arranged symmetrically; The siphon channel A (6-1) and the siphon channel B (7-1) are both curved channels pointing to the center of the microfluidic chip; the top of the curved channel is semicircular; The siphon channel A (6-1) is in communication with the centrifugal zone (5) and the dilution zone (9), and the siphon channel B (7-1) is in communication with the dilution liquid chamber (8) and the dilution zone (9).

6. The microfluidic chip for real-time high-throughput pretreatment of multiple mycotoxins according to claim 3, characterized in that: The sample inlet (3) is provided with a blocking membrane.

7. The microfluidic chip for real-time high-throughput pretreatment of multiple mycotoxins according to claim 1, characterized in that: The bottom of the dilution zone (9) is in an arc-shaped structure.

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