Microfluidic protein chip for allergen detection

Abstract

The application belongs to the field of microfluidic protein chips, and specifically discloses a microfluidic protein chip for allergen detection, which comprises a sample adding area and a microfluidic unit, the microfluidic unit comprises a dilution area, a premixing area, a labeling area, a quantification area and a detection area which are connected in sequence, wherein the sample adding area comprises a first sample adding area and a second sample adding area, the first sample adding area is used for adding a whole blood sample, and the second sample adding area is used for adding a serum or plasma sample, different samples can be distinguished and processed; the first sample adding area is designed ingeniously by setting a capillary channel as a valve; a vibrator is arranged in the premixing area to drive a mixing ball to vibrate, so that the mixing efficiency is improved, the mixing of the sample and the diluent is more sufficient, the mixing time is reduced, and the difference of detection results is reduced; the detection result difference is further reduced through quantification of each sub-quantification area; and the labeling area and the detection area can be coated with multiple antibodies, and detection of multiple to-be-detected substances can be simultaneously performed.

Description

Technical Field

[0001] This application belongs to the field of microfluidic protein chip technology, and relates to a microfluidic protein chip for allergen detection. Background Technology

[0002] Biochips, a novel technological platform developed in the 1990s, offer advantages such as integration, high throughput, and high density. Microfluidics integrates the basic operational units of biological, chemical, and medical analysis processes—including sample preparation, reaction, separation, and detection—onto a single micrometer-scale chip, automating the entire analytical process. Based on the different biomolecules immobilized on the solid-phase support, microfluidic chips can be categorized into DNA chips, protein chips, etc. With further research, microfluidic protein chips have been widely applied in many research fields. Combining the high throughput and integration of microarrays with the high sensitivity and specificity of immunoassays, they enable convenient, rapid, and accurate detection of multiple samples and multiple parameters. They can process and analyze hundreds of samples within minutes.

[0003] In microfluidic protein chips, significant differences in test results between different batches of the same sample or even within the same batch are common problems. This is because blood samples are typically more viscous than diluents. In the tubing of a microfluidic protein chip, capillary force and fluid viscosity are crucial factors affecting flow rate. If the sample and diluent are not thoroughly mixed, inconsistent flow rates between the sample and diluent mixture will result in poor reproducibility of the test results. Although patent CN110026257B discloses a microfluidic chip that uses a premixing chamber to mix the sample and diluent, the problem of insufficient mixing still exists. Summary of the Invention

[0004] The purpose of this application is to provide a microfluidic protein chip for allergen detection, in which the premixed structure has been improved, greatly enhancing the thoroughness of sample and diluent mixing, reducing mixing time, and reducing sample and diluent residue in the tubing.

[0005] To solve the above-mentioned technical problems, this application adopts the following technical solution:

[0006] According to one aspect of this application, this application provides a microfluidic protein chip for allergen detection, including a sample application area and at least one microfluidic unit, said microfluidic unit including a dilution area, a premixing area, a labeling area, a quantification area, and a detection chamber connected in sequence;

[0007] The sample loading area includes at least two sample loading areas, one of which is used to add whole blood samples and the other is used to add serum or plasma samples.

[0008] The dilution zone contains a diluent;

[0009] The premixing zone is used to mix the sample and the diluent. The premixing zone includes a first premixing zone and a second premixing zone. The first premixing zone is provided with a first protrusion, a second protrusion, a third protrusion, a fourth protrusion, a fifth protrusion, a mixing ball, a filter membrane, a vibrator, and a first sensor. The cross-sectional shape of the second premixing zone is Z-shaped.

[0010] The labeled region contains labeled antibodies;

[0011] The quantitative zone is used for quantitative mixing;

[0012] The detection area is equipped with detection antibodies.

[0013] In one embodiment, the labeled antibody is at least one allergen; the detection antibody is at least one allergen.

[0014] In one embodiment, the sample application area is provided with a whole blood filtration membrane.

[0015] In one embodiment, the sample loading area is provided with a first capillary channel, one end of which is connected to the first separation post-region, and the other end is connected to the buffer zone.

[0016] In one embodiment, the first premixing zone is provided with a first partition, a second partition, a third partition, and a fourth partition, which are separated by a filter membrane phase.

[0017] The hybrid sphere can move freely within each zone;

[0018] The first protrusion, the second protrusion, the third protrusion, the fourth protrusion, and the fifth protrusion are fixedly installed.

[0019] In one embodiment, the first premixing zone is provided with a vibrator and a first sensor. The main control module is connected to the vibrator and the first sensor. After receiving the signal from the first sensor, the main control module controls the vibrator to work.

[0020] In one embodiment, the cross-sectional shape of the first protrusion, the second protrusion, the third protrusion, the fourth protrusion, and the fifth protrusion is triangular;

[0021] The first, second, third, fourth, and fifth protrusions are symmetrically arranged about the vertical center line.

[0022] In one embodiment, the quantitative zone includes multiple sub-quantitative zones and multiple second waste liquid zones, with a second sensor provided in the second waste liquid zone.

[0023] In one embodiment, a one-way valve is provided at the connection between the sub-quantitative zone and the detection zone. The main control module is connected to the second sensor and the one-way valve. After receiving the signal from the second sensor, the main control module controls the one-way valve to open or close.

[0024] In one embodiment, the labeling region is provided with a quality control marker, and the detection region is coated with a quality control antibody, which can specifically bind to the quality control marker.

[0025] The advantages of this application are as follows: This application has a sample loading area and multiple microfluidic units. The microfluidic units include a dilution area, a premixing area, a labeling area, a quantification area, a detection area, and a waste liquid area. The sample loading area has a first sample loading area and a second sample loading area. The first sample loading area is equipped with a separation area, a first waste liquid area, and a whole blood filtration membrane. The first sample loading area is used to add whole blood samples, and the second sample loading area is used to add serum or plasma samples. Therefore, different sample loading areas can be selected according to the type of sample solution being added. This application can differentiate and process different samples, making it flexible and convenient to use, and facilitating the rational use of sample solutions based on their properties, thus reducing sample solution waste. The first sample loading area uses a capillary tube as a valve, a clever design that reduces costs. When testing plasma or serum samples, a smaller sample volume is required, reducing sample usage. The premixing area includes a first premixing area and a second premixing area. The mixing effect is improved by mixing balls and protrusions, and the mixing efficiency is further greatly improved by a vibrator, resulting in small differences in test results between different batches of the same sample or within the same batch of the same sample, and good repeatability. Quantification in each sub-quantitative region further reduces the variability in detection results. The labeling and detection regions can be coated with multiple antibodies, enabling simultaneous detection of multiple analytes and high-throughput detection, thus improving efficiency. Placing the quantitative region after the labeling region enhances the accuracy of the results. Additionally, a main control module, sensors, and one-way valves are included, and automation further improves the efficiency and effectiveness of detection. Attached Figure Description

[0026] Figure 1 This is a schematic diagram of the microfluidic protein chip of this application;

[0027] Figure 2 This is a schematic diagram of the sample addition area;

[0028] Figure 3 This is a schematic diagram of a microfluidic unit;

[0029] Figure 4 This is a schematic diagram of the first premixed zone. Detailed Implementation

[0030] The following embodiments provide a more detailed description of this application, but they do not constitute a limitation thereof. Unless otherwise specified, the experimental methods used in the following embodiments are conventional methods. Unless otherwise specified, the reagents used in the following embodiments are commercially available common reagents.

[0031] like Figure 1-4 As shown, this embodiment provides a microfluidic protein chip for allergen detection. The microfluidic protein chip includes a sample application area at the center, eight circumferentially distributed microfluidic units 3, and a shunt channel 14 connecting the sample application area and the microfluidic units 3. It should be noted that in other embodiments, the number of microfluidic units 3 is not limited to eight; for ease of centrifugation, the number of microfluidic units 3 may be two, four, six, ten, twelve, etc.

[0032] Specifically, the microfluidic protein chip includes a base plate 4, a microfluidic unit 3, and a sample application area disposed on the base plate 4. The central part of the microfluidic protein chip is a rotary mounting section with a rotation center 5, which is the rotation center during centrifugation.

[0033] The microfluidic unit 3 includes a dilution zone 16, a premixing zone, a labeling zone 30, a quantification zone, a detection zone, and a waste liquid zone. In a specific embodiment, the microfluidic unit 3 includes a microchannel and the dilution zone 16, premixing zone, labeling zone 30, quantification zone, detection zone, and waste liquid zone disposed on the microchannel. Further, the dilution zone 16, premixing zone, labeling zone 30, quantification zone, detection zone, and waste liquid zone are connected through the microchannel.

[0034] The sample loading area includes a first sample loading area 1, a second sample loading area 2, a buffer zone 6, a first waste liquid zone 8, a separation zone, and a first capillary channel 9. Both the first sample loading area 1 and the second sample loading area 2 are arranged around the rotation center 5. The first sample loading area 1 is connected to the buffer zone 6 via a first pre-separation zone 10 and a first post-separation zone 11. The second sample loading area 2 is directly connected to the buffer zone 6. Direct connection, as described herein, means that the two connected objects are not connected via other cavities, but is not limited to the use of microchannels, capillary channels, or other structures for communication between them. The first sample loading area 1 and the second sample loading area 2 are formed around the rotation center 5. The first sample loading area 1 is used to add whole blood samples, which require centrifugation. The second sample loading area 2 is used to add serum or plasma samples, which do not require centrifugation. Furthermore, the first sample loading zone 1 is directly connected to the first pre-separation zone 10. The first pre-separation zone 10 is further away from the rotation center 5 than the first sample loading zone 1. A filtration zone is provided between the first pre-separation zone 10 and the first post-separation zone 11. The filtration zone contains a whole blood filtration membrane 12. Through centrifugation, the whole blood filtration membrane 12 filters the whole blood. Blood cells are filtered out by the whole blood filtration membrane 12, and the plasma enters the first post-separation zone 11. The first post-separation zone 11 is further away from the rotation center 5 than the first pre-separation zone 10. The first post-separation zone 11 is connected to the buffer zone 6 through the first capillary channel 9.

[0035] The main body of the first capillary channel 9 is V-shaped, and its bend is closer to the rotation center 5. Specifically, the distance between the bend apex of the first capillary channel 9 and the rotation center 5 is less than the overall distance between the buffer zone 6 and the first separation zone 11 directly connected to it and the rotation center 5. In this way, during centrifugation, the sample solution flows with the capillary channel, but because the centrifugal force is greater than the capillary suction, the sample will not flow to the bend apex of the capillary channel. Therefore, during centrifugal separation of the sample, the capillary channel can act as a valve, achieving a closing effect during sample solution quantification and detection.

[0036] The first waste liquid zone 8 is connected to the first pre-separation zone 10 via an overflow channel 13, which is closer to the rotation center 5 than the first waste liquid zone 8 and the first pre-separation zone 10. When the first pre-separation zone 10 is full of sample, the excess sample enters the first waste liquid zone 8 through the overflow channel 13.

[0037] The buffer zone 6 is connected to the diversion channel 14 via a microchannel. The diversion channel 14 is annular and is further away from the rotation center 5 than the buffer zone 6. In this embodiment, the diversion channel 14 is connected to eight microfluidic units 3 via eight channels.

[0038] The microfluidic unit 3 includes a dilution zone 16, a premixing zone, a labeling zone 30, a quantification zone, a detection zone, and a second waste liquid zone 15. Specifically, the dilution zone 16, the premixing zone, the labeling zone 30, the quantification zone, the detection zone, and the second waste liquid zone 15 are disposed on the base plate 4 and connected to each other through microfluidic channels.

[0039] In this embodiment, the dilution zone 16 is connected to the diversion channel 14. The dilution zone 16 is further away from the rotation center 5 than the diversion channel 14. The dilution zone 16 is used to hold the diluent, which is used to dilute the sample. Specifically, the diluent can be pre-placed in the dilution zone 16 or added when needed through a dilution port. In this embodiment, the dilution zone 16 has a circular structure, which facilitates the flow of the diluent and sample away from the rotation center 5.

[0040] In this embodiment, the premixing zone is connected to the dilution zone 16. The premixing zone is further away from the rotation center 5 than the dilution zone 16. The premixing zone is used to mix the sample and the diluent. One end of the premixing zone is connected to the dilution zone 16, and the other end is connected to the labeling zone 30.

[0041] To completely solve the problem of poor repeatability of the same sample across different batches or within the same batch due to uneven mixing, multiple premixing structures are incorporated into the premixing zone to improve mixing efficiency. Specifically, the main factors affecting fluid flow rate are capillary forces and fluid viscosity. Plasma and serum typically have higher viscosity than diluents, resulting in slower sample flow on the microfluidic protein chip compared to the diluent. Consequently, the sample being tested is often unevenly mixed, leading to significant differences in test results and poor repeatability between different batches of the same sample or within the same batch (multiple tests performed with a single sample addition).

[0042] In this embodiment, the premixing zone includes a first premixing zone 17 and a second premixing zone 18. The first premixing zone 17 has a circular structure and is divided into a first partition 19, a second partition 20, a third partition 21, and a fourth partition 22 sequentially from the dilution zone 16 toward the marking zone 30. A first protrusion 24 is fixedly provided on the inner wall of the first partition 19, and a second protrusion 25 is fixedly provided in the middle of the first partition 19. A freely movable mixing ball 29 is provided in the first partition 19. The first partition 19 and the second partition 20 are separated by a filter membrane 23. The filter membrane 23 allows unobstructed passage of the mixture of sample and diluent. The pore size of the filter membrane 23 is smaller than the diameter of the mixing ball 29, preventing the mixing ball 29 from passing through the filter membrane 23 and entering other partitions. A third protrusion 26 is fixedly provided in the middle of the second partition 20, and a freely movable mixing ball 29 is provided in the second partition 20. The second partition 20 and the third partition 21 are separated by a filter membrane 23. The sample and diluent mixture can pass through the filter membrane 23 without obstruction. The pore size of the filter membrane 23 is smaller than the diameter of the mixing ball 29, so the mixing ball 29 cannot pass through the filter membrane 23 to enter other zones. A fourth protrusion 27 is fixedly provided in the middle of the third zone 21. The mixing ball 29 can move freely in the third zone 21. The third zone 21 and the fourth zone 22 are separated by the filter membrane 23. The sample and diluent mixture can pass through the filter membrane 23 without obstruction. The pore size of the filter membrane 23 is smaller than the diameter of the mixing ball 29, so the mixing ball 29 cannot pass through the filter membrane 23 to enter other zones. A fifth protrusion 28 is fixedly provided in the middle of the fourth zone 22. The mixing ball 29 can move freely in the fourth zone 22. The fourth zone 22 and the second premixing zone 18 are separated by the filter membrane 23. The sample and diluent mixture can pass through the filter membrane 23 without obstruction. The pore size of the filter membrane 23 is smaller than the diameter of the mixing ball 29, so the mixing ball 29 cannot pass through the filter membrane 23 to enter other zones.

[0043] In this embodiment, the cross-section of the first protrusion 24 is triangular, and there are 6 first protrusions 24 symmetrically arranged on the inner walls on both sides of the premixing zone inlet. In other embodiments of this application, the shape and number of the cross-section of the first protrusion 24 are not limited. The shape can be circular, semi-circular, elliptical, fan-shaped, etc., and the number can be set arbitrarily, such as 1, 2, 3, 4, etc.

[0044] In this embodiment, the cross-sectional shape of the second protrusion 25, the third protrusion 26, the fourth protrusion 27, and the fifth protrusion 28 is triangular. The vertical center line of the first premixing zone 17 is symmetrically arranged, and the vertical center line is a straight line extending from the inlet to the outlet of the first premixing zone 17 through the center of the first premixing zone 17. In other embodiments of this application, the cross-sectional shapes of the second protrusion 25, the third protrusion 26, the fourth protrusion 27, and the fifth protrusion 28 can be completely identical or completely different. Any two or any three can be identical, and they can be any shape, such as circular, semi-circular, elliptical, or fan-shaped. The number can also be arbitrarily set, such as 1, 2, 3, or 4.

[0045] In this embodiment, the cross-sectional shape of the mixing sphere 29 is circular, and the number is 5-20. In other embodiments of this application, the cross-sectional shape of the mixing sphere 29 is not limited and can be any shape, such as triangle, ellipse, etc. The number can also be set arbitrarily, such as 1, 2...30, 40, etc.

[0046] In this embodiment, the first protrusion 24, the second protrusion 25, the third protrusion 26, the fourth protrusion 27, and the fifth protrusion 28 can achieve a better mixing effect, while reducing the residue of the mixture in the first protrusion 24, the second protrusion 25, the third protrusion 26, the fourth protrusion 27, and the fifth protrusion 28, so that most of it can enter the second premixing zone 18.

[0047] The first premixing zone 17 also includes a vibrator and a first sensor. When the mixture (composed of the sample and diluent) passes through the first premixing zone 17, the vibrator starts to vibrate, causing the mixing ball 29 to move randomly, thereby further achieving the purpose of uniform mixing and avoiding the problem of poor repeatability caused by uneven mixing of the sample and diluent. Furthermore, a filter membrane 23 is provided between the first zone 19 and the dilution zone 16 to prevent the mixing ball 29 from entering the dilution zone 16.

[0048] The microfluidic unit 3 is equipped with a main control module, which is connected to the first sensor and the vibrator to control their operation. Specifically, the main control module controls the vibrator through signals transmitted from the first sensor. When the mixture enters the first premixing zone 17 and reaches a certain amount, the vibrator starts working; when the mixture flows out of the first premixing zone 17 and enters the second premixing zone 18, the vibrator stops working. Furthermore, the connection between the main control module and the one-way valve 32 and the first sensor can be electrical or wireless; the wireless connection can be Bluetooth.

[0049] In a further embodiment, the first sensor is a pressure sensor, located at the center of the first premixing zone. When the mixture flows through the first sensor to a certain amount, the first sensor sends a signal to the main control module, which then controls the vibrator to start working. When the mixture has completely flowed out of the first premixing zone, the first sensor sends a signal to the main control module, which then controls the vibrator to stop working.

[0050] In a further embodiment, the vibrator has a low power and only vibrates the first premixing zone, having little impact on other zones.

[0051] In this embodiment, the first premixing zone 17 is connected to the second premixing zone 18. The cross-sectional shape of the second premixing zone 18 is Z-shaped. Under the action of external force, the mixture can flow to the second premixing zone 18. Since the second mixing zone is Z-shaped, the mixture will slow down its forward speed in the second mixing zone, thereby achieving further mixing.

[0052] In this embodiment, the second premixed region 18 is connected to the labeling region 30, and the labeling region 30 contains a labeled antibody. Under the action of external force, the mixture flows from the second premixed region 18 to the labeling region 30 and combines with the labeled antibody in the labeling region 30 to form a analyte-primary antibody complex. In a specific embodiment, the labeled antibody can be a fluorescein, enzyme, or biotin-labeled antibody, which can specifically bind to specific substances in the sample. The labeled antibodies can be multiple. More specifically, the labeled antibodies can be multiple allergens in allergen testing, such as dairy allergen protein antigens (including cow's milk, goat's milk, etc.); meat allergen protein antigens (including pork, beef, mutton, donkey meat, chicken, duck, goose, turkey, etc.); egg allergen protein antigens (including poultry protein, poultry egg yolk, etc.); nut allergen protein antigens (including almonds, peanuts, walnuts, hazelnuts, pistachios, cashews, Brazil nuts, sesame seeds, etc.); cereal allergen protein antigens (including corn, oats, wheat, buckwheat, barley, rice, millet, etc.); legume allergen protein antigens (including lentils, soybeans, peas, broad beans, etc.); fruit allergen protein antigens (including pineapple, oranges, mangoes, apples, peaches, strawberries, grapefruit, lemons, etc.); and seafood allergen protein antigens (including ribbonfish, yellow croaker, halibut, salmon, carp, etc.). Fish, crucian carp, grass carp, silver carp, shrimp, crab, clams, scallops, oysters, eels, and eels; cockroach allergen protein antigens, including German cockroaches and American cockroaches; mold allergen protein antigens, including Penicillium, Cladosporium, Aspergillus fumigatus, Alternaria alternata, Rhizopus nigricans, Saccharomyces cerevisiae, Ustilago maydis, Mucor, and Ulva prostrata; pollen allergen protein antigens, including those from birch, maple, paper mulberry, and palm trees. The following trees are considered allergens: palm, hazel, cypress, pine, cedar, chestnut, oak, sycamore, poplar, willow, mulberry, elm, artemisia, ragweed, lambsquarters, plantain, cogongrass, burdock, bermudagrass, timothy, reed, cocklebur, cattail, sedge, reed, sunflower, etc.; inhaled mite allergens, including house dust mites, etc.; animal allergens, including cat hair, dog hair, cat dander, dog dander, etc. Other allergens besides those described above may also be considered.

[0053] Furthermore, the labeled antibody is selected from one or more of the aforementioned allergens. When only one is selected, the repeatability of the detection results of this microfluidic unit 3 can be verified. In this application, there are a total of eight microfluidic units 3, and at least eight types of labeled antibodies can be used, enabling at least eight different allergen detections.

[0054] In this embodiment, the microfluidic unit 3 further includes a quantification zone located between the labeling zone 30 and the dilution zone 16. The quantification zone is used to quantify the sample entering the detection zone. The quantification zone has a second waste liquid zone 15; during quantification, excess mixture overflows into the second waste liquid zone 15. A one-way valve 32 is provided between the quantification zone and the detection zone. The one-way valve 32 is closed before quantification is completed, preventing the mixture in the quantification zone from flowing to the detection zone; after quantification, the one-way valve 32 opens, allowing the quantified mixture in the quantification zone to flow to the detection zone. By setting the quantification zone, the amount of liquid entering the detection zone can be determined, resulting in smaller discrepancies in the detection results of the microfluidic unit 3. A second sensor 33 is provided in the second waste liquid zone 15. The main control unit is connected to the one-way valve 32 and the second sensor 33 to control the operation of the one-way valve 32, the second sensor 33, and the vibrator. Specifically, the main control module controls the operation of the one-way valve 32 through the signal transmitted by the second sensor 33. When the sample volume of the mixture in the second waste liquid zone 15 reaches a certain level, the quantification is considered complete, and then the one-way valve 32 is opened, allowing the quantified mixture to enter the detection zone. Furthermore, the connection between the main control module and the one-way valve 32 and the second sensor 33 can be electrical or wireless; the wireless connection can be Bluetooth.

[0055] In this embodiment, the quantitative zone includes a sample flow channel 34 and multiple sub-quantitative zones 31. The sample flow channel 34 is connected to the detection zone and has a V-shaped cross-section. The multiple sub-quantitative zones 31 extend along both ends of the sample flow channel and are symmetrically arranged about the bends of the sample flow channel 34. Each sub-quantitative zone 31 is connected to the sample flow channel 34. Further, the end of the sample flow channel 34 is connected to the second waste liquid zone 15. During quantification, the mixed liquid enters the sample flow channel 34 and enters the sub-quantitative zones 31 one by one. Then, the excess mixed liquid flows along the second waste liquid zone 15 at the end of the sample flow channel 34. Specifically, each sub-quantitative zone 31 has the same volume, and there are four sub-quantitative zones 31, arranged in pairs on both sides of the sample flow channel 34. There are two second waste liquid zones 15, respectively arranged at both ends of the sample flow channel 34. The symmetrical arrangement is beneficial for maintaining balance during centrifugation. The second waste liquid zone 15 is further away from the rotation center 5 than the sub-quantitative zones 31.

[0056] Accordingly, a one-way valve 32 is provided between the sub-quantification zone 31 and the detection zone. The one-way valve 32 is closed before quantification is completed, preventing the mixture in the sub-quantification zone 31 from flowing to the detection zone. After quantification is completed, the one-way valve 32 opens, allowing the quantified mixture in the sub-quantification zone 31 to flow to the detection zone. The one-way valve 32 is connected to the main control module, enabling the main control module to control the operation of the one-way valve 32.

[0057] In a further embodiment, the second sensor is a pressure sensor located at the bottom of the second waste liquid zone. When excess mixed liquid flows into the second waste liquid zone to a certain amount, the second sensor transmits a signal to the main control module, and the main control module controls the one-way valve 32 to open.

[0058] In this embodiment, the detection area includes multiple sub-detection areas 35. Further, the number of sub-detection areas 35 corresponds to the number of sub-quantitative areas 31, with four sub-detection areas 35, each connected to a sub-quantitative area 31. Further, each sub-detection area 35 contains a detection card with multiple detection strips coated with different detection antibodies (secondary antibodies). The type and quantity of the detection antibodies correspond one-to-one with those of the labeled antibodies. In the detection area, the sample-primary antibody complex and the detection antibody on the detection strip form a ternary complex of sample-primary antibody-secondary antibody.

[0059] In this embodiment, there are three detection strips, each coated with a different detection antibody. The detection antibodies coated in each sub-detection region 35 are different, meaning that each microfluidic unit 3 can detect 12 allergens, and each microfluidic protein chip can detect 96 allergens.

[0060] In other embodiments of this application, there is one detection strip, and each detection strip is coated with a detection antibody. The detection antibodies coated on each sub-detection region 35 are different, that is, each microfluidic unit 3 can detect 4 allergens, that is, each microfluidic protein chip can detect 32 allergens.

[0061] In another embodiment of this application, there are two detection strips, each coated with a detection antibody. The detection antibodies coated on each sub-detection region 35 are different, that is, each microfluidic unit 3 can detect 8 allergens, that is, each microfluidic protein chip can detect 64 allergens.

[0062] In another embodiment of this application, there are two detection strips, each coated with a detection antibody. Any two sub-detection regions are coated with the same antibody, meaning that each microfluidic protein chip can detect 32 allergens.

[0063] Furthermore, a quality control labeling area 30 is provided within the labeling area 30, and a quality control line is provided on the detection strip, with the quality control line coated with a quality control antibody. The quality control label and the quality control antibody specifically bind to form a quality control label-quality control antibody complex. The validity of the detection results of the microfluidic protein chip is determined by detecting the quality control label-quality control antibody complex.

[0064] In this embodiment, each sub-detection zone 35 is connected to the third waste liquid zone 7. After the mixed liquid binds to the detection antibody in the sub-detection zone 35, the excess mixed liquid flows into the third waste liquid zone 7.

[0065] When using the microfluidic protein chip, for whole blood samples, first add whole blood samples to the first sample loading zone 1. After loading, start centrifugation. The whole blood sample enters the first pre-separation zone 10, and the excess whole blood sample enters the first waste liquid zone 8. Continue centrifugation. The whole blood sample is filtered through the whole blood filtration membrane 12. The plasma enters the first post-separation zone 11, and the blood cells remain in the whole blood filtration membrane 12. At the same time, some plasma enters the first capillary channel 9. At this time, because the centrifugal force is greater than the capillary force in the first capillary channel 9, the plasma entering the first capillary channel 9 stops flowing when it reaches the highest point of the first post-separation zone 11. It stays in the front section a of the first capillary channel 9. The position of the bend apex of the first capillary channel 9 near the rotation center 5 is closer to the rotation center 5 than the first post-separation zone 11 as a whole. Therefore, the plasma will not cross the bend apex b of the first capillary channel 9, nor will it enter the rear section c of the first capillary channel 9. The first capillary channel 9 acts as a valve during the centrifugation of whole blood samples. After the whole blood sample is centrifuged, the centrifugation is stopped. At this time, the plasma in the first capillary channel 9 will flow along the first capillary channel 9 under the action of capillary force, and finally cross the bend vertex b and enter the buffer zone 6 from the rear section c. Then the centrifugation is continued. The separated plasma sample enters the split channel 14 through the buffer zone 6. After being separated by the split channel 14, the plasma flows into the dilution zone 16 of each microfluidic unit 3. After ultra-low speed centrifugation, the separated plasma sample and the diluent are mixed to form a mixture that flows into the first premixing zone 17. The first sensor receives the signal and sends it to the main control module. The main control module controls the vibrator to work. The vibrator drives the mixing ball 29 to fully mix the mixture. During the ultra-low speed centrifugation, the mixture continues to flow sequentially to the second premixing zone 18 and the labeling zone 30. After the mixture flows out of the first premixing zone 17, the first sensor receives the signal and sends it to the main control module. The main control module controls the vibrator to stop working. In the labeling zone 30, the analyte in the mixture binds with the labeled antibody to form the analyte-antibody complex. During ultra-low speed centrifugation, the mixture continues to flow towards the quantification zone. In the quantification zone, excess mixture flows into the second waste liquid zone 15. The second waste liquid zone 15 is equipped with a second sensor 33. When the mixture in the second waste liquid zone 15 reaches a certain volume, the second sensor 33 sends a signal to the main control module. The main control module controls the one-way valve 32 to open, allowing the mixture after quantification in the sub-quantification zone 31 to flow into the detection zone. In the detection zone, the analyte-primary antibody complex in the mixture forms an analyte-primary antibody-secondary antibody ternary complex with the detection antibody in the labeling zone 30. The content of the analyte in the sample is calculated by detecting the amount of the ternary complex in the detection zone. The mixture that does not bind to the detection antibody flows into the third waste liquid chamber. The quality control marker specifically binds to the quality control antibody on the quality control line, forming a quality control marker-quality control antibody complex. The effectiveness of the microfluidic unit 3 is determined by detecting the quality control marker-quality control antibody complex in the detection chamber.

[0066] For serum or plasma samples, add the sample to the second sample loading zone 2. After loading, start centrifugation. The sample flows into the split channel 14 through the microfluidic channel. The subsequent process is the same as for whole blood samples. There is no need to perform whole blood separation. The serum sample is directly added to the second sample loading zone 2, which reduces the sample volume and shortens the testing process, thus effectively improving the testing efficiency.

[0067] This microfluidic protein chip can detect both whole blood and serum or plasma samples, offering ease of operation and high flexibility. When detecting whole blood samples, separation is achieved through a whole blood filtration membrane 12, resulting in excellent separation and convenient, rapid operation. The design cleverly incorporates capillary channels as valves. Detection of plasma or serum samples requires a smaller sample volume. The chip includes a first and second premixing zone, with mixing balls and protrusions enhancing mixing efficiency, resulting in minimal differences in test results between different batches of the same sample or within the same batch, ensuring good repeatability. Quantification in each sub-quantitative zone further reduces variability in test results. The labeling and detection zones can be coated with multiple antibodies, enabling simultaneous detection of various analytes for high-throughput testing. Positioning the quantification zone after the labeling zone improves the accuracy of the test results. Additionally, a main control module, sensors, and one-way valves are included, leveraging automation to enhance detection efficiency.

Claims

1. A microfluidic protein chip for allergen detection, characterized in that, It includes a sample dispensing area and at least one microfluidic unit, wherein the microfluidic unit includes a dilution area, a premixing area, a labeling area, a quantification area, and a detection area connected in sequence; The central part of the microfluidic protein chip is a rotating mounting part with a rotation center, which is the rotation center during centrifugation. The sample loading area includes a first sample loading area, a second sample loading area, a buffer zone, a first waste liquid zone, a separation zone, and a first capillary channel; both the first and second sample loading areas are arranged around the rotation center. The first sample loading area is connected to the buffer zone through the first pre-separation zone and the first post-separation zone, and the second sample loading area is directly connected to the buffer zone. The first and second sample loading areas are formed around the rotation center. The first sample loading area is used to add whole blood samples; the second sample loading area is used to add serum or plasma samples. The first sample loading zone is directly connected to the first pre-separation zone. The first pre-separation zone is further away from the rotation center than the first sample loading zone. A filtration zone is provided between the first pre-separation zone and the first post-separation zone. The filtration zone contains a whole blood filtration membrane. Through centrifugation, the whole blood filtration membrane filters the whole blood, and blood cells are filtered out by the whole blood filtration membrane. The plasma enters the first post-separation zone, which is further away from the rotation center than the first pre-separation zone. The first post-separation zone is connected to the buffer zone through a first capillary channel. The first waste liquid zone is connected to the first pre-separation zone via an overflow channel; The buffer zone is connected to the dilution zone; The dilution zone contains a diluent; The premixing zone is used to mix the sample and the diluent. The premixing zone includes a first premixing zone and a second premixing zone. The first premixing zone is provided with a first protrusion, a second protrusion, a third protrusion, a fourth protrusion, a fifth protrusion, a mixing ball, a filter membrane, a vibrator, and a first sensor. The cross-sectional shape of the second premixing zone is Z-shaped. The first premixing zone is provided with a first partition, a second partition, a third partition, and a fourth partition, which are separated by a filter membrane. The mixing ball moves freely within each partition. The first protrusion, the second protrusion, the third protrusion, the fourth protrusion, and the fifth protrusion are fixed. The labeled region contains labeled antibodies; The quantitative zone is used for quantitative mixing; The detection area is equipped with detection antibodies.

2. The microfluidic protein chip for allergen detection as described in claim 1, characterized in that, The labeled antibody is at least one allergen, and the detection antibody is at least one allergen.

3. The microfluidic protein chip for allergen detection as described in claim 1, characterized in that, The first premixing zone is equipped with a vibrator and a first sensor. The main control module is connected to the vibrator and the first sensor. After receiving the signal from the first sensor, the main control module controls the vibrator to work.

4. The microfluidic protein chip for allergen detection as described in claim 1, characterized in that, The cross-sectional shape of the first protrusion, the second protrusion, the third protrusion, the fourth protrusion, and the fifth protrusion is triangular; The first, second, third, fourth, and fifth protrusions are symmetrically arranged about the vertical center line.

5. The microfluidic protein chip for allergen detection as described in claim 1, characterized in that, The quantitative zone includes multiple sub-quantitative zones and multiple second waste liquid zones, with a second sensor installed in the second waste liquid zone.

6. The microfluidic protein chip for allergen detection as described in claim 5, characterized in that, A one-way valve is provided at the connection between the sub-quantitative zone and the detection zone. The main control module is connected to the second sensor and the one-way valve. After receiving the signal from the second sensor, the main control module controls the one-way valve to open or close.

7. The microfluidic protein chip for allergen detection as described in claim 1, characterized in that, The labeling area is provided with a quality control marker, and the detection area is coated with a quality control antibody, which can specifically bind to the quality control marker.

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