Microfluidic immunoassay chip and immunoassay method

Abstract

The application discloses a micro-fluidic immune detection chip and an immune detection method, and belongs to the technical field of micro-fluidics. The embodiment of the application combines quantum dot fluorescent microsphere technology and protein microarray technology, realizes quantitative, semi-quantitative or qualitative analysis of immune disease related molecules, greatly shortens detection time and procedure, and thus solves the problem that existing immune detection equipment cannot effectively balance detection efficiency and detection precision.

Description

TECHNICAL FIELD

[0001] The application belongs to the technical field of microfluidics, and particularly relates to a microfluidic immunodetection chip and an immunodetection method. BACKGROUND

[0002] Allergic diseases have become a global health problem, imposing a significant burden on health care resources due to increasing prevalence, severity, chronicity, and associated management costs.

[0003] In the prior art, allergic detection is generally performed by enzyme-linked immunosorbent assay, immunoblotting, chemiluminescence, immunofluorescence quantitative detection, allergen microarray chip method, and immunochromatographic colloidal gold method. However, the above methods have problems such as long reaction cycle, complicated steps, high cost, or poor quantitative effect, that is, they cannot effectively balance detection efficiency and detection accuracy. SUMMARY

[0004] The purpose of the embodiments of the application is to provide a microfluidic immunodetection chip and an immunodetection method, which can solve the problem that existing immunodetection equipment cannot effectively balance detection efficiency and detection accuracy.

[0005] To solve the above technical problems, the application is implemented as follows:

[0006] In a first aspect, the embodiments of the application provide a microfluidic immunodetection chip, comprising a sample cavity, a marker cavity, a sample inlet channel, a washing channel, a reaction detection cavity, and a waste liquid cavity.

[0007] The sample cavity is provided with a sample addition hole for sample addition, and the marker cavity is preloaded with quantum dot microsphere markers. The sample cavity and the marker cavity are connected with a pump port assembly for pumping diluent, and the sample cavity and the marker cavity are connected with one end of the sample inlet channel.

[0008] One end of the washing channel is provided with a third pump port for pumping washing liquid.

[0009] The reaction detection cavity is connected with the other end of the sample inlet channel and the other end of the washing channel, and the reaction detection cavity is provided with a capture microarray.

[0010] The waste liquid cavity is connected with the reaction detection cavity for receiving waste liquid.

[0011] Optionally, in the microfluidic immunodetection chip, the pump port assembly comprises a first pump port, the sample cavity, the marker cavity, and one end of the sample inlet channel are connected in series, and the sample cavity is connected with the first pump port.

[0012] Optionally, in the microfluidic immunoassay chip, the pump port assembly includes a first pump port and a second pump port, the sample dispensing chamber and the labeling chamber are connected in parallel to one end of the sample injection channel, and the sample dispensing chamber is connected to the first pump port, and the labeling chamber is connected to the second pump port.

[0013] Optionally, in the microfluidic immunoassay chip, the quantum dot microsphere marker is a combination of lyophilized quantum dot microspheres and the marker.

[0014] Optionally, in the microfluidic immunoassay chip, the quantum dot microsphere marker is a combination of markers for one or more target items and quantum dot microspheres, and the trap microarray is a microarray of traps for the multiple target items.

[0015] Optionally, in the microfluidic immunoassay chip, the multiple target items include allergens, autoantibodies, and cytokines.

[0016] Optionally, in the microfluidic immunoassay chip, both the sample injection channel and the rinsing channel are provided with anti-backflow structures at their ends.

[0017] Optionally, in the microfluidic immunoassay chip, the anti-backflow structure is a bottle-shaped structure with its opening facing the reaction detection chamber, and the depth of the bottle body is greater than the depth of the sample inlet channel, and the width of the bottle body is greater than the width of the sample inlet channel.

[0018] Optionally, in the microfluidic immunoassay chip, the waste liquid chamber is provided with a water-absorbing substance.

[0019] Optionally, in the microfluidic immunoassay chip, the sample introduction channel is serpentine.

[0020] Secondly, embodiments of this application provide an immunoassay method based on the aforementioned microfluidic immunoassay chip, comprising:

[0021] After the sample is added through the sample well, the diluent is pumped in through the first pump port and the sample is pumped into the labeling chamber to mix with the quantum dot microsphere label;

[0022] After the sample is mixed with the quantum dot microsphere label, the mixture in the label chamber is pumped to the sample inlet channel through the first pump port, and the mixture is bound in the sample inlet channel;

[0023] After the sample is bound to the quantum dot microsphere label, the bound material in the sample inlet is pumped into the reaction detection chamber for incubation through the first pump port.

[0024] After incubation is complete, cleaning fluid is pumped into the third pump port to pump the unreacted material to the waste liquid chamber.

[0025] The reaction detection chamber is analyzed.

[0026] Thirdly, embodiments of this application provide another immunoassay method based on the aforementioned microfluidic immunoassay chip, comprising:

[0027] After the sample is added through the sample well, diluent is pumped into the first pump port and the sample is then pumped into the reaction detection chamber for incubation.

[0028] After incubation is complete, cleaning fluid is pumped in through the third pump port, and unreacted material is pumped to the waste liquid chamber.

[0029] After the reaction detection chamber is cleaned, the mixture in the labeling chamber is pumped to the reaction detection chamber through the first pump port for labeling reaction;

[0030] After the labeling reaction is completed, cleaning fluid is pumped into the third pump port to pump the unreacted material to the waste liquid chamber.

[0031] The reaction detection chamber is analyzed.

[0032] In this embodiment, the microfluidic immunoassay chip includes a sample chamber, a label chamber, an injection channel, a cleaning channel, a reaction detection chamber, and a waste liquid chamber. The sample chamber has a sample loading port for adding samples. The label chamber is pre-loaded with quantum dot microspheres as labels. Both the sample chamber and the label chamber are connected to a pump port assembly for pumping in diluent, and both the sample chamber and the label chamber are connected to one end of the injection channel. One end of the cleaning channel has a third pump port for pumping in cleaning solution. The reaction detection chamber is connected to the other end of the injection channel and the other end of the cleaning channel, and a trap microarray is disposed in the reaction detection chamber. The waste liquid chamber is connected to the reaction detection chamber to contain waste liquid. The aforementioned microfluidic immunoassay chip, because it has quantum dot microspheres pre-loaded in the labeling chamber and a trap microarray in the reaction detection chamber, allows the sample to be mixed with the quantum dot microspheres and then incubated on the trap microarray, or the sample to be incubated on the trap microarray and then washed before being mixed with the quantum dot microspheres. Then, the trap microarray is fluorescently detected using a matching detection instrument. This combines quantum dot fluorescent microsphere technology and protein microarray technology to achieve quantitative, semi-quantitative, or qualitative analysis of molecules related to immune diseases. The detection time and process are greatly shortened, thus solving the problem that existing immunoassay devices cannot effectively balance detection efficiency and detection accuracy. Attached Figure Description

[0033] Figure 1 This is a schematic diagram of the structure of the microfluidic immune detection chip provided in the first embodiment of this application;

[0034] Figure 2 This is an exploded view of the microfluidic immune detection chip provided in the first embodiment of this application;

[0035] Figure 3 This is a schematic diagram of the structure of the microfluidic immune detection chip provided in the second embodiment of this application;

[0036] Figure 4 This is an exploded view of the microfluidic immunoassay chip provided in the second embodiment of this application;

[0037] Figure 5 yes Figure 4 A magnified view of part A in the middle;

[0038] Figure 6 This is a flowchart of an immune detection method provided in an embodiment of this application;

[0039] Figure 7 This is a schematic diagram illustrating the strong positive detection effect provided in the embodiments of this application;

[0040] Figure 8 This is a schematic diagram illustrating the weak positive detection effect provided in the embodiments of this application;

[0041] Figure 9 This is a schematic diagram illustrating the negative test result provided in an embodiment of this application;

[0042] Figure 10 This is a schematic diagram of the detection effect of the control group provided in the embodiments of this application;

[0043] Figure 11 This is a flowchart of an immune detection method provided in another embodiment of this application. Detailed Implementation

[0044] The technical solutions of the embodiments of this application will be clearly described below with reference to the accompanying drawings. Obviously, the described embodiments are only some, not all, of the embodiments of this application. All other embodiments obtained by those skilled in the art based on the embodiments of this application are within the scope of protection of this application.

[0045] The terms "first," "second," etc., used in the specification and claims of this application are used to distinguish similar objects and not to describe a specific order or sequence. It should be understood that such use of data can be interchanged where appropriate so that embodiments of this application can be implemented in orders other than those illustrated or described herein. Furthermore, in the specification and claims, "and / or" indicates at least one of the connected objects, and the character " / " generally indicates that the preceding and following objects are in an "or" relationship.

[0046] The inward-folding electronic device provided in this application will be described in detail below with reference to the accompanying drawings, through specific embodiments and application scenarios.

[0047] Please see Figures 1-4 The aforementioned microfluidic immunoassay chip 100 includes a sample chamber 11, a label chamber 12, an injection channel 13, a cleaning channel 14, a reaction detection chamber 15, and a waste liquid chamber 16. The sample chamber 11 is provided with an injection port 17 for adding samples. The label chamber 12 is pre-loaded with quantum dot microspheres as labels. Both the sample chamber 11 and the label chamber 12 are connected to a pump port assembly 18 for pumping in diluent, and both the sample chamber 11 and the label chamber 12 are connected to one end of the injection channel 13. One end of the cleaning channel 14 is provided with a third pump port 19 for pumping in cleaning solution. The reaction detection chamber 15 is connected to the other end of the injection channel 13 and the other end of the cleaning channel 14, and a trap microarray 151 is provided in the reaction detection chamber 15. The waste liquid chamber 16 is connected to the reaction detection chamber 15 to contain waste liquid.

[0048] The microfluidic immunoassay chip 100 provided in this embodiment of the invention has a microchannel, a pump port, and a micropore. Quantum dot microspheres are pre-loaded in the labeling chamber 12, and a trapping microarray 151 is provided in the reaction detection chamber 15. The sample chamber 11 and the labeling chamber 12 are both connected to a pump port assembly 18 for pumping in diluent, and both the sample chamber 11 and the labeling chamber 12 are connected to one end of the injection channel 13, while the reaction detection chamber 15 is connected to the other end of the injection channel 13. Therefore, in this embodiment, the sample and quantum dot microsphere label can be simultaneously pumped to the injection channel 13 via the pump port assembly 18, and then pumped to the reaction detection chamber 15 after binding, where they are incubated on the captured microarray 151. Then, the unreacted quantum dot microsphere label is flushed away with cleaning fluid pumped in via the third pump port 19, and finally, the captured microarray 151 is fluorescence detected using a matching detection instrument. Alternatively, the sample can be first pumped to the reaction detection chamber 15 via the injection channel 13 using the pump port assembly 18, where it is incubated on the captured microarray 151. Then, cleaning fluid is pumped in via the third pump port 19, followed by pumping diluent into the label chamber 12 via the pump port assembly 18. The quantum dot microsphere label in the label chamber 12 is then pumped to the reaction detection chamber 15 to bind with the sample. Then, cleaning fluid is pumped in via the third pump port 19 to flush away the unreacted quantum dot microsphere label, and finally, the captured microarray 151 is fluorescence detected using a matching detection instrument.

[0049] Quantum dots possess characteristics such as high fluorescence efficiency, resistance to photobleaching, long fluorescence lifetime, narrow and symmetrical emission spectrum, single-unit excitation multi-emission, and simultaneous detection of multiple indicators. Quantum dot microspheres in quantum dot microsphere labeling encapsulate quantum dots, enabling amplification of the fluorescence signal of quantum dots. The coating layer not only stabilizes the quantum dots and improves the biocompatibility and colloidal stability of the microspheres, but also modifies the surface of the microspheres with various functional groups, achieving the binding of the microsphere surface with various labels.

[0050] The capture microarray 151 can be an antigen protein microarray or an antibody protein microarray. It involves coating 0.1–1000 pg of trace protein onto the surface of the microfluidic chip at the reaction detection chamber 15 using a precision spotting device. The surface of the microarray is pre-treated to significantly reduce sample consumption while maintaining detection sensitivity. Optionally, the capture microarray 151 can be constructed by coating 1–50 pg of trace protein onto the surface of the microfluidic chip at the reaction detection chamber 15 using a precision spotting device.

[0051] As can be seen, in the embodiments of this application, the combination of quantum dot fluorescent microsphere technology and protein microarray technology enables quantitative or semi-quantitative analysis of molecules related to immune diseases, which can greatly shorten the detection time and process, thereby solving the problem that existing immune detection equipment cannot effectively balance detection efficiency and detection accuracy.

[0052] In the microfluidic immunoassay chip 100 provided in this application embodiment, the quantum dot microsphere label can be a combination of one or more target item labels and quantum dot microspheres, and the aforementioned trapping microarray 151 is a microarray of trapping substances for each target item. That is, the aforementioned quantum dot microsphere label is a combination of quantum dot microspheres and labeled antibodies or antigens, and correspondingly, the trapping microarray 151 is a trapping antigen microarray or a trapping antibody microarray.

[0053] For example, when the target item is an allergen, the quantum dot microsphere marker is a conjugate of a specific IgE secondary antibody and quantum dot microspheres, and the capture microarray 151 is a microarray of a specific IgE antigen; or the quantum dot microsphere marker is a conjugate of a specific IgE antigen and quantum dot microspheres, and the capture microarray 151 is a microarray of a specific IgE secondary antibody.

[0054] Among them, combining the specificity of antigens and antibodies with the ultrasensitivity of quantum dot microspheres, quantum dot fluorescence immunoassay is realized. It can be used in a variety of biological detection technologies and has the characteristics of high sensitivity, multi-channel, and rapid quantitative detection. It can be used to detect various antigens, antibodies, toxins, antibiotics, pesticide and veterinary drug residues, drugs, etc.

[0055] In practical applications, the excitation wavelength of the aforementioned quantum dot microspheres is between 300 and 450 nm, and the same excitation source can excite one or more quantum dot microspheres with emission wavelengths between 500 and 760 nm. For example, one or more quantum dot microspheres with emission wavelengths of 525±20 nm, 565±20 nm, and 615±20 nm can be selected.

[0056] Accordingly, the detection light source for fluorescence detection of the captured microarray 151 is between 500 and 760 nm, specifically 615 nm.

[0057] The sample amount of the capture microarray 151 can be 0.1 to 100 ng / point, and can include 1 to 400 points, specifically 25 to 225 points. Three to five consecutive array points represent one item, and a total of eight to 100 items can be detected simultaneously. Quality control points can be set at each array point to achieve quantitative detection.

[0058] Optionally, in one embodiment, the quantum dot microsphere marker is a combination of markers for multiple target items and quantum dot microspheres, and the trap microarray 151 is a microarray of traps for each target item, the multiple target items including allergens, autoantibodies and cytokines.

[0059] Optionally, in one embodiment, the quantum dot microsphere label is a combination of freeze-dried quantum dot microspheres and a label, specifically freeze-dried microspheres or freeze-dried powder, that is, the quantum dot microspheres and the label are freeze-dried and then embedded in the label cavity 12. Freeze-drying reduces the amount of liquid reagent, facilitating storage and transportation.

[0060] Of course, the quantum dot microsphere markers mentioned above can also be in liquid form with a volume of 1 to 200 microliters, for example, 50 μL.

[0061] In practical applications, if the labeling chamber 12 is pre-loaded with lyophilized quantum dot microspheres, the lyophilized powder needs to be quickly reconstituted using a liquid sample or diluent before being pumped into the sample inlet channel 13 or the reaction detection chamber 15 for binding reaction.

[0062] Optionally, in one specific embodiment, the freeze-dried quantum dot microsphere label is encapsulated and protected using a protective agent, which is one or more substances selected from mannitol, sucrose, lactose, trehalose, PEG with a molecular weight of 200 to 50,000, and PVP with a molecular weight of 10,000 to 60,000.

[0063] Alternatively, in one implementation, such as Figures 1-2As shown, the pump port assembly 18 includes a first pump port 181, and one end of the sample chamber 11, the marker chamber 12 and the sample injection channel 13 are connected in series, and the sample chamber 11 is connected to the first pump port 181.

[0064] In this embodiment, since the sample chamber 11, the label chamber 12, and one end of the sample inlet channel 13 are connected in series, when a diluted or undiluted sample is pumped through the first pump port 181, the sample must first flow into the label chamber 12 to mix with the quantum dot microsphere label, and then enter the sample inlet channel 13 under the control of the pump. The sample is then mixed and reacted through the sample inlet channel 13 to form a conjugate between the quantum dot microsphere label and the specific capture antibody or antigen in the sample. The conjugate is then pumped into the reaction detection chamber 15 to incubate with the capture microarray 151. Then, the washing solution is pumped in through the third pump port 19 to rinse away the unreacted quantum dot microsphere label, and the capture microarray 151 can be fluorescently detected.

[0065] Optionally, in one embodiment, the sample inlet channel 13 is serpentine to facilitate mixing of the sample with the quantum dot microsphere markers.

[0066] For example, when the target item is allergen-specific IgE, i.e., for the detection of allergen-specific immunoglobulins, the above-mentioned quantum dot microsphere label is a conjugate of allergen-specific IgE antibody and quantum dot microsphere, and the above-mentioned capture microarray 151 is an allergen-specific IgE antigen microarray.

[0067] Alternatively, in another implementation, such as Figures 3-4 As shown, the pump port assembly 18 includes a first pump port 181 and a second pump port 182. The sample chamber 11 and the marker chamber 12 are connected in parallel to one end of the sample injection channel 13. The sample chamber 11 is connected to the first pump port 181, and the marker chamber 12 is connected to the second pump port 182.

[0068] In this embodiment, when pumping diluted or undiluted samples through the first pump port 181, the sample is first pumped through the sample injection channel 13 to the reaction detection chamber 15 for incubation on the capture microarray 151. Then, the cleaning solution is pumped in through the third pump port 19 to rinse the sample. Next, the diluent is pumped into the labeling chamber 12 through the second pump port 182, and the quantum dot microspheres in the labeling chamber 12 are pumped into the reaction detection chamber 15 to bind with the sample. Then, the unreacted quantum dot microspheres are rinsed away with the cleaning solution pumped in through the third pump port 19, and the capture microarray 151 can be fluorescence detected.

[0069] The microfluidic immunoassay chip 100 provided in this application embodiment can be fabricated using traditional micro-injection molding or screen printing. A transparent sealing plate 20 is encapsulated on the chip surface, with higher transparency at the reaction detection chamber 15. The transparent sealing plate 20 is encapsulated by adhesive bonding, thermoforming, or ultrasonic bonding.

[0070] Optionally, the sample application port 17 is disposed on the transparent sealing plate 20 and is positioned opposite the sample chamber 11. In addition, a sample application cap 21 adapted to the sample application port 17 is provided at the sample application port 17. The sample can be added into the sample chamber 11 by removing the sample application cap 21; after the sample is added, the sample application cap 21 can be replaced to start the immunoassay.

[0071] The sample volume is between 1uL and 100uL, for example, 20 to 50uL. The sample can be diluted by pumping diluent into it through the first pump port 181.

[0072] In this embodiment, the sample is driven by a pump, which can control its opening, closing, and dwell time according to the sample's position. The pump can be a positive pressure pump installed at the pump inlets (including the first inlet 181, the second inlet 182, and the third inlet 19), or a negative pressure pump connected to the waste liquid chamber 16. It can drive the sample, diluent, and cleaning solution by creating a negative pressure in the waste liquid chamber 16. The positive and negative pressure pumps can be traditional pumps such as mechanical pumps, ceramic pumps, or peristaltic pumps; the negative pressure pump can also be a paper pump, a sponge pump, etc., to achieve the pumping effect through capillary action. The paper or sponge can be disconnected when the drive needs to be stopped.

[0073] In the chip structure described above, when the microchannels are realized by injection molding, the chip material can be polystyrene, polyester, polymethyl methacrylate, etc.; the chip can also realize the flow channel structure by screen printing, and the microchannels are printed with conductive plasma. Subsequently, the liquid flow position can be determined by sensing electrical signals, thereby achieving precise control of the reaction.

[0074] In the microfluidic immunoassay chip 100 provided in this application embodiment, the other end of the sample injection channel 13 and the other end of the cleaning channel 14 are connected to the reaction detection chamber 15 through the same flow channel.

[0075] Optionally, the microfluidic immunoassay chip 100 provided in this application embodiment is provided with an anti-backflow structure 131 at the end of the sample injection channel 13 and the cleaning channel 14, which can effectively prevent the liquid in the reaction detection chamber 15 from flowing back.

[0076] Specifically, such as Figure 5As shown, the aforementioned anti-backflow structure 131 is a bottle-shaped structure with its opening facing the reaction detection chamber 15. The bottle-shaped structure includes a bottle body 132 and a bottle mouth 133. The depth of the bottle body 132 is greater than the depth of the sample inlet channel 13, and the width of the bottle body 132 is greater than the width of the sample inlet channel 13. The aforementioned anti-backflow structure 131 is first enlarged in a local area of ​​the sample inlet channel 13, and then its depth and width are reduced before it is connected to a normal-sized channel. This structure allows liquid to flow from the sample inlet channel 13 to the reaction detection chamber 15, but prevents it from flowing back from the reaction detection chamber 15 to the sample inlet channel 13, thereby achieving the anti-backflow effect.

[0077] Optionally, the depth and width of the end of the bottle mouth 133 are the same as those of the injection channel 13, and the bottle body 132 is cylindrical and the bottle mouth 133 is conical.

[0078] Optionally, in the microfluidic immunoassay chip 100 provided in this embodiment, the waste liquid chamber 16 is provided with a water-absorbing substance, which can effectively prevent the waste liquid in the waste liquid chamber 16 from flowing back into the reaction detection chamber 15. In addition, a vent 22 for discharging waste gas is also provided on the waste liquid chamber 16 to discharge waste gas in a timely manner and avoid obstructing the pumping of liquid.

[0079] This application provides an immunoassay method based on a microfluidic immunoassay chip, wherein the microfluidic immunoassay chip is as follows: Figures 1-2 As shown, it includes a sample chamber, a label chamber, an injection channel, a cleaning channel, a reaction detection chamber, and a waste liquid chamber. The sample chamber has a sample loading port for adding samples. The label chamber is pre-loaded with quantum dot microspheres as labels. The sample chamber, label chamber, and one end of the injection channel are connected in series, and the sample chamber is connected to a first pump port for pumping in diluent. One end of the cleaning channel has a third pump port for pumping in cleaning solution. The reaction detection chamber is connected to the other end of the injection channel and the other end of the cleaning channel. A microarray of trapping substances is set in the reaction detection chamber. The waste liquid chamber is connected to the reaction detection chamber to contain waste liquid.

[0080] The above methods are as follows Figure 6 As shown, steps 201 to 205 are included:

[0081] Step 201: After adding the sample through the sample well, pump the diluent through the first pump port and pump the sample into the labeling chamber to mix with the quantum dot microsphere label;

[0082] Step 202: After the sample is mixed with the quantum dot microsphere label, the mixture in the label chamber is pumped to the sample inlet channel through the first pump port, and the mixture is bound in the sample inlet channel;

[0083] Step 203: After the sample is bound to the quantum dot microsphere label, the bound material in the sample inlet channel is pumped to the reaction detection chamber through the first pump port for incubation.

[0084] Step 204: After incubation is complete, cleaning solution is pumped into the third pump port to pump the unreacted material to the waste liquid chamber;

[0085] Step 205: Detect and analyze the reaction detection chamber.

[0086] The immunoassay method provided in this application, because it pre-loads quantum dot microspheres with labels in the labeling chamber and sets up a capture microarray in the reaction detection chamber, allows the sample to be mixed with the quantum dot microspheres and incubated on the capture microarray. Then, the capture microarray is fluorescently detected using a matching detection instrument. This combines quantum dot fluorescent microsphere technology and protein microarray technology to achieve quantitative or semi-quantitative analysis of molecules related to immune diseases. The detection time and process are greatly shortened, thus solving the problem that existing immunoassay devices cannot effectively balance detection efficiency and detection accuracy.

[0087] Specifically, when the target item is allergen-specific IgE, i.e., for the detection of allergen-specific immunoglobulins, the aforementioned quantum dot microsphere label is a conjugate of specific IgE secondary antibody and quantum dot microspheres, and the aforementioned capture microarray is an allergen-specific IgE antigen microarray. The immunoassay process for the sample is as follows:

[0088] Add 1uL to 100uL of sample into the sample chamber through the sample loading port. After the sample is added, close the cap and pressurize the first pump port to push the sample downward into the label chamber pre-loaded with quantum dot microspheres-specific IgE secondary antibody. At this time, you can choose to pump in 0 to 5 times the volume of sample diluent from the first pump port to mix with the sample and then enter the label chamber.

[0089] The sample, or diluted sample, passes through the labeling chamber. The lyophilized quantum dot microsphere-antibody powder is rapidly reconstituted within 10 seconds. Then, under the control of the pump, it is mixed through the subsequent serpentine injection channel. After mixing, the pressurization is stopped, and the mixture is allowed to react fully for 1–60 minutes, optionally 10–20 minutes. After the reaction, a conjugate is formed between the quantum dot microsphere-specific IgE secondary antibody and the specific IgE antibody in the sample. The conjugate passes through the anti-backflow zone in the middle and reaches the microarray area in the reaction detection chamber. The conjugate remains in the microarray area for 5–60 minutes, optionally 10–30 minutes. Then, the unbound material after the reaction is pumped into the waste liquid area. Then, the pressurization of the first pump port is stopped, and the pressurization of the second pump port is started. The excess washing solution is pushed into the microarray detection area and kept flowing to wash away the excess unbound quantum dot microsphere-specific IgE secondary antibody. The washing solution also enters the waste liquid area. Then, the reaction detection chamber is slightly dried by blowing or air drying with gas before excitation light detection can be started.

[0090] During the test, if there is no fluorescence at each spot, it is negative. The fluorescence intensity from high to low represents the positive from strong to weak, which indicates different degrees of allergy severity. This can achieve semi-quantitative detection of immunity.

[0091] In addition, if the microarray has quality control points, a standard curve can be made using traceable international standard materials. The obtained fluorescence intensity can be used to calculate the concentration value of the detected item, which can then be substituted into the standard curve to achieve quantitative detection.

[0092] It is understandable that if multiple allergen tests are performed, i.e., the quantum dot microspheres are labeled with multiple secondary antibodies and the microarray is coated with multiple corresponding antigens, if multiple groups of spots are detected simultaneously when excitation light is passed through, it indicates an allergy to multiple allergens. In other words, multiple allergens in a single sample can be detected at once, which is fast, convenient and efficient, and can also achieve semi-quantitative or quantitative detection.

[0093] For example, add 30 μL of serum to the sample well, then cover the sample well. Pump 120 μL of sample diluent into the first pump port at a pressure of 1 mbar. The mixture of serum and diluent is then slowly introduced into the labeling chamber. The liquid rapidly reconstitutes the lyophilized quantum dot microspheres and then slowly passes through the serpentine injection channel for 15 minutes. Within 15 minutes, the specific IgE in the serum binds to the quantum dot microspheres and then enters the reaction detection chamber under the control of the pump. The pump stops after covering the microarray to allow it to fully bind with the antigens on the microarray for 15 minutes. The entire process can be incubated at 37°C. After binding, add washing solution from the second pump port to wash away the unbound reactants. After simple drying, the sample is detected and read to achieve quantitative detection.

[0094] The testing process includes setting blank control points, negative control points, and positive control points.

[0095] The detection effects of strong positive, weak positive, negative and blank control are respectively as follows: Figures 7-10 As shown, each row contains one allergen testing item.

[0096] This application provides an immunoassay method based on a microfluidic immunoassay chip, wherein the microfluidic immunoassay chip is as follows: Figures 3-5 As shown, it includes a sample chamber, a label chamber, an injection channel, a cleaning channel, a reaction detection chamber, and a waste liquid chamber. The sample chamber has a sample loading port for adding samples. The label chamber is pre-loaded with quantum dot microspheres as labels. The sample chamber and the label chamber are connected in parallel to one end of the injection channel, and the sample chamber is connected to a first pump port, while the label chamber is connected to a second pump port. One end of the cleaning channel has a third pump port for pumping in cleaning solution. The reaction detection chamber is connected to the other end of the injection channel and the other end of the cleaning channel. A microarray of trapping substances is installed in the reaction detection chamber. The waste liquid chamber is connected to the reaction detection chamber to contain waste liquid.

[0097] The above methods are as follows Figure 11 As shown, steps 301 to 305 are included:

[0098] Step 301: After adding the sample through the sample well, the diluent is pumped in through the first pump port and the sample is pumped into the reaction detection chamber for incubation.

[0099] Step 302: After incubation is completed, cleaning solution is pumped in through the third pump port, and unreacted material is pumped to the waste liquid chamber;

[0100] Step 303: After the reaction detection chamber is cleaned, the mixture in the labeling chamber is pumped to the reaction detection chamber through the first pump port for labeling reaction;

[0101] Step 304: After the marking reaction is completed, a cleaning solution is pumped into the third pump port to pump the unreacted material to the waste liquid chamber.

[0102] Step 305: Detect and analyze the reaction detection chamber.

[0103] The immunoassay method provided in this application combines quantum dot microsphere labeling in the labeling chamber and a capture microarray in the reaction detection chamber. After incubating and cleaning the sample in the capture microarray, it is labeled with quantum dot microspheres. Then, a matching detection instrument is used to perform fluorescence detection on the capture microarray. This combines quantum dot fluorescent microsphere technology with protein microarray technology to achieve quantitative, semi-quantitative, or qualitative analysis of molecules related to immune diseases. The detection time and process are greatly shortened, thus solving the problem that existing immunoassay devices cannot effectively balance detection efficiency and accuracy.

[0104] It is understood that the above-mentioned immunodetection method provided in the embodiments of this application uses the above-mentioned microfluidic immunodetection chip, which can not only be used to assist in the early screening and diagnosis of allergic diseases and autoimmune diseases, but also to detect samples from various sources such as the environment and food. Moreover, the types of molecules that can be detected are not limited to disease-related molecules. In fact, any antigen and antibody can be detected by this method, that is, its application has universality.

[0105] It should be noted that the various embodiments in this specification are described in a progressive manner, with each embodiment focusing on the differences from other embodiments. The same or similar parts between the various embodiments can be referred to each other.

[0106] Although optional embodiments of the present application have been described, those skilled in the art, upon learning the basic inventive concept, can make other changes and modifications to these embodiments. Therefore, the appended claims are intended to be interpreted as including the optional embodiments as well as all changes and modifications falling within the scope of the embodiments of the present application.

[0107] Finally, it should be noted that in this document, relational terms such as "first" and "second" are used merely to distinguish one entity from another, and do not necessarily require or imply any such actual relationship or order between these entities. Furthermore, the terms "comprising," "including," or any other variations thereof are intended to cover non-exclusive inclusion, such that an article or terminal device that comprises a list of elements includes not only those elements but also other elements not expressly listed, or elements inherent to such an article or terminal device. Without further limitations, an element defined by the phrase "comprising one..." does not exclude the presence of other identical elements in the article or terminal device that includes that element.

[0108] The technical solutions provided in this application have been described in detail above. Specific examples have been used to illustrate the principles and implementation methods of this application. At the same time, for those skilled in the art, there will be changes in the specific implementation methods and application scope based on the principles and implementation methods of this application. Therefore, the content of this specification should not be construed as a limitation of this application.

Claims

1. A microfluidic immunoassay chip, characterized in that, It includes a sample chamber, a marker chamber, a sample injection channel, a cleaning channel, a reaction detection chamber, and a waste liquid chamber; The sample dispensing chamber is provided with a sample dispensing port for sample dispensing. The labeling chamber is pre-loaded with quantum dot microspheres as labels. Both the sample dispensing chamber and the labeling chamber are connected to a pump port assembly for pumping in diluent, and both the sample dispensing chamber and the labeling chamber are connected to one end of the sample inlet channel. The quantum dot microspheres as labels are a combination of one or more target items as labels and quantum dot microspheres. The multiple target items include allergens, autoantibodies, and cytokines. One end of the cleaning channel is equipped with a third pump port for pumping in cleaning fluid; The reaction detection chamber is connected to the other end of the sample injection channel and the other end of the cleaning channel. A microarray of traps is provided in the reaction detection chamber. The microarray of traps is a microarray of traps of the multiple target items. The waste liquid chamber is connected to the reaction detection chamber to contain waste liquid; Both the sample inlet channel and the cleaning channel are equipped with anti-backflow structures at their ends; the anti-backflow structure is a bottle-shaped structure with its opening facing the reaction detection chamber, and the depth of the bottle body is greater than the depth of the sample inlet channel, and the width of the bottle body is greater than the width of the sample inlet channel.

2. The microfluidic immunoassay chip according to claim 1, characterized in that, The pump port assembly includes a first pump port, and one end of the sample dispensing chamber, the marker chamber, and the sample injection channel are connected in series, with the sample dispensing chamber body connected to the first pump port.

3. The microfluidic immunoassay chip according to claim 1, characterized in that, The pump port assembly includes a first pump port and a second pump port. The sample dispensing chamber and the marker chamber are connected in parallel to one end of the sample injection channel. The sample dispensing chamber is connected to the first pump port, and the marker chamber is connected to the second pump port.

4. The microfluidic immunoassay chip according to claim 1, characterized in that, The quantum dot microsphere marker is a combination of freeze-dried quantum dot microspheres and the marker.

5. The microfluidic immunoassay chip according to claim 1, characterized in that, The waste liquid chamber is equipped with a water-absorbing substance.

6. The microfluidic immunoassay chip according to claim 2, characterized in that, The sample inlet channel is serpentine.

7. An immunoassay method based on the microfluidic immunoassay chip of claim 2, characterized in that, include: After the sample is added through the sample well, the diluent is pumped in through the first pump port and the sample is pumped into the labeling chamber to mix with the quantum dot microsphere label; After the sample is mixed with the quantum dot microsphere label, the mixture in the label chamber is pumped to the sample inlet channel through the first pump port, and the mixture is bound in the sample inlet channel; After the sample is bound to the quantum dot microsphere label, the bound material in the sample inlet is pumped into the reaction detection chamber for incubation through the first pump port. After incubation is complete, cleaning fluid is pumped into the third pump port to pump the unreacted material to the waste liquid chamber. The reaction detection chamber is analyzed.

8. An immunoassay method based on the microfluidic immunoassay chip of claim 3, characterized in that, include: After the sample is added through the sample well, diluent is pumped into the first pump port and the sample is then pumped into the reaction detection chamber for incubation. After incubation is complete, cleaning fluid is pumped in through the third pump port, and unreacted material is pumped to the waste liquid chamber. After the reaction detection chamber is cleaned, the mixture in the labeling chamber is pumped to the reaction detection chamber through the first pump port for labeling reaction; After the labeling reaction is completed, cleaning fluid is pumped into the third pump port to pump the unreacted material to the waste liquid chamber. The reaction detection chamber is analyzed.

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