High-sensitivity immunodetection chip and method thereof

Abstract

The invention relates to a high-sensitivity immunodetection chip and a method thereof, the chip comprises a sample adding area, the sample adding area is used for introducing a sample to be tested into the chip, and then the sample enters a reagent pre-embedding area; the reagent pre-embedding area is provided with a plurality of detection antibodies with markers; the detection area comprises a plurality of micro-trap structures arranged in an array, each micro-trap structure comprises a plurality of microsphere clamping grooves and flow channels arranged on the peripheries of the microsphere clamping grooves, each microsphere clamping groove captures and fixes a microsphere, each microsphere is coated with a capture antibody, and the capture antibody is coated with an antibody. A to-be-tested sample and a detection antibody pass through the flow channel and then react with the capture antibody on the microspheres to be fixed, the marker forms an array signal in the detection area, and the residual reagent flows into the waste liquid collection area; the waste liquid collecting area is used for collecting liquid flowing out of the detection area. The microsphere array and the detection antibody are pre-fixed, so that the detection process is convenient and rapid, and the detection sensitivity is high.

Description

Technical Field

[0001] The present application relates to the technical field of immunoassay, and in particular to a highly sensitive immunoassay chip and method thereof. Background Art

[0002] Immunoassay utilizes the specific binding properties of antigens and antibodies. Antigens can be used to perform qualitative or quantitative tests on antibodies, and antibodies can also be used to perform qualitative or quantitative tests on antigens.

[0003] In the existing literature, patents, and products, in conventional POCT microfluidic products used for immunoassays, microchannels are usually used for flow control to achieve the detection process; and the detection area is usually coated with antibody or other methods to directly coat the capture antibody in the detection area. This may lead to problems such as detection signal accumulation, insufficient sensitivity, and high background interference for the complete reaction process.

[0004] Therefore, the prior art still needs to be improved and developed. Summary of the invention

[0005] Based on this, the present application provides a highly sensitive immunoassay chip and method thereof, which is pre-fixed with a microsphere array and a detection antibody, making the detection process convenient and fast with high detection sensitivity.

[0006] In order to achieve the above-mentioned purpose, the present application embodiment provides a highly sensitive immunoassay chip, including:

[0007] A sample adding area, which is used to pass the sample to be tested into the chip and then into the reagent pre-embedded area;

[0008] A reagent pre-embedded area, wherein a plurality of detection antibodies with markers are arranged in the reagent pre-embedded area, and the test sample is mixed with the detection antibody and then enters the detection area;

[0009] The detection area includes a plurality of micro-well structures arranged in an array, and the micro-well structure includes a plurality of micro-ball slots and flow channels arranged on each periphery of the micro-ball slots. Each of the micro-ball slots captures and fixes a micro-ball, and each of the micro-balls is coated with a capture antibody. After the sample to be tested and the detection antibody pass through the flow channel, they react with the capture antibody on the micro-ball and are fixed. The marker forms an array signal in the detection area, and the remaining reagent flows into the waste liquid collection area through the flow channel;

[0010] The waste liquid collection area is used to collect the liquid flowing out of the detection area.

[0011] Preferably, the method of capturing and fixing the microspheres on the microsphere slots is:

[0012] Microspheres of different diameters, shapes and functions are suspended and dispersed through diluent;

[0013] The dispersed microsphere mixture is spotted in the microsphere slots of the microwell structure by an automatic spotter, and only one microsphere falls into each microsphere slot.

[0014] Preferably, the height of the microball slot is 1-1.5 times the diameter of the microball, the perimeter length of the microball slot is 1.2-1.5 times the diameter D of the microball, and the height of the flow channel is 1 / 2 of the diameter of the microball, thereby ensuring that the liquid passes through the flow channel while the microball is fixed in the microball slot.

[0015] Preferably, the capture antibody is coated on the microspheres via carboxyl or amino groups or streptavidin.

[0016] Preferably, the detection antibody reagent is extracted into the reagent pre-embedded area by an automatic spotting machine, and the detection antibody reagent is dried after being evenly dispersed.

[0017] Preferably, the chip comprises a cover plate and a bottom plate, the reagent pre-embedded area, the detection area and the waste liquid collection area are arranged on the bottom plate, the sample addition area is arranged on the cover plate, and a sample addition hole is arranged on the cover plate.

[0018] Preferably, the cover plate and the base plate are fixed together by means of hot pressing bonding, ultrasonic bonding, laser bonding, plasma bonding or gluing.

[0019] Preferably, the marker is a fluorescent marker.

[0020] The present application also provides a method for high-sensitivity immunoassay, which is implemented by the above-mentioned high-sensitivity immunoassay chip, and is characterized in that it includes the following steps:

[0021] Add the sample to be tested into the chip through the sample adding area;

[0022] The sample to be tested passes through the reagent pre-embedded area, and after being mixed with the detection antibody, it enters the detection area;

[0023] After the sample to be tested and the detection antibody pass through the flow channel, they react with the capture antibody on the microsphere. The remaining reagent flows into the waste liquid collection area through the flow channel. The detection begins after five to thirty minutes of reaction.

[0024] Under dark field conditions, the detection area is scanned and positioned, and then a dark field photo is taken. The concentration of the test sample is calculated based on the number of fluorescent points displayed in the dark field photo, or the concentration of the test sample is calculated by calculating the fluorescence intensity and comparing it with the standard.

[0025] Preferably, the sample to be tested flows into the detection area by self-driving of the liquid, or the sample to be tested is added into the chip by a sample addition pump.

[0026] The highly sensitive immunoassay chip provided by the present invention has the following advantages and beneficial effects:

[0027] The detection area is pre-fixed with an array of microspheres with capture antibodies, which can significantly improve the detection sensitivity.

[0028] The reagent pre-embedded area is pre-embedded with detection antibodies. During the test, you only need to add the test sample. The test process is convenient and fast, and you can achieve sample in and result out.

[0029] The chip is easy to manufacture and has low cost. BRIEF DESCRIPTION OF THE DRAWINGS

[0030] Figure 1 This is the overall functional area diagram of the chip for high-sensitivity immunoassay in this application.

[0031] Figure 2 This is a diagram of the micro-well structure of the chip for high-sensitivity immunoassay in this application.

[0032] Figure 3 This is a flow diagram of the chip for high-sensitivity immunoassay in this application.

[0033] Figure 4 This is a rendering of the pre-embedded microspheres of the chip for high-sensitivity immunoassay in this application.

[0034] Figure 5 Flow chart of the highly sensitive immunoassay for this application.

[0035] Figure 6 This is a linear correlation diagram after testing with different IL-6 antigen concentrations. DETAILED DESCRIPTION

[0036] In order to facilitate understanding of the present application, the present application will be described more fully below with reference to the relevant drawings. The preferred embodiments of the present application are given in the drawings. However, the present application can be implemented in many different forms and is not limited to the embodiments described herein. On the contrary, the purpose of providing these embodiments is to make the disclosure of the present application more thorough and comprehensive.

[0037] It should be noted that when an element is considered to be "connected" to another element, it may be directly connected to the other element and integrated therewith, or there may be an intermediate element at the same time. The terms "mounted" and similar expressions used herein are for illustrative purposes only.

[0038] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as those commonly understood by those skilled in the art to which this application belongs. The terms used herein in the specification of this application are only for the purpose of describing specific embodiments and are not intended to limit this application. The term "and / or" used herein includes any and all combinations of one or more of the related listed items.

[0039] In the first embodiment, Figures 1 to 3 As shown, a highly sensitive immunoassay chip is provided, which comprises:

[0040] The sample adding area 100 is used to pass the sample to be tested into the chip and then enter the reagent pre-embedded area 200;

[0041] The reagent pre-embedded area 200 is provided with a plurality of detection antibodies with markers, and the test sample is mixed with the detection antibodies and then enters the detection area 300;

[0042] The detection area 300 includes a plurality of micro-well structures 301 arranged in an array. The micro-well structures 301 include a plurality of micro-balloon slots 302 and flow channels 303 arranged on each periphery of the micro-balloon slots 302. Each of the micro-balloon slots 302 captures and fixes a micro-ball 304. Each of the micro-balloon 304 is coated with a capture antibody. After the sample to be tested and the detection antibody pass through the flow channel, they react with the capture antibody on the micro-ball 304 and are fixed. The marker forms an array signal in the detection area 300. The remaining reagent flows into the waste liquid collection area 400 through the flow channel 303.

[0043] The waste liquid collection area 400 is used to collect the liquid flowing out of the detection area 300 .

[0044] In order to improve sensitivity and reduce signal accumulation and background interference, we adopt the method of pre-embedded microspheres in the detection area to achieve high-density monodispersion of microspheres in the detection area, achieve large-area contact with the reaction reagents, and improve sensitivity.

[0045] In a specific implementation, the method of capturing and fixing the microballs on the microball slot 302 is:

[0046] Microspheres of different diameters, shapes and functions are suspended and dispersed through diluent;

[0047] The dispersed microsphere mixture is spotted in the microsphere slots of the microwell structure by an automatic spotter, and only one microsphere falls into each microsphere slot.

[0048] The specific implementation example is: use microspheres with an average diameter of 3um, dilute with corresponding diluent, and the final concentration is 1mg / ml; use an automatic spotter to extract 1.5ul and add it to the detection area to make the microspheres evenly dispersed and captured, wait for the microspheres to settle for 5 minutes, rinse the excess diluent with a cleaning solution, use an oven at 60°C to dry the cleaning solution and complete the pre-embedding.

[0049] In a specific embodiment, the height of the microball slot 302 is 1-1.5 times the diameter of the microball, the length of the microball slot is 1.2-1.5 times the diameter D of the microball, and the height of the flow channel is 1 / 2 of the diameter of the microball, thereby ensuring that the liquid passes through the flow channel while the microballs are fixed in the microball slot. Preferably, the height of the microball slot 302 can be 4um, the width of the microball slot 302 can be 4.5um, and the height of the flow channel can be 2um.

[0050] In a specific implementation, the capture antibody is coated on the microsphere via carboxyl or amino groups or streptavidin.

[0051] In the specific implementation, the detection antibody is extracted into the reagent pre-embedded area by an automatic spotting machine, and the detection antibody reagent is dried after being evenly dispersed. A specific embodiment is to use an automatic spotting machine to extract 1.5ul of detection antibody reagent and add it to the reagent pre-embedded area. The concentration of the detection antibody reagent is 0.01mg / ml-0.1mg / ml. After the detection antibody reagent is evenly dispersed, it is dried in an oven at 60°C. Figure 4 shown.

[0052] In a specific implementation, the chip includes a cover plate 10 and a base plate 20, the reagent pre-embedded area 200, the detection area 300 and the waste liquid collection area 400 are arranged on the base plate 20, the sample addition area 100 is arranged on the cover plate 10, and a sample addition hole is arranged on the cover plate 10.

[0053] In a specific implementation, the cover plate and the base plate are fixed together by means of hot pressing bonding, ultrasonic bonding, laser bonding, plasma bonding or gluing.

[0054] After the microspheres and reagents are embedded in the chip, the cover plate needs to be bonded to the base plate. Different materials can be bonded in different ways, generally by changing the surface properties of the material, hot pressing bonding, and double-sided tape bonding. When using chips with different bonding methods, the choice of microsphere specifications also needs to be changed accordingly.

[0055] In a specific implementation, the marker is a fluorescent marker or other markers.

[0056] The chip can be made of PDMS, PS, PMMA, glass, quartz or other materials by using methods such as photolithography, etching, nanoimprinting, injection molding, mold replication, mold re-production, vacuum hot pressing, etc.

[0057] In the present application, the diameter of the sample addition hole can be 0.5 to 2 mm, and the optimal diameter is 1 mm; the size of the detection area is 1 mm*1 mm, and there are 20,000 microsphere slots in the entire area.

[0058] The micro-well structure 301 of the chip is surface treated by using CVD chemical vapor deposition (such as APTMS) or physical adsorption (such as titanium dioxide), hydrophilic reagent spraying, coating and other methods, so that the contact angle of the guide groove area is 5 to 40 degrees, and the optimal value is 10 degrees; the modified guide groove has a strong capillary force, which can make the liquid entering the chip pass through the microsphere capture area as quickly and smoothly as possible into the waste liquid area and be collected.

[0059] The present application also provides a highly sensitive immunoassay method, such as Figure 5 As shown, the highly sensitive immune detection chip described above is implemented, characterized in that it includes the following steps:

[0060] S100, adding the sample to be tested into the chip through the sample adding area;

[0061] S200, the sample to be tested passes through the reagent pre-embedded area, and the sample to be tested is mixed with the detection antibody and then enters the detection area;

[0062] S300, the sample to be tested and the detection antibody pass through the flow channel and react with the capture antibody on the microsphere, and the remaining reagent flows into the waste liquid collection area through the flow channel, and the detection begins after the reaction for five to thirty minutes;

[0063] S400, under dark field conditions, scan and locate the detection area and take a dark field photo, calculate the concentration of the test sample according to the number of fluorescent points displayed in the dark field photo, or calculate the concentration of the test sample by calculating the fluorescence intensity and comparing it with the standard.

[0064] The samples to be tested include antigen diluent, serum, plasma, whole blood, lysate, etc.

[0065] In a specific implementation, the sample to be tested flows into the detection area through the self-driving of the liquid, or the sample to be tested is added into the chip through a sample adding pump.

[0066] The steps of self-propulsion through liquid are:

[0067] Slowly drip the sample into the sample injection port and wait for the liquid to flow into the reagent pre-embedded area by self-driving;

[0068] The sample passes through the reagent pre-embedded area, bringing the labeled fluorescent reagent into the detection area to react with the pre-embedded microspheres.

[0069] The remaining liquid carries the unbound reagents into the waste collection area, and detection begins after twenty minutes to half an hour of reaction.

[0070] Under dark field conditions, the detection area of ​​the chip is scanned and a dark field photo is taken after positioning.

[0071] The concentration of the sample can be calculated by the number of fluorescent points displayed by dark field photography; after testing high-concentration samples, the fluorescence intensity can be calculated to determine the concentration and compared with the standard.

[0072] The steps for adding samples using a sample pump are as follows:

[0073] The sample and the reaction reagent are fully mixed, and then sucked into the chip by the automatic sample pump;

[0074] The sample passes through the reagent pre-embedded area, bringing the labeled fluorescent reagent into the detection area to react with the pre-embedded microspheres.

[0075] After the reaction is completed, use a sample pump to add cleaning solution into the chip to flush out excess reagents, and start detection after flushing is completed.

[0076] Under dark field conditions, the microsphere capture area of ​​the microfluidic chip was scanned, and dark field photos were taken after positioning.

[0077] The concentration of the sample can be calculated by the number of fluorescent points displayed by dark field photography; after testing high-concentration samples, the fluorescence intensity can be calculated to determine the concentration and compared with the standard.

[0078] The IL-6 antigen concentration gradient test experiment was adopted, and the test data are shown in the table below.

[0079]

[0080] The test results are as follows Figure 6 As shown, according to Figure 6 It can be seen that the signal of 0pg / ml is less than 100, the sensitivity is as low as 1pg / ml, and it is linearly correlated in the range of 0-500pg / ml, with R2 reaching above 0.99.

[0081] In summary, the present application provides a highly sensitive immunoassay chip and method thereof, wherein the chip comprises: a sample loading area, wherein the sample loading area is used to pass the sample to be tested into the chip and then enter the reagent pre-embedded area; a reagent pre-embedded area, wherein the reagent pre-embedded area is provided with a plurality of detection antibodies with markers, and the sample to be tested is mixed with the detection antibodies and then enters the detection area; a detection area, wherein the detection area comprises a plurality of array-arranged micro-well structures, wherein the micro-well structures comprise a plurality of micro-balloon slots and flow channels arranged on each periphery of the micro-balloon slots, wherein each of the micro-balloon slots captures and fixes a micro-ball, and each of the micro-balloons is coated with There is capture antibody. After the test sample and the detection antibody pass through the flow channel, they react with the capture antibody on the microsphere and are fixed. The marker forms an array signal in the detection area, and the remaining reagent flows into the waste liquid collection area through the flow channel; the waste liquid collection area, the waste liquid collection area is used to collect the liquid flowing out of the detection area, so that the detection area is pre-fixed with an arrayed microsphere array with capture antibodies, which can significantly improve the detection sensitivity; the reagent pre-embedded area is pre-embedded with detection antibodies, and only the detection sample needs to be added during the detection. The detection process is convenient and fast, and the sample can be input and the result can be output. Finally, the chip is easy to make and the cost is low.

[0082] The technical features of the above-described embodiments may be arbitrarily combined. To make the description concise, not all possible combinations of the technical features in the above-described embodiments are described. However, as long as there is no contradiction in the combination of these technical features, they should be considered to be within the scope of this specification.

[0083] The above-described embodiments only express several implementation methods of the present application, and the descriptions thereof are relatively specific and detailed, but they cannot be construed as limiting the scope of the patent application. It should be pointed out that, for a person of ordinary skill in the art, several variations and improvements can be made without departing from the concept of the present application, and these all belong to the protection scope of the present application. Therefore, the protection scope of the patent application shall be subject to the attached claims.

Claims

1. A highly sensitive immunoassay chip, characterized in that: include: A sample adding area, which is used to pass the sample to be tested into the chip and then into the reagent pre-embedded area; A reagent pre-embedded area, wherein a plurality of detection antibodies with markers are arranged in the reagent pre-embedded area, and the test sample is mixed with the detection antibody and then enters the detection area; The detection area includes a plurality of micro-well structures arranged in an array, and the micro-well structure includes a plurality of micro-ball slots and flow channels arranged on each periphery of the micro-ball slots. Each of the micro-ball slots captures and fixes a micro-ball, and each of the micro-balls is coated with a capture antibody. After the sample to be tested and the detection antibody pass through the flow channel, they react with the capture antibody on the micro-ball and are fixed. The marker forms an array signal in the detection area, and the remaining reagent flows into the waste liquid collection area through the flow channel; The waste liquid collection area is used to collect the liquid flowing out of the detection area.

2. The highly sensitive immunoassay chip according to claim 1, characterized in that: The method for capturing and fixing the microspheres on the microsphere slot is: Microspheres of different diameters, shapes and functions are suspended and dispersed through diluent; The dispersed microsphere mixture is spotted in the microsphere slots of the microwell structure by an automatic spotter, and only one microsphere falls into each microsphere slot.

3. The highly sensitive immunoassay chip according to claim 2, characterized in that: The height of the microball slot is 1-1.5 times the diameter of the microball, the perimeter length of the microball slot is 1.2-1.5 times the diameter D of the microball, and the height of the flow channel is 1 / 2 of the diameter of the microball, thereby ensuring that the liquid passes through the flow channel while the microball is fixed in the microball slot.

4. The highly sensitive immunoassay chip according to claim 1, characterized in that: The capture antibody is coated on the microspheres via carboxyl or amino groups or streptavidin.

5. The highly sensitive immunoassay chip according to claim 1, characterized in that: The detection antibody reagent is extracted into the reagent pre-embedded area by an automatic spotting machine, and the detection antibody reagent is dried after being evenly dispersed.

6. The highly sensitive immunoassay chip according to claim 1, characterized in that: The chip comprises a cover plate and a bottom plate, the reagent pre-embedded area, the detection area and the waste liquid collection area are arranged on the bottom plate, the sample addition area is arranged on the cover plate, and a sample addition hole is arranged on the cover plate.

7. The highly sensitive immunoassay chip according to claim 1, characterized in that: The cover plate and the base plate are fixed together by means of hot pressing bonding, ultrasonic bonding, laser bonding, plasma bonding or gluing.

8. The highly sensitive immunoassay chip according to claim 1, characterized in that: The marker is a fluorescent marker.

9. A method for high-sensitivity immunoassay, implemented by the high-sensitivity immunoassay chip according to any one of claims 1 to 8, characterized in that: The steps include: Add the sample to be tested into the chip through the sample adding area; The sample to be tested passes through the reagent pre-embedded area, and after being mixed with the detection antibody, it enters the detection area; After the sample to be tested and the detection antibody pass through the flow channel, they react with the capture antibody on the microsphere. The remaining reagent flows into the waste liquid collection area through the flow channel. The detection begins after five to thirty minutes of reaction. Under dark field conditions, the detection area is scanned and positioned, and then a dark field photo is taken. The concentration of the test sample is calculated based on the number of fluorescent points displayed in the dark field photo, or the concentration of the test sample is calculated by calculating the fluorescence intensity and comparing it with the standard.

10. The highly sensitive immunoassay method according to claim 9, characterized in that: The sample to be tested flows into the detection area through the self-driving of the liquid, or the sample to be tested is added into the chip through a sample adding pump.

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