Digital immuno-chip and method for preparing the same

Abstract

The application provides a kind of digital immune chip and its preparation method, belong to immune detection chip technical field, it can solve the problem of reagent consumption in the existing immune substance detection method, low flux.The digital immune chip of the application, including the first substrate and the second substrate of relative bonding arrangement;The first substrate includes: first base, driving electrode arranged on the first base;First hydrophobic layer is arranged on the side of the layer where the driving electrode is away from the first base;And dielectric layer is arranged between the driving electrode and the first hydrophobic layer;The second substrate includes: second base, immune detection material is arranged on the second base;The immune detection material is arranged on the surface of the side of the second substrate close to the first substrate;The immune detection material includes antigen or antibody.

Description

Technical Field

[0001] This invention belongs to the field of immune detection chip technology, specifically relating to a digital immune chip and its preparation method. Background Technology

[0002] Immunoassay is an analytical method that uses the specific reaction between antigens and antibodies to determine whether a sample contains a potential corresponding antigen or antibody. This method is characterized by high selectivity, high sensitivity, and wide range of applications. After years of application and development, immunoassay has become a powerful tool in clinical diagnosis, biomedicine, and environmental chemistry research.

[0003] Based on the highly specific binding of antigens and antibodies, immunodiagnostics is particularly suitable for detecting trace concentrations of analytes in body fluid samples. Conventional immunoassay involves immobilizing antibodies or antigens on the surface of a well plate, adding the sample to be tested, and incubating for approximately two hours. After the reaction is complete, the immunocomplex is separated from the mixture, and the amount of the immunocomplex detected is used to determine the concentration of the analyte in the sample. However, this method has drawbacks such as complex operation, high reagent consumption, low throughput, and inconsistent results between different operators. Summary of the Invention

[0004] The present invention aims to solve at least one of the technical problems existing in the prior art, and to provide a digital immune chip with high throughput, low reagent consumption and rapid response.

[0005] The technical solution adopted to solve the technical problem of the present invention is a digital immune chip, comprising a first substrate and a second substrate that are bonded to each other.

[0006] The first substrate includes: a first substrate; a driving electrode disposed on the first substrate; a first hydrophobic layer disposed on the side of the layer where the driving electrode is located away from the first substrate; and a dielectric layer disposed between the driving electrode and the first hydrophobic layer.

[0007] The second substrate includes: a second base, and an immunodetector disposed on the second base; the immunodetector is disposed on a side surface of the second substrate near the first substrate; the immunodetector includes an antigen or an antibody.

[0008] Optionally, the second substrate further includes a sample dispensing stage, wherein the immunoassay is disposed on the side of the sample dispensing stage close to the first substrate;

[0009] The second hydrophobic layer is disposed in the same layer as the sampling stage, or disposed on the side of the sampling stage close to the first substrate.

[0010] Further, the second hydrophobic layer is located on the same plane as the surface of the first substrate close to the spotting platform.

[0011] Further, the second hydrophobic layer is located on the same plane as the surface of the first substrate close to the spotting platform.

[0012] Optionally, the spotting platform corresponds to the driving electrode one-to-one, and the orthographic projection of the spotting platform on the first substrate is smaller than the orthographic projection of the driving electrode on the first substrate.

[0013] Optionally, the digital immuno-chip comprises a plurality of spotting areas, and the driving electrode and the immuno-detection object are arranged in each of the spotting areas; the immuno-detection objects in at least part of the spotting areas are different.

[0014] Optionally, the material of the spotting platform comprises silicon dioxide.

[0015] Optionally, the material of the first hydrophobic layer comprises at least one of Teflon and silicon nitride.

[0016] The technical solution adopted to solve the technical problem of the present application is a preparation method of a digital immuno-chip, which is used to prepare any of the above digital immuno-chips, and the preparation method comprises:

[0017] forming a driving electrode, a dielectric layer and a hydrophobic layer on a first substrate in sequence to form a first substrate;

[0018] forming a plurality of spotting platforms on a second substrate through a patterning process;

[0019] forming an immuno-detection object on the spotting platform to form a second substrate;

[0020] bonding the first substrate and the second substrate.

[0021] Optionally, the preparation method further comprises the step of forming a second hydrophobic layer on the substrate; the step of forming the second hydrophobic layer and the spotting platform comprises:

[0022] forming a second hydrophobic film layer and a photoresist layer on the substrate;

[0023] removing part of the photoresist and the corresponding second hydrophobic film layer through an etching process to form a patterned photoresist layer and a second hydrophobic layer;

[0024] forming a spotting platform film layer on the second substrate; part of the spotting platform film layer falls into the hollow area of the second hydrophobic layer, and part of the spotting platform film layer is located on the side of the photoresist layer away from the second substrate;

[0025] The patterned photoresist layer and the spotting stage membrane layer on the side of the photoresist layer away from the second substrate are removed by a stripping process, thereby forming a plurality of spotting stages. BRIEF DESCRIPTION OF DRAWINGS

[0026] Figure 1 Structure diagram of a digital immunochip according to an embodiment of the present application;

[0027] Figure 2 Structure diagram of a first substrate of a digital immunochip according to an embodiment of the present application;

[0028] Figure 3 Structure diagram of a second substrate of a digital immunochip according to an embodiment of the present application;

[0029] Figure 4 Planar structure diagram of a first substrate of a digital immunochip according to an embodiment of the present application;

[0030] Figure 5 Planar structure diagram of a second substrate of a digital immunochip according to an embodiment of the present application;

[0031] Figure 6 Flow chart of preparation of a second substrate in a method for preparing a digital immunochip according to an embodiment of the present application;

[0032] In the drawings: 11, first substrate; 12, driving electrode; 13, first hydrophobic layer; 14, dielectric layer; 15, frame sealant; 21, second substrate; 22, immunodetection substance; 23, spotting stage; 24, second hydrophobic layer; 24A, second hydrophobic membrane layer; 25, photoresist. DETAILED DESCRIPTION

[0033] In order to enable a person skilled in the art to better understand the technical solutions of the present application, the present application will be described in further detail below in conjunction with the drawings and specific embodiments.

[0034] The present application will be described in further detail below with reference to the drawings. In each of the drawings, like elements are denoted by like reference numerals in order to make the present application clearer. Each part in the drawings is not drawn to scale for the sake of clarity. Furthermore, some parts that are well known can not be shown in the drawings.

[0035] Many specific details of the present application are described in the following in order to make the present application more clearly understood, such as the structure, material, size, processing process and technique of components. But as can be understood by a person skilled in the art, the present application can be implemented without these specific details.

[0036] In the embodiment of the present application, the patterning process, such as a photolithography patterning process, comprises: coating a photoresist on a structure layer to be patterned, the coating of the photoresist can be performed by spin coating, blade coating or roller coating; then exposing the photoresist to light using a mask, developing the exposed photoresist layer to obtain a photoresist pattern; then etching the structure layer using the photoresist pattern, and optionally removing the photoresist; and finally stripping the remaining photoresist to form the desired structure.

[0037] In the embodiment of the present application, according to different specific patterns, the sequential patterning process can comprise multiple exposure, development or etching processes, and the formed specific patterns in the same layer can be continuous or discontinuous, and the specific patterns can also be at different heights or have different thicknesses.

[0038] The shapes and sizes of the components in the drawings do not reflect true proportions, and the purpose is only to facilitate the understanding of the content of the embodiments of the present disclosure. Unless otherwise defined, the technical terms or scientific terms used in the present disclosure should be understood as the usual meaning understood by those skilled in the art to which the present disclosure belongs. The "first", "second" and similar words used in the present disclosure do not represent any order, number or importance, but are only used to distinguish different components. Similarly, "one", "an" or "the" and similar words do not represent a quantity limit, but represent the existence of at least one. "Include" or "contain" and similar words mean that the elements or objects before the word cover the elements or objects listed after the word and their equivalents, and do not exclude other elements or objects. "Connected" or "connected" and similar words are not limited to physical or mechanical connections, but can include electrical connections, whether direct or indirect. "Up", "down", "left", "right" and the like are only used to represent relative positional relationships, and when the absolute position of the described object changes, the relative positional relationship can also change accordingly.

[0039] The embodiments of the present disclosure are not limited to the embodiments shown in the drawings, but include modifications of the configurations formed based on the manufacturing process. Therefore, the regions illustrated in the drawings have a schematic property, and the shapes of the regions shown in the drawings illustrate the specific shapes of the regions of the elements, but are not intended to be limiting.

[0040] Embodiment 1:

[0041] As Figures 1 to 5As shown, the embodiment provides a digital immuno-chip which can be used for detecting reagents containing immunological substances. The digital immuno-chip provided by the embodiment comprises a first substrate and a second substrate which are oppositely bonded; the first substrate comprises a first base 11, a driving electrode 12 arranged on the first base 11, a first hydrophobic layer 13 arranged on a side of a layer where the driving electrode 12 is away from the first base 11, and a dielectric layer 14 arranged between the driving electrode 12 and the first hydrophobic layer 13; the second substrate comprises a second base 21 and an immunological detection substance 22 arranged on the second base 21; the immunological detection substance 22 is arranged on a surface of the second substrate which is close to the first substrate; and the immunological detection substance 22 comprises an antigen or an antibody.

[0042] In the digital immuno-chip provided by the embodiment, the immunological detection is combined with the digital microfluidic chip, and the immunological detection substance 22 is arranged on the second substrate of the digital immuno-chip. The digital immuno-chip can transport reagents containing immunological substances and cleaning liquid based on the dielectric wetting technology. The movement of the reagent droplet is driven by controlling the electric signal of the driving electrode 12, so that the reagent droplet can move in the digital immuno-chip. The immunological substance in the reagent can automatically combine with the immunological detection substance 22 on the second substrate, and after being cleaned by the cleaning liquid, the reagent containing the fluorescent direct-labeled immunological detection substance 22 is transported again, the fluorescent direct-labeled immunological detection substance 22 combines with the combined immunological substance again, and then the digital immuno-chip is washed again and placed under a fluorescence microscope for fluorescence detection, so as to realize the detection of the immunological complex and the detection of the immunological substance (such as an antigen).

[0043] In the prior art, the conventional immunological detection analysis method is to fix the immunological detection substance 22 (antibody or antigen) on the surface of a well plate, then add the sample to be detected for incubation for about two hours, separate the immunological complex from the mixture after the reaction is completed, and determine the content of the measured substance in the sample by determining the amount of the detection immunological complex. However, this method has the disadvantages of complex operation, large amount of reagent consumption, low throughput, different results analyzed by different operators, etc. The digital immuno-chip provided by the embodiment can modify the antibody to the glass substrate, accurately control the sample injection of the sample to be detected, and accurately react, so that the detection of the immunological substance in the reagent can be realized by using only a small amount of reagent droplet, and the advantages of high throughput, small amount of reagent consumption, rapid reaction, and less human influence factors are achieved.

[0044] It is understood that the digital immunoassay chip provided in this embodiment is based on the binding characteristics of antigens and antibodies when used for immunoassay. In this embodiment, the immunoassay 22 disposed on the second substrate varies depending on the immunological substance to be detected. For example, when detecting an antigen in a reagent, the immunoassay 22 on the second substrate 21 should be an antibody, and the antigen and antibody should be corresponding immunological substances; when detecting an antigen in a reagent, the immunoassay 22 on the second substrate 21 should be an antibody; when detecting an antibody in a reagent, the immunoassay 22 on the second substrate 21 should be an antigen. The following description uses the example of using the immunoassay 22 on the second substrate 21 as an antibody to detect the corresponding antigen in the reagent.

[0045] The digital immunoassay chip provided in this embodiment can drive reagent transport and flow using a single-plate driving method. For example... Figures 1 to 5 As shown, in the digital immune chip provided in this embodiment, a plurality of driving electrodes 12 are disposed on the first substrate. Specifically, the first substrate includes a first base 11, and the plurality of driving electrodes 12 are bonded and disposed flat on the surface of the first base 11. Figure 2 (the upper surface of the middle).

[0046] The operation process of the digital immunoassay chip provided in this embodiment during immune detection is as follows:

[0047] (1) Add reagent droplets to the digital immunochip and transport them to the desired position via the driving electrode 12. The antigen in the reagent droplets fully recognizes and binds to the antibody coated on the second substrate in the spotting area.

[0048] (2) Add the cleaning solution to the digital immune chip and clean the digital immune chip by controlling the transport of the cleaning solution droplets.

[0049] (3) Add the reagent containing the fluorescent direct labeling antibody to the digital immunochip and transport it to the spotting area by the driving electrode 12 to bind with the already bound antigen and antibody, i.e., double antibody sandwich.

[0050] (4) Add the cleaning solution to the digital immune chip again and clean the digital immune chip by controlling the transport of the cleaning solution droplets.

[0051] (5) The digital immunochip is placed under a fluorescence microscope for fluorescence detection and quantitative analysis.

[0052] In the digital immuno-chip provided in the embodiment, only a small amount of reagent droplet is needed to detect the immune substance component and concentration therein. In the embodiment, sufficient immune detection substance is arranged in the digital immuno-chip, and through sufficient combination of the reagent droplet and the immune detection substance, the antigen corresponding to the antibody in the reagent droplet can be detected, so as to detect the immune substance component in the actual droplet. It can be understood that in the embodiment, when the number of antibodies is sufficient, only a small amount of reagent droplet is needed to detect the immune substance component therein.

[0053] In actual cases, the reagent often contains multiple immune substances (for example, multiple different types of antigens). Alternatively, in the embodiment, the digital immuno-chip includes multiple spotting areas, and the driving electrode 12 and the immune detection substance 22 are arranged in each spotting area. The immune detection substance 22 in at least part of the spotting areas is different. By making the immune detection substance 22 in at least part of the spotting areas different, multiple antigens in the reagent droplet can be detected at one time. Specifically, when the reagent droplet is detected, the driving electrode 12 can be controlled to drive the droplet to the first spotting area first, the antibody (assuming to be the first antibody) in the first spotting area is combined with the antigen (corresponding to the first antigen of the first antibody) in the reagent droplet, and after the combination is completed, the reagent droplet is driven to the second spotting area by controlling the driving electrode 12, the antibody (assuming to be the second antibody) in the second spotting area is combined with the antigen (corresponding to the second antigen of the second antibody) in the reagent droplet, and so on, so that the reagent droplet can be combined with different antibodies in different spotting areas for reaction. Then, the digital immuno-chip is cleaned by using a cleaning liquid, and then the droplet with the fluorescently labeled antibody reagent is transported to the spotting area to recognize and combine with the corresponding antigen adsorbed in each spotting area, so that the detection of different antigens can be realized at one time.

[0054] The droplet with the fluorescently labeled antibody reagent can be a droplet mixed with multiple different antibodies with fluorescent labels, or the droplets with different fluorescently labeled antibody reagents can be transported to the spotting area for reaction. The embodiment does not limit this.

[0055] In the embodiment, Figure 2As shown, the material of the first substrate 11 may include glass. The material of the driving electrode 12 may include a conductive metal. The size of the driving electrode 12 can be set according to the size of the droplet to be controlled and the size of the sample stage 23. A first hydrophobic layer 13 is disposed above the layer containing the driving electrode 12 (i.e., on the side opposite to the first substrate 11) to give the reagent droplet a larger initial contact angle, thereby reducing sample adhesion on the electrode, reducing the resistance to droplet movement, and also helping to reduce the possibility of cross-contamination between samples. The material of the first hydrophobic layer 13 may include Teflon, silicon nitride, etc. A dielectric layer 14 is also disposed between the layer containing the driving electrode 12 and the first hydrophobic layer 13 to prevent the driving electrode 12 from breaking down. Specifically, the material of the dielectric layer 14 may include alumina, tantalum pentoxide (Ta2O5), etc.

[0056] Optional, such as Figure 3 and Figure 5 As shown, in each spotting area, the second substrate further includes a spotting stage 23, and an immunodetector 22 is disposed on the side of the spotting stage 23 near the first substrate. That is, the immunodetector 22 is placed on the second substrate by placing it on the spotting stage 23. It is understood that, similar to the first substrate 11, the second substrate 21 is typically made of materials such as glass. In this embodiment, by providing the spotting stage 23, antibodies are modified onto it, thereby achieving antibody formation on the second substrate 21.

[0057] The material of the sampling stage 23 may include silicon dioxide.

[0058] Optionally, the second substrate further includes a second hydrophobic layer 24, which is disposed in the same layer as the sample dispensing stage 23, or disposed on the side of the sample dispensing stage 23 near the first substrate. In this embodiment, the second hydrophobic layer 24 is used to give the reagent droplets a larger initial contact angle, thereby reducing sample adhesion and residue on the second substrate, reducing the resistance to droplet movement, and also helping to reduce the possibility of cross-contamination between samples. Specifically, the material of the second hydrophobic layer 24 may include Teflon, silicon nitride, etc.

[0059] Optionally, the surface of the second hydrophobic layer 24 near the first substrate and the surface of the sample stage 23 near the first substrate are located on the same plane. That is, the surface of the second substrate near the first substrate (i.e., the lower surface of the second substrate) is planar, thereby ensuring that the lower surface of the second substrate remains smooth and hydrophobic as much as possible, thereby reducing the adhesion and residue of the sample on the second substrate and reducing the resistance to droplet movement.

[0060] Optional, such as Figure 3As shown, the second hydrophobic layer 24 is arranged in the same layer as the sample application platform 23, and has the same thickness. The same layer refers to the same layer in visual perception, that is, the second hydrophobic layer 24 and the sample application platform 23 have the same distance from the second substrate 21. Specifically, in this embodiment, the second hydrophobic layer 24 can be a patterned film layer structure, and the sample application platform 23 can be located in the hollowed-out area of the second hydrophobic layer 24. On this basis, by making the thicknesses of the second hydrophobic layer 24 and the sample application platform 23 the same, the surfaces of the second hydrophobic layer 24 and the sample application platform 23 close to the first substrate can be made into a plane.

[0061] It can be understood that, in this embodiment, the sample application platform 23 can also not be arranged in the same layer as the second hydrophobic layer 24, but can be arranged on the side of the second hydrophobic layer 24 close to the first substrate. Specifically, the sample application platform 23 can be directly arranged on the side of the second hydrophobic layer 24 close to the first substrate, or a groove can be formed on the side of the second hydrophobic layer 24 close to the first substrate, and the sample application platform 23 can be arranged in the groove, so that the surface of the second hydrophobic layer 24 close to the first substrate and the surface of the sample application platform 23 close to the first substrate are located in the same plane.

[0062] Optionally, the sample application platform 23 corresponds to the driving electrode 12 one by one, and the orthographic projection of the sample application platform 23 on the first substrate 11 is smaller than the orthographic projection of the driving electrode 12 on the first substrate 11. In this embodiment, the reagent droplet reaches the corresponding sample application area through the driving of the driving electrode 12 and combines with the antibody on the sample application platform 23 to perform a combination reaction. By making the area of the driving electrode 12 larger than the area of the sample application platform 23, the contact surface of the reagent droplet and the sample application platform 23 can be ensured as much as possible, so that the antigen and the antibody can be combined sufficiently, and the detection precision is improved.

[0063] It can be understood that the digital immuno-chip in this embodiment is also provided with a reagent inlet, a reagent outlet, and a driving circuit and the like, and the specific arrangement mode can refer to related materials of the digital microfluidic chip, which will not be described in detail here.

[0064] Embodiment 2:

[0065] The embodiment provides a preparation method of a digital immuno-chip, which can be used for preparing any one of the digital immuno-chips provided in Embodiment 1. The preparation method comprises the following steps:

[0066] S1, forming the driving electrode 12 and the first hydrophobic layer 13 on the first substrate 11 in sequence to form the first substrate.

[0067] Specifically, this step comprises: forming a conductive layer on the first substrate 11; removing part of the conductive layer material through an etching process to form a plurality of driving electrodes 12; forming a dielectric layer 14 above the driving electrode 12 through a film forming process; and forming a first hydrophobic layer 13 above the dielectric layer 14 through a spin coating, drying and the like.

[0068] S2. Multiple sample stages 23 are formed on the second substrate 21 through a patterning process.

[0069] This step may specifically include: forming a spotting stage 23 film layer on the second substrate 21; removing part of the spotting stage 23 film layer material by etching process, thereby forming one or more desired spotting stages 23.

[0070] Preferably, this embodiment further includes the step of forming a second hydrophobic layer 24 on the substrate. At this time, as... Figure 6 As shown, the steps for forming the second hydrophobic layer 24 and the dotting stage 23 include: forming a second hydrophobic film layer 24A and a photoresist layer 25 on a substrate; removing a portion of the photoresist 25 and the corresponding second hydrophobic film layer 24A by an etching process to form a patterned photoresist layer 25 and a second hydrophobic layer 24; forming a dotting stage 23 film layer on the second substrate 21; a portion of the dotting stage 23 film layer falling into the cutout area of ​​the second hydrophobic layer 24, and a portion of the dotting stage 23 film layer located on the side of the photoresist layer 25 away from the second substrate 21; removing the patterned photoresist layer 25 and the dotting stage 23 film layer located on the side of the photoresist layer 25 away from the second substrate 21 by a stripping process, thereby forming multiple dotting stages 23.

[0071] S3. Modify the sample stage 23 to form an immunoassay 22 to form a second substrate.

[0072] This step may specifically include: activating the surface of the second substrate 21 with hydroxyl groups, then modifying the amino groups on the basis of the hydroxyl groups, then modifying the carboxyl groups on the basis of the modified amino groups, and finally linking the desired antibody to the carboxyl groups and modifying it onto the sample stage 23.

[0073] S4. Bond the first substrate to the second substrate.

[0074] In this step, the first substrate and the second substrate are bonded together using materials such as sealing adhesive 15. The side of the first substrate with the driving electrode 12 faces the second substrate, and the side of the second substrate with the immunoassay agent 22 faces the first substrate.

[0075] It is understood that the above embodiments are merely exemplary implementations used to illustrate the principles of the present invention, and the present invention is not limited thereto. For those skilled in the art, various modifications and improvements can be made without departing from the spirit and essence of the present invention, and these modifications and improvements are also considered to be within the scope of protection of the present invention.

Claims

1. A digital immuno-chip, comprising a first substrate and a second substrate arranged in opposition; characterized in that, the first substrate comprises a first base, a driving electrode arranged on the first base, a first hydrophobic layer arranged on a side of the layer where the driving electrode is away from the first base, and a dielectric layer arranged between the driving electrode and the first hydrophobic layer; the second substrate comprises a second base, a second hydrophobic layer and a spotting stage arranged on the second base, and an immuno-detection object arranged on a side of the spotting stage close to the first substrate; wherein the surface of the second hydrophobic layer close to the first substrate and the surface of the spotting stage close to the first substrate are in the same plane; the material of the spotting stage comprises silicon dioxide; the spotting stage corresponds to the driving electrode one by one, and the orthographic projection of the spotting stage on the first base is smaller than the orthographic projection of the driving electrode on the first base; the immuno-detection object corresponds to the driving electrode, and the immuno-detection object comprises an antigen or an antibody.

2. The digital immuno-chip of claim 1, wherein, The second hydrophobic layer is arranged in the same layer as the spotting stage, and has the same thickness.

3. The digital immuno-chip of claim 1, wherein, The digital immuno-chip comprises a plurality of spotting areas, and the driving electrode and the immuno-detection object are arranged in each of the spotting areas; the immuno-detection objects in at least part of the spotting areas are different.

4. The digital immuno-chip according to any one of claims 1 to 3, wherein, The material of the first hydrophobic layer comprises at least one of Teflon and silicon nitride.

5. A method for producing a digital immunoassay chip, for producing the digital immunoassay chip according to any one of claims 1 to 4, characterized by, The preparation method comprises: forming the driving electrode, the dielectric layer and the hydrophobic layer on the first base in sequence to form the first substrate; forming a plurality of spotting stages on the second base by a patterning process; forming the immuno-detection object on the spotting stage to form the second substrate; bonding the first substrate and the second substrate.

6. The production method according to claim 5, characterized by, Further comprising: a step of forming a second hydrophobic layer on the base; the step of forming the second hydrophobic layer and the spotting stage comprises: forming a second hydrophobic film layer and a photoresist layer on the base; removing part of the photoresist and the corresponding second hydrophobic film layer by an etching process to form a patterned photoresist layer and a second hydrophobic layer; forming a spotting stage film layer on the second base; part of the spotting stage film layer falls into the hollow area of the second hydrophobic layer, and part of the spotting stage film layer is located on a side of the photoresist layer away from the second base; removing the patterned photoresist layer and the spotting stage film layer on a side of the photoresist layer away from the second base by a stripping process, thereby forming a plurality of spotting stages.

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