Micro-fluidic chip for coating biological substance through magnetic force and preparation method of micro-fluidic chip

Abstract

The invention provides a micro-fluidic chip for coating biological substances through magnetic force and a preparation method thereof.The micro-fluidic chip comprises a substrate and a cover plate, the substrate and the cover plate enclose to form a micro-channel, one end of the micro-channel is connected with a sample adding hole formed in the cover plate, the other end of the micro-channel is connected with a waste liquid area, and the waste liquid area has water absorbability; a marking area and a detection area are arranged in the micro-channel, the marking area and the detection area are sequentially distributed in the flowing direction of a sample, a detection point and a reference point are arranged in the detection area, magnetic blocks are arranged below the substrate corresponding to the detection point and the reference point, a marking antibody is arranged in the marking area, and the magnetic blocks are arranged in the detection area. The detection point is provided with a magnetic particle coupled capture antigen / antibody, and the reference point is provided with a magnetic particle coupled anti-species antibody aiming at the labeled region antibody. According to the micro-fluidic chip, the coating mode of biomolecules on the micro-fluidic chip is improved from a traditional chemical coating mode to a coating mode of adsorbing magnetic particles through magnetism, so that the manufacturing process of the chip is simplified.

Description

Technical Field

[0001] The invention belongs to the technical field of in vitro diagnosis and immune detection, and particularly relates to a microfluidic chip for magnetically coating biological substances and a preparation method thereof. Background Art

[0002] Microfluidic chips are the main platform for the realization of microfluidic technology. They can integrate basic operation units such as sample preparation, reaction, separation, and detection in biological, chemical, and medical analysis processes into a very small closed chip. Microfluidic chips have the advantages of being lightweight, using less sample and reagent, fast reaction speed, large-scale parallel processing, and disposable. They have great potential in the fields of biology, chemistry, and medicine. In recent years, they have developed into a new research field that intersects biology, chemistry, medicine, fluids, electronics, materials, and mechanics.

[0003] Existing microfluidic chips generally use chemical coating methods to fix the captured antigens / antibodies on the chip. The coating process requires multiple spotting, incubation, cleaning, and drying. The operation process is complicated and cannot be carried out in large-scale batch production. Summary of the invention

[0004] Aiming at the shortcomings of the existing microfluidic chip for capturing antigen / antibody fixation, the present invention proposes a microfluidic chip for magnetically coating biological substances, which can simplify the operation process of capturing antigen / antibody fixation on the chip and improve production efficiency.

[0005] To achieve the above-mentioned purpose, the technical solution of the present invention is implemented as follows: a microfluidic chip for magnetically coating biological substances comprises a substrate and a cover sheet, wherein the substrate and the cover sheet enclose a microchannel, wherein one end of the microchannel is connected to a sample loading hole provided on the cover sheet, and the other end is connected to a waste liquid area, wherein the waste liquid area is water-absorbent, and a marking area and a detection area are arranged in the microchannel, wherein the marking area and the detection area are sequentially distributed along the sample flow direction, wherein detection points and reference points are arranged in the detection area, and magnetic blocks are arranged under the substrate corresponding to the detection points and the reference points, wherein the marking area is provided with a marked antibody, wherein the detection point is provided with a capture antigen / antibody coupled to a magnetic particle, and wherein the reference point is provided with an anti-species antibody against the antibody in the marking area coupled to a magnetic particle.

[0006] In some embodiments of the present invention, a groove is provided on the lower surface of the cover sheet along its length direction, and the groove and the upper surface of the substrate are combined to form the microchannel.

[0007] In some embodiments of the present invention, the diameter of the magnetic microparticles is 0.1-2.8 μm.

[0008] In some embodiments of the present invention, there are multiple detection points, and magnetic blocks are disposed on the lower surface of the substrate corresponding to the multiple detection points.

[0009] In some embodiments of the present invention, the magnetic block is a neodymium iron boron magnet, a samarium cobalt magnet or other magnets or electromagnets that can reach a magnetic force of 0.2T.

[0010] In some embodiments of the present invention, the magnetic block is rectangular, there are multiple magnetic blocks, each magnetic block is fixed below the detection point and the reference point, the length direction of the magnetic block is the same as the width direction of the microchannel, and the length of the magnetic block is smaller than the width of the microchannel.

[0011] In some embodiments of the present invention, the magnetic block is cylindrical, and there are multiple magnetic blocks, which are distributed in an array of detection points and reference points corresponding to the lower surface of the substrate, and the distance between two adjacent magnetic blocks is not less than the diameter of the magnetic block.

[0012] In some embodiments of the present invention, the water absorption of the waste liquid area is that a water-absorbing material is arranged in the waste liquid area or the waste liquid area is a structure with water absorption function.

[0013] In some embodiments of the present invention, the magnetic block is a plurality of small magnets attached to the substrate coating position, and the magnetic fields between the small magnets do not interfere with each other.

[0014] On the other hand, the present invention also provides a method for preparing a microfluidic chip for magnetically coating biological substances, comprising at least the following steps:

[0015] 1) Coupling the substance to be coated with appropriate magnetic particles;

[0016] 2) suspending the coupled magnetic particles in an appropriate buffer system containing a dispersant and a protective agent;

[0017] 3) Use buffer to adjust the magnetic microparticle solution to a suitable concentration;

[0018] 4) dropping the magnetic particle liquid onto the designated position on the substrate detection area;

[0019] 5) Place the substrate in a dry and clean environment to dry naturally, or dry it at 37°, or by other methods that do not change the position of the liquid;

[0020] 6) The coated substrate is used for subsequent bonding with the cover sheet to form a coated microfluidic chip.

[0021] The microfluidic chip for magnetically coating biological substances and the preparation method thereof obtained by the above technical solution have the following beneficial effects:

[0022] The coating method of biomolecules on microfluidic chips has been improved from the traditional cumbersome chemical coating method to the coating method of magnetic microparticles through magnetic adsorption, which greatly simplifies the chip production process and is suitable for large-scale batch production. In addition, this technical invention adopts a centralized pre-coating method, using magnetic microparticles of a certain particle size as coating carriers, and coating specific antigens / antibodies in advance by chemical coupling. At present, this technology is relatively mature, and the prepared magnetic microspheres are easy to uniformly inspect to ensure the uniformity between each magnetic microparticle, thereby ensuring the uniformity of the coating between each chip, and effectively reducing the batch difference. BRIEF DESCRIPTION OF THE DRAWINGS

[0023] Figure 1 It is a schematic diagram of the structure of the microfluidic chip for magnetically coating biological substances according to the present invention;

[0024] Figure 2 It is a schematic diagram of the side cross-sectional structure of the microfluidic chip for magnetically coating biological substances according to the present invention;

[0025] Figure 3 is a bottom view structural schematic diagram of the substrate in one embodiment of the present invention;

[0026] Figure 4 It is a schematic diagram of the bottom view structure of the substrate in another embodiment of the present invention.

[0027] In the figure, 1, substrate; 2, cover plate; 3, microchannel; 4, sample loading hole; 5, waste liquid area; 6, water-absorbing material; 7, magnetic block; 31, marking area; 32, detection area; 321, detection point; 322, reference point. DETAILED DESCRIPTION

[0028] It should be noted that, in the absence of conflict, the embodiments of the present invention and the features in the embodiments may be combined with each other.

[0029] Unless otherwise defined, the technical terms used in the following examples have the same meanings as those generally understood by those skilled in the art to which the present invention belongs. The test reagents used in the following examples, unless otherwise specified, are all conventional biochemical reagents; the experimental methods, unless otherwise specified, are all conventional methods.

[0030] The present invention generally relates to the field of immunoassay technology, and relates to a microfluidic chip that magnetically coats biological substances. The core of this technical invention is based on microfluidic technology, and uses magnetic adsorption of magnetic particles to replace traditional chemical coating, making the production method of microfluidic chips simpler and convenient for mass production.

[0031] The present invention is further explained below in conjunction with embodiments and drawings. It should be understood that the present invention is not limited to the specific embodiments described.

[0032] like Figure 1-Figure 2 As shown, the present invention proposes a microfluidic chip for coating biological substances by magnetic force, including a substrate 1 and a cover plate 2, wherein the substrate 1 and the cover plate 2 are enclosed to form a microchannel 3, wherein one end of the microchannel 3 is connected to a sample addition hole 4 provided on the cover plate, and the other end is connected to a waste liquid area 5, wherein the waste liquid area 5 is hygroscopic and can absorb the liquid in the microchannel 3, wherein a marking area 31 and a detection area 32 are arranged in the microchannel 3, wherein the marking area 31 and the detection area 32 are sequentially distributed along the sample flow direction, wherein a detection point 321 and a reference point 322 are arranged in the detection area 32, wherein magnetic blocks 7 are arranged under the substrates corresponding to the detection points 321 and the reference points 322, wherein the marking area 31 is provided with a marked antibody, wherein the detection point 321 is provided with a capture antigen / antibody coupled to magnetic particles, and wherein the reference point 322 is provided with an anti-species antibody against the marking area antibody coupled to magnetic particles.

[0033] After the sample is added through the sample addition hole 4, the sample will flow along the microchannel due to the capillary force and the suction of the waste liquid area, and will flow through the labeling area and the detection area in sequence. The antigen / antibody to be tested in the sample will first react with the labeled antibody in the labeling area to form a complex, and then flow to the detection point 321, and be captured by the antigen / antibody coupled to the surface of the magnetic particles at the detection point 321. The remaining liquid will continue to flow to the reference point 322, and the labeled antibody will be captured by the corresponding substance on the surface of the magnetic particles at the reference point 322. The magnetic particles at the detection point 321 and the reference point 322 are kept stationary due to the magnetic adsorption of the magnet under the substrate 1, and the remaining liquid will flow to the waste liquid area 5.

[0034] Specifically, a groove is provided on the lower surface of the cover sheet 2 along its length direction, and the groove and the upper surface of the substrate 1 are combined to form the microchannel 3 .

[0035] Specifically, the diameter of the magnetic particles is 0.1-2.8 μm.

[0036] In one embodiment, the number of the detection points 321 is multiple, and the lower surface of the substrate corresponding to the multiple detection points 321 is provided with a magnetic block 7. The multiple detection points 321 can be suitable for multiple detection of microfluidic chips, such as the combined detection of Mycoplasma pneumoniae and Chlamydia pneumoniae IgM antibodies.

[0037] Specifically, the magnetic block 7 is a neodymium iron boron magnet, a samarium cobalt magnet or other magnets or electromagnets that can reach a magnetic force of 0.2 T, which can effectively absorb magnetic particles.

[0038] like Figure 3As shown, in one embodiment, the magnetic block 7 is rectangular, and there are multiple magnetic blocks 7. Each magnetic block 7 is fixed below the detection point 321 and the reference point 322. The length direction of the magnetic block 7 is the same as the width direction of the microchannel 3. The length of the magnetic block 7 is smaller than the width of the microchannel 3, so that the magnetic particles are not easy to leak out of the microchannel during the reaction.

[0039] like Figure 4 As shown, in one embodiment, the magnetic block 7 is cylindrical, and there are multiple magnetic blocks 7. The multiple magnetic blocks 7 are distributed in an array along the corresponding detection points 321 and reference points 322 on the lower surface of the substrate, and the distance between two adjacent magnetic blocks 7 is not less than the diameter of the magnetic block 7.

[0040] Specifically, the diameter of the magnetic block 7 is 1.0-1.5 mm, the thickness of the magnetic block is 0.5-20 mm, and the distance between two adjacent magnetic blocks 7 in the array below the detection point and the reference point is 1.0-2.0 mm.

[0041] The water absorption of the waste liquid area 5 is that a water-absorbing material 6 is arranged in the waste liquid area or the waste liquid area 5 is a structure with water absorption function. The structure of the waste liquid area 5 itself can absorb water, or there can be external force to absorb water, and it can absorb the liquid in the microchannel 3.

[0042] In one embodiment, the magnetic block 7 is a plurality of small magnets attached to the substrate coating position, and the magnetic fields between the small magnets do not interfere with each other.

[0043] On the other hand, the present invention also provides a method for preparing a microfluidic chip for magnetically coating biological substances, comprising at least the following steps:

[0044] 1) Coupling the substance to be coated (such as antigen or antibody) with suitable magnetic particles;

[0045] 2) Suspending the coupled magnetic particles in an appropriate buffer system (such as PBS), the buffer contains a dispersant and a protective agent (such as BSA and sucrose, etc.);

[0046] 3) Use buffer to adjust the magnetic microparticle solution to a suitable concentration (the suitable concentration refers to the concentration of magnetic beads that can effectively cover the coated area and can be stably fixed in the coated area, such as 1 mg / ml);

[0047] 4) dropping the magnetic particle liquid onto a designated position (detection point or reference point) on the detection area of ​​the substrate;

[0048] 5) Place the substrate in a dry and clean environment to dry naturally, or dry at 37 degrees, or other methods that do not change the position of the liquid;

[0049] 6) The coated substrate is used for subsequent bonding with the cover sheet to form a coated microfluidic chip.

[0050] Example

[0051] Taking Mycoplasma pneumoniae (MP) IgM antibody detection as an example

[0052] 1.MP recombinant antigen coupled to magnetic particles

[0053] Choose the conventional coupling method: first wash and resuspend the magnetic microparticles, then use EDC and NHS to activate the carboxyl groups on the surface of the magnetic microparticles. After sufficient activation, remove the supernatant by magnetic separation. Then mix and incubate the activated magnetic microparticles with the MP recombinant antigen. After the coupling reaction is completed, block the remaining active sites on the surface of the microparticles, wash and resuspend them with the preservation solution, and store them at 4°C for future use. The coupling method of goat anti-mouse IgG antibody and magnetic microparticles is also the same.

[0054] 2. Fabrication of microfluidic chip for MP-IgM antibody detection

[0055] (1) Fabrication of chip cover

[0056] The chip cover is made of PMMA material. The microchannel structure, sample loading holes and other structures on the cover are designed using CAD software and then processed and manufactured using a CO2 laser etching machine.

[0057] (2) Biomolecule coating procedure of chip substrate

[0058] First, place the chip substrate on the chip tray. The chip tray corresponds to the position of the chip detection point and the reference point. A cylindrical magnetic block is placed on the chip tray. The magnetic block has a diameter of 1.5 mm, a thickness of 2.5 mm, and a magnetic force of 0.2 T. Mouse anti-human IgM antibody coupled with fluorescent microspheres is spotted on the labeling area of ​​the chip substrate, MP recombinant antigen coupled with magnetic particles is spotted on the detection area, and goat anti-mouse IgG antibody coupled with magnetic particles is spotted on the reference area. Then, the chip is placed in a clean bench to dry naturally.

[0059] (3) Chip assembly process

[0060] The chip cover containing the microchannel structure is tightly combined with the chip substrate containing the biological molecules, and then bonded with acetone. At this point, the preparation of the detection chip is completed.

[0061] (4) Testing procedures

[0062] First, the sample to be tested is dripped into the sample well. The sample enters the microchannel under the action of capillary driving force. The MP-IgM antibody in the sample first combines with the fluorescent microspheres coupled to the mouse anti-human IgM antibody in the labeling area to form a complex, and then continues to flow to the detection area with the liquid flow, and is captured by the MP recombinant antigen coupled to the surface of the magnetic particles in the detection area. The remaining labeled antibody continues to flow to the reference area and is captured by the goat anti-mouse IgG antibody coupled to the magnetic particles in the reference area. The magnetic particles in the detection area and the reference area remain immobile due to the magnetic adsorption of the magnet under the chip bottom film, and the remaining liquid is collected in the waste liquid area. After the reaction is completed, the chip is placed in a fluorescence detector, and the signal values ​​of the detection area and the reference area are read in the conventional way.

[0063] (5) Test results

[0064] Table 1 MP-IgM test results

[0065]

[0066] This indicates that the capture antigens coated with magnetic microparticles can effectively detect the samples to be tested, simplifying the coating process and allowing subsequent mass production.

[0067] The above technical solutions only reflect the preferred technical solutions of the technical solutions of the present invention. Some changes that may be made to certain parts thereof by technicians in this technical field all reflect the principles of the present invention and fall within the protection scope of the present invention.

[0068] It should be noted that, in the absence of conflict, the embodiments of the present invention and the features in the embodiments may be combined with each other.

[0069] In the description of the present invention, it should be understood that the terms "center", "longitudinal", "lateral", "up", "down", "front", "back", "left", "right", "vertical", "horizontal", "top", "bottom", "inside", "outside" and the like indicate positions or positional relationships based on the positions or positional relationships shown in the accompanying drawings, and are only for the convenience of describing the present invention and simplifying the description, rather than indicating or implying that the device or element referred to must have a specific orientation, be constructed and operated in a specific orientation, and therefore cannot be understood as limiting the present invention. In addition, the terms "first", "second", and the like are only used for descriptive purposes, and cannot be understood as indicating or implying relative importance or implicitly indicating the number of technical features indicated. Thus, features defined as "first", "second", and the like may explicitly or implicitly include one or more of the features. In the description of the present invention, unless otherwise specified, "multiple" means two or more.

[0070] In the description of the present invention, it should be noted that, unless otherwise clearly specified and limited, the terms "install", "connect", "connect", and "set" should be understood in a broad sense, for example, it can be a fixed connection, a detachable connection, or an integral connection; it can be a mechanical connection or an electrical connection; it can be a direct connection or an indirect connection through an intermediate medium, or it can be the internal communication of two components. For ordinary technicians in this field, the specific meanings of the above terms in the present invention can be understood by specific circumstances.

Claims

1. A microfluidic chip for magnetically coating biological substances, comprising a substrate (1) and a cover sheet (2), wherein the substrate (1) and the cover sheet (2) enclose a microchannel (3), wherein one end of the microchannel (3) is connected to a sample addition hole (4) provided on the cover sheet (2), and the other end is connected to a waste liquid area (5), wherein the waste liquid area (5) is water-absorbent, wherein a marking area (31) and a detection area (32) are arranged in the microchannel (3), wherein the marking area (31) and the detection area (32) are sequentially distributed along the sample flow direction, and wherein a detection point (321) and a reference point (322) are arranged in the detection area (32), wherein: A magnetic block (7) is disposed under the substrate corresponding to the detection point (321) and the reference point (322); the labeling area (31) is provided with a labeling antibody; the detection point (321) is provided with a capture antigen / antibody coupled to a magnetic particle; and the reference point (322) is provided with an anti-species antibody against the labeling area antibody coupled to a magnetic particle.

2. A microfluidic chip for magnetically coating biological substances according to claim 1, characterized in that: The lower surface of the cover sheet (2) is provided with a groove along its length direction, and the groove and the upper surface of the substrate (1) are combined to form the microchannel (3).

3. The microfluidic chip for magnetically coating biological substances according to claim 1, characterized in that: The diameter of the magnetic particles is 0.1-2.8 μm.

4. The microfluidic chip for magnetically coating biological substances according to claim 1, characterized in that: There are a plurality of detection points (321), and magnetic blocks (7) are arranged on the lower surface of the substrate corresponding to the plurality of detection points (321).

5. The microfluidic chip for magnetically coating biological substances according to claim 1, characterized in that: The magnetic block (7) is a neodymium iron boron magnet, a samarium cobalt magnet or other magnets or electromagnets that can reach a magnetic force of 0.2T.

6. The microfluidic chip for magnetically coating biological substances according to claim 1, characterized in that: The magnetic block (7) is rectangular, there are a plurality of magnetic blocks (7), each magnetic block (7) is correspondingly fixed below the detection point (321) and the reference point (322), the length direction of the magnetic block (7) is the same as the width direction of the microchannel (3), and the length of the magnetic block (7) is less than the width of the microchannel (3).

7. The microfluidic chip for magnetically coating biological substances according to claim 1, characterized in that: The magnetic block (7) is cylindrical, and there are a plurality of the magnetic blocks (7). The plurality of magnetic blocks (7) are distributed in an array along the corresponding detection points (321) and reference points (322) on the lower surface of the substrate, and the distance between two adjacent magnetic blocks (7) is not less than the diameter of the magnetic block (7).

8. The microfluidic chip for magnetically coating biological substances according to claim 1, characterized in that: The waste liquid area (5) has water absorption property because a water absorption material (6) is arranged in the waste liquid area or the waste liquid area (5) is a structure with water absorption function.

9. The microfluidic chip for magnetically coating biological substances according to claim 1, characterized in that: The magnetic block (7) is a plurality of small magnets attached to the substrate coating position, and the magnetic fields between the small magnets do not interfere with each other.

10. The method for preparing a microfluidic chip for magnetically coating biological substances according to claim 1, characterized in that: Includes at least the following steps: 1) Coupling the substance to be coated with appropriate magnetic particles; 2) suspending the coupled magnetic particles in an appropriate buffer system containing a dispersant and a protective agent; 3) Use buffer to adjust the magnetic microparticle solution to a suitable concentration; 4) dropping the magnetic particle liquid onto the designated position on the substrate detection area; 5) Place the substrate in a dry and clean environment to dry naturally, or dry at 37 degrees, or other methods that do not change the position of the liquid; 6) The coated substrate is used for subsequent bonding with the cover sheet to form a coated microfluidic chip.

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