Disclosure of Invention
Aiming at the defect of the existing method for fixing the capture antigen/antibody of the microfluidic chip, the invention provides the microfluidic chip for coating the biological substances by magnetic force, which can simplify the operation flow of capturing antigen/antibody fixing on the chip and improve the production efficiency.
In order to achieve the above purpose, the technical scheme of the invention is that the microfluidic chip for coating biological substances by magnetic force comprises a substrate and a cover plate, wherein the substrate and the cover plate are enclosed 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, the waste liquid area has water absorbability, a marking area and a detecting area are arranged in the micro-channel, the marking area and the detecting area are sequentially distributed along the flowing direction of a sample, a detecting point and a reference point are arranged in the detecting area, magnetic blocks are arranged below the substrate corresponding to the detecting point and the reference point, the marking area is provided with a marked antibody, the detecting point is provided with a capture antigen/antibody coupled by magnetic particles, and the reference point is provided with an anti-species antibody coupled by the magnetic particles and aiming at the antibody of the marking area.
In some embodiments of the invention, the lower surface of the cover plate is provided with grooves along the length direction thereof, and the grooves and the upper surface of the substrate are enclosed to form the micro-channels.
In some embodiments of the invention, the magnetic particles have a diameter of 0.1 to 2.8 μm.
In some embodiments of the present invention, the number of the detection points is a plurality, and the lower surface of the substrate corresponding to the plurality of detection points is provided with magnetic blocks.
In some embodiments of the present invention, the magnet is a neodymium-iron-boron magnet, a samarium-cobalt magnet, or other magnets/electromagnets capable of achieving a magnetic force of 0.2T.
In some embodiments of the present invention, the magnetic blocks are rectangular, the number of the magnetic blocks is plural, each magnetic block is correspondingly 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 micro-channel, and the length of the magnetic block is smaller than the width of the micro-channel.
In some embodiments of the present invention, the magnetic blocks are cylindrical, the number of the magnetic blocks is a plurality of the magnetic blocks, the plurality of the magnetic blocks are distributed along the detection points and the reference point arrays corresponding to the lower surface of the substrate, and the distance between two adjacent magnetic blocks is not smaller than the diameter of the magnetic blocks.
In some embodiments of the invention, the waste liquid zone has water absorption that is a structure in which a water absorbing material is disposed in the waste liquid zone or the waste liquid zone has a water absorbing function.
In some embodiments of the invention, the magnet is a plurality of small magnets attached to the substrate at the coating location, and the magnetic fields between the small magnets do not interfere with each other.
In another aspect, the present invention also provides a method for preparing a microfluidic chip for coating a biological substance by magnetic force, comprising at least the steps of:
1) Coupling the substance to be coated with suitable magnetic particles;
2) Suspending the coupled magnetic particles in a proper buffer system, wherein the buffer contains a dispersing agent and a protective agent;
3) Preparing the magnetic particle liquid to a proper concentration by using a buffer solution;
4) Dropping the magnetic particle liquid to a designated position on a substrate detection area;
5) Placing the substrate in a dry and clean environment to be naturally dried, or drying the substrate by adopting a 37 DEG drying method or other methods without changing the liquid position;
6) And using the coated substrate for subsequent bonding with a cover plate to form the coated microfluidic chip.
The microfluidic chip coated with biological substances by magnetic force and the preparation method thereof have the beneficial effects that:
The coating mode of biomolecules on the microfluidic chip is changed from a traditional complicated chemical coating mode into a coating mode of magnetic particles through magnetic adsorption, so that the manufacturing flow of the chip is greatly simplified, and the method is suitable for large-scale batch production. In addition, the technology adopts a centralized pre-coating mode, takes magnetic particles with a certain particle size as a coating carrier, coats specific antigens/antibodies in advance in a chemical coupling mode, is mature at present, and the prepared magnetic microspheres are easy to uniformly inspect so as to ensure the uniformity of each magnetic particle, further ensure the uniformity of coating among chips and effectively reduce batch-to-batch differences.
Detailed Description
It should be noted that, without conflict, the embodiments of the present invention and features of the embodiments may be combined with each other.
Unless defined otherwise, technical terms used in the following examples have the same meaning as commonly understood by one of ordinary skill in the art to which the present invention pertains. The test reagents used in the following examples are all conventional biochemical reagents unless otherwise specified, and the test methods are all conventional methods unless otherwise specified.
The invention relates to the technical field of immunodetection in general, in particular to a microfluidic chip for coating biological substances by magnetic force, the core of the invention is based on the microfluidic technology, the traditional chemical coating is replaced by magnetic adsorption of magnetic particles, so that the manufacturing mode of the microfluidic chip is simpler and mass production is facilitated.
The invention is further illustrated below with reference to examples and figures, it being understood that the invention is not limited to the specific embodiments described.
As shown in fig. 1-2, the invention provides a microfluidic chip for coating biological substances by magnetic force, which comprises a substrate 1and a cover plate 2, wherein the substrate 1and the cover plate 2 are enclosed to form a micro-channel 3, one end of the micro-channel 3 is connected with a sample adding hole 4 formed in the cover plate, the other end of the micro-channel is connected with a waste liquid area 5, the waste liquid area 5 has water absorbability and can adsorb liquid in the micro-channel 3, a marking area 31 and a detecting area 32 are arranged in the micro-channel 3, the marking area 31 and the detecting area 32 are distributed in sequence along the flowing direction of a sample, a detecting point 321 and a reference point 322 are arranged in the detecting area 32, magnetic blocks 7 are arranged below the substrate corresponding to the detecting point 321 and the reference point 322, the marking area 31 is provided with a marked antibody, the detecting point 321 is provided with a capture antigen/antibody coupled by magnetic particles, and the reference point 322 is provided with an anti-species antibody coupled by the magnetic particles and directed against the antibody of the marking area.
After the sample is added through the sample adding hole 4, the sample flows along with the micro-channel through capillary force and suction force of the waste liquid area, and flows through the marking area and the detecting area in sequence, the antigen/antibody to be detected in the sample reacts with the marked antibody of the marking area to form a compound first, then flows to the detecting point 321, is captured by the antigen/antibody coupled to the surface of the magnetic particle at the detecting point 321, the rest liquid continues to flow to the reference point 322, the marked antibody is captured by the corresponding substance on the surface of the magnetic particle at the reference point 322, the magnetic particles at the detecting point 321 and the reference point 322 are kept motionless due to the magnetic force absorption of the magnet below the substrate 1, and the rest liquid flows to the waste liquid area 5.
Specifically, a groove is formed in the lower surface of the cover plate 2 along the length direction of the cover plate, and the groove and the upper surface of the substrate 1 enclose to form the micro-channel 3.
Specifically, the diameter of the magnetic particles is 0.1-2.8 μm.
In one embodiment, the number of the detection points 321 is multiple, and the magnetic blocks 7 are disposed on the lower surface of the substrate corresponding to the detection points 321. The plurality of detection spots 321 can be suitable for multi-detection of microfluidic chips, such as joint detection of mycoplasma pneumoniae and chlamydia pneumoniae IgM antibodies.
Specifically, the magnetic block 7 is a neodymium-iron-boron magnet, a samarium-cobalt magnet or other magnets/electromagnets capable of achieving 0.2T magnetic force, and can effectively adsorb magnetic particles.
As shown in fig. 3, in one embodiment, the magnetic blocks 7 are rectangular, the number of the magnetic blocks 7 is plural, 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 smaller than the width of the microchannel 3, so that the magnetic particles are not easy to leak out of the microchannel in the reaction process.
As shown in fig. 4, in one embodiment, the magnetic blocks 7 are cylindrical, and the number of the magnetic blocks 7 is plural, and the plural magnetic blocks 7 are distributed along the array of the detection points 321 and the reference points 322 corresponding to the lower surface of the substrate, and the distance between two adjacent magnetic blocks 7 is not smaller than the diameter of the magnetic block 7.
Specifically, the diameter of the magnetic block 7 is 1.0-1.5mm, and the thickness of the magnetic block is 0.5-20mm, wherein the distance between two adjacent magnetic blocks 7 in the array below the detection point and the reference point is 1.0-2.0mm.
The water absorption of the waste liquid area 5 is that the waste liquid area is internally provided with a water absorption material 6 or the waste liquid area 5 has a water absorption function, the structure of the waste liquid area 5 can absorb water, or the structure can absorb water by external force, and the liquid in the micro-channel 3 can be absorbed.
In one embodiment, the magnetic block 7 is a plurality of small magnets attached under the substrate coating position, and magnetic fields between the small magnets do not interfere with each other.
In another aspect, the present invention also provides a method for preparing a microfluidic chip for coating a biological substance by magnetic force, comprising at least the steps of:
1) Coupling a substance (such as an antigen or antibody) to be coated with a suitable magnetic particle;
2) Suspending the coupled magnetic particles in a suitable buffer system (such as PBS) containing dispersing agent and protecting agent (such as BSA, sucrose, etc.);
3) The magnetic particle solution is prepared to a proper concentration by buffer solution (the proper concentration refers to the concentration of the magnetic beads which can effectively cover the coating area and can be stably fixed in the coating area, such as 1 mg/ml);
4) Dropping the magnetic particle liquid to a specified position (detection point or reference point) on the substrate detection area;
5) Placing said substrate in a dry and clean environment to allow it to dry naturally, or to dry at 37 degrees, or to dry by other means without changing the liquid position;
6) And using the coated substrate for subsequent bonding with a cover plate to form the coated microfluidic chip.
Examples
Taking Mycoplasma Pneumoniae (MP) IgM antibody detection as an example
MP recombinant antigen coupled magnetic particles
The conventional coupling mode is selected by firstly cleaning and re-suspending the magnetic particles, then activating carboxyl groups on the surfaces of the magnetic particles by EDC and NHS, and removing the supernatant by magnetic separation after full activation. The activated magnetic particles are then mixed with MP recombinant antigen for incubation, after the coupling reaction is completed, the remaining active sites on the surfaces of the particles are blocked, washed with a preservation solution and resuspended, and then stored at 4 ℃ for later use. The same applies to the coupling of goat anti-mouse IgG antibodies to magnetic particles.
Preparation of microfluidic chip for MP-IgM antibody detection
(1) Fabrication of chip upper cover
And the chip cover plate is made of PMMA, and micro-channel structures, sample adding holes and other structures on the cover plate are designed by adopting CAD software and then are manufactured by a CO2 laser etching machine.
(2) Coating program of chip substrate biological molecule
Firstly, placing a chip substrate on a chip tray, wherein the chip tray corresponds to the positions of a chip detection point and a reference point, and is provided with a cylindrical magnetic block, the diameter of the magnetic block is 1.5mm, the thickness of the magnetic block is 2.5mm, and the magnetic force is 0.2T. The fluorescent microsphere coupled mouse anti-human IgM antibody is spotted on the mark area of the chip substrate, the MP recombinant antigen coupled with the magnetic particles is spotted on the detection area, the goat anti-mouse IgG antibody coupled with the magnetic particles is spotted on the reference area, and then the chip is placed in an ultra-clean workbench to be naturally dried.
(3) Chip assembly program
The chip upper cover containing the micro-channel structure is tightly combined with the chip substrate containing the biological molecules, and then the chip upper cover is bonded by acetone, so that the preparation of the detection chip is completed.
(4) Detection program
Firstly, dropwise adding a sample to be detected at a sample adding hole, enabling the sample to enter a micro-channel under the action of capillary driving force, combining MP-IgM antibodies existing in the sample with fluorescent microsphere coupling mouse anti-human IgM antibodies of a labeling area to form a compound, continuing to reach a detection area along with liquid flow, capturing MP recombinant antigens coupled to the surface of magnetic particles by the detection area, continuing to flow to a reference area, capturing the residual labeled antibodies by goat anti-mouse IgG antibodies coupled to the magnetic particles of the reference area, keeping the magnetic particles of the detection area and the reference area motionless due to the magnetic force absorption of a magnet below a chip negative film, and collecting the residual liquid into a waste liquid area. After the reaction is completed, the chip is placed in a fluorescence detector, and signal values of the detection area and the reference area are read in a conventional manner.
(5) Detection result
TABLE 1 MP IgM detection results
The capture antigen coated by the magnetic particles can be used for effectively detecting a sample to be detected, so that the coating process is simplified, and the subsequent mass production can be performed.
The above technical solution only represents the preferred technical solution of the present invention, and some changes that may be made by those skilled in the art to some parts of the technical solution represent the principles of the present invention, and the technical solution falls within the scope of the present invention.
It should be noted that, without conflict, the embodiments of the present invention and features of the embodiments may be combined with each other.
In the description of the present invention, it should be understood that the terms "center", "longitudinal", "lateral", "upper", "lower", "front", "rear", "left", "right", "vertical", "horizontal", "top", "bottom", "inner", "outer", etc. indicate orientations or positional relationships based on the orientations or positional relationships shown in the drawings, are merely for convenience in describing the present invention and simplifying the description, and do not indicate or imply that the devices or elements referred to must have a specific orientation, be configured and operated in a specific orientation, and thus should not be construed as limiting the present invention. Furthermore, the terms "first," "second," and the like, are used for descriptive purposes only and are not to be construed as indicating or implying a relative importance or implicitly indicating the number of technical features indicated. Thus, a feature defining "a first", "a second", etc. may explicitly or implicitly include one or more such feature. In the description of the present invention, unless otherwise indicated, the meaning of "a plurality" is two or more.
In the description of the present invention, unless explicitly stated or limited otherwise, the terms "mounted," "connected," and "configured" are to be construed broadly, and may, for example, be fixedly connected, detachably connected, or integrally connected, mechanically connected, electrically connected, directly connected, indirectly connected via an intermediary, or communicate between two elements. The specific meaning of the above terms in the present invention can be understood by those of ordinary skill in the art in a specific case.