Micro-fluidic chip for coating biological substance through magnetic force

Abstract

The micro-fluidic chip comprises a substrate and a cover plate, a micro-channel is defined by the substrate and the cover plate, 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, a water-absorbing material is arranged in the waste liquid area, and the water-absorbing material is arranged in the cover plate. The micro-channel is internally provided with a water-absorbing material, the aperture of the water-absorbing material is 10-25 microns, the micro-channel is internally provided with a marking area and a detection area, the marking area and the detection area are sequentially distributed along the flowing direction of a sample, the detection area is internally provided with a detection point and a reference point, and magnetic blocks capable of adsorbing magnetic particles are arranged below the substrate corresponding to the detection point and the reference point. According to the micro-fluidic chip disclosed by the invention, 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, and meanwhile, the problem of liquid residue in a micro-channel is effectively solved.

Description

TECHNICAL FIELD

[0001] The utility model belongs to in vitro diagnosis and immunodetection technical field, especially relate to a kind of microfluidic chip by magnetic force coating biological matter. BACKGROUND

[0002] Microfluidic chip is the main platform of microfluidic technology, and the basic operation units such as sample preparation, reaction, separation and detection of biological, chemical and medical analysis processes can be integrated on a small closed chip. Microfluidic chip has the advantages of small size, small amount of sample and reagent, fast reaction speed, large-scale parallel processing and disposable use, and has great potential in the fields of biology, chemistry, medicine and other fields. In recent years, it has developed into a new research field of biology, chemistry, medicine, fluid, electronics, materials and mechanics.

[0003] The existing microfluidic chip generally fixes the capture antigen / antibody on the chip by chemical coating method, and the coating process needs multiple spotting, incubation, washing and drying, which is complex and cannot be mass-produced.

[0004] CN108181458B discloses a microfluidic chip based on fluorescence immune combined detection, which comprises a chip substrate and an upper cover plate covering the chip substrate; the chip substrate is provided with a sample adding area, an antibody coating area, a micro-mixing area, at least two detection areas, a quality control area and a waste liquid collecting area connected by capillary micro-channels in sequence; the lower side of the detection area and the quality control area is provided with a magnetic field area provided by a permanent magnet or an electromagnet; the surface of the detection area and the quality control area is provided with a rough structure, which is a downward concave semicircular structure, a sawtooth structure or a concave-convex small rectangular structure. Although this method can replace the traditional chemical coating method by magnetic adsorption, it needs to set rough structure to fix magnetic beads, and process grooves or sawtooth structures in micro-channels, which may cause the following problems: ① uneven fluid flow: rough surface causes turbulent flow, increasing the risk of residue; ② complex process: multiple steps are needed to process micro-channel structure, which is high in cost and difficult to mass-produce. UTILITY MODEL CONTENT

[0005] The utility model aims at the deficiency of the existing microfluidic chip capture antigen / antibody fixing method, and proposes a microfluidic chip for coating biological matter by magnetic force, which can simplify the operation process of capturing antigen / antibody fixing on the chip, improve production efficiency, and effectively solve the problem of liquid residue in micro-channels.

[0006] In order to realize the above-mentioned purpose, the technical scheme of the utility model is such that a micro-fluidic chip for coating biological substances by magnetic force comprises a substrate and a cover sheet, the substrate and the cover sheet enclose a micro-channel, one end of the micro-channel is connected with a sample adding hole formed on the cover sheet, the other end is connected with a waste liquid area, a water-absorbing material is arranged in the waste liquid area, the pore size of the water-absorbing material is 10-25 mu m, a marking area and a detection area are arranged in the micro-channel, the marking area and the detection area are sequentially distributed along the sample flow direction, a detection point and a reference point are arranged in the detection area, and a magnetic block capable of adsorbing magnetic microparticles is arranged below the substrate corresponding to the detection point and the reference point.

[0007] The water-absorbing material is a porous cellulose membrane or a water-absorbing polymer.

[0008] The lower surface of the cover sheet is provided with a groove along the length direction, the groove and the upper surface of the substrate enclose the micro-channel, the upper surface of the substrate is smooth, and the thickness of the substrate corresponding to the detection area position is less than or equal to 1 mm.

[0009] The magnetic block is a neodymium iron boron magnet, a samarium cobalt magnet or a magnet or electromagnet capable of achieving a magnetic force of 0.2-0.3 T.

[0010] The number of detection points is one or more, and a magnetic block is arranged below the lower surface of the substrate corresponding to each detection point.

[0011] The magnetic block is rectangular, one magnetic block is arranged below each detection point and reference point, the length direction of the magnetic block is the same as the width direction of the micro-channel, the length of the magnetic block is less than the width of the micro-channel, and the width of the magnetic block is 0.1-0.5 mm.

[0012] The magnetic block is cylindrical, one magnetic block is arranged below each detection point and reference point, and the diameter of the magnetic block is 1-2 mm.

[0013] The magnetic block is cylindrical, the diameter of the magnetic block is 0.1-0.5 mm, the number of magnetic blocks is multiple, and the multiple magnetic blocks are arranged in a matrix to form multiple magnetic block groups, one magnetic block group is arranged below each detection point and reference point, and the distance between two magnetic blocks in the magnetic block group is not less than the diameter of the magnetic block.

[0014] The magnetic blocks are filled with a magnetic separation material, the magnetic separation material is silica gel or polyimide, and the magnetic permeability of the magnetic separation material is less than 1.

[0015] The micro-fluidic chip for coating biological substances by magnetic force obtained by the above technical scheme has the following beneficial effects:

[0016] The magnetic block is arranged below the substrate, the coating method of the biomolecule on the micro-fluidic chip is changed from the traditional and complicated chemical coating method to the coating method through magnetic adsorption of magnetic microparticles, the manufacturing process of the chip is greatly simplified, and the chip is suitable for large-scale batch production.

[0017] Under the premise that the internal structure of the micro-channel keeps a flat surface, by arranging the water-absorbing material in the waste liquid area and limiting the pore diameter of the water-absorbing material, the liquid in the micro-channel flows to the waste liquid area without affecting the adsorption of the magnetic microparticles, the risk of residue is avoided, and meanwhile, the chip structure is simple, and the cost of batch production is low. BRIEF DESCRIPTION OF DRAWINGS

[0018] Figure 1 is a structural schematic view of the micro-fluidic chip for coating biological substances through magnetic force according to the utility model;

[0019] Figure 2 is a side view structure schematic view of the micro-fluidic chip for coating biological substances through magnetic force according to the utility model;

[0020] Figure 3 is a bottom view structure schematic view of the substrate in one of the embodiments of the utility model;

[0021] Figure 4 is a bottom view structure schematic view of the substrate in another embodiment of the utility model;

[0022] Figure 5 is a bottom view structure schematic view of the substrate in still another embodiment of the utility model;

[0023] Figure 6 is a bottom view structure schematic view of the substrate in still another embodiment of the utility model.

[0024] In the figure, 1, substrate; 2, cover plate; 3, micro-channel; 4, sample hole; 5, waste liquid area; 6, water-absorbing material; 7, magnetic block; 8, magnetic isolation material; 31, mark area; 32, detection area; 321, detection point; 322, reference point. DETAILED DESCRIPTION

[0025] It should be noted that the embodiments in the utility model and the features in the embodiments can be combined with each other without conflict.

[0026] Unless defined, the technical terms used in the following embodiments have the same meaning as generally understood by the person skilled in the art to which the utility model belongs. The test reagents used in the following embodiments, unless otherwise specified, are conventional biochemical reagents; the experimental methods, unless otherwise specified, are conventional methods.

[0027] The utility model relates to the field of immune detection technology, and relates to a microfluidic chip coated with biological substances by magnetic force, and the core of the utility model is based on microfluidic technology, and the traditional chemical coating is replaced by magnetic particle magnetic adsorption, so that the manufacturing mode of the microfluidic chip is simpler and batch production is facilitated.

[0028] The utility model will be explained further in combination with examples and drawings, and it can be understood that the utility model is not limited to the specific implementation.

[0029] As Figures 1-2 shown, the utility model provides a kind of microfluidic chip coated with biological substances by magnetic force, including substrate 1, cover sheet 2, the substrate 1 and cover sheet 2 are enclosed to form microchannel 3, the microchannel 3 one end is connected with the sample addition hole 4 being opened in cover sheet 2, the other end is connected with waste liquid area 5, the water-absorbing material 6 is provided in waste liquid area 5, the pore size of the water-absorbing material is 10 μm-25 μm, the microchannel 3 is provided with mark area 31, detection area 32, the mark area 31, detection area 32 are sequentially distributed along sample flow direction, the detection area 32 is provided with detection point 321, reference point 322, the substrate below corresponding detection point 321, reference point 322 are all provided with magnetic block 7 capable of adsorbing magnetic particle.

[0030] When using, mark area 31 is coated with labeled antigen / antibody in conventional manner, magnetic particle coupled capture antigen / antibody is placed in detection point 321, and magnetic particle coupled anti-species antibody against mark area antibody is placed in reference point 322.

[0031] Among them, the preparation method of antigen / antibody coupled by magnetic particle is as follows:

[0032] 1) the substance to be coated is combined with magnetic particle by carboxyl-amino coupling method;

[0033] 2) the coupled magnetic particle is mixed in PBS buffer solution, and the buffer solution contains 1% BSA, 2% sucrose and 0.01% Triton X-100;Wherein, the function of BSA is to prevent magnetic particle from agglomerating, and the function of sucrose is to form a protective film during drying process and maintain antibody activity.

[0034] 3) adjust the concentration of magnetic particle liquid to 2.0×10 6 ~ 3.0×10 6 / μl to ensure that the coverage of coating area is ≥95%;

[0035] 4) accurately drop 1 μl / point of magnetic particle liquid to the detection point and reference point of substrate detection area;

[0036] 5) place the substrate in 37 DEG C drying for 5 min, and the humidity is less than 30%.

[0037] 6) The coated substrate is used for subsequent bonding with the cover sheet, forming a coated microfluidic chip.

[0038] When the detection is needed, the sample is added through the sample hole 4, and the sample will flow along the microchannel by capillary force and the suction force of the absorbent material 6 in the waste liquid area 5, and then flow through the labeling area 31 and the detection area 32. The antibody / antigen to be detected in the sample first reacts with the labeled antigen / antibody in the labeling area 31 to form a complex, and then flows to the detection point 321. The antigen / antibody coupled to the surface of the magnetic microparticles is captured at the detection point 321, and the remaining liquid continues to flow to the reference point 322. The labeled antibody is captured by the corresponding substance on the surface of the magnetic microparticles at the reference point 322. The magnetic microparticles at the detection point 321 and the reference point 322 are kept stationary by the magnetic force of the magnet below the substrate 1, and the remaining liquid flows to the waste liquid area 5.

[0039] The pore size of the absorbent material 6 directly affects its water absorption capacity. The smaller the pore size of the absorbent material 6, the greater the capillary force.

[0040] However, for the magnetic force coated microfluidic chip, if the pore size of the absorbent material 6 is too small, the water absorption of the waste liquid area 5 is too strong. On the one hand, it will lead to insufficient reaction time of the sample in the microchannel, and on the other hand, it will lead to large shear force generated by the liquid plane flow in the microchannel, causing part of the magnetic microparticles to detach from the original position, ultimately leading to a low test value of the sample to be detected. While selecting a larger pore size of the absorbent material 6, although the capillary force is lower and the magnetic microparticle detachment rate is lower, the insufficient capillary force leads to excessive residual fluorescent microspheres in the microchannel, resulting in an increase in the background signal value, ultimately leading to a low test value of the sample to be detected.

[0041] Therefore, by adjusting the pore size of the absorbent material 6 in the waste liquid area, its water absorption capacity is controlled within a suitable range (10-25 μm), which reduces the residual liquid in the microchannel while making the shear force of the liquid plane flow in the microchannel smaller than the magnetic adsorption force, to ensure the stable fixation of the magnetic microparticles in the original position.

[0042] Specifically, the absorbent material 6 is a porous cellulose membrane or a water-absorbent polymer.

[0043] Specifically, the lower surface of the cover sheet 2 is provided with a groove along the length direction, and the groove and the upper surface of the substrate 1 form the microchannel 3. The upper surface of the substrate 1 is smooth, and the thickness of the substrate 1 corresponding to the detection area 32 is ≤1 mm. 。 By limiting the thickness of the substrate 1 corresponding to the detection area 32, the magnetic field strength of the magnetic block below the substrate 1 can be effectively controlled (the smaller the thickness of the substrate 1, the stronger the magnetic force, which can make the entire thickness of the substrate 1 ≤1 mm, or only the thickness corresponding to the detection area 32 ≤1 mm), so that the magnetic microparticles above the substrate 1 are fixed.

[0044] Specifically, the magnetic block 7 is a neodymium iron boron magnet, a samarium cobalt magnet, or other magnets / electromagnets capable of achieving a magnetic force of 0.2T-0.3T. The diameter of the magnetic particles is 0.1-2.8μm, which can effectively adsorb the magnetic particles. Too small a magnetic field (<0.2T) will affect the adsorption of the magnetic particles, preventing them from being fixed on the substrate. Conversely, too high a magnetic field (>0.3T) may affect the electronic structure of antibody molecules, alter their interactions, or influence their hydrophobic regions, causing the antibody to lose its normal three-dimensional structure, thereby affecting its antigen recognition and binding ability.

[0045] Specifically, the number of detection points 321 is one or more, and each detection point 321 has a magnetic block 7 on the lower surface of the substrate. Setting multiple detection points 321 can be used for multi-detection of microfluidic chips, such as the combined detection of Mycoplasma pneumoniae and Chlamydia pneumoniae I gM antibodies.

[0046] like Figure 3 As shown, in one embodiment, the magnetic block 7 is rectangular, and a magnetic block 7 is set below each detection point 321 and 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, so that the magnetic particles are not easily leaked out of the microchannel during the reaction process. The width of the magnetic block 7 is 0.1-0.5mm.

[0047] like Figure 4 As shown, in one embodiment, the magnetic block 7 is cylindrical, and a magnetic block 7 is disposed below each detection point 321 and reference point 322. The diameter of the magnetic block 7 is 1-2 mm.

[0048] like Figure 5 As shown, in one embodiment, the magnetic block 7 is cylindrical, the diameter of the magnetic block is 0.1-0.5mm, the number of magnetic blocks is multiple, and the multiple magnetic blocks 7 are distributed in a matrix to form multiple magnetic block groups 71 (such as 3×3, 4×4, 5×5, etc.). Each detection point 321 and reference point 322 is provided with a corresponding magnetic block group 71 below it. The distance between two magnetic blocks 7 in the magnetic block group 71 is not less than the diameter of the magnetic block 7.

[0049] like Figure 6 As shown, in one embodiment, the magnetic block 7 is filled with a magnetic shielding material 8, which is silicone or polyimide. The magnetic permeability of the magnetic shielding material 8 is less than 1, so that the magnetic fields between the magnetic blocks 7 do not interfere with each other.

[0050] Example 1: The Influence of Pore Size of Water-Absorbent Material on the Fixation Effect of Magnetic Particles

[0051] 1. Pre-processing of biological raw materials

[0052] (1) MP recombinant antigen conjugated magnetic microparticles

[0053] According to the conventional conjugation method: first, the magnetic microparticles (magnetic microparticle size: 524 nm) were washed and resuspended, then the carboxyl groups on the surface of the magnetic microparticles were activated by EDC and NHS, after sufficient activation, the supernatant was removed by magnetic separation. Then the activated magnetic microparticles were mixed with MP recombinant antigen and incubated, after the conjugation reaction was completed, the remaining active sites on the surface of the microparticles were blocked, and after washing and resuspension with the storage solution (final concentration: 10 mg / mL, magnetic microparticle number: 2.62 x 10 10 The conjugation of goat anti-mouse IgG antibody with magnetic microparticles was also carried out in the same way.

[0054] (2) Biotin labeling of MP recombinant antigen

[0055] First, 10 mM biotin solution was added to the capture antibody solution (molar mass ratio of 20:1), and then the mixture was incubated on ice for 2 h. After the reaction was completed, the reaction mixture was placed in a dialysis cup and dialyzed in PBS overnight to remove free biotin molecules. Finally, the concentration of biotinylated capture antibody was determined by ultraviolet spectrophotometry, and it was stored at 4°C for future use. The biotin labeling of goat anti-mouse IgG antibody was also carried out in the same way.

[0056] (3) Fluorescent microspheres conjugated with mouse anti-human IgM antibody

[0057] According to the conventional conjugation method: first, the fluorescent microspheres were washed and resuspended, then the carboxyl groups on the surface of the fluorescent microspheres were activated by EDC and NHS, then mouse anti-human IgM antibody was added to the resuspended activated product, and incubated at room temperature for 2 h. After the conjugation reaction was completed, 2% BSA was added to block the unreacted sites, and sodium citrate was added to terminate the reaction. After centrifugal washing, it was resuspended in the storage solution and stored at 4°C for future use.

[0058] 2. Coating procedure of chip substrate biomolecules

[0059] (1) Traditional chemical coating

[0060] 1.5 μL of avidin solution was spotted on the detection and reference regions of the substrate and incubated for 1 h; after washing with washing solution 1, 1.6 μL of biotinylated MP recombinant antigen was spotted on the detection region, and 1.6 μL of biotinylated goat anti-mouse IgG antibody was spotted on the reference region, and incubated for 1 h; after washing with washing solution 2, 1.2 μL of fluorescent microspheres conjugated with mouse anti-human IgM antibody was spotted on the labeling region, and dried in a 37°C drying oven for 5 min.

[0061] (2) Magnetic coating

[0062] First, the chip substrate is placed on the chip tray, which corresponds to the positions of the chip detection point and the reference point, and each is placed with a cylindrical magnetic block with a diameter of 2 mm, a thickness of 2.5 mm, a magnetic force of 0.2 T, and the periphery of the magnetic block is filled with a silicone-based material (magnetic permeability <1). 1 μL of fluorescent microsphere-coupled mouse anti-human IgM antibody is spotted on the marker area of the chip substrate, 1 μL of magnetic microsphere-coupled MP recombinant antigen solution is spotted on the detection area, and 1 μL of magnetic microsphere-coupled goat anti-mouse IgG antibody solution (the magnetic microsphere solution needs to be diluted to 1 mg / ml in advance using PBS buffer, and the number of magnetic microspheres is 2.62 x 10 6 Then, the chip is placed in a 37°C drying air box for 5 min.

[0063] 3. Chip assembly procedure

[0064] Four different pore size water-absorbing materials (pore sizes are 5 μm, 10 μm, 25 μm, and 30 μm, respectively) are selected and placed in the waste chamber. Then, the chip cover containing the channel structure is bonded to the chip substrate containing the biomolecules, and the detection chip is prepared.

[0065] 4. Chip detection procedure

[0066] (1) Traditional chip

[0067] First, 35 μL of the sample to be tested is added to the sample well, and the sample enters the microchannel under the action of capillary driving force. The MP-IgM antibody in the sample first binds with the fluorescent microsphere-coupled mouse anti-human IgM antibody in the marker area to form a complex, and then continues to flow to the detection area, where it is captured by the MP recombinant antigen pre-coated on the detection area. The remaining labeled antibody continues to flow to the reference area, where it is captured by the goat anti-mouse IgG antibody pre-coated on the reference area, and the remaining liquid is collected in the waste area. After the reaction is complete, the chip is placed in a fluorescence detector, and the signal values of the detection area (T) and the reference area (R) are read in the conventional manner, T / R is calculated, and the concentration value is converted through the built-in curve.

[0068] (2) Magnetic force-coated chip

[0069] First, 35 μL of the sample to be tested is added to the sample well. Driven by the capillary force, the sample enters the microchannel. The MP-IgM antibody present in the sample first binds to the fluorescent microspheres in the labeled area, coupled with mouse anti-human IgM antibody, forming a complex. This complex continues to flow with the liquid to the detection area, where it is captured by the MP recombinant antigen coupled to the surface of the magnetic microparticles. The remaining labeled antibody continues to flow to the reference area, where it is captured by the goat anti-mouse IgG antibody coupled to the magnetic microparticles. The magnetic microparticles in the detection and reference areas remain stationary due to the magnetic attraction of the magnet beneath the chip substrate. The remaining liquid is collected in the waste liquid area. After the reaction is complete, the chip is placed in a fluorescence detector, and the signal values ​​of the detection and reference areas are read using standard methods. The T / R ratio is calculated, and the concentration value is converted using the built-in curve and compared with the detection results of a traditional chip. Simultaneously, the detachment of the magnetic microparticles before and after the reaction needs to be tested.

[0070] 5. Test Results

[0071] Table 1. Effect of pore size of absorbent material on experimental results

[0072]

[0073] 6. Results Analysis

[0074] (1) The smaller the pore size of the water-absorbing material, the greater the siphon force. For magnetically coated chips, the stronger the water absorption in the waste liquid chamber, the less reaction time the sample has in the microchannel. On the other hand, the shear force generated by the liquid plane flow in the microchannel is large, causing some magnetic particles to detach from their original positions, ultimately resulting in a lower test value for the sample.

[0075] (2) When selecting a water-absorbing material with a larger pore size, although the capillary force is lower and the magnetic particle detachment rate is lower, insufficient capillary force leads to excessive residual fluorescent microspheres in the microchannel, increasing the background signal value and ultimately resulting in a lower test value for the sample. The standard for screening water-absorbing materials is that the detection result of magnetic coating is not less than that of traditional coating. Therefore, the ideal pore size is between 10μm and 25μm, and further refinement is needed based on parameters such as the actual area of ​​the water-absorbing material, liquid viscosity, and magnetic particle size.

[0076] (3) When the traditional chip and the magnetically coated chip detect the same sample, the latter obtains a higher test value. The reasons are as follows: the biomolecules of the traditional chip are coated on the chip substrate layer, which is planar, with limited reaction area and is easily affected by steric hindrance; while the biomolecules of the magnetically coated chip are pre-coated on the surface of magnetic microparticles, with a large specific surface area and high reaction efficiency.

[0077] Example 2: Influence of Magnetic Block Diameter on Magnetic Particle Distribution

[0078] 1. Biomolecular coating program on chip substrate

[0079] Firstly, the chip substrate was placed on the chip tray, which corresponded to the positions of the chip detection points and reference points. A magnetic block was fixed at each detection point and reference point. Three groups of experiments were performed, in which different diameters of magnetic blocks were fixed, i.e. 2 mm, 4 mm and 6 mm. The thickness of the magnetic block was 2.5 mm, the magnetic force was 0.2 T, and the periphery of the magnetic block was filled with a silicone-based material (magnetic permeability <1).

[0080] In each group of chips, 1 μL of fluorescent microsphere-coupled mouse anti-human IgM antibody was spotted on the marker area of the chip substrate, 1 μL of magnetic microsphere-coupled MP recombinant antigen was spotted on the detection area, and 1 μL of magnetic microsphere-coupled goat anti-mouse IgG antibody (which was diluted to 1 mg / ml with PBS buffer in advance, and the number of magnetic microspheres was 2.62 x 1010 / μL) was spotted on the reference area. Subsequently, the chip was dried in a 37°C drying air box for 5 min. 6

[0081] 2. Chip assembly procedure

[0082] A water-absorbing material with a pore size of 15 μm was selected and placed in the waste chamber. Subsequently, the chip cover containing the channel structure was bonded to the chip substrate containing the biomolecules, and the detection chip was prepared.

[0083] 3. Chip detection procedure

[0084] Firstly, 35 μL of the sample to be detected was added to the sample well. The sample entered the microchannel under the action of capillary driving force. The MP-IgM antibody in the sample first combined with the fluorescent microsphere-coupled mouse anti-human IgM antibody in the marker area to form a complex, and then continued to flow to the detection area, where it was captured by the MP recombinant antigen coupled to the surface of the magnetic microspheres in the detection area. The remaining labeled antibody continued to flow to the reference area, where it was captured by the goat anti-mouse IgG antibody coupled to the magnetic microspheres in the reference area. The magnetic microspheres in the detection area and the reference area remained stationary due to the magnetic force of the magnet below the chip substrate. The remaining liquid was collected in the waste area. After the reaction was completed, the chip was placed in a fluorescence detector, and the signal values of the detection area and the reference area were read in the conventional manner. The T / R value was calculated, and the concentration value was converted through the built-in curve. At the same time, the distribution state of the magnetic microspheres in the detection point was observed with a microscope before and after the reaction.

[0085] 4. Detection results

[0086] Table 2 Influence of magnetic block diameter on experimental results

[0087]

[0088] From the above results, it can be seen that when a magnetic block with a diameter of 1-2 mm is selected, the magnetic microspheres have a higher fixation rate due to the concentration of the magnetic field gradient, and the magnetic microspheres can be prevented from detaching from the original position.​

[0089] Effect of magnetic isolation material on experimental results

[0090] 1. Coating procedure of chip substrate

[0091] The pre-prepared chip contains five detection points. First, the chip substrate is placed on the chip tray, which corresponds to the 5 detection point positions of the chip substrate. Five cylindrical magnetic blocks are placed on the chip tray. The diameter of the magnetic block is 2 mm, the thickness is 2.5 mm, the magnetic force is 0.2 T, and the distance between adjacent magnetic blocks is 2 mm (the magnetic blocks are divided into two types, one of which is filled with silicone-based material around the magnetic block, and the other of which is not filled). At the 5 detection points, 1 μL of magnetic microparticles (particle size 524 nm, which needs to be diluted to 1 mg / ml with PBS buffer in advance, and the number of magnetic microparticles is 2.62 x 10 6 The chip is then placed in a 37°C drying air box for 5 min.

[0092] 2. Chip assembly procedure

[0093] A water-absorbing material with a pore size of 15 μm is selected and placed in the waste chamber. Then, the chip cover containing the channel structure is bonded to the chip substrate containing the biological molecules, and the detection chip is prepared.

[0094] 3. Chip detection procedure

[0095] Buffer solution is added to the sample well and enters the microchannel under the action of capillary force. The pre-dried magnetic microspheres are dissolved at the detection point. The magnetic microparticles are kept stationary by the magnetic force of the magnet below the chip substrate. The remaining liquid is collected in the waste area. The distribution of magnetic microparticles is observed under a microscope.

[0096] 4. Experimental results

[0097] (1) When the magnetic blocks are filled with magnetic isolation material, the magnetic microspheres at each detection point are uniformly distributed and arranged in a regular circle.

[0098] (2) When the magnetic blocks are not filled with magnetic isolation material, due to the superposition effect of adjacent magnetic fields, the magnetic microspheres at the detection points are not uniformly distributed and show a phenomenon of gathering at the edge of the magnetic block.

[0099] Example 4: Effect of chip substrate thickness on experimental results

[0100] 1. Chip substrate preparation

[0101] First, three different thicknesses of polymethyl methacrylate (PMMA) material were selected (0.5mm, 1.0mm, 1.5mm). Then, a CNC cutting machine was used to cut the PMMA sheets into the required size and shape (rectangle, 7cm long, 2cm wide), ensuring smooth edges to avoid cracking. Finally, the PMMA surface was modified using plasma treatment to convert its relatively hydrophobic surface to a more hydrophilic one.

[0102] 2. Chip substrate encapsulation program

[0103] First, chip substrates of different thicknesses were placed on a chip tray, corresponding to the positions of the chip's detection and reference points. Magnetic blocks with a diameter of 2 mm, a thickness of 2.5 mm, and a magnetic force of 0.2 T were fixed in place. The magnetic blocks were surrounded by a silicone-based material (permeability <1). 1 μL of magnetic microparticles (524 nm in diameter, pre-diluted to 1 mg / ml with PBS buffer; the number of magnetic microparticles was 2.62 × 10⁻⁶) were then spotted. 6 (pcs / μL). Subsequently, the chips were placed in a drying oven at 37°C for 5 minutes to dry.

[0104] 3. Chip assembly process

[0105] A water-absorbing material with a pore size of 15 μm was selected and placed in the waste liquid chamber. Then, the chip cover containing the channel structure was bonded to the chip substrate containing biomolecules, thus completing the fabrication of the detection chip.

[0106] 4. Chip testing program

[0107] First, 35 μL of the sample to be tested is added to the sample well. Driven by the capillary force, the sample enters the microchannel, where the analyte reacts with the corresponding substances in the labeled and detected areas. The magnetic particles in the detected and reference areas remain stationary due to the magnetic attraction of the magnet beneath the chip substrate, and the remaining liquid is collected in the waste liquid area. After the reaction is complete, the chip is placed in a fluorescence detector, and the signal values ​​of the detected and reference areas are read using standard procedures. The T / R ratio is calculated, and the concentration value is converted using the built-in curve. Simultaneously, the distribution of magnetic particles at the detection points is observed under a microscope before and after the reaction.

[0108] 5. Experimental Results

[0109] Table 3. Effect of chip substrate thickness on experimental results

[0110]

[0111] From the above data, it can be seen that the thickness of the chip substrate has a greater impact on the actual magnetic force of the magnetic particles, that is, the thinner the chip substrate, the closer the distance between the magnetic particles and the magnetic block, and the greater the magnetic force of the magnetic particles, and the higher the fixing rate of the magnetic particles, when the thickness of the substrate is set to be less than 1.0mm, the fixing rate of the magnetic particles is more than 95%, and the magnetic particles can be effectively prevented from separating from the original position.

[0112] The above technical solution only embodies the preferred technical solution of the technical solution of the present application, and some changes made by the person skilled in the art to some parts thereof all embody the principle of the present application and are within the protection scope of the present application.

[0113] It should be noted that the embodiments in the present application and the features in the embodiments can be combined with each other without conflict.

[0114] In the description of the present application, it should be understood that the orientation or positional relationship indicated by the terms "center", "longitudinal", "transverse", "upper", "lower", "front", "rear", "left", "right", "vertical", "horizontal", "top", "bottom", "inner", "outer" and the like is based on the orientation or positional relationship shown in the drawings, and is only for the convenience of describing the present application and simplifying the description, and does not indicate or imply that the device or element must have a particular orientation, be constructed and operated in a particular orientation, and therefore cannot be understood as a limitation on the present application. In addition, the terms "first", "second" and the like are only for descriptive purposes and cannot be understood as indicating or implying relative importance or implicitly indicating the number of the technical features indicated. Therefore, the features limited by "first", "second" and the like can explicitly or implicitly include one or more of the features. In the description of the present application, unless otherwise specified, the meaning of "a plurality of" is two or more.

[0115] In the description of the present application, it should be noted that, unless otherwise specified and limited, the terms "mounting", "connection", "connection", "setting" should be understood broadly, for example, it can be fixedly connected, or it can be detachably connected, or integrally connected, it can be mechanically connected, or it can be electrically connected, it can be directly connected, or indirectly connected through an intermediate medium, and it can be the communication between two elements. For those skilled in the art, the specific meaning of the above terms in the present application can be understood according to the specific circumstances.

Claims

1. A microfluidic chip for magnetically encapsulating biological material, comprising a substrate (1) and a cover plate (2), wherein the substrate (1) and the cover plate (2) enclose a microchannel (3), one end of the microchannel (3) is connected to a sample application hole (4) on the cover plate (2), and the other end is connected to a waste liquid area (5), characterized in that, The waste liquid area (5) is provided with a water-absorbing material (6), the pore size of the water-absorbing material (6) is 10-25 μm, the microchannel (3) is provided with a marking area (31) and a detection area (32), the marking area (31) and the detection area (32) are sequentially distributed along the sample flow direction, the detection area (32) is provided with a detection point (321) and a reference point (322), and the lower surfaces of the detection point (321) and the reference point (322) correspondingly are provided with a magnetic block (7) capable of adsorbing magnetic microparticles.

2. The microfluidic chip for coating biological substances by magnetic force according to claim 1, wherein, The water-absorbing material (6) is a porous cellulose membrane or a water-absorbing polymer.

3. The microfluidic chip for coating biological substances by magnetic force according to claim 1, wherein, The lower surface of the cover sheet (2) is provided with a groove along the length direction, the groove and the upper surface of the substrate (1) form the microchannel (3), the upper surface of the substrate (1) is smooth, and the thickness of the substrate (1) corresponding to the detection area (32) is less than or equal to 1 mm.

4. The microfluidic chip for coating biological substances by magnetic force according to claim 1, wherein, The magnetic block (7) is a neodymium-iron-boron magnet, a samarium-cobalt magnet or a magnet or electromagnet capable of achieving a magnetic force of 0.2-0.3 T.

5. The microfluidic chip for coating biological substances by magnetic force according to claim 1, wherein, The number of detection points (321) is one or more, and the lower surface of each detection point (321) is provided with a magnetic block (7).

6. The microfluidic chip for coating biological substances by magnetic force according to claim 1, wherein, The magnetic block (7) is rectangular, one magnetic block (7) is correspondingly provided below each detection point (321) and 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 less than the width of the microchannel (3), and the width of the magnetic block (7) is 0.1-0.5 mm.

7. The microfluidic chip for coating biological substances by magnetic force according to claim 1, wherein, The magnetic block (7) is cylindrical, one magnetic block (7) is correspondingly provided below each detection point (321) and reference point (322), and the diameter of the magnetic block (7) is 1-2 mm.

8. The microfluidic chip for coating biological substances by magnetic force according to claim 1, wherein, The magnetic block (7) is cylindrical, the number of magnetic blocks (7) is multiple, and the multiple magnetic blocks (7) are arranged in a matrix to form multiple magnetic block groups (71), one magnetic block group (71) is correspondingly provided below each detection point (321) and reference point (322), the distance between two magnetic blocks (7) in the magnetic block group (71) is not less than the diameter of the magnetic block (7), and the diameter of the magnetic block (7) is 0.1-0.5 mm.

9. The microfluidic chip for coating biological substances by magnetic force according to any one of claims 1 to 8, wherein, The outer periphery of the magnetic block (7) is filled with a magnetic shielding material (8), the magnetic shielding material (8) is silica gel or polyimide, and the magnetic permeability of the magnetic shielding material (8) is less than 1.

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