Microfluidic chip based on asymmetric well array and application thereof

Abstract

The application relates to a microfluidic chip based on an asymmetric trap array and application thereof, which comprises a sample inlet (1), a sample inlet shunt guide area (2), a sample buffer area (3), a micro-particle capture area (4), a sample outlet buffer area (5), a sample outlet shunt guide area (6) and a sample outlet (7) arranged in sequence. Through optimization of the capture structure design and the reciprocating motion of the oscillating microfluid, the application not only greatly improves the utilization rate of the microspheres in the microfluidic chip, but also increases the binding efficiency between the magnetic beads and the target molecules, realizes efficient sample processing and analysis in a small volume, greatly reduces the consumption of samples and reagents, and provides an efficient, sensitive and convenient solution for the enrichment and detection of biological molecules such as pathogens and exosomes.

Description

TECHNICAL FIELD

[0001] The application belongs to the field of microfluidic technology, and particularly relates to a microfluidic chip based on an asymmetric trap array and application thereof. BACKGROUND

[0002] Microfluidic technology has the advantages of controllable liquid flow, integration, low consumption, high flux and fast analysis, and has been widely applied to research fields such as biomedical science and environmental science. With the progress and development of microfluidic technology, microfluidic chips can realize transportation, reaction, metering analysis, mixing and sorting of fluid samples through microchannels, reaction chambers and other functional components.

[0003] Micro-particle sensitive elements have been widely applied to related commercial products in the fields of nucleic acid extraction, immune analysis, cell sorting, protein purification and the like. The advantages mainly include: 1) compared with a planar detection carrier, micro-particles show a high surface-volume ratio, thereby enabling rapid analysis; 2) the surface of micro-particles is easily functionalized with various biological molecule types (such as DNA and protein); 3) micro-particles are easy to manipulate and move, thereby allowing the carrier of biological molecules to be transported and transferred.

[0004] Manipulation of micro-particles through microfluidic technology has been widely applied to the biomedical field, including in-vitro blood purification devices, sorting enrichment of biological molecules, high-throughput detection of biological molecules and the like. Passive microfluidic technology is a high-efficiency micro-particle processing technology. Deterministic lateral displacement, inertial microfluidics, extrusion flow sorting and hydrophobic passive technology have been used for various functions, including focusing, capturing, solution transfer and micro-particle separation, without external force. Since there is no external force field, the design, manufacture and implementation of these microfluidic devices are simplified, thereby reducing the yield and cost. However, passive fluid technology is rarely used for portable integrated systems, such as point-of-care diagnostic devices, which may be due to the mismatch of flow formats between the two systems. Passive particle manipulation technology usually uses a continuous flow format, including connected fluid channels, resulting in a decrease in the number of independently controllable fluids and a decrease in the flexibility of the platform. Moreover, in the continuous flow device, it is difficult to meter reagents and collect signals. SUMMARY

[0005] The application aims to provide a microfluidic chip based on an asymmetric trap array and application thereof, which can realize efficient capture of microspheres in combination with oscillatory flow.

[0006] This invention provides a microfluidic chip based on an asymmetric trap array, comprising a sample inlet, a sample inlet diversion guide region, a sample inlet buffer, a microparticle capture region, an outlet buffer, an outlet diversion guide region, and an outlet arranged sequentially; the microparticle capture region includes a first trap array and a second trap array arranged in an alternating manner; the structural units of the first trap array and the second trap array include a first octagonal capture column, a second octagonal capture column, and a conical guide platform base located below, wherein mechanical capture sites are formed between the first octagonal capture column and the second octagonal capture column, which are capture traps.

[0007] The beneficial effects of the design of the sample inlet diversion guide area, sample inlet buffer, sample outlet buffer and sample outlet diversion guide area are to disperse and guide the forward and reverse transport of microparticles, and to provide auxiliary support for the chip cavity.

[0008] Preferably, the first octagonal capture column and the second octagonal capture column are located at both ends of the conical guide platform base.

[0009] Preferably, the first and second well arrays contain a plurality of structural units. The number can be adjusted as needed, and the spacing between each pair of rows is L. Figure 3 As shown, the distance between two adjacent trapping columns is W, the distance between the trapping columns and the trapping bases of the conical guide platform is H, the octagonal trapping columns are mirror-symmetric structures with a long side distance of S and an angle of θ with the horizontal plane, and the distance between adjacent conical guide platform bases is G. When the diameter of the microparticles to be captured is d, the dimension of the distance W between adjacent octagonal trapping columns is between 1.1d and 2.5d, the dimension of the trapping base distance H is between 0.1W and 0.5W, the angle θ between the octagonal trapping columns and the horizontal plane ranges from 30 to 60 degrees, and the distance G between the conical guide platform bases is at least less than 2S cosθ + W + 2H.

[0010] The beneficial effect of using a tapered flow-guiding structure in the structural unit is to provide flow guidance and asymmetric interaction to assist the reverse transport of microparticles such as microspheres in the array chip.

[0011] The advantage of the octagonal capture column design is that it provides a point of action for physical collision and lateral movement of microparticles that are not in the capture fluid, allowing microparticles that are not in the capture fluid to enter the capture flow, thereby increasing the capture efficiency of microparticles in the entire chip.

[0012] Preferably, the length of the bottom edge of the conical flow guide platform base is a, the width is b, and the height of the conical part is c, where a is between 100 and 300 micrometers, and the ratio of b to c is between 0.5 and 1.5.

[0013] Furthermore, the microfluidic chip also includes forward and reverse fluids to form an oscillating flow field. This oscillating flow field efficiently captures microparticles such as microspheres introduced into the microfluidic chip chamber. In addition, after the microparticles are captured by the traps, if the surface of the microparticles is modified with specific capturing molecules, the oscillating flow field formed by the forward and reverse fluids, when a detection sample is introduced into the chip, can enhance the reaction efficiency between the target molecules in the detection sample and the functionalized microparticles captured in the chip within a short time, thereby improving the enrichment and detection efficiency of the functionalized microparticle carrier. The beneficial effects of the oscillating flow field formed by the forward and reverse fluids are: first, it increases the capture efficiency of microparticles such as microspheres in the microfluidic chip based on the asymmetric trap array, thereby improving the utilization rate of microparticles in the chip and reducing reagent consumption; second, it improves the reaction efficiency between the detection carrier and the target molecules in the microfluidic chip, shortening the enrichment and detection time and improving the enrichment and detection efficiency.

[0014] The present invention also provides an application of a microfluidic chip based on an asymmetric trap array in the enrichment and detection of biomolecules.

[0015] Advantages

[0016] This invention, through optimized capture structure design and the reciprocating motion of oscillating microfluidics, not only significantly improves the utilization rate of microspheres in microfluidic chips but also increases the binding efficiency between magnetic beads and target molecules. This enables efficient sample processing and analysis within a small volume, greatly reducing sample and reagent consumption. It provides an efficient, sensitive, and convenient solution for the enrichment and detection of biomolecules such as pathogens and exosomes. Attached Figure Description

[0017] Figure 1 This is a schematic diagram of the microfluidic chip of the present invention.

[0018] Figure 2 This is a schematic diagram of the microparticle capture region of the microfluidic chip of the present invention.

[0019] Figure 3 The dimensions of the adjacent capture units in the microparticle capture region of the microfluidic chip of this invention are shown.

[0020] Figure 4 This is a micrograph of the magnetic bead capture of the microfluidic chip of the present invention.

[0021] Figure 5 This refers to the magnetic bead capture efficiency of the microfluidic chip of this invention.

[0022] Figure 6 This invention provides an evaluation of the sample enrichment efficiency of the microfluidic chip based on NTA testing. Detailed Implementation

[0023] The present invention will be further illustrated below with reference to specific embodiments. It should be understood that these embodiments are for illustrative purposes only and are not intended to limit the scope of the invention. Furthermore, it should be understood that after reading the teachings of this invention, those skilled in the art can make various alterations or modifications to the invention, and these equivalent forms also fall within the scope defined by the appended claims.

[0024] Example 1

[0025] like Figure 1 and Figure 2 As shown, this embodiment provides a microfluidic chip based on an asymmetric trap array, including a sample inlet 1, a sample inlet diversion guide area 2, a sample inlet buffer 3, a microparticle capture area 4, an outlet buffer 5, an outlet diversion guide area 6, and an outlet 7 arranged sequentially; the microparticle capture area 4 includes a first trap array 8 and a second trap array 9 arranged in an alternating manner; the structural units of the first trap array 8 and the second trap array 9 include a first octagonal capture column 10, a second octagonal capture column 11, and a conical guide platform base 12 located below, and a mechanical capture point 13 is formed between the first octagonal capture column 10 and the second octagonal capture column 11, which is a capture trap.

[0026] Chip Fabrication: Microfluidic chips based on asymmetric well arrays have only a single-layer structure and can be fabricated using processes such as soft lithography, hot pressing, and injection molding. Microfluidic chips based on asymmetric well arrays are manufactured using standard soft lithography. The chip structure is designed using CAD 2020, and then transferred to a four-inch silicon wafer using a 20μm thick SU-8 photoresist. First, the silicon wafer is baked at 170℃ for 15 minutes to keep the surface dry. Then, 3025 negative photoresist is spin-coated at 3800rpm for 30s to obtain a 20μm photoresist film. The coated silicon wafer is placed on a 65℃ hot plate and slowly heated to 95℃, held for 15 minutes. After photolithography, post-baking is performed under the same conditions as the previous step. After slowly cooling to room temperature, it is placed in propylene glycol methyl ether acetate (PGMEA) for development. After development, it is placed on a 170℃ hot plate for hard baking for 30 minutes, after which the hot plate is turned off to allow it to cool naturally. Polydimethylsiloxane (PDMS) was prepared by mixing a prepolymer and a corresponding curing agent in a 10:1 ratio. The mixed PDMS was degassed in a vacuum chamber for 1 hour to remove air bubbles. The treated PDMS was poured onto a prepared wafer and cured on a hot plate at 80 degrees Celsius for 90 minutes. Inlet and outlet holes were then drilled at designated locations using a 1.4 mm diameter punch. The PDMS layer was then bonded to a thin glass overlay chip via oxygen plasma treatment.

[0027] Modification of magnetic beads: Take an appropriate amount of streptavidin-modified magnetic bead suspension into a 1.5 mL Eppendorf tube, add 500 μL of PBS solution, and wash using a pipette. Then, place the tube on a magnetic tube rack at an angle and incubate for 5 minutes. Carefully remove the supernatant. Add the corresponding proportion of biotin-modified CD63 antibody solution to the Eppendorf tube, along with 500 μL of PBS solution. Seal the Eppendorf tube and incubate it on a suspension apparatus for 4 hours. Every 0.5 hours, remove the Eppendorf tube and resuspend it using a pipette (to prevent the magnetic beads from adhering to the tube wall or settling at the bottom of the tube). Continue resuspension using the suspension apparatus. After resuspension, tilt the Eppendorf tube containing the magnetic bead-antibody mixture on a magnetic tube rack and let it stand for 5 minutes. Carefully remove the antibody supernatant. Add 5% BSA solution for non-specific site blocking (BSA should be prepared fresh before use). Seal the Eppendorf tube and place it on a resuscitator for blocking for 4 hours, resuspending it every 0.5 hours using a pipette. Continue resuscitation using a resuscitator. After blocking, tilt the Eppendorf tube containing the successfully coated antibody-bSA solution onto a magnetic tube rack and let it stand for 5 minutes. Carefully remove the BSA supernatant, add PBS to the Eppendorf tube, and gently resuspend it once using a pipette. Then tilt the Eppendorf tube onto a magnetic tube rack and let it stand for 5 minutes. Carefully remove the PBS supernatant, add an appropriate amount of 2% BSA solution, and store at 4°C for later use.

[0028] Application of magnetic beads in chip capture: In the initial stage of pathogen enrichment chip operation, ethanol from the reservoir is introduced into the chip via a micropump to pre-treat the chip, ensuring that all small structures within the chip are wetted and preventing small bubble-like structures from interfering with subsequent magnetic bead injection. Subsequently, PBS or PBST is injected into the reservoir to replace the ethanol, and the pump is turned on to perform liquid replacement, completely removing the ethanol. Third, a magnetic bead suspension is added to the reservoir, and the magnetic beads are pumped from the reservoir into the microfluidic chip via the forward fluid 14. A reverse fluid 15 is established based on the time required for the magnetic beads to travel from the reservoir to the chip outlet, thereby achieving uniform distribution of the magnetic beads within the chip. Figure 4 As shown. Simultaneously, statistical calculations were performed on the efficiency of magnetic bead capture in the enrichment chip, showing that the efficiency of magnetic bead capture and utilization can reach 82.32%, such as... Figure 5 As shown.

[0029] Application of magnetic bead enrichment and detection in microfluidic chips: The exosome sample to be tested is introduced into a microfluidic chip based on an asymmetric trap array. Just before the sample solution enters the collection cell, a periodic oscillating flow field is used to repeatedly pass the sample solution through the enrichment chip, enhancing the chip's ability to enrich trace amounts of solution. This periodic oscillation system operates for 10 minutes before the enriched sample solution is pumped into the collection cell. Subsequently, the original sample solution before it passed through the enrichment chip and the enriched solution placed in the collection cell are used as two batches of samples for NTA testing and analysis to detect the content of target exosomes in the two liquids before and after enrichment. Figure 6 As shown, the microfluidic chip based on the asymmetric trap array can achieve enrichment of 85.88% of biomolecules in solution.

Claims

1. A microfluidic chip based on an asymmetric well array, characterized in that: The system includes, in sequence, an inlet (1), an inlet diversion guide area (2), an inlet buffer zone (3), a microparticle capture area (4), an outlet buffer zone (5), an outlet diversion guide area (6), and an outlet (7); the microparticle capture area (4) includes a first trap array (8) and a second trap array (9) arranged in an alternating manner; the structural unit of the first trap array (8) and the second trap array (9) includes a first octagonal capture column (10), a second octagonal capture column (11), and a conical guide platform base (12) located below, with mechanical capture sites (13) formed between the first octagonal capture column (10) and the second octagonal capture column (11), which are the capture traps; the conical guide platform base The base (12) is in the shape of an inverted frustum. Its upper surface is a relatively flat guide surface, and its lower surface gradually contracts downward to form a conical guide profile. The first octagonal capture column (10) and the second octagonal capture column (11) are located above the conical guide platform base (12) and are located on both sides of the conical guide platform base (12) and are in a symmetrical position. The cross-sectional dimensions of the first octagonal capture column (10) and the second octagonal capture column (11) near the conical guide platform base (12) are smaller than the cross-sectional dimensions of the end away from the conical guide platform base (12), so that the first octagonal capture column (10) and the second octagonal capture column (11) present a geometric feature of outward expansion at the top and inward contraction at the bottom.

2. The microfluidic chip based on an asymmetric well array according to claim 1, characterized in that: The number of structural units in the first well array (8) and the second well array (9) is several.

3. The microfluidic chip based on an asymmetric well array according to claim 1, characterized in that: The base (12) of the conical flow guiding platform has a length of a, a width of b, and a height of c, where a is between 100 and 300 micrometers, and the ratio of b to c is between 0.5 and 1.

5.

4. The microfluidic chip based on an asymmetric well array according to claim 1, characterized in that: The microfluidic chip also includes a forward fluid (14) and a reverse fluid (15) to form an oscillating flow field.

5. An application of the microfluidic chip based on an asymmetric trap array as described in claim 1 in the enrichment and detection of biomolecules.

Images