Micro-fluidic chip based on asymmetric well array and application of micro-fluidic chip

Abstract

The invention relates to a micro-fluidic chip based on an asymmetric well array and application of the micro-fluidic chip. The micro-fluidic chip comprises a sample inlet (1), a sample injection shunting guide region (2), a sample injection buffer region (3), a microparticle capturing region (4), a sample discharge buffer region (5), a sample discharge shunting guide region (6) and a sample outlet (7) which are arranged in sequence. By optimizing the design of a capture structure and by means of reciprocating motion of oscillating microfluid, the utilization rate of microspheres in a micro-fluidic chip is increased to a great extent, the binding efficiency between magnetic beads and target molecules is increased, efficient sample treatment and analysis are realized in a tiny volume, and the detection accuracy is improved. Consumption of samples and reagents is greatly reduced, and an efficient, sensitive and convenient solution can be provided for enrichment and detection of biological molecules such as pathogens and exosomes.

Description

Technical Field

[0001] The invention belongs to the field of microfluidic technology, and particularly relates to a microfluidic chip based on an asymmetric well array and applications thereof. Background Art

[0002] Microfluidics has the advantages of controllable liquid flow, integration, low consumption, high throughput and fast analysis, and has been widely used in research fields such as biomedicine and environmental science. With the advancement and development of microfluidics, microfluidic chips can realize operations such as transport, reaction, metering analysis, mixing and sorting of fluid samples through microchannels, reaction chambers and other functional components.

[0003] Microparticle sensors have been widely used in commercial products in the fields of nucleic acid extraction, immunoassay, cell sorting, protein purification, etc. Their advantages mainly include: 1) Compared with planar detection carriers, microparticles have a high surface-to-volume ratio, which enables rapid analysis; 2) The surface of microparticles can be easily functionalized with various types of biomolecules (such as DNA and proteins); 3) Microparticles are easy to manipulate and move, allowing the carriers of biomolecules to be transported and transferred.

[0004] The manipulation of microparticles by microfluidics has been widely used in the biomedical field, including in vitro blood purification devices, sorting and enrichment of biomolecules, and high-throughput detection of biomolecules. Passive microfluidics is an efficient microparticle processing technology. In the absence of external forces, passive technologies such as deterministic lateral displacement, inertial microfluidics, extrusion flow sorting, and hydrophobicity have been used for various functions, including focusing, capture, solution transfer, and particle separation. The absence of external force fields simplifies the design, manufacture, and implementation of these microfluidic devices, thereby reducing production and costs. However, passive fluid technology is rarely used in portable integrated systems, such as point-of-care diagnostic devices, probably due to the mismatch in flow formats between the two systems. Passive particle manipulation techniques usually use continuous flow forms, including connected fluid channels, resulting in a reduction in the number of independently controllable fluids and reduced platform flexibility. Moreover, in continuous flow devices, the metering of reagents and the acquisition of signals are more difficult. Summary of the invention

[0005] The technical problem to be solved by the present invention is to provide a microfluidic chip based on an asymmetric well array and its application, which can realize efficient capture of microspheres in combination with oscillating flow.

[0006] The present invention provides a microfluidic chip based on an asymmetric well array, comprising an injection port, an injection flow diversion guide area, an injection buffer area, a microparticle capture area, a sample output buffer area, a sample output flow diversion guide area and a sample output port which are arranged in sequence; the microparticle capture area comprises a first well array and a second well array which are arranged in an interlaced manner; the structural units of the first well array and the second well array comprise a first octagonal capture column, a second octagonal capture column and a conical flow guide platform base located below, and a mechanical capture site is formed between the first octagonal capture column and the second octagonal capture column, namely a capture well.

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

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

[0009] Preferably, the number of structural units of the first well array and the second well array is several. The number can be adjusted as needed, and the spacing between every two rows is L. Figure 3 As shown, the spacing between the octagonal capture columns between two adjacent capture traps is W, the spacing between the capture traps between the octagonal capture column and the conical flow guide platform base is H, the octagonal capture column is a mirror-symmetrical structure, the distance between its long sides is S, the angle with the horizontal plane is θ, and the spacing between adjacent conical flow guide platform bases is G. When the directness of the microparticles to be captured is d, the size of the spacing W between adjacent octagonal capture columns is between 1.1d and 2.5d, the size of the spacing H between the capture traps is between 0.1W and 0.5W, the angle θ between the octagonal capture column and the horizontal plane ranges from 30 to 60 degrees, and the spacing G between the conical flow guide platform bases is at least less than 2S cosθ+W+2H.

[0010] The beneficial effect of the conical guide structure design in the structural unit is to provide guidance and asymmetric interaction assistance for the reverse transport of microparticles such as microspheres in the array chip.

[0011] The beneficial effect of the octagonal capture column design is to provide a point of physical collision and lateral movement for microparticles not in the capture fluid, so that microparticles not in the capture fluid 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 guide platform base is a, the width is b, and the height of the conical part is c, wherein a is between 100 and 300 microns, and the ratio of b to c is between 0.5 and 1.5.

[0013] Further, the microfluidic chip also includes a forward fluid and a reverse fluid to form an oscillating flow field. Through the action of the oscillating flow field, microparticles such as microspheres introduced into the chamber of the microfluidic chip are efficiently mechanically captured. In addition, after microparticles such as microspheres are captured by the capture trap, when the surface of microparticles such as microspheres is modified with specific capture molecules, after the chip is passed into the test sample, under the action of the oscillating flow field formed by the forward fluid and the reverse fluid, the reaction efficiency between the target molecules in the test sample and the functionalized microparticles captured in the chip can be enhanced in a short time, and the enrichment and detection efficiency of the functionalized microparticle carrier can be improved. The beneficial effects of the forward fluid and the reverse fluid forming an oscillating flow field are: first, the capture efficiency of microparticles such as microspheres in the microfluidic chip based on the asymmetric trap array is increased, thereby improving the utilization rate of microparticles such as microspheres in the chip and reducing the loss of reagents; second, the reaction efficiency of the detection carrier and the target molecule in the microfluidic chip is improved, the enrichment and detection time is shortened, and the efficiency of enrichment and detection is improved.

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

[0015] Beneficial Effects

[0016] The present invention optimizes the capture structure design and, with the help of the reciprocating motion of the oscillating microfluidic, 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, thereby achieving efficient sample processing and analysis in a tiny volume, greatly reducing the consumption of samples and reagents, and providing an efficient, sensitive and convenient solution for the enrichment and detection of biological molecules such as pathogens and exosomes. BRIEF DESCRIPTION OF THE DRAWINGS

[0017] Figure 1 Schematic diagram of the structure of the microfluidic chip of the present invention.

[0018] Figure 2 It is a schematic diagram of the structure of the microparticle capture area of ​​the microfluidic chip of the present invention.

[0019] Figure 3 It is the structural size of adjacent capture units in the microparticle capture area of ​​the microfluidic chip of the present invention.

[0020] Figure 4 This is a microscopic photograph of magnetic beads captured by the microfluidic chip of the present invention.

[0021] Figure 5 It is the magnetic bead capture efficiency of the microfluidic chip of the present invention.

[0022] Figure 6 This is an evaluation of the sample enrichment efficiency of the microfluidic chip of the present invention based on the NTA test. DETAILED DESCRIPTION

[0023] The present invention will be further described below in conjunction with specific embodiments. It should be understood that these embodiments are only used to illustrate the present invention and are not intended to limit the scope of the present invention. In addition, it should be understood that after reading the content taught by the present invention, those skilled in the art can make various changes or modifications to the present invention, and these equivalent forms fall within the scope limited by the appended claims of the application equally.

[0024] Example 1

[0025] like Figure 1 and Figure 2 As shown, this embodiment provides a microfluidic chip based on an asymmetric well array, comprising an injection port 1, an injection diversion guide area 2, an injection buffer area 3, a microparticle capture area 4, a sample output buffer area 5, a sample output diversion guide area 6 and a sample output port 7 arranged in sequence; the microparticle capture area 4 comprises a first well array 8 and a second well array 9 arranged in an interlaced manner; the structural units of the first well array 8 and the second well array 9 comprise 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 site 13 is formed between the first octagonal capture column 10 and the second octagonal capture column 11, which is a capture well.

[0026] Preparation of the chip: The microfluidic chip based on the asymmetric well array has only a single-layer structure, which can be prepared by soft lithography, hot pressing, injection molding and other processes. The microfluidic chip based on the asymmetric well array is manufactured using a standard soft lithography process. Use CAD 2020 to design the structure on the chip, and then use 20μm thick SU-8 photoresist to transfer the designed structure to a four-inch silicon wafer. First, bake the silicon wafer at 170℃ for 15 minutes to keep the surface dry. Then use 3025 negative photoresist to spin coat at 3800rpm for 30s to obtain a 20μm photoresist film. Place the coated silicon wafer on a 65℃ hot plate and slowly heat it to 95℃ for 15 minutes. After the photolithography is completed, post-bake it under the same conditions as the previous step. After it slowly cools to room temperature, put it in propylene glycol methyl ether acetate (PGMEA) for development. After the end, put it on a 170℃ hot plate for hard baking. After 30 minutes, turn off the hot plate and let it cool down naturally. Polydimethylsiloxane (PDMS) was prepared by prepolymer and corresponding curing agent in a ratio of 10:1. The mixed PDMS was degassed in a vacuum chamber for 1 h to remove bubbles. The treated PDMS was poured onto the prepared wafer and cured on a hot plate at 80 degrees for 90 minutes. Then a 1.4 mm diameter punch was used to punch inlets and outlets at designated locations. The above PDMS layer was bonded to the thin glass cover chip by oxygen plasma treatment.

[0027] Modification of magnetic beads: Take an appropriate amount of streptavidin-modified magnetic bead suspension in a 1.5mL Ep tube, add 500ul of PBS solution, use a pipette to blow and wash, then place it on a magnetic test tube rack and tilt it for 5min, and carefully remove the washing supernatant. Add the corresponding proportion of biotin-modified CD63 antibody solution to the Ep tube, and add 500ul of PBS solution at the same time. After sealing the Ep tube, place it on a suspension instrument for 4h incubation. During this period, remove the Ep tube every 0.5h, use a pipette to resuspend it (to prevent the magnetic beads from adhering to the tube wall or settling at the bottom of the test tube), and then continue to use the suspension instrument for suspension. After the suspension is completed, place the Ep tube containing the magnetic bead-antibody mixture on a magnetic test tube rack at an angle for 5min, and carefully remove the antibody supernatant. Add 5% BSA solution to block non-specific sites (BSA is prepared immediately before use), seal the Ep tube and place it on a suspension instrument for blocking, 4h, use a pipette to blow and resuspend every 0.5h. Then continue to use the suspension instrument for suspension. After the blocking is completed, the Ep tube containing the magnetic beads-BSA solution successfully coated with antibodies is placed on a magnetic test tube rack at an angle and allowed to stand for 5min, the BSA supernatant is carefully removed, and PBS is added to the Ep tube. Use a pipette to gently blow and resuspend / 1 time, and then the Ep tube is placed on a magnetic test tube rack at an angle and allowed to stand for 5min, the PBS supernatant is carefully removed, and an appropriate amount of 2% BSA solution is added, and stored in a 4°C refrigerator for use.

[0028] Application of magnetic bead capture in the chip: In the initial stage of the pathogen enrichment chip, the ethanol in the reservoir is introduced into the chip through a micropump to pre-treat the chip to ensure that all small structures in the chip can be infiltrated and to prevent small bubbles from interfering with subsequent magnetic bead sampling. Then, PBS or PBST is pumped into the reservoir to replace the ethanol, and the pump is turned on to replace the liquid until the ethanol is completely replaced. Third, the magnetic bead suspension is added to the reservoir, and the magnetic beads are pumped from the reservoir into the microfluidic chip through the forward fluid 14 in the chip. A reverse fluid 15 is established based on the time required for the magnetic beads to flow from the reservoir to the chip outlet, thereby achieving uniform distribution of the magnetic beads in the chip. Figure 4 At the same time, the efficiency of magnetic bead capture in the enrichment chip was statistically calculated, and the efficiency of magnetic bead capture and utilization can reach 82.32%, as shown in Figure 5 shown.

[0029] Enrichment and detection application of magnetic beads in the chip: The exosome sample to be tested is passed into a microfluidic chip based on an asymmetric well array. When the sample solution flows through the chip and is about to enter the back-end collection pool, the test sample solution is repeatedly passed through the enrichment chip in combination with a periodic oscillating flow field to enhance the chip's enrichment ability for trace solutions. After the periodic oscillation system maintains a working cycle of 10 minutes, the enriched test solution is pumped into the collection pool. Subsequently, the original sample solution that has not yet flowed through the enrichment chip and the solution placed in the collection pool after enrichment are used as two batches of samples for NTA test analysis to detect the content of target exosomes in the two liquids before and after enrichment, such as Figure 6 As shown, the microfluidic chip based on the asymmetric well array can achieve enrichment of 85.88% of the biological molecules in the solution.

Claims

1. A microfluidic chip based on an asymmetric well array, characterized in that: The invention comprises an injection port (1), an injection flow diversion guide area (2), an injection buffer area (3), a microparticle capture area (4), a sample output buffer area (5), a sample output flow diversion guide area (6) and a sample output port (7) which are arranged in sequence; the microparticle capture area (4) comprises a first trap array (8) and a second trap array (9) which are arranged in an interlaced manner; the structural units of the first trap array (8) and the second trap array (9) comprise a first octagonal capture column (10), a second octagonal capture column (11) and a conical flow guide platform base (12) located below; a mechanical capture site (13) is formed between the first octagonal capture column (10) and the second octagonal capture column (11), namely a capture trap.

2. The microfluidic chip based on an asymmetric well array according to claim 1, characterized in that: The first octagonal capture column (10) and the second octagonal capture column (11) are located at two ends of the conical flow guide platform base (12).

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

4. The microfluidic chip based on an asymmetric well array according to claim 1, characterized in that: The length of the bottom side of the conical guide platform base (12) is a, the width is b, and the height of the conical part is c, wherein a is between 100 and 300 microns, and the ratio of b to c is between 0.5 and 1.

5.

5. 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) for forming an oscillating flow field.

6. Use of the asymmetric well array-based microfluidic chip as claimed in claim 1 in biomolecule enrichment and detection.

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