Micro-fluidic chip and method for single particle packaging and magnetic droplet sorting

Abstract

The invention discloses a micro-fluidic chip and method for single particle packaging and magnetic liquid drop sorting, and the micro-fluidic chip comprises a flow channel, and a magnetic bead single-row focusing unit, a liquid drop generation unit and a magnetic liquid drop sorting unit which are sequentially communicated along the flow channel, the magnetic bead single-row focusing unit comprises at least one section of micro-channel for generating an inertial focusing effect and at least one external magnetic field device for applying a magnetic field in a local area of the micro-channel, so that magnetic beads in the magnetic bead suspension liquid in the input flow channel are migrated to a stable balance position under the synergistic effect of magnetic field force and inertial lift force; and the flow channels are arranged in an ordered single row in the flow channel. According to the invention, functional modules of magnetic bead pre-arrangement, single particle packaging, magnetic separation and the like are integrated into a single chip, so that the automation of the whole process of sample introduction, arrangement, packaging and separation is realized, the operation is simplified, and the system reliability is improved.

Description

Technical Field

[0001] This invention relates to the fields of microfluidics and single-molecule detection technology, and more specifically to a microfluidic chip and method for single-particle encapsulation and magnetic droplet sorting. Background Technology

[0002] In cutting-edge research in precision medicine and life sciences, the need for ultrasensitive quantitative detection of biomarkers is becoming increasingly urgent. Single-molecule detection technologies such as digital ELISA, by dividing the reaction system into a large number of independent units (such as microdroplets or microwells), have achieved a leap in detection sensitivity from picomolar to atmolar levels, and have become key means for the detection of trace protein biomarkers.

[0003] However, the practical application of this technology faces two major bottlenecks: First, during the droplet generation process, the solid-phase reaction carrier (usually surface-modified magnetic beads) is difficult to be efficiently and controllably encapsulated individually within the droplet, resulting in low single-bead encapsulation rate, excessively high proportion of multi-bead encapsulation and empty droplets; Second, the large number of generated empty droplets mixed with effective droplets not only occupy detection resources and increase data noise, but also seriously reduce the throughput and accuracy of detection.

[0004] Currently, conventional microfluidic droplet technologies (such as flow focusing and T-junctions) can generate droplets with high throughput, but the encapsulation of particles exhibits a Poisson distribution, with a single encapsulation rate typically below 30%. To improve the single encapsulation rate, researchers have attempted to improve the encapsulation statistical characteristics by pre-arranging particles (e.g., using inertial focusing, acoustic fields, electric fields), but these methods often face challenges such as complex devices, poor compatibility, and difficulty in integrating with downstream sorting modules. Furthermore, existing droplet sorting technologies (such as dielectrophoresis, thermocapillary, and optical tweezers) can achieve droplet manipulation, but they generally suffer from limitations such as low throughput, complex devices, or strict requirements on droplet properties, making it difficult to meet the practical needs for efficient, gentle, and high-throughput sorting of droplets containing magnetic beads. Summary of the Invention

[0005] The technical problem to be solved by this invention is how to improve the single-encapsulation rate of solid-phase reaction carriers in droplets and improve detection efficiency.

[0006] The present invention solves the above-mentioned technical problems through the following technical means: a microfluidic chip for single-particle packaging and magnetic droplet sorting, comprising a flow channel and a magnetic bead single-row focusing unit, a droplet generating unit, and a magnetic droplet sorting unit connected sequentially along the flow channel. The magnetic bead single-row focusing unit includes at least one microchannel for generating an inertial focusing effect and at least one external magnetic field device for applying a magnetic field to a local area of ​​the microchannel, so that the magnetic beads in the magnetic bead suspension in the input flow channel migrate to a stable equilibrium position under the combined action of magnetic force and inertial lift, and arrange themselves into an orderly single row in the flow channel; the droplet generating unit is used to merge the arranged magnetic beads with the oil phase fluid to generate droplets containing magnetic beads or empty droplets; the magnetic droplet sorting unit is used to separate the droplets containing magnetic beads or empty droplets.

[0007] As a preferred technical solution, the magnetic droplet sorting unit includes a flow rate regulation zone and a magnetic field deflection zone. The channel size in the flow rate regulation zone is larger than the channel size not in the flow rate regulation zone. A local magnetic field generating device is provided outside the channel in the magnetic field deflection zone. The local magnetic field generating device can drive the separation of droplets containing magnetic beads and empty droplets without magnetic beads.

[0008] As a preferred technical solution, the flow channel in the droplet generation unit is a flow focusing structure, a T-channel structure, or a coaxial focusing structure.

[0009] As a preferred technical solution, the flow channels in the single-row focusing unit of the magnetic beads include flow channel one, flow channel two, flow channel three, and flow channel four. Flow channel one and flow channel two enclose a rectangular or square oil phase flow channel. An oil phase inlet is provided on the oil phase channel. Flow channel three and flow channel four form an aqueous phase flow channel. The oil phase flow channel and the aqueous phase flow channel intersect perpendicularly at the end of flow channel four.

[0010] As a preferred technical solution, the flow channel four includes a straight channel, an arc channel, and another straight channel connected in sequence. A permanent magnet is provided on the convex surface of the arc channel, and the permanent magnet forms an external magnetic field device.

[0011] As a preferred technical solution, the local magnetic field generating device includes multiple permanent magnets II, which are located in the magnetic field deflection region or on the concave side of the flow channel in the magnetic field deflection region.

[0012] This invention also provides a sorting method for single-particle encapsulation and magnetic droplet sorting, employing a microfluidic chip, comprising: S1. The magnetic bead suspension flows through the magnetic bead single-row focusing unit, so that the magnetic beads are arranged in a single row in the flow channel; S2. The single-row magnetic bead flow output from step S1 is combined with the oil phase fluid in the droplet generation unit to generate droplets containing magnetic beads or empty droplets. S3. The droplets generated in step S2 are introduced into the magnetic droplet sorting unit, causing the droplets containing magnetic beads to deflect and separate from the empty droplets.

[0013] As a preferred technical solution, the single-row arrangement effect of the magnetic beads can be adjusted by adjusting at least one of the following parameters: concentration of the magnetic bead suspension, flow rate, size of the single-row focusing unit channel of the magnetic beads, and magnitude of the applied magnetic field.

[0014] As a preferred technical solution, the uniformity of the generated droplet size and the single magnetic bead encapsulation rate are adjusted by regulating at least one of the following: the flow rate ratio of the water phase to the oil phase, the interfacial tension, and the structural parameters of the droplet generation unit.

[0015] As a preferred technical solution, the separation efficiency and purity of droplets containing magnetic beads and empty droplets can be adjusted by regulating at least one of the magnetic field strength, gradient, direction, and droplet flow rate.

[0016] The beneficial effects of this invention are as follows: (1) In this invention, the functional modules such as magnetic bead pre-arrangement, single-particle encapsulation and magnetic sorting are integrated into a single chip, realizing the full-process automation of sample introduction, arrangement, encapsulation and sorting, simplifying operation and improving system reliability; through the microchannel and the addition of an external magnetic field device outside the microchannel, the magnetic beads in the magnetic bead suspension in the input channel can migrate to a stable equilibrium position under the synergistic effect of magnetic force and inertial lift, and arrange themselves into an orderly single row in the channel, increasing the probability of a single magnetic bead entering the droplet, thereby significantly improving the single encapsulation rate; through the magnetic droplet sorting unit, the droplets containing magnetic beads or empty droplets are separated, improving and utilizing the magnetic response characteristics to actively sort the generated droplets, effectively removing a large number of empty droplets that have no effect on the statistical results, reducing the proportion of empty droplets, greatly improving the effective signal ratio and detection signal-to-noise ratio, thereby improving detection efficiency and accuracy.

[0017] (2) In this invention, the chip adopts continuous flow operation, which is compatible with existing microfluidic systems and is easy to achieve high-throughput processing. Attached Figure Description

[0018] Figure 1 This is a schematic diagram of the chip structure provided in an embodiment of the present invention; Figure 2 This is a schematic diagram of the magnetic bead arrangement provided in an embodiment of the present invention; Figure 3 This is a schematic diagram of the magnetic beads at the flow focusing structure provided in an embodiment of the present invention. In this diagram, the magnetic beads are encapsulated by droplets under the action of oil phase shear force, forming a single particle encapsulating a droplet. Figure 4 This is a schematic diagram illustrating the directional displacement of a single-particle encapsulated droplet generated under the drive of a magnetic field, provided in an embodiment of the present invention. Figure 5 This is a schematic diagram of the magnetic bead-containing droplets prepared according to an embodiment of the present invention, wherein the proportion of magnetic bead-containing droplets is greater than 90%. Reference numerals: 1. Oil phase inlet; 101. Flow channel one; 102. Flow channel two; 103. Flow channel three; 104. Flow channel four; 2. Aqueous phase inlet; 3. Permanent magnet one; 4. Flow focusing structure; 5. Flow rate adjustment zone; 6. Magnetic field deflection zone; 7. Collection zone for droplets containing magnetic beads; 8. Collection zone for empty droplets; 9. Permanent magnet two. Detailed Implementation

[0019] To make the objectives, technical solutions, and advantages of the embodiments of the present invention clearer, the technical solutions of the embodiments of the present invention will be clearly and completely described below in conjunction with the embodiments of the present invention. Obviously, the described embodiments are only some embodiments of the present invention, not all embodiments. Based on the embodiments of the present invention, all other embodiments obtained by those skilled in the art without creative effort are within the scope of protection of the present invention.

[0020] See Figure 1 A microfluidic chip for single-particle packaging and magnetic droplet sorting includes a flow channel and a magnetic bead single-row focusing unit, a droplet generation unit, and a magnetic droplet sorting unit arranged sequentially along the flow channel; in this embodiment, the length or width direction of the chip is used as the setting direction. The inlet of the single-row magnetic bead focusing unit is used to input the magnetic bead suspension, and the outlet of the single-row magnetic bead focusing unit is used to output the magnetic bead suspension. The single-row magnetic bead focusing unit is used to arrange the magnetic beads in the input magnetic bead suspension into a single row in the flow channel and to make the magnetic beads arranged in an orderly manner; in this embodiment, the orderly arrangement can be a linear arrangement. The magnetic bead single-row focusing unit includes at least one microchannel for generating an inertial focusing effect and at least one external magnetic field device for applying a magnetic field to a local region of the microchannel; the geometric parameters of the microchannel are configured such that the flowing magnetic beads are subjected to a significant inertial lift at a specific flow rate. It should be noted that the geometric parameters of the microchannel refer to the length, cross-sectional shape, and size of the microchannel; the external magnetic field device can be a permanent magnet or an electromagnetic coil. An external magnetic field device generates a controllable magnetic field gradient on the side of the microchannel or at other specific locations. Under the combined action of inertial lift and magnetic field gradient force, the magnetic beads are driven and stabilized at one or a few equilibrium positions within the cross-section of the flow channel, thus forming a highly ordered single-row arrangement when flowing out of the single-row focusing unit of the magnetic beads. In this embodiment, the height of each channel is 40 μm. The channels include channel 101, channel 2 102, channel 3 103, and channel 4 104 located in the magnetic bead single-row focusing unit, channel 102 located in the droplet generation unit, and channel 104 located in the magnetic droplet sorting unit. Channel 101 and channel 2 102 enclose a ring-shaped oil phase channel. In this embodiment, the shape of channel 101 and channel 2 102 after enclosing each other is rectangular or square. The inlet of channel 101 and channel 2 102 is the oil phase inlet 1, and the outlet is the inlet of the flow focusing structure 4. Flow channel 101 and flow channel 202 have the same structure and are arranged symmetrically on the chip. Both flow channel 101 and flow channel 202 include three straight channels connected in sequence. The oil phase enters from the oil phase inlet 1 and is divided into two by flow channel 101 and flow channel 202, and then intersects with the aqueous phase flow channel at the end of flow channel 4.

[0021] Flow channel 3 103 and flow channel 4 104 form an aqueous phase flow channel. Flow channel 3 103 is connected to the oil phase flow channel through flow channel 4 104. Flow channel 3 103 is connected to and converges with the outlets of flow channel 1 101 and flow channel 2 102 through flow channel 4 104. Flow channel 3 103 is a straight channel. Flow channel 4 104 includes a straight channel, a quarter-circular arc channel, and another straight channel. Flow channel 4 104 is perpendicular to flow channel 1 101 and flow channel 2 102. An N52 type neodymium iron boron permanent magnet 3 is installed on the convex surface of the quarter-circular arc channel of flow channel 4 104. The radius of permanent magnet 3 is 2mm. Permanent magnet 3 forms an external magnetic field device. The aqueous phase enters the flow channel 3 103 through the aqueous phase inlet 2. Under the action of the external magnetic field device, it turns 180 degrees and enters the flow channel 4 104. By using a roughly rectangular or square flow channel structure, and by using a permanent magnet-3 to drive the magnetic beads to migrate rapidly and stabilize on a specific equilibrium flow line under the combined effect of inertial focusing effect and magnetic field force, a single row of magnetic beads with a tight arrangement and uniform spacing is obtained at the unit outlet, avoiding the use of complex flow channel structures to achieve the single-row orderly arrangement of magnetic beads in the prior art. It should be noted that the channel width at the junction of the oil phase flow channel and the water phase flow channel is reduced to 0 μm to assist droplet generation; all straight channels and arc channels are 30 mm wide and 40 μm high, and the cross-section of straight channels and arc channels is rectangular; of course, it can also be circular or trapezoidal. The droplet generation unit is used to receive the magnetic bead stream arranged in a single row and merge the magnetic bead stream with the oil phase fluid. Through the two-phase flow shearing action, droplets containing magnetic beads (effective droplets) and empty droplets without magnetic beads are generated. The inlet of the droplet generation unit is connected to the outlet of the magnetic bead single-row focusing unit. See Figure 1 The flow channel located in the droplet generation unit is a flow focusing structure 4, a T-channel structure, or a coaxial focusing structure.

[0022] The magnetic droplet sorting unit is fluidly connected to the outlet of the droplet generation unit and is used to process the above-mentioned mixed droplets. In this embodiment, the mixed droplets refer to droplets containing magnetic beads and empty droplets without magnetic beads. The magnetic droplet sorting unit includes a flow rate regulation zone 5 and a magnetic field deflection zone 6 connected in sequence. The flow channels in the magnetic droplet sorting unit include a flow rate regulation zone channel and a magnetic field deflection zone channel. The flow rate regulation zone channel reduces the flow velocity of the droplets by increasing the channel size or other fluid resistance design, thus extending the time window during which the droplets are subjected to the subsequent magnetic field. In this embodiment, the width of the flow rate regulation zone channel is 100 μm. The outlet of the magnetic field deflection zone 6 is connected to a magnetic bead droplet collection zone 7 and an empty droplet collection zone 8. A local magnetic field generating device is provided outside the flow channel of the magnetic field deflection zone. In this embodiment, the local magnetic field generating device includes a permanent magnet 9 or an electromagnetic coil. Multiple permanent magnets 9 are provided and distributed in the magnetic field deflection zone 6 or the concave side of the flow channel of the magnetic field deflection zone. They are used to generate a spatial magnetic field gradient near the sorting point, so that the flow trajectory of the droplets containing magnetic beads is deflected and enters the designated collection branch and the collection area 7 of droplets containing magnetic beads. The empty droplets without magnetic beads are not affected by the magnetic force or are minimally affected. They will flow out along the original main flow direction or another branch and enter the empty droplet collection area 8, thereby achieving efficient separation of droplets containing magnetic beads and empty droplets without magnetic beads. In this embodiment, the magnetic bead concentration is approximately 2 × 10⁻⁶. 7 The suspension is in aqueous phase and fluorinated oil containing 2% FluoSurf surfactant is used as oil phase. This chip is not only suitable for digital ELISA, but can also be extended to single-cell analysis, drug screening and other fields.

[0023] How to use: Surface-modified magnetic microspheres with a diameter of 5 μm were suspended in a buffer solution and injected into the magnetic bead single-row focusing unit at a flow rate of 30 μL / min. Under the combined action of inertial force and magnetic force generated by permanent magnet 3, the magnetic beads formed a stable single-row ordered arrangement before reaching the flow focusing structure 4. The single-row magnetic bead flow meets the fluorinated oil (containing 2% surfactant) injected on both sides at point 4 of the flow focusing structure, and the aqueous phase is sheared to generate monodisperse droplets with a diameter of about 35 μm. The generated droplets enter the magnetic droplet sorting unit, where the flow velocity decreases significantly at the expansion cavity. When the droplets flow near the bifurcation point between the magnetic bead droplet collection area 7 and the empty droplet collection area, the magnetic bead droplets are attracted by the lateral magnetic field, causing their path to deflect and enter the "target droplet" collection channel on one side, i.e., the magnetic bead droplet collection area 7. The empty droplets are not affected by the magnetic field and flow along the original path into the "waste liquid" channel on the other side, i.e., the empty droplet collection area 8.

[0024] The sorting method includes the following steps: S1, Single-row arrangement of magnetic beads: Magnetic beads with biomolecules on their surface are prepared into a suspension and injected into the single-row focusing unit of the chip. By synergistically controlling the physical properties of the magnetic bead suspension, such as concentration, fluid operation parameters such as flow rate, geometric parameters of the focusing unit such as channel size, and externally applied magnetic field parameters such as intensity and gradient, the magnetic beads are rapidly migrated and stabilized on a specific equilibrium streamline under the synergistic effect of inertial focusing effect and magnetic force, resulting in a single-row flow of closely arranged and uniformly spaced magnetic beads at the unit outlet. The density, stability, and flux of the single-row arrangement of magnetic beads can be optimized and controlled by independently or jointly adjusting at least one of the following parameters: concentration and flow rate of the magnetic bead suspension, cross-sectional dimensions of the focusing unit channel such as width, height, and aspect ratio, and magnitude of the applied magnetic field (i.e., current magnitude for electromagnetic coils). For example, the concentration of the magnetic bead suspension is 2×10 7 The magnetic bead density is 1 / ml, the flow rate of the magnetic bead suspension is 30 μL / min, the cross-sectional width of the single-row focusing unit of the magnetic bead is 30 μm, the cross-sectional height of the single-row focusing unit of the magnetic bead is 40 μm, the length-to-diameter ratio of the single-row focusing unit of the magnetic bead is 1:1, and the applied magnetic field is 480 mT. S2, Single magnetic bead droplet packaging: The single-row magnetic bead flow output from step S1 is used as the dispersed phase and transported together with the oil phase (continuous phase) fluid injected from another inlet to the droplet generation unit. By adjusting the flow rate ratio of the water phase to the oil phase and the interfacial tension parameters, the individual magnetic beads and the surrounding solution are wrapped to form monodisperse microdroplets, while empty droplets without magnetic beads are generated. By independently or jointly adjusting the flow rate ratio of the aqueous phase to the oil phase, selecting different surfactants to adjust the interfacial tension, and optimizing at least one of the specific structural parameters of the droplet generation unit, such as the slit width of the flow focusing and the angle of the T-junction, the diameter, monodispersity, and most importantly, the single magnetic bead encapsulation rate of the generated droplets can be precisely controlled. S3, Magnetic Droplet Sorting: All droplets generated in step S2 are introduced into the magnetic droplet sorting unit. All droplets include droplets containing magnetic beads and empty droplets. The droplets first enter the flow rate regulation zone 5, where their flow velocity is reduced. Then, the droplets enter the magnetic field deflection zone 6. Under the action of the magnetic field gradient, the trajectory of the droplets containing magnetic beads is deflected in a controllable manner and enters the preset collection channel, while the empty droplets continue to flow along the original mainstream direction, thus achieving physical separation between the two.

[0025] By independently or jointly adjusting at least one of the following parameters, such as the intensity and spatial gradient distribution of the local magnetic field generating device, the direction of the magnetic field relative to the flow channel, and the flow velocity of the droplets in the sorting area, the sorting process can be optimized to achieve high sorting efficiency or high recovery rate and high sorting purity or low empty droplet mixing rate of magnetic bead-containing droplets.

[0026] The above embodiments are only used to illustrate the technical solutions of the present invention, and are not intended to limit it. Although the present invention has been described in detail with reference to the foregoing embodiments, those skilled in the art should understand that modifications can still be made to the technical solutions described in the foregoing embodiments, or equivalent substitutions can be made to some of the technical features. Such modifications or substitutions do not cause the essence of the corresponding technical solutions to deviate from the spirit and scope of the technical solutions of the embodiments of the present invention.

Claims

1. A microfluidic chip for single-particle packaging and magnetic droplet sorting, characterized in that, The device includes a flow channel and a magnetic bead single-row focusing unit, a droplet generation unit, and a magnetic droplet sorting unit connected sequentially along the flow channel. The magnetic bead single-row focusing unit includes at least one microchannel for generating an inertial focusing effect and at least one external magnetic field device for applying a magnetic field to a local area of ​​the microchannel, so that the magnetic beads in the magnetic bead suspension in the input flow channel migrate to a stable equilibrium position under the combined action of magnetic force and inertial lift, and arrange themselves into an orderly single row in the flow channel. The droplet generation unit is used to merge the arranged magnetic beads with the oil phase fluid to generate droplets containing magnetic beads or empty droplets. The magnetic droplet sorting unit is used to separate the droplets containing magnetic beads or empty droplets.

2. The microfluidic chip for single-particle packaging and magnetic droplet sorting according to claim 1, characterized in that, The magnetic droplet sorting unit includes a flow rate regulation zone and a magnetic field deflection zone. The channel size in the flow rate regulation zone is larger than that in the non-flow rate regulation zone. A local magnetic field generating device is provided outside the channel in the magnetic field deflection zone. The local magnetic field generating device can drive the separation of droplets containing magnetic beads and empty droplets without magnetic beads.

3. A microfluidic chip for single-particle packaging and magnetic droplet sorting according to claim 1, characterized in that, The flow channel located in the droplet generation unit is a flow focusing structure, a T-channel structure, or a coaxial focusing structure.

4. A microfluidic chip for single-particle packaging and magnetic droplet sorting according to claim 1, characterized in that, The flow channels in the single-row focusing unit of the magnetic bead include flow channel one, flow channel two, flow channel three, and flow channel four. Flow channel one and flow channel two enclose a rectangular or square oil phase flow channel. An oil phase inlet is provided on the oil phase channel. Flow channel three and flow channel four form an aqueous phase flow channel. The oil phase flow channel and the aqueous phase flow channel intersect perpendicularly at the end of flow channel four.

5. A microfluidic chip for single-particle packaging and magnetic droplet sorting according to claim 4, characterized in that, The flow channel four includes a straight channel, an arc channel, and another straight channel connected in sequence. A permanent magnet is provided on the convex surface of the arc channel, and the permanent magnet forms an external magnetic field device.

6. A microfluidic chip for single-particle packaging and magnetic droplet sorting according to claim 2, characterized in that, The local magnetic field generating device includes multiple permanent magnets II, which are located in the magnetic field deflection region or on the concave side of the flow channel in the magnetic field deflection region.

7. A sorting method for single-particle encapsulation and magnetic droplet sorting, characterized in that, The microfluidic chip for single-particle packaging and magnetic droplet sorting as described in any one of claims 1-6 includes: S1. The magnetic bead suspension flows through the magnetic bead single-row focusing unit, so that the magnetic beads are arranged in a single row in the flow channel; S2. The single-row magnetic bead flow output from step S1 is combined with the oil phase fluid in the droplet generation unit to generate droplets containing magnetic beads or empty droplets. S3. The droplets generated in step S2 are introduced into the magnetic droplet sorting unit, causing the droplets containing magnetic beads to deflect and separate from the empty droplets.

8. A sorting method for single-particle encapsulation and magnetic droplet sorting according to claim 7, characterized in that, The single-row arrangement effect of the magnetic beads can be adjusted by regulating at least one of the following parameters: concentration of the magnetic bead suspension, flow rate, size of the single-row focusing unit channel of the magnetic beads, and magnitude of the applied magnetic field.

9. A sorting method for single-particle encapsulation and magnetic droplet sorting according to claim 7, characterized in that, The uniformity of droplet size and the single magnetic bead encapsulation rate can be adjusted by regulating at least one of the following: the flow rate ratio of the aqueous phase to the oil phase, the interfacial tension, and the structural parameters of the droplet generation unit.

10. A sorting method for single-particle encapsulation and magnetic droplet sorting according to claim 7, characterized in that, The separation efficiency and purity of droplets containing magnetic beads and empty droplets can be adjusted by regulating at least one of the magnetic field strength, gradient, direction, and droplet flow rate.

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