Multi-fluid effect synergistic three-dimensional single-row focusing micro-fluidic chip and method for single-line focusing of polystyrene microspheres by using micro-fluidic chip

Abstract

The invention relates to the technical field of micro-fluidic chips, and discloses a multi-fluid effect synergistic three-dimensional single-row focusing micro-fluidic chip and a method for single-line focusing of polystyrene microspheres by using the micro-fluidic chip, the micro-fluidic chip comprises a sample liquid inlet, one end of the sample liquid inlet is provided with an inertia focusing link, and the other end of the sample liquid inlet is provided with an inertia focusing link; a tapered flow guide cone structure is arranged between the inertial focusing link and the sample liquid inlet and is used for inhibiting the initial dispersion and diffusion trend of particles at the sample liquid inlet; the bionic gradient height structure is positioned at one end, far away from the tapered flow guide cone structure, of the inertial focusing link and is used for focusing and compressing the particles in the vertical direction; a sheath fluid focusing area is arranged at one end, far away from the inertial focusing link, of the bionic gradient height structure, and a sheath fluid inlet and a chip mixed liquid outlet are sequentially formed in one end, far away from the bionic gradient height structure, of the sheath fluid focusing area. According to the invention, efficient three-dimensional centered arrangement is realized, dynamic convergence of a particle trajectory from a dispersion state to a central single beam is realized, and a digital focusing regulation capability is realized.

Description

TECHNICAL FIELD

[0001] The present application relates to the technical field of microfluidic chips, in particular to a three-dimensional single-line focusing microfluidic chip with multiple fluid effect synergy and a method for single-line focusing of polystyrene microspheres. BACKGROUND

[0002] Microfluidic technology, as a technology for manipulating fluid at the micron scale, has been widely used in biomedical, chemical analysis and material science fields. Precise focusing and arrangement of microspheres or cells in microchannels is the premise for subsequent detection, analysis and sorting. In the prior art, microsphere focusing methods mainly include active (such as acoustic, magnetic, electric, optical, etc.) and passive (such as sheath liquid flow, Dean flow, etc.). Active microfluidic focusing technology includes but is not limited to dielectrophoresis (DEP) cell focusing technology, magnetophoresis (MAP) technology and acoustic focusing technology. Magnetophoresis (MAP) technology usually applies a gradient magnetic field around the microfluidic channel, and the force difference generated according to the magnetic property difference of the cells realizes the precise focusing of the cells; acoustic focusing technology mainly relies on the acoustic radiation force generated by the cells in the flow channel to realize the focusing of the cells. However, without exception, active microfluidic focusing technology needs to introduce an additional field source from the outside to realize focusing, which adds complexity to the design of the chip and the integration of the instrument.

[0003] Passive methods such as simple sheath liquid focusing are difficult to achieve single-line arrangement, and Dean flow focusing has problems such as deviation of the focusing track from the center and uncontrollable spacing between microspheres.

[0004] Therefore, in view of the problems in the related art, there is an urgent need for a three-dimensional single-line focusing microfluidic chip with multiple fluid effect synergy and a method for single-line focusing of polystyrene microspheres. SUMMARY

[0005] In view of the problems in the related art, the present application provides a three-dimensional single-line focusing microfluidic chip with multiple fluid effect synergy and a method for single-line focusing of polystyrene microspheres, which provides a microfluidic chip with simple structure, excellent focusing effect and strong controllability. Through the synergistic effect of inertial focusing and sheath liquid focusing, a stable arrangement of rigid microspheres in a single line, centered and with controllable spacing in the microchannel is achieved, solving the problems of unstable focusing, irregular arrangement and uncontrollable spacing in the prior art.

[0006] To this end, the specific technical solutions adopted by the present application are as follows:

[0007] According to one aspect of the present application, a three-dimensional single-line focusing microfluidic chip with multiple fluid effect synergy is provided, comprising:

[0008] The sample liquid inlet is provided with an inertial focusing link at one end, and a tapered flow guide cone structure is arranged between the inertial focusing link and the sample liquid inlet, which is used to suppress the initial dispersion diffusion trend of particles at the sample liquid inlet; a bionic gradual height structure is located at the end of the inertial focusing link away from the tapered flow guide cone structure, which is used to focus and compress the particles in the vertical direction; a sheath liquid focusing area is arranged at the end of the bionic gradual height structure away from the inertial focusing link, and a sheath liquid inlet and a chip mixed liquid outlet are sequentially arranged at the end of the sheath liquid focusing area away from the bionic gradual height structure.

[0009] Further, the inertial focusing link comprises a plurality of asymmetric curved focusing units connected in series; wherein the asymmetric curved focusing units comprise first and second curved pipes arranged alternately and forming a height difference of zero in the longitudinal direction, the width and the radius of curvature of the first curved pipe are smaller than those of the second curved pipe; wherein the sample liquid completes preliminary focusing under the coupling of inertial lift and Dean drag through the asymmetric curved focusing units, and is synchronously extruded by high-speed sheath liquid on both sides at the cross intersection of the sheath liquid focusing area, realizing central single-line arrangement, trajectory centering and controllable ball-ball spacing; the adjacent asymmetric curved focusing units are connected by a straight channel.

[0010] Further, the sheath liquid focusing area comprises a sample main flow channel and sheath liquid flow channels symmetrically arranged on both sides of the sample main flow channel and having an included angle of ninety degrees.

[0011] Further, the tapered flow guide cone structure is made of hydrophilic polydimethylsiloxane material or adopts a heterogeneous material combination configuration to enhance the interface wettability and structural stability; the tapered flow guide cone structure is designed at an included angle of twenty to sixty degrees.

[0012] Further, the height of the bionic gradual height structure along the liquid flow direction is first increased and then decreased, and the height range of the bionic gradual height structure is forty to sixty microns.

[0013] Further, the bionic gradual height structure forms a continuous and smooth height gradient in the length direction of the channel from the longitudinal section of the microscale channel; the height changes nonlinearly from the inlet section to the outlet section along the flow direction; the nonlinear change is based on the parameters of the initial channel height at the inlet, the maximum channel height, the length of the gradual section and the control slope change.

[0014] Further, a machine learning regression model is established based on the existing CFD data to solve the control slope change parameter, and the objective function of the machine learning regression model is:

[0015] ;

[0016] In the formula, is the focusing width; is the degree of particle deviation from the central trajectory. For the uniformity of particle spacing; To focus the weights of the width target, The weight of the target's deviation from the center trajectory. The weights are for the objective of uniform particle spacing.

[0017] Furthermore, the outlet detection channel of the sheath fluid focusing area is used to provide an optical detection or impedance detection window.

[0018] Furthermore, the substrate of the three-dimensional single-row focusing microfluidic chip is polydimethylsiloxane, and the upper and lower substrates of the substrate are bonded by oxygen plasma. The channel surface of the three-dimensional single-row focusing microfluidic chip is hydrophilized.

[0019] According to another aspect of the present invention, a method for single-line focusing of polystyrene microspheres using a three-dimensional single-row focusing microfluidic chip with synergistic multi-fluid effects is also provided, comprising:

[0020] Microspheres with a preset particle size range are dispersed in phosphate buffer; sample solution is injected into the sample solution inlet using a syringe pump; phosphate buffer is injected into the sheath fluid inlets on both sides;

[0021] Microspheres in phosphate buffer are subjected to the periodic action of Dean's secondary flow and shear lift in the inertial focusing region, achieving symmetrical initial focusing in the outer diameter direction within the channel cross-section;

[0022] At the crossroads of the sheath fluid focusing zone, the microspheres are compressed to the center of the channel by the sheath fluid flow on both sides with high Reynolds shear force, forming a narrow streamline with a stable trajectory. The distance between the microspheres is linearly adjusted with the sheath fluid flow rate ratio to achieve a three-dimensional single-row focusing effect in the detection area.

[0023] The beneficial effects of this invention are as follows:

[0024] 1. This invention integrates a periodically alternating asymmetric curved inertial focusing unit and a cross-shaped sheath fluid focusing structure. By utilizing multi-field coupling of inertial lift, Dean flow, and sheath fluid shearing effect, it achieves efficient three-dimensional centered arrangement of particles within a single-layer planar channel. This invention is the first to introduce an asymmetric curved structure for inertial pre-focusing, constructing a multi-period particle distribution induction mechanism. Combined with the synergistic compression zone formed by the two sheath fluids converging at a 90° angle on both sides, it achieves dynamic convergence of particle trajectories from a diffuse state to a central single beam. The chip structure adopts a modular design, possessing functional expandability compatible with optical / electrical detection windows. It allows for precise control of particle spacing by adjusting the sheath fluid flow rate and possesses digital focusing control capabilities.

[0025] 2. The chip structure of this invention is simple, has good focusing effect, and is highly controllable, making it suitable for high-throughput microsphere manipulation in fields such as material synthesis and biological detection. It possesses good liquid path stability and anti-clogging capabilities, making it suitable for the directional transport and high-throughput analysis of various particle types, including biological particles, functional microspheres, and microalgae. It is widely applicable to material synthesis, cell enrichment, and point-of-care testing (POCT) applications. Attached Figure Description

[0026] To more clearly illustrate the technical solutions in the embodiments of the present invention or the prior art, the drawings used in the embodiments will be briefly introduced below. Obviously, the drawings described below are only some embodiments of the present invention. For those skilled in the art, other drawings can be obtained based on these drawings without creative effort.

[0027] Figure 1 This is a schematic diagram of the overall structure of a microfluidic chip according to an embodiment of the present invention;

[0028] Figure 2 This is a structural diagram of an inertial focusing unit according to an embodiment of the present invention;

[0029] Figure 3 This is a diagram of a cross-shaped sheath fluid focusing structure according to an embodiment of the present invention;

[0030] Figure 4 This is a diagram of a biomimetic gradient height structure according to an embodiment of the present invention;

[0031] Figure 5 This is a structural diagram of the tapered guide cone inlet according to an embodiment of the present invention;

[0032] Figure 6 This is a high-speed photographic image showing the microsphere focusing effect according to an embodiment of the present invention.

[0033] Figure 7 This is a simulation diagram of sheath fluid focusing according to an embodiment of the present invention.

[0034] In the picture:

[0035] 1. Sample liquid inlet; 2. Inertial focusing stage; 21. First bend; 22. Second bend; 23. Asymmetric bending focusing unit; 3. Sheath liquid focusing area; 31. Sample main channel; 32. Sheath liquid flow channel; 4. Bionic gradual height structure; 5. Gradual converging guide cone structure; 6. Sheath liquid inlet; 7. Chip mixture outlet. Detailed Implementation

[0036] To further illustrate the various embodiments, the present invention provides accompanying drawings, which are part of the disclosure of the present invention. These drawings are mainly used to illustrate the embodiments and can be used in conjunction with the relevant descriptions in the specification to explain the operating principles of the embodiments. With reference to these drawings, those skilled in the art should be able to understand other possible implementation methods and the advantages of the present invention. The components in the drawings are not drawn to scale, and similar component symbols are generally used to represent similar components.

[0037] According to embodiments of the present invention, a three-dimensional single-row focusing microfluidic chip with multi-fluid effect synergy and a method for single-line focusing of polystyrene microspheres are provided.

[0038] The present invention will now be further described in conjunction with the accompanying drawings and specific embodiments, such as... Figures 1-7 As shown, according to an embodiment of the present invention, a three-dimensional single-row focusing microfluidic chip with multi-fluid effect synergy is provided, comprising: a sample liquid inlet 1, an inertial focusing link 2 disposed at one end of the sample liquid inlet 1, a tapered guide cone structure 5 disposed between the inertial focusing link 2 and the sample liquid inlet 1, for suppressing the initial dispersion and diffusion tendency of particles at the sample liquid inlet 1; a biomimetic gradient height structure 4, located at the end of the inertial focusing link 2 away from the tapered guide cone structure 5, for focusing and compressing particles in the vertical direction; a sheath fluid focusing region 3 disposed at the end of the biomimetic gradient height structure 4 away from the inertial focusing link 2, and a sheath fluid inlet 6 and a chip mixture outlet 7 sequentially disposed at the end of the sheath fluid focusing region 3 away from the biomimetic gradient height structure 4.

[0039] Using the above-described scheme, this invention achieves efficient three-dimensional centered alignment, enabling dynamic convergence of particle trajectories from a diffuse state to a central single beam, and possesses digital focusing and control capabilities. The chip structure is simple, has good focusing effect, strong controllability, and exhibits good liquid path stability and anti-clogging ability.

[0040] In one embodiment, the inertial focusing stage 2 includes 60-120 repeating, tandem asymmetric bending focusing units 23. Each asymmetric bending focusing unit 23 includes alternating first bends 21 (small bends) and second bends 22 (large bends) forming a zero height difference in the longitudinal direction. The width and radius of curvature of the first bend 21 are smaller than those of the second bend 22. The sample liquid undergoes initial focusing through the asymmetric bending focusing units 23 under the coupling of inertial lift and Dean's drag force, and is simultaneously squeezed by high-speed sheath fluid on both sides at the crossroads of the sheath fluid focusing area 3, achieving precise control of the particle's central single-line alignment, trajectory centering, and sphere-to-sphere spacing. Adjacent asymmetric bending focusing units 23 are connected by a direct current channel. The large bends have a width of 500-600µm and a radius of curvature of 900-1100µm, while the small bends have a width of 250-320µm and a radius of curvature of 280-330µm. The large and small bends are arranged alternately, forming a single-plane channel with zero height difference in the longitudinal direction. The inertial focusing region contains 100 asymmetric curved focusing units 23, which are connected by DC channels. The width of the DC channels is the same as the width of the small curved tube inlet.

[0041] In one embodiment, the sheath fluid focusing region 3 is located downstream of the inertial focusing region and includes a sample main channel 31 and sheath fluid channels 32 symmetrically arranged on both sides of the sample main channel 31 at an angle of 90 degrees. The three channels converge at a crossroads to form an outlet channel. The sample fluid inlet 1, the sheath fluid inlet 6, and the mixed liquid outlet 7 are respectively fluidly connected to the corresponding channels. The sample fluid inlet has a cone angle of 20°–60°, which is used to slow down the initial particle dispersion and promote the sample fluid flow to form a quasi-laminar flow state.

[0042] The sample liquid inlet structure is located in the upstream region of the chip and adopts a tapered flow guide structure.

[0043] In one embodiment, the tapered flow guide cone structure 5 is made of hydrophilic polydimethylsiloxane material or uses a combination of dissimilar materials, such as a glass-silicon composite structure, to enhance interfacial wettability and structural stability. The tapered flow guide cone structure 5 is designed with an included angle of 20 to 60 degrees, which can effectively suppress the initial dispersion and diffusion tendency of particles at the inlet and guide the sample liquid to form a stable flow state that is close to laminar flow, thereby improving the subsequent focusing efficiency.

[0044] In one embodiment, the height of the biomimetic gradient height structure 4 along the liquid flow direction first increases and then decreases, and the height range of the biomimetic gradient height structure 4 is forty to sixty micrometers. For example, it is set to have an inlet section of 45µm, a focusing section that gradually increases to 55µm, and a local contraction to 50µm at the sheath fluid confluence, thereby achieving vertical focusing and compression of particles.

[0045] The biomimetic gradient height structure 4 mimics the "narrow inlet / wide outlet" flow guidance strategy found in insect tracheal systems and plant xylem systems in nature. Starting from the longitudinal cross-section of the microscale channel, it creates a continuous and smooth height gradient along the channel's length. This flow guidance strategy effectively improves the stability and focusing effect of cell focusing, while also reducing the pressure in the sheath fluid entrainment region and enhancing the overall stability of the chip system.

[0046] In one embodiment, the biomimetic gradient height structure 4 starts from the longitudinal section of the microscale channel and forms a continuous and smooth height gradient along the length of the channel; the channel starts at a height of 40µm from the inlet section and gradually transitions to a height of 55µm along the flow direction to the outlet section, with the height change being nonlinear; the nonlinear change is represented by parameters based on the initial inlet channel height, the maximum channel height, the length of the gradient section, and the control slope change.

[0047] Nonlinear changes are represented as:

[0048] ;

[0049] In the formula, The initial height of the entrance channel is 40µm; Maximum channel height, 55µm; Position along the flow direction; The length of the transition segment; The parameters for controlling the slope change are taken in the range of [2, 8], and different biomimetic models are adjusted accordingly; the length of the height change segment is preferably 3–6 mm.

[0050] In one embodiment, a machine learning regression model, centered on a gradient boosting tree or neural network, is constructed based on existing CFD simulation data (covering design parameters such as different flow channel slopes and their corresponding focused performance indicators). This model learns the complex nonlinear mapping relationship between design parameters and performance indicators, forming a high-precision, second-level response performance proxy model. In the design, this model is embedded with optimization algorithms, such as Bayesian optimization, to quickly predict and inversely solve for the optimal flow channel slope parameter combination, thereby achieving an automated closed loop of simulation, prediction, and optimization. Ultimately, this achieves the solution of parameters controlling slope changes, enabling precise adaptive design of the chip structure. The objective function of the machine learning regression model is:

[0051] ;

[0052] In the formula, For focus width; The degree to which a particle deviates from its central trajectory; For the uniformity of particle spacing; The weights of each objective, To focus the weights of the width target, The weight of the target's deviation from the center trajectory. The weights represent the objective of uniform particle spacing. The objective function is to minimize the following: Parameters controlling the slope variation. The setting often relies on experience or fixed values. This invention solves the problem of unstable focusing performance caused by difficulty in adapting to different fluid conditions (such as particle size, flow rate, Reynolds number, etc.) by solving the parameters that control the slope change.

[0053] In one embodiment, the outlet detection channel of the sheath fluid focusing area 3 has a width of 180-220µm and a length of 1-3mm, and is used to provide an optical detection or impedance detection window, which can be used to integrate an optical detection or impedance detection module.

[0054] In one embodiment, the substrate of the three-dimensional single-row focusing microfluidic chip is polydimethylsiloxane, and the upper and lower substrates of the substrate are bonded by oxygen plasma treatment. The channel surface of the three-dimensional single-row focusing microfluidic chip is hydrophilized by polyethylene glycol modification.

[0055] According to another embodiment of the present invention, a method for single-line focusing of polystyrene microspheres using a three-dimensional single-row focusing microfluidic chip with synergistic multi-fluid effects is also provided, comprising:

[0056] Microspheres with a preset particle size range are dispersed in phosphate buffer; the sample solution is injected into sample solution inlet 1 using a syringe pump; the phosphate buffer is injected into sheath fluid inlets 6 on both sides;

[0057] Microspheres in phosphate buffer are subjected to the periodic action of Dean's secondary flow and shear lift in the inertial focusing region, achieving symmetrical initial focusing in the outer diameter direction within the channel cross-section;

[0058] At the cross intersection of the sheath fluid focusing zone 3, the microsphere particles are compressed to the center of the channel by the sheath fluid flow on both sides with high Reynolds shear force, forming a narrow stream line with a stable trajectory. The distance between the microspheres is linearly adjusted with the sheath fluid flow rate ratio to achieve a three-dimensional single-row focusing effect in the detection area.

[0059] In one embodiment, rigid microspheres with a particle size of 10-15 µm are dispersed in PBS solution; the sample solution is dispensed at a rate of 700-720 µL / min using a syringe pump. -1 Inject into the sample inlet; simultaneously at 400-900 µL / min -1PBS solution is injected into the sheath fluid inlets on both sides; the microspheres are first subjected to the periodic action of Dean's secondary flow and shear lift in the inertial focusing zone, achieving symmetrical initial focusing in the outer diameter direction within the channel cross section; then in the cross-shaped sheath fluid focusing zone, the particles are compressed to the center of the channel by the sheath fluid flow on both sides with high Reynolds shear force, forming a narrow stream line with a stable trajectory. The distance between the microspheres can be linearly adjusted with the sheath fluid flow rate ratio, and finally a three-dimensional single-row focusing effect is achieved in the detection area.

[0060] This invention utilizes standard soft lithography to fabricate a microfluidic chip for three-dimensional focusing. The specific process flow is as follows: Using SU-8 1070 photoresist, a positive mold with a channel height of 50 μm is fabricated on a 4-inch single-sided polished silicon wafer using ultraviolet lithography. Subsequently, polydimethylsiloxane (PDMS, Sylgard 184) prepolymer is cast onto the positive mold, and after curing and peeling, a PDMS substrate with a precise microchannel structure is obtained. The PDMS substrate and glass slide are subjected to oxygen plasma surface treatment, and after alignment and bonding, they are permanently encapsulated by bonding on a 75°C hot stage for 3 hours. To improve hydrophilicity, the inner surface of the channel is modified with polyethylene glycol (PEG) to effectively prevent bubble retention and particle adsorption during the experiment.

[0061] In the particle focusing performance test, a 10 μm polystyrene microsphere ethanol suspension with PBS as the solute was used as the sample solution, and PBS solution was used as the sheath fluid. The chip inlet was reliably connected to the injection pump system via a tetrafluoroethylene conduit, and particle motion behavior was recorded in real time on an observation platform equipped with an inverted microscope and a high-speed camera (acquisition frequency no less than 200 fps). Simulation of the sheath fluid focusing process was performed for verification. When the sample solution flow rate was maintained at 710 μL / min (the optimal focusing parameter determined through system optimization) and the sheath fluid flow rate was set to 600 μL / min, a stable focusing flow pattern of microspheres was observed in the microchannel. High-speed image analysis showed that the single-row particle throughput reached 97%, indicating good focusing effect. These quantitative results fully validate that the chip achieved excellent three-dimensional focusing effect under the stated parameters, providing a reliable platform for subsequent cell detection or particle analysis applications.

[0062] In summary, the chip of this invention integrates a periodically alternating asymmetric curved inertial focusing unit and a cross-shaped sheath fluid focusing structure. By utilizing multi-field coupling of inertial lift, Dean flow, and sheath fluid shearing effects, it achieves efficient three-dimensional centered alignment of particles within a single-layer planar channel. This invention is the first to introduce an asymmetric curved structure for inertial pre-focusing, constructing a multi-periodic particle distribution induction mechanism. Combined with the synergistic compression zone formed by the two sheath fluids converging at a 90° angle on both sides, it achieves dynamic convergence of particle trajectories from a diffuse state to a central single beam. The chip structure adopts a modular design, possessing functional expandability compatible with optical / electrical detection windows. It allows for precise control of particle spacing by adjusting the sheath fluid flow rate and possesses digital focusing control capabilities. The chip structure of this invention is simple, has good focusing effect, and strong controllability, making it suitable for high-throughput microsphere manipulation in fields such as material synthesis and biodetection. It exhibits good fluid path stability and anti-clogging capabilities, making it suitable for the directional transport and high-throughput analysis of various particle types, including biological particles, functional microspheres, and microalgae. It is widely applicable to material synthesis, cell enrichment, and point-of-care testing (POCT) applications.

[0063] In this invention, unless otherwise explicitly specified and limited, the terms "installation," "setting," "connection," "fixing," "screw connection," etc., should be interpreted broadly. For example, they can refer to a fixed connection, a detachable connection, or an integral part; they can refer to a mechanical connection or an electrical connection; they can refer to a direct connection or an indirect connection through an intermediate medium; they can refer to the internal connection of two components or the interaction between two components. Unless otherwise explicitly limited, those skilled in the art can understand the specific meaning of the above terms in this invention according to the specific circumstances.

[0064] The above description is only a preferred embodiment of the present invention and is not intended to limit the present invention. Any modifications, equivalent substitutions, improvements, etc., made within the spirit and principles of the present invention should be included within the protection scope of the present invention.

Claims

1. A multi-fluid effect synergistic three-dimensional single-line focusing microfluidic chip, characterized in that, The three-dimensional single-column focusing microfluidic chip comprises: a sample liquid inlet (1), one end of which is provided with an inertial focusing link (2), and a tapered flow guide cone structure (5) is arranged between the inertial focusing link (2) and the sample liquid inlet (1), which is used to suppress the initial dispersion diffusion trend of particles at the sample liquid inlet (1); a bionic gradually changing height structure (4) located at one end of the inertial focusing link (2) away from the tapered flow guide cone structure (5), which is used to focus and compress particles in the vertical direction; a sheath liquid focusing area (3) is arranged at one end of the bionic gradually changing height structure (4) away from the inertial focusing link (2), and a sheath liquid inlet (6) and a chip mixed liquid outlet (7) are sequentially arranged at one end of the sheath liquid focusing area (3) away from the bionic gradually changing height structure (4).

2. The multi-fluid effect synergistic three-dimensional single-channel focusing microfluidic chip according to claim 1, wherein, The inertial focusing link (2) comprises a plurality of asymmetric curved focusing units (23) connected in series; The asymmetric curved focusing unit (23) comprises first and second curved pipes (21) and (22) arranged alternately and forming a height difference of zero in the longitudinal direction, and the width and curvature radius of the first curved pipe (21) are smaller than those of the second curved pipe (22); The sample liquid is preliminarily focused under the coupling of inertial lift force and Dean drag force through the asymmetric curved focusing unit (23), and is synchronously extruded by high-speed sheath liquid on both sides at the cross intersection of the sheath liquid focusing area (3), so as to realize the center single-line arrangement, trajectory centering and controllable ball-ball spacing; The adjacent asymmetric curved focusing units (23) are connected by straight channels.

3. The multi-fluid effect synergistic three-dimensional single-channel focusing microfluidic chip according to claim 1, wherein, The sheath liquid focusing area (3) comprises a sample main flow channel (31) and sheath liquid flow channels (32) symmetrically arranged on both sides of the sample main flow channel (31) and having an included angle of 90 degrees.

4. The multi-fluid effect synergistic three-dimensional single-channel focusing microfluidic chip according to claim 1, wherein, The tapered flow guide cone structure (5) is made of hydrophilic polydimethylsiloxane material or adopts a heterogeneous material combination configuration to enhance the interface wettability and structural stability. The tapered flow guide cone structure (5) is designed to have an included angle of 20 to 60 degrees.

5. The multi-fluid effect synergistic three-dimensional single-channel focusing microfluidic chip according to claim 1, wherein, The height of the bionic gradually changing height structure (4) along the liquid flow direction has a trend of first increasing and then decreasing, and the height range of the bionic gradually changing height structure (4) is 40 to 60 microns.

6. The multi-fluid effect synergistic three-dimensional single-channel focusing microfluidic chip according to claim 5, wherein, The bionic gradually changing height structure (4) forms a continuous and smooth height gradient in the length direction of the microscale channel from the longitudinal section of the channel. From the inlet section, the channel gradually transitions to the outlet section along the flow direction, and the height changes nonlinearly. The nonlinear change is represented based on the parameters of the initial channel height at the inlet, the maximum channel height, the length of the gradient section and the control slope change.

7. The multi-fluid effect synergistic three-dimensional single-channel focusing microfluidic chip according to claim 6, wherein, A machine learning regression model is established based on the existing CFD data to solve the control slope change parameter, and the objective function of the machine learning regression model is: ； wherein is the focus width, is the particle off-centering, is the particle spacing uniformity, is the weight of the focus width target, is the weight of the particle off-centering target, is the weight of the particle spacing uniformity target.

8. The multi-fluid effect synergistic three-dimensional single-channel focusing microfluidic chip according to claim 1, wherein, The outlet detection channel of the sheath liquid focusing area (3) is used to provide an optical detection or impedance detection window.

9. The multi-fluid effect synergistic three-dimensional single-channel focusing microfluidic chip according to claim 1, wherein, The substrate of the three-dimensional single-column focusing microfluidic chip is polydimethylsiloxane, and the upper and lower substrates are bonded by oxygen plasma, and the channel surface of the three-dimensional single-column focusing microfluidic chip is hydrophilic treated.

10. A method for realizing single-line focusing of polystyrene microspheres using the multi-fluid effect synergistic three-dimensional single-channel focusing microfluidic chip according to any one of claims 1-9, characterized in that, The three-dimensional single-column focusing microfluidic chip comprises: The microspheres with a preset particle size range are dispersed in a phosphate buffer solution; a sample liquid is injected into the sample liquid inlet (1) through a syringe pump; and the phosphate buffer solution is injected into the sheath liquid inlets (6) on both sides; The microspheres in the phosphate buffer solution are subjected to periodic action of Dean secondary flow and shear lift force in the inertial focusing zone, and are symmetrically initially focused in the outward radial direction in the cross section of the channel; At the cross intersection of the sheath liquid focusing zone (3), the microsphere particles are compressed to the center of the channel by the high Reynolds shear force of the sheath liquid flow on both sides, forming a narrow beam streamline, the trajectory is stable, and the ball-ball spacing is linearly adjusted with the sheath liquid flow rate, so as to realize three-dimensional single-column focusing effect in the detection area.

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