CN114149893A

CN114149893A - Particle self-clamping flow type micro-fluidic chip, manufacturing method thereof and particle self-dispersion method

Info

Publication number: CN114149893A
Application number: CN202111393286.0A
Authority: CN
Inventors: 马波; 刘沣仪; 徐健
Original assignee: Qingdao Institute of Bioenergy and Bioprocess Technology of CAS
Current assignee: Qingdao Institute of Bioenergy and Bioprocess Technology of CAS
Priority date: 2021-11-23
Filing date: 2021-11-23
Publication date: 2022-03-08
Also published as: WO2023093404A1

Abstract

The invention relates to a particle self-pinch microfluidic chip, a manufacturing method thereof and a particle self-dispersion method, wherein the particle self-pinch microfluidic chip is provided with a particle sample inlet hole and a particle sample outlet hole, and a microfluidic channel is arranged inside the particle self-pinch microfluidic chip and comprises a main channel, a side channel and a pinch structure; the upper end of the side channel is communicated and connected with the upstream of the main channel, the micro-fluid channel is provided with a particle interception structure, the lower end of the side channel is communicated and connected with the downstream of the main channel, and the flow clamping structure is positioned at the communication and connection position of the lower end of the side channel and the downstream of the main channel; the particle sample inlet hole is communicated and connected with a particle inlet of the main channel, and the particle sample outlet hole is communicated and connected with a particle outlet of the entrained flow structure; the chip can disperse the particles which are closely arranged, does not need to additionally separate oil and driving equipment, simplifies the device, can be repeatedly utilized and reduces the cost; the particle self-dispersion method adopting the chip is suitable for self-entrainment of particles with various sizes, and is simple and convenient to operate; the manufacturing method of the chip has high yield.

Description

Particle self-clamping flow type micro-fluidic chip, manufacturing method thereof and particle self-dispersion method

Technical Field

The invention relates to the technical field of micro-fluidic chips, in particular to a micro-particle self-clamping micro-fluidic chip which forms single micro-particle sequential flow with certain intervals by self-clamping flow, a manufacturing method thereof and a micro-particle self-dispersion method.

Background

As is well known, with the progress of modern science and technology, the research on the application field of human beings has been advanced to the micro-scale range, and the research on the micro-nano micromanipulation technology is more and more concerned by scholars at home and abroad. The separation of particles is a key technology in microfluidic manipulation, and is widely applied to various fields such as bioengineering, medicine, nano self-assembly, chemical analysis, material performance evaluation and the like, including the research on single cells, proteins, nano materials, liquid drops and the like. For example, analysis of single cells allows analysis of the differences between individual cells including cell size, growth rate, chemical composition (phospholipids, proteins, metabolites, DNA/RNA), and the cause and mechanism of the differences between cells. The research content relates to the fields of tumor biology, stem cells, microbiology, neurology, immunology and the like. Droplet-based microfluidic chip technology is compatible with a wide range of chemical and biological reagents and "electronic controls" and has good programmability and constructability. In most applications, uniform droplets can ensure constant, controllable and predictable results. The application based on liquid drops mainly focuses on the fields of liquid drop manipulation, liquid drop digital PCR (chip method), including dd PCR (micro drop digital chip method), liquid drop sorting, liquid drop detection and the like.

With the growing interest in the field of microfluidic-based chips, more technologies for controlling, manipulating and functionalizing single cells, proteins, nanomaterial droplets, and other particles have been developed. Wherein the diameter of the particles can be in the order of micrometers to nanometers. The encapsulation of single cells, proteins, nucleic acids, etc. and the synthesis of micro/nanoparticles can be achieved by using microfluidic technology. To operate on a single particle in-line, the particle is first injected into the chip. However, the current particle re-injection technology generally needs to add one or two additional oil phases as sheath fluid to disperse the closely arranged particles. Therefore, a large amount of entrained flow liquid and a large amount of reagents are consumed, and the experiment cost is greatly increased; and the entrained flow liquid needs extra pump driving equipment such as peristaltic pump, leads to equipment structure complicacy, can't accomplish miniaturization and portablely, further increases the experiment cost.

Disclosure of Invention

In view of the above problems, an object of the present invention is to provide a particle self-trapping microfluidic chip, a method for manufacturing the same, and a particle self-dispersing method, by which closely arranged particles can be dispersed to form a single-particle sequential flow with an interval, an additional trapping liquid and a driving device are not required, a cost for re-injecting the particles is reduced, a peristaltic pump and other devices are not required, and simplification of the apparatus is achieved.

In order to achieve the purpose, the invention adopts the following technical scheme:

firstly, the invention provides a particle self-pinch microfluidic chip, wherein at least one microfluidic channel is arranged in the particle self-pinch microfluidic chip, and the microfluidic channel comprises a main channel, at least one section of side channel and a pinch structure;

the upper end of the main channel is provided with a particle phase inlet, and the lower end of the flow clamping structure is provided with a particle outlet;

the upper end of the side channel is communicated and connected with the upstream of the main channel, the microfluidic channel is provided with a particle interception structure for preventing particles from entering the side channel, the lower end of the side channel is communicated and connected with the downstream of the main channel, and the flow clamping structure is positioned at the communication and connection position of the lower end of the side channel and the downstream of the main channel;

the particle self-pinch micro-fluidic chip is provided with a particle sample inlet hole and a particle sample outlet hole, the particle sample inlet hole is communicated and connected with the particle inlet, and the particle sample outlet hole is communicated and connected with the particle outlet.

Preferably, the height of the joint of the side channel and the main channel is higher than the height of the side channel to form a height difference value; the particle interception structure is a particle interception structure arranged at the joint of the side channel and the main channel, or a size difference structure formed by the width of the side channel being smaller than the diameter of the liquid drop, or a size difference structure formed by the height difference being smaller than the diameter of the liquid drop, or a combination of two or three structures.

Preferably, when the particle blocking structure is arranged at the joint of the side channel and the main channel, the particle blocking structure is a micro-sieve array structure.

Preferably, a bubble blocking structure is arranged at the particle sample inlet.

Preferably, the bubble blocking structure is a micro-sieve array structure.

Preferably, at least one serpentine channel is provided between the downstream of the main channel and the pinch structure.

Preferably, at least one branch channel is arranged at the downstream of the main channel, and the downstream of at least one branch channel is communicated with the lower end of the side channel and connected to the flow clamping structure.

Preferably, a positive pressure driving device is provided upstream of the microparticle sampling hole, or a negative pressure driving device is provided downstream of the microparticle sampling hole.

The invention further provides a method for manufacturing the particle self-pinch microfluidic chip, which is used for manufacturing any one of the particle self-pinch microfluidic chips, and comprises the following steps:

preparing a silica gel template:

designing a main channel structure with the main channel and a side channel structure with the side channel through drawing software, and printing;

dripping photoresist on the silicon wafer, throwing the photoresist, covering the side channel structure of the mask with the photoresist, and exposing;

after the exposed silicon wafer is subjected to spin coating again, covering the silicon wafer with a mask to cover the main channel structure, aligning the main channel structure with the side channel structure, and then exposing;

washing the uncured part by a developing solution to obtain the silicon wafer template;

preparing an upper PDMS chip with a channel structure:

mixing PDMS monomer with curing agent to obtain PDMS high polymer;

pouring the PDMS high polymer on the silicon wafer template, and drying to obtain the upper PDMS chip with the main channel structure and the side channel structure;

preparing the particle sample inlet and the particle sample outlet on the upper PDMS chip:

punching the particle sample inlet hole and the particle sample outlet hole in the upper PDMS chip with the channel structure;

adopting a PDMS smooth substrate without an etching pattern for a lower chip to be bonded;

preparing the particle self-pinch microfluidic chip:

and bonding the upper PDMS chip and the lower chip by plasma to obtain the particle self-pinch microfluidic chip, and after the particle self-pinch microfluidic chip is placed, recovering the hydrophobicity of the particle self-pinch microfluidic chip.

The invention further provides a particle self-dispersion method, which is realized by adopting any one of the particle self-pinch microfluidic chips, and comprises the following steps:

and (3) accessing a sample introduction end of the particle phase: connecting the sample introduction end of the particle phase into the particle sample introduction hole;

connecting a pressure driving device: connecting the particle sample inlet hole to a positive pressure driving device, or connecting the particle sample outlet hole to a negative pressure driving device;

separating the particles from the fluid: driving the positive pressure driving device or the negative pressure driving device to enable the droplet phase at the sample feeding end of the particle phase to flow into the particle self-pinch micro-fluidic chip through the particle sample feeding hole; the particle phase separates particles and entrained flow liquid at the lower end of the main channel and the lower end of the side channel respectively;

collecting the microparticles and the entrained flow liquid and dispersing the microparticles: collecting the separated particles and the entrained flow liquid in the entrained flow structure, and self-dispersing the particles through the entrained flow liquid;

obtaining the microparticles after self-dispersion: and enabling the self-dispersed particles to flow out through the particle sample outlet.

Due to the adoption of the technical scheme, the invention has the following advantages:

1. the particle self-pinch micro-fluidic chip and the particle self-dispersion method provided by the invention are suitable for self-dispersion of particles with various sizes, and the height and width of a channel can be adjusted according to the diameter of the particles;

2. the particle self-pinch micro-fluidic chip and the particle self-dispersion method provided by the invention have the advantages that the part of the fluid filtered from the particle phase is taken as the pinch fluid, the consumption of the pinch fluid is reduced, and no additional pump device is needed, so that the particle self-pinch micro-fluidic chip can be miniaturized and portable;

3. the particle self-pinch micro-fluidic chip and the particle self-dispersion method provided by the invention have the advantages that the chip can be repeatedly used, and the operation cost is reduced;

4. the manufacturing method of the particle self-entrained flow microfluidic chip and the particle self-dispersion method provided by the invention are simple and convenient to operate, the yield of the manufacturing method is high, and the product quality can be ensured.

Drawings

Fig. 1 is a schematic structural diagram of a microparticle self-pinch microfluidic chip provided in embodiment 1 of the present invention;

FIG. 2 is a schematic structural view of a flow cell of example 1 including a serpentine channel and a particle-blocking structure and a bubble-blocking structure;

fig. 3 is a schematic view of a particle-blocking structure and a bubble-blocking structure of example 1 of the present invention;

FIG. 4 is a schematic diagram of a side channel of embodiment 1 of the present invention having a bent structure and a main channel width greater than a side channel width at a pinch structure;

FIG. 5 is a schematic diagram of embodiment 1 of the present invention in which two side channels are bent and the width of the main channel is greater than that of the side channels at the position of the pinch structure;

FIG. 6 is a schematic diagram of a side channel of embodiment 1 of the present invention having a meandering structure and a main channel width equal to a side channel width at a pinch structure;

FIG. 7 is a schematic diagram of a flow channel structure with two side channels in a folded configuration, wherein the width of the main channel is equal to the width of the side channels in the pinch flow configuration according to example 1 of the present invention;

FIG. 8 is a schematic diagram of a side channel of embodiment 1 of the present invention having a bent structure and a main channel width smaller than that of the side channel at the position of the entrained-flow structure;

FIG. 9 is a schematic diagram of embodiment 1 of the present invention in which two side channels are bent and the width of the main channel is greater than that of the side channels at the position of the pinch structure;

FIG. 10 is a schematic view showing a side passage of an arc-shaped bent structure according to example 1 of the present invention;

FIG. 11 is a schematic view showing a structure in which two side passages are curved in an arc shape according to example 1 of the present invention;

FIG. 12 is a schematic view showing a side channel of a meandering structure and a main channel provided with two or more branch channels in example 1 of the present invention;

FIG. 13 is a schematic view of a main channel with two side channels of a bent structure and two or more branch channels in the main channel in example 1 of the present invention;

fig. 14 is a flowchart of a method for manufacturing a micro-particle self-pinch microfluidic chip according to example 2 of the present invention;

FIG. 15 is a schematic structural view of a main channel in embodiment 2 of the present invention;

FIG. 16 is a schematic view of a side channel structure according to embodiment 2 of the present invention;

fig. 17 is a flow chart of a method of using the microparticle self-pinch microfluidic chip according to example 3 of the present invention;

FIG. 18 is a diagram showing the effect of self-entrainment of droplets in example 3 of the present invention;

fig. 19 is a flow chart of a method of using the microparticle self-pinch microfluidic chip according to example 4 of the present invention;

FIG. 20 is a graph of the fluorescent signal of a droplet in example 4 of the present invention;

FIG. 21 is a flow chart of a microparticle self-dispersion method of example 5;

FIG. 22 is a flowchart of a microparticle self-dispersion method of example 6;

FIG. 23 is a flow chart of a microparticle self-dispersion method of example 7;

FIG. 24 is a flowchart of a microparticle self-dispersion method of example 8;

FIG. 25 is a flowchart of a microparticle self-dispersion method of example 9;

fig. 26 is a flowchart of a microparticle self-dispersion method of example 10.

Reference numbers in the figures: the device comprises a particle sample inlet 1, a main channel 2, a side channel 3, a flow clamping structure 4, a particle sample outlet 5, a bubble blocking structure 6, a particle blocking structure 7, a snake-shaped channel 8, a main channel structure A and a side channel structure B.

Detailed Description

In order to make the objects, technical solutions and advantages of the present invention more apparent, the technical solutions of the present invention will be clearly and completely described below with reference to the accompanying drawings. It is to be understood that the embodiments described are only a few embodiments of the present invention, and not all embodiments. All other embodiments, which can be derived by a person skilled in the art from the embodiments given herein without making any creative effort, shall fall within the protection scope of the present invention.

In the description of the present invention, it should be noted that the terms "upper", "lower", "front", "rear", and the like indicate orientations or positional relationships based on the orientations or positional relationships shown in the drawings, and are only for convenience in describing the present invention and simplifying the description, but do not indicate or imply that the system or component in question must have a specific orientation, be constructed in a specific orientation, and be operated, and thus should not be construed as limiting the present invention.

In the description of the present invention, it is to be noted that, unless otherwise explicitly specified or limited, the terms "assembled", "disposed" and "connected" are to be construed broadly, e.g., as meaning either a fixed connection, a detachable connection, or an integral connection; can be mechanically or electrically connected; the two components can be directly connected or indirectly connected through an intermediate medium, and the two components can be communicated with each other. The specific meanings of the above terms in the present invention can be understood in specific cases to those skilled in the art.

The invention aims to provide a particle self-pinch type micro-fluidic chip, a manufacturing method thereof and a particle self-dispersion method, wherein the particle self-pinch type micro-fluidic chip can disperse particles which are closely arranged to form single particle sequential flow with a certain interval, no additional pinch liquid and driving equipment are needed, the cost of particle reinjection is reduced, no peristaltic pump or other equipment is needed, and the simplification of a device is realized.

The following describes in detail a particulate self-entrained microfluidic chip provided by an embodiment of the present invention with reference to the accompanying drawings.

Example 1

As shown in fig. 1, in the microparticle self-pinch microfluidic chip provided in this embodiment, at least one microfluidic channel is disposed in the microparticle self-pinch microfluidic chip, and the microfluidic channel includes a main channel 2, at least one section of side channel 3, and a pinch structure 4;

the upper end of the main channel 2 is provided with a particle phase inlet, and the lower end of the flow clamping structure 4 is provided with a particle outlet;

the upper end of the side channel 3 is communicated and connected with the upstream of the main channel 2, the micro-fluid channel is provided with a particle interception structure 7 for preventing particles from entering the side channel 3, the lower end of the side channel 3 is communicated and connected with the downstream of the main channel 2, and the flow clamping structure 4 is positioned at the communication and connection position of the lower end of the side channel 3 and the downstream of the main channel 2;

the particle self-pinch micro-fluidic chip is also provided with a particle sample inlet hole 1 and a particle sample outlet hole 5, the particle sample inlet hole 1 is communicated and connected with a particle phase inlet of the main channel 2, and the particle sample outlet hole 5 is communicated and connected with a particle outlet of the pinch structure 4.

When the particle self-pinch microfluidic chip provided by this embodiment is used, a particle phase enters the main channel 2 of the particle self-pinch microfluidic chip through the droplet inlet 1, because a particle blocking structure is arranged between the main channel 2 and the side channel 3, particles in the particle phase can only flow into the main channel 2 and cannot enter the side channel 3, and a part of fluid in the particle phase enters the side channel 3 as a pinch fluid and finally converges at the pinch structure 4 at the downstream of the main channel 2, so that the pinch fluid filtered into the side channel 3 converges with particles in the main channel 2, and further, the closely arranged particle drops are self-dispersed at the pinch structure 4 under the action of the pinch fluid. As additional entrained flow liquid and driving equipment are not needed, the cost of particle reinjection is reduced, equipment such as a peristaltic pump is not needed, and the simplification of the device is realized. The particle self-pinch microfluidic chip can be applied to cell single wrapping, cell monodispersion, droplet fluorescence signal detection and microorganism droplet signal detection, can also be applied to coupling interfaces of flow sorting and mass spectrometry after droplet enzyme activity screening and microbial component droplet culture and cell sequencing, and is suitable for the microfluidic fields of cell sorting, droplet manipulation, droplet detection, droplet counting, droplet sorting, digital nucleic acid amplification and the like; the method for forming the particle self-entrained flow and sorting further comprises the step of carrying out further operation on the sorted target particles, dispersing the particles which are closely arranged, and forming a single particle sequential flow with intervals, wherein the intervals can be micrometer to meter, and the operation comprises single cell sequencing, single cell morphological analysis, single cell culture and the like.

As shown in fig. 3, in the above embodiment, the particulate blocking structure 7 is a particulate blocking structure provided at the upstream communication connection of the upper end of the side passage 3 with the main passage 2. The particle blocking structure prevents the particles in the particle phase from flowing into the side channel 3, so that part of the fluid in the particle phase enters the side channel 3, and separation of the particles from the fluid is promoted when the particle phase flows through the branch of the upper end of the side channel 3 and the main channel 2.

As shown in fig. 3, in the above embodiments, the particle blocking structure may be a micro-sieve array structure, but is not limited to the micro-sieve array structure. The particle blocking structure of the micro-sieve array structure can further improve the separation effect and separation efficiency of particles and entrained flow liquid when the particles flow through the bifurcation of the upper end of the side channel 3 and the main channel 2. In the above embodiment, as shown in fig. 1, the height of the junction between the side channel 3 and the main channel 2 is higher than the height of the side channel 3 to form a height difference; the particle interception structure 7 is a size difference structure formed by the width of the side channel 3 being smaller than the diameter of the particles, or a size difference structure formed by the height difference being smaller than the diameter of the particles, or a combination of the two structures.

That is, when the inside of the side passage 3 is a circular passage, the size difference structure in which the diameter of the inner passage is smaller than the diameter of the fine particles constitutes the fine particle blocking structure 7; or when the inside of the side channel 3 is a non-circular channel such as a rectangle, the minimum width of the inner channel is smaller than the size difference structure formed by the diameters of the particles to form the particle interception structure 7. Or relative to the connection between the side channel 3 and the main channel 2, i.e. the branch of the side channel 3 and the main channel 2, the height of the connection is higher than the height of the upper inlet end of the side channel 3, i.e. the height of the branch is higher than the height of the upper inlet end of the side channel 3, and the size difference structure with the height difference smaller than the diameter of the particles forms the particle interception structure 7.

It should be noted that, when the interior of the main channel 2 is a circular channel, the diameter of the internal channel is larger than the diameter of the particles; or when the inside of the main channel 2 is a non-circular channel such as a rectangle, the minimum width of the inner channel is larger than the diameter of the particles. Or relative to the junction of the side channel 3 and the main channel 2, namely the branch of the side channel 3 and the main channel 2, the height of the upper inlet end of the main channel 2 is higher than that of the junction, namely the height of the upper inlet end of the main channel 2 is higher than that of the branch, and the height difference is larger than the diameter of the particles.

In sum, the height of the joint of the side channel 3 and the main channel 2 is higher than that of the side channel 3 to form a height difference; the particle stopping structure 7 is a particle stopping structure 7 arranged at the joint of the side channel 3 and the main channel 2, or a size difference structure formed by the width of the side channel 3 being smaller than the diameter of the particles, or a size difference structure formed by the height difference being smaller than the diameter of the particles, or a combination of two or three structures. So that particles can enter through the main channel 2 but not through the side channels 3.

In the above embodiment, as shown in fig. 1, the height and width of the upper end of the main channel 2 are both larger than the diameter of the particles, so as to reduce the flow resistance of the side channel 3 relative to the main channel 2 and prevent the particles from entering the side channel 3.

In the above embodiment, the height or width of the upper end of the main channel 2 is greater than the height or width of the upper end of the side channel 3, the height and width of the upper end of the main channel 2 are both greater than the diameter of the particles, the height or width of the upper end of the side channel 3 is less than the diameter of the particles, and the particle blocking structure 7 at the upstream communication connection of the upper end of the side channel 3 and the main channel 2 is provided for better preventing the particles from entering the side channel 3. It is further possible that the height or width of the lower end of the main channel 2 is greater than the height or width of the lower end of the side channel 3, i.e. the overall height or width of the main channel 2 is greater than the overall height or width of the side channel 3, which serves to promote a reduction in the flow resistance of the side channel 3 relative to the main channel 2, and to better prevent particles from entering the side channel 3.

In the above embodiment, as shown in fig. 3, the bubble blocking structure 6 (e.g., a micro-sieve array structure) is disposed at the particle sampling hole 1, and the bubble blocking structure 6 is used to prevent bubbles from flowing into the main channel 2.

In the above embodiment, the bubble-blocking structure 6 may be a micro-sieve array structure, but is not limited to an array structure. The bubble blocking structure 6 of the micro-sieve array structure can improve the effect and efficiency of preventing bubbles from entering the main channel 2.

In the above embodiment, as shown in fig. 2, at least one serpentine channel 8 is provided between the downstream of the main channel 2 and the pinch structure 4. The serpentine channel 8 can act as a slow flow of particles.

As shown in fig. 1, in the above embodiment, in order to improve the separation efficiency and separation effect of the particles and the fluid in the particulate phase, the number of the side channels 3 may be two or more. In addition, in order to make the fluid in the particulate phase more uniformly and sufficiently separated from the particulates, two or more segments of the side passages 3 may be connected in communication with the main passage 2 in a uniformly distributed manner.

In the above embodiment, the downstream of the main channel 2 may be narrowed, that is, the inner diameter of the downstream of the main channel 2 is smaller than that of the upstream thereof, so as to facilitate the connection of the lower end of the side channel 3 with the downstream of the main channel 2 to form a collecting structure, and to promote the fluid in the side channel 3 to act as an entrained flow to disperse the particles in the main channel 2. Wherein the inner diameter downstream of the main channel 2 may decrease linearly in size.

In the above embodiments, the particle sampling hole 1 enters the through hole of the particle self-sandwich microfluidic chip as a particle phase, and the upstream of the particle sampling hole may be a droplet generation structure, but is not limited to this structure. Namely, the particle sampling hole 1 can be connected with the liquid drop sampling end of the liquid drop generating structure. The oil phase of the liquid drop can be fluorocarbon oil, mineral oil, silicone oil, vegetable oil and the like; the aqueous phase may be pure water, a culture solution, a bacterial solution, a reaction solution, a cultured droplet, or the like.

In the above embodiments, the microparticle sampling hole 1 enters the through hole of the microparticle self-sandwich microfluidic chip as a microparticle phase, and the upstream of the microparticle sampling hole may be a cell suspension, a microsphere, or the like, but is not limited to this fluid. The cell suspension, microspheres, etc. may be dispersed in pure water, culture solution, reaction solution, cultured droplets, etc.

In the above embodiment, as shown in fig. 3, the main channel 2 is used as a particle phase channel, and the front structure connected to the particle sampling hole 1 may be an array structure of the bubble blocking structure 6 to prevent the inflow of bubbles. Upstream of the bubble-impeding structure 6 may be a droplet generation, culture, reaction structure, but is not limited to such a structure.

In the above embodiments, the side channels 3 serve as the entrained-flow liquid channels in the particulate phase, and as mentioned above, an array-structured particulate blocking structure may be added at the interface between the main channel 2 and the side channels 3, but the invention is not limited to this structure. The purpose is to prevent the flow of particles into the side channels. Or the inner diameter of the side passage 3 may be reduced in size relative to the diameter of the particles to prevent the particles from entering the side passage 3.

In the above embodiment, as shown in fig. 1, the entrainment structure 4 is located at the intersection of the main channel 2 and the side channel 3, and the self-dispersion of the densely arranged particles by the entrainment liquid is realized in the structure.

In the above embodiments, the microparticle sampling hole 5 serves as a through hole for the microparticle to flow out of the droplet self-dispersion microfluidic chip, and the downstream thereof may be a signal detection structure, a sorting structure, or the like, but is not limited to this structure. Namely, the particle sample outlet 5 can be communicated and connected with the sample inlet end of the signal detection structure and the sorting structure.

As shown in fig. 13, in the above embodiment, more than two branch passages may be provided downstream of the main passage 2. At least one branch channel is communicated and connected with the lower end of the side channel 3 at the downstream. That is, the microfluidic channel is used as a sample channel and comprises a main channel 2, a side channel 3 and a pinch structure 4, wherein the side channel 3 is separated from the upstream of the main channel 2 and finally collected at the downstream of the main channel 2 or collected at a branch channel of the main channel 2. Since the structure 4 is located at the convergence of the main channel 2 and the side channels 3, when the main channel 2 is provided with branch channels, the structure 4 is located at the convergence of the branch channels of the main channel 2 and the side channels 3. When the main channel 2 has a plurality of branch channels of two or more, each branch channel may match one or more side channels 3, and the number of the pinch structure 4 may be plural. I.e. a plurality of pinch structures 4 are located at the convergence of each side channel 3 of each branch channel with each side channel, respectively.

As shown in fig. 1, the inlet end of the sample channel is connected to a particle inlet 1, and the outlet end is connected to a particle outlet 5.

In the above embodiments, the material of the micro-fluidic chip may be PDMS (polydimethylsiloxane), PMMA (polymethyl methacrylate), quartz, borosilicate glass, or calcium fluoride, but is not limited to the above material.

In the above embodiment, as shown in fig. 3, the inner surface of the microfluidic channel of the microparticle self-pinch microfluidic chip may be a hydrophobic and oleophilic surface. At this moment, as mentioned above, at least one section of serpentine channel 8 is arranged between the downstream of the main channel 2 and the flow clamping structure 4, and the serpentine channel 8 performs slow flow on the particles, so as to better improve the effect and efficiency of separating the entrained flow liquid from the particles. The serpentine channel 8 may be a "C" shaped serpentine channel, an "S" shaped serpentine channel, or a "bow" shaped serpentine channel. The bends of the serpentine channel 8 may be arcuate bends.

In the above embodiment, the particle inlet 1 may be connected to the inlet conduit interface.

In the above embodiment, the inner diameter of the side passage 3 is smaller than the particle diameter, as shown in fig. 1. The inner diameter of the side passage 3 is smaller than the inner diameter of the main passage 2. This further prevents particles from entering the side channel 3 and ensures effective separation of entrained flow from the particle phase.

In the above embodiment, the particle outlet 5 and the fluid-entrapping structure 4 may be connected in communication via a channel, and the fluid-entrapping structure 4 and the main channel 2 may be connected in communication via a channel. The side channel 3 is used to filter the particles in the particulate phase and only allow part of the fluid in the particulate phase to enter; the flow clamping structure 4 is communicated with the channel to form a flow clamping channel, and is used for collecting the fluid of the side channel 3 to be used as a flow clamping liquid, so that the particles tightly arranged in the particle dispersing channel are dispersed.

In the above embodiment, the particle phase inlet of the main channel 2 may be connected to the outlet of the droplet generating structure.

In the above embodiment, the diameter of the liquid droplet may be 50 μm, and the height of the main channel 2 may be 40 to 100 μm, preferably 50 μm; the height of the side channel 3 can be 1-50 μm, preferably 10 μm; the width of a channel between the flow clamping structure 4 and the sample outlet 5 can be 50-100 μm; the maximum width of the main channel 2 can be 50-500 μm, and the minimum width can be 50-100 μm; the maximum width of the side channel 3 may be 50 to 500 μm, and the minimum width may be 10 to 100 μm.

In the above embodiments, the particle sampling hole 1 is connected to the sample outlet of the droplet generation structure, the droplet generation device, or the droplet sample injection device, and the droplet generation structure, the droplet generation device, or the droplet sample injection device may be a gravity-driven adjustment injection device, a syringe, a peristaltic pump, or a syringe pump, but is not limited to the above devices.

In the above embodiments, the microparticle self-pinch microfluidic chip may comprise a droplet generation structure or device, a cell or droplet culture structure or device, and a cell or droplet reaction structure or device, and the droplet generation structure or device, the cell or droplet culture structure or device, and the cell or droplet reaction structure or device should be located upstream of the main channel 2.

In the above embodiments, the microparticle self-pinch microfluidic chip may comprise a sorting structure or device, a detection structure or device, and the sorting structure or device, the detection structure or device should be located downstream of the pinch structure 4.

In the above embodiments, the droplet generation structure or device may include the droplet generation structure in the particle self-pinch microfluidic chip and the droplet generation device outside the particle self-pinch microfluidic chip.

The droplet generation structure in the particle self-pinch microfluidic chip comprises but is not limited to a T-shaped channel, a pinch focusing structure, a confocal structure and the like. After the liquid drop occurs, the micro-particle self-pinch micro-fluidic chip is directly communicated with the main channel 2 or is communicated with the micro-particle sampling hole 1 through a conduit.

The droplet generating structure or device of the particle outside the sandwich type microfluidic chip comprises but is not limited to a droplet generating structure or device which adopts a centrifugal method and an oscillation method and is communicated and connected with the droplet sampling hole 1 through a conduit.

The off-chip droplet sample injection device is selected from, but not limited to: a gravity-driven adjusting sample feeding device, an injector, a peristaltic pump and an injection pump.

Downstream of the entrainment structure 4, there may be, but is not limited to, a detection device and a sorting device, i.e. the particle outlet 5 downstream of the entrainment structure 4 may be connected in communication with a sample inlet of a device, not limited to a detection device and a sorting device. Wherein, the detection device includes but is not limited to a laser-excited fluorescence detection device, a Raman detection device, an optical detection device, a fluorescence detection device, and a conductance detection device.

As shown in fig. 4 to 13, in the above embodiment, the upper portion of the side channel 3 connected to the main channel 2 in the upstream communication manner may be provided with a bent structure, and the bent structure may be a right-angle bent structure, an obtuse-angle bent structure or an arc-shaped bent structure.

In the above embodiment, the lower portion of the side channel 3 connected to the downstream of the main channel 2 may be a bent structure, and the bent structure may be an obtuse-angle bent structure or an arc-shaped bent structure.

At the entrainment structure 4, the width of the main channel may be greater than, less than, or equal to the width of the side channels.

As shown in fig. 12 and 13, the main channel 2 may be provided with more than two branch passages, wherein one branch passage is connected with the lower end of the side channel 3 at the downstream, the flow clamping structure 4 is arranged at the connection position of the branch passage and the lower end of the side channel 3, and the flow clamping structure 4 is connected with one particle sampling hole 5 through the channel; the rest branch passages are directly communicated and connected with another particle sample outlet 5 through a channel. The number of the side channels 3 shown in fig. 4, 6, 8, 10 and 12 is one, and the number of the side channels 3 shown in fig. 5, 7, 9, 11 and 13 is two, and the two side channels 3 are uniformly and symmetrically distributed, or are not uniformly and symmetrically distributed.

In the above embodiment, as shown in fig. 1, the downstream of the side channel 3 may be narrowed, i.e. the width dimension of the downstream of the side channel 3 is smaller than that of the upstream thereof, so as to facilitate the connection of the lower end of the side channel 3 with the downstream of the main channel 2 to form a collecting structure, and to promote the fluid in the side channel 3 to act as an entrained flow to disperse the particles in the main channel 2. Wherein the width dimension downstream of the side channel 3 may be linearly reduced.

As shown in fig. 14, the method for manufacturing the micro-particle self-pinch microfluidic chip provided by the invention comprises the following steps: preparing a silica gel template, preparing an upper PDMS chip with a channel structure, preparing a particle sample inlet hole 1 and a particle sample outlet hole 5 on the upper PDMS chip, and preparing the particle self-pinch microfluidic chip. The method is simple and convenient to operate, high in yield and capable of ensuring product quality.

The following describes in detail a method for manufacturing the microparticle self-sandwich microfluidic chip according to an embodiment of the present invention with reference to the drawings.

Example 2

As shown in fig. 14, the method for manufacturing a microparticle self-pinch microfluidic chip is used for manufacturing the microparticle self-pinch microfluidic chip described in example 1, and comprises the following steps:

s01, preparing a silica gel template:

as shown in fig. 15 and 16, a main channel structure a having a main channel 2 and a side channel structure B having a side channel 3 are designed by drawing software, and printing is performed;

dripping photoresist on the silicon wafer, throwing the photoresist, covering the mask side channel structure B with the photoresist, and exposing;

after the exposed silicon wafer is subjected to spin coating again, covering the mask main channel structure A with the exposed silicon wafer to enable the main channel structure A to be aligned with the side channel structure B, and then exposing;

cleaning the uncured part by using a developing solution to obtain a silicon wafer template;

s02, preparing an upper PDMS chip with a channel structure:

mixing PDMS monomer with curing agent to obtain PDMS high polymer;

pouring the PDMS high polymer on the silicon wafer template prepared in the step S01, and drying to obtain an upper PDMS chip with a main channel structure A and a side channel structure B;

s03, preparing a particle sample inlet hole 1 and a particle sample outlet hole 5 on the upper PDMS chip:

punching a particle sample inlet hole 1 and a particle dropping sample outlet hole 5 on the upper-layer PDMS chip with the channel structure prepared in the step S02;

s04, preparing the particle self-pinch microfluidic chip:

and bonding the upper PDMS chip prepared in the step S03 with the lower chip to prepare the particle self-pinch microfluidic chip, and after the particle self-pinch microfluidic chip is placed, recovering the hydrophobicity of the particle self-pinch microfluidic chip.

In the above embodiment, in the preparation of the silica gel template in the step S01, the main channel structure a having the main channel 2 and the side channel structure B having the side channel 3 may be designed using CAD drawing software.

In the above embodiment, in the preparation of the silica gel template, the film mask printing may be performed at the time of performing step S01.

In the above embodiment, in the step S01, in preparing the silicon template, the cleaned silicon wafer may be dripped with photoresist for spin coating.

In the above embodiment, in the step S01, during the preparation of the silicon template, the SU-8 photoresist may be dropped on the silicon wafer for spin coating.

In the above embodiment, in the preparation of the silica gel template, the exposure may be performed under an exposure machine in performing step S01.

In the above embodiment, in the step S02, in preparing the upper layer PDMS chip having the channel structure, the PDMS monomer and the curing agent may be uniformly mixed in a ratio, where the ratio may be 10:1 by mass.

In the above embodiment, in the step S02, in the step of preparing the upper layer PDMS chip having the channel structure, the PDMS high polymer may be poured on the silicon wafer template, so that the thickness of the PDMS high polymer is 1 to 10 mm.

In the above embodiment, in step S03, the microparticle inlet hole 1 and the microparticle outlet hole 5 on the upper layer PDMS chip are prepared, and the microparticle inlet hole 1 and the microparticle outlet hole 5 are punched out of the upper layer PDMS chip with the channel structure by using a puncher.

In the above embodiment, in the step S03, the thickness of the PDMS smooth substrate is 1 to 10mm in the particle inlet hole 1 and the particle outlet hole 5 on the upper layer PDMS chip.

In the above embodiment, in step S04, the particle self-pinch microfluidic chip is prepared by bonding plasma, and the hydrophobicity of the particle self-pinch microfluidic chip is recovered after the particle self-pinch microfluidic chip is placed at 70 ℃ for 8 to 12 hours.

The particle self-pinch micro-fluidic chip provided by the invention can be applied to cell single wrapping, droplet fluorescence signal detection and microorganism droplet signal detection, and can also be applied to a coupling interface of flow sorting, mass spectrometry and cell sequencing after droplet culture of a microorganism group; and can also be used in the microfluidic fields of cell manipulation, microsphere manipulation, liquid drop manipulation, digital nucleic acid amplification, liquid drop manipulation, liquid drop detection, liquid drop counting, liquid drop sorting and the like.

The following describes in detail a method for using the microparticle self-pinch microfluidic chip provided by the embodiment of the present invention with reference to the accompanying drawings.

Example 3

The present embodiment exemplifies the application of the particle self-pinch microfluidic chip to droplet self-pinch, and provides a use method for realizing the dispersion of closely arranged droplets by a re-injection type particle self-pinch microfluidic chip based on hydromechanics.

As shown in fig. 17, the method for self-dispersing the particles is implemented by using the particle self-pinch microfluidic chip described in example 1, and comprises the following steps:

s11, accessing a liquid drop phase sample introduction end:

connecting the sample introduction end of the liquid drop phase into the particle sample introduction hole 1;

s12, accessing a pressure driving device:

the particle sample inlet hole 1 is connected with a positive pressure driving device, or the particle sample outlet hole 5 is connected with a negative pressure driving device;

s13, separating liquid drops and entrained flow liquid:

driving the positive pressure driving device or the negative pressure driving device accessed in the step S11, and enabling the droplet phase accessed to the sample injection end in the step S11 to flow into the particle self-pinch microfluidic chip through the particle sample injection hole 1; the liquid drop phase is separated into liquid drops and entrained flow liquid at the downstream of the main channel 2 and the lower end of the side channel 3 respectively;

s14, collecting liquid drops, separating oil and self-entrainment of the liquid drops:

collecting the liquid drops separated in the step S13 and the entrained flow liquid in the entrained flow structure 4, and self-dispersing the liquid drops by the entrained flow liquid;

s15, obtaining dispersed liquid drops (as shown in figure 18):

the droplets dispersed in step S14 are discharged through the microparticle outlet 5.

In the above embodiment, in the step S13, in the separation of the droplet and the entrained flow liquid, when the droplet phase connected to the sample injection end in the step S11 flows into the particle self-entrained flow microfluidic chip through the particle sample injection hole 1, the droplet phase intercepts the bubble through the bubble intercepting structure 6, so that the bubble cannot enter the downstream particle self-entrained flow microfluidic chip structure.

In the above embodiment, in the step S13, after the droplet phase connected to the sample injection end in the step S11 flows into the microparticle self-sandwich microfluidic chip through the microparticle sample injection hole 1, in the step S13, due to the microparticle interception structure 7 at the boundary between the main channel 2 and the side channel 3, or due to the small inner diameter of the side channel 3 relative to the main channel 2, or due to the combination of the two structures, the oil phase enters the side channel 3, and the droplet cannot enter the side channel 3, so that the droplet phase can be separated into the droplet and the oil phase at the downstream of the main channel 2 and the lower end of the side channel 3.

In the above embodiment, after the dispersed droplets are obtained in step S15, if the particles are not reused in the microfluidic chip within a certain period of time, the particles may be removed from the microfluidic chip, the main channel 2 is cleaned with absolute ethanol, and the cleaned main channel is dried in an oven and then recycled. Meanwhile, the channels of the side channel 3, the flow clamping structure 4 and the like can be cleaned by absolute ethyl alcohol, namely the whole microfluidic channel is cleaned.

Example 4

In this embodiment, a method for using the particle self-pinch microfluidic chip as a droplet nucleic acid amplification fluorescent signal analysis and statistics device is provided, taking the particle self-pinch microfluidic chip as an example.

As shown in fig. 19, the method for self-dispersing the particles is implemented by using the particle self-pinch microfluidic chip described in example 1, and comprises the following steps:

s21, nucleic acid amplification in liquid drops:

mixing solutions containing nucleic acid, enzyme, buffer solution, primers, fluorescent dye or target and the like in proportion to generate liquid drops, and setting corresponding temperature to amplify target fragments;

wherein, the solutions containing nucleic acid, enzyme, buffer solution, primer, fluorescent dye or target are mixed according to the proportion, for example, in the droplet PCR reaction, the supermix proportion is as follows: the mass percentage of solutes such as nucleic acid, enzyme, buffer solution, primer, fluorescent dye or target to the solution is 1: 2;

wherein, the corresponding temperature can be divided into 95 ℃ denaturation, annealing and 72 ℃ extension in a droplet PCR reaction, for example;

s22, droplet nucleic acid amplification analysis based on fluorescence:

the use of fluorescence-based droplet nucleic acid amplification assays allows for absolute quantification of target fragments, a means of nucleic acid quantification for endpoint counting;

s23, dispersing and counting single liquid drops:

the collected liquid drops are dispersed and counted by using the liquid drop self-dispersion micro-fluidic chip,

firstly, a fluorescence signal acquisition device such as an optical fiber is arranged at a downstream channel of the pinch structure 4, and a fluorescence signal acquisition and analysis system is opened;

secondly, connecting the particle sample inlet 1 with a hose containing liquid drops, and connecting the other end of the hose with a positive pressure driving pump or a syringe or other driving devices, or connecting the particle sample outlet 5 with a negative pressure driving pump or a syringe or other driving devices; thirdly, operating the driving device to enable the liquid drops to flow into the particle self-pinch microfluidic chip and disperse in the pinch structure 4;

the fluorescence signal acquisition software parameters were then adjusted to collect the droplet fluorescence signal and analyzed (as shown in fig. 20).

The method also comprises a step S24 of reusing the micro-particle self-pinch microfluidic chip:

and after the experiment is finished, washing the microfluidic channel of the particle self-pinch microfluidic chip by using absolute ethyl alcohol, and drying the particle self-pinch microfluidic chip for cyclic utilization.

Example 5

In this embodiment, a method for using the micro-particle self-pinch microfluidic chip as a microbiome droplet signal analysis and statistics device is provided, taking the micro-particle self-pinch microfluidic chip as an example of the microbiome droplet signal analysis and statistics device.

As shown in fig. 21, the method for self-dispersing the particles is implemented by using the particle self-pinch microfluidic chip described in example 1, and comprises the following steps:

s31, culturing and amplifying the microbial liquid in the liquid drops:

generating liquid drops by using a selective culture medium containing microorganisms, collecting the liquid drops into an EP (centrifugal tube) tube, and putting the liquid drops into an incubator for culturing for a preset time to ensure that a large amount of target microorganisms are propagated and the liquid drops are filled;

wherein the preset time can be 12 hours;

s32. unmarked growth phenotype droplet sorting based on scattered light:

using a scattered light-based, label-free growth phenotype droplet sorting system, cultured droplets can be sorted, and droplets that overgrow a target microorganism can be sorted out and collected;

s33, dispersion and analysis statistics of single liquid drops:

the collected liquid drops are dispersed by utilizing the particle self-pinch microfluidic chip,

firstly, placing a liquid drop signal collecting and sorting device at a channel of a pinch structure 4, and opening a system;

secondly, connecting the particle sample inlet hole 1 with an injector containing bacterial liquid, or pumping the bacterial liquid from the particle sample inlet hole 1 by using a pump; or the particle sample outlet 5 is connected with a driving device such as a negative pressure driving pump or a syringe;

then, operating a driving device to enable the liquid drops to flow into the particle self-pinch microfluidic chip and disperse in the pinch structure 4;

and finally, adjusting parameters of droplet signal acquisition and sorting software to collect droplet signals, and performing statistical analysis on the droplet signals according to the signals.

The method also comprises a step S34 of reusing the micro-particle self-pinch microfluidic chip:

and after the experiment is finished, washing the microfluidic channel of the droplet self-dispersion type microfluidic chip by using absolute ethyl alcohol, and drying the droplet self-dispersion type microfluidic chip for cyclic utilization.

Example 6

In this embodiment, taking the micro-particle self-pinch microfluidic chip as the coupling interface for flow sorting and cell sequencing after droplet culture of the microbiome as an example, the application method of the micro-particle self-pinch microfluidic chip as the coupling interface for flow sorting and cell sequencing after droplet culture of the microbiome is provided.

As shown in fig. 22, the method for self-dispersing the particles is implemented by using the particle self-pinch microfluidic chip described in example 1, and comprises the following steps:

s41, culturing and amplifying the microbial liquid in the liquid drops:

generating liquid drops by using a selective culture medium containing microorganisms, collecting the liquid drops into an EP tube, and putting the liquid drops into an incubator for culturing for a set time to ensure that a large amount of target microorganisms are propagated and the liquid drops are filled;

wherein, the set time can be 12 hours;

s42, scattered light-based unmarked growth phenotype liquid drop sorting:

s43, self-dispersion of single liquid drop:

the liquid drop self-dispersion micro-fluidic chip is used for dispersing the liquid drop,

firstly, placing a liquid drop signal collecting and sorting device at a channel of a flow clamping structure 4, and opening a system;

secondly, connecting the particle sample inlet 1 with an injector containing bacterial liquid drops, or pumping the bacterial liquid from the particle sample inlet 1 into a bacterial liquid inlet channel by using a pump; or the particle sample outlet 5 is connected with a driving device such as a negative pressure driving pump or a syringe;

thirdly, operating the driving device to enable the liquid drops to flow into the particle self-pinch microfluidic chip and disperse in the pinch structure 4;

then, the parameters of the droplet signal acquisition and sorting software are adjusted to collect the droplet signals, and the droplet signals are sorted according to the signals, so that the required droplets selectively flow into the microparticle outlet holes 5.

S44, obtaining growth condition information of the target microorganisms:

the droplets flowing out of the microparticle outlet holes 5 are collected, and a plurality of items of information of the target microorganism can be obtained through sequencing.

S45, recycling the particle self-pinch microfluidic chip:

and after the experiment is finished, washing the microfluidic channel of the micro-particle self-pinch microfluidic chip by using absolute ethyl alcohol, and drying the droplet self-dispersion microfluidic chip for cyclic utilization.

Example 7

In this embodiment, taking the micro-particle self-pinch microfluidic chip as the coupling interface for flow sorting and mass spectrometry after droplet culture of the microbiome as an example, a method for using the micro-particle self-pinch microfluidic chip as the coupling interface for flow sorting and mass spectrometry after droplet culture of the microbiome is provided.

As shown in fig. 23, the method for self-dispersing the particles is implemented by using the particle self-pinch microfluidic chip described in example 1, and comprises the following steps:

s51, culturing and amplifying the microbial liquid in the liquid drops:

wherein, the set time can be 12 hours;

s52. unmarked growth phenotype droplet sorting based on scattered light:

s53, self-dispersion of single liquid drop:

the dispersion of the liquid drop is carried out by utilizing the particle self-pinch microfluidic chip,

secondly, connecting the particle sample inlet 1 with an injector containing bacterial liquid drops, or pumping the bacterial liquid from the particle sample inlet 1 into a bacterial liquid inlet channel by using a pump, or connecting a negative pressure driving pump or an injector and other driving devices to the particle sample outlet 5;

then, adjusting parameters of droplet signal acquisition and sorting software to collect droplet signals, and sorting the droplet signals according to the signals to make the required droplets selectively flow into the particle sample outlet 5;

s54, mass spectrometry:

and collecting the liquid drops flowing out of the particle outlet holes 5 for mass spectrometry.

The method also comprises a step S55 of reusing the micro-particle self-pinch microfluidic chip:

Example 8

In this embodiment, taking the particle self-pinch microfluidic chip as the flow-type sorting coupling interface after the drop enzyme activity is screened as an example, a method for using the particle self-pinch microfluidic chip as the flow-type sorting coupling interface after the drop enzyme activity is screened is provided.

As shown in fig. 24, the method for self-dispersing the particles is implemented by using the particle self-pinch microfluidic chip described in example 1, and comprises the following steps:

s61, reacting enzyme and substrate in the liquid drops:

generating liquid drops from a solution containing a substrate, an enzyme, a dye and the like to fully react the enzyme, the substrate and the dye;

s62, dispersing single liquid drops and sorting fluorescent liquid drops:

the collected liquid drops are dispersed and subjected to fluorescence detection by using the liquid drop self-dispersion microfluidic chip,

secondly, connecting the particle sample inlet 1 with a hose containing liquid drops, and connecting the other end of the hose with a positive pressure driving pump or a syringe or other driving devices, or connecting the particle sample outlet 5 with a negative pressure driving pump or a syringe or other driving devices;

thirdly, operating the driving device to enable the liquid drops to flow into the particle self-pinch microfluidic chip and perform self-dispersion in the pinch structure 4;

S63, screening liquid drops containing strong enzyme activity;

the droplets flowing out of the particle outlet holes 5 are collected and the enzyme capable of reacting with the substrate is screened.

The method also comprises a step S45 of reusing the micro-particle self-pinch microfluidic chip:

Example 9

The present embodiment exemplifies the application of the particle self-pinch microfluidic chip to cell self-pinch, and provides a use method for realizing the dispersion of the tightly arranged cells by the re-injection type particle self-pinch microfluidic chip based on hydromechanics.

As shown in fig. 25, the method for self-dispersing the particles is implemented by using the particle self-pinch microfluidic chip as described in any one of embodiment 1, and comprises the following steps:

s71, accessing a cell phase sample introduction end:

connecting the sample introduction end of the cell phase into the particle sample introduction hole 1;

s72, accessing a pressure driving device:

s73, separating cells and a flow clamping liquid:

driving the positive pressure driving device or the negative pressure driving device accessed in the step S71, and enabling the cell phase accessed to the sample injection end in the step S71 to flow into the particle self-pinch microfluidic chip through the particle sample injection hole 1; the cell phase is separated into cells and entrained flow liquid at the downstream of the main channel 2 and the lower end of the side channel 3 respectively;

s74, collecting cells, carrying liquid and carrying the cells to self-carry:

collecting the cells separated in the step S73 and the entrained flow liquid in the entrained flow structure 4, and self-dispersing the cells by the entrained flow liquid;

s75, obtaining dispersed cells (as shown in FIG. 18):

the cells dispersed in step S74 are discharged through the microparticle outlet port 5.

In the above embodiment, in the step S73, in the cell and the entrained flow liquid, when the cell phase accessed to the sample injection end in the step S71 flows into the particle self-entrained flow microfluidic chip through the particle sample injection hole 1, the cell phase intercepts bubbles through the bubble intercepting structure 6, so that the bubbles cannot enter the downstream particle self-entrained flow microfluidic chip structure.

In the above embodiment, in the step S73, after the cell phase accessed to the sample injection end in the step S71 flows into the microparticle self-sandwich microfluidic chip through the microparticle sample injection hole 1, in the step S73, due to the microparticle interception structure 7 at the boundary between the main channel 2 and the side channel 3, or due to the small inner diameter of the side channel 3 relative to the main channel 2, or due to the combination of the two structures, the fluid around the cell enters the side channel 3, and the cell cannot enter the side channel 3, so that the cell phase is separated into the cell and the fluid at the downstream of the main channel 2 and the lower end of the side channel 3, respectively.

In the above embodiment, after the dispersed cells are obtained in step S75, if the particles are not reused in the microfluidic chip within a certain period of time, the particles may be removed from the microfluidic chip, the main channel 2 is cleaned with absolute ethanol, and the cleaned main channel is placed in an oven for drying and then may be recycled. Meanwhile, the channels of the side channel 3, the flow clamping structure 4 and the like can be cleaned by absolute ethyl alcohol, namely the whole microfluidic channel is cleaned.

Example 10

The present embodiment exemplifies the application of the microparticle self-pinch microfluidic chip to particle self-pinch, and provides a use method for realizing the dispersion of tightly arranged particles by the re-injection type microparticle self-pinch microfluidic chip based on hydromechanics.

As shown in fig. 26, the method for self-dispersing the particles is implemented by using the particle self-pinch microfluidic chip described in example 1, and includes the following steps:

s81, accessing a particle phase sample introduction end:

the sample introduction end of the particle phase is connected into a particle sample introduction hole 1;

s82, accessing a pressure driving device:

s83, separating particles and entrained flow liquid:

driving the positive pressure driving device or the negative pressure driving device accessed in the step S81, and enabling the particle phase accessed to the sample injection end in the step S81 to flow into the particle self-pinch micro-fluidic chip through the particle sample injection hole 1; the particle phase is separated into particles and entrained flow liquid at the downstream of the main channel 2 and the lower end of the side channel 3 respectively;

s84, collecting particles, carrying liquid and self-carrying flow of the particles:

collecting the particles separated in the step S83 and the entrained flow liquid in the entrained flow structure 4, and self-dispersing the particles by the entrained flow liquid;

s85, obtaining dispersed particles (as shown in figure 18):

the particles dispersed in step S14 flow out through the microparticle outlet 5.

In the above embodiment, in the step S83, in the separation of the particles and the entrained flow liquid, when the particle phase connected to the sample injection end in the step S81 flows into the particle self-entrained flow microfluidic chip through the particle sample injection hole 1, the particle phase intercepts bubbles through the bubble intercepting structure 6, so that the bubbles cannot enter the downstream particle self-entrained flow microfluidic chip structure.

In the above embodiment, in the step S83, after the particle phase connected to the sample injection end in the step S81 flows into the particle self-sandwich microfluidic chip through the particle sample injection hole 1, in the separated particle and the sandwich liquid, due to the particle interception structure 7 at the boundary between the main channel 2 and the side channel 3, or due to the small inner diameter of the side channel 3 relative to the main channel 2, or due to the combination of the two structures, the fluid around the particle enters the side channel 3, and the particle cannot enter the side channel 3, so that the particle phase is separated into the particle and the fluid at the downstream of the main channel 2 and the lower end of the side channel 3, respectively.

In the above embodiment, after the dispersed particles are obtained in step S85, if the particles are not reused in the self-sandwiched micro-fluidic chip within a period of time, the particles may be removed from the self-sandwiched micro-fluidic chip, the main channel 2 is cleaned with absolute ethanol, and the cleaned particles may be recycled after being placed in an oven for drying. Meanwhile, the channels of the side channel 3, the flow clamping structure 4 and the like can be cleaned by absolute ethyl alcohol, namely the whole microfluidic channel is cleaned.

Finally, it should be noted that: the above examples are only intended to illustrate the technical solution of the present invention, but not to limit it; although the present invention has been described in detail with reference to the foregoing embodiments, it will be understood by those of ordinary skill in the art that: the technical solutions described in the foregoing embodiments may still be modified, or some technical features may be equivalently replaced; and such modifications or substitutions do not depart from the spirit and scope of the corresponding technical solutions of the embodiments of the present invention.

Claims

1. The particle self-pinch microfluidic chip is characterized in that at least one microfluidic channel is arranged in the particle self-pinch microfluidic chip, and the microfluidic channel comprises a main channel, at least one section of side channel and a pinch structure;

2. The particulate self-entrained microfluidic chip of claim 1, wherein the height of the junction of the side channel and the main channel is higher than the height of the side channel to form a height difference; the particle interception structure is a particle interception structure arranged at the joint of the side channel and the main channel, or a size difference structure formed by the width of the side channel being smaller than the diameter of the liquid drop, or a size difference structure formed by the height difference being smaller than the diameter of the liquid drop, or a combination of two or three structures.

3. The particle self-pinch microfluidic chip according to claim 2, wherein when the particle blocking structure is a particle blocking structure disposed at a junction of the side channel and the main channel, the particle blocking structure is a micro-sieve array structure.

4. The particle self-pinch microfluidic chip according to claim 1, wherein a bubble-blocking structure is disposed at the particle inlet.

5. The particle self-entrained microfluidic chip of claim 4, wherein the bubble blocking structure is a microsieve array structure.

6. The particulate self-entrained microfluidic chip of claim 1, wherein at least one serpentine channel is disposed between the downstream of the main channel and the entrainment structure.

7. The particulate self-entrained microfluidic chip of claim 1, wherein at least one of the branch channels is disposed downstream of the main channel, and at least one of the branch channels is connected downstream to the side channel in communication with the lower end of the side channel and to the entrainment structure.

8. The particle self-pinch microfluidic chip according to claim 1, wherein a positive pressure driving device is disposed upstream of the particle inlet hole or a negative pressure driving device is disposed downstream of the particle outlet hole.

9. A method of manufacturing a particulate self-entrained microfluidic chip as claimed in any one of claims 1 to 8, comprising the steps of:

preparing a silica gel template:

preparing an upper PDMS chip with a channel structure:

mixing PDMS monomer with curing agent to obtain PDMS high polymer;

preparing the particle self-pinch microfluidic chip:

10. A method for self-dispersing the particles by using the particle self-clamping micro-fluidic chip as claimed in any one of claims 1 to 8, comprising the following steps: