CN117960261A - Ultra-low concentration microparticle enrichment and purification device and operation method thereof - Google Patents

Abstract

The invention relates to a device and a method for enriching and purifying ultra-low concentration particles, wherein the device comprises a micro-fluidic chip, and the micro-fluidic chip comprises a particle inlet, a micro-flow channel and a collecting port which are sequentially connected, and further comprises: the at least one identification and separation part comprises a sheath inflow port, a separation control flow passage and a separation outlet which are sequentially arranged, wherein the sheath inflow port and the separation outlet are arranged on the same side of the micro-flow channel, the separation control flow passage is arranged on the opposite side of the micro-flow channel, a separation area is formed at the junction of the separation control flow passage and the micro-flow channel, the separation outlet is arranged close to the rear end of the separation area, and the front end of the separation area is provided with an identification area. The enrichment and purification efficiency of the microparticles is improved through multistage separation, and the seamless connection design between the first identification separation part and the second identification separation part in the device greatly shortens the length of an invalid flow channel, greatly avoids the loss of the microparticles in the process of flowing through the micro flow channel, and shortens the running time.

Description

Ultra-low concentration microparticle enrichment and purification device and operation method thereof

Technical Field

The invention relates to the field of microfluidic chips, in particular to an ultralow-concentration microparticle enrichment and purification device and an operation method thereof.

Background

As the research of microparticles has been advanced, many studies of ultra-low concentration microparticles have been paid attention to many scientific researches and clinical workers, such as cells, sperm, bacteria, microalgae, microspheres, etc. However, these microparticles often cannot be directly used for research due to the ultra-low concentration in the sample, and thus enrichment of ultra-low concentration microparticles is important. Taking the enrichment of the most widely studied circulating tumor cells at present as an example, there are currently immunocapture methods: immunomagnetic positive and negative enrichment methods, microfluidic immunocapture methods, nanomaterial immunocapture enhancement methods, and related techniques thereof; biophysical property enrichment method: membrane filtration, size-based microfluidic, density-based and dielectrophoresis, and related techniques; other methods: multiple methods combining and secondary separation methods and related techniques.

Prior art immunocapture-based enrichment methods rely on epithelial cell surface markers of biological cells, particularly epithelial cell adhesion molecule (EpCAM), which require a pretreatment of the sample prior to capture enrichment, which can damage the cells, be time-consuming and labor-consuming, and not every target cell can be well labeled. On the other hand, the enrichment method based on the physical characteristics has great requirements on the physical characteristic difference between other microparticles mixed with the target microparticles and the target microparticles, and it is difficult to realize high detection rate and recovery rate of enrichment of the target microparticles in complex samples.

Disclosure of Invention

The invention provides an ultra-low concentration microparticle enrichment and purification device and an operation method thereof, which aim to at least solve one of the technical problems in the prior art.

The technical proposal of the invention is an ultra-low concentration microparticle enrichment and purification device and method, wherein the ultra-low concentration microparticle enrichment and purification device comprises a micro-fluidic chip,

The microfluidic chip comprises a microparticle inlet, a microfluidic channel and a collection port which are sequentially connected, and further comprises:

The device comprises at least one identification and separation part, wherein the identification and separation part comprises a sheath inflow port, a separation control flow passage and a separation outlet which are sequentially arranged, the sheath inflow port and the separation outlet are arranged on the same side of the microfluidic channel, the separation control flow passage is arranged on the opposite side of the microfluidic channel, a separation area is formed at the junction of the separation control flow passage and the microfluidic channel, the separation outlet is closely arranged at the rear end of the separation area, the front end of the separation area is provided with an identification area, the microparticle inlet is connected with the inlet of the first identification and separation part, a plurality of identification and separation parts are connected through the microfluidic channel, and the collection port is connected with the last identification and separation part.

Further, the identification and separation part comprises a first identification and separation part and a second identification and separation part which are sequentially connected, the microparticle inlet is connected with the initial end of the first identification and separation part, and the tail end of the second identification and separation part is connected with the collecting port.

Further, the first identification sorting part comprises a first sheath inflow port, a first sorting control channel and a first sorting outlet, a first sorting area is formed at the junction of the first sorting control channel and the microfluidic channel, the first sorting outlet is closely arranged at the rear end of the first sorting area, and a first identification area is arranged at the front end of the first sorting area.

Further, the second identification and separation part comprises a second sheath inflow port, a second separation control channel and a second separation outlet, a second separation area is formed at the junction of the second separation control channel and the microfluidic channel, the second separation outlet is arranged close to the rear end of the second separation area, and a second identification area is arranged at the front end of the second separation area.

The invention further provides an ultralow-concentration microparticle enrichment and purification device, which comprises the microfluidic chip, and the ultralow-concentration microparticle enrichment and purification device further comprises:

A microscope disposed above the identification zone;

the shooting device is used for shooting a view field image of the identification area at a high speed and is connected with an eyepiece of the microscope;

The image workstation is used for carrying out image recognition target examples on the field-of-view image shot by the shooting device and sending out control signals, and is electrically connected with the shooting device;

the precise pneumatic pump is used for receiving control signals of the image workstation and controlling multi-path pneumatic output, and is electrically connected with the image workstation;

the liquid measuring cylinder group comprises a plurality of liquid measuring cylinders, each liquid measuring cylinder is connected with the air pressure output channel of the precise air pressure pump through an air connecting pipe, and each liquid measuring cylinder is connected with the microfluidic chip through a liquid connecting pipe.

Further, the liquid measuring cylinder group comprises a first liquid measuring cylinder, a second liquid measuring cylinder, a third liquid measuring cylinder, a fourth liquid measuring cylinder and a fifth liquid measuring cylinder,

The inlet of the first liquid measuring cylinder is connected with a first air pressure output channel of the precise air pressure pump through a first air pipe, the outlet of the first liquid measuring cylinder is connected with a microparticle inlet of the microfluidic chip through a first liquid pipe,

The inlet of the second liquid measuring cylinder is connected with a second air pressure output channel of the precise air pressure pump through a second air pipe, the outlet of the second liquid measuring cylinder is connected with a first sheath inflow inlet of the microfluidic chip through a second liquid pipe,

An inlet of the third liquid measuring cylinder is connected with a third air pressure output channel of the precise air pressure pump through a third air pipe, an outlet of the third liquid measuring cylinder is connected with a second sheath inflow inlet of the microfluidic chip through a third liquid pipe,

The inlet of the fourth liquid measuring cylinder is connected with a fourth air pressure output channel of the precise air pressure pump through a fourth air pipe, the outlet of the fourth liquid measuring cylinder is connected with the inlet of a first sorting control channel of the microfluidic chip through a fourth liquid pipe,

The inlet of the fifth liquid measuring cylinder is connected with a fifth air pressure output channel of the precise air pressure pump through a fifth air pipe, and the outlet of the fifth liquid measuring cylinder is connected with the inlet of a second separation control channel of the microfluidic chip through a fifth liquid pipe.

Further, the first liquid measuring cylinder is filled with the microparticle suspension, the first air pipe extends into the first liquid measuring cylinder to a depth higher than the liquid level of the microparticle suspension, and the first liquid pipe extends into the first liquid measuring cylinder to a depth lower than the liquid level of the microparticle suspension;

The depth of the second air pipe extending into the second liquid measuring cylinder is higher than the liquid level of the second liquid measuring cylinder, and the depth of the second air pipe extending into the second liquid measuring cylinder is lower than the liquid level of the second liquid measuring cylinder;

The depth of the third air pipe extending into the third liquid measuring cylinder is higher than the liquid level of the third liquid measuring cylinder, and the depth of the third air pipe extending into the third liquid measuring cylinder is lower than the liquid level of the third liquid measuring cylinder;

the depth of the fourth air pipe extending into the fourth liquid measuring cylinder is higher than the liquid level of the fourth liquid measuring cylinder, and the depth of the fourth air pipe extending into the fourth liquid measuring cylinder is lower than the liquid level of the fourth liquid measuring cylinder;

The depth of the fifth air pipe extending into the fifth liquid measuring cylinder is higher than the liquid level of the fifth liquid measuring cylinder, and the depth of the fifth air pipe extending into the fifth liquid measuring cylinder is lower than the liquid level of the fifth liquid measuring cylinder.

Furthermore, the invention also provides an operation method of the ultra-low concentration microparticle enrichment and purification device, which is applied to the ultra-low concentration microparticle enrichment and purification device, and the operation method comprises the following steps:

S100, setting a microscope, a shooting device and an image workstation, connecting a precise pneumatic pump, a liquid measuring cylinder group and a microfluidic chip, wherein the first liquid measuring cylinder is preloaded with microparticle suspension liquid, and the second liquid measuring cylinder, the third liquid measuring cylinder, the fourth liquid measuring cylinder and the fifth liquid measuring cylinder are preloaded with experimental liquid;

s200, outputting multi-path air pressure by a precise air pressure pump, and respectively controlling a microparticle inlet, a first separation control flow channel and a second separation control flow channel to be high pressure, wherein the first sheath flow inlet is high pressure, and the second sheath flow inlet is sub-high pressure, so that microparticle suspension is input to a microfluidic chip from the microparticle inlet along a first liquid pipe;

S300, enabling the microparticle suspension to flow through a first identification area, continuously shooting field images by a shooting device through a microscope, transmitting the field images to an image workstation, performing image processing on the field images by the image workstation, and judging whether target particles exist or not;

S400, if target particles exist, the image workstation outputs a first control signal for setting a first separation control channel to be low-pressure and maintaining the first control signal for preset time to the precise pneumatic pump, and a fourth output channel of the precise pneumatic pump outputs the low-pressure for preset time, so that the liquid pressure of the first separation control channel is reduced briefly, the target particles are caused to avoid a first separation outlet and continuously flow into a second identification area through a micro-flow channel;

s500, continuously shooting field images by the shooting device through a microscope when the microparticle suspension flows through the second identification area, and transmitting the field images to an image workstation, wherein the image workstation performs image processing on the field images to judge whether target particles pass through or not;

And S600, if the target particles pass through, the image workstation outputs a second control signal for setting the second separation control channel to be low-pressure and maintaining the second control signal for a preset time to the precise pneumatic pump, and the fifth output channel of the precise pneumatic pump outputs the low-pressure for the preset time, so that the liquid pressure of the second separation control channel is reduced briefly, the target particles are promoted to avoid the second separation outlet, and the target particles continue to flow into the collection port through the microfluidic channel.

Further, in the step S400, if the target particles are not present, the first separation control channel maintains a high pressure, and the fine particle suspension containing no target particles flows out from the first separation outlet.

Further, in the step S600, if the target particles are not present, the second separation control channel maintains a high pressure, and the fine particle suspension containing no target particles flows out from the second separation outlet.

The beneficial effects of the invention are as follows:

The ultra-low concentration microparticle enrichment and purification device improves microparticle enrichment and purification efficiency through multistage separation, designs a single-side focusing structure in front of a separation identification area through a sheath inflow port, improves the accuracy of separated microparticles, and greatly shortens the length of an invalid flow channel due to seamless connection design between a first identification separation part and a second identification separation part in the device, thereby greatly avoiding the loss of microparticles in the process of flowing through the micro flow channel and shortening the running time.

Drawings

FIG. 1 is a schematic diagram showing the overall structure of an apparatus for enriching and purifying ultra-low concentration fine particles according to the present invention.

Fig. 2 is a schematic diagram showing the structure of a microfluidic chip in an ultra-low concentration microparticle enrichment and purification device according to the present invention.

FIG. 3 is a flow chart showing the operation method of the ultra-low concentration microparticle enrichment and purification device according to the present invention.

FIG. 4 is a schematic view showing a front-blocking and rear-blocking state in the ultra-low concentration microparticle enrichment and purification device according to the present invention.

FIG. 5 is a simulation diagram showing the state of front and rear plugs in the ultra-low concentration microparticle enrichment and purification device according to the present invention.

FIG. 6 is a schematic diagram showing the state of front-pass and back-plug in the ultra-low concentration microparticle enrichment and purification device according to the present invention.

FIG. 7 is a simulation diagram showing the state of front-pass and back-plug in the ultra-low concentration microparticle enrichment and purification device according to the present invention.

FIG. 8 is a schematic diagram showing a front-pass and rear-pass state in the ultra-low concentration microparticle enrichment and purification device according to the present invention.

FIG. 9 is a simulation diagram showing the front-pass and back-pass states in the ultra-low concentration microparticle enrichment and purification device according to the present invention.

FIG. 10 is a schematic view showing a front-blocking rear-passing state in an ultra-low concentration microparticle enrichment and purification device according to the present invention.

FIG. 11 is a diagram showing a simulation of the state of front plug and rear plug in an ultra-low concentration microparticle enrichment and purification device according to the present invention.

FIG. 12 is a schematic diagram showing a model Yolo v of the operation of a graphics workstation in an ultra-low concentration microparticle enrichment and purification device according to the present invention.

Reference numerals:

100. A microfluidic chip; 110. a first identification and sorting section; 111. a primary sheath inflow port; 112. a first sorting control channel; 113. a first sorting outlet; 114. a first sorting zone; 115. a first identification area; 120. a second identification and sorting section; 121. a secondary sheath inflow port; 122. a second sorting control channel; 123. a second sorting outlet; 124. a second separation zone; 125. a second identification area; 130. a microparticle inlet; 140. a microfluidic channel; 150. a collection port; 200. an image workstation; 300. a precision pneumatic pump; 400. a liquid measuring cylinder group; 410. a first liquid measuring cylinder; 420. a second liquid measuring cylinder; 430. a third liquid measuring cylinder; 440. a fourth liquid measuring cylinder; 450. and a fifth liquid measuring cylinder.

Detailed Description

The conception, specific structure, and technical effects produced by the present application will be clearly and completely described below with reference to the embodiments and the drawings to fully understand the objects, aspects, and effects of the present application. It should be noted that, without conflict, the embodiments of the present application and features of the embodiments may be combined with each other.

It should be noted that, unless otherwise specified, when a feature is referred to as being "fixed" or "connected" to another feature, it may be directly or indirectly fixed or connected to the other feature. Further, the descriptions of the upper, lower, left, right, top, bottom, etc. used in the present invention are merely with respect to the mutual positional relationship of the respective constituent elements of the present invention in the drawings.

Furthermore, unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art. The terminology used in the description presented herein is for the purpose of describing particular embodiments only and is not intended to be limiting of the invention. The term "and/or" as used herein includes any combination of one or more of the associated listed items.

It should be understood that although the terms first, second, third, etc. may be used in this disclosure to describe various elements, these elements should not be limited by these terms. These terms are only used to distinguish one element of the same type from another. For example, a first element could also be termed a second element, and, similarly, a second element could also be termed a first element, without departing from the scope of the present disclosure.

Referring to fig. 1 to 12, in some embodiments, the present invention provides an apparatus and a method for enriching and purifying ultra-low concentration particles, wherein the apparatus comprises a microfluidic chip 100, referring to fig. 1 and 2,

The microfluidic chip 100 includes a microparticle inlet 130, a microfluidic channel 140 and a collection port 150, which are sequentially connected, and further includes:

The at least one discernment separation portion, discernment separation portion is including sheath inflow mouth, separation control runner and the separation export that sets gradually, sheath inflow mouth with the separation export sets up the same side of microfluidic channel 140, separation control runner sets up the opposite side of microfluidic channel 140, separation control runner with the intersection department of microfluidic channel 140 forms the separation district, separation export is closely the rear end setting of separation district, the front end of separation district is provided with the discernment district, microparticle entry 130 is connected with the entry of first discernment separation portion, a plurality of discernment separation portion is connected through microfluidic channel 140 between, collection mouth 150 is connected with last discernment separation portion.

The beneficial effects of the invention are as follows:

The ultra-low concentration microparticle enrichment and purification device improves microparticle enrichment and purification efficiency through multistage separation, designs a single-side focusing structure in front of a separation identification area through a sheath inflow port, improves the accuracy of separated microparticles, and greatly shortens the length of an ineffective flow channel due to seamless connection design between a first identification separation part 110 and a second identification separation part 120 in the device, thereby greatly avoiding the loss of microparticles in the process of flowing through the micro flow channel and shortening the running time.

The invention provides a device and a method for efficiently enriching and purifying a mixed sample of ultra-low concentration microparticles (such as cells, sperms, bacteria, microalgae, microspheres and the like). Target particles and other impurity particles are identified in real time through an image identification technology, and the micro-fluidic chip 100 of a multi-stage channel is used in association for carrying out multi-time particle sorting so as to realize enrichment (enrichment for the first sorting) and purification (hundred percent purification for the second sorting), so that high-speed enrichment and purification of ultra-low concentration particles based on images are possible. Specifically, the multistage sorting microfluidic channel 140 provided by the device can realize high-speed enrichment and purification of the target microparticles through multistage sorting of the target microparticles and the hybrid microparticles, so that the efficiency and speed of sorting based on vision are remarkably improved; the device has a single-side focusing structure of the microparticles, and the sorting accuracy is remarkably improved; the microparticle enrichment and purification structure are in seamless connection, enrichment is realized by first-stage separation, and purification is realized by second-stage separation.

The ultra-low concentration microparticle enrichment and purification device comprises the following technical key points:

1) Multistage sorting micro-channel

And the sorting channels are distributed in multiple stages (two stages and more) under the same microscopic view, and each stage of sorting consists of a sorting intersection, a sorting control channel, a single-side focusing area and a sorting identification area. Multistage sorting is achieved by a specific cooperation between the inlet pressures of a plurality of control channels for sorting. The inlet pressure of the runner is controlled by a precision pressure pump; the hydrodynamic mechanism of focusing and sorting of the microparticles in the chip is analyzed by numerical simulation, and the pressure ratio of the focusing port, the microparticle inlet 130 and the sheath fluid inlet is explored, so that the local flow of the sorting area can be switched at high speed. In addition, the particle velocity in the identification area is extracted, and the optimal parameters for achieving microparticle focusing and microparticle sorting are determined.

2) Microparticle single-side focusing

The unilateral focusing of the particles is realized by unilateral sheath flow action before two sorting intersections on the microfluidic chip 100, so that the particles follow the fluid movement biased to one side in the flow channel, and the particles are prevented from being erroneously sorted at the sorting intersections.

3) And (5) seamless connection of enrichment and purification.

The first stage is separated for large-scale enrichment (concentration), and the second stage is separated for precise purification. That is, the first stage identification area is that a large number of particles in the micro-channel flow side by side at the same time, when one target particle exists in the first stage identification area, all particles in the first stage identification area are driven to the lower stage separation structure, and otherwise, the particles flow to the separation outlet. In the second-stage sorting structure, all particles move along with the fluid at intervals, and at the moment, the second-stage sorting micro-flow field can sequentially identify and sort single target particles.

The second stage flow path is narrower than the first stage flow path, but the design dimensions are limited by the microscope field of view dimensions, so that no specific size range is actually framed. In addition, the ratio of the width of the second-stage flow channel to the width of the first-stage flow channel can have larger fluctuation in the range of 0-1, but the closer to 0, namely the narrower the second-stage flow channel is relatively, the smaller the chip flux is, the more particles which enter the second-stage flow channel are sorted at one time, and the difficulty of the second-stage sorting and purifying is increased, so that the currently adopted ratio is about 6/7.

Microparticles entering the secondary separation are extruded into a narrower laminar flow range by fluid converged by two side channels (a front sheath flow channel and a control separation channel) in the separation region, so that the microparticles basically form a front-back arranged interval flow.

Further, referring to fig. 1 and 2, the recognition and separation part includes a first recognition and separation part 110 and a second recognition and separation part 120 connected in sequence, the fine particle inlet 130 is connected to the start of the first recognition and separation part 110, and the end of the second recognition and separation part 120 is connected to the collection port 150.

In some specific embodiments, the first identification and sorting unit 110 and the second identification and sorting unit 120 are disposed in the same field of view of a microscope, and only one set of microscopic imaging system is used, and a sorting algorithm of parallel processing and a mutually independent liquid driving system are used, so that the technical effects of independent sorting operations of two sorting positions can be achieved.

Specifically, the reference is made to the flow direction of the fluid in the microfluidic chip 100 before and after the separation, that is, before the inlet and after the separation outlet, the positions of the inlet, the positions of the identification areas and the positions of the separation areas where the sheath flows of the first identification and separation part 110 and the second identification and separation part 120 are collected in the microfluidic chip 100 are all distributed from front to back in sequence, and the flow channels connecting the first identification and separation part 110 and the second identification and separation part 120 are both the collection flow channels of the first identification and separation part 110 and the flow channels of the microparticle inlets 130 of the second identification and separation part 120.

Further, referring to fig. 2, the first identification and sorting part 110 includes a first sheath inflow port 111, a first sorting control channel 112, and a first sorting outlet 113, a first sorting area 114 is formed at the junction of the first sorting control channel and the microfluidic channel 140, the first sorting outlet 113 is disposed close to the rear end of the first sorting area 114, and a first identification area 115 is disposed at the front end of the first sorting area 114.

Further, referring to fig. 2, the second identification and sorting unit 120 includes a second sheath inflow port 121, a second sorting control channel 122, and a second sorting outlet 123, where the junction of the second sorting control channel and the microfluidic channel 140 forms a second sorting area 124, the second sorting outlet 123 is disposed close to the rear end of the second sorting area 124, and the front end of the second sorting area 124 is provided with a second identification area 125.

The distance from the identification area to the sorting area depends on the flow rate of the micro-particles in the micro-fluidic chip 100 and the response time of the device (the time interval from identification of the micro-particles to change of the pressure in the sorting control flow channel, so that the movement direction of the micro-particles changes), and the longer the flow rate of the micro-particles is, the longer the response time of the system is, the longer the distance is, and vice versa the distance is about short. The junction point of the sorting outlet flow channel and the collecting port 150 flow channel/lower sorting connecting flow channel should be close to one side of the sorting outlet flow channel, and the front and back positions of the junction point can be adjusted within a certain range without affecting the sorting effect.

The ultra-low concentration microparticle enrichment and purification device verifies the feasibility of setting five inlet pressures through simulation and experiment modes, and synchronously adjusts the five inlet pressures (namely, the pressure of the microparticle inlet 130 is adjusted to be high, the other four inlet pressures are all required to be high, and the other four inlet pressures are positively related but not necessarily linear), and in addition, the relative position of the structure is subjected to certain translation, rotation or certain stretching change of the shape, so that the sorting effect is not influenced as long as the five inlet pressures are adjusted (currently, the adjustment is carried out through an experimental method).

Further, referring to fig. 1, the present invention further provides an apparatus for enriching and purifying ultra-low concentration particles, which includes the microfluidic chip 100, and the apparatus for enriching and purifying ultra-low concentration particles further includes:

A microscope disposed above the identification zone;

the shooting device is used for shooting a view field image of the identification area at a high speed and is connected with an eyepiece of the microscope;

An image workstation 200, configured to perform image recognition on a field image captured by a capturing device and send out a control signal, where the image workstation 200 is electrically connected to the capturing device;

The precision pneumatic pump 300 is used for receiving a control signal of the image workstation 200 and controlling multi-path pneumatic output, and the precision pneumatic pump 300 is electrically connected with the image workstation 200;

The liquid measuring cylinder set 400 comprises a plurality of liquid measuring cylinders, each liquid measuring cylinder is connected with the air pressure output channel of the precision air pressure pump 300 through an air connecting pipe, and each liquid measuring cylinder is connected with the microfluidic chip 100 through a liquid connecting pipe.

Specifically, the microscope lens is aligned to the identification area of the microfluidic chip 100, the high-speed camera connected with the microscope continuously transmits the field image of the microscope to the graphic workstation in a multi-frame real-time manner, the graphic workstation carries on the deep learning and other algorithm models to identify and classify the real-time image, and sends different instructions to the precise pressure pump according to different results to control the inlet pressure of the sorting control flow channel on the multi-stage sorting microfluidic chip 100, the sorting operation of the microparticles is realized through the change of the fluid pressure of the sorting area, the microparticles identified and classified as target microparticles are sorted to the next sorting channel, and the non-target microparticles flow out mostly from the first sorting outlet 113. The particles are screened by the first identification and separation part 110, the concentration of the original ultra-low concentration target particles (the proportion of the number of the target particles to the total number of the particles) is obviously improved, and the adjacent particles in the flow channel flowing to the second identification and separation part 120 are obviously spaced front and back, so that the target particles can be completely separated from the first identification and separation part 110 to flow to the collection port 150 of the microfluidic chip 100, and other particles flow out from the second separation outlet 123, thereby realizing enrichment and purification of the ultra-low concentration particles.

Further, referring to fig. 1, the cartridge set 400 includes a first cartridge 410, a second cartridge 420, a third cartridge 430, a fourth cartridge 440 and a fifth cartridge 450,

The inlet of the first liquid measuring cylinder 410 is connected with the first air pressure output channel of the precise air pressure pump 300 through a first air pipe, the outlet of the first liquid measuring cylinder 410 is connected with the micro particle inlet 130 of the micro-fluidic chip 100 through a first liquid pipe,

An inlet of the second liquid measuring cylinder 420 is connected with a second air pressure output channel of the precise air pressure pump 300 through a second air pipe, an outlet of the second liquid measuring cylinder 420 is connected with the first sheath inflow port 111 of the microfluidic chip 100 through a second liquid pipe,

An inlet of the third liquid measuring cylinder 430 is connected with a third air pressure output channel of the precise air pressure pump 300 through a third air pipe, an outlet of the third liquid measuring cylinder 430 is connected with the second sheath inflow port 121 of the microfluidic chip 100 through a third liquid pipe,

An inlet of the fourth liquid measuring cylinder 440 is connected with a fourth air pressure output channel of the precision air pressure pump 300 through a fourth air pipe, an outlet of the fourth liquid measuring cylinder 440 is connected with an inlet of the first sorting control channel 112 of the microfluidic chip 100 through a fourth air pipe,

An inlet of the fifth liquid measuring cylinder 450 is connected to a fifth air pressure output channel of the precision air pressure pump 300 through a fifth air pipe, and an outlet of the fifth liquid measuring cylinder 450 is connected to an inlet of the second separation control channel 122 of the microfluidic chip 100 through a fifth air pipe.

Further, referring to fig. 1, the first liquid measuring cylinder 410 is filled with the microparticle suspension, the first air pipe extends into the first liquid measuring cylinder 410 to a depth higher than the liquid level of the microparticle suspension, and the first liquid pipe extends into the first liquid measuring cylinder 410 to a depth lower than the liquid level of the microparticle suspension;

The depth of the second air pipe extending into the second liquid measuring cylinder 420 is higher than the liquid level of the second liquid measuring cylinder 420, and the depth of the second air pipe extending into the second liquid measuring cylinder 420 is lower than the liquid level of the second liquid measuring cylinder 420;

the third air pipe extends into the third liquid measuring cylinder 430 to a depth higher than the liquid level of the third liquid measuring cylinder 430, and the third air pipe extends into the third liquid measuring cylinder 430 to a depth lower than the liquid level of the third liquid measuring cylinder 430;

the depth of the fourth air pipe extending into the fourth liquid measuring cylinder 440 is higher than the liquid level of the fourth liquid measuring cylinder 440, and the depth of the fourth air pipe extending into the fourth liquid measuring cylinder 440 is lower than the liquid level of the fourth liquid measuring cylinder 440;

the depth of the fifth air pipe extending into the fifth liquid measuring cylinder 450 is higher than the liquid level of the fifth liquid measuring cylinder 450, and the depth of the fifth air pipe extending into the fifth liquid measuring cylinder 450 is lower than the liquid level of the fifth liquid measuring cylinder 450.

Specifically, the five air outlets of the precision pressure pump are respectively connected with the air holes of the first liquid measuring cylinder 410, the second liquid measuring cylinder 420, the third liquid measuring cylinder 430, the fourth liquid measuring cylinder 440 and the fifth liquid measuring cylinder 450, each liquid measuring cylinder is filled with partial liquid, and two ports are reserved in a sealing way of the liquid measuring cylinders: one opening is that the air pipe hole is connected with an air pipe extending out of the precision pressure pump, and the air pipe opening in the cylinder is higher than the surface of the liquid; the other port is that a liquid pipe is connected with the liquid pipe to convey liquid to the multistage sorting micro-fluidic chip 100, and the liquid pipe opening in the cylinder is below the liquid level. The five air outlets of the precise pressure pump can be independently controlled by a program algorithm to output different pressures, so that the cavity above the liquid in each liquid measuring cylinder forms different pressures, thereby acting on the liquid in the liquid measuring cylinder, and each liquid pipe can independently output the fluid with different pressures.

The microparticle suspension is placed in a liquid measuring cylinder connected with the microparticle inlet 130 of the microfluidic chip 100, and other liquid measuring cylinders are used for placing relevant liquid according to different microparticle requirements. The liquid in each liquid measuring cylinder flows into the micro-fluidic chip 100 under the action of the precision pressure pump,

Further, referring to fig. 3, the invention further provides an operation method of the ultra-low concentration microparticle enrichment and purification device, which is applied to the ultra-low concentration microparticle enrichment and purification device, and the operation method comprises the following steps:

S100, a microscope, a shooting device and an image workstation 200 are arranged, a precise pneumatic pump 300, a liquid measuring cylinder group 400 and a microfluidic chip 100 are connected, a first liquid measuring cylinder 410 is preloaded with microparticle suspension, and a second liquid measuring cylinder 420, a third liquid measuring cylinder 430, a fourth liquid measuring cylinder 440 and a fifth liquid measuring cylinder 450 are preloaded with experimental liquid;

S200, outputting multi-path air pressure by the precise air pressure pump 300, respectively controlling the microparticle inlet 130, the first separation control flow channel and the second separation control flow channel to be high pressure, wherein the first sheath inflow port 111 is high pressure, and the second sheath inflow port 121 is sub-high pressure, so that microparticle suspension is input into the microfluidic chip 100 from the microparticle inlet 130 along the first liquid pipe;

S300, enabling the microparticle suspension to flow through the first identification region 115, continuously shooting field images by a shooting device through a microscope, transmitting the field images to the image workstation 200, performing image processing on the field images by the image workstation 200, and judging whether target particles exist or not;

S400, if target particles exist, the image workstation 200 outputs a first control signal for setting the first sorting control channel 112 to be low-pressure and maintaining the first control signal for a preset time to the precise pneumatic pump 300, and a fourth output channel of the precise pneumatic pump 300 outputs the low-pressure for the preset time, so that the liquid pressure of the first sorting control channel 112 is reduced briefly, the target particles are caused to avoid the first sorting outlet 113, and the target particles continue to flow into the second identification area 125 through the micro-flow channel 140;

s500, when the microparticle suspension flows through the second recognition area 125, the shooting device continuously shoots images of a field of view through a microscope, the images are transmitted to the image workstation 200, the image workstation 200 performs image processing on the images of the field of view, and whether target particles pass or not is judged;

S600, if the target particles are passing, the image workstation 200 outputs a second control signal for setting the second separation control channel 122 to be at a low pressure and maintaining the low pressure for a preset time to the precision pneumatic pump 300, and the fifth output channel of the precision pneumatic pump 300 outputs the low pressure for the preset time, so that the liquid pressure of the second separation control channel 122 is briefly reduced, and the target particles are caused to avoid the second separation outlet 123 and continue to flow into the collection port 150 through the micro-flow channel 140.

Further, referring to fig. 3, in the step S400, if the target particles are not present, the first separation control channel 112 maintains a high pressure, and the fine particle suspension containing no target particles flows out from the first separation outlet 113.

Further, referring to fig. 3, in the step S600, if the target particles are not present, the second separation control passage 122 maintains a high pressure, and the fine particle suspension containing no target particles flows out from the second separation outlet 123.

In some specific embodiments, taking the case that the outlet pressure of the precision pressure pump for controlling the pressure of the micro-particle inlet 130 is set to 20mbar as an example, referring to fig. 4 and 5, the front blocking state and the rear blocking state are the initial states of the microfluidic chip 100 of the device, referring to fig. 5, the initial pressures of the outlets of the precision pressure pumps for controlling the pressures of the micro-particle inlet 130, the first sorting control channel 112 and the second sorting control channel 122 are respectively 20mbar and high pressure (19.1 mbar is set in the figure, the values can be non-unique within a certain range; the high pressure and the low pressure are respectively the pressures before and after the change of the same inlet pressure value and the high pressure (20.3 mbar is set in the figure, the values can be non-unique within a certain range), the pressure values of the outlets of the precision pressure pumps for controlling the first sheath inlet 111 and the second sheath inlet 121, which are not shown in fig. 5, are always constant at 21mbar and 15mbar (suggested values, the non-unique values can be changed appropriately).

When the microparticle suspension flows in from the microparticle inlet 130, the microparticles flowing through the first sheath flow inlet 111 receive the sheath flow thrust action and concentrate on the other side to move forwards, then the microparticles are shot by a camera through the first recognition area 115 and uploaded to a graphic workstation, the algorithm of the graphic workstation classifies and recognizes the microparticles in the picture, if the target microparticles are recognized, the algorithm sends out an instruction for changing the pressure of a precise pressure pump connected with the first sorting control channel 112, the pressure of the precise pressure pump is unchanged for a few milliseconds, the precise pressure pump receives the instruction for changing the outlet pressure to be low (16 mbar, the value can fluctuate in a certain range and is not unique),

Referring to fig. 6 and 7, the state in the microfluidic chip 100 is changed to the front-pass back-plug state, and if no target micro particles exist in the photo identified by the graphic workstation, the outlet pressure is maintained or restored to the initial pressure, and the microfluidic chip 100 is also maintained or restored to the initial state. Referring to fig. 7, a mass of mixed particles containing target particles flows into the second separation zone 124, and at this time, the ratio of the number of target particles from which a plurality of non-target particles are removed in the first separation zone 114 to the total number of particles is greatly increased, i.e., enrichment of target particles is achieved. The difference between the particles in the flow path between the first sorting area 114 and the second sorting area 124 is gradually opened due to the speed distribution difference in the flow path cross section direction, and then flows to the second sorting area 124 one by one, and the particles are focused to one side by the fluid action at the second sheath inflow opening 121, and then flow through the second recognition area 125, the images are uploaded to the graphic workstation, the algorithm recognizes and sorts, and judges whether to send out a command for changing the pressure of the precise pressure pump for controlling the pressure of the second sorting control channel 122 according to whether the target particles exist, if the experiment is changed, the pressure is set to be low pressure (14 mbar, the value can fluctuate in a certain range is not unique),

Referring to fig. 8 to 11, the state of the microfluidic chip 100 is changed to the back-on state at this time, i.e., the target fine particles can smoothly enter the flow path leading to the collection port 150. Similarly, when the graphic workstation algorithm cannot identify that the image uploaded by the second identification area 125 contains the target particles, the pressure of the precise pressure pump for controlling the pressure of the second separation control channel 122 is set to be high (this value is set to 21mbar, and fluctuation in a certain range is not unique), so that the non-target particles flow to the second separation outlet 123, and the process from entering the microfluidic chip 100 to enrichment and finally being precisely separated to the collection outlet 150 is completely presented.

The above process is enrichment and purification of ultra-low concentration particles under ideal conditions, if the system is interfered to cause the particles of the collection port 150 to be mixed with impurities, the particle suspension of the collection port 150 can be introduced into the particle inlet 130 of another same sorting chip again, namely, the purification of target particles is realized through three-level and four-level sorting.

Referring to fig. 12, the deep learning algorithm of the graph workstation of the ultra-low concentration microparticle enrichment and purification device is mainly based on the Yolo v target detection model of the open source.

Specifically, referring to fig. 12, the Yolo v target detection model mainly includes four parts, input, backbone, neck and Prediction, where each part functions as:

input: scaling the size data of the picture and adapting to model training;

Backspace: extracting feature images of different receptive fields of P1-P5 by the model main network through increasing the number of convolution layers, wherein the receptive fields of P1-P5 are gradually increased in sequence, and information in the extracted images is used by a later network;

Neck: the FPN and PAN structures are presented, where FPN (feature pyramid network): detecting targets with different sizes by adopting multiple scales; PAN: bottom-up feature pyramids. In this way, the FPN layer conveys strong semantic features from top to bottom, the feature pyramid conveys strong positioning features from bottom to top, and the two hands better utilize the features extracted by the backstbone to perform feature aggregation on different detection layers from different trunk layers.

Prediction: in the process of P3, P4 and P5, the receptive field is sequentially increased for Head in the model frame and is used for final prediction output, so that the sequential prediction targets are small, medium and large.

The present invention is not limited to the above embodiments, but can be modified, equivalent, improved, etc. by the same means to achieve the technical effects of the present invention, which are included in the spirit and principle of the present disclosure. Are intended to fall within the scope of the present invention. Various modifications and variations are possible in the technical solution and/or in the embodiments within the scope of the invention.

Claims (10)

1. A microfluidic chip (100) comprising a microparticle inlet (130), a microfluidic channel (140) and a collection port (150) connected in sequence, characterized in that it further comprises:

the device comprises at least one identification and separation part, wherein the identification and separation part comprises a sheath inflow port, a separation control flow passage and a separation outlet which are sequentially arranged, the sheath inflow port and the separation outlet are arranged on the same side of a micro-flow channel (140), the separation control flow passage is arranged on the opposite side of the micro-flow channel (140), a separation area is formed at the junction of the separation control flow passage and the micro-flow channel (140), the separation outlet is closely arranged at the rear end of the separation area, the front end of the separation area is provided with an identification area, a microparticle inlet (130) is connected with the inlet of the first identification and separation part, a plurality of identification and separation parts are connected through the micro-flow channel (140), and a collection port (150) is connected with the last identification and separation part.

2. The microfluidic chip (100) according to claim 1, wherein the identification sorting section comprises a first identification sorting section (110) and a second identification sorting section (120) connected in sequence, the microparticle inlet (130) is connected to a start of the first identification sorting section (110), and an end of the second identification sorting section (120) is connected to the collection port (150).

3. The microfluidic chip (100) according to claim 2, wherein,

The first identification sorting part (110) comprises a first sheath inflow port (111), a first sorting control channel (112) and a first sorting outlet (113), a first sorting area (114) is formed at the junction of the first sorting control channel and the microfluidic channel (140), the first sorting outlet (113) is closely arranged at the rear end of the first sorting area (114), and a first identification area (115) is arranged at the front end of the first sorting area (114).

4. The microfluidic chip (100) according to claim 2, wherein,

The second identification sorting part (120) comprises a second sheath inflow opening (121), a second sorting control channel (122) and a second sorting outlet (123), a second sorting area (124) is formed at the junction of the second sorting control channel and the micro-flow channel (140), the second sorting outlet (123) is closely arranged at the rear end of the second sorting area (124), and a second identification area (125) is arranged at the front end of the second sorting area (124).

5. An ultra-low concentration microparticle enrichment and purification device comprising the microfluidic chip (100) according to claim 2, wherein the ultra-low concentration microparticle enrichment and purification device further comprises:

A microscope disposed above the identification zone;

the shooting device is used for shooting a view field image of the identification area at a high speed and is connected with an eyepiece of the microscope;

An image workstation (200) for performing image recognition target examples on field-of-view images shot by a shooting device and sending out control signals, wherein the image workstation (200) is electrically connected with the shooting device;

the precision pneumatic pump (300) is used for receiving control signals of the image workstation (200) and controlling multi-path pneumatic output, and the precision pneumatic pump (300) is electrically connected with the image workstation (200);

The liquid measuring cylinder group (400) comprises a plurality of liquid measuring cylinders, each liquid measuring cylinder is connected with the air pressure output channel of the precise air pressure pump (300) through an air connecting pipe, and each liquid measuring cylinder is connected with the microfluidic chip (100) through a liquid connecting pipe.

6. The apparatus for enriching and purifying ultra-low concentration fine particles according to claim 5, wherein,

The liquid measuring cylinder group (400) comprises a first liquid measuring cylinder (410), a second liquid measuring cylinder (420), a third liquid measuring cylinder (430), a fourth liquid measuring cylinder (440) and a fifth liquid measuring cylinder (450),

The inlet of the first liquid measuring cylinder (410) is connected with a first air pressure output channel of the precise air pressure pump (300) through a first air pipe, the outlet of the first liquid measuring cylinder (410) is connected with a microparticle inlet (130) of the microfluidic chip (100) through a first liquid pipe,

An inlet of the second liquid measuring cylinder (420) is connected with a second air pressure output channel of the precise air pressure pump (300) through a second air pipe, an outlet of the second liquid measuring cylinder (420) is connected with a first sheath inflow port (111) of the microfluidic chip (100) through a second liquid pipe,

An inlet of the third liquid measuring cylinder (430) is connected with a third air pressure output channel of the precise air pressure pump (300) through a third air pipe, an outlet of the third liquid measuring cylinder (430) is connected with a second sheath inflow port (121) of the microfluidic chip (100) through a third liquid pipe,

An inlet of the fourth liquid measuring cylinder (440) is connected with a fourth air pressure output channel of the precise air pressure pump (300) through a fourth air pipe, an outlet of the fourth liquid measuring cylinder (440) is connected with an inlet of a first sorting control channel (112) of the microfluidic chip (100) through a fourth liquid pipe,

An inlet of the fifth liquid measuring cylinder (450) is connected with a fifth air pressure output channel of the precise air pressure pump (300) through a fifth air pipe, and an outlet of the fifth liquid measuring cylinder (450) is connected with an inlet of a second separation control channel (122) of the microfluidic chip (100) through a fifth liquid pipe.

7. The apparatus for enriching and purifying ultra-low concentration fine particles according to claim 6, wherein,

The first liquid measuring cylinder (410) is filled with the microparticle suspension, the first air pipe stretches into the first liquid measuring cylinder (410) to a depth higher than the liquid level of the microparticle suspension, and the first liquid pipe stretches into the first liquid measuring cylinder (410) to a depth lower than the liquid level of the microparticle suspension;

the depth of the second air pipe extending into the second liquid measuring cylinder (420) is higher than the liquid level of the second liquid measuring cylinder (420), and the depth of the second air pipe extending into the second liquid measuring cylinder (420) is lower than the liquid level of the second liquid measuring cylinder (420);

The depth of the third air pipe extending into the third liquid measuring cylinder (430) is higher than the liquid level of the third liquid measuring cylinder (430), and the depth of the third liquid pipe extending into the third liquid measuring cylinder (430) is lower than the liquid level of the third liquid measuring cylinder (430);

the depth of the fourth air pipe extending into the fourth liquid measuring cylinder (440) is higher than the liquid level of the fourth liquid measuring cylinder (440), and the depth of the fourth air pipe extending into the fourth liquid measuring cylinder (440) is lower than the liquid level of the fourth liquid measuring cylinder (440);

The depth of the fifth air pipe extending into the fifth liquid measuring cylinder (450) is higher than the liquid level of the fifth liquid measuring cylinder (450), and the depth of the fifth liquid pipe extending into the fifth liquid measuring cylinder (450) is lower than the liquid level of the fifth liquid measuring cylinder (450).

8. A method of operating an ultra-low concentration microparticle enrichment and purification device according to any one of claims 5 to 7, comprising:

S100, a microscope, a shooting device and an image workstation (200) are arranged, a precise pneumatic pump (300), a liquid measuring cylinder group (400) and a microfluidic chip (100) are connected, a first liquid measuring cylinder (410) is preloaded with microparticle suspension, and a second liquid measuring cylinder (420), a third liquid measuring cylinder (430), a fourth liquid measuring cylinder (440) and a fifth liquid measuring cylinder (450) are preloaded with experimental liquid;

S200, outputting multi-path air pressure by a precise air pressure pump (300), respectively controlling a microparticle inlet (130), a first separation control flow channel and a second separation control flow channel to be high pressure, wherein the first sheath inflow port (111) is high pressure, and the second sheath inflow port (121) is sub-high pressure, so that microparticle suspension is input into a microfluidic chip (100) from the microparticle inlet (130) along a first liquid pipe;

S300, enabling the microparticle suspension to flow through a first identification area (115), continuously shooting field images by a shooting device through a microscope, transmitting the field images to an image workstation (200), performing image processing on the field images by the image workstation (200), and judging whether target particles exist or not;

S400, if target particles exist, the image workstation (200) outputs a first control signal for setting a first sorting control channel (112) to be low-pressure and maintaining a preset time to the precise pneumatic pump (300), and a fourth output channel of the precise pneumatic pump (300) outputs the low-pressure for the preset time, so that the liquid pressure of the first sorting control channel (112) is reduced briefly, the target particles are caused to avoid a first sorting outlet (113) and continuously flow into a second identification area (125) through a micro-flow channel (140);

S500, when the microparticle suspension flows through the second identification area (125), the shooting device continuously shoots view field images through a microscope and transmits the view field images to the image workstation (200), and the image workstation (200) performs image processing on the view field images to judge whether target particles pass through;

S600, if the target particles pass through, the image workstation (200) outputs a second control signal for setting the second separation control channel (122) to be low-pressure and maintaining the second control signal for a preset time to the precise pneumatic pump (300), and the fifth output channel of the precise pneumatic pump (300) outputs the low-pressure for the preset time, so that the liquid pressure of the second separation control channel (122) is reduced briefly, the target particles are caused to avoid the second separation outlet (123), and the target particles continue to flow into the collecting port (150) through the micro-flow channel (140).

9. The method for operating an ultra-low concentration fine particle enrichment and purification apparatus as claimed in claim 8, wherein,

In the step S400, if no target particles exist, the first separation control channel (112) maintains a high pressure, and the fine particle suspension containing no target particles flows out from the first separation outlet (113).

10. The method for operating an ultra-low concentration fine particle enrichment and purification apparatus as claimed in claim 8, wherein,

In the step S600, if no target particles exist, the second separation control channel 122 maintains a high pressure, and the fine particle suspension containing no target particles flows out from the second separation outlet 123.