CN110420672B

CN110420672B - Micro-fluidic chip and application thereof in particle cleaning and liquid changing

Info

Publication number: CN110420672B
Application number: CN201910640344.1A
Authority: CN
Inventors: 郭霄亮; 马洲扬
Original assignee: Beijing University of Chemical Technology
Current assignee: Shenzhen Saiqiao Biological Innovation Technology Co Ltd
Priority date: 2019-07-16
Filing date: 2019-07-16
Publication date: 2020-05-19
Anticipated expiration: 2039-07-16
Also published as: CN110420672A

Abstract

The invention provides a micro-fluidic chip and application thereof in particle cleaning and liquid replacement. The micro-fluidic chip comprises a chip body, wherein the chip body is of a sealed cuboid cavity type structure, the left side surface and the right side surface of the chip body are provided with an upper opening and a lower opening which are connected with the inside of a chip cavity and the outside, and an array structure is formed by uniformly arranging N rows of micro-columns in the chip body cavity, wherein N is an integer more than or equal to 2; the top and bottom surfaces of the microcolumn are tightly connected with the upper and lower substrates of the chip body; the array structure is arranged in the following mode: the (N + 1) th row is obliquely arranged relative to the (N) th row of microcolumns, and the inclination angle is less than 60 degrees; the adjacent cavities among the micro-columns are pore channels through which liquid flows. The micro-fluidic chip can be used for cleaning or changing the liquid of the solution containing the microspheres or cells, the operation is convenient, the effect is good, the microspheres can move in the chip according to the array offset direction, the two liquids can not be mixed due to the laminar flow phenomenon, and finally the microspheres can move from one liquid to the other liquid to realize the liquid change.

Description

Micro-fluidic chip and application thereof in particle cleaning and liquid changing

Technical Field

The invention relates to the technical field of micro-fluidic chip preparation and cell cleaning, in particular to a micro-fluidic chip and application thereof in particle cleaning and liquid replacement.

Background

The current microfluidic chip is widely applied to the medical field by virtue of the advantages of low cost, small equipment and the like. Particularly, the microfluidic technology also has the advantages of small sample consumption, high resolution precision and sensitivity, easy integration and miniaturization, and the like, is widely applied to the research of cell screening, and shows good development prospect.

Cell exchange is one of the essential steps of many experiments, and for example, the operation of cell exchange is very important in experiments such as cell culture and flow cytometry. At present, people often use a centrifugal mode to change liquid, such as suspension cells, and usually need to repeatedly perform the centrifugal-liquid suction-liquid feeding-air beating steps for many times. When the cells are centrifuged, the activity, internal structure, transcription pattern, etc. of the cells are affected to some extent by the centrifugal force and hydrostatic pressure. If the liquid is centrifuged at a high rotating speed for a long time, cells are easy to break, and the cells are easy to die; however, if the liquid is centrifuged for a short time and at a low rotation speed, cells are difficult to settle to the bottom, and the cells are easily lost when the waste liquid is aspirated. Moreover, the operation time of centrifugal liquid change is generally long, the operation steps are complicated, the efficiency is difficult to improve, and the automation is further realized. Meanwhile, the cells are exposed in the air for a long time, and are easy to cause bacterial and mycoplasma pollution.

In addition, a centrifugal machine is needed for liquid exchange in a centrifugal mode, but the centrifugal machine is difficult to integrate in a small-sized working system due to large volume, and can generate great vibration in the operation process, so that the vibration can influence the stability of the whole system device, and even other equipment can not work normally. The micro-fluidic chip has the advantages of low cost, small and exquisite appearance, high plasticity and convenient integration, and in the prior art, the micro-fluidic chip is used for cleaning and replacing liquid, but peripheral equipment such as an ultrasonic method is needed, ultrasonic waves are active, namely an ultrasonic wave source is added on the chip, so that the whole system is complex and high in cost. Therefore, a cell washing and liquid changing device with simple structure, high efficiency, low cost and convenient use is needed.

Disclosure of Invention

Aiming at the defects of the prior art, the invention provides the microfluidic chip which has the advantages of simple structure, low cost, convenient use and high cleaning and separating effects and is used for cleaning and replacing cells or microsphere solution.

The invention provides a microfluidic chip, which comprises a chip body, wherein the chip body is of a sealed cuboid cavity type structure, and the left side surface and the right side surface of the chip body are provided with an upper port and a lower port which are connected with the inside of a chip cavity and the outside, and the upper port and the lower port comprise a left inlet 1, a left inlet 2, a right outlet 1 and a right outlet 2;

in the cavity of the chip body, N rows of micro-columns are uniformly arranged to form an array structure, wherein N is an integer more than or equal to 2; the top surface and the bottom surface of the microcolumn are tightly connected with the upper substrate and the lower substrate of the chip body;

the array structure is arranged in the following mode: the (N + 1) th row is obliquely arranged relative to the (N) th row of micro-columns, the inclination angle is less than 60 degrees, and an obliquely arranged array is formed; the distance between every two microcolumns in each column is the same, and the central connecting lines of every oblique row microcolumn are relatively parallel;

the adjacent cavities among the micro-columns are micro-channels through which liquid flows.

In the microfluidic chip provided by the invention, the height of a micro-channel of the chip is 1-1000 mu m. Further, the height of the micro-channel of the chip is 10-800 μm; preferably, the height of the micro flow channel of the chip is 20-500 μm; more preferably, the height of the micro flow channel of the chip is 50 to 300. mu.m.

In the microfluidic chip provided by the invention, the cross section of the microcolumn is circular, parallelogram, regular polygon or ellipse, and the regular polygon is preferably regular triangle.

When the cross section of the micro-column is circular, the top sectional view of the inner cavity of the chip body is shown in fig. 1.

When the cross section of the microcolumn in the chip body is circular, the radius of the cross section of each microcolumn is the same and is less than 500 mu m; the shortest relative distance D1 between two adjacent microcolumns in each row of microcolumns is 0.01-5000 μm; the shortest relative distance D2 between two adjacent microcolumns of each diagonal microcolumn is 0.01-500 μm; and D1 is always greater than D2.

Preferably, the radius of each circular microcolumn is less than 300 μm, less than 200 μm, less than 100 μm, less than 80 μm, less than 70 μm, less than 60 μm, less than 50 μm, less than 40 μm. More preferably, the radius of each circular microcolumn is 5 to 30 μm, 8 to 25 μm, 10 to 23 μm, 12.5 to 20 μm.

Preferably, the shortest relative distance between two adjacent microcolumns in each column of microcolumns is 1-1000 μm, 10-1000 μm; the shortest relative distance between two adjacent micro-columns of each diagonal micro-column is 1-100 μm, and the inclination angle of the two is less than 60, less than 30 °, less than 20 °, and less than 10 °.

More preferably, the radius of each circular microcolumn is 5-30 μm, 8-25 μm, 10-23 μm, 12.5-20 μm; the shortest relative distance between two adjacent microcolumns in each row of microcolumns is 40-500 mu m; the shortest relative distance between two adjacent microcolumns of each diagonal microcolumn is 3-10 mu m; and the inclination angles of the two are more than 0.5 degrees and less than 10 degrees.

When the cross section is a parallelogram, the side length of the parallelogram is less than 500 mu m, and the acute included angle of the parallelogram is less than 60 degrees; the shortest relative distance D1 between two adjacent microcolumns in each row of microcolumns is 0.01-5000 μm; the shortest relative spacing D2 of two adjacent microcolumns per diagonal microcolumn is 0.01 μm to 500 μm, and D1 is always greater than D2.

Preferably, the sides of the parallelogram are less than 100 μm, less than 80 μm, less than 70 μm, less than 60 μm, less than 50 μm, less than 40 μm, the acute included angle of the parallelogram being 40 °; the shortest relative distance D1 between two adjacent microcolumns in each row of microcolumns is 1-1000 μm and 10-1000 μm; the shortest relative distance D2 between two adjacent microcolumns of each diagonal microcolumn is 1-100 μm;

more preferably, the side length of the parallelogram is 25-35 μm, and the acute included angle of the parallelogram is less than 30 °; the shortest relative distance D1 between two adjacent microcolumns in each row of microcolumns is 35-45 μm; the shortest relative distance D2 between two adjacent microcolumns of each diagonal microcolumn is 20-30 μm.

When the cross section of the micro-fluidic chip provided by the invention is a regular triangle, the side length of the regular triangle is less than 100 mu m; the shortest relative distance D1 between two adjacent microcolumns in each row of microcolumns is 0.01-5000 μm; the shortest relative distance D2 between two adjacent microcolumns of each diagonal microcolumn is 0.01-500 μm;

preferably, the side length of the regular triangle is 10-100 μm; the shortest relative distance between two adjacent microcolumns in each row of microcolumns is 1-1000 μm and 10-1000 μm; the shortest relative distance between two adjacent microcolumns of each diagonal microcolumn is 1-100 mu m;

more preferably, the side length of the regular triangle is 20-40 μm; the shortest relative distance between two adjacent microcolumns in each row of microcolumns is 40-500 mu m; the shortest relative distance between two adjacent microcolumns of each diagonal microcolumn is 3-10 μm.

Specifically, in one embodiment of the present invention, when the cross section of the microcolumn in the chip body is a regular triangle, the side length of the regular triangle is 16 to 26 μm; the distance between the central connecting lines of two adjacent microcolumns in each row of microcolumns is 40-60 mu m; the difference of the vertical distance of the central points of two adjacent micro-pillars of each diagonal micro-pillar relative to the width of the chip body is 48-52 μm, and the difference of the vertical distance of the central points of two adjacent micro-pillars of each diagonal micro-pillar relative to the length of the chip body is 2-4 μm.

When the cross section of the microfluidic chip is oval, the horizontal axis length of the oval is less than 100 micrometers, and the vertical axis length of the oval is less than 100 micrometers; the shortest relative distance D1 between two adjacent microcolumns in each row of microcolumns is 0.01-5000 μm; the shortest relative distance D2 between two adjacent microcolumns of each diagonal microcolumn is 0.01-500 μm; and D1 is always greater than D2;

preferably, the horizontal axis of the ellipse is 10-90 μm long, and the vertical axis is 10-90 μm long; the shortest relative distance between two adjacent microcolumns in each row of microcolumns is 1-1000 μm and 10-1000 μm; the shortest relative distance between two adjacent microcolumns of each diagonal microcolumn is 1-100 mu m;

more preferably, the horizontal axis of the ellipse is 20-50 μm long and the vertical axis is 20-40 μm long; the shortest relative distance between two adjacent microcolumns in each row of microcolumns is 40-500 mu m; the shortest relative distance between two adjacent microcolumns of each diagonal microcolumn is 3-10 μm.

In one embodiment of the present invention, when the cross-section of the microcolumn in the chip body is elliptical, the horizontal axis length of the ellipse is 50 to 70 μm, and the vertical axis length is 25 to 35 μm; the distance between the central connecting lines of two adjacent microcolumns in each row of microcolumns is 40-60 mu m; the difference of the vertical distance of the central points of two adjacent micro-pillars of each diagonal micro-pillar relative to the width of the chip body is 50-70 μm, and the difference of the vertical distance of the central points of two adjacent micro-pillars of each diagonal micro-pillar relative to the length of the chip body is 15-25 μm.

Furthermore, the left inlet 1, the left inlet 2, the right outlet 1 and the right outlet 2 of the chip body of the microfluidic chip provided by the invention are respectively connected with a pipeline capable of flowing liquid, and the acute angle included angle between the pipeline and the length of the chip body is 0-30 degrees; and/or the inlet 1 and the inlet 2 are connected with a micro pump, and the micro pump enables the flow rate of liquid passing through the inner cavity of the chip body to be 10-20 mu L/min.

When the microfluidic chip disclosed by the invention is used, the chip in the figure 1 can be rotated by 90 degrees clockwise, and when a solution is added, the solution enters from the two inlets on the upper side (left side) and exits from the two outlets on the lower side (right side) by virtue of gravity. The micro-pump can be horizontally arranged according to the example of fig. 1, and is connected with two inlets, and the micro-pump can make the solution added from the inlets flow in the opposite direction.

The material of the chip of the invention can be selected from Polydimethylsiloxane (PDMS), PMMA, glass, silicon chip, metal and the like, but in consideration of cost and experimental convenience, PDMS is adopted as an experimental material in the embodiment, and other materials can be considered when being applied to the field of mass production.

The invention provides application of the microfluidic chip in cleaning or replacing a solution containing particles. The particles include, but are not limited to, cells or microspheres.

The invention provides a method for cleaning or replacing a solution containing particles, wherein the particles are cells or microspheres, and the microfluidic chip is adopted for cleaning or replacing the solution containing the particles.

The particles of the present invention have a diameter of less than 20 microns.

In the above method, in actual operation, the diameter of the particle to be exchanged and cleaned is smaller than the shortest relative distance between every two adjacent micro-pillars in the micro-pillar array, and the diameter of the particle to be exchanged and cleaned is larger than the shortest relative distance between every two adjacent micro-pillars in each diagonal line in the micro-pillar array (see the column and diagonal line at the position of fig. 1).

The microfluidic chip provided by the invention has the capability of transferring cells or particles in one solution of cells or particles smaller than 20 microns to another solution, and simultaneously can separate and collect the two solutions again. In practice, both solutions, regardless of concentration, can transfer particles to solution B at the same time as solution A, as long as there are cells or other particles in solution B. The transfer rate of the two collected liquid cells or particles is more than 95%.

The specific operation process is as follows: and respectively injecting the solution A and the solution B (5-20 micron cells or particles) into the flow channels of the two inlets at the same side at the same speed, respectively flowing out and collecting the two liquids from the outlet 1 and the outlet 2 of the flow channel at the other side through the micro-column array structure in the cavity region of the chip body, and collecting the solution A (the cells or particles smaller than 20 microns) and the solution B.

The invention adopts the microcolumn array to change the liquid for the microscopic particles, and has important function in the application of the microfluidic sorting technology to cell liquid change and cleaning due to the advantages of cleanness, simplicity, convenience, continuous separation and the like. The sorting device consists of micro-columns which are regularly arranged, and each row of micro-columns is slightly staggered with the previous row to form an obliquely arranged array. When the fluid flows among the micro-columns, the inclined main flow channel and the transverse sub-flow channel are naturally formed. When the diameter of the particles is significantly smaller than the shortest relative distance between every two adjacent columns in the microcolumn array, and the particles to be changed and cleaned are directly larger than the shortest relative distance between every two adjacent columns in each diagonal row in the microcolumn array, the particles will shift, so that the particles move along the inclined main flow channel. The transfer rate of the particles is more than 95 percent, and the method is convenient to operate and low in cost.

Drawings

Fig. 1 is a top sectional view of the internal structure of the chip body of the microfluidic chip of the present invention.

Fig. 2 is a top view of the shape and size of the chip body of the microfluidic chip according to example 1 of the present invention.

Fig. 3 shows the shape and size of a circular array in the inner cavity of the chip body of the microfluidic chip of the present invention.

Fig. 4 shows the shape and size of a parallelogram array in the inner cavity of the chip body of the microfluidic chip of the invention.

Fig. 5 shows the shape and size of a regular triangle array in the inner cavity of the chip body of the microfluidic chip of the present invention.

FIG. 6 shows the shape and size of an oval array in the inner cavity of the chip body of the microfluidic chip according to the present invention.

FIG. 7 is a diagram showing the effect of the mask in example 2.

Fig. 8 is a schematic diagram of the round microcolumn arrangement in the inner cavity of the microfluidic chip body manufactured in example 2.

FIG. 9 is a diagram showing a simulation of the flow channel in example 3.

FIG. 10 is a graph showing the concentration simulation in example 3, wherein the left side shows a high concentration and the right side shows a low concentration.

FIG. 11 is the results of the simulation in example 3, the concentration profile at the outlet.

FIG. 12 is an under-the-eyepiece view of the laminar flow phenomenon of example 3.

Fig. 13 is a view of the laminar flow phenomenon under the camera in example 3.

FIG. 14 is an experimental graph of microsphere replacement with two solutions in example 4, showing the trace of fluorescent microspheres from inlet to outlet, and ABCD in the graph indicates four different time node frame images from inlet to outlet, respectively, showing that the beads enter from the bottom right and flow out from the top left by offset.

Detailed Description

The following detailed description of embodiments of the present invention is provided in connection with the accompanying drawings and examples. The following examples are intended to illustrate the present invention, but not to limit the scope of the invention, which is defined by the claims.

Example 1 structure and internal dimensions of microfluidic chip

Fig. 2 is a top view of the chip of this embodiment, and fig. 1 is a top cross-sectional view of the inner cavity of the chip body. The micro-fluidic chip comprises a chip body, wherein the chip body is of a sealed cuboid cavity type structure, and the left side surface and the right side surface of the chip body are provided with an upper port and a lower port which are used for connecting the chip cavity with the outside and comprise a left inlet 1, a left inlet 2, a right outlet 1 and a right outlet 2; in the cavity of the chip body, N rows of micro-columns are uniformly arranged to form an array structure, wherein N is an integer more than or equal to 2; the top surface and the bottom surface of the microcolumn are tightly connected with the upper substrate and the lower substrate of the chip body; the array structure is arranged in the following mode: the (N + 1) th row is obliquely arranged relative to the (N) th row of micro-columns, the inclination angle is less than 60 degrees, and an obliquely arranged array is formed; the distance between every two microcolumns in each column is the same, and the central connecting lines of every oblique row microcolumn are relatively parallel; the adjacent cavities among the micro-columns are pore channels through which liquid flows.

The chip comprises a chip body, wherein a left inlet 1, a left inlet 2, a right outlet 1 and a right outlet 2 of the chip body are respectively connected with a pipeline capable of flowing liquid, and an acute included angle between the pipeline and the length of the chip body is 0-30 degrees; and/or the inlet 1 and the inlet 2 are connected with a micro pump, and the micro pump enables the flow rate of liquid passing through the inner cavity of the chip body to be 10-20 mu L/min.

TABLE 1 parameters of micro-channel region in inner cavity of chip body corresponding to FIG. 2

H in Table 1 refers to the height of the micro-channel in the inner cavity of the chip body, which is 10-500 μm.

The cross section of the microcolumn in the inner cavity of the chip body is circular, parallelogram, regular polygon (preferably regular triangle) or ellipse.

1. When the cross section of the microcolumn is circular, the radius of the cross section of each microcolumn is the same and is 12.5-16 mu m; the distance between the central connecting lines of two adjacent microcolumns in each row of microcolumns is 40-60 mu m; the distance between the central connecting lines of two adjacent microcolumns of each diagonal microcolumn is 30-40 μm, and the inclination angle of the two microcolumns is 3.2-60 deg. See fig. 3, table 2. The shortest relative distance between two adjacent microcolumns in each row of microcolumns is 40-500 mu m; the shortest relative distance between two adjacent microcolumns of each diagonal microcolumn is 3-10 μm.

TABLE 2 respective parameters of the circular microcolumn in the chip body corresponding to those in FIG. 3

2. When the cross section is a parallelogram, the side length of the parallelogram is less than 100 μm, less than 80 μm, less than 70 μm, less than 60 μm, less than 50 μm and less than 40 μm, and the acute included angle of the parallelogram is less than 40 °; the shortest relative distance D1 between two adjacent microcolumns in each row of microcolumns is 1-1000 μm and 10-1000 μm; the shortest relative distance D2 between two adjacent microcolumns of each diagonal microcolumn is 1-100 μm;

The side length of the parallelogram is 25-35 μm, and the acute angle included angle of the parallelogram is less than 60 degrees; the distance between the central connecting lines of two adjacent microcolumns in each row of microcolumns is 40-60 mu m; the difference of the vertical distance between the central points of two adjacent micro-pillars of each diagonal micro-pillar relative to the width of the chip body is 35-45 μm, and the difference of the vertical distance between the central points of two adjacent micro-pillars of each diagonal micro-pillar relative to the length of the chip body is 20-30 μm. See fig. 4, table 3.

TABLE 3 respective parameters of the parallelogram microcolumns in the chip body corresponding to those in FIG. 4

3. When the cross section is a regular triangle, the side length of the regular triangle is less than 500 mu m; the shortest relative distance between two adjacent microcolumns in each row of microcolumns is 0.01-5000 microns; the shortest relative distance between two adjacent microcolumns of each diagonal microcolumn is 0.01-500 mu m; and D1 is always greater than D2;

Specifically, the side length of the regular triangle of the microfluidic chip is 16-26 μm; the distance between the central connecting lines of two adjacent microcolumns in each row of microcolumns is 40-60 mu m; the difference of the vertical distance between the central points of two adjacent micro-pillars of each diagonal micro-pillar and the width of the chip body is 48-52 μm, and the difference of the vertical distance between the central points of two adjacent micro-pillars of each diagonal micro-pillar and the length of the chip body is 2-4 μm; see fig. 5, table 4.

TABLE 4 respective parameters of regular triangular microcolumns in the chip body corresponding to those in FIG. 5

4. When the cross-section is an oval shape,

the horizontal axis length of the ellipse is less than 100 μm, and the vertical axis length is less than 100 μm; the shortest relative distance between two adjacent microcolumns in each row of microcolumns is 0.01-5000 microns; the shortest relative distance between two adjacent microcolumns of each diagonal microcolumn is 0.01-500 mu m; and D1 is always greater than D2;

In the embodiment, the horizontal axis length of the ellipse is 30-50 μm, and the vertical axis length is 25-35 μm; the distance between the central connecting lines of two adjacent microcolumns in each row of microcolumns is 40-60 mu m; the difference of the vertical distance of the central points of two adjacent micro-pillars of each diagonal micro-pillar relative to the width of the chip body is 50-70 μm, and the difference of the vertical distance of the central points of two adjacent micro-pillars of each diagonal micro-pillar relative to the length of the chip body is 15-25 μm. See fig. 6, table 5.

TABLE 5 parameters of the elliptical microcolumns in the corresponding chip body in FIG. 6

Variables of	a	b	c	d	e
						Parameter(s)	40 to 60 μm	50 to 70 μm	25 to 35 μm	50 to 70 μm	15 to 25 μm

All the parameters in the embodiment are obtained by repeated screening through a large number of experiments, and by adopting other parameters except the parameters, the separation, cleaning and liquid change of the cells and the microspheres can not be well realized.

Example 2 preparation of microfluidic chip

1. Principle for determining diameter of liquid-changing and cleaning particles

As shown in fig. 2, when the diameter of the particles is significantly smaller than the shortest relative distance between every two adjacent columns in the micro-column array, and the diameter of the particles to be replaced and cleaned is larger than the shortest relative distance between every two adjacent columns in each diagonal row in the micro-column array, the particles are shifted, so that the particles move along the inclined main flow channel. When the diameter of the particle is smaller than the shortest relative distance of the diagonal rows of the micropillar array, the particle will basically keep the original traveling direction, so the diameter of the particle for liquid exchange and cleaning should be smaller than the shortest relative distance of each column (longitudinal direction in fig. 1) of the adjacent micropillars in the micropillar array, and the diameter of the particle for liquid exchange and cleaning should be larger than the shortest relative distance of each two adjacent micropillars in the diagonal rows (diagonal direction in fig. 1) in the micropillar array.

2. Mask pattern drawing

According to the invention, a silicon wafer and SU-82015 photoresist are used as templates of the microfluidic chip, and an optical mask is required to be manufactured. The optical mask is a graphic master mask used by a common photoetching process of a micro-nano processing technology, a mask graphic structure is formed on a transparent substrate by an opaque shading film, and graphic information is transferred to a product substrate through an exposure process. The mask effect pattern is shown in fig. 7.

In fig. 7, the white area becomes transparent after being processed into a film, and is a light-transmitting area, and the black area is a light-shielding area after being processed into a film. According to the principle of deterministic lateral migration, the circle of the invention has a diameter of 25 microns, a longitudinal centre distance of 50 microns, a lateral centre distance of 37 microns, and an inclination angle of 23.962 °, and is schematically shown in fig. 8. The micro-column array designed by the method can separate 15-micron particles.

3. Lithography

According to the invention, a 4-inch silicon wafer is used as a die substrate of a chip, the silicon wafer is cleaned firstly, plasma treatment is carried out for 2 minutes, and the used SU-82015 photoresist is heated by a water bath at 30 ℃. The photoresist was then poured into the center of the wafer and a spin coating (500rpm for 10s, 1300rpm for 30s) was performed.

After standing for 10 minutes, soft baking was carried out at 95 ℃ for 4 minutes and 20 seconds, exposure was carried out for 14 seconds, and after baking was carried out at 95 ℃ for 5 minutes and 20 seconds. And developing the silicon wafer for 4 min and 20 sec, cleaning, and hardening at 150 ℃ for 10 min to finish the preparation.

Because negative glue is used for manufacturing, a region irradiated by ultraviolet rays forms a convex structure, and a black circular array can form a groove array on the convex structure.

4. Microfluidic chip fabrication

The microfluidic chip required to be manufactured by the method can be widely used in materials such as Polydimethylsiloxane (PDMS), a plastic and laser bonding technology, a 3-D printing technology and the like.

In the examples, PDMS was selected as the substrate of the microfluidic chip. PDMS is a high molecular organic silicon compound, has optical transparency, is inert under common conditions, is non-toxic, harmless and non-flammable, and is widely applied to the field of micro-channel systems in the bio-micro electro-mechanical systems. The unheated PDMS is in a viscous liquid state, 20g of PDMS is taken from a laboratory, a hardening agent is added according to the proportion of 10:1, the mixture is fully stirred for 3 minutes, bubbles in the mixture are floated to the surface and broken in a vacuumizing mode, and then the mixture is poured into a mold made of a silicon wafer. Air blowing was used to blow off bubbles from the surface and then the PDMS covered mold was placed on a heating table at 120 ℃ until the PDMS solidified to a solid state. Thereafter, the slide glass was cleaned with ultrasonic waves.

And combining PDMS with the glass slide by using a vacuum oxygen plasma bonding method, so that a plate-shaped structure containing a pore channel and a micro-column array and wrapping well is formed. At this time, the present invention has completed the production of a microfluidic chip having cell washing and liquid exchange functions.

Example 3 performance testing of microfluidic chips

COMSOL Multiphysics simulation test

Simulation test was performed on the micro-column array structure of the microfluidic chip prepared in example 2 using COMSOL Multiphysics software in a Windows system, and it was observed whether the chip structure could generate a laminar flow phenomenon and deterministic lateral migration of particles.

Firstly, drawing a structure in a body cavity of the microfluidic chip as shown in figure 1, wherein the whole microfluidic chip comprises tens of thousands of micro-column structures, and simulation software cannot simulate the whole structure, so that the embodiment simplifies the treatment, reserves the most basic column structure and parameter proportion, and ensures that a fluid flow channel is similar to an actual experiment.

In the flow channel simulation diagram of fig. 9, the white dots are columnar structures, and the gray parts are hollow channels, so that liquid can flow in from the lower part and flow out from the upper part. The inlet 2 is fed with a high concentration liquid and the inlet 1 is fed with a low concentration liquid, and the outlet receives the mixed liquid flowing through the whole mold. Fig. 10 is a graph of concentration simulation, fig. 11 is a graph of outlet concentration, which shows that the left outlet in fig. 11 is high concentration, the right outlet is low concentration, and the middle curve represents the junction of the two concentrations, which is in the middle area of the chip, and it is proved that the two liquids are not obviously mixed, and if mixed, the concentration is close to be consistent at the whole outlet.

As can be seen from fig. 9-11, two liquids of different concentrations simultaneously pass through the array structure with the inclined angle, and they do not have obvious mixing, but rather have laminar flow. I.e. the concentration boundary at the outlet is still in the middle of the model. With such simulation results, we can speculate that good phenomena should occur in the actual experimental results.

2. Observation experiment of laminar flow phenomenon

The present embodiment uses red and blue inks to verify laminar flow.

The microfluidic chip prepared in example 2 was first placed horizontally on an electron microscope, water was passed through the chip from the left, the gas in the chip was purged, and water was removed from the right.

Then, the two channels on the left side are inlets, red ink and blue ink are respectively introduced by using a needle tube built on the micropump, and the ink waste liquid flowing through the chip is led out by using the needle tube on the right side. The observation under microscope is shown in FIGS. 12 and 13. As can be seen from the experimental phenomena shown in the figure, the laminar flow phenomenon is very obvious, that is, the two liquids are not obviously mixed, which is very effective for cleaning cells and exchanging liquid for cells.

Example 4 application of microfluidic chip in ion cleaning and liquid exchange

The microfluidic chip prepared in example 2 was used to perform a fluid change treatment on fluorescent microspheres of 15 μm.

First, the microfluidic chip is placed horizontally on an electron microscope, water is introduced from the left side, the gas in the chip is exhausted, and the water is discharged from the right side.

Then, the two channels on the left side are inlets, the needle tube built on the micropump is used for respectively introducing water and microsphere mixed liquor, and the right side is an outlet for leading out two kinds of liquid flowing through the chip. The observation under a microscope is shown in FIG. 14.

According to the experimental phenomenon, the microspheres move in the chip according to the array offset direction, the two liquids cannot be mixed due to the laminar flow phenomenon, and finally the microspheres move to the other liquid from one liquid, so that the liquid changing operation is realized, photographing monitoring and video monitoring are carried out in the experimental process, and the separation rate of the fluorescent microspheres from the silkworm chrysalis chip disclosed by the invention is estimated to be about 95% by visual inspection.

The method of the embodiment can be also suitable for cleaning and replacing the cells, the liquid replacing operation steps of the centrifugal method are complicated, the use condition requirement is higher, the operation steps are greatly reduced by the cell cleaning and replacing method based on the microfluidic chip, the application range of the miniaturized chip model is wider, and systematic and integrated cell processing integrated equipment can be designed more favorably. The invention adopts a physical method to separate, change liquid or wash the particles (cells or microspheres), and has another remarkable advantage that the damage to the cells in the liquid changing process can be reduced to the minimum, and the experimental result proves that the large-diameter particles can be completely changed liquid, so the integrity of the particles and the thoroughness of the liquid changing are ensured to a great extent.

Although the invention has been described in detail hereinabove with respect to a general description and specific embodiments thereof, it will be apparent to those skilled in the art that modifications or improvements may be made thereto based on the invention. Accordingly, such modifications and improvements are intended to be within the scope of the invention as claimed.

Claims

1. A micro-fluidic chip is characterized by comprising a chip body, wherein the chip body is of a sealed cuboid cavity type structure, and the left side surface and the right side surface of the chip body are provided with an upper port and a lower port which are used for connecting a chip cavity with the outside and comprise a left inlet 1, a left inlet 2, a right outlet 1 and a right outlet 2;

the adjacent cavities among the microcolumns are micro channels through which liquid flows; the cross section of the microcolumn is circular, parallelogram, regular polygon or ellipse;

when the cross section of the microcolumn is circular, the radius of the cross section of each microcolumn is the same and is less than 500 mu m; the shortest relative distance D1 between two adjacent microcolumns in each row of microcolumns is 0.01-5000 μm; the shortest relative distance D2 between two adjacent microcolumns of each diagonal microcolumn is 0.01-500 μm, and the inclination angle of the two microcolumns is less than 60 degrees; and D1 is always greater than D2; or

When the cross section is a parallelogram, the side length of the parallelogram is less than 500 mu m, and the acute included angle of the parallelogram is less than 60 degrees; the shortest relative distance D1 between two adjacent microcolumns in each row of microcolumns is 0.01-5000 μm; the shortest relative spacing D2 of two adjacent microcolumns per diagonal microcolumn is 0.01 μm-500 μm, and D1 is always greater than D2; or

When the cross section is a regular triangle, the side length of the regular triangle is less than 500 mu m; the shortest relative distance D1 between two adjacent microcolumns in each row of microcolumns is 0.01-5000 μm; the shortest relative distance D2 between two adjacent microcolumns of each diagonal microcolumn is 0.01-500 μm; and D1 is always greater than D2; or

When the cross section is an ellipse, the horizontal axis length of the ellipse is less than 100 μm, and the vertical axis length is less than 100 μm; the shortest relative distance D1 between two adjacent microcolumns in each row of microcolumns is 0.01-5000 μm; the shortest relative distance D2 between two adjacent microcolumns of each diagonal microcolumn is 0.01-500 μm; and D1 is always greater than D2.

2. The microfluidic chip according to claim 1, wherein the height of the micro flow channel of the chip is 1-1000 μm.

3. The microfluidic chip according to any of claims 1-2,

the radius of each circular microcolumn is less than 100 μm, or less than 80 μm, or less than 70 μm, or less than 60 μm, or less than 50 μm, or less than 40 μm; the shortest relative distance D1 between two adjacent microcolumns in each row of microcolumns is 1-1000 μm or 10-1000 μm; the shortest relative spacing D2 between two adjacent microcolumns in each diagonal microcolumn is 1-100 μm, and the two are inclined at an angle of less than 60 °, or less than 30 °, or less than 20 °, or less than 10 °.

4. The microfluidic chip according to claim 3, wherein the radius of each circular microcolumn is 5-30 μm, or 8-25 μm, or 10-23 μm, or 12.5-20 μm; the shortest relative distance D1 between two adjacent microcolumns in each row of microcolumns is 40-500 μm; the shortest relative distance D2 between two adjacent microcolumns of each diagonal microcolumn is 3-10 μm, and the inclination angle of the two microcolumns is more than 0.5 DEG and less than 10 deg.

5. The microfluidic chip according to any of claims 1-2,

the side length of the parallelogram is less than 100 μm, or less than 80 μm, or less than 70 μm, or less than 60 μm, or less than 50 μm, or less than 40 μm, and the acute included angle of the parallelogram is less than 40 °; the shortest relative distance D1 between two adjacent microcolumns in each row of microcolumns is 1-1000 μm, or 10-1000 μm; the shortest relative distance D2 between two adjacent microcolumns of each diagonal microcolumn is 1-100 μm.

6. The microfluidic chip according to claim 5, wherein the side length of the parallelogram is 25-35 μm, and the acute included angle of the parallelogram is less than 30 °; the shortest relative distance D1 between two adjacent microcolumns in each row of microcolumns is 35-45 μm; the shortest relative distance D2 between two adjacent microcolumns of each diagonal microcolumn is 20-30 μm.

7. The microfluidic chip according to any of claims 1-2,

the side length of the regular triangle is 10-100 mu m; the shortest relative distance D1 between two adjacent microcolumns in each row of microcolumns is 1-1000 μm or 10-1000 μm; the shortest relative distance D2 between two adjacent microcolumns of each diagonal microcolumn is 1-100 μm.

8. The microfluidic chip according to claim 7, wherein the side of the regular triangle is 20-40 μm; the shortest relative distance D1 between two adjacent microcolumns in each row of microcolumns is 40-500 μm; the shortest relative distance D2 between two adjacent microcolumns of each diagonal microcolumn is 3-10 μm.

9. The microfluidic chip according to any of claims 1-2, wherein the elliptical shape has a horizontal axis length of 10-90 μm and a vertical axis length of 10-90 μm; the shortest relative distance D1 between two adjacent microcolumns in each row of microcolumns is 1-1000 μm or 10-1000 μm; the shortest relative distance D2 between two adjacent microcolumns of each diagonal microcolumn is 1-100 μm.

10. The microfluidic chip according to claim 9, wherein the elliptical shape has a horizontal axis length of 20-50 μm and a vertical axis length of 20-40 μm; the shortest relative distance D1 between two adjacent microcolumns in each row of microcolumns is 40-500 μm; the shortest relative distance D2 between two adjacent microcolumns of each diagonal microcolumn is 3-10 μm.

11. The microfluidic chip according to any of claims 1-2, wherein the left inlet 1, the left inlet 2, the right outlet 1, and the right outlet 2 of the chip body are respectively connected to a fluid conduit; and/or a micropump is connected to the inlet 1 and the inlet 2.

12. A method for cleaning or replacing a solution containing particles, wherein the particles are cells or microspheres, and the method is characterized in that the microfluidic chip of any one of claims 1 to 11 is used for cleaning or replacing the solution containing the particles.

13. The method of claim 12, wherein the diameter of the liquid-changed and washed particles is smaller than the shortest relative distance between every two adjacent microcolumns in the microcolumn array, and the diameter of the liquid-changed and washed particles is larger than the shortest relative distance between every two adjacent microcolumns in each diagonal row in the microcolumn array.