CN113751091B

CN113751091B - Micro-fluidic chip for efficient drug screening

Info

Publication number: CN113751091B
Application number: CN202111151239.5A
Authority: CN
Inventors: 刘松柏; 常宏
Original assignee: Suzhou Vocational Health College
Current assignee: Suzhou Vocational Health College
Priority date: 2021-09-29
Filing date: 2021-09-29
Publication date: 2022-10-11
Anticipated expiration: 2041-09-29
Also published as: CN113751091A

Abstract

The invention discloses a micro-fluidic chip for efficient drug screening, which comprises: the micro-channel structure comprises a concentration gradient generation unit located at the upstream of the chip body, a cell sampling unit located at the downstream of the chip body, a mixing unit communicated with the downstream of the concentration gradient generation unit, and a cell culture unit communicated between the mixing unit and the cell sampling unit. According to the invention, through the design of the flow channel volume, the flow rate of the candidate drug in unit time has a certain gradient, the preparation liquid keeps the same amount, and the two solutions can form a series of candidate drug solutions with concentration after being mixed; the invention does not need to consider the uniform mixing of two solutions in the process of forming the flow gradient of the candidate drug, so the invention is not limited to the traditional mode of increasing layer by layer, and the number of structural layers of the flow channel can be greatly reduced.

Description

Micro-fluidic chip for efficient drug screening

Technical Field

The invention relates to the field of drug screening, in particular to a micro-fluidic chip for efficient drug screening.

Background

In the process of developing new drugs, drug screening is to adopt a proper method for substances possibly used as drugs to detect the possible pharmacological activity of the substances, so as to provide test data for developing new drugs, and a proper drug screening strategy can improve the screening efficiency and shorten the development cycle of the new drugs.

The high-throughput drug screening technology based on the microfluidic chip is widely used for primary screening of drugs at present due to the characteristics of rapidness, high efficiency and the like. The microfluidic chip has a plurality of unique advantages in the aspect of cell level drug screening, for example, the microfluidic chip has little cell amount and drugs required by operation, and is suitable for the research of cells with scarce sources or drugs with high price; the microfluidic chip can conveniently generate various concentration gradients through the design of the flow channel structure, so that the relation between the concentration and the effect of the medicine is researched. Compared with the traditional experiment operation in which drugs with various concentrations need to be manually configured, the method for generating the concentration gradient by adopting the microfluidic chip has remarkable advantages.

The concentration gradient generating structure commonly adopted in the current micro-fluidic chip is a Christmas tree structure and a deformation structure thereof, and the basic principle is as follows: when two fluids with concentration difference pass through the designed microchannel network, the solutions are repeatedly separated and mixed for many times, each branch comprises the original solution with different proportions, and finally, a concentration gradient is formed at the bottom of the Christmas tree-shaped network structure. For example, a microfluidic chip integrating concentration gradient and bacteria detection and a design method thereof disclosed in patent CN113171807a, a solution gradient generation and cell culture microfluidic chip disclosed in CN206502830U, an integrated microfluidic chip for drug screening and an application method thereof disclosed in CN105080627a, all of which adopt a christmas tree structure or a deformed structure thereof to generate the concentration gradient. However, this structure has some drawbacks: on one hand, the formation of a plurality of concentration gradients is realized through a plurality of layers of 'Christmas trees', each additional layer is added with 1-2 flow channel units, so that 1-2 concentration gradients are increased, for example, the first layer is generally 3 flow channel units, so that the concentration gradient is 3, the second layer is 4-5 flow channel units, so that the concentration gradient is 4-5, the concentration gradient number is increased by adding the flow channel units, but the structure principle is that a plurality of concentrations are newly added by mixing a plurality of solutions which cannot be concentrated on the upper layer, in order to ensure more uniform concentration gradients, the number of the added flow channel units on each layer cannot exceed a certain number, and therefore, more layers of layers are needed for obtaining more concentration gradients. For example, when 8-9 concentration gradients are required, at least 4 layers are typically required (e.g., patent CN113171807a includes 4 layers and can generate 6 concentrations; patent CN206502830U includes 4 layers and can generate 8 concentrations; patent CN105080627a includes 6 layers and can generate 8 concentrations). This results in lengthy flow paths and inefficient concentration gradient generation. On the other hand, in each flow channel unit, a tortuous flow channel structure (such as an S-shaped flow channel structure) is usually adopted to uniformly mix two liquids, the number of layers is high, the number of flow channel units is large, so that the number of tortuous flow channel structures exists in the whole flow channel structure, the flow channel resistance is greatly increased, the length of the flow channel is increased in multiples, the flow velocity is further reduced, the formation efficiency of concentration gradient is reduced, and the loss of the medicine caused by the adhesion of the medicine to the flow channel wall is increased.

Therefore, there is a need to improve the existing solutions to provide more reliable microfluidic chips suitable for drug screening.

Disclosure of Invention

The technical problem to be solved by the invention is to provide a microfluidic chip for efficient drug screening, aiming at the defects in the prior art.

In order to solve the technical problems, the invention adopts the technical scheme that: a microfluidic chip for high-efficiency drug screening, comprising: the micro-channel structure comprises a concentration gradient generation unit positioned at the upstream of the chip body, a cell sampling unit positioned at the downstream of the chip body, a mixing unit communicated with the downstream of the concentration gradient generation unit and a cell culture unit communicated between the mixing unit and the cell sampling unit;

the concentration gradient generating unit comprises a gradient generating structure and a concentration configuration structure, wherein the gradient generating structure comprises a medicine inlet, a first distribution cavity communicated with the medicine inlet, a first distribution channel group communicated with the first distribution cavity and a plurality of second distribution channel groups communicated with the first distribution channel group.

Preferably, the first distribution channel group includes N first distribution channels sequentially arranged at intervals along the Y direction, the first distribution channels are parallel to the X direction, all the first distribution channels have the same length, and the channel volumes of the first distribution channels sequentially increase from the first to the nth;

each of the first distribution passages is communicated with one of the second distribution passage groups downstream, each of the second distribution passage groups includes M second distribution passages communicated with one of the first distribution passages through one of the second distribution chambers, the second distribution passages are parallel to the X direction, all the second distribution passages have equal lengths, and passage volumes of the second distribution passages sequentially increase from a first one to an M one in the same second distribution passage group;

the mixing unit comprises N-M mixing structures which are sequentially arranged at intervals along the Y direction, and the cell culture unit comprises N-M cell culture chambers which are arranged in an array along the Y direction;

the concentration configuration structure comprises a configuration liquid inlet and N x M configuration liquid channels which are communicated with the configuration liquid inlet and are arranged in an array along the Y direction, the length and the channel volume of each configuration liquid channel are equal, one configuration liquid channel and one second distribution channel are communicated with one mixing structure together and then communicated with one cell culture cavity;

n, M is not less than 2, the candidate drugs enter through the drug inlet, and then flow distribution is performed sequentially through the first distribution channel and the second distribution channel, so that the flow of the candidate drugs entering the mixing structures in unit time is sequentially increased from the first to the Nth, and then a certain amount of preparation liquid is conveyed to the N-M mixing structures through the preparation liquid channel at a medium flow, and finally the concentration of the candidate drugs in the first to the Nth mixing structures is sequentially increased.

Preferably, the first distribution cavity comprises a first V-shaped cavity section and a first rectangular cavity section which are communicated with each other, the first V-shaped cavity section is communicated with the medicine injection port, and the first rectangular cavity section is communicated with the upstream of the first distribution channel;

the second distribution cavity comprises a second V-shaped cavity section and a second rectangular cavity section which are communicated with each other, the second V-shaped cavity section is communicated with the downstream of the first distribution channel, and the second rectangular cavity section is communicated with the upstream of the second distribution channel.

Preferably, the chip body comprises a main chip body and an auxiliary chip body arranged on the main chip body, the gradient generating structure, the cell sampling unit, the mixing unit and the cell culture unit are all formed in the main chip body, and the concentration configuration structure is formed in the auxiliary chip body.

Preferably, the mixing structure comprises a first conical mixing cavity, a first mixing channel arranged in the middle of the upper end of the second mixing cavity, a second conical mixing cavity communicated with the first mixing cavity through the second mixing channel, a third mixing channel communicated with the middle of the upper end of the second mixing cavity, and a fourth mixing channel tangentially communicated with the periphery of the upper end of the second mixing cavity, the outlet ends of the fourth mixing channels are converged to the third mixing channel, and the outlet end of the third mixing channel is communicated to the cell culture cavity.

Preferably, the outlet end of the second distribution channel is tangentially communicated with the periphery of the upper end of the first mixing cavity, and the outlet end of the preparation liquid channel is communicated with the first mixing channel;

the inlet end of the second mixing channel is tangentially communicated with the periphery of the lower end of the first mixing cavity, and the outlet end of the second mixing channel is tangentially communicated with the periphery of the lower end of the second mixing cavity;

the second distribution channel, the second mixing channel and the fourth mixing channel are on the same side of a reference line parallel to the X-direction and passing through both the center line of the first mixing chamber and the center line of the second mixing chamber.

Preferably, the end of the third mixing channel has a first flared section which communicates with the side of the cell culture chamber;

and a one-way valve is arranged in the third mixing channel and is positioned between the outlet end of the fourth mixing channel and the first flaring section.

Preferably, the check valve comprises two valve plates which are symmetrically arranged, each valve plate comprises an arc-shaped elastic part connected to the inner wall of the third mixing channel and a linear closed part connected with the elastic part, and the elastic part is far away from the cell culture chamber relative to the closed part;

when no liquid flows in the third mixing channel or the flowing direction of the liquid is from the closed part to the elastic part, the closed parts of the two valve plates are kept in contact with each other, and the two valve plates close the third mixing channel;

when the flowing direction of the liquid in the third mixing channel is from the elastic part to the closed part, the elastic parts of the two valve plates bend, the closed parts of the two valve plates are separated from each other, and the one-way valve is opened.

Preferably, one of the surfaces of the two closed parts of the two valve plates is provided with a saw-toothed protrusion, and the other is provided with a saw-toothed groove matched with the saw-toothed protrusion.

Preferably, let us say that the channel volume of the ith first distribution channel is U _i Jth second distribution channelHas a channel volume of V _j Wherein i =1,2,.., N, j =1,2,.., M;

the channel volume of the first distribution channel and the channel volume of the second distribution channel are set such that:

after deformation: v _m *U _i-1 <V ₁ *U _i 。

Preferably, the cell sample introduction unit comprises a cell sample introduction port, a cell main channel communicated with the cell sample introduction port, and N × M cell branch channels communicated with the cell main channel, and each cell branch channel is correspondingly communicated to one cell culture chamber through one second flaring segment.

The beneficial effects of the invention are:

according to the invention, through the design of the flow channel volume, the flow rate of the candidate drug in unit time has a certain gradient, the preparation liquid keeps the same amount, and the two solutions can form a series of candidate drug solutions with concentration after being mixed; in the invention, a large number of concentration gradients can be obtained through one layer or two layers of flow channel structures, and the concentration gradients are formed by forming the flow gradients of the candidate drugs, so that the uniform mixing of two solutions is not required to be considered in the process of forming the flow gradients of the candidate drugs, the traditional mode of increasing layers by layers is not limited, and the number of layers of the flow channel structures can be greatly reduced;

according to the invention, the candidate medicine with the flow gradient and the preparation liquid distributed in equal amount are mixed by adopting the independent mixing unit, and the mixing structure can realize three-time mixing of two fluids, so that the two fluids can be fully mixed, the phenomenon that the two fluids adopt lengthy and tortuous flow is avoided, the flow velocity of the fluids can be greatly improved, the mixing efficiency is improved, the generation efficiency of the concentration gradient can be finally improved, and the loss of the subsequent medicine can be reduced;

the micro-fluidic chip disclosed by the invention can quickly generate various concentration gradients, can improve the drug screening efficiency and reduce the drug waste.

Drawings

FIG. 1 is a schematic structural view of a micro flow channel structure on a main chip body according to the present invention;

FIG. 2 is a schematic structural view of a micro flow channel structure according to the present invention;

FIG. 3 is a schematic structural diagram of a main chip body according to the present invention;

FIG. 4 is a schematic structural diagram of a sub-chip body according to the present invention;

FIG. 5 is a schematic structural diagram of a second distribution channel group according to the present invention;

FIG. 6 is a theoretical concentration gradient curve in example 1 of the present invention;

fig. 7 is a concentration gradient curve of the meropenem solution produced in example 1 of the present invention;

FIG. 8 is a schematic sectional view showing a micro flow channel structure in example 2 of the present invention;

fig. 9 is a schematic structural view of a hybrid structure in embodiment 2 of the invention;

fig. 10 is a top view of a hybrid structure in embodiment 2 of the present invention;

FIG. 11 is a schematic view (closed) showing the structure of a check valve in embodiment 3 of the present invention;

fig. 12 is a schematic structural view (open) of a check valve in embodiment 3 of the present invention;

fig. 13 is a schematic structural view (closed) of another check valve in embodiment 3 of the invention;

fig. 14 is a schematic structural view (open) of another check valve in embodiment 3 of the present invention.

Description of reference numerals:

1-chip body; 10-main chip body; 11-sub-chip body;

2-micro flow channel structure;

3-a concentration gradient generating unit;

30-gradient generating structure; 300-medicine inlet; 301 — a first distribution chamber; 302-a first distribution channel set; 303 — a second distribution chamber; 304 — second group of distribution channels; 3010 — first V-shaped cavity section; 3011 — a first rectangular cavity segment; 3020 — first distribution channel; 3030-a second V-shaped cavity section; 3031-a second rectangular cavity section; 3040 — a second distribution lane;

31-concentration configuration structure; 36-make-up fluid inlet; 37-configuring liquid channels;

4-a mixing unit; 40-mixed structure; 41-a first mixing chamber; 42 — a first mixing channel; 43 — a second mixing channel; 44-a second mixing chamber; 45-third mixing channel; 46 — a fourth mixing channel; 47 — first flared section; 48-a one-way valve; 480-valve plates; 481-elastic portion; 482-closure; 483-jagged protrusions; 484-saw tooth like recesses; 485 sealing sheets; 486-a limiting block;

5-cell culture unit; 50-cell culture chamber; 51-a second flared section; 52-waste liquid port;

6-cell sample introduction unit; 60-cell sample inlet; 61-Total channel of cells; 62-cell branching channel.

Detailed Description

The present invention is further described in detail below with reference to examples so that those skilled in the art can practice the invention with reference to the description.

It will be understood that terms such as "having," "including," and "comprising," as used herein, do not preclude the presence or addition of one or more other structures or combinations thereof.

Referring to fig. 1 to 5, the microfluidic chip for efficient drug screening of the present embodiment includes: the micro-channel structure 2 comprises a chip body 1 and a micro-channel structure 2 formed in the chip body 1, wherein the micro-channel structure 2 comprises a concentration gradient generation unit 3 positioned at the upstream of the chip body 1, a cell sampling unit 6 positioned at the downstream of the chip body 1, a mixing unit 4 communicated with the downstream of the concentration gradient generation unit 3 and a cell culture unit 5 communicated between the mixing unit 4 and the cell sampling unit 6;

the concentration gradient generating unit 3 comprises a gradient generating structure 30 and a concentration configuration structure 31, wherein the gradient generating structure 30 comprises a medicine inlet 300, a first distribution cavity 301 communicated with the medicine inlet 300, a first distribution channel group 302 communicated with the first distribution cavity 301, and a plurality of second distribution channel groups 304 communicated with the first distribution channel group;

the first distribution channel group 302 comprises N first distribution channels 3020 arranged at intervals in the Y direction, the first distribution channels 3020 are parallel to the X direction, all the first distribution channels 3020 have the same length, and the channel volumes of the first distribution channels 3020 increase from the first to the nth in turn;

a second distribution channel group 304 is communicated downstream of each first distribution channel 3020, each second distribution channel group 304 includes M second distribution channels 3040 communicated with one first distribution channel 3020 through one second distribution cavity 303, the second distribution channels 3040 are parallel to the X direction, all the second distribution channels 3040 have the same length, and the channel volume (i.e., the cross-sectional area) of the second distribution channels 3040 increases from the first to the mth in the same second distribution channel group 304;

the mixing unit 4 comprises N x M mixing structures 40 arranged in sequence at intervals along the Y direction, and the cell culture unit 5 comprises N x M cell culture chambers 50 arranged in an array along the Y direction;

the cell sample introduction unit 6 comprises a cell sample introduction port 60, a cell main channel 61 communicated with the cell sample introduction port 60, and N × M cell branch channels 62 communicated with the cell main channel 61, wherein each cell branch channel 62 is correspondingly communicated to one cell culture cavity 50 through one second expansion port section 51.

The concentration configuration structure 31 comprises a configuration liquid inlet 36 and N × M configuration liquid channels 37 which are communicated with the configuration liquid inlet 36 and are arranged in an array along the Y direction, the length and the channel volume of each configuration liquid channel 37 are equal, one configuration liquid channel 37 and one second distribution channel 3040 are communicated with a mixing structure 40 and then communicated with a cell culture cavity 50;

the N, M is not less than 2, the candidate drugs enter through the drug inlet 300, and then flow distribution is sequentially performed through the first distribution channel 3020 and the second distribution channel 3040, so that the flow rate of the candidate drugs entering the mixing structures 40 in a unit time is sequentially increased from the first to the nth, and then a certain amount of the preparation liquid is delivered to the N × M mixing structures 40 through the preparation liquid channel 37 at an equal flow rate, and finally the concentration of the candidate drugs in the N × M mixing structures 40 is sequentially increased from the first.

In the present invention, the flow rate of the candidate drug dispensed from the first dispensing chamber 301 into the N first dispensing channels 3020 is directly proportional to the channel volume of the first dispensing channel 3020, i.e., the larger the channel volume, the more candidate drug flow rate dispensed; similarly, for the same second distribution channel group 304, the flow rate of the drug candidate distributed by second distribution chamber 303 to M second distribution channels 3040 is also in direct proportion to the channel volume of second distribution channel 3040, with the larger the channel volume, the more the drug candidate flow rate is distributed.

Theoretically, the larger the cross-sectional area, the greater the flow rate at a given differential pressure, so the greater the channel volume, the greater the flow rate for the same length of channel. Considering factors such as channel size, unit pipe length resistance and the like, the distribution of the flow in a plurality of branch channels can be calculated in various ways, but the flow and the channel volume can be in a direct proportion relation.

In one embodiment, first and

second distribution channels

3020 and 3040 may both be circular in shape or both may be rectangular in shape.

In this embodiment, let U be the channel volume of the ith first distribution channel 3020 _i And jth second distribution lane 3040 has a lane volume V _j Wherein i =1,2,.., N, j =1,2,.., M;

the channel volume of first distribution channel 3020 and the channel volume of second distribution channel 3040 are arranged such that:

after deformation: v _m *U _i-1 <V ₁ *U _i 。

The purpose of the above conditions is: the candidate drug flow rate in the first second distribution lane 3040 in the next second distribution lane group 304 (1 st in the ith second distribution lane group 304) is made greater than the candidate drug flow rate in the last second distribution lane 3040 in the previous second distribution lane group 304 (M in the (i-1) th second distribution lane group 304), so that the candidate drug flow rate in all second distribution lane groups 304 that satisfies the requirement of the 1 st desired nth by M second distribution lane 3040 is kept sequentially increasing.

Referring to fig. 1, in the present invention, the channel volumes of N first distribution channels 3020 from top to bottom are sequentially increased, so that the drug candidate flow entering each first distribution channel 3020 is sequentially increased from top to bottom in a unit time, which is the first distribution of the drug candidate flow;

in each second distribution channel group 304, the channel volumes of the M second distribution channels 3040 from top to bottom also sequentially increase, so in the same second distribution channel group 304, the candidate drug flow entering each second distribution channel 3040 also sequentially increases from top to bottom, which is the second distribution of the candidate drug flow;

therefore, by setting the sizes of the channel volumes of the first distribution channel 3020 and the second distribution channel 3040, the flow rates flowing out of the N × M second distribution channels 3040 from top to bottom can be sequentially increased in a unit time, and the N × M distribution liquids can be delivered at equal flow rates through the N × M distribution liquid channels 37, so that N × M drug candidate solutions with sequentially increasing concentrations from top to bottom can be obtained in the N × M mixed structures 40, and the generation of a drug candidate with a constant concentration gradient can be realized.

For the sake of understanding, the principle of the formation of the concentration gradient will be described below by using a specific formula.

For convenience, the input concentration of the candidate drug is 1 unit concentration (e.g., 1 mol/L), the concentration of the candidate drug in all the second distribution channels 3040 is the same, and the flow rate flowing out of the k-th second distribution channel 3040 per unit time is Q _k The flow rate of the flow from the (k + 1) th second distribution passage 3040 is Q _k+1 ,Q _k+1 ＝Q _k +Δ _k Since the flow rate is increased in sequence, it is apparent that Δ _k ＞0；

The flow rate of the preparation liquid flowing out of each preparation liquid passage 37 per unit time is V _p ；

Then, in the unit time, in the fully mixed state, the concentration of the candidate drug after the candidate drug flowing out from the k-th second distribution channel 3040 is:

recording as the concentration of the candidate drug in the K group;

the concentration of the candidate drug after the candidate drug flowing out of the (k + 1) th second distribution channel 3040 and the preparation liquid are mixed uniformly is:

recording as the concentration of candidate drug in group K + 1;

q above _k 、Q _k+1 、V _p The units are the same;

it can be seen that it is necessary to satisfy: c _k+1 ＞C _k (plus a positive number Δ in the numerator and denominator _k The score value becomes large);

the specific derivation procedure is given below:

that is, the concentration of the candidate drug in group K +1 is greater than that of the candidate drug in group K, so that the concentration of N × M candidate drugs in the N × M mixed structures 40 can be increased from top to bottom.

It is understood that the concentration generation process is a dilution process, and therefore the resulting concentration of the candidate drug is between 0 and 1 unit concentration. That is, if the concentration of the candidate drug to be infused is C ₀ And then the concentration C of the candidate drug is obtained as follows:

so 0 < C ₀ 。

And wherein the preparation solution is used for diluting the candidate drug to prepare the candidate drug with a certain concentration gradient. And by adjusting the delivery of the preparation liquidDischarge quantity V _p And candidate drug solutions with different concentration gradient ranges can be obtained.

The preparation solution may be the same solution as the solvent of the candidate drug, or a cell culture medium solution, or deionized water, or physiological saline, or other solutions that do not affect cell culture and do not interfere with the candidate drug.

The concentration gradient generating structure 30 commonly used in the current micro-fluidic chip is a Christmas tree structure and a deformation structure thereof, and the basic principle is as follows: when two fluids with concentration difference pass through the designed microchannel network, the solutions are repeatedly separated and mixed for many times, each branch comprises the original solution with different proportions, and finally, a concentration gradient is formed at the bottom of the Christmas tree-shaped network structure. This structure has some drawbacks: which generates a concentration gradient through a plurality of "christmas tree" structures or their deformed structures. However, this structure has some drawbacks: on one hand, the formation of a plurality of concentration gradients is realized through a plurality of layers of 'Christmas trees', each additional layer is added with 1-2 flow channel units, so that 1-2 concentration gradients are increased, for example, the first layer is generally 3 flow channel units, so that the concentration gradient is 3, the second layer is 4-5 flow channel units, so that the concentration gradient is 4-5, the concentration gradient number is increased by adding the flow channel units, but the structure principle is that a plurality of concentrations are newly added by mixing a plurality of solutions which cannot be concentrated on the upper layer, in order to ensure more uniform concentration gradients, the number of the added flow channel units on each layer cannot exceed a certain number, and therefore, more layers of layers are needed for obtaining more concentration gradients. For example, when 8-9 concentration gradients are desired, at least 4 layers are typically required; this results in lengthy flow paths and inefficient concentration gradient generation. On the other hand, in each flow channel unit, a tortuous flow channel structure (such as an S-shaped flow channel structure) is usually adopted to uniformly mix two liquids, so that the number of layers is high, the number of flow channel units is large, the number of tortuous flow channel structures exist in the whole flow channel structure, the resistance of the flow channel is greatly increased, the length of the flow channel is increased in multiples, the liquid flow speed is further reduced, the formation efficiency of concentration gradient is reduced, and the loss of the medicine caused by the adhesion of the medicine to the wall of the flow channel is increased.

On one hand, the invention adopts different principles to form concentration gradient, in particular, the invention ensures that the flow rate of the candidate drug in unit time has certain gradient and the preparation liquid keeps the same amount through the design of the flow channel volume, and the two solutions can form a series of candidate drug solutions with concentration after being mixed. In the invention, a large number of concentration gradients can be obtained through one layer or two layers of flow channel structures, and the concentration gradients are formed after the flow gradients of the candidate drugs are formed, so that the uniform mixing of two solutions is not required to be considered in the process of forming the flow gradients of the candidate drugs, the method is not limited to the traditional 'layer-by-layer increase' mode, and the number of layers of the flow channel structures can be greatly reduced. For example, in this embodiment, only two layers are required, and 9 concentration gradients can be formed, thereby greatly reducing the length of the flow channel and improving the efficiency.

In the invention, the first distribution channel 3020 with different volumes is formed through the first distribution channel group 302 to perform the first distribution, and the second distribution channel 3040 with different volumes is formed through the second distribution channel group 304 to perform the second distribution, so that the length, the width and the thickness of the chip body 1 can be fully utilized, more and more uniform concentration gradients can be obtained, and the design requirement of the flow channel size can be relaxed. For example, in the first distribution channel group 302, since the number of channels is small (3 channels) in the width direction, the channel volume can be increased mainly by gradually increasing the width; in the second distribution channel group 304, since the number of channels in the width direction is larger (9), the channel volume can be increased by gradually increasing the width and the depth at the same time, so that the size design of the channels can be more flexible.

On the other hand, the independent mixing unit 4 is adopted to mix the candidate medicine with the flow gradient and the preparation liquid distributed in equal quantity, a tortuous flow passage structure is not needed, the uniform mixing efficiency and the concentration gradient generation efficiency can be greatly improved, and the loss of subsequent medicines can be reduced. In the examples which follow, further description is given.

The foregoing is a general idea of the present invention, and more detailed examples are provided below to further illustrate the present invention. And in the following embodiments, N =3, m =3 (i.e., 3

first distribution channels

3020 and 9 second distribution channels 3040, which can generate 9 concentration gradients) is taken as a specific example for convenience of description.

Example 1

In this embodiment, the width of the channels in the first distribution channel group 302 ranges from 50 μm to 1500 μm, and the depth ranges from 2 μm to 20 μm; the channels of second distribution channels 3040 may have a width in the range of 30-200 μm and a depth in the range of 2-25 μm.

In one embodiment, numbered sequentially from top to bottom, the 1 st to 3 rd first distribution channels 3020 have a volume of U ₀ 、3U ₀ 、9U ₀ The volume of the 3 second distribution channels 3040 connected behind each first distribution channel 3020 is V from top to bottom ₀ 、1.5V ₀ 、2V ₀ That is, 3 second distribution passages 3040 in each second distribution passage group 304 have a volume ratio of

Wherein, U ₀ And V ₀ Each may represent a unit volume; also in this embodiment, the lengths of the channels in the first distribution channel set 302 and the second distribution channel set 304 are equal, so the volume ratio is also equal to the area ratio (deposition of depth and width).

The flow rate of the drug candidate entering the first dispensing chamber 301 per unit time is 13Q ₀ Then, theoretically, the flow rate distributed into the 1 st to 3 rd first distribution channels 3020 is sequentially: q ₀ 、3Q ₀ 、9Q ₀ The flow rates distributed to and entered from the 1 st to 9 th second distribution lanes 3040 are, in order:

it can be seen that the flow is sequentially increasing; wherein Q is ₀ Represents a unit flow rate;

it is further assumed that the initial concentration of the injected drug candidate is C ₀ Preparing a liquidThe concentration of the candidate drug in (1) is 0, and the flow rate of the preparation liquid outputted from each preparation liquid channel 37 per unit time is Q ₀ The concentration of the candidate drug solution in the 1 st group (theoretical concentration of the candidate drug from the first dispensing channel 3020 mixed with the first dispensing fluid from the first dispensing channel 37) is then generated

The concentrations of the selected drug solutions of groups 2-9 obtained by the same method are as follows in sequence:

the group number of the candidate drug solution is used as the abscissa and the corresponding unit concentration is used as the ordinate, so as to obtain a theoretical concentration gradient curve, as shown in fig. 6, it can be seen that the concentration is in a gradient increasing trend.

Further, in a more specific embodiment, first distribution channel 3020 and second distribution channel 3040 are each rectangular in shape and the dimensions of 1 st to 3 rd first distribution channel 3020 are as follows in table 1:

TABLE 1

The dimensions of 3 second distribution channels 3040 from top to bottom of each second distribution channel 3040 are as follows:

TABLE 2

Infusing liquid with precision micro pump, wherein the drug candidate is meropenem, and the initial concentration is C ₀ 40mg/L, the preparation solution is culture solution, the concentration of candidate drug is 0, the injection amount of candidate drug per unit time (which can be 0.01-0.5 uL/min) is 13 times of the preparation solution, and 9 kinds of candidate drug are generated in a stable stateThe concentrations of (a) are shown in table 3 below:

TABLE 3

Meropenem solution group number	Meropenem solution concentration mg/L
		1	6.9
2	9.4
		3	12.2
4	15.0
		5	21.0
6	24.5
		7	28.0
8	31.2
		9	33.9

The group number of the candidate drug solution is used as the abscissa and the corresponding unit concentration is used as the ordinate, and a curve is drawn, as shown in fig. 7, it can be seen that the concentration of the generated 9 groups of meropenem solutions tends to increase in gradient, and the trend of the curve is close to that of fig. 1, which shows that the invention can realize the generation of the concentration gradient.

Example 2

Referring to fig. 8-10, in this embodiment, the first distribution chamber 301 includes a first V-shaped cavity section 3010 and a first rectangular cavity section 3011 that communicate with each other, the first V-shaped cavity section 3010 communicating with the drug injection port, and the first rectangular cavity section 3011 communicating with the upstream of the first distribution channel 3020;

the second distribution chamber 303 includes a second V-shaped chamber segment 3030 and a second rectangular chamber segment 3031 that communicate with each other, the second V-shaped chamber segment 3030 communicating downstream of the first distribution passage 3020, and the second rectangular chamber segment 3031 communicating upstream of the second distribution passage 3040.

Through the arrangement of the V-shaped cavity section, the solution in the distribution cavity can be uniformly distributed in the whole rectangular cavity section, so that the solution can be better distributed to a downstream distribution channel according to the size of the inlet section.

In this embodiment, the chip body 1 includes a main chip body 10 and an auxiliary chip body 11 disposed on the main chip body 10, the gradient generating structure 30, the cell sampling unit 6, the mixing unit 4 and the cell culturing unit 5 are all formed in the main chip body 10, and the concentration configuration structure 31 is formed in the auxiliary chip body 11. The sub-chip body 11 is fastened to the main chip body 10, and the concentration configuration structure 31 is communicated to the mixing unit 4.

In this embodiment, the mixing structure 40 includes a first mixing cavity 41 in a conical shape, a first mixing channel 42 disposed in the middle of the upper end of the second mixing cavity 44, a second mixing cavity 44 in a conical shape communicating with the first mixing cavity 41 through a second mixing channel 43, a third mixing channel 45 communicating with the middle of the upper end of the second mixing cavity 44, and a fourth mixing channel 46 tangentially communicating with the periphery of the upper end of the second mixing cavity 44, the outlet ends of the fourth mixing channels 46 are collected to the third mixing channel 45, and the outlet end of the third mixing channel 45 is communicated to the cell culture cavity 50.

Wherein, the outlet end of the second distribution passage 3040 is tangentially communicated with the upper end periphery of the first mixing chamber 41, and the outlet end of the distribution liquid passage 37 is communicated with the first mixing passage 42;

the inlet end of the second mixing passage 43 tangentially communicates with the lower end periphery of the first mixing chamber 41, and the outlet end of the second mixing passage 43 tangentially communicates with the lower end periphery of the second mixing chamber 44;

second distribution passage 3040, second mixing passage 43, and fourth mixing passage 46 are all on the same side of a reference line parallel to the X-direction and passing through both the centerline of first mixing chamber 41 and the centerline of second mixing chamber 44.

The candidate drug delivered by the second distribution channel 3040 enters the first mixing cavity 41 tangentially, meets the dispensing fluid delivered by the first mixing channel 42 and entering the first mixing cavity 41 vertically downward, the candidate drug entering tangentially generates a rotational flow (counterclockwise as viewed from the top in the drawing), the dispensing fluid generates a vertical flow from top to bottom, the two flows meet each other to generate a violent collision, so that the whole fluid in the first mixing cavity 41 moves downward while rotating, and the two flows are fully mixed during the movement;

then tangentially flows out from the bottom of the first mixing cavity 41 to a third mixing channel 45, and then tangentially enters the second mixing cavity 44, because the periphery of the upper end of the second mixing cavity 44 is tangentially communicated with a fourth mixing channel 46, and the middle part of the upper end is vertically communicated with the third mixing channel 45, under the guidance of the two channels, the mixed fluid tangentially entering from the third mixing channel 45 rotates and rises in the second mixing cavity 44, and in the process, the two fluids are secondarily mixed; then a part of the mixed fluid is discharged from the third mixing channel 45 at the top, and another part is discharged from the fourth mixing channel 46 at the side;

finally, the fluid exiting the fourth mixing channel 46 is collected in the third mixing channel 45, during which the two fluids are mixed for a third time. Wherein the fourth mixing channel 46 cooperates with the third mixing channel 45 on the one hand to create a swirling flow in the second mixing chamber 44 and on the other hand to increase the mixing of the primary fluid again.

Can realize the cubic of two kinds of fluids through above-mentioned mixed structure 40 and mix to can guarantee two kinds of fluid intensive mixings, avoid adopting the lengthy tortuous flow simultaneously, can improve the fluid velocity of flow greatly, improve mixing efficiency, thereby finally can improve concentration gradient generation efficiency, and can also reduce the loss of follow-up medicine.

Example 3

Referring to FIGS. 3, 11-14, the end of the third mixing channel 45 has a first flared section 47, the first flared section 47 communicating with the side of the cell culture chamber 50; by providing the first flared section 47, rapid access of fluid to the cell culture chamber 50 can be facilitated.

A one-way valve 48 is arranged in the third mixing channel 45, the one-way valve 48 being located between the outlet end of the fourth mixing channel 46 and the first flared section 47. The check valve 48 plays a role in stopping reverse flow, and by arranging the check valve 48, the candidate drug solution with the configured concentration can smoothly enter the cell culture chamber 50, and the culture solution or cells in the cell culture chamber 50 cannot reversely flow towards the upstream of the chip body 1.

Referring to fig. 11-12, in one embodiment, the check valve 48 includes a sealing plate 485 and a limiting block 486, the upper end of the sealing plate 485 is rotatably or elastically connected to the inner wall of the third mixing channel 45, and the limiting block 486 is located below and on the left side of the sealing plate 485 and can prevent the lower end of the sealing plate 485 from rotating to the left. When the fluid in the third mixing channel 45 flows from right to left, the lower end of the sealing sheet 485 is in contact with and clings to the limiting block 486, so that the sealing sheet 485 and the limiting block 486 can seal the third mixing channel 45 to prevent the fluid from flowing from right to left. When the fluid in the third mixing channel 45 flows from left to right, the sealing sheet 485 rotates to the right or bends to the right (counterclockwise), the third mixing channel 45 is opened, and the fluid can flow through the third mixing channel 45. The sealing piece 485 and the limiting block 486 can be made of anticorrosive metal or plastic materials.

Referring to FIGS. 13 to 14, in another embodiment, the check valve 48 includes two valve plates 480 symmetrically arranged, the valve plates 480 include an arc-shaped elastic part 481 connected to the inner wall of the third mixing channel 45 and a linear closing part 482 connected to the elastic part 481, the elastic part 481 is farther from the cell culture chamber 50 than the closing part 482;

in a further embodiment, the surfaces of the enclosing parts 482 have inclined surfaces, and the two enclosing parts 482 form a tip shape when they are in contact.

When no liquid flows in the third mixing channel 45 or the flow direction of the liquid is from the closed part 482 to the elastic part 481, the closed parts 482 of the two valve sheets 480 are kept in contact, and the two valve sheets 480 close the third mixing channel 45;

when the liquid in the third mixing channel 45 flows from the elastic part 481 toward the closed part 482, the elastic part 481 of the two valve plates 480 is bent by the force of the liquid, the closed parts 482 of the two valve plates 480 are separated from each other, the check valve 48 is opened, and the liquid can flow through the gap between the two valve plates 480.

In a more preferred embodiment, the two closing portions 482 of the two valve sheets 480 are provided with serrations 483 on one surface and serrated recesses 484 to be engaged with the serrations 483 on the other surface.

The elastic part 481 of arc shape makes two valve plates 480 can draw close each other at no exogenic action messenger to the closing 482 through two lineages keeps closely laminating, realizes sealedly, when liquid reverse flow (from right side left), liquid extrusion elastic part 481 can make two closing 482 more tend to draw close, the setting of the inclined plane on the closing 482 simultaneously, make liquid can make two closing 482 hug closely more to the pressure energy on closing 482 surface, can guarantee fine sealed effect. And further, when the elastic part 481 generates a tendency of moving leftwards due to the liquid pressure, the two valve plates 480 are combined through the zigzag structure, so that the two valve plates do not slide with each other and can be attached more closely.

When the liquid flows in the forward direction (from left to right), the elastic portion 481 is pressed by the liquid and bent so that the two closed portions 482 are away from each other, thereby opening, and the liquid can pass through the gap between the two closed portions 482. In this embodiment, since the two closed portions 482 can both be bent, a larger gap can be obtained between the two closed portions after the two closed portions are opened, and the efficiency of passing liquid can be improved.

It will be appreciated that the shape and size of the one-way valve 48 in both embodiments need to be matched to the third mixing channel 45 in which it is located to ensure that the one-way valve 48 is able to close the third mixing channel 45. In one embodiment, the check valve 48 is generally rectangular in shape, and the third mixing channel 45 in which the check valve 48 is located is also rectangular in shape.

In a preferred embodiment, a one-way valve 48 is also disposed in the cell branch channel 62, so that the flow in the cell branch channel 62 can be reversed only from the cell sample inlet 60 toward the cell culture chamber 50; the structure of the check valve 48 may be any of those described above.

In the preferred embodiment, the upper portion of the cell culture chamber 50 is provided with a waste port 52. In a more preferred embodiment, the size of the waste liquid port 52 is smaller than the size of the cells or other samples used in the application of the microfluidic chip, so as to prevent the cells or other samples from being discharged from the waste liquid port 52.

Example 4

The embodiment provides an application method of the microfluidic chip for efficient drug screening, which includes the following steps:

1. injecting cells through the cell injection port 60 by using an injection pump, uniformly distributing the cells to each cell culture cavity 50 through the cell branch channel 62, and stopping static culture after each cell culture cavity 50 is injected with a proper amount of cells (for example, 2/3) or the cell culture cavities are filled with the cells;

2. after the cells adhere to the wall, injecting a certain amount of culture solution through the cell sample injection port 60 for continuous culture for a certain time;

in the above process, due to the function of the check valve 48, the cells or the culture solution do not enter the upstream mixing unit 4, and the waste solution is discharged from the waste solution port 52;

3. the cell sample inlet 60 is sealed (if the cell branch channel 62 is also provided with the one-way valve 48, the cell sample inlet 60 can not be sealed, and the cell or the candidate drug solution can be prevented from entering the cell branch channel 62 through the one-way valve 48), so that the candidate drug solution or the cell can be prevented from entering the cell branch channel 62 as much as possible under the action of air pressure;

injecting candidate drugs through the drug inlet 300, keeping the injection pressure constant during the injection process, and injecting the preparation solution through the preparation solution inlet 36, so that the generated candidate drug solutions with different concentrations respectively enter each cell culture cavity 50 to continue culturing for a certain time; in the process, the candidate drug solution can be continuously injected or only injected for a period of time;

4. the apoptosis condition of the cells in each cell culture cavity 50 is detected, so that the apoptosis rate of the cells under different concentrations of the drugs is evaluated. In the process, the apoptosis condition can be directly observed by adopting a microscope, or the fluorescence staining is firstly carried out, and then the apoptosis rate is calculated by fluorescence detection.

It is to be understood that the microfluidic chip of the present invention can be used not only for cell-based drug screening, but also for drug screening based on other test bodies. For example, in one embodiment, useful for nematode-based antimicrobial drug screening, the following method can be used:

1) Injecting the nematodes and the culture solution through a cell sample inlet 60, allowing the nematodes and the culture solution to enter a cell culture cavity 50, and culturing for a period of time;

2) Injecting target bacteria with subsequent drug action into the cell culture cavity 50 through the cell sample inlet 60 to infect nematodes;

3) Injecting culture solution through the cell sample inlet 60 to wash the cell culture cavity 50 and remove the residual target bacteria;

4) Injecting candidate drugs through the drug inlet 300, keeping the injection pressure constant during the injection process, simultaneously injecting a preparation solution through the preparation solution inlet 36, so that the generated candidate drug solutions with different concentrations respectively enter each cell culture cavity 50, injecting the candidate drugs through the drug inlet 300, keeping the injection pressure constant during the injection process, and simultaneously injecting the preparation solution through the preparation solution inlet 36, so that the generated candidate drug solutions with different concentrations respectively enter each cell culture cavity 50, and continuously injecting for a certain time;

5) After culturing for a period of time, observing the survival condition of the nematodes under a microscope, calculating the survival rate, obtaining the survival rate of the nematodes under different candidate drug concentrations, and representing the antibacterial effect of the nematodes, thereby obtaining the optimal concentration of the candidate drug for the target bacteria.

While embodiments of the invention have been disclosed above, it is not intended to be limited to the details shown in the description and the examples, which are set forth, but are fully applicable to various fields of endeavor as are suited to the particular use contemplated, and further modifications will readily occur to those skilled in the art, since the invention is not limited to the details shown and described without departing from the general concept as defined by the appended claims and their equivalents.

Claims

1. A microfluidic chip for efficient drug screening, comprising: the micro-channel structure comprises a concentration gradient generation unit positioned at the upstream of the chip body, a cell sampling unit positioned at the downstream of the chip body, a mixing unit communicated with the downstream of the concentration gradient generation unit and a cell culture unit communicated between the mixing unit and the cell sampling unit;

the concentration gradient generation unit comprises a gradient generation structure and a concentration configuration structure, wherein the gradient generation structure comprises a medicine inlet, a first distribution cavity communicated with the medicine inlet, a first distribution channel group communicated with the first distribution cavity, and a plurality of second distribution channel groups communicated with the first distribution channel group;

the first distribution channel group comprises N first distribution channels which are sequentially arranged at intervals along the Y direction, the first distribution channels are parallel to the X direction, the lengths of all the first distribution channels are equal, and the channel volumes of the first distribution channels are sequentially increased from the first to the Nth;

each of the first distribution channels is communicated with one of the second distribution channel groups downstream, each of the second distribution channel groups includes M second distribution channels communicated with one of the first distribution channels through one of second distribution cavities, the second distribution channels are parallel to the X direction, all the second distribution channels are equal in length, and in the same second distribution channel group, the channel volumes of the second distribution channels sequentially increase from a first one to an M one;

n, M is not less than 2, the candidate drugs enter through the drug inlet, then flow distribution is performed sequentially through the first distribution channel and the second distribution channel, so that the flow of the candidate drugs entering the mixing structures in unit time is sequentially increased from the first to the Nth, and then a certain amount of preparation liquid is conveyed to the N-M mixing structures through the preparation liquid channel at a medium flow rate, so that the concentration of the candidate drugs in the first to the Nth mixing structures is sequentially increased;

the first distribution cavity comprises a first V-shaped cavity section and a first rectangular cavity section which are communicated with each other, the first V-shaped cavity section is communicated with the medicine injection port, and the first rectangular cavity section is communicated with the upstream of the first distribution channel;

2. The microfluidic chip for high efficiency drug screening according to claim 1, wherein the chip body comprises a main chip body and a sub-chip body disposed on the main chip body, the gradient generating structure, the cell sampling unit, the mixing unit and the cell culture unit are all formed in the main chip body, and the concentration configuration structure is formed in the sub-chip body.

3. The microfluidic chip for high efficiency drug screening according to claim 1, wherein the mixing structure comprises a first conical mixing cavity, a first mixing channel disposed in the middle of the upper end of the first mixing cavity, a second conical mixing cavity communicated with the first mixing cavity through a second mixing channel, a third mixing channel communicated with the middle of the upper end of the second mixing cavity, and a fourth mixing channel tangentially communicated with the periphery of the upper end of the second mixing cavity, wherein the outlet end of the fourth mixing channel is converged to the third mixing channel, and the outlet end of the third mixing channel is communicated to the cell culture cavity;

the inlet end of the second mixing channel is tangentially communicated with the periphery of the lower end of the first mixing cavity, and the outlet end of the second mixing channel is tangentially communicated with the periphery of the lower end of the second mixing cavity.

4. The microfluidic chip for high efficiency drug screening of claim 3, wherein the outlet end of the second distribution channel tangentially communicates with the upper end periphery of the first mixing chamber, and the outlet end of the distribution channel communicates with the first mixing channel;

5. The microfluidic chip for high efficiency drug screening according to claim 4, wherein the end of the third mixing channel has a first flared section, and the first flared section is communicated with the side of the cell culture chamber;

6. The microfluidic chip for efficient drug screening according to claim 5, wherein the check valve comprises two valve plates symmetrically arranged, each valve plate comprises an arc-shaped elastic part connected to the inner wall of the third mixing channel and a linear sealing part connected to the elastic part, and the elastic part is farther away from the cell culture chamber than the sealing part;

when no liquid flows in the third mixing channel or the flow direction of the liquid is from the closed part to the elastic part, the closed parts of the two valve plates are kept in contact, and the two valve plates close the third mixing channel;

7. The microfluidic chip for high efficiency drug screening according to claim 6, wherein one of the two sealing portions of the two valve plates is provided with saw-tooth protrusions, and the other is provided with saw-tooth grooves matching with the saw-tooth protrusions.

8. The microfluidic chip for high efficiency drug screening of claim 1, wherein the channel volume of the ith first distribution channel is denoted as U _i The channel volume of the jth second distribution channel is V _j Wherein i =1,2,.., N, j =1,2,.., M;

after deformation: v _m *U _i-1 <V ₁ *U _i 。

9. The microfluidic chip for high efficiency drug screening according to claim 1, wherein the cell sampling unit comprises a cell sampling port, a cell main channel communicated with the cell sampling port, and N x M cell branch channels communicated with the cell main channel, each cell branch channel being correspondingly communicated to one of the cell culture chambers through a second flared section.