Microfluidic chip and application thereof

Abstract

The application provides a microfluidic chip and application thereof, and the microfluidic chip comprises a main structure layer, a micro-valve film layer and a micro-valve control layer; the microfluidic chip is provided with a sample adding hole, a sample outlet hole and a waste liquid hole; the main structure layer is provided with a first processing unit, a second processing unit and a third processing unit in parallel; a first liquid flow channel is connected between an input end of the first processing unit and an input end of the second processing unit; a second liquid flow channel is connected between the input end of the second processing unit and an input end of the third processing unit; the first processing unit, the second processing unit and the third processing unit each have a first output end and a second output end; and the first processing unit, the second processing unit and the third processing unit are provided with a liquid gradient mixing area. One technical effect of the application is that not only can drug-resistant cells be efficiently screened, but also different drug sensitivity test processes can be observed simultaneously.

Description

TECHNICAL FIELD

[0001] The present application belongs to the technical field of gene detection, and particularly relates to a microfluidic chip and application thereof. BACKGROUND

[0002] Whole genome sequencing (WGS) is to sequence the whole genome of different individuals or populations by using high-throughput sequencing platform, and to perform bioinformatics analysis at the individual or population level. WGS can fully mine genetic variations at the DNA level, and provide important information for screening pathogenic and susceptible genes of diseases, studying genetic mechanisms of diseases, human evolution and human population genetics, etc.

[0003] The commonly used WGS whole genome detection technology for cell clusters is manually operated by an experimenter, and the cell clusters are processed and then subjected to WGS whole genome detection test by using a kit. The whole test process includes the following main steps: digestion, sedimentation, cell lysis, lysis termination, DNA amplification and machine test of the cell clusters. Years of laboratory and clinical tests have proved the effectiveness of this method. However, there are still the following three problems:

[0004] 1. When the sample amount is small, the consumption and errors caused by manual operation will have a great influence on the results of WGS whole genome detection. 2. It is relatively complex and difficult to perform targeted gene detection on drug-resistant cells. 3. It is difficult to perform in-situ observation and other characterization of the cell clusters to be detected.

[0005] The emergence of microfluidic technology provides a new means for WGR detection, but the most urgent needs in clinical practice at present are: 1. Detection of drug-resistant cells to detect low-frequency genes in a targeted manner; 2. In-situ observation and other characterization of the cell clusters before gene detection to enrich the data for subsequent analysis. Although microfluidic technology has shown excellent performance in various aspects, there is still no chip that can meet the above two clinical needs at the same time. SUMMARY

[0006] The present application aims to at least solve one of the technical problems existing in the prior art, and provides a new technical solution of a microfluidic chip and application thereof.

[0007] According to a first aspect of the present application, a microfluidic chip is provided, comprising a main structure layer, a micro-valve membrane layer and a micro-valve control layer; wherein the micro-valve membrane layer is arranged between the micro-valve control layer and the main structure layer, and the adjacent two layers are sealingly matched;

[0008] The microfluidic chip is provided with a sample adding hole, a sample outlet hole and a waste liquid hole which are penetrated from the upper surface of the micro valve control layer to the main structure layer; wherein the sample adding hole comprises a first sample adding hole and a second sample adding hole;

[0009] The main structure layer is provided with a first processing unit, a second processing unit and a third processing unit on one side of the micro valve thin film layer; the first processing unit, the second processing unit and the third processing unit are used for processing cell groups; a first liquid flow channel is connected between the input end of the first processing unit and the input end of the second processing unit, and the first sample adding hole is in communication with the middle part of the first liquid flow channel; a second liquid flow channel is connected between the input end of the second processing unit and the input end of the third processing unit, and the second sample adding hole is in communication with the middle part of the second liquid flow channel; the first processing unit, the second processing unit and the third processing unit each have a first output end and a second output end; the first output end is used for communicating with the sample outlet hole, and the second output end is used for communicating with the waste liquid hole; the first processing unit, the second processing unit and the third processing unit each are provided with a liquid gradient mixing area at one end close to the sample adding hole; the liquid gradient mixing area is formed by a liquid flow channel which is bent for multiple times;

[0010] The first drug is introduced into the first sample adding hole, and the second drug is introduced into the second sample adding hole; through the mixing of the liquid gradient mixing area, the first drug is introduced into the first processing unit, the mixture of the first drug and the second drug is introduced into the second processing unit, and the second drug is introduced into the third processing unit, so as to simultaneously perform different drug sensitivity tests on the cell groups in the first processing unit, the second processing unit and the third processing unit.

[0011] Optionally, the first processing unit, the second processing unit and the third processing unit each have a functional area, and the functional area comprises a cell group capturing and culturing area, a cell division area, a division termination area and a DNA amplification area;

[0012] The liquid gradient mixing area, the cell group capturing and culturing area, the cell division area, the division termination area, the DNA amplification area and the sample outlet hole are sequentially connected and arranged in a linear form;

[0013] The cell groups introduced from the sample adding hole can be captured, in-situ cultured, in-situ drug sensitivity tested and in-situ observed in the cell group capturing and culturing area, can be divided in the cell division area, can be terminated in the division termination area, can be amplified in the DNA amplification area, and can be outputted as test products through the sample outlet hole.

[0014] Optionally, the liquid gradient mixing area, the cell cluster capturing and culturing area, the cell division area, the division termination area, the DNA amplification area, and two adjacent ones of the waste liquid holes are each provided with a micro-valve control area for controlling the reaction of the respective functional area.

[0015] The micro-valve control layer is provided with a plurality of gas flow channels on the side facing the micro-valve membrane layer, each of the gas flow channels being matched with the micro-valve membrane layer to control the opening or closing of the corresponding micro-valve control area.

[0016] Optionally, each of the gas flow channels is connected with a micro-valve gas inlet for controlling the gas pressure in the gas flow channel, the micro-valve gas inlet penetrating the micro-valve control layer.

[0017] Optionally, the device further comprises a waste liquid flow channel.

[0018] The flow channel connecting the cell cluster capturing and culturing area and the cell division area is connected with a first input end of the waste liquid flow channel, the flow channel connecting the cell division area and the division termination area is connected with a second input end of the waste liquid flow channel, and the output end of the waste liquid flow channel is connected with the waste liquid hole; the first input end and the second input end of the waste liquid flow channel are each provided with a micro-valve control area.

[0019] When the micro-valve control area between the cell cluster capturing and culturing area and the cell division area is closed, the micro-valve control area of the first input end of the waste liquid flow channel is opened, and the micro-valve control area of the second input end of the waste liquid flow channel is closed, the waste liquid in the cell cluster capturing and culturing area can be discharged through the waste liquid hole by the waste liquid flow channel.

[0020] When the micro-valve control area of the first input end of the waste liquid flow channel is closed, the micro-valve control area of the second input end of the waste liquid flow channel is opened, the micro-valve control area between the cell cluster capturing and culturing area and the cell division area is opened, and the micro-valve control area between the cell division area and the division termination area is closed, the waste liquid in the cell division area can be discharged through the waste liquid hole by the waste liquid flow channel.

[0021] Optionally, the gas flow channels include a first gas flow channel, a second gas flow channel, a third gas flow channel, a fourth gas flow channel, a fifth gas flow channel, and a sixth gas flow channel.

[0022] The first gas flow channel is connected with a first micro-valve gas inlet, and the first gas flow channel is used to control the opening or closing of the micro-valve control area of the first input end of the waste liquid flow channel.

[0023] The second gas flow channel is connected with a second micro-valve gas inlet, and the second gas flow channel is used to control the opening or closing of the micro-valve control area between the cell cluster capturing and culturing area and the cell division area.

[0024] The third gas flow channel is connected with a third micro valve gas inlet, and the third gas flow channel is used for controlling opening or closing of a micro valve control area of the second input end of the waste liquid flow channel;

[0025] The fourth gas flow channel is connected with a fourth micro valve gas inlet, and the fourth gas flow channel is used for controlling opening or closing of a micro valve control area between the cell division area and the division termination area;

[0026] The fifth gas flow channel is connected with a fifth micro valve gas inlet, and the fifth gas flow channel is used for controlling opening or closing of a micro valve control area between the division termination area and the DNA amplification area;

[0027] The sixth gas flow channel is connected with a sixth micro valve gas inlet, and the sixth gas flow channel is used for controlling opening or closing of a micro valve control area between the DNA amplification area and the sample outlet.

[0028] Optionally, the cell cluster capturing and culturing area comprises a plurality of rows of cell cluster capturing units; the number of cell cluster capturing units in each row is a plurality, and the cell cluster capturing units in adjacent rows are staggered.

[0029] The first opening and the second opening are respectively arranged on two sides of each cell cluster capturing unit, and the width of the first opening is greater than the width of the second opening.

[0030] The first opening is directed to the liquid gradient mixing area, and the second opening is directed to the cell division area.

[0031] Optionally, the cell division area is provided with a single cell capturing unit.

[0032] A plurality of single cell capturing units are distributed along the width direction of the cell division area, and a gap of 5 microns is formed between adjacent single cell capturing units.

[0033] Optionally, the plurality of single cell capturing units form a V-shaped structure.

[0034] According to a second aspect of the present application, an application of a microfluidic chip is provided, which adopts the microfluidic chip according to the first aspect, and comprises:

[0035] Patient-derived cell clusters are respectively introduced into the first processing unit, the second processing unit and the third processing unit through the first sample inlet and the second sample inlet for culturing, and waste liquid in the culturing process is discharged through the waste liquid outlet.

[0036] The first medicine is introduced into the first sample adding hole, and the second medicine is introduced into the second sample adding hole; the first medicine is introduced into the first processing unit, the mixture of the first medicine and the second medicine is introduced into the second processing unit, and the second medicine is introduced into the third processing unit through the mixing of the liquid gradient mixing area, so that different drug sensitivity tests are simultaneously performed on the cell groups in the first processing unit, the second processing unit and the third processing unit, and the waste liquid in the drug sensitivity test process is discharged through the respective waste liquid holes;

[0037] After the drug sensitivity test is completed, the test products in the first processing unit, the second processing unit and the third processing unit can be discharged through the respective sample discharge holes for sample test research.

[0038] One technical effect of the present application is that:

[0039] In the embodiment of the present application, the cell groups can be added into the first processing unit, the second processing unit and the third processing unit respectively before the drug sensitivity test, so that the cell groups can be characterized in situ before gene detection, thereby enriching the data for subsequent analysis.

[0040] When the drug sensitivity test is performed, the first medicine is introduced into the first sample adding hole, and the second medicine is introduced into the second sample adding hole; the first medicine is introduced into the first processing unit, the mixture of the first medicine and the second medicine is introduced into the second processing unit, and the second medicine is introduced into the third processing unit through the mixing of the liquid gradient mixing area, so that different drug sensitivity tests are simultaneously performed on the cell groups in the first processing unit, the second processing unit and the third processing unit, thereby enabling detection of drug-resistant cells, significantly improving the efficiency of the drug sensitivity test, and enabling observation of the drug sensitivity test process of different medicines on the cell groups at the same time, and enabling accurate screening of drug-resistant cells. BRIEF DESCRIPTION OF DRAWINGS

[0041] Figure 1 A structural schematic diagram of a microfluidic chip according to an embodiment of the present application;

[0042] Figure 2 A structural schematic diagram of a main body structure layer of a microfluidic chip according to an embodiment of the present application;

[0043] Figure 3 A structural schematic diagram of a microfluidic chip according to an embodiment of the present application; Figure 2 An enlarged detail view of position A in FIG. 1;

[0044] Figure 4 A structural schematic diagram of a microfluidic chip according to an embodiment of the present application; Figure 2 An enlarged detail view of position B in FIG. 1;

[0045] Figure 5 A bottom view of a micro valve control layer of a microfluidic chip according to an embodiment of the present application;

[0046] Figure 6 A perspective view of a microfluidic chip according to an embodiment of the present application.

[0047] In the figure: 1, main structure layer; 11, first processing unit; 12, second processing unit; 13, third processing unit; 101, first liquid flow channel; 102, second liquid flow channel; 103, waste liquid flow channel; 100, liquid gradient mixing area; 200, cell cluster capture and culture area; 300, cell division area; 400, division termination area; 500, DNA amplification area; 600, microvalve control area; 2, microvalve membrane layer; 3, microvalve control layer; 31, first gas flow channel; 311, first microvalve gas inlet; 32, second gas flow channel; 321, second microvalve gas inlet; 33, third gas flow channel; 331, third microvalve gas inlet; 34, fourth gas flow channel; 341, fourth microvalve gas inlet; 35, fifth gas flow channel; 351, fifth microvalve gas inlet; 36, sixth gas flow channel; 361, sixth microvalve gas inlet; 41, first sample addition hole; 42, second sample addition hole; 43, sample outlet hole; 431, first sample outlet hole; 432, second sample outlet hole; 433, third sample outlet hole; 44, waste liquid hole; 441, first waste liquid hole; 442, second waste liquid hole; 443, third waste liquid hole; 5, cell cluster capture unit; 51, first opening; 52, second opening; 6, single cell capture unit; 61, gap. DETAILED DESCRIPTION

[0048] Various exemplary embodiments of the present application will now be described in detail with reference to the accompanying drawings. It should be noted that the relative arrangement of the components and steps set forth in the embodiments, numerical expressions, and numerical values, unless specifically stated otherwise, do not limit the scope of the present application.

[0049] Embodiments of the present application will be described in detail below, examples of which are shown in the accompanying drawings, wherein the same or similar reference numerals represent the same or similar elements or elements having the same or similar functions throughout. The embodiments described below by reference to the accompanying drawings are exemplary and are for the purpose of explanation only, and are not to be understood as limiting the present application. Based on the embodiments in the present application, all other embodiments obtained by those of ordinary skill in the art without creative labor are within the scope of protection of the present application.

[0050] The terms "first", "second" in the description and claims of the present application can explicitly or implicitly include one or more of the features. In the description of the present application, unless otherwise specified, the meaning of "a plurality of" is two or more. In addition, "and / or" in the specification and claims means at least one of the connected objects, and the character " / ", generally means that the front and rear associated objects are in an "or" relationship.

[0051] In the description of the present application, it needs to be understood that the orientation or positional relationship indicated by the terms "center", "longitudinal", "transverse", "length", "width", "thickness", "upper", "lower", "front", "rear", "left", "right", "vertical", "horizontal", "top", "bottom", "inner", "outer", "clockwise", "counterclockwise", "axial", "radial", "circumferential" and the like is based on the orientation or positional relationship shown in the drawings, and is only for the convenience of describing the present application and simplifying the description, and does not indicate or imply that the device or element referred to must have a particular orientation, be constructed and operated in a particular orientation, and therefore cannot be understood as a limitation to the present application.

[0052] In the description of the present application, it needs to be understood that unless otherwise explicitly specified and limited, the terms "mounting", "connection", "connection" should be understood broadly, for example, it can be fixed connection, or detachable connection, or integral connection; it can be mechanical connection, or electrical connection; it can be direct connection, or indirect connection through intermediate medium, or internal communication of two elements. For those skilled in the art, the specific meaning of the above terms in the present application can be understood according to the specific circumstances.

[0053] According to the first aspect of the present application, referring to Figures 1 to 6 , a microfluidic chip which can carry out integrated experiment of cell cluster culture, drug sensitivity detection and gene detection is provided.

[0054] Specifically, the microfluidic chip comprises a main structure layer 1, a micro valve film layer 2 and a micro valve control layer 3; wherein the micro valve film layer 2 is arranged between the micro valve control layer 3 and the main structure layer 1, and the adjacent two layers are sealingly matched; the microfluidic chip is provided with a sample adding hole, a sample outlet hole 43 and a waste liquid hole 44 which are penetrated from the upper surface of the micro valve control layer 3 to the main structure layer 1; wherein the sample adding hole comprises a first sample adding hole 41 and a second sample adding hole 42, the first sample adding hole 41 and the second sample adding hole 42 are used to realize the inflow of sample and reagent into the main structure layer 1, the waste liquid hole 44 is used to realize the outflow of waste liquid, and the sample outlet hole 43 is used to realize the outflow and collection of test product;

[0055] The main structure layer 1 is provided with a first processing unit 11, a second processing unit 12 and a third processing unit 13 on one side of the micro valve film layer 2, and the first processing unit 11, the second processing unit 12 and the third processing unit 13 are used for processing cell groups; a first liquid flow channel 101 is connected between the input end of the first processing unit 11 and the input end of the second processing unit 12, and the first sample adding hole 41 is in communication with the middle part of the first liquid flow channel 101; a second liquid flow channel 102 is connected between the input end of the second processing unit 12 and the input end of the third processing unit 13, and the second sample adding hole 42 is in communication with the middle part of the second liquid flow channel 102; the first processing unit 11, the second processing unit 12 and the third processing unit 13 are respectively provided with a first output end and a second output end, the first output end is used for communicating with the sample outlet hole 43, and the second output end is used for communicating with the waste liquid hole 44; the first processing unit 11, the second processing unit 12 and the third processing unit 13 are all provided with a liquid gradient mixing area 100 near one end of the sample adding hole, and the liquid gradient mixing area 100 is formed by a liquid flow channel which is bent for multiple times;

[0056] The first drug is introduced into the first sample adding hole 41, and the second drug is introduced into the second sample adding hole 42; after mixing in the liquid gradient mixing area 100, the first drug is introduced into the first processing unit 11, the mixture of the first drug and the second drug is introduced into the second processing unit 12, and the second drug is introduced into the third processing unit 13, so that different drug sensitivity tests are simultaneously performed on the cell groups in the first processing unit 11, the second processing unit 12 and the third processing unit 13.

[0057] In the embodiment of the application, before the drug sensitivity test, cell groups can be respectively added into the first processing unit 11, the second processing unit 12 and the third processing unit 13, so that the cell groups can be in-situ observed and characterized before gene detection, so as to enrich the data for subsequent analysis.

[0058] During the drug sensitivity test, the first drug is introduced into the first sample adding hole 41, and the second drug is introduced into the second sample adding hole 42; after mixing in the liquid gradient mixing area 100, the first drug is introduced into the first processing unit 11, the mixture of the first drug and the second drug is introduced into the second processing unit 12, and the second drug is introduced into the third processing unit 13, so that different drug sensitivity tests are simultaneously performed on the cell groups in the first processing unit 11, the second processing unit 12 and the third processing unit 13, thereby the drug-resistant cells can be detected, the low-frequency genes can be detected in a targeted manner, the efficiency of the drug sensitivity test is significantly improved, the drug sensitivity test processes of different drugs on the cell groups can be simultaneously observed, and the drug-resistant cells can be accurately screened out.

[0059] In one embodiment, referring to Figure 2 The width of the flow channel of the liquid gradient mixing zone 100 is 150 microns. For example, the liquid gradient mixing zone 100 is composed of six long flow channels and two short flow channels arranged in parallel, the six long flow channels are sequentially connected, and the two short flow channels are respectively located on the opposite sides of the six long flow channels and are parallel to the long flow channels. The short flow channels are parallel to the corresponding long flow channels, and the spacing between adjacent flow channels is 150 microns. The length of the long flow channel is 2500 microns, the length of the short flow channel is 1250 microns, and the adjacent flow channels are connected by a circular channel.

[0060] Optionally, the first processing unit 11, the second processing unit 12 and the third processing unit 13 each have a functional area, and the functional area includes a cell cluster capture and culture area 200, a cell division area 300, a division termination area 400 and a DNA amplification area 500. The liquid gradient mixing zone 100 and each functional area can be formed by etching grooves on the surface of the main structure layer 1.

[0061] The liquid gradient mixing zone 100, the cell cluster capture and culture area 200, the cell division area 300, the division termination area 400, the DNA amplification area 500 and the sample outlet hole 43 are sequentially connected and arranged in a linear manner.

[0062] The cell cluster entering from the sample inlet hole can be captured, cultured in situ, tested for drug sensitivity in situ and observed in situ in the cell cluster capture and culture area 200, can be divided in the cell division area 300, can be stopped from dividing in the division termination area 400, can be amplified in the DNA amplification area 500, and can output the test product through the sample outlet hole 43.

[0063] In the above embodiment, the liquid gradient mixing zone 100, the cell cluster capture and culture area 200, the cell division area 300, the division termination area 400, the DNA amplification area 500 and the sample outlet hole 43 are arranged in a reasonable manner, which is conducive to the realization of the processes of cell cluster capture, cell cluster culture, drug sensitivity detection and gene detection.

[0064] For example, the width of the inlet of the cell cluster capture and culture area 200 is 150 microns (the narrowest), the width of the middle part is 2800 microns (the widest), and the width of the outlet is 250 microns. The width of the cell division area 300 is 1100 microns, and the length is 1500 microns. The width of the division termination area 400 is 1000 microns, and the length is 1500 microns. The width of the DNA amplification area 500 is 3500 microns, and the length is 3500 microns.

[0065] Optionally, the liquid gradient mixing area 100, the cell cluster capture and culture area 200, the cell division area 300, the division termination area 400, the DNA amplification area 500, and two adjacent ones of the sample outlet holes 43 are each provided with a micro-valve control area 600 for controlling the reaction of each functional area; wherein the micro-valve control area is formed by arranging a groove on the surface of the main structure layer, when the micro-valve control area is closed, the pressure of the corresponding gas flow channel is increased to drive the corresponding micro-valve membrane layer to move into the groove of the micro-valve control area, so as to close the micro-valve control area; when the micro-valve control area is opened, the pressure of the corresponding gas flow channel is reduced, and the corresponding micro-valve membrane layer moves to the micro-valve control layer, so as to open the micro-valve control area.

[0066] The micro-valve control layer 3 is provided with a plurality of gas flow channels on the side facing the micro-valve membrane layer 2, and each gas flow channel cooperates with the micro-valve membrane layer 2 to control the opening or closing of the corresponding micro-valve control area 600.

[0067] In the above embodiment, the micro-valve control area 600 can quickly realize the connection or disconnection of the connected functional areas, and the operation is simple and fast.

[0068] It should be noted that the three groups of functional areas of the first processing unit 11, the second processing unit 12, and the third processing unit 13 are added through two sample addition holes (i.e., the first sample addition hole 41 and the second sample addition hole 42), and the added samples are divided into three concentration gradients by the three liquid gradient mixing areas 100 and sequentially introduced into the cell cluster capture and culture area 200.

[0069] The cell cluster capture and culture area 200 is a large chamber, and a U-shaped cell cluster capture unit 5 is arranged in the chamber for cell cluster capture, in-situ culture, in-situ drug sensitivity experiment, and in-situ observation. The cell cluster capture and culture area 200 is connected with a waste liquid flow channel 103, and the output end of the waste liquid flow channel 103 is connected with a waste liquid hole 44 for discharging waste liquid.

[0070] For example, the first input end of the waste liquid flow channel 103 connected with the cell cluster capture and culture area 200 is provided with a micro-valve control area 600, and the opening or closing of the waste liquid flow channel is controlled by the opening or closing of the micro-valve control area 600. When the first input end of the waste liquid flow channel is opened, the waste liquid generated by the cell cluster capture and culture area 200 is discharged through the first input end of the waste liquid flow channel and the waste liquid hole in sequence.

[0071] In one embodiment, a single cell capturing unit 6 is provided in the cell division zone 300 for capturing the single cells after digestion in the cell cluster capturing and culture zone 200, and the cell division is achieved in the cell division zone 300 by the combination of the introduction of reagents and the opening or closing of the microvalve control zone 600. Meanwhile, the cell division zone 300 is connected with a waste liquid flow channel 103, and the waste liquid is discharged through the waste liquid hole 44.

[0072] For example, the microvalve control zone 600 is provided on the second input end of the waste liquid flow channel 103 communicating with the cell division zone 300, and the opening or closing of the waste liquid flow channel is controlled by the opening or closing of the microvalve control zone 600. When the second input end of the waste liquid flow channel is opened, the waste liquid generated in the cell division zone 300 is discharged in sequence through the second input end of the waste liquid flow channel and the waste liquid hole.

[0073] Further, the neutralization and division reagent is introduced to push the liquid in the cell division zone 300 into the division termination zone 400 for reaction to achieve the termination of the cell division process. Then, the amplification DNA enzyme is introduced to push the liquid in the division termination zone 400 into the DNA amplification zone 500 to achieve the amplification of DNA in this DNA amplification zone 500. After a certain time and temperature reaction in the DNA amplification zone 500, the sterile water is introduced to push all the test products out of the sample hole 43 and collect them for subsequent detection and experiment. For example, the thickness of the main structure layer 1 is 5000 microns, and the depth of the liquid flow channel is 70 microns-100 microns.

[0074] In one embodiment, referring to Figure 5 The microvalve control layer 3 is provided with a groove on the side facing the microvalve membrane layer 2 to form a gas flow channel. For example, the thickness of the microvalve control layer 3 is 5000 microns, and the depth of the gas flow channel is 100 microns-150 microns. The length of the microvalve control layer 3 is 1000 microns, and the width is 600 microns.

[0075] For example, the thickness of the microvalve membrane layer 2 is 30 microns-50 microns.

[0076] Optionally, each of the gas flow channels is connected with a microvalve gas inlet for controlling the gas pressure in the gas flow channel, and the microvalve gas inlet penetrates the microvalve control layer 3. This makes the control of the gas flow channel very simple.

[0077] Optionally, referring to Figure 2 and Figure 6 The microfluidic chip further comprises a waste liquid flow channel 103;

[0078] The flow channel connecting the cell mass capturing and culturing area 200 and the cell division area 300 is connected with the first input end of the waste liquid flow channel 103, the flow channel connecting the cell division area 300 and the division termination area 400 is connected with the second input end of the waste liquid flow channel 103, and the output end of the waste liquid flow channel 103 is connected with the waste liquid hole 44; the micro valve control area 600 is arranged on the first input end and the second input end of the waste liquid flow channel 103;

[0079] When the micro valve control area 600 between the cell mass capturing and culturing area 200 and the cell division area 300 is closed, the micro valve control area 600 of the first input end of the waste liquid flow channel 103 is opened, and the micro valve control area 600 of the second input end of the waste liquid flow channel 103 is closed, the waste liquid in the cell mass capturing and culturing area 200 can be discharged by the waste liquid flow channel 103 through the waste liquid hole 44;

[0080] When the micro valve control area 600 of the first input end of the waste liquid flow channel 103 is closed, the micro valve control area 600 of the second input end of the waste liquid flow channel 103 is opened, the micro valve control area 600 between the cell mass capturing and culturing area 200 and the cell division area 300 is opened, and the micro valve control area 600 between the cell division area 300 and the division termination area 400 is closed, the waste liquid in the cell division area 300 can be discharged by the waste liquid flow channel 103 through the waste liquid hole 44.

[0081] In the above embodiment, the waste liquid in the corresponding functional area can be quickly discharged through the waste liquid flow channel 103, avoiding the influence of the waste liquid on the reaction process, and better ensuring the functional integrity of each functional area.

[0082] Optionally, referring to Figure 5 and Figure 6 The gas flow channel includes a first gas flow channel 31, a second gas flow channel 32, a third gas flow channel 33, a fourth gas flow channel 34, a fifth gas flow channel 35, and a sixth gas flow channel 36.

[0083] The first gas flow channel 31 is connected with a first micro valve gas inlet 311, and the first gas flow channel 31 is used for controlling the opening or closing of the micro valve control area 600 of the first input end of the waste liquid flow channel 103;

[0084] The second gas flow channel 32 is connected with a second micro valve gas inlet 321, and the second gas flow channel 32 is used for controlling the opening or closing of the micro valve control area 600 between the cell mass capturing and culturing area 200 and the cell division area 300;

[0085] The third gas flow channel 33 is connected with a third micro valve gas inlet 331, and the third gas flow channel 33 is used for controlling the opening or closing of the micro valve control area 600 of the second input end of the waste liquid flow channel 103.

[0086] The fourth gas flow channel 34 is connected with a fourth micro valve gas inlet 341, and the fourth gas flow channel 34 is used for controlling the opening or closing of the micro valve control area 600 between the cell division area 300 and the division termination area 400.

[0087] The fifth gas flow channel 35 is connected with a fifth micro valve gas inlet 351, and the fifth gas flow channel 35 is used for controlling the opening or closing of the micro valve control area 600 between the division termination area 400 and the DNA amplification area 500.

[0088] The sixth gas flow channel 34 is connected with a sixth micro valve gas inlet 361, and the sixth gas flow channel 36 is used for controlling the opening or closing of the micro valve control area 600 between the DNA amplification area 500 and the sample outlet hole 43.

[0089] In the above embodiment, the opening or closing of the corresponding micro valve control area 600 is controlled by different gas flow channels, so that the corresponding reaction can be carried out in each functional area of the microfluidic chip according to the demand, and the control is very convenient.

[0090] Optionally, referring to Figure 2 and Figure 3 The cell cluster capture and culture area 200 comprises a plurality of rows of cell cluster capture units 5; the number of cell cluster capture units 5 in each row is a plurality, and the cell cluster capture units 5 in adjacent rows are staggered.

[0091] The two sides of each cell cluster capture unit 5 are respectively provided with a first opening 51 and a second opening 52, and the width of the first opening 51 is greater than the width of the second opening 52.

[0092] The first opening 51 faces the liquid gradient mixing area 100, and the second opening 52 faces the cell division area 300.

[0093] In the above embodiment, the cell cluster capture unit 5 can realize the accurate capture of the cell cluster.

[0094] For example, the cell cluster capture unit 5 is U-shaped.

[0095] For example, the cell cluster capturing and culturing area 200 is provided with 24 cell cluster capturing units 5 arranged in staggered rows. The distance between two rows of cell cluster capturing units 5 is 1200 microns, and the distance between adjacent cell cluster capturing units 5 in the same row is 800 microns. The length of the widest part of the cell cluster capturing and culturing area 200 is 4800 microns, and the overall length is 6800 microns.

[0096] In one specific embodiment, the cell cluster capturing unit 5 has a height of 100 microns and a width of 50 microns, and is composed of two circular arc structures and two cuboid structures. The inner radius of the two circular arc structures is 100 microns, and the angle of the circular arc is 10 degrees to 90 degrees. The length of the cuboid structure is 100 microns, and the width is 50 microns.

[0097] Optionally, referring to Figure 2 and Figure 4 , the cell division area 300 is provided with single cell capturing units 6;

[0098] The plurality of single cell capturing units 6 are distributed along the width direction of the cell division area 300; and a gap 61 of 5 microns is formed between adjacent single cell capturing units 6.

[0099] In the above embodiment, the single cell capturing unit 6 can quickly achieve accurate capture of single cells.

[0100] Optionally, the plurality of single cell capturing units 6 form a V-shaped structure. For example, the cell division area 300 is provided with 102 single cell capturing units 6 arranged in a V-shaped structure at an angle of 45 degrees. The arrangement of the V-shaped structure can also make the cells flow to both sides, ensuring that the cells do not accumulate and block the entrance at the entrance.

[0101] For example, the single cell capturing unit 6 is composed of a rectangular structure with a height of 50 microns, a length of 20 microns, and a width of 15 microns. Each rectangular structure is spaced 5 microns apart and arranged in a V-shaped structure at an angle of 45 degrees in the cell division area 300.

[0102] In one specific embodiment, the micro-valve control layer 3, the micro-valve membrane layer 2, and the main structure layer 1 are all made of PDMS material.

[0103] The liquid gradient mixing area 100 can realize uniform mixing of two sample reagents. If the first sample hole 41 is connected to reagent A and the second sample hole 42 is connected to reagent B, the reagent concentrations in the cell cluster capturing and culture area 200 of the first processing unit 11, the second processing unit 12, and the third processing unit 13 are 100% reagent A, 50% reagent A and 50% reagent B mixed liquid, and 100% reagent B, respectively. If the first sample hole 41 and the second sample hole 42 are simultaneously connected to the same reagent or cell suspension, the liquid gradient mixing area 100 can realize simultaneous connection of the same reagent or cell suspension into the first processing unit 11, the second processing unit 12, and the third processing unit 13.

[0104] The cell cluster capturing unit 5 in the cell cluster capturing and culture area 200 can capture the cell cluster when the cell suspension is connected, and the cell cluster outside the cell cluster capturing unit 5 can be blown out by the connection of the culture medium, avoiding the occlusion of the line of sight for subsequent observation. Since the cell cluster will not move during the subsequent connection of the culture medium, in-situ culture is realized; when the sample port is connected to a drug, in-situ drug sensitivity test can also be realized; when the sample port is connected to a staining agent, in-situ staining test can also be realized. Since the chip is made of transparent material, in-situ observation of the cell cluster in the cell cluster capturing and culture area 200 is easy. Therefore, the microfluidic chip can realize in-situ real-time observation of the culture, drug sensitivity, and fluorescent staining process of the cell cluster. In addition, since the waste liquid flow channel 103 is provided behind the cell cluster capturing and culture area 200, the opening or closing of the microvalve control area 600 can be controlled to realize the flow of waste liquid through the waste liquid hole 44, so as not to pollute the subsequent functional areas of the cell cluster capturing and culture area 200.

[0105] The spacing of the cuboid columns in the cell division area 300 is 5 mm, which can capture the cells after digestion of the cell cluster capturing and culture area 200, and the V-shaped arrangement can also make the cells flow to both sides, ensuring that the cells will not accumulate and block the inlet. There is also a waste liquid flow channel 103 behind the cell division area 300, which can be controlled by the opening or closing of the microvalve control area 600 to realize the flow of waste liquid through the waste liquid hole 44, so as not to pollute the subsequent functional areas of the cell division area 300.

[0106] The cell division area 300 and the division termination area 400 each correspond to a microvalve control area 600 for on-off control, which can realize control of liquid flow and also realize mixing of liquid in the liquid flow channel or functional area through rapid opening and closing of the switch during the experiment.

[0107] The division termination area 400 and the DNA amplification area 500 each correspond to a microvalve control area 600 for on-off control, which can realize control of liquid flow and also realize mixing of liquid in the liquid flow channel or functional area through rapid opening and closing of the switch during the experiment.

[0108] In the embodiment of the present application, the cell cluster capturing unit 5 can realize the capture of cell clusters, thereby ensuring the in-situ drug sensitivity test of the cell clusters. Compared with the traditional technology, the cell cluster capturing and culture area 200 enables the cell clusters to be arranged regularly, and the same cell cluster can be observed for a long time. Meanwhile, the cell cluster capturing and culture area 200 can also observe the growth and death state of the cell clusters in the drug sensitivity test process for a long time, and can characterize and analyze the state of the cell clusters in the drug environment. Meanwhile, after the fluorescent reagent is introduced, the cell cluster capturing and culture area 200 can also perform in-situ fluorescence observation on the cell clusters.

[0109] Further, the microfluidic chip can first perform preliminary screening on the cell clusters through the drug sensitivity test, so as to screen out drug-resistant cells, and make the subsequent gene detection process more targeted. The traditional gene detection can only identify high-frequency genes, but if the drug sensitivity test is performed before the detection, the subpopulation of low-frequency genes can be identified, and this advantage also conforms to the trend that the direction of gene detection gradually shifts to low-frequency genes.

[0110] Moreover, the microfluidic chip realizes the precise gene detection of a small amount of sample in a closed environment, the setting of the waste liquid flow channel 103 can avoid the pollution of the early-stage waste liquid, the structure with fixed volume can realize the precise control of the introduction of drugs, and the rapid opening or closing of the micro valve control area 600 can realize the mixing of the sample. The gene detection process of the traditional method is performed in an open environment, and problems such as sample consumption, operation error and environmental pollution may occur. The structure of the microfluidic chip of the present application is small, precise control and closed, which avoids the above problems.

[0111] In addition, the microfluidic chip realizes high-throughput and rapid processing of samples. The degree of automation is high, and the whole process from sample injection to gene detection is automated. The automatic process includes but is not limited to liquid exchange, drug addition, digestion, cell lysis, lysis stop, DNA amplification and the like. This greatly reduces manual operation and saves a lot of manpower and time. Meanwhile, the microfluidic chip has a wide range of applications. The size of the cell cluster capturing unit 5 and the single cell capturing unit 6 can be adjusted according to different cells, and the arrangement structure can be adjusted according to different cell suspension concentrations, which is highly flexible and does not increase the cost.

[0112] According to a second aspect of the present application, a microfluidic chip is provided, which comprises:

[0113] The patient-derived cell clusters are introduced into the first processing unit 11, the second processing unit 12 and the third processing unit 13 through the first sample addition hole 41 and the second sample addition hole 42 for culture, and the waste liquid in the culture process is discharged through the waste liquid hole 44;

[0114] The first drug is introduced into the first sample inlet 41, and the second drug is introduced into the second sample inlet 42; through mixing in the liquid gradient mixing area 100, the first drug is introduced into the first processing unit 11, the mixture of the first drug and the second drug is introduced into the second processing unit 12, and the second drug is introduced into the third processing unit 13, so that different drug sensitivity tests can be simultaneously performed on the cell clusters in the first processing unit 11, the second processing unit 12, and the third processing unit 13, and the waste liquid in the drug sensitivity test process can be discharged through the respective waste liquid outlets 44, for example, see Figure 2 The waste liquid in the drug sensitivity test process of the first processing unit 11 is discharged through the first waste liquid outlet 441; the waste liquid in the drug sensitivity test process of the second processing unit 12 is discharged through the second waste liquid outlet 442; and the waste liquid in the drug sensitivity test process of the third processing unit 13 is discharged through the third waste liquid outlet 443.

[0115] After the drug sensitivity test is completed, the test products in the first processing unit 11, the second processing unit 12, and the third processing unit 13 can be discharged through the respective sample outlets 43 for experimental research on the samples.

[0116] For example, see Figure 2 The test product of the first processing unit 11 is discharged through the first sample outlet 431; the test product of the second processing unit 12 is discharged through the second sample outlet 432; and the test product of the third processing unit 13 is discharged through the third sample outlet 433.

[0117] In the above embodiment, different drug sensitivity tests can be simultaneously performed on the cell clusters in the first processing unit 11, the second processing unit 12, and the third processing unit 13, so that drug-resistant cells can be detected, the efficiency of the drug sensitivity test is significantly improved, and the drug sensitivity test processes of different drugs on the cell clusters can be simultaneously observed, and drug-resistant cells can be accurately screened out.

[0118] In a specific embodiment, patient-derived organoids or cell line-derived cell clusters are diluted to a concentration of 10 4 After the cell clusters are captured and diluted, the micro valve control area 600 between the cell cluster capture and culture area 200 and the cell division area 300 is closed, and the second input end of the waste liquid flow channel is closed, the micro valve control area 600 of the first input end of the waste liquid flow channel 103 is opened, and the first sample inlet and the second sample inlet 42 are simultaneously introduced into the microfluidic chip at a speed of 10 ml / min. After the cell clusters are captured, the culture medium is continuously or periodically introduced to realize long-term culture of the cell clusters, and the waste liquid in the culture process is discharged from the waste liquid outlet 44 through the waste liquid flow channel 103.

[0119] After 5 days of culture, the cell clusters are observed to have grown significantly. On the 1st day and the 3rd day of the subsequent experiment, the test drug is introduced, and the medium is introduced at other times. The process of the 4-day drug sensitivity test is completed, and the waste liquid is discharged from the waste liquid hole 44 through the waste liquid flow channel 103. During this process, the growth state of the cell clusters in the drug environment can be observed and recorded.

[0120] Then, the micro valve control area 600 of the first input end of the waste liquid flow channel 103 is closed, the micro valve control area 600 between the cell cluster capture and culture area 200 and the cell division area 300 is opened, the micro valve control area 600 of the second input end of the waste liquid flow channel 103 is opened, and the micro valve control area 600 between the cell division area 300 and the division termination area 400 is closed. Digestion solution is introduced into the microfluidic chip, so that the single cells after digestion flow into the cell division area 300 through the gap between the cell cluster capture units 5, and are captured by the single cell capture unit 6. Then, a cell division reagent for gene detection is introduced into the microfluidic chip, the micro valve control area 600 of the second input end of the waste liquid flow channel 103 is closed, and the reaction is carried out at a preset temperature for a certain time to achieve cell division.

[0121] Then, the micro valve control area 600 between the cell division area 300 and the division termination area 400 is opened, and a division termination reagent for gene detection is introduced into the microfluidic chip. The micro valve control area 600 between the division termination area 400 and the DNA amplification area 500 and the micro valve control area 600 between the cell cluster capture and culture area 200 and the cell division area 300 are closed, and the mixing of the liquid in the microfluidic chip is achieved by multiple incomplete switching of the micro valve control area 600 between the cell division area 300 and the division termination area 400. After a certain time of reaction at a preset temperature, the termination of cell division is achieved.

[0122] Then, the micro valve control area 600 between the cell cluster capture and culture area 200 and the cell division area 300 and the micro valve control area 600 between the division termination area 400 and the DNA amplification area 500 are opened, and a DNA amplification reagent for gene detection is introduced. The micro valve control area 600 between the cell division area 300 and the division termination area 400 and the micro valve control area 600 between the DNA amplification area 500 and the sample outlet hole 43 are closed, the mixing of the liquid in the microfluidic chip is achieved by multiple incomplete switching of the micro valve control area 600 between the division termination area 400 and the DNA amplification area 500, and the amplification of DNA is achieved after a certain time of reaction at a preset temperature.

[0123] Finally, the micro-valve control region 600 between the cell division region 300 and the division termination region 400 and the micro-valve control region 600 between the DNA amplification region 500 and the sample outlet hole 43 are opened, sterile water is introduced into the micro-fluidic chip, the reaction reagents in the micro-fluidic chip are pushed out through the sample outlet hole 43, the sample is collected, and subsequent experimental research is carried out.

[0124] It can be understood that the above embodiments are only exemplary embodiments for illustrating the principles of the present application, and the present application is not limited thereto. Various modifications and improvements can be made by those of ordinary skill in the art without departing from the spirit and essence of the present application, and these modifications and improvements are also considered to be within the protection scope of the present application.

Claims

1. A microfluidic chip, characterized by, It includes a main structure layer, a microvalve film layer, and a microvalve control layer; wherein the microvalve film layer is disposed between the microvalve control layer and the main structure layer, and adjacent layers are sealed together. The microfluidic chip is provided with a sample dispensing port, a sample outlet port, and a waste liquid port extending from the upper surface of the microvalve control layer to the main structure layer; wherein, the sample dispensing port includes a first sample dispensing port and a second sample dispensing port; The main structural layer has a first processing unit, a second processing unit, and a third processing unit arranged side-by-side on the side facing the microvalve film layer. These three units are used to process cell clusters. A first liquid channel connects the input ends of the first and second processing units, and the first sample dispensing port communicates with the middle of the first liquid channel. A second liquid channel connects the input ends of the second and third processing units, and the second sample dispensing port communicates with the middle of the second liquid channel. Each of the three processing units has a first output end and a second output end. The first output end communicates with the sample outlet, and the second output end communicates with the waste liquid outlet. A liquid gradient mixing zone is provided at the end of each of the three processing units near the sample dispensing port. This liquid gradient mixing zone is formed by a liquid channel that has undergone multiple bends. A first drug is introduced into the first sample well, and a second drug is introduced into the second sample well. After mixing in the liquid gradient mixing zone, the first drug flows into the first processing unit, a mixture of the first and second drugs flows into the second processing unit, and the second drug flows into the third processing unit, so as to simultaneously perform different drug sensitivity tests on the cell clusters in the first, second, and third processing units.

2. The microfluidic chip of claim 1, wherein, The first processing unit, the second processing unit, and the third processing unit each have a functional region, which includes a cell cluster capture and culture region, a cell division region, a division termination region, and a DNA amplification region. The liquid gradient mixing zone, the cell cluster capture and culture zone, the cell division zone, the division termination zone, the DNA amplification zone, and the sample outlet well are sequentially connected and arranged in a straight line. Cell clusters entering through the sample wells can be captured, cultured in situ, subjected to in situ drug sensitivity tests, and observed in the cell cluster capture and culture zone; they can divide in the cell division zone; they can terminate division in the cell division termination zone; they can amplify DNA in the DNA amplification zone; and the test products can be output through the sample wells.

3. The microfluidic chip of claim 2, wherein, The liquid gradient mixing zone, the cell cluster capture and culture zone, the cell division zone, the division termination zone, the DNA amplification zone, and the sample outlet wells are all provided with microvalve control zones between adjacent pairs to control the reactions of each functional zone. The microvalve control layer has multiple gas channels on the side facing the microvalve film layer. Each gas channel cooperates with the microvalve film layer to control the opening or closing of the corresponding microvalve control area.

4. The microfluidic chip of claim 3, wherein, Each of the gas channels is connected to a micro-valve inlet for controlling the gas pressure within the gas channel, and the micro-valve inlet penetrates the micro-valve control layer.

5. The microfluidic chip of claim 4, wherein, It also includes a waste liquid flow channel; The flow channel connecting the cell cluster capture and culture area and the cell division area is connected to the first input end of the waste liquid flow channel; the flow channel connecting the cell division area and the division termination area is connected to the second input end of the waste liquid flow channel; the output end of the waste liquid flow channel is connected to the waste liquid orifice; a micro-valve control area is provided on both the first input end and the second input end of the waste liquid flow channel. When the microvalve control area between the cell cluster capture and culture area and the cell division area is closed, the microvalve control area at the first input end of the waste liquid flow channel is open, and the microvalve control area at the second input end of the waste liquid flow channel is closed, the waste liquid in the cell cluster capture and culture area can be discharged through the waste liquid flow channel via the waste liquid hole. When the microvalve control area at the first input end of the waste liquid flow channel is closed, the microvalve control area at the second input end of the waste liquid flow channel is open, the microvalve control area between the cell cluster capture and culture area and the cell division area is open, and the microvalve control area between the cell division area and the division termination area is closed, the waste liquid in the cell division area can be discharged through the waste liquid flow channel via the waste liquid hole.

6. The microfluidic chip of claim 5, wherein, The gas flow channels include a first gas flow channel, a second gas flow channel, a third gas flow channel, a fourth gas flow channel, a fifth gas flow channel, and a sixth gas flow channel; The first gas flow channel is connected to a first micro valve inlet, and the first gas flow channel is used to control the opening or closing of the micro valve control area at the first input end of the waste liquid flow channel; The second gas channel is connected to a second microvalve inlet. The second gas channel is used to control the opening or closing of the microvalve control area between the cell cluster capture and culture area and the cell division area. The third gas flow channel is connected to a third micro valve inlet, and the third gas flow channel is used to control the opening or closing of the micro valve control area at the second input end of the waste liquid flow channel; The fourth gas flow channel is connected to a fourth micro-valve inlet, and the fourth gas flow channel is used to control the opening or closing of the micro-valve control area between the cell division zone and the division termination zone. The fifth gas channel is connected to a fifth microvalve inlet, and the fifth gas channel is used to control the opening or closing of the microvalve control area between the division termination region and the DNA amplification region. The sixth gas channel is connected to a sixth microvalve inlet, and the sixth gas channel is used to control the opening or closing of the microvalve control area between the DNA amplification region and the sample outlet well.

7. The microfluidic chip of claim 6, wherein, The cell cluster capture and culture area includes multiple rows of cell cluster capture units; each row contains multiple cell cluster capture units, and the cell cluster capture units in adjacent rows are staggered. Each of the cell cluster capture units has a first opening and a second opening on both sides, and the width of the first opening is greater than the width of the second opening. The first opening faces the liquid gradient mixing zone, and the second opening faces the cell division zone.

8. The microfluidic chip of claim 7, wherein, The cell division zone is equipped with a single-cell capture unit; Multiple single-cell capture units are distributed along the width of the cell division zone; a 5-micrometer gap is formed between adjacent single-cell capture units.

9. The microfluidic chip according to claim 8, characterized in that, Multiple single-cell capture units form a V-shaped structure.

10. An application of a microfluidic chip, characterized in that, The microfluidic chip described in any one of claims 1 to 9 includes: Patient-derived cell clusters are introduced into the first processing unit, the second processing unit, and the third processing unit through the first and second sample feeding wells for culture, and waste liquid during the culture process is discharged through the waste liquid hole. A first drug is introduced into the first sample well, and a second drug is introduced into the second sample well. After mixing in the liquid gradient mixing zone, the first drug flows into the first processing unit, a mixture of the first and second drugs flows into the second processing unit, and the second drug flows into the third processing unit, so as to simultaneously perform different drug sensitivity tests on the cell clusters in the first, second, and third processing units, and discharge the waste liquid during the drug sensitivity test through their respective waste liquid holes. After the drug sensitivity test is completed, the test products in the first processing unit, the second processing unit and the third processing unit can be discharged from their respective outlets for sample testing.

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