Microfluidic chip for constructing tumor microenvironment based on multiple markers and application thereof

Abstract

The application discloses a kind of microfluidic chip based on multiple markers constructs tumor microenvironment and application thereof.The microfluidic chip of the application includes upper structure and double-layer chip;Upper structure includes common reagent sample inlet, several independent reagent reservoirs, inlet and outlet and reagent outlet;Common reagent sample inlet is communicated with each independent reagent reservoir by independent pipeline;Double-layer chip includes air valve layer and detection layer;Air valve layer includes through hole corresponding to independent reagent reservoir, inlet and outlet and reagent outlet, and air valve in the form of air bag structure;Detection layer includes several independent detection microchannels, detection microchannel is communicated with corresponding independent reagent reservoir by independent reagent sample inlet, and all detection microchannels are finally gathered together and communicated with reagent outlet.The microfluidic chip of the application improves staining efficiency, can realize process standardization, accurate liquid volume and time, flow rate adjustment, can be used for staining result quality control, effectively saves antibody reagent and tissue sample.

Description

TECHNICAL FIELD

[0001] The present application relates to the field of microfluidic technology, in particular to a microfluidic chip for constructing tumor microenvironment based on multiplex labeling and application thereof. BACKGROUND

[0002] Malignant tumor is one of the most important health problems worldwide, which seriously affects people's quality of life. The occurrence and development of tumor is an evolutionary ecological process, and the continuous interaction between tumor microenvironment (TME) plays a decisive role in the progression, metastasis and response to treatment of tumor. Therefore, in order to deeply understand the mechanism of tumor disease and develop corresponding treatment plan, it is the key to recognize the occurrence and development of tumor and improve the efficacy of immunotherapy to systematically analyze the phenotype of different cells in tumor microenvironment. Conventional research methods such as flow cytometry and gene detection technology can qualitatively and quantitatively analyze the target cell population in the tumor region, but cannot obtain the morphological information of the tumor region and the relative spatial distribution characteristics between different markers. The conventional tissue section chemical staining technology and immunohistochemical technology have been developed to quantitative analysis, and these studies can be used to estimate the immune frequency and cell state in tumor microenvironment, but they lack information related to actual cell proportion, cell heterogeneity and deeper spatial distribution.

[0003] At present, multiplex immunofluorescence technology is often used to quantify cell subpopulation in tumor, its functional state and spatial arrangement in microenvironment, which can obtain complete morphological information of tissue and perform in situ analysis, and is the core technology for detecting immune status of tumor microenvironment. However, under the conventional multiplex staining process, manual operation for multiplex labeling often needs several days, the reaction efficiency in static solution is limited, and it is difficult to realize the uniformity of experimental conditions, which has large human error and low reproducibility, and it is also difficult to present in situ information of multiple cell populations on a single slice. The automated staining platform and equipment are expensive, and require a large amount of slices and reagents, as well as human and material resources for equipment maintenance, etc. The dilution of antibodies, the matching between antibodies and tyramide fluorescein, and the determination of staining order all need to be further optimized and adjusted after repeated experiments. In view of the limitations of current multiplex staining technology, it is essential to develop new rapid, high-throughput and resource-saving staining technology and scheme. SUMMARY

[0004] The purpose of the present application is to provide a new microfluidic chip for constructing tumor microenvironment based on multiplex labeling and application thereof.

[0005] The present application adopts the following technical solutions:

[0006] The first aspect of the present application discloses a microfluidic chip for constructing tumor microenvironment based on multiple markers, comprising an upper structure and a double-layer chip, which are sequentially stacked when in use; the upper structure comprises at least one common reagent inlet, a plurality of independent reagent reservoirs, an air inlet and outlet, and a reagent outlet; the common reagent inlet is in communication with each independent reagent reservoir through an independent pipeline; the double-layer chip comprises a valve layer and a detection layer which are sequentially stacked; the valve layer comprises through holes corresponding to the independent reagent reservoirs, the air inlet and outlet, and the reagent outlet, and a valve in the form of an air bag structure, the through hole corresponding to the air inlet and outlet is arranged on the valve for inflating and deflating the valve; the plurality of independent reagent reservoirs correspond to a plurality of through holes, i.e. independent reagent inlets; the detection layer comprises a plurality of independent detection microchannels corresponding to the independent reagent reservoirs, the detection microchannels are in communication with the corresponding independent reagent reservoirs through the independent reagent inlets, and then all the detection microchannels are finally gathered together and in communication with the reagent outlet; when in use, the upper structure is in contact with the valve layer, the detection layer is in contact with the slice to be detected, the independent reagent reservoirs are communicated with the corresponding detection microchannels through the through holes on the valve layer; when detecting independently, the valve is in a positive pressure mode by inflating through the air inlet and outlet, the top of the detection layer is a closed membrane, the valve covers the upper end of the membrane on the top of the detection layer, and pressure is applied downward to press the detection layer and the slice closely, at the same time, the slice to be detected closes the lower end of the detection microchannel, thereby forming an independent detection channel; when panoramic labeling is needed, the valve is in a negative pressure mode by deflating through the air inlet and outlet, the membrane on the top of the detection layer is inflated upward, the detection microchannels of the detection layer are communicated from the lower face in contact with the tissue, and a large chamber is formed on the surface of the tissue slice, thereby the same panoramic labeling is performed on the tissue in the coverage area of the large chamber.

[0007] Preferably, the upper structure is further provided with an observation window, and the valve is arranged at a position corresponding to the observation window.

[0008] Preferably, the valve layer and the detection layer are respectively prepared by using polydimethylsiloxane, and when assembled, the sample inlet of the detection microchannel is aligned with the independent reagent inlet of the valve layer, the reagent outlet is aligned, and the valve is directly opposite to the detection microchannel at the upper part thereof, thereby forming a complete double-layer chip.

[0009] Preferably, the upper structure is prepared by using a light-cured resin material.

[0010] The other aspect of the present application discloses a microfluidic detection system using the microfluidic chip of the present application.

[0011] Preferably, the microfluidic detection system of the present application comprises the microfluidic chip of the present application, and a common reagent module, a reagent control module, a positive pressure control module, an external air path switch, a negative pressure module, and a waste liquid collection module; the common reagent module comprises a plurality of independent common reagent pools, each common reagent pool is in communication with an inlet end of the reagent control module through an independent pipeline; the reagent control module is used for independently controlling and selecting a corresponding common reagent pool, and an outlet end of the reagent control module is in communication with a common reagent sample inlet of the microfluidic chip; the positive pressure control module is an external air source, is in communication with an air inlet and outlet of the microfluidic chip through the external air path switch, and is used for inflating the air valve; the negative pressure module is independently in communication with the air inlet and outlet of the microfluidic chip, and is used for exhausting the air valve; and the waste liquid collection module is in communication with a reagent outlet of the microfluidic chip, and is used for collecting waste liquid.

[0012] Preferably, the waste liquid collection module is in a closed state, and the negative pressure module is in communication with the waste liquid collection module through an independent pipeline, and is used for independently controlling the waste liquid collection module to keep a negative pressure state.

[0013] Preferably, a first flow meter is arranged on a communication pipeline of the reagent control module and the common reagent sample inlet of the microfluidic chip.

[0014] Preferably, a second flow meter is arranged on a communication pipeline of the waste liquid collection module and the reagent outlet of the microfluidic chip.

[0015] Preferably, the microfluidic detection system further comprises a computer control end, the computer control end is in signal connection with the reagent control module, the positive pressure control module, the external air path switch, and the negative pressure module, and is used for controlling specific parameters of pressure, a switching sequence of fluid, and a time of each process through a program.

[0016] Still another aspect of the present application discloses an application of the microfluidic chip of the present application or the microfluidic detection system of the present application in multiple immunofluorescence detection.

[0017] Still another aspect of the present application discloses a multiple immunofluorescence detection method, which comprises performing multiple immunofluorescence labeling on a same slice sample by using the microfluidic chip of the present application or the microfluidic detection system of the present application.

[0018] Preferably, the multiple immunofluorescence detection method of the present application comprises inflating the air valve to a positive pressure mode through the air inlet and outlet, marking in each detection microchannel by using different antibodies respectively; and then exhausting the air valve to a negative pressure mode through the air inlet and outlet, so as to make the detection microchannels communicate, and form a large chamber on the surface of the tissue slice, and the same panoramic marking is performed on the tissue in the large chamber coverage area.

[0019] Preferably, the multiplex immunofluorescence detection method of the application further comprises taking out the section sample after panoramic labeling, and post-processing or continuous staining thereof.

[0020] The application has the beneficial effects that:

[0021] The microfluidic chip and the multiplex immunofluorescence detection based on the microfluidic chip of the application improve the staining efficiency, can realize the standardization of the process, the adjustment of the precise liquid volume and the time and flow rate, can be used for the quality control of the staining result, effectively saves the antibody reagent and the tissue sample, and provides a more rapid, simplified and portable multiplex labeling scheme and way for the tumor microenvironment research. BRIEF DESCRIPTION OF DRAWINGS

[0022] Figure 1 is a structural schematic diagram of the microfluidic chip and the section assembled in the embodiment of the application;

[0023] Figure 2 is a structural schematic diagram of the upper layer structure of the microfluidic chip in the embodiment of the application;

[0024] Figure 3 is a planar structural schematic diagram of the double-layer PDMS chip in the embodiment of the application;

[0025] Figure 4 is a working state schematic diagram of the double-layer PDMS chip in the embodiment of the application;

[0026] Figure 5 is a structural schematic diagram of the microfluidic detection system in the embodiment of the application;

[0027] Figure 6 is a partial structural schematic diagram of the microfluidic chip assembled in the embodiment of the application;

[0028] Figure 7 is a process schematic diagram of the conventional immunohistochemistry in the embodiment of the application;

[0029] Figure 8 is a uniformity characterization result diagram of the strip type immunohistochemistry labeling in each detection microchannel in the embodiment of the application;

[0030] Figure 9 is a control result diagram of the panoramic type immunohistochemistry labeling (b) and the conventional immunohistochemistry result (a) in the embodiment of the application;

[0031] Figure 10 is a result diagram of the parallel labeling of multiple different markers of the tonsil tissue section in the embodiment of the application;

[0032] Figure 11 is a process schematic diagram of the TSA multiplex immunofluorescence staining based on the microfluidic in the embodiment of the application;

[0033] Figure 12 Figure 1 is a result chart of TSA multiplex immunofluorescence staining of tonsil slices using the markers screened in the embodiments of the present application. DETAILED DESCRIPTION

[0034] In view of the limitations of current multiplex staining techniques, the present research aims to develop a new type of fast, high-throughput, and resource-saving staining technique and scheme.

[0035] The present research takes the analysis of tumor microenvironment as the goal, and aims to solve the technical problem of difficult realization of multi-marker analysis on a single tissue slice. The present research combines microfluidic technology to perform multiple parallel rapid immunolabeling on a single pathological tissue slice using high-throughput microchannel of the chip, and screens markers according to the labeling results. Then, the microchannel is sucked up by the air valve of the chip to form a complete large chamber, and a full-slice staining mode is performed. In combination with the multiplex immunofluorescence technology, multiple rounds of fast staining based on microfluidics are performed to show the relative spatial distribution information between different tumor microenvironment markers, which is suitable for the data analysis needs of visualizing multiple interrelated markers in situ on a tissue slice. The present research first uses the flow path design of the microchannel in the microfluidic chip to perform rapid screening of multiple biological markers on a specific tissue slice. Then, the air valve device is used to separate the microchannels and the slice in the microfluidic chip, and reagents are continuously introduced to perform rapid labeling of multiple immunofluorescence on the same slice. Finally, the multiplex immunofluorescence technology based on the TSA tyramide signal amplification principle is used to further amplify the signal and improve the staining efficiency.

[0036] Specifically, the present application first develops a microfluidic chip, as shown in Figure 1 , which comprises an upper structure 1 and a double-layer chip 2, which are sequentially stacked when in use. The upper structure 1, as shown in Figure 2 , comprises at least one common reagent inlet port 11, a plurality of independent reagent reservoirs 12, an air inlet and outlet port 13, and a reagent outlet 14. The common reagent inlet port 11 is in communication with each independent reagent reservoir 12 through an independent pipeline. The double-layer chip 2, as shown in Figure 1 and Figure 3 , comprises a valve layer 21 and a detection layer 22 which are sequentially stacked. The valve layer 21 comprises through holes corresponding to the independent reagent reservoirs 12, the air inlet and outlet port 13, and the reagent outlet 14, and a valve 211 in the form of an air bag structure. The through hole corresponding to the air inlet and outlet port 13 is arranged on the valve 211 for inflating and deflating the valve 211. The plurality of independent reagent reservoirs 12 correspond to a plurality of through holes, i.e., independent reagent inlet ports 212. The detection layer 22 comprises a plurality of independent detection microchannels 221 corresponding to the independent reagent reservoirs 12. The detection microchannels 221 are in communication with the independent reagent reservoirs 12 through the independent reagent inlet ports 212. Then, all the detection microchannels 221 are finally gathered together and in communication with the reagent outlet 14.

[0037] In use, the upper structure 1 contacts the valve layer 21, and the detection layer 22 contacts the test slice 3. The independent reagent reservoir 12 is connected to the corresponding detection microchannel 221 through the through-holes in the valve layer 21. For independent detection, such as... Figure 4 As shown, air is injected through the air inlet / outlet 13, putting the air valve 211 in positive pressure mode. The air valve 211 covers the upper end of the membrane at the top of the detection layer 22, squeezing the detection layer 22 downwards to fit tightly against the test slice 3, thus sealing the upper and lower ends of the detection microchannels 221 and forming independent detection channels. When panoramic marking is required, air is extracted through the air inlet / outlet 13, putting the air valve 211 in negative pressure mode. The membrane at the top of the detection layer 22 bulges upwards, separating the detection layer 22 from the tissue. Each detection microchannel 221 connects from the surface in contact with the tissue below, forming a large chamber on the surface of the tissue slice, thereby performing the same panoramic marking on the tissue within the area covered by the large chamber.

[0038] Based on the microfluidic chip of this application, this application further developed a microfluidic detection system, such as... Figure 5 As shown, the microfluidic chip 01 of this application, along with a common reagent module 02, a reagent control module 03, a positive pressure control module 04, an external gas path switch 05, a negative pressure module 06, and a waste liquid collection module 07, are included. The common reagent module 02 includes several independent common reagent pools, each of which is connected to the inlet of the reagent control module 03 via an independent pipe. The reagent control module 03 is used to independently control and select the corresponding common reagent pool, and its outlet is connected to the common reagent inlet 11 of the microfluidic chip 01. The positive pressure control module 04 is an external gas source, connected to the inlet and outlet 13 of the microfluidic chip 01 via the external gas path switch 05, and is used to inflate the gas valve 211. The negative pressure module 06 is independently connected to the inlet and outlet 13 of the microfluidic chip 01 and is used to evacuate the gas valve 211. The waste liquid collection module 07 is connected to the reagent outlet 14 of the microfluidic chip 01 and is used to collect waste liquid.

[0039] Further improvement plans, such as Figure 5 As shown, the waste liquid collection module 07 is in a closed state. The negative pressure module 06 is connected to the waste liquid collection module 07 through an independent pipe and is used to independently control the waste liquid collection module 07 to maintain it in a negative pressure state. A first flow meter 081 is installed on the connecting pipe between the reagent control module 03 and the common reagent inlet 11 of the microfluidic chip 01; a second flow meter 082 is installed on the connecting pipe between the waste liquid collection module 07 and the reagent outlet 14 of the microfluidic chip 01. The microfluidic detection system also includes a computer control terminal 09, which is connected to the reagent control module, the positive pressure control module, the external gas circuit switch, and the negative pressure module. The computer control terminal 09 controls the specific parameters of the pressure application, the fluid switching sequence, and the time of each process through a program.

[0040] The following technical problems are solved by the present application:

[0041] ①Based on microfluidic, high-throughput and rapid immunolabeling staining technology is realized on the same slice. In this study, the staining throughput of a single slice is improved by designing multiple microchannels on the chip. The use of laminar flow of fluid in the microfluidic system is proposed to promote the diffusion of antibody molecules, thereby breaking through the limitations of antigen-antibody reaction kinetics under static reaction system, and realizing automatic and precise flow regulation through external control system.

[0042] ②For analyzing tumor microenvironment, continuous marker screening and multiple immunofluorescence labeling are realized on a single slice. In this subject, multiple consistent parallel microchannels are adopted to ensure consistent fluid flow rate in each channel, and gas valve layer is used to flexibly regulate the two staining states between microchannel layer and slice, i.e. regional multi-band parallel staining and panoramic staining. Continuous steps from biomarker screening to multiple immunofluorescence verification can be carried out. The automatic device can strictly control the staining time and variables during the experiment when multiple markers are screened in parallel, and can also ensure consistent staining conditions in each round of staining in panoramic multiple staining experiment, so that the results have higher repeatability and comparability.

[0043] Compared with the prior art, the microfluidic chip and detection system of the present application have the following advantages:

[0044] ①The antibody labeling time in immunohistochemistry or immunofluorescence of tissue samples is shortened from more than 1 hour or even overnight to within 10 minutes, and the entire immunolabeling experiment process is shortened from 1-2 days required in conventional laboratory to about 1 hour.

[0045] ②Through switching between multi-channel single staining and full-slice multiple staining, the utilization rate of single tissue slice and the marker throughput are significantly improved, and even multiple staining can be continued in the strip-shaped single microchannel to further improve the throughput.

[0046] ③The manual operation of macroscopic laboratory is integrated into the microfluidic system, and automatic labeling is realized after sample loading.

[0047] In summary, the present application greatly improves the staining efficiency and realizes the standardization of the process. Precise liquid volume and time and flow rate adjustment can be used for quality control of the staining results, effectively saving antibody reagents and tissue samples, and providing a faster, simpler and lighter multiple labeling platform for pathologists to study tumor microenvironment.

[0048] The present application will be further described in detail through specific embodiments. The following embodiments are only further descriptions of the present application and should not be understood as limitations of the present application.

[0049] Unless otherwise specified, all materials and reagents used in the following examples are commercially available, and all experimental methods used in the following examples are conventional methods.

[0050] Example

[0051] I. Design and fabrication of multifunctional microfluidic chips

[0052] 1. Design and fabrication of microfluidic chips

[0053] Microfluidic chips, such as Figure 1 As shown, it includes an upper structure 1 and a dual-layer chip 2, which are stacked sequentially during use; the upper structure 1, as... Figure 2 As shown, it includes at least one common reagent inlet 11, several independent reagent reservoirs 12, air inlets and outlets 13, reagent outlets 14, and observation windows 15; the common reagent inlet 11 is connected to each independent reagent reservoir 12 through independent pipes; the double-layer chip 2, as shown... Figure 1 and Figure 3 As shown, the system includes a valve layer 21 and a detection layer 22 stacked sequentially. The valve layer 21 includes through holes corresponding to independent reagent reservoirs 12, inlet / outlet ports 13, and reagent outlets 14, as well as a gas valve 211 with an airbag structure. The through holes corresponding to the inlet / outlet ports 13 are provided on the gas valve 211 for charging and decharging the gas valve 211. Several independent reagent reservoirs 12 correspond to several through holes, i.e., independent reagent inlets 212. The detection layer 22 includes several independent detection microchannels 221 corresponding to the independent reagent reservoirs 12. The detection microchannels 221 are connected to their corresponding independent reagent reservoirs 12 through the independent reagent inlets 212. Then, all the detection microchannels 221 are finally gathered together and connected to the reagent outlet 14. The through holes on the valve layer 21 corresponding to the inlet / outlet ports 13 are the gas valve inlet / outlet ports 213. The openings of the detection microchannels 221 of the detection layer 22 corresponding to the independent reagent inlet 212 are the sample inlet 222 of the microchannels. The openings of all the detection microchannels 221 that eventually converge and connect to the reagent outlet 14 are the sample outlet 223 of the microchannels.

[0054] Fabrication of upper-layer structures for microfluidic chips based on 3D printing:

[0055] In this example, the upper structure is a 3D printed part with internal channels, and the lower double-layer chip is a chip with two layers of PDMS (polydimethylsiloxane) bonded together. Fluid is injected from the common fluid inlet (i.e., common reagent inlet 11) of the 3D printed part or from the open reservoir, flows from the internal channels to the multiple inlets of the lower PDMS chip, enters the microchannel, and reacts with the surface of the tissue section at the bottom.

[0056] 3D printed parts such as Figure 2The 3D printed piece has 16 open reservoirs on one side (the number of reservoirs is consistent with the number of detection channels in the PDMS chip), namely, independent reagent reservoirs 12, for adding different reagents to each reaction channel independently, for example, adding detection of different protein primary reagents to each reservoir with a pipette or other tool. Each reservoir has an internal pipe 16 below it for transporting the liquid in the reservoir to the 16 reservoir reagent outlets leading to the next layer of the PDMS chip. After the common fluid enters the inlet, it first enters a total pipe, and then is divided into 16 by the total pipe 1, connected to the internal pipe outlet below each reservoir, so that the common reagent can be distributed into different microchannels of the next layer of the PDMS chip. The internal pipes of the 3D printed piece are distributed symmetrically left and right, and the pipe cross-sectional diameter is 300 μm, which is small enough to allow the fluid in the pipe to be affected by a larger capillary force, which can to some extent resist the hydrostatic pressure when each open sample port is connected to the atmospheric pressure. When different reagents are added to each reservoir, the fluid in the pipe is affected by the capillary force, and the flow of the fluid in the 1 / 16 common reagent pipe to the adjacent reservoir and pipe is very small, and only a slight diffusion occurs at the connection of different pipes. At this time, a certain positive pressure is applied to the fluid at the common reagent sample port, so that the fluid in the 1 / 16 pipe dilutes the independent reagent at a very slow speed, which can effectively prevent the reagent crosstalk caused by the connection between different sample ports. In order to facilitate installation, a chip and slice placement groove is designed at the bottom of the upper structure.

[0057] Design and manufacture of the flow path of the PDMS chip:

[0058] According to the morphology of the pathological tissue section on the glass slide, the design can adjust the flow path according to the shape of the actual tissue and the tumor region rich in detection target cells, so as to ensure that when multiple independent antibody detections are performed on a single sample, enough tissue area can be covered, the one-time detection probability of multiple markers is improved, and the influence of tissue heterogeneity is reduced.

[0059] The PDMS chip is composed of two layers of PDMS bonded together, the first layer is the air valve layer, and the second layer is the detection layer adjacent to the tissue section. The manufacturing process is to first manufacture the corresponding single-layer SU-8 photoresist mold of the two layers of chips, then cast and cure PDMS in the mold, and then obtain it after degassing. The surfaces of the two PDMSs to be bonded are treated by plasma, and then the two layers of PDMS chips are bonded, so that the 16 reagent sample inlets of the upper and lower layers are aligned, the reagent sample outlets are aligned, and the air valves are opposite the detection channels located on the upper part, that is, a complete PDMS chip is formed. The planar structure of the two layers of chips is shown in Figure 3 .

[0060] The air valve layer has an air valve covering all the microchannels, 16 reagent injection ports corresponding to the 16 reagent injection ports of the lower detection layer, and 16 reagent outlets of the upper 3D printed part, and the reagent is injected from top to bottom into the 16 microchannels of the detection layer after the reversible sealing of the clamp, as shown in Figure 1 The air inlet of the air valve is connected with the positive pressure control module and the negative pressure control module. The surface of the detection layer has 16 parallel microchannels, each with a width of 300 pm and a height of 80 pm, and the length and spacing of each microchannel are consistent, and the spacing between the channels is 200 pm. The surface is used to adhere to the tissue section.

[0061] The working state of the double-layer PDMS chip is shown in Figure 4 Because PDMS has a certain elasticity, and the detection layer is relatively thin (about 700 pm), and the upper part of the air valve layer is relatively thick (PDMS thickness of 2 mm, air valve chamber height of 300 pm), the PDMS detection layer in the lower layer can be sucked up by vacuumizing the air valve. When the air valve layer is in a positive pressure state, the lower detection layer is pressed to adhere to the section, and at this time, the biomarker rapid screening can be carried out; when the negative pressure is applied, the lower detection layer is separated from the chip, forming a complete large chamber with a top arch, and at this time, multiple immunofluorescence staining of the whole section can be carried out for tumor microenvironment analysis. The second layer is a multi-channel biomarker detection layer, which is in close contact with the tissue section under positive pressure, forming several microchannels without mutual leakage and color mixing, and the third layer is a tissue section, which only needs to use one section throughout the process.

[0062] 2. System construction of microfluidic device

[0063] Based on the conventional labeling steps of multiple immunolabeling experiments, a pressure-driven microfluidic system is designed and constructed, and the structure of the entire microfluidic system is shown in Figure 5 The entire system provides positive and negative pressure to the control module through a desktop positive pressure source and a vacuum source. The pressure has two functions: pushing and pulling the fluid, and precise pressure control by the control module, and the purpose of controlling the flow rate of the fluid is achieved by adjusting the pressure; controlling the air valve to make the air valve in a positive pressure or negative pressure state, thereby changing the working state of the chip.

[0064] The whole microfluidic system comprises: a microfluidic chip 01, and a common reagent module 02, a reagent control module 03, a positive pressure control module 04, an external air path switch 05, a negative pressure module 06, and a waste liquid collection module 07; the common reagent module 02 comprises a plurality of independent common reagent pools, and each common reagent pool is in communication with an inlet end of the reagent control module 03 through an independent pipeline; the reagent control module 03 is used for independently controlling and selecting a corresponding common reagent pool, and an outlet end of the reagent control module 03 is in communication with a common reagent sample inlet 11 of the microfluidic chip 01; the positive pressure control module 04 is an external air source, is in communication with an air inlet and outlet 13 of the microfluidic chip 01 through the external air path switch 05, and is used for inflating the air valve 211; the negative pressure module 06 is independently in communication with the air inlet and outlet 13 of the microfluidic chip 01, and is used for exhausting air from the air valve 211; the waste liquid collection module 07 is in communication with a reagent outlet 14 of the microfluidic chip 01, and is used for collecting waste liquid. The waste liquid collection module 07 is in a closed state, the negative pressure module 06 is in communication with the waste liquid collection module 07 through an independent pipeline, and is used for independently controlling the waste liquid collection module 07 to keep a negative pressure state. A first flow meter 081 is arranged on a communication pipeline of the reagent control module 03 and the common reagent sample inlet 11 of the microfluidic chip 01; and a second flow meter 082 is arranged on a communication pipeline of the waste liquid collection module 07 and the reagent outlet 14 of the microfluidic chip 01. The microfluidic detection system further comprises a computer control end 09, the computer control end 09 is signal connected with the reagent control module, the positive pressure control module, the external air path switch, and the negative pressure module, and specific parameters of given pressure, switching sequences of fluid, and time of each process are controlled through a program.

[0065] The positive pressure control module is two positive pressure control modules in series, the negative pressure module is two negative pressure modules in series, the negative pressure module is signal connected with the reagent control module, one positive pressure control module, one negative pressure control module, and one reagent control module are used to realize sequential sampling and fluid exchange of multiple fluids; one positive pressure control module and one negative pressure control module are used to realize positive and negative pressure mode switching of air in the air valve; the external air path switch is used to shut off the communication of gas between the positive and negative pressure control modules. The control of all modules is connected to the computer control end, and specific parameters of given pressure, switching sequences of fluid, and time of each process are controlled through a program.

[0066] The common reagent module is a common reagent to be used by a sample, which can include PBS buffer, 3% H2O2, 3% BSA, HRP labeled goat anti-rabbit secondary antibody, HRP labeled goat anti-mouse secondary antibody, etc., the amount of reagent is adjusted according to specific experimental needs, is loaded in a sealed liquid storage tube, is connected to a multi-way reversing valve (i.e. the reagent control module) through a liquid path, is connected to a positive pressure module through an air path, is driven by positive pressure to the reagent control module, and then enters the microfluidic chip.

[0067] The assembled microfluidic chip part, specific details are as followsFigure 6 The only outlet of the valve is connected to the 3D printed piece on the chip, after the common reagent enters from the common fluid inlet of the 3D printed piece, it is simultaneously distributed into different microchannels inside the 3D printed piece. Flow meters, one at each inlet and outlet of the chip, are used to monitor the flow rate in real time. A sealed waste pipe, with its gas path connected to a negative pressure module and its liquid path connected to the reagent outlet of the chip, provides negative pressure to draw the reacted fluid from the chip to the waste pipe. The chip and tissue section are reversibly sealed by the clamp 4 under pressure and fixed on the microscope stage 5, and the 3D printed piece and the clamp have an observation window at the tissue position, so that the microfluidic system has the ability to move to different imaging systems for real-time imaging observation of the staining state.

[0068] 3. The working process of tissue section immunostaining based on the microfluidic device

[0069] Tissue section pretreatment and loading. Taking paraffin sections as an example, the sections are treated by dewaxing and hydration, and antigen repair to make the antigen markable, and then loaded in the order of 3D printed upper structure-PDMS chip-tissue section, and fixed on the customized stage with the clamp. After starting the pump valve, the specific instructions can be set in the control software according to the experimental needs, such as the switching sequence of the valve, the reaction time of different reagents, and the pressure parameters of positive and negative pressure. Different reagents will be injected into the device in sequence to realize automatic experiment.

[0070] Tissue section and microchannel wetting. The purpose is to create a wet environment for the tissue section to be immersed in the fluid throughout the process, and to ensure that all liquid paths in the front end of the chip and the chip part do not enter the air. The operation process is as follows: switch the valve to connect the PBS buffer solution storage tank, and give a certain positive pressure in the positive pressure control module to control the fluid. The PBS buffer solution will be injected from the valve outlet into the common reagent inlet of the 3D printed piece, fill the internal pipeline for 1 minute and 16 seconds, and enter each reservoir pipeline from the reservoir reagent outlet. Finally, it enters the 16 open reservoirs. Without any negative pressure acting on the outlet end of the chip to draw the fluid, the fluid injected into the 3D printed piece by positive pressure will preferentially enter the open reservoirs connected to the atmospheric pressure, rather than directly entering the flow path of the PDMS chip. After the open reservoirs are filled, negative pressure is drawn from the outlet end of the chip part to make PBS enter the PDMS chip and enter all the detection microchannels, until the 3D printed piece pipeline and all the microchannels of the PDMS are filled with fluid without air bubbles.

[0071] Endogenous peroxidase and non-specific binding sites of the closed tissue are blocked. A 3% H2O2 solution is used to react with the tissue slice surface in a slow flow state for 10 min, then switch to PBS buffer to wash away the reagent in the previous step, and then switch to a 3% BSA solution to react for 10 min in a slow flow state. Both reagents are common reagents. The operation process is as follows: the negative pressure module controls the air valve to a certain negative pressure, so that the top of the detection layer is arched, and the fluid is filled in each part of the chip at this time. A large cavity without air bubbles is formed on the surface of the tissue slice, and the blocking reaction of the tissue surface in all areas of the cavity can be carried out. The negative pressure control module responsible for pumping away the fluid returns to 0, the change-over valve is switched to the reagent tank containing the 3% H2O2 solution, a certain positive pressure is given, the 3% H2O2 solution flows from the outlet of the change-over valve into the common reagent sample inlet, and is injected into the chip independent open reagent pool. A sufficient amount is filled in the reagent pool to maintain the subsequent flow reaction for about 10 min, then the change-over valve is closed to stop the positive pressure control of the fluid to prevent the fluid at the positive pressure from being pulled to cause excessive or insufficient reagent. A small negative pressure is given to the outlet end of the chip to make the reagent flow through the surface of the tissue slice at a slow speed. The reaction time is much shorter than the reaction time of the conventional laboratory operation. The conventional laboratory static state needs 0.5 h to 1 h of steps, and the flow state reaction only needs 5 to 10 min. The same applies to all subsequent reaction steps. After the end, switch to PBS for washing, and load the 3% BSA solution into each open reagent pool in the same way, and start the negative pressure reaction.

[0072] One and two antibody labeling. The air valve in the chip can achieve two modes of reaction. When it needs to be labeled independently in 16 microchannels, the air valve is in positive pressure mode, the inside of the air valve is full of gas, and the top of the detection layer is pressed down, while the fluid in the PDMS chip is controlled by negative pressure. At this time, 16 independent microchannels are formed. Since each channel may need to be used for labeling different biomarkers, the one antibody is a specific antibody against a certain protein, so different one antibody solutions need to be loaded into 16 independent open reagent reservoirs. At this time, the common reagent sample port does not work, and the 1 / 16 pipe of the 3D printed part is filled with the remaining fluid when injecting PBS buffer for flushing, which can give a small amount of positive pressure to counteract the hydrostatic pressure of each reservoir, ensuring that the reagents between each reservoir do not diffuse excessively. After the one antibody reaction is completed, switch to PBS flushing. The two antibodies are used to specifically bind to the one antibodies, and HRP-labeled goat anti-mouse IgG or HRP-labeled goat anti-rabbit IgG is used for unified labeling, which is loaded in a sealed reservoir connected to a reversing valve and injected through a common fluid distribution pipe. The working mode of the common reagent is the same as when it is closed, as described above. When panoramic labeling is needed, the air valve is in negative pressure mode, the inside of the air valve is in a vacuum state, and the top of the detection layer is sucked up to form a large chamber. At this time, the same biomarker is labeled for all tissue regions in the large chamber, so the one antibody reagent at this time belongs to the common reagent, which can be loaded in a sealed reservoir connected to a reversing valve and injected into each reservoir for temporary storage through a common fluid distribution pipe. A small negative pressure is applied to the outlet end of the chip during the reaction to incubate the antibody at a slow flow rate. After the two antibodies are completed, switch to PBS flushing, which completes an immunolabeling process in the microfluidic device. Next, DAB or fluorescent dye can be added for color development with HRP on the two antibodies, and a dye for staining the cell nucleus can be added.

[0073] Post-processing or continuous staining of tissue sections. If all the required markers have been labeled, the positive and negative pressure pumps are returned to 0, the positive and negative pressure sources are turned off, the clamp and chip are removed, the tissue section is removed, and post-processing such as dehydration and transparency is performed. The section is observed and stored after being sealed. If more than one round of TSA staining is required, the panoramic labeling process is repeated after one round of labeling, the air valve is kept in negative pressure mode, and multiple rounds of immunolabeling are continued.

[0074] II. Rapid micro-biomarker screening and multiplex staining based on microfluidics

[0075] 1. Characterization and optimization of immunolabeling experiments

[0076] Laboratory immunolabeling of tissue sections is very common, all have the same principle, different laboratories and different antibody labeling workflow may exist some differences, but the main steps are very fixed. Mainly can be divided into two kinds of technology of immunohistochemistry (bright field observation), immunofluorescence labeling (fluorescence observation). The process generally includes tissue section preparation (can make paraffin section or frozen section), section through fixation, dewaxing, hydration, antigen repair and other pretreatment, static incubation of the surface of tissue section of primary antibody working solution (specific binding to a certain marker on the tissue), static incubation of HRP labeled secondary antibody specific binding to primary antibody, bright field observation generally uses DAB color development, fluorescence observation generally uses the secondary antibody labeled with fluorescein, TSA labeling is used tyramide fluorescein to covalently bind to the tissue after the incubation of HRP labeled secondary antibody. The conventional immunohistochemical process is shown in Figure 7

[0077] A typical laboratory routine immunohistochemical process is described as follows:

[0078] Baking: put the section on the section holder, bake in the oven at 60℃ for 1h, so that the wax film on the surface melts and flows down.

[0079] Paraffin section dewaxing to water: put the section into the environmental protection type dewaxing solution I 10min-environmental protection type dewaxing solution II 10min-environmental protection type dewaxing solution III 10min-anhydrous ethanol I 5min-anhydrous ethanol II 5min-anhydrous ethanol III 5min-distilled water.

[0080] Antigen repair: medium fire for 8min, stop fire for 8min, low fire for 7min. During this process, the buffer should be prevented from excessive evaporation, and the section should not be dried. After natural cooling, put the slide in PBS (PH 7.4) and shake on the decoloring shaker for 3 times, 5min each time. Among them, the repair solution and repair conditions are determined according to the tissue.

[0081] Blocking endogenous peroxidase: put the section into 3% hydrogen peroxide solution, incubate at room temperature for 25min, put the slide in PBS (PH 7.4) and shake on the decoloring shaker for 3 times, 5min each time.

[0082] Serum blocking: add 3% BSA in the histological circle and evenly cover the tissue, block at room temperature for 30min. The primary antibody in this example is goat derived, which is blocked with rabbit serum, and other sources are blocked with BSA.

[0083] Add primary antibody: shake off the blocking solution, add the primary antibody prepared with PBS in a certain proportion on the section, incubate at room temperature for 1-1.5h, or put the section flat in the wet box and incubate overnight at 4℃.

[0084] ​Add secondary antibody: the slide was washed in PBS (PH 7.4) on a decoloring shaker for 3 times, 5 min each time. After the slice was slightly dried, the corresponding secondary antibody (HRP labeled) of the species of the primary antibody was added to cover the tissue, and incubated at room temperature for 50 min.

[0085] DAB color development: the slide was washed in PBS (PH 7.4) on a decoloring shaker for 3 times, 5 min each time. After the slice was slightly dried, freshly prepared DAB color developing solution was added in the circle, and the color developing time was controlled under a microscope. The positive was brownish yellow, and the color development was terminated by washing the slice with tap water.

[0086] Counterstain the cell nucleus: hematoxylin counterstaining was performed for about 3 min, then washed with tap water, differentiated with hematoxylin differentiation solution for several seconds, washed with tap water, returned to blue with hematoxylin return blue solution, and washed with running water.

[0087] Dehydration and mounting: the slice was sequentially placed in 75% alcohol for 5 min, 85% alcohol for 5 min, absolute ethanol I for 5 min, absolute ethanol II for 5 min, n-butanol for 5 min, xylene I for 5 min, and then taken out from xylene, slightly dried, and mounted with mounting medium.

[0088] Microscopy: placed under a white light microscope for result interpretation.

[0089] The conventional experimental procedure usually takes 9 hours to two days to be manually operated by the experimenter. If multiple markers need to be labeled by immunohistochemistry, only one marker can be labeled on each slice (the color developing agent used in bright field is DAB, and two or more markers cannot be distinguished), so if multiple markers need to be labeled, multiple slices need to be labeled. Immunofluorescence can label different emission spectrum fluorescent dyes on the secondary antibody, which can overcome this limitation to some extent. However, for immunofluorescence experiments, if multiple markers are labeled by the primary antibody at the same time, different species of primary antibodies (usually derived from mice or rabbits) are needed, and antibodies from other sources are less common and have limited species, so there are species limitations. Another way is multi-round labeling, that is, after one round of labeling, the influence of the previous round is removed by bleaching the fluorescence or washing out the antibody complex, and the next round of new marker labeling is continued. This method needs to be imaged and registered once for each round of shooting area, which is very cumbersome, and the chemical reagents for elution and multiple rounds of repetition can also cause damage to the tissue and antigen sites. The TSA multi-plex immunofluorescence technology can overcome the limitations of antibody species, and after labeling the secondary antibody, tyramide fluorescein is added, which will be covalently bound to the amino acid residues on the surface of the tissue slice under the catalysis of HRP. Microwave treatment can remove the antibody complex on the surface of the tissue slice, leaving the labeled fluorescent signal. The next step is to label other markers with other waveband fluorescent signals by repeating the labeling steps, and the cycle is repeated multiple times, and finally only one-time imaging is needed. The main disadvantage of this technology is the long time of multiple rounds of labeling. The multi-channel mode of the chip we designed can improve the throughput of one-time labeling to 16 markers, and in the panoramic staining mode, this technology can not only realize the switching of multi-channel to full-slice staining, but also realize the continuous iterative staining in the device, saving staining time and improving work efficiency.

[0090] To prove the stability and staining ability of the immunostaining results in the chip experiment under the microfluidic system, the conventional immunolabeling experiment and the microfluidic labeling experiment manually operated by the experimenter were carried out respectively, the staining results were compared, and the stability, uniformity and repeatability of the staining between each channel were verified, such as Figure 7 . And verify the uniformity of the chamber when staining the whole slice, such as Figure 8 . Figure 8 The results show that the strip immunohistochemical labeling has good uniformity in each channel.

[0091] A set of tissue sections with known markers were used as an example, human tonsil sections (from Haikou People's Hospital, which has passed the hospital ethics review) were used as an example, because they have multiple germinal centers and are evenly distributed throughout the section, rich in various immune cell groups, so they are very suitable as model samples for exploring antibodies. In addition, this study uses a multiple immunofluorescence technology based on the TSA tyramide signal amplification principle that can cycle multiple rounds of staining and simultaneous multi-color imaging, requires TSA dye labeling experiments for each marker in each emission spectrum, and performs TSA cycle labeling under conventional labeling conditions. Optimize the labeling effect, explore the influence of different antigens on stability under conditions such as cycle elution.

[0092] Take CD20 labeling on tonsil sections as an example, the detailed steps of microfluidic labeling experiment are as follows:

[0093] Tissue section pretreatment

[0094] Baking the section: Place the tonsil section on the section holder and bake it in the oven at 60°C for 1h to melt the wax film on the surface.

[0095] Dewaxing the paraffin section to water: Place the section in the following order: environmental-friendly dewaxing solution I for 10min - environmental-friendly dewaxing solution II for 10min - environmental-friendly dewaxing solution III for 10min - anhydrous ethanol I for 5min - anhydrous ethanol II for 5min - anhydrous ethanol III for 5min - distilled water.

[0096] Antigen repair: Medium fire for 8min, stop fire for 8min, low fire for 7min. During this process, the buffer should not be over evaporated, and the section should not be dried. After natural cooling, place the slide in PBS (PH 7.4) and shake it on the decolorizing shaker for 3 times, 5min each time. The repair solution and repair conditions are determined according to the tissue, and the tonsil section uses trs-EDTA pH=9 antigen repair solution.

[0097] Reversible assembly of microfluidic chip

[0098] Place the bonded PDMS chip and the pretreated tonsil section into the grooves of the 3D printed upper structure in order, align the hole positions, and attach the microchannels to the surface of the hydrated tissue section. Use clamps and screws to fix the chip on the stage. The pressure should not be too high, otherwise the chip may leave indentations on the tissue section. This assembly method is reversible, and after the microfluidic experiment is completed, the section can be removed for subsequent processing and conventional mounting and storage. The whole process is carried out at room temperature.

[0099] Microfluidic labeling process

[0100] Reagent loading: Add 3% hydrogen peroxide solution, 3% BSA solution, PBS buffer, and HRP-labeled goat anti-rabbit secondary antibody working solution into the closed reservoir connected to the reversing valve.

[0101] Wetting of the channels and tissue slice surface: Switching the valve to PBS buffer, controlling the positive pressure of the liquid circuit to 800 mbar and the negative pressure to 200 mbar, so that the buffer can quickly enter and fill the open reservoir, and ensure that all the gas in the 3D printed pipe and PDMS microchannel is removed by negative pressure. After the liquid fills each channel, the negative pressure of the gas circuit is controlled to 300 mbar to make the gas valve vacuum, and a large bubble-free chamber is formed on the surface of the tissue slice. The positive pressure returns to 0, and the valve is closed. The negative pressure continues to remove the PBS in the open reservoir, leaving a little at the bottom. If gas enters the internal pipe and causes blockage, it will lead to inconsistent flow rate of each channel. The whole process keeps each microchannel and tissue slice surface in a liquid environment.

[0102] Blocking endogenous peroxidase: Switching the valve to 3% hydrogen peroxide solution, controlling the positive pressure of the liquid circuit to 1000 mbar, and loading about 32 μL in the open reservoir within a few tens of seconds. Return to 0, close the valve, and the negative pressure is 25 mbar. Flow at a slow speed for 8 min. After the end, switch the PBS to the positive pressure of 1000 mbar, load an appropriate amount in the open reservoir, and then the negative pressure is 200 mbar. Flush at a large flow rate for 1 min.

[0103] Serum blocking: Switching the valve to 3% BSA solution, controlling the positive pressure of the liquid circuit to 1000 mbar, and loading about 40 μL in the open reservoir within a few tens of seconds. Return to 0, close the valve, and the negative pressure is 25 mbar. Flow at a slow speed for 10 min. After the end, switch the PBS to the positive pressure of 1000 mbar, load an appropriate amount in the open reservoir, and then the negative pressure is 200 mbar. Flush at a large flow rate for 1 min.

[0104] Addition of primary antibody: Switching the valve to PBS, the positive pressure of the liquid channel is 5 mbar, and the flow meter at the common reagent inlet shows about 1 μL / min (no backflow and very slow injection). Mode one: multi-channel staining mode. The positive pressure of the gas channel is 50 mbar, and the air valve is filled with air, so that the detection layer is in contact with the surface of the tissue section, and the 16 microchannels do not leak. Add 40 μL of rabbit anti-CD20 primary antibody working solution to each open reservoir with a pipette, and each reservoir can be replaced with an antibody of another marker. Here, CD20 antibody working solution is added to verify uniformity, and the negative pressure is 25 mbar, with a slow flow rate for 8 min. After the end, switch to PBS with a positive pressure of 1000 mbar to fill the open reservoir, and control the negative pressure of the gas channel to 300 mbar to make the air valve vacuum, the negative pressure is 200 mbar, and the large flow rate is flushed for 1 min. Mode two: whole section staining mode. The negative pressure of the gas channel is 300 mbar to make the air valve vacuum, and the detection layer rises to form a large chamber. Add the same rabbit anti-CD20 primary antibody working solution to each open reservoir with a pipette, and flow at a slow flow rate for 10 min. After the end, switch to PBS with a positive pressure of 1000 mbar to fill the open reservoir, and control the negative pressure to 200 mbar to flush at a large flow rate for 1 min.

[0105] Addition of secondary antibody: Switching the valve to HRP-labeled goat anti-rabbit secondary antibody working solution, the positive pressure of the liquid channel is 1000 mbar, and about 40 μL is loaded into the open reservoir within a few tens of seconds, and then it is returned to 0. The valve is closed and stopped, the negative pressure is 25 mbar, and the flow rate is slow for 10 min. After the end, switch to PBS with a positive pressure of 1000 mbar to fill the open reservoir, and control the negative pressure to 200 mbar to flush at a large flow rate for 1 min.

[0106] Unloading of the section and subsequent processing

[0107] Unloading of the section: After the secondary antibody incubation and PBS flushing, if only one round of labeling is performed, the clamp can be unloaded, and the section can be subjected to subsequent color development and mounting for storage. If real-time observation is required within the chip, the working solution of DAB or TSA dye for color development is added to the reservoir to enter the chamber for reaction and observation of the labeling under a microscope.

[0108] Multiple cycles of labeling: If multiple rounds of labeling are required after the end of the first round of labeling, such as TSA multiple immunofluorescence experiments, the tissue section needs to be subjected to antigen retrieval to remove the existing CD20 primary antibody-secondary antibody complex (the fluorescence signal of the previous round is still retained due to covalent binding), and the above microfluidic labeling process is repeated.

[0109] The results are shown in Figure 9 , and Figure 9Figure 1 shows the results of conventional immunohistochemistry, and Figure 1b shows the results of panoramic immunohistochemistry. Figure 9 The results show that the microfluidic chip and system of this example can effectively replace conventional immunological techniques.

[0110] 2. Microfluidic multi-channel marker screening

[0111] Different antibody reagents were introduced into multiple microchannels, and multiple markers were detected simultaneously on the same slice, as shown in Figure 2a. Figure 7 Images were collected, and the average staining intensity was calculated by extracting the image gray value. The images obtained from the conventional labeling experiment were compared and analyzed. Markers that were positive in the preliminary screening can be used in subsequent TSA cycle multiple staining experiments. The results of parallel labeling of multiple different markers in tonsil tissue slices are shown in Figure 2b. Figure 10 Figure 10 In Figure 3, from bottom to top, they are CD20, CD3, Ki67, CD4, CD5, CD8a, c-Myc, CD10, BCL-6, BCL-2, MUM1, and CD68.

[0112] 3. Microfluidic multiple immunofluorescence staining

[0113] By controlling the on-off state of the air valve, the mode of fluid passing through the slice is adjusted to form a microcavity, achieving the purpose of multiple staining of the entire tissue slice. Selecting the positive biomarker antibody reagents from the screening results, and according to the marker morphology of the imaging results, TSA dyes with different brightness intensity and fluorescence waveband are selected for rapid and automated cycle multiple labeling process in the microfluidic device, as shown in Figure 4a. Figure 11 The multi-color sample is prepared, and finally imaging is performed. The microfluidic application steps focus on sealing, incubation, and staining, effectively shortening the reaction time of each round. As shown in Figure 4b, a 7-color immunofluorescence was constructed using tonsil slices as model samples. Figure 12 Figure 11 Figure 5 shows the TSA multiple immunofluorescence staining process based on microfluidics. Figure 12 Figure 6 shows the TSA multiple immunofluorescence staining of tonsil slices using the selected markers.

[0114] Figure 12 The results show that the markers obtained by screening in this example can effectively perform TSA multiple immunofluorescence staining on tonsil slices, and the results are consistent with the expected results.

[0115] ​​In view of the problems of complex process, time-consuming, waste of human and material resources, poor accuracy and reproducibility of staining when multiple immunofluorescence staining of tissue sections is used to show tumor microenvironment, the microfluidic technology is creatively introduced, and a microfluidic chip capable of realizing two modes of multi-strip single staining and whole section staining is independently designed. The chip has a multi-channel array and a gas valve layer device inside, which can realize continuous multi-marker staining screening on a single section and panoramic multi-staining based on whole section. Next, the staining steps are highly integrated to realize the automatic processing flow of multiple immunofluorescence labeling of tissue sections, the laminar flow principle of microfluidics is used to break through the kinetic limitation of antigen-antibody reaction, shorten the labeling time, fast staining, and save reagents and samples. The external control system built can control the sequential switching of multiple different reagents, the open antibody sample port can freely add different reagents, and the capillary force of the internal pipeline can resist the hydrostatic pressure at the sample port, so as to ensure that the reagents between adjacent channels do not leak, and the parallel and accurate automatic staining is carried out.

[0116] The above is a further detailed description of the present application in combination with specific embodiments, and cannot be regarded as limiting the specific implementation of the present application to these descriptions. For ordinary skilled persons in the technical field to which the present application belongs, some simple deductions or substitutions can be made without departing from the concept of the present application.

Claims

1. A microfluidic chip based on multiple labeling to construct a tumor microenvironment, characterized in that: It includes an upper structure (1) and a double-layer chip (2), which are stacked sequentially during use; The upper structure (1) includes at least one common reagent inlet (11), several independent reagent storage tanks (12), air inlet and outlet (13), and reagent outlet (14). The common reagent inlet (11) is connected to each independent reagent storage tank (12) through an independent pipe; The dual-layer chip (2) includes a valve layer (21) and a detection layer (22) stacked sequentially. The valve layer (21) includes through holes corresponding to the independent reagent storage tank (12), the inlet and outlet (13) and the reagent outlet (14), and a gas valve (211) in the form of a gas bag. The through holes corresponding to the inlet and outlet (13) are provided on the gas valve (211) for charging and decharging the gas valve (211); several independent reagent storage tanks (12) correspond to several through holes, namely independent reagent inlets (212). The detection layer (22) includes a number of independent detection microchannels (221) corresponding to the independent reagent storage pool (12). The detection microchannels (221) are connected to their corresponding independent reagent storage pools (12) through independent reagent inlets (212). Then, all the detection microchannels (221) are finally gathered together and connected to the reagent outlet (14). In use, the upper structure (1) contacts the valve layer (21), and the detection layer (22) contacts the test slice (3). The independent reagent storage tank (12) is connected to the corresponding detection microchannel (221) through the through hole on the valve layer (21). During independent detection, air is supplied through the air inlet and outlet (13) to put the valve (211) in positive pressure mode. The top of the detection layer (22) is a closed membrane. The test slice (3) closes the lower end of the detection microchannel (221). 11) Cover the top of the membrane on the top of the detection layer (22), squeeze the detection layer and the slice to fit tightly, thus forming an independent detection channel; when panoramic marking is required, draw air through the air inlet and outlet (13) to put the air valve (211) in negative pressure mode, the membrane on the top of the detection layer (22) bulges upward, connecting each detection microchannel of the detection layer (22) from the bottom to the surface in contact with the tissue, forming a large chamber on the surface of the tissue slice, so as to perform the same panoramic marking on the tissue within the area covered by the large chamber.

2. The microfluidic chip according to claim 1, characterized in that: The upper structure (1) is also provided with an observation window (15), and the air valve (211) is located at the position corresponding to the observation window (15).

3. The microfluidic chip according to claim 1, characterized in that: The valve layer (21) and the detection layer (22) are respectively prepared using polydimethylsiloxane. During assembly, the sample inlet of the detection microchannel (221) is aligned with the independent reagent inlet (212) of the valve layer (21), the reagent outlet is aligned, and the valve (211) is located above the detection microchannel (221), thus forming a complete double-layer chip (2).

4. The microfluidic chip according to any one of claims 1-3, characterized in that: The upper structure (1) is made of a photocurable resin material.

5. A microfluidic detection system employing the microfluidic chip according to any one of claims 1-4.

6. The microfluidic detection system according to claim 5, characterized in that: Includes the microfluidic chip (01), as well as a common reagent module (02), a reagent control module (03), a positive pressure control module (04), an external gas circuit switch (05), a negative pressure module (06), and a waste liquid collection module (07); The public reagent module (02) includes several independent public reagent pools, and each public reagent pool is connected to the inlet of the reagent control module (03) through an independent pipe; The reagent control module (03) is used to independently control and select the corresponding public reagent pool. The outlet end of the reagent control module (03) is connected to the public reagent inlet (11) of the microfluidic chip (01). The positive pressure control module (04) is an external air source, which is connected to the air inlet and outlet (13) of the microfluidic chip (01) through an external air circuit switch (05) and is used to inflate the air valve (211). The negative pressure module (06) is independently connected to the air inlet and outlet (13) of the microfluidic chip (01) and is used to evacuate the air valve (211); The waste liquid collection module (07) is connected to the reagent outlet (14) of the microfluidic chip (01) and is used to collect waste liquid.

7. The microfluidic detection system according to claim 6, characterized in that: The waste liquid collection module (07) is in a closed state. The negative pressure module (06) is connected to the waste liquid collection module (07) through an independent pipe and is used to independently control the waste liquid collection module (07) to keep it in a negative pressure state.

8. The microfluidic detection system according to claim 7, characterized in that: A first flow meter (081) is installed on the connecting pipe between the reagent control module (03) and the common reagent inlet (11) of the microfluidic chip (01).

9. The microfluidic detection system according to claim 8, characterized in that: A second flow meter (082) is installed on the connecting pipe between the waste liquid collection module (07) and the reagent outlet (14) of the microfluidic chip (01).

10. The microfluidic detection system according to claim 9, characterized in that: The microfluidic detection system also includes a computer control terminal (09), which is connected to the reagent control module (03), the positive pressure control module (04), the external gas circuit switch (05), and the negative pressure module (06) via signal connection. The specific parameters of the pressure applied, the switching sequence of the fluid, and the time of each process are controlled by the program.

11. A multiplex immunofluorescence detection method, characterized in that: This includes using the microfluidic chip as described in any one of claims 1-4 or the microfluidic detection system as described in any one of claims 5-10 to perform multiple immunofluorescence labeling on the same slice sample.

12. The multiplex immunofluorescence detection method according to claim 11, characterized in that: The process involves first inflating the gas through the inlet / outlet (13) to put the gas valve (211) in positive pressure mode, and then labeling each detection microchannel (221) with different antibodies. Then, the gas is evacuated through the inlet / outlet (13) to put the gas valve (211) in negative pressure mode, so that the detection microchannels are connected and a large chamber is formed on the surface of the tissue slice. The tissue within the area covered by the large chamber is then labeled in the same panoramic way.

13. The multiplex immunofluorescence detection method according to claim 12, characterized in that: This also includes removing the sliced ​​samples after panoramic marking and performing post-processing or continuous staining.

Images