A microfluidic chip

Abstract

The present application relates to a kind of microfluidic chip, for simultaneously detecting CAR-T cell and multiple cytokines in the peripheral blood of patient, comprising: from top to bottom sequentially connected valve layer, pipeline layer and substrate layer;Pipeline layer includes: sample mixing module, CAR-T cell capture detection module, concentration gradient generation module, cell filtration module and cytokine detection module are sequentially connected;The microfluidic chip of the present application can simultaneously detect CAR-T cell and cytokine in the peripheral blood of cancer patient receiving CAR-T cell therapy, without needing to transfer whole blood sample to flow cytometer for detection, different concentrations of sample can be simultaneously detected in microfluidic chip, effectively improve the detection range, microfluidic chip reaction pipeline is closed environment, for CAR-T cell and cytokine detection provides relatively stable detection environment;And the blood sample amount required for detection is 50 microliters, less than the sample amount required by existing detection technology, the amount of corresponding reagent is also reduced.

Description

Technical Field

[0001] This invention relates to the field of biomedical detection technology, and in particular to a microfluidic chip. Background Technology

[0002] Chimeric antigen receptor T-cell immunotherapy (CAR-T therapy) is a novel, precise, targeted therapy for cancer, reshaping treatment options for patients with advanced cancer, especially those with limited treatment choices. It has shown significant efficacy in treating relapsed and refractory B-cell non-Hodgkin's lymphoma and has the potential to be extended to other solid and liquid tumors. It is considered a rapid and accurate new tumor immunotherapy with the potential to cure cancer, bringing new hope to cancer treatment. However, CAR-T therapy is also accompanied by a series of life-threatening adverse reactions, such as cytokine storm (CRS) and neurotoxicity (NE). Studies have found that the aggravation of NE is associated with high levels of CAR-T cell replication and cytokine levels in the body. Therefore, after CAR-T cell infusion, close monitoring of CAR-T cell replication and cytokine levels in peripheral blood is crucial to predict the severity of adverse reactions and implement timely interventions. However, current CAR-T cell detection primarily relies on flow cytometry. This instrument suffers from drawbacks such as high cost, large blood sample volumes required for each test, and the inability to simultaneously detect cells and multiple cytokines. Considering the patient's health condition, such intensive, large-volume blood sample collection is impractical. Therefore, the existing detection system cannot effectively manage potentially life-threatening adverse events associated with CAR-T therapy. In conclusion, there is an urgent need to develop new methods for timely and rigorous monitoring of CAR-T cells and cytokines in CAR-T therapy.

[0003] In the past decade or so, with the rapid development of microfluidics technology and its integration with other technologies, detection technologies based on microfluidics have experienced vigorous growth. Microfluidic chips, with their advantages of low cost, small volume, rapid reaction, and ease of automation, have provided new ideas for CAR-T cell detection. Microfluidics is a technology that integrates macroscopic laboratory instruments and biochemical reactions and operations onto microfluidic chips that are only a few centimeters in size. Through photolithography, 3D printing, and other techniques, dozens or even hundreds of micro- and nano-scale microfluidic channels with different functions can be fabricated and detection can be achieved within these reaction fluid channels. Heating modules and optical modules can also be integrated with microfluidic chips to achieve fully automated, integrated, portable, and rapid micro-volume detection. This technology is expected to be widely used in modern medical testing and disease analysis, effectively solving problems in these fields.

[0004] Currently, the detection of CAR-T cells and cytokines in China mainly relies on flow cytometry and traditional ELISA methods. The use of microfluidic technology for CAR-T cell and cytokine detection is still in its early stages, with no products available yet. Continuous technological breakthroughs and methodological innovations are needed to drive the development of this field. Summary of the Invention

[0005] The purpose of this invention is to address the shortcomings of existing technologies by providing a microfluidic chip.

[0006] To achieve the above objectives, the technical solution adopted by the present invention is as follows:

[0007] A microfluidic chip is provided for simultaneously detecting CAR-T cells and multiple cytokines in a patient's peripheral blood, comprising: a valve layer, a conduit layer, and a base layer connected from top to bottom;

[0008] The pipeline layer includes: a sample mixing module, a CAR-T cell capture and detection module, a concentration gradient generation module, a cell filtration module, and a cytokine detection module connected in sequence by pipelines;

[0009] The sample mixing module includes: an "S"-shaped sample mixing pipe; the sample mixing pipe has a first inlet, a second inlet and a third inlet at its inlet, and a first outlet at its outlet.

[0010] The CAR-T cell capture detection module includes: at least one first reaction chamber, the inlet of the first first reaction chamber is connected to the outlet of the sample mixing pipe, the inlet of the subsequent first reaction chamber is connected to the outlet of the preceding first reaction chamber, the inlet of the sample outlet pipe is connected to the outlet of all the first reaction chambers respectively; each of the first reaction chambers except the first one is independently provided with a fourth sample inlet, and the outlet of the sample outlet pipe is provided with a second sample outlet;

[0011] The concentration gradient generation module includes: at least one stage of concentration gradient generation pipelines, the first stage having three concentration gradient generation pipelines, the subsequent stage having one more concentration gradient generation pipeline than the previous stage, the inlet of the first stage concentration gradient generation pipeline being connected to the outlet of the last first reaction chamber and the outlet of the first sample inlet pipeline, respectively, the inlet of the subsequent stage concentration gradient generation pipeline being connected to the outlet of the previous stage concentration gradient generation pipeline; a fifth sample inlet is provided at the inlet of the first sample inlet pipeline;

[0012] The cell filtration module includes at least three filtration chambers, the number of which is the same as the number of concentration gradient generating pipes in the last stage. The inlets of all the filtration chambers are respectively connected to the outlets of the corresponding concentration gradient generating pipes in the last stage and the outlet of the second sample inlet pipe. A sixth sample inlet is provided at the inlet of the second sample inlet pipe.

[0013] The cytokine detection module includes: at least three cytokine detection channels, the number of which is the same as the number of the filtration chambers; the inlet of each cytokine detection channel is connected to the outlet of the corresponding filtration chamber and the outlet of the third sample inlet channel; each cytokine detection channel has an independent third sample outlet at its outlet, and the inlet of the third sample inlet channel has a seventh sample inlet.

[0014] Preferably, the valve layer and the pipe layer are both made of PDMS material; the base layer is made of silicon dioxide material or gold-plated glass sheet material.

[0015] Preferably, the valve layer includes: a semi-permeable valve located at the outlet of the filter chamber and full valves located at other inlets / outlets.

[0016] More preferably, when the semi-permeable valve is closed, only liquid can pass through the pipeline, while solids cannot; when the full valve is closed, neither liquid nor solids can pass through the pipeline.

[0017] Preferably, the height of the pipe layer is 25μm-50μm; the width of the pipe layer is 100μm-500μm.

[0018] Preferably, the bottom surface of the sample mixing pipe is provided with a fishbone structure along the pipe direction.

[0019] More preferably, the fishbone structure is an asymmetrical "V" shaped structure, the width of the fishbone structure is 5μm-10μm, and the distance between two adjacent fishbone structures is 5μm-15μm.

[0020] Preferably, the inner surface of the first reaction chamber is uniformly modified with T cell capture antibodies, and the bottom surface of the first reaction chamber is provided with staggered cylindrical structures.

[0021] More preferably, the diameter of the cylindrical structure is 3μm-5μm, and the center distance between two adjacent cylindrical structures is 1.5μm-6μm.

[0022] Preferably, the cytokine detection conduit includes: at least one second reaction chamber, the inlet of the first second reaction chamber is connected to the outlet of the filter chamber via a pipeline, and the inlet of the subsequent second reaction chamber is connected to the outlet of the preceding second reaction chamber via a pipeline.

[0023] The present invention adopts the above technical solution and has the following technical effects compared with the prior art:

[0024] The microfluidic chip of this invention can simultaneously detect CAR-T cells and cytokines in the peripheral blood of cancer patients receiving CAR-T cell therapy, eliminating the need to transfer whole blood samples to a flow cytometer for detection. The microfluidic chip can simultaneously detect samples of different concentrations, effectively improving the detection range. The reaction channel of the microfluidic chip is a closed environment, providing a relatively stable detection environment for CAR-T cell and cytokine detection. Furthermore, the blood sample volume required for detection is only 50 microliters, which is less than the sample volume required by existing detection technologies, and the corresponding reagent consumption is also reduced. Attached Figure Description

[0025] Figure 1 This is a schematic diagram of the microfluidic chip in Embodiment 1 of the present invention;

[0026] Figure 2 This is a schematic diagram of the module division of the microfluidic chip in Embodiment 1 of the present invention;

[0027] Figure 3 This is a schematic diagram of the fishbone structure in Embodiment 1 of the present invention;

[0028] Figure 4 This is a schematic diagram of the cylindrical structure in Embodiment 1 of the present invention;

[0029] Figure 5 This is a schematic diagram of the structure of the filter microbeads in Embodiment 1 of the present invention;

[0030] The reference numerals in the figures include:

[0031] First injection port 1; Second injection port 2; Third injection port 3; First outlet port 4; Fourth injection port 5; CAR-T cell capture detection module 6; First reaction chamber 7; Concentration gradient generation module 8; Sample mixing module 9; Third outlet port 10; Fifth injection port 11; Sixth injection port 12; Cell filtration module 13; Seventh injection port 14; Cytokine detection module 15; Second reaction chamber 16; Filtration chamber 17; Second outlet port 18; Full valve 19; Cylindrical structure 20; Semi-permeable valve 21; Filter microbeads 22; Fishbone structure 23. Detailed Implementation

[0032] The technical solutions of the embodiments of the present invention will be clearly and completely described below with reference to the accompanying drawings. Obviously, the described embodiments are only some embodiments of the present invention, and not all embodiments. Based on the embodiments of the present invention, all other embodiments obtained by those skilled in the art without creative effort are within the scope of protection of the present invention.

[0033] It should be noted that, unless otherwise specified, the embodiments and features described in the present invention can be combined with each other.

[0034] The present invention will be further described below with reference to the accompanying drawings and specific embodiments, but this is not intended to limit the scope of the invention.

[0035] Example 1

[0036] This embodiment provides a microfluidic chip (length × width × height is 10cm × 8cm × 1cm) for simultaneously detecting CAR-T cells and multiple cytokines in the peripheral blood of patients, including: a valve layer, a channel layer and a base layer connected from top to bottom;

[0037] Wherein, the pipe layer is as follows Figure 1-2 As shown, it includes: a sample mixing module 9, a CAR-T cell capture and detection module 6, a concentration gradient generation module 8, a cell filtering module 13, and a cytokine detection module 15 connected in sequence by tubing;

[0038] The sample mixing module 9 includes: an "S"-shaped sample mixing pipe; the sample mixing pipe has a first inlet 1 for injecting a dilution solution, a second inlet 2 for injecting a fluorescent antibody solution, and a third inlet 3 for injecting a venous blood sample at its inlet, and a first outlet 4 at its outlet.

[0039] The CAR-T cell capture detection module 6 includes: at least one first reaction chamber 7, the inlet of the first first reaction chamber 7 is connected to the outlet of the sample mixing pipe, the inlet of the subsequent first reaction chamber 7 is connected to the outlet of the preceding first reaction chamber 7, the inlet of the sample outlet pipe is connected to the outlet of all the first reaction chamber 7 respectively; all the first reaction chamber 7 except the first one are independently provided with a fourth sample inlet 5, and the outlet of the sample outlet pipe is provided with a second sample outlet 18;

[0040] The concentration gradient generation module 8 includes: at least one level of concentration gradient generation pipeline, the first level having three concentration gradient generation pipelines, the subsequent level having one more concentration gradient generation pipeline than the previous level, the inlet of the first level concentration gradient generation pipeline being connected to the outlet of the last first reaction chamber 7 and the outlet of the first sample inlet pipeline, respectively, and the inlet of the subsequent level concentration gradient generation pipeline being connected to the outlet of the previous level concentration gradient generation pipeline; a fifth sample inlet 11 for injecting a dilution solution is provided at the inlet of the first sample inlet pipeline;

[0041] The cell filtration module 13 includes at least three filtration chambers 17, the number of which is the same as the number of concentration gradient generating pipes in the last stage. The inlets of all filtration chambers 17 are connected to the outlets of the corresponding concentration gradient generating pipes in the last stage and the outlet of the second sample inlet pipe, respectively. The inlet of the second sample inlet pipe is provided with filter beads 22 (such as...) for sample introduction. Figure 5 As shown, the sixth inlet 12 has a diameter of 500nm-10μm and a quantity of ~200 per pipe;

[0042] The cytokine detection module 15 includes at least three cytokine detection channels, the number of which is the same as the number of filter chambers 17. The inlets of all the cytokine detection channels are connected to the outlets of the corresponding filter chambers 17 and the outlet of the third sample inlet channel, respectively. Each of the cytokine detection channels has an independent third sample outlet 10 at its outlet, and the inlet of the third sample inlet channel has a seventh sample inlet 14.

[0043] In a preferred embodiment, both the valve layer and the pipe layer are made of PDMS material; the base layer is made of silicon dioxide or gold-plated glass sheet material.

[0044] In a preferred embodiment, the valve layer includes: a semi-permeable valve 21 located at the outlet of the filter chamber 17 and full valves 19 located at other inlets / outlets.

[0045] In a preferred embodiment, the height of the pipe layer is 40 μm; the width of each pipe in the pipe layer is 200 μm, the width of each chamber is 1000 μm, and the length of each chamber is 2000 μm.

[0046] In a preferred embodiment, the bottom surface of the sample mixing pipe is provided with, along the pipe direction, as shown in the image. Figure 3The fishbone structure 23 shown is an asymmetrical "V" shaped structure. Four adjacent fishbone structures 23 are grouped together. Two groups of adjacent fishbone structures 23 are axially symmetrical along the pipe direction. The width of the fishbone structure 23 is 5μm, and the distance between two adjacent fishbone structures 23 is 10μm.

[0047] In a preferred embodiment, the number of the first reaction chambers 7 is eight, the inner surface of the first reaction chambers 7 is uniformly modified with T cell capture antibodies, and the bottom surface of the first reaction chambers 7 is alternately arranged as shown in the figure. Figure 4 The cylindrical structure 20 shown has a diameter of 3μm, a height of 5μm, and a center-to-center distance of 3μm between two adjacent cylindrical structures 20.

[0048] In a preferred embodiment, the concentration gradient generating pipeline has three stages, that is, the last stage has five concentration gradient generating pipelines.

[0049] In a preferred embodiment, the cytokine detection pipeline includes twelve second reaction chambers 16. The inlet of the first second reaction chamber 16 is connected to the outlet of the filter chamber 17 via a pipeline, and the inlet of the subsequent second reaction chamber 16 is connected to the outlet of the preceding second reaction chamber 16 via a pipeline. The inner surfaces of the twelve second reaction chambers are uniformly modified with specific capture antibodies for L-2, IL-4, IL-5, IL-6, IL-8, IL-10, IL-17A, IFN-γ, TNF-α, TNF-β, G-CSF, and GM-CSF.

[0050] Example 2

[0051] This embodiment provides a method for simultaneously detecting CAR-T cells and multiple cytokines in a patient's peripheral blood, the steps of which include:

[0052] One drop (50 μL) of peripheral blood sample, 50 μL of fluorescent antibody solution, and 50 μL of PBS dilution solution were injected into the sample mixing module 9 of the microfluidic chip as described in Example 1 through the third inlet 3, the second inlet 2, and the first inlet 1 at a flow rate of 5 μL / s, respectively. In the sample mixing pipeline, the peripheral blood sample was diluted, and the CAR-T cells therein bound efficiently to the fluorescent antibody in the fluorescent antibody solution.

[0053] In the CAR-T cell capture detection module 6, the peripheral blood sample diluted 3-fold was used to capture T cells and CAR-T cells with T cell capture antibodies, thereby enabling the simultaneous detection of fluorescent CAR-T cells and T cells and the calculation of the proportion of CAR-T cells.

[0054] Most of the T cells and CAR-T cells captured in the peripheral blood sample are mixed and diluted in the concentration gradient generation module 8 with PBS diluent injected at the same flow rate through the fifth injection port 11 to form a blood analysis sample with a concentration gradient.

[0055] Polystyrene filter beads 22 are injected through the sixth inlet 12 to form a microfiltration column of polystyrene filter beads 22; thus, in the cell filtration module 13, only the plasma portion without blood cells can pass through the semi-permeable valve 21 in the above-mentioned blood analysis sample.

[0056] The plasma fraction is incubated for 2 minutes in each of the second reaction chambers 16 of the cytokine detection module 15. PBST washing solution and specific fluorescent detection antibody complex solution are then injected through the seventh injection port 14 to complete the fluorescence detection of each cytokine.

[0057] A single test can obtain the number and proportion of CAR-T cells and the concentration of various cytokines in 50 μL of peripheral blood from a patient. The test results for a patient are shown in Table 1.

[0058] Table 1

[0059]

[0060]

[0061] In summary, the microfluidic chip of this invention can simultaneously detect CAR-T cells and cytokines in the peripheral blood of cancer patients receiving CAR-T cell therapy, eliminating the need to transfer whole blood samples to a flow cytometer for detection. The microfluidic chip can simultaneously detect samples of different concentrations, effectively improving the detection range. The reaction channel of the microfluidic chip is a closed environment, providing a relatively stable detection environment for CAR-T cell and cytokine detection. Furthermore, the blood sample volume required for detection is only 50 microliters, which is less than the sample volume required by existing detection technologies, and the corresponding reagent consumption is also reduced.

[0062] The above description is merely a preferred embodiment of the present invention and does not limit the implementation and protection scope of the present invention. Those skilled in the art should realize that any equivalent substitutions and obvious changes made based on the description and illustrations of the present invention should be included within the protection scope of the present invention.

Claims

1. A microfluidic chip for simultaneously detecting CAR-T cells and multiple cytokines in a patient's peripheral blood, characterized in that, include: The valve layer, pipe layer, and base layer are connected sequentially from top to bottom; The pipeline layer includes: a sample mixing module (9), a CAR-T cell capture and detection module (6), a concentration gradient generation module (8), a cell filtering module (13), and a cytokine detection module (15) connected in sequence. The sample mixing module (9) includes: an "S"-shaped sample mixing pipe; the sample mixing pipe has a first inlet (1), a second inlet (2) and a third inlet (3) at the inlet, and a first outlet (4) at the outlet. The CAR-T cell capture detection module (6) includes: a first reaction chamber (7), the inlet of the first first reaction chamber (7) is connected to the outlet of the sample mixing pipe, the inlet of the next first reaction chamber (7) is connected to the outlet of the previous first reaction chamber (7), and the inlet of the sample outlet pipe is connected to the outlet of the first reaction chamber (7); all the first reaction chambers (7) except the first one are independently provided with a fourth sample inlet (5), and the outlet of the sample outlet pipe is provided with a second sample outlet (18). The concentration gradient generation module (8) includes: at least one level of concentration gradient generation pipeline, the number of the first level of concentration gradient generation pipeline is three, the number of the subsequent level of concentration gradient generation pipeline is the number of the previous level of concentration gradient generation pipeline plus one, the inlet of the first level of concentration gradient generation pipeline is connected to the outlet of the last first reaction chamber (7) and the outlet of the first sample injection pipeline respectively, the inlet of the subsequent level of concentration gradient generation pipeline is connected to the outlet of the previous level of concentration gradient generation pipeline; a fifth sample injection port (11) is provided at the inlet of the first sample injection pipeline. The cell filtration module (13) includes at least three filtration chambers (17), the number of which is the same as the number of concentration gradient generating pipes in the last stage. The inlets of all the filtration chambers (17) are connected to the outlets of the corresponding concentration gradient generating pipes in the last stage and the outlet of the second sample inlet pipe, respectively. A sixth sample inlet (12) is provided at the inlet of the second sample inlet pipe. The cytokine detection module (15) includes: at least three cytokine detection channels, the number of which is the same as the number of the filter chambers (17); the inlet of each cytokine detection channel is connected to the outlet of the corresponding filter chamber (17) and the outlet of the third sample inlet channel; each cytokine detection channel has an independent third sample outlet (10) at its outlet, and the inlet of the third sample inlet channel has a seventh sample inlet (14). The inner surface of the first reaction chamber (7) is uniformly modified with T cell capture antibodies, and the bottom surface of the first reaction chamber (7) is provided with alternating cylindrical structures (20).

2. The microfluidic chip according to claim 1, characterized in that, The valve layer and the pipe layer are both made of PDMS material; the base layer is made of silicon dioxide or gold-plated glass sheet material.

3. The microfluidic chip according to claim 1, characterized in that, The valve layer includes: a semi-permeable valve (21) located at the outlet of the filter chamber (17) and full valves (19) located at other inlets / outlets.

4. The microfluidic chip according to claim 1, characterized in that, The height of the pipe layer is 25μm-50μm; the width of the pipe layer is 100μm-500μm.

5. The microfluidic chip according to claim 1, characterized in that, The bottom surface of the sample mixing pipe is provided with a fishbone structure (23) along the pipe direction.

6. The microfluidic chip according to claim 5, characterized in that, The fishbone structure (23) is an asymmetrical "V" shaped structure. The width of the fishbone structure (23) is 5μm-10μm, and the distance between two adjacent fishbone structures (23) is 5μm-15μm.

7. The microfluidic chip according to claim 1, characterized in that, The diameter of the cylindrical structure (20) is 3μm-5μm, and the center distance between two adjacent cylindrical structures (20) is 1.5μm-6μm.

8. The microfluidic chip according to claim 1, characterized in that, The cytokine detection pipeline includes at least one second reaction chamber (16), the inlet of the first second reaction chamber (16) is connected to the outlet of the filter chamber (17) via a pipeline, and the inlet of the subsequent second reaction chamber (16) is connected to the outlet of the preceding second reaction chamber (16) via a pipeline.

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