Cell local chemical stimulation and secretion detection microfluidic device and operation method thereof

Abstract

The invention discloses a cell local chemical stimulation and secretion detection microfluidic device and an operation method thereof. The cell local chemical stimulation and secretion detection microfluidic device comprises: a multi-axis positioning platform; the micro-fluidic probe is arranged on the multi-axis positioning platform, and a first suction flow channel, a first injection flow channel, a second injection flow channel and a second suction flow channel are arranged on the micro-fluidic probe; a first detection electrode assembly and a second detection electrode assembly, the first detection electrode assembly is arranged in the first suction flow channel, and the second detection electrode assembly is arranged in the second suction flow channel; a secretion measuring device; a fluid driving device; a cell sample platform; a microscope objective. According to the cell local chemical stimulation and secretion detection micro-fluidic device provided by the embodiment of the invention, chemical stimulation can be carried out on a cell local area, secretions in different areas of the cell can be respectively detected, and the device has the advantages of high response speed, high sensitivity, strong applicability, small cell mechanical damage and the like.

Description

Technical Field

[0001] The present invention relates to the field of microfluidics, and in particular to a microfluidic device for localized cell chemical stimulation and secretion detection, and an operating method of the microfluidic device. Background Art

[0002] Single cells, as living units, can respond to external chemical stimuli. Studying their stimulus-responsive behavior can provide insights into cellular properties. Stimulating localized regions of single cells can reveal deeper properties. Stimulations cause changes in cell metabolism, and analyzing cellular secretions can reveal cellular properties and understanding the mechanisms of intercellular communication without damaging the cells.

[0003] Compared with mass spectrometry and fluorescence imaging, electrochemical detection methods have the advantages of fast response, high sensitivity, high temporal and spatial resolution, and continuous measurement.

[0004] The electrochemical detection method for cell chemically stimulated secretions in related technologies can monitor and quantify the instantaneous biochemical molecules released from cells or tissues in real time, but it is difficult to detect the dynamics of biochemical molecule release in different regions of the cell after the local area of a single cell is stimulated. Summary of the Invention

[0005] The present invention aims to solve at least one of the technical problems existing in the prior art. To this end, the present invention provides a microfluidic device for localized chemical stimulation of cells and detection of secretions. This microfluidic device can chemically stimulate a localized region of cells and detect secretions from different regions of the cell separately. It has the advantages of fast response, high sensitivity, strong applicability, and minimal mechanical damage to cells.

[0006] The present invention also provides an operating method of the microfluidic device for local cell chemical stimulation and secretion detection.

[0007] To achieve the above objectives, according to an embodiment of the first aspect of the present invention, a microfluidic device for localized cell chemical stimulation and secretion detection is provided, the microfluidic device comprising: a multi-axis positioning platform; a microfluidic probe, the microfluidic probe being disposed on the multi-axis positioning platform, the microfluidic probe being provided with a first suction channel, a first injection channel, a second injection channel, and a second suction channel arranged in sequence and spaced apart in a horizontal direction; a first detection electrode assembly and a second detection electrode assembly, the first detection electrode assembly being disposed in the first suction channel, and the second detection electrode assembly being disposed in the second suction channel; a secretion measuring device, the secretion measuring device being electrically connected to the first detection electrode assembly and the second detection electrode assembly, respectively; a fluid driving device, the fluid driving device being connected to the first suction channel, the first injection channel, the second injection channel, and the second suction channel, respectively; a cell sample platform, the cell sample platform being located below the microfluidic probe and being suitable for planting cell samples; and a microscope objective lens, the microscope objective lens being located below the cell sample platform.

[0008] The microfluidic device for local cell chemical stimulation and secretion detection according to the embodiment of the present invention can chemically stimulate local areas of cells and detect secretions from different areas of cells separately. It has the advantages of fast response speed, high sensitivity, strong applicability, and little mechanical damage to cells.

[0009] In addition, the microfluidic device for local cell chemical stimulation and secretion detection according to the above embodiment of the present invention may also have the following additional technical features:

[0010] According to one embodiment of the present invention, the distance between the first injection channel and the first suction channel is equal to the distance between the second injection channel and the second suction channel.

[0011] According to one embodiment of the present invention, each of the first detection electrode assembly and the second detection electrode assembly includes a counter electrode, a working electrode and a reference electrode, and the counter electrode, the working electrode and the reference electrode are arranged at intervals and are electrically connected to the secretion measuring device through multiple wires.

[0012] According to one embodiment of the present invention, the fluid driving device includes: an injection injection pump; two injection syringes, the two injection syringes are respectively connected to the first injection channel and the second injection channel, and the two injection syringes are arranged on the injection injection pump; a suction injection pump; two suction syringes, the two suction syringes are respectively connected to the first suction channel and the second suction channel, and the two suction syringes are arranged on the suction injection pump.

[0013] According to one embodiment of the present invention, the multi-axis positioning platform is a three-axis positioning platform having three degrees of freedom: up and down, left and right, and front and back.

[0014] According to one embodiment of the present invention, the cell sample platform includes: a temperature-controlled stage, which is movable in two degrees of freedom, left and right and front and back, and can adjust the temperature; a culture dish, which is placed on the temperature-controlled stage and is suitable for holding a protective solution and for planting cell samples.

[0015] According to one embodiment of the present invention, the first injection channel and the second injection channel are suitable for injecting a phorbol ester solution and a protective solution, respectively.

[0016] According to one embodiment of the present invention, the first detection electrode assembly and the second detection electrode assembly are configured to be suitable for detecting the concentration of hydrogen peroxide.

[0017] According to one embodiment of the present invention, the length directions of the first injection channel, the second injection channel, the first suction channel, and the second suction channel are all oriented in the up-down direction.

[0018] According to an embodiment of the second aspect of the present invention, a method for operating the microfluidic device for localized cell chemical stimulation and secretion detection according to an embodiment of the first aspect of the present invention is provided, comprising the following steps:

[0019] Under observation through the microscope objective lens, adjusting the microfluidic probe to above the target cell sample through the multi-axis positioning platform so that the midlines of the first injection channel and the second injection channel span the target position of the target cell sample;

[0020] injecting a protective solution and an irritant solution into the first injection channel and the second injection channel respectively by using the fluid driving device, and simultaneously sucking solutions from the first suction channel and the second suction channel;

[0021] detecting the concentration of target secretions in the first suction channel and the second suction channel using the first detection electrode assembly and the second detection electrode assembly;

[0022] The secretion measuring device is used to monitor the detection values of the first detection electrode assembly and the second detection electrode assembly in real time, and analyze the substance release dynamics of different subregions of the target sample cells.

[0023] According to the operating method of the microfluidic device for local chemical stimulation and secretion detection of cells according to the embodiment of the present invention, by utilizing the microfluidic device for local chemical stimulation and secretion detection of cells described in the embodiment of the first aspect of the present invention, it is possible to chemically stimulate the local area of the cell and detect the secretions of different areas of the cell separately, which has the advantages of fast response speed, high sensitivity, strong applicability, and little mechanical damage to the cell.

[0024] Additional aspects and advantages of the present invention will be set forth in part in the description which follows and, in part, will be obvious from the description which follows, or may be learned by practice of the present invention. BRIEF DESCRIPTION OF THE DRAWINGS

[0025] The above and / or additional aspects and advantages of the present invention will become apparent and readily understood from the following description of the embodiments with reference to the accompanying drawings, in which:

[0026] Figure 1 3 is a schematic structural diagram of a microfluidic device for local cell chemical stimulation and secretion detection according to an embodiment of the present invention.

[0027] Figure 2 3 is a schematic structural diagram of a cell sample platform of a microfluidic device for local cell chemical stimulation and secretion detection according to an embodiment of the present invention.

[0028] Figure 3 3 is a schematic diagram of the partial structure of a microfluidic device for local chemical stimulation of cells and secretion detection according to an embodiment of the present invention.

[0029] Figure 4 4 is a flow chart of an operating method of a microfluidic device for local cell chemical stimulation and secretion detection according to an embodiment of the present invention.

[0030] Figure 1: Microfluidic device for detecting local chemical stimulation and secretions of cells 1, microfluidic probe 10, first suction channel 11, first injection channel 12, second injection channel 13, second suction channel 14, base layer 15, first detection electrode assembly 20, counter electrode 21, working electrode 22, reference electrode 23, second detection electrode assembly 30, cell sample platform 40, temperature-controlled stage 41, culture dish 42, microscope objective 50, wire 60, cell sample 2, protective solution 3, stimulant solution 4. DETAILED DESCRIPTION

[0031] This application is based on the inventor's discovery and understanding of the following facts and problems:

[0032] The electrochemical detection method for cell chemically stimulated secretions in related technologies can monitor and quantify the instantaneous biochemical molecules released from cells or tissues in real time, but it is difficult to detect the dynamics of biochemical molecule release in different regions of the cell after the local area of a single cell is stimulated.

[0033] Specifically, in some related technologies, microfluidic chips are used to stimulate cells locally, but they can only stimulate cells and observe the cell response behavior through a microscope, and lack the ability to detect secretions from different local areas of cells.

[0034] The following describes embodiments of the present invention in detail. Examples of the embodiments are shown in the accompanying drawings, wherein the same or similar reference numerals throughout represent the same or similar elements or elements having the same or similar functions. The embodiments described below with reference to the accompanying drawings are exemplary and are intended only to explain the present invention and are not to be construed as limiting the present invention.

[0035] In the description of the present invention, it should be understood that the terms "center", "longitudinal", "lateral", "length", "width", "thickness", "up", "down", "front", "back", "left", "right", "vertical", "horizontal", "top", "bottom", "inside", "outside", "clockwise", "counterclockwise", "axial", "radial", "circumferential" and the like indicate orientations or positional relationships based on the orientations or positional relationships shown in the accompanying drawings, and are only for the convenience of describing the present invention and simplifying the description, rather than indicating or implying that the device or element referred to must have a specific orientation, be constructed and operated in a specific orientation, and therefore cannot be understood as limiting the present invention. In addition, features defined as "first" or "second" may explicitly or implicitly include one or more of such features. In the description of the present invention, unless otherwise specified, "multiple" means two or more.

[0036] In the description of the present invention, it should be noted that, unless otherwise expressly specified or limited, the terms "mounted," "connected," and "connected" should be understood in a broad sense. For example, they may refer to fixed, detachable, or integral connections; mechanical or electrical connections; direct or indirect connections through an intermediate medium; and internal communication between two components. Those skilled in the art will understand the specific meanings of the above terms in the present invention based on specific circumstances.

[0037] The following describes a microfluidic device 1 for local cell chemical stimulation and secretion detection according to an embodiment of the present invention with reference to the accompanying drawings.

[0038] like Figure 1-Figure 4 As shown, the microfluidic device 1 for local chemical stimulation and secretion detection of cells according to an embodiment of the present invention includes a multi-axis positioning platform (not shown in the figure), a microfluidic probe 10, a first detection electrode assembly 20, a second detection electrode assembly 30, a secretion measuring device (not shown in the figure), a fluid driving device (not shown in the figure), a cell sample platform 40 and a microscope objective 50.

[0039] The microfluidic probe 10 is arranged on the multi-axis positioning platform, and is provided with a first suction channel 11, a first injection channel 12, a second injection channel 13 and a second suction channel 14 arranged in sequence along the horizontal direction (the up and down, left and right and front and back directions are shown by the arrows in the figure, and the horizontal direction is perpendicular to the up and down direction). Figure 1 and Figure 2 1 shows an embodiment in which the first suction channel 11, the first injection channel 12, the second injection channel 13, and the second suction channel 14 are arranged in sequence from left to right. In other words, the first injection channel 12 and the first injection channel 12 are located between the first suction channel 11 and the second suction channel 14.

[0040] The first detection electrode assembly 20 is disposed in the first suction channel 11, and the second detection electrode assembly 30 is disposed in the second suction channel 14. The secretion measuring device is electrically connected to the first detection electrode assembly 20 and the second detection electrode assembly 30, respectively.

[0041] The fluid driving device is communicated with the first suction channel 11 , the first injection channel 12 , the second injection channel 13 and the second suction channel 14 respectively.

[0042] The cell sample platform 40 is located below the microfluidic probe 10 and is suitable for planting the cell sample 2. The microscope objective lens 50 is located below the cell sample platform 40.

[0043] Specifically, the multi-axis positioning platform has multiple degrees of freedom to enable movement of the microfluidic probe 10. The first detection electrode assembly 20 and the second detection electrode assembly 30 are adapted to detect the concentration of a target secretion, and the secretion measurement device is adapted to monitor the detection values of the first detection electrode assembly 20 and the second detection electrode assembly 30 in real time. The fluid drive device is adapted to inject fluid into the cell sample platform 40 through the first injection channel 12 and the second injection channel 13, and to aspirate fluid from the cell sample platform 40 through the first aspiration channel 11 and the second aspiration channel 14. The microscope objective 50 is adapted to observe the cell sample 2 on the cell sample platform 40.

[0044] Reference below Figure 1-Figure 4 The operation process of the microfluidic device 1 for local cell chemical stimulation and secretion detection according to an embodiment of the present invention is described.

[0045] Under observation through the microscope objective lens 50, the microfluidic probe 10 is adjusted to above the target cell sample 2 through the multi-axis positioning platform so that the center line a of the first injection channel 12 and the second injection channel 13 spans the target position of the target cell sample 2. It should be understood here that the "center line" refers to an imaginary straight line passing through the center point of the imaginary line connecting the first injection channel 12 and the second injection channel 13 and perpendicular to the imaginary line. The figure shows an embodiment in which the center line a is oriented along the front-to-back direction and is located at the center of the first injection channel 12 and the second injection channel 13 in the left-right direction. Those skilled in the art can adjust the "target position" according to experimental requirements.

[0046] The fluid driving device is used to inject the protective solution 3 and the irritant solution 4 into the first injection channel 12 and the second injection channel 13 respectively, and at the same time, the solutions are sucked from the first suction channel 11 and the second suction channel 14. Figure 2 As shown, the protective solution 3 and stimulating solution 4 injected through the first injection channel 12 and the second injection channel 13 respectively form closed solution micro-regions. The intermediate boundary between the two solution micro-regions remains stable due to the symmetrical fluctuation offset effect, so that the boundary is formed at the center line a, thereby forming the boundary at the target location of the cell sample 2. By positioning the target cell sample 2 on both sides of the boundary, one side of the cell sample 2 is protected by the protective solution 3 and the other side is stimulated by the stimulating solution 4, thereby stimulating a localized area of the single cell sample 2. At the same time, the fluid within the cell sample platform 40 is drawn back to the microfluidic probe 10 through the first suction channel 11 and the second suction channel 14, carrying the secretions generated by the stimulation of the cell sample 2 back to the microfluidic probe 10.

[0047] The first detection electrode assembly 20 and the second detection electrode assembly 30 are used to detect the concentration of target secretions in the first suction flow channel 11 and the second suction flow channel 14 .

[0048] The secretion measuring device is used to monitor the detection values of the first detection electrode assembly and the second detection electrode assembly in real time, and analyze the substance release dynamics of different subregions of the target sample cells.

[0049] For example, the dynamic changes in the concentration of target secretions released by the single-cell sample 2 can be tracked and monitored in real time based on the current signals detected by the first detection electrode assembly 20 and the second detection electrode assembly 30, and the dynamic information of the concentration of target secretions released in the local areas on both sides of the cell sample 2 can be analyzed to analyze the stimulation response behavior of the single-cell sample 2 in different local areas and the "one move affects the whole body" regulation mechanism.

[0050] According to the embodiment of the present invention, the microfluidic device 1 for local chemical stimulation and secretion detection of cells is provided with a microfluidic probe 10, a first detection electrode assembly 20, a second detection electrode assembly 30 and a secretion measuring device. The first detection electrode assembly 20, the second detection electrode assembly 30 and the secretion measuring device can be used to detect and monitor the secretion concentration of the cell sample 2 in real time while the microfluidic probe 10 injects and aspirates the solution into the cell sample platform 40. Compared with the microfluidic devices in the related art that can only stimulate cells, the microfluidic probe 10 can not only stimulate cells, but also track and monitor the dynamic changes of the target secretion concentration in real time, and has a fast response speed and high sensitivity.

[0051] Furthermore, by providing a first suction channel 11, a first injection channel 12, a second injection channel 13, and a second suction channel 14 on the microfluidic probe 10, the protective solution 3 and the stimulating solution 4 injected into the first injection channel 12 and the second injection channel 13 can each form a closed solution microregion, thereby protecting one side of the cell sample 2 with the protective solution 3 and stimulating the other side with the stimulating solution 4, thereby stimulating a localized region of the single-cell sample 2. Simultaneously, the secretions produced by the stimulated cell sample 2 will flow back into the first suction channel 11 and the second suction channel 14 under suction, enabling the first detection electrode assembly 20 and the second detection electrode assembly 30 to respectively detect the concentration of target secretions in different localized regions. Compared to microfluidic devices in related technologies, this facilitates analysis of the substance release dynamics of different subregions of the cell and analysis of the dynamic information of the target secretion concentrations released from localized regions on both sides of the cell, thereby analyzing the stimulation response behavior of single cells in different localized regions and the "one-touch-the-whole" regulatory mechanism.

[0052] In addition, the fluid distribution is controlled by the microfluidic probe 10. On the one hand, there are no requirements for the stimulation substances and cell types, and it is applicable to a variety of different research experiments. For example, damaging substances or neurotransmitters can be used to stimulate different areas of cells, so as to analyze the material changes during cell repair and regeneration, intercellular communication and regional differences of single cells. It has strong applicability. On the other hand, the microfluidic probe 10 does not directly contact the cells, and the mechanical damage to the cells is relatively small. Moreover, the first detection electrode assembly 20 and the second detection electrode assembly 30 can select a suitable electrochemical measurement system according to the properties of the target secretions, including but not limited to the detection of hydrogen peroxide, dopamine, acetylcholine, lactic acid, and uric acid, further improving the applicability of the microfluidic device 1 for local cell chemical stimulation and secretion detection.

[0053] Therefore, the cell local chemical stimulation and secretion detection microfluidic device 1 according to the embodiment of the present invention can chemically stimulate the local area of the cell and detect the secretions of different areas of the cell separately, and has the advantages of fast response speed, high sensitivity, strong applicability, and little mechanical damage to the cell.

[0054] The following describes a microfluidic device 1 for local cell chemical stimulation and secretion detection according to a specific embodiment of the present invention with reference to the accompanying drawings.

[0055] In some specific embodiments of the present invention, Figure 1-Figure 4 As shown, the microfluidic device 1 for local chemical stimulation of cells and secretion detection according to an embodiment of the present invention includes a multi-axis positioning platform, a microfluidic probe 10, a first detection electrode assembly 20, a second detection electrode assembly 30, a secretion measuring device, a fluid driving device, a cell sample platform 40 and a microscope objective lens 50.

[0056] Advantageously, as Figure 1 and Figure 2 As shown, the distance between the first injection channel 12 and the first suction channel 11 is equal to the distance between the second injection channel 13 and the second suction channel 14. This allows the four channels to be symmetrically arranged in pairs, and the four channels are symmetrically distributed around the center line a, so that the injected protective solution 3 and irritant solution 4 form a boundary at the center line a between the first injection channel 12 and the second injection channel 13.

[0057] Specifically, if Figure 1 and Figure 3 As shown, each of the first detection electrode assembly 20 and the second detection electrode assembly 30 includes a counter electrode 21, a working electrode 22, and a reference electrode 23. The counter electrode 21, the working electrode 22, and the reference electrode 23 are spaced apart and electrically connected to the secretion measurement device via multiple wires 60. This forms a three-electrode structure, facilitating the detection electrodes to detect changes in the concentration of the target secretion.

[0058] Specifically, the microfluidic probe 10 may have a base layer 15. The electrodes may be prepared on the base layer 15. Figure 3 As shown, the counter electrode 21, the working electrode 22, and the reference electrode 23 can be fixed to the surface of the substrate 15 by inkjet, screen printing, or other methods using conductive ink, and then the substrate 15 except for the electrode reaction area and the contact pad area is covered with an insulating coating. The electrode contact pad area is connected to the wire 60 via a conductive carbon paste and fixed with epoxy glue. The modified substrate 15 is bonded to the rest of the microfluidic probe 10. This makes it easier to prepare the detection electrode on the microfluidic probe 10.

[0059] More specifically, the fluid drive device includes an injection syringe pump, two injection syringes, a suction syringe pump, and two suction syringes. The two injection syringes are connected to the first injection channel 12 and the second injection channel 13, respectively, and are mounted on the injection syringe pump. The two suction syringes are connected to the first suction channel 11 and the second suction channel 14, respectively, and are mounted on the suction syringe pump. This allows the syringe pump to drive the syringes, further driving the fluids, achieving simultaneous injection into the two injection channels and simultaneous suction into the two suction channels, thereby facilitating the formation of a stable boundary between the protective solution 3 and the irritant solution 4 at the midline a.

[0060] Optionally, the multi-axis positioning platform is a three-axis positioning platform with three degrees of freedom: up and down, left and right, and front and back. This allows the microfluidic probe 10 to be adjusted in three directions, such as up and down, left and right, and front and back, making it easier to move the microfluidic probe 10 to a desired position.

[0061] Furthermore, if Figure 1 and Figure 2 As shown, the cell sample platform 40 includes a temperature-controlled stage 41 and a culture dish 42. The temperature-controlled stage 41 is movable in two degrees of freedom, left and right and front and back, and the temperature is adjustable. The culture dish 42 is placed on the temperature-controlled stage 41. The culture dish 42 is suitable for holding the protective solution 3 and for planting the cell sample 2. Specifically, the cell sample 2 is planted at a density of 100-10,000 cells per square centimeter. This makes it easy to move the target cells to a position suitable for observation by the microscope objective 50, and also makes it easy to control the temperature of the culture environment and adjust the temperature required for the experiment, for example, maintaining a culture environment of 37 degrees Celsius.

[0062] In some embodiments, the first injection channel 12 and the second injection channel 13 are respectively adapted to inject a phorbol ester solution and a protective solution. Specifically, the protective solution 3 may be a culture medium. In this way, the phorbol ester can activate specific signaling pathways of the cell, generating excess hydrogen peroxide as a final product that is released into the extracellular environment. A redox reaction occurs at the electrode, generating a change in the current signal. By observing the change in the current signal of the electrode in real time, the dynamic information of hydrogen peroxide release in the localized regions on both sides of the cell can be analyzed, thereby analyzing the stimulus response behavior between different regions of the single cell.

[0063] In some embodiments, the first and second detection electrode assemblies 20 and 30 are configured to detect hydrogen peroxide concentrations. For example, metal nanoparticles can be modified through electrochemical deposition to enhance the electrodes' electrochemical sensing performance for hydrogen peroxide. Using the chronoamperometry program of the secretion measurement device, a range of hydrogen peroxide standard concentrations can be sampled to establish a linear relationship between hydrogen peroxide concentration and measured current values. This facilitates targeted detection of target secretions.

[0064] More advantageously, if Figure 1 As shown, the length directions of the first injection channel 12, the second injection channel 13, the first suction channel 11, and the second suction channel 14 are all oriented in the vertical direction. This can avoid the generation of components tilted in the vertical direction during fluid injection and suction, and facilitate the formation of a stable boundary at the center line a of the first injection channel 12 and the second injection channel 13.

[0065] The following describes an operating method of the microfluidic device 1 for localized cell chemical stimulation and secretion detection according to the above embodiment of the present invention, comprising the following steps:

[0066] Under observation through the microscope objective lens 50, the microfluidic probe 10 is adjusted to above the target cell sample 2 through the multi-axis positioning platform so that the center line a of the first injection channel 12 and the second injection channel 13 spans the target position of the target cell sample 2;

[0067] The fluid driving device is used to inject the protective solution 3 and the irritant solution 4 into the first injection channel 12 and the second injection channel 13 respectively, and simultaneously aspirates the solutions from the first suction channel 11 and the second suction channel 14;

[0068] Detecting the concentration of target secretions in the first suction channel 11 and the second suction channel 14 using the first detection electrode assembly 20 and the second detection electrode assembly 30;

[0069] The secretion measuring device is used to monitor the detection values of the first detection electrode assembly 20 and the second detection electrode assembly 30 in real time, and analyze the substance release dynamics of different subregions of the target sample cells 2.

[0070] According to the operating method of the microfluidic device 1 for local chemical stimulation and secretion detection of cells according to the embodiment of the present invention, by utilizing the microfluidic device 1 for local chemical stimulation and secretion detection of cells according to the above-mentioned embodiment of the present invention, it is possible to chemically stimulate the local area of the cell and detect the secretions of different areas of the cell separately, which has the advantages of fast response speed, high sensitivity, strong applicability, and little mechanical damage to the cell.

[0071] Specifically, before adjusting the position of the microfluidic probe 10, the following steps may be further included:

[0072] Under the observation of the microscope objective lens 50 , the position of the temperature-controlled stage 41 is adjusted to move the target cell sample 2 to a desired position.

[0073] After adjusting the horizontal position of the microfluidic probe 10 , the distance between the microfluidic probe 10 and the cell sample 2 in the vertical direction may also be adjusted, preferably 10-100 microns.

[0074] Other configuration examples and operations of the microfluidic device 1 for localized cell chemical stimulation and secretion detection and its operation method according to the embodiment of the present invention are well known to those skilled in the art and will not be described in detail here.

[0075] Throughout this specification, reference to terms such as "one embodiment," "some embodiments," "illustrative embodiments," "examples," "specific examples," or "some examples" means that a specific feature, structure, material, or characteristic described in conjunction with that embodiment or example is included in at least one embodiment or example of the present invention. In this specification, illustrative uses of the above terms do not necessarily refer to the same embodiment or example. Furthermore, the specific features, structures, materials, or characteristics described may be combined in any suitable manner in any one or more embodiments or examples.

[0076] While embodiments of the present invention have been shown and described, it will be appreciated by those skilled in the art that various changes, modifications, substitutions, and variations may be made to the embodiments without departing from the principles and spirit of the invention, and that the scope of the invention is defined by the claims and their equivalents.

Claims

1. A microfluidic device for local chemical stimulation of cells and detection of secretions, characterized in that: include: Multi-axis positioning platform; A microfluidic probe, the microfluidic probe being arranged on the multi-axis positioning platform, and being provided with a first suction channel, a first injection channel, a second injection channel, and a second suction channel, which are arranged in sequence and spaced apart in a horizontal direction; a first detection electrode assembly and a second detection electrode assembly, wherein the first detection electrode assembly is disposed in the first suction flow channel, and the second detection electrode assembly is disposed in the second suction flow channel; a secretion measuring device, the secretion measuring device being electrically connected to the first detection electrode assembly and the second detection electrode assembly respectively; a fluid driving device, the fluid driving device being in communication with the first suction channel, the first injection channel, the second injection channel, and the second suction channel, respectively; a cell sample platform, located below the microfluidic probe and suitable for planting cell samples; A microscope objective lens is located below the cell sample platform.

2. The microfluidic device for local cell chemical stimulation and secretion detection according to claim 1, characterized in that: A distance between the first injection flow channel and the first suction flow channel is equal to a distance between the second injection flow channel and the second suction flow channel.

3. The microfluidic device for local cell chemical stimulation and secretion detection according to claim 1, characterized in that: Each of the first detection electrode assembly and the second detection electrode assembly includes a counter electrode, a working electrode and a reference electrode. The counter electrode, the working electrode and the reference electrode are spaced apart and electrically connected to the secretion measuring device through a plurality of wires.

4. The microfluidic device for local cell chemical stimulation and secretion detection according to claim 1, characterized in that: The fluid driving device comprises: Injection syringe pump; Two injection syringes, the two injection syringes are respectively connected to the first injection channel and the second injection channel, and the two injection syringes are arranged on the injection syringe pump; Aspiration syringe pump; Two suction syringes are respectively connected to the first suction flow channel and the second suction flow channel, and the two suction syringes are arranged on the suction syringe pump.

5. The microfluidic device for local cell chemical stimulation and secretion detection according to claim 1, characterized in that: The multi-axis positioning platform is a three-axis positioning platform with three degrees of freedom: up and down, left and right, and front and back.

6. The microfluidic device for local cell chemical stimulation and secretion detection according to claim 1, characterized in that: The cell sample platform comprises: A temperature-controlled stage, which is movable in two degrees of freedom, left-right and front-back, and has adjustable temperature; A culture dish is placed on the temperature-controlled stage, and the culture dish is suitable for containing a protective solution and for planting cell samples.

7. The microfluidic device for local cell chemical stimulation and secretion detection according to claim 1, characterized in that: The first injection channel and the second injection channel are suitable for injecting phorbol ester solution and protective solution respectively.

8. The microfluidic device for local cell chemical stimulation and secretion detection according to claim 1, characterized in that: The first detection electrode assembly and the second detection electrode assembly are configured to detect the concentration of hydrogen peroxide.

9. The microfluidic device for local cell chemical stimulation and secretion detection according to claim 1, characterized in that: The length directions of the first injection flow channel, the second injection flow channel, the first suction flow channel, and the second suction flow channel are all oriented in the up-down direction.

10. A method for operating a microfluidic device for localized cell chemical stimulation and secretion detection according to any one of claims 1 to 9, characterized in that: The following steps are involved: Under observation through the microscope objective lens, the microfluidic probe is adjusted to above the target cell sample by the multi-axis positioning platform so that the midlines of the first injection channel and the second injection channel cross the target position of the target cell sample; injecting a protective solution and an irritant solution into the first injection channel and the second injection channel respectively by using the fluid driving device, and simultaneously sucking solutions from the first suction channel and the second suction channel; detecting the concentration of target secretions in the first suction channel and the second suction channel using the first detection electrode assembly and the second detection electrode assembly; The secretion measuring device is used to monitor the detection values of the first detection electrode assembly and the second detection electrode assembly in real time, and analyze the substance release dynamics of different subregions of the target sample cells.

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