A microfluidic chip, chip-mass spectrometry coupling system and experimental method

Abstract

The application discloses a microfluidic chip, a chip-mass spectrometry coupling system and an experimental method, relates to the technical field of microfluidics, and the microfluidic chip comprises a cell culture cavity used for culturing cells; a flushing channel is used for guiding a dispersed phase to the cell culture cavity; a collection channel is used for collecting cell secretions generated from the cell culture cavity; a droplet forming unit receives the cell secretions collected by the collection channel, and the droplet forming unit is connected with a continuous phase with a first flow rate, so that the cell secretions received by the droplet forming unit form droplets, wherein the first flow rate is adapted to the flow rate of the dispersed phase. The application realizes a non-contact collection mode of cell secretions by arranging the cell culture cavity; the droplet forming unit makes the cell secretions form more than five droplet samples per second, realizes a time resolution of 200 ms and below, and facilitates researchers to accurately detect and analyze the process change of recovered and degraded neurotransmitters in real time.

Description

Technical Field

[0001] This application relates to the field of microfluidics, and in particular to a microfluidic chip. Background Technology

[0002] The microfluidic chip-mass spectrometry coupling system is an advanced technology that combines the low sample consumption and high throughput advantages of microfluidic devices with the high sensitivity of mass spectrometry. Microfluidic devices, due to their superior cell culture conditions, low-volume systems, and precise control over the extracellular environment, can construct stable in vitro cell analysis models, making them suitable for studying neurotransmitter release. Simultaneously, the advantages of mass spectrometry allow for qualitative and quantitative analysis of multiple neurotransmitters, making it suitable for real-time analysis of neurotransmitter signals.

[0003] The low concentration of neurotransmitters in samples is a common problem. To improve sensitivity and analytical efficiency, microdialysis systems are often used to enrich samples before analysis. However, traditional microdialysis methods have low time resolution, typically ranging from several minutes to tens of minutes, which is detrimental to studying the release process of neurotransmitters. Therefore, a microfluidic chip-mass spectrometry coupling system has been successfully used for real-time dynamic acquisition and analysis of neurotransmitter signals. Based on microchannel design and combined with low-flow electrospray ionization-tandem mass spectrometry (ESI-MS / MS) technology, microfluidic chip-mass spectrometry can successfully acquire samples at a rate of several samples per second, achieving a time resolution of 8-11 seconds, for detecting real-time dynamic changes in neurotransmitters, realizing neurotransmitter detection with a time resolution of seconds. To maintain the normal function of neurons, the level of neurotransmitters released in the intercellular space is naturally regulated by neurotransmitter recycling and degradation mechanisms. Depending on the type of neuron and neurotransmitter, this regulatory mechanism can recycle or degrade released neurotransmitters within milliseconds to tens of seconds.

[0004] However, the current time resolution and analysis efficiency of microfluidic-mass spectrometry technology can only achieve a time resolution of 8-11 seconds. For neurotransmitters whose recovery and degradation occur within a few seconds or milliseconds (for example, the average recovery time of dopamine is 2 seconds, and the degradation or recovery of neurotransmitters such as acetylcholine, glutamate, and γ-aminobutyric acid are at the millisecond level), researchers find it difficult to accurately detect and analyze the process changes of these neurotransmitters. Summary of the Invention

[0005] This application aims to address at least one of the technical problems existing in the prior art. To this end, this application proposes a microfluidic chip capable of real-time mass spectrometry analysis of neurotransmitters whose recovery and degradation occur within seconds or milliseconds.

[0006] This application also proposes a chip-mass spectrometry coupling system and experimental method using the above-mentioned microfluidic chip.

[0007] A microfluidic chip according to one embodiment of this application includes: a cell culture chamber for culturing cells; a flushing channel having a first inlet at one end and the other end connected to the cell culture chamber; the first inlet for introducing a dispersed phase into the flushing channel; the flushing channel for guiding the dispersed phase to the cell culture chamber; a collection channel connected to the cell culture chamber and for collecting cell secretions generated from the cell culture chamber; and a droplet forming unit connected to the collection channel and for receiving the cell secretions collected by the collection channel. The droplet forming unit includes an outlet and a continuous phase inlet, the continuous phase inlet for introducing a continuous phase having a first flow rate, so that the cell secretions received by the droplet forming unit form droplets, and the droplets can be discharged to the outside of the microfluidic chip through the outlet.

[0008] According to one embodiment of this application, the flushing channel includes multiple columns, which connect to the cell culture chamber. The multiple columns are arranged on the cell culture chamber for simultaneously introducing the dispersed phase into various locations of the cell culture chamber. The cell culture chamber includes a second inlet and a third inlet for introducing cell suspension into the cell culture chamber.

[0009] According to one embodiment of this application, the collection channel includes a main channel and multiple sub-channels. The multiple sub-channels are connected to the cell culture chamber and are arranged on the cell culture chamber for simultaneously receiving cell secretions generated at various locations in the cell culture chamber. One end of the main channel is connected to the droplet forming unit, and the other end is connected to the sub-channels. The main channel is used to receive cell secretions from the sub-channels and to introduce cell secretions into the droplet forming unit.

[0010] According to one embodiment of this application, the droplet forming unit includes a continuous phase channel and an outlet channel. The continuous phase channel is connected to the continuous phase inlet and is used to introduce the continuous phase. Cell secretions introduced into the droplet forming unit through the main channel form droplets under the action of the continuous phase. The outlet channel is used to receive droplets and lead to the outlet.

[0011] According to one embodiment of this application, there are two continuous phase channels and one outlet channel. The extension direction of the outlet channel is the same as the extension direction of the main channel. The extension directions of the two continuous phase channels are the same, and the extension directions of the two continuous phase channels are perpendicular to the extension direction of the outlet channel.

[0012] A chip-mass spectrometry coupling system according to another embodiment of this application includes: the microfluidic chip described above; a first micro-injection pump connected to a first inlet of a flushing channel for introducing a dispersed phase into the flushing channel; a second micro-injection pump connected to a continuous phase inlet of a droplet forming unit for introducing a continuous phase into the droplet forming unit; an electrospray ionization device for ionizing droplets flowing out of the droplet forming unit to form a spray; a third micro-injection pump connected to the electrospray ionization device for introducing a methanol solution into the electrospray ionization device; and a mass spectrometer for detecting the spray formed by the electrospray ionization device.

[0013] According to another embodiment of this application, the electrospray ionization device includes a double-layer capillary structure, which includes an inner capillary and an outer capillary, with the inner capillary nested inside the outer capillary. One end of the inner capillary is used to receive droplets generated by the droplet forming unit, and the inner wall of the other end is etched and modified with C-18. The outer capillary is used to receive methanol solution introduced by a third micro-injection pump and to merge the methanol solution with the droplets passing through the inner capillary.

[0014] According to another aspect of this application, the experimental method of the chip-mass spectrometry coupling system using the above-mentioned chip-mass spectrometry coupling system includes the following steps: S1, sealing the second and third inlets and the continuous phase inlet located in the cell culture chamber, opening the first inlet and the outlet, and introducing PBS solution into the first inlet using a first micro-injection pump to fill the microfluidic chip with PBS solution; S2, sealing the first inlet, the continuous phase inlet and the outlet, and opening the second and third inlets located in the cell culture chamber; drawing out the PBS solution from the cell culture chamber from the third inlet and adding the cell suspension from the second inlet; S3, blocking the second and third inlets and performing adherent culture of the cells in the cell suspension; S4, introducing a dispersed phase with a second flow rate into the first inlet using a first micro-injection pump, and introducing the second micro-injection pump... S5. A continuous phase with a first flow rate is introduced into the continuous phase inlet by a jet pump; S6. Potassium chloride solution is added into the cell culture chamber through the third inlet to stimulate the cells; the dispersed phase enters the cell culture chamber through the flushing channel to remove cell secretions, and enters the droplet forming unit through the collection channel. The second micro-injection pump is turned on to introduce the continuous phase into the droplet forming unit to compress the dispersed phase and form droplets; S7. The electrospray ionization device and the third micro-injection pump are turned on. The third micro-injection pump introduces methanol solution into the electrospray ionization device; the droplets are introduced into the electrospray ionization device, and after the polar components are removed by the etched and modified C-18 inner capillary, they are combined with the methanol solution. The combined solution forms a spray under the action of the electrospray ionization device and enters the mass spectrometer for detection; S8. The above steps S1-S6 are repeated for repeated detection.

[0015] According to another aspect of this application, the dispersed phase is an ammonium formate solution with a concentration of 140 mmol / L, and the continuous phase is a volatile droplet-generating oil containing 2% perfluorinated surfactant; the second flow rate of the dispersed phase is 900-41400 μL / min, and the first flow rate of the continuous phase is 450-41400 μL / min.

[0016] According to another embodiment of this application, the cell suspension is a PC-12 cell suspension with a cell density of 2 × 10⁻⁶ cells. 6 cells / mL.

[0017] The microfluidic chip according to the embodiments of this application has at least the following beneficial effects: a cell culture chamber is set in the microfluidic chip, and a dispersed phase is introduced into the cell culture chamber to realize a non-invasive and non-contact collection method for cell secretions; by introducing a continuous phase into the droplet forming unit, more than 5 droplet samples of cell secretions are formed per second, thereby achieving a time resolution of 200 ms or less, which facilitates researchers to accurately detect and analyze the process changes of neurotransmitters with fast recovery and degradation rates in real time.

[0018] Additional aspects and advantages of this application will be set forth in part in the description which follows, and in part will be obvious from the description, or may be learned by practice of this application. Attached Figure Description

[0019] The present application will be further described below with reference to the accompanying drawings and embodiments, wherein:

[0020] Figure 1 This is a microfluidic chip according to one embodiment of this application;

[0021] Figure 2 This is a chip-mass spectrometry coupling system according to another embodiment of this application;

[0022] Figure 3 This is a partial structural diagram of the electrospray ionization device and mass spectrometer of a chip-mass spectrometry coupling system according to another embodiment of this application;

[0023] Figure 4 This is a partial internal structural diagram of the electrospray ionization device of a chip-mass spectrometry coupling system according to another embodiment of this application.

[0024] Figure 5 The mass spectrum of a PBS solution mixed with a 1:9 methanol solution for testing dopamine using an nESI ion source.

[0025] Figure 6 The mass spectrum of the PBS solution used to test dopamine in this application embodiment.

[0026] Figure label:

[0027] 1000, Microfluidic chip; 2000, Chip-mass spectrometry coupling system;

[0028] 100. Cell culture chamber; 110. Second inlet; 120. Third inlet;

[0029] 200. Flushing channel; 210. First inlet; 220. Column; 230. L-shaped channel; 240. Straight channel;

[0030] 300. Collection Channel; 310. Main Channel; 320. Sub-channel;

[0031] 400, Droplet forming unit; 410, Outlet; 420, Continuous phase inlet; 430, Fourth capillary; 440, Outlet channel; 450, Continuous phase channel;

[0032] 500. First micro-injection pump; 510. First capillary tube;

[0033] 600. Second micro-injection pump; 610. Second capillary tube;

[0034] 700. Electrospray ionization device; 710. Inner capillary; 711. C-18 etched modification layer; 720. Outer capillary;

[0035] 800. Third micro-injection pump; 810. Third capillary tube;

[0036] 900. Mass spectrometer. Detailed Implementation

[0037] The embodiments of this application are described in detail below. Examples of these embodiments are shown in the accompanying drawings, wherein the same or similar reference numerals denote the same or similar elements or elements having the same or similar functions throughout. The embodiments described below with reference to the accompanying drawings are exemplary and are only used to explain this application, and should not be construed as limiting this application.

[0038] In the description of this application, it should be understood that the orientation descriptions, such as up, down, front, back, left, right, etc., indicate the orientation or positional relationship based on the orientation or positional relationship shown in the accompanying drawings. They are only for the convenience of describing this application and simplifying the description, and do not indicate or imply that the device or element referred to must have a specific orientation, or be constructed and operated in a specific orientation. Therefore, they should not be construed as limitations on this application.

[0039] In the description of this application, "several" means one or more, "multiple" means two or more, "greater than," "less than," and "exceeding" are understood to exclude the stated number, while "above," "below," and "within" are understood to include the stated number. The use of "first" and "second" in the description is merely for distinguishing technical features and should not be construed as indicating or implying relative importance, or implicitly indicating the number of indicated technical features, or implicitly indicating the order of the indicated technical features.

[0040] In the description of this application, unless otherwise expressly defined, terms such as "setup," "installation," and "connection" should be interpreted broadly, and those skilled in the art can reasonably determine the specific meaning of the above terms in this application in conjunction with the specific content of the technical solution.

[0041] In the description of this application, the terms "one embodiment," "some embodiments," "illustrative embodiment," "example," "specific example," "some examples," etc., refer to specific features, structures, materials, or characteristics described in connection with that embodiment or example, which are included in at least one embodiment or example of this application. In this specification, the illustrative expressions 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 one or more embodiments or examples.

[0042] The microfluidic chip-mass spectrometry coupling system is an advanced technology that combines the low sample consumption and high throughput advantages of microfluidic devices with the high sensitivity of mass spectrometry. Microfluidic devices, due to their superior cell culture conditions, low-volume systems, and precise control over the extracellular environment, can construct stable in vitro cell analysis models, making them suitable for studying neurotransmitter release. Simultaneously, the advantages of mass spectrometry allow for qualitative and quantitative analysis of multiple neurotransmitters, making it suitable for real-time analysis of neurotransmitter signals.

[0043] The low concentration of neurotransmitters in samples is a common problem. To improve sensitivity and analytical efficiency, microdialysis systems are often used to enrich samples before analysis. However, traditional microdialysis methods have low time resolution, typically ranging from several minutes to tens of minutes, which is detrimental to studying the release process of neurotransmitters. Therefore, a microfluidic chip-mass spectrometry coupling system has been successfully used for real-time dynamic acquisition and analysis of neurotransmitter signals. Based on microchannel design and combined with low-flow electrospray ionization-tandem mass spectrometry (ESI-MS / MS) technology, microfluidic chip-mass spectrometry can successfully acquire samples at a rate of several samples per second, achieving a time resolution of 8-11 seconds, for detecting real-time dynamic changes in neurotransmitters, realizing neurotransmitter detection with a time resolution of seconds. To maintain the normal function of neurons, the level of neurotransmitters released in the intercellular space is naturally regulated by neurotransmitter recycling and degradation mechanisms. Depending on the type of neuron and neurotransmitter, this regulatory mechanism can recycle or degrade released neurotransmitters within milliseconds to tens of seconds.

[0044] However, the current time resolution and analysis efficiency of microfluidic-mass spectrometry technology can only achieve a time resolution of 8-11 seconds. For neurotransmitters whose recovery and degradation occur within a few seconds or milliseconds (for example, the average recovery time of dopamine is 2 seconds, while the degradation or recovery of neurotransmitters such as acetylcholine, glutamate, and γ-aminobutyric acid are at the millisecond level), researchers find it difficult to accurately detect and analyze process changes.

[0045] To address the aforementioned issues, this application provides a microfluidic chip 1000, which can be used, but is not limited to, for detecting and analyzing process changes in neurotransmitters. The microfluidic chip 1000 includes a cell culture chamber 100, through which a dispersed phase is introduced to achieve a non-invasive and non-contact acquisition method for cell secretions. By setting the microchannel size of the microfluidic chip 1000 and introducing a continuous phase with a flow rate adapted to the dispersed phase flow rate into the droplet forming unit 400, more than five droplet samples of cell secretions are formed per second at the droplet forming unit 400, thereby achieving a time resolution of 200 ms or less. This facilitates researchers in accurately detecting and analyzing process changes in neurotransmitters with rapid recovery and degradation rates in real time.

[0046] Please see Figure 1 and Figure 2 This embodiment uses Figure 1 and Figure 2 The first direction is the A direction of the coordinate axis, and the second direction is the B direction of the coordinate axis. The first and second directions are perpendicular to each other.

[0047] Please see Figure 1One embodiment of this application provides a microfluidic chip 1000, which includes a cell culture chamber 100, a flushing channel 200, a collection channel 300, and a droplet forming unit 400. The cell culture chamber 100 is used for culturing cells. A flushing channel 200 has a first inlet 210 at one end and is connected to the cell culture chamber 100 at the other end. The first inlet 210 is used to introduce a dispersed phase into the flushing channel 200. The flushing channel 200 is used to guide the dispersed phase into the cell culture chamber 100. A collection channel 300 is connected to the cell culture chamber 100 and is used to collect cell secretions generated from the cell culture chamber 100. A droplet forming unit 400 is connected to the collection channel 300 and is used to receive the cell secretions collected by the collection channel 300. The droplet forming unit 400 includes an outlet 410 and a continuous phase inlet 420. The continuous phase inlet 420 is used to introduce a continuous phase with a first flow rate so that the cell secretions received by the droplet forming unit 400 form droplets. The droplets can pass through the outlet 410 to the outside of the microfluidic chip 1000.

[0048] like Figure 1 As shown, the microfluidic chip 1000 has a flushing channel 200, which is formed as an L-shaped channel 230 and a straight channel 240. The L-shaped channel 230 has a long straight channel and a short straight channel, with the long straight channel arranged along a first direction and the short straight channel arranged along a second direction. The first end of the long straight channel has a first inlet 210, and the second end of the long straight channel is connected to the first end of the short straight channel. The straight channel 240 is arranged along the first direction, and the second end of the short straight channel is in the middle of the straight channel 240. In this embodiment, the angle between the long straight channel and the short straight channel is 90°, and the angle between the short straight channel and the straight channel 240 is 90°. The cell culture chamber 100 forms a straight channel, which is parallel to and connected to the straight channel 240 of the aforementioned flushing channel 200. The cell culture chamber 100 is also connected to a collection channel 300, which is connected to a droplet forming unit 400. Calculations show that the long straight channel of L-shaped channel 230 is 25 mm long, the short straight channel of L-shaped channel 230 is 3 mm long, the straight channel of L-shaped channel 240 is 50 mm long, 1.2 mm wide, and 0.3 mm deep; the cell culture chamber 100 is 1 mm wide, 50 mm long, 0.6 mm deep, and has a volume of 30 μL.

[0049] The dispersed phase is introduced through the first inlet 210, then into the flushing channel 200, and continues into the cell culture chamber 100. The dispersed phase carries away cell secretions from the cell culture chamber 100, further converging into the collection channel 300. The collection channel 300 then guides the dispersed phase into the droplet forming unit 400. The droplet forming unit 400 is equipped with an outlet 410 and a continuous phase inlet 420. The continuous phase, at a flow rate adapted to the dispersed phase, is introduced into the droplet forming unit 400 from the continuous phase inlet 420, causing the dispersed phase to form droplets. The droplet forming unit 400 then guides the droplets through the outlet 410 to the outside of the microfluidic chip 1000, facilitating further processing of the droplets by other experimental equipment or detection and analysis devices, including mass spectrometers, to detect and analyze the process changes of cell secretions.

[0050] Please see Figure 1 In some embodiments of this application, the flushing channel 200 includes a plurality of columns 220, which connect to the cell culture chamber 100. The columns 220 are arranged on the cell culture chamber 100 to simultaneously introduce the dispersed phase into various locations within the cell culture chamber 100. The cell culture chamber 100 includes a second inlet 110 and a third inlet 120, which are used to introduce cell suspension into the cell culture chamber 100. That is, as... Figure 1 As shown, the straight channel 240 in the aforementioned flushing channel 200 is connected to the cell culture chamber 100 via multiple pillars 220. All pillars 220 are arranged along the second direction, with their ends connected to the parallel cell culture chamber 100 and the straight channel 240, respectively. The pillars 220 are evenly distributed at equal intervals on the cell culture chamber 100. When the dispersed phase is introduced into the flushing channel 200, the multiple pillars 220 simultaneously guide the dispersed phase into the cell culture chamber 100, ensuring uniform flow of the dispersed phase into the cell culture chamber 100. In the embodiment of this application, a total of 16 pillars 220 connect the cell culture chamber 100 and the straight channel 240. Calculations show that the width of each pillar 220 is 0.15 mm, its length is 0.9 mm, and its depth is 0.3 mm.

[0051] Please see Figure 1 In some embodiments of this application, the collection channel 300 includes a main channel 310 and multiple sub-channels 320. The multiple sub-channels 320 are connected to the cell culture chamber 100 and are arranged on the cell culture chamber 100 to simultaneously receive cell secretions generated at various locations in the cell culture chamber 100. One end of the main channel 310 is connected to the droplet forming unit 400, and the other end is connected to the sub-channels 320. The main channel 310 is used to receive cell secretions from the sub-channels 320 and to introduce cell secretions into the droplet forming unit 400.

[0052] In other words, such as Figure 1 As shown, the collection channel 300 includes a main channel 310 and branch channels 320. The branch channels 320 are arranged along the second direction and are presented in a tree-branch shape. In this embodiment, the branch channels 320 include 16 primary collection channels, 8 secondary collection channels, 4 tertiary collection channels, and 2 quaternary collection channels. The first end of the 16 primary collection channels is connected to the cell culture chamber 100, and the second end of two adjacent primary collection channels is connected to a secondary collection channel. Among the 8 secondary collection channels, two adjacent secondary collection channels are connected to a tertiary collection channel, and so on. Finally, two quaternary collection channels are connected to the main channel 310. The structural design of the main channel 310 and the branch channels 320 can achieve optimal flow rate uniformity of the collected dispersed phase. Specifically, it can ensure that all channels of the microfluidic chip 1000 pass through the liquid in a laminar flow manner, that is, ensure that the Reynolds number is consistent at every point. To achieve this effect, the corresponding dimensions of the primary, secondary, tertiary, and quaternary collection channels in channel 320 were calculated: the primary collection channel has a width of 0.15 mm and a length of 1.2 mm; the secondary collection channel has a width of 0.3 mm and a length of 1.2 mm; the tertiary collection channel has a width of 0.6 mm and a length of 1.2 mm; and the quaternary collection channel has a width of 1.2 mm, a length of 1.2 mm, and a depth of 0.3 mm.

[0053] The main channel 310 includes a crossflow channel arranged along a first direction and a vertical flow channel arranged along a second direction. The main channel 310 is used to connect the branch channel 320 to the droplet forming unit 400. One end of the vertical flow channel is connected to two four-stage collection channels, and the other end is connected to the crossflow channel. The crossflow channel is formed into a narrow opening at the tail end connected to the droplet forming unit 400 to accelerate the flow rate of the dispersed phase into the droplet forming unit 400. Calculations show that the width of the vertical flow channel is 2.4 mm and the length is 5 mm, the width of the crossflow channel is 2.4 mm and the length is 25 mm, and the width of the crossflow channel gradually narrows to 1.6 mm at the tail end, with a depth of 0.3 mm.

[0054] Furthermore, the droplet forming unit 400 includes a continuous phase channel 450 and an outlet channel 440. The continuous phase channel 450 is connected to the continuous phase inlet 420 and is used to introduce the continuous phase. Cell secretions introduced into the droplet forming unit 400 through the main channel 310 form droplets under the action of the continuous phase. The outlet channel 440 is used to receive droplets and lead to the outlet 410. There are two continuous phase channels 450 and one outlet channel 440. The extension direction of the outlet channel 440 is the same as the extension direction of the main channel 310. The extension directions of the two continuous phase channels 450 are the same and perpendicular to the extension direction of the outlet channel 440.

[0055] In other words, such as Figure 1 As shown, the droplet forming unit 400 has two continuous phase channels 450, and correspondingly, two continuous phase inlets 420. Both continuous phase channels 450 are arranged along the second direction. The droplet forming unit 400 has one outlet channel 440, and correspondingly, one outlet 410. The outlet channel 440 is arranged along the first direction. The two continuous phase channels 450, the outlet channel 440, and the main channel 310 of the collection channel 300 together form a cross-channel structure, i.e., a flow focusing structure. This structure has the following advantages: it can generate smaller droplets, the generated droplets have higher temporal resolution and monodispersity, and the geometric versatility of the structure design makes it more adaptable to complex application requirements.

[0056] It is understood that the number of continuous phase channels 450 in the droplet forming unit 400 can be one. When there is only one continuous phase channel 450, the continuous phase channel 450, the outlet channel 440, and the main channel 310 of the collection channel 300 together form a T-shaped channel structure. This structure can also be used to form droplets. In order to achieve better beneficial effects, this embodiment specifically sets two continuous phase channels 450, which can generate smaller droplets and achieve droplet generation with higher time resolution.

[0057] Please see Figure 2 Another embodiment of this application provides a chip-mass spectrometry coupling system 2000, including a microfluidic chip 1000 as described above, a first micro-injection pump 500, a second micro-injection pump 600, a third micro-injection pump 800, an electrospray ionization device 700, and a mass spectrometer 900. The first micro-injection pump 500 is connected to the first inlet 210 of the flushing channel 200 and is used to introduce a dispersed phase into the flushing channel 200; the second micro-injection pump 600 is connected to the continuous phase inlet 420 of the droplet forming unit 400 and is used to introduce a continuous phase into the droplet forming unit 400; the electrospray ionization device 700 is used to ionize the droplets flowing out of the droplet forming unit 400 to form a spray; the third micro-injection pump 800 is connected to the electrospray ionization device 700 and is used to introduce a methanol solution into the electrospray ionization device 700; and the mass spectrometer 900 is used to detect the spray formed by the electrospray ionization device 700.

[0058] like Figure 2As shown, a first micro-injection pump 500 is connected to the first inlet 210 of the flushing channel 200, and is used to introduce the dispersed phase into the flushing channel 200 through the first capillary 510. A second micro-injection pump 600 is connected to the continuous phase inlet 420 of the droplet forming unit 400, and is used to introduce the continuous phase into the droplet forming unit 400 through the second capillary 610. The continuous phase merges with the dispersed phase in the droplet forming unit 400 and compresses the dispersed phase to form droplets. A third micro-injection pump 800 is connected to the electrospray ionization device 700, and is used to introduce methanol solution into the electrospray ionization device 700 through the third capillary 810. The droplets formed by the compression of the continuous phase flow to the outlet 410 under the guidance of the outlet channel 440 of the droplet forming unit 400. After flowing to the outlet 410, the droplets are then introduced into the electrospray ionization device 700 through the fourth capillary 430. The electrospray ionization device 700 guides the incoming methanol solution and droplets, causing them to merge. The methanol solution is used to assist in the ionization of the droplets. The DC voltage in the electrospray ionization device 700 causes the merged methanol solution and droplets to form a spray, which is then received and detected by the mass spectrometer 900.

[0059] Please see Figure 3 and Figure 4 In the aforementioned chip-mass spectrometry coupling system 2000, the electrospray ionization device 700 includes a double-layer capillary structure, which includes an inner capillary 710 and an outer capillary 720. The inner capillary 710 is nested inside the outer capillary 720. One end of the inner capillary 710 is used to receive droplets generated by the droplet forming unit 400, and the inner wall of the other end is etched and modified with C-18. The outer capillary 720 is used to receive the methanol solution introduced by the third micro-injection pump 800 and to merge the methanol solution with the droplets passing through the inner capillary 710. In other words, the double-layer capillary structure of the electrospray ionization device 700 includes an inner capillary 710 and an outer capillary 720. The first end of the inner capillary 710 is connected to a fourth capillary 430 to receive droplets, and the second end of the inner capillary 710 leads to the mass spectrometer 900. A C-18 etching modification layer 711 is etched and modified on the inner wall of the second end of the inner capillary 710. The C-18 etching modification layer 711 is used to adsorb polar components such as salts and proteins in the droplets to improve the sensitivity and accuracy of subsequent mass spectrometer 900 analysis. The first end of the outer capillary 720 is connected to a third capillary 810 to receive methanol solution, and the second end of the outer capillary 720 leads to the mass spectrometer 900. The distance between the second end of the outer capillary 720 and the inlet of the mass spectrometer 900 is approximately 5. mm; the inner capillary 710 is nested inside the outer capillary 720, and the droplets flow out from the inner capillary 710 and merge with the methanol solution located in the outer capillary 720.

[0060] In view of this, another embodiment of this application discloses an experimental method for a chip-mass spectrometry coupling system, using the chip-mass spectrometry coupling system 2000 as described above, including the following steps:

[0061] Step S1: Block the second inlet 110 and the third inlet 120 and the continuous phase inlet 420 located in the cell culture chamber 100, open the first inlet 210 and the outlet 410, and introduce PBS solution into the first inlet 210 by the first micro-injection pump 500 so that the PBS solution fills the microfluidic chip 1000.

[0062] Step S2: Seal the first inlet 210, the continuous phase inlet 420, and the outlet 410, and open the second inlet 110 and the third inlet 120 located in the cell culture chamber 100; slowly aspirate the PBS solution from the cell culture chamber 100 through the third inlet 120, and add the cell suspension through the second inlet 110;

[0063] Step S3: Block the second inlet 110 and the third inlet 120 to allow the cells in the cell suspension to adhere to the culture vessel.

[0064] Step S4: The first micro-injection pump 500 introduces a dispersed phase with a second flow rate into the first inlet 210, and the second micro-injection pump 600 introduces a continuous phase with a first flow rate into the continuous phase inlet 420.

[0065] Step S5: Add potassium chloride solution into cell culture chamber 100 through third inlet 120 to stimulate cells; dispersed phase enters cell culture chamber 100 through flushing channel 200 to remove cell secretions, and enters droplet forming unit 400 through collection channel 300; turn on second micro-injection pump 600 to introduce continuous phase into droplet forming unit 400 to squeeze dispersed phase to form droplets.

[0066] Step S6: Turn on the electrospray ionization device 700 and the third micro-injection pump 800. The third micro-injection pump 800 introduces methanol solution into the electrospray ionization device 700. After the droplets are introduced into the electrospray ionization device 700 and the inner capillary 710 modified with C-18 is etched to remove polar components, they are combined with the methanol solution. The combined solution forms a spray under the action of the electrospray ionization device 700 and enters the mass spectrometer 900 for detection.

[0067] Step S7: Repeat steps S1-S6 above for repeated testing.

[0068] Specifically, in step S1 above, the PBS solution is a phosphate-buffered saline solution; in step S2 above, the PBS solution in the cell culture chamber 100 can be slowly aspirated from the second inlet 110, and the cell suspension can be added from the third inlet 120, wherein the cell suspension is PC-12 cell suspension with a cell density of 2 × 10⁻⁶ cells / mL. 6The dispersed phase is an ammonium formate solution with a concentration of 140 mmol / L, and the continuous phase is a volatile droplet-forming oil containing 2% perfluorinated surfactant. The second flow rate range of the dispersed phase is 900-41400 μL / min, and the first flow rate range of the continuous phase is 450-41400 μL / min. In this embodiment, the second flow rate is 900 μL / min, and the first flow rate is 450-1200 μL / min. μL / min; In step S5 above, PC-12 cells are stimulated by potassium chloride solution to secrete neurotransmitters; In step S6 above, the droplet enters the inner capillary 710 of the electrospray ionization device 700 through the fourth capillary 430, and the droplet passes through the C-18 etching modification layer 711 of the inner capillary 710. The C-18 etching modification layer 711 removes polar components such as salt and protein from the droplet; After desalting, the droplet enters the outer capillary 720 and merges with the methanol solution. The methanol solution assists the droplet in ionization. Under the DC voltage of the electrospray ionization device 700, the merged droplet and methanol solution form a spray that enters the mass spectrometer 900 to achieve dynamic detection and analysis of neurotransmitters.

[0069] In step S3 above, when external stimulation such as potassium chloride solution is applied to PC-12 cells to promote neurotransmitter separation, the dispersed phase with an initial flow rate of the second flow rate enters the cell culture chamber 100 through the flushing channel 200. The dispersed phase flushes the cell culture chamber 100 with liquid flow, and the neurotransmitters secreted by PC-12 cells are flushed through the collection channel 300 to the outlet 410 of the microfluidic chip 1000. Since the amount of neurotransmitter secretion has the characteristic of dynamic change with stimulation time, the concentration of neurotransmitters in the droplets continuously flowing out of the outlet 410 will have gradient differences within the same time interval. By injecting the continuous phase into the droplet forming unit 400 and setting the flow rate of the continuous phase to match the flow rate of the dispersed phase, 5-10 droplets can be generated in 1 second for subsequent detection and analysis by the mass spectrometer 900, achieving a time resolution of 100-200 ms. This facilitates researchers to detect and analyze the dynamic changes of neurotransmitters with fast recovery and degradation rates.

[0070] like Figure 5 and Figure 6 As shown in the figure, the dopamine signal intensity and signal-to-noise ratio obtained using the nESI ion source are much lower than those obtained using the microfluidic chip-mass spectrometry test.

[0071] The embodiments of this application have been described in detail above with reference to the accompanying drawings. However, this application is not limited to the above embodiments. Within the scope of knowledge possessed by those skilled in the art, various changes can be made without departing from the spirit of this application. Furthermore, unless otherwise specified, the embodiments and features described in the embodiments of this application can be combined with each other.

Claims

1. A microfluidic chip, characterized in that, include: Cell culture chamber, used for culturing cells; A flushing channel, wherein a first inlet is provided at one end of the flushing channel and the other end is connected to the cell culture chamber; The first inlet is used to introduce the dispersed phase into the flushing channel; the flushing channel is used to guide the dispersed phase into the cell culture chamber; A collection channel, connected to the cell culture chamber, is used to collect cell secretions generated from the cell culture chamber; A droplet forming unit is connected to the collection channel and is used to receive cell secretions collected by the collection channel. The droplet forming unit includes an outlet and a continuous phase inlet. The continuous phase inlet is used to introduce a continuous phase with a first flow rate so that the cell secretions received by the droplet forming unit form droplets. The droplets can be directed to the outside of the microfluidic chip through the outlet. The flushing channel includes multiple columns, which connect to the cell culture chamber. The multiple columns are arranged on the cell culture chamber to simultaneously introduce the dispersed phase into various locations within the cell culture chamber. The cell culture chamber includes a second inlet and a third inlet, which are used to introduce cell suspension into the cell culture chamber.

2. The microfluidic chip according to claim 1, characterized in that, The collection channel includes a main channel and multiple sub-channels. The multiple sub-channels are connected to the cell culture chamber and are arranged on the cell culture chamber to simultaneously receive cell secretions generated at various locations in the cell culture chamber. One end of the main channel is connected to the droplet forming unit, and the other end is connected to the sub-channels. The main channel is used to receive cell secretions from the sub-channels and to introduce cell secretions into the droplet forming unit.

3. The microfluidic chip according to claim 2, characterized in that, The droplet forming unit includes a continuous phase channel and an outlet channel. The continuous phase channel is connected to the continuous phase inlet and is used to introduce the continuous phase. Cell secretions introduced into the droplet forming unit by the main channel form droplets under the action of the continuous phase. The outlet channel is used to receive droplets and lead to the outlet.

4. The microfluidic chip according to claim 3, characterized in that, The number of continuous phase channels is two, the number of outlet channels is one, the extension direction of the outlet channel is the same as the extension direction of the main channel, the extension directions of the two continuous phase channels are the same, and the extension directions of the two continuous phase channels are perpendicular to the extension direction of the outlet channel.

5. A chip-mass spectrometry coupling system, characterized in that, include: The microfluidic chip as described in any one of claims 1 to 4; A first micro-injection pump, which is connected to the first inlet of the flushing channel, is used to introduce a dispersed phase into the flushing channel; A second micro-injection pump is connected to the continuous phase inlet of the droplet forming unit and is used to introduce the continuous phase into the droplet forming unit; An electrospray ionization device is used to ionize the droplets flowing out of the droplet forming unit to form a spray; A third micro-injection pump, which is connected to the electrospray ionization device, is used to introduce methanol solution into the electrospray ionization device; A mass spectrometer is used to detect the spray generated by the electrospray ionization device.

6. The chip-mass spectrometry coupling system according to claim 5, characterized in that, The electrospray ionization device includes a double-layer capillary structure, comprising an inner capillary and an outer capillary, with the inner capillary nested inside the outer capillary. One end of the inner capillary receives droplets generated by the droplet forming unit, and the inner wall of the other end is etched and modified with C-18. The outer capillary receives the methanol solution introduced by the third micro-injection pump and merges the methanol solution with the droplets passing through the inner capillary.

7. An experimental method for a chip-mass spectrometry coupling system, characterized in that, The chip-mass spectrometry coupling system as described in claim 6 includes the following steps: S1. Block the second and third inlets and the continuous phase inlet located in the cell culture chamber, open the first inlet and the outlet, and introduce PBS solution into the first inlet to fill the microfluidic chip with PBS solution. S2. Block the first inlet, the continuous phase inlet, and the outlet, and open the second inlet and the third inlet located in the cell culture chamber; The PBS solution in the cell culture chamber is aspirated from the third inlet, and the cell suspension is added from the second inlet. S3. Block the second inlet and the third inlet to allow the cells in the cell suspension to adhere to the culture vessel. S4. The first micro-injection pump introduces a dispersed phase with a second flow rate into the first inlet, and the second micro-injection pump introduces a continuous phase with a first flow rate into the continuous phase inlet. S5. Potassium chloride solution is added to the cell culture chamber through the third inlet to stimulate the cells; the dispersed phase enters the cell culture chamber through the flushing channel to carry away cell secretions, and enters the droplet forming unit through the collection channel. The second micro-injection pump is turned on to introduce the continuous phase into the droplet forming unit to squeeze the dispersed phase to form droplets. S6. Turn on the electrospray ionization device and the third micro-injection pump, and the third micro-injection pump introduces methanol solution into the electrospray ionization device; After the droplets are passed into the electrospray ionization device and the inner capillary, which is etched and modified with C-18 to remove polar components, they are combined with the methanol solution. The combined solution forms a spray under the action of the electrospray ionization device and enters the mass spectrometer for detection. S7. Repeat steps S1-S6 above for repeated testing.

8. The experimental method for the chip-mass spectrometry coupling system according to claim 7, characterized in that, The dispersed phase is an ammonium formate solution with a concentration of 140 mmol / L, and the continuous phase is a volatile droplet-generating oil containing 2% perfluorinated surfactant; the second flow rate of the dispersed phase is 900-41400 μL / min, and the first flow rate of the continuous phase is 450-41400 μL / min.

9. The experimental method for the chip-mass spectrometry coupling system according to claim 7, characterized in that, The cell suspension was a PC-12 cell suspension with a cell density of 2 × 10⁻⁶ cells / mL. 6 cells / mL.

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