CN115400804A - Micro-fluidic chip for pretreatment of single-cell proteome sample and cell treatment method - Google Patents

Abstract

The invention relates to the technical field of cell sorting, and discloses a micro-fluidic chip for pretreatment of a single-cell proteome sample and a cell processing method. Based on above-mentioned structure, need not to adopt the liquid-transfering device to remove to the reaction solution of different processes, all processes of cell separation, albumen degeneration, reduction, alkylation and enzymolysis all can be accomplished in this micro-fluidic chip, and the integration degree is higher, and the reaction is more thorough, improves the efficiency of sorting.

Description

A microfluidic chip and cell processing device for single-cell proteome sample pretreatment rationale

technical field

The invention relates to the technical field of cell sorting, in particular to a microfluidic chip and a cell processing method for single-cell proteome sample pretreatment.

Background technique

With the development of chromatography and mass spectrometry, the combination of nanoliter ultra-high liquid phase and ion mobility-time-of-flight 4D mass spectrometry or orbital well ultra-high resolution mass spectrometry can achieve in-depth analysis of 1000 proteins in a single cell. Due to the limited amount of protein in a single cell, mass spectrometry-based single-cell proteome analysis relies heavily on whether the pre-processing step can maximize protein extraction efficiency and reduce sample loss during processing, thus requiring single-cell processing.

The existing microfluidic chip for cell sorting has a relatively simple structure and only has a simple sorting function, and a subsequent sorting operation needs to be performed by a pipetting device, and the efficiency of cell sorting is low.

Contents of the invention

The purpose of the present invention is to provide a microfluidic chip with high integration and high sorting efficiency, which can perform single cell sorting, protein denaturation and reduction, enzymatic hydrolysis and labeling on the chip.

In order to achieve the above object, the present invention provides a microfluidic chip for single-cell proteome sample pretreatment, which includes a chip body, the chip body has a liquid injection hole, a first flow channel, a second Flow channel, capture chamber, third flow channel and collection hole, the liquid injection hole is set in multiples, the number of the first flow channel and the liquid injection hole are equal and one-to-one correspondence, the first flow channel and the A first air valve is arranged between the second channels, the capture chamber is provided with reaction microholes, and the reaction microholes are used for sorting out single cells, and the third channel is provided with a plurality of serpentine sections, A second air valve is arranged between any two adjacent serpentine segments.

In some embodiments of the present application, there are multiple second flow channels, the first flow channel communicates with multiple second flow channels, the capture chamber, the third flow channel and the The number of the second runners is equal and corresponding to each other.

In some embodiments of the present application, it also includes a fourth flow channel and a first waste liquid hole connected to the fourth flow channel, the fourth flow channel is connected to the second flow channel and is located at On the downstream side of the first flow channel, a third air valve is provided between the fourth flow channel and the second flow channel, and the second flow channel is provided with a valve located between the fourth flow channel and the first flow channel. A fourth air valve between the first flow channel, the third flow channel is provided with a fifth air valve between the serpentine section and the capture chamber.

In some embodiments of the present application, it further includes a fifth flow channel and a second waste liquid hole connected to the fifth flow channel, the fifth flow channel is connected to the third flow channel and is located at On the upstream side of the fifth air valve, a sixth air valve is provided between the fifth flow channel and the third flow channel.

In some embodiments of the present application, the capture chamber includes an entry section and an outflow section, the entry section and the outflow section are oppositely arranged, the entry section communicates with the second flow channel, and the outflow section In communication with the third channel, the reaction microhole is arranged between the inlet section and the outflow section and communicates with the two.

In some embodiments of the present application, buffer tubes are provided on both sides of the inlet section of the reaction microwell, and at least part or all of the buffer tubes are arc-shaped, and the buffer tube is close to the outflow section. The positions of the reaction micropores are connected.

In some embodiments of the present application, the buffer pipe includes a first buffer section, a second buffer section, a third buffer section, a fourth buffer section, and a fifth buffer section connected in sequence, and the first buffer section and The inflow section is connected, and the first buffer section is perpendicular to the fluid flow direction of the inflow section, the second buffer section is opposite to the fluid flow direction of the inflow end, and the third buffer section is opposite to the fluid flow direction of the inflow end. The fluid flow direction of the inflow section is the same, the fluid flow direction of the fourth buffer section is opposite to that of the inflow section, the fluid flow direction of the fifth buffer section is the same as that of the inflow section, and the fifth buffer section It communicates with the position of the outflow section close to the reaction micropore.

In some embodiments of the present application, it also includes a sixth flow channel communicated with the collection hole, the number of the sixth flow channel is set to two, the number of the collection hole and the sixth flow channel is equal and one In a one-to-one correspondence, some of the third flow channels communicate with one of the sixth flow channels, and the rest of the third flow channels communicate with the other sixth flow channel, and the sixth flow channel communicates with the sixth flow channel. The end connected with the third flow channel has a buffer chamber.

In some embodiments of the present application, the chip body includes a base layer, a gas valve layer, a fluid control layer, and a fluid entry and exit layer that are sequentially stacked, and the gas valve layer has the first gas valve and the second gas valve. A valve, the fluid control layer has the second flow channel, the capture chamber and the third flow channel, and the fluid inlet and outlet layer has the liquid injection hole, the first flow channel and the collection hole.

Another object of the present invention is to provide a cell processing method, which includes the following steps:

S1. Add 0.1% BSA solution from the injection hole, and incubate at room temperature for 1 hour;

S2, add PBS solution from the injection hole to rinse for 10 minutes, and dry;

S3. Close all the first air valves and the second air valves;

S3. Close the fifth air valve and the sixth air valve, add the cell suspension from one of the injection holes, and open the corresponding first air valve;

S4, add cell lysate from the first described injection hole, add the TEAB solution containing 100ng/μL trypsin from the second described injection hole, add concentration 5% from the third described injection hole For the hydroxylamine solution, add 5% FA solution from the fourth injection hole, add TEAB solution from the fifth injection hole, and add the acetonitrile solution for sample labeling to the other injection hole;

S5. Open the first air valve corresponding to the first liquid injection hole, open the fifth air valve, heat the chip body to 70° and keep it for 40 minutes, and cool to room temperature;

S6. Open the first air valve corresponding to the second liquid injection hole, open the first second air valve along the fluid flow direction of the third channel, heat the chip body to 37° and keep for 12h;

S7. Open the first air valve corresponding to the injection hole containing the acetonitrile solution, open the second second air valve along the fluid flow direction of the third channel, and let stand for 1 h;

S8. Open the first air valve corresponding to the third liquid injection hole, open the third second air valve along the fluid flow direction of the third channel, and let stand for 15 minutes;

S9. Open the first air valve corresponding to the fourth liquid injection hole, and open the fourth second air valve along the fluid flow direction of the third channel;

S10. Open the first air valve corresponding to the fifth liquid injection hole, open the fifth second air valve along the fluid flow direction of the third channel, and collect samples from the collection hole.

The present invention provides a microfluidic chip for the pretreatment of single-cell proteome samples. Compared with the prior art, the beneficial effects are as follows:

The microfluidic chip provided by the present invention includes a chip body, which has a liquid injection hole, a first flow channel, a second flow channel, a capture chamber, a third flow channel and a collection hole arranged in sequence, and the liquid injection holes are set in multiples. The number of the first flow channel and the liquid injection hole are equal and one-to-one correspondence, a first air valve is set between the first flow channel and the second flow channel, and the capture chamber is provided with a reaction microhole, which is used to sort out a single cell, The third flow channel is provided with a plurality of serpentine sections, and a second air valve is arranged between any two adjacent serpentine sections. Based on the above structure, this microfluidic chip can be specially used for the pre-treatment of single-cell proteome mass spectrometry detection. The single cell can be captured into the reaction microwell through the capture chamber. The setting of multiple injection holes can make the solution added in each process flow It is stored in the liquid injection hole before the reaction to prevent mixing before the reaction. Through the opening and closing of the first air valve, various reaction reagents can be added in sequence according to the sorting process, and the opening and closing of the second air valve can make different reaction reagents The reaction is carried out in different serpentine sections, without using a pipetting device to move the reaction solution in different processes, and all the processes of cell sorting can be completed in this microfluidic chip, which has a higher degree of integration, more thorough reaction, and improved Improve the efficiency of protein/peptide recovery from single cells.

In addition, the present invention also provides a cell processing method, which is simple and fast, and has low operation difficulty.

Description of drawings

1 is a schematic diagram of the overall structure of a microfluidic chip according to an embodiment of the present invention;

Fig. 2 is a partial schematic diagram of area A in Fig. 1;

Fig. 3 is a partial schematic diagram of area B in Fig. 1;

Fig. 4 is a partial schematic diagram of area C in Fig. 1;

FIG. 5 is a partial schematic diagram of area D in FIG. 3 .

In the figure: 100, chip body; 110, fluid inlet and outlet layer; 111, liquid injection hole; 112, first flow channel; 113, collection hole; 114, fourth flow channel; 115, first waste liquid hole; 116, fifth Flow channel; 117, second waste liquid hole; 118, sixth flow channel; 119, buffer chamber; 120, fluid control layer; 121, second flow channel; 122, third flow channel; 122a, serpentine section; 130 , valve layer; 131, the first valve; 132, the second valve; 133, the third valve; 134, the fourth valve; 135, the fifth valve; 136, the sixth valve; 137, the first valve Seven air valves; 200, capture chamber; 210, reaction micropore; 220, entry section; 230, outflow section; 240, buffer tube; 241, first buffer section; 242, second buffer section; 243, third buffer section ; 244, the fourth buffer segment; 245, the fifth buffer segment.

Detailed ways

The following will clearly and completely describe the technical solutions in the embodiments of the present application with reference to the accompanying drawings in the embodiments of the present application. Obviously, the described embodiments are only part of the embodiments of the present application, not all of them.

It should be understood that in the description of this application, the terms "center", "upper", "lower", "front", "rear", "left", "right", "vertical", "horizontal", The orientations or positional relationships indicated by "top", "bottom", "inner", "outer", etc. are based on the orientations or positional relationships shown in the drawings, and are only for the convenience of describing the application and simplifying the description, rather than indicating or implying References to devices or elements must have a particular orientation, be constructed, and operate in a particular orientation and therefore should not be construed as limiting the application. The terms "first" and "second" are used for descriptive purposes only, and cannot be understood as indicating or implying relative importance or implicitly indicating the number of indicated technical features, that is, limited to "first", "second Two" features may explicitly or implicitly include one or more of these features. Also, unless otherwise specified, "plurality" means two or more.

It should be noted that, in the description of this application, unless otherwise clearly stipulated and limited, the terms "installation", "connection", and "connection" should be understood in a broad sense, for example, it can be a fixed connection or a detachable connection. Connected, or integrally connected; it can be mechanically connected or electrically connected; it can be directly connected or indirectly connected through an intermediary, and it can be the internal communication of two components. Those of ordinary skill in the art can understand the specific meanings of the above terms in this application in specific situations.

As shown in Figures 1 to 5, the embodiment of the present invention provides a microfluidic chip for single-cell proteome sample pretreatment, which includes a chip body 100, which has a liquid injection hole 111, a second The first flow channel 112, the second flow channel 121, the capture chamber 200, the third flow channel 122, the collection hole 113, and the liquid injection hole 111 are set to be multiple. In this embodiment, the liquid injection hole 111 is set to sixteen. The flow channels 112 and the liquid injection holes 111 are equal in number and correspond to each other. A first air valve 131 is provided between the first flow channel 112 and the second flow channel 121. The capture chamber 200 is provided with a reaction micropore 210. The reaction micropore 210 is used for For sorting single cells, the third channel 122 is provided with a plurality of serpentine sections 122a, and a second air valve 132 is provided between any two adjacent serpentine sections 122a. It should be noted that the liquid injection hole 111 may also be filled with gas.

Based on the above-mentioned structure, this microfluidic chip can be used exclusively for the pre-treatment of single-cell proteome mass spectrometry detection. A single cell can be captured into the reaction microwell 210 through the capture chamber 200. The setting of multiple injection holes 111 can make each process flow add The solution is stored in the liquid injection hole 111 before the reaction to prevent mixing before the reaction. Through the opening and closing of the first air valve 131, various reaction reagents can be added sequentially according to the sorting process, and through the opening and closing of the second air valve 132 Different reaction reagents can be reacted in different serpentine sections 122a, and there is no need to use a pipetting device to move the reaction solutions of different processes. All the processes of cell sorting can be completed in this microfluidic chip, and the integration degree is even higher. High, the reaction is more thorough, and the efficiency of sorting is improved.

Optionally, as shown in FIGS. 1 to 3 , in this embodiment, multiple second flow channels 121 are provided, the first flow channel 112 communicates with multiple second flow channels 121 , the capture chamber 200 , the third flow channel 121 The number of flow channels 122 and the second flow channels 121 are equal and corresponding to each other. In this way, the reaction solutions with different labels can be reacted in different serpentine sections 122a.

Optionally, as shown in Figures 1 and 3, in this embodiment, the microfluidic chip further includes a fourth flow channel 114 and a first waste liquid hole 115 communicating with the fourth flow channel 114, the fourth flow channel The channel 114 communicates with the second channel 121 and is located on the downstream side of the first channel 112. A third air valve 133 is provided between the fourth channel 114 and the second channel 121. The second channel 121 is provided with a A fourth air valve 134 is provided between the fourth channel 114 and the first channel 112 , and the third channel 122 is provided with a fifth air valve 135 located between the serpentine section 122 a and the capture chamber 200 . Based on this, the fourth air valve 134 and the fifth air valve 135 can intercept the reaction waste liquid between the capture chamber 200 and the second flow channel 121 and the third flow channel 122, so that the operator can drain the capture chamber 200 as needed The waste liquid, the operation is more flexible. The third air valve 133 is used to open and close the fourth channel 114 .

Optionally, as shown in Figures 1 and 3, in this embodiment, the microfluidic chip further includes a fifth flow channel 116 and a second waste liquid hole 117 communicating with the fifth flow channel 116, the fifth flow channel The channel 116 communicates with the third flow channel 122 and is located upstream of the fifth air valve 135 , and a sixth air valve 136 is provided between the fifth flow channel 116 and the third flow channel 122 . In this way, the waste liquid can be drained from the upstream side and the downstream side of the capture chamber 200 respectively, and the operation is more convenient.

Optionally, as shown in FIG. 5 , in this embodiment, the capture chamber 200 includes an entry section 220 and an outflow section 230, the entry section 220 and the outflow section 230 are arranged oppositely, and the entry section 220 communicates with the second flow channel 121, The outflow section 230 communicates with the third channel 122 , and the reaction microhole 210 is arranged between the inflow section 220 and the outflow section 230 and communicates with the two. Specifically, the reaction micropore 210 is set to be 4-10 microns. In this way, after the cell suspension enters the capture chamber 200 , single cells can be preliminarily sorted through the reaction microwell 210 .

Considering that if only the reaction micropore 210 with a small diameter is provided, the local pressure of the capture chamber 200 will be too high, which is not conducive to the flow of the cell suspension and the reaction. Optionally, as shown in FIG. 5, in this embodiment The inlet section 220 is provided with buffer tubes 240 on both sides of the reaction microwell 210 , the buffer tubes 240 are at least partly or entirely arc-shaped, and the buffer tubes 240 communicate with the outlet section 230 near the reaction microwell 210 . In this way, the cell suspension part flows between the inflow section and the outflow section 230 through the buffer tube 240 , so that the capture of single cells is smoother.

Optionally, as shown in FIG. 5 , in this embodiment, the buffer pipe 240 is in the shape of a "T", which includes a first buffer section 241, a second buffer section 242, a third buffer section 243, The fourth buffer section 244 and the fifth buffer section 245, the first buffer section 241 communicates with the inflow section, and the first buffer section 241 is perpendicular to the fluid flow direction of the inflow section, and the second buffer section 242 is connected to the fluid flow direction of the inflow end On the contrary, the fluid flow direction of the third buffer section 243 is the same as that of the inflow section, the fluid flow direction of the fourth buffer section 244 is opposite to that of the inflow section, the fifth buffer section 245 is the same as the fluid flow direction of the inflow section, and the fifth buffer section 245 It communicates with the position of the outflow section 230 close to the reaction micropore 210 .

Optionally, as shown in FIG. 4 , in this embodiment, the microfluidic chip further includes a sixth flow channel 118 communicating with the collection hole 113, the sixth flow channel 118 is set to two, and the collection hole 113 and the sixth flow channel The number of the six flow channels 118 is equal and corresponds to one by one, part of the third flow channels 122 communicates with one of the sixth flow channels 118, and the remaining third flow channels 122 communicate with the other sixth flow channel 118, that is, some of the sixth flow channels 118 communicate with each other. The sample liquid in the three flow channels 122 is mixed and flows out from the first collection hole 113, and it flows out from the other collection hole 113 after being mixed with the sample liquid in the third flow channel 122; the sixth flow channel 118 is connected to the third flow channel 122 One end of the channel has a buffer chamber 119. In this way, the samples with different labels can be mixed and collected from the collection hole 113 , and the setting of the buffer chamber 119 makes the flow of the samples flowing out from the third flow channel 122 relatively gentle.

Optionally, as shown in FIG. 1 , in this embodiment, the chip body 100 includes a base layer, a gas valve layer 130 , a fluid control layer 120 , and a fluid entry and exit layer 110 that are sequentially stacked. The gas valve layer 130 has a first gas valve 131, the second air valve 132, the third air valve 133, the fourth air valve 134, the fifth air valve 135 and the sixth air valve 136, the fluid control layer 120 has the second flow channel 121, the capture chamber 200 and the third flow channel 122, the fluid inlet and outlet layer 110 has a liquid injection hole 111, a first flow channel 112, a fourth flow channel 114, a fifth flow channel 116, a sixth flow channel 118, a first liquid waste hole 115, a second waste liquid hole 117 and Collection hole 113 .

Optionally, as shown in FIG. 2 , in this embodiment, the valve layer 130 is further provided with a seventh valve 137, and the seventh valve 137 is arranged on the second channel 121, and part of the first channel 112 is located on the second channel. On the upstream side of the seventh air valve 137 , the rest of the first channels 112 are located on the downstream side of the seventh air valve 137 . The seventh air valve 137 can open and close the second channel 121 , so that the addition of the reagent on the upstream side and the addition of the labeling solution on the downstream side can be distinguished.

Optionally, as shown in FIG. 1 , in this embodiment, the volume of the pipeline between the fourth air valve 134 and the seventh air valve 137 is 50 nanoliters. In this way, there is no need to use a nanoliter pipetting device or a liquid adding device, and only need to fill up the pipeline to ensure that the addition volume of various reagents is 50 nanoliters.

The embodiment of the present invention also provides a cell processing method, which includes the following steps:

S1. Add a 0.1% BSA (Bovine serum albumin, bovine serum albumin) solution from the injection hole A, and incubate at room temperature for 1 hour, so as to reduce non-specific adsorption on the surface of the chip body 100;

S2. Add PBS (phosphate buffered saline, phosphate buffered saline) solution from the injection hole to rinse for 10 minutes, and then dry;

S3, closing all the first air valves 131 and the second air valves 132;

S3. Close the fifth air valve 135 and the sixth air valve 136, add the cell suspension from one of the injection holes CELL, and open the corresponding first air valve 131. At this time, the cell suspension will flow from the first flow channel 112 and The second channel 121 flows to the capture chamber 200 for cell capture;

S4. Add cell lysate from injection hole A, add TEAB (Tetraethylammonium bromide, tetraethylammonium bromide) solution containing 100 ng/μL trypsin from injection hole B, and add 5% hydroxylamine from injection hole D Solution, add 5% FA (fatty acid, fatty acid) solution from injection hole E, add TEAB solution from injection hole F, and label 128N with acetonitrile solution for sample labeling, that is, 5 μg/μL TMT-11plex , 128C, 129N, 129C, 130N, 130C, 131N, and 131C acetonitrile solutions were added to the injection holes C1-C8;

S5. Open the first air valve 131 corresponding to the liquid injection hole A, open the fifth air valve 135, the reaction liquid flows to the first serpentine section 122a, heat the chip body at 100° to 70° and keep it for 40 minutes, and cool to room temperature, thereby perform cell lysis;

S6. Open the first air valve 131 corresponding to the liquid injection hole B, open the first and second air valves 132 along the fluid flow direction of the third channel 122, and the reaction liquid flows to the second serpentine section 122a to heat the chip Body 100 to 37° and keep for 12h;

S7. Open the first air valve 131 corresponding to the liquid injection hole C1-C8 containing the acetonitrile solution, open the second second air valve 132 along the fluid flow direction of the third flow channel 122, and the reaction liquid flows to the third Serpentine section 122a, stand still for 1h;

S8. Open the first air valve 131 corresponding to the liquid injection hole D, open the third second air valve 132 along the fluid flow direction of the third flow channel 122, and the reaction liquid flows to the fourth serpentine section 122a, and let stand 15min;

S9, open the first air valve 131 corresponding to the liquid injection hole E, open the fourth second air valve 132 along the fluid flow direction of the third channel 122, and the reaction liquid flows to the fifth serpentine section 122a;

S10, open the first air valve 131 corresponding to the liquid injection hole F, open the fifth second air valve 132 along the fluid flow direction of the third flow channel 122, and collect the sample from the collection hole 113; specifically, part of the sample liquid is The sixth flow channel 118 flows out from one of the collection holes 113 after being mixed, and the rest of the sample liquid flows out from the other collection hole 113 after being mixed in the other sixth flow channel 118 .

To sum up, the embodiment of the present invention provides a microfluidic chip, which is mainly composed of a chip body, which has a liquid injection hole, a first flow channel, a second flow channel, a capture chamber, a third flow channel and There are multiple collection holes and liquid injection holes, the number of the first flow channel and the liquid injection holes are equal and one-to-one correspondence, the first air valve is arranged between the first flow channel and the second flow channel, and the capture chamber is provided with reaction micropores, The reaction microhole is used for sorting single cells, the third flow channel is provided with multiple serpentine sections, and a second air valve is provided between any two adjacent serpentine sections. Compared with the prior art, the microfluidic chip has the advantages of high integration and high sorting efficiency.

In addition, the embodiment of the present invention also provides a cell processing method, which can realize the extraction of protein from a single cell, denature reduction, enzymatic hydrolysis, labeling and other steps to recover the sample. Compared with the prior art, the cell processing method The utility model has the advantages of being simple and fast, and having low operation difficulty.

The above is only a preferred embodiment of the present invention, it should be pointed out that for those of ordinary skill in the art, without departing from the technical principle of the present invention, some improvements and replacements can also be made, these improvements and replacements It should also be regarded as the protection scope of the present invention.

Claims (10)

1. The utility model provides a micro-fluidic chip for unicellular proteome sample pretreatment, a serial communication port, includes the chip body, the chip body has notes liquid hole, first runner, second runner, catches room, third runner and the collecting hole that communicates the setting in proper order, it establishes to a plurality ofly to annotate the liquid hole, first runner with annotate liquid hole quantity and equal and one-to-one, first runner with be provided with first pneumatic valve between the second runner, it is provided with the reaction micropore to catch the room, the reaction micropore is used for sorting out single cell, the third runner is equipped with a plurality of snakelike sections, arbitrary adjacent two be provided with the second pneumatic valve between the snakelike section.

2. The microfluidic chip of claim 1, wherein:

the number of the second flow channels is multiple, the first flow channels are communicated with the second flow channels, and the trapping chambers, the third flow channels and the second flow channels are equal in number and are in one-to-one correspondence.

3. The microfluidic chip of claim 1, wherein:

the waste liquid trap further comprises a fourth flow channel and a first waste liquid hole communicated with the fourth flow channel, the fourth flow channel is communicated with the second flow channel and is located on the downstream side of the first flow channel, a third air valve is arranged between the fourth flow channel and the second flow channel, a fourth air valve located between the fourth flow channel and the first flow channel is arranged on the second flow channel, and a fifth air valve located between the snake-shaped section and the trap chamber is arranged on the third flow channel.

4. The microfluidic chip of claim 3, wherein:

the waste gas recovery device further comprises a fifth flow channel and a second waste liquid hole communicated with the fifth flow channel, the fifth flow channel is communicated with the third flow channel and is positioned on the upstream side of the fifth gas valve, and a sixth gas valve is arranged between the fifth flow channel and the third flow channel.

5. The microfluidic chip of claim 1, wherein:

the capture chamber comprises an inlet section and an outlet section, the inlet section and the outlet section are arranged oppositely, the inlet section is communicated with the second flow channel, the outlet section is communicated with the third flow channel, and the reaction micropores are arranged between the inlet section and the outlet section and communicated with the inlet section and the outlet section.

6. The microfluidic chip of claim 5, wherein:

the entering section is located both sides of reaction micropore all are equipped with the buffer tube, the buffer tube is at least partly or whole to be the arc, the buffer tube with the outflow section is close to the position of reaction micropore is linked together.

7. The microfluidic chip of claim 6, wherein:

the buffer tube is including the first buffer segment, second buffer segment, third buffer segment, fourth buffer segment and the fifth buffer segment that are linked together in proper order, first buffer segment with the inflow section communicates, just first buffer segment with the fluid flow direction of inflow section is mutually perpendicular, the second buffer segment with the fluid flow direction of inflow end is opposite, the third buffer segment with the fluid flow direction of inflow section is the same, the fourth buffer segment with the fluid flow direction of inflow section is opposite, the fifth buffer segment with the fluid flow direction of inflow section is the same, the fifth buffer segment with the outflow section is close to the position of reaction micropore is linked together.

8. The microfluidic chip of claim 2, wherein:

the number of the collecting holes is equal to that of the sixth flow channels, the collecting holes correspond to the sixth flow channels one by one, part of the third flow channels are communicated with one of the sixth flow channels, the rest of the third flow channels are communicated with the other sixth flow channel, and one end, communicated with the third flow channel, of each sixth flow channel is provided with a buffer chamber.

9. The microfluidic chip of claim 1, wherein:

the chip body comprises a base layer, an air valve layer, a fluid control layer and a fluid inlet and outlet layer which are sequentially stacked, wherein the air valve layer is provided with a first air valve and a second air valve, the fluid control layer is provided with a second flow channel, a capture chamber and a third flow channel, and the fluid inlet and outlet layer is provided with a liquid injection hole, a first flow channel and a collection hole.

10. A method of cell processing, comprising the steps of:

s1, adding BSA solution with the concentration of 0.1% from a liquid injection hole, and incubating for 1h at room temperature;

s2, adding a PBS solution from the liquid injection hole, washing for 10min, and drying;

s3, closing all the first air valves and all the second air valves;

s3, closing the fifth air valve and the sixth air valve, adding the cell suspension from one of the liquid injection holes, and opening the corresponding first air valve;

s4, adding cell lysate from the first injection hole, adding a TEAB solution containing 100 ng/muL pancreatin from the second injection hole, adding a hydroxylamine solution with the concentration of 5% from the third injection hole, adding an FA solution with the concentration of 5% from the fourth injection hole, adding the TEAB solution from the fifth injection hole, and adding an acetonitrile solution for sample marking into the other injection holes;

s5, opening a first air valve corresponding to the first liquid injection hole, opening a fifth air valve, heating the chip body to 70 degrees, keeping the temperature for 40min, and cooling to room temperature;

s6, opening a first air valve corresponding to the second liquid injection hole, opening a first air valve along the fluid flowing direction of the third flow channel, heating the chip body to 37 degrees, and keeping the temperature for 12 hours;

s7, opening a first air valve corresponding to the liquid injection hole containing the acetonitrile solution, opening a second air valve along the fluid flowing direction of the third flow channel, and standing for 1h;

s8, opening a first air valve corresponding to the third liquid injection hole, opening a second air valve in the third fluid flowing direction of the third flow channel, and standing for 15min;

s9, opening a first air valve corresponding to the fourth liquid injection hole, and opening a fourth second air valve along the fluid flowing direction of the third flow channel;

and S10, opening a first air valve corresponding to the fifth liquid injection hole, opening a fifth second air valve along the fluid flowing direction of the third flow channel, and collecting a sample from the collection hole.

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