Microfluidics-based single cell mass spectrometry detection system and method

Abstract

The invention belongs to a mass spectrometry technology, and particularly provides a single cell mass spectrometry detection system and method based on microfluidics, and the single cell mass spectrometry detection system comprises a microfluidic chip and a recognition device; the bearing unit is provided with a plurality of single cell bearing positions distributed in a matrix manner and a waste liquid pool; the driving unit is used for realizing relative movement between the bearing unit and the micro-fluidic chip, so that single cells discharged from the micro-fluidic chip enter a selected bearing position, or liquid without single cells enters the waste liquid pool; the controller controls the driving unit according to a result of the identification device, and the identification device is used for identifying whether a single cell exists in a liquid flow of a single cell channel of the micro-fluidic chip or not. The device has the advantages of controllable droplet size and the like.

Description

Technical Field

[0001] The present invention relates to single-cell detection technology, and particularly to a microfluidics-based single-cell mass spectrometry detection system and method. Background Art

[0002] Cells are the most basic units that make up the human body. Cells have significant differences in morphology and size in different organs of the human body. Even the same type of cells within the same organ also have differences, which is the heterogeneity of cells. Therefore, it is necessary to analyze cells at the single-cell level. Single-cell metabolomics can provide single-cell metabolite information related to phenotypes and reveal the metabolic processes within single cells. Mass spectrometry is one of the most commonly used techniques for detecting cell metabolites. Mass spectrometry has advantages such as high resolution and high sensitivity, and responds to most molecules. Mass spectrometry detection methods combined with traditional methods have developed very mature commercial products, such as gas chromatography-mass spectrometry and liquid chromatography-mass spectrometry. However, these techniques usually perform mass spectrometry analysis on population cells, and the mass spectrometry analysis of single cells still remains at the research stage.

[0003] After three or four decades of development, microfluidics technology has become a very mature tool for studying cells and droplets at a microscale and has developed rapidly in the fields of biology, medicine, chemistry, chemical engineering, etc. In the field of analytical chemistry, microfluidics technology has many advantages over other similar benchtop instruments. The miniaturized microfluidic chip greatly reduces the amount of sample and reagent volume used, thus saving analysis costs. Another advantage of the microfluidic chip is that multiple analysis processes can be concentrated on a single platform. In recent years, the microfluidics-mass spectrometry coupling technology has developed rapidly in the direction of cell analysis. The microfluidics-mass spectrometry coupling technology can quickly obtain information on cell metabolites and has been widely applied in the field of mass spectrometry analysis. However, most of the existing microfluidic single-cell-mass spectrometry coupling devices use oil as the continuous phase, which limits their mass spectrometry application scenarios, is not suitable for open direct ionization mass spectrometers, and cannot accurately obtain the metabolite information of single cells. Summary of the Invention

[0004] To solve the deficiencies in the above-mentioned prior art solutions, the present invention provides a microfluidics-based single-cell mass spectrometry detection system.

[0005] The object of the present invention is achieved by the following technical solutions: A microfluidics-based single-cell mass spectrometry detection system includes a microfluidic chip and an identification device; the single-cell mass spectrometry detection system further includes: A carrying unit, the carrying unit having a plurality of single-cell carrying positions distributed in a matrix and a waste liquid pool; A driving unit, which is used to realize the relative movement between the carrying unit and the microfluidic chip, so that the single cells discharged from the microfluidic chip enter the selected carrying positions, or the liquid without single cells enters the waste liquid pool; A controller, which controls the driving unit according to the result of the recognition device, and the recognition device is used to recognize whether there are single cells in the liquid flow of the single cell channel of the microfluidic chip.

[0006] The purpose of the present invention also lies in providing a single cell mass spectrometry detection method based on microfluidics, and this invention purpose is achieved through the following technical solutions: The single cell mass spectrometry detection method based on microfluidics includes the steps of: In the single cell channel of the microfluidic chip, single cells pass through in sequence; The recognition device detects whether there are single cells passing through the single cell channel; If the result is yes, the controller sends a movement instruction to the driving unit, and the selected carrying position of the carrying unit is located below the outlet of the single cell channel; If the result is no, the controller sends a movement instruction to the driving unit, and the selected waste liquid pool of the carrying unit is located below the outlet of the single cell channel; The selected carrying position carries single cells, or the selected waste liquid pool receives the liquid.

[0007] Compared with the prior art, the beneficial effects of the present invention are:

[0008] 1. The microfluidic single cell printing system uses air flow as the continuous phase, which greatly expands its application scenarios and is applicable to most mass spectrometers; 2. Under the condition of a certain sample flow rate, the symmetric air flow can control the size of the generated droplets, which is suitable for the sorting of cells of different sizes; 3. Different types of cells are sorted by using the AI cell recognition algorithm based on the microhole array and deep learning, and single cells can be accurately printed and mass spectrometry analysis can be carried out. Description of the Drawings

[0009] Referring to the attached drawings, the disclosure of the present invention will become easier to understand. It is easy for those skilled in the art to understand that these drawings are only used to illustrate the technical solutions of the present invention and are not intended to limit the protection scope of the present invention. In the figures: Figure 1 is the structural schematic diagram of the single cell mass spectrometry detection system of the present invention; Figure 2 is the structural schematic diagram of the microfluidic chip of the present invention; Figures 3 - 7 is the structural schematic diagram of the carrying unit of the present invention; Figure 8It is the mass spectrometry graph of bladder cancer cells. Detailed implementation manners

[0010] Figures 1 - 8 The following description and explanation describe alternative specific implementation manners of the present invention to teach those skilled in the art how to implement and reproduce the present invention. To teach the technical solutions of the present invention, some conventional aspects have been simplified or omitted. Those skilled in the art should understand that variations or substitutions derived from these specific implementation manners will fall within the scope of the present invention. Those skilled in the art should understand that the following features can be combined in various ways to form multiple variations of the present invention. Thus, the present invention is not limited to the following alternative specific implementation manners, but is only defined by the claims and their equivalents. Example 1

[0011] This example is a microfluidic-based single-cell mass spectrometry detection system, as Figure 1 shown. The single-cell mass spectrometry detection system includes: A microfluidic chip 11, an identification device 30, and a mass spectrometry device 61.

[0012] As Figures 3 - 7 shown, the carrier unit 41 has a plurality of single-cell carrier positions 42 distributed in a matrix and a waste liquid pool 43.

[0013] The driving unit is used to realize the relative movement between the carrier unit 41 and the microfluidic chip 11, so that the single cells discharged from the microfluidic chip 11 enter the selected carrier positions 42, or the liquid without single cells enters the waste liquid pool 43.

[0014] The controller controls the driving unit according to the result of the identification device 30, and the identification device 30 is used to identify whether there are single cells in the liquid flow of the single-cell channel 16 of the microfluidic chip 11.

[0015] As Figure 2 shown, the microfluidic chip 11 has a sample channel 15, a single-cell channel 16, and a plurality of auxiliary channels 13. The sample inlet 14 is sequentially connected to the asymmetric bent sample channel 15 and the single-cell channel 16. The fluid inlet 12 is sequentially connected to the plurality of auxiliary channels 13 and the single-cell channel 16. The outlets of the plurality of auxiliary channels 13 uniformly surround the single-cell channel 16. The fluid flow rate and flow volume entering the single-cell channel 16 from the plurality of auxiliary channels 13 are the same.

[0016] As Figure 2 shown, the auxiliary channel 13 is in an S shape, and two auxiliary channels 13 are symmetrically arranged on both sides of the sample channel 15.

[0017] As Figure 1As shown, the identification device 30 includes a light source 31, an imaging unit 34, and an identification unit 35. The emitted light of the light source 31 is incident on the single-cell channel 16.

[0018] As Figures 3 - 7 shown, the waste liquid pool 43 is strip-shaped and is arranged outside the loading position 42 and / or between adjacent loading positions 42.

[0019] The substrate of the microfluidic chip 11 is made of transparent organic glass, and the upper-layer microfluidic main structure is made of two layers of polydimethylsiloxane (PDMS). The first layer of the main structure is fabricated by soft lithography technology. After manufacturing the microfluidic mold by soft lithography technology, the PDMS prepolymer and curing agent are uniformly mixed in a ratio of 10:1 and then poured onto the microfluidic mold. After vacuum degassing treatment, it is baked in an oven at 60 °C for 1 hour. The second layer of the main structure is fabricated by a spin coater. The above-mentioned PDMS mixed liquid after vacuum treatment is poured into the middle of a polished silicon wafer or copper plate, and evenly spread on the spin coater at a rotation speed of 300 - 1000 rpm, and then baked in an oven at 60 °C for 1 hour. Punch holes in the two inlets (sample inlet 14 and fluid inlet 12) of the cured first layer of PDMS. After the two layers of PDMS are treated with oxygen plasma, the two layers of PDMS are bonded to each other. After being placed in the oven at 60 °C for another half hour, the excess PDMS at the outlet is cut off to expose the printing outlet. Then, after being treated with oxygen plasma, it is bonded to the transparent organic glass to obtain the microfluidic chip 11. The fabricated microfluidic chip 11 is baked at 120 °C for 2 hours to hydrophobize the PDMS surface in the microchannels. The microchannels in the microfluidic chip 11 have a rectangular cross-section, with a width of 100 - 400 μm and a depth of 40 - 100 μm; the diameter of the microcolumn array 17 is 50 - 200 μm, and there are a total of 64 - 172.

[0020] The loading unit 41 uses a microporous array chip, and the manufacturing method is the same as that of the microfluidic chip. First, use lithography technology or 3D printing technology to fabricate a microporous array mold, and then pour the PDMS mixed liquid after vacuum treatment, bake it in an oven at 60 °C for 1 hour to cure, and after uncovering, bond it to the transparent organic glass through oxygen plasma to obtain the microporous array chip. The diameter of the single-cell micropores of the microporous array chip is 200 - 500 μm, the center distance between the micropores is 400 - 1000 μm, the total number of loading positions (single-cell micropores) 42 is 16 - 400, and the number of waste liquid pools 43 is 2 - 64.

[0021] The single-cell mass spectrometry detection method based on microfluidics in the embodiment of the present invention, that is, the working method of the single-cell mass spectrometry detection system in this embodiment, the single-cell mass spectrometry detection method includes the steps: Within the single-cell channel 16 of the microfluidic chip 11, single cells pass through in sequence; The recognition device 30 detects whether there is a single cell passing through the single-cell channel 16; If the result is yes, the controller issues a movement instruction to the drive unit, and the selected bearing position 42 of the bearing unit 41 is located below the outlet of the single-cell channel 16; If the result is no, the controller issues a movement instruction to the drive unit, and the selected waste liquid pool 43 of the bearing unit 41 is located below the outlet of the single-cell channel 16; The selected bearing position 42 bears single cells, or the selected waste liquid pool 43 receives liquid.

[0022] In order to confirm the single cells in the single-cell channel 16, within the microfluidic chip 11, the cell suspension passes through the sample inlet 14, the asymmetric bent sample channel 15, and the single-cell channel 16 in sequence, and the fluid passes through the fluid inlet 12 and a plurality of auxiliary channels 13, and enters the single-cell channel 16 at the same fluid flow rate and the same flow rate. Within the single-cell channel 16, the cells pass through one by one.

[0023] As Figure 2 shown, the auxiliary channels 13 are in an S shape, and two auxiliary channels 13 are symmetrically arranged on both sides of the sample channel 15.

[0024] As Figures 3 - 7 shown, the waste liquid pool 43 is strip-shaped and is arranged outside the bearing position 42 and / or between adjacent bearing positions 42. Example 2

[0025] Application example of the microfluidic-based single-cell mass spectrometry detection system and method according to Embodiment 1 of the present invention in bladder cancer detection.

[0026] As Figure 1 shown, the injection pump 21 is used to extract and eject the suspension of bladder cancer cells, and the outlet is connected to the sample inlet 14 of the microfluidic chip 11 through a PTFE capillary. The gas provided by the air pump 22 enters the fluid inlet 12 after passing through the flow meter 23.

[0027] The recognition device 30 includes a light source 31, a convex lens 33, an objective lens 32, and an imaging unit 34 arranged in sequence, wherein the convex lens 33 and the objective lens 32 are respectively on both sides of the single-cell channel 16. The recognition unit 35 is used to recognize single cells in the output image of the imaging unit 34, and the recognition result is sent to the controller. The imaging unit 34 can be a high-speed camera.

[0028] The mass spectrometry device 61 uses an open direct ionization mass spectrometer. The capillary 71 is used to extract and eject the cell extract, and to extract the extraction substance of bladder cancer cells, and send it to the mass spectrometry device 61.

[0029] The driving unit adopts a three-dimensional moving platform for driving the three-dimensional movement of the bearing unit 41.

[0030] As Figure 2 shown, the sample inlet 14 is sequentially connected to the asymmetric bent sample channel 15 and the single-cell channel 16. The fluid inlet 12 is sequentially connected to two auxiliary channels 13 and the single-cell channel 16. The two auxiliary channels 13 are in an S shape and are symmetrically arranged on both sides of the sample channel 15. The outlets of the two auxiliary channels 13 uniformly surround the single-cell channel 16, and the fluid flow rate and flow volume entering the single-cell channel 16 from the multiple auxiliary channels 13 are the same. Micro-column arrays 17 are arranged at the sample inlet 14 and the fluid inlet 12, the micro-column diameter is 50 - 200 μm, and there are a total of 64 - 172.

[0031] The single-cell mass spectrometry detection method based on microfluidics in the embodiment of the present invention, that is, the working method of the single-cell mass spectrometry detection system in this embodiment, the single-cell mass spectrometry detection method includes the steps: Sample injection: The prepared suspension of bladder cancer cells with a cell density of 10 5 cells / mL is placed in a 1 mL syringe and injected into the microfluidic chip 11 through the PTFE capillary 71 by the injection pump 21 at a flow rate of 1 μL / min and connected to the sample inlet 14 of the microfluidic chip 11. The air pump 22 is connected to a high-purity nitrogen cylinder through a pressure reducing valve and is injected into the microfluidic chip 11 at a pressure of 200 mbar through the gas flow meter 23 and the PTFE capillary 71 and connected to the fluid inlet 12 of the microfluidic chip 11.

[0032] Cell orderly arrangement: According to the principle of inertial focusing of micron-sized particles, micron-sized cells are arranged into an orderly single-cell sequence by the Dean flow acceleration effect in the asymmetric S-shaped sample channel 15.

[0033] Single-cell recognition: The sorted single cells are captured by the high-speed camera 34 in the cell recognition area of the droplet generation structure, and the recognition unit 35 uses the AI cell algorithm for recognition.

[0034] Single-cell droplet generation: Droplets containing single cells are generated at the liquid generation structure and then ejected from the droplet printing outlet.

[0035] Single-cell printing: The micro-hole array chip is as Figure 3As shown, there is a waste liquid pool 43 at both the upper and lower parts. The default position of the droplet printing outlet (the outlet of the single-cell channel 16) is at the waste liquid pool 43 in the upper left corner, and printing is performed from left to right. When a droplet containing a single cell is recognized, it moves downward to the first single-cell micro-well in the upper left to receive the printed droplet. If the droplet does not contain a single cell or contains multiple cells, the micro-well array chip is moved to the position where the waste liquid pool 43 is located. In this way, single cells are printed on the micro-well array in an orderly manner. When single cells are printed to the lower half of the micro-well array chip ( Figure 3 below the center dash line), droplets that do not contain a single cell or contain multiple cells will be printed into the waste liquid pool 43 below the micro-well array chip.

[0036] Single-cell metabolite extraction: When all single-cell micro-wells of the micro-well array chip are filled, printing stops. The micro-well array chip is moved to the single-cell extraction position, and the capillary 71 drips a droplet containing the extraction solution into the first single-cell micro-well, waits for 1 second, and then the capillary 71 sucks the droplet of the extraction solution containing the metabolite.

[0037] Mass spectrometry analysis: The capillary 71 containing the extraction solution is moved into the open direct ionization mass spectrometer for mass spectrometry analysis, and the obtained single-cell mass spectrometry diagram of bladder cancer is as Figure 8 shown.

[0038] During the above process, if the recognition device 30 recognizes a single cell, the controller controls the driving unit so that the selected bearing position 42 of the bearing unit 41 is located below the outlet of the single-cell channel 16.

[0039] In order to accurately print the droplet containing a single cell onto a single micro-well array, it is necessary to satisfy Ld / Vd > ta + Lm / Vm.

[0040] t a has a value range of 10 - 20 ms, L d has a value range of 3 - 5 cm, V d has a value range of 1 - 2 m / s, L m has a value range of 300 - 500 μm, V m has a value range of 90 - 750 mm / s.

[0041] The recognition time of the recognition device 30 is t a , the distance between the microfluidic chip 11 and the micro-well array is L d , the droplet velocity is V d , the moving distance of the micro-well array is L m , and the moving velocity is V m .

[0042] The parameters of this embodiment are as follows: L d = 4 cm, V d= 1.5 m / s, L d / V d = 26.7 ms. t a = 15 ms, L m = 400 μm, V m = 200 mm / s, L m / V m = 2 ms. It satisfies: L d / V d > t a + L m / V m . Example 3

[0043] An application example of the microfluidics-based single-cell mass spectrometry detection system and method according to Embodiment 1 of the present invention in bladder cancer detection, different from Embodiment 2: As Figure 4 shown, each waste liquid pool 43 contains a row of single-cell microholes above and below. The default position of the droplet printing outlet (the outlet of the single-cell channel 16) is the position of the left-upper first waste liquid pool 43, and printing is performed from left to right. When a droplet containing a single cell is recognized, it moves to the left-upper first single-cell microhole to receive the printed droplet. If the droplet does not contain a cell or contains multiple cells (i.e., a waste droplet), the microhole array chip moves to the first waste liquid pool 43. In this way, the single-cell droplets will be printed onto the microhole array in an orderly manner. When the first row of single-cell microholes is filled, the position of the droplet printing outlet is on the right-upper first single-cell microhole at this time. If a new droplet is generated, the microhole array chip moves to the position of the first single-cell microhole from the right in the second row on the right-upper for single-cell printing, and the waste droplets are still printed into the first waste liquid pool 43; when the second row of single-cell microholes is filled, the microhole array chip moves to the third row of microholes on the left-upper for printing, and the waste droplets at this time are printed into the second row of waste liquid pools 43; and so on, until all single-cell microholes contain single-cell microdroplets. Example 4

[0044] An application example of the microfluidics-based single-cell mass spectrometry detection system and method according to Embodiment 1 of the present invention in bladder cancer detection, different from Embodiment 2: As Figure 5As shown in the figure, there is a waste liquid pool 43 at each of the upper, lower, left, and right positions. The default position of the droplet printing outlet is the position of the first waste liquid pool 43 in the upper left corner, and printing is performed from left to right. When a droplet containing a single cell is recognized, it moves to the first single-cell micro-well in the upper left corner to receive the printed droplet. If the droplet does not contain a cell or contains multiple cells (i.e., a waste droplet), the micro-well array chip moves to the first waste liquid pool 43. In this way, single-cell droplets will be printed onto the micro-well array in an orderly manner. Waste droplets are printed according to the principle of proximity, that is, the upper part of the two diagonals is printed into the upper waste liquid pool 43, the lower part of the diagonal is printed into the lower waste liquid pool, the left part of the diagonal is printed into the left waste liquid pool 43, the right part of the diagonal is printed into the right waste liquid pool 43, and the single-cell micro-wells on the diagonal are printed into the upper or lower waste liquid pool 43 according to the principle of proximity. Example 5

[0045] An application example of the microfluidics-based single-cell mass spectrometry detection system and method according to Embodiment 1 of the present invention in bladder cancer detection is different from Embodiment 2 in that: As Figure 6 shown in the figure, there is a waste liquid pool 43 at each of the upper, lower, left, and right positions outside the micro-well array, and there is a waste liquid pool 43 every two rows of single-cell micro-wells. The default position of the droplet printing outlet is the position of the first waste liquid pool 43 in the upper left corner, and printing is performed from left to right. When a droplet containing a single cell is recognized, it moves to the first single-cell micro-well in the upper left corner to receive the printed droplet. If the droplet does not contain a cell or contains multiple cells (i.e., a waste droplet), the micro-well array chip moves to the first waste liquid pool 43. In this way, single-cell droplets will be printed onto the micro-well array in an orderly manner. Waste droplets are printed according to the principle of proximity, that is, if the micro-well is in the outermost circle (outside the Figure 6 dash line), the waste droplet 43 is printed into the four waste liquid pools 43 at the upper, lower, left, and right positions outside the micro-well array; if the micro-well is in the inner circle (inside the Figure 6 dash line), the waste droplet is printed into the nearest three internal waste liquid pools 43. Example 6

[0046] An application example of the microfluidics-based single-cell mass spectrometry detection system and method according to Embodiment 1 of the present invention in bladder cancer detection is different from Embodiment 2 in that: As Figure 7As shown, there is a waste liquid pool 43 in the middle of the microhole array, and a waste liquid pool 43 is added every two rows of single-cell microholes outward. The default position of the droplet printing outlet is the position of the waste liquid pool 43 in the upper left outer part. When a droplet containing a single cell is recognized, it moves to the first single-cell microhole in the upper left to receive the printed droplet. If the droplet does not contain cells or contains multiple cells (i.e., waste droplet), the microhole array chip moves to the waste liquid pool 43 outside. Droplet printing is from left to right and from outside to inside, that is, from left to right and clockwise to the outermost microholes, then the second outermost microholes, until all microholes are printed full. Waste droplet printing is done according to the principle of proximity, that is, the outermost waste droplets are printed into the waste liquid pool 43 in the outermost layer, the waste droplets in the second and third layers are printed into the waste liquid pool 43 in the second layer, and so on.

Claims

1. A single-cell mass spectrometry detection system based on microfluidics, comprising a microfluidic chip, an identification device, and a mass spectrometry device; characterized in that, The single-cell mass spectrometry detection system further includes: a carrier unit having a plurality of single-cell carrying positions distributed in a matrix and a waste liquid pool; a driving unit configured to effect relative movement between the carrier unit and the microfluidic chip, such that single cells discharged from the microfluidic chip enter selected carrying positions, or liquid without single cells enters the waste liquid pool; a controller that controls the driving unit according to the result of an identification device, the identification device being configured to identify whether there are single cells in the liquid flow of the single-cell channel of the microfluidic chip.

2. The single-cell mass spectrometry detection system according to claim 1, wherein The microfluidic chip has a sample channel, a single-cell channel, and a plurality of auxiliary channels. A sample inlet is sequentially connected to an asymmetric bent sample channel and the single-cell channel. A fluid inlet is sequentially connected to the plurality of auxiliary channels and the single-cell channel. The outlets of the plurality of auxiliary channels uniformly surround the single-cell channel. The fluid flow rate and flow volume of the fluid entering the single-cell channel from the plurality of auxiliary channels are the same.

3. The single-cell mass spectrometry detection system according to claim 2, wherein The auxiliary channels are S-shaped, and two auxiliary channels are symmetrically arranged on both sides of the sample channel.

4. The single-cell mass spectrometry detection system according to claim 1, wherein The identification device includes a light source, an imaging unit, and an identification unit, and the emitted light of the light source is incident on the single-cell channel.

5. The single-cell mass spectrometry detection system according to claim 1, wherein The waste liquid pool is strip-shaped and is arranged outside the carrying positions and / or between adjacent carrying positions.

6. The single-cell mass spectrometry detection system according to claim 1, characterized in that, The mass spectrometry device includes an open direct ionization mass spectrometer.

7. A microfluidics-based single-cell mass spectrometry detection method, characterized in that, The single-cell mass spectrometry detection method includes the steps of: in the single-cell channel of the microfluidic chip, single cells pass through in sequence; the identification device detects whether there are single cells passing through in the single-cell channel; if the result is yes, the controller issues a movement instruction to the driving unit, and the selected carrying position of the carrier unit is located below the outlet of the single-cell channel; if the result is no, the controller issues a movement instruction to the driving unit, and the selected waste liquid pool of the carrier unit is located below the outlet of the single-cell channel; the selected carrying position carries single cells, or the selected waste liquid pool receives the liquid.

8. The single-cell mass spectrometry detection method according to claim 7, wherein In the microfluidic chip, the cell suspension sequentially passes through the sample inlet, the asymmetric bent sample channel, and the single-cell channel. The fluid sequentially passes through the fluid inlet and the plurality of auxiliary channels and enters the single-cell channel at the same fluid flow rate and the same flow volume. In the single-cell channel, the cells pass through one by one.

9. The single-cell mass spectrometry detection method according to claim 8, characterized in that The auxiliary channels are S-shaped, and two auxiliary channels are symmetrically arranged on both sides of the sample channel.

10. The single-cell mass spectrometry detection method according to claim 7, wherein: L d / V d >t a + L m / V m The recognition time of the recognition device is t a The distance between the microfluidic chip and the microhole array is L d The droplet velocity is V d The moving distance of the microhole array is L m The moving velocity is V m 。

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