CN113337451B - Accurate patterning method for single cells in multi-shear force microfluidic chip - Google Patents
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Abstract
The invention discloses an accurate patterning method for single cells in a multi-shear force microfluidic chip, which comprises the following steps: step one, dripping protein liquid on one side with a pattern of the pattern seal, incubating in a dark place, dipping the pattern seal, and drying; pressing the pattern stamp on the substrate glass slide and standing to obtain the substrate glass slide with a plurality of protein micro-pattern areas; putting the substrate slide into a culture dish, injecting cell suspension into the culture dish, putting the culture dish into an incubator for culture, and obtaining cell patterns growing adherent to a plurality of protein micro-pattern areas; step four, pasting the substrate slide on the bottom of the PDMS chip, and communicating the PDMS chip to the air suction hole of the substrate slide and the negative pressure vacuum channel at the bottom of the chip to perform vacuum negative pressure sealing so as to seal the substrate slide and the PDMS chip; and fifthly, adding liquid into the sample adding hole of the PDMS chip, shunting the liquid, then allowing the liquid to enter the plurality of reaction chambers, and performing fluid shear force stimulation on the cell pattern from one side of the reaction chambers to the other side of the reaction chambers.
Description
Technical Field
The invention relates to a cell culture technology, in particular to a method for accurately patterning single cells in a multi-shear force microfluidic chip.
Background
From the seventies of the twentieth century, patterning technology began to be used for biomolecules and cells. Cell patterning techniques are largely divided into two categories: one is to form a pattern of cell adhesion growth selectively by surface modification to form a patterned area of cell adhesion growth; another type is cell patterning by confining the cells to growth in a patterned area by a removable physical barrier.
In the prior art, methods of cell patterning are mainly divided into photolithography and soft lithography. Photoetching utilizes ultraviolet light to transfer geometric patterns on a mask to a substrate, firstly, a layer of photosensitive polymer photoresist is paved on the substrate, then, the substrate is exposed and developed under the coverage of the photomask to form patterns, after a surface modification material is adsorbed on the surface of the substrate, the substrate is immersed into an organic solvent to elute the photoresist, thus, a pattern of surface modification protein is formed on the bottom surface, cells can be adhered and grow according to the modified pattern, and the cell patterns are formed.
The soft etching technology forms a stamp on the patterned base film by using a high molecular polymer, such as Polydimethylsiloxane (PDMS), so as to achieve the purpose of copying a micron or even nano-scale structure. Commonly used are micro-contact printing (μ CP), microfluidic patterning, and template patterning. The traditional micro-contact printing can only form one or two molecular patterns, and can form various molecular patterns only by using a multi-stage stamp, but the operation process is relatively complex. Microfluidic patterning is a process related to micro-contact printing, but differs from micro-contact printing in that the bottom surface of the PDMS template has a network of micro-channels. Since microfluidic patterning uses solutions of different molecules to form patterns, the pattern of each molecule is generally continuously distributed over the surface, which limits the diversity of pattern shapes. Template patterning is a method that can perform cell patterning without chemically modifying a substrate, but is easily applicable to the preparation of simple patterns such as circles and squares.
The soft lithography patterning represented by microcontact printing μ CP and microcontact lift-off, the patterning by photolithography, or the patterning by microchannel and micropore confinement can realize precise control at single cell level. However, the realization of single cell patterning in the micron-scale channel of the microfluidic chip has not been solved effectively.
In 2006, the journal Lab-on-chip reports that plasma bonding and protein patterning of a microfluidic chip are completed in one step by using a PDMS stamp as a protective layer, but the control scale of a cell pattern can only reach 50-100 μm, single cell control cannot be realized, and the single cell control precision is 1-30 μm. Therefore, the advantages of the micro-fluidic chip in simulating the biophysical, chemical and mechanical environments of cells cannot be satisfied. In the last two decades, on the basis of realizing single cell patterning, when mechanical stimulation, medicament stimulation and the like are applied to cells, only a traditional mode is adopted to construct a flow chamber with a sandwich structure, and a pressing sealing or negative pressure sealing mode is adopted.
Therefore, the invention provides a simple and rapid method for accurately patterning single cells in the microfluidic chip, and provides a new research technology and strategy for the research of the fields of cell space positions, multi-condition microenvironments and cell interaction.
Disclosure of Invention
The invention designs and develops a method for accurately patterning single cells in a multi-shear force micro-fluidic chip, and solves the problem of control precision of the single cells in the chip, wherein the control precision reaches 1-30 mu m.
Another technical purpose of the invention is to realize the problems of one-step realization of patterning, fluid stimulation and vacuum packaging in a chip; the problem of multi-pattern and multi-shear synchronous stimulation is solved.
The technical scheme provided by the invention is as follows:
step one, pretreating a substrate slide; dripping protein liquid (Fibronectin or Collagen or the like) for promoting cell adherence on one side with the pattern of the pattern seal, incubating in a dark place, washing the pattern seal, and drying;
the pattern stamp comprises a plurality of columnar bulge pattern areas, each columnar bulge pattern area comprises a plurality of columnar bulges which are arranged at equal intervals, and the cross section size of each columnar bulge is approximately equal to the size of a single cell;
pressing the pattern stamp on the pretreated substrate glass sheet, standing, and transferring the protein on the stamp onto the substrate glass sheet to obtain a substrate glass sheet with a plurality of protein micro-pattern areas;
putting the substrate slide with the protein micro-patterns into a culture dish, injecting cell suspension into the culture dish, putting the culture dish into an incubator for culture, and obtaining cell patterns growing adherent to the plurality of protein micro-pattern areas;
step four, pasting the substrate slide with the cell pattern at the bottom of the PDMS chip, communicating the PDMS chip to the air suction hole of the substrate slide and the negative pressure vacuum channel at the bottom of the chip for vacuum negative pressure sealing, and sealing the substrate slide and the PDMS chip;
the bottom of the PDMS chip is provided with a plurality of reaction chambers, and the protein micro-pattern areas and the reaction chambers are arranged in a one-to-one correspondence manner;
and fifthly, adding liquid into the sample adding hole of the PDMS chip, shunting the liquid, then allowing the liquid to enter the plurality of reaction chambers, and performing fluid shear force stimulation on the cell pattern from one side of the reaction chambers to the other side of the reaction chambers.
Preferably, the cross-sectional shape of the columnar protrusion is circular, rhombic or elliptical; the distance between the axes of two adjacent columnar bulges ranges from 25 micrometers to 200 micrometers.
Preferably, the pattern stamp further comprises a plurality of strip-shaped raised pattern areas;
the strip-shaped protruding pattern area comprises a plurality of strip-shaped protrusions which are arranged in parallel at equal intervals, the cross section of each strip-shaped protrusion is in a rectangular strip shape, the width of each strip-shaped protrusion is 1-3 mu m, and the width of a gap between every two adjacent strip-shaped protrusions is 1-3 mu m.
Preferably, the height of the columnar projections is 10 μm; the height of the strip-shaped bulges is 1 mu m.
Preferably, in the first step, the method for pretreating the substrate slide comprises:
immersing the substrate glass slide into potassium dichromate concentrated sulfuric acid pickling solution for more than 12 hours, washing away residual acid solution with running water, washing with deionized water for multiple times, immersing into 70-75% alcohol solution, ultrasonically cleaning for 25-30 minutes, cleaning with deionized water for multiple times, and drying or blowing by nitrogen.
Preferably, in the first step, the protein solution is Cy5 fluorescently-labeled fibronectin or type II collagen; the concentration of the protein solution (Fibronectin) is 100 mug/mL, and the dropping amount is 20 mug L.
Preferably, in the second step, the pattern stamp is allowed to stand on the pretreated substrate slide for 12 hours to obtain the substrate slide with the protein micro-pattern.
Preferably, in the second step, the method further includes:
the slide with the protein micro-pattern is gently washed 1-2 times by PBS, passivated by 5% F127 for 45-60 minutes and then washed by PBS for a plurality of times.
Preferably, in the third step, the culture dish is injected with the cell suspension, and CO is added 2 Culturing in an incubator, incubating for 20-30 minutes, and sucking out cell suspension; washing the substrate slide with PBS for multiple times to obtain patterned and accurately positioned cells; after the culture medium is supplemented into the culture dish, CO is added 2 Culturing for 4-10 hours in an incubatorCells are grown in a patterned adherent manner.
Preferably, the PDMS chip includes: a first layer and a second layer;
the sample distribution channels are arranged in the first layer, and the inlet ends of the sample distribution channels are respectively communicated with the outlet ends of the sample adding holes;
wherein the inlet end of the sample adding hole is formed on the upper surface of the first layer;
the sample distributing holes are arranged in one-to-one correspondence to the sample distributing channels, inlet ends of the sample distributing holes are communicated with outlet ends of the sample distributing channels, and outlet ends of the sample distributing holes are arranged on the lower surface of the first layer;
the reaction chamber liquid inlet holes are through holes formed in the second layer and are communicated with the sample separation holes in a one-to-one correspondence manner;
a plurality of reaction chamber liquid outlet holes which are through holes arranged on the second layer;
the reaction chamber liquid inlet hole and the reaction chamber liquid outlet hole are respectively positioned at two ends of the reaction chamber.
The invention has the beneficial effects that:
(1) And meanwhile, the precise control of the four shearing forces, the cell spacing and the cell direction is realized.
(2) The patterning control of various single cells in the chip is realized, and four channels can realize four different cell patterns, such as different cell distances and different cell directions.
(3) And simultaneously, a composite microenvironment of fluid shear force and different cell space factors is constructed.
(4) The vacuum negative pressure channel is simple to package and is disposable.
Drawings
FIG. 1 is an exploded view of a PDMS chip and a substrate slide according to the present invention.
Fig. 2 is a perspective view of the bottom (lower) side of the first layer according to the present invention.
Fig. 3 is a perspective view of the upper surface side of the first layer of the present invention.
Fig. 4 is a bottom view of a second layer according to the present invention.
Fig. 5 is a perspective view of the upper surface side of the second layer in accordance with the present invention.
FIG. 6 is a schematic view of a substrate slide of the present invention.
FIG. 7 is a schematic view of the structure of the pattern stamp of the present invention.
Fig. 8 is an enlarged view of the pillar bump pattern region 211 according to the present invention.
Fig. 9 is an enlarged view of the patterned stud bump area 212 according to the present invention.
Fig. 10 is an enlarged view of the strip-shaped protrusion pattern region 213 according to the present invention.
Fig. 11 is an enlarged view of the raised stripe pattern 214 according to the present invention.
Detailed Description
The present invention is further described in detail below with reference to the attached drawings so that those skilled in the art can implement the invention by referring to the description text.
The invention provides a single-cell accurate patterning method of a multi-shearing force micro-fluidic chip, which is implemented by matching the multi-shearing force micro-fluidic chip with a pattern stamp.
As shown in fig. 1, the multi-shear force microfluidic chip comprises a PDMS chip and a substrate slide, wherein the PDMS chip comprises two layers of microchips, a first layer 110 is a sample-adding layer, and a second layer 120 is a reaction layer; wherein the first layer 110 is disposed above the second layer 120 and the base slide 130 is disposed below the second layer 120.
As shown in fig. 2-3, the first layer 110 is a sample adding layer, and the first layer 110 has a sample adding hole 111, two sample adding channels 112, four sample dividing channels 113, four sample dividing holes 114, four air vent holes 115, and an air vent hole 116. The inlet end of the sampling hole 111 is arranged on the upper surface of the first layer 110, and the outlet end extends into the first layer 110; the sample addition channels 112 are formed in the first layer 110, and the outlet ends of the sample addition holes 111 are simultaneously communicated with the inlet ends of the two sample addition channels 112. The sample distribution channels 113 are opened inside the first layer 110, and the outlet end of each sample application channel 112 is simultaneously communicated with the inlet ends of the two sample distribution channels 113. The sample distribution holes 114 are arranged in one-to-one correspondence with the sample distribution channels 113, inlet ends of the sample distribution holes 114 are located inside the first layer 110 and communicated with outlet ends of the sample distribution channels 113 corresponding thereto, and outlet ends of the sample distribution holes 114 are arranged on the lower surface of the first layer 110. The four exhaust holes 115 and the suction holes 116 are all through holes in the first layer 110.
As shown in fig. 4-5, the second layer 120 is a reaction layer for reaction of the sample and vacuum sealing of the chip. The second layer 120 has four reaction chambers 121, a negative pressure passage 122 and a pumping hole 123. The reaction chamber 121 is a groove formed in the bottom (lower surface side) of the second layer 120, and two ends of the reaction chamber 121 are respectively provided with a reaction chamber liquid inlet hole 121a and a reaction chamber liquid outlet hole 121b, wherein the reaction chamber liquid inlet hole 121a and the reaction chamber liquid outlet hole 121b are through holes formed in the second layer 120. The liquid inlet holes 121a of the reaction chamber and the outlet ends of the sample separation holes 114 are correspondingly and coaxially arranged one by one and communicated, and the liquid outlet holes 121b of the reaction chamber and the exhaust holes 115 are correspondingly and coaxially arranged one by one and communicated. The negative pressure passage 122 is a groove opened at the bottom (lower surface side) of the second layer 120, the negative pressure passage 122 encloses a rectangular passage around each reaction chamber 121, and the negative pressure passage 122 communicates with the suction hole 123. The air suction hole 123 is a through hole formed in the second layer 120, and the air suction hole 123 is coaxially disposed and communicated with the air suction hole 116.
As shown in fig. 6, the base slide 130 is a cover glass having a large area, and the base slide 130 is thinner than the first layer 110 and the second layer 120.
As shown in fig. 7 to 11, one side of the pattern stamp 210 has four raised pattern regions including two pillar-shaped raised pattern regions 211, 212 and two bar-shaped raised pattern regions 213, 214. The columnar bump pattern regions 211 and 212 respectively comprise a plurality of columnar bumps arranged at equal intervals, and the cross-sectional size of each columnar bump is approximately equal to the size of a single cell, so that the single cell of the cell can be accurately controlled. The cross section of each columnar bulge can be circular, rhombic or elliptical, and the distance between the axes of two adjacent columnar bulges ranges from 25 micrometers to 200 micrometers. In the present embodiment, the columnar projections in the columnar projection pattern regions 211 and 212 have a uniform circular cross section, a diameter of 25 to 38 μm, and a height of 5 to 10 μm, and can realize accurate control of single cells of osteoblasts similar-sized cells (15 to 30 μm). If the cross-sectional diameter of the stud bump exceeds 45 μm, multiple cells will adhere, which is not favorable for precise control of single cells. The strip-shaped protrusion pattern regions 213 and 214 respectively comprise a plurality of strip-shaped protrusions which are arranged in parallel at equal intervals, the cross section of each strip-shaped protrusion is in a rectangular strip shape, the width of each strip-shaped protrusion is 1-3 micrometers, and the width of a gap between every two adjacent strip-shaped protrusions is 1-3 micrometers. In the present embodiment, the stripe-shaped protrusions in the stripe-shaped protrusion area 213 are arranged along the axial direction of the stamp, the direction of the stripe-shaped protrusion in the stripe-shaped protrusion area 214 is perpendicular to the direction of the stripe-shaped protrusion in the stripe-shaped protrusion area 213, and the heights of the stripe-shaped protrusions in both areas are 1 μm.
In practical applications, the pattern areas are not limited to four, nor to the shapes given in this embodiment, and may be determined according to specific situations. The number of the pattern areas is the same as that of the reaction chambers, and the positions correspond to one another.
The flow of the method for accurately patterning the single cell in the multi-shear force microfluidic chip provided by the invention is mainly divided into three parts, the cell patterning is realized on the substrate glass slide by using a micro contact technology, the PDMS chip is in contact sealing with the substrate glass slide, and the shear force stimulation is realized by adding fluid into the PDMS chip. The specific implementation process is as follows:
1. the substrate slide 130 is immersed into potassium dichromate concentrated sulfuric acid pickling solution (the concentration of sulfuric acid is over 95 percent) for over 12 hours, the residual acid solution is washed away by running water, then the substrate slide is washed by deionized water for 3 times, immersed into 70 to 75 percent alcohol for ultrasonic cleaning for 25 to 30 minutes, and then the substrate slide is cleaned by deionized water for more than 3 times, and dried at 37 ℃ or dried by nitrogen for standby.
When in use, the PDMS chip (the combination of 110 and 120) is first plasma bonded to the substrate slide 130 by using a plasma cleaning apparatus. Selecting a proper pattern stamp 210, and dripping about 20 mu L of protein liquid with the concentration of 50-150 mu g/mL on the side of the pattern stamp 210 with the convex pattern. Wherein the protein solution is Cy5 fluorescence labeled Fibronectin (Fibronectin) or type II collagen. And (4) taking care of keeping out of the sun when the protein solution is dripped, and putting the solution in a culture dish tightly wrapped by tin foil paper after the completion of the light-tight incubation for 1 to 1.5 hours. After 1-1.5 hours, the protein liquid on the surface of the pattern stamp 210 is absorbed, the pattern stamp 210 is clamped by tweezers, and the pattern stamp is firstly dipped and washed once by PBS and then dried by nitrogen, and then dipped and washed by ultrapure water and dried by nitrogen.
Lightly pressing the pattern stamp 210 on the previously processed substrate slide 120, standing for 12 hours to transfer the proteins on the pattern stamp 210 onto the substrate slide 130, and forming a plurality of protein micro-pattern regions (corresponding to the pattern regions on the pattern stamp 130 one by one) on the substrate slide 130.
Osteoblasts were digested and centrifuged, the supernatant discarded, and the medium was added to the suspension for further use. The base slide 130 with the multiple protein micropatterned areas was washed 3 times with PBS in a clean bench, treated with 5% F127 for 45-60 minutes to passivate the areas not covered by protein to prevent cell adhesion growth, and then the base slide 130 was washed at least 3 times with PBS. Thereafter, the base slide 130 is placed in a 35mm small petri dish.
Injecting the blown cell suspension into a small culture dish with a substrate slide 130, and adding CO 2 Culturing in an incubator; wherein the density of the cell suspension is 1500K/mL-2000K/mL. After incubation for 20-30 minutes, the cell suspension was aspirated and washed several times with PBS (care was taken to avoid cell attachment areas, the procedure was gentle), and the patterned precisely-positioned cells were observed under a microscope. Supplementing 1-2 mL of culture medium, and slightly shaking the small culture dish to ensure that the cells are uniformly distributed; then adding CO 2 Culturing in an incubator for 4-10 hours, and taking out to observe the cell patterned adherent growth.
2. The glass slide with the cell pattern is taken out by tweezers and is attached to the bottom of the PDMS chip, and the PDMS chip is communicated with the air extraction holes 116 and 123 of the substrate glass slide 130 and the negative pressure channel 122 on the second layer 120 through the first layer 110 and the second layer 120 for vacuum negative pressure sealing treatment, so that the substrate glass slide 130 with the cell pattern is sealed with the PDMS chip. Wherein, the cell pattern regions on the substrate slide 130 correspond to the reaction chambers 121 one by one.
3. Fluid shear force stimulation is applied, liquid is added through the sample adding holes 111 of the first layer 110, the liquid is divided into two parts and enters the two sample adding channels 112 of the first layer 110, the two parts are divided into four parts and enter the four sample dividing channels 113, the liquid enters the four reaction chambers 121 through the 4 sample dividing holes 114 and the reaction chamber liquid inlet holes 121a of the second layer 120, and the fluid shear force stimulation is carried out on cell patterns from one end to the other end of the reaction chambers 121, so that the problem of synchronous stimulation of multiple patterns and multiple shearing is solved. After the liquid flows to the other end of the reaction chamber, the redundant liquid is discharged from the exhaust hole 115 after passing through the liquid outlet hole 121b of the reaction chamber; the air vent 115 is also used to vent air bubbles trapped in the liquid. In this embodiment, the cell pitch is controlled to be 50 μm and 100 μm, respectively, and the cell direction is controlled to be horizontal and vertical by providing four protrusion pattern regions on the stamp.
While embodiments of the invention have been described above, it is not limited to the applications set forth in the description and the embodiments, which are fully applicable in various fields of endeavor to which the invention pertains, and further modifications may readily be made by those skilled in the art, it being understood that the invention is not limited to the details shown and described herein without departing from the general concept defined by the appended claims and their equivalents.
Claims (10)
1. An accurate patterning method for single cells in a multi-shear force microfluidic chip is characterized by comprising the following steps:
step one, preprocessing a substrate slide; dripping protein liquid on one side with the pattern of the pattern seal, incubating in a dark place, and drying the pattern seal after being stained and washed;
the pattern stamp comprises a plurality of columnar bulge pattern areas, each columnar bulge pattern area comprises a plurality of columnar bulges which are arranged at equal intervals, and the cross section size of each columnar bulge is approximately equal to the size of a single cell;
pressing the pattern stamp on the pretreated substrate glass sheet, standing, and transferring the protein on the stamp onto the substrate glass sheet to obtain a substrate glass sheet with a plurality of protein micro-pattern areas;
putting the substrate slide with the protein micro-patterns into a culture dish, injecting cell suspension into the culture dish, putting the culture dish into an incubator for culture, and obtaining cell patterns growing adherent to the plurality of protein micro-pattern areas;
step four, pasting the substrate slide with the cell pattern at the bottom of the PDMS chip, communicating the PDMS chip to the air suction hole of the substrate slide and the negative pressure vacuum channel at the bottom of the chip for vacuum negative pressure sealing, and sealing the substrate slide and the PDMS chip;
the bottom of the PDMS chip is provided with a plurality of reaction chambers, and the protein micro-pattern areas and the reaction chambers are arranged in a one-to-one correspondence manner;
and fifthly, adding liquid into the sample adding hole of the PDMS chip, shunting the liquid, then allowing the liquid to enter the plurality of reaction chambers, and performing fluid shear force stimulation on the cell pattern from one side of the reaction chambers to the other side of the reaction chambers.
2. The method for accurately patterning single cells in a multi-shear force microfluidic chip according to claim 1, wherein the cross-sectional shape of the columnar protrusions is circular, rhombic or elliptical; the distance between the axes of two adjacent columnar bulges ranges from 25 micrometers to 200 micrometers.
3. The accurate patterning method for single cells in a multi-shear force microfluidic chip according to claim 2, wherein the pattern stamp further comprises a plurality of strip-shaped raised pattern regions;
the strip-shaped protruding pattern area comprises a plurality of strip-shaped protrusions which are arranged in parallel at equal intervals, the cross section of each strip-shaped protrusion is in a rectangular strip shape, the width of each strip-shaped protrusion is 1-3 mu m, and the width of a gap between every two adjacent strip-shaped protrusions is 1-3 mu m.
4. The method for accurately patterning single cells in a multi-shear force microfluidic chip according to claim 3, wherein the height of the columnar protrusions is 10 μm; the height of the strip-shaped bulges is 1 mu m.
5. The method for accurately patterning single cells in a multi-shear force microfluidic chip according to claim 4, wherein in the first step, the method for pretreating the substrate slide comprises:
immersing the substrate glass slide into a potassium dichromate concentrated sulfuric acid pickling solution for more than 12 hours, washing the residual acid solution with running water for many times, immersing the substrate glass slide into a 70-75% alcohol solution, ultrasonically cleaning for 25-30 minutes, cleaning with deionized water for many times, and drying or blowing by nitrogen.
6. The method for accurately patterning single cells in a multi-shear force microfluidic chip according to claim 5, wherein in the first step, the protein solution is Cy5 fluorescently-labeled fibronectin or type II collagen; the concentration of the protein solution is 100 mug/mL, and the dropping amount is 20 mug L.
7. The method for accurately patterning single cells in a multi-shear force microfluidic chip according to claim 5 or 6, wherein in the second step, the pattern stamp is allowed to stand on the pretreated substrate slide for 12 hours to obtain the substrate slide with the protein micro pattern.
8. The method for accurately patterning single cells in a multi-shear force microfluidic chip according to claim 7, wherein in the second step, the method further comprises:
the slides with the protein micropattern were washed multiple times with PBS, treated with 5% F127 for 45-60 minutes, and then washed multiple times with PBS.
9. The method for accurately patterning single cells in a multi-shear force microfluidic chip according to claim 8, wherein in the third step, the culture dish is filled with the cell suspension, and CO is added 2 Culturing in an incubator, incubating for 20-30 minutes, and sucking out cell suspension; washing the substrate slide with PBS for multiple times to obtain patterned and accurately positioned cells; after the culture medium is supplemented into the culture dish, CO is added 2 IncubatorCulturing for 4-10 hours to make the cells grow in a patterned adherent manner.
10. The method for accurately patterning single cells in a multi-shear microfluidic chip according to claim 9, wherein the PDMS chip comprises: a first layer and a second layer;
the sample distribution channels are arranged in the first layer, and the inlet ends of the sample distribution channels are respectively communicated with the outlet ends of the sample adding holes;
wherein the inlet end of the sample addition hole is formed on the upper surface of the first layer;
the sample distributing holes are arranged in one-to-one correspondence to the sample distributing channels, inlet ends of the sample distributing holes are communicated with outlet ends of the sample distributing channels, and outlet ends of the sample distributing holes are arranged on the lower surface of the first layer;
the reaction chamber liquid inlet holes are through holes formed in the second layer and are communicated with the sample separation holes in a one-to-one correspondence manner;
a plurality of reaction chamber liquid outlet holes which are through holes arranged on the second layer;
the reaction chamber liquid inlet hole and the reaction chamber liquid outlet hole are respectively positioned at two ends of the reaction chamber.
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