TWI429909B - Biochip, manufacturing method thereof and sensing method for cells - Google Patents

Description

Cell detection wafer, manufacturing method thereof and cell detection method

The present invention relates to a biodetection wafer, and more particularly to a cell detection wafer, a method of manufacturing the same, and a method of detecting cells.

Cancer has been the leader of the top ten causes of death in China for 29 years. In 1998, the number of cancer deaths among Chinese people was 39,917, accounting for 28.1% of all deaths. Cancer occurs when cells become abnormal and continue to divide themselves to form more mutant cells. The reason why cancer is terrible is that the current treatment is still not perfect and is often regarded as an incurable disease.

In 1973, American scientists discovered that in the human immune system, a group of cells can effectively kill cancer cells, and named it Nature Killer Cell (NK Cell). Since then, natural killer cells have been used to activate other white blood cells (including T lymphocytes (T cells) and phagocytic cells (phagocytic cells)) and how the immune system responds to a wide range of infections. It has also been established. Therefore, natural killer cells are also widely regarded as beneficial for fighting cancer and infection.

Traditional chemical anticancer drugs have a strategy of inhibiting cell growth, often leading to drug resistance or side effects. Therefore, in novel anti-cancer therapies, the strategy adopted is to induce the patient's own natural immunity to reduce side effects.

Most of the "specific T cells" or "natural killer cells" in cancer patients are less robust. Natural killer T cells are equivalent to vanguards. Once the pioneers are active, the next specific T cells (like the special forces) will naturally drive and attack the cancer cells.

Natural killer cells have the ability to kill cancer cells spontaneously (also known as poisoning ability). Unlike antibody responses and T cells, natural killer cells can directly contact tumor cells to produce a toxic effect, and they are not specific and antibody-dependent, so natural killer cells can be said to be the first line of defense against cancer.

For anti-cancer research, the current screening and matching of cells is still carried out in traditional biological experiments. However, traditional biological experiments require more cell samples, and it is impossible to accurately observe the cell-to-cell reaction at a specific ratio, and the cost is high. Therefore, the ability to kill natural killer cells cannot be effectively monitored.

In view of the above problems, the present invention provides a cell detecting wafer, a method of manufacturing the same, and a cell detecting method, thereby solving the problems of the prior art.

In one embodiment, the cell detection wafer includes a main channel, a first trench, a second trench, a first secondary runner, and a second secondary runner.

The main channel includes a first end, a second end, and a conduit. The first end and the second end are opposite each other. The piping is connected between the first end and the second end.

The first groove is disposed on one side of the pipe of the main flow path. The second groove is disposed on the other side of the conduit of the main flow channel with respect to the first groove.

The first secondary flow channel is disposed on the other side of the first groove relative to the main flow channel, and a side of the first secondary flow channel is coupled to the first groove.

The second secondary flow channel is disposed on the other side of the second groove relative to the main flow channel, and the side of the second secondary flow channel is coupled to the second groove.

In another embodiment, the cell detection wafer further includes a first pneumatic valve and a second pneumatic valve.

The first pneumatic valve is disposed on the main flow channel on the same side of the first groove and the second groove. The second pneumatic valve is disposed on the main flow path of the first groove and the second groove with respect to the other side of the first pneumatic valve.

In one embodiment, the cell detection method comprises: simultaneously injecting a first cell sample, a first buffer solution, and a second cell sample to a main channel; and comparing a second buffer solution and a third buffer solution to a mainstream The flow rates of the same flow and different from the main flow are respectively injected into the secondary flow channels; in the first cell sample, the first buffer solution and the second cell sample are in the main flow channel and the second buffer solution and the third buffer solution are After flowing for a while in some of the secondary flow passages, the communication between the introduction zone and the mixing zone and the communication between the lead-out zone and the mixing zone are blocked, and the second buffer solution and the third buffer solution are opposite to each other in the opposite main flow channel. The flow direction is separately injected into the secondary flow path; and after the secondary flow path has the opposite flow direction for a period of time, the mixed area is observed.

The mainstream track includes a mixing zone, a lead-in zone and a lead-out zone. The lead-in area is connected to the left side of the mixing area, and the lead-out area is connected to the right side of the mixing area. The first cell sample and the second cell sample flow from the introduction zone through the mixing zone to the lead-out zone under the separation of the first buffer solution.

Wherein, a side of one of the flow channels communicates with an upper side of the mixing zone via the first groove, and a side of the other of the secondary flow paths communicates with a lower side of the mixing zone via the second groove.

In one embodiment, the method for manufacturing a cell detection wafer includes: forming a first-order channel layer; combining the channel layer with a surface having a main channel, two grooves, and two sub-channels with a transparent substrate; forming a surface having a two-chamber a pneumatic valve layer; and combining the flow channel layer with a surface having a two chamber with respect to the surface having the main flow path, the groove and the secondary flow path and the pneumatic valve layer.

Wherein, the surface of the flow channel layer is formed with a main channel, two grooves and two secondary channels. One side of one of the flow passages communicates with one side of the main flow passage through a groove, and one side of the other auxiliary flow passage communicates with the other side of the main flow passage through the other.

Wherein, one surface of the pneumatic valve layer is formed with two chambers, and the other surface of the pneumatic valve layer is formed with at least one injection port communicating with the two chambers.

Moreover, after the pneumatic valve layer is combined with the flow channel layer, the two chambers of the pneumatic valve layer are respectively located on the main channels corresponding to the two sides of the two grooves.

Here, the cell detecting wafer of one embodiment is a sample solution which simultaneously flows into two cells using the sheath flow principle, and does not react with each other during transport. The microfluidic channel structure is then used to capture cells in the sample solution. The captured cells are then confined in a fixed space, and eddy currents are generated to sufficiently mix and react the two cells in a fixed space. Finally, it can be directly observed using an optical display system. Alternatively, the respective detection values of the state of the cell reaction are further calculated.

Among them, the structure of microchannel channels of different lengths can be designed to capture cells of a specific ratio.

Also, the micropneumatic valve can be used to confine the captured cells to a fixed space.

Furthermore, by providing adjacent two-channel solutions in different flow directions, eddy currents are generated to sufficiently mix the cells in the fixed space.

Figure 1 is a schematic illustration of a cell detection wafer of one embodiment. Fig. 2 is a schematic bottom view showing the center line frame A corresponding to Fig. 1.

Referring to Figures 1 and 2, the cell detecting wafer includes a main channel 110, two sub-channels (hereinafter referred to as a first sub-channel 132 and a second sub-channel 134, respectively) and two grooves (hereinafter referred to as respectively It is a first trench 152 and a second trench 154).

The main channel 110 is a micro flow channel. The first secondary flow channel 132 and the second secondary flow channel 134 may also be micro flow channels. The micro flow channel means that the flow field state in the central region of the flow channel is laminar, that is, the Renolds number is less than 2300.

The main flow channel 110 includes a first end 110a, a second end 110b, and a conduit 110c. The first end 110a and the second end 110b are opposite to each other, that is, the two ends of the main flow channel 110. The line 110 is then connected between the first end 110a and the second end 110b.

The first groove 152 is disposed at one side of the pipe 110c of the main flow path 110. The first secondary flow channel 132 is disposed on the other side of the first trench 152 with respect to the main flow channel 110, and the side of the first secondary flow channel 132 is coupled to the first trench 152. In other words, the side of the line 132c of the first auxiliary flow path 132 and the side of the line 110c of the main flow path 110 are respectively engaged with the opposite sides of the first groove 152, so that the tube of the first auxiliary flow path 132 is caused. The path 132c communicates with the line 110c of the main flow path 110 via the first groove 152.

The second groove 154 is disposed on the other side of the line 110c of the main flow path 110 with respect to the first groove 152. The second secondary flow path 134 is disposed on the other side of the second groove 154 with respect to the main flow path 110, and the side of the second secondary flow path 134 is coupled to the second groove 154. In other words, the side of the line 134c of the second auxiliary flow path 134 and the side of the line 110c of the main flow path 110 are respectively engaged with the opposite sides of the second groove 154, so that the tube of the second auxiliary flow path 134 The path 134c communicates with the line 110c of the main flow path 110 via the second groove 154.

That is, the first sub-flow path 132, the first groove 152, the main flow path 110, the second groove 154, and the second sub-flow path 134 are sequentially adjacent to each other side by side. Also, the two flow paths (132, 110/110, 134) are in communication with the grooves (152/154).

In an embodiment, the first trench 152 and the second trench 154 have different lengths L1, L2, that is, the lengths L1, L2 of the sides of the pipeline 110c connecting the main channel 110 are different. For example, the length L1 of the first trench 152 may be smaller than the length L2 of the second trench 154.

In an embodiment, the height of the two grooves (152, 154) is less than the height of the main flow channel 110. The height of each groove (152/154) may also be less than the height of the adjacent secondary flow path (132/134).

Figure 3 is a schematic perspective view of the center frame B corresponding to Figure 2; Referring to Figure 3, the microchannel layer 10 can be an integrally formed layer of material having a surface structure. The surface of the microchannel layer 10 has a surface structure of the main flow path 110, the auxiliary flow paths (132, 134), and the grooves (152, 154). Wherein, the microchannel layer 10 can be an integrally formed material layer having a surface structure.

Referring to FIG. 3, the main flow path 110, the auxiliary flow paths (132, 134), and the grooves (152, 154) may be disposed on the surface 20a of the transparent substrate 20. The height H1/H2 of the surface 20 of the transparent substrate 20 with respect to the top of each trench (152/154) is smaller than the height H3 of the surface 20a of the transparent substrate 20 with respect to the top of the main channel 110. Moreover, the height H1/H2 of the surface 20a of the transparent substrate 20 with respect to the top of each of the grooves (152/154) may be smaller than the secondary flow path of the surface 20a of the transparent substrate 20 with respect to each of the grooves (152/154). The height of the top of (132/134) is H4/H5.

In one embodiment, referring back to Figures 1 and 2, the conduit 110c of the main flow channel 110 includes a lead-in area 112, a mixing zone 114, and a lead-out zone 116.

The upper side and the lower side of the mixing zone 114 are respectively provided with a first groove 152 and a second groove 154. The lead-in area 112 communicates with the left side of the mixing zone 114, and the lead-out area 116 communicates with the right side of the mixing zone 114. In one embodiment, the conduit 110c can be a straight-lined pipeline that is sequentially connected to the lead-in zone 112, the mixing zone 114, and the lead-out zone 116. Wherein, under the top view cell detection wafer, the mixing zone 114 can be a rectangular chamber, or a circular chamber, or an arbitrary polygon. The lead-in area 112 and the lead-out area 116 are oppositely disposed on both sides of the mixing area 114. The two grooves (152, 154) are opposite to the two sides of the line connecting the lead-in area 112 and the lead-out area 116.

In one embodiment, the first end 110a of the main flow channel 110 has three solution inlets E1, E2, E3, and a side sheath flow channel 122, an intermediate flow channel 124, and an edge sheath flow channel 126 are sequentially connected. The second end 110b of the main flow channel 110 is a solution outlet.

One end of the side sheath flow channel 122 has an injection port 122a, and the other end of the edge sheath flow channel 122 engages the solution inlet E1 of the first end 110a of the main channel 110.

The intermediate flow passage 124 has an injection port 124a at one end, and the other end of the intermediate flow passage 124 engages the solution inlet E2 of the first end 110a of the main flow channel 110.

One end of the side sheath flow channel 126 has an injection port 126a, and the other end of the side sheath flow channel 126 engages the solution inlet E3 of the first end 110a of the main channel 110.

Figure 4 is a schematic illustration of a cell detection wafer of another embodiment. Fig. 5 is a bottom view showing the structure of the center frame C corresponding to Fig. 4.

In one embodiment, referring to Figures 4 and 5, the cell detection wafer may further include a first pneumatic valve 172 and a second pneumatic valve 174.

The first pneumatic valve 172 is disposed on the line 110c of the main flow path 110 on the same side of the first groove 152 and the second groove 154. That is, the first pneumatic valve 172 can be disposed in communication with the mixing zone 112 and the mixing zone 114, and the first pneumatic valve 172 can control communication between the lead-in zone 112 and the mixing zone 114.

The second pneumatic valve 174 is disposed on the line 110c of the main passage 110 of the first groove 152 and the second groove 154 with respect to the other side of the first pneumatic valve 172. That is, the second pneumatic valve 174 can be disposed in communication with the mixing zone 116 and the mixing zone 114, and the second pneumatic valve 174 can control communication between the lead-out zone 116 and the mixing zone 114.

Fig. 6 is a schematic perspective view showing the center line frame D corresponding to Fig. 5. Referring to Figure 6, the surface of the pneumatic valve layer 30 has the surface structure of the chamber. Wherein, the pneumatic valve layer 30 can be an integrally formed material layer having a surface structure.

The first pneumatic valve 172 and the second pneumatic valve 174 may be chambers above the main flow channel 110 on either side of the first trench 152 and the second trench 154, respectively. In other words, the chamber as the first pneumatic valve 172 is located above the junction of the lead-in zone 112 and the mixing zone 114, while the chamber as the second pneumatic valve 174 is located above the junction of the lead-out zone 116 and the mixing zone 114.

Referring to Fig. 7, in the manufacture of the cell detecting wafer, the first-order layer 10 and a pneumatic valve layer 30 may be formed first (step S510), and then the pneumatic valve layer 30, the flow channel layer 10 and the transparent substrate 10 are sequentially laminated. Combined (step S530).

Referring to the third or sixth drawing described above, the surface of the flow channel layer 10 is formed with a main flow path 110, two grooves (152, 154) and two secondary flow paths (132, 134), and a secondary flow path (132). One side communicates with one side of the main channel 110 through a groove (152). The other side of the other flow path (134) communicates with the other side of the opposite groove (152) of the main flow path 110 through the other groove (154).

Referring to FIG. 4, one surface of the pneumatic valve layer 30 is formed with two chambers (172, 174), and the other surface of the pneumatic valve layer 30 (ie, the outer surface of the cell detecting wafer) is formed with a communicating two chamber. At least one injection port 172a, 172b of (172, 174). Here, although the two chambers (172, 174) have their respective injection ports 172a, 172b as an example, this is not a limitation of the present invention. For example, in actual production, it is also considered to design a two chamber (172, 174) to share an injection port.

In step S530, the flow channel layer 10 is bonded to the transparent substrate 10 with the surfaces of the main flow path 110, the grooves (152, 154), and the auxiliary flow paths (132, 134). Also, the flow channel layer 10 has chambers (172, 174) with respect to the other surface having the surfaces of the main flow path 110, the grooves (152, 154) and the secondary flow paths (132, 134) and the pneumatic valve layer 30. The surface is combined. That is, the chambers (172, 174) are respectively located on the line 110c corresponding to the main flow path 110 on both sides of the grooves (152, 154).

Among them, the pneumatic valve layer 30, the flow channel layer 10 and the transparent substrate 10 can be treated by plasma treatment to cause the three to be sequentially joined. Here, the plasma treatment can use oxygen plasma. Further, the parameters of the plasma treatment can be set to 500 mTorr, 30 mW and performed for about 60 seconds. For example, the surfaces of the various layers (the pneumatic valve layer 30, the flow channel layer 10, and the transparent substrate 10) are first treated with oxygen plasma, and the three are sequentially laminated and joined immediately after the treatment. After bonding, the entire structure can be placed on a hot plate at about 120 ° C for about 30 minutes to enhance the bond strength.

In step S510, the flow channel layer may be formed by micro-extrusion or formed by a template.

Referring to Fig. 8A, first, a template 40 having a surface structure 412 corresponding to a main flow path, a groove, and a secondary flow path is formed. Referring to Fig. 8B, the flow path layer 10 is formed by using the template 40. Referring to Fig. 8C, next, the mold release process of the template 40 and the flow path layer 10 is performed to obtain the flow path layer 10.

In addition, the pneumatic valve layer 30 may be first bonded to the other side surface of the flow channel layer 10 relative to the template 40, and then the mold release process of the template 40 and the flow channel layer 10 may be performed.

For example, the sputum oil may be applied prior to the surface of the stencil 40 to facilitate stripping of the formed flow channel layer 10. Next, the prepared transparent material is poured onto the stencil 40, and the stencil 40 (having a transparent material thereon) is placed in the vacuum chamber for about 30 minutes to remove air bubbles in the rubber material. After the bubbles were removed, the template 40 (having a transparent material thereon) was placed on a hot plate at about 120 ° C for about 15 minutes to cure the transparent material thereon. The cured transparent material is the flow channel layer 10, and the formed flow channel layer 10 is peeled off from the template 40.

Alternatively, the surface of the material of the flow path layer 10 is directly imprinted on the surface of the template 40 to form the surface structure of the micro flow path.

In step S512, the template 40 can be formed by a two-dimensional lithography process.

Referring to Figures 9A and 9B, a mask 420 identical to the outer contours of the main channel 110, the grooves (152, 154) and the secondary flow paths (132, 134) is formed on the surface of a substrate 410.

The surface of the substrate 410 is patterned under the masking of the mask 420.

Referring to Figures 10A and 10B, after patterning, the mask 420 is removed to obtain a surface structure 412 having contours corresponding to the main channel 110, the grooves (152, 154) and the secondary runners (132, 134). Substrate 410.

Referring to Figures 11A and 11B, mask 422 is again formed on the surface of substrate 410 having surface structure 412. Here, the mask 422 exposes the surface 410a, 410b at the location of the substrate 410 corresponding to the trenches (152, 154).

Next, the surfaces 410a, 410b of the substrate 410 are etched under the masking of the mask 422. Also, the etching depth h1 is smaller than the thickness h2 of the surface structure 412 as shown in FIGS. 12A and 12B.

After patterning, the mask 422 is removed to obtain a template 40 having surface structures 412 corresponding to the main runners, trenches, and secondary runners.

The material of the flow channel layer 10 and the pneumatic valve layer 30 may be a material (ie, a transparent material) having good optical properties (enough to provide an optical observation system for observation). The material of the transparent substrate 20 may be glass, or a material having good optical properties (enough to provide an optical observation system for observation) (ie, a transparent material).

Transparent materials such as resins, gelatin and enamel materials. Rubber such as PDMS (polydimethylsiloxane; polydimethylsiloxane), PMMA (polymethyl methacrylate), PBA (polybutylene succinate), ABS (Acrylonitrile Butadiene Styrene; acrylonitrile-styrene-butyl Diene copolymer), parylene, PC (Polycarbonate; polycarbonate resin), acrylic, and the like.

The material of the template 40 can be a coffin. Also, the thickness (height) of the transparent substrate 20 may be about 170 μm. The thickness (height) of the template 40 may be about 30 μm.

Referring to Fig. 13, and referring to Figs. 3-6, the use of the above cell detecting wafer will be described below.

First, the first cell sample S1, the first buffer solution Buf-1, and the second cell sample S2 are simultaneously injected into the main channel 110 by the sheath flow principle (step S610).

Referring again to FIG. 14, in step S610, the first cell sample S1 may be injected by the injection port 122a located at one end of the sheath flow channel 122. The first buffer solution Buf-1 may be injected by an injection port 124a located at one end of the intermediate flow path 124. The second cell sample S2 can then be injected by an injection port 126a located on one end of the side sheath flow channel 126.

After the first cell sample S1, the first buffer solution B1, and the second cell sample S2 are injected, the first cell sample S1 and the second cell sample S2 are flowed from the introduction zone 112 through the mixing zone 114 under the separation of the first buffer solution B1. To the lead-out area 116.

And, the second buffer solution Buf-2 and the third buffer solution Buf-3 are respectively injected into the two secondary flow paths (132, 134) in the same flow direction as the main flow channels 110 and different from the flow rate of the main flow path 110 (steps) S620).

In step S620, the second buffer solution Buf-2 may be injected from the injection port located at one end 132a of the first sub-flow channel 132, different from the flow rate of the fluid in the main flow channel 110, so that the first sub-flow channel 132 The second buffer solution Buf-2 has the same flow direction but different flow rates as the fluid in the main flow channel 110. That is, the second buffer solution Buf-2 flows from one end 132a of the first sub-flow path 132 to the other end 132b.

The third buffer solution Buf-3 is also injected from the injection port located at one end 134a of the second auxiliary flow path 134 at a flow rate different from that of the fluid in the main flow path 110, so that the third buffer solution in the second auxiliary flow path 134 Buf-3 has the same flow direction but different flow rates as the fluid in the main flow channel 110. That is, the third buffer solution Buf-3 flows from one end 134a of the second sub-flow path 134 to the other end 134b.

At this time, the cell cell-1 in the first cell sample S1 flowing through the mixing zone 114 is caught in the first groove 152 due to the pressure difference generated by the first secondary flow path 132 and the main flow path 110 due to different flow rates. in. At the same time, the cell cell-2 in the second cell sample S2 flowing through the mixing zone 114 is trapped in the second trench 154 due to the pressure difference generated by the second secondary flow channel 134 and the main flow channel 110 due to different flow rates. in.

Here, the first buffer solution Buf-1, the second buffer solution Buf-2, and the third buffer solution Buf-3 may employ the same buffer solution. Furthermore, the first buffer solution Buf-1 may also be the same as the base solution of the first cell sample S1 and the second cell sample S2.

After the solutions injected into the main flow path 110 and the secondary flow paths (132, 134) are simultaneously circulated for a while, the communication between the introduction zone 112 and the mixing zone 114 and the communication between the lead-out zone 116 and the mixing zone 114 are blocked. Further, the second buffer solution Buf-2 or the third buffer solution Buf-3 is changed to be injected into the auxiliary flow paths (132, 134) with respect to the flow directions opposite to each other with respect to the main flow path 110 (step S630).

In step S630, in an embodiment, gas may be injected from the injection ports 172a, 172b into the two chambers as the first pneumatic valve 172 and the second pneumatic valve 174 to form downward (toward the flow channel layer 10). The pressure causes the main flow path 110 to deform the line 110c at the communication between the lead-in area 112 and the mixing zone 114 and the junction of the lead-out area 116 and the mixing zone 114. That is, pressure is applied to the chamber (172, 174) to apply pressure to the junction of the inlet region 112 and the mixing zone 114 and the junction of the lead-out zone 116 with the mixing zone 114, thereby flattening the two chambers (172, 174). The line 110c below.

Referring again to Fig. 15, at this time, the second buffer solution Buf-2 is still injected from the injection port located at one end 132a of the first sub-flow path 132. However, the third buffer solution Buf-3 is injected from the injection port located at the other end 134b of the second auxiliary flow path 134, so that the third buffer solution Buf-3 in the second auxiliary flow path 134 and the first auxiliary flow path The fluids in 132 have different flow directions.

Here, by blocking the communication between the lead-in area 112 and the mixing area 114 and the communication between the lead-out area 116 and the mixing area 114, the cell cell-1 captured by the first trench 152 and the second trench 154 can be grasped. The cell-2 is limited to the mixing zone 114. Further, by the opposite flow direction of the fluid in the first sub-flow passage 132 and the second sub-flow passage 134, eddy currents can be generated in the mixing zone 114, so that the cells cell-1 and cell-2 are sufficiently mixed.

After the vortex is generated and mixed, the reaction state of the cells cell-1 and cell-2 in the mixing zone 114 can be observed by an instant optical observation system (step S640).

In Fig. 13, although the step S610 is performed first, and then the step S620 is performed, the order of execution is not a limitation of the present invention. For example, step S610 and step S620 may be performed simultaneously with the actual operation state, or step S620 may be performed first, and then step S610 may be performed.

Here, by designing the first trench 152 and the second trench 154 of different lengths, different ratios of cells cell-1 and cell-2 can be captured.

Taking the poisoning ability for monitoring natural killer cells as an example, a cell detecting wafer using three different first trenches 152 and second trenches 154 (hereinafter referred to as a first wafer, a second wafer, and a third wafer, respectively) ).

The first cell sample S1 is a solution having natural killer cells (NK92) as an effector cell.

The second cell sample S2 is a solution having cancer cells (K562) (as target cells).

The first buffer solution Buf-1, the second buffer solution Buf-2, and the third buffer solution Buf-3 were both treated with PBS (phosphate buffer solution).

The first wafer has a first trench 152 having a length of about 100 μm and a second trench 154 having a length of about 180 μm.

The second wafer has a first trench 152 having a length of about 200 μm and a second trench 154 having a length of about 360 μm.

The third wafer has a first trench 152 having a length of about 400 μm and a second trench 154 having a length of about 720 μm.

That is to say, the length of the groove for capturing effector cells is smaller than the length of the groove for capturing the target cells.

The width (slit gap) of the first trench 152 and the second trench 154 is about 5 μm, which width means from the side adjacent to the main flow channel 110 to the other side adjacent to the auxiliary flow path (132/134). The distance between them.

The width of the mixing zone 114 is about 560 [mu]m, which is the distance from the side adjacent the first trench 152 to the other side adjacent the second trench 154.

The width of the conduit (132c/134c) of the secondary flow passage (132/134) adjacent the groove (152/154) is about 280 μm, which width refers to from the side of the adjacent groove to the opposite side. the distance between.

In the example, the first cell sample S1 and the second cell sample S2 were injected into the main flow channel 110 using a syringe pump at a flow rate of 2 μl/h. The second buffer solution Buf-2 and the third buffer solution Buf-3 were injected into the secondary flow path (132, 134) at a flow rate of 4 μl/h using a syringe pump.

Here, the entire experimental procedure was recorded using an instant observation instant optical observation system, and the experiment was carried out under an appropriate temperature and gas mixture (about 37 ° C, 5% CO ₂ ). After the mixing reaction, the cells were stained with a cell stain (for example, PhiPhiLux) for about one hour. The lysed K562 cells were observed and counted using a fluorescence microscope.

On the first wafer, the ratio of the total engaged effector cells to the target cells (tcE/T) was calculated to be 2.1.

On the second wafer, the ratio of total engaged effector cells to target cells was calculated to be 2.3.

On the third wafer, the ratio of total engaged effector cells to target cells was calculated to be 2.3.

Moreover, the average cytotoxicity analysis obtained for the three wafers was higher than 0.6. The toxic average values obtained for the three wafers were 0.85, 0.72, and 0.63, respectively.

On the first wafer, the ratio of the effector cells to the target cells (cE/T) in the four junction states in which 1, 2, 3 or 4 effector cells are ligated on the target cell are 0.75, 0.8, 1 and 1.

On the third wafer, cE/T in the four bonding states were 0.63, 0.33, 0.75, and 0.67, respectively.

In summary, this is to provide a miniaturized, low-cost cell detection wafer that can more accurately observe the results of cell-to-cell reactions at specific ratios. This cell detection wafer is used in the screening and matching of transplanted cells, which can reduce the cost of cell detection, shorten the cell capture time, and improve the reliability of cell detection wafers, which is very competitive.

10. . . Microchannel layer

20. . . Transparent substrate

20a. . . surface

30. . . Pneumatic valve layer

40. . . template

110. . . Mainstream road

110a. . . First end

110b. . . Second end

110c. . . Pipeline

112. . . Lead-in area

114. . . Mixed area

116. . . Export area

122. . . Edge sheath flow channel

122a. . . Note entry

124. . . Intermediate flow channel

124a. . . Note entry

126. . . Edge sheath flow channel

126a. . . Note entry

132. . . First secondary flow channel

132a. . . One end

132b. . . another side

132c. . . Pipeline

134. . . Second secondary flow channel

134a. . . One end

134b. . . another side

134c. . . Pipeline

152. . . First groove

154. . . Second groove

172. . . First pneumatic valve

172a. . . Note entry

174. . . Second pneumatic valve

174a. . . Note entry

410. . . Base

410a. . . surface

410b. . . surface

412. . . Surface structure

420. . . Mask

422. . . Mask

A. . . Center wireframe

C. . . Center wireframe

B. . . Center wireframe

D. . . Center wireframe

L1. . . length

L2. . . length

H1. . . height

H2. . . height

H3. . . height

H4. . . height

H5. . . height

E1. . . Solution inlet

E2. . . Solution inlet

E3. . . Solution inlet

H1. . . depth

H2. . . thickness

S1. . . First cell sample

S2. . . Second cell sample

Buf-1. . . First buffer solution

Buf-2. . . Second buffer solution

Buf-3. . . Third buffer solution

Cell-1. . . cell

Cell-2. . . cell

Figure 1 is a schematic illustration of a cell detection wafer of one embodiment.

Fig. 2 is a schematic bottom view showing the center line frame A corresponding to Fig. 1.

Figure 3 is a schematic perspective view of the center frame B corresponding to Figure 2;

Figure 4 is a schematic illustration of a cell detection wafer of another embodiment.

Fig. 5 is a bottom view showing the structure of the center frame C corresponding to Fig. 4.

Fig. 6 is a schematic perspective view showing the center line frame D corresponding to Fig. 5.

Fig. 7 is a flow chart showing a method of manufacturing a cell detecting wafer of an embodiment.

8A, 8B to 8C are flowcharts showing a method of forming a flow path layer of an embodiment.

9A, 10A, 11A to 12A are flowcharts showing a method of forming a template of an embodiment.

Fig. 9B is a cross-sectional view taken along line I-I of Fig. 9A.

Fig. 10B is a cross-sectional view taken along line II-II of Fig. 10A.

Fig. 11B is a cross-sectional view taken along the line III-III in Fig. 11A.

Fig. 12B is a cross-sectional view taken along the line IV-IV in Fig. 12A.

Figure 13 is a flow chart showing the cell detection method of one embodiment.

Figure 14 is a schematic illustration of an embodiment of a cell capture procedure.

Figure 15 is a schematic illustration of an embodiment of performing a cell mixing procedure.

110. . . Mainstream road

110c. . . Pipeline

112. . . Lead-in area

114. . . Mixed area

116. . . Export area

132. . . First secondary flow channel

132c. . . Pipeline

134. . . Second secondary flow channel

134c. . . Pipeline

152. . . First groove

154. . . Second groove

172. . . First pneumatic valve

174. . . Second pneumatic valve

C. . . Center wireframe

D. . . Center wireframe

L1. . . length

L2. . . length

Claims (12)

A cell detecting wafer comprising: a main channel comprising a first end and a second end opposite to each other and a pipe connected between the first end and the second end; a first groove, set a side of the pipeline of the main flow channel; a second groove disposed on the other side of the pipeline of the main flow channel opposite to the first trench; a first sub-flow passage, The first trench is opposite to the other side of the main flow channel, and the side of the first auxiliary flow channel is connected to the first trench; and a second secondary flow channel is disposed on the second trench opposite to the second trench The other side of the main flow path, and the side of the second auxiliary flow path is connected to the second groove; wherein the first groove is a common pipe wall of the pipe and the first auxiliary flow path Opening to capture cells by a flow pressure difference between the main flow path and the first auxiliary flow path; and the second groove is an opening on a common pipe wall of the pipe and the second auxiliary flow path And capturing the cells by the flow velocity difference between the main flow channel and the second auxiliary flow channel. The cell detecting wafer of claim 1, further comprising: a first pneumatic valve disposed on the main channel on the same side of the first groove and the second groove; and a second pneumatic valve disposed at the The first groove and the second groove are on the main flow path of the other side of the first pneumatic valve. The cell detecting wafer of claim 1, wherein the pipeline comprises: a mixing zone, the first side and the second side of the mixing zone are respectively provided with the first groove and the second groove; , connecting the left side of the mixing zone: and a lead-out zone connecting the right side of the mixing zone. The cell detecting wafer according to claim 3, further comprising: a first pneumatic valve disposed at a communication between the lead-in area and the mixing area to control communication between the lead-in area and the mixing area; A pneumatic valve is disposed at a communication between the lead-out area and the mixing zone to control communication between the lead-out zone and the mixing zone. The cell detecting wafer of claim 1, further comprising: a sheath flow channel connected to the first end of the main channel; and an intermediate flow channel between the edge sheath flow channels, and Connected to the first end of the main channel. The cell detecting wafer of claim 1, wherein the first trench and the second trench have different lengths. A cell detecting method comprises: simultaneously injecting a first cell sample, a first buffer solution and a second cell sample into a main channel by using a sheath flow principle, wherein the main channel comprises a mixing zone and is connected to the mixing zone. a lead-in area on the left side and a lead-out area connecting the right side of the mixing area, and the first cell sample and the second cell sample flow from the lead-in area through the mixing area under the separation of the first buffer solution to a lead-out area; a second buffer solution and a third buffer solution are respectively injected into the second side flow path at a flow rate different from the flow direction of the main flow path and different from the main flow path, wherein one of the auxiliary flow channels The side of the auxiliary flow channel communicates with the upper side of the mixing zone via a first groove, and the side of the other of the secondary flow channels communicates with the lower side of the mixing zone via a second groove After the first cell sample, the first buffer solution, and the second cell sample are in the main channel, and the second buffer solution and the third buffer solution are simultaneously flowed in the sub-flow channels for a period of time, blocking the guide The communication between the zone and the mixing zone and the communication between the lead-out zone and the mixing zone, and the second buffer solution and the third buffer solution are respectively injected into the opposite flow directions of the main flow channel to the respective a secondary flow channel; and viewing the mixed zone after the secondary flow paths have reverse flow for a period of time. The cell detecting method according to claim 7, wherein the blocking step comprises: respectively blocking communication between the lead-in area and the mixing zone and communication between the lead-out zone and the mixing zone by using two pneumatic valves. The cell detection method according to claim 7, wherein the blocking step comprises: applying pressure to the junction of the introduction zone and the mixing zone, and the communication zone of the lead-out zone with the mixing zone, so that the main channel is introduced into the introduction The intersection of the zone and the mixed zone And the pipeline is deformed at the intersection of the lead-out zone and the mixing zone. The cell detecting method according to claim 7, wherein the step of separately injecting the second buffer solution and the third buffer solution in opposite flow directions with respect to the main flow channel to the auxiliary flow channels comprises: changing the first One of the two buffer solutions and the third buffer solution is in a corresponding flow direction in the secondary flow channel. A method for manufacturing a cell detection wafer, comprising: forming a first-order channel layer, wherein a surface of the channel layer is formed with a main channel, two grooves, and two secondary channels, and one side of one of the auxiliary channels passes through the holes One of the trenches is in communication with one side of the main channel, and the other of the other of the subchannels passes through the other of the trenches and the other side of the main channel opposite the side The sides communicate, and each of the grooves is an opening in one of the auxiliary flow channels and a common pipe wall of the main flow path, so that each groove is between the main flow path and the corresponding auxiliary flow path The flow rate differentially captures the cells; the flow channel layer is combined with the transparent substrate by the surface having the main flow channels, the grooves and the auxiliary flow paths; forming a pneumatic valve layer, wherein the pneumatic valve layer The surface is formed with two chambers, and the other surface of the pneumatic valve layer is formed with at least one injection port communicating with the chambers; and the flow channel layer is opposite to the main channel, the grooves, and the The other surface of the surface of the secondary flow channel and the pneumatic valve layer have the chambers Binding surface, wherein the plurality of chambers are located at the two sides of the main track corresponding to the plurality of trenches. The method of manufacturing a cell detection wafer according to claim 11, wherein the step of forming a first-order layer comprises: forming a template, wherein the template comprises a surface corresponding to the main channel, the trenches, and one of the sub-channels Structure; forming the flow channel layer by using the template; and performing a demolding process of the template and the flow channel layer to obtain the flow channel layer.