CN218435714U - Single cell transfer structure based on micro-fluidic and high-power microscopic visual identification - Google Patents

Abstract

The utility model discloses a unicellular transfer structure based on micro-fluidic and high-power little visual identification that shows, including end chip, end chip top surface rigid coupling pneumatic valve layer, pneumatic valve layer top surface rigid coupling micro-fluidic chip, pneumatic valve layer area is unanimous with end chip, micro-fluidic chip area is less than the pneumatic valve layer, micro-fluidic chip one side is the alignment state with the pneumatic valve layer, pneumatic valve layer top surface is located two gas inlet pipes of micro-fluidic chip outside fixed scarf joint, gas channel one and gas channel two are seted up to pneumatic valve in situ portion, gas channel one and gas channel two communicate two gas inlet pipes respectively, the fixed scarf joint cell liquid of micro-fluidic chip top surface advances appearance pipe, three visual identification runner is seted up respectively to the inside gas channel one that is located of micro-fluidic chip and gas channel two tops. The utility model discloses the using-way is simple and convenient, can not pollute adjacent cell, also need not to use more cell, reduces the cell consumption, also need not to use magnetic field, and is simple to use, and environmental adaptability is strong.

Description

Single cell transfer structure based on micro-fluidic and high-power microscopic visual identification

Technical Field

The utility model relates to a micro-fluidic chip technical field specifically is a unicellular transfer structure based on micro-fluidic and high-power little visual identification that shows.

Background

The micro-fluidic chip technology integrates basic operation units of sample preparation, reaction, separation, detection and the like in the chemical, biological and medical analysis process into a micron-scale chip, and automatically completes the whole analysis process. The technology has been widely researched and applied in the fields of biology, chemistry, medicine and the like.

Most of the existing finished products separate or sort cells based on a laser microdissection method and optical tweezers single cell extraction, and the principle is as follows: 1. laser microdissection principle: a mechanical arm in the laser microdissection system suspends and controls a plastic cap coated with a thermoplastic film and puts the plastic cap on a target part on a dehydrated tissue slice. And selecting target cells under the direct vision of a microscope, emitting laser pulses, and instantly heating to locally melt the EVA film. The molten EVA film penetrates into the microscopic interstitial spaces on the section and solidifies rapidly within a few milliseconds. The tissue adheres to the membrane more strongly than to the slide, so that the target cells can be selectively transferred. The laser pulses typically last for 0.5 to 5.0 milliseconds and may be repeated multiple times across the plastic cap surface, allowing for rapid isolation of large numbers of target cells. The molecules of interest can be isolated for experiments by placing plastic caps on centrifuge tubes containing buffer and transferring selected cells to the centrifuge tubes. 2. Optical tweezers single cell extraction: according to the size and the refractive index of target cells, in a light interference measurement mode, laser is gathered to form a light trap, a tiny object is restrained at the light trap under the action of light pressure, and the light beam is moved to enable the tiny object to move along with the light trap, so that the target cells can be captured and sorted. The control position of the capture beam is positioned on the microsphere between the two single fibers, the position of the microsphere determines the coupling degree between the two fibers and determines whether light is focused or deflected between the two fibers, and then the generated light signal is coupled with electrolyte particles in the microchannel, and finally the electrolyte particles are captured by electric field gradient to capture target cells. 3. Immunomagnetic cell sorting (MACS): the method for separating cells by the immunomagnetic bead method is based on the fact that cell surface antigen can be combined with specific monoclonal antibody connected with magnetic beads, in an external magnetic field, cells connected with the magnetic beads through the antibody are adsorbed and retained in the magnetic field, and cells without the surface antigen have no magnetism because the cells cannot be combined with the specific monoclonal antibody connected with the magnetic beads and do not stay in the magnetic field, so that the cells are separated. These approaches all have the following limitations:

1. adjacent cells are easy to be polluted by laser microdissection, and the integrity of the cells can be damaged in the processes of tissue fixation and laser cutting, so that the damage to nucleic acid of the cells is large, and the subsequent amplification of genetic materials is influenced; 2. these methods require the use of a relatively large number of cells, typically on the order of 10^5 to 10^6, resulting in higher sample consumption and higher requirements for killing after use of the apparatus, however, in many studies, cultured cells are not easily accessible to this order. In a small number of cell sorting, both methods face significant challenges; 3. the immunomagnetic cell sorting (MACS) requires the use of a magnetic field, and therefore, the cells need to be magnetically labeled, which is likely to cause damage to the cells, and also requires a large amount of samples, resulting in a low cell utilization rate; moreover, the cells need to be eluted after being separated under the action of a magnetic field, so that the time is consumed; and the above methods can only be applied to high-throughput cell streams.

Therefore, we propose a single cell transfer structure based on microfluidics and high-power microscopic visual recognition to solve the above problems.

SUMMERY OF THE UTILITY MODEL

An object of the utility model is to provide a unicellular transfer structure based on micro-fluidic and high power microscopic visual identification to solve the problem that provides among the above-mentioned background art.

In order to achieve the above purpose, the utility model provides a following technical scheme: a single cell transfer structure based on micro-fluidic and high-magnification microscopic vision recognition comprises a bottom chip, wherein the top surface of the bottom chip is fixedly connected with a pneumatic valve layer, the top surface of the pneumatic valve layer is fixedly connected with a micro-fluidic chip, the area of the pneumatic valve layer is consistent with that of the bottom chip, the area of the micro-fluidic chip is smaller than that of the pneumatic valve layer, and one side of the micro-fluidic chip is aligned with the pneumatic valve layer;

the utility model discloses a three visual identification flow channel, including pneumatic valve layer, gas channel two, three visual identification runner, pneumatic valve two intercommunication visual identification runner, pneumatic valve layer top surface is located two gas inlet pipes of micro-fluidic chip outside fixed scarf joint in position, the inside gas channel and the gas channel two of seting up respectively of pneumatic valve layer, gas channel one and gas channel two communicate two gas inlet pipes respectively, the fixed scarf joint cell sap of micro-fluidic chip top surface advances appearance pipe, three visual identification runner is seted up respectively to the inside gas channel one that is located of micro-fluidic chip and two tops of gas channel, gas channel is located three visual identification runner under the position rigid coupling three pneumatic valve one respectively, pneumatic valve two intercommunication visual identification runners, the visual identification runner is located pneumatic valve two and is seted up unicellular visual identification detection zone in the position between the pneumatic valve one.

Preferably, an inlet flow channel I, an inertial focusing flow channel and a single-side sheath fluid focusing main flow channel are formed in the micro-fluidic chip, the gas inlet pipe is communicated with the inlet flow channel I, the inlet flow channel is communicated with the inertial focusing flow channel, and the inertial focusing flow channel is communicated with the single-side sheath fluid focusing main flow channel.

Preferably, the top surface of the micro-fluidic chip is positioned at the end part of the unilateral sheath fluid focusing main flow channel and fixedly embedded with a waste fluid outlet, the three visual identification flow channels are communicated with the unilateral sheath fluid focusing main flow channel and are vertical to the unilateral sheath fluid focusing main flow channel, the top surface of the micro-fluidic chip is positioned at the end part of the three visual identification flow channels and fixedly connected with three single cell outlet pipes, and the visual identification flow channels are communicated with the single cell outlet pipes.

Preferably, two entrance flow channels two are formed in the micro-fluidic chip, the entrance flow channel two is parallel to the single-side sheath fluid focusing main flow channel, one of the entrance flow channel two is close to one end of the single-side sheath fluid focusing main flow channel and is vertically communicated with the sheath fluid converging flow channel, the sheath fluid converging flow channel is communicated with one end of each of two sheath fluid inlet ports, the other end of each of the sheath fluid inlet ports is communicated with the single-side sheath fluid focusing main flow channel, the other end of the entrance flow channel two is close to one end of the single-side sheath fluid focusing main flow channel and is communicated with one end of each of the sheath fluid inlet ports, the other end of each of the sheath fluid inlet ports is communicated with the single-side sheath fluid focusing main flow channel, the top surface of the micro-fluidic chip is located at one end, far away from the single-side sheath fluid focusing main flow channel, of the micro-fluidic chip is fixedly connected with two acellular culture fluid inlet pipes, and the acellular culture fluid inlet pipes are communicated with the entrance flow channel two.

Preferably, the inertial focusing flow channel is a structure in which a plurality of arc-shaped flow channels are communicated with one another, and the size of the space between the single cell visual identification detection regions is larger than the size of a single cell and smaller than the size of two cells.

Compared with the prior art, the beneficial effects of the utility model are that:

the utility model discloses the using-way is simple and convenient, can not pollute adjacent cell, also need not to use more cell, reduces the cell consumption, also need not to use magnetic field, and is simple to use, and environmental suitability is strong.

Drawings

FIG. 1 is a schematic view of the present invention;

FIG. 2 is an enlarged schematic view of the explosion structure of the present invention;

FIG. 3 is an enlarged schematic view of a sectioning structure of a microfluidic chip of the present invention;

FIG. 4 is a schematic view of a cutting structure at the position of the middle pneumatic valve layer of the present invention;

fig. 5 is a schematic view of the communication structure between the runners according to the present invention.

In the figure: 1. a bottom chip; 2. a pneumatic valve layer; 3. a microfluidic chip; 21. a gas inlet tube; 22. a first gas channel; 23. a second gas channel; 24. a first pneumatic valve; 25. a second pneumatic valve; 31. a cell sap sampling tube; 32. entering a first flow channel; 33. an inertial focusing flow channel; 34. a unilateral sheath fluid focusing main flow channel; 35. visually identifying the flow channel; 36. a single cell visual recognition detection area; 37. a single cell outlet tube; 38. a waste liquid outlet; 39. a cell-free culture solution inlet pipe; 311. entering a second flow channel; 312. a sheath fluid converging flow channel; 313. a sheath fluid inlet I; 314. and a second sheath fluid inlet.

Detailed Description

The technical solutions in the embodiments of the present invention will be described clearly and completely with reference to the accompanying drawings in the embodiments of the present invention, and it is obvious that the described embodiments are only some embodiments of the present invention, not all embodiments. Based on the embodiments in the present invention, all other embodiments obtained by a person skilled in the art without creative work belong to the protection scope of the present invention.

Example 1:

referring to fig. 1-5, the present invention provides a technical solution: a single cell transfer structure based on micro-fluidic and high-magnification microscopic vision recognition comprises a bottom chip 1, wherein the top surface of the bottom chip 1 is fixedly connected with a pneumatic valve layer 2, the top surface of the pneumatic valve layer 2 is fixedly connected with a micro-fluidic chip 3, the area of the pneumatic valve layer 2 is consistent with that of the bottom chip 1, the area of the micro-fluidic chip 3 is smaller than that of the pneumatic valve layer 2, and one side of the micro-fluidic chip 3 is aligned with the pneumatic valve layer 2;

the top surface of the pneumatic valve layer 2 is located two gas inlet pipes 21 fixedly embedded at the outer side position of the micro-fluidic chip 3, a gas channel I22 and a gas channel II 23 are arranged inside the pneumatic valve layer 2, the gas channel I22 and the gas channel II 23 are respectively communicated with the two gas inlet pipes 21, the top surface of the micro-fluidic chip 3 is fixedly embedded with the cell sap sample inlet pipe 31, the inside of the micro-fluidic chip 3 is located above the gas channel I22 and the gas channel II 23 and is respectively provided with a three visual identification flow channel 35, the gas channel I22 is located under the three visual identification flow channel 35 and is respectively fixedly connected with a three pneumatic valve I24, the pneumatic valve I24 is communicated with the visual identification flow channel 35, the gas channel II 23 is located under the three visual identification flow channel 35 and is respectively fixedly connected with a three pneumatic valve II 25, the pneumatic valve II 25 is communicated with the visual identification flow channel 35, the visual identification flow channel 35 is located between the pneumatic valve II 25 and the pneumatic valve I24 and is provided with a visual identification detection area 36.

Example 2:

referring to fig. 3 and 5, an inlet flow channel 32, an inertial focusing flow channel 33 and a main unilateral sheath fluid focusing flow channel 34 are formed in the microfluidic chip 3, the gas inlet tube 21 is connected to the inlet flow channel 32, the inlet flow channel 32 is connected to the inertial focusing flow channel 33, and the inertial focusing flow channel 33 is connected to the main unilateral sheath fluid focusing flow channel 34.

Referring to fig. 3 and 5, the top surface of the microfluidic chip 3 is located at the end of the main channel 34 for focusing the sheath fluid on one side and fixedly connected with the waste liquid outlet 38, the three visual recognition channels 35 are communicated with the main channel 34 for focusing the sheath fluid on one side, the visual recognition channels 35 are perpendicular to the main channel 34 for focusing the sheath fluid on one side, the top surface of the microfluidic chip 3 is located at the end of the three visual recognition channels 35 and fixedly connected with the three single cell outlet tubes 37, and the visual recognition channels 35 are communicated with the single cell outlet tubes 37.

Referring to fig. 3 and 5, two inlet channels two 311 are formed in the microfluidic chip 3, the inlet channels two 311 are parallel to the single-side sheath fluid focusing main channel 34, one end of one inlet channel two 311 close to the single-side sheath fluid focusing main channel 34 is vertically communicated with the sheath fluid converging channel 312, the sheath fluid converging channel 312 is communicated with one end of two sheath fluid inlets one 313, the other end of the two sheath fluid inlets one 313 is communicated with the single-side sheath fluid focusing main channel 34, one end of the other inlet channel two 311 close to the single-side sheath fluid focusing main channel 34 is communicated with one end of a sheath fluid inlet two 314, the other end of the sheath fluid inlet two 314 is communicated with the single-side sheath fluid focusing main channel 34, the top surface of the microfluidic chip 3 is located at one end of the inlet channel two 311 far from the single-side sheath fluid focusing main channel 34 and is fixedly connected with two cell-free culture fluid inlet tubes 39, the cell-free culture fluid inlet tube 39 is communicated with the inlet channel two 311, when in use, the cell sample fluid is introduced from the cell fluid inlet tube 31 through the external pump, the disorderly inertial focusing flow channel 33 is introduced, the cells are primarily focused to the right middle of the flow channel, the distance between the cells is primarily enlarged, in addition, the air of all the flow channels in the micro-fluidic chip 3 can be discharged in the step, the cell culture solution is filled in the air, meanwhile, certain air is introduced into the pneumatic valve 24 along the air channel I22 in the air inlet pipe 21, the pneumatic valve I24 is in a closed state, the acellular culture solution enters the single-side sheath solution focusing main flow channel 34 from the acellular culture solution inlet pipe 39 through the internal flow channel through the external peristaltic pump, the high-speed cell flow in the single-side sheath solution focusing main flow channel 34 is decelerated and further increased in distance through the reverse sheath solution, the single cells enter the visual identification flow channel 35, the single cells in the visual identification flow channel 35 are shot by a micro-camera which is vertical to the right upper part of the chip, after the signal is transmitted back to the single chip microcomputer, the single chip microcomputer controls the air pump to pump air into the pneumatic valve layer 2 from the second air channel 23, so that the second pneumatic valve 25 is closed, thereby single cells are trapped in the small space in the visual identification flow channel 35, after short time delay, the single chip microcomputer controls the air pump to reversely pump air in the first air channel 22, the first pneumatic valve 24 is opened again, the peristaltic pump connected with the 37 part of the single cell outlet pipe reversely runs at the moment, meanwhile, the second pneumatic valve 25 is opened, negative pressure is generated at the 37 part of the single cell outlet pipe, thereby the single cell is sucked out, and finally the purpose of transferring single cells is achieved.

Referring to fig. 3 and 5, the inertial focusing channel 33 is a structure in which a plurality of arc channels are interconnected, and due to the special design of the inertial focusing channel 33, when suspension containing cells passes through the portion, the cells therein will be arranged in a row to pass through due to focusing, and the distance is also controlled within a certain distance, so that the blockage caused by the accumulation of the cells in the main channel can be effectively prevented, the distance size of the single cell visual recognition detection area 36 is larger than the size of a single cell and smaller than the size of two cells, and a plurality of cells are prevented from entering the single cell visual recognition detection area 36.

Example 3:

the utility model discloses when using, make cell sample liquid enter from cell sap sampling tube 31 through external peristaltic pump, unordered inertia focus runner 33 that lets in, the cell is by focus on to runner positive centre and intercellular distance is drawn greatly by preliminary, in addition, this step can discharge the air of all runners in the micro-fluidic chip 3, make it be full of cell culture liquid, let in certain gas to pneumatic valve 24 along gas channel 22 in the gas inlet pipe 21 simultaneously, make pneumatic valve 24 be in the closed condition, make acellular culture liquid pass through internal flow path entering unilateral sheath liquid focus runner 34 from acellular culture liquid inlet pipe 39 through external peristaltic pump, through reverse sheath liquid, make the high-speed cell stream in unilateral sheath liquid focus runner 34 slow down, further increase the distance, make single cell get into in the visual identification runner 35, the single cell in the visual identification flow passage 35 is shot by the micro-camera which is vertical to the top of the chip, after the signal is transmitted back to the single chip machine, the single chip machine controls the air pump to pump air into the pneumatic valve layer 2 from the air passage II 23, so that the pneumatic valve II 25 is closed, and the single cell is trapped in the small space in the visual identification flow passage 35, after a short time delay, the single chip machine controls the air pump to reversely pump the air in the air passage I22, so that the pneumatic valve I24 is reopened, at the moment, the peristaltic pump connected with the single cell outlet pipe 37 reversely runs, and the pneumatic valve II 25 is opened at the same time, so that the single cell outlet pipe 37 generates negative pressure, and the single cell is sucked out, and finally the single cell is transferred. The environmental adaptability is strong.

Although embodiments of the present invention have been shown and described, it will be appreciated by those skilled in the art that changes, modifications, substitutions and alterations can be made in these embodiments without departing from the principles and spirit of the invention, the scope of which is defined in the appended claims and their equivalents.

Claims (5)

1. The utility model provides a unicellular transfer structure based on micro-fluidic and high power microscopic vision discernment, includes end chip (1), its characterized in that:

the top surface of the bottom chip (1) is fixedly connected with a pneumatic valve layer (2), the top surface of the pneumatic valve layer (2) is fixedly connected with a micro-fluidic chip (3), the area of the pneumatic valve layer (2) is consistent with that of the bottom chip (1), the area of the micro-fluidic chip (3) is smaller than that of the pneumatic valve layer (2), and one side of the micro-fluidic chip (3) is aligned with the pneumatic valve layer (2);

pneumatic valve layer (2) top surface is located two gas inlet pipe (21) of the fixed scarf joint of micro-fluidic chip (3) outside position, inside gas channel (22) and two (23) of gas channel of seting up of pneumatic valve layer (2), three visual identification runner (35) are seted up respectively to gas channel (22) and two (23) of gas channel, the fixed scarf joint cell sap of micro-fluidic chip (3) top surface advances appearance pipe (31), micro-fluidic chip (3) inside is located gas channel (22) and two (23) tops of gas channel and sets up three visual identification runner (35) respectively, gas channel (22) are located three visual identification runner (35) under the position three pneumatic valve one (24) of rigid coupling respectively, pneumatic valve one (24) intercommunication visual identification runner (35), gas channel two (23) are located three visual identification runner (35) under the position three pneumatic valve two (25) of rigid coupling respectively, two (25) of visual identification runner (35) intercommunication visual identification runner (35), visual identification runner (35) are located between two pneumatic valve (25) and the visual identification single cell detection area (36) are seted up.

2. The single-cell transfer structure based on microfluidics and high-power microscopic vision recognition according to claim 1, wherein: the micro-fluidic chip (3) is internally provided with a first inlet flow channel (32), an inertial focusing flow channel (33) and a single-side sheath fluid focusing main flow channel (34), the gas inlet pipe (21) is communicated with the first inlet flow channel (32), the first inlet flow channel (32) is communicated with the inertial focusing flow channel (33), and the inertial focusing flow channel (33) is communicated with the single-side sheath fluid focusing main flow channel (34).

3. The single-cell transfer structure based on microfluidics and high-power microscopic vision recognition according to claim 2, wherein: micro-fluidic chip (3) top surface is located the fixed scarf joint waste liquid export (38) of unilateral sheath liquid focus sprue (34) tip position, and is three vision identification runner (35) intercommunication unilateral sheath liquid focus sprue (34), vision identification runner (35) are perpendicular with unilateral sheath liquid focus sprue (34), micro-fluidic chip (3) top surface is located three unicellular outlet pipe (37) of three vision identification runner (35) tip position rigid coupling, vision identification runner (35) intercommunication unicellular outlet pipe (37).

4. The single-cell transfer structure based on microfluidics and high-power microscopic vision recognition according to claim 3, wherein: two entrance flow channels two (311) are arranged inside the micro-fluidic chip (3), the entrance flow channel two (311) is parallel to the single-side sheath fluid focusing main flow channel (34), one of the entrance flow channel two (311) is close to one end of the single-side sheath fluid focusing main flow channel (34) and is vertically communicated with the sheath fluid converging flow channel (312), the sheath fluid converging flow channel (312) is communicated with one end of a first sheath fluid inlet (313) and one end of a second sheath fluid inlet (313), the other end of the first sheath fluid inlet (313) is communicated with the single-side sheath fluid focusing main flow channel (34), the other end of the second sheath fluid inlet is communicated with the single-side sheath fluid focusing main flow channel (34), the top surface of the micro-fluidic chip (3) is positioned at one end of the entrance flow channel two (311) which is far away from the single-side sheath fluid focusing main flow channel (34) and is fixedly connected with two acellular culture fluids (39), and the acellular culture fluid inlet pipe (311) is communicated with the two entrance flow channels (39).

5. The single-cell transfer structure based on microfluidics and high-power microscopic vision recognition according to claim 2, wherein: the inertial focusing flow channel (33) is of a structure that a plurality of arc-shaped flow channels are communicated with one another, and the space size of the single cell visual identification detection area (36) is larger than the size of a single cell and smaller than the sizes of two cells.

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